Document EmjL6a0bqJKmGm67dMb31wYqR

i S- im *- * ''ili ^''QraAt:-', ' /, / 9 r ^ M i CTKBOO ASO TQZIOLOGICL APflUTAlOF \ PSCDOCTS BVOX.VP IB iE 2th,5TRTCHMBDKiaiOmcair C ACID STNT3SGI3 SJ&ITTKD BT TK KGKSAJTTO QIICAl CCKPAMY, SITBO, W. ZQ S O Sfr.t> General Ob.leotlve - A* b d e t e r '*... m ine ; 'th e 'ppootteennt t ia l cn ^ttilliy tfnd Ni S O O ~CJvJ 0/ . o r CG ' {.;'^:k<Kr ; . '-:vAx-"5`-'*' - -' ' ' .>; -.11. " ' 'T1'- ;= iS:: , o fc rth a p - x p lo r* th e p a th o e n e sis o f o c c u p a tlo a a l w o * , ad t i y">f?:>iyndpi which r s u ite d ro n eapoouro t o opesvt.io.as oonc'ood w ith 2 5 3.'th e production p f ,b , -T* deseribod in throo ( ) p roviso0 rep o rta - 7 T* - j i >; V. ,, ' v c ^ f r a a . t h l * Laboratory r y . - . , . V... r:; --i- -.. ... w ith p a r i a e n t a l work perfora d on r a t f a i t e , Adatas e t a l* d is c u s s e d ^ ;r * b b i i s tp oowpound* r e p o r te d t W * c a v e e d *CM rrv ;- . r y " 1 ^fcarm U tl* in . aen ;Thay .w a lo d o d t h a t t h e i r a v id o # fagottot b i r `i - - : >. . acnefona d e rm a titis to be tba v is ib le r& sronsa o f the Sirin to n " a c tin g taw a i t fSroa th e t e r 1er Cnd t h a t t h i s r t i p a M ;..r. : C',KV1//J:V'V--.''; "r..--.'.'.y'yy. th e fona of e p ith e lia l hyperplasia, neoondory i n f l`a m a to A r.y ani : . , vy,4 .. ^f-JflQgaartlve changes, and fin a lly , roc^neratlve processes" The technique dsaorlbed by Adara and hie eo-vorkerc, uas aaplay in AxGi^r.r- .*jw4`:.,v r . V ~- - ......,.. ., ... ... . . .. :j.. -'?V,-iV;Wi-^v>^y`f`v5ljii-ia lab...o. ratory In connection w ith another v a lu a tio n in which knoiw- n : "4- -w-- - ......... ' . . r'. ..... *.' ii-.-r_>>ir..'i.-r ' ^ a a n ware im p lied ih ti p r o s o r iW stumer and th a lr jgaan lta were i s 146 / rreproseed "^sleaeteed was therefore ragardid aa aaaSee w '*rr~zfr- -- -^vrr;- " INAriAtj*ZS-iF>^- tifateli 3>j<r*iyr- DOW 751749 \y\j; 'J<i . " : - 2- The o h ssrratlo n a o f P a rn e ll on the p o s tn a ta l developasot o f the . i- aobaceans fie n d s o f th e r a t e ap p eared to prowj.de * rewme o f e v a lu a tin g in th is xidnsl;- P e m rll dicribed ..K\ ft, 0 to day 171 ; : 'glan<J* B T . atMsd lo K l]r and dcrtsal f a t uiareesee. ^i":fc-.':. ' *',: ' B* flay 18 to day 27 * - . ' ,. i. - C. la y *7 t o day 28* Sebaceous c o ll u a rg a e and f i l l s with cebua a t the nepenae of d erm l fat Behan co lls disappear in the secretory phase 'i 8';Bflay 2288 t o day 21* " . S tr a t u s g a r a im tiv u * i s a c tiv a te d and ;'`t i & i i 8 th a deeper l a y e r s o f ,th e c e l l v a H .. :. ( '. ; j ^ r . i ? .- -. -'*r/g-/T 8',~*;.--.\?.-,r' ., .>', .a^.s--r-r.. .. t, .-*ev?v w * . . ; *>: * .*- ",,v . / . . /,..* f:>a- .' -",,i *.1* V . - - v ' ^ `7 *i** ^ i n * V * ^ * * * a--! ' than voa a toown p a tte rn In sebaceous gland devolcjw ent hereby ' p lic a tio n o f s suspected acnoEoalc M aterial In th e proper U se . p h a a i' C o f th e Cycle and p e rh a p s l e a d t a h ase es - . . Ji. **." . ... ' y: Btrtenl e t h e r re a so n * o a p p o rto d th e s e l e c t i o n o f tlw r a t a s a n e a p e r i - ' . . ' ' .**}>. ' f ' ^ siantal a c tu a l. F irst# the h a ir cycle and oonooedtant epidaerm l ohanees .ii..JLn th e r a t w r e v e i l knovn S eco n d ly , V*sebaceous gland o f th e r a t ~ a r e w e ll d e fin e d ar.d e a s i l y re o o g n ira b lo h i s t o l o g i c a l l y . T h ir d ly , th e r e 16147w ere rrm ila b le d a ta on th e e f f e c t o f ap p ly in g v a rio u s e h e n le e l au bstanoes to the epidensla o f the r a t 'Xg^Sr^^-r ?p ^ -r*t: ?~v. v-1v^r^ i thh 'i if i' hriiwinsrf|a'ii'' r`'t r i r .V* > * -..:.>' ..W...V- ,.7^.-^.:<i;..j i V * ; $lir. *'TV'---r...*fc..<oi-f- Kp.brlcen-ts?.'t.e-r.b. f ' tu r. "axu swp*ioTtst>i'iwteuri1s!-g e ii ;- ^ ,<-; ***?****' " ' :". -< -- 'r'-' ' -*-.--* - . . ... .. .1. !Ti. ..va - 3- o OW 7517'JO ' _ (In consideration o f P a rz o ll'a d c a c rip tio n o f sebaceous gland TdanfeXopaeat)* Vhon oocrpletely negative reeulta were observed In thews I t w d dool<fed to apply the ''an aabetance* In various of ib h a ir 0ytt3e*;' Xt has bece reported by one ixiveetigetcr : beta arOKor* -:'n^r.iV :the *rovthph*. - jtootJt*i*rfetig*tfir *ee af. the. opinion th a t ..., :>-v\ vr.^; 'O ' ^'.. ^ ' ': --c* * * 7 - ' ' ' - I n" . bo. naa-.ne `produced aaxiwat . .*; eatanaou# damage hen the hair vae in the telogen phaaa or re stin g * , iienco in eseond group o f experim ents th e r a te r e tre a te d In vario u s sta g e s o f th e h a ir cycle* X U " f o r p o e e o f P r e s e n t B x p erl en ta : ,;--ri.J'. 'th e " :a ffe o te 'o f j ^ B S i r i T p r W d i i h r f * ^ ^ ^ ^-r^f a knosn:aoWB<Bi*,tta tb s 'd ev alo p lsg 'seb ao eo u--a J ': w d f r r t r /ib lX lc lo o f .yoonc ra t .:'u.:^Y .7Y ..^.v^^ - . . . r.-.o - .. v ' V c - - : . > -.v . -v. . . 5:; . U . ; i . v - . - - ftf .the product subalttod a s v e lla s known .. f "'."UJ^`Vr.-r r.*=: - v-t ->r-; i aenetpeos vhen a p p lie d d a rin g ^ o c i f l c otagea o f th e h a ir c y c le , V*c*e eneffK^.-^elflewu v-To itudy iha affeote of the ropoatod applioation o f the {voduete twaatttcNl and known aono^on to tho wkln o f tamm volunteer w ith particu lar rofertnoo to the pilosebaoeoua u n it. i -'V:';vPa T-?1* Obtain any Information ro^wding the paroutaneoue tecd oity e f I r '7-;>..-. i ;- """%>" eubetanesa subnlttod, when they are applied to ra t or bumn 16148 j u .Vv r `^. /Sio i - I: - o* ' rv* K atnriala and Methods <1 A Experimental Animals? CJl , k-` U-* -% The r a t s w ere b re d in t h i s L ab o rato ry ft*'*- e a altadUX> t r a i n o b - h-4 <1 cn tain ed from Carworth Faran, Kocklond, Sew York The re s u lta n t 'V'-vv" > ` y-:" .. - * ' - .-i`- . ' ... . >iV ? " : *- '. V ' : ! l itte r s averaged 1 0 - 1 2 a n lm la . .` S*5, i . . .. ' -*'. -.-ft *. : . . .. . . - ' - . . i i f i ' l V 1' -...< 37 Weaning was c a rrie d e a t a t th e - vi-^c '~-. ^ . . . - V i ttood o f thl r e e (3 ) weeks R a ta wore s o t r t o r e d f e r a t l e a s t one ^ r _Ml _on4tUh f^o llo w in g w eaning. .^.U: : ,::1 '/ experim ental work V : .- Only s a le r a t a war used th ro u g h o u t th e .; -: B* -' Cfasnicals Saployod .f ' 1* ttichloroaniB ola ,r-y:v..'_r .Vi. i \ l '; f 2 Pentaohloroonieole . S" ' V " add K a W ia ls frocsL th e F "lfrtntser Cairo* v V -i l :: *r*Wr.0*792. W . :. : h >--... '- . ^ -: V - - " - : : :-L 7 "Jlftloiax Ib llj .; ... f % &oim'aetogahs,, a llo y e d a oohtrols ? :-v;: ep- J.>Jr--C>--;V.'.,'' ..< r ^:' The t e s t s o lu tio n s used f o r Rj p lio a tlo n t o th e s k in w are p re p a re d a s $i w eig h t I n a c e to n e s a lu t io n ax co p t i n th e c a se o f "W ringer Cake11 which was d io eo lv o d i n v a tn r . C Techniques* 1 A nlnal K xperinents A skin te s t s its ensuring apfroK inately 1 on in d isa ste r, and 6149 w ith I t s a n t e r i o r adga a t th e 'S b so ld ar-X s* * !. O t b s ^ ^ - q t ^ ^ i v ; - o f .the a d a o l mo.u sed . - . " " iaans o f a a i l e f i t r l e U p p e r, a n d 't& U 1rdeEti^6ilr:w .... . ......... .............. ... Sk* -S lrl* ~*" ` * *r+r . t.'t s r ,' i ' v' 1 ; >gg^s . ^ r V j v:`- <. * TO DERGENT OR!GfNAI DOW 75175 m5 I lo a a th a n 2 aai n lnnfrth d u rin g th e e x p e r ln s n t. ' .-.Aninala wore p o in te d d a ily w ith a f u l l y w e tted c an a l* s h a ir ^?.c\ ?* V'% ?.***r--'?.. *i.`.#*;"iv r' .--*-- ..-. *, -1 .\.*r.V>/* ,.*.-v-. * ' ***** ( f c i l * 3 ^ o type oeasl*s h a ir b rash ^ nrtftefceeher, t'C f i nal l y . * ' d a l l y < K Wrts t a t i s t thro : (3 ) : -v -w .v v -,.-^v : :. t V-"-' ^ t t t t h . a fu lly w ettad brush* v ia to r l a the . coarse o f tha eaqperlasnte, fiv e (5) f u lly v e tte d ap p licatio n s - * b ru s h met aad o each d a y . th a t o t a l dose p a r tre a tm e n t i ; V ^ fo r each substance a p p lie d was c a lc u la te d and reco rd ed . D ally - A p a in tin g o f t h e e n i a a l a was c o n tin u e d f o r a n a p p ro p ria te ly . r-'-t* y' ~V. / ^ C v * p l * a n * d le n g th . o f t i a o . (Seo gabion f a r t o t a l dose a p p lie d i n ;. x p d ja ca t'' 4 .d n raU d a. a f-.a a p o a w a ,)'-^ J b l l a i ^ t h i S f / t h e v-iO- .__ ..na^r**.-'} iudUMhlja .war' a n s e th e tia e d 1tttbh tStanabbuutUallRn*, gs Uivvena inttpraanp<er*it&o -. '` ijW'X--"' ` 'j ..'L * . - . ' *` * ... .' .'. ." ... A~^' ir v - f e iS ^ Mar* .end necropsiad. .................... - - liilllflp ii i|ftl^ ^ *-4 * -in tho iavQ aiig^iiona w ith trioblorcA aljolc aod th e Ih ln v c M :. .... pariod df varying 'duatlon" Ware 'Bpioyed^and' . .<?'*n*J ^ in i tia ta d in <ttffarcnt phases o f the r a t h air eyola* tha atidml aoeptrlnontatlon mo oorrtrolled by treatin g a l i t t e r w *? --.. i t x ,. m ta with the wahlela uaed as a solvent fo r the ebaoloal being * ' - - ;-* ... '^ appraised. O ther ocntrols ware used in d iffe re n t phases of tha to ta l sxperlnental process^ and were as follows) a . Untreated. ..... ' "' *' ~ th e afcln o f th ^ A d ^ T xmWmt^rikwA ->`*;A -*- . ** Jj ' i' j '- T."- * -< / i '. yfijaagata ** mompad f a r 20 tla e a * ' - .+'*. ' ^ ': ' ^ " DOW 751753 V!T ../'* ' ' M r - 6- 0* Brushed* the sk in o r tha o n im l wos stroked or ta-ushcd w ith 1a d ry easel* h air brush fo r $ tla e s * d* P a tro lsta a j tho sk in o f tl a n ie a l cub rubbed w ith w h its p etrolstm * ' ' ' . <f. -;-.J `i"' *.`'y_/A4" -V ';7o r.-. A t f d n t e s t * i t s wosarin g sppreudLm tely two (2 ) la c h e s i n r , ; d i s a s t e r And lo c a te d error th e c e n te r o f th o s c a p u la was u se d . The sk in a re s was p a in te d d a ily u ith oao o f th e se v e ra l M aterials under study fo r varying periods of tia s a s in d i cate d i n T ables 17 and ? The t o s t a i t s was observed a t : re<Suant In te rv a ls a n d a t tb o c e s s a tio n . o f tre a tra ao t* a : . .. . . ' :: :...... " v . e le c t r ic punoli* . tx a ise d ttssu e s v e re fix e d 0 Suewary o f F x p e ria e n ta l Procedures -- t, - . ** H tsa^aefV aflr. wJ**i T'.tf ---_v Ths d s t a i l s o f each e x p o rla e n t a r e re c o rd e d I n T ab les I VI and a a y bo sunm ariaod a s follow s* .*jrs-r vus*. S * * r 7 - v i : ^ w .- J V - : XT jti^9iaea%9ffX 6(Table 1/ Sections Asnd'Q)*' "~zfjrr-.*./e *** x - : -j,.., , trlchloranlsolo and the control substances wars applied repeatedly to tl skin of yoicv: rats* These applications 1615 ~ * * * ^ ,? te *- ,?wam m w T*_T1jv -V*' r- ' '* ^ _ -T'4*v " - * ?"y`:- *VrTr-,",-****`l{7*' and M a in ta in e d 'fo r -p e rio d o f v e ry in * d n re tle n fMnr' f t i ,4V v i r t>B,* 'r,` r r i J--rniiiU iU c iiU li'rtf l ^ a #! m m M m I *--- - ------------------ - --------- - * ------------A - A -------^ 1 U--f < - H ^ T >1^ * 4 ** .r. a --v wi. -- ---- *t -*< r . . ; -- .}xr^~,..:-rz . DOW 751754 t/.:- ?- At ^ x p c rin e n ta jfT - 8 (T ab le I I ) * V, %,Ut$tr ie h lo r a n is o lQ and th o c o n tr o l su b stan ces w ere a p p lie d i'dWJ; \r * . -* *T*"J* 'K~+r't` ' ..................................... rep eated ly to tho ekin o f youny ra ta during sp e c ific phases o f th e h a ir e y c ln . Tho f i r s t gro;.-p o f an im als v a s t r e a t e d d u rin g (3) s u e e e a i& ia nagan phaaoa an d '1!}'1te a n d group d u rin g 'V:*d 1**" " **vV'V* ' S-'t:;Vu..r'V :Vr- 5.- ' '. v.K'7- *~.t+hiWr eVe9(3 ) aae e ea a iv t a i n gen p h a se s o f th e r a t h n ir e y o le * Y I>XWp eir<iw m t #> (T ab la l i t ) . ' .-;'V ,!** trlcM oropfcencnyaco t i e a d d , p a n ta c lilo ro a n ia o le and **<ringer Coke" w ere a p p lie d re p e a te d ly to r a t a k in f o r 60 a lly ap p licatio n in an a t t e s t to determine tho e ffe c t of p ro lo n g e d ex p o su re i n anim al p r i o r to a p p lie s t i o n o f th e r. ineo.ftphawan experim enta #1 #9 In c lu siv o th a anim als wore c a re fu lly to x ic ity * i* *.<*:. 'kin':-' -- .4, . j . -. " ' I'Tiirttgtiyr.r.r.irf V lo ili^ a ^ to o v n aengsn, 'was applied rep e a ted ly ' t o ' ' " ~ 'v^ ' th e s k in O f fo u r (1) human a u b je c ta w ith a p p a re n tly norm al .'';.'v(rtdn f o r a p e r io d o f 87 days* $ Experiment f l l (Tabla 7)* C ' P a n to e lilo ro a n is o le and Z,kt$-T:ware a p p lie d r e p e a te d ly t o th a i <t a k in o f tv o (E) groups o f human s u b je c ts (to o i n each group) w ith a p p a re n tly norm al k in , f a r a p e rio d o f 59 day* 2,U ,5 j *"*f h , > ^ e a a a p p lie d to th e normal akiaaC tw e 16152 -.- A - r '.49t:^3Ty:'"!.-<'. ':- .._ . .. ,- . - r--., -.. f.. DOW 75175U [ - 8,-* ^.Y J goswary o f Findings J U f t w ttrpoated d a ily a p p lic a tio n t o th e s k in o f young, n s lo r a t s **g s o lu tio n o f t r l c b lo r o a n is o lo f o r v a ry in g p e rio d s up to 3 1 'd ay i produced n e lth o r @ro<3 o r p a th o lo g ic a l changes ini th e >-. : , ; t : sere in itia te d In ^ - . , v , ^ iS.-;1,';f;P,: '1i '*Uhw*r* t*h**e" " *n to3e' n I* " o f **" .* !'. * j t u M -i l i " * V - *?k t ' *rr,`. \ * t ** ig&Svf. * c a r r ie d o u t i n th r e e ( 3 ) # u c c e s iv e p e rio d s .-r-r.:;: . ., . .. . . .::-^r. " period o f 31..to 39 days* .. A.y.* v * _v ' ;.*.! ' _ >- v. ,, for a to tal appUea & ( r f rilta '.* w a ftts' il application to : ,v-.-y..v. <;=....-vry th ;;. e rtdh of -.r - young, m le ra ts .-.-.--. .--. of a ____ jj V : - 1 _ - ; va. - ~ ^ v i r ~ , s - s * a . _ : - r * ; -.-* .?. Vs&itv' .reTj~ rrr9 r? ' niriLn^'C ake" jfor a period of Q'dfcy* f tlls d 1* produce !^^hor':c lin ic a l or pathological ehangas in the dkin or other Vy^r] ^ 4* * pa35^iD^rybTT.(T^t 'n v . a - _k ifV SA1 . o f m l a f a t f o r poriocinzpr t o ,31 day f a i l e d i;*.v'- " p .7 ^ . . ; y ft. * 'i :*'_ / - * . 'g ra ta * o llnidal or pathologioal ebangso in tho skin or otlwr "K'' V ,jr *. V>' '.-. - .....r-a n o y sta . .- = / : ' ;:fhs.'i|p p lU a iio n o&5% o lu tio n o f 2,1^$.'t r l e h l o r o a n i s o l s to . 'r',`'` iasnan s k in , extended ov er a th ro o -o a n th p e rio d , produced no Ji ' ' ' c lin ic a l o r p a th o lo g ic a l change i n th o akin* '',J-. ;-: - |r ^ ,5 Jt s p l u t t o n ^ r ponti^ih^ ^"yoriod c f ts6 "t* 5.T** * `^--,' ` j,v;',ife m fli *..,'* * Aft .'* . a ^ft a . V ft .. A k ia j^ ; < < * ) * ? .; ' %"* tv" y_: 16153 A ... ? }''j. ' aft ir./ *_.- : , .... - 9- . 7v G* The a p p lic a tio n o f a $%a o lu tio n o f ijalowax ld U * 1 t o bom n a k in over a four-week perio d pro dueod no c lin ic a l or p a th o lo g ic al " 1,4 t h e akin* *. to ta rp re ta tlo n e ad Ornaidorat i no fo r T a ta ra In v a a tlflatten - l'I . ; s ,. _ Zt l a f a ir ly ev id en t Arca tho r e s u lta o f tte s a a sp a rlm n ta th a t n e ith e r Cutaneous chaufjw o r th s y s ts a lc e f f e c t s n o te d i n th d l n i o a l ; X hav* been reproduced m th fo u r (it) o f th e suspected m te r la ls :>-:. ;r: . o r l t h th a k n o ra acn e geos ouch a s "lialowax IODI* an d *9CLamx201h* V.-y**- ;ifc '>V -- ` 1 J ' v` ^ ' r ' ' " ' . - ondar th e aasp eriw m tal c o n d itio n s eft$>lojnsd* I n r a t a , a a e a c h a s 372 *3* t f tr l o h le r o a n t s o la and th Salowuoas o re a p p lie d o v e r a p e rio d DOW 75175,; . P p |a S f ^ a p p lie d I n 6 0 d a i l y t r e e V a r a t e v i tfaqut aqy \ / /.v XL, AfltAaeable e f f e c t* a n d 1*5 epao f iA in 'a r Cake11 w ar a p p lie d d u rin g . . " ' P - I n hnBato# a k p o sc ra t o G*i6, ga 6 f ' ^ pplleaU ona^ * 0 .spe p f .xMihr-\ 53 d a ily a p p l i c a t i o n s , l.O gtaa o f 2,lx,!rT i n 53 h ily a p p lic a tio n s , a s e l i a a 1#38 9 a f K ,b,5 tric h lo ro a n is o la i n a eerlo a o f 98 d a ily ; , ap p lisatlcn a, produced no clln ic a! or p ath o lo slo al effects* r'.- ' - r- I t i s a p p a re n t t h a t th e o iro u a s ta n c a a vfrLcb b ro u g h t b o at th e q l i n l o a l prolan in the p la n t exposure vera n o t approaahed in e ith e r the a n im i DOW 751757 i ! N. 'A df.>-t- "j;# - ... - - <55^.1;.;:. ' . . ; :;Vr B --.-"L-* *x-. -t.^ i;. ;C*-./A#v***1,, *.|t-*.*. C, * io * i - --'V V ..... - - / . .: uuuera to t h e <pjeatlena va way be d d td in to th e v e rt ;-e V ttp erin an tal approachao to ih ia p r o b i bioh should b* considered fo r . .'''.the c o n tin u a tio n f t i d a i n v e s t i roU to plrograat the r a t akin appear to have renained n alterad w ith 'v l- - V * <-* - -- 1- o o n co atratioa$ used cd tb a Manner o f ospoaure aployad, ' i ' ' ' J - r . ;* ^ o o w io ta 'o < w io trtU c rt i o f monogena a c i b a r "" w * :*t>wpnmai*. J io w r th tle s a , t v a r i e t y |f > t b e r > a n s a la a p p ra isa l ' - j B l d casi Icg& pl t o w$U ;. V V X '.< X r `T V . . : a n la a la vhlah apontatlaoualy dovalo p , or in t&ieh ther* stay *,?> , ji. :*. 'v . . . '. .- '^0'. .sV- r, r-.v ;;t ' - ' *. * '., * . U f " * bo indueod, fo llic m la r in fla m a tio a ouch oa in t dog or eat I t wmld bo v o ll to connidor ciao poclfic s ite a i n xporinental an s a la in vtdah tiw re io an abundanca a f la rg a aabtoaoiaa glanda ich a l t e i a th octomai a u d ito ry c a n ti o f th e r a b b it (n o t ib a akin o f th e a u ric le tfiioh una aaployed by Jdm a and b ia . .-<,V- oo-wjrfcoro) Theuaefulnosa o f th ia a r a t f b r iacatl0Ktiffii ' if?-??&.: is E d iJ Mn f u^tB on od*nrf m M m )nYi--ri tiirrm a ta d h ir Unfitimo? ' ^ r vl,-w- ,v' DOW 751758 troaood t o mal i t t l a ma01 I t 1 a la o p o s s ib le t h a t 5; 12 DOW 751759 ' lik e ly th a t cutaneous ab so rp tio n o f atmesons under p e r i - a a n ta l co n d itio n s s y be H a lte d * To onbance such a b so rp tio n 'KH^W^'rC*.'"! .. tb s a d d itio n o f jra rfh e ta n ts to th e a p p lie d a o la tto n sw a y bo _______ tr ia d * . or, id''' T$ * p o s s ib le 'th a t "tone Or i l l o f tho Ittcuorr $} substances ...<*..-=$ '" * ' tr im t h i 2 ,li,'x T i* e te rln la cboaon f o r t h s 1i n i t i a l s tu d y a r e " Bot acnepenio* Sovurtholees sin ce th e netboda o f a p p ra isa l produced n e g a tiv e r e s u l t s w ith known acnogans* th o s e fo u r (la) J. 5s .. . . Z. - a w e ll a s th e reo& inder o f tlw n a t e r i a l a aufcsitted., should be investigated la future studies* S t. JS" t i f i l a : : . llssepHsnili.tions /:; phase of the investLsatLon which has b e riled o a t withc.;";'V-'' '. ^y^Zy%:^0-'$orwabos a t e r i a l s and known aen sp en s sh o u ld bs re g a rd e d a s qcplora- *>r*r`***- o o iid sra tla jafobipc ia .in c p e x ^ le v el and th a t ItrrostL -, s a tir e a c tiv ity should be c a rrie d o u t s in u ltenuously in se v e ra l d ire c - tlo n s I f p r o p e a s i s t o b e mda. aiWlcif T h at new o a se s a r i s e c u r r e n tly i n t i 2,1*, 5 - s y n th e s is , dos p i t e Wte-efnl re o rg a n is a tio n o f th e p ro cess and r i g i d hygionlo p re c a u tio n s , in d ic a te a th a t aeravano a ro s t i l l beinrr ovolved and th a t tlse p o te n tia l hasard i s s t i l l present* 16157 ^ x^wyA1:s*.v--m - * > ; ' ; -V,7... ,,,. .. x r <-><-. j .-a v ~ ,-7 T ,: r T . -,,****w^wa*S8HN^ta:^;vi^,yr< ; -v ; *~i ~ 111 f ii'ii 11 i*iin*ii a n* n 'ii Hm h i i r a r ^ v w i W i s a t t r M i t i . r <^w*'.v *.v^xwe#* I V - * - ' - .,, K - .rv ^ 'ii^S i ' ,'-?*.*` w *. >*.7'. "'*!TOarpi?vr^Y,* t r ^ t ^ s^ DOW 7517U0 V* - V. \ *- *' H -13- I t eat im p erativ e, tiaerofo r , t h a t a fundam ental in v e s tig a tio n o f V'.*'".-1* *x't'r y4*,`;"*V ` . th o p a t i o janea i s o f ^chloracno^ and a d e ta i le d a p p r a is a l o f th e ma t e r i a l s ah io h gave r i s e t o th o "h i t r o Syndrome"# be c a r r ie d out Kotioda o f etuc^r m a t be developed before the m ajor o b je c tiv e s can 4iqp*j^^u^ ' I t >dllr e q u ir e `tbo o m b in o d s k ill en d ^ p ^ a n c e o f '' * r ' -t-;V:; . ; *> s,t e \ veral e ,\ .' ..... d e n tifle d is c ip lin e s to m ke eoh Methods iof Utudy and the a p p ra is a l o f th e su sp ected acnegana, p o s s ib le . The s tu d y w i l l re q u ire '/; ' , ' >. aVJ* ; t ' a lo n g term p ro a re a s y s te m a tic a lly o rg a n ise d , I n Which th e problem s aay be a ssa iled fren sev eral d ire c tio n s dLaultaneously. This sta d f Should be oonsidared in term s o f n o t lo se then a throe- to fir e - y e a r progrs. ' '-m.v. r'r**^* 7 *-*^v ' it*.'!'>/ y>%*./;.>:. :>:--,..`flie jxrogran f o r th e n e x t two (2 ) y e a rs sh o u ld in c lu d e t h e fo llo w in g . xpw lw ajtsi in high concentrations and over largo cutaneous areas to a ". v a r ie ty o f e x p e rim e n ta l an im als in c lu d in g d o g s, o a t s , r a t s , e t c . B* fie te m in o tho cutaneo u s and sy stem ic o f f e a t s o f known acnogona ^Trvr^od chem ical m a te ria ls from th e ft, h, 5-4 p ro c e ss when e d h in ie to re d . '""''*&?.'?*"*" _ - ----' ...... >- ~........ "re p e a te d ly over an extended period by In h alatio n and by o ra l feeding A v a rie ty o f experim ental s n in a ls should bo u sed . i l I 0 -* l'-u V :''^ ::.'j' i C. U tilis e the sebaceous-rich akin of the e x te rn a l au d ito ry canal t *.-*---y ........` - - .V**'*.*--v^,we1*Vo-^>.S.-,, t .:. rea fbiwd t*s' ao d* d.u...t..e..r..n..i..n..e.....t.h. a a f--f e c t o f- acoof-flsns q^.ftKib-iiraM MMWU ` fn*' }^e-rvttfve; , -,-- --------- - . - ^ > '. ><. .t iaft - D O W 7 5 7 6 'i 1U- 3*v ; 1(C'W D* C o a t t i h u m a x p a riM R ts l a which leosa acnogens and cbaaical a tn ria ls fro the procaoj aro to bo applied " ` ropeatecy l a higb ooncm tratianfl to araaa oomQ&^ fr*otadf V' .v-v.-.-.! :-; -v . ; . - - ........ r- - . aucb a a la r erdaencoo, proauricular aro as, a la noai and to v .v * o f t b o baokSorifcco activ o abanta noy ba aaed to ; .y^> j^ _ ^ in a ro a a a partnitaneou* abaorptloo ai* th* aanogm a aad onbanca }; Uhan a **tbod o f inducinn: p ilosobacsoua c h a n c o l a a n s a la oouparable h':l- ''.' ' . ....to aan l a tfcvolopad* th o c y to lo g ic an d c y to ch o e d c al a p p r a ia a l o f auob a h ic h a y bal in d u c ed by in d iv id u a l nongono h ould ba a r r ia d a l ^ t a f t l u d a t h o .ocnaidar& tioa e f llp id a , ^phoaphatwwai;. :: w`V*1ar a#trata#;'XyogDn, and k o ratln u dotaral& ad h lsto d u w d c ally . . ,jT * . / l i ' > . ' 5Vr"* I -^T .. ..... . ...... .. iV .T r-r*! V ........W.' _1' - . tha lattcrag laboratory, in ti Cepartnoat f Provaitlv o f K ediclrw , tT n iv eraity o f G ln c in n a ti* v/': ' Appro*/nd - Jtobort A. Kahoa, D irector Z9TS I ':' v r 1 -V- ' - .. . I* S*H.f m a h , 0*D*, s p t t w r , HC*, and S0t#V ?S. Tho J " . ^ B*Bponaaof R abbit Skin t o Coaxoamia il e p o r t c d t e S a v e Cmoad r-.i " * ' . 5: - . j ... - ' Aaoafora B arstitiii* X adotrlal .. t .. u-' ^ -V * ?* ' s g p s a s ^ ^ ^ ?<^ ih -..- ;V :v-V ^a:',,/ .'..J..- - . .;. " : -V. -:: . S e b a ea tt* COftnds l a th e R a t. ,=A** Jf0*mU, Biil, l9i9. .... 0.: ` V" . h fortc h e r, g0 i h e f a i r C ycles l a th A lbino B a t* ' t a a t . JRecord* 61j5, 193U,-. -: V>' ' ' ; |** .B atoher^ g*oi B e S T feo ta o f A p p U o etio n a fo trlo aa ftibotanoo* f v': ' V : ' '\> V - ^ Hontagaa W r ..to r s o c a l n o w w n io aU o a* r-;.: . ...-. v ;'.'.V j&Ka>w',':&** i _ a fc ^ t k foV n<l ila n k , K* >, A 8 la KooJit T p r th o Stu<$r o'f S k ia v ; ~ __ >,tv v. - -' ^ Jp ia ia tlfla ti'w i S a ra a to lo g y , jfoe Z9# '' DOW 7517GJ -. : ADUC X :7 - -Y ->. Ttm Vffoota o f Ropaated A pplication o f 2, 1,5 Trt.chlnrcsuu.aola, "Bslowsjc lXU*, and Control TToatnont to th a SJdJ^of Jovmg Rota, I n itia te d Poring th a 'R n t Hnth o f U fo * v: *i: '.f Swwary af. B qpartem tal Condition* a n d o tsw natlaaa .y ' ;V .A ' * fo.', -i >' ^ ihj ___________________________________ :____________ :______________________ 7 i t e r i ton a) * ixparlnnnt* * i ~ :/ 3a ; r r.1^ >* i- J\ No of , -% Aga j Start Ana Cow !*' i:r' . iXirailon p la tlo n : n - ' - Bo*f ; o f T o rti, f' ;- , ,"**4iV'-'-**-, .} Animi ` f o f 11 (m l* Hat *f \ la s t of Tast Ac; a t ; A lla~ Applica Vaoropsif Uooa ' ti ona Katerlai ' ; ' Control rats) B irth ' l.lr n m Y . (Haya) tQWl (D iri)' (n a y ) Vh : Untreated 1 1W 1-53 ^ ' t a/* if'i m V kintre a te d 1 *. * a J o' < a* lintrou tod ` 1 T 'J "* - ' 7 . '` V - " iialowax 10Q." ni r so ted - Aootono 1 i.' * - l l *' a- '' 1 ' - 3 ' id u . 7 . 8 (5i by v t . In : aootono) 2,li,S trlcULoroanlaola (5f ty r tV In aeatono)> .... "llalovax lOCd* 2, 1,5 T richlor0 ; * ; . f !; fi1 Unir*tod Aaatona 1 . ; . i.irir i.nVi - 1 1 1 *K \-.ir1 . .;, " 3 ' 3 1 .________ ID 10 31 3l n '' n r.18 :' 36 i-V .;-7 ' -t, <j a. :5 i V ;^ W ffi - 8 8 29 29 anisla r 1 f' *Kaiowax '1 ,l,5 T richloro- an is la * ' %.S , Untreated Acatena Vr..V**<; ,.* '' 1 1 1 1. QT ; - .^v(*-P; . . t 7v :,.r .'`'Jv'-'-' r ?: V '^i :3 31 V - -- 7 7 a-l 7 __________ 31 3l .<& . r i f,.T,V!v'*<-". -- 31.. V ; ..:fg . . . . , C' ff . ; ^1 . 29 29 25 4 apgt.-Tv ' Fg-,'*,V r . r P j ,;' *jj-V l it o t a l flta*- '< ltf m l^ ." | Appllad n a t> 1 r ' Wyt!? `i- W i !'f itiw Cram Iflf _ i ' m ? \* o.1.oau im m t \ - ' 1 J i 7 Jr *?. * Jj .,. a . ' ETfoa h m . VI 0, 08li t 1 .<r ). M - # t; * 0.200 0.208 Neos Nona r . 'v i Nona i 'C M ` * av. * Ar. ~ m ^____i___t^ v a 0.252 -- ^TY 042$2.;_ Nom '.if t-, > K B . "<&VH i V -iii:: - s ' ? ; .............. .............. "7 -7 7 7 S 7 mm iM]y- ..o:;, IM: i ! yi MJ :<i' I7'-: imi:;} 16164 V l '3if1ijIi OQW MJ : .">>. fcj' %o. % .V/-i- : Tho . .vic":-?r -8 EfXeoto o t'jto p v ito d Kjw'foLi.n =?'" Ampliotlona ocf 2t U,? !!*sIh JrichJJac J^t*ohloroaninol^n(^f>idO (tB r Cota* io Ow . ;.>0! i :"- iXinnary d f DqjBrinqntal Condilan ai V v:v - - ' j . / ;v. -fm; WVtkfcrrimnt' H?\-\ ? ' VS. :>.'f nd Controla ir-': no. ot Anim i 'A ppllf- -;i JftzsL Mm!ph'r. /: Hi V | ajli.S TrtdOofo571!ph^n.niaxj,ot-to aoic^ mocilc Add. ngra 1 j 1/ i ; :V ;` ji: ;f !Pm tachloroanlaolf * "/M agar Cblee* " ii ( 0M dlooolvod `fa in loo pia ; ';j_ d latlllad uator) ' - J . j U :;r ^ r .^ ; \ & h'^7.!-7n-- l-i,: ; ; i t : - ' sm. . ':r:V 4 '| T "c'.-".t;.:.m:.':`iA-/si)j -60; ' fi ^ j iv-i; :;iir a ^ 1 sfeL^. .'IHi ! !h ` f i -i ! );(.i jlj.iwi l ;<: I1 V'tI il1 J:t'. ir .f :f :l{v |! :1- pli : t 1 " i.fe fl: OOW 751767 ; : !' f il ' i i l )! f . ;i 3 3 .p l !ii q f lavi rmt '"<>:V! t x m trvf t MJV .tfHi DOW;75W y K:-..r t- .' Jlb Sffixrta af.Rcpoatod Appli,catione ^ IPfepchlcyoftnl^oW'r.^^ rle^lar^rii*t>toygt.U>i <4.4 j ; Hi; f^ ^ M' '' -: ^ '^^ f: r; ivis !'v i ,;>!? ;< Kr-*:.' f.fJ` ; !I .;!:>!rv>W. .I1.J(rarm^ijo'!) j-. <? i | . . ' i - ' / . r _ . i V m * ^ : ) .: t m i i D O W 749666 1957 "P ath o g en esis of C hem ically Induced A cne" The objectiv es of th is study a r e : (1), to c o m p a re q u an titativ ely the re s p o n s e s of h u m an skin w ith ra b b it skin to c h e m ic a l a c n e ig e n s, and (2), to study in detail the stru c tu ra l pathophysiologic and bactriologie changes w hich o ccu r during the evolution of ex p erim en tally induced hum an d ise a se . a. The subjects w ill be healthy adult m ale p ris o n e rs - - inm ates of the P h iladelphia County P ris o n at H olm esburg, P h ilad elp h ia. b. acneigens - 2 su b stan ces of d iffere n t potency w ill be evaluated: 1. h e x a c h lo rd ip h # n y l e th e r 2. tetrach lo ro d ib en zo l dioxane c. p ro c e d u re s : - The initial studies w ill be c a rrie d out on the back a p p ly in g . 05 m l. of th e a n e ig e n in a s u ita b le s o lv e n t to a 2" s q u a re of sk in . The a re a w ill be covered w ith a sq u are of gauze held in place by strip s of adhesive. The applications will be m ade once daily. The principle variables w ill be concen tra tio n and total num ber of application's. The aim h e re w ill be to e sta b lish th re s h old concentrations for v ario u s n um b ers of exposure. This w ill re q u ire , of co u rse, a considerable num ber.pf range finding ex p erim en ts. Both aneigens w ill be applied to the skin of the sam e su b ject in the final te s ts . M oreover, te sts of liv e r function w ill be included as follow s: SGOT, BSP. , B ilirubin, Alkaline P hosphatase, and Cephalin Flocculation. -1- 16169 996PI A\tt<l f d. p ath o g en esis - The skin w ill be e x a m in e d fo r clin ica l sig n s of change. At appropriate tim es, punch biopsies will be rem oved and stained as follows: H it E , O r c e in - G ie m s a , H ale, M a llo ry an d A lk alin e P h o s p h a ta s e . T h ese p ro c e d u re s w ill give detailed in fo rm atio n of the effect on v a sc u la tu re , the sy n th esis of m u co p o ly sacch arid es, collagen and elastin , and the g en eral a r c h i tectu re. Enzym e content w ill be evaluated by histocham ical p ro ced u res for A T P 'a se and succinic dehydrogenase. As re g a rd s th ree dim ensional view s of th e le sio n s, the ep ith eliu m w ill be s e p a ra te d by so d iu m b ro m id e and m o u n ted in tact as a sheet. e. bacterio lo g y - C om edones w ill be re m o v e d at v ario u s stag es and the contents sam pled qualitatively and quantitatively for bacterial contents. F or com parison, a sim ilar study will be conducted on com edones derived fro m patients w ith acne vulgaris. L esions which becom e inflam m ed will be sam pled for th eir b a c it e r i a l c o n t e n t , e i t h e r b y b i o p s y o r by a s p i r a t i n g f lu i d . f. horny la y e r - The h o rn y la y e r fro m tre a te d site s w ill be s e p a ra te d as a sheet and studied stru ctu rally , physically and chem ically. V alues for norm al h o rn y la y e r have been obtained. We w ish to know w h eth er h y p e rk e ra to tic sa m p les re p re se n t an in c re ase in m itotic activity or w hether the ep id erm is is pathologically a lte re d at the sam e tim e. The rate of d esq u am atio n of the horny la y e r w ill be estim ated by m easu rin g the tim e req u ire d for the d isap p earan ce of a flu o resce n t dye which has been a p p ro p ria tely used to stain the above horny la y e r. -2- C LO VER LABO RATO RIE S. INC. P. O. BOX 7S8S P H IL A D E L P H IA I. P E N N S Y L V A N IA EVcnonccN 6 *3 4 5 8 D ecem ber 21, 1964 To: Dr. V. K. Rowe Biochem ical R esearch L aboratory 1701 B uilding The Dow C hem ical Company M idland, M ichigan F r o m A lb e rt M. K lig m an , M. D. Clover L aboratories, Inc. , P. O. Box 7686 Philadelphia, Pa. F o r: Study w ith A cnnigenic su b stan ces Help and O verhead at P riso n Subjects: A pprox. 100, av erag e cost @ $15.00 per subject = B acterio lo g y Histology and H istochem istry M iscellaneous: M aterials, Photos, H orm ones, etc. Total U n iv ersity O v erh ead 20% TOTAL $2, 000. 00 1, 5 0 0 . 00 1,000.00 1,000.00 500.00 $6,000.00 $1,200.00 7 ,2 0 0 .0 0 G ran t to be m ade out to T ru s te e s of The U n iv e rsity of P e n n sy lv an ia 9996U 16171 D O W 7496y g. P re v e n tio n an d T r e a tm e n t - It is know n th a t x - r a y s , e s tr o g e n s , and k erato ly tic agents a re helpful in acne v u lg a ris. The beneficial effects of th ese m odalities will be evaluated. h. n tiscellan eo u s studies - L im ited a tte m p ts w ill be m ade to evaluate the effect of age, sex, and body a re a on the developm ent of e x p erim en tal acne. Respectfully subm itted, AM K/a A lb e rt M. K ligm an, M. D, 3- - 16172 0 I - DOW 471 026 JP E C T R U M that have had implants. A spokesman for the Department of Agriculture told Environment t h a t r e s i d u e s of DES ar e no t o rdi na r il y found in animals with implants because the levels of DES are below the sen s itiv it y of the an aly tica l techniques used, but that there were undoubtedly some re s id u e s . The spokesman als o explained that the small farmer will be h ur t by a p ar ti al ban on DES b e c a u s e w h e r e a s large farm operations can simply switch to implants, the small farmer does not have the equipment needed for implantation. A SHARP INCREASE in the i n c i d e n c e of a particular type of liver cancer in North Vietnam may be related to herbicide spraying programs, according to Dr. Ton That Tung of the University of Hanoi. Dr. George Perera who visited Dr. Tung on behalf of the American Friends Service Committee told Environment that Dr. Tung had s ee n a greater than five-fold increase in liver hepatoma cases between the periods 1955-61 and 1962-66. Perera said that Dr. Tung su spects that dioxin, a contaminant of the herbicide 2 . 4 . 5 - T which causes birth defects, can cause liver cancer. Human liver samples s u s p e c t e d of containing small amounts of dioxin have been sent to the U . S . for a n a ly sis, but techniques to test for low quantities of the chemical are still in the developmental stage. Although no herbicidespraying was conducted over North Vietnam, Dr. Tung claim s that there is a gre at deal o i movement between North and South, so that his patients in Hanoi may have been affected by 2 ,4 ,5 -T in the South. 2 . 4 . 5 - T is again in the courts. A federal district court judge in Arkansas issued a preliminary injunction June 23 a g a i n s t the Environmental Protection Agency (EPA) plan to hold public hearings concerning the fate of the registration of the herbicide for use on rice c r o p s . In a c o s e Drought by Dow Chemical Company, a manufacturer of the herbicide, the judge ruled that William Ruckclshaus, a d m i n i s t r a t o r of i'.PA, must make a c l c u r r u i d e c i s i o n on whether or not to ban 2 , 4 , 5 - T for the specified use boloro hearings are hold. Last summer Ruc ke ls h au s f<mnd t ha t foil-- wing n----------- -----------------r n i f ii--witfarT -iii^ 24 the report of an advisory committee on 2,4,5-T, he had insufficient evidence to make a final decision and .dered a hearing. A spokesman for EPA e x p l a i n e d to Environment t h a t t he administrator's order was sufficient, and said that a notice of appeal had been filed. When asked if Dow was using 2 , 4 , 5-T as a test c a s e , a spokesman for the company told Environment that "We've got to stop this ridiculous mess somewhere; who else is going to stand up in a case like this?" A CORRELATION may e x i s t b e t w e e n a i r 3 pollution and what is known as the sudden * infant death syndrome, in which an apparently healthy infant is found dead in o o i t s c r i b . Prel iminary a n a l y s e s made by Dr. *""* Bertram Carnow of the University of Illinois ^ Medical School showed that when sulfur ^ dioxide and particulate air levels were high, there was an associated rise in the incidence of sudden infant deaths. However, Dr. Carnow told Environment that further analyses have yet to back up the preliminary work. Dr. Carnow theorizes that the sudden deaths are actually due to a constriction of the throat opening caused by acute tracheobronchitis, which is aggravated by high air pollution levels. A NEW PRICING regulation by the Federal Power C o m m i s s i o n (FPC) may spur e x pl o r a t i on and development of new natural gas resources in the U .S . The FPC, which now regula tes gas prices, will allow gas producers to sell in the interstate market without being restricted to current area price ceilings at the wellhead, reports Coal N ew s, August 4. However, the new ruling applies only to gas from r e s e r v e s d i s co v er ed after April 6 or gas diverted from intras tate m arkets. The Commission retained the right to ensure that gas prices will remain "in the public interest." Natural g a s , one of the c lea n est fuels, has been in short supply, a_fact which Coal News says is attributable to "restrictive FPC price regulation of gas at the w ellhead." A NEW WAY of cont rol li ng m o s q u i t o e s i s being studied by Clyde Umphlett of Clemson University. A report in BioScience, July, states that Umphlett is working with a microscopic fungus that attacks and kills mosquito larvae. If the fungus is found to be without unacceptable side effects, it could eliminate the need for spraying p esticid es or oil on mosquito breeding grounds. The fungus is extremely effective on several s p e c i e s of i iuvtronfTifcfil. Vol M. ho 7 19 JO DOW 381612 THE DOW CHEMICAL COMPANY MIDLAND June 21, 1968 H. Ferguson cc : M. G Wiltse J. R. Ridner C. A. Bryant 0. H. Hammer k. J. Watson W. J. McCoy RECEIVED JUN 2 1 1968 . BJOPRODUCTS DEPT.*I Your letter to Andy Watson has beeu passed to me for ans wering. I Enclosed are several pieces of literature dealing with the toxicological aspects of silvex. A good summary of the effects of silvex ester and salts on fish has been assembled by Hammer. The summary by Mullison lists several references that we could supply if you feel this is necessary. The size of the market you mention in your letter was a bit surprising but perhaps this includes some other areas than New Zealand. I have sent a copy of this letter to W. J. McCoy. He will advise you on his thoughts regarding tne world market for silvex. Let us know if we can be of further help. Plant Science Research and Development Agricultural Products Center bk end 16174 2008 . (jO s r t S 3 / / f S 8 ^ NATIONAL AGRICULTURAL CHEMICALS ASSOCIATION '.`."TV. . .*..4: " i TO: Mr. H itc h n e r O ffice Correspondence Vi i: ' t PROM: Jack D rees s en //w ,J SUBJECT: J u l y 28 C o n fe re n c e w ith USDAf^annSs 2 ,4 -D and 2,4 5-T on g r a z i n g r&Qd n the use of v r , USDA a r r a n g e d f o r a J o i n t c o n f e r e n c e b e t w e e n on J u ly 28 in th e o f f i c e o f Mr. R. S. Roe, D i r e c t ^ , O Od t h e USDA of B i o l o g i c a l and P h y s i c a l S c i e n c e s , PDA. A t t e n d i n g t h w ere^ i v ' ' ?*.' V,\v ;r .-i' 1 *.y -V- . v -J ; ' V' H. L . H a l l e r - A g r i c u l t u r a l R e s e a rc h S e r v i c e , USDA W. B . E n n i s , - J r . - Weed S e c t i o n s , USDA W. C. Shaw - Weed S e c t i o n s , USDA D. L. K lingm an, Weed S e c ti o n s USDA J . T. Coyne - P e s t i c i d e R e g u la tio n S e c t i o n , USDA E. A. W alker - P e s t i c i d e R e g u la ti o n S e c t i o n , USDA W. L ig o n - P e s t i c i d e R e g u la tio n S e c t i o n , USDA J . Cummings - P e s t i c i d e R e g u la tio n S e c t i o n , USDA R. S . Roe - PDA A. J . Lehman - FDA P . V orhes - FDA P . J . M cF arland - FDA J . A. Noone - NAC J . D re e s s e n - NAC CO O -3 y j \ c0n0 The w r i t e r had m et w ith*U r* W. B. E n n is , J r . , i n a d v an c e o f th e m e e tin g and t o g e t h e r we w orked o u t a s u g g e s te d ag en d a w h ic h i n t u r n was fo rw a rd e d to a l l p a r t i c i p a n t s by D r. H a l l e r who p r e s id e d a t th e co n feren ce. The fo llo w in g i s th e su g g ested agenda as developed by Ennis and m yself. 1. Purpose o f M eeting - To dete rm in e th e in fo r m a tio n needed jto con - t in u e recom m endations on th e u s e ~ b f~ 274- D a n d " 2 ,4 ,5 "T on p a s tu r e s and r a n g e la n d s . ........ H. L . H a lle r (5 m in.) 2. S ta tu s o f r e g i s tr a ti o n o f 2 ,4 -D and 2 ,4 ,5 - T on g ra z in g la n d s . J . T . Coyne, PFCD, ARS (5 m in.) 3 . What I n d u s tr y i s d o in g to comply w ith M ille r Amendment w ith re fe re n c e to use o f 2,4-U and 2 ,4 5-T on p a s tu re s and ra n g e la n d s. J a c k D r e e s s e n , NACA (5 m i n . ) li. N e c e s s i t y f o r c o n t i n u i n g u s e s o f 2 , 4 - D . D. L. Klingman,ARS, (5 m in .) A n a ly sis o f e x is tin g d a ta on r e s id u e s o f 2 ,4 -D , 2 ,4 ,5 -T and MCPA when u se d on g r a z i n g l a n d s . J . T . C oyne, PPCD, ARS (5 m in .) ?" 161 t2 6. A nalysis f e x is tin g d a ta on th e pharm acology of 2,i|-D and 2 ,i* ,5 -T . , x A. J . Lehman, FDA (5 m in .) t. 7. D iscussion of questio n s ra is e d concerning r e g is tra tio n . (a ) On June 9 1955 Mr G. B. Lynn w r o te t o M r. W. B. R a n k in and th e fo llo w in g "excerpts are tak en from th a t l e t t e r : " I am w r i t i n g t o be s u r e t h a t Mr. S u n d e rla n d and I h a v e a c le a r understanding of the conclusions reached w ith re s p e c t to 2,^-D and th e re la te d compounds in our m eet- ing w ith you on June 3 . . - .; . " r . r . ^ "W e_ ex p lo red _ at some l e n g t h th e p ro b le m o f r e s i d u e s o f 2,ij.-D ''and 2 , ij., T _snd.`M CPin~ r t~ Xt~w s'"o r""`p i n i o n t h a t "the b u lk o f th e s p ra y in g o n r a n g e l and_and,,pasr tu r e s was c a r r ie d _ o u t_ in _ th e _ sp rin g 'o r" e a r l y su mmer. A O 'Stock g razin g _ o n _ su ch la n d s joe e jn o t s la u g h te re d f r "ij.'~to''6 m onths l a t e r . A h ig h p e r c e n t a g e o f ""this s to c k "Is" l o t ' f e d a f t e r th e y a r e rem oved fro m th e ra n g e and CO -3 b e fo re th e y a r e .s la u g h te r e d . Thus i t d id n o t seem CD lik e ly to us th a t e a rly tra n s ie n t exposure to these OO chem icals would c o n s titu te a re s id u e problem in sto ck s la u g h te re d s e v e ra l m onths l a t e r . You took t h i s CO m atter up w ith your co lleag u es and gave us th e opin io n t h a t i t ap p e a re d th e re _ w a s no p o s s i b i l i t y o f ...a..r e s id u e problem under""these c irc u m sta n ce s and th a t you would n o t ask "u s to o b ta in d a ta on t h i s p a r t i c u l a r p ro b le m ." Mr. R ankin r e p lie d to Mr. L y n n 's l e t t e r on June 1$ as follow s: " T h is a c k n o w le d g es r e c e i p t o f y o u r l e t t e r o f Ju n e 9 1955 which r e f le c ts a c c u ra te ly our T inderstanding o f th e q u e stio n s d isc u sse d a t our co n feren ce on June 3 . "In rea d in g th e l e t t e r I r e a liz e th a t th e re is one a d d i t i o n a l comment w hich need3 bo b e made a b o u t th e problem o f re s id u e s of h e rb ic id e s on fo ra g e . Jlf^ h e rb i c id e re s id u e s a re p re s e n t .on fo ra g e , o th e r th a n a l f a l f a , w hich moves i n . i n t e r s t a t e commerce, ,,on b a le d h a y " f o r " exam ple',"'t o l e r a n c e s o r ex em p tio n s w ould n eed to be e s ta b lis h e d for__them ." ...... .......... The s l t u a t i o n c o n c e r n i n g 2,1}.-D,. 2 ,i* ,5 -T jand MCPA a p p li c a tio n s where b e e f c a ttle 'm a y be g ra z in g have hot_changed^ s i n c e 1955 i . e T / th e 2,!j.-D," 2 , i |,5 - T and MCPA'ar'e n o rm a lf y applied e a rly in the grazing season, a p p lic a tio n ra te s a r e 'r e l a t i v e l y . low and g e n e ra lly o n ly one a p p lic a tio n p e r y e a r i s made. I f th e same c o n c lu sio n s b e 'd r a w n 'to d a y 'a s in" 1955 th e n th e re i s no p o s s i b i l i t y o f a re s id u e problem under th ese circum stances and recom m endations oan be con tin u e d . , (b) How much and w h at k in d o f d a t a a r e n e e d e d t o d e te r m in e w hether th e re a re re s id u e s in m ilk? 16176 3 ( c ) How s e n s i t i v e a m e th o d i s r e q u i r e d i n r u n n i n g r e s i d u e s tu d ie s on m ilk? W ill d a ta o b ta in e d by chem ical, b io - "'.i.iir'-' l o g i c a l a n d r a d i o l o g i c a l m e th o d s b e e v a l u a t e d o n a n e q u a l ' )> b asis? I n a l e t t e r d a te d A p r i l 1 2 , 1955* from W. B. R a n k in t o Mr. L. S. H ltc h n e r th e fo llo w in g s ta te m e n ts w ere made: "The t o x i c i t y d a ta on 2,lj.-D w ould n o t i n o u r o p in io n ju s tif y th e estab lish m en t o f a to le ra n c e h ig h er th an 5 p a rts p er m illio n fo r th is h erb ic id e. v: "The m e th o d u s e d t o d e te r m in e r e s i d u e s o f 2*lj.-D s h o u ld d e te c t r e s id u e s o f 0 .5 ppm and a t a le v e l o f 5ppm sh o u ld b e a c c u r a t e t o p l u s o r m in u s _ 0 .5 p p m .................. v O (d) Can d a ta a c c u m u la te d on r e s id u e s fro m one e s t e r b e to o th e r e s t e r s ? One s a l t t o a n o th e r? OW 376844 (e) I f re sid u e s are found, are th e re adequate p h arm aco lo g ical d a ta to e s ta b lis h a to le ra n c e f o r m ilk? (f) I f re s id u e s a re found to o ccu r in m ilk from an im als g ra z in g tre a te d fo rag e and i t i s found th a t th e se re s id u e s a re no lo n g er p re v a le n t w ith in x number o f days a f te r tre a tm e n t w ill r e g is tr a tio n be co n tin u ed by in c lu d in g on th e la b e l a s ta te m e n t t o th e e f f e c t "Do n o t a llo w m ilk a n im a ls t o g r a z e o n p a s t u r e s t r e a t e d w i t h 2 ,4 - D w i t h i n ________________ days a fte r tre a tm e n t." 8. `Sum m arization The v a rio u s ite m s on th e agenda w ere d is c u s s e d and th e fo llo w in g a re my im p r e s s io n s a s t o s ta te m e n ts m ade b y v a r io u s p e r s o n s a t t e n d i n g th e conference. 1 . D r, H a lle r m erely ex p ressed a d e s ire to tr y to develop w hat is ,lj.,5-Tr e q u i r e d f o r t h e D e p a r tm e n t o f A g r i c u l t u r e t o c o n t i n u e re c o m m e n d in g th e u s e o f 2,1*-D an d 2 on p a stu re s and ran g e la n d s. 2 . M r. Coyne s t a t e d t h a t th e s t a t u s o f r e g i s t r a t i o n o f 2,1^-D and 2,lj.,5**T o n g r a z in g la n d s h a s n o t c h a n g e d a s f a r a s h i s s e c t i o n w as c o n c e rn e d . He in d ic a te d th a t l e t t e r s w hich h av e b e e n s e n t to r e g i s t r a n t s i n r e f e r e n c e to c l e a r i n g th e u s e o f 2,ij.-D on ra n g e and p a stu re la n d s had been m ailed to p u t th e In d u stry on n o tic e th a t such u ses had n o t been c le a re d in th e S e c tio n 's o p in io n b u t th a t r e g i s t r a t i o n s w ere n o t b e in g c a n c e lle d . I t was h is o p in io n t h a t th e b a iic p ro d u c e rs ' a c tio n in rem oving th e u se o f 2 ,^-D on ra n g e and p a s tu r e la n d s had b e e n done a t th e I n d u s t r y 's own v o l i t i o n . He w ent on t o s t a t e t h a t_ t h a u s e o f 2.U-D fg g --ra n g e and p a s .tu r f t- la n d s would c o n tin u e t o be r e g ia t'era d Z u n t^ '^ o m a -. o th e r c^qurse' of ac tio n i s I n d i c a t e d b y a d m i n i s t r a t i v e s u p e r i o r s and V ta te ^ '" t& a t <J S lti< ^ d ..t h e In d u s t r y ' memb e r s ' ^ ea^ b m lt^ aT ^ ia b el'^ 'b e i^ in ig th e s e - u s e s ,^ i t- - w o u l d ' be a c c e p ted." A ttached as e x h ib it 1 is a copy of a l e t t e r to one o f our memBer co m p an ies s i m i l a r to th o s e t h a t have b e e n s e n t to o t h e r r e g i s t r a n t s o f 2,lj.-D i n r e f e r e n c e to t h i s p ro b le m . 1617/ I DOW376845 - 4- 3 . I commented t h a t th e In d u s try rem oved th e u se o f 2 ,4 -3 on p a stu re and range lan d s from th e ir la b e ls based on l e t t e r s th e re g i s t r a n t s h a d r e c e i v e d from TTSDA and w h ic h w ere commented oh by Mr. Coyne. I s ta te d th a t I t was th e r e g i s t r a n t 's o p in io n th a t th e co st of c o lle c tin g data to c le a r th e suggested uses under the M iller Amendment w ere n o t j u s t i f i e d on th e p a r t o f I n d u s tr y b a s e d on th e expected co st of c o lle c tin g such d a ta v ersus th e expected p r o f it from the continued sa le o f 2 ,4 -3 fo r such u s e s . These statem en ts w ere made b a se d on d is c u s s io n s a t a m e e tin g o f th e H e rb ic id e T ech n ic a l Subcom m ittee l a s t f a l l and te le p h o n e c o n v e rsa tio n s w ith members o f th e Subcom m ittee p r i o r to th e c o n fe re n c e . 4 . D r. Kllngman p re se n te d a v ery c o n v in cin g c a s e ,,, p o in tin g ..o u t th e n e c e M i ty .'/f o r 'jth e 'c o n tin u e d u s e * j5 f " 2 - 4 3 and 2 ,4 > 5 -T on~ g r a z i n g la n d s . A copy .o f Dr .^K lingm ah's sta te m e n t i s a tta c h e d as E x h ib it 2. D r. K lin g n ia n 's s ta te m e n t b ro u g h t t o t h e a t t e n t i o n 'o f FDA t h e n e c e s s ity fo r continuing the use o f 2 ,4 -3 on p a stu re lan d s. 5 . Mr. Coyne r e f e r r e d t h i s ite m to D r. W alker and o th e r s o f h i s s t a f f who re p o rte d " t h a t th e y h ad I n s u f f i c i e n t d a t a t o c o n c lu s iv e ly d e te r m in e w h e th e r 2 ,4 -3 2 ,4 5 -T and MCFA le a v e r e s i d u e s when u s e d on grazing lan d s. Ih e y .a lso s ta te d th a t they d id not have conclu siv e d a ta as to w hether any o f th e se p ro d u cts were tra n s m itte d to meat and/or m ilk, 6 . D r. Lehman s ta te d t h a t i f r e s id u e s o f 2 ,4 -3 a n d /o r 2 ,4 5 -T o ccu r in meat o r m ilk th a t f u r th e r p h arm aco lo g ical s tu d ie s would have to be conducted w ith th ese h e rb ic id e s . I t was p o in te d out by r e p r e s e n t a t i v e s o f FDA t h a t i f r e s i d u e s o f any c h e m ic a l o r i t s m e t a b o l i t e s a re p re s e n t, th a t pharm acology o f th e se end p ro d u c ts would have to b e d e t e r m i n e d b e f o r e FDA c o u l d p a s s o n t h e i r s a f e t y . 7. The v a rio u s to p ic s l i s t e d u n d e r ite m 7 w ere co v ered d u rin g th e course of the conference and d e f in ite answ ers were n o t o b tain ed in a ll cases. ta ) I t was g e n e ra lly agreed th a t th e s i t u a t i o n c o n ce rn in g 2 ,4 -3 , 2 ,4 5 -T and MCPA a p p l i c a t i o n s w h ere b e e f c a t t l e may b e g r a z i n g h av e n o t ch an g ed s i n c e 195>5 ( r e f e r t o t h i s ite m i n t h e above a g e n d a ) . However, based on p rev io u s sta te m e n ts, th e P e s tic id e R e g u la tio n S ection had continuously accepted tn ese chem icals fo r th is use and c o u ld s e e no r e a s o n why su c h r e g i s t r a t i o n s h o u ld n o t b e c o n ti n u e d . ( b ) ' T h is q u e s tio n was n o t a d e q u a te ly an sw ered . ( c ) No s p e c i f i c a n s w e r w as g i v e n t o t h e q u e s t i o n r a i s e d i n t h i s i t e m . FDA i n d i c a t e d t h a t t h e n a t u r e o f r e s i d u e s , p h a r m a c o l o g y o f r e s id u a l m a te r ia l and many o th e r f a c to r s w ould have to be c o n sid e re d b e fo re th e s e n s i t i v i t y o f a m ethod c o u ld be p a sse d upon. (d ) FDA i n d i c a t e d t h a t th e y w ere o f t h e o p in io n t h a t t h e v a r i o u s e s te r s and amine s a l t s sh o u ld be c o n sid e re d as d i f f e r e n t c h em ic als -and th a t i f d a ta o r pharm acology on re sid u e s i s accum ulated f o r one or more e s t e r s enough work sh o u ld b e done to in d ic a te t h a t t h i s same d ata could be tra n s la te d to o th er fo rm u la tio n s. In d ic a tio n s are t h a t FDA and th e P e s t i c i d e R e g u la tio n S e c ti o n w ould a c c e p t a l i m i t e d \ J. am ount o f d a ta on some e s t e r s and am ine s a l t s i f I t can be shown r th a t th e ir p ro p e rtie s are s im ila r to o th e r fo rm u latio n s on which more com plete d a ta has been accum ulated. (e) The answ er to t h i s q u e s tio n was No. ( f ) I t seemed to be th e o p in io n of th e group th a t la b e l s t a t e m e n ts t o th e e f f e c t ,rDo n o t a ll o w m ilk a n im a ls t o g r a z e on p a s t u r e s t r e a t e d w ith 2,ij.-D w i t h i n _______ d a y s a f t e r t r e a t m e n t " w ould be accep tab le provided s u ffic ie n t d a ta had been accum ulated to con c lu s iv e ly in d ic a te th e in te r v a l betw een a p p lic a tio n and g ra z in g w h ic h w ould r e s u l t i n no r e s i d u e s o c c u r in g i n m i l k . .'a ' The above r e p r e s e n ts some o f th e an sw ers to s p e c i f i c q u e s tio n s r a i s e d i n t h e s u g g e s te d a g e n d a . I w ould l i k e t o e m p h a siz e t h a t FDA was q u ite p o s itiv e in s ta tin g th a t th e y d id n o t have enough pharma c o lo g ic a l d&za c o n c e rn in g 2,ij.-D to e s t a b l i s h to le r a n c e s sh o u ld resid u es be found in meat or m ilk. DOW 376846 Jack D reessen 2037 KURON TOXICOLOGICAL INFORMATION AND SUGGESTIONS REGARDING TREATMENT FOR PERSONS SWALLOWING KURON O O FIRST AID CO o o C a ll a p h y s ic ia n im m e d ia te ly . Keep th e p e rs o n warm. I Do n o t a t t e m p t t o i n d u c e v o m i t i n g . TO THE PHYSICIAN K u r o r t c o n t a i n s 65. 3# o f 2 - ( 2 , 4 , 5 - t r i c h l o r o p h e n o x y ) p r o p io n i c a c i d , p ro p y le n e g l y c o l (C^HgO t o C ^ H ^ O ^ ) b u t y l e t h e r e s t e r s an d 35*5? o f i n e r t p e tro le u m o i l s an d e m u ls if y in g a g e n t s . K uron h a s a low a c u t e o r a l t o x i c i t y . The LD^Q v a l u e s fo r v ario u s anim al sp ecies a re : r a t , 1070 mgAg; guinea p ig , 850 m g/kg; r a b b i t , 850 mg/lcg; m ouse, 2 1 4 0 m g/kg; and c h i c k s , 2000 m g/kg The a d v i s a b i l i t y o f a tte m p tin g to em pty th e sto m ach by em esis o r g a s tr ic lav ag e is c o n tro v e rs ia l s in c e a s p ir a tio n o f a petroleum so lv e n t is to be c a r e fu lly a v o id e d . R e sp ira to ry em bar ra ssm e n t c au sed by pulm onary edema and pneum onia i s th e p rim a ry d an g er from sw allo w in g th e p e tro le u m s o lv e n t. In su ch a c a s e , th e a d m in is tra tio n o f oxygen p ro b a b ly w ill be b e n e f ic ia l. C hem otherapy w ould have no e f f e c t on th e s o - c a lle d "ch e m ic a l pneum onia" b u t i t may have some b e n e f i c i a l e f f e c t i n p r e v e n tin g o r c o n t r o l l i n g p o ssib le subsequent b a c te ria l In v asio n . D e p re s s io n o f th e c e n t r a l n e rv o u s sy ste m way a l s o be a problem . O il la x a tiv e s o th e r th an m in e ra l o i l should be a v o id ed . DOW 323410 2 L iv e r damage may be m in im ize d by p ro m p tly p la c i n g th e p a t i e n t on a d i e t low in f a t a n d h ig h in p r o t e i n . The u s e f u l n e s s o f l i p o tr o p ic drugs has n o t been in v e s tig a te d . K idney in v o lv em en t is ra re ly s u ffic ie n t to m erit sp e c ia l tre a tm e n t and fo r th o se ra re c a se s w here th e k id n e y s a re s e r io u s ly in v o lv e d , th e tre a tm e n t sh o u ld be th e same a s f o r to x ic n e p h r i t i s o f o th e r e tio lo g y . NOTICE T his in fo rm atio n is g iven in good f a i t h , b u t w h ile i t is b e lie v e d to be c o r r e c t, no w a rra n ty i s m ade. NOTE: I n case_ o f a n y q u e s t i o n s , c a l l The Dow C h e m ic a l C om pany a t M idland, M ichigan, on phone M E lro s e 6-2376 ( T o x i c o l o g y L a b o r a t o r y ) M E lro s e 6-2243 ( M e d ic a l D e p a r t m e n t ) B iochem ical R esearch L ab o rato ry T he Dow C h e m ic a l Com pany May 28, 1959 <3 ) 203S DOW32330 4 B iochem ical R esearch L ab o rato ry The Dow C h e m ic a l Com pany RESULTS OP RANGE FINDING TOXICOLOGICAL F ile T 2.30-237-1 ! TESTS ON AGRICULTURAL CHEMICAL FORMU K No. Sam ple 1 * LATION M -2121 CONTAINING 5< # SILVEX Chg. 127^-26 2- EQUIVALENT R ef. GH3 - 6 7 7 - I 3 7 c S u b . by G. S c o le s R e p t.b y K. J . O iso n NN.' -s. *Vi T 2 ,,30-237-1 DISTRIBUTION The Dow C h e m ic a l Co i , M id la n d R. H. Boundy CRI H. H. M c In ty re R. P. P erk in s A. H a r t N. W r ig h t G. E. Lynn (2 ) G. D. J o n e s K. B a r r o n s (2 ) . G. S c o le s M idland D iv is io n H. H. Gay W. L . T i s d a l e E. N. Luce L . M. G re e n e S. A. S h r a d e r W. J . F a l k e n s t e i n L ouisiana D iv isio n CRI Texas D iv isio n CRI D. J . K i l i a n R. W. C o lb y W estern D iv isio n CRI D. E ls h e re J . F . Kagy James R iv er D iv isio n CRI THIS REPORT IS THE PROPERTY OF THE DOW CHEMICAL COMFANY 16182 DOW32337 Biochemical Research Laboratory The Dow Chemical Company RESULTS OP RANGE FINDING TOXICOLOGICAL TESTS ON AGRICULTURAL CHEMICAL FORMULATION M-2121 CONTAINING 50# SILVEX EQUIVALENT PROBLEM A sample of M-2121 was submitted to the Biochemical Research Laboratory for toxicological testing and definition of industrial handling hazards. The material is a pelletized formulation containing Silvex Acid and Silvex Potassium Salt on Attaclay. The formulation is designed for use as a brush-killer in the undiluted dry granular form. CONCLUSIONS M-2121 possesses a low acute oral toxicity and is only slightly irri tating to the eye as well as to intact and abraded skin. Excessive skin exposure to the moist material, particularly if confined on the surface, might result in some skin redness. Such exposure should be avoided. It is anticipated that no problems should arise from handling the subject material under conditions where reasonable care and clean liness are practiced. SUMMARY OF RANGE FINDING TOXICOLOGICAL DATA Acute Oral Toxicity Animal Rat Preparation Fed 5# in corn oil Dose (gAg) 0.50 No.Died No. Fed Response-Remarks 1/2 Animal died four days after feeding, pathology ess. negative. Rat 5# in corn oil 1.0 0/2 Pathology ess. negative. Eye Contact - Rabbit Material Treatment Undiluted Washed and unwashed Response-Remarks SI. conjunctivitis subsided in one week, Skin Contact - Rabbit Condition Material of Skin Undiluted (Dry) Intact Undiluted (Dry) Abraded N o .of Appi. Site Response-Remarks 10 Eelly Ho irritation observed. 3 Belly No irritation observed. SI scar in 21 days. 16183 DOW323308 2 Skin Contact - Rabbit (Cont'd.) Condition No. of Material of Skin Appi. Site Undiluted (Wet) Intact 10 Belly Undiluted (Wet) Abraded 3 Belly Response-Remarks SI. to mod. hyperemia with si. swelling and si. exfol. Skin normal in 21 days. Ess. the same as above with si. burn following 3rd appl. Skin healed with si. scar in 21 days NOTE: When applied wet to the rabbit skin under a bandage, the material adhered to the skin which probably resulted in the irritation. Skin Absorption There is no Indication, from the skin irritation tests conducted, that this material is absorbed through the skin in toxic amounts. i 6184 DOW323309 EYE CONTACT SKIN CONTACT INHALATION INGESTION COMMENTS* nad FIRST AID MEASURES If the eye are c o u u n io n ed , they hould be flushed immediately with copioua amount of flowing water for at lease l j minute. u Medical attention should be obtained if irritation persists or develops aiier washing oi the eyes. Medical attention should be obtained. MEDICAL ATTENTION SHOULD THEN BE OBTAINED WITHOUT DELAY. Any injuries or irritstions which may develop should receive medical attention. Contaminated clothing and shoes should be removed and not re-used until thoroughly denned. Wash contaminated skin with soap and plenty of water. Contaminated clothing, including ahoe^ should be removed sad the affected akin area should be washed thoroughly with soap and plenty of water. Medical attention should then be obtaioed. Contaminated clothing and shoes should not be re-used until thoroughly cleaned. All contaminated clothing, including shoes, must be removed immediately and the affected akin res flushed thoroughly with water from a safety shower, or other suitable device and cleansed with soap and plenty of water. MEDICAL ATTENTION MUST THEN BE OBTAINED AS RAPIDLY AS POSSIBLE. Contaminated clothing including shoes, must not be re-used until thoroughly cleaned cr must L* discarded. If a person should experience any noticeable ill effects from breathing the vvpor or fumes of this material, medical attention should be obtained promptly. If a person should be overcome from breathing this material, he should be removed to fresh air at.once, be made to rest, kept wans, and MEDICAL ATTENTION SHOULD BE OBTAINED IMMEDIATELY. If breathing stops, artificial respiration should be administered. If appreciable amounts of material are swallowed, vomiting should be induced by tickling the back of the tongue with the finger or by giving an emetic such as 2 tsblespooofuls of table salt in a glass of w ins water. Medical attention should then be obtained. If the material is swallowed, vomiting must be Induced by tickling the back of the tongue with the finger or by giving an emetic such as 2 tsblespoonfuls of table salt in a glass of warm water. MEDICAL ATTENTION SHOULD THEN BE OBTAINED WITHOUT DELAY 16185 20 i0 DOW 277379 /V/rr* IV. RESPONSE OF HUMANS TO HURON AND OTHER FORMULATIONS -e* OF SILVEX. (V) o vO -N 00 1. Wolf, M. A. 1959, Results of Human Skin Irritation and Human Skin Sensitization Studies on Huron. Dow Internal Report No. BC T2.30-40-3. 2. Wolf, M. A. 1961, Results of Human Skin Irritation and Sensitization Tests on Kurosal SL (M-2033). Dow Internal Report No. T 6 0 .14-39291-2. 9 m Z M O < l \\ \ \ 1TUC P/iItiY O / T i ^ i l , ' i l i n n ` iii i y , Iti tun mini 2 / , I u i'.i , p u i;u 2 - -a. . i: ity NYl.A riiu .'.'i; nil/r,i ; li. r S e]ln,<irni;:iio,o[-d:.ist;ni-f;oI<ri-;siUu>v:i.l-!,,,.d: fmi.-dijlil.i/oSnil jd'nuvi-- 1 lac:; fur dama;;: i tu jpia-ii.',, I; JiliidvMaciV-nviiili'i.i*il;iiraela'sjmuUlit..eL-d|i>diolupftii-oiidl:yiilldil':imi.Uyi- i " In ad Idli.a v.c csp/vt la (Ilo 1! tadc)-hangsaiijn'iil.1i.iLIII'(-fuUr.Sm. yFnouroesutfMSeerl-- vice for Uatncijd lo the entire on- ! .viio:m!ei;l," says the spokesman ioiifg2 fdoap dsii-.i.,1" ohi.f rb'J residents cla im- j1vC!nn..nll.S.l'cl'i.iju'-jirelinh.s-':'y-a,i..s:sl;i11Ir:MkcbahenitertilsiaiUrcs..nio'itu.lcVsSirvJyf:..i!odllt1vuhiY`igOeeiJS.VtihinliMSueSiii:z:tuh!paScnSlcatiOo'ecnirlf,im:V:sJRivfiliiiCtnoa--, aaVmnrieHodrniJpeKi'a:pmurStrjj,o.:csg.-,acf,rly.aaasmlcs--loulo-Mt.bl.iil!-ai);i',,alol Sruraiit!;v*u/Jo:.inalfgiiv---oTe- (pev.l'i in the Final Moiii.tsiin foothills, says Robeii Coialu.y of 1`iucnA, Temo National fo r esi S!!;>:1ViaD:'. 'Jlie .Sail irr'igelicni poj:ct be came isivuk.esi fc-a-enr.'.;- n.h xc il brush c, ver incruairs water ilu"WThiies'.slparaCylTOisii'd. cfmitvly c-a- K:ytu:-: to l::!! shiwbs mid brush," Ci-srlr,. y y iii. p:x!.:.b'.v vpolHa. ionl dmi.:e."o.r.inl most bioadlca -vsin r.cr.rby Kus fed sel! Gisivli a:.d Kellner Canyon agree that lire spray.!,sas, indeed, ; f- fed Lroa!-k-aru;l plants -- not ic iwaistion fruit ami si,sale lives, birds, annuals, people uic! lira vvsiler supply. " I call il (Tento Nsd.iossnl Fcr- CfijvI)e U!..v..Rc.r."vTyhRj sdi'eita,"dlsya"yssprMayrss. :.e-usi dimislale iba (p-m.-lh of lire i....,1 arai kill Ilio l-vir.g'.'' (M l sue cklotiunUd hydruar- bone, said do recidale (.rovelli.) mfJeaJacSVtpcclbl(btplioljsoieil..ludii/i.liaIleoceu.ailnllyoniseins-l"umdlreCiiv`(lM/emfTvslrcvolNlai;`meioi;muluyii.i,hAnilliat.i,UllUasj"u.tiimrdll-iolpo-a:'cti;i:uiSiehtsozuniit;iboolTjvbiliby-laliellTe,uithleaissojclinoiUit-Ipivn.lijisline(rifpblr-loli;i;ses.e.hol.eilclsl:lal,"e-iev.npliysli|:o.a>covdiulatiNri",uisD,fnllfeTolmtomyrjllinriap;cfi.:te>ualwrtth'jiai,hioh,erol.(rd.iiiil:sa.siu.isUleeasl-isns|otr';wa.ivlnIhii.indiItaisldii,ibfd.:elabinvulbuiityw''i.wslaYuipAM.eldae.whrFussslvMmliIympieiuerlnoi:rvseri!o,lsaloblhiuVvie;erv"iSi)|llllz'a;ptiss..uorsulrik.:lnli.;cl.itrllacador...ltu/)yi,eMcsine;ilnudvyb`idilji!.oiliorml\Mducs'oy,fyauebIivoIaCvl,n:uIiiriel,!ms-et'ti.,rlrc,:-.,lna.dn.v:S.seioo.r.t;n,uocl-e.lfi-;>s.nli;ylenlv.s'.u.,rirvlpiluc,l-.L"l)sueinv,.`r,.pnis^v'..lu:sJu!mSu..::li.Vi.zs;n..,::.n,.us.pi!uiv;a.-e,.ou.i.,g:i-:si,*'*u-i*..',--;wl,-.,.*..l"-11,.*.Irjj,|JKtI;!eb<m>;s;:iG!sci'\Hc;c.i|'lUct.s*siv>lIV>..*.ni-a-jialdsus*,lAi'ukla.y/oH\A:u;usu.inrid1--Si':,yjabMivssnr:!se;i.u*u.l,bl.ol;5is,.l<eeneCi-lt!i'1r.pi'ic`VloIU(ptlUil-,u'W!iiC-;/.v.lbviitinKui:r'.t.;-vl'il.tih,S!.\,v-.s.iica:ty.,nr'JV,l*bntri.'!-,el.crTl.M,owdc.i,ad.!;'urs:D-inti-.yu.K'l,Svi::ueL'J;f',isihjjlM..litu."-c'viiwtniribrcr!-,fer!a.l:ooo7i...<i!.!e0uJg-ev:irImuvii:yrlvn:I.linm;oed-it.swl;mssile;eci-;lS/n-.:orul:a.,ao:suo-.'..n.::Mwoni.y.i.vs(rrij:*i!lt'vstrhrc.p.cnstn-..!l.!--ea'sbo;-'b1.s1ci;-auisD.s"bss(ehsttceirl1Cy.`aiIogtlioitubvi,aiCerjrt-nvhhsiUhj.aa.p:-o.iii:its..Mu,ntaaeociItavI'nl>nvp.1otJl)tMnLd.it'!ha.osO:5dMryou:rsri,c'lorrss:-;elioal"ue<oisi-o!.ri,.t-D:ino,.f.ui!-pJ,Kefrsix:s.h;v:-iliCcl.oU..uiuy.eriepiedmi.--g.illtRe.biW.sni,l,u.iMSnlJ"cmlsdIidi..l:aytolInit-.cnltt.osnn'.:bb-t-.Fibs'ih;at,oiba..ehed..1iJhm.ob-ioilma;ua"ent'evMwbuau.lrirHlf5cerlnd.sdn,eyrd'.rIyyjtttr*.,t,s-t-.t,,-. During list past sevcsvl -- ' i n , i ; : d H I. c u e h - u e l . l . u '- rnyr.tbs be lias been coll-eciiiig in I V t!..c-s v. hub In-! siiovvn lop 'ar(c.lpi'idiepeUasA.rFe1isnnhltuaoly.ldssuopnioiosasllbiutositsnse!odfarimelslabodlylitariphspleaslranbl-ltosisthMe aarretian. fa-ls that the Forest leaves in ctnisainii'i.scsg. 's-.d geuse nu-l : day f;1. .1'iSwt.-gi'i' Stan'-! ai: 1 J.thil a ,, ,i h abila: gani'.iulol ufi. Cibi il v:r grji;j up all the Sendee has used a treatment, wrohimosieesstot taarlec-uftn'clc-sbnsovonsti. li"sIat emsiaviytake i;s decades lo find cist," h-crFitirceiez.e' so. f pollution w ill coi re toa-es. Tiie fY,,l 1.acame bici: iniin-'uis-i-.-P \r a St'.U 0? 3 died d-.aii.;; many as la our w.'.'.i vi o--or l r .so..: aJudias y ;'.S;* (Iullune.lu"!i^IIW" iiu`b'l`"ljauiins.livilunuu.Mali,.naI.nliln/iii,i,.!,,.f dsiiiisifc. lint, viewed as a pwhre<t/!tey, ntlhseinynpiiai;i;n."t tu suiudhiug pi il-aDniil/l-'evsiaacyl,i,n1)ll1i,,)1 IdIbnei,-fmu.nvpihlielldiilimtmi Innid,i;ead1;ead A pine Ileo, Nel,lui,I uud u,n-. mal on one side, bad conus but nGolobnoeegdalersdeonn, stheeveroatlhelor.w;I;n oaf chiles were chile-green ou one side. On the other, they were bind: and decomposed. And l-eaebcs rotted before they fruited fully. Conili natura! diseases have esun.ed all tiiis? Dr. Slsuiley M. Alenili, a UA plant pnlli'.)!,-;;!..!, iliiii!-..-: imt, Assays wout.l have In be made to Lu muu, 1,.: s:i)s, but lie believes it la 1.2 unlikely tli.U any one virus would siffeel so many different plants in the same way. To understate their mood, the Ffolarmese'.d Sbeyrvwichiiatir.onseomsteawffhamteimn ber lersnod "not 1-iO I.meli ral. in,,,aioeieob.i ii.h"o part of some people litt"lMe aysbcearfeodlk,s" asreaysvanAni.ng 5ba Kdvivuisnioton oc'* thraengFeoreiisutmSaegrevmicuen':i; iti V.'r.sliinglosi. Jk-f abded out that "there's a ey to thro-.v a rope .v. O'.md IS'CLtbUt'ilOC d.uuiiie.s sPdeizosptliloer.s ranba,hiest plo!isulieihior , h-.-i Incides au-.l D D T." thaf\lheessf,cdluais'atl gmuuvennlhimonn--l..lnn-o.ntiedIlio i-.-: of ?, -1, S-T of!o r lenniiii!; !ii: [ j.-,;:}- be dangerous lo am il lull 1ie. Uh'.ass the Foe.! ;.,,.l lhvy, '.d m ir .iilii- v ;il.! i:.ii, > si " snfo legal tiileianue. in and an foods, * the U.S. Ikparimont <( ! ' A(;iicullme will b.m the u->e of k61t7 DOW271217 trJ.kdiL *^ k~- t2, 4, 5-V on*food crops effective * 'J .211 1, Jj/0. In a prepared statement, D r.j .co A. Puliudge, presidejitiiil science adviser, pointed out thut; l" tl,e relationship of die effects 'of 2 ,4 ,4-T on laboratory animals to effects in man are nut entire-, ' ly clear at this tim e .". j Kot so, says the National Health Federation^ a. nonprofit foundation with headquarters in ' Monrovia, Calif. Since the fedet-' alien began an investigation last July of the Clobe spraying, it has " gathered enough material fVuircec. PSerensaitdeeinntvHesotwigaardtioCn.,"Losnagys. Although the project in the: Pinats was approved by the Federal Committee oil Pesticide Control, die Forest Service has . "delayed future herbicide treat ment," admitting there v,as "so.'V.e drift damage." tho" Wroeugdholyfeienlvetshtaigt avtered',veapll rethttey fcnov.kdga.blc complaints,"' i Kvaiitm said last v. re!;. , jLAi.-;. .a (-,-vij- h<>| g..V f ' " ' WV t Vs i t . i . *> - 1. v. W - . VA ' - V ' fr : r . ^ vWihs'/- - ''' .s' !V;: VCs-. : C ^ . ' y . " y \ . 'fV f -vv. ............. : fC-/ V 'dv- 1 a1- ^ v-rjt \. ... "V . / , V -6-,V- h 'tp S ' . : k ^ ,; r ~ ` '" V '7 . . ( *s: . tn :T . I. J npnJ--* >*l'l aPo 1? . IT1 'ono-pai. < r.i sr. a, ry ii J poo fj LEJi.*ji onc pnn tpr fi- ipttopovrj C/O noonf-)>1 5uOT* vc r P 0u3 nCOrrjL"J. M o t--:i I-* K-? o cP1M-avo* ' /f (J l\ . . r' ..... r- - lV i C*- - V . T H ' * | 1 * r v . s :/* //'* i Kf ", rv' ' s, V\ Pi l H i V '.*<***' / .x' >- . 1\ * . , TESTIMONY OF A. T. TALCOTT SHOECRAFT ET AL. VS. DOW 2723 T_ - ' ? 7- 6 ; D O W f 469-660 I am A. T. Talcott, administrator of Regulations and Labeling in the Quality Assurance Department of The Dow Chemical Company. I have a Bachelor of Science degree in chemistry from Alma College, Alma, Michigan, and have done graduate work at Michigan State University but have no advanced degree. I joined The Dow Chemical Company, Midland, Michigan, in 1957 as a chemist in their analytical laboratories and moved through a series of technical promotions in the analytical chemical field. In 1965 I moved to the Labeling/ Product Registration section of the Legal Department, becoming supervisor of that section in 1975. In 1976 I became manager of Product Safety Compliance in Dow's Quality Assurance Department, which included the functions of labeling, material safety data sheets, and product registration. In 1979 I assumed my present position where I am responsible for labeling policies on Dow products. I am a member of the American Conference on Chemical Labeling, the Labeling Standard Revision Committee of the Chemical Manufacturers Association, a member or chairman of several committees dealing with regulations and labeling within the Chemical Specialties Manufacturers Association, a former member of the Labeling and Precautionary Information Committee of the Chemical Manufacturers Association, and the Labeling Committee of the National Paint and Coatings Association. I was also one of two industry representatives participating with EPA to put together their National Symposium on Pesticide Labeling in 1974. I will testify on the history and procedures used in the development of labels by The Dow Chemical Company and to the registration requirements for labeling used by the Pesticide Registration Division of EPA which was part of the USDA in 1968 prior to the formation of EPA. I will also testify to -2 - Che relationship of the labeling policies used by Dow and independent standards as & well as how the regulations promulgated by the Pesticide Registration Division which-apply to the product KURON* weed and brush killer. /I A'S-i i-A The procedures used at Dow allow us to get the input from our experts such as health professionals, technical specialists, and legal and regulatory counsel in areas, pertinent to such a product. The labeling used on KURON weed and brush killer in the-time~period~1967 ~ through 1969 was in"*part specified in^regulations for pesticides which were called economic poisons under the statute in effect, at~that timev Theselrequired the product to bear a label containing the name of the product, the name and address of the manufacturer or registrant, the net contents, an ingredient statement which was prescribed in a special manner, a warning or caution statement as appropriate to regulations prescribing same, and directions for use-- which if complied with, would be adequate for the protection of the public. These elements of a label were further prescribed in a certain manner by the regulations and interpretations promulgated by the Pesticide Registration Division and required a preclearance or acceptance by that Division before they could be used on the package of the pesticide. This labeling was also submitted to the state of Arizona and other states for review as part of the state registration of the product required prior to sale. The product name KURON, the Company name, and certain other elements such as the registration number required to appear on the label are for the most part self-explanatory. For precautionary labeling purposes, pesticides were placed in one of four dJLMUtA different categories based on certain toxicity data that would be i* animal tests provided to the Pesticide Registration Divisions These were calfed- categories < c' one, two, three, and four^ category one being materials that were highly toxic and requiring the word "poison" and the skull and crossbones to be used on *Trademark of The Dow Chemical Company 16131 3- - Z9969H MW! the label. The other categories moved in decreasing toxicity to' category four r- which was for products determined not likely to cause injury under any reasonably foreseeable conditions of use. Each of the second and third categories were ten fold decreases in toxicity from the lower-numbered category. During this time precautionary statements used on pesticides were set forth in a document called "Interpretation 18, Revision 2" published in the Federal Register of March 9, 1962 as an interpretation of 7 CFR, Part 362.There were two ways in which this interpretation was used. . One related to signal words and statements of hazard that were used on pesticides^/containing specifically-listed active ingredients and the o t h e r s e t forth general procedures based on the toxicity categories previously mentioned which- was applicable to KURON weed and brush killer. Applying the principles stated in Interpretation 18, one comes up with the precautionary labeling which was accepted by the government as adequate to protect @ the public.,and used by Dow on. its product KURON weed and brush killer. Quoting from section 362.116(b)(2)(III), "Labels of products in the third category should carry the word "caution" in statements indicating the means of avoiding the principal hazards of use. Use of the skull and cross bones, the word "poison,rt and antidote statements are not necessary for these products." Another part of the label which may need more explanation is the ingredient statement. Ingredient statements are required to be made on all pesticide products and must be stated in certain ways. The constituents of a product must be characterized as either active or inert ingredients. The active ingredients must be described by their chemical name. The two ways in which ingredient statements can be listed were specified in section 362.7 of the regulations. -These>w*e to list each active ingredient and its percentage and the total of the inert ingredients or to list the total of the active ingredients and each individual active ingredient in descending order of percentage.without the actual percentage of each ingredient.plus the 161S2 LJt*i tn n T n ' ^ l T J T .M ff" ti <wIt T jtb iju O O 4- - >J total inert ingredients. Equal prominence is required to be given to all of the ingredient statements and these ingredient statements were required to be placed on the front panel of pesticide products. In other words, only those materials that were considered active ingredients from a pesticidal standpoint were allowed to be listed in the ingredient statement even though the product contained other materials that comprised the total. It has been suggested that should have been listed on the label of this product. Such a listing would not have been acceptable from the government's . standpoint since this is not an active pesticidal constituent; additionally, the initials TCDD do not represent a common chemical name acceptable to the federal government so such listing would again be prohibited. The other reason for not listing TCDD as an ingredient is that c:;e limits of analytical detection at the time of production of the KURON in question were such that TCDD was not found in this product. There are other sections of the label that are, of course, pertinent to the allegations made concerning the product KURON weed and brush killer. Among these are the instructions under a section titled "Warning" that "applications by airplane, ground rigs, and hand dispensers should be carried out only when there is no hazard from drift." Other instructions which are meaningful to a user of such products are the instructions that "coarse sprays are less likely to drift" as well as the instructions on application rates and times for specific applications of the product. The procedures followed for labeling pesticidal products are an application of the principles that are included for labeling of chemicals set forth in the American National Standard for Precautionary Labeling ANSI Z129.1. ` This is a :t consensus standard which was not published until 1976, howeverT~does-have the benefit of review of such organizations as the American Society of Agricultural Engineers, the National Safety Council, the American Society of Safety Engineers, the Society of Toxicology, AFL-CIO, the American Conference of Governmental - 16133 5- - Industrial Hygienists, and numerous other government, industry, and independent organizations with an interest in conveying precautionary information. These principles prescribe a signal word, statement of hazard, precautionary measures, and as appropriate, instructions in case of contact or exposure as procedures for providing precautionary labeling. The labeling used by Dow on KURCN weed and brush killer demonstrates these principles. The procedures used within Dow to develop labeling in 1967- involved the initial labeling being developed from a use standpoint by a technical specialist from our Agricultural Products Department along with the individual responsible for submitting registration applications to the federal government, and precautionary information coming from a labeling specialist like myself. This draft copy then would be typed and sent for review by the numerous specialists within the Company with input on the health and safety aspects coming from toxicologists, industrial hygienists, medical doctors, individuals from our quality function in connection with our manufacturing people providing input on the composition of the product, trademark and patent comments coming from our attorneys, and the original three persons reviewing these inputs and those of our marketing personnel. Once these inputs had been composited, the labeling was sent to the federal government for their acceptance or suggestions for change if they-did-not accept it. " Upon return of a copy of the accepted labeling, the labeling specialist would initiate the actions responsible for getting the labels or the preprinted container copy to our production people for application on containers of the final product. It is important to remember when developing or reviewing labeling for a product that the labeling must be based on the product as a whole; consequently, the labeling must be viewed in its entirety, not just in parts-- since it is the whole product that is used, not a part. UUW 1469665 6- I have compared the labeling used by other pesticide producers of similar products and find the- labeling used, which also was required to be submitted to the Pesticide Registration Division before being used, to be essentially the same where applicable as that used on the product KURON weed and brush killer. Based on my education, training, and experience, it is my opinion that the label used on the product KURON weed and brush killer in the time period 1967through 1969 met or exceeded all regulations and standards of care pertaining to such products; and further the directions and precautions if followed were adequate to prevent accident or injury with the handling and application of the product. ISli5 % i 2732 r bwocaow AJO Immomtt. Das. ISIS; p. U1P-U34 Cwyngtn O 1>7< American Sed efy (pe Mlarahielngy Ex. 118 V iU tN ll fir it f . /-S-A. 0102000001020100010200 DO W 1369171 Increased Susceptibility to Bacterial Infection as a Sequela of Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J. E. THIGPEN,* X. E. FAITH, E. E. McCONNELL. u ra J . A. MOORE National Intm utt of Eiurironmnial Btallk Stinnett. Xneank TriantU Park. North Carolina XTTCB Received for publication 12 August 1973 The affects of subdiaical 1avals of 2,3,7,8-Utrachlonxiiban2o^>-dioxLn (TCDD) on tha rasponsa of mica to infection with either SalmantUa barn or H trptseirtu tuts, also known as pseudorabies virus, are reported. TCDD is a contaminant of certain commercially useful chemicals, such as chlorinated phenols or herbi cides. It has been shown to cause thymic strophy and to suppress cell-mediated immunity in laboratory animals. Sublethal levels of TCDD were used: 0.2, 1. 5, 10, or 20 Mgfkg, given through a gastric tube once weekly for 4 weeks. A significant decrease in weight gain compared with control mica occurred at the 20-pg doeage. Dose schedules of 1 Mg or more, followed by salmonella infection, resulted in significant increases in mortality and decreases in the time from infection to death. However, TCDD had no significant effect on mortality in the pseudorabies-infected mice. The moat Important finding in this study is that extremely low levels of TCDD, which do not produce clinical or pathological change, still have the capacity to affes.;. host defense. O O' SO -M < Various environmental chemicals have been munity in rats, mica, and guina pigs (28. 29). It shown to affect immune responses or host resist is not knpwn whether TCDD-expoaed animals ance to infectious agents (Friend and Trainer art more suscsptible to infectious agents. These ( [6], Gainer [8], Gainer and Pr7 [10], Hamphill et studies ware designed to determine the effects si. [11], Jonas et al. [14], Holler and Thigpen of subdiaical levels of TCDD on tha response of [19], Holier end Kovadc [18], Holler [IT], Ver- mica subsequently infected with either Salmo schuuren et al. [2T], Vos and van Gendertn nella barn or pseudorabies virus (PRV). The [30], Voa et aL [29], Voa and Moore [28], Wasaer- criteria used to measure these effects were clini mann t al. (32D- These reports generally in cal appearance, weight gains, mortality rates, volve obeerved defects in antibody raoponae (14, rime from infection to death, and pathological 17, 18, 19, 27, 30,32) or on call mediated immu examination. nity (28, 29, 30), although several (8. 8. 10, 11). also describe suppressed host resistance to infec tion. MATERIALS AND METHODS 2,3,7,8-Tetrachlorodibenso-p-diaxm (TCDD) can occur as a highly toxic contaminanrin the production of some chlorinated phenols or chem icals synthesized from chlorinated phenols such aa the herbicide 2.4,5-trichlorophenoxyacetic add and thus could be unknownly distributed in tha environment. TCDD as an environmen tal contaminant can present health problems, both in man and other animals. It is asmciated with chioracne in man (16) and chick edema disease in chickens (5). More recently TCDD and 2,4.5-trichlorophenol ware implicated as tha probable cause of toxidty in e group of horses in eastern Missouri. In addition to the horses, insects, birds, and rodents were poi soned, and there were several cases of human illness attributed to TCDD in this inddant (3). TCDD has bean observed to cause profound thymic atrophy and suppress cell-mediatad im- Animal. Four-week-old male CSTSUCJfh (JS7) sped fit .pathogen-free mice were purchaeed from Jackson Laboratories. Bar Harbor. .Me. The mice were caged individually under Altar tope (Envirogard. Lab Products. Garfield. N.J.) following t rigid sanitary regimen end given food (Stenlizabie LabBlos. Allied Mille. Inc.. Chicago. I1L) end water ad libitum. TCDD. TCDD, greater than 99% purity (lac 331- 144-0). was a gift from Dow Chemical Company, Midland. Mich. It was dissolved in reagent-grade acetone and subsequently diluted with et least 6 pans of corn oil. The exact volume of corn oil was adjusted to provide each mouse with e dose volume of approximately 0.2 mi. In/ecrioua agents. The b ecunum used wee a 5 . born isolate from a naturally infected opossum. This organism was grown overnight at 38 to 37 C on Trypticaae toy agar containing 3% sheep blood. Bac terial dilurions were prepared in sterile phyetelngieel saline, and the number of bacteria was quaau- 1 1319 r 1320 THIGPEN ET AL. Iktott. Immuh. ! !"! !"! !"!"!"! ! ! !"!"!"!"!"! tacad tarbidimefricaily with a Caiman modal 5(20 ipectiophotomatar at 540 urn. la addition, iba numbar of total viable battan a in all dilutions used for aaim al inoculanoo was confirmad by conventional local placa count procedures. Tha rira l i f t s t uaad waa H trp n o irtu saie (PRV) nation wwn Axed ia 10% neutral buffered formalin, amboddad in paraffin, sectioned at ( am . aad rou tinely stained with hematoxylin aad eosia. The criteria used to compare tha effects of sublet tha! levels of TCOO ia mica subsequently infected with 5 . bem or PRV w en clinical appearance, (9). PRV waa produced ia 75-em' Falcon flaaka can weipht faiaa. mortality rates, time from infection to taiainp RX-13 monolayers. Afear a 4-piua cyupatbo- death, and pathological examination. (oaic effect waa ahaerved. tha medium from each Statistical aaalyses. For the mortality data, doaa flaak waa cantrihipad at MO / to ram ora large response tn n d s were analysed by the Barthelemew cellular dobria. The rapam acanta from *acb (leak teat (2). aad pair-wise comparisons were mads by tha w en pooled, thoroughly m isad, aliquota! ia 2-ml Fisher exact teat for 2 x 2 tables (12). For the other n aia, and acarad a t -7 9 C until uaad. At tha tim e of . variables, the sipniAcaaca of doaa response m arls inoculation tha oficinal suspension contained ap was determined by the Joackheen cast (13). and proxim ately 5.0 x io ' uaxuo cuitara jneeave doaaa/ treatment-control differeacaa w e n evaluated by tha ml. The pooled n ra i nipension waa cultura u p - two-eided Mann-Whitney casts (12). o ra for mycoplasma ta d bacteria. Prallm iaary acody. Lethal dosa response curras RESULTS w an astafaUahad for 5 . barn aad PRV to determine Effects of TCDO on weight pain, mortality challanpa daaea (0.2 oil intraperttoosally (Lp.D that would produca 20% mortality in t-week-oid normal mica. All mica w an ehaarrad (or 14 days. Dead mica w on examined by both microbioioficai and hieio- potholopical procedure to verify infection. Experim ental doaipn. Two different experiments w an performed. In each experiment- 4-weak-old mica w e n randomly divided into four group. Each rat*, and time from infection to death. There was eaaenuaily no difference in mean group body weighta prior to TCDO doting. By week 4, the high-dose group (20 mb of TCDD per kg) gained significantly <P < 0.01) less weight than did the controls. The 10 mb ofTCDD per kBdose group also gained leu weight; ail ocher TCDD mouaa waa weiphed and doaad by pavapa ooca each group body weights w en comparable to the weak for 4 consecutive weeks. Mica ware doaad with group which received acetone-corn oil (Table 1). either TCDD in acaune-oers oil or anth acatoneeora oil. In. tha f im experiment, each mouaa received either 0. 5, 10. or 20 up of TCDD/kp per week. Two daya aitar tha last dse of TCOO. whan tha mica w en 5 weeks old. each froup was randomly divided into three subgroups. Mica in ona aubproup aran injected Lp. with 10* viable 5 . barn orpaniama At a given level ofTCDD there were no signifi cant differences in the week 4 weights among the mice assigned to the bacteria, virus, or control groups. There was little association be tween body weight at the time of infection and subsequent survival. Mortality rates in the bscteria and virus con (LOw,. dosel, cha aacand aubproup received 10*TCID trol groups that had not received TCDD were u of PRV (LBwm doaa). and the third subgroup (con anR apa cad baaed on tha L D ,. preliminary trol) eras injected arith a lin e only. (LO h i* the number of bacterial or viral apante required to kill approximately 20% of the mica inoculated within a 14-day observation period.! The second experiment waa conducted in a man- study (Table 2). The groups of mice exposed to 1. 5, 10, or 20 mb of TCDD per kB and given 5 . b tm showed a significant IP < 0.05 to 0.01) increase in mortality rates. The mortality m e par identical to the first except th a t each mouaa racaived either 0. 0.5. 1. or 5 Mg of TCSD/kp par a sk and tha PRV aubproup waa olitainatad. TaSLX 1. EffectofTCDD on tight gain of> -outr a 3 -iattk exposure period Both experiments w e n performed as blind i n d ias, and the order of inoculation waa raudomitad. Neither tha bacterium nor tha virus appeared to leee or pain potency durinp tha utae of administration. Weakly TCDD daee* (aprkg ef body wu No. ef tatuai wt igr* Wt gaia at chailanpa i*P The level of mortality teen in the Anal 2S% of tha animala inoculatad waa not R atuticxily differnc from th at observed in tha 3 n t 25% or tha middle 50%. Therefore, tha order of inoculation was vir tually im la v a n t to subsequent survival. After bacterial and viral ehallonpe. tha animalo E x p tl 20 10 5 0 1S.SI : 0.21 15.71 s 0.22 SO 11.41 X 0.22 15.55 x 0.19 2.0 x 0.20* 5.21 x 0.17 5.7T x 0.19 5.55 X 0.17 w an observed at least tenca daily for 14 day. Body weights w a n recorded at death or at termination of the study. Necropsias w on performed on i t loan three mica from each lubfroup, and elected tissues w en obtained for histopatholopical and microbiolog- 171 n 5 i .a 0 15 20.51 x 0.22 23 21.05 x 0.22 25 20.57 x 0.24 25 21.13 x 0.2* 4 . s 0.17 4.30 s 0.17 4.06 x 0.19 4.29 x 0.14 ical examination. Mico from subpnupa with lasa than three deaths were sacrificad and aeempsied on a u individuai body --Pt basis. day 14. * Mean : standard sner. Tha tissues seiactad for histopatholopical esami- P < 0.01. V 3101000102010001020100 DOW 1 363173 ( Voi. I. lf7S TCCD-DECREASED HOST RESISTANCE TO INFECTION 1321 in tbs 0.5 ( if of TCDD per kg Som group * u identical to tho control groop; therefore. the 'no rfect" lovol baaod on tfao pori motor of this stady would bo somewhere botwoon 0J sad 1.0 Mg of TCDD por kg (Toblo 2; Fig. 1). The mortality ratos in tho groups that rs- coivcd virus wort not soon to differ significantly with variation in tho dost of TCDD. T han was a significant IP < 0.01) date-re lated decrease in time from infection to death in the 5 . bern-challenged mice (Table 3; Fig. 1). Ta s u 2. Com parison o f 14-day m orta lity ratm o f m ies exposed to eu U eth a i le v e ls o f T C D D a n d _____ ehailenped w ith eith er S . bem o r P R V ______ Weekly TCDD iOM4 et bady wtl M anality ratea <) la a a n a i V ini challenge* Cao- wel* C a p ti 20 10 1 0 M IlSiMI" tu)* a ito 10(100) 0 (0120) is ( u n 11(3/30) 0(0/30) i y it) 0(W30) orano Cape Q S 1 0.S 0 10U4/20>* ND* o r a n o t u r a * ND IO A 3) 8(5/30) ND 0 (0 0 0 am o) ND 0 (0 0 0 *TCDD ia lin i mrn aii a m a r via p a r e innibanan m ea individual W y weight b a . B a c a n ti cbaileae >LDw_J r f S . h w y ia Lp. ianctioa a bay I tlU r the a weakly 4em rfTCDD. * Vira] d ia lk n s t i U J cf TRV via t-p. tiycrntn a Say S altar (Sa 4th weakly bate af TCDD. * Central aa ia a is w en kneeled Lp. with ae rile teline e a Say 2 after the eh weekly 4oee of TCDD. ( The a ila l t r e ia panathaeae ia d ir n e the a a a b a r ct aie* dying per the tecai m ia b a fV m ic e ia the (roup. <0.01. A'P A -f Tune leer) F ie. 1. C a r e la n tie m orta lity rates a n d tim e from k m . r - < 7 v / V ND. Net Sene. V V N V in fection to death in m ice exposed to ta h ieth a l le v e ii o f T C D D a n d tubeequentiy in fected w ith 3 . hern . Weekly TCDD daae* l a r k s et bedy wti T A S U 3. A seroge tim e from in jection to death idaye) a e u ria l rhbllens* V ini Ne. ef aeaeapnrera* H ate io Saetti* Ne. et eeaaurnvan* U n a te bearti* Eapt I 20 10 5 0 19 2 J 6 s 0 .4 ? 13 5.59 3 0.67 13 4.54 s 0.54* 5 1.40 = L59 2 9.00 s 5.00 0 3 1.33 3 L31 3 4.57 s 0.33 Eapt H S.0 LO 0.5 0 14 2.56 = 0 .3 ? 13 6.08 = 0.92 3 9.00 s 1.95 S 7.40 r 1.91 D* Nb ND > .N D * TCDD ia acetone-ara oli carrier via gasate intubatimi oa sa individuai body tig h t basta. * Batteria! cballenge `LD,,.) of S . ssi via i.p. rajeenon on day 2 after th 4th e e k ly dose of TCDD. Virai challeaga LDW4) of PRV via i.p. injecnoa an day 2 after th 4eh weeidy dose of TCDD. 4 The aumber of aoasurvivon aa th numer of ninni dyisg ith ia 14 daya after bacienti or virai cballenge. *i z standard error. l P < 0.01. * P < 0.05. * * ND. Noe dona. { 16138 1322 THIGPEN ET AL. I m e i . botoN . Pathological b H m tio iu . Poetmortem ex- rata of weight gain, and showed no histopath- miniann of mice treated with TCDD only re ological evidence of toxicity. This demonstrates vealed dose-dependent lesions in the liver ta d the moat important aspect of this study. Le.. thymus similar to those described by Voa at a l that extremely low levels ofTCDD which do not (31). The thymus Grom mica in tbs 20 jtgfkg produce clinical or pathological change still group a s amailar than normal. Microscopic have the capacity to afreet host defense. Thaee examm adon revealed cortical atrophy duo to findings are significant in that Iw w may bs loaa of small lymphocytes. Thymic atrophy was exposed to environmental contaminants inch much lass apparant and often equivocal in ths aa TCDD m minute quantities and thereto* 10 Mfritg group and was not obaarvad at the may be rendered more suaeapebie to certain lower doaas. Interestingly, no lesions ware types of infectious agents. found in tha spleen. Tha act that TCDD did not increase mortal Tba liven from tha 20 pgfkg- group ware ity in mice challenged with PRV snggeefe e lighter in color than normal and ware mottled. difference in _tha pathogenesis of these two This was lass evident in tha 10 jtgfrcg animals, agents. Ths ressonfs) or this difierenca is not and no macroscopic lesions ware visible in the understood. liven from mica in tha 5.1. or 0.5 jig of TCDD There are several host defense factors which par kg dose groupe. Histopathological examina could account for the increased niscapcibility to tion of liven from taro high-dose groups re a facultative intracellular organism such as 5 . n a l ad a nodular surface with variable degrees iem . These include: (i) e defect in call-me of hepatocellular cytomagaly and occaaioral diated immunity either through lose of mediat tod of angle call necrosis. A few inflammatory ing cells or by inhibition of call Unction; (ii) a cells (predominantly mononuclear) w en found defect on the humoral system; (iii) e defect in in the areas of necrosis. In addition, th e n w en one or more of tha steps of phagocytosis: and often intracellular accumulations aflipsd in bap- (tv) a reduction or loss'of phagocytic cells. atocytes, especially in midxonal an a s. Lesions Cell-mediated immune reactions as rri- w en not observed at lower doees or in other deneed by delayed hypersensitivity have been organs. shown to play an important role in immunity Ths lesions described w en still apparent in and recovery from infections with salmonella the mice from the two highest does groupe inoc and other fhcultativ* intracellular organisms ulated with virus or bacteria (both survivors (4. 15. 20. 22. 23, 25). Delayed hypersensitivity and nonsurvivors). In addition, mice in the bac is thought to be mediated through production of terial groups that died showed macro- and mic tympholrines by specifically activated T calls in roscopic lesions typical of salmonellosis (1. 26). response u bacterial antigens (4.20, 22.23,25). T h an was widespread infiltration of polymor TCDD has been shown to suppress delayed by- phonuclear leukocytes, particularly in visceral ptrsensirivity reaction* end respeeaea ofT cells organs. Focal necrotic hepatitis resembling mi- to activation by th* mitogens ccncsnavaiin A croinfjsrcoon was attributed to adjacent areas and phytohamaggiuonin (28, 29). These studies of vasculitis and thrombosis. Lymphoid necro did not investigate th* influence of TCDD on sis. thrombotic vasculitis, and suppuration lymphekine production, but it would be surpris w en also observed in the spican. Necrotic enter ing if TCDD did not also suppress their produc-. itis. purulent peritonitis, end occasionally sup tion since it has such s profound afreet on other purative meningitis w en also noted. T-csll Unction. In th e virus portion of the experiment, le Humoral (acton do not appear to be domi sions attributable to PRV were not observed in nant in impaired host resistance to salmonella any of the mice examined. PRV m laboratory infection in mice since ftill protection from chal rodents is known to product few lesions at the lenge appears to depend upon the immune re light microscopic lave! (21). sponse that leads to deiayed-cype hypersensitiv ity, rather than to that which rasults in area lac DISCUSSION ing antibody and trthus sensitivity (4). Tba The data presented in this study clearly estab afreet of TCDD on numeral antibody production lished that mica exposed to four weekly doses of in mica is not known. Secant studies by one of l. J. 10. or 20 ug of TCDD per kg and subse th* authors (R. E. Faith, unpublished data) quently infected with an L D m dose of S . b tm indicates that TCDD has no afreet on humoral have e significantly higher r u t of mortality antibody production to bovine gamma globulin with a shortened incubation period. Further- in rata. In contrast, th* guinea pig humoral m on. the mice exposed to 1or 5 ug ofTCDD per response to tetanus toxoid was obsarved to bs kg appeared clinically noitnal. had a normal impaired (Vos et al. [29D- ___ CD O CO 03 ^ *** v_ a. in s tccd-decreased host RESISTANCE TO INFECTION 1323 Alterations in til ftinctions of phegocyuc Ilf. idler polymorphonuclear or mononu clear. may be involved. This could result from neemsis at these calls and/or a decrease in pro duction or blockage of functional abilities of these cell*. A recent Kudy by Weissberg ami Zinkl '33> indicated no significant change in peripheral blood monocyte numbers in rata treated with TCDO. In addition, these authors report an elevation in peripheral blood neutro phil counts after TCDD treatment. Thu would, suggest that the defect in host rwsistancs is not the result of dastroction of circulating phago cytes. However, this dots not rula out the possi bility of death or destruction oftissue-fixed mac rophages. The number ofcirculating monocytes is small in relation to those that are tissue fixed, and the study of drculating monocytes does not ntcassarily rafiect tbs stats of produc tion or Auction of modonuclear phagocytes. In this light, it is of interest to noto the results of two studies (T. id) which show that treatment with agents that interfere with monocyta pro duction also suppress host resistance to beicte* riai infactioa . Studios designed to inveetigste the possibili ties of alteration in functional abilities ofphago cytic calls and production of monocytes in rela tion to dioan exposure are being undertaken in this laboratory. In addition, possible defectls) in cooperation between T calls tn d phagocytic ceils are being investigated. The results of these studies should lend insight into the mech anist si of suppressed host resistance to infec tion induced by exposure to TCDD. ACKNOWLEDGMENTS V w uh a (has* Mary Clamases. M anila W. Ham a, sad ia a a t 0 . Allas far axcallsat technics! an iata-- tad J . A ffaaa--ar far w sn sn tsl asaltea a. L H X IA T U IE CTTED L Baken. L . asd T. X . Vagriasag. 1*70- The yerbaga i T galesallm tyfkimmnom iatarqaa ia a j e a A n a PtaheL MieraWat. S e a . 27*1-40. X larchaiam av. 0. J . IMS. A taat t hamagasaity Car aran d altam adaas. Haniarriha I 1 J I tS. X C aar. C. D_ X 0 . K iatauugh J . A. Liadla. A E. Cliaa. A A Zack. Ja.. V. P. StnfcaL A A to a alar, sed P. A Phillips. 1BTI. Tatnabieredibeasadiema: i c codociaJ pnianning astasda ia haraa arana 3an a lUtTSS-MO. 4. Calima P. A . asd G. A Mackaaaaa. 19*4. Balayad h y p a n a n e a c itr aad anhus reacomty ia n la u a a ia fcsac i w a n n t ia S aim oM iU taiecud mica. J. l a s a - L etuno-us. X fln araa 0 . I C I Eualagy d d tid t adama iliaaaaa. Estoco. Baaith Pu spa . 1:31. I. Priead. A . aad 0 . O. Trataar. 1970. Pslychlsnaaaed Mpbesyi: iateracaea n O daca Srpauua n m . 3 a. t e s t 17*0314-131. T. fl.dahw x. l V J . i t RSadaa. aad 3. 0. L an as. 1 W . Iba aO baafi dataaa t - I Italad HBA aatihtdy -- r 1 hy tfctci caiU. l u v a i i i i y flt-T O . X GaiBar. J. A ISTX lam aaad oartaU ty ia la ta p h tlim y acardtaa tos Infartad a k a caaaumiag aabalt aaL fcu : n a raa ata rratit i a d w W t Am. X Vea. la s . ix30d7-nnx X Gaiaer. J . A . J. Laa. J r .. P. A HilL sed W. L Capea. I t t i . laactnraoae af che psaudanhia* Kwa bydkbia- thm aai. Vfaeiagy 4XS1-10O. 18. Gaiaar. i . A a b ST. W. Pry. I T I A fcm ssfsiu m im lt vm.a rira l infim a na ia sa. Am. J . Vea. R a a J W S S - U. BampdiU. P. A . A L. Kaabaria, asd W. A SwcA IPIL Laad sappramnm ai laaitae raaiataaea aa gtlw eaalie f f iii a i n a a . Saiwire 172U021-1Q3X IX Hallandar, A . aad 0 . A Walla. 1S7X Wasptram atrir aaiistirai mathad, Jaba WUey aad Seas. New Yard. IX lasah h ttra. A A 1M4. A H w nhnnaa frm Ir templa taat ag siast ardarad aitaraaaraa. Ilia ta rn lia I1U33. 14. Janas, A A . A L. Williams. aad A A Jaoaa. 1>TL E S a cu af haary n at al a s (ha mmuaa remanas. Prw bm insy flsdiafa b r cadmium is rats. frac. See. Exp. BiaL Mad. 137:1331-124. A Cantar. P. A 13TX Ufecoea. asargy ad cali wadlefad immunity. N. Eagi. J. Mad. 292*29-04. A Kimmig. J .. aad A A Safcuis. 1MT. W W liOia Akaa tsag. chlwehwat d s h alariene raw aneaba tyfc- liadas atkar. Dww iatalfiaa 111:340. 17. Callar. L. 0 . IS73. Immuaaauppteawao praduaad by laad. radmmm aad eiwaiity. A s . J . Ve. la a . H-J4S7-UU. l*. Kailar. L 0 .. ta d S. K erads. 1974. D im aaad aadbody ' tarm asse ia a la s axpaaad Ja laad. Natura llandas) 238:148-180. A Kailar. L D.. aad J . A riu fp a a . 197X ladriennn af aadbady w parirlarabias rtru s ia palrcUariaaSad bipneayl-espeiaed rabfatu. Am. i . Vaa. laa. 24UHS- 1*04. A lan a. F. C . aad A A Uaaaua. 197X lapuiram ant t dtrauia (Ti Ijwipliurirtm Car raainanra ia Baaan aait J. E n . Mad. U X U O t-U lX 2X M c fa rn a . J. B^ aad C. Qaw. itTO. Esparimaetal Aujaatfcy't tiaaaaa (paaadsrabtaai la rata. Br. Vat. J. 111:172-17*. 2X Mackaaaaa G. B. M . T ia inflnanct t iam uaolagi- tally raaanunad lymphrad c u lt on aucraphpga actv- dy la m m. J. Esp. Mad. I2T 3-**X & MauaL J . 1174. CU-madiaiad immunt laerhanwnn m bactanai aad imitasti ia /dcoont . P n g . Irttnannl U 4:10S-IM U . S a n A A J. 1970. SupntM-ion t cail-mad lttad iauau- aity is ia<tniaa by t a l a d a i l t d i drag. J . L p . Mad. 133^38-443. S . Baaton. J ,, J . MdsmaJa aad J. X Baaaagme. 1*4*. Stadias sa Uw sciam ani a f w a r n s la phyiagdaaacaily di ana mnacalliiJar argam aan J . Immu nol. 143^82-284. A Sm uh. A V. ISTX Tba taouaa. Aanam d. Baa. 19:11- 13. 27. Vatacnauraa. H. G.. A J . Ruitaabatg, T. Paataam. P W Hailaaiaa. ad G. J. *aa Eacd. 1970. Idlluanaa ot aipbaarltua r1a9a:4p0o0n--aa4a10t.a aCclaausswaapsiclsy.mTpahsacuacLciAaapupai.aaPdhairmaimaausrLa a . Vaa. J . a ., aad J. A Masra. 1974. Slips rasaiaa aTcallu- lar immiuucy is rata asd suca by o a ta rsa i uuatm ant with X J.T.Xiausdilatadibaiisn p dian n 1st. Aiub. Allergy 47:777-794. A Vaa. J. G.. J . A Moara. aad J. G. S ak i. 19TX Efltat o f 2J.7,-iagacilaradibanTa p diaain ae A a immuaa 01480102010001020000010 02000002020001010200539 ( 1324 THIGPEN ET AL. n<?ac!tiUtfOU-lfCca.nurp Binala la v in a . Haalth Par* 30- Va. i . G_ mmi H. t u fjadTn 1*13. T im ln p in l apaoa in n i i im pp; n . p. 527- M . In P m i- oda o r i ih* m n a a u a u a rnnuiw m y. J y B B M I Sm m IUm t f i a i i 31. V . J . G,, j T u m ! and i . fi. ZiaU. lftd . Taaidty t tA T J^ airaih U n v IitiO T p Hiann iTCOO) m C37BL1 Mia. TaucN. Appi. Pharmaaai. 2tl22P-2Al. In fc c t. Im m u n . a. W m nann. M.. D. V i a m t a a . Z. ( M m aad L. Zallaraayar. UW. ESacu t f n rp in a th lw in iaaacti- m n i mid d n a bady da& an tjra u aa. A aa. N.Y. A m i. S a . S . Wataafcarg. J .' i . ad J. 0 . ZiakL 1973. ESacta ot 7J.74.tm iilnndihaain p diaria apa banawaaia aad htam alnfic faacoaa ia tha rat. S avina. Haafth Panpact SOit-133. 0001020200010201000102010 00W 1 369176 ( 2740 O2019535 Shoecraft et al v. The Dow Chemical Company LABORATORY TEST RESULTS* OF KAUKO SATAMA -.i BLOOD COUNT HGB HCT WBC 23 or RBC MCV MCH MCHC PTT Control SR (Sed Rate) PRO. TIME Control PL. CT. DIFF. SEGS Lymphs Mono EOS Baso. Band 15.2 (3) 45.0 (3) 04 .1 _li> 4.92 (3) 091 (3) 30.6 (3) 33.7 (3) 7 (3) 255 ,000 60 (3) 35 (3) 2 (3) 2 (3) 1 (3) TESTS ORDERED BY PLAINTIFFS' COUNSEL: IgD FSH/RIA igE PLASMA ESTRADIOL TSH URINALYSIS: Color Appearance Specific Gravity pH Protein Glucose Ketones Occult Blood Bilirubin Casts/LPF WBC/HPF RBC/HPF Epithelial Cells Bacteria Crystals Mucus yellow iLLt--- 1--3-- 1J __ ill or^islunal BLOOD CHEMISTRIES Albumin Globulin 'A/G Ratio Total proteins Alkaline Phosphatase Bilirubin LDH SGOT SGPT GGT (GGTP) co2 Sodium (NA) Potassium (K) Chloride (CL) Calcium Triglycerides Creatinine Blood Urea Nitrogen Uric Acid T3 T4 Free Thyroxine Index Cholesterol HDL Cholesterol Total Cholesterol/ HDL LDL Cholesterol LDL/HDL VDRL VLDL 4.0 J J 2.3 (3) 1.7 (3) 6.3 (3) 76 (3) 0.5 (3) 254 (3) 41 (3) 43 (3) 23 (3) 32__ i l l 140 (3) 4.4 (3) 101 (3) 9.1 (3) 75 (3) 1.1 (3) 23 (3) 4.6 (3) 27. 1 (3) 7.6 (3) 2.06 (3) 201 (3) 57 (3) 3.53 (3) 129 (3) NR II OTHER/COMMENTS: GLUCOSE PHOSPHORUS 93 3.5 DIRECT BILIRUBIN 0.1 INDIRECT BILIRUBIN 0.4 i * (1) /3) Gila County General Hospital; 1 tl 1 ^ H r -aH o -a-HrNvi.-ie (I2d)i Diagnost,icandLabo5)ratOotrhye;r 16202 _ . , ---- - Sbitivoat-HmSei*d--icaL r-es-e'arch 1 25778 u u II fm\J i f 0 w>..jv _ * pj Avr - /iJ7 J (3wt~ w o o Nvy^cJL- Urcu- '^ v J ^X o (^^>C^AXlt, U^Lvyr^JX . ow p Y\OV p<j >k4 UTr+^atS&k-j Y1 / ^ 1 " S ? * ' , M.P.H. 2750 imuini in ir ihi -iff /* -*sr*:^ "P*"' REVIEW OF TRANSLATED ARTICLE; KARDELL, L. AND SANDSTROM, A. CASE-CONTROL STUDY. MALIGNANT MESENCHYME SOFT TISSUE TUMOUR AND EXPOSE TO PHENQXY ACIDS OR CHLOROPHENQLS. LAKARTIDNINGEN, ^'V0L:'75`, NO. 40, 1978, (sic) This was a confusing article to review: the Swedish to English trans lation was loose; references were hot included; the data was incomplete; and, there were multiple inconsistencies or contradictions incorporated into the body of the article. In this paper the authors addressed some interesting epidemiologic problems.; confounding, recall, selection, consistency, and causation. I am not convinced they solved any of them satisfactorily. In addition, the methodology they used, case-control while rapid and flexible is prone to greater bias than the more rigorous cohort approach. Although phenoxy acids or chlorophenols may be associated with malignant mesenchyme soft tissue tumors, this study does not prove causation. It simply points out the need for "continued investigations," Cases were drawn from patients hospitalized at the Oncologic Clinic in Umea, Sweden, 1970-1977. Males with any malignant mesenchymal soft tissue tumor (fibrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, osteosarcoma, etc.) were included. Each malignancy has a different age distribution of occurrence. Unless cases and controls were matched within fairly tight age ranges, the results were potentially con- founded. $ The selection factors leading to hospitalization at this, dlinipcwere not discussed. If foresters or pulp workers were more apt to be seen at this center, the implications are obvious, i.e., the relative risks were inflated. UU0703H DOW 1257786 16204 -..Their matching criteria were not adequately presented. As a rule of thumb, the reader should be able to replicate the investigation using the article as a guide. I would be hard pressed to do that in this case. For example, I don't know what they mean by "otherwise matching" on page 2. In addition, they apparently used different matching criteria for live and dead cases. They also chose 8 controls for each live case and 10 controls for each dead case, and then limited their analysis to a 4-to-l match. Why? They excluded suicides from the control group. Why? They made a point of selecting controls who had worked up to 2 years prior to death. Why? One matches to control confounding. Having matched in the design, one has to match in the analysis. They did not. The statistical methods they quote are Miettinen's. His methods do not have complete acceptance by biostatisticians and epidemiologists. What do they mean on page 3, under Statistical Methods, by "effect of s k"? What percent participation did they have? They suggest 100% among cases and 99.23% among controls. This degree of success is difficult to believe. Unless other investigations by the same authors demonstrated a high degree of epidemiologic sophistication (which this analysis does not), I would suspect a fair amount of selection bias has been incorporated into this study. Since they had such poor environmental measurements, they were really testing the risk of being a wood product worker. The chemicals tiey ; s y ` >?' i'- arbitiarily choose as labels were merely surrogates for the Workers' total exposures. One has to ask, what were the workers other chemical exposures, 05 16205 Was there a dose response in any sense? . For me, the study did a better job of presenting questions than answers. What is a Swedish municipality? What are the problems asso ciated with the register of population (RTB)? How did they use the register of dead persons? What was their definition of exposed? Was it greater than ''totally 1 day's length"? Why were "all persons in the region with the studies tumor diseases ... not investigated at the clinic (sic)"? In 60% of the cases and controls, exposure histories were obtained from a member of the "bereaved family"; can your spouse list your exposures of twenty years ago? R. R. Cook, M.D. 14 November 1978 DOW 1257788 DOW CHEMICAL U.S.A. 2757 November 10, 1976 / MIOLAND, MICHIGAN 48640 N. B. Crummett - 574 B. B. Holder - 607 R. L. Johnson - 2040 M. J. Mintz - 1710 R. D. Olson - 607 J/ E. S. Parsey - 834 B. A. Schwetz - 1803 W. W. Muelder - 9001 W. B. Seward - 9001 CHLORINATED DIOXIN ISSUE MANAGEMENT TEAM MEETING, November 17, 1976, 1:15 P.M., Planning Conference Room, Agricultural Products Department, 9008 Building. AGENDA M. J. Mintz Progress in the process for minimizing the TCDD in 2,4,5-T and silvex products. E. S. Parsey Appropriate timing for announcing new TCDD product spec. B. A. Schwetz Review of NIEHS meeting. B. B. Holder Comments gathered from conversations with those concerned with TCDD problems - Italy, etc. R. L. Johnson Status of studies to determine how the chlorinated phenolic Dowicides are used and their fate in the enviro:iment. i . fMdlf A N O PERA TIN G UNIT OP TH e O O W CH EM ICA L C O M PA N Y IC I OOW 1515900 W. B. Crummett et al Page two November 10, 1976 R. D. Olson a) Current status of employer monitoring study. b) Status of soil monitoring studies for TCDD around and in the Plant - including a protocol of theYstudy. W. B. Crummett Current status of EPA Dioxin Implementation Plan. W. W. Muelder Status of investigations on occurrence of chlorinated dioxins and furans in Dow products, W. B. Seward, J. H. Davidson Status of legislation and P.R. efforu to support phenoxy herbicides. J./H. Davidson Agricultural Products Department nac 62C8 DOW 1515901 AGRICULTURAL EXPERIMENT STATIONS U n iv e r s it y o f F l o r id a E v e r g l a d e s E x p e r im e n t S t a t io n 2799 December 23> 1964 B C LL K O U O C , FLO RID A CC Dr. Lawrence Southvick Agricultural Chemical Development The Dov Chemical Company Abbott Road Buildings Midland, Michigan Dear Larry The following comments are in response to Chuck Lichy's phone request concerning silvex (Kuron) for sugarcane in Florida. $ rc oo CO GO G. GO Information on the performance of silvex under our conditions has been sent Dov on several occasions in the fora of mimeo reports, data summaries and letters. Continued, long-time use of 2,4-D in parts of our cane area has per mitted the development of broadleaf weed populations resistant to 2,4-D. Prop erly timed. 2,4,5-T applications may control some, but not all, of these species effectively. These to which I refer are dogfennel, ground cherry, nightshade, purslane, ragweed, and wild, lettuce. Other low-growing, less competitive winter annuals which are less important in cane culture may not be controlled by 2,4-D and/or 2,4,5-T. The weeds which I listed are amenable to control'With silvex. I think that silvex is a needed and useful tool in cane culture which I would recommend if it were cleared. My use suggestions would be for postemer gence application of 1 - 1^ lb./A ai (and not over 2 lb./A in extreme cases) silvex to the species named after establishment of plant cane or ratooning of stubble cane up until the cane is closed-in. I would stress that the rate of application should be adjusted to weed maturity. Please contact me if you wish further information. Sincerely yours, JRO:sdt J. Ry^ORSENIGO Associate Horticulturist 162.09 2821 f Reprinted from B ioSci cnre The Public Health Implications of Widespread Use of the Phenoxy Herbicides and Picloram Julius E. Johnson DOW This paper is concerned primarily Table I shows a condensed version of Martin and Duggan, 1968;Corneliussen. with 2,4-D, 2,4,5-T, silvex, and tolerances proposed for 2,4-D, 2,4,5-T, 1969). picloram-- the phenoxies because of MCPA, silvex, and picloram. Those Air and water are potential sources widespread use; picloram, 2,4,5-T, and marked with an asterisk are for toler of exposure of man to these herbicides. 2,4-D more specifically because of their ances at a permissible level for 2,4-D in Manigold and Schulze (1969) have re use as military defoliants. The purpose rice and flax seed and for picloram in ported on pesticide residues in selected 01 this discussion is to explore possible forage grasses and meat.-The remainder streams in the West such as the Missouri, public health consequences of wide are negligibleresidue tolerances. Yellowstone, Colorado, Arkansas, Rio spread use. This presentation will be Table 2 demonstrates the infre Grar.de, Snake, and others. Phenoxy wonfined primarily to information as it quency of residues of phenoxy herbi herbicides were detected as shown in Table 3. Of the 320 samples taken, fractional parts per billion concentra TABLE 1. T olerances proposed to FDA tions were reported in 78 samples. The 2,4-D 2.4.6-T MCPA Silvex Pici oram measurement of picloram residues in water has been reported following direct Fruits XX X spraying o f a static pond in Texas Berries XX (Hoffman et al., 1969) and a -watershed Grains XXX X experiment was conducted in North Rice X* X X X Carolina using 2,4-D, 2,4,5-T, and pic Flax Seed X* X loram (Sheets and Lutz, 1969). The Potatoes Sugarcane Hay Forage Grasses Meat X results of these studies are summarized XX X in Table 4_,lt is noted that picloram XX X* persisted longer than 2,4-D or 2,4.5-T. X* Air samples collected in spring and Milk X summer from two wheat-growing areas (All negligible residue tolerances except th o se m arked*) in the State of Washington were ana lyzed for phenoxy herbicides by Bames- berger and Adams (1966). The results may translate to the health and safety cides in foods asconfirmed by (he FDA are summarized in Figure 1. Phenoxy of man. The data presented will empha market basket survey of pesticides in herbicides are widely used for early size exposure, toxicity, and chemistry. composites of 12 categories of food postemergence control of weeds in these First, Iwill comment on exposure to stuffs. Of the 1548 samples analyzed, crops. There were detectable levels of food, w;:er, and air. There isvery little only 36 positive identifications were 2,4-D and 2,4,5-T in the air up to 0.06 exposure of man to the phenoxy herbi made. All but one was less than 0.2 iig/m3.Man will inhale, about 30 m J of cides through food. The principal uses ppm. 2,4,5-T was found in three of the air per day. At a level of 0.06 vg/m 3 , of these herbicides in growing food surveyed samples (Duggan et al., 1967; this would amount to an exposure of crops were originally registered on a no-residue basis. A change in policy now requires these registrations be supported TABLE 2. FDA m arket basket survey pesticide residues 1965-68 by a negligible residue tolerance. An industry task force has formally re Number of Semples with Residues of 1548 Samples Analyzed quested FDA to establish these toler ances. (The request for 2,4,5-T has been temporarily withdrawn without preju dice to future filing.) The a u th o r is V ice-President and D irector o f Research Dow Chemical Company. Presented at ihe Sym posium on Possible Public H ealth Im p lications of W idespread Use of Herbicides (Chairm an: Dr. Arthur Galston) Am erican Institute of Biological Sciences Bloomington. Indiana, 26 August 1970. 2,4-D MCPA 2.4.5-T Silvex PPM 0.001 - < 0.1 0.1 - < 0.2 23 4 21 21 20 0.4 0 1 0 0 Total 27 4 3 2 Pesticide M onitoring Journal. 1967, 1968, 1969 Duggan, R. E., H. C. Barry, an d L. Y. Jo h n so n , 1967. Pestic. M onit. J. 1(1): 2-12. M artin, R. J ,, and R. E. Duggan. 1968. Pestic. M onit. J. 1(41: 11-20. C orneliussen, P. E. 1969. Pestic. M onit. J. 2 (4 ): 140-152. ^ 0 j *f0 h- rc CD CO CO % S ep tem b er 1, 1971 899 D O W 7.12333 1.8 *ig phenoxy herbicide per day per TABLE 3. Rssiduea in w ater trom 20 Commercial 2,4,5-T containing leu man. For a 70-kg man, this would be 0.025 >ig per kilogram body weight per day of exposure. A variety of pesticides were reported in dust trapped in rain collected in Cincinnati in 1965 (Weibel etal., 1966). The dust was created by high winds over the southwestern United States and m onitoring sites in 16 w ettern states 1965-68 2.4-D 2.4.5-T Silvex No. Positive par 320 Sample 36 28 14 Paris per Billion 0.01 - 0.36 0.01 - 0.07 0.01 - 0.21 than 1 ppm of 2,3,7,8-tetrachiorodibenzo-p-dioxin was administered by gavage togroups of pregnant female rats on days 6 through 15 of the gestation period. The doses of 2,4,5-T adminis tered were 0 (control), 1,3, 6, 12, and 24 mg/kg/day. No clinical or gross pathological signs of adverse chemical moved in a cloud in a northeasterly [Pattkida M onitoring Journal. Menigold and effects were observed in treated dams direction to Ohio where the precipita Schulze, 1969) during the period of treatment or gesta tion occurred. An amount of 0.04 ppm of 2,4.5-T was determined to be present based on the airdried weight of the dust in the rain. No value was given for the amount of dusi in the water. If it were as much as 10%, the water could have contained a 4 ppb of 2,4,5-T. The significance of these findings are open to question, however, because of the (1) difficulties in analyzing for such small amounts, and (2) the alleged presence of a newer pesticide, ronnel, which isused in relatively small amounts as an addi benzo-p-dioxin in amounts which might have been a complicating factor. The sample employed in the NCI study was analyzed and found to contain about 27 ppm of 2,3,7,8-tetrachlorodibenzo-pdioxin. Teratology tests with 2,4,5-T and the impurity were conducted in Dow laboratories. Results of the first experiments were observed by represen tatives of the Department of Health, Education & Welfare. tion. Fetuses were harvested by cesarean section on day 20 of gestation. Visual and histopathologic*! examination of the fetuses failed to reveal any terato genic or embryotoxic effects (Emerson etal., 1970). In another test, commercial produc tion grade 2,4,5-T, given to rats at a dose of 50 mg/kg/day from days 6-15 of pregnancy, produced a minimal amount of fetotoxicity with one fetus in 203 having intestinal hemorrhage. When tive to cattle feed and asa topical spray on cattle. The results are summarized in TABLE 4. Residues in water Table 5 Direct skin contact together with vapor and mist exposure are encoun Type of Treatment tered in varying degrees by applicators and employees :;i manufacturing plants. Direct pray on pond Tile exposure of an infrequent user is luch less than professional pest control operators or those engaged in manufac Spray on 25% of watershed turing 5 days a week. A medical evalua tion of the effect of 2,4-D and 2,4,5-T has been made of men engaged in their manufacture. The v/orkmen were given extensive physical examinations includ ing a battery of at least 20 laboratory Herbicide Lb/Acre Pictoram A 2,4-0 2 .4 .5-T Picloram 4 4 4 PPB in W ater 2400 700 150 6 1800 40 680 90 4200 340 Days A fter Spray <1 1 23 180 2 21 2 21 2 21 tests. The results of these studies are given in Tables 6 and 7. No meaningful Following is a description of the commercial grade 2,4,5-T was given at differences were noted when the clinical series of experiments concerned with 100 mg/kg/day, a high incidence of results on these workers were compared the teratology question. maternal deaths occurred, and of the to those obtained on a control popula pregnant animals that survived, most tion of 4600 men not exposed to 2,4-D had complete earlyresorptions. No fatal or 2,4.5-T. An important observation anomalies were observed as a result of was that there were no chromosomal the administration of the commercial effects.1 production grade 2,4,5-T to rats. Like In the fall of 1969, considerable wise, no abnormalities were noted m apprehension was created in the minds litters of treated dams which were of the public when an announcement allowed to deliver and raise theiryoung was made of tests sponsored by the through lactation. Treatment had little National Cancer Institute reportingthat effect on the survival of pups through 2,4,5-T was teratogenic. Following that this time period.1 announcement, imade special efforts to trace the identity ol the samples used Rabbits (New Zealand White) were and reported to representatives of the used in another experiment to deter National Cancer Institute and the Mrak Commission that the test sample proba- p Par i iCui l i t V VAPOR mine the embryotoxicity or teratogen icity of commercial 2,4,5-T containing eontained 2,4,7,8-tetrachlorodi- lessthan 1 ppm of 2.3.7,8-tetrachlorodi- Pig. I. Phenoxy herbicide* in air (Bamca- 1U npublished dala, 1970. Dow Chemical Cu. berger and Adams, 1966). 2 U npubiished d a ti, 1970, Dow Chemie! Co. 900 BioScience Vol. 21 No. 17 DOW 712940 2.4.5-T 0.04 2,4.5-T, and (2) pure 2,4,5-T to which solvent. Although the p u n 2.4.5-T was specific amounts of the 2,3,7,8-tetra- reported to have shown teratogenicity chlorodibenzo-p-dioxin were added. The dosages of 100 mg/kg were required ti benzo-p-dioxin. Groups of pregnant resuits of these srudies show (1) that a produce the cleft palates oi kidnes female rabbits were given the compound dose of 50 mg/kg/day of 2,4,5-T from involvement.'4 by gavage from day 6 through 18 of day 6 thtcu.,.. 15 of pregnancy Steinfeld5 also reported terata :r gestation. The doses of 2,4,5-T adminis (maximum tolerated dose for our strain hamsters injected with commerua- tered were 0 (control), 10. 20, ar.d 40 of rats)did not cause teratogenic effects 2.4,5-T containing < 1 ppm 2..'-.7 * mg/kg/dav. Fetuses were harvested by in the younz. and i2) that when a toxic tetrachlorodibenzo-p-dioxin at a dose o' cesarean section performed on day 29. dose of the 2.3.7.8-tetrachiorodibenzo- 100 mg/kg/day. Terata were ai.vn ob No clinical or gross pathological signsor p-dioxin was superimposed upon this served in hamsters injected with 2o mg adverse chemical effects were observed dose o; 2.4.5-T, cleft palates were of 2,4,5-T containing 45 ppm of the durin the period of treatment or gesta observed in a few fetuses in addition to cetrachlorodibenzo-p-dioxin per kg/dav tion. ::*ainnation of the fetuses showed no Jose-related teratogenic or embryotoxic .fleer (tmerson eta!.. 1970). TABLE 6 . Industrial health experience--m an u factu re of 2,4-D th e D ow Chem ical Com pany In order to check the effect of the I. contaminant itself, a group of oregnant No. Men Exposed Work Experience Range of Exposure Results rats were admmistered 2.3,7.3-tetrachlorodihenzo-c-dioxin by gavage on days 6 through 15 of gestation. The dosages used were 0 (control), 0.03, 0.125, 0.5, 2.0 and 8.0 M g /kg /b o d y weight/day. The fetuses were harvested on Jay 20 of gestation. No differences were observed in the fetuses taken from 220 0.5-22 years 30-40 mg/da No differences wher compared to rnniro p o p u latio n of 4 6 0 0 me> II. 10 m en exposed to 2 .4 -0 w ere k ary o ty p ed . Result. No efleci on structural integrity or rearrangem ent of the genetic m aterial of the lymphocyte chromosomes. dams treated at the dosage of 0.03 Mg/kg/day and those taken from dams that received the solvent vehicle only. At the 0.125 Mg/kg/day dose level, there was evidence of intestinal hemorrhage and subcutaneous edema in some of the fetuses. At the 0.5 and 2.0 Mg/kg/day doses, there were a substantial number ot fetal deaths. Intestinal hemorrhage in the fetuses was frequent. The 8 Mg/kg/day dosage was toxic to the dams. There were no viable fetuses in the dams which were examined on day 20 of gestation. All resorptions occurred early and no evidence of fetal tissue was the typical toxic manifestations of the 2,3,7,8-teiras hlorodibenzo-p-dioxin when given ai.ir.c However, it is very important i.- note that when no effect levels of 2.3.'.h-tetrachlorodibenzo-pdioxin and 50 mg of pure 2,4,5-T were combined, no teratogenic and only minimal tetmovic results were ob served.3 The results ate summarized in Table 8. ^Unpublished dia. 1470. Dow Chemical Co. Verrett has reported that 2.4-D 2.4,5-T and silvex produce terata and chick edema syndrome following injec tion into the yolk sac. The extreme ^Testim ony before the U.S. Senate Com m it tee on C om m erce. S u b c o m m itte e on Energy Water, Natural Resources and the Environ* m ent. IS April 1V70. Dr. Jesse Steinfeld pages 167-149. Report ol the Committee Serial 91-60 (1970). ^Testimony before the U.S. Senate Commit tee on C ommerce. Sub-Committee on Energy Water, Natural Resources, and the Environ m e n t. 18 J u n e 1970. Dr. Jesse S te in fe ld found. Skeletal examinations revealed delayed ossification of some stemebra and skull bones. This occurred generally tliroughout the various groups, includ TABLE 7. Industrial health experience--m a n u fa c tu re of 2.4.5-T , th e Dow C hem ical C om pany N um ber o t Djvs E xposed to 2-8 mg 2.4.5-T Daily 1. Number of Men Exam ined Results ing controls, and was not considered to be of practical significance. The results of this experiment indicated a high level of maternal and fetal toxicity to be associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Its presence in 2,4,5-T 1 - 59 6 0 -1 1 9 120 - 239 240 - 479 480 - 959 960 + 24 \ 15 1 160 1( 61 31 No differences when com pared to control population of 4600 men at levels of 278 ppm could well have accounted for the observations reported and attributed to 2,4.5-T in the original NCI study conducted by the Bionetics Laboratories (Sparschu et al.. 1970). II. 52 men exposed to 2.4.5-T were karyotyped. Result: No effect on structurel integrity or rearrangem ent of the genetic m aterial of the lym phocyte chromosomes. September 1.1471 901 ;1 DOW 712941 sensitivity of this test procedure makes TABLE 9. T eratology study of 2,4,5-T w ith (heap it difficult to interpret the meaning of the results from a public health point of view.6 Compound Amount Fed No. of Ewea Number Pregnant Results Bmns of Utah has tested the effects of ^.S-T exposures on the 14th to 36th day of gestation of sheep, the period in which they are most suscep tible to teratogenic effects from the plant V e r a tr u m c a l i f o m i c u m 7 *. No anomalies were noted as a result of the exposure to 2,4,5-T(seeTable 9). A pilot study to determine the toler None 2,4,5-T Dow Prod'n. PQBE eater of 2,4,5-T Dow Prod'n. None 20 100 mg/kg (a) 100 mg/kg (a) 11 11 15 10 9 l1a5mebws.e1i bore tw in live, . norm al 1 ewe eborted 28 days fetus normal. 10 ewws bore live, norm al lamba. 7 awes bore live, normal lambs, 1 twin. 1 awe died 36 days, ance ot pregnant female ratstosilvex in aspirated feed. Fetus prcpaiation to conducting a conven norm al. tional teratology study in rats has been completed. Silvex was fed to groups of pregnant rats (5 to 6 per group) by 1 awa died 35 days, profuse diarrhea from 7th day. Fetus normal. eavagi on days 6 through IS of the W. Binrts. Poisonous Plant Research L ab o rato ry , USD A , Logon, Utah gestation nenod. Doses given were 100, ` C om pound given by stom och tube..m ixad w ith 0 .5 lb. ground alfalfa. Dosed 14-36 days of ;.'.C. 200. and 300 mg/kg/day. There gestation. Varatrum c a lifo m icu m fed this p eriod induces cyclopia, sh o rt m etatarsals and wet no eltects on either the dams or m etacarpals, harelip and cleft palate. letu-.es harvested on day 20 by cesarean section at ihe dose level of 100 mg kg/day. At 150 mg and above there was loxicity noted in the dams with an csing amount of maternal mortality with increasing dosage. No teratology was ol'served by gross examination in fetuses at these levels; however, there :-r 'om.' resorptions.3 1 eiitology experiments have also 'cert conducted with 2,4-D. To date we avc concluded studies on pregnant rats with 2,4-D acid and with 2,4-D propyl ene glycol butyl ether ester. In this study, involving about 320 rat litters, wc administered com oil as a control, the acid form of 2,4-D at dosage levels of I2.5, 25, 50, 75, and 87.5 mg/kg/day and the ester form at dosage levels of 20, 40, 81. 122, and 142 mg/kg/day. About one-half of thelitterswere taken by cesarean section on day 20 of gesta tion for examination for soft tissueand skeletal abnormalities and the other half were allowed to go to term. The off spring were observed for 21 days, after which they were sacrificed and exam ined. Fetal weights were reduced at a dose of 50 mg of 2,4-D acid/kg/day, and at a dose of the esterat 121 mg/kg/day. No teratogenic effects were noted. Among litters delivered naturally, treat ment with 2,4-D during pregnancy had no effect on neonatal growth and sur vival.9 viability, lactation, by body weight, and by teratologicalexamination offetuses.10 A pilot study has been concluded to determine the tolerances of pregnant female rats to picloram in preparation for a conventional teratology study. Picloram was administered to groups of pregnant rats (5 to 6 per group) by gavage on days 6 through 15 of the gestation period. The doses.given were 250, 500, 750, 1000, and 2000 mg/kg/day. There was no adverse effect on the dams, nor to fetuses taken at day 20 of gestationatthe levelsof 250, 500, and 750 mg/kg/day. At the 1000 mg/kg/day dosage, there was no adverse effect on the dams, but there was a Groups of male and female ratswere small increase in the absorptions. The T v.'im on y before the U.S. Senate C om m it tee on ('iimmerce, Sub-Committee on Energy, Water. Natural Resources, and the Environ m en t. lit June 1970, Dr. Jacqueline Verrett, paees 190-203, Report of the Committee, Serial 91-60 (1970). maintained on diets containing 0, 3000, 1000, or 300 ppm picloram through a three-generation reproduction study without evidence of adverse effect as fetuses harvested showed no teratogenesis. At the 2000 mg/kg/day level, only one dam survived to the 20th day of gestation. Eleven fetuses were har 7 llinns . W. 19 70. P o is o n o u s P la nt R es e ar ch L aboratory , U.S.D.A., Logan, Utah. Unpub lished data. ^Unpublished data, 1970. Dow Chemical Co. judged by indexes of fertility,gestation. 9Unpublished data, 1970, Dow Chemical Co. vested from this dam all of which were viable and appeared normal by grqss observation.10 Since October 1969, great interest T A B L E a. T eratology stu d y io rats 2 >3 .7 i8 -tetrach :arcd ib en zo -p -d io x in added to 50 mg pure 2.4.5-T/kg body w t/dav has been created in 2,3,7,8-tetrachlorodibenzo-p-dioxin as a possible serious and continuing contaminant in the Treatm ent No. of Litters No. of Fetuses Litters with U tters with environment, it is important to empha Cleft Paiate Intstni. Hem orr. size that the tetrachlorodibenzo-p- C ontrol (vehicle) 50 mg pure 2.4,5-T per kg +0.01 ug TCDBD +0.03 ug TCDBD + 0 .0 6 M9 TCDBD 125 pg TCDBD 17 17 15 1177 15 194 0 172 0 150 0 173 0 155 1 134 0 0 0 0 dioxin is no longera significant contam inant and probably never was in carefully prepared commercial 2,4,5-T. 0 When it did occur-in 1964 and 8 1965-the contaminant carried over 11 from the intermediate 2,4,5-trichloro- ug TCDBD ^ . .0 tJQ TCDBD 14 76 4 13. 15 46 5 8 10Unpublished dan, 1970. Dow Chemical Co. 90 : BioSckncc Voi. 21 No. 17 was established to ensure that no more than minimal amounts would occur in the product (the best method available had a sensitivity of 1 ppm). Improved assay methods now show the actual contaminant is less than 0.5 ppm. ______ 0 .0 4 7 a / 100 ml 7 g /l o o mi dioxin in the environment are exreedingly small. Concern has been expressed that the combustion of 2.4,5-T (even though the + 2 NoCl 24 C (75 F). This evidence of degrada tion is so slow it wQI require further observationsto quantify.11 2,3,7,8-tetrachlorodibenzo-p-dioxin does not move in soil. It is strongly adsorbed on clay and organic matter and ispractically insoluble in water (0.2 parts per b iliio n ). First studies on up take by plants indicate none from water and very little, if any, from soil. 12 o parent material contained no tetracnlorodibenzo-p-dioxin) would cause dioxin formation. A laboratory experi ment was conducted wherein agent Orange (a defoliant containing equal quantities of the n-butyl estersof 2,4,-D and 2,4,5-!') was applied to 18.5-cm filter paper at rate of 10.9 kg per 0.41 hectares. This isequal toapproximately 4.5 kg of 2,4,5-T acid equivalent per 0.41 hectares. The paper was burned and combustion products plusairdrawn through ar. absorber packed with glass TABLE 11. Solubilities O (g/100 ml o f so lu tio n a t 25 C) ------------------------ ---- Solvent W ater 8enzene Xylene Picloram 0.043 0.02 - P icloram , Potassium S a-ltv T_ 40.00 h - 4 0-.01 O CJD beads cooled with liquid nitrogen. The combustion products plus the ash was solvent extracted and analyzed by gas chromatography (Fig. 4). The burning Hg. 3. Effect of radiation from G.E. sunlam p a t d istan ce o f one m eter on 12 p p m aohition of 2,3,7,8-tetnichlorodibenzo-p-dioxin. temperatures encountered were as follows: *F *C Paper surface 600 -8'JO 300 -400 Body of flame 1000-1200 550-650 Incidentally, the 2,4,5-trichlorophenol Front of flame 1300-1400 700- 750 submitted by the National Cancer Insti There was no 2,3.7,8-tetrachlorodi- tute in 1964 to be tested by the benzo-p-dioxin detected in the combus Bionetics Laboratory d id n o t ca u se a tion products at a sensitivity of 5 ppm. tcratological response. This was a A similar experiment was tried using sample procured from Coleman wood as a substrate, but there were Matheison Bell who got it from substances in the combustion products McKesson Robbins who got it from which interfered with the analysis. Dow. Speculative claims are widespread that The following information is known 2,4,5-T residues on vegetation might be about the stability and properties of the converted to 2,3.7.8-ietrachlorodiben- 2,3,7,8-tetrachiorodibenzo-p-dioxin: (a) zo-p-dioxir. ifthe dead foliage isourned. in solution it is rapidly degraded by All available evidence to date indicates ultraviolet light at wavelengths that this conversion does not occur. The appear in the spectrum of the sun (Fig. alleged precursor is in dilute form on 3);(b) h e t e t r u dioxin has one-fifth the the substrate, the reaction isbiomolecu- solubility of DDT in water and one- lar-the molecules must be forced close sixteen hundredth the solubility of DDT together to react. in benzene (Table 10). Thus there is Concern has also been expressed that probably less tendency to concentrate traces of preformed dioxin contami in fat, but the question is really aca nating 2,4,5-T sprayed into the environ demic because the quantities of tetra ment would accumulate. The evidence Fig.4. Silvex is also made with 2,4,5-trichlorophenol as a starting material. A total of eight lots of silvex has been analyzed, obtained from 1967, 1968. and 1969 production. No detectable amounts of 2,3,7,8-tetrachlorodibenzop-dioxin have been found using a method having a sensitivity of 0.1 ppm. Before leaving the subject of contami nating chlorodibenzo-p-dioxins, it is noteworthy that no detectable dibenzop-dioxins (including the 2,3.7,8-tetrachloro- or the 2.7-dichloro-) have been found in 2,4-D samples analyzed. The sensitivity of the analytical method used was 1 ppm.13 ^U n p u b lish ed data. 1970, Dow Chemic.il Co. 1 3 Private c o m m u n i c a t i o n f r o m Dr. P. C. Kearney, 1970, USDA, Beltsvillc, Md. 13Unpublished data, 1970, Dow Chemical Co. S eptem ber 1, 1971 903 10214 OVV 712941 TABLE 12. A cut oral toxicity, ung* oral dote potential to occur as residues in foods 2.4-D 2.4,5-T S lv e x Picloram DDT P2.a3ra.7th.8io-tnB trachlorodibanzo-p-dioxin LD50 8` Milligram! par kg Body Weight R at G uinea Pig 3 7 6 .0 - 666. 1000.0 500.0 380.0 500.0 850.0 8200.0 3000.0 1 5 0 -8 0 0 .0 * 400.0 1 .7 -3 0 .0 * 9.3-32.0* 0.022 - 0.045* 0.0006 and water or to come in contact with nontarget organisms. Degradation of herbicides is accelerated by increased temperature, soil microflora, and or ganic matter. A comparison of degrada tion rates in soils has been published by Kearney et al. (see Fig. 5) (Gunther, 1969). It is true that picloram is more persistent. Unlike DDT, however, aV a rin according to vehicle or tax. picloram forms salts that are water soluble. The solubilities are shown in Table 11. Picloram does not concentrate The formation of 2,7-dichlorodi- over, 2,7-dichlorodibsnzo-p-dioxin is in fat. Moreover, the-mammalian tox benzo-p-dioxin does not occur in the more difficult to form than the icity of picloram when compared with manufacture of 2,4-dichJorophenol 2,3,7,8-tetrachlorodibenzo-p-dioxin most other herbicides is low. The po since itismade by directchlorination of even when reaction conditions are favor tential hazards from picloram will result phenol and not by alkalinehydrolysisof able. primarily from effectson sensitiveplant a trichlorobenzene. Reaction conditions The persistence of pesticides in soil species. Up to the present time the do not favor biomolecular condensation and water isan important factorintheir bioassay is more sensitive than chemical to form a chlorodibenzodioxin. More- T A B LE 13. N o-effect level (m g/kg/dey) Species 2 .4 -0 2.4,5-T Silvex Picloram R eproduction and Frtil ity Rat 25 - 150 2-Year Dietary Feeding Toxicity Rat 62 - 5 150 Dog 12 - 5 150 TABLE 14. Grass p ro d u c tio n --Panam a Tons of Vegetation/A cre 15 M onths Deferred Grazing Treatm ent Jaragua Grass None 0.6 lb. picloram + 2.4 lb. 2.4-0 Per acre 1.80 13.55 Swezey and M ontano, 1968 (26) Other Grasses 0.28 0.29 Live Brush 12.65 0.50 T o ta l 14.43 14.34 techniques for determining the presence of picloram. MONTHS b ^,. o. Persistence o f herbicides in sol. Length o f bar represent! tin e required fo t 75 to 100% lot* o f the co m p o u n d (after K earney, 1969). The acute oral toxicities of 2,4-D, 2,4,5-T, silvex,and picloram are given in Table 12, in comparison with DDT, parathion, and the 2.3,7,8-tetrachlorodibenzo-p-dioxin. It is evident that this dioxin is highly toxic, in fact one of the most toxic substances known. This alone isa good and sufficient reason to keep this impurity at an absolute minimum. By contrast, the low order of acute toxicity of picloram is also of interest. The no-effect levels attained in long-term dietary feeding studies also indicate a low order of toxicity of picloram. Concentrations were not tested above 150 mg/kg/day (see Table 13). The reproduction and fertility tests were conducted through three genera tions of rats receiving continuous feeding of the test compounds. No birth anomalies or other adverse effects were observed with continuous intakes of 25 mg/kgyday of 2,4-D or with 150 mg/kg/day of picloram, respectively. Some of the benefits of using herbi cides are illustratedinTables 14 and I5. The resulting increases in beef produc tion are important to nutrition and 904 BioScience Voi. 21 No. 17 < .. : ( TABLE 15. G rau p ro d u c tio n --O klahom a A p p lication Per Acre Weed None 2 lb .-2.4,5-T 3 'b.-2.4.5-T 4 lb.-2.4.5-T 4 lb .-2.4,5-T Oaks Oaks Oaks Osks Oaks Eiw ell. 1964 (27) M onths Deferred Grazing 6 6 6 6 6 G rau, Lb./Acre Wet Years Dry Years 862 2487 2266 2917 * 348 2200 2063 2083 2750 health of man (Gunther, 1969; Swezey important factor-particularly the and Montano, 1968). chlorodibenzo-p-dioxins-but these can In conclusion. 1set forth the opinion be controlled by proper manufacturing that the widespread use of phenoxy techniques. As always, care in applica herbicides has produced no demonstra tion isan important part of safe practice ble evidence of potential harm to man. both from the standpoint of man and The herbicides used most widely (2,4-D the ecosystem in which these tools are ami 2.4,5-T) are degraded and do not used. bioeoncentrate. Moreover, the compara tive toxicity shows these materials to be w ell tolerated in a variety of test sys tems. Man is not exposed to harmful c u n centrations. Impurities can be an References B am esberger, W. L., and D. R. A dam s. 1966. O rganic pesticides in th e environm ent. Advan. Chem Ser C o rn eiiu u en , P. E. 1969. Pesnc. M onit. J 2(4): 140-152. D uggan, R. E ., H. C. Barry, and L. Y. Johnson. 1967 Peslic. M onit. J.. 1(1): 2-12. Elwell, H. M. 1964. A gron. J. 5 6 : 4 1 1 --415 Emerson, J. L.. D. J. T hom pson, R 1 Strebing, C. G. Gerbig, and Robinson. 1970 M anuscript subm itted for publication to F ood and Cosmetics Toxicology. G unther, F. 1969. Residue Reviews. 29, Springer-Verlag, New York. H offm an, G. O ., E. D. R obison, and M. G. Merlcle. 1969 WSSA ABST. 75. Las Vegas, Feb. M anigold, D. B., and J. A. Schulze. 1969 Pestic. M onit. J.. 3: '124-135. M artin, M onit. R. J. and J.. 1(4): 1R1.-2E0.. Duggan. 1968. J'erric S heets, T . J .. and V. F L utz. 1969. B ull N. C. E xp. Sea. S panchu, G. L ,, F L. Dunn, and V. K. Rowe. 1970. M anuscript subm itted for publication to F ood and Cosmetics Toxicology. Sw ezey. A. W.. and A. M ontano. 1968. D ow n to Earth. Summer, p. 6-9. W eibel, S. R ., R . B. W eidner, J. M. C o h en , and A. G. Christianson. 1966. J. A m e r Water Works Assoc. 58: 1075-1084. o O Ci* t * r 16216 2832 t pie tnd Retpir. IL r. 73, : to 111 deity in PtrftoL, >0 from nocteie i Appi. ractioBS '76L tin and Ihini in i ty luat, in stsaa in CjS C (j& n C A c . xo/fru X s /f / o *j Co <-C>G<J. 7 0*177} 7 IMMUNE SUPPRESSION AS RELATED TO TOXICOLOGY H oc& Ji ft> > 8 l-6 Z > 4. I Author: J. G. Vos laboratory of Pathology National Institute o f Public Health Bflthoven, The Netherlands pA/tTl+t, 7CVT- AJt-V___ i tr*-t-.. W, ' lUfarM: John A. Moon N atio u l Instituta of EnTiranniantal Haalth Sdaaeas Kasaarcti Triangla Park. North Carolina oO O "NJ ro -3= ro i O *c*o oCoO oCO CD & r 8 V kr INTRODUCTION Aberrant or defective immune responses are known to be the resuit of primary immuno deficiency diseases, e.g., in the DiGeorge syndrome (thymic hypoplasia) in man or in homozygous mice with the mutation " nude" (nu/nu), which are hairless and suffer from congenital thymic hypoplasia (Figure 1). Immune deficiency can also be secondary, e.g., a result o f undernutrition, irradiation with y- -or X-rays, neonatal thymec tomy, treatment with antilymphocyte serum, treatment with certain chemicals, and during in fe c tio u s diseases. R egarding chemical compounds, reiativeiy much information is available on the small number of immuno suppressive agents used clinically for immune suppression, e.g., some thiopurines and alkylating agents (reviewed by Bach).1 Surprisingly, only a limited number o f chemicals have been shown in toxicity studies to have immunosuppressive properties, and particu larly the cell-mediated imrmmity has been poorly studied. A likely reasotTfor this fact is that current procedures for toxicity testing1"* underestimate the importance of the immune system: lymphoid Organs and in general the immune system have, been poorly examined. Therefore, in-depth investigations were performed* with only a few chemicals as to their effect^ on the immune response. This raises the question o f whether these chemicals represent only the tip o f an iceberg, a question which can only be resolved by adequate toxicity testing that includes careful examination o f the immune system both morphologically and functionally. In this review, U will be stressed that Imowlegde from the expansive growing immunology discipline should be incorporated into the field o f toxi cology. First, the immune system and Jie immunological response wiQ be introduced. Next, attention will be given to the interdependence of the immune system and the macrophage system. Of special importance in toxicology is the discrimination between direct effects on the immune system and indirect effects such as nutritional deficiencies, pathogenic organisms, and changes in the endocrine balance. In this light, procedures are presented to detect immuno suppression in routine toxicity studies. Further more, function studies will be discussed, o f the cell-mediated immune system, the humoral immunity, and the macrophage system: these function tests are necessary in order to gain insight into the mode o f action of the chemical and to determine the functional significance * ' | May 1977 i - `V ffce-V.J;" i-V - fc.v <?' S fympitoid organs, tint increase (he susceptibility to infection, or that impair the immunological responsiveness. These parameters are considered important in the evaluation o f the toxicity o f these chemicals. Chemicals for which in depth information is available (including data on an indirect or a direct action o f (he chemical) will be discussed first. In addition, chemicals whose effects require further investigations will be reviewed. 0 2^,7,8"TetTaciilorodibcnzo*p-dioxut (TCDD) H ie compound 2 ^ ,7 ,S*tetrachtorodibcnzo-p- dioxin (Table 3) is a highly toxic impurity that may be formed during the production o f 2,4,5-tridilorophenol. Buu-Hoi et al.*3 were the ibst to describe, in addition to hepatotoxicity, severe thymus atrophy in rats exposed to TCDD (Figure 3). Since this first report, several studies with rats, mice, and guinea pigs have confirmed thd extended this finding.* 3,13 7 In contrast to the aduil animal, rat and mouse pups that are. exposed during the perinatal period by maternal . treatment *do not show major liver pathology, while, severe effects arc seen in the lymphoid organs, particularly in the thymus.*7 Liver lesions are mUd in the adult guinea pig when compared with the severe atrophy o f thymus and . peripheral lymphoid organs.* * As shown by Moore, and Faith13 (Figure 7), rats exposed during the pre- and/or postnatal period by maternal treatment with TCDD had significantly lower body and thymus weights it weaning age. At an age o f 39 days, pups exposed only postnatally gained more weigh! than animals exposed during the gestation and nursing period. After 145 days, the body and thymus weights o f the former group did not differ significantly from TABLE 3 The Effect of 1 ^,7WcinCUoeotoaioy d to d n on SusceptMBty to Infection and on Varions Immunologic P u o N ttn j g CD 00 CD O In vivo or hi vitro Sped* trainitat Bat Mouse Guinea pig . Bat In vivo Invivo Invivo Inviso Mom Guinea pig Moose In vivo Invho In vivo B at. Guinea pig Bat Moose Bat Mouse Guis *1pig Bat Mourn Bat Invho Invho Invho Invho . Invho lu vivo , Invho Invho Invho Invitto la vitro faametm Effect* Ret 9 Thymus atrophy Thymus atrophy 44* 32,53. < 1.93-95,97-99 44 52.61,94-96 - Thymus atrophy 44 61,94,95 Seram e-foctoprotein - Peripheral lymphocyte count - Peripheral lymphocyte count Peripheral lymphocyte count *b J o o 4( Peripheral monocyte count - 96 Baduetion seram a, ff, and y giobuUas 96 Suaeeptibility Stlmondla here infection 444 101 SuaecptibUity.peeudoiabies iafection - 101 Delayed-type hypersensitivity Deiayed-type hypeneaaitivitr b * 53.61 4a Graft vs. host activity 4 52 Graft s t hoat activity Rejection of akin allografts * b 4 32.61 4 52 Rejection of skia allografts 4 52 Antibody response to tetanus toxoid -/* .61 Antibody response to bovine pinma globulin -h * 53 Lymphocyte transformation by I'llA and Con A 4 52.53 Lymphocyte tsansfonnalion by IT!A Lymphocyte transformation by PHA 4/-< 52 - 52 Lymphocyte transformation by PHA and Con A - 52 1Desiole* th Incteaaed suscqitlbflitir to iafection and th (oppressive effect oo Immunologteai parmmeters: , iHght; ++, moderate; *, slrong; - , no cilcct. *Thymus atmpfay alio: occnned in adrenaleetosniicd and hrpophfsectanihod rais and in mie hjccled idi thymk Twnuoqt" (Usymasin). <i *1 ysmng aduli mia and in rais cxpoecd during th periata! period by materna! treatment, ropectirety. X 4|a ynung and aduli animali, icipectirety. *On th printary and aecondary autibody (espanse, Rspeethrcty. May 1977 11 :.8 monocyte counts, but scrum concentrations o f a. P, and 7 globulins were reduced by TCDD ex posure. A very sensitive parameter o f TCDD exposure is the resistance to Salmonella bent Inlcction.11 * Intubation o f mice, once weekly far 4 wivLs with l a dose as low as i itg TCDD per kilogram body 7w ei|ftl. sicnificantiv increased the mortality rate and decreased the lime from infection to death in animals infected with SaimoncHa bent. Tim dose o f TCDD was one order o f magnitude lower than the dose that caused atrophy o f the thymus, in contrast, TCDD had no significant effect on mortality in mice infected with pseudorabies rinis. The UlCftaied susceptiouity of TCDD-treated mica to Salmonella infection is probably due to the endotoxin content o f the bacteria, sines recent results have shown that oral intubation, once weekly for 4 weeks with SO pg TCDD per kilogram body weight (a dose that gave a moderate thymus atrophy), rendered mice SO times more sensitive to endotoxin (L P S , colt).* 7 FIGURE 7. Body and tfajrnms weights ( parentis* of eontreb) of fenula Fhher rets that re exposed to . 2*3.7,S>totrachlofodibeiuo-p-dioxM potnatally via snataniai dorinj (5 m/kg body weight) oa days 0 ,7, and 14 and pro- and postnatally i s . a a m a l dating oa gestation day 18 and on days 0 .7 , and 14. (Adapted from Moore and Faith).** O f the different parameters o f the cellular immunity, the ddayed-type hypersensitivity to tuberculin was reduced in guinea pigs but not in young adult rets.** The delayed-type hyper sensitivity to oxazoione was reduced when rats were exposed during the perinatal period by maternal treatment with TCDD; this reduction was even present in rets evaluated at 4 4 5 days o f the control, whereas severe growth stunting and . age.** The graft vs. host activity o f spleen cells o f thymus atrophy were present in the Utter group. Pto- and postnatal maternal treatment in mice can rats exposed . to TCDD during the postnatal period*3, and o f young mice** was significantly also produce a wasting syndrome in pups.*3 These reduced, but no reduced activity was seen when findings dearly demonstrate the severe con spleen cells o f adult mice were used.*3 In both seq u en ces o f TCDD exposure during the rats and mice, perinatal exposure to TCDD developmental phase. ' prolonged the rejection o f skin transplants.* 3 The eiTect on the thymus is not indirectly Regarding the thymus-dependent antibody mediated by the pituitary or adrenals, since response, treatment o f niinea pigs with TCDD thymus atrophy occurred in TCDD-ecposed rats reduced the secondary response to tetanus toxoid, that were adrenalcctomized or. hypophysecto- but no effect was seen on the primary response.* * mized.** In addition, the influence o f TCDD on In the rat, perinatal exposure to TCDD did not the thymus is not. caused by reduced food reduce the primary and secondary antibody intake.** Alsu, recent results have shown that response to bovine gamina globulin.*3 serum -fetoprotein levds were not elevated in rats The mitogenic response o f lymphocytes from with TCDD-induccd thymus atrophy.*7 Finallyv*^ thymus and spleen o f rats exposed during the preliminary results indicate that injections o f perinatal period was suppressed on a cdl-fur-cdl thymic "hormone" (thymosin) did not protect basis and in particular on an organ-for-organ TCDD-exposed mouse pups from developing basis/ 3 **3 The transformation o f spleen cells by ,,thymus atrophy/ 7 TLDD did reduce the number^ ` PHA was reduced in young mice, but not in adult o f peripheral lymphocytes in guinea pigs and mice, animals.* 3 but not in rats.1** In another study with mice,** The responsiveness o f mouse spleen cdls and n o effect was seen on peripheral lymphocyte and SZ CRCOHkatReriratinTtukokmy rat thymus cd ls to the mitogens PIIA or Cun A w:i! Tt: nm yet lot an* Pit nu an hy b*. Ill in. tit tii oo . 1] a! oM cn >1 Co rt CD W O0 1 03 l i i \7 ii ws f I S v* was not reduced wlietr cultuicd in the presence o f TCDD at concentrations up to 0 .0 2 pg/ml medium.*1 liven the presence' o f 0 J 2 pr TCDD per milliliter medium has been reported not to be toxic for human lymphocytes.101 However, in another study, TCDD at a concentration o f 0.48 pg/tnl medium was cytotoxic for several mam malian cell types.103 whereas concentrations that ate tw o orders o f magnitude lower induce aryl hydrocarbon hydroxylase activity in human lymphocytes. 104 th ese results make it unlikely that TCDD needs metabolic activation, but indicate that the lymphocyte depletion o f the thymus and the thymus-dependdnt lymphoid tissue is not caused by a direct cytotoxic action o f TCDD on lymphocytes. In conclusion, TCDD causes thymus atrophy in all mammalian species' studied. Regarding the biological significance o f this eflect, it has been shown In several experiments that TCDD exposure results in functional impairment o f the cellular immunity, but with quantitative different effects on subpopulations o f T cells. In particular, T-heiper cells, possibly with the exception o f those in the guinea pig, seem less compromised. In addition, it seems that the suppression o f the c e ll-m e d ia te d immunity b art age-refatetf phenomenon, at least in the mouse and rat where . TCDD exposure during ontogenesis o f the immune system seems to be prerequisite; in this respect, TCDD mimics the effect o f neonatal thymectomy. As discussed earlier,1 3 it is tempting to speculate to what degree depressed cell-mediated immunity in young rats and mice and in adult guinea pigs contributed to their deaths, since severe effects were observed in the absence of- major liver i pathology found in adult mice and rats. Regarding | the m ode o f action o f the TCDD-mduced atrophy ' j f the thymus, it b probably not caused by a direct eytotoxic action on lymphocytes, neither , indirectly by. an effect on pituitary, adrenals, or : production o f thymic "hormone," nor via in duction o f a-fetoprotcin or reduced food intake. However, as discussed by van Logten e l al. 00 it b still possible that TCDD may impair the ab sorption and transportation o f nutrients. An alternative hypothesu that needs investigation may be through an effect o f TCDD on bone marrow stem cells as precursors o f lymphocytes in the Thym us. The Increased susceptibility o f mice to infection with SahttnieUa bent is remarkable. Host resistance to this type o f infection priiium ^ d ep e n d s upon cell-mediated immunity;10V however, since increased mortality occurred at dose levels o f TCDD that do not produce thymus atrophy, another mode o f action seems likely. This increased susceptibility b very probably due to the endotoxin content o f the bacteria, which has also been demonstrated to be the cause o f the greatly enhanced susceptibility to R coli infection o f rats exposed to lead.100 Regarding litis sensitizing effect o f TCDD for endotoxin, it would be o f interest to investigate whether TCDD impairs the endotoxin detoxifying properties o f macrophages. , Di-o-octyitindichloride (DOTC) and Dt-o-butyltindichloride (DBTC) In-depth investigations were carried out on fleets on the immune system o f the chemicals di-n-octyltindichloride (DOTC) and di-n-butyltindichloride (DBTC) (Table 4). As shown by Seinen and Willems,1-1 the main effect o f DOTC in rats b on the thymus. Hbtologically, lymphocyte deple tion was observed in- thymus and thymusdependent areas o f spleen and lymph nodes. In cell suspensions o f the thymus, a dose-related decrease was seen in the number and viability o f thymocytes; the effect on spleen cell count and* ceO viability was less pronounced, whereas no efleet was found on bone marrow count and viability and* on peripheral lymphocyte and monocyte numbers. As shown in Figure 8 , in a series o f dialkyltin compounds tested, both DOTC and DBTC pro duced a severe thymus atrophy (which was revers ible upon discontinuation o f exposure), whereas a less pronounced effect occurred with diethyltindichloride and di-n-propyltiadidiloridc. (n contrast, tka other dialkyltin compounds (dimethyltindichloride, di-n-dodecyitindibromide, end di-noctadecyltindibromide) as well as mono-noctyltintrichloridc, tri-n-octyitinchioride, and tetraoctyltn did not cause atrophy o f the thymus.14 The selective effect o f DOTC on thymus is not indirectly caused by stress-related release o f glucocorticoids, since a similar eflect on thymus was present in adrcnaleetomizcd rats.1 1 A strong argument in favor o f a direct action o f DOTC and DBTC on rat thymus b their in vitro cytotoxicity for thymocytes o f rats, but not o f guinea pigs and mice; in these latter two species, thymus atrophy d o e s not occur after 'exposure to these chemicals.14 . In function studies o f Use thymus-dependent immunity,, the delayed-type hypersensitivity to May 19T7 U T -- -- -- -- -- -- -- -- -- -- -- i-- ilwrtwstMMmwrrr oo o CD I 00 i CD \ Ot I .6220 EXHIBIT E KURON Complaints: 1. E. L. Hand - Lake Jackson, Texas 2. John Norton - Glendale, Arizona O o rozz. o co l\> ro CO CO o M 16221 AGRICULTURAL DEPARTMENT ^ Copies to: DSM * '" 4 ' / Product Sales Manager Product Technical Specialist Salesman PRODUCT COMPLAINT QUESTIONNAIRE (PLANTS) Information given here should be factual unless indicated otherwise. a O Written by: R. W, Swart___________________________________ Date: 8/4/71 Exact Name, Address and Telephone Number of Complainant: E. L. Hand,______________ Highway 332, Lake Jackson, Texas 77566________________________ ro 00 CD ~C T +-- *- Telephone No. 7 1 3 - 7 9 8 - 2 5 9 0 County Brazoria_________ state______ Texas___ Detailed locationofdamaged and non-damaged area(locate on Geological Survey, highway, SCS or irrigation map): Buffalo Camp Bayou - north of Highway 332 - west of Lake Jackson, T e x a s __________________ Name, addressand telephone number of person receivingcomplaint: W, S. McGregor,__________ Agricultural Department, Dow Chemical U.S.A., Lake Jackson, Texas Who requested Dow assistance:_____ Mr, Hand____________________________________ Name and address of Dow and other personnel involved in investigation: D. M. Turner, Dow Texas Division Water Supply Dept., Freeport____ R. W. Swart. Dow Agricultural Dept.. Lake Jackson________________ Date of complaint: ____ July 12 , 1971 Product and Application: Product: Huron June 21, 24, 25 Within Date ofApplication 1971________ Date of purchase: 6 Mo . Proofof puchase:_ Distributor or Dealer: Sprayco, direct from Dow Condition ofcontainer immediately priorto use: _______________ Lot number 5 Q - 1 2 - 2 9 _________ Amount of Product Used 35 Gal.______ Area Treated- Rate/Acre Spot Spray V01.Spray/Aere 5 Gal, Plyac Emulsifier Wind velocity and direction at time ofapplication:__________________________ None_________ Obtain originalcontainer with suspected contents:______________________Spray water source: Prioruse ofsprayer:____None_________ Other pesticidesused in tank mix: _____ None Identification,exact name, telephone number and addressof: Contractor: Page 1 Soravco Systems. 1327 Judiwav. Box 1002S. Houston Mr, M. W, Held - 713-686-0598. Texas. 7 7 0 1 8 Sub-contractor: Contract applicator: Weed commissioner: o O ro ee CD Farmer applicator: _ t\i Other: _____ Equipment used- method ofapplication(ground,air,boom or gun, nozzle, pressure,chisel,tarp,etc.): P r e s s u r e - method of mixing (nurse tank or singlebatch):____ Spray from Boat___________ Isequipment used foraipplicationofother pesticides? Application Site: Acres Utility ROW: () Highway ROW: ( ) . Railroad ROW: ( )______ Cropland Planted: Fallow: Grassland Crop: _ Future _Crop _ Grass -Species_ Acres Ind. Plant Site: ( ) Other: ( ) (Acresor sizeofstructure): Buffalo Camn Bayou_r_s_pot_s_praxJLQT_cj2Qt:.rol of aquatic weeds Organism Affected: Human*: Livestock: Cattle____ Wildlife: Ornamental: Crop:_____________Specific identification:________________________________________________ Number (plants,acres,animals, etc.): Abortions______ Dollar value claimed:_____ None__________ Proximity to treated area:_____ Cattle drln'.C from bayou_____________________________ " N o t i f y D o w M e d ic a l D ir e c to r [D r. //.L . G o r d o n ): 2 0 3 0 D o w C e n te r, T h e D o w C h e m ic a l C o m p a n y , M id la n d , M ic h 4 3 6 4 0 ' P h o n e ( 5 1 7 ) 6 3 6 - 2 1 0 5 Suspected Mechanism ofMovement: ---------------- :---- 40,000 gal./ Drift:____________ Volatility:___________ Water movement:__ minute__Soil movement:_______________ _ Crop residue:________________ Manure:_______ Faulty Appln. Equip.:____________ Other:_________ Field History (previous years): Suspected pesticide:_________ X___________________________________ Date Applied:_______________ Other pesticides:____________ ^__________________________________ Date Applied:____________ ;__ Disease, insect or other pests:________________________________________________________ y Fertilizer(analysis):__________ ___________________________ Source:___________________ Source ofdrinking water, ifsick or dead animals involved Bayou where weeds were Sprayed Previous crops grown in field____~____________________________________________________ 7S o i l t y p e _ _ ___________________________ - _________________________ T y p e o f i r r i g a t i o n e m p l o y e d _________ ____________________ Page 2 23 Summary ofPossibleContributing Factors Not Covered Above (including competence ofapplicator,other pesticidesapplied inarea,and opinion ofwriter asto cause): _____ Mr. Hand Is a retired Dow employee, 11 vine; on 5^ acres adjacent Q Qto Buffalo Camp Bayou. He has 16 cows and 1 bull that pasture on the area and drink from the bayou. The bayou carries water to the ^ canal that supplies water to the Dow Plant, It is a practice of the Water Supply Department of Dow to spot spray weeds In this____ bayou each s u m m e r , _______________________________________________ _____Mr. Hand stated he has had ^ or 5 calves die soon after birth ro 00 CO 00 CO over a several year period, and had 3 abortions this year. 1970 - Bayou sprayed on June 29 and 30. There was 1 calf died soon after birth in the fall, several months after spraying. 1971 - Spraying was; done on June 21, 2 b , and 25. Calves lost: 1 . June 5 lived short while 1 June 27 about-6 m o . 1 July S.____ If 1! I discussed the happenings with Mr. Hand and thanked him for reporting to us . However. I do not think Kuron was the cause of his problem and Mr. Hand really doesn't either. Various causes of abortion were discussed, and it was suggested that he consult his veterinarian with the possibility of checking blood samples. He agreed to do this and thanked us for our interest, and will report their findings to us later. Page 3 16224 SKETCH (to include the following inrespect to area showing injury): (1) Location oftreated area showing damaged and non-damaged areas. (2) Direction ofsurface water drainage. (3) Location and flow ofnearby streams and/or drainageditches(withdistances). (4) Prevailingwind direction. (5) Include location ofother sensitive crops in the area and indicate whether or not affected. (6) Have photographs taken, ifappropriate,showing overall effect and closeup ofalleged plant disorder. o O CO 00 CO C3 Page 4 16225 Summary of Disposition of Claim or report: Samples (samples to be taken only as a last resort). Ifindependent laboratory, obtain: Name________________________________ Address__________________ Taken Analized Analytical results:_____________________________ Soil ____________________________________________________ Water ____________________________________________________ Plant ____________________________________________________ Animal ____________________________________________________ Wildlife ______ _____________________________________________ Settled, closed:____________________ Nature of settlement: None______ Complaint active:__________________ Recommended action: None o rcoe----- co ^ 3 --x n ___ Mr. Hand, being a former Dow employee, said he thought we would be interested in checking any possible relation of our product to his problem. I assured him we were interested and again thanked him for calling us. Page 5 16226 9 ? !7 8 N e w S c i e n t i s t 1 3 A p r i l 1 9 7 8 Chloracne: the chemical disease Chloracne is caused only by contact with chlorinated organic chemicals--it is truly a disease of modern civilisation. Although sometimes disfiguring, it is, in itself, not serious. But it could be a vitr indication that sufferers have been exposed to chemicals with other, much more serious, effects Dr Kenneth Crow One cannot usefully discuss chlor- the eyes (the malar crescent) and behind the ears. The.1 isconsultant dermatologist at Princess Margaret Hospital,Swindon and isa leading world authority on chloracne aen'e without considering the chemistry, toxicology, and even the environmental impact of the chemicals which cause the disease. Chloracne is a cutaneous eruption which is generally symptomlcss areas are frequently affected when every other part of th skin isnormal. The remainder of the face, neck, shoulder genitalia, chest and lower trunk can also be involved (i that order). The hands, feet and legs are rarely involve and then only in the worst cases. In cases of exception severity unusual findings may be encountered. F< but sometimes disfiguring. Its sig example, every single "hair" follicle in affected areas mn nificance is as a marker, an indicator of the presence of a become a blackhead or acne cyst (above, right). In ran chemical which islikely to be toxic in other.ways and may cases the entire body surface may be covered with smn well have environmental effects. When chloracne has horny spines like a nutmeg grater and in others generalise sounded its warning, our whole attention should be hyperpigmentation may occur to such an extent that th directed to these other, more serious, problems. They may patient's racial origin may be in doubt. not be present-- indeed generally are not-- but until there There is every reason to believe that the response r isconclusive proof to the contrary, chloracne should always the human skin to the same dose of causative chemical ma be regarded as a sign of potential systemic poisoning and differ greatly in different areas of the body but this h. environmental hazard. Chloracne is produced by skin con never been tested experimentally. In mild cases, chloracu tact, ingestion or inhalation of certain chlorinated aromatic may closely resemble adolescent acne but the curioi hydrocarbons. All known '`chloracneigeniclL.chemical_s.,(;nn concentration of blackheads and cysts round the eyes an pass, through, the..intact skin,, but in doing,so their ioxic on the ears is specific to chloracne. Us duration depem effect is.les.s.cned^SQ.that systemic poisoning-from skin con- greatly on severity-- the worst cases may still have acti\ tact alone is usually accompanied by very severe chloracne. skin lesions 15 years after contact with the chloiacneigcn Clinically, chloracne isan eruption of blackheads, usually chemical has ceased. Mild cases, however, clear with accompanied by small pale yellow cysts which, in all but months; after three years all but a "hard core" of 20 p> the worst cases,are from pinhead to lentil size (above. Id:). cent of cases are likely to have resolved themselves. Tl In severe, cases there may be inflammatory papules .md only permanent skin effect is scarring, which may n even pustules (a papule is a spot, a pustule is a papule occur at all in mild cases but in the worst is very sevei containing pus). The distribution of chloracne over (be Apart from the human subject, chloracne (or somethir body is interesting: the disease has a predilection for the very like it) has been produced in only three experiment skin of the face, especially in a crescent outside and under animals; the rhesus monkey (on the face, with toss of h. 16227 80 /V ' N e w S c ie n tis t ij Apr uo0 w 2 8 1 0 5 7 cause the initial damage was due to chemical the airborne dust consisting of particles of the released by the accident. This dust produced t and swelling seen in so many of the cases after sion. As the burns faded, so the chloracne ar almost every case it has been mild. Contact wit these cases appears to have been mainly exit firmed by the fact that (apart from the classic f~ already described) only externally exposed arc body were affected. All chloracncigcns appe absorbed through the skin, although the extent tion varies greatly. Some, systemic level of the oo invariably occur. But such systemic concentratio are unlikely to cause problems, except possibly i. term, when much depends on rate of metabolism. There is no possibility of consumer poisoning r mild,chloracne from manufactured products, b \.l' If' -V (>' :7 <Z>~-<J>^'Tzr~ O --O -- .-- ^ No available hydrogen (topright), asymmetry (second from top. right), mashing of the reactive bridge by chlorine atoms the Chemical industry the risk of chloracne although much diminished as awareness of the increases and'as diagnosis (especially in mild c comes more accurate. From the environmental si the risk of low dose, long-term effects-- in particul. ( third from top, right), and unadjacent chlorine atoms ( bottom and damage to genetic material (mutagenicity)-- right) all make the chemical relatively non-toxic ably more important. Poorly metabolised materials ahazard in the long term even when the dose issm (causing steric hindrance) and the absence of adjacent chlorines all result in the relative non-toxicity ot the compounds on the right of Figure 3 "Usually short- and medium-term toxic effects arc only seen in heavily poisoned subjects with severe chloracne Experimental data on the long-term effects c acneigcns on animals arc limited to the rat and When fed to rodents in large doses throughout th time, PCBs produce liver tumours. The results of . study in which varying amounts of TCDD were fed and, with few exceptions, have been produced by poly throughout their life revealed that tumours were chlorinated dibenzodioxins and polychlorinated dihcnzo- only by a dose large enough to produce other evid furans. The worst incident of poisoning by chloracneiccnic chronic toxicity in various organ systems. Lower chemicals was the Yusho incident in Japan in 1968, when which did not appear to affect the health of the a over 1000 people were accidentally poisoned by consuming produced no tumours. This suggests a dose-rclatec a rice-based cooking oil contaminated with a poly Of further interest is a careful follow-up of 70 mi chlorinated biphenyl (PCB) which contained an abnormally severely poisoned by TCDD 24 years ago in an inc high amount of polychlorinated dibenzofurans. It is now accident. So far there has been no evidence of ex believed that the dibenzofurans were largely responsible or unusual malignant disease. In short, it seems p for the toxic effects. The symptoms of severe poisoning are that TCDD and PCBs may be carcinogenic to maiT'c known in detail only for TCDD and the PCDF/PCB mixture high doses. There remains the'possibility ol adOit in the Yusho accident. The two chemicals differ little in synergistic effects between mild carcinogens, as w their target organs. Chloracne, disturbances of the central co-carcinogenesis, when a non-carcinogen can be act and peripheral nervous systems, wasting, hepatic damage by interaction with other chemicals. As yet noth and hyperlipaemia (high blood lipid levels) have been most known about these phenomena among chloracneigcr prominent. Recent experimental work revealing that .these arc under intensive scrutiny following the recet TCDD, although known to cause birth defects, is in fact covery of PCDFs in a wide variety of materials. far more directly toxic to the fetus, is borne out by the history of pregnant Yusho victims: there was an excessive Controlling the risks incidence of still-births in this group but no congenital mal- What can the chemical industry do to reduce the formations caused bv exposure to the dioxin-like PCDFs. of chloracncigenic chemicals? One priority must be t< PCBs a~re"known to be fctotoxicbut not teratogenic. lish and disseminate toxicological information \ An interesting toxic manifestation is porphyria-- a meta throughout the industry. Otherwise costly and le bolic abnormality in which the pigment porphyrin is re research would be wasted. Secondly, because acne tained in the tissues. For years porphyrin mss been known potential in a chlorinated or brominated aromatic to be associated with severe TCDD poisoning in man and stance appears to imply various other toxicities whicl can be produced in experimental animals by TCDD and be serious and of environmental significance, Ibeliev PCBs-- even pure (PCDF-free) PCB isomers, especially the the simple, but extremely sensitive, rabbit car test : 3,4.5.3'.4',5' hcxachlorobiphcnyl. It now seems most likely become part of the standard battery of toxicologica that porphyria may be due to an effect on the enzyme applied to every new substance (especially herbicide dclta-aminolaevulinic acid synthetase, the enzyme that pesticides). If a contaminant is responsible for the a limits the rate of porphyrin production. This enzym*' is it may be possible to eliminate it altogether or reriuc strongly induced in vitro by TCDD and itseems likely that negligible levels (providing that long term low dose the PCB isomers which strongly induce A H H also induce have been adequately assessed). The headlong ri the synthetase enzyme. produce new chemicals after the Second World War Mild chloracne, such as that suffered after the much- that a great many new substances were inadeq publicised Sevcso accident in Italy in July 1976, docs not examined toxicologically. Increasing awareness on tt \ . appear to be accompanied by short-term systemic effects of the major manufacturers and government rescarc! and surveys of the Seveso population one year after the tutions, especially in the US, makes such problems le ' accident confirm this hypothesis. The absence of terato less likely to recur.________________ _ genic effects at Seveso is as expected in view of the finding already quoted. The Seveso cases were most confusing be- * Vh<% *n<U tt baattf on 0<(vcttioAtl Hyfi t *# So<cy. <***. wk < th a N e w S cie n tist 13 A pril 1978 DOW 2 S 1 0 5 6 disruption of glands in the eyelids-- Meibomian glands In the human subject); the hairless mouse, and, most Inportant of all, on the inner surface of the rabbit's ear. y (j atIt It not possible to produce chloracnc on any other part the rabbit's skin. Indeed, even systematic absorption of thloracncigens by the rabbit produces chloracnc only on the innersurfaces of the cars. Thus there isa clear parallel Mo the curious sensitivity of the malar crescents and ears in 'the human subject. The rabbit's car appears to be the most attieniitivc biological surface known, reacting to a total dose the most potent chloracneigenic chemicals as small as one microgramme. Thus the rabbit's ear affords a simple, cosy and relatively cheap method of identifying cliloracaelgcnic materials. The biological basis of chloracne, and b particular of its predilection for certain areas of the AZOD1MZOO ">f: .- r How carbon atom s i in arom atic L hydrocarbons ards num bered jbody, is quite unknown. Investigation of the disease is chlorine content somewhere between maximum and m i ,:ihampered by the lack of an analytical method accurate m u m produces the greatest toxicity. For example, t w^.nd sensitive enough to estimate quantitatively chlor- dioxins can have from one to eight chlorine atoms. Li Kneigcnic chemicals which may be present in blackheads than four, or more than six, chlorine atoms in_a diox jjtadcysts as opposed to other skin tissues. molecule greatly reduce toxicity: between tour and six y t The chi:.'inatcd aromatic hydrocarbons which are known dTTrp highly rnvir m mpmmri.s ITowevcr. the most imnnrt,-i tflhave produced chloracne in humans are cfiemical property determining toxicity is isomerism, th ./.chlornaphthalcnes commercial polychlorinated biphenyls (PCBs) polychlorinated dibenzofurans (PCDFs) polychlorinated dibenzodioxins (PCDDs) 7Vp`.lz-eetn-rc(a(cThTrlCaoA/r-O\onBa)>z.obenzene (TCAB) and tetrachloroazoxyben- is, not just the number of halogen atoms but their exa position in the molecule. By convention these positions a allotted a number (see Figure 1). Thus we may ha 2.3,7.8 and 1,2,3,4 tetrachlorodibenzo-p-dioxins or, similar: 3,4.3',4' and 3,5,3',5' tetrachloroazobenzene. The fox potential o f these icnmnr pairs varies sn greatly that- v must be able to distinguish them. The detailed study . With the exception of the chlornaphthalcnes and PCBs, as many isomers as possible is essential. Successful wo: 'ail these compounds are contaminants formed accidentally in this field depends entirely on the chemist's ability 'during the manufacture of other chemicals. For example, synthesise the various isomers in pure form. Once the .although the PCBs have been shown to have a toxicity of arc available as standards, modern instrumentation pr j!'their own, in their commercial form they are always con- vidcs a means of relatively rapid, accurate and sensitii laminated by varying amounts of the much more toxic analysis, not only qualitative but also quantitative. n .PCDFs. Again, the manufacture of 2,4,5 trichlorophcnol Vproduces as a contaminant the most toxic and chlor- Sim ple test-tube test acncigcnic small molecule known, 2,3,7,8 tetrachlorodi- Ironically, many toxic chemicals initiate their ow [benzop-dioxin (TCDD) of Seveso fame. Crude pentachloro- destruction-- they can activate (or "induce") colln!. phenol production, as recent research has revealed, leads enzymes whose function is to metabolise and detoxil ;.to the formation of a range of I'CDF impurities. The latter them. These enzymes can be separated from liver cell ;havc also recently been discovered in crude hcxachlnr- a particularly rich source, and used for in v it r o tests < benzene. The contaminants TCAB and TCAOB occur during toxic potential. Arylhydrocarbon hydroxylase (AHH) i the chemical reduction of dinitrochlorbcnzcnc to the cor the most useful for assessing acncigcnic chemicals. Tli responding diphenyl hydroxylamine and dichloroanilinc, degree and duration of enzyme induction has proved i .both industrially significant intermediates. TCAB and parallel closely the acncigcnic and systemic toxicity of a TCAOB arc never found in the final products and exposure rhlorocncigcns so far studied, giving us an elegant an therefore occurs only in the chemical industry. Little is simple method for distinguishing toxic from less tox1 known of the detailed chemistry of the commercial chlor- isomers. On the other hand, in vitro tests will not reve. ,naphthalenes. Whether their toxicity is due to contamin the sometimes great differences in susceptibility betwee ants or the pure chlornaphthalcnes isunknown. species Thus animal studies are still necessary but onl The acncigcnic potential of a chemical appears to he for a very small number of selected compounds. As a resu1 directly related to its overall toxicity. The importance of of recent intensive research with the enzyme inductio this fact is the implication that chloracne in an experi technique we are now able to make some generalisation mental animal can provide a very important screening about the relationship between structure and toxicity. test for systemic (not only skin) toxicity. This appears to lateral symmetry in the molecule, the chlorination of iiv hold true for all known chloracncigcns with the exception ad iacent carbon atoms,the presence of available, hydrogen of 3,4,3',4' tctradiloroaznbcnzcne and possibly azoxybrn- aif3 1he absence of stcric hindrance (where the spati.' zcnc. Recent work reveals a vast diffrence between the arrangement of a molecule "hides" reactive, atoms, pn oral and cutaneous toxicity of these chemicals, presumably venting certain reactions) all enhance toxicitv. Bccen .because of the well-known susceptibility of the azo bond work indicates that all diloracneigcnic compounds at. todestruction in the mammalian gut. either exactly or approximately isostcric (that is. have tli- Certain basic elements of their chemical structure deter same distributions of electrons over the outside of th- mine the toxicity of chloracncigcns. The crudest of all molecule-- sec Figure 2). The absence of free hydrogen, la: factors is_the_degree of chlorination. It appears that a oral 'asymmetr` y, chlorination next^ to the diphenyl' Lbr- i!dJg- 'y. Models show that the distribution of electrons (shading) is practically identical in all chloracneigens 1.4.lie TCTZACnUJRAZCKYBCNZSM 1.1,7. TrTIUCnLCRDIBCNZO-r-OICCUH* 1.3.7. a TETACHL0RDQIO4Z0rU1lAH 1.1.3*.I* TETRACHUXUllPHEV' 2885 U . S. D E P A R T M E N T O P A C R i t U L T U R E AGRICULTURAL RESEARCH SERVICE P E S T IC ID E S REG U LA TIO N DIVISION WASHINGTON, O.C. 20280 FORM APRROVEO 'f B U D G E T B U R E A U N O . 4 0 --R 1 7 4 5 ,,APPLICATION FORIaMENDEDIREGISTRATION OF ECONOMIC POISONS (Under the Federal Insecticide, Fungicide, and Rodenticide A ct) n IMPORTANTI R E A D I N S T R U C T I O N S O N R E V E R S E o1. D A T E OR A P P L I C A T I O N : ae*>noTfj l/'Ttu? - -.o :jM.iS a m i r y ' l O , 1 9 7 3 2 , N A H l O P ECONOMIC POI SON (Must be same product name as on label--do not list active ingredients J ^ . ^ KHEiL (Include Zip Code| n a m e s m a i l i n c a d d r c s s o p r e c i s t r a n t ) 4 , R E G I S T R A T I O N NO. ---V----.---i--!--- - Dow C h e m i c a l U . S . A . *' Ag-Organics Department P . 0 . Box 1706 . r. . . ... M idland, Michigan 48640 .ir;,r , i f . / *'* '*. *, j*';. o ' / ; it?.' . ' r t ? ....4 6 4 - 1 6 2 - r o < PROPOSED EFFECTIVE DATE Q Q OF CHANGE -_ uen; TtiO.-i.r : , r . v : t n . < T '-D -,r\. 'i^'';: :u * :-.i t O . . J . A t t i c __ B r - P . Ra.-Mu l l i s cq________1____ : .. ..rr. - TrPTrorHaf-gl y 6 . n a t u r e o f r e v i s i o n (Check applicable item and give details in item 7, when required) 00 CvT 9G E N E R A L R E V I S I O N O F L A B E L I N G c h a n c e IN f o r m u l a t i o n (Give description of exact change , .. in item 7) OTHER O 1 -t*l ' (Specify in item 7) . --e : ADDITIONAL USES ADDED TO LABELING Q ( List new recommendations in item 7 ? - ' C M A N G E 'J4 f r o o u c t ' n a m e _ . . . - .... -.j. ' ''(Give old name and new name . 1 w" V. 1 it tiitn iitteemm 77)) .`f r : - ' . ' J r . -U - ! - - - -'l--1 7 . d e t a i l s r e q u i r e d b y r e v i s i o n c h e c k e o i n i t e m a (Attach additional sheets i f more space is needed) T his a p p lic a tio n f o r amended r e g i s t r a t i o n in v o lv e s a g e n e ra l la b e l r v is io n . The recommendations have been re o rg a n iz e d in to fo u r major headings: I n d u s t r i a l and Non-Cropland A reas, A quatic Need C o n tro l, " Pasture and Rangeland, and Uses in C roplands. The p recau tio n ary s t a t e ments have been expanded and stren g th en ed . . . No r e a l l y new u s e s h a v e b e e n a d d e d b u t t h e l a b e l d o e s h a v e s e v e r a l r e c o m m e n d a tio n s w h ic h a r e p r a c t i c e s t h a t h ave b e e n I n common u s e b u t w e re n o t p re v io u sly s ta te d on th e la b e l. These a re : f o r e s t c o n if e r r e le a s e , b a sa l bark and stump, powered knapsack b a sa l tre a tm e n t, and m odified b asal treatm ent. These recommendations are a l l fo r I n d u s tr ia l and.Non- Cropland areas. - i-- " < The A q u atic s e c tio n has been m odified s l i g h t l y to make i t s h o r t e r and le ss complex. I I CONTINUED ON ATTACHMENT T H E F O L L O W IN G M U S T B E S U B M IT T E D Y/ITH THIS A P P L I C A T I O N F iv e (5) c o p ie s o f re v is e d la b e lin g , in c lu d in g any p rinted or g ra ph ic motto? w h ich may accom pany the sa le of th is product* C o p ie s must be c le a rly le g ib le and id en tical. C If o ch an ge in form u lation is in v o lv e d , fiv e (5) c o p lo s o f a statem ent or re v ise d form ula sh ow ing the p re c is e name and p recentago of eoc^ a c tiv e and eoch Inert ingredient, (This information is treated confidentially) When a p p ro p ria te , th ree (3) c o p ie s o f S u p p o rtin g D ata. 9. SIGNATURE OF A U TH O R IZED FIRM R E PR E SE N T A T IV E IN ANY C O R R ES PO N D EN C E ON TH IS PRO D U CT R E F E R TO . R E G IS T R A T IO N NO IN IT EM 4 , A B O V E . vt: . >iu lu.t i t l e | l , D A TE SIGNEO --Recels t r a t i o n -Gpo c i a l i s t -------- ------------------------- P R FORM 9 -1 9 8 `EX ISTIN G S T O C K O F P R F O H N V W 198 lA U G . 190) MAV 1969 WILL S E USED U N TIL EX HAUSTED APPLICANT'S COPY DOW 289283 t ITEM INSTRUCTIONS FOR PR FORM 9 -1 9 8 ..V- ^ - 7 c ~ \ >n r: r:r.e.\ ij.n;;.n'*wrk.*n*ia '*V, i e y q Applications should be submitted as far In advance as possible prior to desired registration date? The'time required to process applications may vary depending on the extent of. review required. Applications which require consultation with other governmental agencies will take.a longer time to process. . ^ . * no ^ H O irsuH Tini o i j n 2 "`The name of the economic poison shown In the application must'be the'same jjrWuct name as thal'shownVtTT tthhPe lIan bh eelliinnpg- sCuI bl hmm iftntpeHd. Do."I nnnoft ll liostf tf hh ae 0a/c1 tHiv1 me Iinn igrrreo dr fileann tf se . i ' 3. ---The name of the firm or person and address shown in your application is the person or firm Z' ~ ' r to whom reg--- istratlon will be issued. If you are acting in behalf of another party, you' must'submit'authorization"from * that party to act for them in registration matters. The address given in item three will be the mailing ad d r e s s permanently on record unless changed by the registrant. ^ Iavti-'-ot" arn*"'j^i'' !tr The registration number assigned must appear on the label. The number must be the same as that appear ing on the notice of registration and shall be preceded by the phrase "USDA Registration Number" or the phrase "USDA Reg. No," (Refer to Sec. 362.6(F) of the Regulations).. ,!_i_ . / ___ ____________ ( Y'S"'ii,vv> >:'it i.- .-j-.; vnn rrii: ni^.'r.iV'uqt ,c .b 5. Changes in the labeling or changes,in the formula.must be submitted in advance of-the proposed effective date. IV-j I tu ?.;-:i_. ' " u HI . 3 0 H A H 3 6. Nature of revision -- The .r$gislrant^must d'escrlbelhe exact changes desired and tJpdn' request"must sub mit a description of test resuits to justify such changes. Minor changes in formulation may be handled as revisions of existing registrations. This might include a change in the percentage of active ingredients or ___ a ..change in inert-ingredients..However, any basic change in formulation such as a. change.in the principal __ active ingredients would require a "separate registration1as anew product with a1different-product'name: 7. ..Self Explanatory. . - ; .[ Vi,.' i -j ?._v--v.-^ v.-~ 7 or. nn.Vr l r-T 8. Labeling -- All copies of labeling must be clearly legible and identical. Photo copy of lithograph labels should be submitted. Do not send containers. Your supplier should be able to furnish photo copies of the *'screen print. Submit reduced photo copies of labeling for dry agricultural fertilizer - insecticide, or her bicide bags. Any label smaller than half the size of a sheet of paper 8 inches by 10 inches should be stapled to a sheet of paper 8 inches by 10 Inches before attaching to application. . -- , -x- ,, I .` .- ;\ .'* \ - -*. ; t - j * . ^r' * Formulation -- If a change in formulation is involved, five copies of a separate statement listing the cor-, `rect name and percentage by weight of each active and each inert ingredient must be submitted with each application. This statement is treated confidentially. It cannot be used to support a customer's (formulator) application for subsequent registration without written authorization from the registrant. If necessary - information on the formula is not available to the applicant (formulator or packer), he should; (a) Obtain the necessary information from the basic supplier and submit five copies of the statement with the appli cation or (b) Request the basic supplier to furnish the necessary Information to the Division along with a statement specifically authorizing the use of such information to support separate registration for the applicant. The statement from the basic supplier must clearly identify the applicant's product to which It applies. "1 " ATTENTION: The submission of an <ipp!icr''or does not constitute registration. Comment or notice of registration will be sent after examination by the Pesticides Regulation D lv i-,. , sion of the information submitted. , .i.'-'ro-r, lo ' >*<n-*. "- 1 * . ui- r... *. ">'Ru'r- 16231 WILLARD AND BILLEE SHOECRAFT VS. THE DOW CHEMICAL COMPANY EXHIBIT C 2 ,4 ,5 -T rich lo ro p h en o l A n aly tical and Shipment Record 2 , 4 , 5-TRICHLOROPHENOL ANALYTICAL AND SHIPMENT RECORD MICHIGAN D I V I S I O N , DOW CHEMICAL U . S . A . FOR .. ALL LOTS PRODUCED DURING THE PERIOD MAY 2 7 , 1 9 6 7 AND MAY 2 , 1 9 6 8 THAT WERE USED IN THE MANUFACTURE OF HURON. 00 i--1 c A<D G A<U 1 0N 0a G G CU rH 0 H 0c 0 C A0) A<D g Aa) a 0 P a 0 P a. 0 0H P x; 0 rAH 0 fAo0H o H 4H-> H H *0 'O r1r 1 1 in* * m V CM CM CM H rH Aa) 0 0a G a) jC a 0 p G Aa) a o P 0 p iA0u-H A0VH H rA0H A3 PP O *H -ap) pH 4pJ I1D i ID Si 1 in * in cn h co S CM CM CM A0 P ma 1 m | 0p A0H 0H i TT K CM XI X Jp0Z m 0u o g 1 (N | AtVri Q acy o p pp --h o QJ A P 1c o CO.x r0 in co -a s| cm a LOT DATE BLDG CAR % % % % % % % % (PPM) 05277 06017 06067 06077 06127 06137 06147 06167 06237-1 CO CO 5/27/67 6/1/67 6/6/67 6/7/67 6/12/67 6/13/67 6/14/67 6/16/67 6/23/67 489 309 489 313 267 314 489 313 489 314 489 313 48 9 3 0 9 <( 489 314 267 314 <0.5 <0.5 <0.5 0.4 0.4 - 0.2 0.2 . 0.2 0.3 <0.5 i 0.4 0.3 0.3 0.3 .5 0.2 0.4 0.2 0.6 0.5 0.6 0.8 91.5 1.7 89.7 . 0.2 2.5 89.7 <0.5 2.8 89.2 <0.5 3.0 88.1 0.2 3.2 90.0 3.4 89.2 3.2 90.8 0.3 0.3 CM O 2.2 0.3 1.5 0.2 2.2 <0.5 2.4 <0.5 2.0 0.4 2.1 0.9 2.2 1.1 2.8 0.3 09T8T0/AOQ <1 <1 <1 <1 <1 <1 <1 SHIPMENTS -d ichlorophenol LOT 06267 06287 06287-2 06307 07067 07087 07117 07137-1 07267-2 C3 0 8 1 0 7 - 2 w 0815-7 ^ 08157-1 DATE . BLDG CAR 6/26/67 6/28/67 6/28/67 6/30/67 7/6/67 7/8/67 7/11/67 7/13/67 7/26/67 8/10/67 8/15/67 8/15/67 489 309 489 313 489 314 489 309 489 313 489 314 267 309 489 313 267 309 489 309C 267 314 489 313 rr (VJ % <0.5 0.4 0.4 < 0.5 0.3 < 0.5 < 0.5 OF TRICHLOROPHEHOL rH 0 c rH 0c r-H rH 0c 0c 0) a) jCCu X cu rH 0c X0) cu a) xr0000Hd4 o X 0u 0M 0 u x0d Cu 0 r0H r0H r0H <n r0H jC u iH T) in1 Xr0H. Xu H Xu H HM 4H-1 4HJ 1 tt* l1> 1 in fc. rok. n TP +j 14a01J) * in m V CM CM CM CM CM %%%%% r0H r0H c, G Xa) Xa) N01 a Cu c >1 0X >1 X<Dd 0 5 <u s1 in 1 00 r0H x0 H SaE1) r0000H14 x0rdi OVh O x0: aa V(0TJ --uH 04<10Ju -rcH -X '1 TP T1J in nr~^ *TOrH3 CM -1 tT CM a % % (PPM) 0.4 0.5 3.1 91.0 2.6 0.4 0.5 0.6 2.9 90.5 2.8 0.4 0.4 0.6 3.3 90.0 2.8 0.4 <0.3 0.4 3.0 92.7 <0.2 2.5 0.3 0.1 0.1 0.3 0.2 <0.1 < .1 '0.4 0.4 0.6 0.3 <0.5 < .5 3.1 90.3 2.8 89.9 2.2 92.6 . 6 88.5 1.7 87.9 1.7 89.2 <0.2 0.2 <0.2 0.2 <0.2 0.2 2.3 2.2 1.8 2.1 1.8 2.1 0.1 0.1 0.4 0.3 <1.0 <1.0 TilTRFO MOO <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 oOooo oooo VD VO VO VD VO vo vo GO 00 to f--1 O ct> -o1I h-* o o o to to 1--* M Ln CD vo 00 1 - J -o f--1 t-* P> VO VO VO vo VD vo VO 00 00 \\\\ to M M t-* \Ln \\ CD to \ to o o\ \ CT \er \ o l-* \ \ Ol CTl -O o\ -o \er vo \CT 00 er -o 'O oo 00 00 to to h-1 *u -o o CO ' M CT *11sj to 00 00 00 \\\ to to M t-* o \ \cr> \ CT> \er -O t-* o -3 > 1-3 w .u ro .* to CD CD 00 CTi 00 CD <T\ CD 00 VO VO VO -O VD VO O VO vo t&h 00 CO 00 vo vo vo CJ CO CO to OJ Co CO CJ Co M h-* K* vo H-* h-4 O O M LO CO U> VO vo *U CJ CJ CJ H- o CJ VD CD tr* o o > SHIPMENTS OF TRluHLOROPHENOL DOW 018162 A o o oo o 1111 u> to Ln Ln /\ /\ A ooo Ln Ln Ln I--* o o o o o o I-* o M er to to to t--1 to Ln /\ oo Ln I- ooooooooo Ln Ln CJ CJ Ln -J A oo -J Ln U1 cp2 , 4 - d i c h l o r o p h e n o l dP2 , 5 - d i c h l o r o p h e n o l <#>2,3,4 - t r i c h l o r o p h e n o l to t--1 to to to to to CJ to M CD LO vo o\ Ln CT 00 to to 1--* o to CJ vo vo vo VD CD CD CD 00 vo o (-* u> CJ vo vo vo vo to o to Ln o er o o 00 vo vo vo M to o Ln CJ 2 ,3,6 - t r i c h l o r o p h e n o l 2 ,4 ,5-trichlorophenol ooo oo o to to CJ to to oo to CJ ooo CJ CJ to ** 2 , 3 , 5 , 6 - t e t r a c h l o r o p h e n o l to ro to to NJ ro to CJ to -o <T NJ o o h-> ro o a \ to to ro cr> ro AA oo CJ Ln A /\ /\ o oooo CJ J Ul CJ CJ A A /N I-1 r--* M o cp2 , 4 - d i c h l o r o - 5 - m e t h o x y p h e n o l ** 4 , 5 - d i c h l o r o - 2 - m e t h o x y p h e n o l /N / \ /N A M Mi H (D 3 A A A A \ M ri t--1 t--* h-1 H* t--* t) ti) M M 3M OI D *3 ^ 2 , 3 , 7 , 8-tetrachlorodibenzo- '-p-dioxin (TCDD) SHIPMENTS OF TRICHL0R0PHEN0L H* H* H* o O O O O o O o O O O vo VO VO VO VO VO O o O O O tO to to to to to CT er cn Ln U) VD CD CT tO to o 11 i -o i ii i1 Ii iO -O -O -o 11 u> to tO H* H* i-- H* J--1 to * I--* 1--1 t--1 VO VO vo VO VO VO o o o o o\ \ \ \ \ \ \ \ \\ CTl ov CTl U1 LO VtOo to CD to tO ctv u> to to tO o \ CTl \ CTl \OV \ ov \ ov \ o\ \ a^ \ CTl \\ CTV o\ \ CTV -J -o ^1 o -o -J -J -J t-< o 1-9 > 3 to .fc* to to J* *tk to to er 00 CTl CTV 00 CD CD CO CD er er -o VO ~o VO vo VO vo VO -o -o to LO to ' to LO u> to to to to to vo o VO VO t--1 t--* -* t--1 M vo vo VO io A to lU 111111 11 D o vO n n o o to t-* o o o > 50 A A. A A A A A AA o o o o o o oo o o H* 1--' M M t-* i--1 1--* M M M dP 2, 4 - d i c h l o r o p h e n o l oo CT to o Oo CD Ln -o oo CT o f--1 I-1 M vo O o O <*>2,5 - d i c h l o r o p h e n o l ooooOoooo oO cn LO 00 co to U ) Ln to t--1 dP2,3, 4 - t r i c h l o r o p h e n o l u> u > to to to to to to to to I-* to to - 1--* ~o GO vo - o vo 00 tn VO VO VO 00 VO vo vo vo vo vo vo vo o o VO 1--1 o to to to to to t--1 t--' o VO o o to to to cn H* 2 ,3 ,6-trichlorophenol <*' 2 , 4 , 5 - t r i c h l o r o p h e n o l oooo to to CO M oo o 1--* .fcfc to o oO to o I--1 0,5 2,2,5,6 - t e t r a c h l o r o p h e n o l to to to to to to t--1 to to to to o to -o t-1 to -o to cn VO CTV A / \ A A /s A o o o Oo o o o oo o to to to to to to to to to U) to dP 2 , 4 - d i c h l o r o - 5 - m e t h o x y p h e n o l #>4 , 5 - d i c h l o r o - 2 - m e t h o x y p h e n o l DOW 018163 A A A /V / \ A A A A A /V t--1 t-> (- t-1 t-* -* y-1 t-* t-- t-* (--1 2,3,7,8-tetrachlorodibenzo3 p - d i o x i n (TCDD) - t- SHIPM ENTS OF -dichlorophenol LOT DATE BLDG CAR 10097-1 10097-2 10127-1 10137-1 10167-1 10187-1 10217-1 10237-1 10257-1 10287-1 M03S 1 7 -1 D 10/9/67 10/9/67 10/12/67 10/13/67 10/16/67 10/18/67 10/21/67 10/23/67 10/25/67 10/28/67 10/31/67 489 313-C 267 294-D 267 309-C 489 314-C 489 313-C 489 314-C 489 313-C 489 309-C 267 314-d 489 313-C 489 309-C % <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 TRICHLOROPHENOL rH 0 c rH a> 0 si ca oo Ak a0 0 rH k x: 0o rH H X! k uA H l 'd *r i in n fc. CM CM %% 0.5 0.5 0.5 0.3 0.6 0.3 0.7 0.4 0.7 0.3 0.6 0.3 0.4 0.2 0.3 0.2 0.4 0.4 0.6 0.4 0.6 0.3 rH rH 00 cc Q) <D Si A aa 0o kk 00 pH rH si x: 0u H H kk 4J P l1 IO in V ro CM CM %% 2.7 89.6 2.9 92.1 2.4 90.0 2.5 91.2 2.9 90.5 2.7 90.5 2.3 91.8 2.7 90.8 3.4 89.7 3.6 88.9 4.0 88.3 00 GG i <1) <D o rH A A N 0aa c G a) O >i XI x : X X H a 0 0 d 0 si A 0 kAA k o 0) 0> 0 rH e e rH x ; i i A -- u in CM u a <Tj i 1 <o a k 0 0 ku 4J k In -P En a) 0 0 01 P rH rH P i x: X 1 G *X) 0 0 00 -<H H H -X in K T) 1 d i r- 0 m m cn d < 1 CM CM cm a % % % (PPM) 0.2 2.0 0.2 1.9 0.1 1.8 0.2 2.2 0.2 2.4 0.2 . 2.1 0.2 2.4 0.2 2.3 0.3 2.3 0.3 2.0 0.3 2.4 <0.3 <0.3 <0.3 0.3 0.3 0.3 <0.3 <0.3 <0.3 <0.3 <1 <1 <1 <1 <1 <1 <1 <1 <1 't>9T8T0 VvOa SHIPMENTS OF TRICHLOROPHENOL 8S29I tO HO* i--i1* to OVOo 1 M I--* to o0'MO1!0 to o -hoI1-1 HH** to f--I11 (t--** to tn -J K* H* H-* to -MJ11 HH** KU*) 1 1M--1 OVO O 1-* Mh-1 OCTv f--111 M HO* -t11oo to oOOa o o Tl a a hJ M t--1 t--1 M t-* t--J I-1 I-* l--* to to to to M 1- t-- H* t-* t-1 \\ \ \\\\\\ M VD 00 to to to h-> VO cn to O \ -J Ln to \ \ \ \ cn cn CTi \ \ \ cn cn cn cn -j ~o CTl o\ Ch CTl -J -o -o -o -o -o tfc*. C* to to cnCD CD CO CD CD CD CD CTi CD CD VD VD VD VD VO VO VO VO VO u> to to to to to to to to to to M o M t--1 h-> o o t--1 O H* to vo to vo vo to VO to 1 r1 1 1 1 t1 1 1 1 Oo n o n n o n OOO f1 O -3 5> 3 to tr< O <n n > 50 <0. <0. <0. <0. 1 1I I 11 1 1 H * t--* t--' t--' <#> 2 , 4 - d i c h l o r o p h e n o l oOoo o o o o o oo cn cn cn cn cn cn to Ul t--1 cn oooo o oo o ooo CO co co co co CO LO cn tn co to to to to to to to CO tn tCfc- J* cn co cn CO vo VD vo vo VO CD. vo vo vo CO CD M -o o o o VO o t--1 t-- vo VO co tn tn to CD -J o CD -J cn t*2 , 5-dichlorophenol ^ 2,3,4-trichlorophenol *>2 , 3 , 6 - t r i c h l o r o p h e n o l dp 2,4,5-trichlorophenol CO u> to i 1 1 1 1 1 1 1 cn cn CD oooo o oo oo oO to to CO CO to to to to CO to to to to to to to to to to to to CO M to CO to o H* CO M CO <0. oooo o oo o CO CO CO CO to CO to tn CO <*2,4,6 - t r i c h l o r o p h e n o l dp 2,3,5,6-tetrachlorophenol dp2 , 4-dichloro-5-methoxy phenol ^ 4,5-dichloro-2-methoxy phenol /N ' S /\ t--* M A /N A M H /V A M M |o 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o 2 p-dioxin (TCDD) DOW018165 9 oo O to o-CoO oO00J 1i t--* o o t--1 o1--' to to V0D0 -0o0 i M I-1* Oo o H* to M00 (--1* D oOO t00--00* 1--1 0a0t hi- '* 0C0J H* i-i* 1--1* ao o t(--* to 00 1 t-> D t-1 tt--o* t--1' D to -o \ to \ LO \ h-1 \to VD \~tt-o>J M \It--o1 t-> \ 0t-0> i--* \t-> at t--1 \tC-*J I--* \l-> to H* to \H 0o0\ CcDn \(CTD> \\ <0T0l 000V \ <00J\ \ a0t0 \a00t \ o00\ Ov tur* -3 >D t-3 w GVOO 00 vo .u V0D0 00 VO to a -o V0D0 V0tD0* 00 VD 00 VO 00 VO LO VoD i u> M 1 LJ CJ tC--J' o VO i1 u> U> LJ CJ t--* t-- to t--1 oVD 11 CJ IC-*J t CJ M 1 oo nnoOoooo tfl f o <D n > A AA A o M V i o o i i 1 o at Ul ai dP 2 , 4 - SHIPMENTS OF TRICHLOROPHENOL Oo ooo o o oo o OV 'O -J 00 o -o o\ o\ dP 2 , 5 - o o oooo o ooo U1 o cn o a* 'O o -o CJ dP 2 , 3 , to to to to to LO U) to to to ao -a\ 00 00 00 LO o VO vj VD i-* VD VD I--* o VaO> VO M vo o 00 VD VO o VO o 0VD0 ~~i 00 o CJ to at CJ to to 00 dP 2 , 3 , dP 2 , 4 , o o o o Oo o o o o to to to to to to to to to CJ ddPP 22,. 33.,5,6-tetrachlorophenol DOW018166 to to to to to to to to to to to M OJ CJ to LJ LO CJ CJ A o Oo ooOo o o o t-* it* LO CJ (jJ to LJ CJ CJ d<#P> 22 ,, 44-- dichloro-5-methoxy phenol d#P> 44 ,, 55-- dichloro-2-methoxy phenol A t-* AA t--* t-* A. A t-- A H* AA i--1 AA t-* (--1 a 0 2 ,3 , 3: p-di - L- Z9T oO O O OO o OJ LO Lo OJ OJ L J to oLO to t o to to to O CD ro O VO 00 00 CD CD CD 00 CO CD 1i i1 I 1 1I 11 M M M H t-- M M *3 3 oo oo to to to to to o CTv M O co co oo oo III I-- ' i-- I-- Lo to to to to to to \\\ \ \\\ LO to to to to VO to O CD (J\ to o CO \ \ \ cn \ cn o \ cn er <n 00 o \ oo 00 00 00 00 CO to to to to \\ \ to M M 1--1 o cn H- O \\\\ G\ cn CTv CTV CD 00 00 00 ot-< H3 4k u 4k 4k 4k. to 4k 00 00 00 00 CO a \ 00 VO VO VO VO VO 'O vo 4k 4k. to 4k 00 00 CTV 00 VO VO VO to to to LO to to to cj C i u> cj co M Lo h-* o ' 4k vo 4 o to vo 4k. O vo >t-3 M I--1 t--* . co ^ IX O W t"< O CD SHIPMENTS OF TRICHL0R0PHEN0L AA AA o oooO I-1 1--* t--J i--* <#* 2,4-dichlorophenol o O O ooo Oo o o <#> 2,5-dichlorophenol CTV CTV cn CTV CTV cn on CTV cn o O O "o o O o o o o vo cn tn tn t-> cn cn cn 'J on #> 2,3,4-trichlorophenol to to to to to b J to to to to 4k co tJ M cn to 4k. 0J to to vo vo vo vo VO vo vo vo vo vo to to to ro M to t-* to t-* to o to Jk vo CTV Ln vo to o o *> 2 , 3,6-trichlorophenol dp 2,4,5-trichlorophenol o o ooOo oo oo to LO to to to H* to t-* h-` t--* <#> 2,3,5,6-tetrachlorophenol DOW018167 to to to to to to to to I-* to CO o to to o LO to t-* vo o <*> 2 , 4-dichloro-5-methoxy phenol o o o ooo oo O o dP 4,5-dichloro-2-methoxy phenol to u> 4k tJ 0J 0J 0J 0J OJ CJ /\ A M /\ A /\ A K* 1-- /\ A t--* t- ' /\ t--1 M 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o 2 p - d i o x i n (TCDD) 8 O Ln o nj WV !- 00 1 h-J O OOOOO o OOOO UO1 K ) h-> VO nCTj* N>J NJ U M 00 M-sJ H* H* U O vo OLn *U oNJ 00 CD 00 00 CO 00 00 00 00 OO 11 11 1 I- H* M h-* M H* M M H* H* O DO Ln U1 iN t t *>. \N> \CT 00 \ M \Ch 00 \N> \NJ KD CT* \CCTO* \C0T0* \NJ NJ \CT* 00 \ CO \CT* 00 \ H* \ O* 00 \ M M \CT* CD KD \CT* 00 Ln \CT* 00 NJ \CT* 00 t-< o -3 O > 1-3 W nCTj -J \ LJ H* U) fc 00 00 NCT>* C* 00 NJ CT* * 00 NCTJ* 00 U 00 CO vo VO *-J KD KD KD vo KD siQw O> M I--*t-3 LJ O KD LJ LMJ LJ 'J O LJ LJ M et o> t--'1-3 Lt--J* H* LJ O VO LMJ HC-h* X I-* X 00 CO t* O O O > 0 SHIPMENTS OF TRICHLOROPHENOL 8 9 1STO f A O a op 2,4-dichlorophenol o OooOO ooooo Ch CT* <T* a s Ln ^1 a s dp 2,5-dichlorophenol o OOp oOooooo <T* CT* CT* CT* CT* a s Ln CT CT* CT* CT* dp 2,3., 4-trichlorophenol f--1 NJ N) NJ NJ NJ NJ NJ NJ NJ N) VO NJ -NJ NJ m Ln Ln LJ LJ NJ >N VO LJ vo NO VO o vo LJ vo LJ vo h-> vo LJ KNDJ vo vo (-- M CO VO 00 M (h LJ NJ '- t CT* Ln vo ** 2,3,6-trichlorophenol dP 2,4,5-trichlorophenol o Oo oOOoo oOo M M 1-* t--1 NJ NJ NJ NJ NJ H* t- 1 <*> 2,3,5,6-tetrachlorophenol NJ NJ NJ NJ NJ NJ NJ NJ NJ NJ NJ (-* NJ NJ en LJ Ln iN a s Ln LJ <#> 2,4-dichloro-5-methoxy phenol O o o o o o o o oOo k. 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' 2778 -IF 0 3 0 8 3 - I S244 Pi/- - 0.2 0.6 .6 :Jr*- f C /C 1.2 95.5 ?25 .f 2 .0 i 0.5 - i 0.1 ! 2 ? i 0 .5 < 0.6 0.3 / 5 92.5 ; i .* / t 2 . / i i- 0.6 * -- 2.2 % 7 r '; i */ ' 1 /i-n 0.5 C./ 2/ r--,--,o y i ! ^/ 1/ i -I 4 1! C T>/ T P C M O n 297 Reprinted from WEED SCIENCE Yol. 13, No. 6, Xo\ember. 1**70 16249 Effects of Herbicides on Water and Its Inhabitants1 W . R. M u l u s o n 2 vj <T> oAnuobrmstiwrcaactote.rrTsinhudepurpeslietrisisalilnitutlaseamgoeeuvinadtrescnctroeeactcahhuasinteghaeorprboicalilducectisuomnfruoplamrtoinbaglegmrion. Fish tolerance to weed killers varies with their size and species as well as with differences in the aquatic site. There may be variation to different forms of the active ingredient. Other components in a formulation may he more toxic than the herbicide itself. Therefore, the LD^, of different formulations scitthhisrecouutoemldfxfseitccabitntesyceodtsfeotiehnwrevmarobtihenicreiinddgie.nshaIanpobpnitlaiacdfnaisdtthsiio,t.inopOnlo,aufnrtakhtceohunermrr,beainacntnidndeeknromotawhaneyledrdcghwoaeianlitgeoeerrf in surface water must be evaluated. The accumulatio: of herbicides, if it occurs, by plankton and water nnimai could start the biological magnification process first a. cTlheuasrl,ythdeeirmeofnfescttraotnedfishw,ipthlantkhteoni,nasnedctiwciadteer DcrDusTtace(6a8n is of considerable consequence. There are several general references dealing with tlu toxicity of chemicals, particularly herbicides, to fish sAivpeplefigshatetocxticaitly. (5) and Lawrence (73) both have exten data. Three reports from the Fish anc. Wildlife Service (135. 136, 137) are excellent summaries of investigations carried out by the U.S. Department oi inhabitants indicate that harmful effects with our present herbicides, when such exist, are onlv temporarv. Available tthoexicIintytedriaotra. fTorhemaHneyrbwiceieddekillHerasn.dbook (62) has fish, ewviitdhenhceerbsicuigdgeess.ts there is no biological magnification problem There base been relatively few investigations on the effect of herbicides on plankton. Usually the work re ported occurs as a brief note in conjunction with a T In t r o d u c t i o n o d a y there is much professional and lav interest in our environment, particularly with respect to pollution- problems. The unfavorable subtle effects dichloro dipheml trichlorocthnne (DDT) on certain organisms and :ts practtcallv tmiioianl distribution in our environment have stimulated questions concerning the possibility of similar effects from other agricultural chemicals. Herbicides arc common today and their use is growing rapidly. Increasing concern wiil be directed toward the effects of weedkillers on non-target organisms ns well ns the ultimate fate of these chemicals in our environment. Our anahtical methods are becoming incrcn.xinglv sensi tive and trace amounts of various herbicides are likelv to befound in more and more places. However, interpreting the importance of such small quantities as parts per ...illion and even parts per billion of the weed killers when found in our environment is the real problem. It appears timely, therefore, to survey the possible occur herbicidal study of an aquatic weed problem. As the study of herbicides is becoming a more mature science and wehave realized the possible existence of a biological of magnification emphasis and problem studv will throngh herbicidal use, be placet! on this aspect more of the subject in developing new products. This is not a review of herbicide! effectivesec<: os a(1q3u),ataicndwemedoteprorebcleemntsly. KthliengNmaatnion(6a8l ).RFesiveearrcahntCi oEuvnacnisl o(8f6 ) in their books base good sections various herbicides for aquatic weed on the application control. A general review on this subject and a good history of also is given bv the development Hoofuasequcat tica l .w(e3e7d) control is given bv Timmons (182). Instead, tin'spublica tion is concerned with the ultimate fate of hci biridcs after use and also their effects on non-taiget organisms. In this paper, the organization has been according to the common names of herbicides taken up in alpha betical order. In certain instances, for convenience, more than one may Ire discussed under one heading. rkennocweleodfgehecrobniccideernsinign twheaitrereffaencdts oton sfiusmh,mpalarinzektopnre, saenndt othTehrewraetseidrudawl eelflefercst. of herbicides in the ecosvstcm that involves water supplies is of great importance. Drainage from watersheds and other areas treated with herbicides might result in secontlnrv effects involving the health of domestic animals, wildlife, aquatic animals, and even man. It has been estimated (90) that more than 80% of the population will depend upon surface water for drinking bv 1080. Thus, the prevention of water con tamination is of the utmost direct importance to man. aWsitwhellthaesicnrcorpelaasnindg, tuhseesoigfnhifeicrabnicciedeosf otnhenironoc-ccruorprelanncde 'Recehcil for publication October 21. IfiiO. H erbicides Avec rrvo pp!> l teoi nx.ic Rurdick to fish. for fmgerling Tcht eacl.he(1m7i)carlelpiaosrtead21c-hhart brown trout i S a h n n tr u ttr . acrolein TLL.).,,1anoilf is 760 pwSpueFbeddeasrnfgo.aurnsPbothilnuvsetncogattiiolllsasxli.(c(IiL(ht3vci8Up)sontmuumdsieisaedsmmaiaacetDrroi2ool5cemhinapirnhutiaslola5Rcr0io.a)n.ptpsrpompl .)ashqinouwatehtidec minor or no injure to furrow-irrigated crops, but Horn! irrigation of vegetable seedlings at 15 ppm was toxic. Effective downstream carriage of acrolein tens tlemon- Nirntcti for a distance of 1 mile. The snails were 08 to `.".i'",', killed and all submersed aquatic weed- were desLt'rtoiryaeud.ct al. (133). experimenting with acrolein in 'Agricultural Department. The Dow Chemical Company. Mid land, Michigan. 'Median tolerated limit. 7 3 8 Volume IS, Issue 6 (November). 1970 16250 MULLISON : HERBICIDE EFFECTS ON AQUATIC ORGANISMS PMPQ M O "gypt, also found it very effective against snails as well sithineiarrqiguaattiiocncuilriltyclliecasf. pTohnedwHOeetod jiOP opt|i)ninn ocgonnt ocnenetrnastpiouns equired lor weed control had no adverse effect on local crops. Field tests demonstrated the importance of water control during the application period. oA fr sseondiciu. mMaacrskeennittelu.tnDu(7ri7n)gretpheortpeadston10thveenlros,ngH-ItSe,r0m00uslbe of AS-jOj have been applied along a lake shoreline: -uniform distribution of tin's quantile over the top inch of bottom mud or hydrosoil would amount to 3SUppm. Mud samples analyzed in the fall had -18 to 154 ppm .and the following spring 10 to S'J. Five species of fish 3 or 4 years old were analyzed for arsenic: highest con centration found in the fish flesh was 0.07 ppm in yellow ipnertchhe(oPrge raenas(wL ai vsc s0c.2c7n spMpm.). inHisgkhinesat ncdonsccaelenstraotfiobnluefoguilnlsd. Water samples from the outlet were analyzed for 10 weeks starting in June and contained from 0.073 to 0.225 ppm of arsenic. Ball (9) used 74As-tagged sodium arsenite in a study of its effects on pond ecology. Treatment was at 8 ppm, the standard commercial rate. The sodium arsenite was taken up rapidly bv the plants anti recycled to the water and soil bv decay and sedimentation of the plants within 5 days. another One one nlga (Chur ( n S psipr.o)g \arbasosrbpe.)d was the killed whereas herbicide and appeared to be unaffected. The tagged material per sisted in the water for tlie duration of the experiment about one-half the concentration of the applied nount. The invertebrates (microcrustacea) showed great ariation from vc--sensitive to very resistant in response o the herbicide. The arsenite moved into tiic soil anti reached a depth of 3 inches in the hydrosoil during a 60-day period. General effect on the conummitv metab olism of the pond as shown by the diurnal 0_. curve indicates that the herbicide had a drastic effect. It raises serious questions as to the advisability of using sodium arsenite in fish-producing ponds due to its deleterious effects on several levels ol the fish loud chain. Gilderhus (-15) studied the use of sublethal concen trations of sodium arsenite on bluegills and pond in vertebrates. Applications that totaled 4 ppm reduced the survival and growth of the fish: immature fish ,.^re more affected than adults. Substantial quantities of arsenic were found in the water, hydrosoil, anil the organs and flesh of bluegills at the end of the experiment. Bottom fauna and plankton populations were reduced or in hibited in several pools with the highest concentrations of herbicide. Lawrence (71) and Surlier and Meehean (123) both used 4 ppnt arsenic as a herbicide for filamentous algae and found it to be toxic to desirable food organisms. Lawrence also noted that it decreased blucgill produc tion. Aamsitturdoyl e in(3 -Oa mreignoo n-S rotrnia zan lcs)t.reaMmarsdtroaninirnt gala. (SI) made municipal watershed. An air application was made at 2 lb A to nxtirmolumthecosnaclemnotrnabteiornrvo(fR1n5b5n ps pbU iwcca's.ahfoiluisndPuinrslwi)a. teAr rnples Iff) min later, decreasing to 20 ppb at the end . 2 hr anil none in 6 dais. At l.S miles below the Volume 18, Issue 6 (November), 1970 sprayed area, no amitrole wasdetected at anv time. Water sampled during their study probably would have been safe for use in home irrigation systems. There was not sufficient amitrole present at anv time to cause toxicitv to wai m-blooiled animals. In other tests with amitrole for salmonberrv control. Tarrant and Norris (126) made aerial applications 2 successive years using Hlb A in 1Ugal of water. Level of amitrole in samples of stream water at the spray- site was 400 ppb in samples taken 5 min after spraying; 10 hr later, it hail decreased 100-t'old to 4 ppb. Ami trole was not detected after 3 days. also treated HG0 acres with amitrole Naton2aslbctA.a l.Af(t9e3r) wards, stream water contained 42 and 45 ppb of ami trole which totally disappeared in 48 hr. Dreacloagj>noiznedf 2t,h2a-dticdhalloarpoopnr o phai so n ai c vearcvid )lo. wItdegisreegeonfcrtaollxv icitv to sTihu iscga su r a(f1ius3ths0.)LI.ta)ncadafutes.MerdaHgIzeeehroraomnfdoerxCtpaoolihstvnureetroa(gt7o91l;0d0fsishphopwm( Ceda(6mHist)s-. rapid decomposition in soil. Chancellor anil Ripper (22) indicated that yellow p3R0e.()r1,chpa,pnmdno, pratuhltmehropnukignfhastchaeetda4d0i L0mc ppinponmmoiws50g%(iPbibmoofcspnthhsea lLcb.sr)opwtornloemrtareotleaudst were killed. The sodium salt of dalapon apparently did not R.) aatffe3c,0t0th0epLpamk.eDErmageornald11vshminmerp(hXso l ( ropi O do s n a at th a csrpi.n)owi decres not killed bv 2,000 ppm and several insects that normal ly arc eaten bv fish were unaffected by dosages between 400 to 800 ppm. JacorosSi (65) in investigations on controlling weeds in canals reported no toxic effects on seven hsh species exposed for 3 tlavs to dalapon at concentrations far ex ceeding those likelv to be obtained in field practice. Kochkin (69), in an organoleptic study of dalapon. concluded that 2 ppm is the maximum permissible con centration. D i c h l o b c n i l ( 2 , 6 - d i c h I o r n b c n z n n i t r i l c ) . Frank c t i.f. (41) investigated the effects of canal-bottom plots treated with dichlobcnil and (2.3.6-trichlorophenyI)acctic acid (fenac) on irrigation water flowing over them. Concentrations of dichiobenil and fenac initiallv were 8.6 and 8.8 ppm. respectively. The bulk of the herbicides lost Iroin the jslots due to the water flow occurred quicklv and safe levels were reached after I hr of water How. Van Valin (139) applied dichlobcnil at 10. 20. and 40 ppm to ponds. Residues in both water anil fish were highest within 3 davs after treatment. Concentration in water 11 davs after treatment was about 2% of that after 3 davs. The residue in fish, whole bodv, dropped ncarlv asfast but could still be detected after 112 days. Taylor (12S. 129) reported that the LD-,> of dichio benil for most species of fob was 15 to 20 ppm: the usual recommended rate of 0-5 ppm did not adversely affect spawning. It had a low degree of toxicitv to species such as vir'iinicn sGh.r)i:mppla(nRkcton nnc utos lesrpa.t)edan1d0 popymste.rsMetCd.iarans sloedthrcaal concentration to the fiesh water crustacean D u p L n n i sp.. a common iood chain oigunNm, was approximately 10 ppm. Walker (112) reported that concentrations of 12 to 739 16251' W EED SCIENCE 22 ppm with a 9(3-hr exposure perioil produced 50% mortality in four warm water fish species. tndCoCpoeloerl aadlo. .(2A.1)t reported on pond studies in the former site, only 3% of Oklahoma the dichlo- benil applied at 10, 120, ami -10 ppm remained in the wafer after 11 days; even with *10 ppm, only 1.5 ppm remained after (JOdays. At that time, the same amount was left in the mud. lint fish reproduction was alfccted adversely by the -10 ppm. and there were some patho logic effects in the fish organs. There was no immediate mortality, but survival after 16 weeks was 60% for the controls and 20, 5, and 2% in the ponds with the in creasing dosages. Increase in weight was 67% for the controls but 169, 169, and -1S2%, respectivelv, for the other treatments mentioned above, presumable owing to a more abundant food supply. In Colorado, 0.5S ppm of dichlobenil. resulted in maximum concentration of 0.152 and 0.13 ppm of the herbicide in the water after 3 weeks. After 63 davs. it declined to 0.17 ppm when there was 1.98 ppm in the mud. Xo fish mortality occurred outside the cages al though a lack of oxygen killed all the caged fish at the bottom. andD i q u a t 6 , 7 - d i h y d r o d i p x r i d o 1 (2 - a :2 ', l '-c p x r a z i n e d i i u m ion IJ '-d im e th y l-f J'-bipxridiniurn ion (paraquat Allen 1 hr pertoadlu. c(3e)dfosuonmde that the useof diquat at prophvlactic effect on 2ppm for adult and juvenile affected D cnnocy bsctyhi dintihoueomkpsespar.nlmicoionu<s0dnisreoarshe\ nccahuussedts hbavi ctxhtes c hfuanVgu.s) "'aBrneqauslae:yaent ti a1l .(1p0p)mfooufndvhqau.mit ainximuvudmrosoofus7.9whpepremthoef >oi.s had been treated 4 vears previousiv -with 0.3 lb A. quat anti paraquat will persist in hvdrosoils for long periods of time. Blackburn and Weldon (12) reporred that diquat, originally applied at 2.5 ppm, persisted in water 8 and II clays. Xo toxicity to fish was apparent. It appeared to have little effect on natural fauna and plankton. Coats et quat anti paal.ra(q2u3)atinwvehsetnigaatpepdliethde disappearance of di at 0.5 and 0.1 ppm in plastic pools and ponds. Paraquat in plastic ponds in water alone took 31 weeks to disappear and diquat Id weeks. In contrast, neither was found in pond water after 15 days. This work shows the strong sorptive power of soil for these compounds. that water analysis revealed onlv a Dalv trace oc tf apla. r(a3q1u)aftouanndti diquat 21 hr after thev had been applied to control eurasian watennilfoil { M x r i o p h y l l u m ap ic a tu r n L.). Frank ct al.* in comparing the residual effect of para quat. an ester of (2.1-dichlorophenoxy)acetic acid (2.4-D). dichlobenil. anti fenac in pond water and soils, found paraquat was the least persistent in water; it was not detected 12 davs after application. But the butoxyeth- anol ester of 2.4-D took 21 davs to disappear. Dichlo benil and fenac persisted for 150 days anti traces of fenac were still present in the water 1 vear after appli cation. After 85 davs. the top inch of the hvdrosoil contained 10 ppm paraquat anti no 2.1-D. Fenac in `Frank. P. A.. R. D. Comes, and E. B. Hollinesworth. 106/5. visicnee of herbicides in pomied water and soils. Weed Sci. Spc. mer. Abstr. p. 3. the hvdrosoil dropped from 12.1 ppm at 21 days to 0.26 ppm after 160 days; dichlobenil went from 10.6 ppm initially to 1.3-1 ppm after 120 days. Later, Frank and Comes (-12) studied herbicide resi dues in pond water anti hydrosoil. They applied dichlo benil at 0.58 and 0.-10 ppm, fenac at i and 1.56 ppm, 2,-1-D at 1.33 ppm. 7-oxabicyclo (2.2.1)heptane-2,3-di- carboxylic acid (endothall) at 1 ppm, paraquat at 1.14 prepsmid,ueasntpiedrsiiqsuteadt iant s0o.6il2apnpdmw. aFteernafcoramndoredicthhalonbe1n6i0l days. Low amounts of 2,-f-D were found in water for 24 o o days and in disappeared. the hydrosoil for 55 days after Endothall, paraquat, and diquat wwehriechlesist * peisistent anti were not found in the water after 21. 8. 5317 48 and 4 days, respectively. Paraquat and tliquat persisted at high concentrations in the hydro.voil for 85 and 160 days, respectivelv. Cilderhus (46j exposed fingerling and adult bluegills to tliquat at various concentrations and frequencies of treatment in outdoor pools for 24 weeks. Xumbers among bottom fauna and plankton were unchanged. Treated fish did not show any changes that could be attributed to diquat. The herbicide was toxic to a fresh wanaGdterzrpeacnrrdauaqsutaeatcte,aal .na,p(5p0Cli)leacddoomcaeptraa2re.5dsp.pthpempeinrsispteonncde of diquat water, to fenac at 4.0 ppm and nmitrole at 1.0 ppm. Both para quat and diquat disappeared from the water in less than 1month. Soil sediment in the water increased the rate of their disappearance; they did not appear to be desorbed from bottom clays. The paraquat and diquat treatments were repeated with esseminllv rhe ante re sults. Fenac was reduced to 2.1 ppm by 202 -Jays .md nmitrole to 0.13 ppm by 151 days after treatment. He concluded that fenac appears to have the least potential I for use in sources of potable water because of its long persistence. paRraoqduraigtucozn-Kathbeanagroewt thal. (117) studied the effect of of the pathogenic fungus. SInc l etrhoet i ufomrmrenrl,f s igiroSwatchc.. in was liquid culture and in soil. decreasetl at all concentra tions. In soil experiments, evolution of C02was lowered at all concentrations but greatly reduced at only the 0.5 ppm and I ppm iates. Yeo (150) studied the effects of diquat and paraquat in water on fish. In reservoirs, diquat applied at 1000 ppb dissipated to 9 ppb after 12 days. In fish-growing pools, diquat at 1000 ppb dissipated to 900 ppb or less. Paraquat was slower to disappear athnadBteegasxolhledivbfisitehetdpalalu.cnep(d1re0i)dn,itcoutaswbinlaegtefrrluaccdotiuonaatatciiotnivninesg. d8iqpupamt. found diquat for 72 hr and then into fresh water for 10 days had radioactivity in jhe flesh and various organs in a negli gible amount except in the gastrointestinal tract. Gil- derhus (16) investigated the effects of diquat on blue- gills and their food organisms. He reported that diquat disappeared from the water at about the same rate dur ing spring or summer. Residues were highest about 10 davs after treatment and disappeared 6 to 12 weeks after treatment. Diquat at 1 and 3 ppm did not affect adult or fingerling bluegills. Xo hematological or histo- pathological effects were noted. It was quite toxic to the freshwater crustacean. C la d o c e ra sp., but these returned Volume IS, Issue 6 (Xovember), 1970 16252 MULLISOX : HERBICIDE EFFECTS OX AQUATIC ORCAXISMS ffOW 531749 to their nonnal numbers with the disappearance of di- 'uat. Holz (50) used paraquat for aquatic weed control in ditches. He reported that fish, frogs ( R a t io sp.), and aquatic insects appeared to be unharmed by paraquat. Cattle drinking the ditch water after treatment (how long not specified) showed no toxic effect. Riemcr (110) used diquat at 0.31, 0.53. and 0.73 ppm to control inLbtaolactdedeidddpcerodnwneo)ot,rlaatprfigfc(eeUkcetIrmreithcloueuflrtayhsrpiiaEabwassosnpsxi.n).sfgrpyX.)o.o(fMopraiupcymprooaupprnketgicnnrtsyueeseelflofdsewaclstmsupnoewfiridceschhres;. Bottom duelling invertebrates, primarilv chironomids, were unaffected. Plankton were present in an insufficient amount to make valid comparisons before and after treatment. Blok (13), using paraquat and diquat at 0.4 to 0.8 ppm to control filamentous algae, noted no effect on fish or fish footl organisms. Used at 0.8 ppm for control of filamentous algae. 3-.3.4-dichIorophcnyl)-l.1-dimethyl- urea (diuron) had no deleterious effects on fish: in shal low ditches, no fish were killed. He also noted that one species of rotifiers was present in large numbers and tphhayttoopnleanskpteocnieswoefreD acphhien filay increased in unicellular numbers. flagellates The and bVeonl vzoaxmidaeu r e(hues reEinhar.fteDr icrelifleorbreedniltoanasd 2.6-dichlorothiochlorthiantith at I ppm were equally well tolerated bv the fauna. In another trial with them. C.rustaceac. especiallv copepods and bottom fauna, increased in number, but the pltvto- slankton were restricted to flagellates and isolated dia- oms. Cassie (21) used dinuar at an estimated 0.5 ppm in New Zealand and noted adecreased number of diatoms and algae. The decrease in algae might have been as sociated with anaerobic conditions caused by the de composing aquatic weeds. Lawrence (7-1) gave a numerical and graphic presen tation of aquatic weed tlata on nine different combina tions of treatments over a 2-vear period. He used paraquat, diquat. fenac. 2-chloro-1.fi-bisfethvlamino)-s- tria/inc (sima/inc). and 3-(3.1-dichlorophenvT)-l.-methoxy- l-methylurca (linuronj in various combinations. This reviewer interpreted the tlata as showing that plankton were unaffected bv these treatments with the exception of the paraquat-Iinuron combination. This caused a 50% reduction in plankton tite first sear and a slight reduction the following vear. Fish production appeared to have been adversely affected in all treatments with perhaps the exception of the fenac treatment at the end of the first year. Newman (87) gave data on tite use of diquat and paraquat as it affected population changes in tire small er arthropods. There was a two to three-fold increase in earthworms; a residual amount of the herbicide was found in them which was rapidly lost upon their trans fer to untreated soil. Amounts found even following high application rates seere negligible. In aquatic situa- :ons no drastic effects on anv group of animals (insects, istnecne, mites, mollusca. worms, leeches, and flat vorms) were found. He iS`J) also reported on the eco logical effect of paraquat and diquat at 0.5 to 1.0 ppm at 10 different sites. Xo direct toxic effects to fish were noted and in one case an increase in Dipterous larvae, inainlv Chiromedae. occurred which was associated with the bieakdown of killed weeds. Xewman and Way (88) reported no direct effects of diquat and paraquat on aquatic invertebrates present in their experiments. But thev noted severe owgen de pletion at one location following decay of the treated aquatic weetls. This resulted in some mortalitv among Hirudinea. Jsopoda. Odonata, Coleptera. Trichoptera, Gastropoda, Lambellebranchiata. anil captive trout al though free-living coarse fish and trout were generally unaffected. These herbicides were absorbed bv the vegc: tation and the bottom mud which in one case con tained 36% of the original dose in the top 2 inches 5of\Apamroanqtuhast aafntedr dtrieqautamteantt.0T.5hetov 1a.l0sopprempoartte1d0odniffeeffreecntst sites. Xo toxic effects to fish were noted. An increase in dipterous larvae, mainly Chironomidac. associated with the breakdown of ilead weeds occurred once. (etPliyielr-caeminclo)-af>l.-(is(1o0p9r)opyusleadminfeon)a-ci-.irisai/minaezin(ea.tra2z-icnhcl)o,ro2-,11-bis(isop)opylamino)-G-metInhuercapto-s-triazine (prometryne), diquat, and paraquat for aquatic weed control. The triazines were effective for control of filamentous algae. Microcvstic blooms were removed and rapidly re placed with more desirable plankton algae when aira/iue was used at u.i)8 anil 0.16 ppm. Diquat and paraquat used at 0.25 to It) ppm had no adverse effect on fish, fish footi oiganisim. or plankton. Tatum and Blackburn (127) used diquat at 0.5 ppm rinaritlvwoaffseucbt-etdi opimiiuml ciplioaniedslv. PbulnttorpcclaonvketiOeiiii rwaap-.iiihte.mUpoi-ganic matter from dcraviug vegetation appeared to significamlv increase certain benthic organisin'. Wile (118) studied the effect of diquat, fenac. and sima zine on phytoplankton. Applications were at 1 and 2 lb--A. In two ponds where diatoms were dominant met the flagellates and in two where blue green algae were dominant, there was no reduction. There was a sub stantial reduction (80',',) in one pond whiilt had a distinct bloom at the time of spraying. In agreement with Hilscnholf (55). no effects were found on bottomdwelling organisms examined in dredge samples. Xo diquat was detected in the water 5 and 7 dais later. Fenac was used at 1 to 3 ppm. In three ponds, there was no effect on the phvtoplankton. In three others, the numbers dropped, but total recovery was observed in 6 to 10 weeks. In one pond, the population was re duced 75% anil remained there for the rest of the sea son. X'o effect was noted on bottom-dwelling organisms. Simazine was applied at 0.5 to 3.0 ppm to control aquatic weeds, but phvtoplankton studies were not made. It has potential in control of filamentous algae. E n d o th a l l. Lindabcrrv (75) reported that although the normal dosage of endothall is 1to 2 ppm. 5 ppm endo thall completelv disappears in -1days. He also reported no residue in fish which were placed in water con taining 6 and 12 ppm. filucgill* tolerated, with no mortalitv. Iffff ppm for 21 davs. There was no morialitv in three species of minnows (C.\ p r i t u d n r sp.) at Iff ppm. Salmon ( S a l m o sp.), rainbow trout ( S a l m o atril- Volume IS, Issue 6 (November), 1970 00ATPO U' E E D S C I E N C E intyc r iait R.), anil 10 ppm. bass (M i c r o p t c r u s sp.) sulfcretl no mortal Sinies (1-0) applied endothall at 5 ppnt in ponds or trout-rearing raceways in a Pcnnsvlvania lish hatchery and noted no indication of distress by the fish. Steuckc (123, 12-1) reported that 2 ppm of endothall in fish hatchery ponds did not atlcct the fish population--large mouth bass, blucgills, goldfish, or fish lood organisms. Walker (HI) reported that it took 25 days for disappear ance of residue from fish flesh exposed to 1 to 3 ppm of endothall; some lesidues were found in fish food or ganisms 3 weeks later. F1e ntoa c .3 Pierce ppm. (104, 105, 10G) There was no applied fenac to ponds at eilect on plankton, small invertebrates, frogs, fish were present. turtles (C ltc lo n a sp.;, and fish. Young P a r a q u a t. Sec section on diquat. 2P,-h1e-Dn oixny airlri pighaattiiocna cwiad steirn lu s v u l g a r i s L.) has been ornweparoteerdrte. dMAebsxytiucEadnyninobsfeat(nh1se5)ie.PffhWeacshtceoon-f the herbicide at 2 lb A in the water was applied to beans in the seedling stage, root systems were severely in jured; recovery began within 1Gdays. When the dosage was increased to 6 lb A, yield was reduced -10%. The 2 lb-rate, when applied in the bloom stage, caused no significant reduction in yield; but the loss at 6 lb/A was 29%. Aldhous (2) used the nonvl ester of 2.-1-Dat 4 lh/ae 12 dgv(Buaraollginnwagera.id)tserCaSbtaoaorlruit.sr)teba.3;f.toaarcWfrcoeaosret,nestvrtrvooelslreaSomifttpaklksaeecsosn,ptcrbhfurecofemhoeriePaitahcscpeardraisty{ciiCtnhcaghlcltnauhsnnaidast up to 4 weeks. Analysis showed 0.0 ppm after 28 days. Aly anil Faust (4) found that 2,4-D disappeared in 35 days from lake bottom soil that had been previously treated; on untreated hvdrosoil. it took G5 days. Averitt (G) in one field test applied an amine formu lation of 2,1-D (Mart.) Solms). to At w4altber2.h1y-DaciAn,th10t Epi cphbh owr en riea fcoruans sdip cast 3 weeks and O.Gppb at 20 weeks. In another field test, when 4 lb 2,4-D A were injected into the water at two different locations, he found 689 and 967 ppb 1 day atter application; 31 days later, he found 11 and 19 ppb. In other tests, when the original applications were 4 and 5 Ib/A, he found 4 and 15 ppb after 29 days. Averitt5 also applied the mcthvl amine salt of 2,-1-D at 4 lb.'A. He found St)2 ppb the second flay, 44Gthe third, 74 the fourth, and then a gradual decline until 102 flays when no 2.4-D was detected. Similar results were produced by 2-(2,4,5-trichlorophenox\ipropionic acid (silvex) and a polvpropvlene glvcol butvl ether ester of 2,4-D. glyBcoailleybutcyt l aeItPherapepsliteerd to ponds the of silvex at S polypropylene lb 'A. Half of the ester was hydrolyzed to the acid in 5 to 7 hr anil 99% in 33 to 49 hr. There was absorption of both aciil `Averitt, W. K. 1068. A summary of a studv of itic persistenev and residues of some herbicides in surface waters. Weed Sci. Sue. Amcr. Abatr. p. 63. Bailee, G. W., J. D. Pope. Jr., and D. R. Cochrone. 1968. The degradation kinetics and persi'tence of silvex under iiH|K>uiuled tndiiions. Weed Sci. Soc. of Amcr. Abair. p. 83. and ester upon the bottom sediments with complete dis sipation of both after 5 weeks. Cochrane et al. (24) ap plied 8 lb/A of the same ester of silvex to three plastic ponds, each with a dilterent soil type at the bottom. In 2 weeks the ester hydrolyzed almost totallv to the acid which gradually dissipated over 19 weeks. Absorp tion of both ester and acid apparently occurred on the hydrosoil, followed by gradual diminution. There is a Q possibility that silvex acid and/or adegradation product an may be desorlsed anil re-enter the water. Therefore, they concluded that it should not be applied directly to ^ water supplies for human consumption. ^ There have been lew reports dealing with the effects of herbicides applied to watersheds and reservoirs where the water is ilc-siined for human consumption. One U.S. Government publication (134) lists 0.1 ppm for 2,4-D, 2,4,5-T, and silvex as permissible criteria for surface water for public water supplies. No such criteria are given for other herbicides. The same reference has a very useful table giving the likely concentration of herbicides in irrigation water resulting after a typical application rate and also the crop injury threshold in ppm. ityFaasusatlicetnaeld. (35. bv 37) investigated drinking water qual 2.-1-D and 2,4-ilichlorophcnol (lierein- uher relencd to as 2.I-DGP). The latter is a most potent ollcndcr in water as it causes an objectionable medicinal taste at 8 ppb and has an odor even at 2 ppb. In lab- orntorv r.irlxjv tests using 2.1-D, 9.5 and 16.7 ppb of 2,-1-DCI' were released in 7 days. Maximum concentra tions ot 2H.7 ppb developed in 218 days. When the car- bois were aerated and ir. the presence of small amounts of uigjinc m.utcr. this phcuoi disappeared rapidly. Un der acid toiidiiions and water unfavorable for biological oxidation, alxmt half of the 2,4-DCP persisted. estFearuoift 2c.t1-uDl. w(3h6i1chincolanttearinfeiedld2,t4e-sDtsCaPpapsliaend itmhepuisroitoyc.tyItl w.is applied at 20 lb A to control eurasian watermil- foil. I Ins ipse a theoretical concentration in the lake of 131Uppb 2,1-D and 4.3G ppb of 2.4-DCP. This did not i.uisc an odor. There were 13 ppb of 2,4-DCP al though it did not appear to accumulate as the result of biulogu al degradation of 2.1-D. Maximum level of 2.4-D was pi j ppb. dropping to 13ppb after 132davs. Flunk .uni Comes (42) introduced into a pond 1.33 uppmmicnu ltra2i.i1o-Dn fiom was a 20% granular formulation. 0.024 ppm. The maximum. Initial 0.067 ppm. octimcd after IS days anil fell to 0.019 ppm after 21 davs. This formulation persisted longer in the G11vn11b1.dumioiiiI b,u|St|croeupldortneodt obeil dtheetecctoeniltathmeirneaatioftnero5f5wdaaytes.r in a reservoir with the butyl ester of 2,4-D which rendered it unfit for use by organoleptic criteria. No cases of poisoning were reported. Gircnda (19). in a public health report on weed con trol in potable water supplies, gave data concerning six common herbicides and their persistence in water after varying periods of time. Application was a maximum of 1 ppm. After some days, no more than 1 ppm was found. For the dimethyl amine salts of 2.4-D. 62 days after treatment, the amount hail dropped to 0.001 ppm. Orntckowski (47) and Tarrant and Norris (126) did aerial forest spraying with a commercial 50-50 formu- 742 Volume 18, Issue 6 (November). 1970 16254 M l'LLISON : HERBICIDE EFFECTS ON A Ql'A TIC ORGANISMS TG/.TCG AAnn 'ation of low volatile esters of 2.4-D and 2.4.5-T diluted Tth diesel oil and applied at 2 lb A. This gave regi es of 0.2 to 70 ppb in the water, l.'suallv the level v .ell'in a few davs to 0.2 ppb. Maximum time for total disappearance of 2.I-D was 2days at one test station and 17days at another. Krammes and Wellets iTO) sprayed a brush-covered area on a steep slope to encourage conversion to grass. Application rate was 11n lb 2.4-D and lb A 2.4.5-T as low volatile esters in a 2i>-gal mixture of water plus M gal of diesel oil. A follow-up hand treatment of brush was made with i 7gal of 2 lb each 2.-1-Danti 2.4.5-T 100 gal of water that contained 1gal of diesel oil. No herbi- "cide was found in surface water. In soil samples, small amounts of herbicide were detected 8 days later. None was found after 11.) months. Noll and Burdick (92) applied to a lake an ester of silvex at 2 ppm. When heated before treatment, the water had an eatthv. niuitv odor. After treatment, a kerosene-like odor developed. This odor lessenetl con siderable in 1 week although it was still detectable 8 weeks after treatment. The lake was treated in two sec tions; a number of large ami small fish were killed when the second half was treated although this was not noted for the first half. A second lake was treated at a concentration of 1ppm anil no fish were killed. bruNsohrriksiller tr nfol .rmr9u3la) tiaopnpltieod-47I*t>s helicopter A at a rate a phenoxv of 1 lb of 2,4-D and 1lb of 2.1.5-T 10 gal of oil A. Another area .. was spot-treated in 25 locations resulting in 4i)7 treated res. A stream drained each area. Samples taken from rib streams were analvzed. Measurable quantities of i. rbicide were not found in nm samples although a ^ total of 3t o to 512. inches of rain had fallen over the treated area. Norris (94) cites work by Linden and Muller (7(>> who used diesel oil at rates in exress of 500 gp.a. which was followed hv leaching with 4 in ''of`rain water. A maximum of `2 ppm of diesel oil was found in asands loam soil at a depth hclow 2i 7 inches. It was concluded that application of diesel oil at rates of 50 gp.a to soil surfaces presented no threat to ground water quality. Reigncr along iwo scttrean ml . fill) discuss treatment of vegetation hanks with the usual amounts of aiid and ester formulations of 2.1.5-T. Water samples were taken for analvsis periodicallv for 3 weeks. Odor tests were conducted. Thev concluded that 2.4.5-T. if applied with normal precautions, could he used on municipal watersheds without creating anv water contamination. Reinhart (115) applied 1325 gal (740 gal of diesel oil plus 52 gal of an ester of 2.4.5-T containing ti ih gal) on 30 A of a timber watershed. This was a basal spray applied to all uces 1inch dbh or larger. Tin's treatment is heavier than would be toed in a riparian area and diesel oil normallv would not be used. An odor panel found no contamination in numerous water samples taken from tIre stream draining the treated area. Schwam (119) reported a lahoratorv stttdv of aqueous solutions of 2.4-D under conditions favorable for mi- ^obiological activitv. At least t)'i*", of die 2.4-D persisted '3 to ti months. .unith and Isom (121) applied a 20r" formulation of e butoxv ethanol ester of 2.1-D to control Eurasian watennilfoil. Application of SSS tons of the 2.4-D was made to SiItHi infested acres in seven TVA reservoirs at rates of -in to |no lb A. Analvses showed little up atallkemnuld2s.a4m-Dplebsv fish hut some hv mussels showed some 2.4-D. Eight o .t\ ln\ tini hei s sp.); water treatment plants showed less than 1ppb of 2.4-D in the water. The ninth had its water intake directly under a 2.4-D application plot and its highest concentration was 1 and 2 ppb. Thev also reported that mosquito slaurrvvaivee(dA 2n .o4p-Dh e lteres a<tm/unetniltsir nantn Ui lKn)l up. spmL.:) in the adults laboratory from these larvae were carried to the F._.. No difference in hardi ness or reproductive ability could be detected. Thev concluded that high rates of 2,4-D applietl in TVA reservoirs have not produced adverseeffects upon aquatic fauna or water qualitv. ofWanerisohnaizwatciot nnlc. o(n1s4t7a>ntresptmorlvtedof o2n.4-aD.liVtearraiotuursearuetvhioerws showed 2.4-D could persist for periods up to f> months in natural waters. Winston and Riley (149) discussed tiie fate of phenoxv herbicides when applietl to a water shed. At least eight species of soil bacteria use phenoxv- nliphntic herbicides as an energv source. Thev concluded that the plienoxvalipiintic herbicides are decomposed into carbon dioxide, inorganic chloride ions, and water, and when properlv applied to watershed areas will not create a water pollution problem. ccsPlaiadhgmecendsonffxo.yMv1as-yAtlnei prtsnihd.rnrebtnwtlncuaretiaenccrirLdap.sb;lo.sntasninCddsneftlfilissiulnhip.r ncBtteoueasncr.voteshnnnetproicedletunesatuel.rRra(asa1itn1aad)nbtohiwnuaa)dt,- termdfoi!. The butoxvethanol ester of 2.4-D w*a.s used at 30. fiO. and IjO lb A. TV- two latter ticatments were rep licated three times. When normal aerobic conditions were maintained, treatments at 30 lb 'A resulted in no mortalitv to the native microfaima and the valuable native plants. Butler (20) exposed ovsters continuously for 7 davs to flowing sea water containing 0.1 ppm of the butoxv ethanol ester of 2.1-D. No mortalitv occurred. The ovsters had accumulated IS ppm 2.4-D acid. Half tlte treated ovsters then were flushed with normal sea water for 7 davs at which time thev had no 2.4-D residue. In other experiments no effects of 2.1-D were noted on pigtoe given fmorusesfefelscts( P loefn r3n7h edniaffecroerndtn thuemrbicGidreeesn)o. nDaotvastcairsc. shrimp, fish, and phvtoplankton. He concluded that herbicides in general are less toxic than most other pesticides. Hughes (fid) and Hughes and Davis ((>!) reported on variations of different formulations of 2.4-D. 2.4.5-T. silvex. and 2-i 2.-l-dichloroDhcnoxv)propionic acid (di- chloroprop) to blucgills. The number of different for mulations varied from three with dichlorpiop to 21 for 2.4-D. Differences in toxicity of various formulations of tlie same herbicide were great ami nnv he greater than the difference in toxicitv between different herbi cides. Amine salts were generally less toxic than esters. The actual formulation must be evaluated to determine the toxicity. Lawrence c t nl. i72) ran experiments for aquatic weed control with 2.4-D acid and silvex both at 5 ppm. Volume 18. Issue 6 (NovemI>er). 1970 WEED SCIENCE cliqut at 2 ppm. cliqut at I ppm plus 3 ppm <(-l-cliloroo-tolyl) ox\) acetic acid (MCIW). eiulotliall at 2 ppm. and nmitrole at 1.5 ppm -- 3 ppm MCJ'A. Tlicv placed fathead minnows and redear sunfish i L r p n n u s m i c r o t o p h u s G.) in their plastic pools. \o deaths up to 9 months after treatment occurred with either fish: in several instances thev had offspring: Mackcntluin (78) noted in using 2.1-D granules to control aquatic weeds in fish hatchery ponds that applications ol 25 to 30 lb 2.1-D A had no harmful ellccts. Rao and Murtv (112) found that using the sodium salt of 2.1-D at 8.8 lb/A in 100 gal of water to control I p o m c a r e p t a u s L. caused no fi'h mortality. Rawls (113) worked with blue crabs, eastern ovsters, soft shell clams, and various fish species in conjunc tion with herbicidal experiments. The following herbi cides were applied at these rates as lb A: 2,1-D acetamide-- 20; pohpropslenc glycol butyl ether esters of 2.1-D-- 20: silvex-- 10-, butoxy ethanol ester of 2.-1-D -- 20 to 120: anti isooctyl ester ol 2,1-D-- 20 to 60. At the end of 1 month, he concluded that only the 2,1-D acetamide appeared dangerously toxic. Thomas and Dulfv M3I) investigated both in labora tory and field the effect of the buioxveihanol ester of 2,1-D for control of eelgrass (Z o s t c r a m a r i n a L.). Thev quote Mount and Stephen (8-1) who stated that con tinuous exposure to 0.3 ppm 2.-1-D had no effect on reproduction of fathead minnows. Rassis (113) also is quotcil as sasing that fi ppm 2.1-D had little effect on oyster egg development: there is evidence that 2.-1-D residues in shell fish are eliminated in 10 months. Iiutlcr (18. 19) also indicates that continuous exposure to 3.75 ppm 2.-1-D reduced osster she!! growth, bv 50p': 1 ppm for a 1-hr exposure period reduced phytoplankton pro ductivity by jr>n',. In their own laboratory experiments. Thomas and Duffy found that with application of -10 lb/A, the highest concentrt ion of 2.1-D detected in the water was 0.1 ppm: the last detectable amount occurred 20 days after treatment. In the field experiments at 30 lb'A and in water samples taken for a consecutive weeks, no 2.-1-D was detected after 21 (lass. Highest field con centration was l.-l ppm. In the laboratory experiment, highest 2,1-D residue detected in oysters was 3.07 ppm: no 2.1-D residue was detected 17 days after its disap pearance from the water. Henthos samples from the field showed generally increasing fauna biomass during 1 months following treatment: total number of animals also rose. No mortality of natural fauna or flora was observed and relative percentage of species remained un changed. Tschirley (133) discussed the effects in Vietnam of using both phenoxvaliphatic weed killers and 1-amino3,5,6-trichloropicolinic acid (picloram) applied over more extensive areas anti at higher dosages than normally used on crops. Statistics on the fish caught oser a 3-vear period, including fresh anti salt svatcr fish and also cuttle fish ( S e p i a sp.), mollusks. shrimp, and crabs, indicated that the aquatic food chain had not been seriously dis turbed. Estes (31) applied to poh props lenc glvcol butsl ether esters of .silvex at 8 lb A to control aquatic needs. Within 30 min after sprasing where the average depth ol water was 3 ft. some fish seemed distressed and Mime pickerel 7-14 a*n o (Evi.v sp.;died. In a second area, no distressed fish were observed. Anahscs of fish after the treatments showed less than 0.01 ppm silsex: there was no change in the benthic population. Gavlur and Houser (Hi reported on 3 sears' work with silvex used lor aquatic weed con trol. Results arc given for eight species of aquatic weeds with good results lor three ol the most troublesome. No dead fish were observed, although the fish hunted new habitats when their old plant cover was destroyed. Houser and C.aylor (58) used einloihall and silvex for control of water weeds. These herbicides caused no fish mortality, nor were there reports of ofT-davor in the fish caught by many anglers in that area. Houser (39) reports that three species of fish in two ponds treated x with aihex experienced a weight loss in the 2 vears r following application. It is not known whether this was tlirectlv due to the silvex or indirectly through the C destruction of the weed beds. Mathews (S3) reported f that in New Zealand, trout ( S a l m a sp.) were unallccted bv 5b ppni silvex. Mullison (S5) reviewed the silvex literature and gave toxicological data for rats, guinea pigs (C az-ia p o r c c l l u s L.), rabbits, mice, chickens ( G u l l i t s ga l i u s LA, dogs I C a n is f a m i l i a r i s L.). and two species of fish. Silvex apparently does not constitute a hazard to game birds such as bobwhite quail ( C o l i n u s v i r g i n i a n u s L.). mallard ducks ( A n a s p l a t \ r l n ; n r h o LA. and ring-neck pheasants ( P h a s i a n u s c o l c h i c i t s LA. There seemed to'lie fish species varia tion in regard to tolerance of ester formulations of silvex. but in most situations where a silvex ester was used, fish populations were not seriously affected. Nonethe less. the potassium salt of silvex was less toxic to fish than the ester of silvex. Effects of '.fie ester of silvex on plankton were variable: some injury was observed in fresh water, but this was temporary and populations soon were back to normal. P f i r n o . w a l i p h a t i r a c i d s a n d p l a n k t o n . Duller (20) re ported physiological data-on decrease in carbon fixation of a natural salt water phv toplankton community, chieilv dinollagcllatcs and diatoms. It was given a 1-hr expo sure to 1 ppm of various herbicidal formulations. There was a wide variation in response even among members of the same group of herbicides such as different esters of 2.1-D. The sarnv. paper contains much EG-,,' data on effects ol herbicides on oysters, shrimp, and fish. Cowell (28) used silvex at concentrations of 2 ppm in a study on aquatic vegetation and plankton in farm ponds. It gave excellent control of P n t a m n a c t o n sp. and L c m n a sp. but not satisfactory control of the alga. C.hara >p. There were no adverse ellccts on the phytoplankton and zooplankton: no fish mortality was observed. Cowell (29) reported that neither silvex nor sodium arsenitc. at the dosages used, had anv effect on phv toplankton: 2 ppm of silvex were not too toxic to zooplankton, but sodium arsenitc at 4 ppm drastically reduced zooplankton and heavilv decreased roiificrs. copcpods. and cladocerans. Silvex treatments had no such effects. Crosby and Tucker (30) studied the effect of various herbicides on D n p h n i a m a g n a I., after a 2*i-hr exposure. Thev reported 2.1-D and M C P A were innocuous, hut :.\n enviiisifialilL- concentrate which causes response in iinlivuiuah. of t h e Volume IS. Issue 6 (November). 1970 ML'LLLSOX : HERBICIDE EFFECTS OX AQUATIC ORGANISMS 2,3-dichtoro-1.-l-naphthnquinone (hereinafter referred to ns diclilonej. S-cthvl hcxnhydro-1H-.t/cpme-1-carbothio- i (molinnte). JTl'-ilichioropropionanilide (propanil). -de.ur,l fdieilcdhlcoobnedniitliocnosulidf upsreedseantt enxcdeasussiveer rtaoteDs.n p lu iiu un Dc Oliveira (33) reported that a Brazilian lake sprayed with an amine of 2.1-D teniporarilv increased in plank ton. This was attributed to a better food supply caused by the dying weeds. Harp and Campbell (51) studied the effects of the potassium salt of sihex on benthos "organisms of a farm pond, it was applied at 2.S to -1.(3 ppm and the pond was sampled at 2S-day intervals for 3 months. The benthos population doubled. There teas an increase in tendipedids. aligochaetes. anti chnoborus correlated with an increased food supplv from the de caying plants. C.haoborus and libellidids increased greatly within I sp.), and cyeoaernaagfrtieornittlrseaatmppeenat.redSnauinlsa,lfeleneccdhe.sL(aHcei rwuidnog flies (C h r y s o p s sp.) decreased quickly but reappeared in smaller numbers during the last 2 months. Petruk (97; applied 2.1-D and trichloroacetic acid (TCA) on saprophvtic microorganisms in ponds. The second or third dav after the sodium salt of 2,1-D was administered, the population of microorganisms in the pond increased aljout 50^, but it gradually diminished after 12 to 11 days and approached its original value by 25 davs. Similar results occurred with TCA. Varia tions of the microorganism population, particularly with 2,-1-D, strongly suggested that the herbicides were being used as food. Pierce (9S, 99, 100) has studied in depth the poly- -optlcne gylcol butvl ether esters of silvex. Plankton .s represented bv about 50 species in tiic following asses. Annelida. Chiorophyceae, Crustacea. Dcsmida- ceae, Dictomaceae, Flageilata, Myxophyceac. Xematoda. Rotifcra, and other protozoa. Benthic organisms were rcpresenteil bv die following groups: Amphipoda. Anne lida, Diptcra. Epberncrida. Gastropoda, l'clccypoda, and Odonata. Also present were large aquatic vertebrates, such as fish, frogs, anti turtles. Silvex at 1.2 ppin tempo rarily decreased the plankton population, but it was back to normal within 2 weeks. The benthic organisms, fish and other larger aquatic vertebrates were unaffected. Pierce (103) compared two formulations of the potassium salt of silvex at 2.2ppm and observed no effects on plank ton or benthic organisms. Fish of all ages, frogs, anil turtles were present throughout the summer and were unaffected. Pierce (101. 102) made another series of in vestigations using granular 2.-1-D at rates of 1.5 to fi.2 ppm. No effect on the benthic or plankton population was noted, although in one trial there might have been a temporary reduction in plankton which returned to normal in 3 weeks. Pierce (107) determined the effects of a number of herbicides on plankton and other pond organisms. Di- chlobenil. feme. 2-methoxv-3.li-dichIorobenzoic acid (ili- camba), endothall. the potassiumsalt of silvex. and 2.3-D acid in a granular formulation were used at dosages of 1 to 2.5 ppm. There were no significant changes in the number of individuals or species in the plankton. Neither the benthic organisms, consisting chiellv of chiro- mid larvae and gnmmarids. nor anv other ot the pond llers howed am injtirv. Pierce (10m continued her ecological investigations using a combination of 2.1-D i7.2f,(',) and endothall ij.GQ.) to give a concentration of 2 ppm. This had no deleterious elfects on plankton, large and small fish. i:ul|>oles. frogs, turtles, and water snakes ( X a t r i x sp.;. Walker (HO) reported that IxMtom-dwelling organisms in fish ponds were siguificanilv reduced in number bv 2.1-D, 1-butvI-3-f3.1-dithlorophenvD-l on), sodium aisenite. ami copper -smidefta1t1cyIudreuariningebtuhre- sear following treatment but were not affected by en dothall and simnzine. Dosages were not stated except as "reasonable concentrations". He discussed the possi bility of sublethnl dosages changing the species composi tion and how that would affect pond ecologv. Whitney* studied the effects on aquatic fauna of the polypropvlene glycol but\l ether ester of sihex applied at (3 lb A. Fish deaths were observed in half the gsiillvlse,x a.anpdpliIcaargtieomnso:utghizzbaarsds swhearde (tDheo r opsroinmcaipaspl .i.spbelcuiees- affected. Highest value found was 19 ppm 21 hr after treatment in a forage fish. This decreased to a negligible amount 1 to 3 weeks later. Benthic organisms increased significnntlv in number after treatment. The normal phytoplankton consisting of numerous species was dc- stroved and replaced bv E u ^ l r n a sp. and dinoflagellatcs. It did not return in 3 weeks. Rotifers and crusiacca also decreased following application but regained their normal numbers within I to 3 weeks. P icloram e t a t. (32), (9.3 lb'A a( 4ft-earmui nsion-gj .5p/icj -loi rriarmh l oarto pic an o h n i c a c id ) . Davis unusually high rate in a pellet formulation) on a watershed area, found detectable amounts of picloram in the stream after rains. Highest concentration fount! in the streamwas 0.37 ppm. After 1Gmonths, during which a total of 10 inches of rain fell, no picloram was detected in the stream. Butler (20) using a formulation of picloram and 2.1-D at I ppm. reported no effect on oysters, shrimp, fish, anil plmoplankton. Kenaga (f>7) studied and reviewed the effect of picloram on 15 fish species anil three game birds. This work indicated that it presents an extremely low potential hazard, it anv. to such wildlife. Hardv (53) showed that picloram at I ppm did not retard the growth of algae or D a p /m ia . Guppies were grown in water with 1 f.p.'.- picloram and fetl D a p h n ia reared in such treated water. The guppies were normal in appearance, also grew anil rbeephraovdiuocre, danndornenparollvduwctiitohn.noTheevidDeanpchen ioaf biological magnification of the picloram in them. SMoadt.isuumguPchCiI '(ifsilo)d iruemporpte'-dr ilathcah lto rthoep hsc on adliuc )m. IsshailztaowfaTaCnPd mixed with soil at a rate of 1200 ppm stimulated bac Atemriamlognriofiwcnthti.onP s eaunddo miroonna sresdpu. cbtieocnomwienrge predominant. depressed at concentrations greater than 120ppm. The Okubos (95) reported that an extensive number of fish and clams were killed in 1`Jfil and I9G2 oil the coast of the Ariake .Sea. Kyushu. A sodium PCP applica tion had been followed at once by a heavy rain. They concluded that the resistance of a native clam( 'c n e r u p is AVhitnev. E. U . 1063. The toxicity of silvex to aquatic fauna. Weed soc. of A m ., A ljslr. p. 6J. Volume 13, Issue 6 (November), 1970 7-15 531753 C y.*Wi :7 WEED SCIENCE sp.) to PCP decreases to one-tenth its normal value under stress of the diluted sea water. Saito e t id . (IIS) used the sodium salt of PCP as a 25% granular material applied at fi.ii ]h A on rice ( O i y z a s n t i v u L.) paddy fields. Samples of river water were collected at 21 different spots near the treated fields. Maximum quantity of PCP detected was 50 ppb: thev concluded there was no danger front this use of the herbicide. s- T r i a z i n e s and a A / i - l T i f i u o r o - 2 , 6 - d ' m i t r o - X \ - d i p r o p x b p l o l u i d i n e ( t r i f l u r a l i n ) . Axe e t a l . (S) studied the residual effect in farmers' fields of atrazinc, 2-chloro-4.6-bisiisopropylamino)-s-triazine (propazine), and trifluralin. Onlv 1-1% of the tiilluralin was found in the soil after 3 months. 20% propa/ine after 5 weeks, and 33% of the atrazinc after 5 davs. Highest concentrations of herbicide in the runoff water were 0.0-1, 0.05. and 0.01 ppm for airazine, propa/ine. and trifluralin. respectively. It was assumed that much of the herbicides must have been on or in the plants. Avraman (7; studied the effect of prometryne on the taste of water anti its toxicitv to mammals. The herbi cide, within its solubilitv limits, did not affect the color or odor of the water. The taste threshold was 1.2 ppnt. The short-term LD-,n value for mice ( M u s m t i s c u l u s L.) was 9.3 to 20.0 gr-lba and 19.2 to 2-1.0 gr lb0 for rats ( R n t t u s n o i - e r p c m L.). Dailv doses of -1.3 X 10- -*gr lbr` in long-term studies had no significant effect on rabbits ( O x y c t o l a g u . % c u n i c u l u s L.) and rats. He concluded that a level of 1 ppm would lie the maximum permissible concentration in water. Martynyuk and G/hegotskii (821 investigated simazine. atrazinc. propa/ine, 5-amino-l-chloro-2-phcnyl-3 ( 2 H )pyridazinone (pyra/on). sodium TCA, and diuron in water. Water containing 0.1 to 5.0 ppm ol these herbi cides did not differ from control samples in its organo leptic properties even alter heating to I5S F. T C A excepted, increasing the concentration of these herbi cides to 50 to 2()<l ppm markedly changed the organo leptic properties of the drinking water, particularly for pxrazon. Diuion excepted, ground water containing not) to 1000 ppm of these herbicides stored in bottles lor 3 to 20 months at temperatures of -13 to 71 F showed no drop in concentration. Ground water containing 50 to 100 ppm of these herbicides was treated with 30i) :< It)-- 3 gr'qt of calcium hypochlorite: this completely decon taminated the water. Weldon ami Blackburn (145) reported that 2-fcthslamino)-4-(isopropy]amino)-fi-(medix hhio)-j-triazinc (ainetrync) persisted in plastic-lined pools nearly unchanged for 32 days after application. In the field, however, no residues were found under treated water hyacinth 1 day after application. Snow (122) used simazine at 2 ppm in two ponds. In one pond, adult redear sunfish were killed although no small fish died; none died in the other. Another pond was treated at 3 ppm and no fish died. The phytoplankton developed slowlv after 4 to 6 weeks. Fish production was low to axerage. but the evidence was inconclusive as to the cause. Walker (113) experimented with simazine and other 'Avoirdupois units. 746 r-tria/incs in fish habitats. Sima/ine was less toxic than propa/ine. 2,1-bis(iopiops lamino)-li-mcthoxv-s-t riazin e (prometonei. or atra/ine to fish. Lahoratorv tests on hottom oiganisms showed simazine had an LD-, ol 23 ppm: howexer. field observations did not demonstrate a m serious reproduction problc-ni'. Airazine is mote toxic V. than simazine to bottom organisms, but thev recovered in -1 to u months. Prnmcione and propazine xx-erc not critically examined, bu; no fish mortality was observed. Flanagan (39) reported simazine as non-toxic to fi'h C at 3 ppm. Guse (51). in a summary of reports on aquatic C xx-ced control, mentioned that ametrvnc at -1 anil 5 ppm caused irritation or death to some fish. Prowse dll) noted that T C A at H>u ppm caused no distress to fish c after several days'contact. He aho noted that simn/.inc y. at 120 ppm caused 7"% mortality in -1 hr: after 12 hr the tanks were no longer toxic. Wellborn (Ilfii in vestigated, the fish toxicitv of diquat. diuron. and sima zine and found simazine to be the most toxic to bass ( R o c a n sp.) with an LC.-,0 of 0.25 ppm. T r i f l ' . t r n U n . Cope (25; stated the LC-,, concentration of trifiuniiin for a 21-hr exposure of 2 to 1-inch rainbow trout was 14 to 2IH ppb and for smaller bluegills it xvas 23 to 120 ppb. Increasing the temperature increased die toxic response: e.g.. trifluralin once xvas 130 times as toxic at 85 as at -15 F. In some cases, there was greater toxicitv in soft than hard water anti in other cases there xvas no difference. Cope (2l>) later reported the EC-,,, xalucs of tiilluralin to be 11 to 19 ppb for rainbow trout anil bluegills. L a m once10 incorporated tiilluralin in soil 7 dnxs bet ore putting it in waivr-fulcd plastic ponds. T lie p o n d s then were planted xviih several species of water weeds and stocked with fathead minnows. There xvas no mor tality to die fish during 4 xvceks after Hooding: thexreprodneed satisfactorily. Parka and Worth ddb) found that trifluralin had an I.C-,,, of 5S.2 ppb to bhiegilis: when put on soil and this soil added to water. IS to 227 times more xveic required to obtain an I.C-,,. In a simulated field test using swim ming pools, trilluralin was spraxed at the rate ot 1 11 A on soil and tilled into it. It then xvas gixen 10 inches ol simulated rainfall: water draining from the treated area xv.is not toxic to bluegills. Analxsis of tlie sediment sliouci! 55 ppb tiitluialin. Tliis chemical has a water solubile.x of 50 ppb. is strongly absorbed b\ soil, and leaches xerv little exen on sandy soils. S u b m i t : i c d m e n s . Walker f!44) found momiion. diuion. Icm uroii. neburon. and mixtures of them with T C A elfectixelx controlled certain aquatic weeds. Monuron and femiron were lC'S toxic than diuron and neburon to fish. These urea herbicides mixed with T C A xvore somexxiiat more toxic than the urea herbicides alone. Species differences xvere manifested. Fingei lin g s xvere more sensitix-e than adults. Fish iooil organisms were noticeable reduced in the treated areas. Frank >Oh studied the disappearance of innnuion and nchmnn in an aquatic environment. Significant loss xlid not oxxur until alter S xx-ceks for moniinni and It) xvecks for neburon. The rate of loss then accelerated :"[.2u .vmcc. |. M. IP**, r.ifcct* of <*1 nppiitii (riil4i:niii on fi-h prrxiuuioii in poiuls. Won! Sc*c. of Airur. pp. s P-`.'0. Volume IS. Issue 6 (November). 1970 MULLISOX : HERBICIDE EFFECTS OX AQUATIC ORCAXISMS umil about 50'". of the herbicides disappeared, niter which it continued ut a decreasing rate. Alter 128 weeks 'pproxiinatclv 15`J. of the monition and 39' '0 of the - neburon remained. Bnngenbcrg de Jong (16) used diurem at concentra tions up to li.-I ppm in fish .spawning ponds: it did not inhibit the growth of pike. First anil second vear carp suffered some growth inhibition attributed to the re tarded development of fish foot! organisms. Hnmbric (521 used diuron at 2 lb A or 0.2 lb A foot of water on two lakes in Texas anil no fish were killed. Maloney - (80) used monuron at concentrations of 0.01)4 to 32.0 p p m to control algae. Toxicitv data are given for 32 species of algae with exposure periods of 7 and 2S days. H e also noted that ponds treated at 1 and 1.6 ppm were not toxic to fish. Pierce (IIOi used diuron at 0.25. 0.33, and 0.5 ppm to control common duckweed t L c m n a m i n o r L.) and noted light to moderate fish mortality at all concentrations. He attributed this to a low ox\gc-n supply and not to the herbicide. Xo plankton bloom developed in these ponds within 6 weeks. Alabaster (1) in a study involving 161 different chem icals, many of them herbicides, pointed out that the toxicity of formulated pesticides can not be predicted from knowing the toxicity anil percentage of the active ingredient. It is essential to test the actual formulation under consideration because manv additives are marked ly toxic, some more so than the active pcsticidal ingre dient. Xishiuchi and Hashimoto fJI) studied three kinds of fresh water fish anil two kinds of fresh water D a p / m i a anil their susceptibility to 76 different pesticides. Alhougli some hrrhj< idc-s were toxic to the species tested, icy concluded most herbicides were non-toxic to both ^ fish and D a p h n i a . D iscussion - This literature survey presents a very considerable mass of evidence concerning the fate in our aquatic environment of various herbicides. Evidence at hand shows few signs of cither a contamination problem or an accumulation in our waters ifour presently available weed killers are used according to label directions. T h e toxicitv of herbicides to fish is a complex prob lem. There is the usual biological variation in size. age. and species as well as physical and chemical differences at the aquatic site. There also may be variation with different forms of the active ingredient: for example, the esters of phcnoxvaliphntic herbicides are more toxic than sodium or amine salts. Additives to a formulation may be markedly toxic, some more so than the active pesticide ingredient. Therefore, the specific formulation should be evaluated to determine its LD-n. In addition, the L D 3n may not indicate the actual hazard to fish because of the circumstances involving the herbiciilal application. For example, trilluralin when applied to the soil is stronglv adsorbed and moves little. This cre ates no real hazard to fish except for accidental con tamination, even though the compound p e r sc is extremely toxic to them. Plankton studies indicate that our present herbicides, lulling those used in aquatic sites, have at most only temporary harmful effect. Volume 18, Issue 6 (Xovember), 1970 LIOW 531755 There is no evidence of a biological magnification problem with herbicides in the looil chain organisms. Theoretical!;., the intrinsic fact that herbicides kill plants which then deiav and are not generallv eaten should prevent biological contamination at one begin ning pari of the iuod chain, the plankton. Johnson c t n l . font discussed the studies on pesticide residues being carried out cooperative!'- bv several gov ernment departments. He found that of the manv pesticidal chemicals now in me the most important to fish, wildlife, anil estuaries are DDF. its metabolites, and other chlorinated hydrocarbon insecticides. Xot a single herbicide is on hi> list. Brown and Xishioka (14) dis cussed the result-, oi their monitoring program. Eleven streams in the western part of the United States were checked month!' for nine insecticides and 2,4-D, 2,4,5-T, and sihex. Xo iieibiciile residues were found. Most hei bitides now in common use are low in toxicitv to man ,iml animals, and tbey break down in soil anil disappear lrom water. In addition they are applied in such a way that they normally do not accumulate to create a Ji.i/ard in our environment. Xoneiheless. com placent' is not justified. Continued careful study of each new herbicide is essential with the express pur pose ot pieventmg environmental contamination in the future. M a n ' s tool s frequently interfere with nature, and hcihiiidcs arc a powerful tool. It is the wisdom witli vshuh tilts extremely useful agricultural tool is used that will determine whether it has a beneficial or lunnlul eioiugiial cliect. LITERATURE. CniD 1. \L'UIII I. S 1969. Survival of fish in 104 herbicides. irw'riM nils, tmiqicidcs. wettins .'igfiits anil miscellaneous suljMjim. Ini IVst C o m r. 11:c." v-- ">. 2. Alduoi i . I R. I'.'oT. 2.4-D residues in water following actiai I'UMiie III a Scottish forest. U'eeil Res. 7:239-2-11. 3. Atux. R I . T. K. Menus. G. It. I'ucrv. and M. I'.vt'L H jins*.! l'G-s. Mortality niunuq cliino.sk salmon uvsn- riai.J v.u!i tlie funqus D e r m o n u i i i m m . I. Fish Res. Board Gan 3 2Pi?-g173. -f \i.v. () M. and s. D. Faust. 19GI. .Studies on the fate of 3) li and i.iir derivatives in natural surface waters. J. Vqr !*! l hem. 12:541-346. j. Srrm.vn. \ C.. J. W. Hovrri l. A. E. H vll, and M. A. S'iiiii 1937. Toxicitv of 4.346 chemicals to larvae, lainl*tis> and li-hes. Fish and Wildlife Serv., l."_S. Pep. of lot \vjshmgiun. D.C. Si>cc. Sci. Rep. Fisheries 207. PI 0 win it U k. 1007. An evaluation of the persistence of 2.1 IJ amine in sinface waters in the state of Louisiana. I'nx So U s e d Conf. 20:342-317. 7 \vhvmvs f V. IPOs. Maximum pcimissiblc concentration nl ii.im iiin' in bodies of water. Gigicna i Sanit. Hvg. x-imi " 10.1-107. S. \\r. J. A V C. M vtiiers and A. F. 3Vif.se. 1900. Disappearaim- oi airj/me. propazinc. and irillnraiin from soil and vvjicr. Tiivc. So. Weed Conf. 22:307. fAbstr.) 9. B u t. R C 1900. I'sc of :,As-tasgcd scxliutn arsenite in a stmlv of idiets of a herbicide on pond ceoloqv. 1'ioc. of Simp. On 23-29. Ipfl7>. Vienna. Austria, pp. 149-10:1. 10. Bcvsirv. P G . J. M. Laurence, and II. H. Funderburk. Jr. l'.'li'. The absorption and distribution ot (T'-labclcd diqnai in the c'ldlish i C n o m u s a i r t a i u s L.). Proc. So. Weed C o n f. IS:3'I. '1\htr.l 11. Be a u x , 0 . F.. C. k. R awls, and G. F.. Beckfet. 1962. Field obseu jtious upon rstuarinc animals exposed to 2.1-14. Proc. No. last. Weed Conir. Coot. 10:419--l`S. 12. B i. vckbikx. R. I). and L. W. W iloon. 1903. Results of tliue 'ears testing diipiat as an :u|iiatic herbicide in Florida. Proc. So. Weed Conf. 10:303. (Ahstr.) 747 59 WEED SCIENCE 13. Blok, F.. 1960. Experiments with herbicides in fishing waters 1961-1967. Weed Alwr. 18:117. No. 746. H . Brown, E. and Y. A. N isiiiok.a. 1%7. Pesticides in selected western streams-- A contribution to the national program. Pest. Moni. J. 1:33-11. 15. Bruns, V. F. J93I. The response of certain crops to 2.T-ilichlorophcno.xvacciic add in irrigation water. Weeds 1:359376. 16. Buncenbire. oe Jonc. C. M. 1969. The use of diuton in pond pisciculture. Weed Ahstr. 18:113. No. 753. 17. B lrdrick, C. E.. H. J. Dt.cN. and . J. H arris. 19*5-4. Toxicitv of aqualin to fingcrling brown trout and blttegills. N Y. Fish Came J. II.106-111. 18. B utllr. P. A. 1963. Commercial fisheries investigations. L'.S. Fish and Wiltllife Serv. Cite. 167:11-35. 19. B utler, P. A. 1961. Commercial Itsherics investigations. L'-S. Fish and W ildlife Serv. Circ. 199:5-23. 20. Butler, P. A. 1905. Elfects of herbicides on estuatine fauna. Proc. So. Weed Conf. 13:576-530. 21. Cassie. V. Effects of sptasing on phstonlankton in Lake Rotorua. Proc. Rotorua Seminar on Water Weeds. *]>onsored bv L'.Ext. Setv. Lniv. Auckland, New Zealand. Oct. 15. 1966. 31-40. 22. Chancellor, R. J. and W. E. Rin-rR. 1960. Control of reetls and other emergent water weeds in drainage ditches, ponds and water courses. The effects of dalj|>ou on fish. Weed Abstr. 9:No. 696. 23. Coats, G. E.. H. H. Funderburk, J r.. J. M. L awrence, ami D. E. D ams. 1964. Persistence of iliquat and paraquat in pools and ponds. Proc. So. Weed Conf. 17:30-*--.11214. 24. Cochrane. D. R.. J. D. Porr. | r.. H. P. N icholson, and C. W. Bailey. 1967. The persistence of ' ib e x in water anil hydrosoil. Water Reviur. Res. 3:517-52325. Core, O. II. 1965. Some responses of fresh water fish to herbicides. Proc. So. Weed Conf. 13:I3``--145. 26. Core, O. II. 1966. Contamination of the fresh-water eco system by pesticides. |. Appl. F.col. 3iSuppleinenti:33--44. 27. Cove. O. li.. J. P. McCk-srun, and L. Eller. 1969. Effects of dichlolreiiil on two lisli |>ond environments. Weed 3ci. 17:158-165. 28. Cowell, 11. C. 1965. T h e effects of silvex on aquatic vegetation anil plankton in central New Yoik farm ponds. Down to Earth `Jill x 2r.19-20. 29. Cown.L, B. C. 1965. The effects of sodium arsenite and silicx on the plankton populations in farm ponds. Trans. Am. Fish. Soc. 94:371-377. 30. Crosby, D. G. and R. K. T ucker. 1966. Toxicitv of aquatic herbicides to D a /t/in ia m n ^ n a . Science 154:239--291. 31. D aly, R. W.. H. 11. F vndirulkk. Jr., ami J. M. L awrence. 1963. Persistence of diquai. paraquat, ami 2.1-DrdlL when used for eiirasian wateimilfoil (M y i u p i . x i t u m s p i e a t u m L.) control. Proc. So. Weed Conf. 21:299. 32. D avis. E. A., P. A. Ixrrr.ii. and C. P. P.ase. 1903. Effect of a watershed treatment with picloram on water qualitv. U.S. Forest Res. Note KM-100. Roikv Mountain Forest ami Range Experiment .nation. Fort Collins. Colorado. 4 p. 33. D lO ijveira E. Su va. S. L. 1961. Inllucnrc of 2.4-1) amine on plankton of marshv areas fprcliminarv notci. Weed Ahstr. 10:No. 1080. Not paged. 34. Estes. R. D. I960. Some preliminarv olwcnations and findings on effects of silvex on the aquatic cm irouiiicni. Proc. So. Weed Conf. 19:116--419. 35. Faust. S. D., R. J. T ltkir. anil O. M. Alv. 1901. A pre liminary report on the effect of some aquatic herbicides on water qualitv. Pioc. No. East Weed Contr. Conf. 15: 546-548. 36. F aust. S. D,, R. R. Youx'r.r*. J. T. T icker, and O. M. A lv. 1962. Observations on the occurrence and pcisistence of 2.4-D and 2.4-ilichlornplieiiol in lake water. Proc. No. Fast Weed Contr. Conf. It:159--465. 37. Faust. S. D. and O. M. At v. 1963. Some effects of 2.4-D and 2.4-DC.P on drinking water qualitv. Proc. No. East Weed Contr. Conf. 17.Itin--470. 38. FiRr.tsoN. F. F.. F.. K. lh u n o n . anil R. BLONor.vu. 1965. Preliminarv field trials of acrolein in ihe Sudan. World Health Oig. Hull. 32:213-218. 39. Flvn'.ve.vn. J. H. I960. review of dmarinc for ariuatic weed control. Proc. No. East Weed Cou.t . Coot. 14:502- 505. 748 40. Flank. P. A. IOtiti. Persistence and distribution of monuron ami neburoii in an aquatic cmiionmcnt. Weeds 14:219- *>O 41. Frank. P. A.. R. H. H odoson. R. D. Coaies. 1967. Residues of two hetLieiiles in water iirigation canals. Weeds 15:353--355. 42. Frank. P. A. anil R. D. Coaies. 1967. Flcrhiciilal residue: in pond water and hvilrosoil. Weeds 15:210-213. 43. Fra ir . J. D. anil S. A. Evans. I9li.s. Wcevl Control Hand' book, Yol. I. Piinciples. Blackwell Scientific Publications Oxford and FidinInirgh. 494 p. 44. G.vvlor, J. anil A. H olslr. 1962. Three vears' results with silvex for aquatic plant control in Oklahoma. Down to Earth IS(3}:2-4. 45. Gilderiils. I*. A. 1966. Some effects of sublcthal concentra tions of sodium arsenite on bluegills and the aquatic en vironment. Trans. Am. Fish. Soc. 95:289-296. 46. CiLDrRtils. P. A. 1967. Effects of iliquat on bluegills and their food organisms. Prog. Fi'll Cult. 29:67-74. 47. G r a t i k o w s k i , H. 1965. Project 3: L'mlcsirablc woihJ v plants. Res. Comm. Prog. Rep. IW't. Weed Contr. Cnuf. 21:35-50. 48. Gribanov, O. I. 1967. CoutamiiMiism of a surface water suppls source of the Shortamlv Station with heilinide 2.4-D and buisl ester. Gig. Sanit. 32:97-98. 49. Crzlxda, A. R. 1963. Public health aspects of tveed con trol in potable water supplies. Pioc. So. Weed Conf. 16: 42(1--424. 50. Crzenoa. A. R.. H. P. N ichoi-son. and W. S. Cox. 1965. The persistence of four herbicide in pond water. Proc. So. Weed C.onf. 18:521-525. 51. G ist.. L. R. 1961. Aquatic weed control research in Indiana. Res. Rep. No. Cent. Weed Contr. Conf. 18:102. 52. H aaibbic. R. N. 1969. Testing diuron as an economical herbicide in iccrcation lakes. Pioc. So. Weed Coni. 22: 321-323. 53. H arda .J. L. 1966. Effect of Toulon herbicides on aquatic chain organisms. Down to Fiith 22(21:11-13. 51. H are.G. L. ami R. S. C.v.vircULL. 1961. Elfects of the herbi cide silvex on benthos of a farm pond. J. Wildlife Manage. 28:308-317. lIiiAE.NiiOFF. W. 1966. Effect o f di-'|ii:t on 3-'6*-~*:c ii'iS-TCt? and related animals. J. Econ. Entomol. 59:1520-1521. 56. H ole. W. 1!H33. Chemical weed coiittol in ilitclies: trials on the control of submerged plants. Weed Abstr. 12:ItlO. No. 507. Not paged. a i . H ouse. W. B.. 1.. FI. C oooso.n . H. M. G.AnRFRRv, ami K. W. D ockter. 1967. Assessment of ecological effects ot extensive or rc|>caied u-o of herbicides: final repott. Midwest Res. Inst.. Kansas l.itv, Missouri. 369 p. 53. Hoi sFR. A. ami |. Y. G.aat.or . 1962. Results of etidoiball and two foiiiiiit.ninns of silvex lor the control ot aquatic plant in Oklahoma. Proc. Weed Conf. 15:211-255. 59. HotsfR. A. 1963. Loss in weight of sunb-li billowing aquatic vegetation control using ttic herbicide silvex. Proc. Okla. Acad. Sci. 13:232-237. 60. Hir.ms. J. S. 1962. The toxicitv of Fish of different formu lations of 2.4-5-1". 2(2.4-DP) and silvex. Pioc. So. Weed Conf. 15:265-2l>6. 61. Hur.nrs, J. S. and [. T. D avis. 1963. Variations in toxicitv to hlucgill sunlish of pheuoxy herbicides. Weeds II: 50- 53. 62. Hull, H. M. (Chm.). 1967. Heibiciilc Hanilbook. Weed Soc. of Anier. 293 p. 63. Huxr, E. G. 1966. Biological Magnification of Pesticides Scientific Aspects of Pest Control 251-262. Nat. Acad. Sci., Washington. D.C. 470 p. 61. I siiieaaa a . S. and T. M atsu c u c iii. 1966. Effects of pesticides and herbicides tqioii iiiicrooiganisms in soil ami water under wateilogged conditions. Bull. Nat. Imi. Agr. Sci. ifapan). Scr. B. Hi:1-90. 65. J acorossi, F. 1965. The toxicological problems of controlling weeds in canals. Weed Abstr. 14:267-268. No. 150i>. 66. Johnson. R. FI.. T. C. C arver, ami F.. II. D ustman. 1967. luiiicator species near top ot bawl chain chosen for assess ment of pesticide base loscis in lish and wildlife-- clams, owrr. and sediment in estuarine environment. Pest. Monit. f. 1:7-13. 67. KtNvr.v. F.. F.. 1969. Tordon heihicidc*-- Evaluation of safety to lish and biids. Down to E.irtli 25yl):5-9. Volume IS. Issue 6 (November), 1970 MULLISOX : HERBICIDE EFFECTS OX AQUATIC ORGANISMS 68. Ku.Nr.MiN. G. C. 196*. Weed Control: As a Science. John Wiley i- Vine. Jnc.. \e' York. 42I ;>. 69. KociiKiN. V. I*. 1967. Experimental data for the hvcicuic standardization of the dalj[ion level in bodies of water. Gig. Nunit. 7--11. 70. Krammes, J. S. and D. B. W illets. 1964. Effects of 2.4-D ' and 2.4,5-T on water qualm after spraying (rcatnient. L'-S. forest Sen. Re-. Note PxW 32:1-4. 71. L aurence,J. M. H'jT. Recent investigalions on the tic of sodium arsenite as an algacidc and its cilccts on fish pro duction in ponds. Southeast. Assoc. Game and Fish Com missioners 11:251-256. 72. Laurence. J. ,\L. R. D. Blvckburn. and P. G. Bfvsley. 1961. * Aquatic herbicide esaluation tests using plastic pools. . Proc. So. Weed Conf. 14:309-316. 73. Lawrence, J. M. 1962. Aquatic herbicide data. Agr. Hand book No. 231. Agr. Res. Serv.. L.S. Dep. of Agr. -171 p. 7- L L aurence. J. M. 1963. Graphic presentation of aquatic herbicide data. Proc. No. Weed Conf. ls:566-573. 75. L indaberry, H. L. I'.Hil. Considerations regarding the use of aquatliol in potable watersheds. Proc. No. East Weed Contr. Conf. 15:181--13-1. 76. Linden, G. and A. .Miller. 1963. Further investigations on the effect of diesel oil on the soil. Allg. Forstzeitschr 18: 791-793. 77. Mackentiiin, K. M. I960. Some limnological investigations on the long-term use of sodium arsenite as an aquatic herbicide. Proc. No. Centr. Weed Contr. Conf. 17:30-31. 78. M ackentiiin. K. .M. 1961. Observations on the u>c of 2.4-D granules in fish hatchery ponds. Res. Rep. No. Ccntr. Weed Contr. Conf. 18:163. 79. M ac.ee. L. A. and A. R. Colmer. 1959. Decomposition of 2J2-dichJoropropionic acid by soil bacteria. Can. J. .Micro biol. 5:255--260. 80. M ai-ONF.y, T. F.. 1958. Control of Algae with chlorophcnvl dimethyl urea. J. Amcr. W ater Woi k< \oc. 50:417--422. 81. M arston, R. B.. D. W. Sciiilts, T. Siiiroyama. and L. V. -S.wntR. 1965. Amitroie ... ...................... in -creek waters downsircam front an aerially spraveti watershed snb-basin. Pest. Monit. J. 2:123-128. 52. M arts nvik.V. /. anti S i. I.G/ulliiiskii. 967. Toxic chem ical level in water ami soil treated with ciiiornganic hcinicitles. Gig. Sanit. 32:96-98. 83. M athews, L. J. I960. Aquatic weed control. Proc. New Zealand Weed Contr. Conf. 13:53-61. 8- 4. M ount,D. I. and C. E. Steepen. 1967. A method for cstahJishing acceptable toxicant limits for fish-nialathion and the luitowethanol ester of 2,-4-D. Trans. Amcr. Fish Soc. 96:185-193. 85. M pluson,W. R. 1966. Some toxicological aspects of silvex. Proc. So. Weed Conf. 19:42(5--435. 86. N ational Acvdemy of Sciences-- N vtiox vl Res. Council. 1966. Principles of Plant and Animal l'ei Control. \'ol. 2. Weed Control. Nat. Acad. Sci.. Washington. DC. 471 p. 87. N e w m a n .J. 1'. I9<35. Methoils of assessing wildlife hazards. Proc. Biit. Invest, anrl Fungic. Conf. 3:342-357. 88. N e u m a n , J. F. ami J . M. Way. 1966. Nome ecological obser vations in the use of paraquat and diqtiat as aquatic herbicide. Proc. Brit. Weed Contr. Conf. 532-533. 89. N e w m a n , J. F. 1968. The ecological ctfccts of paraquat and diqtiat when used to control aquatic weed. Weed Abstr. 17:361. No. 2211. 90. N icholson. H. P. and J. R. T homan. 1963. Pesticide per sistence in public water, their detection and removal, pp. 181-189. In Research in Pesticides. Academic Press. Jnc.. Mew York. 91. Xtsiiiuciir. Y. and Y. H vshimoto. 1967. Toxicitv of pesti cide ingredients to some fresh water organisms. Bull. Inst. Insect Contr. 32:5-11. 92. N oll, D. C. and R. Bi rdrick. 1961. Interim report on aquatic weed control of recreational waters above potable water. Proc. No. East Weed Contr. Conf. 15:470--473. 93. N orris,L. A.. M. N ewton,and J. Z witkovvki. 1967. Stream contamination with amiiroly Iroin forc-i sprav operaImils. Res. Prog. Rep.. West. Weed Contr. Corif. pp. 33-35. 94. N orris. I.. A. IPiV*. Stream contamination bv herbicide* after fall rains in forest land. Res. Ping. Rep.. West. Noe. of Weed Sci. pp. 33-51. O kuto, K. and T. O n bo. 965. Influence of diluted sea Volume IS, Issue 6 (November), 1970 water on the ph'sjological actiiitv of habv-neck clam. l ' e n e r u f u i t c T ' o n t m , and the t o x i c effect of an herbicide PCP, pentachioioplitnate. Bull. Tokai. Reg. Fish Res. Lab. 44:31-10. 96. Parka, S. J. and II. M. Worth. 1965. The effects of triIIura1in on hsh. Proc. No. Weed Conf. 15:469--473. 97. Petri K. C. F. 1965. Effect of herbicides on heierotrophic microorganism of ponds. Wctxi Abstr. 14:330. No. 1537. 98. Purge.M. . 1955. The effect of the weediciilc Kiirou upon the flora and fauna of two experimental areas of Long Pond. Dutches Countv, New Yoik. l'roc. No. East Weed Contr. Conf. 12:355-343. 99. Pierce. M. E. 1939. Further studv of the effect of the weedicide Kuron upon the Hora and fauna of Long Pond. Dutchess County, New York. Proc. No. East Weed Contr. Conf. 13:310-314. 100. Pierle. M. E. 1960. Progress report of the effect of Kuron upon the biota of Long Pond. Dutchess County. New York. Proc. No. East Weed Contr. Conf. 14:472--475. 101. PitJtcE.M. E. 1960.A studv of the effect ofthe weedkiller. 2.4- D Granular, on three experimental plots of Long Pond, Dutchess Countv. New York. Proc. No. East Weed Contr. Conf. 14:4-53-- 45. 102. Pitrce,M. E. 1961.A studv of the effect ofthe weedkiller 2.4- D AquaGranular on six experimental plots of Long Pond. Dutchess Countv, New York. Proc. No. East Weed Contr. Conf. 15:539-544. 103. Pierce. M. . 1902. A comparative studv of the application of three vvccdiciilcs. Kurusai C. K m oval NL and 2.4-D ester to three areas in Long Pond. Dutchess County. Proc. No. East Weed Contr. Conf. 16:435-441. 104. Pierce, M. E. 1963. Progress report of the application of fenac granular to six small plots in Long Pond. Dutchess Countv. New York. Proc. No. East Weed Contr. Conf. 11 :4.I-- 155. 105. Pierce. M. E. 1966. Application of fenac to four small ponds on Vavsar Campus. Proc. No. East Weed Contr. Conf. 20:4<0-- b5. I0G. Piirce, .W. E. 1967. Studv of a second vear application of fenac to two small ponds on Yassar Campus. Proc. No. East Weed Contr. Coni. 21:530-553. 07. ptiRfx. M. E. tutiN. The effect <>t several herbicides on eight test areas in Nobska Pond. Woods Hole. Massachu setts. Proc. No. East Weed Contr. Conf. 22:195-203. 108. Pierce. M. E. Ieoh. The effect of a combined formulation of 2.4-D and endothal] iqxvn a small pond. Proc. No. East Weed Contr. Conf. 23:376-379. 109. Pierce, P. C-. J. E. Frey, and H. M. Yawn. 1965. Field evaluations of newer aquatic herbicides. Proc. So. Meed Conf. 13:497-506. 110. Pierce. P. C. 1966. Results of field testing newer aquatic herbicides with emphasis on spot treatment applications. Proc. So. Weed Coni. 19:374-375. 111. PROvvsr. G. A. 1960. Notes on the toxicitv to fish of some common agricultural insecticides and wccdicidcs. Weed Abstr. 9:No. 1665. N m paged. 112. R ao, K . N. and M. S. M vrty. 196S. Control of I p o m n e a r e p la n s Poir with herbicides in Andhra Pradesh. Weed Abstr. 17:364. No. 2233. 113. R awls, C. K. 1965. Field tests of herbicide toxicitv to certain estuarine animals. Chesapeake Sci. 6:150-161. 114. Rnr.NER, L C.. W. F.. Nt'prER. and R. R. Johnson. 1965. Will the use of 2.424-T to control strcninside vegetation con taminate public water supplies? [. Forest. 66:914-913. 115. R einhart. K. G. 1963. Herbicidal treatment of watersheds to increase water yield. Proc. No. East Weed Contr. Conf. 19:546-551. 116. R ie m e r , D. N. 1964. Observations on some acid water lakes treated with diquat. Proc. No. East Weed Contr. Conf. 18:489-493. 117. Roorii.iez-K V8vn v, R.. E. A. C url, and H. H. FvxofRBCRK. Jr. 1967. Effe-ct of paraquat on growth of S r l a o t i u m io//ni in liquid culture and soil. Phvtopathologv 57:911-915. 118. Sum. M.. M. Kirvvvvn. and T. Kitsitakv. 1965. Studies on the prevention of |>oisnniug bv agricultural chemicals:12. Iil111u lice of heihitidc isodium peinaihlnrophenol.spread on paddvtield regions tvitli river water. Rep. Hokkaido Inst. Pub. Health 17:1<>:>--107. 119. Nchvvariz, H. G.. Jr. (967. Microbial degradation of pesti- 749 DOW 5317137 16261 w rsn .< W E E D S C I E N C E cities in aqueous solution. J. Water Poll. Contr. Fed. 39: 1701-14. 120. Simes, J. C. 1901. Control of the ponilwccd. P o t m - . n ^ c i o n c r iifn ts in both Mowing ami static situations with cndutliall. Proc. No. East Weed Contr. Coni. 15:53o-539. 121. Smith, C . E. a n d B. C. Isom. 1907. In v e stig a tio n o f effects of large-scale applications of 2.1-1) upon aquatic tauna and water qttalitv. Pest. Monit. J. 1:16-21. 122. Snow. J. R. 1963. A preliminary report on the control of Pitltophora with sitnatine. Proc. So. Weed Conf. 16:329335. 123. Steickt. E. W. 1961. Observations on the use of divwiiutn cntlothall in fi>h hatchery ponds. Res. Rep. No. Centr. Weed Contr. Conf. 18.104. 12-1. Stelckf. E. W. 1 j . Observations on the use of tlisotiittm endothall in fish hatchers ponds. Weed Abstr. 12:197. No. 1036. 125. Slrber. E. W. and O. L. M eehea.v. 1931. Lethal concentra tions of arsenic for certain aquatic organisms. Trans, of Antcr. Fisheries soc. 61:225-239. 126. T arrant, R. F. and L. A. N orris. 1967. Residues of herbi cides anti diesel oil carriers in forest water. Proc. symp. Herb, in Veg. Manage. pp. 81-66. 127. T atum, W. M. anti R. D. Blvckiurn. 1965. Prelitninarv studv of the effects e.f diquat on the natural bottom fauna and plankton in two subtropical ponds. Weed Abstr. 14:26. No. 144. 128. T a y l o r , J. L. 1906. Casoron a new aquatic herbicide. Hvacinth Contr. |. 5:20-21. 129. T aylor, J. L. I960. Dichlobcnil aquatic herbicide for use in non-flowing water. Proc. So. Weed Conf. 19:376-377. 130. T hiecs. B. J. 1955. The .-lability of dalapor. in soils. Down to Earth IW2i:l--1. 131. T homas, M. L. H. and ]. R. Drirv. 1968. Butowothanol ester of 2.4-D in :hc control of Ei-lqmss (X o sleia u .r.rtnn L.i and its effects on ossters (C r a u o s t r e n v i r z i m e a Gmelin) and other benthos. Proc. No. East Weed Contr. Conf. 22:186-193. 132. T immons. F. L. 1962. Present and future of aquatic weed control. Proc. No. East Weed lontr. Conf. 16:1-6. 135. TsattRLEv. F. H. lJo9. Deluliation in Vietnam. The- eco logical consequences of the defoliation program in Vietnam are assessed. Science 163:779-786. 134. U.S. Dr.p. of Int. 1965. Report of the Committee on Water Quality Criteria. Fed. Water Poll. Contr. Admin.. Wash ington, D.C. 234 p. 133. L..S. D fp.of 1st. In63. Fish and Wildlife Service. PesticideWildlife Studies. Review of Fi-li and Wildlife Service in vestigation dining 1961 and 19i>3. Circ. IG7. 109 p. 136. L'_S. I)li\ of Int. 1961. Fish and Wildlife Service. PesticideWildlife Studies, Review of fi'h anti Wildlife Service in vestigations during calendar vent 1963. Circ. 199. 130 p 137. L'.S. Di.t1.of Int. IPOS. Fish ami Wildlife Service. Pesticide Wildlife Studies. Circ. 226. 77 p. 138. U nrvu. G. O.. M. F vrooo, K. Dvuoon. L. M ic.cel, anti B D.vzo. 1903. Field trials in Eg'pi with acrolein herbicide molliisciciilc. World Hcaltli Org. Bull. 32:219-2tiO. 139. Van Valin,C. C. 1966. Persistence of 2.6-dichlorobciwonitrili in Aquatic Environments in Organic Pesticides in die En vironment. A Svm. Amcr. Chem. Soc. Advances in Chem isiry Series 60:271-279. 140. W alker, C. R. 1961. Toxicolocical effects of several herbi cides to bottom dwelling fish-food organisms in Missour ponds. Res. Rep. No. Centr. Weed Contr. Conf. 13:10-4--10"> 141. W alkfr.C. R. 1963. Endothall derivatives an aquatic herbi cide in fisherv habitats. Weeds 11:2215-232. 142. W vlkfr, C. R. 1904. Dichlolicnil as a herbicide in fish habitats. Weed 12:267-269. 143. W vlkfr, C. R. 1964. Simazine and other s-triazinc com pounds as aquatic herbicides in fish habitats. Weeds 12: 134-139. 144. W alker. C. R. 1965. Diuron, fcmiron, monuron. neburon, and T C A mixtures as aquatic herbicides in fish habitats. Weeds 13:297-301. 145. W eldon,L. W. anti R. D. Blvckbern". 1967. The control of floating aquatic weeds with ametrvne. Proc. So. Weed Conf. 20:312-318. 146. \Vji.ldorn.T. L,, Jr. 1S69. The toxiciiv of nine therapeutic and herbicitlal compounds to striped bass. Prog. Fish-Cult. 31:27-32. 147. W erxiiaw. r . L.. M. C. G olddcrc.. anti D. J. Pinckney 1967. The determination of the ionization constant of 2,4-D in water. Water Rcsour. Res. 3:511-310. 143. W i l t . I. 1903. Aquatic plant coniiol with diquat. fcnac. and simazine in Ontario farm ponds. Proc. No. East Weed Conn. Coiif. 22.173-IS5. 1ID. W insion, A. W.. k.ami P. M. R;ii\. iCui. What 'nappe:.} to phenoNv hcrliiciiles when applied to a watershed area? Proc. No. East Weed Contr. Conf. 13:390-- 101. 150. Yro. R. R. 1907. Dissipation of diquat and paraquat, nml effects on aquatic weeds and fish. Weeds 15:42-46. 162627 5 0 Volume 18, Issue 6 (November), 1970 322. 29i MNO 5 ?107. 0 DOW 1428521 ,h,SPROGRESS REPORT OH THE USE OF KURON, 2,li-D and 2 TP GRANULES AS AQUATIC HERBICIDES ' by . Roy R. Younger l/ Assistant Fisheries Biologist s. N. J. Division of Fish and Game The purpose of this report is to summarize the results of three year's experimentation using Kuron, and one year's progress in using 2,lt-D and 2,14-5 TP granules as aquatic herbi cides , Kuron The first experimental work with Kuron was conducted at Ramapo Lake, Passaic County in 1956. 2/ The following year thirteen lakes and ponds were treated. The results of these experiments have been presented in a previous paper, 3/ At Ramapo Lake, the herbicide was applied at a con centration of 2.5 p.p.m. to control water milfoil, Myriophyllum heterophyllum; fanwort, Cabomba caroliniana; and white water lily, i'lumphaea odorata. No further treatment was necessary in 1957, however, in 1956 the treated areas were completely rein fested with the same noxious weeds. Good control of the most predominant species - water milfoil, iiyriophyllum sp., yellow water lily, Huphar sp,,, and white water lily, Hymphaea sp,, was obtained in five ponds re ceiving total applications of Kuron in 1957 at concentrations ranging from 0.5 to 2.5 p.p.m. In 1958 these ponds needed no further treatment because the water milfoil had not reappeared and the water lily stands were greatly reduced. Considering the size and extent of the lily root systems it is not diffi cult to understand why total control in one application is not possible. Plant reinfestation seems most likely to occur, at least in New Jersey, from seeds and plant fragments entering from untreated lakes upstream, rather than from the germination of seeds after the application of Kuron, There is some evidence T f The author is now employed as a technical representative of Halco Chemical Company, Kenilworth, H. J, 2/ Huckins, Robert K, 1956. Job completion report, Project F-l-R, Aquatic Weed Control, unpublished. 3/ Younger, Roy R, 1957. Preliminary studies using Kuron as an aquatic Weed Control Conference, New York, January, 1958, All this work has been conducted under federal aid in Fish & Wildlife Restoration Acts, New Jersey project numbers F-10-R L F-lh-D. . 6263 V' . j 324. DOW 1428523 reduced prior to treatment greater plant destruction was achieved, Therefore, it is recommended that lakes and ponds receive total treatment and that the lake volume be reduced; to insure sufficient contact time. The concentration recommended to obtain adequate control with Kuron is 2.0 to 2,5 p.p.m. The Dow Chemical Toxicological Laboratory has determined the L.D, $0 for the emerald shiner* Hotropis atjierinoides, is 7,0 p.p.m. The recommended concentration is far below the toxic level and fishkills should not occur, providing other chemicals are not used before or after, treatment. Furthermore, damage to shoreline vegetation should be negligible at this-concentra tion, 5/ 3. Bottom application i Spraying the exposed bottom Tri.th Kuron at the rate of 4 gallons per acre has proven successful in one instance; It might be worthwhile to more fully explore this technique, thereby reducing the cost and making it possible to spray at an earlier period in the spring. Conclusions The results of three years of experimentation would seem to indicate that longer periods of control are obtained with Kuron than with other herbicides. 2,4-D and 2,4,5 TP granules As a result of Dr. Grigsby's 6/ work in Michigan, the New Jersey Division of Fish and Gamo became interested in further field testing of 2,4-D and 2,4,5 TP granules. Thirty-six plots containing 2,500 square feet (.057 acres) were laid out to evaluate the effectiveness of 2,4-D and 2,4,5 TP granules of either 15/30 or 8/15 mesh size. The "TP" granules were tested in three plots and the "D" in thirty-three plots. There have been some fishkills in the past, In one instance the fishkill vras caused by the addition of copper sulfate for algae control prior to treatment with Kuron. Another fishkill in a farm pond was attributed to the rinsing of spray equipment previously used to apply Halathionr \4vthird fishkill-has never been explained ... due to so many extraneous factors which occurred either, bj prior to or immediately following application, Grigsby, B, H, and H, 'H, Hamilton, 1958, New techniques ^ in the application of herbicides for control of.aquatio plants, ,Presented.at the.1958 meeting of the Weed/ Society of America, Memphis, Tenn,^^uary^l^ 8,1 `:-r Conclusions: The advantage of using granules is ease of dis tribution since no cumbersome spray equipment is required. This makes it possible for lake front property owners to treat their portion of the lake. If the mechanics of the granules can be improved, and if the rates of application can be determined, they will definitely have a place in aquatic weed control. DOW I 4285 ro or U U f t ) 4 2 B 5.2.7... T a b i I Contd. SUttiART OF 2,U-D and 2,U,$ TP 7? tZHuriTS IM it,V X ro d 1958 <jO CO Vt C> vH Area treated Applegate's Pond Monmouth County Type of vegetation Fanrort, Cabomba sp. SDatterdoek, ilupiiar sp* Control None Medford Lakes Camden County Musconetcong Lake Morris County Water milfoil, Myriophyllum sp. White water lily ^.^haea sp. Water milfoil, hyriophyllum sp6 None 2,h-D control only with 12 and 18 lbs/acre. 2 , k , $ TP control with 9 lbs/acre l / Hopatcong Sussex County Water milfoil, hyriophyllum sp. Spatterdock, . duphar sp. Pond weed Potmegeton sp, Good control at all rates 2/ C-rovers Mills Mercer County Esterille Lake Atlantic County Saxtons Falls Warren County Fanwort, Cabomba sp. Bladdyi-wor t Utricularia sp. Water milvoil MyriopiTyllum sp. None 3/ None 3/ None 3/ l/ At time of application no Vallisneria was obsarved. After treatment and destruction o f Hyriophyllum, the Vallisneria took over tha treated area. Z ! Regardless of rates of application, good control was had for a short period of time since weed fragments from untreated areas settled to the bottom, putting out advantitious roots, thus reinfesting the treated area. 3/ In these plots the 2,h-0 was sprayed on soft attaclay which brolce down immediately upon contact with water. G -irH H 4-> .C C * EH ocn 0 o TD r rc 4J 0end S=oo o *> er ra P 60 0 > G -P k h ra ra X00) ol3 0G x: ra O (ouu 4ou->*^ S; , ' 00 x: x: P -P B ou Voh <M T> 0 -4-> G ufH Ka0 ... h i : o-.-V- 'j': -. ' *?.* h.!':" ! ::! .t'` 3 2 a. D O W 288954 2919 J* ACD File No. HA-8 REPRINT OP PAPER PRESENTED AT THE TWELFTH ANNUAL MEETING OP THE NORTHEASTERN WEED CONTROL CONFERENCE j Hotel New Yorker New York City January 8, 9, 10, 1958 332 PRELIMINARY STUDIES USING KURON AS AN AQUATIC HERBICIDE^ by Roy R. Younger Assistant Fisheries Biologist New Jersey Division of Fish and Game In the summer of 1956 the New Jersey Division of Fish and Game, under Dingell-Johnson Project No. F-l-R, Aquatic Weed Control, experi GO 'Z - mented with Kuron on one-acre plots in two northern Hew Jersey lakes. 28895 The chemical called Kuron - 2 (2,ij.,5-Trichlorophenoxy Propionic Acid - is a hormone-type growth regulating compound which Is absorbed by the leaf and translocated to other parts of the plant. The above mentioned plots were heavily infested with water milfoil, Myriophyllum hetero- ciphyllum; fanwort, Cabomba caroliniana. and white water lily, Nymphaea odoraia. One week after application good control of the submerged species was evident: the emergents were not materially affected, (Huckins, 1956, ms.) It was felt that no further treatment was neces* sary after examination of the plots in the spring of 1957 The results did seem to indicate that further evaluation was ~ , desirable and a testing program was initiated in the summer of 1957-- ' Methods and procedures Lakes and ponds, In order to be included as part of this testing program, had to meet certain requirements: 1. As a group they had to repiysent a cross-section of existing lakes and ponds throughout the state with regard to weed species, both emergent and submerged, alkalinity and hydrogen-ion concentration. 2, They had to have a woody shoreline so that the effect of Kuron on woody plants could be evaluated. In treating a pond the procedure was to calculate the active ingredient in Kuron required to produce the predetermined concentration in parts per million. To determine the effectiveness of Kuron at various concentrations the herbicide was tested at 0 .5 p.p.m., 1 .0 p.p.m., 2 .0 p.p.m., and 2.5 p.p.m. One reclaimed lake In southern New Jersey was treated at 3.5 PjP.m. The herbicide was applied by a Bean Estate Sprayer, skid mounted, with split intake lines and the by-pass was used for circulation and agitation. A spray boom was devised and a thirty-gallon drum served as a spray tank. The entire rig was mounted on a 11.5 foot aluminum boat arkL was operated by two men. The herbicide was applied in rows with a alight overlap between them. 1/ Wise. Report*#21, New Jersey Fisheries Laboratory, Division of Fish and Game, Mllltown, New Jersey. 2/ This program would not have been possible without the assistance of Dr. E, Evaul and Neal Munch of the Soil Conservation Service who obtained ponds for testing; Dr. Mark Wiltse of the Dow Chemical Company supplied the Kuron. 333. After the ponds had been treated water saunples were taken and forwarded to the Dow Chemical Company for Kuron analyses. The water samples were taken from two to twenty-four days after application. (Table I) Ponds were checked at seven day intervals for the first month with monthly checks thereafter. Findings During the course of the summer eight lakes received total appli cations and five lakes received plot treatments of a half-acre in area. A summary of these applications is presented in Table II. The concentration of 0.5 p.p.m. was effective on white water lily Nymphaea. The submerged species were only slightly affected. At 1 p.p.m. good control of water weed, Anacharls sp., yellow water lily, Nuphar. and mud plaintain, Heteranthea sp., was obtained. Complete G control of water milfoil, Hvriophyllum, and bladderwort, Utrlcularia. was achieved with concentrations of 2,0 p.p.m. and 2.5 p.p.m. From these data there is every indication that adequate control of most ro species can be obtained at 2 p.p.m. however, further testing of the oo effects of Kuron on pondweed, Potamogetn, is necessary since none of CO the concentrations employed controlled this species. Duckweed, Lemna CO minor, turned brown forty-eight hours after application of Kuron and t n then recovered fully. As to the effects of Kuron on trees, either in the water or urrounding the lake, no damage was evident on such species as silver maple, black gum, pitch pine, willow ./and white cedar. Since this herbicide contains phenol the possibility of its tainting fish flesh was considered. Fish were collected from a treated area and taste tests revealed that Kuron did not taint in any manner. As mentioned earlier, measurements of pH, alkalinity and tempera ture were recorded. None of these water characteristics appeared to affect the herbicidal effects of Kuron. Observations suggest that Kuron is slow to act. Seven days after treatment submerged plants appeared wilted and emergents were curled. Fourteen days after treatment open areas were visible; submerged.vege tation had started to rot, while the emergents continued to curl. It was not until twenty-one days after treatment that the submergent weeds were completely disintegrated and leaf and stem systems of the emergent vegetation Had broken free from the roots. One month after application root systems of both typos of weeds were observed in windrows along the shore, Since the affected plants did seem to break down quite slowly, there was some suggestion that no serious oxygen lag occurred. However, this cannot be substantiated since an insufficient number of D . 0. determinations was taken. Observations also suggested the absence of lgae blooms, sometimes characteristic of certain herbicidal applica'*ons, caused by the release of essential nutrients from the rapid eakdown of plant tissues. However, this lack of algae blooms could .Iso indicate possible algacidal effects of Kuron. 11269 ' 334. Further evidence of the slow breakdown of Kuron was indicated by the water samples which were forwarded to the Dow Chemical Company for Huron analyses. In the total applications the concentration of Kuron remained fairly constant while in plot applications the concentrations were affected by dilution, or a build-up in stream application. Resi dual effects cannot be determined until next spring when the viable seeds, if any, will germinate. Plot applications were not as effective as total applications since reinfestation was possible when shoots and seeds from untreated areas floated into treated areas. The possibility also exists that once the vegetation has been destroyed other aquatic species, either desirable or undesirable, may over-run the treated area. Total applica tions were most effective since reinfestation was less likely to occur. Summary and conclusions The experiments conducted to date have more or less been designed to determine the herbicidal effects of Huron on aquatic vegetation. Success has been obtained on the most predominant weed species in the thirteen lakes and ponds so treated, using a concentration of 2.0 p.p.m. The period necessary for total plant destruction is normally twenty-one days. The usual algal blooms were not observed following treatment with Kuron; preliminary data suggest no serious oxygen lag. This could be attributed to the slow breakdown of the plant tissues but the apparent scarcity of phytoplankton also suggests the possibility of algacidal properties. None of the concentrations employed have affected the trees either in the water or along the shoreline. Neither pondweed, Potamogeton, nor duckweed, Lemna minor, has been effectively controlled by any con centrations of Kuron. There are many important questions still to be answered regarding the possible use of Kuron as an aquatic herbicide. These questions include; At what time of year will application achieve the most effective control of weeds? Does this herbicide have any undesirable side effects on certain micro fauna and flora important to game and pan fish species? Will it control filamentous algae? Does it have a residual effect? Will Its cost affect its use? The results to date have been promising and the proposed questions are in the process of being investigated. In the meantime Huron can be considered as one of the mo3t important aquatic herbicides on the market since the development of sodium arsenite. Oo O 00 CO CD UT Table I. AMOUNT OF KURON DETECTED FROM FIVE TREATED PONDS 2 TO 24 DAYS AFTER APPLICATION (Analyses conducted by Dow Chemical Company) Name of Pond Rte. No. 1 Golomb's Pond Keswick Pond New Waywayanda Panther Pond i i . CM i i Kuron applied p.p.m. 2.5 2.0 2.0 2.0 No. days sample taken after application 2 days 24 days 14 days 3 days 8 days Kuron detected . p.p.m. 2.0 1.5 1.8 2.1 -Less than 0.5 u> U) VJ1 896882 Jo -a Tab l e II SUMMARY OF KURON APPLICATIONS - 1957 Name of ialce Kuren applied Tot .alle. or pond p lum PH . p.p.m. Temp. Dominant weed species Tavlor Pond Mohawk Lakt Bell Lake Ludlum Lake Cape May Fark Pond Barker Pond l.C 1.0 3.5 2.5 (plot) 3.0 0.5 7.2 13.0 8.4 82.0 8.3 105.0 6.4 14.C 6.6 12.0 7.3 6.0 86 Heteranthera dubia Anacharis canadensis Potamogetn crispus 78 Typha latifolia icirpus americanus Sparganium sp. Lemna minor Nuphar variegatum Anacharis canadensis 68c Nuphar variegatura Eriocaulon septan81 gulare Myriophyllum heterophyllum Vallisneria americana Fymphaea odorata Potamogetn sp. 79 Anacharis canadensis Utricularia purpurea Myriophyllum 78 heterophyllum Applegate Pond 0.5 7.0 27.0 Nymphaea odorata Potamogetn sp. Vallisneria americana 80 Cabomba caroliniana Myriophyllum heterophyllum Tegree cf Control Complete Excellent control of submergents, no control of eraergents except those individual plants which came in contact with spray Excellent control of both species Excellent control Reinfestation Excellent control In doubt. Pond was previous ly treated with sodium arsenite Only species affected was water lily Table II Cont. Oelomb Pond 2.0 6.7 12.0 Foo Lake Keswick Col ony Lake Panther Lake New Wayuay- anda Lake MacDowell Pond 0.5-plot 0.1-plot 2 ,0-plot % 2.5 2.5 2.0. 2.0 5.4 53.0 5.6 2.0 8.8 103.0 8.8 107.0 6.8 76.0 o o ! Myriophyllum 81 heterophyllum Nuphar variegatum Scirpus sp. iiuphar variegatum Caborr.ba caroliniana Myriophyllum Lemna minor Anacharis canadensis Utricularia purpurea Nymphaea odorata 78 Focamogeton sp. Myriophyllum 74 heterophyllum Nymphaea odorata Myriophyllum 76 heterophyllum Scirpus americanus 80 Potamogetn sp. Excellent control Plot p i only Nuphar. Plot -$2 and Plot #3 good control No control of Lemna Excellent control except for Pctamogetcn Excellent control Excellent cortrol Only Scirpus was controlled h-4 CD ro ~v3 G 0762> 2> T?< pW O Q 12u2 SUMMARY OF TOXICOLOGICAL AND ANALYTICAL INFORMATION ON THE CIILOKACNEGEN IMPURITY FOUND IN SOME SAMPLES OF 2,-1,5-T OCCURRENCE AND IDENTITY Several impurities can occur in 2,4,5-T arising from their presence in 2.4, 5- t'richlorophenol. They can be minimized by propei' operating procedures. The most important impurity which causes the chloracne problem is: symmetrica 1 tetrachlorodibenzo-p-dioxin Cl Cl C2R oCO CO TOXICOLOGY p N o t e . All doses given as MICROGRAMS (10~ grams) Skin Contact Human 1. Known to cause severe chloracne by skin contact. Very small amounts can cause the lesions. 274 Ra b b i t 1. Inner surface of rabbit ear treated with various levels of the chloracnegen in 100 microliters (ul) e.'-;1 of a test solution in acetone benzene or chloroform on an area of 1 to 2 sq. in. (5 animals per dose): Total Dose Applica tions repeated = no effect CO _1 oo -vi o to i; 0.5 ug repeated marginal effect* 1-2 ug 4-8 ug )/sometimes from"*1)V = usually causes an effec t [one application) = almost always causes an effect, usually severe, but not lethal 2. Shaved belly of rabbits treated v/ith 10% solution in acetone, 5 animals per dose. LD,-q by skin absorption = 275 (142-531) ** ug/kg body v/t . Mortality generally occurred 12-22 days after dosing. S [For parathion, NO g-;' -0-P-(OCgHg) g , the LD_Q by skin absorption lias been reported to be: rabbits = 40,000 rats (.1) = 21,000 ug/kg rats(F) = 6,800 ug/kg"! ug/kg body w t . Intraperitoneal When administered as a 10 mg % suspension in corn oil to rabbits (5 animals per dose level): I.P. LD50 = 283 (114-703)-* ug/kg body wt . Follicular prominence and/or epithelial thickcuiing begins 1 to 3 weeks after exposure. * * 95'/ confidence l im i t s . [For parathion, the intraperitonea1 LD r e p o r t e d t o be: has been rat = 5,500 ug/kg guinea pig - 12,000 ug/kg dog = 12,000-20,000 ug/kg Acute Oral (single dose) Rabbit C.V5 CO o CO CO When administered as a 10 mg % suspension in corn oil (5 animals per dose level): Acute oral L D j-q = 100 (29-389)** ug/kg. Two rabbits died at 32 ug/kg, 1 died at 16 ug/kg. Typical clinical picture was severe liver and kidney injury (histopathologic examination) at doses approaching L D j-q . Have seen histopathology at single dose of 3 ug/kg. Dose of 500-1000 ug/kg was always fatal; sometimes took 10-20 days for death to occur. iFor parathion, the acute oral LD<-q in the rabbit = 10,000 ug/k|J Rat 5 mg % in 1:1 corn oil/acetone (5 animals per dose): Acute LD_q , female rat = 45 (36-65)** ug/kg male rat = 22 ug/kg [For parathion, the acute oral LD.^ is: rat(M) = 5,000-30,000 ug/kg rat(F) = 1,750-5,000 ug/kg! Data for parathion taken from "Evaluation of The Toxicity of Pesticide Residues in Food". FAO/Vi'HO Joint Expert Committee on Pesticide Residues. FAO Mtg. Rept. No. PL/1965/10/1. * * 5D ',n c o n f i d e n c e l i m i t s . 16278 o s o e i t i r.()0 Guinea Pi- 50 mg % in corn oil (5 animals per doso) Acute oral LD-^, female guinea pigs = 0.6 (0.4-0.9) [For parathion, the acute oral LD,-q is 9,30032,000 ug/kg for guinea pigs] ug/kg ANALYTICAL METHODS Vapor Phase Chromatography can detect 1 ppm. Need 100 g. for assay. (Method attached.) Liquid Chromatography can quantitate 1 ppm in a 2 g. sample. Bloassay (see under TOXICOLOGY, Skin Contact, Rabbit) . Can detect the chloracnegen in smaller samples and should be used to verify absence or presence of "dioxin" in 2,4,5-T used in toxicity tests. DOSE RELATIONSHIP OF 2,4,5-T/"DI0XIN" WHERE "DIOXIN" PRESENT AT 10 PPM IN 2,4,5-T 2 , 4 , 5-T mg/lcg 1 10 50 100 h-l O O o o 2,4,5-T ug/kg 1, 000 50,000 100,000 10 ppm "Dioxin" ug/kg______ 0,.01 0 ,.1 0 ,.5* 1,.0 *Equal to the acute oral LD.^ for the "dioxin" to female guinea pigs and 1/6 of the single oral dose which causes histopathology in liver of rabbits. * * 95',. c o n f i d e n c e l i m i ts . h U l.Ji' 1 - * W> V I DEFiCiEMT. ORIGINAL -.1., 1204 ( MN012332 F reeeat: II..C . y ~ 21. G o r d o n L. K-Udioro B . H older L. B o l l inger C. K r a m e r R. btcwurt R. Kblaios A. Leake K. Coail rot at- K. Z r J' l e A. '...r.;...'-.i- . J i i L. S ilv e r ste in K. O lson The c a s t i n g w as c a lle d to o r d e r a t r.p?rc:ahv,?.to!y iR r to bp D r. le .r r y Mathews p r e s e n te d a l i t c r a t u r n r e v ie w on o h lc r a o u e . Fa indicate- th a t the tro a trcn b o r ch loracn c i s tun g r e a te s t o f th e p u s s ie s to ha solve-?.. 7 - o i . o a o l y , a s w i t h a n y S iy a a o o 'c r t h i n t y n c . b r e a t r s n t c c r s i s t n c f r e m o v in g t h e p a t i e n t f r o t h e a :/20.. Kcvrevcr o i t o p r e m o v a l t h e l e s i o n s ccrztirauo t c a p p ea r fo r up to tr o y o s r a . i-buy m ethods h ave too:' cr/; e r e b e i n g u s e d : n t h e l e s i o n s th c.o -.a X v o s i n c l uc iu .5 r a d i a t i o n , a n t i b i o t i c s s t e r o id ., e::pr c so i.c n o f ocin odc-eo and Ir en eto iy t .lc~ e 9 & ts. F r o s e n tly , h o r a i d ; u e a r o t r e a t i n g o u r p a t i e n t s v r ith e n p r e:ssssiieeru. oc f t' h e e o r a d e a a s a n d f r e q u e n t br:Lc;.: r o o h i n g o f t h e s r p o s c : a r e a r'aibt h nr eea p ;an d a rev. ah w ash c l o t h . S t e r o i d t h e r a p y i s *30 11:2; t r i a d a t sni:-.1r-o in u fte*i.'t v u i e r s b u t n o r e p o r t a o f s u c c e s s o r f a i l u r e Ima a s y e t b e e n r e p o rrtteedd,. Dv.tuifc-3 a l l a t t e m p t s ., t h e d v.r-c.tlo n o f t h e eh icr-i; cn a a p p e a r s to" hb oe unocci h a n g ed , u n t i l m ore s u i t a b l e iu o a sv r a u o f t r e a t m e n t e r e f o u n d . t;bIieo p r oe v;; e dnittiioo n c f e:q;o:v.irc i s th b o n ly s a t i s f a c t o r y r a y o f c li? ": r, >> p r c b l a n . L . S i l v e r s t o i r i p r e s e n t a c h r o n o l o g i c a l chart o f t h e p r o c e s s change.*:. a to .$ r e l a t i n g cucl> c h a r g e s t o th e ohlor-nono p r o b le m . S ilv e r sv e in in d ica ted th a t tho o i l draw -off op era tio n in tho p r e se ts is th e r e s t v u ln e r a b le e p o t f o r e::p03i:re to r o n o fe m p rod u cers;. A f lo '.i - n l e e e t c h a r t o f t h e p r o c e s s ;-e;.a p r -e o r v te d unci d i s c u s s e d b y Re;; R o l r e o , Hay : ie p h a c is e J t h a t h e row 13 l:*J:o to h a r e a n a n a l y t i c : ! ra th :-:: v :h ic h c o u ld bo c o r r a l s t a d w i t h t h e a e n s f o r .. r a t o r i a l s i n o r d o t t o h a sten the clean -u p werk. V . K. Hone r e v ie w e d t h e w ork d o n e b y t h e i e r n e n s . He p v t t i c s tr -.v c tu r n l form ula cn th e board fo r te tr a c h le r o b sr sa d ic a sn e w hich has b can In crim in ated as b ein g a t le a s t one c f the r e n t p o ten t o f th e - v te r ie is w h ic h p r o d u c e s t h e n e n e fe r m d e r m a t i t i s and p e r h a p s t b s s ;.;t o . - a: . : .t o . X t i s c u r u n d a r e t a n d in p t h a t Kan Lrr.llcy is" i n t h e p r o c e c : v e p iv . a i r i n g t h is m a te r ia l tih ich l e i l l be S3::t to fic-f'cT .;. fo r E tu i:;. The f o ll.c n in p e ec o rr esa d a tio n s v:cre a e r e e f upon by the grou p : 1 . S u b s t a n t i a l c le a n u p o f o p e r a t i v e a r e ... t o o l s and e q u ip n o n e , Pay K oine2 p i-o c ed u r e . has He c o n ta cte d in d ica ted D ow ell p erso n n el fo r a d esire to sta r t on acivic.-j -cr. S a tu rd a y , th e Aug clc ust m2 r9c. p Thu reon w i l l 'r e a r r u b b e r s u i t s f o r t h e j o b . 2 . R eview p e r s o n a l h y g ie n e p r a c t ic e s and c lo t h in g p o l i c i e s w ith r::a in 199 b u ild in g . 16278 c Tito .CL' T O'J'.!C'`r* ".. i,.Uf.` t. e ci' r.\: v* ;':;v? cj p e r i c l c P h r L Cealc.. V.l v.i 7. "j. -*-7_; Lv. o te: .> *<?c v l o r s tho th o o ln : p:.:os o u t e t-.be f o r m u l a i.*o ir- report:: c bolnp C.-7 O/ '* / , :v;c-:.:::l b l e t V *...' :' :c a f o u e t t e : .! prsr.cut; i:-zi:io6olo32lr coada .>icO 3.r. L-Iocloul. Thlr. ;i:::.:;:;;h''cr ;?lo.clc:'uu e h c u l b s r a s o l v s i . '.Cn crdc::* to ce t h i o B ic c lix : . b a s o f i ivod Uthlevs- uu :n: oe ci' tbo CerJLho:? Ce un t e r . rePRAfi CC The n a t t e r o.f accu rn u la tir^ nornuX v a lu e s f o r c o r a rea v:aa d ic c u n so CXoC.12:3.V-3ly. loyic; l i s t e d a nur^hcr o f ir.aheralr; tha: n lp h t e f f o e b ttie honsviee e ::p c s u r e s , Ko a l s o :a?tieat?.ci th u t b on son e oxpoaaro l e a b a c a i`c l o v o l :'.n th o plc.r.t and 1;h" S th a MOV v a lu e s couclactoc! or. r.,?.ay c f tlvcr.e p e u p le .iuv r e p r s s a n t a r.orr.al v a lu e In co fu r a s our a r e s p orscu n el arc oaaceruecl. C 16279 aao n T i7.fc-12-13 Sample Ho. 1 2 3 5 S' o 7 'MATEREADS TRSt ED (Data in Table I) Process Stage Coil Reactor Decanter* 5teaming-out Drying column Finishing column Materials Blag, i; A composite of all mate rials in reactor. 159 .Oil material scanted and disposed of in burner. 199 Condensate from top cf column. Contains chloroaniaolo. ehlorcethorj and methyl alcohol. 199 Tops stripped from the* product from drying column s'contains chicr-ophcnol concentrate). Tope consisting essen tially of 2-4-o.ichlsrc~ phenol. Reeldue from distillation of 2 JiT-5"trichlorcphen,oi. Contains chlorinated diphonylethcr. chlorinated resorcinols., ssthozy dichlorophenolu, c-tc. Goar, to tvasto disposal. 3 b-9 3^9 3^9 R0& - 16280 /--N - 1.7 s z m CONTACT 2R?.ffj?jVgIP3 - RAB3XT - 2,4,5-'.? O H K - 2 7 7 7 - 5 5> Mar. 23, 19-5*0 T17.4-12-13 1 o --c >,~p.^r>;,;.':,T_ % :ii x k ?m Amina 1 No, ns P 1X5 S'. Condition of Skin Intact Intact No, of ctvplie af.ione Site 1 R. ear 4 L. ear* Re epona s-Hetep.rks Node-rate orythetia, edsnia and bur-n . Animal died ifi one week, Slight erythema after 3rd clay; follicles >rcr*inent. {sawr) animal as bove) i D?K 0.25# y??5 0.25# D?3: 164 M. 555 1?. 555 F. Intact Intact Intact 10 ear Slight erythema only. Follicles prominent, Animal died`aftor 10th applicatitn. IS e a r Follicles prominent. Afilinal died after the 12th applica tion. 12 bel.ly Slight exfoliation, no hyperplasia, Same animal as above. o j c -; I)??-!: 930 r-i. ! Intact 14 ' ear {17 days) only Moderate folliculitis, 0, OJ.;j jJj.'ii 940 K, Intact 14 ear (17 daya) only Very slight folliculitis. K4 05 t\3 00 ^ r'P r f p m (O<i O H4 Co to <x K material {X > i n 3'-l?rl 5J$ i n D P M 5?i i n E K - I 1 :l.r D P I 4 !> i n DPI-I 0.25# in D?M 0 . 2 3 ^ i n D?:-i O . l f J i n n:.;M 0 . 0 1 ^ i n X)?M rAsrs ~ix T17.4-12-14 SlOT CONTACT IIIRITATIOH - RAE33* ^ , 4 , 5 ~ T O i l fT I 7 . 4 - 1 2 - 1 4 , - 2 7 7 7 - 5 5 , A p r i l s r , i $ S 4 ) 'f L^y-tp - u l'C - 'fuiiS -- 4-<" r-*)^ Animal No. 37 5 M. Condition o f S;:ir Intact 1 Mo. or Anp Xicatlcns 3 Site ear Reracnso-Ronatfks E s s e n t i a l l y rib i r r i t a t i o n . Follicles prominent. 3 75 M. Intact 5 belly E x t e n s i v e h y p e r e m i a ail cl swelling after last appli cation. Animals sacrificed. 375 M. 7 5*. Abraded Tin t a c t 3 belly E s s e n t i a l l y t h e s a m e fea dtfb* 1 5 ear Ho irritation, no folliculi^i A n d i e d aftex* l a s t a p p l i cation. 557 F. Intact 5 belly Saiv;e e s a b o v e , n o l j y p e i p l a f i i i v 603 M. 60S M. lilt a c t Intact 6 ear No irritation, follicles prominent after last appli cation , Animal died. 6 belly Same as above} no liypar-told h i 61 932 M. 1. 934 M. J.tl CctG ii Intact 14 < 1 7 (2ayr>) ear only 14 (17 d a y s ) ear only * 'Moderate f o l l i c u l i t i s . S l i g h t fo.Uicttl.iti3! t1 t* 966 MOQ 'jApia__ X REACTION IV Tils*. RARBICT EAR TO PROCESS SAMPLES 'S 1 `7, Saiiiple Ho, Material Teste-ri 1 9 cfA CtXi7 \jUL<LxJCf m ii ; , | 2 1> ater 1Cp in water 3 /vye?a. 4 10jy in waters Unf2.i3.uted 5 3^7W<j 1C$ in 3>PM D^ ) i ^ 5 W . 6 "3 ^ ^ < 10$ in vinter k f - ________________ j7 ip^ in.DPM .195) Very slight fol?i.:lculits (no thickening of ear) 3 eve re folliculitis '(0 (3 S thickening of ear) Ho foil ;iou1it2.3 (no thickening of car) Ho folliculitis (no thickening of ear) Ho folliculitis (no thickening of ear) No folliculitis (no thickening of ear) Very alight folliculitis (2 X thickening of ear) 1S6H (Hay So) Severs folllculiti-; 0 (5 X thickening of e;:r > SsVore f'o1::.1cmlii;is (! X thickening ox"* earl Essentially no folliculi;, (no thickening of ear) Ho Sarnie loderete to c o v e r s j.\j1 . Aat..',r. (?; X thickening of s5asr ) /ory slight folliculitis ('2 X thickenifjy of oar) Very a1iaht fc11ic\.1it:'. (slight thickening of ear died after Cth applies tr- Potent acneforai producers. Okc.. n < R O .U 16283 8EG6fi MOC1 c\ c - ( -f ~ t ~ i ~ htcvi" -Q. ri 'P .'; Vlipo Tents .f 323S"3:s iiu lld in r ; 7 / 2 7 / 6 '! Chlor-ac.no Potential Sample jbirhes D;cc rind icn Laboratory 2ic:;<! - table vCp ..*0. r>:,s nvi* Oi+ Laboratory bench tcoa. *> Laboratory vl`3 0 UC--/ "*i-'-c jLiuncn R o o m - *#-*}* --}^y*- W ...... *> w * / "'-.y,*1 .**!'* *"" r*'>.'1 - " * *' * -* w -A table tope. 5 Looker Aeon * benches. 6 Stripper and Methanol Pump Area vt-.i.J *. iZcwiiCiJ-vi- +- i 44-t' 7 0:11 drsv:off - valves and -chains. 8 Oil Bcispster and pipe-- in oil. spills. -H-:- 9 Taylor's shorn - 6-3 non els old. v v 1 0 Tool bones ana Taylor's tcol3. 1?. Operator's deck and chair at panel. 12 Caustic - methanol pv.rps - valves. j-v- -f--I--I--i' * <--- 5-5.JwJ. * 4 13 14 T^ 16 17 j 18/ Roof - rai.VJ.ro arcani-i stripper columns (error vent), Roof ~ ^X- (tehore Fliocni: Reef - rallies , operator trail: stands. <>oa**ar& -ts/ Caustic - r.mbba: CSuBtlG screens Taylor1s sieves off oil cn 7/St/S4. Control slaves. 3 4 -h h -;- . CO_ A*`r..v-w'7--^* 71)--;--v.->y'~*<*?, r*. ^ -7r> *? r\^ able o: Caustic insoluble ell^ `par, 15, 19: Caustic insoluble oil}(April it, 1S: .Ivorstain and 9. Garland, 11.14 :;ta A. F. .Lucclc on 7/27/64. Anir.dIl sacrificed after 8th a-*c"-olicatior>. Bleedinr*c* from both cars. 12. Animal prostrate and autopsied after 5 applications and 15 caps. 3. Anir-r.-v prostrate and autopsiccl after 3 applications and 15 days. Aninai died on 10th day uith 3 applications. itw.L.i* ivfelte rlcvcs vrare contaminated by v:ipin;; the finsrrtips on the .:u.. ^ U.;^ -- ..s*--< the plcves ana extracted uith bcnncr.e and adjusted to 1C cc vol. 0.2 cc cf the extract uac applied to ties rabbit ear for 7 applications; 0.1 cc 1 day therafter. 16284 Rit'ZS8 M O a * i 2 \ j T) M S R C U L S S I X C G 7 L 11-- ' rr> avi is:LjJ-N S Y N T H E T I C S D E P A R T M E N T W I L M I N G T O N . D E L A W A R E 1 9 8 9 9 T E L E P H O N : 3 0 2 - 6 5 5 - 9 3 11 N o v e m b e r 27, 1967 Dr. Harry Kays, Director Pesticide Regulation. Division U. S. D. A. Washington, D. C. Dear Mr. Hays: I a m writing to request .that the effective date for possible cancella tion of the" registrations for use of phenoxy herbicide viz. , 2,4-D, 2, 4, 5-T. M C P A , and Silvex, on range and pasture grass be extended for at least one year beyond the D e c e m b e r 31, 1967 date established for cancellation of no-residue registrations. The N A C A Industry Ta s k Force on Phenoxy Herbicide Tolerances has a s s e m b l e d petitions requesting tolerances to cover uses previously registered on a "no-residue" basis.. T h e D e p a r t m e n t of Agriculture has cooperated with this group in several ways. Dr. L. L. Danielson w a s assigned the function of D e p a r t m e n t liaison with our group, and has provided information and data f r o m U S D A sources. Dr. Danielson and other m e m b e r s of the D e p a r t m e n t h a v e also consulted with us at various stages in the preparation of our petition. O n N o v e m b e r 17, representatives of the T a s k F o r c e m e t with you and other U S D A officials in the office of Dr. Rob e r t J. A n d e r s o n to inform the group of progress in the preparation of petitions. T h e status of range grass and pasture grass as ra w agricultural commodities and tolerances whi c h might be n e c e s s a r y for t h e m w e r e also discussed. Pursuant to the results of that discussion, w e are deleting f r o m our petitions requests for the 20 p p m tolerances for grasses that h a d been considered, and requesting that an extension be granted so that data n e c e s s a r y for tolerances at a level higher than 20 p p m m a y be developed. Continuation of the registrations for use of phenoxy herbicides on grass is in the best interests of both the agricultural industry and the public. T h e usefulness of these materials in recovering and improving of grass lands and pasture for livestock production w a s e m p h a s i z e d by U S D A r e p r e sentatives at our m e e t i n g on N o v e m b e r 17. T h e r e is no h a z a r d to animals, as evidenced by the extensive use of these materials for m o r e than a decade, I 16285 D r. H arry Hays -2 - N o v e m b e r 27, 1967 MOa nor is the presence of residues in m e a t or m ilk a regulatory problem. Evidence f r o m feeding studies concerning residues in m e a t and m i l k is included in the petitions being submitted in support of negligible tolerances for no-residue uses. W e have requested a cooperative p r o g r a m with U S D A to resolve the matter of residue tolerances for p henoxy herbicides used on grass, and w h e r e necessary, for m e a t products.' ' W e intend to pursue this by a series of conferences to establish a p r o g r a m that will m e e t the needs and desires of the various agencies concerned with the agricultural use of p henoxy . herbicides, as well as those of the regulatory agencies. A m e e t i n g with Pesticide Regulation Division to further discuss such a p r o g r a m will be scheduled in the near future. Petitions seeking negligible tolerances for uses of 2,4-D, 2,4, 5-T, M C P A , and Silvex presently registered on a no-residue basis (except for range and pasture grasses) are being submitted by Hazleton Laboratories, .Inc. , in behalf of the Industry T a s k Force. T h e s e should be in your hands within the next week. CCD:alc V e r y truly yours, C-'o/ / C. L. Dunn, C h a i r m a n N A C A Industry T a s k F o r c e on Phenoxy Herbicide Tolerances cc: Dr. G e o r g e W . Irving, Administrator Agricultural Research Service U.S.D.A. Washington, D. C. Dr. R. J. Anderson, Deputy Administrator Regulatory and Control P r o g r a m s Agricultural Research Service : U.S.D.A. Washington, D. C. ( \ N Dr. L. L>. Danielson AgOricultural R e s e a r c h Service Beltsville, M a r y l a n d '*<*.miteni: -- il*' >" v. 16286 DOW852471 d e p a r t m e n t o f h e a l t h ,e d u c a t i o n ,a n d w e l f a r e FOOD AND DRUG ADMINISTRATION WASHINGTON. D.C. 30304 l 2 or, Pesticide Petition Ho. 8F0G75 April 18, 1800 Mr. C. L, Dunn, Chairman HACA Industry Task Force on Phenoxy Ilerbicide Tolerances 1155 Fifteenth Street, H.H. Hashington, D.C., 20005 Dear llr, Dunn: i This refers to Pesticide Petition Ho. 8F0675 requesting tolerances for negligible residues of the herbicide silvex at 0.2 ppm on apples, pears, prunes, rice, and sugarcane. He have completed our review of the petition and find that we are unable to establish the tolerances as requested for the reasons listed below. Our conclusions with regard to the residue data are as follows: 1. The metabolism and degradation of silvex and its ester and salt formulations are not adequately delineated. Data are also required to identify and qunntitate the major toxic metabolites which may be present in plants at normal harvestj of particular concern ore conjugates of (y i1 silvex such as those reported by Meagher, HJl., J . A g r . Food Chem. 14, 374-6 (1966). If such metabolites are found to constitute a significant If ^ residue at harvest, an analytical method for their determination will be required. 'y x - V .>1 v,r` /V-'\ ` fcv 2. The methods of analysis outlined in the Pesticide Analytical Manual, Vol. I, 6.11, 6.21, 6.31(A), and 6.33(A), would not be expected to deter mine the metabolites mentioned above. 3. The present residue data are not sufficient to conclude whether the proposed tolerances are adequate to cover all toxic residues, including the metabolites mentioned above. In addition, residue data (including toxic metabolitee if found present) are required (a) for apples reflecting the concentrated spray applicationj (b)-for dried prunesj (c) for rice bran and straw; (d) for sugarcane bagasse and sugarcane products (syrup, molasses, sugar) derived from cane bearing residues of 0.2 ppm. (The present study of residues in refined products does not indicate whether residues wore found on the cane from which the products were derived). The application rate and PHI used in the present residue data for prunes should be furnished. The additional residue data should represent appli cation of the most persistent esters or suits of silvex* If residues are -1 1 (Mr. C. It, Dunn) 2 concentrated in any of the above processed foods, appropriate food addi tive tolerances should be proposed. DOW 8 4. Depending on the level of residues found in livestock feed items (rico bran, rice straw, bagasse, and molasses), we may need additional tissue storage data as a basis for concluding whether or not residues would transfer to meat and milk. We are unable to draw final conclusions with regard to the adequacy of the toxicity data until the necessary information is received. You may amend the petition or you may withdrew it and refile when the necessary Information is available. Otherwise it may be necessary to establish a tolerance of zero for residues of silvex on the crops in the petition. Please let us have your reply within 30 days. C7T j\^ Sincerely yours, co Pesticides Regulation Division a h a - nsDA Triiliam Stokes Petitions Control Branch Bureau of Science 162B8 Cop' ed to: M . L. Leng ITFPHT File National Agricultural Chemicals A ssociation Mr. Drev M. Baker, Jr. 1 Petitions Control Branch Bureau of Science Department of Health, Education, and Welfare Food and Drug Administration Washington, D. C. 2020^ THE MADISON 8UIL0INS f1155 FIFTEENTH STREET. N .W .^ W a s h ingt on, d.c .. 2000s .1 ,2'j { T iltphon; 296*1585 Are Code 202 July 11, 1968 o -, O ^< orNJ O 00. o 03 fo * C/T 05 Dear Mr-. Baker: I am writing in regard to Pesticide Petition Nos. 8FC669 (2,^,5-T); 8FO67O (2,^-0); 8FCo75 (Silvex); and 8FO676 (MCFA). Letters detailing ob jections, primarily concerned with questions concerning metabolic products, were sent to us in April, 19 6 8 . On April 25 representatives of our Task Force met with you, Mr. Cummings, and Mr. Ramsey to discuss these objections. At the meeting we stated that we would prepare an additional section for the petition reviewing the scientific literature with respect to metabolism of phenoxy herbicides and emphasizing the application to residues. Work on that project was started shortly after our meeting, and I wish to report on its status at this time. Publications dealing with the metabolism of phenoxy herbicides in plants and animals as well as recent residue studies have been screened. From this material, about 60 references and H review articles have been selected as being pertinent to this review. Documents have been obtained, examined in detail, and information tabulated. In some cases authors have been written or called for additional information or clarification of their research findings. The writing of various portions is currently in progress, and it-is estimated that the draft will be completed in about two weeks. Revision and reproduction of this somewhat bulky document will require an additional several weeks, and we are aiming at submission of the document by about August 15. DECEIVED * JUL 1 5 1968 ii** l aiOPRODUCTS DEPT. 16289 lx. Drew M. Baker, Jr -2- July 1 1 , 1568 An outline of the section that is being prepared is as follows: Table of Contents Summary Statement Glossary of Terras I. Metabolism in Plants A. Introduction B. Tabulation of Conditions Used in Metabolic Studies in Plants, C. Metabolism of 2,U-D 1. Hydrolysis of Esters and Salts 2. Decarboxylation 3. Conjugation (a) Formation of Water Soluble Unknown 1 (b) Formation of Water Soluble Unknown 3 Transformation to a Different Ether Soluble Compound D. Metabolism of 2 } k , 5 - T and 2,U,5-TB E. Metabolism of MCPA and MCPB F. Metabolism of Silvex (2,^',5-TP) II. Metabolism in Animals III. Recent Residue Studies by Newer Methods IV. Significance of Metabolites as Residues in Foods and Feed Please let me know if there are further questions about our activity in the execution of our commitment to prepare this additional information discussed at our April 25 meeting. Very truly yours, C. L. Dunn, -Chairman NA.CA Industry Task Force on Phenoxy Herbicide Tolerances CLD:mea cc: USDA Pesticides Regulation Division f r c K y. i CHL0RA CNE Dow E x p e r ie n c e 12 0 O CN *S4 00 O ro 29T9 TCBD2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n -- c OH C 2 ',4 ,5 -T R IC H L O R O P H E N O L ~ TCP 1: ; ; ;* -n - CHLORACNE IN HUMANS 1 . L ik e t e e n a g e a c n e , b u t more b l a c k h e a d s . ! 2 . Ap p e a r s f i r s t on f a c e , m ay s p r e a d to t h e b a c k an d OTHER PORTIONS OF THE BODY. 3 . Long- ter m -- 2 to 5 y e a r s to r e c o v e r , w ith or WITHOUT TREATMENT. 4 . Us u a l l y n o t d i s a b l i n g b u t . m a y b e f a t a l . 5 . L iv e r in ju r y -- do cu m en ted in Eu ro pean e x p e r ie n c e . AND R A B B I T T E S T S , NOT D E T E C T E D I N DOW CASES BY L I V E R F U N C T I O N T E S T S , ONE B I O P S Y ON A S E VE R E C A S E . ^3 02 <3 0022 6 . Ma n y o t h e r s y m p t o m s c l a i m e d -- l i s t l e s s n e s s , t i r e MORE E A S I L Y , E TC . 18232 DOW EXPERIENCE - 1964 TO PRESENT --1 9I19A1 MOa 1. 100Ab o u t cases, 2, 60 TCPAb o u t in in c id e n t d esc r ib ed below . 3 . Mo s t o f r e m a i n d e r i n Do w i c i d e s p r o d u c t i o n , 4 . Some c a s e s in la b o r a t o r y r e s e a r c h , 5, Th ree ex tr em ely s e v e r e c a s e s / about f if t e e n s e v e r e . CASES/ ABOUT THREE "A C U T E " CASES/ ABOUT FOUR PEOPLE. LOSTWS, T,W 6 , Mo s t c a s e s a r e m i l d / m a n y w e r e f o u n d i n h e a l t h in v e n t o r y a c t i v i t y b y " s e n s i t i z e d " M.D.'s , ft 1 ^7168 SIGNIFICANT CASES ~ DOW 193A-1936 PDo w i c i d e pro ductio n -- EMPLOYEES, s e v e r e outbreaks among ONa SODIUM TETRACHLOROPHENATE O-CHLOROPHENYLPHENOL 1930,s La b o r a t o r y c a s e f r o m " c h l o r i n a t e d d i p h e n y l e n e o x id e " DIPHENYLENE O X ID E / ALSO DIBENZOFURAN 34 1941 n - D, D. E,I r i s h a n d M ,-A dams P U B L IS H E D PAPER ON R A B B IT EAR T E S T FOR P R ED IC TIN G ACNEGENS! S T I L L THE MOST S E N S IT IV E T E S T A V A IL A B L E . 1 94 0'S 1966 DOW CUSTOMERS CONTRACTED CHLORACNE FROM THEN DOW ICIDE 3 (NOW Do w i c i d e 31 & 32/ h i g h p u r i t y t h a n Do w ic id e 3 ). OH C1 M IX TU R E OF 4 - AND 6 - C H L 0 R 0 - 2 - P H E N Y L PHENOL Pr o d u c t a nd pr o c e s s were c l e a n EX C E P T FOR TA R . I62S5 OU" V67169 DOW 1 767170 1942 Benzene research lab - - one severe TCPCASE FROM REC YCL ING R E S I D U E IiN GLYCOL SO LVE NT, AN E X O T I C MOLECULE WAS I D E N T I F I E D BY I R AND H Y P O T H E S I Z E D AS C U L P R I T , BUT NOT CONVICTED BY THE EVIDENCE, Clx \ c-c OH OH /-- 1950's TO 1960's Dow 6X PRODUCTION " AT LEAST ONE SEV E RE C A S E / SOME M I L D C A S E S . DOW T E R M I N A T E D P R O D U C T I O N BECAUSE I T WAS LOW-VOLUME/ L O W -P R O F IT I T E M / NOT WORTH THE COST OF R E - E N G I N E E R I N G TO PREVENT FURTHER INJURY, Cl. . Active 4-6 Clg Inactive 162S6 1963-1964 C H E M IC A L -P H Y S IC S LAB -- SEVERAL CASES, AT L EAS T ONE S E V E R E , FROM REC YC LIN G CAUSTIC INSOLUBLE O IL IN ALCOHOLIC H Y D R O LY S IS OF TETRACHLOROBENZENE, AT LEAST ONE EXPLO SIO N OCCURRED. 1965 WESTERN D I V I S I O N TORDON PROCESS ~ FOUR M I L D CASES. R A B B IT TESTS F A I L E D , S U G G E S T I N G NEW F A M I L Y OF ACNEGENS I N CHLORINATED PYRIDINE CHEMISTRY. 4 - am i no- 3 , 5 , 6 -t r ic h l o r o p ic o l in ic a c id I62b7 n 1966 DOW C O R N I N G - RESEARCH LAB C H E M I S T TCPH E A T E D FOR RESEARCH PURPOSE U N RE L A T E D TO DOW P R O C E S S E S , I S O L A T E D TCBD AS AN UNKNOWN BUT I N T E R E S T I N G BY" PRODUCTt__BECAME ONE OF OUR BETTER SOURCES CASUAL DISPOSAL OF LAB WASTES RESULTED IN ONE M ILD CASE -- THE JAN ITO R i 16288 1966 FORT SASKATCHEWAN/ DOW CANADA ~ ABOUT TEN CASES RELATED TQ DoW IC ID E PRODUCTION/ PROBABLY DOWICIDE G DRYER AND HIGH TEMPERATURES (WITNESS HAD SEEN AT LEAST THREE FIR E S IN THE DRYER), PLANT WAS CLEAN BY THE TIM E WE GOT THERE TO WIPE TEST? <5 90 i 1966 1968 Un i v e r s i t y o f I owa -- 2 ,3 / 7 ,8 - TETRABROMODIBENZO-P-DI OXIN APPEALS TO TCBD,BE AS POTENT AS AT LEAST ONE SEVERE CASE; D ID NOT RELATE I T TO H IS RESEARCH U N T IL ASKED FOR A SAMPLE AND DESCRIBED THE PROBLEM, 2 ,3 ,7 ,8 - tetr a b r o m o d ib en z o - p - d io x in ONE M ILD CASE RESULTED FROM DICHLOROPHENOL t a r s FROM 3 4 9 B U IL D IN G . THE TAR WAS USED IN A PUMP TESTING SET UP AT 5 6 2 B U IL D IN G , POSSIBLE HAZARD HAD BEEN MENTIONED BUT NOT ' EMPHASIZED TO USERS, V IC T IM DISMANTLED CONTAMINATED PUMPS WITH NO PRECAUTIONS. 16300 1930'S OTHER EXPERIENCE n MANY SEVERE CASES INCLUDING L IV E R INJURY RESULTED FROM CHLORINATED NAPHTHALENES/ THREE CHLORINE EQUIVALENTS OR MORE, /WW 1767175 1950'S FRENCH -- MANY SEVERE CASES, REVISED PROCESS, WERE VERY SE C R E TIV E . USED MOUSE TEST IN PLACE OF R A B B IT / APPEARED TO BE IGNORANT OF MANY IMPORTANT ASPECTS OF PROBLEMS, 1955 1957 1954 GERMAN S E V E R E CA SES INCLUDING SOME SU SP EC TED F A T A L I T I E S , SHUT DOWN. AND RESEARCH ED NEW P R O C ES S , SHOWED AGAIN THAT R A B B IT EAR AND L IV E R ARE MOST SEN SITIV E TESTS. SEN T A REM ARKABLE L E T T E R TO A L L KNOWN TCPPRODUCERS OF DESCRIBIN G PROCESS PARAM ETERS N EC ESSA R Y TO AVOID CHLORACNE HAZARD -- L E T T E R WAS F I L E D AND FORGOTTEN? GERMANS WERE Q U IT E KNOWLEDGEABLE A N D . V ER Y C O O P E R A T IV E . DOW BOUGHT PROCESS IN FO R M A TIO N , c K C 16302 THE ANILINE MOUNTAIN STORY - 199 BUILDING 1946 1952 1957 1962 BATCH PROCESS STA R T UP/ RAN U N T IL 1952. BEGAN CONTINUOUS C O IL REACTOR PROCESS, 10%C . I , O IL AT --------- ^ FO LLIC U LIT IS , SEVERE . ^5 73% 24%EXPERIM EN TED WITH AND C A U S T IC TO R E P U C E F U K E , 10%C , I , O IL AT ----------^ - F O L L I C U L I T I S , NO D IF F E R E N C E IN S E V E R IT Y BETWEEN 24%F U K E PRODUCED AND C A U S T IC PRODUCED O IL S , I7G 7177 16303 -n ~ 1963 JULY NEW EQUIPM ENT STA R T U P / . . PLUGGED L IN E S PROBABLY , PRODUCED MORE EX P O SU R E. NOVEMBER pu sh ed pr o c ess to in c r e a s e PRODUCTION/ R A ISED 195 CTEMPERATURE FROM TO 205-225 C. MADE MORE O IL / BEGAN SAMPLING I T . 81U 9H MOI} 1964 FEBRUARY S U P E R V IS O R S / LAB MAN AND OPERATORS WITH CHLORACNE, PHOENIX S P R IN K L E R MEN MOVED IN . REDUCED EXPOSURE BY EQ U IP MENT AND PROCEDURE CHANGES. DOW J 7 6 7 1 7 9 APRIL 1 APRIL 15 APRIL 20 MAY JULY C . f, O IL AT 0.25% K IL L E D R A B B IT S PH O EN IX MEN MOVED OUT. 0,25%C . I . O IL AT K ILLED R A B B ITS. 1 1 10%C , I . O IL AT ------ =* S E V E R E FO LLIC U LIT IS . PH O EN IX MEN WITH CHLORACNE. n - m id -1964 MANAGEMENT ASKED TWO QUESTIONS OF M EDICAL/ IN D U STRIA L HYGIENE AND S A F E T Y : (1) CAN PLANT BE OPERATED SAFELY AT ALL? (2) WHAT IS.NEEDED TO ENSURE SAFETY? ANSWER TO (1) WAS YES, ANSWER TO ( 2 ) COST MANY $ 1 0 0 / 0 0 0 's AND MUCH MANPOWER, Vi 05 f-- k. 00 o PROCESS CONTINUED 1964-1966 WITH NO NEW CA SES OR A D V ERSE E F F E C T S ON OLD ONES, 1966TWO NEW CASES IN -- D ID NOT FOLLOW RULES, 17 6 7 18 1 DISPOSITION OF 199 BUILDING P R IZ E WINNING SUGGESTION ~ "BU R Y I T AND MAKE A S K I RESORT CALL IT A N IL IN E MOUNTAIN," 1968DISMANTLED -IN -- NO IN J U R IE S , 05 **vl CO to 16308 NEW PLANT -- 804 BUILDING C , I , O IL PRODUCT " H - l l*i 7 ^ O { P s z c r s s 30-50 ppm TCBD cH^ee-S/wj ALWAYS L E S S THAN 4-mt W IPE TESTS CLEAN BOW \ 7 6 7 1 8 3 16309 OOW / 7 6 7 l84 COMPOUNDS KNOWN ACNEGENS -n - ACTIVITY "V ery Ac t i v e " DATA NOT CONCLUSIVE C1l- x UNKNOWN A C T IV IT Y C1l- 2 C l3-6 C18 UNKNOWN A C T I V IT Y SOME V ER Y A C T IV E NOT A C T IV E C l4-6 C l8 SOME A C T IV IT Y NOT A C T IV E C l 3 + SOME A C T I V IT Y 16310 MATERIALS POSSESSING UNEXPLAINED CHLORACNE ACTIVITY OH SOME MIDLAND BATCH ES ARE M ILDLY A C TIV E MANY C A N A D IA N .BA TC H ES ARE M ILDLY R EA CTIVE NaOH DOWICIDE G A LL SLUDGES ARE A C TIV E A LL DECOMPOSITIONS ARE A CTIV E \767185 SOME HAVE A C T IV IT Y -n - COMPOUNDS KNOWN TO BE INACTIVE V 1/3/7/8-TCBD AT LEAST 10"^ LESS A CTIVE than 2,3,7,8-TCBD ` 2/7-DlCHLOROBENZODIOXIN OCTACHLOROBENZODIOXIN DOW T e t r a c h l o r o b e n z e n e DOW 2 A 5 - T C P & T Ac id s 03 -k co C3 DOWICIDE 9 4-C hloro- 2 - c y c lo p en ty l pheno l Ca r b o n T e t r a c h l o r i d e Ac c u s e d in L it e r a t u r e ! 16312 SU9UM0I! I SUMMARY 1 . PLATITU D ES 2, 150* C,AVOID TEM PERATURES GREATER THAN 3. BEWARE OF TARS AND O IL S ''''V , LABORATORY SCA LE IS HAZARDOUS 5, USE R A B B IT T E S T EA RLY 6 , COMMUNICATE -- IN D U S T R IA L H Y G IEN E/ M ED ICAL *'. /T <1 X DC X THE DOW CH EM ICAL CO M PAN Y MIDLAND, Michigan August Id , 1966 C. A. Kighhill 2,4-D Plant 489 Building cc: E. C. Staehllng, Org. Chem. Frcdn., 258 Building P. C. Amstutz, Herbicide Section, 441 Building F. I. Chase, 2,4-D Products, 489 Building B. B. Holder, M.D., Medical Department, 607 Building A. W. Wilson, Safety Department, 401 Building V . K. Rowe, Biochemical Research Laboratory O o vP>* cn on tc cz I would like samples from ycur first run with 2,4,5trichlorophenol in the direct ester process. The animal test will detect the presence of chloracne-producing compounds. Please send me a sample of the final product and any intermediates that you think should be tested. If activity is detected, the intermediate may tell us where the trouble starts. Because of the concern about chlor3cne, I think we should run animal tests on 2,4,5-T esters for a time to prove the lack of hazard, even if the first tests are negative. L. G. Silverstein Biochemical Research Labora toe 1701 Building LGSrsjl 1 o Dow herbicide ce O cc traced ~vj Co ''w .4-. p'.'.!.'M..dM1tn1knihm.wdo6tltoattortMdIwItoehe-loudooiittnavnrvvoponmuia**tcodcaxWumCanreoIoIioon1tr4aoaedhocnhlrmlaothacxmtnoctd9wmptZdo.a,hlyh.>f,peuheeio*t2niwlaau.thm.4ia1.flMt4llg.dlmoa-odiao,daficamP.at.ctu.rlta49t.caoon.aaa5nMrthttmUcCraoiaha.*xd.3daod-rnwtwacoiDodtlcoTohaoutlrcsIhac-dnta.nkddaotnhTnfihhaoorioen-cat.ntouDtDnoopwraobl2entowiitdvdocowtillj-tcaaaptr'ficod,rrdmoofpbdnoimwtattorohivrattnlwepxbul-dwt0ooayvcrc*otrii,dntagoctattamwhohi-lnrca(trwnatbctcnari*haDdaouagcidlodo7ooiogttloooaobJTcuciaomiIToaonttteltoltgnrrpootththajaCacotaCdvaubxiirnTtplanmi1ntuoboltpooaob.tpaoproi9UDoeDimsenlannoInofooladlnaJtoolo*ielieabmmnnovHngurae4iDtaDyontoompiotuadtoD.impiiaenndead-rehllatpao.atd*ydatpmitxDgoHooghDabhn,aodrkllet(*ntotfiioy1deonooeitooBloialirbat-ofhnonty'kg*tsn.ofcoxwgiorEowDetpaoe.ugotttxMa9-c.hittdhtaiwna--optaoT'v0tDPhoTraal.oplMno1r.CuU*oomwmCamu2-livohaoADborT*orrrbooohr.lbwintcDaairdObeb4xoAnyhhort.otbfddka<t.tcahhTpoat3nCtboDoguwecgoomIdiDdaaixd.mCioa-liolantcciclbbtrT4rtoaietIoyhool*ooo*iater"UpptbDiltcoy.odra*htSnvowwoaw.mtoxouaeuoroimduiotaoeciDdaaaomdoTilodo'nao'itao--t.lt-l--rttir-_-"'-.''~fwaw:cITrbpawotwukaCatt'.nRpecnoyCfaDpoMwpnaootnbmMchWJlbgapropiorlnlopeotaagDadyotDiuiaA"UlBdclamlarayemaap.nw-bha*h1ovaiyDAaahnodmurccenplnIaeecyat.mtwngaliwptaionnyDTrt*nnadforUeeua-abloticmtdpwCdonwaedemitt'n,maat.eeont*boo(tmieoCrvrebhlrSaDoeitaghlnpuwokhwcyrtfaet.a.lhoeeaonornrahiaafDdDaeDcnouieoenrnataaime'tflnuoachuiebllahhraeuorletdeta.lyo*rl.ai(yemr*Iuetfspaowgamdatto,xliat2teoewiw*nma,moaorfhuvta'Uarad.arutir9cdOia4bnrtahaaatfcae.barhb9obnt"e-xl1imnl3uor,yefolcaafeyimAntlga9riadchpato-araopmatt7mla,Tiitbw.argetceama1cecfuccreolmaatdh2Da-xaTIaoaoatma*rhlboncypiinpwilooarhCopcayaDoa3lnlnawloaaipio-3coniontw-nMltmubuteDa,pangaIwoyaoepusgSgtpplvxtifwielrctteniDxioablhalcnep.coreudaetlhptaedo*hpdbiirhotdlcfectpihwdyelbrao*nimabanbamanr2oda'mitndgdatpoue,vtobtntelrlel.aidelnte.oooi4ioea.radrrnoonvyHTfremamneettPoffanan3nd^adpfenetCpadctlarntlriait-ab'baoomrrc*mtoloaTr.doyDar,2.ae"ccncDr(ilt-dra.ddeyeema-.eutDahtfla4hr3utittecoaotveehuihxdem.orlv10ocJaaoclcekcextrmlanLeor0t,-I-tnto--.-uf--dK-nTanak.'1e-'.--li.''<,''-:1 .V* C hem ical te rm s d efin ed *v`psAmtJttpp>wpinttsocDUfiethc(pihepcgahc(Ohm1iietsyuvlnaapirHue1oaoeeeerloelhceoDaiwaacUevOec0onmemmbbPepieueuinopvleomlfhyeseshhievx0olooaeloUdgHbnoes.e.amtommetenderfii.*nUoxsx*nio)tschoaxp0)oiueurr-ada,skuouiioo.qiamn0rsosnatKnpkglcwsrm'lirh)onx0le()bwmgSclm,toleat.-ul(ietllh2,rlo--paeatyoiofeibkloedevlehctora--taeisooicui.hXvis*crre*4rrteenierwneicfpbplle*cadhseo.llJrehre.yigds2lsrattt5en.eocic--.iteomehrlsTfuhcJToh(e)e-tttcarxuciogtti-icuTo,.gbtacilerhalqye7atddneohphkaiirahfm*UnoS--re#eft,uAcmrAkiirfeeemsebsortsam--epree-yi.cneraeatoalgdto-urrewsreocheoterTeelbhlnepsanfenemul.sdeceAkuliexeictCoxsil)Tooremenuiteoeoa7ftnc*nta,Tbfhm.iibo(unoxrb.5ltD,orptlleeCosiloroemtcnilhe,oldt(tooMnpeyo-stteetph.pbharDtapmedUotuerDeeh(dapiuoeenpwhctsetsppes-otedmaV,ebymogdDdkareesdreceirhmeh-earxe^toccioatnaeetoaho'iotmsifsiotcfg-a--fr)wlrUeadto>perdiaexmfa)eiunoxretr,nnoaxh.ccinlhtiosiefnmccl-&obmhtm'eelfisradeeae(htncct1e.ostyttmuaeWueesefdor.tmloeioootTstoctarntl<.ctferehdrlosoweeiutadmffguxoxuuoioehitlpnieprAcffehcenanrahoraareotdxeceiixrongacrttlteaotrhmrLlyihI)oots*ioersfa-etec>--*,fe.-.l.-*.f..'.'-.-*'./owoMaewatcstsmOstO'mmabctccOedtafhathsrhhhioounauroahcreoaohmtcherpJoieohfiTedeToeIeeeahaermPouhrersedaxe.raelxueeaiom7eomerrtlanlisf.haotmrryeefmehcycbseenypchocJpsrtxrhHert,a1amabacoiFaami*des,nuatU.ibpiiniiefiaureccxoTo4ctockcoohy-iulotbhmcmhleynpestaeoekIroecaG-neoaetflfnseeiilrDeahxncclapd-ragaeaxleaometl--crmuaduLrl.vffDreycbdichkyqbd.tthlnba,oeocifs.e--tiiaotaopettoiaeiphucyiDwmoeepehooyuhnncwrtwabooOmnxaoTcasapisrbomavexidiiceh.duldbcauecohdesiOsl.naha1litccoaeiamsoeinitaeetaiiuiirrdeerr:nntgitodecoanTiolb1ssyirstoudtes)adehoe,ctigyahndlse,etgahsnwpnh.Titd--ah'oaMnorectfe't*aAceertcaptseshncmemyaubeahteranroioJtibwtobeUdedieaeealhsghbTsle(trtsrfonJpmefalttsumseuflgveteeeh.ihohc*witteaaesrdfwa1hbetstqchofrsewttnkratedorfirheu.Wclpesentepeueeime4etsahdwdqosdptuToh'atmtt-tshadrm.rla,atascmaAttbtdtueisirnuerrffhnahbi^t,otlmafeobvhrateeei-lloioeetabenecshideegTtooicciidtyhkslfnqrflnmxohtsogxprer,fierpesotthaen~ieftgncliu2nyemareecraoc.oemwsigsselonco3msegaao-,engstwnvlPrsxeppltogedtpcmfltvaoriuwtcrsoilfetorpltbhdislaieehabioeanhgfnfruceWiedloeopsrtuoore--eoeiatedooenredesepshayl>sxl^rir*-*-**rr-,t;r'4.;-.%s. l * Q o'->Cb 9<;6TZ0NW OCCUPATIONAL DISEASES y d iZ t U U * --- t f - Y f A Guide To Their Recognition Rewritten and Enlarged Edition of Occupation Hazards and Diagnostic Signs W. M. GAFAFER, D. Sc., Editor Division of Occupational Health C.S. DEPARTMENT OF HEALTH, EDI CATION, AND WELFARE Puliiic Health Service- 2fi occur/ 'NAL DISEASES may hr damaged by maceration, mechanical or chemical trauma, and certain internal diseases. Lipid-soluhle and nonionizahle (organic) substances are able to bypass the harrier layer and enter by way of the transfollicular path way. Gases, except for carbon monoxide, are readily exchanged through the epidermis. Protection against actinic stimuli is afforded by increased melanin pro duction and a concomitant thickening of the keratin layer. An antimicrobial action is attributed to certain fatty acids in the surface lipid film, and the 6uhcornc.al harrier layer also offers some protection against microorganisms. Primary Irritants Primary irritation causes most of the cases of contact dermatitis in in dustry. A primary irritant is an agent which will cause dermatitis hy direct action on the normal skin at the site of contact if it is permitted to act in sufficient intensity or quantity for a sufficient length of time. Thus, the normal skin will almost always react to a primary irritant if the necessary conditions are present. Irritants can act in several ways to injure the skin. Several examples have already been given, such as removal of lipid him, denaturation of keratin, and interference with Ihe subcorneal harrier layer. Other examples o f pri mary irritation include dehydration hy inorganic acids, anhydrides, and alkalis; protein precipitation hy heavy metal salts and tanning agents; and oxidation by blenches, chlorine compounds, and per- salts. Sensitizers Ordinarily, no more than 20 percent of contact dermatitis in industry is caused hy allergens. However, certain of the strong sensitizers may affect main workers. Almost any chemical can be a sensitizer, hut the mode of action usually differs from that exhibited by primary irritants. The cutaneous sensitizer, therefore, does not necessarily cause demonstrable cutaneous change on first contact, hut may effect such specific changes in the skin so that after 5 to 7 days or more, further contact on 'he same or other parts of the body may cause dermatitis. The difference between the irritant and the sensitizer, therefore, is generally a matter of time as well as mode of action. The irritant will usually act within a matter of minutes to a few hours, whereas the sensitizer requires at least $ to 7 days because sensitization must build up during the period of incubation. Low-grade irritants such as soap may require prolonged or repealed contact before a dermatitis appears, and this prolonged reaction ime may he confused with allergic incubation. secondary Effects After the cutaneous defenses have been broken down and a contact dernnlilis is present, the oozing or fissured surface offers ideal conditions for he entrjm6e and growth of bacteria. Thus, it is not unusual for a contact 03 CO OCCUPATIONAL DERMATOSES 29 dermatitis to lie secondarily infected. Another secondary effect commonly seen in contact dermatitis is over-treatment hy irritating or sensitizing medication. Diagnosis Diagnosing an occupational dermatosis is generally contingent upon satis fying certain criteria. The disease should look like a contact dermatitis or one of the other clinical types of occupational dermatoses. It should be located on sites of exposure, and the time of nppearance of the eruption as well as periods of remisson and exacerbation should correlate with the history of work exposures. When the patient does not pet well following complete withdrawal from the suspected contact agent, stimuli of nonoccupational origin should be reinvestigated. Multiple factors not associated with the occupation can perpetuate a chronic dermatosis. Patch tests are helpful in differentiating between a primary irritation dermatitis and an allergic contact dermatitis. A nonirritating concentra tion of the suspected allergen is applied to the normal skin of a patient for 24 to 48 hours in an attempt to reproduce an eczematous dermatitis beneath the covering patch. Classification of Lesions The clinical lesions seen among occupational exposures are multiple, varying from the mildest erythema to lesions of neoplastic nature. How ever. occupational skin disease can be classified as follows: (1) Acute contact eczematous dermatitis characterized by erythema, edema, papules, vesicles, or bullae, crusts and finally desquamation. These effects are generally the result of contact with either a primary irritant or a sensitizer, or with both. (2) Chronic contact eczematous dermatitis characterized by erythema, lichenificalion, and Assuring of the skin, usually resulting from contact with dehydrators, fat solvents, soaps, and detergents. (3) Folliculitis and acneform types, including chloracne, characterized by plugged sebaceous follicles and suppurative lesions. Chloracne also shows numerous straw-colored cystic lesions. These forms of occupational derma toses are usually caused by contact with oils, tars, waxes, and certain chlorinated hydrocarbons. (4) Neoplastic ( benign and malignant! types, characterized by keratoses, papillomata and epitheliomata of the exposed areas, usually caused by petro leum products, coal tar and certain derivatives, sunlight, and ionizing radiation. (5) Pigmentary disturbances characterized by increase or decrease of pig ment in the epidermis. Increased pigmentation can result from contact with ro CO cn CHEMICAL HAZARDS 117 o-Dichlorobenzene workers Dry cleaners Dye makers Fumigant workers Greasemakers Gum makers Heat transfer workers Hide processors Insecticide workers Lacquerers Lacquer workers Metal degreasers Metal polish makers Organic chemical synthesizers Paint remover workers Paint workers Polishing compound makers Resin makers Rubber makers Solvent workers Stainers Stain makers Sulfur processors Tannery workers Tar makers Tar remover workers Termite exterminator workers Varnish makers Varnish remover workers Wax makers Wood preservative workers Wool processors p-Dichlorobenzcne Deodorant makers p-Dichlorohenzene workers Disinfectant workers Drug makers Dye makers Insecticide workers Moth hall makers Soil fumigalors T nchlorobcnzenvs Dye makers Electric equipment makers Heat transfer workers Insecticide workers Lubricant makers Trchlorobenzene workers Hcxachlorobenzcnc Fungicide, workers Ifexach loro benzene Workers Organic chemical synthesizers Seed disinfectors References i i o l l i s c m v o r t i i , ii. L.: o w e . v. k .; ovf.n , E.; HOYLE, II. It.. AND Sl'ENCEII, II. C. : Toxicity of pur.iclirliiorulirM/L-ne; <jri<Tniin;in* lib <>n cxjH 'fiinriil.il ii iu naii a n d ti n m an auhjrdb. A .M .A . A rch. Indust. H ealth I T. Kill, I95d. IH ll.LI NLSWi >HT11, It. L . ; HttWE, V. K . ; OYKN. r . ; ToitKJ-.i.bu's, T. i:., a m i a iia u >, k. m .: Toxicity of o-fliclilorobcnzenc; studies on uni m a is a n d in du>tria l <*Nprriri *. Arch. httluu. Health 17: 180, 1958. (48) Chlorinated Diphenyls anil Naphthalenes Harmful Effects Local Prolonged contact with fume or the cold wax leads on exposed skin to comedones, sebaceous cysts and pustules, know n as cldoracne. Routes of Entry Inhalation of fume or vapor; percutaneous absorption of liquid. System ic Acute or chronic exposure can produce varying degrees of liver damage depending on amount of chlorine in compound and preexisting <=3 ro CO CJ1 U cn i . i*2cL -11'7\ '****: .-o-* `-S'- 4. . 118 OCCUPATIONAL DISEASES state of liver. Symptoms include jaundice, anorexia, nausea, indigestion, abdominal pains, and edema. Death from acute yellow atrophy of liver has occurred. Special Diagnostic Test None. Recommended Threshold Limit Chlorodiphenyl (42 percent chlorine), 1 milligram per cubic meter ofair. Should be reduced when also absorbed percutaneous!)'. Chlumdiphen-d ( 5 1 percent chlorine), 0.5 milligram per cubic meter of air. Should be reduced when also absorbed percutaneous!)'. Chlorinated diphenyl oxide, 0.5 milligram per cubic meter uf air. Pcntachloronuphlhalene, 0.5 milligram per cubic meter of air. Should be reduced when also absorbed percutaneously. Trichloronaphlhalene, 5 milligrams per cubic meter of air. Should be re duced when also absorbed percutaneously. Potential Occupational Exposures Aniline dye makers CaLle coalers Carbon removers Chlorinated diphenyl workers Condenser impregnators Crankcase oil additive makers Dye makers Electric equipment makers Electricians Electroplaters Flameproofers Gutn processors Heal transfer workers Herbicide workers Ink makers Insecticide workers Insect proofers Lacquerers Lacquer makers Eight fixture makers .Machinists Metal degreacrs Mineral oil processors Moisture proofers Paint makers Paper treaters Petroleum refinery workers Plasticizer makers Plastic makers Rayon makers Resin makers Rubber workers Solvent workers Stainers Stain makers Textile flameproofers Transformer workers Upper cylinder oil makers Varnish makers Vegetable oil processors Wax makers Wire coalers Wood preserves Reference MF.JCS, J . W . ; A U iO M , J. ASO KARTIN, to arocldor. J. Am. ited . Assoc. IS4 .U 1 7 , a. L.: i95-i Chloracnc from an uniiMial Axposur T Harmful Effects (- CSZ Local Extreme irritation of sion of teeth. Route of Entry Inhalation of Systemic Acute respiratory pain, dyspnea, and cyanosis, nia, and pulmonary edema ma Special Diagnostic Test None. CO Cl Recommended Threshold . C 1 part per million parts of a of air. Potential Occupational Ex Aerosol propellant makers Alkali salt makers Aluminum purifiers Benzene hexachloride maker Bleachers Bleaching powder makers Bromine makers Broom makers Carpet makers Chemical synthesizers Chloride of lime makers Chlorinated solvent makers Chlorine workers Color makers DDT makers Disinfectant makers ) Dye makers j Ethylene glycol makers i Ethylene oxide makers ii \ Hour bleachers 'Freon makers Casoline additive workers t> Cold extractors Ink makers j Iodine makers j Iron detinners Iron dezinkers 1 H crhicutcx H "bicides, or weed killer;, may lie classified as pesticide chemicals. They cfl; .1 plants on contact, or they can be translocated; that is, absorbed by one part of the plant and carried to other parts where they exert their primary toxic effect. Most of the commonly used herbicides--ammonium sulfamate, dalapon, phenoxyacctic acid derivatives, carbamate derivatives, petroleum oils, sodium borate, Crag'1 herbicide-- have a low toxicity and have caused little difficulty among users. Some herbicides pose more serious problems; for example, the central nervous system effects of maleic hydrazide or the methemoglobinemia and central nervous system depression of sodium chlorate. I'entachloropbcnol, a metabolic stimulant, has been responsible for several deaths because of hyperthermia. Amino triazol has produced cancer in experimental animals, but there have been no untoward effects reported in man. Herbicides with cutaneous effects include trichloroacetic acid, a corrosive irritant of the skin aud mucous membranes; maleic hydrazide, a producer of allergic contact dermatitis; pentachlorophenol, a producer of a primary irritant type of contact dermatitis; and creosote, a primary irritant and pholosensitizer. Reference may be made to chemicals in '.he section on Chemical Hazards for the toxicity of the following herbicides: Arsenic trioxide and sodium arsenate (see Arsenic), copper sulfate fsee Copper and Compounds), creosote compounds (see Cresol and Phenol), dinitrophenols (see Dinilrophenol), keresine. and phen\Irnercuric acetate (see Mercury and Compounds). Threshold limit values, in milligrams per cubic meter of air, have been recommended for the following herbicides as shown. Ammonium sulfainate (Animate")____ 1ri mg per cu. m. Crag" herbicide____________________ 15 mg. per cu. m. 2,41) (2,4-dichlorophenoxyacetic acid) _ 10 mg. per cu. m. Peniachlorophennl ( P C P )____________ 0.5 mg. per cu. m.* Phenylmercuric acetate fP M A )_______ 0.01 mg. per cu. m.* ffor organic mercury) 2,4.5-T (2,4,5-trichlornphenoxyacetic 10 mg. per cu. m. (tentative) acid). 'Should be reduced when alsoabsorbed percutaneously. KCT4i C*3 EOO Fumigants Fumigants are pesticides which may be applied in the solid, liquid, or gaseous state. A combination of high volatility with high pest toxicity is generally desired; however, compounds with low volatility may he preferred for soil fumigation. The possibility of excessive exposures exists wherever houses. bums. ships, mills, freiy111cars, aiul greenhouses. Each of tlic following coin|ioinuls lias found use as a fum._ .ii. Ueca.iist- they have oilier industrial a|>|>l1 .11i>.ns ns well, they are discussed individually in the section on Chemical Ila/anls. Acrvlonilrile Carhon Disulfide Carbon Tetrachloride p-Dichlorobcnzene (see nated Benzenes) Dioxane Ethylene Dihromide Ethylene Dicliloride Ethylene Oxide Hydrogen Cyanide Chlori Methyl Bromide (see Bromine and Compounds) Methylene Chloride Methyl Formate Naphthalene Perchloroethylene Propylene Dichloride Sulfur Dioxide Tetrachloroethane T ricldoroethylene References ARTERBERnY, J.D.;DIRHAM, w. r.;ELLIOTT, J. W.. AND WOLFE, H. R.: Exposure 111 parathion. .Measurement by blood clinIino-trase level ami urinary p-nitrophcnnl excrribiii. A r c h . E m i r o n . H e a lth 3: 476. 1061. ASSOCIATION OF AMERICAN I'll I1(11)1 CONTROL OFFICIALS: P e s t i c i d e C h e m i n t s Oj- ficiat C o m p e n d iu m . A. I). Jlragy. Cnieersity Post Office, College Park, Mil., 1962. boose-leaf senire. c o m m ittee on PESTICIDES: Outlines<1/information on pesticides. Pari 1. Agricultural fungicide*. J. A m . M e d . A s s o i .157:237. 1955. c om m ittee on toxico lo cy: Ocriipulinnal dii-ldriii poisoning. J. A m . M e d . A ssoc . 172: 2077. I960. C*MMi' nicaf;lf. disease CENTER. l` i ill.ic h eal th ser vic e: Clinical memoranda on economic poisons. Pub. Health Service Pub. No. 476. U.S. Government Printing Office, Washington, D C.,1956. division of hiolocy and ACniciT.Tiair, n at io na l research co u nc il - n i t i o n i l a ca de my OE SCIENCES: H a n d b o o k of T oxicolo gy. Vol. 3, Insecticides. Vol. 5, Fungicides. W. It. Saunders Co., Philadelphia, 1959. Du rham , w. r.: c a n e s , t. B., AND HAVES, w. J. JR.: Paralylic and relaied effects of certain organic phosphorus compounds. A . 'I . A . A rch . In d u st. H e a lth 13: 326, 1956. d i r h a m , w f . and h a y e s ., w. j ., j li.: Organic phosphorus poisoning ami its therapy; ivilh special reference lo modes of action and compounds lhat reactvale inhibited cholinesterase. A r c h . E n v iro n . H ealth 5: 21, 1962. FREAII, D. E. II. ( editor) : P esticid e I n d e x . College Science Publishers, Slate College, Pa., 1961. C aines . T. H.: The acute toxicity of pesticides lo rals. T o x ic a l. A p p l . P h a r m a c o l . 2: Rfl, I960. enu, li. 11.aNil SHAFFER, c. IL: T o xicological I n jo r m a tio n on C y nn a rn id t In se c tic id e s . American Cyanamiil Co.. New 5urk, I`*o0. h a y e s , w. j., jii.: The toxicity of duddrin lo man; report on a survey. H ull. If H O 20: H91. 1959. HAYES, w. j., jn.: Pesticides in relation lo public health. A n n . R ev. Entorno!. 5: 379. 960. 2 ih-ooy o - o n - 1 ' LVSCSI VWI I 0OW 537307 Section C DOW 53 7308 Petition for the Establishment of Tolerances for the Pesticide Chemical, 2,4,5-Trichlorophenoxyacetic Acid on Raw Agricultural Commodities SECTION C Full Reports of Investigations Made with Respect to the Safety of the Pesticide Chemical, 2,4,5-Trichlorphenoxyacetic Acid December 8 , 1967 * .Tv Acute Studies DOW537309 Oral Administration - Rats, Mice, Guinea Pigs, and Chicks The acute oral LD50 of 2,1+,5-trichlorophenoxyacetic acid for young adult male and female albino rats, mice, and guinea pigs and for young chicks, as reported by Rowe, et al. (l), was as follows: Species U)50 Confidence Limits (mg/kg) Rats 500 391-6UO Mice Guinea Pigs 389 381 2^5-619 307-^72 Chicks 310 211-1*56 Slope 1.1*9 1.8 0 1.2 1 1 .1+0 The material was administered as a 3*0 or 10$ suspension in olive oil. Gross toxic signs consisted of ataxia and myotonia, particu larly at the higher doses. Oral Administration - Dogs According to Drill and Hiratzka (2 ), the acute oral LD50 ? 2 ,1+,5 -trichlorophenoxyacetic acid for adult mongrel dogs is probably in the range of, but slightly higher than, 10 0 mg/kg of body weight. Only 25 % mortality occurred "t this level. Gross toxic signs consisted of anorexia, body weight loss, slight or moderate stiffness in the frinid' 1 cfc' legs, and ataxia. Histologic changes included some necrosis and inflammation of the intestinal mucosa, moderate diffuse hepatic necrosis, and a mild degree of renal tubular degeneration. C2 Acute Oral Administration - Rats, Mice. Ouinea Pips, Rabbits, Chicks, and Steers Rowe and Hymas (3 ) reported the acute toxicity of various for mulations of 2,*+,5-trichlorophenoxyacetic acid and mixtures of 2,*+dichlorophenoxyacetic acid and 2,*+,5-T. The reported LD^ values and/or range of toxicity are as follows: DOW 537310 Esteron 2*+5 (old formulation containing 33-3$ isopropyl ester of 2,*+,5-T and 12.1$ mixed amyl esters of 2,*+,5-T) Adult female albino rats - Estimated LD,_q of 1000 rag/kg of body weight (range 300-2000 mg/kg). Esteron 2*+5 (new formulation containing 65-3$ mono-, di-, tripropylene glycol butyl ether esters of 2.,*+,5-T) Adult male albino rats - Estimated LD^q 800 mg/kg of body weight (range 600-1000 mg/kg). Isopropyl ester of 2,*+,5-T Male and female albino rats - LD^q *+95 mg/kg of body weight (confidence limits *+20 -58*+ mg/kg). Male albino mice - LDcjq 551 mg/kg of body weight (confidence limits 380-799 mg/kg) Male guinea pigs - LD^q *^*9 mg/kg of body weight (confidence limits 38 2 -5 5 7 "mg/kg). , Mixed Butyl esters of 2,*+,5-T " :i , 'V *'*. ' .' Male rats - LD,_o *+8l mg/kg of body weight (confidence limit 3 313-739 mg/kg). C3 Male rabbits - Estimated IDso 712 mg/kg of body weight (range 500-1000 mg/kg). Male mice - ID50 9^0 mg/kg of body weight (confidence limits 67^-1312 mg/kg). Guinea Pigs - Estimated LD50 750 mg/kg of body weight (range 500-1000 mg/kg). Mixed Amyl ester of 2 , k , ^ - T Female rats - Estimated LD^q 750 mg/kg of body weight (range 500-1000 mg/kg). Brush Killer T (52.2$ butyl esters of 2 , b , 5 ~ T ) Female rats - LD^q 1200 mg/kg of body weight (confidence limits 7 8 3 -I85O mg/kg). Female guinea pigs - LD50 1^10 mg/kg of body weight (confidence limits 875-2290 mg/kg). Female mice - LD50 12 3 0 mg/kg of body weight (confidence limits 9 38 -16 20 mg/kg). Male rabbits - LD50 8^9 mg/kg of body weight (confidente limits 604-1190 mg/l:g). Chicks - LD50 2000 mg/kg of body weight (confidence limits 1350 -29 0 0 mg/kg). Brush Killer 50-50 (formulation containing 27-2$ butyl esters of 2,1-D and 26. 5$ butyl esters of 2 ,^,5-T). ,. .; .$ , 'y Female rats - ID50 1070 mg/kg of body weight (confidence limits 70 0 -16 50 mg/kg). DOW 537311 1 '6325 OOW 537312 cu Female guinea pigs - LD^q 1160 mg/kg of body weight (confidence limits 8 2 0 -16 3 0 mg/kg). Female mice - LDcq COO mg/kg of body weight (confidence limits 6 2^ -1070 mg/kg). Female rabbits - Estimated LD^q 1^20 mg/kg of body weight (range 500-2000 mg/kg). Chicks - ID50 ^000 mg/kg of body weight (confidence limits 2700-5900 mg/kg). Esteron Brush Killer (old formulation containing 25.6$ isopropyl ester of 2,k-V and 2b.k% isopropyl esters of 2,b,5-T) fiale rats - Estimated LD^q 1000 mg/kg of body weight (range 3OO-3OOO mg/kg). Esteron Brush Killer (new formulation containing mono-, di-, and tripropylene glycol butyl ether esters of 2,^-0 3^*8% and 2,i+,5~T 33-0%) Male and female rats - 860 mg/kg of body weight (confidence limits 800-930 mg/kg). Male guinea pigs - LD^q 1220 mg/kg of body weight (confidence limits 1040-1^30 mg/kg). Female guinea pigs - LD^q l600 mg/kg of body weight (confidence ' limits 13 9 0 -18^0 mg/kg). Male and female rabbits - LD^q 90 mg/kg of body weight (confidence limits 790"ll60 mg/kg). Chicks - Estimated LD n 2000 mg/kg of body weight (range 1000- r* *.i- 3000 mg/kg). vf 4 , , Steers - Acute oral LD^q greater than 1000 mg/kg of body weight. 16326 Eye A p p lic atio n - R abbits According tc a report by the Dow Chemical Company (k), a 10% solution of 2,U,5-T in propylene glycol produced severe conjunctivitis and moderate corneal damage which persisted for longer than five days. Acute Oral Administration - Rats; Acute Dermal and Eye Application-Rabbits The results of acute studies on Nu Amine D/T, a formulation containing 1,3-propylene diamine salts of 2,b-D (ll.95%) and 2,L,5-T (11.85%) was reported to Diamond Alkali Company February 23, 1962 by Hazleton Laboratories, Inc. (5 ). The acute oral LDcq of Nu Amine D/T for adult male albino rats is 708/^/kg of body weight with confidence limits from l+li7 "1 1 2 0 iA->'/kg. Deaths occurred within 1 to lU days, and toxic signs were mainly characterized by depression, weight loss, labored respiration, ataxia, abnormal gait, circling movements, depressed or absent reflexes, and hematuria. A single application of 0.05 ml. of Nu Amine D/T to the eyes of albino rabbits produced marked irritation, in eliding corneal damage, which did not completely subside within seven days. However, no evidence of systemic toxicity from mucous membrane absorption of the test material was observed. The estimated acute dermal LD50 undiluted Nu Amine D/T for albino rabbits is 1.2 ml/kg of body weight. A corrosive-type skin reaction ' .^ was produced at dosage levels of 0 .1 , 0 .5 > and 2 .0 ml/kg of body- j/piglvtc'' ' iAt 2.0 ml/kg, toxic signs included depression, labored respiration.,' depressed righting and placement reflexes, incoordination of hindquarters, abnormal gait, prostration, sluggish pupillary response to light, absent 5373 l3 163 A DOW 537314 pain response in hind limbs, running movement oi'hind limbs, and coma. Necropsies revealed definite liver and kidney changes at the 2.0 ml/kg level and possible liver and/or kidney damage at the two lower levels. 'dubacute Studies Oral Administration - Dogs According to Drill and Hiratzka (2) oral ingestion of 2,^,5-T by adult male and female mongrel dogs for 1 3 weeks at levels of 2 .0 , 5 .0 , and 10 mg/kg of body weight produced no adverse effects. At a dosage level of 20 rag/kg, total mortality occurred between days 1 1 and 7 5 , and gross toxic signs included weight loss, weakness, slight hind leg stiff ness, difficulty in swallowing food, and bleeding from the gums. Dietary Feeding - Rats The results of a 9-day feeding study involving Dovarol 97B (ester of 2,b,5-l) was reported by Biochemical Research Laboratory of (\ the Dow Chemical Company (&). v. Ingestion of Dowanol 97B by young male and female albino rats for 90 days produced no adverse effects at dietary levels of 100 and 300 ppm. At 1000 ppm, increased kidney weights were noted in the male animals and histologic alterations were noted in the livers of both sexes and in"the kidneys of the females. At 3000 ppm, growth suppression and increased' v liver and kidney weights were noted only in the male rats, while gross and microscopic changes in the livers and kidneys and increased terminal serum alkaline-phospbatase values were noted in botli male and female animals. 1 C7 Oral Administration - Steers Repeated oral administration of Esreron Brush Killer (formulation of ester of 2,^-D and 2,14,5-1) to steers, one per level, was also reported by Rowe and Hymas (3)- Results of these studies were as follows: DOW 537315 1. Fifteen (15) consecutive daily doses of Esteron Brush Killer at a level of 100 mg/kg/day produced no gross toxic effects. How ever, necropsy performed hours after the last dose revealed some intestinal irritation and histologic alterations were noted in the liver and kidneys. 2. Two (2) consecutive daily doses at 500 mg/kg/day produced slight toxic signs 'k hours after the last dose but apparent recovery within an additional 2k hours. 3. Three (3 ) consecutive daily doses at 500 mg/kg/day produced no gross toxic signs. 1*. Three (3 ) consecutive daily doses at 1000 mg/kg/day produced death (three days after the last dose). Consequently, assuming field usage at two quarts/acre, a staer grazing on sq. ft. of treated forage would consume a maximum of 100 mg/kg of the formulation which, as shown above, was not lethal 'after 15 daily doses. -.i, Oral Administration - Sheep and Cattle Palmer and Radeleff (7 ) reported the following results of re peated oral studies in sheep and cattle (one animal per level). I C8 The propylene glycol butyl ether ester of 2,^,5-T was lethal to sheep following 369 consecutive doses at a daily dosage level of 100 ing/kg of body weight and to sheep and cattle following seven consecutive doses at a daily dosage level of 250 mg/kg. The triethylamine salt of 2,4,5-T produced no toxic effects in one sheep after 48l daily doses at a dosage level of 100 mg/kg of body weight. Repeated Dermal Applications - Rabbits The results of repeated dermal applications of Nu Amine D/T was also contained in the report to Diamond Alkali Company (5), and a supplemental report (5a) dated December l6, 1965 presented findings from microscopic examination of bone marrow from three animals. Aqueous emulsions of Nu Amine - D/T at field strength (1+ oz/gal. or 3*1 $) and at three (12 oz/gal. or 9*M O and five (20 oz/gal. or I5.61/) times field strength were applied at a constant daily dose of 1 .0 ml/kg/ animal five days per week for 20 applications to intact abdominal skin. Moderate dermal irritation and evidence of systemic toxicity from percutanious absorption of the test material were produced at all con centrations. Toxicity was characterized by depression, labored respiration, sprawling of the limbs, weakness, and body weight loss; prostration, incoordination or partial loss of use of hind limbs, depressed reflexes, clonic convulsions, and death was also noted in one animal at each concentration. ,, .4'n." ,,. ' Depressed hemoglobin values, increase^.percentage of segmented neutrophils, and increased urinary bilirubin values were noted in each test group. Histologically, evidence of compound effect was noted in DOW 537316 16331 C9 liver, kidney, and skin at field strength and at five times field strength (middle level tissues not examined). No distinctive bone marrow effect was noted at three and five field strength. OOW 537317 Fish and Marine Life Toxicity Toxicity studies on 2,4,5-trichlorophenoxyacetic acid and its various derivatives in fish and other marine species have been performed by The Bureau of Commercial Fisheries and reported in Fish and Wildlife Service Circular No. Ic7> No. 199, and No. 226. Data pertaining to 2,4,5-T has been excerpted and is summarized below. Oysters - 0.l4 ppm of the polyglycol butyl ether ester of 2,4,5-T caused 50$ decrease in oyster shell growth (96- hour EC50) with re covery within one week (8); 2.0 ppm of the acid of 2,4,5-T produced no toxic effect (9). Brown Shrimp - 1 . 0 ppm of the polyglycol butyl ether ester of 2,4,5 -T produced 10 $ mortality after 24-hours exposure and 20$ mortality after V 48-hours exposure, and 1.0 ppm of the acid of 2,4,5-T produced no effect after 48-hours exposure (1 0 ). Fish - 50 .0 ppm of the acid of 2,4,5-T produced no effect in Longnose ( Killifish after 48-hours exposure (9); 1-0 PPra f the pcjlyglypol butyl ether ester of 2,4,5-T produced 0.32$ mortality in Spots after 2^- and 48-hour e x p o su re (1 0 ). ) OOW 5 3 7 3 1 8 Cio The potency ratio of 2,J|,5-T derivatives for Eluegill relative to that of DOT was quoted in Table 2 (11) as 0.10. Although no specific studies or mortality data on only 2,^,5-T for wildlife were found in the above Fish and Wildlife Service Circulars, the potency ratio of 2,U,5-T derivatives for Bobwhite Quail, Ring-Necked Pheasants, and Mallard Ducks relative to that of DDT was quoted in Table 2 (ll) as approximately 0.50. 1633 DOW 5 3 7 3 1 9 Conclusion Cll Although considerably more toxicity data, partiqularly regard ing subacute toxicity is available on 2,4-D than on 2,4,5-T, the available toxicity data indicates that the two materials are comparable. The acute oral toxicity of 2,4-D and 2,4,5-T and their various derivatives for labora tory animals is in the same range, 300-1000 mg/kg of body weight. Short-term (90-day) dietary feeding of esters of 2,4,5-T to rats at 100 and 300 ppm level, and short-term (13-weeks) oral administration of the acid of 2,4,5-T to dogs at level of 2.0, 5-0, and 10 rag/kg of body weight produced no adverse effects. Sheep tolerated 48l doses of the salts of both 2,4-D and 2,4,5-T at a dosage level of 100 mg/kg of body weight, while the ester of 2,4,5-T was lethal to both sheep and cattle at levels of 2,4-D which were only slightly toxic or non-toxic. However, 389 doses of the ester of 2,4,5-T at a level of 100 mg/kg of body weight were administered to one sheep before death occurred. The toxicity of 2,4-D and 2,4,5-T for fish and wildlife appears to be comparable. Particularly, since the long history of usefulness has not produced evidence of unusual toxicity. 1833 C12 .....| BIBLIOGRAPHY 1. Rowe, V. K. , McCollister, D. D., and Spencer, K. C., The Acute Oral Toxicity of 2,*+,5-Trichlorophenoxyacetic Acid to Rats, Mice, Guinea Pigs, and Chicks; Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan, 1950. 2 . Drill, V. A. and Hiratzka, T., Toxicity of 2 , *4-Dichlorophenoxyacetic Acid and 2,*+,5-Trichlorophenoxyacetic Acid, A Report on Their Acute and Chronic Toxicity in Dogs; Industrial Hygiene and Occupational Medicine, 7:61-67, 1953* 3- Rowe, V. K. and Hymas, T. A . , Summary of Toxicological Information on 2,*+-D and 2,*t,5-T Type Herbicides and an Evaluation of the Hazards to Livestock Associated with Their Use; American Journal Vet. Res. 15: 622-629, 195^ *+. Results of Toxicological Tests on 2, k , 5-Trichlorophenoxyacetic Acid, Reported by Biochemical Research Department, The Dow Chemical Company, Midland, Michigan, August, 1950. 5- Toxicity Tests on Nu Amine D/T (l,3-propylene diamine esters of 2,*+-D and 2,^,5-T; Report to Diamond Alkali Company dated February, 1962 by Hazleton Laboratories, Inc. 5a. Microscopic examination of bone marrow sections from the above repeated dermal study, Report to Diamond Alkali Company, dated December lb, 1965 from Hazleton Laboratories, Inc. 6 . Results of 90-day Dietary Feeding Studies of Dowanol 97B (Ester of 2,^,5-T) in Rats, Report by Biochemical Research Laboratory, The Dow Chemical Company, dated November 27> 1961. ,7. Palmer, J. S. and Radeleff, R. D . , The Toxicologic Effects of Certain Fungicides and Herbicides on Sheep and Cattle, Annals 01 N. Y. Acad, of Sc. Bol. Ill, (2 ); 729-732, 196*4. 8. Butler, P. A . ; Commercial Fishery Investigations, Acute and Chronic Toxicity Studies, U. S. Department of the Interior, Fish anu Wildlife Service Circular 199, 5-8 and 2b. 196*). 9- Butler, P. A . ; Commercial Fishery Investigations, Laboratory Studies and Toxicology, U. S. Department of the Interior, Fishapd?i:- v Wildlife Service Circular 17, 11-15, 22 and 2*:, 1963*' \ J DOW 537320 18334 DOW 5 3 7 3 2 1 Cl 2 10. Butler, P. A . ; Commercial Fishery Investigations, Acute Toxicity (Biological Laboratory, Gulf Breeze, Florida), U. S. Department of the Interior, Fish and Wildlife Service Circular 226, 69-73, ' 1965. 11. George, J. L., Recommendations for Minimizing Dangers of Pest Control and Pesticides to Fish and Wildlife, U. S. Department of the Interior, Fish and Wildlife Circular 16?, 101-105, 1963- 1 R O B E R T E. J E N K I N S , M. D. 9311 N. MERIDIAN ST. INOIANAPOU6 0. INDIANA W IN T H R O P 2 S O O June 7, I95I4- Dr. Harold H. Gay Medical Director, The Dow Chemical Co. Midland, Mich. Dear Dr. Gay; Mr. James R. Mayfield has recently brought in several letters from you and other inter ested parties regarding an alleged contact dermatitis he may have sustained in August 1953 from the use of 2,4,5 something* I have seen Mr. Mayfield on several different occasions since his first visit here on Aug ust the l4th 1953. At that time he presented a severely excoriated dermatitis of the peri anal area. Prom the history at that time he was stated to have had pruritis ani for ten years. There was some dermatitis of the el bow extensor then but I was not clinically im pressed with the dermatitis venenata picture* He has been treated with various topical med icaments and with some success but still has some dermatitis. I also had him seen by Dr. R. Kierland at the Mayo Clinic and they felt much as I do, namely that there might have been an element of contact early but that would not explain the pruritis ani of long duration except from the "over-treatment" standpoint. UUW J 278634 12 iS 1-6.336 T T H E D O W C H E M I C A L C O M P A N Y M IDLAND M ICHIOAN April 12, 195* 278633 Dr. J. S. vandemark Abb latent Professor of Horticulture Purdue Bbiversity Agriculture1 Extension Servios Department of Horticulture Lafayette, Indians Dear Dr. vandemarki your letter to our Nr. V. K. Howe 1b now In my hands for oanaent. As Mr. Rowe has told you, this Is oertainly not a reaction typical of 2,4,5-T contacts. I am assuming that Mr. Mayfield has had no subsequent oontacts with 2,4,5-T since the original back In August, and if this 1b true then it follows apriori that the densatoils which he now has, la not the result of contact with 2,*,5"T because contact dermatoses by definition are cleared oertainly within two months after discontinuance of the contact. If oontaot has continued however slnoe that tine, then the first thing that wust be done la to remove him from easy further contacts, and It must be remembered that contact does not necessarily mean direct contact with the material. It may be contact through re-wearing of clothing which are Impregnated with the substance. I hesitate to offer any suggestions for therapy without knowing more than the fact that "this area turned red In the field and now remains rather red and irritated.* I think the most effective way for me to offer any help would be to talk to some physician who has recently seen the akin condition. If you could give me the name of the physician who last saw him, I should fee glad to call and talk with him. Very sincerely, Harold H. Qey, K.D. M edical D ire c to r HHQrEW CC: V. K. Rowe, E io ch em . Rea D e p t., 280 B ld g . 11  I THE DOW C H E M IC A L C O M P A N Y M IDLAND M ICHIGAN June 5 i 1953. V. K. Rowe BIO-CHEMICAL LAB 2-280 Bldg. cc: H. L. Smith SUBJECT: REPORT OF CALL BY CLOFT, BOSTON OFFICE ON LUCAS TREE EXPERT COMPANY Attached you will find a copy of a report of call from Harry Cloft wherein he requests some information describing the effect of a 4# solution of 2,4,5 -T in kerosene on eyes. Perhaps some action has already been taken on this matter; however, Hillard Smith will be in town on Monday, June 8. Could you call Hillard Monday to decide what action,if any, should be taken at this time? Yours very t: Clyde A.. Bryant \ J Agricultural Chemical Sales Ext 8804 rms -COPY- I 2oO PURDUE UNIVERSITY A g ric u ltu ra l E xtension Service L afay ette, Indiana Departm ent o f H o rtic u ltu re March 24, 1954 Mr. V. K. Rowe Dow C h em ical R e s e a rc h D e p a rtm e n t Dow C h em ical Company M idland, M ichigan c-sr.i ct CJ D ear Mr. Rowe: I am inquiring as to whether you or anyone in your or ganization might know of a treatment for an apparent allergy developed by 245 T. One of our growers in this state received severe toxi cological reaction appearing to be a burn over a portion of his body while spraying fence rows with 245 Y. This occured last August. This area turned red in the field and now remains rather red and irritated. Mr. Mayfield was quite concerned over this and local physicians were not able to give him any satisfaction and he went up to Mayo Clinic last January. They didn't seem to know any treatment for this type of Bkin injury. Could you possibly suggest what might be done, if anything, or refer us to anyone who is more familiar with this type of a toxicological problem. Very tr u ly y o urs, JSV /pf /s/ Joe J . S . Vanderoark Assistant Professor in Horticulture 16339 Sl.i Forrn G. R. 30-- 150M--fi-.i.l COPY 125 EUGENE A. EA1JD, M.D. Diseases of the Skin.. 211-12 Bearinger Bldg. .Saginaw, llichigan June 2, 1951* John T. v/ocdruff Consupers Power Company Bay City.. Michigan _. EE: Richard Ouiliet vs. Consumers Power Company iO ^ cr: M CT" uear Sir: Phis mar. returned to ay office 5- c U - 5 k for the paten nests as you requested. The results are as follows: 2b hrs ^8 hrs Crude oil-------------- b plus b plus Ectron 2bp------------- negative negative TInber Tar-- ------------- u plus negative Poison Ivy--------->-----1 plus -1 plus These teste would indicate the cause cf the recent diffuse dermatitis was the crude ail'used as a vehicle for the Bstrcn 2b5 'veea killer. I think it iE only fair tc give this pan another trial at this came job before transferring hip. He vac instructed on how to avoid the Hoping this information will be cf service to you,.I remain, Sincerely yours, Eugene A. Hand (SigDed) EAHuues:cd Eugene A. Hand M. B. CO N FID EN TIA L DOWI 27861 125S iT T H E D O W C H E M IC A L C O M P A N Y M IDLAND M ICHIGAN July 12, 1955 D r. Andrene G. doe s i D aniel-H a rrell C lin ic M edicai A rts B u ild in g 3 17 S ta te L ine Avenue T exarkana, Aricanaas-Taxaa Dear Dr. G oesli Mr. R. A. C r a n d a ll o f o u r A g r i c u l t u r a l C h em ical D i v i s i o n a t S t. L ouis has aalced a e to r e p ly to your l e t t e r o f Ju n e 2 5 th addressed to th a t o ffic e . I am v e ry s o r r y t o h e a r t h a t you h a v e a p a t i e n t who i s s u f fe rin g from m ild h e p a titis fo llo w in g use o f our E stero n 245. Our s tu d ie s o f th e to x ic o lo g y o f t h i s m a te r ia l have been con ducted on la b o ra to ry anim als and liv e s to c k , and w hile m ild h e p a tic symptoms have o c c u rre d , th e d o sag es in a l l c a se s have been ra th e r m assive and by th e o ra l ro u te . F urtherm ore, e f f e c ts upon th e h e p a tic o r re n a l system s seemed to ap p ear only a f t e r r a th e r la rg e doses w ere g iv en and a f t e r o th e r more d ra s t i c e f f e c ts such a s m yotonia, a n o rex ia, and g a s t r i t i s were ex p e rie n c e d . Thus, i f th e p a tie n t had sw allow ed th e m a te ria l in s u b s ta n tia l am ounts, i t would n o t be u n reaso n ab le to ex p e c t some t r a n s i e n t h e p a tic and p e rh a p s k id n e y in ju r y to fo llo w o th e r prim ary symptoms. I t i s re c o g n iz e d , o f c o u rse , th a t such e x p erim en tal work i s s u g g e s tiv e b u t n o t c o n c .u s iv e a s f a r as human s u b je c ts a re concerned. As you a r e p ro b a b ly a w a re , we h a v e m a n u fa c tu re d an d s o l d E s te r o n 245 and re la te d m a te ria ls fo r se v e ra l y e a rs . M illio n s o f g a l l o n s have b e en u s e d and many p e o p le hav e b e e n e x p o s e d . To our k n o w le d g e, t h e r e h a s n e v e r beer, a c a s e o f s y s te m ic t o x i c i t y attributed to t h e s e m a t e r i a l s e x c e p t p e rh a p s w here i t h a s b e e n swallowed a c c i d e n t a l l y o r w ith s u i c i d a l i n t e n t , hr. o c c a s i o n a l ., c a s e of d e r m a t i t i s h a s b e en r e p o r t e d , b u t i n m o st o f t h e s e c a s e s , the diluent o i l ( f u e l o i l o r k e r o s e n e ) h a s b e e n shown t o be th e causative a g e n t . The s ig n ific a n c e o f th is p la n t end f ie ld ex p erien c e i s n o t ap p r e c ia te d u n t i l one r e a l i z e s t h a t th e r e a re many custom s p ra y in g firm s in t h i s c o u n try and ab ro ad whose em ployees do l i t t l e DOW1275616 Dr. Andrew G. Goesl -2 July 12J 1955 e lse but apply th ese m a te ria ls. I t 1b not unusual fo r th ese men to have t h e i r c l o t h i n g w et w ith t h e m a t e r i a l f o r h o u rs a day, day a f t e r day. Ve, o f c o u rse , do n o t recommend such p ra c tic e and do our b e s t to d isco u rag e i t , b u t n e v e rth e le s s , i t does occur. I n view o f o u r l a b o r a t o r y f i n d i n g s an d t h e e x p e r i e n c e we h a v e h a d , we d o u b t v e r y much i f t h e a p p l i c a t i o n o f E s t e r o n 2^5 by your p a tie n t co u ld have been in any way r e l a t e d to h is h e p a ti tis . I am Bending a oopy o f y o u r l e t t e r t o g e t h e r w i t h a co p y o f t h i s re p ly to D r. H. H. Gay, D ire c to r o f o u r M edical D epartm ent, and th e re b y w ill a sk him to c o n ta c t you d i r e c t l y w ith h is o p in io n s in the m atter. I f we c a n be o f an y f u r t h e r a s s i s t a n c e , p l e a s e do n o t h e s i t a t e to contact us. S incerely yours, V. K. Rowe B iochem ical R esearch D epartm ent 2-280 B uilding YKR/bb enc. P .S . I am e n c l o s i n g a r e p r i n t e n t i t l e d , "Summary o f T o x ic o lo g i c a l In fo rm a tio n on 2 ,1-D and 2 ,4 ,5 -T Type H e rb ic id e s and E valuation o f T heir Hazards to L ivestock A ssociated w ith T h eir U se." R ecognizing th a t th is in fo rm a tio n does n ot a p p ly d i r e c t l y t o human b e in g s , we b e l i e v e i t i s i n d i c a tiv e o f th e ty p e o f th in g which can be e x p e c te d . V .K .R . D r. H. H. Gay ^ R. A. C r a n d a ll H. L. S m ith G. J . W illia m s 16342 1259 2/ RESULTS OF TOXICOLOGICAL TESTS ON 2,4,5-TRICIILOEGPIIZXOXYACETIC ACID Biochemical Research Department The Dow Chemie al Company Midland, Michigan August, 1950 DOW 668872 PROBLEM The widespread handling of 2,4,5-trichlorophenoxyacetic acid made it desirable for us to have a significant amount of toxico logical information.. Data on the oral toxicity was necessary for presentation-to the F.D.A. Hearing on residue tolerances. ' MATERIAL Name : 2,4,5-Trichiorophenoxyacetic acid Formula: Structural: > Empirical: CgH-Cl^O^ EXPERIMENTAL RESULTS The acute oral toxicity of 2,4,5-trichlorophenoxyacetic acid ior rats, mice, guinea pigs and chicks is given in the attached summary which was presented in evidence at the F.D.A. Hearing. In .concluding our testimony on this material we stated, 16343 *> DOW 668873 "Because oi tho similarity in chemical structure and in toxicity and physiological effect between 2,4-D and 2,4,5-T, it sco rn s that a residue tolerance for the latter on fresh fruits and vegetable..should be the same as that established for 2,4-D; it would seem that a residue of 20 p.p.m. would provide a wide margin of safety" . *- Zye Irritation Y.'hen 2,4,5-trichlorophenoxyacetic acid as a 10% solution in propylene glycol was introduced into the eye of a rabbit, it produced marked pain and slight conjunctival irritation immediately. 7ive hours later, severe conjunctivitis and moderate corneal damage was apparent. This picture remained about the same for two days before healing began. Tive days after the exposure mild conjunc tivitis and corneal damage was still apparent. Skin Irritation T/hen 2,4,5-trichlorophenoxyacetic acid as a 10% solution in butyl.carbitol acetate was applied repeatedly to the rabbit ear and bandaged repeatedly to the shaven abdomen, a slight simple irri tation developed in both instances. SUMMARY The "toxicological properties of 2,4,5-trichlorophenoxyacetic acid may be summarized as follows: 1. The material is moderate in acute oral toxicity for xhe four species, rat, mouse, cavie, and chick. Of these species the rat seems to be the more resistant and the chick the most susceptible. 2. The material is markedly irritating to the eyes. 3. The material is slightly to moderately irritating to xhe skin upon prolonged exposure. ,n o, 'i 1 DOW 668874 HANDLING HAZARDS AND PRECAUTIONS EOR SATE HANDLING ^a llo w in g It docs not .seem likely that 2,4,5--trichlorophenoxyacctic acid can be swallowed accidentally in amounts dangerous to life, although this might be possible if concentrated solutions of the material were available to children. If substantial quantities of the material'were to- be swallowed by anyone, serious ill effects could be anticipated. If the material should be swallowed, induce vomiting by giving i common emmetic such as 2 tablespoonfuls of table salt in a glass }f warm water. Call a doctor. es The 2,4,5~trichlorophenoxyacetic acid is capable of causing .erious damage to the eyes. Precautions should be taken when .anaiing this material to prevent possible eye contacts. It is omnended when handling strong solutions of 2,4,5-trichiorophenoxycetic acid that goggles, face shield, or other similar device be orn to prevent accidental splashes in the eye. It is believed hat the handling of the solid does not present a hazard simply ecause of its insolubility in aqueous medium. If the material in any form should get into the eyes, they ^>uld be promptly washed with flowing water for at least 15 Lnutes and medical attention obtained. (in Prolonged and repeated contact with the skin may result in rif ation. Occasional contacts for short periods are not expected > cause irritation. Strong solutions are no doubt more likely to use difficulty than the dry solid. In case of contact wash the exposed area with soap and water, not wear clothing or shoes which have been contaminated with soluons of the material. DOW 668875 i'C C 'O Inhalation Jio studios of toxicity upon inhalation of 2,4,5-trichlorophenoxyacexic acid dust have been made. V/e believe, however, that in view of the similarity with this material to that of 2,d-dichlorophenoxyacctic acid, that it is unlikely that toxic concentrations nx xho dust would.be inhaled, primarily because of the irritation it will produce upon the upper respiratory passages. > 16346 C-T -- 'in . i:y ;lc. 7 : 6 1 , ibJi J .;i)w Occur tlo.V 1 : ; DOW668 TOXICITY OF 2,4-DICHLOROPHENOXYaCETIC ACID AND 2,4,5-TRICHLOROPHENOXYACETIC ACID A Report on Their Acute and Chronic To*icity in Dogi VICTOR A. DRILL, Ph D., M.D. AND TOMIHARU HIRATZKA, M.D. B DETROIT cn OTH 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-tnchloro]>hrnoN\acetic cn acid (2,4,5-T) may act as plant hormones and herbicides *123and are now being fairly widely used for the control of certain types of weeds on farm Icr.ds. Thc\ are being sprayed from airplanes on many acres of specialized farms and grazing land and are present in trademark preparations sold a: retail for home gardener.-. With the widespread use of these herbicides, possible toxic effects are important, as accidental ingestion may occur or residues may be inadvertently encountered in food. In an earlier report, 2,4-D was mentioned as being nontoxic to animals and man when administered orally.'* * However, the injection of iarge doses of 2,4-D in animals produced symptoms similar to those seen in clinical myotonia.J More detailed observations were reported by Hill and Carlisle who studied ihe acuteand subacute effects of 2,4-D in various species of animals.1There ha\e beer, no reports concerning the toxic properties of 2.4,5-T. The present study concerns the acute oral toxicity and chronic oral toxicity of 2.4-D and 2,4,5-T in dogs. EXPERIMENTAL PROCEIKRES Adult mongrel dogs of both sexes were used. Most of the animals were housed in the laboratory for a period of tv.i to three months before the study was started During this control period they were immunized against distemper with the Green vaccine. The dogs were fed a standard stock diet (Friskies) ad libitum. Both 2.4-D and 2.4.5-T were commercial materials, with a purity of 93.5 and 93.9% and freezing points of 132.8 and 150.6 C., respectively In the acute studies, the calculated dose of 2.4-D or 2.4.5-T was administered a3 a single oral dose in capsules. The dogs were observed for a period of 14 days, at which time the sur- This study was supported by a grant from the Dow Chemical Company From the Department of Physiology and Pharmacology and the Department of Pathology. Wayne University College of Medicine. 1. (c) H i'ueLiiiid, E. M .: War on Weeds. Science 103:465-468, 1946. (6) van OverbeeW, J., and Velez, I.: Use of 2,4-Dichlorophenoxyacetic Acid as a Selective Herbicide in the Tropics, Science 103:472-473, 1946. (c ) Smith, F. G .; H:.mner. C. L-, and Carlson, R F.: Control of Ragweed Pollen Production with 2,4-Dichlorophenoxyacetic Acid. Science 1(13:473-4/4, 194n (if) Marth,- P, G , and Mitchell, J. W .: 2,4-Dichlorophenoxyacetic Acid as ia Differential Herbicide, Bot. Gaz. 100:224-232, 1944. - 2. Bucher, N. L. R .: Effects of 2,4-Dichlorophenoxyacetic Acid pn EtTp^rirriilrKa!1 Animals, Proc, Soc. Exper. Biol. &: Med. 63:204-205, 1946. 3. Hill, E. V., and Carlisle, H .: Toxicity of 2,4-Dichlorophenoxyacetic Acid for Experi mental Animals, J. Indust. Hyg. ic Toxicol. 29:85-95, 1947. 61 16347 62 i x h u s t k i a i . in c if . x r . a x d o c c u '.iT to x .u \u n ta x i- . vivur> were autop-icti. lu the clirmuc >1 mlics. the 2,-tD or 2,-1,5-T wa< administered m ill; in capsules five day, a week over a U week period. Each capsule wav imbedded in a 4 to pu pieve of commercial canned dog food, which the animals consumed readily. thus avoiding the continued trauma of the stomach tube. During the study the animals were weighed twne a week. Control blood counts were taken before the administration of the drug, on the JOtli <>u . and on the 90th day at the completion of the study. Hemoglobin was deiii.imned with photoelectric colorimeter.* L pon death or at the completion of the study, the animals were autopsied. Tissues were taken from the lung, heart, liver, kidney, adrenals, spleen, thyroid, and ovary or testes, fixed in lUi formalin, and stained with hematoxylin and eosin. T able 1.--E f f e c t of Single O r a l Doses of 2,f-DirAf<wo/>/irno.r_voivrir A e i d a n d 2 jt j- T r ic h lo r o p h c n o x y a c t lic A c id in Dogs DOW 6 6 8 8 5 b D o* X.> i r ....... jit.... I P ..... 4 31.... 4 M ..... P ....... 1 T ....... 8 F ....... p ..... 10 F ....... n f ..... 12 M___ iiv .,.. 14 31___ 15 F ....... 16 F ....... 17 F ....... UP..... UM ... a p..... n y.... Po. s i* ./k 'e . 100 100 ....... 25 ..... 25 ....... ....... ........ ........ ...... ...... ...... ...... ...... 250 100 urn 100 loo 50 50 50 50 W eight Loss, Ke. n -D --O 1 --0,6 -1 .8 --2.0 --0.1 --5 5 -5 0 -1 7 --2 1 00 +02 + 0 . J.I.S-T --1.1 -1 . --0 5 --LO --1.0 --0.4 +0.3 --+o2.3i --0.6 Sym ptom s) + ++ +++ ++ ++ + 0 + 0 0 0 0 + + 0 0 0 0 0 0 0 0 D eath D r I 3 8 7 4 8 9 9 S 8 S a 2 3 S 78 a aaa 9 M ortality tn 3/3 2, t 0/3 3/3 Wl l/< 0,4 1F lu tbl!i colum n mean* irm ale; 51, mal* ' ColUIQD Indicate* r^cra 1 M vcnty of sy m p to m * as dlK U attd In te x t. 0 m euns no chanye from n o n ; 8 lo tbl<colum n m eans survived U day te s t period (ttlS L 'L T S /. A c u t e O r a l T o x i c i t y .--(a) Mortality: The etTect of single oral doses of 2,1P or 2,4,5-T on survival for 14 days is shown in Table 1. The deaths were delayml and occurred two to nine days alter the compounds were administered. Hie or;:l L-D.jo of 2,4-D was approximately 100 mg. per kilogram of body weight; for 2,4,5-T it was in the rangeof 100mg./kg. or higher. ' (>) Body Weight: The large oral doses of 2,4' D or 2,4,5-T produced a decrease in body weight (Table 1). Such animals' deyejopld various degrees of anorexia, and those that died refused even canned dbg food toward the end of their survival.4 4. Evelyn, K. A.: A Stabilized Photoelectric Colorimeter with Light Filters, J. Biol. Cbcm. 115:63-75, 1936. 1634 6581899 MOO D M L l . - l l l l \ ' A T Z K . - l -- T O X IC I ! 1' O T I I l - . K H i a I ` l . \ (y\ (f) Symptoms: In dogs that tlictl the effect produced 11\ 2,4-1) \;uiol inmi :i mild ataxia and stiffness in the hind legs to adefinite imotnnin. Initial sign- were often notedsix hours alter theoral admiuisitaiion of 24-P. At this lime the animal- were more>|uict tlian nnrinal, and aslight ataxia, was occasionally jirecut. The hind legs were always alTecied hr>t; the forelegs later m not at all. '1here wa- uu;illv a progressive increase in spasmin the hind legs associated with incrvamg atai:i andsonic-dogs spontaneously extended thehind limbs in aspastic mmi-mcni I:miu;; afewseconds. 1heknee jerk was cither normal or liy|>eracti\ e. In the early stages of acute poisoning with 2.1-1), the animals were uii.dlv quiet. If the animal was lifted and madeto walk, thes[>asmand ataxia won cleailv noticed, but after it hadwalked for awhile, thespasmand ataxia tended tn dwriae In thelater stagesof intoxication, the animal, when placedon its feet, \ un.ihlc n stand and would then sit or lie in an awkward position with spastic limb marko particularly by spasmof thehind legs. Other dogs stumbled and rolled >mthe tbv.i in attempts to right themselves. An occasional dog showed evidence ni irritation m pain when the skin over the back of the neck was grasped. Some dog, when held up by the shoulders, would show extension and crossing of the Imid limb- \- tin toxic effects of the drug progressed, the (logs eventually refused the various tvpe. of food offered. Occasion;.liy, sneezing, rubbing of the eyes, and diarrhea wen observed, but not vomiting. Such signs were also seen in one dog that survived a lower dose of 2.4-1) for 14 days (Table 1). In this instance the changes were relatively mild and were limited to spasmof the hind legs and aslight ataxia. Marked effects were not observed in the (logs receiving 2,4.5-T. Ihi- vlumgi were limited to aslight nr moderate stiffness in the hmd legs, with the dcvelopmen' of ataxia in the two dogs receiving the highest doses. No symptoms were evident in theonedog that died after asingle doseof 100mg/kg. (Table 1) (d ) Pathology; Changes observed were limited to the gastroiiiiO'imd tr.nt and lungs. Four dogs (1, 2, 4, and ft; receiving 2.4-D and two (logs i 1.1and 17i m! 2.4.5- T showed mild todiffuse rednessof the mucosa in thesmall inie-tmc In -nine dogs pneumonia was present. Histologically, the above (logs showed some m-ermiand inflammation of the intestinal mucosa. One dog (1.3) had a moderate. ditiu <hepatic necrosis. Two dogs (13 and 16) had amild degree of renal tubular degrn eration. In most dogs that died the findings were limited to nonspecific Chang-, suchashepatic congestion, in many cases death was apparently due to pneumonia, which followed the development of anorexia, weight loss, and myotonia. //. C h ro n ic O r a l T o .v ic ily .--(a) Mortality; All dogs receiving 2. .>. or 10mg of 2,4-D or 2,4,5-T per kilogram of body weight survived the 90-dav ui period The dogs receiving 20 mg./kg. of these compounds died during the studv. Tinadministration of 20 mg./kg. in divided doses twice a da)' produced death m both dogs receiving 2,4.5-Tand in onedog receiving 2,4-F) (Table 2 ). (h ) Body Weight: The only significant change in body.weight occurred in tinanimals that failed to survive the study (Table 2). Loss of weight in the-c dogbegan7to 12days before the deathof the dog. ' -./_ ,ji (c) General Symptoms: Dogs,thal survived the'oral administration oi 2.4-Dor 2.4.5- T for 90 days were free of any symptoms. u w IXDLX'FFI.U. HVi.lFXF AX ft OCCFF.H IOWU. U il'U I'.l The tlirec ilngs licit dice! while receiving the highest ilu-e ut 2.41) \lmwcd -i, differing somewhat iroat tliose oteerved in the mute studio. The wnr iju:1. weaker, and less rcsputiMve than normal. Mu>cle tillin'. ;b higher in the him! i>. particularly on passive extension. The him} legs were held mure stilllv than u a.! when walking, and a slight ataxia was present, Paring the last two tu three il.ivof their survival two oi the dogs showed difficultv in chewing or 'wallowing a; I eventually even refused a small bolus of canned dog food (usually rcadilv sumed). There was also some oozing of blood from the gums and Imccal nm.i.... T a b l e 2.-- Body Weight and Sur-Xtol of Dogs Fed iJ-DicM orofheno i em etic .1, i,l ,m j 2Aj - T rUhl&roplu'iujsyai'ctic Acid for 90 days DOW 668860 Do* Xo.* ar..,, 24 F . . . 25 U .. . M F .... 7 51... tar.... 5* M ... 10 11.. n it... s i.. s r-- U M .., 15 M . m r.... rr ii.. M F... so H 4 0 F ... 41 31.. r... u y.. 44 F ... till.. UT. 47 K... 4111.. Dos*. 9 In itial. U g ./K e. K*. I.l-D 14.9 9.5 AS 15.2 6 9.5 6 12 0 14.7 10 11 A 10 17.2 10 13.0 101 9 8 20 12 2 50 13 4 1.4,5-T 7.3 12 t t UJ 9.9 6 S.A 12 3 10 11.1 10 130 10 9.S to t 11.8 sot 10.5 y> It 9 20 10.9 W etihl F ln.l, Kg. 1A 4 10 9 9.5 13.S 9.5 12.5 14.8 11.9 17.S 13.7 59 10 1 9.7 7.7 14 5 12. 10.0 84 12 .7 11.5 12.2 9.2 1! T.9 12 7 8.2 Dirt.. Kg. -t; 7 -its 4 2.7 --1.4 00 405 401 40 3 -J <1A +0 7 --3 9 - 2l -3 7 +0 4 42 t 4 1-4 4 It --0.2 402 -0 8 -0 8 --0 6 --76 --2 A --92 - 27 rw th Day s. s K s s 6 s Sft 3 4* 18 5. ft S s ft 3ft 8 Sft 49 7;'. U 50 * T kn Ibis colurna ra ra n i letn alr; M. mole i Daily dose *n>l Im inM frM o nr hut! in a m . an<! oa-hui( in p. m ; S In Ibis colum n m e a m survive'! Vi day le st period. In dogs that died while receiving 2,4,5-T, the prominent effects were weakue--, slight stiffness in the hind legs, difficulty in swallowing iood, and, in one dug, bleeding front thegums. (d ) Blood Count: The administration of 2,4-1) or 2,4,5-T did not have am significant effect on the hemoglobin, red cell count, or total white ceil count ut animals that survived or died during the study. Thedifferential bloodcount remained normal in the surviving animals. In three animals that dit-d a terminal (all in the percentage ot lymphocytes was observed (Table 5).-. _^ vi ( t ) Organ Weights: There, was no significant'change-`in the weight oi tinthyroid gland, adrenal gland, heart, liver, or kidney in animals that survived the 90-day period of study. In twoof the threedogs that died during the administration  W 6688G1 DKiLl.-HIh'ATZKA--TOXIC IT V O f Hf.KHIClDfS <> of 2,4-D, there was a slight increase in heart and kidney weight. A similar clung-' was noted in two of the four dogs that died asaresult of 2,4,5-T. (/) Gross Pathology: Two dogs receiving 2,4-1) (2(i and 34) showed aro.r- of redness in the duodenum. With the administration of 2,4,5-T tliei e developed in dog 42 adiffusely reddened duodenumand jejunum, while in dog IK, aiiio[>sied mi the 59th. day, ageneralized icterus was demonstrated. T a b l e 3.-- E f f e c t o f 2 , 4 - D a n d 2 , 4 ^ - T o n H e m o g l o b i n , T o t a l B l o o d , a n d Differential Count \ Do* No.* Do m , U i./k 'c. d *t U T ............................... Noo> U T ............................... N o m M I ............................... N one P M.............................. Nooc 0 so 90 0 80 90 0 90 90 0 ao 90 Hh. Gm, % 13.20 13 83 13.70 12.80 14.72 lift* 960 10.75 12.71 12 ? IS 31 13.95 RHQ, MU. WBO C ootiol Do* 6 72 17,800 5.93 15.600 09 13,850 01 10.900 666 13,150 6 16 7.100 608 18,100 6 63 13,500 6? 14.700 6 53 14..400 6.78 14.900 4.47 6.400 DlfTtreatial C ount . .f a u t . , L ym ph., Moo B t" Kftiiu % %%% W 3* 0 0 6 64 29 0 0 7 27 2 0 0 n86 11 3 0 79 12 7 U 2 79 21 0 0 0 67 26 1 0 6 56 81 it; 18 20 10 f3t 53 3*1 2 0 4 63 26 4 0 7 48 51 l 0 0 .4-P B U .............................. t 0 10 43 SOT 13.800 61 27 50 4 6so 12.44 6 10 16,400 66 2t 40 4 90 II .V? 6 35 12,100 70 28 00 It id 6Mln r _ ............................ tut 0 13 H 14.8-V h4 41 2 0 3 so h AS 9.700 47 40 4 0 3 49 U3n 5 67 16,600 79 14 7 00 U N ............................ 0 13 20 5.77 15,200 59 44 4 ft .1 N .............................. ft0 1196 6 47 13.150 76 11 1 ft 13 11.59 6.34 9,100 68 27 4 l tsM ............................... 1..................... sot a t ..................... to U N .................... to 0 30 40 0 K 0 a0o so 18126M5-14 1249 14 M l4 M 12.4H 1? 11 97 t,4> T 533M5 16.400 16,456 1.07 6,9/0 7.10 682 5.73 6A8013 4 14 18.050 11.200 13^01 16.800 14.650 7J75 72 77 4 63 71 03 51 78 92 12 13 4 29 28 32 44 18 9 *J la thie colum n ra^aan t DaQy 4 qm divided tori p.M, ran)* oo< h a lt i a. nt. Ann one n ail la m. T o ohacnr epaca, only d a ta o n co o tro l <Jogi anil the highest dose* ace given. 60 23 0 0 20 00 40 20 14 0 0 in 7 0 61 1 300 (g ) Microscopic Changes: 1he liL'art, lungs, th) roid adrenal, ovar), n r if* did not demonstrate ;iny Mgnif'caut changes. Three of the rn riving 2.4.s I and two receiving 2,4-D.showed an occasional area^of focalTnecrrisis in the h\ei This finding, however, was not T r ia le d to either the dose of the' c o m p o u n d s or th e death of any animal (Table 4) and was judged to be of no significance. I h r d im denumof dogs 26, 34, 42, and 48 showed varjing degrees of hyperemia a n d sumr early infiltration of cells in the mucosa. The snbiiutcosa and serosa were nornul 1635 * r- i D O W 668862 66 IXmSTRI.d. lYClliXh .-XI) OCCUl'.li IOX.II. M l . h U I X l - A slight increase in the number of casts in kidi) sections was noted m mum- .1.. (Table -4), but again this was unrelated to dosageand was of doubtful signim-m-. COM U K N T In the acute studies, single large oral doses of 2,4-D a dm ini ste re d to dog m produce signs characteristic of clinical myotonia. This effect ot 2,1 D w a s su.,.:. to that observed after various experimental species had received ' 'injection.. h addition, sonic dogs showed signs of irritation and pain when the skin of the b u t the neck was grasped, which may indicate meningeal irritation. T h e extension crossing of the hind limbs, when lifted by the shoulders, may point to a spin..; -i T able 4.--Histological Changes in Liver and Kidney of Dogs Fed 2,4-D or 2,4^-T for *>i> .. Dog No. Do*. M i-g 'K e . n ................................ . 24................................ . 5 ................................ M ................................ 27................................ 2................................ . 29................................ 20................................ n ................................ ................................ <2................................ 5................................ None None * 5 9 JO 10 10 20................................ >7................................ ........................ I ........................ 40.............................. ........................ c ........................ a .............................. 44.............. ........ 4 5 ........................ 44........................ 40........................ * * 10 D tftth Do C o n gestion 2,4 D 0 0 0 0 1+ 1+ 0 0 1+ 0 49 1+ 25 * + 2.4 > T 0 0 0 0 0 0 t-t 0 1+ 40 1 + 75 I t 60 t + ----- ---- s khlnry Tocl r ~ Necrosl* C u ts Tuti 000 000 00n 0 i+ 0 i+ 0 0 0 1+ H 0 0 < 000 000 000 1+ 0 00 0 0 Q 0 0 0 0 1+* 0 0 0 1+ 1+ 0 0 0 1+ 0 0 T 1+ 0 0 0 1+ 0 II 0 0 O 0 11 0 0 0 0 0 0 * M U o 1+ Obroslj. TUauea c rtd td oa bails of 0, 1-f, 2 + , H*. nod 4+. central lesion. Other effects occasionally observed were sneezing, rubbing 01 m- eyes, and diarrhea. The effects of 2,4,5-T, previously studied, were not as so-., i- being limited to stiffness in the hind legs and ataxia. The higher doses or compound produced anorexia and weight loss. Although initial effects may be m. six hours after the compounds are given, the syndrome developed slovl> in tie. ,, dogs, and death was delayed, occurring several days after the administration oi tie- compounds. .y In the chronic studies, the dogs survived doss'oL.?; '5p'on:d0 mg. of 2.4-1' 2,4,5-T per kilogram of body weight without apparent ill1'effects. However, t!.. daily oral administration of 20 mg./kg. of 2,4-D produced death in three of u.m dogs within 18 to 49 days. This delayed death may indicate a cumulative effect of 2,4-D. The signs observed in animals dying from the 20 mg./Wg. dose of 2,4 1' 16352 OOW668863 DMLL-HtKATZKA--TOXICTV OT libKlUCltA.s 67 differed somewhat from those seen in the acute studies. With the cliiunic mhnitiD tration, the animals exhibited chiefly a stiffness of the hind legs and ataxia, weak ness, difficulty in chewing or swallowing, and occasionally bleeding from the gumAll tour dogs which received 20 mg./kg. of 2,4,5-T succumbed between the llih and the 75th day of the study. Toxic effects in these animals consistently included slight muscle spasmand difficulty in swallowing food. Thus, the chrmiic oral toxic dosetor 2,4,5-T in dogs seems to be in the samerange as that obtained with 2,4 l 1 With bothdrugs, there was aterminal loss in body weight. Dogs surviving the chronic doses of 2,4-D or 2,4,5-T did not show any signifi cant change in hemoglobin, total blood counts, or differential blood count. In direr of the dogs (64, 57, and 105) which died while receiving 20 mg./kg. of 2,4-D nr 2.4.5- T, there was a definite decrease in the percentage of lymphocyte in tin peripheral blood. Hill and Carlisle1have observed a fall in the polymorphonuclear leucocyte count in one dog and a decrease in the per cent of lymphocytes in tun dogsto which 50mg./kg. of 2,4-D was administered intravenously each day for six days. In mice which received chronic injections there was no alteration in the peripheral blood picture.1 Large dosesof 2,4-Dadministered intravenously or, in the present study, orally occasionally produced some necrosis in the liver. However, the administration ni chronic oral doses produced only aslight and inconsistent focal necrosis ot doubtiii! significance (Table 4). Deaths during the chronic oral administration ot 2,4-D or 2.4.5- T were not correlated with significant lesions in the liver, kidney, nr other organs examined. s u m st A R Y The acute oral L.D.1() of 2,4-dichlorophenoxyacetic acid (2,4-D) in d>>g> waapproximately 100tng. per kilogramof body weight. Doses in this range or higher producedadefinite myotonia accompanied by anorexia and weight loss. The acute oral L.D.jofor 2,4,5-trichlarophenoxyacetic acid (2,4,5-T) was in the range of 100 mg. per kilogram or higher. Such toxic doses produced only signs of a mild spasticity. All dogs survived the oral feeding of 2, 5, or 10 mg. per kilogram of 2.4 f) or 2.4.5- T 5 days a week for a period of 90 days. These doses did not produce any symptoms or changes in body weight, organ weights, or blood count. Threeof the four dogs receiving repeated dosesof 20mg. per kilogram of 2,4-D died, and all four dogs receiving 20 mg. per kilogram of 2.4,5-T died during the study. Toxicity to 2,4-D at the dosage level wasaccompanied by bleeding from tingums, necrotic changes in the buccal mucosa, and some difficulty in chewing ami swallowing, but there was only little evidence of clinical myotonia. A terminal fall in thepercentageof lymphocytes was observed in three of the animals. Death during the repeated administration of 2,4-D or 2,4,5-T was not related to pathological changes in the live.-, kidney, or other organs examined. X* . . . . r a s ACUTE ORAL TOXICITi" OP 2 , 4 , 5 5RICHL0ROPHEKO2YACETIC ACID TO HATS, MICE, `GUINEA PIGS `AND CHICICS 1261 1 OOW66S852 V . K . R ov/g D. D, K cC o lIister and H. C. Spencer * E lo ch en ical Reecaro . L aboratory ' The Dow C h e m ica l C oapany M id la n d , I-lich ea.i 1950 f!SSS99MOCI -1 - S inglc o ra l doses of 2 ,4 ,5 "brichlorophonoxyacctic acid no a a 0 pcrcc**c xn o^xvc O xl v.^r%-* ucxmxnxsccrcd sy sionx-cn uusc to m ixed so x g ro u p s ox* r a t s , m ic e an d g u in e a p i g s . A l i q u o t s o f a jj p e rc en t s o lu tio n In o liv e o i l w ere a d m in iste re d to chicles. The v.hito r s t s and th e g u in ea p ig s x .-ere'selected from th e sto ck c o lo n ic s o f th is l a b o r a t o r y . W hite m ic e w ere o b ta in e d from th e C arv/orth R a m s an d hav/ har.pshirQ Red c h ic k s w ere p u rch ased from a com m ercial h a tc h ery * n a tu re , young a d u lt anim als were used in each in stan c e except fo r th e chicks which were ap p ro x im ately th re e weeks of ag e. T able I sum m arizes th e c o n c e n tra tio n s and volum es o f o liv e o i l s o l u t i o n s c o n t a i n i n g 2 , 1r , 5 * - t r ic h lo r o p h e n o x y a c e t i c a c i d a d m i n i s t e r e d to each sp e c ie s. TABLE I SINGLE ORAL ADMINISTRATION 0? 2 ,4,5-TRICHLR0P?IEN0NACETIC ACID Snecies P ercen t M a te ria l in o liv e o il R ats ' 10 l-h.ee Guinea P igs 10 10 . C h ick s 5 Volume Given by Stom ach Tube Minimum ( c c . '> Maximum ( c c . ) '0 .7 ' 0 .0 5 1 .0 0.5 2 .9 0 .3 2 .9 5 .8 M o rta litie s r e s u ltin g from th e v ario u s sin g le o ra l dosages f 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic a c id to the experim ental anim als are G iv en i n T a b le I I . A l l s u r v i v i n g a n im a ls w ere o b s e r v e d u n t i l It was c e r ta in t h a t th e y had f u l l y re c o v e re d ( u s u a lly a b o u t two w e e k s). / ``nxptoas o f p o i s o n i n g , p a r t i c u l a r l y w i t h t h e h i g h e r d o s e s , w e re v-* * ax ia a n d m y o to n ia . TABLE II MORTALITIES RESULTING FROM THE ADMINISTRATION 0? SINGLE ORAL DOSES 0? 2,4,5-TRICHM?.0?;iEHO:ffACETIC ACID ? HATS, MICE, GUINEA PIGS AIviD CHICICS ->?.r o Rats Mice' Guinea ?irr~ Chick;: -- .. a .) N o .fed No .died Mo .fed No'.died ?:o.fedTTo ciled N o .fed No .diecl 0 H 0.1 0.2 0.3 0.4 0 .5 .7 10 10 10 - 10 0 . 3 8 10 3 6 6 6 6 0 2 4' . 6 6 5 3 5 5 0 4. 4 5 6 0. 60 6 4- 6 5 66 33 The acute oral LDso values with their 1 9 / 2 0 .confidence limits vere determined for each species by use of the method described by L itc h fie ld and W ilcoxon . The r e s u l t s o f th e s t a t i s t i c a l tre a tm e n t of the dosage-response d ata a re presented- in Table I I I . TABLE III > ACUTE ORAL LDso VALUES FOR 2,4,5-TRICIIL0R0PHZN0XYACETIC ACID WHEN ADMINISTERED TO RATS, MICE, GUINEA PIGS AMD CHICKS LD50 (19/20 confidence limits) Species . g m ./kgn. Slope A 'Function** Klee Guinea Pigs Chicks . O.5OO (O.3 9I - 0.640) O .389 (0.245 -- 0 .6 1 9 ) 0 .3 8 1 (O.307 - O.472) 0 .3 1 0 (0 .2 1 1 - O.45S) 1.49 1 .8 0 1.2 1 1.40 A the fold change in dosage required to produce one unit standard deviation in response along the line. 1 . L itch field , J.-T Jr., and V/iIcoxon, J. Pharm, and Expt. The?. 06: 9 9 , 1949. DOW 66S855 -> The Litchfield end Uilcoxon tecta for pare.lie-11a::, of tv;o "lines end estimate of relative potency revealed that the 2 ,^,5-trichlorophenoxyacetlc ecld was signlficantly (19/20 probability) more toxic to the chicks than to the rets. There were no significant oiff-erences in either parallelism or relative potency as a result of the following comparisons: rats versus mice, rats versus guinea pigs, d e e versus guinea pigs, nice versus chicks and guinea pigs versus chicks. > 12 4 4& R r*` ! -opy Blcc'.'.i mlcal Research l.jpart:nout Thf Dow Ciemlcal Company 5S233 RESULTS OP SKIN TESTS ON: Pile 1. CONCENTRATED IMPURITIES IN D O W S tf=S* 2.4.5- TRICHLOROPIiENCL (K 2777-40) K No. 2. CONCENTRATED IMPURITIES IN 2,4,5- TRICHLOROPHENOJ, MADE FROM KOOKEU'S 12,3>4-TETRACHL0R0EENZEKE (K 2777-41) 3 . CONCENTRATED IMPURITIES FROM 2,4,5TRICKLOROPHENOL FROM DCW'S 1,2,3*4- TETRACHLOROEENZENE (K 2777-42) Che. Rept. Ey 4. CONCENTRATED IMPURITIES FROM HOOKER'S 2.4.5- TRICHLOROPHENOL (K 2777-43) 5. IMPURITIES FROM DOW'S TETRACHLOROBENZENE (K 2696-5) 6 . IMPURITIES FROM HOOKER'S TETRACHLORO- EENZENE (K 2696-6 ) T16.4-60-4 T17.4-12-9 2777-40 2777-41 2777-42 2777-43 1219-2 Mark A. Wolf 00 THIS REPORT IS THE PROPERTY OP THE DOW CHEMICAL COMPART THE DOW CHEMICAL COMPANY J u l y 1 2 , 1 9 ?6 MIIM-&MB MICMIAAM iv n 453182 Mr. R. Reese Biaphenol Plant 349 Building SUBJECT: SKIN IRRITATION OP SOI-E IMPURITIES IN 2,4,5t r i c h l o r o p k e :k)L a n d t e t r \c k l o r o e e n z e n e p r o m VARIOUS SOURCES The akin testa on the impurities In 2,4,5-trichlorophenol made from various sources have been completed. These tests were undertaken to help explain, If possible, the differences in Bkin responses produced by various sam ples of 2,4,5-trichlcrophenol. Previous tcst3 have shown that only the impurities found in Hooker's 2,4,5-trichlorophenol were capable of causing skin responses which were unusual. Other possible impurities were shown to be similar in their effects or the 3kin to 2,4,5-trichlorophenol. The material?: submitted for testing were as fol lows : 1. Concentrated impurities from Dow's 2,4,5Urichlorophenol (K 2777-40). 2. Concentrated impurities from 2 ,4 ,5-trichlorophenol cade from Hooker's 1,2,3 ,4-tetrachlorobenzene (JC 2777-41). 3. Concentrated impurities from 2,4,5-trichlorophenol from Dow'a 1,2,3,4-tetrachlorobenzene (K2777-42). BOW'1453183 Hr. R. Rccac - c July 12, 195-5 4. Concentrated impurities from Hooker's 2,4,5* trlchlorophenol (K 2777-43). All of t.^.cse materials were applied to the lntaet rabbit's skin as a 10^6 oolutlon In Dowanol 50E. When tested In this manner, all of these materials gave essenf tlally the Bame response lr. that they were slightly irri tating to the rabbit's skin. This response was character ized by alight reddening of the skin and slight exfoliation or scaliness. This is considered typical of the response caused by 2,4,5-triohlorop:-.er.:l at this concentration. These results show that the iiapuritiea, as ob tained in these sarplea, should not contribute aignifioantly to the irritating properties of 2,4,5 -trichlorophenol regard less of source of the raw material. This finding is not in accord with those reportsd earlier in which it was suggested that an isrpurity of Hooker's 2,4 ,5-trichlorophenol may cause. acneform dematitlo In humans (See letter of January 27, 1956). Two possible explorations for this difference in response were suggested. T le first was that the "acneform" effect wan weak in m;uro ;nd may have been overlooked ?n this oerlort of tcc:o ailf.-.rugh they were repeated on several rebbits with, r.o signifies, t change in response. T..e tccor.t possible explanation was that there nay have been differences In the oiginal aanplea used even 16360 D O W "14 5 3 18 4 Hr. R Resse -3- July 12, 1956 though the l.Tpurltlea were concentrated In a similar manner. Consequently, the samples were analyzed by infrared scan ning wlthtr.e following resulto. ANALYSES OP CONCSriTRATZD IiTRITIES 2,4,5-tri- chlorophenol made from Dow'3 2 ,4 ,5- Hooker1s trlchloro- tetrachloro- Dhenol \ benzer.e 59 3 75 + 3 12 + 2 1.3 + -5 <.0.5 0.6 +_ 0.5 12 2 <0.5 1.7_ .5 13 2 8 2 6 2 2,4,5-trichlorophenol made from. Dow's tetra- chlorobenzene 9 + 3 7 +1 <0.5 5 +1 10 . + 2 6 +2 Hooker *s 2,4,5-tri- Identification chloroober.oi Of Constituents SI + 2 1.5 + ^0.5 3+1 ^ 2,4,5-triuiiloro- phenol % 2,3 x-trlehlcro- phenol * 2 ,4-dichlorophenol :* 2,5"dlohicrophenol <1 i 2,i)-dichloro-5methorypehrol (r-nurlty A) <1 % 4,5-dlchloro-2- raethaccyphenol (impurity B) The rc-cnly.irg naterials lr. tl.c hevo oanpleo aie not ldentified but nrc r.irrdlar in ntruebure to tha methoxy dichloro phenol'.' ar,.i di'.Chrxy dic.-.loro bcnzenec. Tnlo would indicatc t e pass 1b' 1 r . c / ':*ut'.'xy chlore phcnols ano dichloro cc* c and cichltrn ':-so;T.lnol. Unfort.i... j ..ly r. oi.-.ijr.r nnalyaiP was not perfenaod on t);0 prevlouajy repo:'cd "conce r.fnt.ed lmruvtt..^ l'mi ioo^er'a 2 .U . -tri c: lerr ;>orr.c] . 1t la :.fynro- , .v.wevcx-, that f e aa-ipl* report^d c;i :> t : n * . -.-ve her- eure.:". *ated ir/rrK T , D0W"145318 n? . R. Reese July 12, 1956 from a asterl?..1 o: a hlSh on'er of purity, using 1 + 2 f c f 2,4,5-trichlcrr.phenol. I`. is reasonable to assume that any impurities that were In the submitted sample were present only In ninor amount in the criminal product thus reducing the possibility of its producing the skin effects observed in the first sample. The skin irritation tests on the two samples of impurities from tetrachlorcbentene have been completed also. The samples submitted were: K 2696-5 Residue from Dow 1,2,4,5 tetrachloro- benaene. K 2696-6 Residue from Hooker 1,2,4,5-tetra- chlorobensene, When applied reportedly to '.he rabbit's skin, botn of these materials, z-. a 5# solution in Dtwanol 50E, caused only very alight irritation and slight to moderate exfoliation. A similar response was obtained when the Hooker l,2,4,S-`oli lorcbenzenc residue was applied as a 1J solution. This x-osetnae is typical cf 2,4,5 -trl- chiorcp: Ci.^1 t-:a level. Hence the impurities, as reprenerted *r. t..e; c two ".spies, should not affect mate rially the t07.lcc.l1 gical .-Japcr.se. of 2,4 ,5-trichlorophenol. 1 hep.': information proves uaeful to you. If 1 can be of any fui ; Ip, pleure let me know. 07 - T r tx ,x J c _ Mar',; A. Wolf < J ^ ' 5ioc.`emical Research. Department 12-634 fcoildlng KAW/mc Mr. R. Rwb* UNIT INTEX . . . . .. ' V-- V. Rffeota of akin teats of various lrgw ritlss fro* 2,4,5-trichlcrophenol and 1,2,4,5-tetrabenzene fX*os various souroes are given. SSjj:^ INTEX HEADINOS C.R.I. Name: Phenol! 2 .4 ,^ -trio h lo ro 7 laporlfcl-- from, Benzene 1 .2 .4 .5 -te tra o h lo ro -, TripimitihW from 2 ,4 ,5 -trlo h lc ro * -phenol Im purities from 1 ,2 ,4 ,5 -t-etrachlorobcnzane DISTRIBUTION Directon? of Research - R. Boundy Central Research Index Western Central Research Pil* Tfcxas Contral Research Index Blsphanol Plant - R. Reese Spectorscopy Laboratory - K. Bradley -S3 >3Vi I3 Vs It O A B U t A D O R M S D O W eH D IC O KAM CH M i n O P F IO B New Y O R K C I T Y SA N F R A N C ISC O P H IL A D E L P H IA LO S ANOCLCS W A SH IN G T O N SA IN T L O U IS C LK V SLA N O H OUSTON C H IC A G O SC A T T L K D C T R O IT BO STO N The Dow Chem ical Company MIDLAND MICHIGAN December 9 19*9 Dr. V. R. Veasey iUln Office K J Building VoJ1 mm* OB ^ 0*--3 Ofct o cn o cn Subjects Request for authorisation aot and sub-aot* toxicity tasta on dogs with 2,4-D and 2,4,5-f. (1) In preparing toxioologloal information sultabls for prasantatlon at tha FDA haarings on agricultural ehaaloals, we be lieve it would ba daslrabla to have lnforaatlon on tha acuta and sub-acute toxicity of 2,4-D and 2,4r5-T on dogs. This work will ba In addition to siallar work which wa have oonductaa on saallar laboratory animals and in addition to that which appears In tha literature. Tha advisability of conducting this work has bean dis cussed with representatives of tha Agricultural Salas Department and tha Legal Department. I (2 ) Wa have discussed this problem with Dr. V. A. Drill, sad of tha Department of Pharmacology, School of Medicine, Wayne diversity, Detroit, and have received word from him that he is willing to undertake this work. It Is our feeling that a fair price for the work we have in mind would ba about three thousand dollars (#3,000.00). (3) Wa therefore request that an authorisation for three thousand dollars be set up to oover the acute and sub-acute toxicity studies on dogs with 2,4-D and 2,A,5-T. (4) If you have any questions regarding this work we will be happy to discuss them with you. Sincerely yours. YIR>g sci W. W. Allen M. I. Putnam V. K. Rmwe Bloohemld* department 16364. DOW 797G69 i * 12oS < r i I T i rt 4. Acute and Semi-Chronic Toxicity of Silvex to Laboratory Rats 3 a Biochemical Research Department I The Dow Chemical Company L i i L L L L L L [_ I 1' 6'365  \ AOTTE AND SEMI -C"?.ONIC TOXICITY OF \ lXegrXi'CjRV RA' 'Biochemical Research' Department The Dow Chemical Company . Midland, Michigan D O H.t C n j i MATERIAL C < "Silvex" is a trade name that has been coined for the material known chemically.as 2(2,4,5 -trichlorophenoxy) propionic "" acid. The test substanceused in these studies was a 3 odium salt preparation'in the form .of a brown, rosin-like,'hygroscopic 3olid. c This material assayed 90$ acid equivalent and was shown to con-'. tain one molecule' of water of hydration. . `. SINGLE ORAL ADMINISTRATION ' Procedure . The toxicity of Silvex was determined when administered in single oral doses to young adult white rats o f both sexes from . the stock colonies of this laboratory. Aliquots of a 10$ solu tion of the acid (as the sodium salt) in water were fed by intuba tion. Eight animals (4 of each sex)' were used at each of 4 dosage levels differing.in quantity by a geometric ratio of 2. All sur viving animals were observed for about 2 weeks or until they had fully recovered from any loss of weight and'were gaining normally. ' .. Results _ The dooage-respon3 e data were treated statistically ac cording to the method of "moving-averages" described by Thompson. The LD5 0 for rats was found to be 0.50 g./kg. with 19/20 confi dence limits of 0 . 3 9 to'O . 6 5 g./kg. \ .. ' '. -- 16366 7 9 7 G7 1 SEMI-CHRONIC DIETARY FEEDING '' Procedure 'All-of .the rats used in'this work were progeny of stock obtained by thl3 laboratory from the Wistar Institute in 1938. ' These animals were maintained-on the stock diet from the time of O weaning until they were 45 days of age, when they were divided o according t o .body weights into well-matched groups o f 10 rats of each sex and. started on the control or experimental diets, 'The control and.basic ration used in .this investigation .was the usual stock-diet, employed in this laboratory.,- The .follow ing ingredients .are-used in the preparation of. this- stock .diet:-.' .Percent by Weight Whole wheat, freshly ground .' ;A 55.o Dried whole milk '. /. Dried extracted liver -: 26.5 12.0 Dried brewers' yeast Iodized table salt . Calcium carbonate '5.0. ' 0 : 5 1.0 The experimental diets were prepared by thoroughly mixing ' . , the finely ground test material with the .stock -diet'in amounts calculated to.give diets containing the equivalent of 1.0, '0.3, 0.1, 0.03, or 0.01.percent 2(2/4,5-trichlorophenoxy) propionic acid. . = ' Five rats of the same sex were caged together in wire . N. . bottom cages and were allowed, free' access to food and water at -.. - - i t - \ A . : ' , / , ' / / A ; ,.. " A-."'. 1 8 3 6 7 DOW 797G72 -3- ail tinea. All of the rate were-weighed twice a week for the first 30 days, and weekly thereafter throughout the remainder of ' * * a 9 0 -day experimental feeding period. Records were kept of body . weight, general appearance, and average daily food consumption 1 per animal. Whenever possible, failing'animals were sacrificed 1 when moribund and examined in an effort to ascertain the cause of impending death. At the end of the 90-day experimental period, representa tive groups of female rats were selected for hematological examlna tion. All of the surviving rats were then fasted overnight, weighed, killed by decapitation, and examined. . The lungs, heart, liver, kidneys, and spleen from all the rats.and the.testes from the male rats were weighed. Hematoxylln-and-eosin sections 0 ^ these organs, ,as well as from the pancreas and adrenals., were prepared for histologic examination.- 1 . 0 Percent ~ Results The diet containing the equivalent of 1% Silvex was not acceptable to the groups, of male-'and female rats that received it. Pood consumption for each sex averaged but 5.3 grams per rat per day compared to 'l6 . 0 g. for the control male rats and 1 2 - . 3 g. for the control femalcn . T.r.: became *rough and u n k e m p t ' in appearance. The"`average body weight records (see charts) showed a severe depression. Within 32 days six of the ten male rats and two of the ten female rats had died. `At this time, the surviving 16388 7 9 7 C7.3- I p. [ | animals were sacrificed and examined. Histopatholo%gic findings ware as follows: , (a) Male and Female Rats. Heart - Cloudy swelling of muscle fibers. Liver - Generalized cloudy swelling and granular degenera tion of parenchymal cell3 together with foci of necrotic cells especially portally. . :O $ . Kidney.- Cloudy swelling of tubular epithelium and vacuola- tion or epithelium cells of the convoluted (proximal and di::tal) tubules. .. . (b) Male Rats. Testes - Degeneration of the tubules with necrosis of germinal epithelium. . Histopathological examination of tissues from the control animals revealed no abnormalities. . G.R Percent The groups op rats chat received the diet containing 0.3 Silvex for 90 days exhibited a marked retardation of growth. Food consumption- records indicated that the animals were accepting the ration'but eatinr.; slightly less (12.1 g./rat/day 'for the males; L . 10.9 g. for the females) than the animals receiving the control diet. L' ' Four of the ten male rats and three-of the ten female L rats failed t'o survive the bf-day experimental`period. These deaths were believed' to be due`to spontaneous infections of-the L lungs or middle ear. The incidence of this type of illness was similar in the control and experimental groups. 16369 -5- o o w 797074 Terminal hematological values were normal (Table l). Organ weight studies (Table 2) revealed a large Increase in the average weight of the livers and kidneys for both sexes. H l s t o - ' pathologic examination revealed: (a) M a l e 'and Female Rats.- Heart - Slight to moderate fatty degeneration of the tsele fiber. Liver - Marked generalized cloudy swelling of the parenchymal cells. Foci of necrosis in the portal areas involv- . ; -ing 3 to 12 cells. Kidney - Degeneration of numerous uriniferous tubules. Vacuolation and cloudy swelling of the epithelium ' cells of many remaining convoluted and collecting tubules with accumulation of debris in their lumen . but without cast formation. Degeneration.of several glomeruli.. (b) ' Male Rats. Testes - Numerous degenerated tubules and degenerated germinal 'epithelium cells in other tubules but there was still evidence of spermatogenesis 0.1 Percent ' ,. -' . : -t. .The groups' of male and female rats ,maintained for'90; days on t h e .di e t .containing 0.1^ Silvex seemed normal as far as gross appearance, behavior, hematological values (Table l), and food consumption records were concerned. However, the male-rats ex- hiblted a moderate depression of growth (see chart). A suggestive, but not statistically significant, depression of growth was ob- served for-the :female-rats . Liver and kidney weights were in- creased significantly.' Histopathologic examination revealed: 16370 HOW 797G75 I r.' C 1 [ t" -I - 6- (a) Male and Female Rats. Heart - Very slight cloudy swelling and fatty degeneration in seme of the animals. Liver - Marked generalized cloudy swelling of the parenchymal cells. Foci of necrosis in the portal areas involv- ing 3 to 12 cells. Kidney - Degeneration of numerous uriniferous tubules. Vacuolation and cloudy swelling of the epithelium cells of many remaining convoluted and collecting tubules with accumulation of debris in their lumen but without cast formation. Degeneration of several glomeruli. 0.03 Percent The group of male rats that received the diet containing 0.03% Silvex exhibited a slight depression of growth, but the fe male rats grew essentially as well as the control animals (see charts). Liver and kidney weights each were increased signifi cantly in .the males, but liver weight only was affected in the female rats. Microscopic examination of the tissues revealed: (a) Male and Female Rats. Liver - Marked generalized cloudy swelling of the parenchymal cells. Foci of necrosis in the portal areas involv ing 3 to 12 cells. Kidney - Degeneration of numerous uriniferous tubules. Vacuolation and cloudy swelling of the epithelium, cells of many remaining convoluted and collecting tubules with accumulation of debris in their lumen but without cast formation. Degeneration of several glomeruli. 0.01 Percent The groups of male and female rats that received the diet containing 0.01^ Silvex for 90 days showed no evidence of adverse 16371 r>OW797G76 -6- (a) Male and Female Rata. Heart - Very, slight cloudy swelling and fatty degeneration . i n some of the animals. Liver - Marked generalized cloudy swelling of the parenchymal cells. Foci of necrosis in the portal areas involv ing 3 to 12 cells. Kidney - Degeneration of numerous uriniferous tubules. Vacuolation and cloudy swelling of the epithelium' cells of many remaining convoluted and collecting tubules with accumulation of-debris in their lumen . ' but without cast formation. .Degeneration of several, glomeruli. 0.03 Percent The group of male rats that received the diet containing 0.03^ Silvex exhibited a slight depression of growth, but the- fe-. male rats grew essentially.as well as the control animals (see ' charts). Liver and kidney weights each were Increased slgnifi- -. . cantly in the males, but liver weight only was affected i n 'the female rats.' Microscopic examination of the tissues revealed: (a) Kale and Female Rats. ) Liver - Marked generalized cloudy swelling of the parenchymal .. cells.' Foci of necrosis-in the, portal areas involv- . lng 3 to 12 cells. 'Kidney - Degeneration of numerous uriniferous tubules: Vacuolation and cloudy swelling of the epithelium . cells of many rer.u, i.r.ir.r*, convoluted and collecting . `tubules w i u . . . o ' . . . `..ton of debris in their lumen . buz w i t ; - . . . . . V ..zioo . Degeneration of several . j 1*wj* --. 0.01 Percent '.1 ' ; The groups of male and.female rats that received the diet containing. 0.01^ Silvex for 90 days showed' no-evidence of adverse 16372 OOW 797G77 O . . . '. effects as judged by growth (see charts), gross appearance, and behavior, mortality, or food consumption records. Organ'weights (Table 2) were normal except for slight increases in the average weight of the liver from the female rats and of the kidneys from the male rats. Klcroscopic examination revealed: (a). Kale and Female Rats. Liver - Very'slight oloudy swelling of some of the parenchymal cells in the periphery of the lobe, together with foci of necrosis of cell3 in a few portal areas. Kidney ^Male Rats)'- Very slight cloudy .swelling and vacuolatlon of epithelium cells of numerous convoluted tubules together with ac- .'cumulation of debris in the lumens of their tubules but without.case formation. SUMMARY ' Kale and female rats (10 of each sex per group) were maintained for 90 days on diets containing the equivalent of 0.0 (control), 0.-3, 0.1, 0 .0 3 ,. and 0.01 percent 2(2,^,5-trichlorophen- oxy) propionic acid (Silvex)'. The actual test material was in the form of the aonohydrate sodium salt. A similar group of rat3 that were offered a diet containing 'Vjo Silvex did not accept the ration satisfactorily and this portion of the experiment was terminated after 3 2 days.- Growth was retarded for the male rats at all levels ex- cept 0.0l, but for the females retardation occurred only at the 0 .3 ^ level. . -Terminal hematogical values were determined on groups of 5 female rats each from' the 0.3 and 0.l levels 'and normal results were obtained. 810i6iM O -a- ' QrSan weight studies showed significant increases in the average weights oi* the livers and kidneys from the male rats that received dietary concentrations of 0.3,-O.i, or 0.032. Kidney weights were increased also for the 0.01$ male animals. For the female rats, the average weights of the liver were increased for all groups, including the 0.01$ level, hut kidney weights were elevated only for the female rats on' the 0.3 or 0.1$ diets. Histopathologic examination revealed evidence of adverse effects, appearing predominantly in the liver'and kidneys, from rats of both sexes and at all levels of dietary concentration, . except.that the kidneys from the female rats .fed the lowest dietary concentration (0.01#) were not affected. .' . 16374 1 ' ': -9 - 1 '. l. 1 AVERAGE HEMATOLOGICAL VA LUE3 FROM FEMALE RATS THAT RECEIVED DIETS CONTAINING'SILVEX FOR A .* ' . 'PERIOD 0? 90 DAYS Determination ' Average Value Per Grouo of 5 Rat3 Control , 0.3^ ' O.K T o t a l 'rythrocytes , (X10) 3.93 ..8.99 ..V. r -8.57 I .1 J 11- 1^ Total Leucocytes ; ' 18.8 ... (X103) Hemoglobin (g./lOO c c .) 13.2 . Diffe-rential Count Neutrophiles % Lymphocytes ' % Monocytes Eosinophiles .2 1 . 8 ' . .74.4' " 1 .2 2.6 17.1.;, . 19.1 ' 13.1 : ' 1.3. C'. i 1 3 . 8 . ; 27.4 84.8 ' 70; 0 0 .8 ' -: 0.8 " 'o.6' ' : '1 . 8 I:,.; - ] -, J-.; L. .. . .. l o ; CD O <E '.CD'- 'L I 16375 N- 1 L 1, l l 9 . TABLE 1 . .' ALTERAGE HEM ATOLO GIC AL VALUES PROM PEMALE RATS THAT RECEIVED DIETS CONTAINING'SILVEX FOR A 'PERIOD OP 90 DAYS' , Determination 1~ ' " " '' ' Average Value Per Group of 5 Rata Control 1: 1 ~0.3"^ 1 I . 0.1^ T o t a l 'rythrocytes (X10) 3.93 8.99 .. ; -8.57 Total Leucocytes > . . 18.8 ( x io 3 ) _ . Y.-' 17.1 ' 19.1 Hemoglobin . (g./lOO c c .) 13.`2 ' '13.1 - 13.0 . : Differential Count . , . 'V /. , Neutrophiles V .21.8 ' % Lymphocytes 74.4 Monocytes " 1.2 v % .Eosinophiles 2.6 .13.8 . 84.8 0.8 0.6 V;. _ . 27.4 . 70;0 . : 0.8 1.8 oJ -GE CD -si ct 00. " ., / 1I c J- > -..io - TABIJ? 2 AV/OiAOK FINAL BODY AHI) (iRfl/iH v:2ICrKTS F RGtl MALET ARD V X l AIAi M T S .. THAT K crCKIViD M E T S CO;iTAijiiija S3CL72X V O R A F2 KI0 V) OF 9 0 LAYS O *O1-' Percent in Diet .i Ruto . Sex.>'>Jvft;.bViftf..Bod(y.) Ore;.';') Vs X t t i ( & 2 S * L J L . body ut. ) Lun Heart Liver I i a y Spio v'ivi 'To ate-s Contro]. : 0.3 . 0.1 0.03 .0 .0 1 \ .-Control .0.3 " " 0.1 0.03.' 0.01 6 6 '9 9 .9 . H. K : K. - Fi H '.9 ' ;F . 7 ' F ' . 8 .> 10 * .. F-. 10 . F /; 359 . :;`.?oi, 263 3 1 0 *. 3^2 208 ; ISS I9 2 .' I96 209 0.50 0.69 0.61 0.58 0.51 . O.31 0.38 .0.37 O.31 ; O .30 0.66 0 . 3 6 0 . 7 8 0.i*3 0.62 O.30 0 . 7 0 . 0.35 . 7 1 0 . 3 6 2.45 5.98 5.3 '0 .62 ;o . 8 9 0.78 3.15 . : 0-75 2.46` .0.72** 2.46 5.72 4.78' 3.19 2.70* O .69 '0 . 8 1 0 :7 6 * 0.12 0.72 .- ; .. b ;r p **.0.01 to 0.0p - ': p <0.01 j as calculated by "tV test. 0.24 . O.32 0.22 0.31 O .26 .0.83 I.32 1.02 '0 . 9 1 '0.67 0.30 0.30 .0,29 0.29 0.27 - ai ^ as 1 8 9 6 y ^ o o CO -asr \I : . , /. -- :'I'. 'i ' :. f: I *: : t: ;. :i ........................ ................ .."..t...".:.:.!" . ATESAOS OD3T i-jsiGH2:'cD?.VES= FOR'GROUPS ' i U L l U i U | :0>;: : ii.-i:!-:o?.m a l e ;e a t s ';t h a t .vrEio :f o :V-5 0 :'d a y s ';:i...-i - r a f r : ----------O--l-----D--I--E---T---S---;-C---C---E--I--A---I--I--J--I-M----S------S---Z----L---l--^----_S O;__D__I_L__a:__S__A_L__T i- ---- : : ! .... i i- : i :M v . : .! j : ;' -, : : ; " ," : , ;- v r :- 7 - T .7 r .v ;.r ;;* * ;:::;^ :' V - u " - * r : r T r ; ' : : ^ I *U *j .. ' *J **' j * . . , ' * 1* 1 . i * .u i:; i . : '. . . L iU ::l- u -i-":: O i : 1t;'!: j;;r;^ ir;Z 1:j! :1: f ;!:jj'" ;v :i~ j'j.U l ;:.v;.j;; :J. :..j i :j |;;^.T.:; [::: !f ........t I-- ...--- -------1U .- 1 .1 * -- * ***-- .-- ..i-- ; ` --- n iq i'u UvHUR:ilFrS':;TF-::]V:!^ *i;: V-'-':iuu ;;-! ?' ;'j; ` ';*"* !".: ! ': !'-,j : []2.}lvL!I1:?:^1::IlKILIi_''i :i!';V:::i , i:* :i . 1*t ,* "!-} tiI liv:n:rd- ic:Jv^T-Ci: lu ir u t. !; \y..r _uV--> i,ir-J..r,,s.l : "Ie : Ii.PI P. i jHTf's:in!t* * ; :r * r |T;r'Fi:*:r:jTr.nincfHrrr'T*pi*; . : r in?rl-;I p q l a^ jfe o l i:S1k?inrH%r<d/f$>H';;iS Pj--jp LULL!!: b X .. &* y I ; v j-v \ y : L U - ' - ' l ' - v ! : : : j i, ; T- : : i . ' !: 1 -. I ........ U r ;200 . !:: !' : :!iui !IIS ;!':ii5:ii!:!i!:::' :i:'ii. U ^U lS O i:' ! ' - / V L -!; *'I i " i ' 1 ' : 1' 11 ...i.-.. LU*:,, y.,C- V. f ' I I 160 l- !.. t*A .j.: I< r " '| ^ ^ ^ p i p T - ` ! ; U ;u ':^ * ; ! i*: r r ~ r . __il jrsl--a ::-rPYEonRnCT?E\iNn%T ?. i r -EGEND -TT^illjTlu-j U ; A -- ^Control-- -r- ---- i-- -i 1; !i*: :*| i iv:j;);-Lr:;l:!f:L;:l zUiu!1 ; ' "h o' | : i<22o0 . ! "ICO : \ - : I l U m ''U'^'U i'...'::. u !E i .:'!u.'.! . 1i "j. Ti-''.U;'ii:':rTi--ii:1K!`r-J-'-;:Tli|';^!'r:I'aT;;;u:::;3.i!-1:::::^1T*;;;''::-'1:.;;L1}i5*-`':A-.";!I:^*tUk!!}1i_:::iT:;i-:::!I`LL:i*u-":d.!!1`til?`i.:i.'^':'i!:';u:ui ;i~!I.: i O" I .O ;`'5 C>n :rU:. -'U :.U.l.r 5: - '^V!7 7" o .j:;: .0 -** 6<V?T*.2***VT-: vVi ) pll" ! p T F P - T :'-'A;?^ 6 3 7 8 16379 ( ] I r 3 .1 1- - r 1 L 1 L L L 2. Results of 90-Day Dietary Feeding of Propylene Glycol Isobutyl Ether Ester of Silvex (Dowco 171) to Rats. Biochemical Research Laboratory. The Dow Chemical Company, Midland, Michigan. June 19, 1962 D0 \y 797684 Toxicology. 9 (1978) 7 5 -8 6 O Elsevier/North-Holland Scientific Publishers Ltd. W -' j a i STUDIES ON 2,3,7,8-TETRACHLORODIBENZO-n-niOXIN-TNnnrEn IMMUNE SUPPRESSION AND DECREASED RESISTANCE TO INFECTION: ENDOTOXIN HYPERSENSITIVITY, SERUM ZINC CONCENTRATIONS AND EFFECT OF THYMOSIN TREATMENT J.G . VOS, J.G . KREEFTENBERG, H.W.B. ENGEL, A. MINDERHOUD and L.M. VAN NOORLE JANSEN National Institute o f Public Health, P.O. Box 1, Bilthoven (The Netherlands) (Received August 23rd, 1977) (Accepted October 3rd, 1977) UN0 7D202W83O686 133 SUMMARY Sublethal doses of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) cause thymus atrophy in several species and induce suppres sion of cell-mediated immunity as measured by different parameters. The selective effect on thymus is not likely caused by a cytotoxic effect on lymphocytes or by an effect on pituitary or adrenals. In the present study, mice received daily injections with thymosin in order to study whether reduced production of thymic hormones could be involved in the atrophy. Also, serum zinc concentrations were measured. As thymosin injections did not increase thymus weight and mitogenic responsiveness of thymocytes, and zinc levels were not depressed, the mode of action of TCDD-induced thymus atrophy remains unknown. It has been reported that TCDD marked ly decreased resistance of mice to infection with Salmonella bern. In this study, data are presented that indicate that the increased susceptibility is likely due to the endotoxin content of the bacteria: pre-treatment of mice with single or repeated doses of TCDD markedly enhanced their suscepti bility to endotoxin even at dose levels that did not produce thymus atrophy. Finally, possible effects of TCDD on macrophage functions were studied. As treatment with TCDD did neither impair non-specific killing and phagocyto sis of Listeria monocytogenes, nor macrophage reduction of nitro-blue ttra zolium, it seems likely that the immunosuppression is only due to a, to date unknown, effect on T-lymphocytes, and not due to a combined effect on both T-cells and macrophages, and thal the endotoxin hypersensitivity is not the result of alteration in phagocytic function of macrophages. INTRODUCTION 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a chemical that can be 75 D0WO686 134 formed in small, but significant, amounts in the production o f some chlo rinated phenols or chemicals synthetized from chlorophenols, such as the herbicide 2,4,5,-trichlorophenoxyacetic acid. It is one of the most toxic chemicals known [1 ], it is a teratogen [2 ,3 5 ], it is associated with chick edema disease in chickens [4] and with several industrial incidents resulting in cases of chloracne in man [5 ,6 ]. TCDD exposure has been shown to induce thymus atrophy in rats, mice, guinea pigs [7--12] and monkeys [McConnell and Moore, in press]. Thymus atrophy appeared to be one of the most consistently sensitive parameters o f TCDD exposure. Since lympho cytes derived from the thymus (T-cells) play a central role in cell-mediated immunity, several funciton studies of the thymus-dependent immunity have been carried out in mice, rats and guinea pigs. TCDD exposure did result in functional impairment of diffferent parameters of cellular immunity (delay ed type hypersensitivity, allograft rejection, graft-versus-host activity and responsiveness of lymphocytes to the mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) [11,13, Moore and Faith, in press] but the suppression o f the cell-mediated immunity seems to be an age-related pheno menon, at least in the mouse and the rat where TCDD exposure during the ontogehesis o f the immune system seems to be pre-requisite in order to obtain immune suppression. TCDD mimics in this respect the effect of neonatal thymectomy. Since the TCDD-induced atrophy of the thymus can be explained in many ways and no clear-cut mode of action has been described, we were interested to examine whether a reduced production of thymic hormones by thymus-epithelial cells could be involved. Therefore, in the present study, the effect of daily administration of thymosin on TCDD-induced thymus atrophy was studied. As shown by Thigpen et al. (fl4j), TCDD exposure markedly decreased the resistance of mice to infectionWith Salmonella bern, whereas TCDD has no significant effect on mortality in pseudorabies virusinfected mice. In the present study, data are presented that indicate that the increased susceptibility to Salmonella infection is very likely due to the endotoxin content o f the bacteria. Since on the one hand zinc deficiency can result in severe atrophy of lymphoid organs [15,16] and on the other hand parenteral administration o f zinc increases the resistance of mice to endo toxin [1 7 ], serum zinc concentrations were also measured in TCDD-exposed mice. Finally as macrophages play an important role in the immune response and in the detoxification of endotoxin, function studies o f macrophages (non-specific phagocytosis and killing of Listeria monocytogenes and reduc tion of nitro-blue tetrazolium by macrophages) were also included. MATERIALS AND METHODS TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin (purity 98.6%, Lot No. 851:144-11, 76 16382 kindly provided by Dow Chemical Company, Midland, Mich., U.S.A.) was dissolved in reagent grade acetone at a concentration of 50 /ig/ml. For the animal experiments, this stock solution was diluted with at least 9 parts of arachis oil to provide the different concentrations. Mice were intubated orally with 0, 1.5, 5 ,1 5 , 50 or 100 fig TCDD/kg body weight. Animals Specific pathogen-free outbred Swiss mice were reared at the Institute. They received food and water ad libitum. Because of the high toxicity of TCDD, all animal experiments were carried out under non-sterile conditions in plastic Trexler isolators, and all contaminated material was incinerated at temperatures over 800 C (N.V. Roteb, Rotterdam, The Netherlands). Zinc analysis For the determination of zinc in serum, an atomic absorption spectro meter with acetylene-air slot burner and zinc hollow-cathode lamp was used. Fifty ^1 o f serum was diluted with 500 fil of distilled water. After mixing thoroughly 100 il o f the sample was aspirated into the flame and the result ing atomic absorption signal was recorded. Peak heights were compared with results obtained from the aspiration of aqueous standard solutions under the same conditions and zinc concentrations were calculated. Experiment with thymosin The effect o f thymosin injections on TCDD-induced thymus atrophy was studied in mouse pups that were exposed to TCDD by postnatal maternal treatment. At birth the number of neonates was standardized to 6. Groups of 5 nursing mice received 0 or 10 fig TCDD/kg body weight (in a volume of 0.1 ml/30 g), postnatally on days 1, 4, 8, 11, 15 and 18. Calf thymosin (fraction 5, Lot No. BPM 390, kindly provided by Dr. A.L. Goldstein, Galveston, Tex., U.S.A.) was dissolved in sterile saline at a stock concentra tion of 15 mg/ml, passed via Millipore bacteria filter and aliquots stored at --20 C. Just prior t<^ injection, a dilution (1 mg/ml) was made in sterile phosphate-buffered saline (PBS). In the first week, groups of 2 pups per litter received daily subcutaneous injections of a high (50 fig) or low (15 fig) dose of thymosin. In addition, 3 pups per control litter received PBS. In the second and third week, the doses were doubled and tripled respectively. The endotoxin content o f the thymosin (1 mg/ml) solution was estimated with the Limulus assay [18] and contained between 12.5 and 62.5 ng endotoxin/ ml. After the 22-day experimental period, the animals were killed. Zinc concen tration was measured in serum. The thymus of half of' the number of mice was removed under sterile conditions. Cell suspensions were made by using a glass homogenizer, the viability was determined with a dye exclusion test and the final suspension 77 D0W06861 was adjusted to a concentration of 3 X 106 viable nucleated cells/ml in minimum essential medium (MEM, Gibco F14) buffered with Tris (0.025 M) and supplemented with 20% inactivated fetal calf serum, streptomycin (100 Mg/ml) and penicillin (100 IU/ml). A total volume of 1 ml was cultured in polystyrene tubes (Nunc, Denmark) and 12.5 pi reconstituted phytohemag glutinin (PHA, Wellcome Labs., Beckenham, Kent, U.K.), 6 pg concanavalin A (Con A, Pharmacia, Uppsala, Sweden) or 50 pi reconstituted pokeweed (PWM, Gibco) were added. After 24 h cultivation, 10 pCi of [6-3H]thymidine (specific activity 600 mCi/mmole) was added. Twenty-four hours later the cells were harvested on glass fiber filters using a multiple cell culture harvester (Skatron, Lierbyen, Norway). After drying, the filters were placed into scintillation vials and 2 ml of scintillation fluid were added. Counting was done in a Packard liquid scintillation counter. Results (median value of triplicate cultures) were expressed as the difference between stimulated and non-stimulated cultures (in counts/min (cpm) per 3 X 104 cells). Experiments with endotoxin In 3 experiments, the effect of TCDD on the susceptibility of mice to endotoxin was studied. Since there is no major difference in the biological activity of endotoxins of Salmonella and E. coli species, E. coli endotoxin was used in the present study. Endotoxin (lipopolysaccharide, E. coli, O 127: B 8, Difco Labs, Detroit, Mich.) was dissolved in sterile saline. In two studies, 3--4 week-old male Swiss mice were intubated orally, once a week for 4 weeks, with 0, 1.5, 5, 15 or 50 pg TCDD/kg (0.1 ml/10 g body weight). Two days after administration of the final dose of TCDD, the mice were injected intravenously with 10, 20, 100, 250 or 500 pg endotoxin in 0.1 ml. The mortality due to endotoxin shock was scored after 48 h. Surviving animals were killed and examined macroscopicaRy. In the third experiment, 3--4 week-old female mice received a single oral dose of 0 or 100 pg TCDD/kg body weight (0.2 ml/10 g). Animals were challenged with endotoxin 5 days after intubation. In the second and third experiment, additional animals were intubated and killed to determine the effect of TCDD on body weight and on the weight of thymus and spleen. Also, serum zinc concentrations were measured in the third experiment. CO CD Clearance o f Listeria monocytogenes The resistance of Listeria monocytogenes is a combination o f non-specificphagocytosis and cell-mediated immunity: non-specific phagocytosis and killing can be measured shortly (within 2 days) after the intravenous inocula tion of Listeria monocytogenes [19]. Male Swiss mice (3--4 weeks old) were intubated orally, once a week for 4 weeks, with 0 or 50 pg TCDD/kg body weight. Four days after the last intubation the animals were inoculated intra venously with 2.4 X 103 Listeria monocytogenes organisms (Strain L 242/73 type 4b). One or two days after the injection, the spleens were weighed. 78 1638( DOW0686137 homogenized and serial dilutions were plated to determine the viable counts of Listeria [2 0 ]. The number of bacteria was expressed as 10log/spleen. Reduction o f nitro-blue tetrazolium Lymphokine activated macrophages show increased glucose oxidation via the hexose-monophosphate pathway; an increase that can be quantitated by the reduction o f soluble nitro-blue tetrazolium (NBT) to insoluble formazan. Krueger et al. [21] demonstrated a correlation between delayed type hyper sensitivity skin tests and NBT reduction by lymphokine-activated macro phages. Similarly Engel and Sekhuis (unpublished data) showed an increase in NBT reduction in macrophages of mice that were injected with Corynebacterium parvum, without the addition of lymphokines to the culture chamber. In the present experiment, the effect of TCDD on NBT reduction of macrophages was determined in order to study an effect on the baseline activity of non-activated macrophages. For that purpose, 3--4-week-old male Swiss mice were intubated orally, once a week for 4 or 5 weeks with 0 or 50 pg TCDD/kg body weight. Five days after the last intubation, peritoneal macrophages were harvested in 2 washings with 2.5 ml Eagle's Basal Medium (Gibco, G 13) supplemented with 20% inactivated calf serum, 40 mM L-glutamine and 5 U heparin/ml. Of this suspension 0.5 ml was added to an 8-chambered tissue culture slide and incubated at 37 C in 10% CO? in air for 2 h. At this time chambers were washed 3 times to remove non-adherent cells. Of each mouse, one chamber was used for the identification of macro phages (Giemsa stain); the other was used for the NBT assay according the technique described by Krueger et al. [2 1 ]. One hundred cells were indi vidually scored at 1000 X on the quantity of formazan precipitate as a measure of NBT reduction (no precipitate = 0; slight precipitate = 1; mode rate precipitate = 2; strong precipitate = 3). Results were expressed as the sum of the scores per 100 cells. Statistical analysis Student's t test was used to make one-sided treatment--control compari sons. RESULTS The effect of daily thymosin injections on body and thymus weights of 22-day-old mice is given in Table I. Body and thymus weights of the 3 TCDD groups were significantly lower when compared with the control group. Treatment o f TCDD-exposed pups with thymosin significantly reduced body weight, but did not alter thymus weight. Serum zinc concentrations were the same in control and TCDD-exposed pups. The responsiveness of thymus cells to the mitogens PHA, Con A and PWM is presented in Table II. In general, the response o f the TCDD groups to these mitogens was approximately half 79 D0W0686138 TABLE I BODY AND THYMUS WEIGHTS' AND SERUM ZINC CONCENTRATIONS OF 22DAY-OLD TCDD-TREATED MICE THAT RECEIVED DAILY INJECTIONS WITH THYMOSIN* Dose of TCDD (Mg/kg) 0 10 10 10 T hym osin treatm ent 0 0 low doses* high doses* Body weight (g) 13.1 t 0.9 12.4 * 0.8b 11.2 1.0*-d 11.2 i 1.2*,d Thymus weight (8) 85 5 60 t 10* 60 t 10* 58 t 7* Zinc (mg/1) 1.1 0.2 1.2 --0.6 * Mean values t S.D. (b o d y w eight 8--10 animals, thym us weight and zinc analyses o f 4--5 animals). Mice were exposed to 0 o r 10 Mg TCDD/kg by m aternal treatm en t postnatally on days 1, 4, 8 ,1 1 , 15 and 18. 11 Significantly (P < 0.05) d ifferen t from control group (no TCDD and no thym osin). * Significantly (P < 0.001) different from control group (no TCDD and no thym osin). d Significantly (P < 0 .0 2 5 ) d ifferen t from thym osin control group (10 Mg TCDD and no th ym osin). * For schedule see text. o f the response seen in the control group. Since the results are expressed on a cell-for-cell basis and thymus weight was reduced by TCDD treatment, the total response per thymus is likely even more decreased. Thymosin treat ment of TCDD-exposed mice did not result in a significant increase in the mitogenic responsiveness of the thymus cells. The results o f the first two experiments with endotoxin are given in Table III. Pretreatment of mice with TCDD markedly enhanced their susceptibility TABLE II MITOGENIC RESPONSE OF THYMUS CELLS FROM 22-DAY-OLD TCDD-TREATED MICE THAT RECEIVED DAILY INJECTION WITH THYMOSIN* Dose of TCDD (Mg/kg) T hym osin treatm ent A PHA (cpm) A Con A (cpm) A PWM (cpm ) 00 19,001 10,774 20,895 t 6,517 6,494 t 2,647 10 0 8,097 1 2,731b 10,779 6,345b 2,108 * l,879b 10 low doses* 9,985 i 8,248 14,616 4,989 3,714 t 1,485 10 high doses* 11,878 t 4,677 11,244 i 2,595b 2,542 i l,1 3 5 b * Mean values S.D., 4--5 animals per group. Mice were exposed to 0 o r 10 Mg TCD D /kg by m aternal treatm en t po stn atally on days 1, 4, 8, 11, 15 and 18. Results are expressed as the difference betw een stim ulated and non-stim ulated cultures (In cpm /3 X 10* cells). b Significantly (P < 0.05) different from control group (no TCDD and no thym osin). e For schedule see text. 80 1638S DOW0686 139 TABLE UI EFFECT OF 4 WEEKLY DOSES OF TCDD ON THE SUSCEPTIBILITY OF MICE TO ENDOTOXIN Dose of TCDD (Mg/kg)* 0 1.5 5. 15 50 0 1.5 50 Incidence o f m ortality11 10 Mg endotoxin 250 Mg endotoxin 0/4 0/5 0/4 1/5 0/4 6/6. 1/4 6/6 2/4 NDC 10 Mg endotoxin 100 Mg endotoxin 0/6 0/6 0/6 1/6 2/5 5/6 500 Mg endotoxin 2/63/6 6/6 1 In tw o experim ents, male mice (3--4 weeks o f age) were intubated orally, once a week for 4 weeks, with TCDD in acetone-arachis oil carrier. b Intravenous injection with endotoxin (E. coli. LPS) tw o days after last intubation. Mor tality scored after 48 h. 6 ND =not done. to endotoxin. After the injection of 10 Mg endotoxin, 2 out of 4 animals died in the 50 Mg TCDD group and 1 in the 15 Mg TCDD group. Injection of 250 /ig endotoxin was lethal for all mice in the 5 and 15 Mg TCDD groups and caused one death in the 1.5 Mg TCDD group, whereas all controls surviv- TABLE IV EFFECT OF 4 WEEKLY DOSES OF TCDD ON BODY AND ORGAN WEIGHTS OF MICE* Dose of TCDD (m g/kg) Body weight (g) Thymus weight (mg) Spleen weight (mg) 0 30.7 i 2.2 66 - 20 106 r 13 1.5 28.7 : 2.5 65 : 12 97 s 23 50 26.0 r 2.3C 34 t 10 89 t 8b * Mean values r S.D., 6 animals per group. Male mice, 3--4 weeks o f age, were intubated orally, once a week for 4 weeks, with TCDD in acetone-arachis oil carrier. b P < 0.025. c P < 0.005. 81 16387 TABLE V EFFECT OF A SINGLE ORAL DOSE OF TCDD ON BODY AND ORGAN WEIGHTS AND ON SERUM ZINC CONCENTRATION OF MICE*, AND ON THE SUSCEPTIBI LITY TO ENDOTOXIN Dose of TCDD Org/kg) 0 100 Body weight* (g) 19.3 i 1.8 18.7 t 2.4 Thym us weight* (mg) 70 t 15 51 * 14b Spleen weight* (mg) 120 i 23 97 t 19b Zinc* (mg/1) 1.0 t 0.2 1.4 t 0.5 Incidence o f mortality * 20 Mg endotoxin 100 ug endotoxin 500 Mg endotoxin 0 NDd 100 5/5 0/5 5/5 5/5 ND * Mean value i S.D., 7 animals per group. Female mice, 3--4 weeks of age, were intubated once orally w ith TCDD in acetone-arachis oil carrier and killed after 5 days. b P < 0.05. c Intravenous injection with endotoxin (E. coli, LPS) 5 days after intubation. M ortality scored after 48 h. d ND = not done. ed. In the second experiment, animals were given 0, 1.5 or 50 g TCDD/kg body weight and were challenged with 10, 100 or 500 fig endotoxin. The 10 fig dose of endotoxin caused 2 deaths out of 5 animals in the 50 fig TCDD group, and a similar mortality (2 out of 6) was scored in the controls injected with 500 fig endotoxin. Thus, the susceptibility to endotoxin was approximately 50-fold enhanced in the 50 fig TCDD group when compared with the controls. Even at the 1.5 fig TCDD level there was a slightly increas ed mortality after injection of endotoxin. This latter dose of TCDD being more than one order of magnitude lower than the dose that caused thymus atrophy at gross inspection. As shown in Table IV, body weights and weights of thymus and spleen o f mice of the 50 fig TCDD group were significantly decreased, whereas these parameters in the group intubated with 1.5 fig TCDD/kg body weight did not differ from the controls. A single oral dose of 100 fig TCDD/kg body weight significantly reduced thymus and spleen weight o f female mice, killed after 5 days; but body weights and serum zinc levels were not altered (Table V). Also, this treatment enhanced the suscepti bility to endotoxin: a 100% mortality occurred in the TCDD-exposed animals after injection with 20 fig LPS, whereas a similar mortality was seen in controls receiving 500 fig LPS. The results of the study of the effect of TCDD on non-specific phagocyto sis and killing of L. monocytogenes are given in Table VI. The number of viable Listeria organisms, determined one or two days after inoculation, of this 82 D0W0686140 j DOW0686H 1 TABLE VI EFFECT OF 4 WEEKLY DOSES OF TCDD IN MICE ON BODY AND SPLEEN WEIGHTS AND ON DAY 1 AND DAY 2 SPLEEN COUNTS OF LISTERIA MONOCYTOGENES* Dose of TCDD (^g/kg) Body weight (g) Spleen weight (mg) Listeria countb ( lolog/spleen) Day 1 p.i. 0 50 / Day 2 p.i. 0 50 23.4 i 1.4 21.7 * 0.6C 21.7 1.5 21.2 t 2.4 140 i 10 96 18d 178 23 124 29d 4.89 0.40 4.58 0.24 5.13 0.31 5.08 0.27 * Mean values t S.D., 5 animals per group. Male m ice,3--4 weeks o f age, were intubated orally, once a week for 4 weeks w ith TCDD in acetone-arachis oil carrier. b Animals were inoculated intravenously with 2.4 x 101 Listeria monocytogenes organisms 4 days after the last intubation; the num ber of viable Listeria organisms per spleen was determined after 1 and 2 days. L' p < 0.05. d P < 0.01. organism, was the same in spleens from treated and control mice. Spleen weights were significantly lower in TCDD-exposed animals. In addition, body weights of mice killed one day after inoculation were significantly reduced in the TCDD group. Finally, as shotvn in Table VII, in two experi ments TCDD did not alter the number of peritoneal macrophages, nor did it cause a significant effect on the macrophage reduction of NBT. TABLE VII EFFECT OF 4 O R 5 WEEKLY DOSES O F TCDD IN MICE ON NUMBER OF PE R I TONEAL MACROPHAGES AND ON MACROPHAGE REDUCTION OF NBI* Dose of TCDD (pg/kg) No. of peritoneal m acrophages (x 10*) Reduction of NBTb F500o u r doses 1.94 >. 0.68 2.23 s 0.35 32.2 * 22.2 37.0 > 8.9 0Five doses 50 1.81 - 0.54 2.08 -. 0.31 23.8 17.1 57.0 -14.0 " Mean values S.D., 4 --5 anim als p e r group. Male mice, 3-- l weeks o f age, were in t u b a t ed orally, once a week for 4 or 5 weeks w ith T CD D in acetone oil carrier. h For details see text. 83 -7 Vlyl ?0'Q MQ'Q.- I DISCUSSION From the present results it can be concluded that thymosin treatment of mice at the employed doses does neither protect the animals from thymus atrophy nor from a reduced mitogenic responsiveness of thymocytes. The presence of a minor quantity of endotoxin in the thymosin solution could not conceal a possible increase, since treatment of mice with E. coli LPS has been shown to increase the PHA and Con A responsiveness of thymo cytes [2 2 ]. Thus, it seems unlikely that the depletion of lymphocytes in the thymus, as induced by TCDD, is caused by a reduced production o f thymic hormones, since calf thymosin has been shown in several studies to potenti ate various parameters of thymus-dependent immunity [2 3 ]. Neither seems zinc deficiency to be a cause for the thymus atrophy. A direct cytotoxic action of TCDD on lymphocytes is also unlikely, since the mitogen respon siveness of mouse or rat lymphocytes was not reduced when cultured in the presence of TCDD at concentrations up to 0.02 Mg/nil medium [13]. Even the presence of 0.32 fig TCDD/ml medium has been reported to be not toxic for human lymphocytes [2 4 ], whereas concentrations that are two orders of magnitude lower do induce aryl hydrocarbon hydroxylase activity in human lymphocytes [2 5 ]. In another study [26] a cytotoxic action of TCDD at a concentration o f 0.48 fig/ml was reported for several mammalian cell types. These results make it unlikely that TCDD does need metabolic activation, but they do indicate that the lymphocyte depletion of the thymus and the thymus-dependent lymphoid tissue are not caused by a direct effect on lymphocytes, but rather by an indirect way. There are several conditions that can cause thymus atrophy [2 7 ]. Regarding the selective effect of TCDD on thymus, it is not likely caused by an effect on pituitary, on adrenals or by reduced food intake, since thymus atrophy did occur in TCDD-exposed rats that were hypophysectomized or adrenalectomized and in rats that were placed on a restricted diet [Van Logten, Gupta and Moore, in preparation] though it is still possible that TCDD may impair the absorption and trans port of certain nutrients. In addition, serum a-fetoprotein levels are not elevated in TCDD-exposed rats [Vos and Boekestein, unpublished data], thus the observed immunosuppression is not due to a a-fetoprotein induc tion. In conclusion, the mode of action of the TCDD-induced thymus atrophy remains unknown. The results o f the present experiments with endotoxin show a marked in crease in susceptibility to endotoxin of mice treated with a single or with repeated doses of TCDD. This sensitizing effect of TCDD for endotoxin offers a logical explanation for the decreased resistance of TCDD-exposed mice to infection with (endotoxin-containing) Salmonella bacteria as observ ed by Thigpen et al. [1 4 ]. Another explanation for the increased suscepti bility to this type of infection, which is primarily dependent upon cellmediated immunity [28] is by general suppression of the thymus-dependent immunity. This explanation is, however, less likely since increased mortality by Salmonella occurred at dose levels of TCDD that did not produce thymus 84 163H0 atrophy. The endotoxin hypersensitivity, which has to date only been studi ed in mice, is a phenomenon of particular interest when considered in connection with: (1) terminal hemorrhages that were seen in TCDD-exposed rats and guinea pigs [8,29] and mice [1 2 ,3 0 ], (2) thrombocytopenia,, observed in rats and guinea pigs [10,31], and (3) presence of thrombi in blood vessels of heart and lung in the rat after a lethal dose of TCDD [7,8]. These lesions are also seen in endotoxin shock and since TCDD, at least in the mouse, also induces endotoxin- hypersensitivity, one has to consider endotoxin shock as a possible cause of death. Regarding the mechanism of endotoxin hypersensitivity, several possibilities exist (see review of Cook et al. [32]). With regard to the endotoxin hypersensitivity produced by TCDD it would be worthwhile to investigate the role of the enzyme glutathione-S-transferase B, a cytosolic glutathione-conjugating enzyme, since sulfhydryi containing enzymes are necessary for endotoxin inactivation. TCDD has been reported to increase the activity o f both hepatic and renal glutathione-S-transferase B [3 3 ]. Finally, as treatment o f mice with TCDD did neither impair the non specific killing and phagocytosis of L. monocytogenes nor the macrophage reduction o f NBT, it seems likely that the TCDD-induced suppression of the cell-mediated immunity is only due to a, to date unknown, effect on T-lymphocytes and not due to a combined effect on both T-cells and macro phages. Similarly, the results of the experiment with L. monocytogenes indicate that the endotoxin hypersensitivity is not the result of alteration in phagocytic function of macrophages but, as discussed in the previous paragraph, perhaps due to impaired endotoxin detoxifying properties of macrophages. REFERENCES 1 B.A. Schw etz, J.M. Norris, G.L. Sparschu, V.K. Rowe, P .J. Gehring, J.L . Em erson and C.G. Gerfaig, Environ. H lth Perspect., 5 (1973) 87. 2 K.D. C ourtney and J.A . M oore, Toxicol. Appl. P harm acol., 20 (1 9 7 1 ) 396. 3 D. N eubert, P. Zens, A. R othenw allner and H.J. M erker, Environ. Hlth Perspect., 5 (1973)87. 4 D. Firestone, Environ. H lth Perspect., 5 (1973) 59. 5 K.D. Crow , Trans. St. Jo h n 's D erm atol. Soc., 56 (1970) 7 9 . 6 J. Kim mi g and K.H. Schulz, D erm atologica, 115 (1 957) 54 0 . 7 N.P. Buu-Hoi, P.H. Chm.ii, G. Sesqufe, M.C. A zum -Gelade and G. Saint-R uf, N atur- wisa., 4 (1972) 174. 8 B.N. G upta, J.G . Vos, J.A . M oore. JG . Zinkl and B V. Bullock, Environ. H lth Per spect., 5 (1973) 125. 9 M.W. Harris, J.A. M oore, J.G . Vos and B.N. G upta, Environ. Hlth Perspect., 5 (1973) 101. 10 R.J. Kociba, P.A. Keeler, C.N. Park and P.J. Gehring, Toxicol. Appl. Pharm acol., 35 (1976)553. 11 J.G. Vos, J.A. M oore and J.G . Zinkl, Environ. Hlth Perspect., 5 (1973) 149. 12 J.G. Vos, J.A. M oore and J.G . Zinlci, Toxicol. Appl. Pharm acol., 29 (1975) 229. 13 J.G. Vos and J.A . Moore, Int. Arch. Allergy Appl. Im m unol., 47 (1974) 777. 85 DOW0686 144 14 /J.E . T higpen, R.E. F aith , E.E. McConnell and J.A. M oore, Infect. Im m un., 12 (1975) --/ 1319. 15 W. G roth, ZbI. Vet. Med. A, 23 (1976) 31. 16 B.L. O 'Dell, P.M. N ew berne and J.E . Savage, J. N utr., 65 (1958) 505. 17 S.L. Snyder and R.I. Walker, Infect. Im m un., 13 (1 9 7 6 ) 9 98. 18 J.G . K reeftenberg, H.G. Loggen, J.D . van Ram shorst and E.C. Beuvery, Develop. Biol. S tand., 34 (1977) 15. 19 S.P. T ripathy and G.B. Mackaness, J. Exp. Med., 130 (1969) 1. 20 E.J. R uitenberg and L.M. van Noorle Jansen, ZbI. B akt. Hyg., I. Abt. Orig. A, 231 (1975) 197. 21 G.G. Krueger, B.E. Ogden and W.L. W eston, Clin. Exp. Im m unol., 23 (1976) 517. 22 L. A dorini, L. Ruco, S. Uccini, G. Soravito de Franceschi, C.D. Baroni and G. Doria, Im m unology, 31 (1975) 225. 23 A. White, Thym us horm ones: a new field in endocrinology and its significance for host im m unological com petence. In: D.W. van Bekkum (Ed.), Activity o f Thym ic Hormones, Kooyker Scientific Publications R otterdam , 1975, p. 17. 24 P.W. B eatty, K .J. Lem bach, M.A. H lscher and R.A. Neal, Toxicol. Appl. Pharm acol., 31 (1975) 309. 25 R.E. K ouri, H. R atrie, S.A. Atlas, A. Niwa and D.W. N ebert, Life Sei., 15 (1374) 1585. 26 A. Niwa, K. Kumaki and D.W. N ebert, Mol. Pharm acol., 11 (1975) 399. 27 J.G. Vos, Crit. Rev. Toxicol., 5 (1977) 67. 28 F.M. Collins and G.B. Mackaness, J. Im m unol., 101 (1968) 830. 29 K.S. Khera and J.A. R uddick, Adv. Chem. S ir., 120 (1973) 540. 30 G. Jones and J.B. Greig, Experientia, 31 (1975) 1315. 31 J.G. Zinki, J.G . Vos, J.A . Moore and B.N. G upta, Environ. Hlth Perspect., 5 (1973) 111. 32 J.A. Cook, N.R. DiLuzio and E.O. Hoffman, Crit. Rev. Toxicol., 3 (1975) 201. 33 R. Kirsch, G. Fleischner, K. Kamisaka and I.M. Arias, J . Clin. Invest., 55 (1975) 1009. 86 DW0631545 Lennart Harcip.il*: Malignant mesenchymal tumors and exposure to phenoxy acids - A clinical observation [Maligna mesenkymala ^ tumorer och exposition for fenoxisvror - en klinisk obser- 0 vation]. ^ Lakartidningen, 74_, (33), pp. 2753-2754, 1977. ^ 0O0' > *Dept. of Oncology, Regional Hospital, Umea. AGRICULTURAL PRODUCTS DEPARTMENT INDEX M. J. Traynor - 2030 B. B. Holder - 2030 H. C. Scharnweber - 2030 R. J. Kociba - 1803 B. A. Schwetz - 1803 P. J. Gehring - 1803 V. K. Rowe - 1803 / J. G. Gleeson - 2030 / V. B. Robinson - I N / H. C. Spencer - A Z /' x M. L. Leng D~. D. McCollister FR OM: John H. Davidson 1 1 / 2 3 / 7 7 Translated from Swedish by the Ralph McDlcoy Co., Custom Division, 2102 Rio Grande, Austin, Texas 73705 USA Code: 101-8715 U0W063 1540 MALIGNANT MESENCHYMAL TUMORS AND EXPOSURE TO PHENOXY ACIDS - A CLINICAL OBSERVATION S c'ca c::scs t ni;iii-."v.iru rr.-.-'OiC.iym..i tu mours observed ;it [t-.e depi of oncology in Ui:i-c.\ u'-r.r.g :hj v Tii -- 7f>^nd with expo uresure to phenoxy .icids oser r. period of 0-20 years described. Tli_ exposure wjs direct and cump.iraiivdy .T-osao in ti\e of the casei. The latent period i.s in agreement with that assumed for eficimc.il carcinogenesis. The distribution between the sexes in this material, of S7 >.itiei:(s in all. deviates from the national average, with a dominance of males. No defi nite conclusions concerning a possible causal connection can he drawn Yen these cases: this would call for more extensive investiga tions. 001 Osi O' 03 > The possibility that phenoxy acids (for example, hormoslyr) and the contaminants that occur in them may have harmful effects in humans and animals has been the object of an intensive debate. No cases of carcenogenesis in man have been reported so far. In this article we shall describe seven patients suffering from malignant mesenchymal tumors observed at the oncological clinic in Umea during the years 1970-76. In these patients the exposure to phenoxy acids had occurred 10-20 years before the diagnosis of the tumors. These cases do not, naturally, prove any possible connection between such an exposure and the malignant tumors; to prove or disprove such a connection extensive epi demiological studies are required. However, we believe that adequate evidence is available to report the observations in a casual form. O During the years 1970-76, the oncological clinic in Umea received (and checked) 87 patients who had malignant mesenchymal tumors. 32 of these were women (37Z) and 55 were men (63 Z ) . 43 of the men have been employed; occupation is missing, however, in one case. Nine were forestry workers (20.9x1), four worked in farming and forestry (9.3?.) and six in saw mills or the pulp indus try (14.0%). DOW0631547 Page 2 Cases Case 1 . A 62 year old man, forest worker until retirement in 1971; presenting symptoms, diarrhea and pains on the left side of the stomach. Smoker. Treated for tuberculosis in 1959. Other wise, previously basically healthy. In this work he had sprayed *, the pher.oxy acid 2,4,5-T roughly one week in 1963 and 1964 , >tvo weeks in 1965, one month in 1966, one month in 1967, and two weeks in 1968. Since August 1976 increased pain in the right fossa iliaca and also swelling in the right leg. Laparotomy revealed a tumor in the region of the small of the back, attached to the wall of the hip bone. DIAGNOSIS: Rather highly differentiated leiomyosarcoma. Case 2 . A 57 year old forest worker, nensmoker. High blood pressure since 1960. Treated for postinfectious arthritis in 1974. In the 1950's and 1960's, for three or four summers each, and for each summer for three weeks he had worked using a mixture of 2,4-D and 2,4,5-T for spotting and to a lesser extent for back spraying. Since August 1976 ventral tumor growth, on the proximal portion of the right thigh. DIAGNOSIS: Mesenchymal tumor, pro bably relatively highly differentiated fibroid liposarcoma. Case 3 . A 49 year old man, workshop owner. For many years gastric symptoms, but otherwise healthy. On his workshop premises, from 1961 to 1972, he had stored thousands of liters of phenoxy acids for a forestry company during the nenspraving season, about 11 months per year. The acid was stored partially in open containers. The patient had come in contact with the substance, which emitted a constant odor in the shop. In August 1976 melena and stomach pains. In November again acute stomach symptoms which led to laparotomy. This revealed a tumor in the region of the small of the back and involvement of the rectum, urinary bladder and over growth to the small intestines. DIA.GNOSIS: Mesenchymal tumor, more specifically, r.eurosarcof ibroma . U0W063 1548 Page 3 Case 4 . A 60 year old forest worker, moderate smoker. In 1938 appendectomy, but otherwise healthy. During the years 1961-66 for 20 workdays per year he had sprayed with phenoxy acids. In August 1971 saw a doctor for defecation trouble. Palpation of the rectum and sphincter muscle revealed submucous tumor growth. DIA.GNOSIS: Sarcoma recti (leiomyosarcoma) . A Case 5 . A 44 year old man, who had previously been in perfect health. During the years 1945-46 for 3 weeks each year he had been exposed to herbicides while doing farm work in Denmark. Employed in the oil-industry in-1954-68. During the summers of 1960-68 for about two weeks each year he had "rather carelessly" handled phenoxy acids both with a sprayer and for spotting. Visited a doctor in 1974 with complaint of increasing peripheral resis tance of palm and right underarm. DIAGNOSIS: Rhabdomyosarcoma. Case 6 . A 76 year old man, who had been healthy until 1972, when he was operated on for a perforated ulcus ventriculi. Reported some exposure to phenoxy acids for 4-5 summers in the 50's, at which time he had cut and collected grass along roadsides, which had been sprayed by the highway department. The patient also reported that he had worked in forests which had been sprayed with phenoxy acids. Visited a doctor for trouble with numbness and pain in right arm. The palpation showed a 1 decimeter long tumor on the inside of the right upper arm. DIAGNOSIS: Myxo fibrosarcoma with suspected lipoblastic differentiation. Case 7 . A 67 year old forest worker with three earlier cases of pneumonia and gastric anamnesis. The patient had worked in the forests until 1970. According to his own account, had done cutting and clearing from 1956 to the beginning of the 1960's within a phenoxy-spraved area. Indicated work in spraying for a few years after this time. Since June 1969 he had noticed a peripheral 16396 00^0631549 Page 4 resistance in the left underarm. Visited a doctor in March 1970. DIAGNOSIS: Polymorphocellular sarcoma, possibly a rhab domyosarcoma. In addition to these seven patients, among whom the exposure to the phenoxy acids had been direct, at least in the first .* five cases, there are also a couple of patients - both forestry workers - whose places of work in the 1950's and 1960's have been occasionally located in proximity to sprayed areas. In one case, harvesting berries after spraying was also reported. DIAGNOSIS: Rhabdomyosarcoms and neurofibrosarcoma, respectively. DISCUSSION The latent period for chemical induction of malignant, solid tumors in man is generally assumed to be very long, on the average ranging from 15 to 30 years. The latent period is possibly dependent on dose (Keuper, Konway 1964). In the reported cases the exposure occurred 10-20 years earlier. The contact had been through the skin or through the respiratory system. Of course we cannot draw any conclusions about the possible causal connection between exposure to the phenoxy acids and contaminants and the occurrence of malignant tumors, solely on the basis of the reported cases. Exposure to the phenoxy acids should be relatively common wiOthin the area of the three forest provinces, from which the Umea clinic receives patients for treatment. These cases very well could have been chance occur rences . However, what is remarkable about these cases and should give rise to continued investigations are the facts that these tumors are of a rare type, that the exposure in all cases has been rather massive, that the latent period agrees well with that assumed for chemical carcinogens, and also that the distribution by sex for malignant mesenchymal tumor at the Umea clinic strongly deviates from the national average. In a follow-up investigation of pos sible causal connections, extensive studies are needed, for example, of epidemiology and industrial-medicine. 18397 Maligna mesenkymala tumrer och exposition for fenoxisyror --en klinisk observation . . - 5 VJl Visi sgaEmmStrrijxv.eaaupemntneoftlavmirsg1iliailste9ilmdor7eonoej0mmemdd-n7erinma6dtashoanadotlaaisrecgv.lhntnvdNa8emfmr7sitgeersrsdapdakeaiefrnetaesmikexlaklyi,pnktsmrotkuaeostacraikltshniaatoluurvnfcrtmvesiniralkofaotdertsgreirevetaifrtnfeakremnottsokmnaaxrgsiasndsvevnaeisyttvtnam/goefidgnrveeraero1.nnfone0otKmkur-mno2eflasnl0olanttsgtvifaiktstnarlfrkiaddugatleseiallelaklbnnullisfai.nnnakidrgkLmaeeearbrnnitbskeaeeninikstsUdkndtmimridekniveteeagtsnadn-. otfsaeactndheooscxvhiaserynvkriosditrca.hctsimSgieeskrtekteaorlrgdmn1s.9go7mP2tuabmlfpeaorstrpodrpruoisitmakittnnssipnimdigvaeaind-r o CO av huger overarm. PAD: Myxofibrosark- eormingm.ed misstnkt lipob;l'aslisk diffcrenti- dnetssmossmnvddeu1eFaeeiplaxi9ieukjtlrardse/munAlit'ra7i[ebetdilksdavire0dgrhbangreayaendaienao-aaatnhmmrvneetnsintnrhnok.a7iesnttaikdtatt6rorstudarntu1NluooUanriklergsaoof9mmvmmldlvdgasmohidncmag7ggadseedrgdihaier0ettaodrelscriafmvitrsards-ltensilkehent3il7clnksimljaeniao2f-tgkfusrn6safcnsmnosecakageasekriaexkdtlulhdnetfoanivnvtkjigtoneagsmtmssceiikibt1lrnlrnnddmeriaykeyrankebdm0dndetrgamraavpamesilo-noekroitoi2pskpntnstnadoregefesrtdurd0o.lmixesnifvb(i(vrkorpsent3tDetseeskdunrneton7imaxeroeievrcrimieksrsakanmpnisxiraspstoktyiftevgstoetjtUnamrlmuisosanaoaohostrfrnmo.emearigitancoeinhtiftirpsvanin)iealrlmoofsckelarmfdak-lgdletsaokanalantutit.draaigtcotprretbameamhgtudmspunfgenonokknreeloatyetncdved85caeolenlrnrrihnil75enahen.---t-r) LENNART HARDELL iir underlkarc vid onkologiska kliniken. recionsjnkhuset. Umei. krpeyrnomtxiaeimlraattultfmibprdor,shetrgraoenlridgIeednrlbipeomns.eadPrekAolhDm:g.tNidesifefen-MdFngadllrig3a. g49a-sdtrriitgsymmatonm, vmereknstafdrsiigvarrige.t cfl1irati9es6rk111.a-7vIm2sdfiennunanodvdeexeirrsrkdysicrrtloakigrdeesfnlbo.erksfasplkrrvouhagtanrsdaibnetoglhtsauasgsnsesndortrnaekglns-. mnnaiendgh.inmFkeadrrv.leaPtr.iantUgieepnnpteghnaavdheasdtdeenaldvviigsenslukhkeattntdpisdkpaaptrs-bctsplatsen. Augusti 1976 melena oeh esvbnyiudgmkasptgomdemvmisratsaonodrmge. sIafntvuroamvrneeolekmrdtubidmeelirl,lladaunpbryaionrocbaktlodekmsunateia.t sHbmaumekrd--t SmgfapntateiidrmaoilfnobiitrtkgFneoertfa1edfetxracrea9srridlese7lt.fnlyis0le7ire.Prgana.n1ntadbHp9ar6stee5neriySle7en6usseg-na.npitudgsnttrareimrSkteilueininogseitongmardbsncmsdah,nitkenaaepirslojdrrdimaasjgeuntbvson1eajamsela9iisrnaernb7vrpbsmel10deiretg9.n6duettta6t0aetrP9rn-tneeueAtiagn.thngtiDlagaemdes:dsnetrAetktaerirPdniolabinuitocghrdapomtaeelaonrpymevnin--.--l myosarkom. dssetdyspUeelnkottoutmtraormestgniirpbvtnyflsiidicaenoosgarslgtnsalro1epbaedr9ntreernetu5ekitsh0latofisn--armeaiiernrb6ttdgse0df-reejs-eeutnppfssasfaorsplopeeurruxemraentnitaktksoidtpeohytmifiveandmraeottrfvietsmbreoinrtsnsmha.ietirtan.fehpruardforlriroPddvsolnaceAallre-gikvnbbrDnndii.ertl,:iakotndbsa1sgfiRpiiarngelaedaranegkkebxntfse-t--t-- om. e(snmy2kalrl0akoese,ngr9dssmvo(bp6ecrra3uokrskkcspsi)aar,(eoi9mnftc,tyd3m)ru.faapaslArt;laro.vircbNy()1emri4kotoaae,c0dnhsvenbapseerrienontsexcokc)hmov.kaigndrjsoina4srgr3ddbg-evvstoaaeacrrrkheik-t Kasuistik framF a ltlili Ip. en6s2io-dnreigringma1n9.71skpodggsarurbnedtaarev diarre och smtirtor tili vnster i buken. mvaeirtvumxtrt.ilsi ntaurnansttanremu.roPfibArDo:saMrkeosme.nkyfhertFsravlklriag4r.te.f6r0iAs-kdr.rig1A9rs3ek8nogas1pa9pr6eb1ne-dt6ae6rket,hoammdiedtmthliaegnncffceaknao2t0xioisnaysrrbboeert.ssvdSarg.katPer apilpeaarutogdrurissbktiet s1ip9r7reu1ktatfutmrmdiened-nanfr sfinktern submukost vxandc tu mr. PAD: Sarcoma recti (Iciomyosark- Diskussion il(lLnLailgaolalaunltmtelecealmnsnnp.nsselsiittngtroiitdd.hglieeedeaKnntavroevftnsxuaitwipromrraoarklnyeeremmkmers1ryiion9sejhrl6gkikdog4eense)in.tninnmdII1cduie0idnkndn-egtn2oiois0rs1onebkc5dleaah-rar3toveab0eirnaamnskcdddcdearees.-t i tiden. Kontakten har skett via hud och Rkare. Vdrdad fr tbc 1959. Fr vrigt khtalv2niMulvicaau.apde4ndsdgcad.eceu5avretnso-1elTttht9licnsohi6gakdam7kg1emohnn9tnireta7sdrp6ape1ibsdfmfp9nrvfevi6etttussik5srklpeea.taltninrvnndieualddettntdiivaseedbctvmirgccaaevcknadvtckmanrckreltaeekhuno.dd1ivmro9igmI61eg13h9fry9grcs66oeino6igb8tsncol.te.eiah.lxrlenraaiSnks.Pfr1eoyobbA9mdrVsme6aDasicdt4d.nane-:-. okl6hl3aaUarrrhdr-Fetepao.a2olcei,hslh4Hnat,2iyn5nu.vp-fnaTeae5drkn7rjefcttv-oirarnrssvniigofatiamcrsdsekremtkcnndroriiaatangbnrgsl1taaur9eInor7n9bcd4d6eeh.n0etllaiP.rcn/|r$^ItgenB^lj-lcj1aeyin9vhirc5advak0rcn2e-cscd.o4oklruamc-oDtdh---r au^asktniin1g976ftrillvrayxgagnhdcesptruumtifiuri,j.venSterdaaltn, om). o6hhmtstvr8pveeeda.arirtFurbvrkStsaicav.fcalorceilimdkmosAc5oeeknktm.hrr".ossa4truiArt4mnitflrd-ilodeddJe;irrnderhlmiigirgadder1ojfaoemeen9lvnjrn4eadh5ofinbanhi--1cxnradkiu9ssn1uone6k6ydsmisx0rnstpauo-rkg6ionrrta.b8inddteseSebrigetrhrdoaanadktrvdteegeiase1ari9gDvndmI5hyasc4afeakrr7ndaina-4tr fr tilltagandc ressteos volare hger un derarm. PAD: Rabdomyosatkom. cf1eu9ns7Fo2vaxdelisldnytrIr.roiacr7nid6uoi-.dnprdAeigernergmraa4adv-ne5vssisos.fxmormexvprpaaeorrrifntoperdferiiirns5.kgi0t-fttuaillr-i llPeant.tgiesvnavtrevnigdruehpnapangrassvloamgdietsvosaeughtvoetmirlikveaatltrabhteaasncpi!raugrtbracts-. oslfadsmasvsfksskisenraaoataaaetamtyidntatnemmnnrmlNVrgs.ndalsirocmdvdaakadtteedrxmefddtmgE.aanrnfuptmeeoraskolixtbaoalnosgmlrtcsayplaastvoegviritmlvon>svtimivhstgricekdl,sfiviaoeekuohrnkreitdiltrattdLnaaeatmmigsdgdnoot'evdraamtaefsneitcasmrnostnurtfaleslfkcnlikluno.rmeaasednidlkrmmsistnrdg-aneseiakfrnpkenanorrfnteledegaimilatmmsdnknnxnadrnrrmnteyociipitenkdmmlnvmisesyiounnfeggdeiesietansdodinetaengilnkiJgdlrgdestnseekeaoitUeeefroysnvnatexnrnnmtueoiltssfitlfelnusvartpnmaemesgfyvmtrrpktmetolsrsauumtnrors-avrnmremahamikekirrengitlfadenltaolnerirgntatnetrlgtmtginaiiarlonvginikamrskoggorkarrgygfaanledncneaaixrpaiasvufhnniadeanilt---.rri L A K A flT IO N IN G E N - V O L Y M 74 NH 33 - 1977 D O W 0 6 34 I > fg . , ' Ammoniumnitratdamm och nitrit . faSbtFugreroontnburdrdg,dedeseoitetkeesrar,uslbgsonoleosiomnkkllcmanshskoinnatfmdlaiigrtkerderasanmedrothehmiaoubncrnaodrhditemvrteeeneebsndirsnattsooraititiivtrntttea.eisosnatiamdsinndaoisnsenmvlstaaithtsniruvtavsuttbroitorrirmvtountnakreaceevrtkdnttrteasodfvjceemahrlbrvtegtaasdnitsentdgaasistessplnaaipnrfonerrgodamasnbrsmkaflveosmrsmtcaeivhd.irelsaaarftEnaroenpdrroasioarlvie.tveeenSpprnktaiiddvdadeaeeetaxvtmmnepdeaiihooorniascllsgooinkttagguisdogiidvsesncaikklersstaaa--r- nicMllcictinudogflorbbincstrmsneidnagnav19u7m6iinmforndgdveidr skalinreisttkett. keBmodisekna. labvoreratnooriremsalgCrenntsralflarhmeemihoegmloobginloebt.in r*"2 proc av det totala rHfyrg:ieniskt grnsvrde fr dricksvatten nitrit 0,02 mg/liter, dbmIelaaLnnKadvAsaetB9d5m:asnepgdrIo9ru5c6v0ponreriottrcititMsfdpriaiertltsmneabglomemlrjmeagd.eoetnDlaieubntmevskntanitledlraanest-r Arbetet utgir frin LKAB. Malmbergct. dar SE1JA ABRAHAMSSON ar fretagsskterska, LEIF KAGSTRM skyddsingenjor, olittejnVrinitdrfliagrt1t0v30su0omxmnmgalg.inv/ilstirtmearet dpfeelrr. barn och 50 mg/ liter anses vatten hhoAodrrhsmTdsfiiylactnrneaNerlohnSNlgmlgrrinksndnfdaognoiAma,tfeo.mivirrrssNlirieasisndslrodukIaatgt.kaerttnirsccbmvhthopslttkadfnaaravbkmiesnrdrllfreirrtaletcautdaurtlhtrlenkntrnaetvisndtrdtevibaoiddrtinlgadrgutiirycnealsr.geg.lfghitskkvtadfbagsrMfbnekrametamldasaaaraarmjnehesrvelnnmlsdadavfbeskdteseraoitnonohaagxbrnrimtsatdptitlrrAtilrhunoyrlomprpoNfsccmijsrhtnauseiaglkkFat.igirnlnpomltvmOuitaiHenterntibagifrrldri.siolretelavnlssiekrmiteoniasrogksfhnrpagvekm.iatarnsnrinsunddmlsrmdebohvotiteiekaugenicmaomlnitas-t----.trt reuoincvkhedeneta.trusrboeFekllttnsrimknaegeunadsriacfloisnanasvsmkabtktataenxrudatkrekterpdiriinv.dsienmginigoalovegniesdtket Litteratur HUEi'F.R W C. CONWAY W D: Chemical Carcinogenesis and Cancers. Springfield. Ill: Thomas 1964 AaMnadClilgiEnnxiacpanoltsuOMrebessteoernvic'ahhteyiomnnoa.xlyTAumcidosu-rs LENNART HARDFLL. MD. Dept of Onco logy. Regional Hospital. S-901 85 Umei LAKARTIDNINGF.N 74: 2 7 5 3 -'754 1977 Seven cases of malignarjt mesenchymal tu mours observed at the (Jepl of oncology in U m ei uunng the years l*J7jll -76 and with expo sure to phenoxy acids n h r a period of 10-21) years are described. ThJ exposure w js direct and comparatively ma-isAe in tisc of the cases. The latent pcriiHU i.4 in agreement wirh that assumed for chcimcjil J.ircinocenesis. "Ilie distribution between [lic/se^es in this material, of 87 patients in all. deviates from the national average, with a dominance y f males. No defi nite conclusions conccjningla possible causal connection can be dr:lwn ^om these cases: this would call for mure extensive investiga tions. ' j1 KARL GORAN S:T CLAIR RENARD fretagslkare och KURT STENLUND skydds ingenjor. I anikeln redovisade analyser av meihemoglobin r uitrda vid kliniskt kemiska laboraloriet, Centrallasarettet. Boden. aaogaelarCdsdtvniraecnamtmamnshnaoihnAddkmrisgrimijivtaaennenNi-kmsasrefecnc-atfrtnarrrobseaaindd.r.itvpnitfntu5ijfrlitfrtidkRnrDiscedrtotsavonaraycpiresesnilnsir.srteankkeRgdtoasvnmtgeoameceamaemdnemrnmirpnicvnninninpsaflfmsdis.fjanneeefmlpereerc,krArefrituurhrirnaennoovcntelkiacrnoigitadsglnrhnordlcmagoelvdasneimh.rsmoarreensucavtcrsstos.navahl1htalkabd0emnoiaelriarudscllnvnCimdcvhlpttscarjiuayr-etpir1apsrdsseosohditteest4mmknoe.kokrdlirm-msaaaecumeaISsarrUSlrrncilcuvtnedmrhiatdsnr(oonfanteenkaComrmilrdainlai--.-rri lneirtHriHtserird.arendoavvispardoeblesmtuedti.o r inriktad pa oosgOanNsoetpbmknsNmcAnlocrkvcrtoliiieimonnrieeeettOhemhtNMlrirmddrgdtbusrctiarvaoeattseio.neiiibkatthnlunorbratekt-rriioidaunnnhivnoimlnmbjiDanfndcntenekdkgeedmoelndahmadeonjabilarmresrxcibisorrvlknnlaaalarthkrejiasuinraadnoangtrmepkvotnoagnsdeiinalpymstsimkcladmltse.nnreoitlorkmivfttcamlnaaldibhrirtvbmullnngtiajagiitcartilralvgaa.lni-gtd.emednb.ekitnsetssrdkaktDiidbltfoglnoionisuttiltllaeklrmnarcalgdereneviurnmkfnitsteclcnltaxko-inoellrvjltngeckiitdbracnnmnii-istxsrnhinissathntgdonnauptgviagIcldrpiirmm.traoprrltisueramscAserpioestusivuiivBvrktnhenktikoreieinamentiaegnnitlinigngvulrhsaerosttm.bsaide.loerdAlolonntlieaijnnnnmnsbtbonumslbueivuvdioiadeilgdannilaincngmenjtlgllnn.gadoMr.a.y.naisncrsbnpbnnBnembirimmnoDiiaibgntiAnmnnmalrlerteieceaeidiregnakkiaotnnlnsnlstla.-t--trl.l-l fSApEooxNrpsft-koe(uAnsipsiiNtenpiondgFnrOasSgtm-a)vglacaitantdnvsidnkungpSinpDtgife.tcrtoilssnkcipknrfoobrBsPlckcrmenrglieintmengkinnhvisraiadkr dNeHls,pNOfilj-temrotnchingdaerlshpar ngijotrratst ipvadttemnfmas, gobhgllpkrsMmiaorcoeckjnouoehmorUnEmgrdgpdlpttamemnoeipsnididgnsta)mtcud(.neikdlnnolSoealpitnrilenns.bckrriSadenrilgkiadsibnttoegguaitKrnkodeumtpaetraiiaernnneraralrl.bgdadikkavailperfyutNtdierduiardl1ldslteHiere9emja.oarre7nlrm,ulaamrdm5axndnvbve)apNe.dkeanvothO(utenvreosnkitrdr,tibfmet1ltliisi2lxgojlrsgoevlmpdarnssaarrmoesevksdlrbdesmdeitaeljeivtrjtaelarii(gaalokrttadvjsnei1vnotdtg9fiirengnMehd7jtoirrgtryn6nuoamerngigdpntnlhraemeeeppggosaltnns-)-.---r ddroattNeCiruacdn(.MhSrmFi)ge`.tnim(torv^ioxlrllcclierkpbNrhdankaeeOueresrctlnlt.;aagpvdndaroaa1ndac9vdBnbuhemOoignonsdNrdrkgauretcasiHopniruarbpssc,vnr.eoeb.ecgrkrSenenSsntenat1esienr9tprikrh7ibgeelan6aaelitglul1s2astprlk-e1bf2oarn-oa0nr4esrvd.ata2laievl.dt.lsgoaeSNrsialnnuOamaagmd,tv--. samSalamts.liglar iiandsdtraurketioonchdagbeonrralorere hmaadteaulnanpidndpdgnrauamrrtentgaasd.sevkSsiyd.admd.Maiiianibnndcteetckrsosmmkkeundllienvfguesneresnknkvfosilrineedotnaiinnsg(aeaannuv acivn:mtgtnigdnrsioonsnlkisuiarlmtkaeniAvrit.rlreiasgtia.tr(sksNkitiitlltltaararndt,iphssopepecnnreaufditnl.atdrenoj!i)a)svkouacllthet IroI'icrrnooh!Ervviliseselreenrrrnraipieablasg.bkcr2euIcihI1lv)rta-san4nmiduid2acltltsaesvdhtaeeiolenestmvbonlgseoase5ddnd0eaa0likbnllvvam.sare2tkun0uni.uJdfmo5keiy--rs2hlesj3eonk.prAa0dad5p.eoktcltiShndiaspymkgalntt-iikdeceingemttneiorslomkpgaAseGltatpbtreoollkivrvaneatirosrkenptei.pilmirniifsa^Bstlootfptdd'eieeirinisiQ.noonlama-l 2754 LAKA^riONINCEN.VOUVM74-NS,1.1977 ECOLOGICAL BULLETINS No 27 2446 M No 0 1 6 6 5 D0WG44243 Chlorinated Phenoxy Acids and Their Dioxins Mode of Action, Health Risks and Environmental Effects C. Ramel (ed.) 1978. Ecological Bulletins. SwedishNatural ScienceResearchCouncil. ISBN91-546-0234-3 Productionanddistribution: NFREditorial Service LF/ALLF20977002 PrintedinSwedenby Berlings, Lund 1978 Copies of the bulletins can be ordered from the Editorial Service of NFR. Box 23136, S-10435 Stockholm, Sweden. The Editorial Service, which provides financial and practical assistance for the publishing of scientific works, is responsible for the distri bution of all NFR publication. Report from a conference arranged by the Royal Sw.edish Academy of Sciences Stockholm, Sweden, 7-9 February 1977 LIST OF CONTENTS Editor's preface C. Ramel CONCLUSIONS AND RECOMMENDATIONS / li CHEMISTRY Summary Chemical background of the phenoxy acids and dioxins C. Rappe Analysis of 2,3,7,8-tetrachlorodibenzo-paru-dioxin in the Seveso area P. Aliamoli, E. Angeli, G. Bandi, A. Bertolotti, E. Bianchi, L. Boniforti, I. Camoni, F. Caitabeni, G. Colli, M. Colombo, C. Corradi, L. De Angelis, G. De Felice, A. Di Domenico, A. Di Muccio, G. Elli, R. Fanelli, M. Fittipaldi, A. Frigero, G. Galli, P. Grassi, R. Gualdi, G. Invernizzi, A. Jemma, L. Luciano, L. Manaro, A. Marinella, F. Merli, S. Nicosia, F. Rizzello, C. Rossi, G. Rossi, G. Salvatore, A. Sampolo, G. P. Schmidt, F. Taggi, G. Tebaldi, E. Zaino & G. Zapponi. 31 The 2,3,7,8-tetrachiorodibenzo-para-dioxin problem: a review D. Firestone 39 '.I I / Analysis of 2,3,7,8-tetrachlorodibenzo-para-dioxin in environmental samples 53 W 0C * J. D. McKinney f t i Gft % 2,3,7,8-Tetrachlorodibenzo-/wa-dioxin decontamination F. Pacchiaci 67 y j Confirmatory tests for pesticide residue analysis G. Widmark 71 y / Photochemical reactions of phenoxy acids and dioxins B. Akermark n& Q ! PLANT PHYSIOLOGY Summary The herbicidal effects of phenoxy compounds B. Aberg & L. Eliasson TOXICOLOGY Summary The phenoxy acid problem in Sweden J. Bckstrm / Phenoxy acids: effects and fate in mammals P. J. Gehring & J. E. Betso v Toxicity of 2,3.7,8-tetrachlorodibenzo-para-dioxin J. A. Moore 2,3,7,8-Tetrachlorodibenzo-para-dloxin and enzyme induction A. Poland & E. Glover Phenoxyacetic acids: sublethal effects P.-O. Sjdn & U. Sderberg Ji&adZ 2,3,7,8-Tetrachlorodibenzo-parii-dioxin: effects and mechanisms J. G. Vos 83 ECOLO 85 O Summary 86 o Chemical G G. E. Blui -P'' -Fro The use o modes o f . o U. S. M. 101 103 -F' The use oi < n B. Gransi 108 Phenoxya considra 122 B. Hggi c e . ' l * Effects of 1^4 O Dfle.C in Sweden T. Ingelu 145 Bloaccum A. R. Isi> 149 Effects of L . T orsti Kcologicn with 2,3.' A. H. GENETICS y Summary Genetic effects of phenoxyacetic acids in animals C. Ramel List of p; List of ol List of at 177 i?a Genetic effects of chlorophenoxyalkanoic acids on plants L. Ehrenberg 186 / Mutagenicity tests of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlo- 190 phenoxyacetic acid in genetically stable and unstable strains of Drosophila melanogaster B. Rasnmson & H. Svahlin / Genetic effects of phenoxy acids on microorganisms G. Zetterberg 193 ECOLOGY AND ECONOMY Summary Chemical weed control in the tropics G. E. Blackman & J. D. Fryer 205 207 210 The use of phenoxy acid herbicides in Swedish forestry: amounts, types and 219 modes of application U. S. M. Barring The use of phenoxyacetic acid herbicides in Swedish agriculture B. Granstram 231 Phenoxyacetic acid herbicides as a tool in forest management: silvicultural 235 considerations B. Hiiggsirom Effects of the silvicultural use of phenoxy acid herbicides on forest vegetation in Sweden T. Ingelog Bioaccumulation of 2,3,7,8-tetrachloradibenzo-^ariJ-dioxin A. R . Iseusee 240 C c ts 255 DA L Effects of phenoxyacetic acid herbicides on soil organisms L. Torstensson 263 Ecological considerations regarding massive environmental contamination 285 with 2 ,3 ,7 ,8 -tetrachlorodibenzo-pwfl-dioxin A. H. IVesting List of participants List of observers List of additional authors D0WO442Q4S 7 1 8 403- fm D 3m sm BIBBSS3BaBBBS Ramel. C. (cd.) 1978. L'hlonnaiedPhenoxyAcidsandTheir Dioxins Ecol. Bull. (Stockholm) 27:13-16. CHLORINATED PHENOXY ACIDS AND THEIR DIOXINS: CONCLUSIONS AND RECOMMENDATIONS l 'r Q2770M00 i Conclusions The phenoxy acid herbicides are not formed naturally in the environment. There is no evidence that dioxins are formed from phenoxy acid herbicides in the en i vironment. The only dioxin of environmental concern with respect to the phenoxy acid herbicides is 2,3,7,8-tetrachiorodibenzo-pnrn-dioxin (TCDD), the dioxin found in the herbicides, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5- trichlorophenoxypropionic acid (2,4,5-TP). TCDD is found neither in 2,4-di- chlorophenoxyacetic acid (2,4-D) nor in 4-chloro-2-methylphenoxyacetic acid (MCPA). Levels commonly encountered in soil after normal applications of phenoxy acid herbicides are 1-2 parts per million (ppm); due to heterogeneity of soils and irregularity of application, these levels could be somewhat higher or lower locally within sprayed areas. The half-life of the phenoxy acid herbicides is of the order of 3-4 weeks; that of 2,4,5-T is up to 10 weeks. The rate of degradation generally increases with increasing microbiological activity and decreases at i lower pH, especially below pH 4. Repeated applications of 2,4-D and MCPA result in somewhat faster microbiological degradation. The level of phenoxy acid herbicides in the green parts of target vegetation shortly after normal spraying is 200-500 ppm. After 10 weeks, this level decreases to 20-50 ppm. During the cold winter period this value seems in some cases to be nearly constant, although in other cases it decreases. Injected phenoxy acid i Ii herbicides may persist for 4-6 years within the woody parts of vegetation, however, the ecological consequences of such persistence in wood are probably not very great. I Photolysis is the primary mode of degradation of TCDD on environmental t surfaces; microbiological degradation occurs slowly in soil. Its halflife in soil has i been estimated in the United States to be 1-1.5 years (on the basis of studies in i which high levels of dioxin-contaminated phenoxy acid herbicides were used). v / Wind-drift of sprayed phenoxy acid herbicides may reach non-target areas. To avoid such risks, strict regulations with regard to weather conditions during spraying and with regard to spraying techniques are required. There appears to be virtually no downward movement of TCDD in the soil: i it can be estimated that no more than \ % is transported from the site of applica tion annually. Movement in the air would occur largely via airborne particles. Vegetation takes up very little or no TCDD from soil. 13 i D0W0U2048 There is no evidence of bioaccumulation of phenoxy acids; bioaccumulation of TCDD resulting from normal use of 2,4,5-T is insignificant. The fate of phenoxy acids in plant tissues, changes in plant composition induced by these compounds and their mechanism of toxic action are understood only imperfectly. It can be concluded that further studies in this field would be of importance for understanding the response of crop plants to phenoxy acids and for the development of more selective and efficient methods of herbicide usage. Such studies would also provide important information about aspects of herbi cide toxicity at the cellular level that may be relevant to their toxicity in animals. Possible levels of exposure to phenoxy acids and/or dioxins vary among different populations, such as: employees in factories where phenoxy acid herbi cides and/or trichlorophenol are made; persons who prepare and apply the herbicides, and members of the general public who enter sprayed areas and/or consume treated vegetation. People representing potential risk groups, pregnant women and individuals with chronic liver disease, should be excluded from activities involving high exposures, such as manufacturing and occupational spraying. Only very low levels of phenoxy acid herbicides are normally found in food stuffs. Levels in Swedish wildlife, fish and waters are also low. The residue levels in woodland berries from recently sprayed areas might be higher, of the order of 10 ppm. A daily intake of as much as 1 kg of such berries would correspond to a dose of 0.2 mg k g'1day'1 of phenoxy acids for a person weighing 50 kg. This level probably represents the same degree of exposure as that realized by herbi cide sprayers. Calculations based on extreme conditions (high exposure from food and lowest dose of 2.4,5-T that caused teratogenic effects in mice) still provide a safety factor of at least 100. If the levels of TCDD in phenoxy acid herbicides remain below 0.1 ppm. considerations of health risks associated with these products should be based primarily on the potential toxicity of the phenoxy acids per se and not on that of its contaminants. In evaluating the risk associated with the presence of TCDD in phenoxy acid herbicides, potential exposure to compounds that have similar toxic effects, i.e., other halogenated dibenzodioxins, dibenzofurans, naphthalenes and azoxybenzenes, should also be considered. Studies to assess the tumorigenicity of phenoxy acids and TCDD provide no defintive indication that these agents are or are not carcinogenic. Additional, more extensive evaluations are underway. The only statistically significant demonstration that phenoxy acids induce mutations transmissible to the offspring was obtained in the recessive lethal test in Drosophila melanogaster. Work on microorganisms sustains the conclusion that the phenoxy acids are mutagenic under the test conditions used. Two in vestigations on 2,4,5-T and one on 2.4-D, both using pure acids, showed an in creased number of recessive lethals in Drosophila. From a tentative comparison of the radiation dose that would give a similar effect, the mutagenic effects of 2,4,5-T and 2,4-D were concluded to be weak. Somatic mutations have been observed in Drosophila, and chromosomal aberrations have been shown to be induced in plants and mammals. The relevance of these latter findings to a risk of mutation from phenoxy acids (associated or not 14 DQW0442049 with heritable damage) requires further studies. Observation in one study of a possible role of the emulsifier and/or solvent show the necessity of including these constituents of phenoxy acid formulations in future studies. In view of positive mutation tests in bacteria and findings of chromosomal aberrations mono mammal and in one plant species withTCDD, it must be con cluded that this compound is mutagenic. The fact that other tests of a similar kind were negative may have many explanations, such as problems in keeping the compound in solution. Such difficulties make it impossible at present to quantify genetic risks from TCDD. With regard to behavioral effects of the phenoxy acids in mammals, it was concluded that further studies of dose-response relationships are necessary before it would be possible to make an evaluation of similar effects, if any, of the doses of phenoxy acids to which humans could be expected to be exposed in connection with normal herbicide usage. No counterpart to the physiological mechanism involved in the major herbicidal effects of phenoxy acids in plants is known to exist in animals. In plants, high concentrations of phenoxy acid compounds cause less specific effects, e.g., the uncoupling of oxidative phosphorylation; this effect appears to be weak in mammalian organisms. Recommendations 1. The stability and bioaccumulation of TCDD should be studied in realistic situations, under normal field conditions, in various types of climates and soils. ~ 2. Risks associated with exposure to TCDD could be greatly reduced if manu facturers would utilize processes, currently available, that minimize the formation of TCDD from trichlorophenol. Manufacturers should also use techniques that minimize potential contamination of the surrounding environ ment by TCDD in the event of industrial accidents. In addition, strict precau tions should be taken to ensure safe disposal of TCDD-containing residues. 3. Failure of manufacturers to utilize techniques that minimize TCDD formation or contamination should lead to a re-evaluation of the feasibility of continued use of trichlorophenol or of products derived from it, such as the phenoxy acid herbicides, 2,4,5-T and 2,4,5-TP. 4. The use of other synthetic processes that eliminate formation of TCDD as a contaminant should be investigated. 5. At present, the mechanism for the toxicity of TCDD is unknown. A further understanding of how TCDD and related compounds act is necessary. 6. Further sutdies of the mutagenic activity of the phenoxy acids are important and should comprise all constituents of formulations and their impurities. In order to make quantitative estimates of risk to man. it is essential that the mechanisms for the genetic effects observed be clarified. 7. Studies of mutagenicity should also aim at safe-guarding plant genotypes. S. It is recommended that epidemiological investigations be made of human groups exposed to phenoxy acid herbicides and dioxins and that these com prise all effects that have a possible genetic mechanism. 9. In forest use of herbicides, it is recommended that consideration be taken ^ i/ 15 of non-target plants within target areas, especially those of recreational and scientific interest. Information about the effects of herbicides on non-target * flora is insufficient, and further research within this field is recommended. 10. It is recommended that special consideration be given to the ecological importance of field borders and other ecotones in any spraying program. 11. In choosing herbicides, their selectivity for intended target species and other species within target areas should always be considered. 12. The ecological importance of habitat, including both food and cover, to vertebrate and other fauna was recognized; it is recommended that this factor be taken into consideration in the use of herbicides and in other manipulations of habitat. Further research on the possible significance of alterations to wild life habitats is recommended. Both vertebrates and invertebrates should be considered. 13. The conference stressed the importance of establishing and maintaining reference areas representative of major ecological types. With respect to herbicide use, it is recommended that areas are set up in which herbicides would not be employed and in which other management operations are strictly controlled. DOW CHEMICAL U.S.A. 24-Jl S8Z8POMQQ0 9008 Building May 21, I97U J. H. Davidson, Manager 2,1*,5-T Project 9008 Building MIDLAND. M ICHIGAN 4 8 8 4 0 DC c, en EO. Ai Ml. 4a-4a-78, e INJUNCTION DOW/EPA AGREEMENT 9-7 ' cc: E. H. Blair M. C. Feigelson G. E. Lynn D. D. McCollister IJ. M Thei a j 9 M. J, Traynor M. R. Wessel C. S. Williams H SILVEX M D ERBON - REGISTRATION AND TOXICOLOGY On May 16, EPA published a notice of intent to broaden the 2,U,5-T hearing to include all registered uses of herbicides derived from 2,^,5-trichlorophenol, including silvex and erbon (Federal Register 39 No. 9 6 , p. 17^66). Dow is the principal manufacturer of silvex and the only manufacturer of erbon. Current Dow registrations for silvex are : KURON Low Volatile Brush and Weed Herbicide(EPA Reg. No. k 6 h ~ l 6 2 ) Silvex Acid for Manufacturing Use Only (EPA Reg. No. U61*-l*55) Silvex Butoxvpropyl Esters for Manufacturing Use Only (l*6U-l*6l) Silvex Isooctyl Esters for Manufacturing Use Only (U6U-U36) Registration for other Dow silvex products was cancelled in October 1966 when Dow indicated they would not renew their registrations as required every five years. GARLON Grass and Weed Killer (1*6U-217) KUROSAL SL Aquatic Herbicide (U6U-277) KUR0SAL8 G Aquatic Herbicide (l*61*-276) COLORSET for control of preharvest drop in apples (U6U-267). Other products similar to KURON include: SILVI-RHAF LV-UTP (formerly Hercules, now Transvaal) WEEDONE 2U5-TP (Amchem) Silvex 1*TP (Diamond-Shamrock) Silvex 1*L (Chipraan Division, Rhodia, Inc.) DED-WEED Silvex LV (Thompson-Hayward). Salt formulations used for aquatic purposes are registered by Pennwalt, as mixtures with endothall (e.g. AQUATHAD3). Dilute formulations of amine salts are used as growth regulators in fruit crops (e.g. Arcadian STA-SET UOO, EPA Reg. No. 218-1*92, Allied =1/ Chemical Corporation), A large portion of the estimated A N OPERATING UNIT OP THE DOW CHEM ICAL COM PANY 16408 J . H. Davidson - 2 - May 21, 1974 1 .5 million pounds of silvex produced annually is used in the home and garden market (e.g. WEED-B-GONE, Chevron). With regard to erbon, we have three registered products: ERBON 4 Non Selective Herbicide (formerly BARON, 464-172) NOVEGE Weed and Grass Killer (U6U--255) ERBON R for Manufacturing Use Only (U6U--335)- The latter contains 42? erbon and 15? related chemicals which are not designated but are claimed as active ingredients. It is sold to many formulators for registration under their labels for home and garden use. Examples include: Greenfield Contact Killer (1471-33, Elanco or Eli Lilly) Best Liquid Edger (7001-RIA, Occidental) Best Ded n' Gon or All-Out Weed Killer (7001-88-AA, Occidental) Four Seasons Edge pr Trim Grass and Weed Killer (7393-UE, Wonder) Ferti-Lome New Improved Perma Trim (Voluntary Purchasing Groups) Weed and Grass Killer No. 1 and No. 2 (407-xx, Imperial) Green Light Liquid Lawn Edger (869-6 1 ) Sears Liquid Edger (539-254) Uses, rates of application, toxicology, residues, and status of regis tration are discussed individually below for silvex and erbon. Additional details can be provided as necessary. Silvex is the common name for 2-(2,4,5-trichlorophenoxy)propionic acid. (Note that it is a mixture of D and L forms of the chemical due to the presence of an asymmetric carbon on the side chain.) According to the current EPA Summary of Registered Agricultural Pesticide Chemical Uses, it is registered for use as an herbicide at rates up to 4 lb/A in pasture and rangeland, 1.5 lb/A in rice, 5 lb/A in sugarcane (Florida, Louisiana and Hawaii), and 40 lb/A in lakes and ponds. It is also registered for use as a growth regulator applied postharvest at 7-5 ppm on pear trees and preharvest at 20 ppm in apples and prunes, with a maximum rate equi valent to 60 grams per acre. Uses for control of brush in industrial and other non-cropland areas include rates up to 16 lb/A as approved by EPA in October 1973 for KURON. KURON contains 6 9 * 2 % propylene glycol butyl ether esters of silvex, or 45.8? silvex acid equivalent, which amounts to 4 lb a.e. per gallon. We have considerable toxicology data for silvex, including reports on subacute 90-day studies with the sodium salt and the PGBE ester in rats and with the potassium salt in dogs. We also have chronic 2-year studies with the potassium salt in rats and dogs, and recent teratogenic studies with the acid and the PGBE esters in rats. The results of these are summarized in the attached excerpts from Section C of Pesticide Petition No. 8F0675. EPA has indicated that they are satisfied with the studies but that a multigeneration reproduction study would be needed, for toler ances greater than 0 .1 ppm (negligible residue) in food crops. J . H. Davidson -3 - May 21, 197b ) Q 7 Q h H Aa All registered uses for silvex in food crops are covered by PP 8F0675 first submitted in December 1967 by the Industry Task Force fdr Phenoxy Herbicide Tolerances (formally disbanded in April 197**)- Amendments to the petition were submitted by Dow in September 1968, December 1970, October 1971 and January 1973, for a total of nine volumes to date. The petition is currently being held in abeyance until 11/ 1 / j b pending com pletion of.a study on persistence of silvex in soil and of another feeding study in cattle. The residue pattern for silvex in milk and meat resembles that for other phenoxy herbicides in some cases but not in others. For example, residues were not detected in milk of cows fed silvex, 2,^-D or MCPA until the level in total diet was raised to 1000 ppm, but residues of 2 , b , 5 - T and/ or 2 ,U,5-trichlorophenol occurred in milk when the cows were given as little as 100 ppm 2,lt,5-T in the diet. Residues in meat tissues, parti cularly liver and kidney, were much higher for silvex than for 2,i+-D and MCPA and were substantially higher than for 2 , k , 5 - T at levels of 300 to 2000 ppm in the diet of. cattle and sheep. However, residues of the 2,U,5-trichlorophenol metabolite were low or non-detectable in milk and meat of animals fed silvex in contrast to relatively high levels of the phenol in milk, liver and kidney of animals fed 2,U,5-T. Additional feeding studies are currently being conducted at reasonable levels of 300 ppm in the diet, to determine the preslaughter intervals required to reduce residues of silvex (or of 2,^,5-T plus trichlorophenol) to below the 0.1 ppm negligible residue level in liver and kidney. We have proposed limiting the rate of use to 2 lb/A in pasture and rangeland, with no cutting of hay during the season of application of silvex or 2,1+,5-T. Interim tolerances were established in late 1972 for many pesticides formerly registered on a no-residue basis in food crops. Included was silvex at 0.1 ppm in or on apples, plums (prunes), rice and sugarcane. (Federal Register 3J_, No. 1 6 9 , p. 1775, August 30, 1972). However, a subsequent order proposing additional interim tolerances, including 2,1*-D and MCPA in grass, stated that interim tolerances could not be established for 2,U,5-T in any crop, nor for silvex in or on grasses (pasture and rangeland), grass hay, and in water. The reason given was because the data in petitions and otherwise available data were judged inadequate to rule out the likelihood of injury to consumers from residues from these uses (Federal Register 37., No. 1 7 8 , p. I8565-I8566, September 13, 1972). Dow appealed this for silvex in grass but the appeal was denied. In February 197^ we renewed the request for an interim tolerance for silvex in grass but EPA replied on April 5, 197*+ that data from the additional feeding study were needed before they could finalize the usage on grass, i.e. application rates, withdrawal times, etc.; hence they could not establish interim or permanent tolerances for grass at this time. Use of silvex as a growth regulator in pears was cancelled by EPA in March 1972 when they denied our request for exemption from the need for tolerances. We had hoped they would consider it a non-food use since the 7.5 ppm spray is applied to the trees after harvest of the pears, J . H. Davidson - t4 . - 4 - May 21, 1971* i.e. a year before harvest of the next crop. However, EPA ruled that this use would reasonably be expected to result in small residues in or on harvested food or feed. Subsequently, we provided all available data on silvex in pears for submission by Inter-Regional Project No. 4 on behalf of the State of Washington. Their Pesticide Petition No. 3E1339 was deemed adequate by EPA and a permanent tolerance of 0.05 ppm was established for silvex in pears (Federal Register 3_, No. 165, p. 22894, August 27, 1973, 180.340 of the Code of Federal Regulations). Uses of silvex as an aquatic herbicide are covered by Pesticide Petition No. 1E1012 submitted by the U.S. Corps of Engineers in conjunction with the Bureau of Reclamation, USDI. However, EPA has repeatedly found their petition inadequate. We have supplied them with information for incor poration in their petition and have authorized EPA to consult our petition 8F0675 on their behalf. EPA has raised questions about residues of meta bolites in fish, and the possibility that TCDD might be formed by cooking of fish containing residues of trichlorophenol. Their last amendment was submitted in November 1973 and their cover letter included the statement: "We understand that a hearing will be held by the Environmental Protection Agency in April 1971*- If this hearing includes the aquatic use of silvex, we would like to defend our position on the basis of Technical Report No. 5 - Aquatic Use Patterns for Silvex." This report dated October 1973 is 1/2 inch thick. A copy was sent to M. Leng by Dr. E. 0. Gangstad of the Corps of Engineers as part of the inter-communication between Dow and the petitioners for tolerances in water and fish. The first sentence of the Foreword states that an announce ment by Dr. Lee A. DuBridge in October 1969 listed "actions being taken by governmental agencies to restrict the use of the herbicide 2,4,5trichlorophenoxyacetic acid (silvex)". Apparently they don't know the difference between the various compounds since they also reported finding 2,4,5-T as a metabolite of silvex in birds. (Dick Otten of Amchem is a member of the Registration Work Group of the Interagency ad hoc Committee on Use of Herbicides in Aquatic Sites.) Erbon is the common name for the herbicide chemical 2-(2,4,5-trichlorophenoxy)ethyl 2,2-dichloropropionate. It is the alcohol form of 2,4,5-T combined as an ester with Dalapon. It degrades slowly in soil, presumably to 2,4,5-T and Dalapon which kill broadleaf weeds and grasses, respect ively. When used at high rates, it eradicates all vegetation. Dow's original product was called BARON Non Selective Herbicide, which was reregistered in 1971 as ERBON 4 Non-Selective Herbicide under the same registration number (464-172) after we sold the trademark BARON for other purposes. It contains 30.5? erbon and 10.5? related compounds for a total of 4 lb/gal. It is recommended for use in industrial areas such as storage and parking areas, tank farms, rail sidings, substations, outdoor theaters, drainage ditch banks, fence lines, pole yards, lumber yards, elevators, equipment storage-areas, around farm sheds, and in non-crop agricultural land. It is usually applied in late May or early June in northern areas or in early winter or mid-winter in California. Recommended rates are 3 to 4 quarts per thousand square feet or 30 to 40 gallons per acre. This amounts to a maximum of l60 lb/A including 40 lb/A "related products". 1S41.1 6839t'fV'i\Oa J . H. Davidson -5 - May 21, 1971* Dow also has a private label product called NOVEGE Weed and Grass Killer (EPA Reg. No. U65-255)* This contains 9% erbon and 3% related compounds for a total of 1 lb/A. It is recommended for use in driveways, walkways, parking areas, under fences, around garages and other buildings, and in similar areas where vegetation is not wanted, as well as for edging various areas. The rates are described as 1 qt in 2 gal of water applied to 250 sq ft, or to 100 sq ft when extended control is desired. This amounts to a maximum of l/L pound per 100 sq ft or about 110 lb/A including about 30 lb/A "related products". Presumably, other products formulated from ERBON R { \ 2 % erbon, 15# related products) are recommended for use at similar rates. Acute toxicity data on erbon and ERBON U indicate an acute oral LD50 of 1 to 2 g/kg. Precautionary labeling indicates it may cause skin irritation and eye irritation. Toxicity studies have been conducted in fish (2/28/63) and in birds (submitted to EPA on 9/26/73)* According to Warren Crummett, analyses for TCDD are complicated by the high content of other materials in our technical erbon. Marguerite L. Leng Government Registration Health & Environmental Research enc :3 P. S.. See attached article on ERBON ^ plus KUR0N in the latest issue of the Dow publication "Industrial Vegetation Management" (Vol. 6, No. 1, 197*+) by Juell Johnson of the South Dakota Department of Highways. Newly approved uses of HURON are listed in an excerpt from the same magazine as an addendum to an Update on the Status of 2,^,5-T by L. Southwick. rr arutw:j - silvex - Section G.l, October 1971 Toxicology : A summary of known information on the toxicology of silvex was included in the December 1967 submission, and was repeated as Sections C 0.1 and C .0.2 in the amendment submitted in ' December .1970 . Additional reports on toxicity studies in c cattle and sheep were included in Section C.l in December 1970.G r `.i. Data presented in these submissions show that the oral LD^q ^ of silvex and its various formulations is in the range of 500 to 1000 mg/kg of body weight for various species of animals. A no-effect level of 7 mg silvex per kilogram per day was found in 90-day feeding studies with the sodium salt and the propylene glycol butyl ether ester in rats, and with the potas sium salt in dogs. The no-effect level in 2-year feeding studies was essentially 2.6 mg/kg/day in both species. No significant adverse effect was produced in livestock given salts and esters of silvex orally at levels of 25 or 50 mg/kg/da However, 100 mg/kg/day was lethal to cattle and sheep after 29 and 11 daily doses, respectively, and 250 mg/kg/day produced death in cattle after five doses. Chickens showed reduced weight gain after ten doses of 100 or 250 mg/kg/day. The lethal dose of the butoxyethanol ester of silvex was >9000 mg/kg of body weight for pheasants and young Bobwhite quail. Mortality was produced in 50% of young Bobwhite quail given the ester at 5000 ppm in the diet for less than 10 days, in young pheasants at 5000 ppm for less than 100 days, and in adult Mallard ducks at 2500 ppm for less than 100 days. 16413 I Report anc cate Bio-Test Labs 6/19/62 Dow Chemical 10/26/54 Dow Chemical 10/30/53 Dow Chemical 3/17/65 Dow Chemical 4/S/65 Summary of Toxicological Data on Silvex in Rats and Dogs Formulation used Dowco 171* (PGBE ester) Sodium salt Kurosal SL (K salt) Kurosal SL (K salt) Kurosal SL (K salt) Test animal Rats Duration of test 90 days Rats 90 days Dogs 89 days Rats Dogs 2 years 2 years No-effect level given in report 10 mg/kg/dav 0.01% in diet ' 0.03% in diet 100 ppm in diet 190 ppm in diet mg/kg/cay silvex ecuiv. 6.8 5-10 (calculated) 7 (based on daily food intake) 2.6 2.6** "Propylene glycol butyl ether esters of silvex (67.5% silvex acid equivalent). KUF.CN'C v:eed and brush killer contains approximately 6.8% PGBE ester of silvex (45% silvex acid equivalent) . **A slight histopathological effect was noted in male dogs but not in female dogs at 2.6 mg/kg/cay silvex equivalent after two years of continuous feeding. Prepared by Marguerite Leng, Agricultural Department/ The Dow Chemical Company, December 29, 1966 Revised September 10, 1970 ca> : T r> - p r <' < 1 Z6S8P0M -'***$*9?; Section C.2.0 Summary oC Teratology Studios with Silvex in nr.tr;, January 1973 . Numerous teratology studies were conducted with phenoxy herbicides by various government and industrial groups following disclosure on October 29, 1969, by Dr. Lee A. DuBridge, Science Adviser to the President, that studies conducted in 1965-1963 by Bionctics Laboratory in Falls Church, Virginia, indicated teratogenic responses were produced in mice and rats given high doses of 2,4,5-T. The significance of these and other studies conducted at exaggerated levels by non-representative routes of administration is still under discussion by experts in the field of teratology. As part of this program, studies were conducted by The Dow Chemical Company on silvex and its propylene glycol butyl ether (PGBE) esters by oral administration in rats. The two studies were reported in Sections D.2.1 and D.2.2 of this amendment to PP 8F0675 for silvex. Groups of Sprague Dawley rats were given silvex at doses of 0, 50, 75 or 100 mg/kg/day on days 6 through 15 of gestation, or from day 6 of gestation through lactation. The tost compound was administered orally once daily as a corn oil suspension. Additional groups were given silvex at 25 to 150 mg/kg/day or its PGBB ester at 35 to 200 mg/kg/day, on days 6-15, 7-9 or 9-11 of gestation as described in rhe reports. Certain groups wore utilised for teratologic studies while others were used.for postnatal studies. The following parameters, where applicable, were examined in those studies with silvex and its FGBE esters: clinical observations; body weights; food consumption; gross visceral 18.415 examina Lion of clams; position of Ictuses in utero; numbers of live fetuses, resorptions and corpora lutea; individual pup weights (days 0, 5, 21) and sc;:; external skeletal airci visceral examinations on teratology litters; histologic examination of selected tissues; examination-of weanling rats to determine degree of skeletal ossification. Conclusion ` No overt clinical signs of toxicity, major visceral or skeletal anomalies or undesirable effects on other repro ductive parameters resulted from treatment with silver at 25 mg/kg/day or silvex PGBE esters at 35 mg/kg/day (equiv-' alent to 23 mg of silvex per kilogram of body weight per day) on days 6 through 15 of gestation . Doses of 50 mg/kg/ day resulted only in fetal toxicity as reflected by decreased fetal weight and minor skeletal variations, but no major abnormalities were observed at this dosage level. Maternal and fetal toxicity and fetal anatomical alterations were observed at levels of 75 mg/kg/day or more for silvex and at 100 mg/kg/day for silvex PGBE esters. Maternal death occurred when silvex was administered at 1.00 mg/kg/day for 10 days during gestation. DOW CHEMICAL U.S.A. 9008 Building August 10, 1976 M IO LAN O . M ICHIG AN 48640 L M. W. Sauerhoff Health & Environmental Research 1803 Building cc: E. H. Blair, 2020 G. E. Blau, 1707 M. B. Chenoweth, 607 J. H. Davidson, 9001 P. J. Gehring, 1803 H. L. Gordon, 2030 J. E. LeBeau, 1803 D. D. McCollister/Reg. B. A. Schwetz, 1803 ' Section____ \ REPORT ON SILVEX STUDY IN HUMANS Thank you for sending me a copy of your report on Fate of Silvex Following Oral Administration to Humans, dated May 21, 1976. It represents a tremendous amount of work and should be very helpful in resolving some of our problems with this herbicide. However, as we discussed by telephone, a somewhat different treatment of the data could provide even more useful information on rate of excretion of silvex by humans, for comparison with 2,4-D and 2,4,5-T. First of all, I believe the half-life should be based on total excretion of silvex plus silvex conjugate in urine and feces rather than on only free silvex in urine. This would take care of variations in proportion of silvex in free and conjugated form in urine of the various subjects, ranging from a ratio of 5.19 for subject 1 to 0.66 for subject 4 (page 12). Would It be possible to have Gary Blau run the computer analysis of data for silvex plus silvex conjugate for successive 24 hr (or 12 hr) interval? in urine? It would provide better comparison with rates of excretion of 2,4-D and 2,4,5-T in humans where only small amounts, if any, were in conjugated form. Secondly, fecal samples should be analyzed for longer than the first two days after dosing to see whether fecal excretion was delayed, at least for those subjects where total recovery in urine was less than /5% (subjects 5 and 6). It is possible that silvex taken up by the second compartment in the body would end up in feces. However, the first two fecal samples may have been taken as early as 0.5 and 24.5 hr after dosing which doesn't allow much time for uptake, possible conjugation in the liver or elsewhere, and delayed elimination as conjugate(s) in faces. Thirdly, Figure 2 for excretion in urine should be based on silvex pin? silvex conjugate to represent total rate of elimination of the chemical from the body. Differentiation should be made between "metabolism"^in A N O P E R A T IN G `J N I T O F T H E D O W C H E M IC A L C O M P A N Y *ou M. W. Sauerhoff -2- August 10, 1976 which Che parent chemical can be regenerated unchanged from the conjugate, and true metabolism in which the parent molecule is irreversibly altered (such as by hydroxylation of the ring, cleavage of the ether link to release the trichlorophenol, etc.). Similarly, hydrolysis of silvex ester(s) to give silvex and silvex conjugate(s) is not comparable to true metabolism/degradation of the herbicide molecule. t It will be interesting to see whether reanalysis of the data for silvex plus silvex conjugate will take care of the biphasic pattern of elimination developed by the computer. This pattern is not readily apparent from the plot of silvex excretion by the eight subjects (Figure 2). Perhaps total excretion will fit a one compartment model as found for 2,4-D and 2,4,5-T. Were any studies done on ease of dissociation of the conjugate(s)? As far as I can see, the samples were simply adjusted to pH 1 or 13 and refluxed to hydrolyze any and all conjugates. On the other hand, a slight change in pH might have been adequate to release the silvex. If the conjugates are very labile, this could account for the variability in proportion of free vs conjugated silvex in the urine among the various subjects. The apparent difference between silvex and 2,4-D and 2,4,5-T may also be due to the fact that the silvex administered was a racemic mixture of optically active isomers. Perhaps one isomer forms soluble salts like 2,4-D and 2,4,5-T whereas the other isomer may preferentially form a conjugate with some optically activ amino acid. Only one isomer of silvex has herbicidal properties according to Loos in a book issued recently (Kearney et al. 1976). As we discussed, I would greatly appreciate having recalculated half-live(s) based on total excretion of silvex in urine as soon as possible. I am currently finalizing a paper on comparative metabolism of phenoxy herbicides in animals for presentation at the ACS meeting in San Francisco on September 2. One slide deals with the three studies conducted by Dow in humans, comparing the excretion pattern and rates of elimination for 2,4-D, 2,4,5-T and silvex following single oral doses of 5, 5, and 1 mg/kg, respectively. It would help if the half-lives were all based on total excretion. Joe LeBeau has suggested that we meet soon to discuss the data generated in this study. Milt Getzendaner Is commenting separately on the need for more actual analytical data, such as on analyses of individual samples. We are also concerned about inclusion of a paragraph on page 16 which postulates that "the fraction of silvex not recovered in some subjects may represent metabolism of silvex and excretion in urine or bile of a heretofore unidentified metabolite(s)." The report also suggests that studies without a tag are of limited value. Such statements should not be made in reports which may be submitted to government agencies or used in court actions. s '] /} '] a-w y .iJ m.'.r Marguerite L. Leng Government Registration Health and Environmental Research abc e'98Z0jft'o DOW CHEM ICAL U.S.A November 8 , 1978 M ID LA N D , M IC H IG A N 48640 Irvin g J . S e lik o f f , M.D. Environmental Science Laboratory Mt. S in ai School o f M edicine 100th S tr e e t and 5th Ave. New York, N.Y. 10029 Dear Dr. S e lik o f f , I t was good to ta lk to you y e ste r d a y . This l e t t e r is to confirm w ith you th a t Dow s c i e n t i s t s are p lea sed to a ccep t your in v ita tio n to come to Mt. S in ai and p resen t a seminar on phenoxy h erb icid es on Wednesday, December 20, 1978. I t i s my (our) understanding th a t you wish us to p resen t the s c i e n t i f i c data we have on 2 ,4 -D , 2 ,4 ,5 -T and r e la te d su b sta n ces. The purpose i s to acquaint you and your s t a f f w ith the data so th a t you w ill be b e tte r ab le to plan and execu te th e proposed ep id em io lo g ic study in the P a cific Northwest. The people we have s e le c te d to p r e se n t th e seminar are: John Davidson, T echnical A dvisor to the D irecto r o f Research and Development, A gricultural Chemicals Department. Bernard Schwetz, D irecto r o f Dow's T oxicology Research Laboratory in Health and Environmental R esearch. Richard Kociba, Head o f Pathology in th e T oxicology Research Laboratory. Benjamin Holder, Medical D ir ec to r fo r Dow USA and D irecto r o f Biomedical Research in H ealth and Environmental Research Warren A. Crummett, T echnical Manager o f A n a ly tica l Labora to r ie s. V. K. Rowe, D irecto r o f T o x ic o lo g ic a l A f f a ir s , Health and Environ mental S cien ces (I may come along as moderator or w h a tev er). You asked i f we had any o b je c tio n to your in v it in g a few s c i e n t i f i c c o lle a g u e s from such p la ces as NIEHS and NYU. C erta in ly n o t. AN OPERATING UNIT OF THE DOW CHEMICAL COMPANY 028638 I. J. Selikoff November 8, 1978 I mentioned to you th at Walter Melvin Jr. M.D., M.P.H. and Sc.D. o f the I n s titu te o f Rural Environmental Hefih, Colorado State U niversity at Fort C ollin s might well be w illin g ta co n trib u te. Dr. Melvin is well informed and has had a wealth of hum experience in the phenoxy herbicide area. He served with th e Air Forcef&r many years and has been d ir e c tly involved in V iet Nam, with the dispoal o f the Air Forces' co n tro v ersia l inventory o f Agent Orange, with theiSveso in cid en t and p r esen tly is engaged in an environmental or ep id a io lo g ica l study o f some s o r t in the Oregon-Washington area. You asked what papers your people cod'd b e n e fit from reading before the seminar. I suggest the following: 1. Toxicology o f Phenoxy Herffcides and Hazard Assessment of th e ir Use in R eforestation by Fnak N. Dost. Ph.D. 2. Long-term Hazards o f Polydforinated Dibenzodioxins and Poly chlorinated Dibenzofurans. WHO - IARC Technical Report No. 78/001, a report o f the Odht NIEHS/ARC Working Group. 3. Chlorinated Phenoxy Acidsaad Their D ioxins. Ecology B u lletin No. 27 from Royal SwedishAtademy o f S cien ces, C. Ramel (ed ). 4 . Embryo Problems Posed by tie Seveso A ccident. H. Tuchmann. D u p lessis, M.D., LeConcomssMedical No. 44, Nov. 2 6 ,.1 9 7 7 . I have included copies of the T itleqage of the f i r s t three o f these so as to help in id e n tific a tio n . Thereare, o f course, hundreds o f papers but th ese reviews w ill g iv e .a good o e r a ll p ersp ectiv e and referen ces to s p e c if ic s . I am enclosing a copyof a tra n sla tio n o f number 4 because I did not expect th at you had seen tto r might not be able to g e t i t in tim e and I do think i t is important. I hope I have answered a ll the quesfons you asked. I f you have s p e c if ic item s you would lik e to have discussal, l e t me know and I wi l l do our b e s t to Include them. With b est personal regards, V. K. Rowe, Sc.D. D irector, Toxicological Affairs Health and Environmental Science 1803 Building (517) 636-2376 Enclosure krr bcc; E.Blair WLCrummett J_Davidson J.DonaIds P. Gehring B. Holder R. Kociba B. Schwetz P .5 , Asof now we are planning to take the Dow plane .9te*Neywou York and more d e back tails on December later. 20. I will S84, TTOOmOQ 25-l O TOXICOLOGY OF PHENOXY HERBICIDES AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXLN Frank N. Dost, D.V.M. Department of Agricultural Chemistry and Environmental Health Sciences Center Oregon State University Corvallis, Oregon 97331 February 1978 16421 TOXICOLOGY OF PHENOXY HERBICIDES AND 2,3,7,8-TETRACHLQRODIBENZO-p-DIOXIN Introduction ................................................ 1 Toxicology of T C D D ....................................... . . History of Human Exposure ............................... General Toxicity in LaboratoryAnimals ................... Pathologic Morphology Resulting From TCDD Intoxication . . . Effects of TCDD Upon Reproductive Functions ............... Carcinogenic and Mutagenic Potential of TCDD ............. Absorption, Distribution, Metabolism, and Excretion of TCDD . Behavior of TCDD in the Physical Environment and in Submammalian Species .................................. Induction of Microsomal Enzymes .......................... 2 2 4 7 9 11 12 14 18 2 , 4 , 5 - T .................................................... General Toxicity in Laboratory Animals .................. Effects of 2,4,S-T on Reproduction ...................... Carcinogenic and Mutagenic Potential of2,4,5-T ............ Absorption, Distribution, Metabolism, and Excretion of 2 , 4 , 5 - T ........................................... Behavior of 2,4,5-T in the Environment and Effects on Submammalian Species .................................. 22 22 25 28 29 32 2 , 4 - D ...................................................... Toxicity to H u m a n s ..................................... General Toxicity to Laboratory Animals .................. Effect of 2,4-D on Reproductive Function ................. Carcinogenic and Mutagenic Potential of2,4-D .............. Absorption, Metabolism, Tissue Distribution, and Excretion of 2 , 4 - D ............................................. Behavior of 2,4-D in the Environment and Effects on Submammalian Species .................................. 35 35 36 38 39 40 42 Silvex...................................................... Biological Effects of Silvex and Its Derivatives ......... Effect of Silvex on Aquatic and Invertebrate Species . . . . Metabolic Fate of Silvex in Mammals....................... 44 44 46 48 References 51 TOXICOLOGY OF PHENOXY HERBICIDES AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN Frank N. Dost, D.V.M. Introduction The phenoxy herbicides 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), 2.4- dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxypropionic acid (silvex) are important agents for reforestation site prepara tion and for release of conifer plantings. To be used, the compounds must be inserted into a segment of the environment that may be occupied by humans or may have a potential for migration to points of human con tact or consumption. This document is a review of the pertinent litera ture describing biological effects and environmental behavior of the three herbicides, and of 2,3,7,8-tetrachlorodibenzo-p-dioxin, an extremely toxic contaminant found in small quantities in 2,4,5-T and silvex. For various reasons, the greatest public attention has been focused on 2,4,5-T and its unique contaminant, TCDD. Since 1969 public and sci entific attention to TCDD has overshadowed the specific concerns about 2.4- D, which has no TCDD, and about 2,4,5-T and silvex, which are con taminated. The unusual nature of the TCDD contaminant has made a special case of the herbicide 2,4,5-T, and has made it the subject of an unusually vehement public, political, and scientific controversy. An enormous scientific effort has evolved in an attempt to learn about the unique character of the compound, and to provide a rational basis for regula tory decisions. Hopefully, this effort to bring the existing literature into focus will aid in the decision-making process. As matters presently stand, there is a division in the collective public and scientific minds on the safety and benefits of use of the phenoxy herbicides. As in any controversy, both views include unreason able people, but the bulk of the argument is among sincere citizens and competent scientists on either side. It is my hope that the reviews and assessments in this document are dispassionate and without bias; other wise it will be of limited utility. In preparing this review, emphasis has been placed on information about the toxic properties of the respective agents. An assessment of hazard necessarily considers along with biological effects, factors govern ing the probability of exposure, and the probability that the chemical once contacted will enter the organism. With the exception of the section on TCDD, I have chosen to touch only lightly upon the environmental behavior of the compounds. This information about phenoxy herbicides is well established and is much less complex than the behavior of TCDD. A more thorough treatment of the environmental behavior of TCDD is necessary here because: 1) this contaminant is clearly the most important of the four compounds under 1 study; 2) the high toxicity demands thorough understanding of the com pound as it exists outside the target of toxic effect; 3) the environ mental chemistry of TCDD is incompletely studied and subject to some controversy, whereas that of th herbicides themselves is well estab lished. The organization of this review and assessment includes sections re viewing the scientific literature on TCDD, 2,4,5-T, 2,4-D, and 2,4,5-TP (silvex), an assessment of the hazard potential of their distribution in the environment, recommendations on some aspects of methods of use of the herbicides and recommendations of needed research. A shortened ver sion of the review and assessment is also attached. Organization within the review differs slightly among the chemical sections because of differ ences in research emphasis or the character of the agent itself. In cer tain instances, for example in considering the enzyme induction capacity of TCDD, an area may be dealt with that is not entirely germane to the issue of hazard but of considerable importance in understanding the biology of the compound. A number of useful reviews of interaction of phenoxy herbicides or TCDD with animal systems have been or are being published. The most recent one by Leng (1977), discussing metabolic fate of phenoxy acids in domestic animals, Mercier (1976) in response to the Seveso accident which spread large amounts of TCDD through several Italian communities, and McQueen et al. (1977) refuting alleged association between herbicide use and fetal abnormalities in humans. McKenzie et al. (1975), Norris et al. (1972), Buhler (1977), and the EPA position paper arising from the Dioxin Working Group (presently in draft) are also all of considerable value. TOXICOLOGY OF TCDD History of Human Exposure Probably the first clinical description of human TCDD exposure arose from the study by Kimmig and Schulz (1957) of workers in chlorophenol factories in Germany. The principal symptom was a persistent skin lesion (chloracne; so-called because it is characteristic of a number of related compounds and was at one time thought to be caused by free chlorine. The active agent was established as TCDD, and animal studies showed that the same lesions appeared on rabbit ears after application of as little as 0.002% TCDD (Kimmig and Schulz, 1957). High topical doses and oral doses of 50 ug/kg or greater caused liver necrosis. A similar in dustrial intoxication in France was reported by Dugois and Colomb (1957). Pathology of the skin lesions is well described by Kimbrough (1974). A study of 29 phenoxy herbicide workers by Bleiberg et al. (1964) disclosed chloracne and a high frequency of uroporphyrinuria, and tissue deposition of porphyrins related with hyperpigmentation and skin fragility. This assemblage of symptoms is commonly called porphyria cutanea tarda. The excessive production and excretion of porphyrins apparently results from a hyperactivity of the enzyme 6-amino levulinic acid (ALA) synthetase, the first and rate limiting step in porphyrin formation. Porphyrins are 2 3................................. DoWOOll 789 eventual components of heme proteins, such as hemoglobin and cytochromes. Effects on ALA synthetase and other enzyme systems will be discussed in another section. Poland et al. (1971) examined the same factories later, after somewhat more satisfactory industrial hygiene measures were estab lished. Among the 73 male workers observed, no clinical porphyria was apparent even though some degree of chloracne still persisted in most of the subjects. There was also apparently a significant psychological effect as measured by the Minnesota Multiphasic Personality Inventory. The absence of porphyria and persistence of chloracne in the later study has led Poland et al. (1971) to conclude that while both may be caused by TCDD, the mechanisms are probably dissimilar. Several severe accidental exposures to TCDD have occurred. In several cases, runaway chlorophenol plant reactions caused widespread exposures within the factory or in the surrounding community. An accident in Germany in 1953 exposed a large number of workers, said to have suffered liver, kidney, splenic, eye, cardiovascular, and nervous symptoms. A worker who apparently was in extensive contact with a contaminated seg ment of the plant five years after the accident became ill and later died. An explosion in a Dutch plant in 1963 released 30-200 g of TCDD into the main gallery of the factory exposing SO people. In the subsequent two years, four died, but no clear association with TCDD intoxication could be shown (Hay, 1976). An explosion in a British plant in 1968 resulted in 79 cases of chloracne (May, 1973). In Czechoslovakia a pentachlorophenate production stream overheated under pressure, producing large amounts of TCDD (Jirazek et al., 1974). (It should be noted in this and some ear lier German work a different numbering convention was used. 2,3,6,7-TCDD and 2,3,7,8-TCDD are the same compound.) Of 80 exposed workers, 76 devel oped chloracne, porphyria cutanea tarda, disorders of plasma lipids, hepatic damage, neural lesions, and neurasthenia. An explosive reaction in a plant at Seveso, Italy, in 1976 scattered TCDD, apparently in kilogram amounts, over a wide area that included sev eral communities. Details are still not widely disseminated but great numbers of animals were killed and many people were made ill. A review of the incident was prepared by Mercier (1976) a few months after the incident but the slow onset and persistent effects did not permit a de tailed assessment of the damage. Since that time the Seveso disaster has been given unprecented publicity and as might be expected this tragedy has been sensationalized in press coverage, in part because there may have been an effort to minimize or cover up the extent of the problem. There were apparently defects in the administration of the plant that may have resulted in the accident itself, and management of public warn ing and information early after the accident was questionable. Further contributing to the extensive public attention and sensationalizing of the incident is a virtual absence of objective reporting in the scien tific literature. As far as I am aware, there has been no scientific evaluation of the aftermath in publication since the review by Mercier (1976), shortly after the accident. In the U.S., TCDD extracted from hexachlorophene production was stored in oil, then mistaken for waste lubricating oil and sprayed on several horse arenas and farms in Missouri, for dust control. The 3 16425 incidents occurred in spring and summer of 1971. On one horse breeding farm, 62 of 85 horses exercized in the sprayed area became ill and 48 died, the last in January, 1974, two and a half years later. Hundreds of birds and rodents died, along with a number of dogs and cats. Several children were exposed through play in the arena soil. One developed a severe hemor rhagic cystitis and others showed clear evidence of chloracne. All re covered. The TCDD concentration in the soil was later established at more than 30 ppm, a truly massive amount. (This concentration amounts to 120 lb per acre per foot of depth, assuming 4 x 10^ lb soil/acre-foot.) The de tails of the latter event are described in detail by Carter et al. (1975) and Kimbrough et al. (1977). All of these exposures were to a mixture of TCDD with other agents. Possibly the only published account of exposure to pure TCDD is the des cription by Oliver (1975) of laboratory contact by three individuals as sociated with synthesis experiments. The individuals all had utilized routine protective procedures but sufficient contact developed nonetheless. The exposures were not sufficient to cause porphyrinuria or liver damage but serum cholesterol was raised. In two cases personality changes and neural disorders developed two years after exposure. The amount of TCDD contacted is not known. General Toxicity in Laboratory Animals This section is a review of data on effects of TCDD other than repro duction and teratology, carcinogenesis, and its enzyme inducing properties upon mammals. These will all be considered independently, as will studies of tissue distribution and metabolism. Schwetz et al. (1973) have determined lethalities for several species. The guinea pig is exceedingly sensitive, with an LD50 of only 0.6 yg/kg; rats are less sensitive; LD50 for males is about 20 yg/kg and for females the LD50 is in excess of 40 yg/kg. Rabbits are still less sensitive at more than 100 yg/kg. Dogs were found to require even higher effective doses. In these studies, responses of mice were highly erratic and were not included. McConnell et al. (1977a) have determined a single 30 day LD50 fot mice of 284 yg/kg, and 2 yg/kg for guinea pigs. An important characteristic of TCDD is a very long delay before lethality, often 2-3 weeks and occasionally 7-8 weeks. The pathology associated with TCDD toxicity may include thymic atrophy liver necrosis, and degeneration in the kidney and thyroid. The guinea pig is unique in that it suffers limited hepatic damage, even at doses that cause substantial thymus depletion (Gupta et al., 1973). The degree of thymic atrophy is generally dose dependent; the studies of Gupta et al. (1973) did not establish a zero effect dose, but the lowest dose in the rat of 1 yg/kg/day for 31 days caused moderate thymus and liver damage, and kidney and thyroid degeneration. Guinea pigs treated with 0.2 yg/kg weekly for eight weeks had limited thymus damage and no other pathology. Studies of guinea pigs at lower doses suggest a no observed effect level at 0.008 yg/kg/weekly for eight weeks, a total of 0.064 yg/kg, although there was a slight decrease in lymphocytes at that dose (Vos et al., 4 u o il 7^1 1973). Animals g iv e n te ta n u s to x o id a t th e fo u rth week to m easure humoral im m unity w ere found to have somewhat low er thymus w e ig h ts th a n c o n tr o ls , ' a lth o u g h th e d if f e r e n c e was n o t s i g n i f i c a n t . A ccording to th e d a ta o f Kociba e t a l . (1976) th e no observed e f f e c t l e v e l i n r a t s g iv e n TCDD in th e d i e t 5 d ay s a week f o r 13 weeks i s 0 .0 0 1 PgA g/ day, to ta lin g 0.065 yg/kg, alm ost id e n tic a l to th e value found in g u in ea p ig s . A g ain , thymus w eight ap peared to be th e most s e n s i t i v e param e t e r . The Kociba stu d y d e a lt w ith a very broad range o f c l i n i c a l , ch em ical, and h em ato lo g ic a l m easurem ents. At a dose o f 1 .0 y g /k g /d a y , 2 o f 10 r a t s d ie d and w eight g a in d ecre ase d s h a rp ly , e ry th ro c y te num bers and volume d ecreased , re tic u lo c y te s in creased in m ales. In fem ales throm bocytes d ecreased and to t a l leu k o cy tes in c re a se d . D if f e r e n tia l counts were n o t a l t e r e d . U r in a r y p o r p h y r i n s , c r e a t i n e and 6 am ino l e v u l i n i c a c id (ALA) a l l i n c r e a s e d . Serum b i l i r u b i n , BUN and a l k a l i n e p h o s p h a ta s e a l s o w ere e le v a te d . At an in ta k e o f 0 .1 y g /k g /d ay re d c e l l num bers and volume w ere s t i l l s l i g h t l y d e c r e a s e d i n m ales and p o r p h y r i n , ALA, b i l i r u b i n , and a l k a l i n e p h o s p h a ta s e was e l e v a te d i n f e m a le s . No c h a n g e s i n b lo o d p a r a m e te r s o ccurred below th e se le v e ls . In a s i m i l a r s tu d y Z in k l e t a l . (1973) fo u n d no ch a n g e s i n SGPT, b i l i r u b i n , o r e r y t h r o c y t e s a f t e r 31 d ays o f tr e a t m e n t a t 1 .0 y g /k g /d a y and no ch an g e in SGOT, c h o l e s t e r o l , o r serum p r o t e i n a t 0 .1 y g /k g /d a y . T h e re was a m odest d e p re s s io n in blood g lu c o se a t th e low er dose r a t e . Mice tr e a te d w ith 1 .0 yg/kg/w eek f o r s ix weeks w ere found to have d e p le te d thym us w e ig h t (Vos e t a l . , 1974) b u t no ch an g e s w ere e v id e n t a t th e low est dose, 0 .2 yg/w eek. H em atological changes occu rred only a t a d o se o f 25 y g /k g /w e e k , f o r s i x w eeks. Some c h a n g e s i n seru m p r o t e i n s d id occur a t an in ta k e o f 0.2 yg/kg/w eekly. U n fo rtu n a te ly , th e re is no d a ta su g g estin g th e m agnitude o f th e n o -e ffe c t sin g le dose in any s p e c ie s. S tu d ie s in p r im a te s h a v e been l i m i t e d . M cN ulty a d m in is te r e d TCDD a t a r a t e o f 20 p a r t s p e r b i l l i o n in th e d i e t to a s i n g l e monkey and c a u s e d i t s d eath in a few d ay s. A 1 0 -fo ld low er c o n c e n tra tio n p e rm itte d s u rv iv a l f o r ab o u t two m onths. T o ta l in ta k e was e s tim a te d a s ab o u t 6 y g /k g (M cNulty, 1 9 7 7 ). A s tu d y o f e i g h t fe m a le R hesus monkeys f e d 500 p a r t s TCDD p e r t r i l lio n has r e c e n tly been com pleted in th e la b o ra to r y o f J .R . A lle n . The anim als showed d e r m a titis and m en stru al i r r e g u l a r i t i e s w ith in s ix months a f t e r i n i t i a t i n g t r e a t m e n t . One monkey d ie d p r i o r t o th e s e v e n th m onth and by n in e months o f ex p o su re, a l l su rv iv o rs ex p erien ced a se v e re anem ia and le u k o p e n ia . Food in ta k e was norm al b u t a l l o f th e a n im a ls l o s t w e ig h t. A f t e r 11 m o n th s, f i v e o f t h e e i g h t monkeys d i e d , a l l w ith s e v e r e an em ia and d e s tr u c tio n o f blood form ing t i s s u e s . The t o t a l dose was e stim a te d a t about th re e yg/kg (A llen e t a l . , 1977a, 1977b) or about .3 yg/m onth. A number o f q u e s tio n s a r is e from th e se d a ta . The monkey s tu d ie s a re lim ite d , b u t th ey do su g g est th e p o s s i b ili ty th a t th e le th a l dose is s im ila r re g a rd le s s o f th e tim e over which i t i s a d m in is te re d . The im p l i c a t i o n s a r e q u i t e i n t e r e s t i n g . I f i t i s t r u e t h a t TCDD d o s a g e i s tim e in d e p e n d e n t in th e m onkey, th e n th e e f f e c t i s t r u l y c u m u la tiv e . No c h e m i c a l i s known to h av e su c h p r o p e r t i e s , and i f TCDD b e h a v e s in t h i s f a s h i o n , 5 -J. . any e stim a te o f a c c ep tab le dose must assume a d d itiv e e ffe c ts over long p e r i o d s . TCDD d o e s n o t rem ain in th e b o d y o f monkeys (Van M i l l e r e t a l . , 1976) o r o th e r s p e c ie s (Van M ille r e t a l . , 1976; F r ie s and M arrow, 197S; Rose e t a l.., 1976). A cu m u lativ e e f f e c t , i f i t e x i s t s , must th e n e n t a i l irre v e rs ib le e ffe c ts rem aining a f te r th e m olecule leaves a c tiv e s i t e s . TCDD i s a p p a r e n tly n o t m e ta b o liz e d to a s i g n i f i c a n t e x t e n t (Rose e t a l . , 1976; V inopal and C a sid a , 1973; Van M ill e r e t a l . , 1976) so i t i s c o n c e iv able as w ell th a t a sin g le m olecule could in te r a c t se v e ra l tim es b efo re le a v in g th e body. A p o s s i b l e m echanism may be i n t e r c a l a t i o n o f th e p l a n a r TCDD m o le c u le i n t o DNA (H u s s a in , 1 9 7 3 ), p r o d u c ti o n o f m is in f o r m a tio n and d e p a r tu r e o f th e a g e n t t o i n t e r a c t a t a n o t h e r DNA s i t e . D ata in o th e r s p e c ie s , e s p e c ia lly th e g u in e a p ig do n o t su p p o rt th e id ea o f ex ten siv e cum ulative e f f e c t. A dose r a te o f 0.2 U g/kg/w eekly f o r e i g h t weeks p r o v id e d a t o t a l d o se a lm o s t t h r e e tim e s th e LD50, but no d eath s occu rred and organ changes, w hile m easu rab le, were n o t s e v e re (Vos e t a l . , 1 9 7 3 ). R ats a ls o a c c e p te d a b o u t t h r e e tim e s th e LD50 o f 20 u g /k g o v e r 13 w eeks, w ith o u t l e t h a l i t y . Some m ic e , on th e o t h e r h a n d , d ie d a t a t o t a l d o se l e s s th a n th e a c u t e LD50, d i s t r i b u t e d o v e r a f o u r week p e r io d (Vos e t a l . , 1 9 7 4 ). T h e re a r e a few o t h e r b i o l o g i c a l r e s p o n s e s t o TCDD t h a t a p p e a r a f t e r h ig h d o ses o f from 10-25 U g/kg. One o f th e more i n t e r e s t i n g i s a d e p re s s e d c a p a c i t y f o r b i l i a r y e x c r e t i o n . TCDD s h a r p l y d e c r e a s e s rem o v al o f PCBs fro m th e l i v e r v i a th e b i l e (Yang e t a l . , 1977) and d e c r e a s e s plasm a c le a ra n c e o f ou ab ain in t o b i l e (Hamada and P e te rs o n , 1977) . In the l a t t e r work, s te r o id a l in d u cers o f m icrosom al enzymes re v e rse d th e TCDD e f f e c t . W hether t h i s e f f e c t h a s p o t e n t i a l s i g n i f i c a n c e i n th e response to secondary to x ic ity can only be sp ecu la ted upon. In any case th e doses req u ire d fo r e f f e c t are q u ite high and p ro b ab ly n o t a tta in a b le under r e a l i s t i c environm ental co n d itio n s. B ecause TCDD c a u s e s in c r e a s e d m icro so m al f o r e i g n compound m e ta b o l i z i n g a c tiv ity in th e kidney c o rte x (Fowler e t a l . , 1975, 1977), Pegg e t a l . (1976) e v a lu a te d proxim al tu b u la r fu n c tio n in in to x ic a te d r a t s . They found l i t t l e change a t any b u t la rg e doses (25 and 50 u g /k g ). T h ere h av e been few o b s e r v a ti o n s o f m e ta b o lic c h a n g e s c a u s e d by TCDD. In c o rp o ra tio n o f ^H-sodium a c e ta te in to l i v e r l i p i d f r a c tio n s seems to be im p a ire d a t TCDD d o s e s ab o v e 0 .1 Ug/kg (C unningham and W illia m s , 1 9 7 2 ), but the sig n ifican ce o f th is e ffe c t has ap p aren tly not been explored fu r th er. E v id en ce o f th y m ic a tr o p h y a f t e r TCDD t r e a t m e n t h a s s t i m u l a t e d m ore d i r e c t s tu d y o f e f f e c t s upon th e immune r e s p o n s e s . Vos and M oore (1 9 7 4) and Vos e t a l . (1973) fo u n d ly m p h o c y tic d e p l e t i o n in th e c o r t e x o f t h e thymus and d ep ressed c e l l u l a r im m unity in r a t s and mice exposed d u rin g g e s ta tio n and th e p o s t-n a ta l p e rio d . G uinea p ig s were s im ila r ly a ffe c te d (Vos e t a l . , 1 9 7 3 ). T higpen e t a l . (1975) a tte m p te d to t r a n s l a t e th e e f f e c t in to term s o f i n f e c t i v i t y and found th a t d oses o f 1 ug/kg o r more in creased m o rta lity and tim e to death of mice in fe c te d w ith S alm o n ella b ern . Course and s e v e r ity o f d ise a se due to p se u d o ra b ie s v iru s was n o t a l t e r e d by TCDD. W ith a l l th e s tu d y o f TCDD e f f e c t s , no p a t t e r n o f 1S428 path o lo g y , tis s u e d is tr ib u tio n , and biochem ical o r p h y sio lo g ic a l change has emerged th a t i s c o n s is te n t enough to su g g est a mechanism o f le th a l e f f e c t . M cC onnell e t a l . (1977a) q u o te p a p e r s in p r e p a r a t i o n by Van L ogten e t a l . w hich show no a s s o c i a t i o n o f d e a th in TCDD i n t o x i c a t i o n w ith a d re n a l o r p i t u i t a r y hormones o r m a ln u tritio n . a O O OC H*' ^ 'V* P a th o l o g ic M orphology R e s u lt in g From TCDD I n t o x i c a t i o n Much o f th e m ore p ro n o u n c e d t i s s u e ch an g e r e s u l t i n g from TCDD h as been observed fo llo w in g high acu te doses o f th e compound. There is some q u e s tio n w h eth er th e a c u te damage seen r e p r e s e n ts t h a t o b se rv e d in a low le v e l lo n g e r term ex p erim en t o r in a f i e l d ex p o su re . B e fo re TCDD a s su c h becam e an i s s u e N orback and A lle n (1969) s t u d i e d th e membrane changes in l i v e r fo llo w in g in g e s tio n o f h e x a c h lo ro h e x a h y d ro p h en a n th re n e , a c o n s titu e n t o f s o -c a lle d " to x ic f a t . " The le s io n s found u n d er e le c tro n and li g h t m icroscopy a n tic ip a te d th e damage l a t e r to be fo u n d a f t e r TCDD. M ost s t r i k i n g was a g r e a t i n c r e a s e in t h e sm ooth en d o p lasm ic re tic u lu m (SER), w ith re o rg a n iz a tio n in to a c o n c e n tric a rra n g e m e n t, and th e ro u g h ER becam e f o ld e d and r o l l e d o v e r a p e r i o d o f d ay s t o form dense a rra y s o f c o n c e n tric s e p a ra te , n o t s p ir a lin g , p a ire d membranes. The a u t h o r s s p e c u l a t e t h a t SER p r o l i f e r a t i o n in r e s p o n s e t o i n t o x i c a t i o n i s r e l a t e d to a c c e p ta n c e o f enzym es s y n th e s iz e d by th e RER. The i n c r e a s e d membrane mass may a ls o be a means o f s e q u e s te r i n g c h l o r i n a t e d h y d ro carb o n in th e l i p i d p h ase o f th e membrane in c lo s e p ro x im ity to fo re ig n compound m e ta b o liz in g enzym es. (T his p a p e r c o n ta in s v ery u s e fu l sch em atic r e p r e s e n ta tio n s o f th e membrane developm ent se q u e n c e .) The same a u th o rs l a t e r re p o rte d s im ila r le s io n s in r a t s fo llo w in g a d m in is tra tio n o f 1 yg TCDD/kg/day f o r 21 d a y s (N orback and A lle n , 1 9 7 3 ). They fo u n d h y p o p la s i a o f lymph t i s s u e and bone marrow w ith p r o g r e s s iv e anem ia and le u k o p e n ia . The r e s u lta n t reduced r e s is ta n c e caused s u b s ta n tia l lo s s e s due to in f e c t i o n . Monkeys and c h ic k e n s in th e same s tu d y w ere much more s e n s i t i v e in th e l a t t e r p aram eters than r a ts . They a lso developed e x te n siv e flu id accum ulation in a l l body c a v itie s , probably due to decreased osm otic a c tiv ity o f blood due in tu rn to serum p ro te in d e c re a se s. (R e la tiv e s e n s itiv ity of chickens has also been noted by G reig e t a l . (1973).) Monkeys w ere s u b je c t to e x te n s iv e s k in le s i o n s , a p p a re n tly c o rre s p o n d in g to th e ch lo ra cn e o f humans, b u t r a ts and ch ick en s d id n o t s u f f e r sk in damage. Monkeys and c h ic k e n s a ls o e x h ib ite d d e g e n e ra tio n o f th e t e s t e s and decreased a c tiv ity o f sem iniferous tu b u les (see a lso A llen e t a l ., 1975). L arg e d o s e s o f TCDD ( s i n g l e d o s e s o f 50 o r 100 y g /k g ; 16 t o 31 d a i l y d o ses o f 10 y g/kg) t o th e r a t caused se v e re l i v e r and thym us dam age, w ith ic te r u s and d issem in ated hem orrhages, e s p e c ia lly in th e m yocardium . The liv e r c e lls increase in siz e and c e llu la r reg en erativ e attem pts occur. R e n a l t u b u l e c e l l s becam e v a c u o l a t e d . No p a t h o l o g i c a l ch an g e o c c u r r e d a t d o s e s o f 0 .1 y g /k g /d a y o v e r 31 d a y s , a t 1 .0 y g /k g w e e k ly f o r 6 w eeks o r 5 yg/kg in a s in g le dose (Gupta e t a l . , 1973). Fow ler e t a l . (1973) stu d ie d th e p r o g r e s s i v e c h a n g e s in r a t s o v e r 28 d a y s f o ll o w i n g a s i n g l e d o se o f 5 o r 25 yg TCDD/kg. I n c r e a s e d SER, ro u g h ly dose r e s p o n s iv e , was e v id e n t by d a y t h r e e , e s p e c i a l l y in c e l l s a d j a c e n t t o b i l i a r y d r a i n a g e . B o th RER and SER w ere g r e a t l y i n c r e a s e d by day 9, SER was a lm o s t a t n o rm a l l e v e l s by day 7 TTOO M O G -16, and b o th were in d is tin g u is h a b le from th o s e o f c o n tr o l a n im a ls by day 28. Jones and B u tle r (1974) c a r r ie d o u t a s im ila r experim ent w ith r a ts 'g iv e n a s i n g l e 200 y g /k g t r e a t m e n t . (A m a ss iv e d o s e , c o n s id e r i n g th e m in imum e f f e c t i v e d o s e .) M ost change was fo u n d i n c e n t r i l o b u l a r c e l l s , w ith d e g e n e ra tio n and n e c r o s is o f parenchym al c e l l s e v id e n t w ith in a week a f te r tre a tm e n t. They suggest th a t th e m u ltin u c le a te c e lls seen in in jjv c r e a s i n g num bers th r o u g h two months a f t e r tr e a tm e n t a r e form ed th r o u g h membrane d is tu r b a n c e and c o a le s c e n c e o f h e p a t o c y t e s . By 10 weeks some re c o v e ry was e v id e n t b u t f i b r o s i s o f c e n t r a l v e in s and s in u s d i l a t i o n s t i l l p r e v a i l e d . No m e n tio n o f l e t h a l i t y was m ade, a lth o u g h th e d o se was on th e o r d e r o f f i v e (fe m a le ) t o t e n (m ale) tim e s th e LD50. L o n g er term trea tm e n t o f r a ts (0 .0 0 1 -1 .0 y g /k g /d ay , S days w eekly x 13) caused alm ost com plete thymus in v o lu tio n a t a dose r a t e o f 1.0 y g /k g /d a y , somewhat d ecreased c o r tic a l thym ocyte and lymphoid c e l l numbers a f t e r 0 .1 y g /k g / day. S p le n ic m orphology was e s s e n ti a lly unchanged a t 1 .0 y g /k g /d ay . The l a t t e r dose a ls o cau sed some g ro ss edem a, and one o f f i v e m ales had decreased sperm atogenic a c tiv ity . Females a t th e h ig h e s t dose tended toward form ation o f cu b o id al e p ith e liu m in th e u te ru s and d ecreased s iz e and numbers o f c o rp o ra lu te a . O varian t i s s u e was a ls o abnorm al. At a l l lo w er d o s e s no e f f e c t s w ere o b s e rv e d (K o cib a e t a l . , 1 9 7 6 ). Two y e a r c o n tin u o u s f e e d in g s t u d i e s a t TCDD i n t a k e s o f 0 .0 0 1 t o 0 .1 y g /k g /d a y h a v e ju s t been te rm in ated and h is to p a th o lo g ic e v a lu a tio n is not com plete. Gross ex am in atio n showed t h a t in some r a t s g iv e n 0 .1 o r 0.01 yg TCDD/kg/ day th e re were in c re a se d numbers o f n o dules in th e li v e r (R .J. K ociba, Dow C hem ical Company, p r e li m i n a r y s t a t u s r e p o r t ; p e r s o n a l c o m m u n ic a tio n ). L esions g e n e ra lly s im ila r to th o se seen in r a t s were found in mice g iv e n s i n g l e o r a l d o se s up to 200 y g /k g and w eek ly d o s e s up to 25 yg (Vos e t a l . , 1974). At a dose r a te .1 yg/kg/w eek f o r 6 weeks d ecre ase d thymus w eig h t o c c u r r e d . A t 25 y g /k g /w e e k , serum g l o b u l i n s w ere d e c r e a s e d , and porphyria and b ile duct e p ith e lia l p r o lif e r a tio n occurred. S im ilar changes were a l s o r e p o r t e d .f o llo w in g tr e a tm e n t o f m ice w ith an LD50 (30 day) TCDD (McConnell e t a l . , 1977a). McConnell e t a l . (1977b) tr e a te d rh e su s monkeys w ith heavy s in g le d o se s o f TCDD ( in e x c e s s o f 70 y g /k g ) w h ich w ere f a t a l in e v e r y c a s e . Integum ental changes were pronounced, anem ia and lym phopenia and an i n creased n eu tro p h il count follow ed tre a tm e n t. L iv er, ad ren al and kidney a p p e a re d t o in c r e a s e i n w e ig h t r e l a t i v e to b o d y w e ig h t, b u t t h i s e f f e c t may have been due to g e n e ra l w astin g . W ith th e sk in h y p e rp la s ia , s im ila r changes o c c u rre d in th e e p ith e liu m o f some hollow o rg a n s . The m a jo r d i f fe re n c e from e f f e c ts in s im ila r ly t r e a te d r a t s was th e absen ce o f s i g n ific a n t liv e r lesio n s. C h ro n ic low le v e l in to x ic a tio n (500 p p t in d i e t s , ab o u t 0 .0 1 y g / kg/day) o f monkeys u n ti l d eath a t 7-12 months caused anem ia and p a n c y to p e n ia , w ith e x te n s iv e hem orrhage. Bone marrow and ly m p h atic t i s s u e s were h y p o p la stic , and e p ith e lia l s tr u c tu re s were h y p e rp la s tic (A llen e t a l ., 1977). The p a t h o l o g i c a l ch an g e s r e s u l t i n g from a b s o r p t i o n o f TCDD by horses and c a ts during th e M issouri horse arena episode have been re c e n tly d e s c rib e d in d e t a i l by Kimbrough e t a l . (1 9 7 7 ). The most pronounced and 18430 8 o O c o n s is te n t fin d in g s were th e g e n e ra liz e d h e p a tic f ib r o s is and n e c ro sis and g a s t r i c u l c e r a t i o n . T h ere was an a p p r e c ia b le in c id e n c e o f t u b u l a r d eg en eratio n and ab scesses in th e kidney and c a p su la r th ic k e n in g o f th e sp leen . Lesions in c a ts were g e n e ra lly s im ila r. T tO O E f f e c t s o f TCDD Upon R e p ro d u c tiv e F u n c tio n s O f th e many t o x i c r e s p o n s e s t o TCDD, th e p o t e n t i a l f o r t e r a t o g e n i c i t y has re c e iv e d perh ap s th e g r e a te s t n o to r ie ty . The e a rly fin d in g s o f 2 ,4 ,5 -T te ra to g e n ic ity ra ise d a co n sid era b le p u b lic consciousness and th e fin d in g t h a t th e h e r b i c i d e was c o n ta m in a te d by h ig h l e v e l s o f TCDD, a l s o fo u n d t o cause f e t a l m alfo rm atio n , focused a d d itio n a l a tte n tio n on th e c o n tam in a n t. In e v a l u a t i n g t h i s a s p e c t o f TCDD h a z a r d i t s h o u ld b e n o te d t h a t t h e te r a to g e n ic p o t e n t i a l o f a to x ic a n t can be c o n s id e re d in two w ays. The a b s o l u t e t e r a t o g e n i c d o se o f TCDD t o th e p r e g n a n t a n im a l on a b o d y w e ig h t b a s i s i s v e r y low , a s i s t r u e o f a l l TCDD e f f e c t s . A more r e a l i s t i c way o f r e la tin g th e te ra to g e n ic p o te n tia l, however, is by com parison w ith th e dose req u ire d to cause g e n e ra l to x ic e f f e c ts in th e m other. For exam ple, i n th e g u in e a p i g , TCDD t e r a t o g e n e s i s h a s n o t b een s t u d i e d b e c a u s e t h e te ra to g e n ic dose is ap p aren tly higher than the le th a l dose. -si - CaDt T h is s e c t i o n i s l i m i t e d t o s t u d i e s o f r e l a t i v e l y p u r e TCDD upon rep ro d u ctiv e fu n ctio n in experim ental anim als. There is re feren c e e ls e where to re p ro d u c tiv e s tu d ie s o f 2 ,4 ,5 -T and s ilv e x , which c o n ta in a t le a s t some TCDD. T e r a to g e n ic d e f e c t r e s u l t i n g from TCDD a r e r a t h e r s p e c i f i c i n e x p e r i m ental anim als; th e p rin c ip a l e ffe c ts a re in c re a se d frequency o f c l e f t p a la te (reported o n ly in m ice) and an ab n o rm a lity in which th e c e n tr a l c o lle c tio n reg io n o f th e kidney becomes e n la rg e d , w ith an a s s o c ia te d f lu id accu m u latio n (N eu b ert e t a l . , 1 9 7 3 ). L im ited lim b d e fo rm a tio n may be seen in a few anim als (S parschu e t a l . , 19 7 1 ). I n t e s t i n a l hem orrhage i s a ls o seen; th is e ffe c t is not terato g en ic but ra th e r is d ire c t to x ic consequence o f f e t a l e x p o s u re t o TCDD. F a t t y d e g e n e r a tio n o f f e t a l l i v e r s i s in th e same c a te g o ry (B ecker, 1974) as i s su b cu tan eo u s edema and d e la y e d o s s i fica tio n . In te ra to g e n ic ity stu d ie s of rodents the usual procedure is to ad m in is te r in to x ic a n t a t some p o in t d u rin g , o r th ro u g h o u t days 6-15 o f p re g nancy, w hich covers th e organ form ing p e rio d . S parschu e t a l . (1971) found t h a t th e lo w e st e f f e c t i v e dose o f 1.25 y g /k g /d a y cau sed some f e t a l m o rta lity , re so rp tio n s, and in te s tin a l b le ed in g . S k e le ta l ab n o rm a lities were lim ite d to delayed o s s if ic a tio n . Renal d e fe c ts ten d ed to o ccu r a t ab o u t th e same freq u en cy re g a r d le s s o f d o se. These changes a re a l l a r e s u lt o f d ire c t to x ic ity to th e fe tu s and are not developm ental. In te s tin a l hem orrhage has been rep o rte d in r a t fe tu s e s a t m aternal dose ra te s o f 0.25 yg/kg/day (Khera and R uddick, 1973). At h ig h e r doses up to 8 y g /k g /d ay th e same k in d o f e f f e c t s o c c u rre d w ith g r e a te r fre q u e n c y ; 0.5 yg/kg/day caused d ecreased m aternal w eight gain and h ig h e r doses resu lted in severe to x ic ity . C o u rtn ey and Moore (1971) exam ined r a t s and th r e e mouse s t r a i n s and found t h a t a dose o f more th a n 1 y g /k g /d ay was re q u ire d to p ro d u ce k id n e y 9  <75 IN d e fe c ts in r a t s , and th e c l e f t p a la te and kidney changes in mice appeared a t a b o u t 3 y g /k g /d a y , o v e r d a y s 6-1S o f g e s t a t i o n . The C57B1 mouse s t r a i n r i was much more s e n s i t i v e th a n th e o t h e r s t e s t e d . A somewhat l a r g e r gToup H o f NMRI m ice show ed a c l e f t p a l a t e fre q u e n c y o f a b o u t 3% (N 'eubert and D illm an , 19 7 2 ), s i m i l a r to th e low fre q u e n c y g ro u p s o f C o u rtn e y and M oore. o The p e r io d o f maximum s e n s i t i v i t y f o r TCDD i s a t th e e l e v e n th d a y o f g e s ta tio n , as d is tin g u is h e d from dexam ethasone, fo r exam ple, which is m ost e f f e c t i v e a t d ay 13 (N eu b ert e t a l . , 1 9 7 3 ). 0 C ontinuous a d m in is tra tio n o f 0.001 y g /k g /d ay to male and fem ale r a t s Q f o r 90 days p r io r to m ating and through w eaning o f o f f s p r in g produced no e ffe c t on f e r t i l i t y (Murray e t a l . , 1977). The stu d y con tin u ed through th re e g e n e ra tio n s , w ith each p receed in g g e n e ra tio n s a c r if ic e d a t weaning o f i t s o f f s p r in g ; th e t h i r d g e n e ra tio n was m a in ta in e d u n t i l two y e a rs from th e beginning o f tre a tm e n t o f th e f q g e n e ra tio n . T o x ic ity and poor l i t t e r su rv iv a l d ic ta te d te rm in atio n o f feed in g a t 0.1 yg/kg/day; 0.01 yg/kg/day caused d ecreased f e r t i l i t y in g en era tio n f j and f 2 but not f(). f 2 and $ l i t t e r s w ere sm a lle r and growth and s u rv iv a l were decreased a f t e r tre a tm e n t a t 0.01 y g /k g /d a y , b u t no p a th o lo g ic a l change was o b serv ed in liv e r , kidney, o r thym us. More r e c e n tly , S m ith e t a l . (1976) have t r e a t e d CF-1 m ice a t d o ses ranging from 0.001 yg to 3.0 yg/kg/day through days 6-15 o f g e s ta tio n . T here was no m a te rn a l t o x i c i t y a t any d o s e . C le f t p a l a t e ap p e a re d a t 1 y g , r e n a l d i l a t a t i o n a p p e a re d a t 3 y g /k g /d a y . No s i g n i f i c a n t e f f e c t s occurred a t 0.1 yg. A re c e n t r e p o r t by C ourtney (1976) d e sc rib e d r e s u lt s o f tr e a tin g CD-I m ice w ith h ig h d o s e s o f TCDD (2 5 -4 0 0 y g /k g /d a y ) on d ay s 7 -1 6 o f g e s ta tio n . These v ery h ig h doses caused no m atern al d eath s d u rin g th e g e sta tio n p erio d , b u t re s u lte d in high f e ta l m o rta lity . The n a tu re of o b s e rv e d t e r a t a w ere t y p i c a l o f TCDD i n t o x i c a t i o n , b u t th e enorm ous doses ren d er th e s tu d y u s e le s s fo r hazard e v a lu a tio n . However, th e s u b s ta n tia lly g re a te r in cid en ce o f te r a ta a f te r subcutaneous a d m in istra t i o n , com pared w ith t h a t f o llo w in g o r a l TCDD s u g g e s ts t h a t h e p a t i c m e tab o lis m may a f f e c t TCDD more th a n h a s been b e l i e v e d . A d o se r a t e o f 25 y g / k g /d a y o r a l l y c a u s e d 3% in c id e n c e c l e f t p a l a t e ; th e same d o se s u b c u ta n e o u s ly c a u s e d 82% i n c i d e n c e o f c l e f t p a l a t e . T h e re h a s b e e n l i t t l e r e s e a r c h on e f f e c t s o f TCDD on p r im a te r e p r o d u c tio n . E xperim ental anim als a re in lim ite d su p p ly and th e numbers o f o ffs p rin g a re so low th a t a s a ti s f a c to r y study, i s alm o st im p o ssib le . A lle n e t a l . (1 9 7 7 ) f e d TCDD t o e i g h t fe m a le r h e s u s monkeys a t a co n c e n tr a tio n o f 500 p p t in th e d i e t , which p ro v id e d an in ta k e o f about 0 .3 U g/kg/m onth. Skin le s io n s , en d o crin e, and m en stru al changes o ccu rred in a l l o f th e s u b je c ts by 3 m onths. S ix fem ales o f th e e ig h t w ere bred a f t e r 7 months o f tr e a tm e n t; one monkey d ie d b e fo re m ating and a n o th e r was e x cluded from th e re p ro d u c tiv e s tu d y . T hree o f th e an im als c o n c e iv e d ; two a b o r te d , and one c o m p le te d a n o rm al p r e g n a n c y . Two o f th e t h r e e b a r r e n anim als were b red f o u r tim e s , th e o th e r was b red tw ic e . T h is dose r a t e is on th e o rd e r o f 0 .01 yg/k g /d ay and com parison w ith th e r a t stu d y o f M urray e t a l . (1977) s u g g e s ts t h a t r e p r o d u c tiv e t o x i c i t y in monkeys may be some what g r e a te r th an in r a t s , b u t is by no means p ro p o rtio n a l to th e le th a l dose. 10 DOW O C a rc in o g e n ic and M u tag en ic P o t e n t i a l o f TCDD Van M ille r and A lle n (1977) have e v a lu a te d p a th o lo g ic a l ch an g es in r a t s f e d TCDD i n d i e t a r y c o n c e n tr a ti o n s r a n g in g fro m 1 p p t t o 1000 p p b , b eg in n in g a t anim al w eig h ts o f about 60 g . E stim a te d w eekly in ta k e s o f TCDD ra n g e d from 0 .0 0 0 3 y g /k g a t a d i e t a r y l e v e l o f 1 p p t to 2 y g /k g a t S p p b . A c tu a l i n t a k e o f TCDD a t d i e t a r y c o n c e n t r a t i o n s o f 50-1000 ppb was n o t c l e a r . At 65 w eeks th e abdom inal v i s c e r a o f s u r v iv in g a n im a ls were exam ined s u r g ic a lly and any observed tum ors were b io p s ie d . The d i e t w ith TCDD was th e n c o n tin u e d th ro u g h th e 7 8 th week and a l l s u r v i v i n g an im a ls s a c r i f i c e d a t 95 w eeks. '' T h ere w ere 10 a n im a ls a t each dose ra n g e ; a l l th o s e a t 50-1000 ppb TCDD d ie d by th e f o u r t h w eek. In th e g roup g iv e n 1 p p t t h e r e w ere no tu m o rs, b u t o f th o s e f e d 5 , SO, 5 0 0 , 1000, and 5000 p p t 23 o f 50 h ad d e v e lo p e d tu m o rs o f s e v e r a l t y p e s . No c o n t r o l a n im a ls f o r t h i s o r a p a r a l l e l experim ent developed neoplasm s. The g re a t v a r ie ty o f tum ors s u g g e s te d t o th e a u t h o r s th e p o s s i b i l i t y t h a t TCDD i s a p ro m o te r r a t h e r th an in d u c e r o f n e o p la s tic a c t iv it y , in view o f th e u su a l narrow sp e c trum o f tu m o rs c a u s e d by many c h e m ic a l c a r c in o g e n s . G e n e ra l p a t h o l o g i c changes have been describ ed in a previous se c tio n . Two s t u d i e s o f c a r c in o g e n e s is i n r a t s h av e u t i l i z e d c o n tin u o u s fe e d in g o f TCDD o v e r a tw o -y e a r p e r i o d . One p ro g ra m h a s been c o m p le te d but th e a n a ly s is o f h isto p a th o lo g y and gross tum or in c id e n c e have not been c o m p le te d . The o t h e r s tu d y , by Dow C h em ical Company, h a s b een c o m p le te d and a p r e l i m i n a r y r e p o r t f i l e d w ith EPA. D i e t a r y l e v e l s w ere 21 , 210, and 2200 p p t TCDD. The maximum d o se c a u s e d i n c r e a s e d tu m o r in c id e n c e in some o rg a n s and d e c re a se d in c id e n c e in o th e r s . T h ere was s u b s ta n tia l m o r ta lity r e s u ltin g from g e n e ra l to x ic e f f e c t s . The i n t e r m ediate dose caused m oderate system ic to x ic ity b u t no tum ors and th e lo w est d ose was w ith o u t e f f e c t o th e r th a n s l i g h t l i v e r changes such as induction o f in creased drug m etabolizing c a p a b ility . These l a t t e r changes were seen o nly in fem ales (J. D avidson, p e rso n a l com m unication) . The r e a s o n s f o r d i f f e r e n c e s b etw een th e Dow and Van M i l l e r s t u d i e s i s n o t a p p a r e n t ; i t i s p a r a d o x i c a l t h a t th e Dow work had a h i g h e r in c i d e n c e o f tum ors in c o n tro l an im als, w hile showing a low er in c id e n c e o f e f f e c t in tre a te d anim als. C y to g e n e tic s t u d i e s o f TCDD by G reen and M o relan d (1975) s u g g e s te d th a t th e d io x in does n o t have p o te n tia l f o r p ro d u cin g chromosomal ab n o r m a l i t i e s in bone m arrow . The d o se s c h e d u le p r o v id e d up to 15 y g /k g / d a ily f o r S d ay s, w ith s a c r i f i c e on day 5. A s in g le in je c tio n e x p e r i ment w ith s a c r i f i c e a t fo u r weeks was a ls o n e g a tiv e . Khera and Ruddick (1973) w ere a ls o u n ab le to fin d m utagenic a c t i v i t y o f 2 ,4 ,5 -T . S in c e i t i s n o t p o s s ib le to o b ta in an a b s o lu te ly TCDD-free p r e p a r a t i o n o f 2 , 4 , 5 - T , th e s t u d i e s o f th e h e r b i c i d e may a l s o be o f v a l u e i n c o n s i d e r i n g TCDD e f f e c t s . E f f e c t s o f 2 ,4 ,5 - T and TCDD on d i v i d i n g A f r i can blood l i l y endosperm c e lls are not e x p lic itly p e rtin e n t to anim al s tu d ie s , b u t th e r e l a t i o n shown betw een h e r b ic id e and c o n tam in a n t i s o f i n t e r e s t . H ig h ly p u r i f i e d 2 ,4 ,5 -T a t 10~4 m was n o t e f f e c t i v e , b u t 0 .2 * 16433 ii b o w 0G^ 1 yg TCDD/L w ith o r w ith o u t 2 ,4 ,S - T , and co m m ercial 2 , 4 , 5-T a l l c a u s e d d ra m a tic m ito tic i n h i b i t i o n and chromosom al a b e r a tio n s (Jackson, 1972) (0 .2 yg/L = 200 p p t ) . H u ss a in (1972) fo u n d t h a t TCDD was so m e w h a t'm u ta g e n ic u s in g th e Ames p r o c e d u r e a t d o s e s t h a t w ere i n t r i n s i c a l l y v e r y t o x i c t o th e o r ganism s. The a u th o rs su g g e ste d t h a t th e lim ite d m utagenic a c t i v i t y was by i n t e r c a l a t i o n o f TCDD i n t o DNA. S e i l e r (1973) h a s a l s o o b ta in e d d a t a s u g g e s tin g t h a t TCDD i s m u ta g e n ic i n th e S_. ty p h im u riu m TA 1532 s t r a i n . T h ere was no i n d i c a t i o n o f th e c o n c e n tr a tio n s n e c e s s a r y to produce e ffe c ts . A b s o r p tio n , D i s t r i b u t i o n , M e ta b o lism , and E x c r e tio n o f TCDD The b io lo g ic a l a c tio n o f a f o r e ig n compound i s h ig h ly d ep en d en t on i t s l o g i s t i c s . A chem ical must o b v io u sly be ab so rb ed from th e environm ent in o r d e r to i n t e r a c t , and i t m ust be c a r r i e d in b lo o d to c e l l s and move to o r a c ro s s c e l l membranes to s i t e s o f r e a c tio n . The ag e n t in i t s o r i g i n a l form may i n t e r a c t w ith s e n s i t i v e s y s te m s , o r i t may be c o n v e rte d to an a c tiv e d e riv a tiv e a f t e r e n te rin g an organism . Such co n v ersio n u s u a lly ta k e s p l a c e in th e l i v e r . A g iv e n o rg a n o r c e l l ty p e may h a v e a p a r t i c u l a r a f f in i ty o r s e n s itiv ity f o r a to x ic a n t and th e re b y be sin g le d o u t fo r a t tack . The chem ical n a tu re of the agent or i t s pro d u ct w ill a lso d ic ta te th e e x te n t and r a te o f e x c re tio n in u rin e o r b ile o r r e s p ir a to r y g ases. Even w ith an a c tiv e e x c r e tio n mechanism such f a c t o r s as s e n s i t i v i t y o f b la d d e r e p ith e liu m o r an a c tiv e e n te r o h e p a tic c i r c u l a t i o n may r e s u l t in to x ic e f f e c ts a f te r e lim in a tio n seems to have been accom plished. In s p ite o f th e enormous c a p a c ity fo r in d u c tio n o f mixed fu n c tio n o x id a s e s ( d is c u s s e d b elow ) TCDD i s m e ta b o liz e d t o a v e r y l i m i t e d e x t e n t . R a ts g iv e n a s i n g l e d o se o f 50 yg/TCDD -U -l4C /kg by sto m ach tu b e e x c r e te d a b o u t 30% i n f e c e s d u r in g th e two d a y s f o llo w in g t r e a t m e n t , and th e n 1-2% d a i l y o v e r th e 19 d a y s f o ll o w i n g , t o a t o t a l o f 53%. U r in a r y - ^ 4C was a b o u t 13% and r e s p i r a t o r y e x c r e t i o n t o t a l l e d 3.2% . A f t e r th e i n i t i a l 2 d a y s , t o t a l c l e a r a n c e h a l f - t i m e (T /2 ) was a b o u t 17 d a y s . R e s id u a l TCDD a t 3 , 7, and 21 d ay s was g r e a t e s t in l i v e r and f a t ( P i p e r e t a l . , 1 9 7 3 ). The long re sid e n c e tim e and lim ite d r e s p ir a to r y and u rin a ry e x c re tio n su g g ested t h a t TCDD was e s s e n t i a l l y un ch an g ed b u t t h i s was n o t v e r i f i e d . As a g e n e ra l r u le lip o p h ilic compounds are e ith e r se q u e ste re d in f a t , e lim in a te d as c o n ju g ates in b il e , o r co n v erted to a w ater s o lu b le form and e x c re te d by kidneys. Absence o f u rin a ry e x c re tio n th e re fo re im p lie s lim ite d co n v er s i o n . In a l a t e r s tu d y o f r a t s g iv e n a lo w e r s i n g l e d o se o f 1 y g /k g , TCDD was i d e n t i f i e d in fe c e s b u t n o t -u rin e , and a g a in , l i v e r and f a t c o n ta in e d th e h i g h e s t c o n c e n t r a t i o n s . T /2 was 31 d ay s (Rose e t a l . , 1 9 7 6 ). In th e same in v e s tig a tio n , re p e a te d doses 5 days a week f o r 7 weeks r e s u lt e d in some u r i n a r y l o s s , b u t m ost TCDD was e x c r e t e d i n t o f e c e s . In t h i s p ro g ra m o f e s s e n t i a l l y c o n tin u o u s in t a k e , T /2 was c a l c u la te d as 24 d a y s . The r e s i d u a l r a d i o a c t i v i t y i n th e l i v e r was i d e n t i f i e d a s u n ch an g e d TCDD. The r e p o r t o f V in o p al and C a s id a (1973) i s s u g g e s t i v e t h a t TCDD d o es n o t m e ta b o liz e o n ly because a f t e r g iv in g a dose o f 130 yg TCDD-%/kg to m ice, no m ention was made o f t r i t i u m in th e u r in e . A lle n e t a l . (1975) found t h a t o v e r a 25 day p e r i o d a b o u t 4.5% o f a d o se o f 50 yg TCDD/kg a p p e a r e d in u rin e . The d a ily f r a c tio n o f to ta l in ta k e e x c re te d g ra d u a lly in c re a s e d , which seems in d ic a tiv e o f an u n u su a lly s ta b le m o lecu le , b ecau se a t le a s t a modest exchange of tritiu m w ith body w ater should be ex p ected . 12 D O W OOjLl.799 The v a r i e t y o f i n d i r e c t e v id e n c e i n d i c a t i n g t h a t TCDD i s n o t m e ta b o lic a lly a lte re d must be considered in th e p e rs p e c tiv e o f o th e r in d ire c t e v i d en ce t h a t m e ta b o lic ch an g e may o c c u r . B e a tty and H eal (1975) h av e shown th a t p re tre a tm e n t w ith p h n o b a rb ita l d ecre ase s and c a s tr a tio n in c re a s e s TCDD t o x i c i t y . The fo rm e r tr e a tm e n t i n c r e a s e s and th e l a t t e r d im in is h e s drug m etab o lizin g c a p a c ity . The te ra to lo g y stu d y o f C ourtney (1976) a ls o i s s u g g e s tiv e t h a t l i v e r m e ta b o lism o f TCDD o c c u r s . An o r a l d o se o f 25 y g /k g /d a y th r o u g h th e o r g a n o g e n e s is p e r i o d c a u s e d o n ly 3% c l e f t p a l a t e among th e e x p e r im e n ta l f e t u s e s , b u t s u b c u ta n e o u s a d m i n i s t r a t i o n o f t h a t d o se r e s u l t e d i n 82% c l e f t p a l a t e s . In v iew o f th e a p p a r e n t a f f i n i t y o f TCDD f o r th e i n c r e a s e d e n d o p la sm ic r e t i c u l u m f o llo w in g in d u c tio n (Van M ille r e t a l . , 1976; Poland e t a l . , 1976), i t i s p o s s ib le th a t in d u c tio n m e re ly i n c r e a s e s th e c a p a c i t y f o r s e q u e s t r a t i o n o f TCDD in a t e m p o r a r i l y n o n -activ e system , but th e rem arkable d iffe re n c e seen by Courtney is d if f ic u lt to ascrib e to d is trib u tio n d iffe re n c e s only. The s e l e c t i v e d i s t r i b u t i o n o f TCDD i n t o l i v e r and f a t h a s a l s o b een shown by F r i e s and Marrow (1 9 7 5 ), A lle n e t a l . ( 1 9 7 5 ) , and Van M i l l e r e t a l . (1976) in r a t s and by Van M ille r e t a l . (1976) in p r im a te s . The l i v e r TCDD a p p e a re d t o be s e q u e s te r e d on sm ooth e n d o p la sm ic r e t i c u l u m (SER), p r in c ip a lly th a t induced by p re se n c e o f th e to x ic a n t (A llen e t a l . , 1 9 7 5 ). W hile l i v e r s t o r a g e o f TCDD in monkeys was much lo w e r th a n i n r a t s , the d iffe re n c e appeared to be due to th e c h a ra c te ris tic le s s e r p r o lif e r a t i o n ' o f SER i n th e monkey t h a t h a s b een d e s c r i b e d by Van M i l l e r e t a l . (1 9 7 6 ). C o n s id e r in g th e d i f f e r e n c e b etw een s p e c i e s in t o t a l in d u c e d ER membrane, TCDD a p p e a re d t o b in d t o e n d o p la s m ic r e t i c u l u m t o a b o u t th e same e x te n t in b o th s p e c ie s (Van M ille r e t a l . , 1 9 7 6 ). U nlike th e behavior o f o th e r c h lo rin a te d hydrocarbons in fa ste d or u n d e rfe d a n im a ls , TCDD i s n o t l o s t from a d ip o s e t i s s u e w ith f a t m o b i l i z a t i o n (A lle n e t a l . , 1 9 7 5 ). In th e c a s e o f PCB, f o r e x a m p le , th e f a t so lu b le c h lo rin a te d hydrocarbon is lib e r a te d w ith m ob ilized f a t and even t u a l l y may re a c c u m u la te in th e l i v e r o r o th e r t a r g e t o r g a n s . The re a s o n f o r th e d i f f e r e n c e i s n o t c l e a r , b u t i t seem s p o s s i b l e t h a t TCDD may n o n s p e c ific a lly p a r titio n in to f a t, then s e le c tiv e ly bind to adipose c e ll membrane p r o t e i n . L iv e r TCDD r e s i d u e s do d e c r e a s e d u r in g f a s t i n g , p o s s ib ly because in c re a se d g lu co n eo g en esis makes demands on a v a ila b le la b ile p r o t e i n in c l u d i n g th e e n d o p la sm ic r e t i c u l u m t o w h ich t h e TCDD may be b o u n d . H epatic s u b c e llu la r d is tr ib u tio n has been fu rth e r d efin ed by Poland and h is a sso c ia te s (Poland and G lover, 1974, 1975; Poland e t a l . , 1976; N ebert e t a l . , 1975); and by Chhabra e t a l . (1974) in s tu d ie s in which th e g e n e ti c a l l y d e te rm in e d s u s c e p t i b i l i t y f o r TCDD in d u c e d b e n z p y re n e h y d r o x y la s e i n d u c tio n was fo u n d t o c o r r e l a t e d i r e c t l y w ith th e e x t e n t o f TCDD b in d i n g in liv e r. The T /2 f o r TCDD r e s i d e n c e in t h e body h a s ra n g e d from a b o u t 1 6 -2 0 days fo r sin g le dose experim ents (P ip er e t a l ., 1973; A llen et a l ., 1975), a lth o u g h Rose e t a l . (1976) a r r i v e d a t a f i g u r e o f 31 d a y s . When TCDD was a d m in is te re d in th e d i e t o v e r 12 days a t d o se r a t e s o f a b o u t 0 .5 and 1 .5 y g /k g /d a y , T /2 was fo u n d t o be 12 d a y s f o r m a les and 15 f o r fe m a le s ( F r i e s and Marrow, 1 9 7 5 ). Rose e t a l . (1976) c a l c u l a t e d a h a l f - t i m e o f 24 d ay s a f t e r a seven week fe e d in g a t dose r a t e s o f 1 - 0.01 y g /k g /d a y . 16435 13 0 0 8 TTOC^ The c o n c e n t r a t i o n o f TCDD r e a c h e s a maximum a t a g iv e n d o se r a t e . F rie s and Marrow (1975] found t h a t a f t e r 6 weeks o f a d m in is tr a tio n r e t e n tio n approached a s te a d y s ta t e which would be 10.5 tim es th e d a ily in ta k e . Rose e t a l . (1976) found s im ila r ly in 7 weeks t h a t a s te a d y s t a t e con c e n t r a ti o n o f a l i t t l e more th a n 10 tim es th e d a i ly in ta k e c o u ld be p r e d ic te d , a g re e in g re m a rk a b ly w ell w ith th e F r ie s and Marrow e s tim a te . o The d iffe re n c e in d is trib u tio n between p rim ates and r a ts is o f in Q t e r e s t . Seven days a f t e r e q u iv a le n t d o ses o f TCDD-%, l i v e r s o f a d u lt monkeys c o n ta in e d 0.09% o f t o t a l dose/gm t i s s u e , r a t s c o n ta in e d 4.54% (Van M ill e r e t a l . , 1 9 7 6 ). In a d ip o se t i s s u e th e r e l a t i o n s h i p was monkey, 0.16, r a t 3.46, suggesting th a t sto rag e or re te n tio n is not s t r i c t l y r e la te d to b in d in g on in d u c ib le membranes. In term s o f t o t a l body d i s t r i b u ti o n , t h e r a t l i v e r c o n ta in e d 40% o f th e d o se and t h a t o f th e monkey, 10%. T h is i s a much c l o s e r r a t i o ( 4 /1 ) th a n t h a t o f l i v e r c o n c e n t r a t i o n s ( 4 .5 4 / .0 9 ) . S k in as a w hole s to r e d a much h ig h e r p e r c e n ta g e o f a d m in is t e r e d TCDD, b e c a u s e o f th e much h ig h e r am ount o f a d ip o s e t i s s u e , in s p i t e o f a c o n c e n tra tio n r a t i o (3 .5 /0 .1 6 ) t h a t was a ls o q u it e u n fa v o ra b le . W hether th e se d iffe re n c e s b ear any r e la tio n to d iffe re n c e s in s e n s i t i v i t y to to x ic e f f e c t s is n o t known. The d if f e r e n c e in l i v e r is a t le a s t a s s o c ia te d in amount and tim e w ith th e much g r e a t e r c a p a c ity f o r in d u c tio n in th e r a t . T h ere i s no d a ta a v a ila b le to i n d i c a t e w h eth er th e thymus o f s p e c ie s l i k e th e g u in e a p i g , w hich i s s u b j e c t t o more e x t e n s i v e TCDD i n duced thymus damage, has a s e le c tiv e a f f i n i t y f o r th e to x ic a n t as does th e l i v e r in th e r a t . The thymus o f in f a n t monkeys i s ex p ec ted to be h ig h ly a c tiv e , b u t appeared le s s re te n tiv e compared w ith o th e r tis s u e s , w hile th e a d u lt r a t thymus accu m u lated more TCDD/unit w eig h t th a n m ost o rg a n s (Van M ille r e t a l . , 1976), f u r th e r co n fu sin g th i s is s u e because li v e r damage is thought to be th e prim ary le sio n in the r a t. Each o f th e s in g le a d m in is tra tio n experim ents u t i l i s e d heavy doses o f TCDD, t a k i n g a d v a n ta g e o f th e lo n g d e la y i n o n s e t o f symptoms and d e a t h to o b ta in improvement in rad io ch em ical d e te c tio n c a p a b ility . S tu d ie s in w hich 400 p g /k g ( a b o u t 20 x LD50) w ere a d m in is te r e d (Van M i l l e r e t a l . , 1976) produced l i v e r c o n c e n tra tio n s o f about 3.6 ppm. Kociba e t a l . (1976) caused d is c e m a b le b u t minor h e p a tic le sio n s a t a t o t a l dose o f 0.65 pg/kg g iv e n o v e r se v e n w e e k s. The e x p e c te d c o n c e n t r a t i o n o f TCDD in l i v e r a t t h i s in ta k e i s on th e o rd e r o f 20 ppb (Rose e t a l . , 1 9 7 6 ). A t e n - f o l d lower dose caused no h isto p a th o lo g ic damage. I t is n o t s u rp ris in g th e r e fo re t h a t th e b each m ice s tu d ie d by Young e t a l . (1974) w hich accu m u lated 300-500 p p t TCDD i n t h e i r l i v e r s , d id n o t show l i v e r m o rp h o lo g ic l e s i o n s . B eh av io r o f TCDD in t h e P h y s ic a l E n v iro n m en t and in Submammalian S p e c ie s The e x tre m e ly h ig h t o x i c i t y o f TCDD and th e v e r y s m a ll am ounts o f th e agent p re s e n t in 2 ,4 ,5 -T and in th e environm ent p re se n t a problem th a t is d if f e r e n t le s s in p h ilo so p h y th an in s c a le . There is a tendency on th e one hand t o d is m is s TCDD a s a p o l l u t a n t b e c a u se th e am ount a v a i l a b l e a t any one p o in t o r even s e c to r i s m in u te, and on th e o th e r to assum e th a t any amount in excess o f tru e zero re s id u e is c a ta s tro p h ic , -because o f th e enorm ous t o x i c i t y o f t h e m a t e r i a l . The am ount o f TCDD d i s t r i b u t e d on v eg etatio n a f te r a ty p ic a l treatm ent is so sm all as to defy d ire c t a n a ly sis and th e q u e st f o r re s id u e s in b io lo g ic a l m a te ria ls has fo rced a n a ly tic a l 14 16436 I now s e n s itiv ity c lo se r to zero than has been th e case w ith any o th e r p o llu ta n t. In s p i t e o f th e s e u n iq u e c h a r a c t e r i s t i c s , TCDD m ust be c o n s id e r e d in th e same way a s any o t h e r c h e m ic a l: How much i s p r e s e n t i n th e human e n v iro n m e n t? How p e r s i s t e n t i s i t ? Can i t move to u n in te n d e d t a r g e t s ? I f so , w ill th e amount a s s im ila te d by humans o r o th e r organism s re a c h harm ful p ro p o rtio n s? P r e s e n t m a n u fa c tu re o f 2 ,4 ,5 - T c o n t a i n s a b o u t 0 .0 2 p a r t s TCDD p e r m illio n p a r ts 2 ,4 ,5 -T , a lth o u g h m a n u fa c tu re rs claim o n ly a b o u t 0 .0 5 ppm. Some l o t s c o n ta in o n ly a b o u t 0 .0 1 ppm. A two pound p e r a c r e a p p l i c a t i o n o f 2 ,4 ,5 -T w i l l , th e r e f o r e , d i s t r i b u t e a l i t t l e le s s th a n 20 yg TCDD/acre. I t i s n e c e s s a r y t o p r e d i c t w hat may be e x p e c te d t o h appen to t h a t m a te r ia l by exam ining e x is tin g la b o ra to ry d a ta . The e n v iro n m e n ta l p e r s i s t e n c e o f TCDD i s h i g h l y d e p e n d e n t on t h e c o n d i t i o n s t o w hich i t i s e x p o s e d . When m ixed i n t o s o i l , d e g r a d a t i o n was found to be v e ry slo w , w ith a h a l f tim e o f one y e a r (K earney, 1972) . B ecause th e m a te r ia l was in tro d u c e d as an a c e to n e s o l u t i o n th e r e has b een some q u e s tio n ab o u t p o s s ib le c r y s t a l l i z a t i o n and lim ite d a c c e s s o f TCDD t o s o i l o rg a n is m s . H ow ever, TCDD i n 2 ,4 ,5 - T d e p o s i t e d on le a v e s and exposed to d i r e c t s u n lig h t was found to d eg rad e w ith a h a l f - t im e o f a l i t t l e more th a n an h our (Crosby e t a l . , 1977). The l a t t e r stu d y shed a good d e a l o f l i g h t on e a r l i e r s t u d i e s i n w hich p u r e TCDD was c a r e f u l l y co ated on c le a n g la s s s l i d e s , w a te r, o r dry s t e r i l e s o i l f o r exposure to s u n lig h t, and found to be alm ost in s e n s itiv e to p h o to d eco m p o sitio n (Plim mer e t a l . , 1973; Kearney e t a l . , 1972). Crosby e t a l . n o ted th a t re d u c tio n would p roceed in m ethanol, and th a t th e re a c tio n slow ed s ig n if i c a n tl y in h ig h ly p u r if ie d re a g e n t. The fin d in g th a t benzene a c c e le ra te d th e d eg ra d a t i o n o f TCDD in a q u e o u s s y ste m s (P lim m er e t a l . , 1973) s u g g e s te d t h a t a good hydrogen donor was e s s e n ti a l to p h o to d e c o m p o sitio n . T his c o n c lu sio n has been v e r if i e d in more re c e n t work (C rosby e t a l , , 197 7 ). In th a t stu d y TCDD w ith 2 ,4 ,5 - T was a l s o a p p l i e d cm s o i l , and a h a l f - t i m e o f 5 0 -5 5 h o u rs c o u ld be in f e r r e d from th e d a ta . T his fin d in g p ro v id e s some s u g g e s tio n about th e re a c tio n tim e in in d ire c t lig h t, because th e rough s u rfa c e w ill p r o v id e s h a d e d a r e a s w here r e a c t i o n m ust be s lo w e r . As y e t , s t u d i e s o f th e e f f e c t o f v ario u s p a r ts o f th e spectrum em erging from lig h t tr a n s m itte d th ro u g h le a v e s, o r r e f le c te d from v a rio u s s u rfa c e s has n o t been d o n e . The a b s o r p t i o n maximum f o r TCDD i s known (C ro sb y e t a l . , 1973) b u t th e r e l a t i v e s e n s i t i v i t y to d e g ra d a tio n a t th a t wave le n g th a p p a re n tly is n ot. A c c u m u la tio n o f TCDD in th e b i o t a h a s b een s t u d i e d b o th in t h e la b o r a t o r y and in th e f i e l d , b u t th e r e a re s t i l l many gaps in th e n eed ed d a ta . In s p i t e o f th e l i m i t e d s o l u b i l i t y o f TCDD ( 0 .2 p p b ) , i t c a n accum u l a t e in a q u a tic organism s to th e e x te n t i t i s a v a ila b le on sed im e n ts o r o th e r r e s e r v o ir (Matsumura and B en ezet, 1973). In t h e i r e x p e rim e n ts, TCDD was d e p o s i t e d on s a n d , i n s o l v e n t w h ich was e v a p o r a te d t o le a v e th e d io x in as a film on sand p a r t i c l e s . The sand was p la c e d in a s m a ll co n fin e d s t a t i c aqueous system and v ario u s aq u a tic organism s added. Brine shrim p reac h ed a c o n c e n tra tio n o f 157 p p b , m osquito la rv a e c o n c e n tra te d TCDD t o 4150 ppb and s i l v e r s i d e f i s h a c c u m u la te d v i r t u a l l y no TCDD. I t may b e assum ed t h a t th e c o n c e n t r a t i o n o f TCDD in w a te r was m a in ta in e d th ro u g h o u t th e experim ent by th e excess d io x in re s id u e on sand. 15 16437 I o r 00 't H voO > Q Q When s u f f i c i e n t TCDD to r e p r e s e n t 162 ppb in th e aq u ariu m was in g e s t e d by a lg a e which were then p la c e d in an aquarium w ith Daphnia and w ith Ost r a c o d s , th e l a t t e r s p e c i e s a c c u m u la te d TCDD t o c o n c e n t r a t i o n s o f 879 and 279 ppb, re s p e c tiv e ly . They a lso showed th a t raosquite f is h c o n c e n tra te TCDD t o a l e s s e r e x t e n t th a n do t h e b o tto m f e e d in g l a r v a e on w hich th e y fe e d . I t is c le a r th a t accum ulation does o ccu r, b u t depends on m a in ta in in g s a tu ra tio n o f th e am bient w ater. M atsum ura and B e n e z e t (1975) a l s o exam ined movement o f TCDD w hich h ad been bound to san d , to a sandy loam s o i l (in o rg a n ic to o rg a n ic s o i l ) . V ery l i t t l e TCDD w a s - t r a n s l o c a t e d by a slo w l e a c h in g w ith w a te r . S u b s e q u e n tly , a more complex food ch ain system o f s o i l , w a te r, a lg a e , duckweed, s n a i l s , d ap h n id s, gam busia, and c a t f i s h was assem bled (Is e n se e and J o n e s , 1975). 14C-TCDD bound t o s o i l i n c o n c e n t r a t i o n s v a r y in g from 0 .0 0 0 1 to 7 .4 5 ppm was p la c e d in a q u a r ia . The re s id u e s found in th e v a rio u s s p e c ie s a f t e r 33 days e x p o su re te n d e d to p eak a t th e D aphnia s t a g e . Gam busia w ere added a t th e end o f th e exposure p e rio d w ith access to w ater and o th e r organism s fo r 3 days. C a tfish fin g e rlin g s were exposed fo r 6 days, beginning a f te r a l l o t h e r o rg a n is m s w ere rem o v e d . The c a t f i s h a c c u m u la te d TCDD t o r o u g h ly th e c o n c e n tra tio n found in th e f i r s t b io lo g ic a l s ta g e , th e a lg a e . None t h e l e s s , a t a w a te r c o n c e n t r a t i o n o f 7 p p t TCDD a t e r m in a l a c c u m u la tio n r a t i o o f more th a n 1 0 ,0 0 0 was fou n d in c a t f i s h , r e p r e s e n t i n g TCDD co n c e n tr a tio n s o f ab o u t 100 ppb (Is e n se e and J o n e s , 1975). The work a lso shows, however, th a t as s o il o r w ater co n c e n tra tio n s d ecre ase , so also do c o n c e n tra tio n s in th e organism s o f th e system . T o x ic ity was n o t s tu d ie d alth o u g h th e h ig h e r t i s s u e accu m u la tio n s exceeded mammalian to x ic d o se s, assum ing uniform d is tr ib u tio n o f dosage. The a u th o rs p o in t o u t th a t w ith th e lo n g l a t e n c y o f TCDD t o x i c i t y , th e f i s h may n o t hav e had tim e to develop le sio n s and d ie . Ward (1976) exam ined th e p e r s i s t e n c e o f TCDD in la k e w a te r and s e d i m e n ts . As e x p e c te d , TCDD i s a lm o s t e n t i r e l y bound t o la k e s e d im e n ts and is s u b je c t to v ery lim ite d b u t n o n eth eless r e a l m etab o lic d eg rad atio n when m icro o rg an ism s w ere p r e s e n t. The slow m ic r o b ia l a c t i v i t y i s enhanced by p r e s e n c e o f n u tT ie n t s . Ward s u g g e s te d t h a t w a te r-m e d ia te d e v a p o r a tio n o f TCDD o c c u r r e d t o a l i m i t e d e x t e n t . T o x i c i t y o f TCDD t o coho salm on h a s b e e n a s s a y e d by M i l l e r e t a l . (1 9 7 3 ). TCDD was a d m in is te r e d in th e d i e t and d o s a g e e x p r e s s e d a s ng TCDD p e r gram o rg a n is m m ass. At 1 3 .1 n g /g o n ly a b o u t 30% o f th e e x p e r i m en tal group su rv iv e d f o r 80 days p a s t ex p o su re , and a t 5 .4 n g /g alm ost h a l f th e f is h d id n o t s u rv iv e . D eaths o fte n d id n o t o ccu r u n t i l 10 days a f t e r th e end o f e x p o s u r e , and some l e t h a l l y a f f e c t e d f i s h s u r v iv e d 60 days o r more. In s tu d ie s o f rainbow tr o u t th e re w ere no ap p aren t e f f e c ts a t i n t a k e s o f 6 .3 ng TCDD/g o r g r e a t e r . M o sq u ito l a r v a e p u p a te d a t a n o r mal r a t e i n w a te r c o n t a i n i n g 200 p p t TCDD; s n a i l s r e p r o d u c e d n o rm a lly and o lig o c h a e te worms s u f f e r e d some r e p r o d u c tiv e d e f i c i t a t th e same c o n c e n tra tio n . B eatty e t a l . (1976) found Rana c a te s b ia n a ta d p o le s to be rem arkably r e s i s t a n t , w ith no ap p aren t le th a l e f f e c t by i n t r a p e r i to n e a l d o se s o f 1000 yg TCDD/kg. Doses up to 500 y g /k g d id n o t a f f e c t a d u lt fro g s, and no h is to p a th o lo g ic a l le sio n s ap peared. 16438 16 F i e l d s t u d i e s o f f i s h and mammals h a v e p r o v id e d e v id e n c e b o th s u p p o r t i n g and c o n t r a d i c t i n g c la im s o f TCDD a c c u m u la tio n in h ig h e r a n im a ls . An e v a l u a t i o n o f b i o t a in th e a r e a u sed by USAF f o r t r a i n i n g bom ber crews in a p p lic a tio n o f m ilita ry d e fo lia n ts showed l i t t l e accum ulation o f TCDD i n f i s h l i v i n g in th e s tre a m s o f t h e a r e a . The a r e a was l i t e r a l l y s a tu r a te d w ith 2 ,4 ,S -T , re c e iv in g on th e o rd e r o f 1000 lb / a c r e d u rin g th e p e a k y e a r , c o n t a in in g up t o 2-10 ppm TCDD. M ice in th e a r e a accum u la te d tis s u e burdens o f se v e ra l hundred p p t as measured a t th e end o f th e sp ra y p ro g ram , when th e san d y s o i l had acc u m u la te d r e s id u e s up t o 700 p p t. T hese an im a ls d e m o n stra te d no p a t h o lo g ic a l changes (Young e t a l . , 1 9 7 4 ), w hich i s n o t s u r p r i s i n g when e x t r a p o l a t i n g th e d a ta o f Rose e t a l . (1976) and Kociba e t a l . (1976) to r e l a t e t i s s u e le v e ls and e f f e c tiv e dose r a te s in r a t s . Given th e extended p e rio d o f ex p o su re, i t seems p o s s ib le th a t th e mouse p o p u la tio n could ad ap t th ro u g h s u r v iv a l o f n o n -resp o n d in g s t r a i n s o v e r th e e s tim a te d 30 g e n e r a tio n s o f e x p o s u re s . I t may be a ls o t h a t f i s h s t r a i n s h av e e v o lv e d s i m i l a r l y a s n o n - a c c u m u la to r s o f TCDD, a l though such a d a p ta tio n seems u n lik e ly . a O .o ( \ry\ .q F ish have a ls o been analyzed in d ra in a g e s from area s in Texas and A rkansas which had been e x te n siv e ly tr e a te d w ith 2 ,4 ,5 -T (S hadoff e t a l . , 1977). The T exas sam ples Were from a pond w h ich c o l l e c t e d r u n - o f f from a w a te rsh e d on w hich th e h e r b ic id e was u sed f o r b ru sh c o n t r o l . The A rkansas s i t e was a pond in t o which a d ja c e n t r i c e f i e l d s were d ra in e d and from w hich i r r i g a t i o n w a te r was o b ta in e d , in e s se n c e r e c y c lin g any c o n ta m in a n ts . T reatm en t was 1.2S lb 2 ,4 ,5 - T / a c r e , 4-8 weeks p r i o r to f lo o d i n g . The c y c le had b een in u se f o r 18 y e a r s . F is h from t h e Texas s i t e d id n o t c o n t a in TCDD, a t d e t e c t i o n l i m i t s o f 5 -7 p p t . Mud a t a d e te c tio n lim it o f 3 p p t and w ater a t 0.1 p p t were a lso n e g a tiv e . Six human m ilk sam p les o b ta in e d from th e San A ngelo a r e a n e a r th e T exas s i t e w ere a l s o n e g a t i v e a t a d e t e c t i o n l i m i t o f 3 p p t . No TCDD was fo u n d in th e A rkansas f is h sam ples. M eselson and O'K eefe (co rresp o n d en ce to Rep r e s e n ta tiv e James W eaver, 1977) have p re s e n te d p re lim in a ry fin d in g s o f ab o u t 1 -2 p p t TCDD i n human m ilk from th e San A n g elo a r e a , and from an a r e a in w estern Oregon. Cows m ilk s a m p le s from 2 , 4 , 5 - T - t r e a t e d a r e a s o f O klahom a, A r k a n s a s , and M is s o u ri w ere fo u n d to be n e g a t iv e f o r TCDD by M ahle e t a l . (1977) w ith d e t e c t i o n l i m i t s on th e o r d e r o f 1 p p t . TCDD in b e e f f a t was u n d e te c ta b le in two s e r i e s o f anim als w hich had g raze d on 2 ,4 ,5 -T t r e a te d p a s tu re s in Oklahoma, T exas, and M isso u ri. In a th i r d group o f seven anim als confined in an e n tire ly sprayed p a s tu re , th re e sam ples were p o s i t i v e a t t h e d e t e c t i o n l i m i t o f 3-4 p p t (K ocher e t a l . , 1 9 7 7 ). TCDD h a s been found, how ever, in se v e ra l b eef c a t tle m aintained in a f ie ld e x p e ri ment on f ie ld s w hich had re c e iv e d 1, 2, 3, o r 4 lb s 2 ,4 ,5 - T /a c r e . A verage TCDD i n f a t was 2 0 .3 p p t in th e 4 l b / a c r e g ro u p , b u t t h e r e w ere o n ly t h r e e sam ples, one o f w hich was ex trem ely h ig h . At 3 lb / a c r e th e a v e ra g e was 12.8 p p t, a g a in d e riv e d from some h ig h v a lu e s and many n e g a tiv e s . I n t e r p r e t a t i o n o f t h i s d a t a by EPA i s s t i l l in p r o c e s s , and i s s u b j e c t t o some argum ent. A number o f w ild anim al sam ples were o b ta in e d in tr e a te d f o r e s t a re a s o f w estern O regon, s e v e ra l o f which co n ta in e d u n u su a lly h ig h c o n c e n tra tio n s o f TCDD. The u n iq u e c h a r a c t e r o f th e d a t a r a i s e s some q u e s t i o n s , s i n c e 1843.9 17 rji O GO H- Tf-j' O o> o; a t h e r e seem s t o be few o r no sa m p le s c o n t a in in g TCDD c o n c e n t r a t i o n s i n te rm e d ia te betw een th e low background le v e l and th e 100-200 p p t v a lu e s in a few an im als. An i s s u e h as been r a i s e d a b o u t f o r m a tio n o f TCDD d u r in g c o m b u stio n o f 2 ,4 ,5 -T , and subsequent d is tr ib u tio n in a i r . S teh l and Lamparski (1977) ig n ite d g ra s s tr e a te d w ith 2 ,4 ,5 -T in a system w here com bustion was s e l f s u p p o rte d and fo u n d l e s s th a n 0.0002% c o n v e r te d t o TCDD. C om bustion o f 2 ,4 ,5 -T and tric h lo ro p h e n o l on f i l t e r p ap er led to p ro d u c tio n o f even le s s TCDD. I f th e c o n v e r s io n o f a f i e l d a p p l i c a t i o n w ere a t t h i s r a t e , a b o u t 2 p a r ts o f a v a ila b le 2 ,4 ,5 -T p e r m illio n would be c o n v e rte d , w hich i s con s id e ra b ly more th a n th e r e s id u a l co n tam in an t l e v e l . I t sh o u ld be remem b e re d , how ever, t h a t b u rn in g would n o t be done u n t i l f o lia g e d r i e s , and w ith a 1 -2 week h a l f tim e i t i s d o u b tf u l w h e th e r much h e r b ic id e would be a v a i l a b l e f o r c o n v e r s io n . Even t h i s maximum l e v e l o f c o n v e r s io n i s probably n o t o f g r e a t co ncern, s in c e th e p ro d u c t would be d ilu te d w ith com bustion gases and a i r in m illio n s o f cu b ic m eters o f atm o sp h ere. The e ffe c t o f exposure to lig h t on decom position w hile in th e atm osphere is unknown. M asking o r w av ele n g th s h i f t s may have an i n h i b i t o r y in f l u e n c e on p h o to d eg rad atio n , b u t th e d u ra tio n o f atm ospheric su sp en sio n and lig h t exposure should enhance the re a c tio n . In d u c tio n o f M icrosom al Enzymes TCDD h a s been fou n d t o be a re m a rk a b ly p o t e n t i n d u c e r o f many enzym es which a c t on fo r e ig n c h e m ic a ls. The e f f e c t i s a p p a re n tly dep en d en t upon sy n th esis o f a d d itio n a l enzyme, r a th e r th an a c tiv a tio n o f p a r ti a ll y sy n th e s i z e d o r o th e r w is e i n a c t i v e c o m p le te p r o t e i n . TCDD a l s o i n c r e a s e s a c t i v i t y o f S -a m in o le v u lin ic a c id s y n th e ta s e in some s p e c i e s , r e s u l t i n g in an accum ulation o f p o rp h y rin s in tis s u e s and e x c re to ry r o u te s . The p o ten cy o f TCDD f o r in d u c in g some enzymes i s b etw ee n f o u r and f i v e o r d e r s o f mag n itu d e g re a te r th an th a t o f such c la s s ic a l inducing agen ts as 3-m ethyl c h o la n th r e n e o r p h n o b a r b i t a l . A c c o rd in g to Hook e t a l . (1 9 7 5 a) TCDD in d u c e s cy to ch ro m e P -450 a t d o s e s o f fe w e r m o le c u le s o f TCDD th a n t h e r e are o f P-4S0 in th e liv e r . T his p ro p e rty is im p o rtan t because th e induced enzymes may in c re a s e c o n v e rsio n o f o th e r o rg a n ic in t o x ic a n t s to n o n - to x ic p r o d u c ts , o r c o n v e r s e ly may in c r e a s e c o n v e r s io n o f n o n - to x i c m a te ria ls to h ig h ly re a c tiv e in te rm e d ia te s which e x e rt profound to x ic e f f e c t s . Many c a r c in o g e n s a r e a c t i v a t e d in t h i s way. The c h a n g e s in "d ru g -m etab o lizin g " enzymes a lso have been used to d e s c rib e profound g e n e tic d if f e r e n c e s among an im al s t r a i n s , and t h e i r r e s p e c t i v e re s p o n s e to in to x ic a tio n . Enzyme in d u c t i o n may a l s o s e r v e a s a p r e d i c t o r o f g e n e r a l t o x i c i t y , sin ce th e in d u c tio n potency seems w ell c o rre la te d w ith l e th a l, a c n e g en ic, and te r a to lo g ic p o te n tia l (Schwetz e t a l . , 1973; Poland and G lo v er, 1 973a). The enzymes w hich o x id iz e fo re ig n ch em icals have ev o lv ed in re sp o n se to r e a c tiv e g ro u p in g s on m o lecu les found in n a tu re . W hile n a tu r a l and s y n t h e t i c c h e m ic a ls may r e p r e s e n t an a lm o s t i n f i n i t e v a r i e t y o f s t r u c tu re s , th e b u ild in g blocks a re th e same, and th ey e x is t in r e la tiv e ly lim ite d numbers. C onsequently, organism s have n o t had to develop an in f i n ite number o f enzymes to s u rv iv e , b u t r a th e r have developed en zymes s p e c i f i c f o r r e a c tiv e g ro u p s. 18 16440 The e f f e c t s on th e s e system s a r e among th e more s e n s i t i v e changes fo u n d t o be c a u s e d b y TCDD. To s i m p l i f y p r e s e n t a t i o n th e f o l l o w i n g t a b l e i d e n t i f i e s s p e c i f i c enzym es and th e TCDD d o s e s a t w hich r e s p o n s e s h av e been o b serv ed . U n fo rtu n a te ly , few e x p lo ra tio n s o f th e e n tir e dose r e s ponse cu rv e have been made; th e "no observed e f f e c t" in ta k e has u s u a lly not been determ ined, nor has th e re been an adequate stu d y o f th e im pact o f low le v e l c h ro n ic s tu d ie s on th e m icrosom al enzym es. Most o f th e d a ta in th e ta b le r e f e r s to r a t s . R e la tiv e ly few s tu d ie s o f mice have been made, and in th e guinea p ig th e e f f e c tiv e doses approach th e le th a l d o se, and o b serv ed ch an g es a r e m in o r. Hook e t a l . (1975b) a d m in is te re d 0 .1 7 5 u g A g to guinea p ig s and found a s lig h t in c re a se in liv e r b ip h en y l-4 -hydroxylase and b ip h e n y l- 2 -h y d ro x y lase, and no change in a ry l hydrocarbon h y d ro x y lase o r UDP g lu c u r o n y l t r a n s f e r a s e . O o S e v e ra l g e n e r a liz a tio n s appear a p p lic a b le to th e enzyme in d u c tiv e p r o p e r t i e s o f TCDD. W hile m o st m icro so m al enzym es a r e i n c r e a s e d a f t e r TCDD, th e enzym es i d e n t i f i e d a s d e m e th y la tin g a m in o p y r in e , b e n z p h e ta m in e and morphine a re d ecreased (L ucier e t a l . , 1973). Fem ales seem t o be m ore s e n s i t i v e t o t h e in d u c in g e f f e c t s o f TCDD ( L u c ie r e t a l . , 1 9 7 5 b ). W hile e f f e c t s on UDP g lu c u r o n y l t r a n s f e r a s e s a re s u b s ta n tia l, s te r o id g lu cu ro n y l tr a n s f e r a s e s a re n o t changed. The bulk o f in d u c tio n occurs in liv e r but a s im ila r but u su a lly le s s e r in c re a s e can o c c u r in th e k id n ey and some o th e r t i s s u e s (see ta b le ) . In kidney, a c t iv it y seems co n cen trated in th e " o u te r s tr ip e " o f th e m edulla and in th e c o rte x . T his re g io n c o n ta in s th e te rm in a l s tr a i g h t segm ents o f th e p ro x im al tu b u le s , which ap p ear to be th e o n ly s i g n i f i c a n t l y i n d u cib le c e lls in th e kidney (Fowler e t a l . , 1977). The i n d u c t i o n o f a r y l h y d ro c a rb o n h y d r o x y la s e (AHH) by TCDD h a s b een very u se fu l in e x p la in in g g e n e tic v a r i a b i l i t y in re c e p to r and e f f e c to r m echanism s. TCDD i s a b o u t 3 x 104 tim e s m ore p o t e n t th a n 3 - m e th y lc h o la n th re n e , p ro d u c in g h a l f maximal s tim u la tio n in th e r a t l i v e r a t 0.85 n m o le s/k g and i n th e C3H/HeN mouse ( fe m a le ) a t a d o se o f 0 .4 2 n m o les TCDD p e r kg (P o la n d and G lo v e r , 1 9 7 4 ). W hile t h e s u s c e p t i b i l i t y to AHH i n d u c t i o n i s a b o u t th e same i n th e s p e c ie s t e s t e d (EDg = 0 . 4 - 1 . 2 n m o l e A g ) , and th e LDsos d i f f e r by a b o u t two o rd e rs o f m a g n itu d e , t h i s d if f e r e n c e does not d e tra c t from th e v alu e o f the system in stu d y in g th e g e n e tic s o f drug response and th e g re a t problem s o f non-hom ogeneity o f exposed p o p u la tio n s. T here i s known to be a pro fo u n d d if f e r e n c e in th e re s p o n s e s o f v a rio u s s t r a i n s o f m ice t o AHH in d u c t i o n by 3 - m e t h y lc h o la n th r e n e (3-M C), l e a d i n g to a d e s ig n a tio n o f "re sp o n siv e " and "n o n -re sp o n siv e " s t r a i n s . Even a t h ig h d o s e s , 3-MC f a i l s to c a u s e in d u c t i o n in t h e n o n - r e s p o n s iv e a n i m a ls . TCDD, h o w e v e r, w i l l e l i c i t an a m p lif ie d enzyme a c t i v i t y in s u c h m ice (P o la n d and G lover, 1975; Chhabra e t a l . , 1974). Poland and G lover (1975) and N eubert e t a l . (1975) have shown t h a t th e g e n e tic a lly n o n -re sp o n siv e mice have th e g e n e tic a p p a ra tu s n e c e ssa ry fo r expression of the in d u cib le a c tiv itie s and suggest th a t the d iffe re n c e a ris e s from a m utant fo rm o f in d u c er-b in d in g re c e p to r w ith a d im inished a f f in i ty f o r aro m atic hydro carb o n s. The in d u c tio n p ro c e ss is a p p a re n tly an i n c r e a s e i n de_ novo s y n t h e s i s o f enzym e, r a t h e r th a n a c t i v a t i o n o f p r e e x is tin g p r o te in o r d e c re a se d d e g ra d a tio n o f p r o te in (Haugen e t a l . , 19 1844 T 18442 Induction of Foreign Compound Metabolizing Enzymes by TCDD Enzyme TCDD Dose (pg/kg) Extent of In itia l In d u c tio n (x norm al) p-N itrophenol-U D P glucuronyl tran sferase 3 3 25 5 0.2 14 7 6 5 2.5 Benzpyrene (a ry l hydrocarbon) hydroxylase K) o 3 3 2.5 2.5 0.2 0.2 1.0 25 25 10 10 10 .08 14 2 14 100 7 8 1.5 3 >70 180 10 100 3 NADPH d ia p h o ra s e (d e h y d ro g e n a se ) 90 13 A niline hydroxylase 5 2 0.2 1.2 Cytochrome P-450 1.0 1.6 C ytochrom e Bg 1.0 1.4 B iphenyl-2-hydroxylase 25 25 13 18 05 40' , f* bow Q Oil 806 (1) D uration of Increased A ctiv ity >34d >34d 73d >28d -- >2 1d -- ---- -- -- --- -- -- -- >15d >15d -- -- -- w-- R eferences Remarks ( l i v e r unless other tissu e sp ecified) L ucier e t a l ., 1975a L ucier e t a l ., 1975a L ucier e t a l . , 1975b Hook e t a l . , 1975b L u cier e t a l . , 1973 pregnant r a ts ( 2) n eo n ate, (3) male r a t L ucier e t a l . , 1975a L ucier e t a l . , 1975a B erry e t a l . , 1976 B erry e t a l . , 1976 L u c ie r, 1973 Hook e t a l . , 1975b Hook e t a l . , 1975b Hook e t a l . , 1975b Hook e t a l . , 1975b Poland and G lo v er, 1974 Poland and G lo v er, 1974 Poland and G lover, 1974 Poland and G lo v er, 1974 pregnant r a ts (2) n eo n ate, (4) p reg n an t r a t , (5) fe ta l liv er male r a t s (6) fem ale r a ts (7) male (7) male r a t l i v e r m ale r a t k id n e y ( 8) r a t kidney r a t lung ra t in testin e C3H/HeN mouse B eatty and N eal, 1976 male r a t l i v e r (9) L ucier e t a l . , 1973 L ucier e t a l . , 1973 L u cier e t a l . , 1973 m ale, 3 day L u cier, 1977 m ale, 3 day Hook e t a l . , 1975b Hook e t a l . , 1975b rat liv er r a t kidney (11) Induction of Foreign Compound Metabolizing Enzymes by TCDD (cont) Enzyme B iphenyl-4 -hydroxylase TCDD Dose (pg/kg) 25 25 E xtent of In itia l In d u c tio n (x norm al) 2 40 D uration o f Increased A ctiv ity R eferen ces Hook e t a l . , 1975b Hook e t a l . , 1975b Amino l e v u l i n i c a c id sy n th e ta se 25 25 1.5 ng/egg none 2 2 Woods, 1973 G o ld ste in , 1973 Poland and G lover, 1973c Remarks ( li v e r unless o th er tis s u e specified^ ra t liv er ra t kidney mouse ( 1 1 ) ch ick embryo *This ta b le i s s e l e c t i v e and i s in te n d e d to show th e v a r ie ty o f work d o n e, th e dose ra n g e s s tu d ie d , and d u r a tio n o f e f f e c t s . In most cases th e low est dose p ro d u cin g e f f e c t has been shown and d a ta f o r h ig h e r doses o m itte d . The re fe re n c e s no ted sh o u ld be c o n su lte d f o r g r e a te r d e t a i l . 2 P re g n a n t r a t s , t r e a t e d on day 10 o f 23 day g e s t a t i o n , s a c r i f i c e d 21 d ay s p o s t p a rtu m . 3 M other t r e a t e d a t day 5 o f g e s t a t i o n ; a c t i v i t y m easured a t d ay 21 p o s t n a t a l . No in c r e a s e i n a c t i v i t y b e f o r e p artu ritio n . 4 Same as 2 e x c e p t m easured a t day 8 p o s t n a t a l . ^TCDD a t d ay 17 o f g e s t a t i o n , s a c r i f i c e d a t day 20. ^Male r a t s , s a c r i f i c e d 3 d ay s a f t e r TCDD. 7 Male r a t s d id n o t resp o n d a t t h i s d o se; th e r e was a sm all b u t s i g n i f i c a n t (P < 0 .0 5 ) in c r e a s e in am ino p y r in e d e m e th y la se and cytochrom e P-450 in fe m a le s a t 0 .2 |jg TCDD/kg. 8R e s tin g a c t i v i t y b elo w d e t e c t i o n l i m i t . 9M aximal a c t i v i t y in c y t o s o l a t 7 d a y s , s t i l l r i s i n g m m icrosom es a t 15 d a y s . ^ 5 pg/kg produced no e f f e c t. ^ R e s tin g a c t i v i t y v e ry low in th e s e t i s s u e s ; f a c t o r o f in c r e a s e a p p ro x im a te . O? t& 0 8 TTOOVmocj 1 9 7 6 ). W hether th e e f f e c t o f TCDD on i n d u c i b l e enzymes h a s i m p l i c a t i o n s on o th e r a s p e c ts o f p r o te in s y n th e s is can o n ly be s p e c u la te d upon. Poland and G lover (1975) showed about a 1 0 -fo ld d iffe re n c e in th e dose n ecessary to cause in d u c tio n . Through th e fin d in g th a t heterozygous o f f s p r in g o f C57B1/6J ( r e s p o n s iv e ) and DBA/2J ( n o n -re s p o n s iv e ) s t r a i n s are in te rm e d ia te in s e n s itiv ity , they su pport the c o n te n tio n o f Poland and G lover (1975) th a t th e d if f e r e n c e is a r e c e p to r s i t e m u ta tio n . L a te r stu d ie s have strengthened th a t prem ise in fin d in g th a t h e p a tic accum ula t i o n o f l^C-TCDD a d m in is te r e d i n t r a p e r i t o n e a l l y was g r e a t e r in r e s p o n s iv e th a n n o n -resp o n siv e s tr a i n s (Poland e t a l . , 1976). In th e same study, in v itro exam ination of binding in th e so lu b le fra c tio n o f liv e r O c e lls showed th a t s p e c if ic b in d in g s i t e s e x i s t , and th a t t h e i r a f f i n i t y Q c o rre la te s w ith th e g en etic c a p a b ility fo r in d u c tio n . Furtherm ore, te s ts o f an extended s e rie s o f halogenated dio x in s and dibenzofurans showed th a t c y to s o lic b in d in g corresponded c lo s e ly w ith th e in d u c tio n potency o f the chem ical. Niwa e t a l . (1975) h av e m easu red AHH in d u c tio n by TCDD in a v a r i e t y o f c e l l c u l t u r e s . They used 10 e s ta b l is h e d c e l l l i n e s , human lym pho c y te s , and prim ary f e t a l c u ltu re s from c h ic k , r a t , r a b b it, h a m ste r, and f o u r s t r a i n s o f m ic e . G e n e r a lly , k i n e t i c s o f TCDD in d u c tio n in each c e l l ty p e i s s i m i l a r t o t h a t o f 3-MC, and th e i n d u c t i o n was i n h i b i t e d by actinom ycin-D and cyclohexam ide, which a re in h i b ito r s o f p r o te in syn th e s is . They found no r e la tio n between in d u c ib il ity and c y to to x ic ity o f TCDD, w hich i s f u r t h e r e v id e n c e t h a t TCDD i t s e l f i s n o t m e ta b o liz e d . The s e n s i t i v i t y o f some c e l l li n e s i s such t h a t th e a u th o rs su g g e st use o f H -4 -II-E c e l l s (d e riv e d from. Reuber hepatom a H-35 in r a t s ) as a b io a s s a y , w ith a s u g g e s te d s e n s i t i v i t y below 1 pMole ('t 0 .0 0 0 3 pg) in 3 ml o f c u l tu r e medium. The same k in d o f g e n e tic d if f e r e n c e s have been s tu d ie d in human lym phocyte c u ltu r e s , which re p re s e n t th e g e n e tic background o f th e c e ll donor and a re th e re f o r e u s e fu l in making r e l a t i v e l y n o n -in v a s iv e s tu d ie s o f p o t e n t i a l human re s p o n s e to i n t o x i c a t i o n . The s e n s i t i v i t y t o TCDD in d u c tio n o f AHH i s a b o u t 5 0 - f o ld g r e a t e r th a n t o 3-MC i n d u c t i o n , co n s id e ra b ly le s s th an th e 3 0 ,0 0 0 -fo ld d iffe re n c e in re sp o n siv e mice (K ouri e t a l . , 1974). A tla s e t a l . (1976) have c a rrie d th e se s tu d ie s forw ard w ith ly m p h o cy tes from human t i s s u e s , b u t h a v e a s y e t d e a l t o n ly w ith 3-MC, n o t TCDD. P a r t o f th e s tim u lu s f o r e x am in in g human c e l l s l i e s in th e s u g g e s t i o n t h a t e x t e n t o f in d u c t i o n a t c o n t a c t s i t e s ( i . e . , s k i n , lu n g ) may r e la te to th e p r o b a b ility o f can cer i n i t i a t i o n by a c tiv a te d carcin o g en s (K e lle rm a n e t a l . , 1 9 7 3 ). A lth o u g h TCDD i s a s y e t n o t e s t a b l i s h e d as a c a rc in o g e n such r e s e a r c h may p ro v id e s u g g e s tio n s a b o u t th e e x te n t o f e x p e c te d v a r i a t i o n s in o t h e r TCDD r e s p o n s e s i n hum ans. 2 ,4 ,5 -T G eneral T o x ic ity in L ab o ra to ry Animals A pparently the f i r s t published account o f 2 ,4 ,5 -T acute to x ic ity was t h a t o f D r i l l and H ir a tz k a (1 9 5 3 ). The LD50 f o r dogs was a b o u t 100 rag /k g J 'the p r i n c i p a l symptom was a m ild i n c o o r d i n a t i o n . When fe d f i v e days w eek ly f o r 90 d a y s , d o s e s up t o 10 m g /k g /d a ily w ere w ith o u t e v i d e n t 22 16445 DOW 0Q11 80S e f f e c t , b u t 20 m g /k g /d a y was l e t h a l t o a l l 4 t r e a t e d a n im a ls b etw een 11 and 75 days a f t e r th e f i r s t d o se . Symptoms w ere l i m i t e d to m uscle tw itc h in g and im paired sw allow ing. A f ie ld stu d y by G rigsby and F arw ell (1950) [a s q u o te d by Rowe and Hymas (1 9 5 4 )] e x p o se d a v a r i e t y o f s to c k on p a stu re im m ediately a f te r sp ray in g 2 ,4 ,5 -T a t 2-4 tim es u su al le v e ls , w ith no e f f e c t. Rowe and Hymas (1954) sum m arized th e a v a i l a b l e d a t a a t t h a t tim e and estim a te d th e a c u te o ra l median le th a l dose fo r male r a t s to be about 500 m g/kg, m ale m ice 589 m g/kg, and g u in e a p ig s 381 m g/kg. LDsqs f o r v ario u s e s te rs were hig h er th an fo r th e a c id . A sheep fed th e propyl g ly c o l b u ty l e s t e r o f 2 ,4 ,5 -T d ie d a f t e r 369 d a ily d o ses o f 100 mg/kg and a n o th e r sh eep and a cow d ie d a f t e r se v e n d a i l y d o se s o f 250 m g/kg (Palm er and R a d e le ff, 1964). The tr ie th y la m in e s a l t o f 2 ,4 ,5 -T a t 100 mg/kg c a u s e d no o b s e r v a b le e f f e c t a f t e r 481 d a y s o f t r e a t m e n t a t 100 mg d a i l y . As s in g le anim al o b serv atio n s th e se can be co n sid ered rough estim a te s on ly , b u t th ey convey th e g e n e ra lly high doses n e c e ssa ry to cause harm in rum i n a n ts . 2 ,4 ,5 -T h as been shown to cause d e c re a se d v o l a t i l e f a t t y a c id p ro d u c tio n in v itr o a t c o n c e n tra tio n s o f 500 yg/m l o r g r e a te r (K utches e t a l . , 1970); th is c o n c e n tra tio n is p ro b ab ly g r e a te r th a n can be main ta in e d by a su rv iv ab le d a ily dose o f 2 ,4 ,5 -T . S ubchronic (90 day) feed in g o f male and fem ale r a t s caused no e f f e c t s below 30 m g /k g /d ay , b u t body w eig h t and fo o d in ta k e w ere d e p re s s e d a f t e r t r e a t m e n t a t 100 m g /k g /d a y . A lk a l in e p h o s p h a ta s e and SGPT w ere e le v a te d and e ry th ro c y te co u n t and hem oglobin was d e c re a s e d . The h i s to p a th o lo g ic fin d in g s were lim ite d and in c o n s is te n t (M cC o llister and K ociba, 19 7 0 ). R ats w ere a b le to t o l e r a t e 186 m g/kg/day tre a tm e n t w ith mixed mono-, d i- , and trip ro p y le n e g ly c o l b u ty l e th e r e s te r s o f 2 ,4 ,5 -T o v e r 90 days b u t d e v e lo p e d some i n d i c a t i o n s o f t o x i c i t y . At a do se o f 1 8 .6 m g/kg no e v id e n c e o f t o x i c i t y was o b s e r v e d (Dow C h em ical Company, 1961). A two y e a r s tu d y o f r a t s g iv e n 3 to 30 mg 2 , 4 ,5 - T / k g / d a y h a s v e r y r e c e n t l y b een c o m p le te d by Dow C h em ical Company. Much o f th e a n a l y s i s has y e t to be com pleted, in c lu d in g m orphologic p a th o lo g y . A nim als th a t r e c e iv e d 30 mg 2 ,4 ,5 - T / k g /d a y w ere fo u n d to h av e i n c r e a s e d u r i n a r y p o r p h y rin e x c re tio n a f t e r 4 months o f tre a tm e n t, and t h i s change c o n tin u e d th r o u g h th e e n t i r e 2 y e a r p e r i o d . No c h a n g e s w ere fo u n d i n an y o t h e r hem atologic, u rin a ry , or c lin ic a l chem istry measurement a t th a t r a te o f i n t a k e , and th e in c r e a s e d p o rp h y rin e x c r e t i o n d id n o t o c c u r a t 10 o r 3 m g /k g /d a y ( R .J . K o cib a, Dow C h em ical Company, p r e l i m i n a r y s t a t u s r e p o r t ; p erso n al com m unication). A feed in g stu d y o f re in d e e r was prom pted by a lle g a tio n s th a t a h ig h in c id en ce o f d eath and a b o rtio n o ccu rred in 1970 in a h erd a f t e r use o f phenoxy h erb icid es the previous y ear. F ifte en o f th i r ty pregnant re in d e e r were fed b irc h leav es which had been sprayed w ith a 2 ,4 -D /2 ,4 ,5 -T m ix tu re , th ro u g h a 1.5 month p e rio d l a t e in g e s ta tio n . The d a ily dose o f phenoxy a c i d was a b o u t 1 m g /k g /d a y . No c l i n i c a l c h e m ic a l c h a n g e s c o u ld be d e te c te d , nor d id any changes appear a t au to p sy e i th e r in th e a d u lt anim al or th e f u ll term fe tu s (Erne, 1976). . 23 D O W 0011 810 The to x ic e f f e c t s o f 2 ,4 ,5 -T in ro d e n ts have been s tu d ie d in much more d e t a i l th a n th e g ro ss t o x i c i t y s tu d ie s a lr e a d y d e s c r ib e d . Highman e t a l . (1976a, 1976b) e sta b lish e d th a t le th a l doses in pregnant o r nonpreg nant m ice caused m y o card ial le s io n s , bone marrow a p la s ia , and lym phocytic d e p le tio n in thuraus, s p le e n , and lymph n o d e s, and t h a t a n im a ls w hich r e mained a p p a re n tly h e a lth y d e s p ite s im ila r doses d id n o t s u f f e r th e same s e v e rity o f le s io n s . A m ild hem olytic anem ia d id occur in such an im als. P regnancy d id n o t augment th e to x ic e f f e c t . I t was a ls o c l e a r th a t m ajor d i f f e r e n c e s e x i s t i n th e r e s p o n s e o f s p e c i f i c s t r a i n s . Many NCTR m ice w ere s e r i o u s l y a f f e c t e d a t d o s e s below 60 mg 2 ,4 ,5 - T / k g / d a y (6 -9 d ay s o f t r e a t ment w h ile m ost CRBL m ice rem a in ed u n a f f e c t e d a t d o s e s as h ig h a s 9 0 -1 2 0 m g/kg/day a t th e same tim e. The h is to p a th o lo g y , hem atology, and blood c h e m is try changes i n t r e a t e d m ice w ere a ls o s tu d ie d . In many m o n ito re d an im a ls, some m y o c a rd ia l f ib e r s were found to be p a le and s w o lle n , w ith lo n g itu d in a l s t r i a t i o n s . The c o n d itio n was r a r e l y seen in tr e a te d ap p a re n tly n o n -in to x ic a te d anim als. N ecro tic changes were a lso o fte n seen in th e o u te r m yocardium . High doses caused thym ic a tro p h y , w ith alm ost no lym phocytes in th e c o rte x and in c re a se d numbers in th e m edulla ( th is e f f e c t was enhanced in l a t e p regnancy by th e norm al te n d e n c y to c o r t i c a l in v o lu tio n in p reg n an t m ice). Spleens were o fte n a tro p h ie d , and th y ro id f o l l i c l e s were en larg ed w ith en larg ed e p i th e li a l c e l l . In th e li v e r , glycogen was o f te n d e p le te d . Blood c h e m is try changes a p p ea red n o t to be m arked. 2 ,4 ,5 -T has been found to cause in c re a se d liv e r w eight in r a ts a t doses o f 167-334 mg/kg over a tw o-day p e rio d , a p p a re n tly thro u g h stim u l a t i o n o f p r o t e i n an d RNA s y n t h e s i s , b u t n o t a s n ew ly form ed 2 ,4 ,5 - T m e ta b o liz in g enzym es. The change re v e rs e s a f t e r w ith d raw al (Chang e t a l . , 1974; Rip and C h erry , 1976). L iv er n u c le i is o la te d from tr e a te d an im als w ere more a c t i v e in RNA s y n t h e s i s th a n th o s e from c o n t r o l s . 2 ,4 ,5 - T a t a c o n c e n t r a t i o n o f 4 rcmolar i s c a p a b le o f s h a r p l y i n h i b i t i n g in_ v i t r o i n c o rp o ra tio n o f m ev alo n ate-^ 4C in to n o n -s a p o n ifia b le li p id s by r a t l i v e r (O lson e t a l . , 19 7 4 ). I f d a ta o b ta in e d in m ice by Nony e t a l . (1976) can be a p p lie d , t h i s c o n c e n tra tio n is ap p ro ach ab le a t s in g le doses o f 50 to 100 m g/kg, w hich a r e s u r v i v a b l e by m ost s p e c i e s . K oschier and B erndt (1976a, 1976b, 1976c) have d escrib ed th e e f f e c t of 2 ,4 ,5 -T upon re n a l physiology. Large doses of 2 ,4 ,5 -T appear to im pair s e c re tio n o f i t s e l f by d ecreasin g th e a c t iv it y o f th e o rg an ic a c id tr a n s p o r t system in th e p ro x im al tu b u le . O rg an ic b a se tr a n s p o r t was also d ep ressed . Sm all doses o f 2 ,4 ,5 -T were ex cre ted very ra p id ly , but la rg e d a ily doses le d to re n a l d ep ressio n and re te n tio n o f th e compound. The a u th o rs c o n s id e r th e d a ta to su p p o rt th e c o n c lu sio n th a t tr a n s p o r t o f phenoxy h erb ic id es is an a c tiv e p ro cess. The psychopharm acology o f 2 ,4 ,5 -T does n o t seem to be a p o p u la r f ie ld o f stu d y . In th e one stu d y found, s in g le doses o f up to 100 mg/kg were g iv e n on day 7, 8, o r 9 o f p reg n an cy . The m ale o f f s p r in g o f fem a les given th e h ig h e s t dose e x h ib ite d more e x p lo ra to r y open f i e l d b e h a v io r, but no d iffe re n c e was found in fem ales. The h ig h e s t doses caused d e crea sed l i t t e r s i z e b u t no in c re a s e in m a lfo rm atio n s (S joden and S o d e rb e rg , 1972). 24 P o u ltry seem r e l a t i v e l y in s e n s it iv e to 2 ,4 ,5 -T . W hitehead and P e ttig re w (1972) found th a t a s in g le o r a l dose o f 900 mg/kg to 4 week o ld c h ic k s c a u s e d 40% l e t h a l i t y . H owever, f e e d i n g o f 1000 mg 2 ,4 ,5 - T p e r kg d ie t/d a y fo r th re e weeks to ch ick s, begin n in g a t one day o f age caused o n ly some slow ing o f grow th; 5000 mg/kg d i e t was l e t h a l . At le v e ls n o t c a u sin g g ro ss to x i c i t y , plasm a calcium and magnesium were n o t a f f e c te d . Given a c h o ic e , th e b ir d s r e je c te d th e tr e a te d d ie t in fa v o r o f nonc o n ta m in a te d fo o d . T u rk ey s fe d 2 ,4 ,5 - T a t a r a t e e q u i v a le n t t o 62 mg 2 ,4 ,5 - T a c i d d a i l y f o r 11 d ay s w ere u n a f f e c t e d (R o b e rts and R o g e rs , 1 9 5 7 ). B jorklund and Erne (1971) in tro d uced v ario u s 2 ,4 ,5 -T d e riv a tiv e s in to w ater and feed of chickens, q u a il, p h easan ts, and ducks. In w ater the LCioO t h e tr i e t h a n o l a m i n e s a l t f o r c h ic k e n s o v e r a 29 week p e r i o d was 1000 ppm a c i d e q u iv . (a b o u t 200 m g /k g ). In t h e d i e t o f o t h e r s p e c i e s o v e r a 7 -d a y p e r i o d , th e LC50 was i n e x c e s s o f 2000 ppm. Kenaga (1975) h a s e x te n siv e ly review ed av ian to x ic ity and s a fe ty o f b ird s in areas tr e a te d w ith h e r b ic id e s and co n clu d es th a t th e no o b serv ed e f f e c t le v e ls a re s u b s ta n tia lly above amounts th a t m ight be c o n ta c te d in a f i e l d a p p lic a tio n . E ffe c ts o f 2 ,4 ,5 -T on R eproduction The te ra to g e n ic p o te n tia l o f 2 ,4 ,5 -T has re c e iv e d wide p u b lic ity s in c e 1969 when a l le g a t io n s w ere made t h a t i t s u se as a m i l i t a r y d e f o l i ant had caused f e ta l m alform ations in the V ietnam ese p o p u la tio n . A succession o f stu d ie s over the next th re e years confirm ed the te r a to g e n ic ity o f 2 ,4 ,5 -T , but the im p lic atio n of the h e rb ic id e in any in c re a se in b i r t h d e fe c ts has n o t been su p p o rte d . The i n i t i a l r e p o r t o f 2 ,4 ,5 -T te ra to g e n ic e f f e c t (C ourtney e t a l . , 1970; B io n etics R esearch L a b o ra to rie s, 1970) d e s c rib e d c l e f t p a l a t e and c y s t i c k id n e y s a t d o ses o f 46 and 113 rag /k g /d ay on days 6 th r o u g h 14 o f g e s t a t i o n . Of th e two l e s i o n s , c y s t i c kidney app eared to be a more s e n s iti v e in d i c a to r . The 2 ,4 ,5 -T u sed in th e s tu d y was fo u n d to c o n ta in 30 ppm 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - - d i o x i n (TCDD), and th e r e s p e c t i v e t o x i c i t i e s o f t h e two a g e n ts w ere s t i l l u n c l e a r a t t h a t tim e . The d a ta was a ls o c r i t i c i z e d f o r an u n u s u a lly h ig h and v a ria b le in c id e n c e o f em b ry o to x icity in c o n tro l anim als (N eubert and D illm an, 1972). Emerson e t a l . (1971) e v alu ate d a com m ercial 2 ,4 ,5 -T w ith le s s th a n 0 .5 ppm TCDD and fo u n d t h a t 24 and 40 m g /k g /d a y th r o u g h d a y s 6 -15 o f g e s ta tio n caused no te ra to g e n e s is in r a t s , Sparschu e t a l . (1971) then found t h a t SO mg was a l s o n o n t e r a t o g e n i c , a lth o u g h t h e r e was a s l i g h t in c re ase in in cid en ce o f delayed s k u ll o s s if ic a tio n . (Such a lte r a tio n in developm ent is not consid ered te ra to lo g ic , because th e abnorm ality d is a p p e a rs w ith a g e . One c r i t e r i o n o f t e r a t o g e n i c e f f e c t i s i r r e v e r s i b i l i t y . ) T reatm ent a t 100 m g/day f o r days 6-10 caused g e n e ra liz e d m atern al t o x i c i t y , w ith o n ly 4 s u r v i v o r s o f 25 t r e a t e d a n im a ls . I n t e s t i n a l h e m o rrh a g e , c o n s id e r e d a common f e t o t o x i c b u t n o t t e r a t o l o g i c l e s i o n , was fo u n d in only one pup in th i s s tu d y . Khera and McKinley (1972) found a s im ila r dose response. In m ic e th e e f f e c t i v e d o se i s s i m i l a r t o t h a t in r a t s . At 50 mg 2 ,4 ,5 -T /k g /d ay , through days 6-15 o f g e s ta tio n , th e frequency of c le f t p a l a t e s was i n c r e a s e d from 4.7% t o 20% and r e s o r p t i o n f r e q u e n c y i n c r e a s e d . The i n c i d e n c e was r a i s e d t o 73% by a d o se o f 110 m g /k g /d a y w ith a d e c r e a s e 25 Ci rH 00 r* <h : O o O Q in f e t a l w eight (Bage e t a l . , 1973). The high dose a lso caused a tw o -fo ld in c re ase in rib and v e rte b ra l m alform ation, but only m arginal in crease in d ila te d re n a l p e lv is . N eubert and D illm an (1972) found in c re a se d c l e f t p a l a t e i n NMRI m ice a t d o s e s o v e r 200 m g /k g /d a y th r o u g h d a y s 6 -1 5 , in c re a s e d embryo l e t h a l i t y a t d o ses o v e r 45 m g /k g /d ay r e d u c tio n in f e t a l w e ig h t above 15 m g /k g /d a y . S t r a i n d if f e r e n c e s in m ice a r e s i g n i f i c a n t . The d o se c a u s in g a t l e a s t one c l e f t p a l a t e i n 50% o f l i t t e r s was 25 mg/ k g /d a y i n th e m ost s e n s i t i v e o f 5 s t r a i n s t e s t e d and 105 m g /k g /d ay in th e le a s t s e n s itiv e (G aines e t a l . , 1975). R oll (1971) found d e te c ta b le te ra to g e n e s is a t d oses above 35 m g/kg/day through days 6-15 o f pregnancy. The n o - e f f e c t l e v e l w ith r e s p e c t to te r a t o g e n e s i s was e s t a b l i s h e d a s 20 m g/kg/day. H am sters a re l e s s s e n s i t i v e to c l e f t p a l a te , b u t do have a somewhat h ig h e r in c id en ce o f d elay ed head o s s i f ic a t io n which is n o t g e n e ra lly d efin e d as a te r a to lo g ic m a n ife s ta tio n (C o llin s and W illiam s, 1971). The d o se s ran g ed from 4 0 -1 0 0 mg/kg and th e r e was a c o n s id e r a b le d if f e r e n c e among sam p les o f 2 , 4 ,5 - T o f d i f f e r e n t s o u r c e s and l e v e l s o f TCDD c o n ta m i n a tio n . F e ta l r a b b its a r e a p p a re n tly u n a f f e c te d by m a te rn a l d o ses up .to 40 mg 2 , 4 , 5 -T /k g /d a y th r o u g h d ay s 6 -1 8 . Ruminants a re a p p a re n tly a ls o q u ite r e s i s t a n t to te ra to g e n ic e f f e c ts o f 2 ,4 ,5 - T . B inns an d B a l ls (1971) f e d 100 mg 2 ,4 ,5 - T / k g t o 11 ewes from t h e 1 4 th t o 3 6 th d ay o f g e s t a t i o n , and f e d 100 mg 2 ,4 ,S -T (P G B E )k g to a n o th e r gToup d u r in g t h e same p e r i o d . No e v id e n c e o f d e f o r m ity a p p e a r e d i n any o f th e la m b s. The TCDD c o n t e n t was 1 ppm. The no ap p aren t e f f e c t le v e ls in ro d en ts re p o rte d in th e p receed in g stu d ie s c o n tra st sh arp ly w ith d ata in a b s tra c ts o f re p o rts by K onstantinova (1 9 7 4 a, 1 9 7 4 b ), who i s s a i d t o h a v e fo u n d t h a t 4 .2 m g /k g /d a y o f t h e b u t y l e s te r o f 2,4,5-T through th e e n tire g e sta tio n p erio d caused em bryotoxicity, nervous and hem atologic change and h isto p ath o lo g y in the m other. A dose r a t e o f 0 .4 2 mg/kg a f f e c t e d grow th and d evelopm ent, n e rv o u s, l i v e r and k id n e y f u n c tio n s and lo w ered f e r t i l i t y . The s u c c e e d in g g e n e r a tio n was found to have m inor changes in organ w eights. (D irect tr a n s la tio n s o f th e se p ap ers a re n o t a v a ila b le ; d a ta quoted is o b ta in e d from Chem ical A b stracts.) There has been lim ite d study o f th e te ra to g e n ic e ffe c t o f 2,4,5-T in p rim a te s. D ougherty e t a l . (1975) a d m in iste re d 0 .5 , 1 .0 , and 10 mg/ k g /d a y t o groups o f 10 p re g n a n t rh e s u s monkeys from day 22 th ro u g h 38 o f g e s t a t i o n . The h ig h d o se was e s t a b l i s h e d a f t e r f i n d i n g t h a t 12 mg o r m ore 2 ,4 ,5 - T /k g d a i l y f o r 18 d ay s c a u se d v o m itin g and w e ig h t lo s s in monkeys o f b o th s e x e s . No t e r a t o g e n e s i s o c c u r r e d , a lth o u g h t h e r e w ere 1 o r 2 ab o rtio n s, prem ature b ir th s , or neonatal deaths in a ll groups, including th e c o n tro l. The im p o rta n c e o f TCDD i n th e t e r a t o g e n i c i t y o f 2 ,4 ,5 - T was ex am in ed soon a f t e r th e c o n ta m in a tio n was re c o g n iz e d . C o u rtn ey and Moore (1971) t e s t e d 2 ,4 ,5 - T w ith 0 .5 an d 0 .0 S ppm TCDD, 2 ,4 ,5 - T an d TCDD t o g e t h e r and 26 o CT JTpO TCDD a lo n e i n m ic e . I t was e v i d e n t t h a t th e c o n c e n t r a t i o n s u s e d o r a d d i t i o n o f 1 yg TCDD/kg/day th ro u g h days 6-15 d id n o t a l t e r th e a p p a re n t te ra to g e n ic ity o f 2 ,4 ,5 -T . N eubert and D illm an (1972) reached a s im ila r c o n c lu sio n , su g g estin g th a t to p o te n tia te 2 ,4 ,5 -T e f f e c t on p reg n an t m ice a t l e a s t 1 .5 ppm TCDD i s n e c e s s a r y . C o l l i n s and W illia m s (1 9 7 1 ) o b s e rv e d a d d i t i v e e f f e c t s o f 2 .9 an d 45 ppm TCDD c o n ta m in a tio n o f 2 , 4 ,5 - T a d m in is te r e d to h a m ste rs, b u t many o f th e changes ap p eared c h a r a c t e r i s t i c o f p r i m ary TCDD i n t o x i c a t i o n . N u tritio n a l s ta tu s seems to have l i t t l e in flu e n c e on th e e f f e c t o f 2 . 4 . 5 - T on r e p r o d u c t i v e f u n c t i o n s . H a ll (1972) fe d 250 and 1000 ppm 2 . 4 . 5 - T w ith d i e t s c o n t a i n i n g 20% an d 60% c a s e i n . R e s o r p tio n and s t i l l b ir th in cid en ce and l i t t e r s iz e were s im ila r in a l l groups, although f e ta l organ and p la c e n ta w eights were d ecreased in th e high 2 ,4 ,5 -T , high p ro t e i n gro u p . E f f e c ts o f low p r o te in in ta k e w ere augm ented by th e hig h concentration of h erb ic id e. Incubating eggs have been considered to be p a rtic u la rly v u ln e ra b le to h e rb ic id e s because th e y rem ain in one p la c e , and because th e e g g sh ell is p o ro u s. A number o f s tu d ie s o f 2,4-D e f f e c t on eggs have appeared sin c e 1967, w ith c o n f lic tin g r e s u lts (see s e c tio n on 2 ,4 -D ), b u t 2 ,4 ,5 -T has n o t re c e iv e d much a t t e n t i o n . Somers e t a l . (1973) sp ray ed hen eggs w ith a fo rm u latio n c o n ta in in g 2 ,4 ,5 -T and 2,4-D in a tre a tm e n t e q u iv a le n t to 11.2 k g /h e c ta re (10 tim es norm al f ie l d a p p lic a tio n r a te s ) p r io r to in c u b a tio n w ithout e f f e c t on h a tc h a b ility o r e a rly s u r v iv a b ility o f c h ic k s. They then tr e a te d eggs o f th e p h e a sa n t, as a g e n e tic a lly more h etero g en eo u s s p e c ie s , in th e same way, w ith o u t ad v erse e f f e c t . E n try o f h e rb ic id e in to th e egg was v e r i f i e d a n a l y t i c a l l y . The tre a tm e n t d id , how ever, in c re a s e w eig h t g ain in m ale ch ick s d u rin g th e f i r s t fo u r weeks o f l i f e (Somers e t a l . , 1 9 7 4 ). The LD50 o f 2 , 4 ,5 - T i n DMSO, i n j e c t e d d i r e c t l y i n t o th e a i r s p a c e o f c h ic k e n eggs a t d o s e s up to 125 m g/kg has b een c a l c u l a t e d to be 62 mg/ k g , and i n a c e to n e c a r r i e r , 133 m g/kg ( S tr a n g e e t a l . , 1 9 7 6 ). No t e r a to g e n ic e f f e c t was e v id e n t. The s o lv e n t t o x i c i t y was shown to be s u b s ta n t i a l , b u t i t seems c le a r th a t a c q u is itio n o f an e f f e c tiv e dose o f 2 ,4 ,5 -T by an egg in th e f ie ld is h ig h ly u n lik e ly . In f a c t , im m e rsio n o f h en eggs i n 1% 2 , 4 ,5 - T was i n e f f e c t i v e , and a 5% s o l u t i o n was o n ly m o d e r a te ly e f f e c t i v e . The eg g s w ere im m ersed f o r 10 seconds, th e n re tu rn e d to in c u b a tio n (Gyrd-Hansen and D algaard-M ikkelsen, 1974). I n s e c ts may be m ore s e n s i t i v e ; D av rin g and S u n n er (1971) and D avring (1975) h av e shown t h a t w h ile D ro so p h ila a r e h ig h ly r e s i s t a n t to l e t h a l e f f e c t s o f 2 ,4 ,5 - T e s t e r (LD50 = 4700 ppm i n th e d i e t ) , 1 ppm c a u s e d d is tu rb e d egg f o l l i c l e developm ent and chromosomal d e fe c ts in developed o o c y te s. A s tu d y on a s p e c ie s o f k i l l i f i s h h as shown t h a t c o n c e n tra tio n s o f 20 ppm h av e s u b s t a n t i a l t e r a t o g e n i c e f f e c t . The em bryos d e v e lo p e d s e v e r a l c a r d io v a s c u la r a n o m a lie s , and o c c a s io n a l eye and s p le n i c d e f e c t s . The 20 ppm t r e a t m e n t r e d u c e d h a t c h a b i l i t y t o l e s s th a n 50%, an d 25 ppm a llo w e d l e s s th a n 5% t o h a t c h . No a n o m a lie s w e re fo u n d a f t e r t r e a t m e n t w ith 14 ppm 2 ,4 ,5 - T , and t h e h a t c h was re d u c e d o n ly 4% ( S c h r e iw e is and M u rray , 1976). 27 18450 DOW 0011 dii C arcin o g en ic and M utagenic P o te n tia l o f 2 ,4 ,5-T Probably th e most im portant issu e about any chem ical in tro d u ced in to th e en v iro n m en t by human a c t i v i t y i s th e p o s s i b i l i t y t h a t i t may in c r e a s e th e in c id e n c e o f c a n c e r in th e human p o p u la tio n . The p r o b a b i l i t y o f m uta genic a c tiv ity is o f alm ost eq u iv alen t concern, both because o f the p o s s i b i l i t y o f g e n e tic a l t e r a t i o n and b e c a u se m u ta g e n e sis may be a u s e f u l p r e d ic to r o f carcin o g en ic a c tiv ity . The re la tio n s h ip is by no means c o n s ta n t. H ow ever, McCann e t a l . (1975) h av e a sse m b le d d a t a from a num ber o f la b o r a t o r i e s u s in g m i c r o b ia l sy ste m s ("Ames t e s t " ) and f in d t h a t 85% o f known c a r c in o g e n s a r e p o s i t i v e , 10% o f n o n - c a r c in o g e n s a r e a c t i v e . T h is i s n o t to say th a t alm ost every ag en t id e n tif ie d as a mutagen w ill be c a rc in o g e n ic , b u t th e t e s t should become an a id in d ecid in g p r i o r i t i e s f o r d i r e c t sc re e n in g . Only a lim ite d number o f ex p erim en tal s tu d ie s o f 2 ,4 ,5 -T c a rc in o g e n i c i t y h av e been m ade. A s c r e e n in g p rogram f o r 120 p e s t i c i d e s and i n d u s t r i a l ch em icals was re p o rte d by Innes e t a l . (1 9 6 9 ). E leven o f th e a g e n ts caused an elev ate d incidence o f tum ors; none of the phenoxy h e rb ic id e s, in c lu d in g 2 ,4 ,5 -T , caused in c re a se d tumor form atio n a t a dose p re v io u sly determ ined to b e t h e maximum t o l e r a b l e d a i l y d o se o v e r 20 d a y s , w ith o u t l e t h a l i t y (2 1 .5 m g/kg/day f o r 2 ,4 ,5 - T ) . M uranyi-K ovacs e t a l . (1976) t r e a t e d two s t r a i n s o f m ice w ith 80 ppm 2 ,4 ,5 - T (TCDD c o n t e n t 0 .0 5 ppm) i n th e d i e t f o r m ore th a n 500 d a y s. D aily dosage was ab o u t 12-15 m g/kg. In one s t r a i n s u r v iv a l tim e was s i g n i f i c a n t l y d e c re a se d in m a les, in th e o th e r s i g n i f i c a n t l y in c re a s e d i n f e m a le s . An a p p a r e n t l y s i g n i f i c a n t s m a ll i n c r e a s e i n tum or i n c i d e n c e o ccu rred w ith th e in c re a s e in l i f e sp an . The au th o rs a re concerned th a t a l t e r e d l i f e span may confound th e a n a l y s i s , b u t c o n s id e r t h a t 2 ,4 ,5 - T can n o t y e t c o n fid e n tly be co n sid ered n o n -carcin o g en ic , and re q u ire s f u r th er analysis. A tw o y e a r s tu d y o f r a t s g iv e n up to 30 mg 2 ,4 ,5 - T / k g /d a y h a s j u s t b e e n te r m in a te d by Dow C hem ical Company, b u t th e p a t h o l o g i c a l e v a l u a t i o n had n o t been com pleted a t the tim e o f th is p re p a ra tio n . One e p id e m io lo g ic a l s tu d y h as been made o f tum or in c id e n c e in a group o f Sw edish r a i l r o a d em ployees. I t was p o s s ib le to i s o l a t e gro u p s who had h a n d le d p h en o x y a c id s e x c l u s i v e l y , and a c o h o r t o f 207 p e r s o n s w ith a t l e a s t 45 d ay s o f e x p o s u r e , t o t a l i n g 1747 p e r s o n - y e a r s . No i n c re a se in tum or in c id e n c e could be d e te c te d in th e se w orkers alth o u g h in creased incidence appeared in groups exposed to o th e r h erb ic id es (A xelson and S u n d e ll, 1974). M utagenic assessm en ts have been c a rrie d out on a number o f sy stem s. B u selm aier e t a l . (1973) w ere n o t a b le to show 2 ,4 ,5 - T in d u c ed m u ta tio n s i n S a lm o n e lla ty p h im u riu m G46 h i s - , and S e r r a t i o m a rc e s c e n s a21 L eu - and a31 h is - in a h o s t m ediated a ssay in m ice. Serum from an im als tr e a te d o r a lly w ith 2 ,4 ,5 -T a ls o d id n o t induce m utants in S. typhim urium h is ( S t y l e s , 1 9 7 3 ). A n d erso n e t a l . (1972) wex*e u n a b le to d e m o n s tr a te any m utagenic p r o p e r tie s o f 2 ,4 ,5 -T o r o th e r phenoxy h e rb ic id e s when te s t e d a g a in st e ig h t h is tid in e re q u irin g m utants o f S. typhim irium . 28 O O Sex lin k e d l e t h a l t e s t s o f a d u lt m ale D ro so p h ila showed 2 , 4 , 5-T to be n e g a tiv e , a lth o u g h f e r t i l i t y was d e c re a se d (Vogel and C h a n d le r, 1 9 7 4 ). Majumdar and G o lia (1974) w ere a b le to show an in c re a s e in sex lin k e d r e c e s s i v e l e t h a l m u ta tio n s a f t e r IS day f e e d i n g on 1000 ppm 2 , 4 , 5 -T . A n o n - s i g n i f i c a n t i n c r e a s e was a p p a r e n t a f t e r f e e d in g 2S0 ppm. In g e r b i l s s i x - d a y f e e d i n g o f up t o 150 mg 2 ,4 ,S - T /k g p ro d u c e d no in c re a s e in ch lo ro so m al a b n o r m a litie s , b u t h ig h e r doses d id c a u s e some in c re a s e (Majumdar and H a ll, 19 7 3 ). J e n s s e n and Renberg (1976) t e s t e d 2 ,4 ,5-T f o r m u ta g e n ic ity by seek in g m icT onuclei in e ry th ro c y te s o f mouse bone marrow, which is a h ig h r e s o lu tio n d e te c tio n system . The system was c y to g e n e tic a lly n e g a tiv e , a lth o u g h some m i to ti c d e p re s s io n was d e t e c t e d . I t was n o te d t h a t o n ly a b o u t 5% o f c i r c u l a t i n g 2 , 4 , 5-T fo u n d i t s way i n t o th e c e l l s , w hich was s u g g e s te d a s i n d i c a ti n g a lim ite d h a z a rd due to lack o f access. A l i m i t e d s u r v e y o f p e s t i c i d e a p p l i c a t o r s who h ad b e e n i n c o n t a c t w ith a v a r ie ty o f a g e n ts showed some in c re a s e in chrom atid gaps and b re a k s d u rin g th e sp ray seaso n (Yoder e t a l . , 1975). R ecovery in th e o f f seaso n reduced frequency below th a t o f c o n tro ls , su g g e stin g an augm ented r e p a ir system . 2 ,4 ,5-T was th e le a s t f re q u e n tly used a g e n t, and i t is n o t lik e ly th a t any observed e ffe c t can be a ttrib u te d to i t . Dow C hem ical Company h a s m a in ta in e d a p ro g ram o f c o n t in u in g h e a l t h m onitoring o f 2 ,4 ,5-T p ro d u ctio n w orkers. C y to g en etic e v a lu a tio n s o f 52 men in e a r l y 1970 w ere r e p o r te d t o be n e g a tiv e (Jo h n so n , 1 9 7 1 ), and a second a n a ly s is in 1974 a ls o found no ev id en ce o f a b n o rm a lity ( K ilia n , 1 9 7 5 ). An i n t e r e s t i n g f i n d i n g , h o w e v e r, was t h a t th e f r a c t i o n o f ea c h exposed g ro u p fo u n d t o have no ab n o rm al c e l l s was ab o v e 80%, w h ile o f th e c o n t r o l g ro u p o n ly 74% h ad no ab n o rm a l c e l l s . A b sorption, D is tr ib u tio n , M etabolism , and E x c re tio n o f 2 ,4 ,5-T 2 , 4 , 5-T h a s a r a t h e r s h o r t r e s id e n c e tim e in a l l s p e c i e s . A cow g iv e n 450 mg o f 2 , 4 , 5-T a c id i n f o u r d a i l y d o s e s e x c r e te d th e e n t i r e amount in u rin e in s ix days (S t. John e t a l . , 1964). Erne (1966a, 1966b) a d m in is te r e d 100 rag o f th e am ine s a l t / k g t o r a t s and sw in e and fo u n d plasm a h a l f tim e f o r th e r a t to b e th r e e h o u rs , and 10 h o u rs f o r p ig s . Kidney, li v e r , lu n g s, and sp le e n c o n c e n tra tio n s o f 2 ,4 ,5-T co u ld o cca s io n a lly be fo rced above plasm a le v e ls , b u t a p p a re n tly none o f th e m a teria l en tered th e b ra in or adipose tis s u e . T issue h a lf tim es ranged from 5 -3 0 h o u r s . When th e a g e n t was a d m in is te r e d r e p e a t e d l y p la sm a l e v e l s tended to le ss e n , w ith in c re a se d e x c re tio n . Of th e 2 ,4 ,5-T in b lood, about 20% was i n e r y t h r o c y t e s . 2 , 4 , 5-T i s e x c r e te d m ore s lo w ly b y m ic e th a n b y r a t s , a t a r a t e o f 1-4% o f th e o r i g i n a l d o se p e r h o u r ( Z i e l i n s k i and F ishbein, 1967). By o v e r lo a d in g s h e e p ( f o u r 250 mg d o s e s ) i t i s p o s s i b l e to p r o d u c e r e s i d u e s i n t i s s u e s . Maximum a c c u m u la tio n s w ere a b o u t 100 ppm i n f a t and m uscle. In each case re sid u e s were found in the acid form re g a rd le s s o f th e form in w hich i t was fe d (C la rk e t a l . , 19 7 0 ). The l a t t e r fin d in g i s a t some v a r i a n c e w ith C la r k e t a l . (1 9 7 1 ) , i n w h ich t h e PGBE e s t e r o f 2 , 4 , 5-T was r e p o rte d to rem ain as th e e s t e r in u r in e and t i s s u e s . C a t t l e fe d 0 .1 5 -O' Hy Cr\ 29 DOW OQll S is and 0 .7 S rag 2 , 4 , S-T p r o p y le n e g ly c o l o r b u ty l e t h e r e s t e r s / k g / d a y f o r 52 weeks, produced no in c re a s e o v er background r e s id u e . C lark e t a l . (1971) l a t e r r e p o r t e d f e e d in g sh eep 2000 ppm 2 ,4 ,5 - T i n th e d i e t and f i n d i n g 1 .0 ppm 2 , 4 , S-T in m u s c le . No r e s i d u e c o u ld be d e t e c t e d 7 days l a t e r . D eer brow sing on land tr e a te d f o r r e f o r e s ta tio n were found not to c o n ta in s i g n if i c a n t ti s s u e r e s id u e s ; m easu rab le 2 , 4 , S-T was found in stom ach c o n te n ts , u r in e , and fe c e s up to 43 days a f t e r h e r b ic id e a p p lic a tio n (Newton and N o rris, 1968). The im p lic a tio n , a g a in , is th a t su sta in e d f ie ld in ta k e w ill not cause d etecta b le tissu e resid u es. Grunow e t a l . (1971) found ab o u t 50-70% o f a d m in is te re d 2 ,4 ,5 - T in u rin e , and found th a t a s u b s ta n tia l f r a c tio n emerged as d e r iv a tiv e s , one o f w hich was id e n t i f i e d as N (2 ,4 ,5 -tric h lo ro p h e n o x y a c e ty l) g ly c in e . The ta u r in e c o n ju g a te and 2 ,4 ,5 - tr ic h lo r o p h e n o l have a lso been id e n tif i e d (Grunow and Bohrae, 1 9 7 4 ). Nony e t a l . (1976) have m easured 2 ,4 ,5 - T , i t s g ly c in e amide and a lk a lin e h y d ro ly z a b le c o n ju g a te s in b lo o d , u r in e , and f e c e s . The p ro p o r tio n o f m e ta b o lite s i s q u i t e low in b lo o d , and in c r e a s e s s u b s ta n tia lly in u rin e from 24-72 hours a f te r a d m in is tra tio n o f th e h e r b i c id e . In th e fe c e s th e g ly c in e amide is found in o n ly lim ite d am ounts, but o th e r c o n ju g a te s may a c c o u n t f o r a s much a s 25% o f th e t o t a l r e c o v e r y . R esearch w ith la b e le d 2 ,4 ,5 -T in r a t s showed th a t th e h a lf-tim e (T/2) o f plasm a c le a r a n c e was ab o u t 4 .5 h o u rs and e s s e n t i a l l y d o se in d e pendent below d o ses o f 50 mg/kg b u t in c re a s e d as dosage in c re a s e d to 1 9 .4 hrs a t 100 mg/kg and 2 5 .2 h r a t 200 mg/kg. The T/2 o f a 5 mg/kg dose in dogs was much lo n g e r , 77 h o u r s . A bout 90% o f th e 2 ,4 ,5 - T in b lo o d was re v e rs ib ly bound to plasm a p ro te in , ov er a wide range of c o n c e n tra tio n s . I t was found a ls o t h a t as th e do se was e le v a te d , a sm all amount o f f e c a l e x c r e tio n took p la c e . D e te c ta b le , m inute amounts o f *J C emerged in th e r e s p ir a to r y gases (P ip e r e t a l . , 1975). Fang e t a l . (1973) a ls o showed a n c n -d o se d ep en d e n t T /2 a t lo w er d o s e s . Nony e t a l . (1976) found in e x ce ss o f 10% o f th e t o t a l e x c r e te d m a t e r i a l i n f e c e s . The e x t e n t o f p r o t e i n b inding has been f u r th e r d efin e d by K olberg e t a i . (1973); bovine serum alb u m in b in d s 13-1 4 m oles 2 ,4 ,5 -T /m o J. BSA. As s u g g e s te d by t h e s lo w e r T /2 , t h e p e r c e n ta g e o f d o s e e x c r e t e d / d a y decreased sharply a t the higher doses, in d ic a tin g a lim it in ex creto ry cap a city by th e kidney. Rat kidney tis s u e s lic e s can c o n c e n tra te 2 ,4 ,5 -T a b o u t 15 tim e s , s l i c e s from dogs c a n c o n c e n t r a t e a b o u t 9 t i m e s . B e m d t and K oschier (1973) found an energy dependent uptake o f phenoxy h e rb ic id e s by re n a l c o r tic a l ti s s u e . M etabolic in h ib ito r s d ep ress th e p ro c e s s . A cetate and la c ta te as su b stra te s enhance the accum ulation. In creased 2 ,4 ,5 - T i n th e medium slow s th e c o n c e n tr a tin g c a p a c ity o f th e k id n e y . The above work was l a t e r c a r r ie d fo rw ard by r e s o r t i n g to in tr a v e n o u s in je c tio n of 2 ,4 ,5 -T in o rd er to tak e advantage of u se fu l pharm acokinetic models (S a u e rh o ff e t a l . , 1976). Doses o f 5 mg/kg and 100 mg/kg w ere used,and sa m p le s w ere ta k e n th ro u g h 36 h o u rs (5 mg) and 72 h o u rs (100 m g). A f te r 5 m g/kg r a t s e x c r e te d a b o u t 50% o f t h e body b u rd e n e v e ry 12 h o u r s , in s p ite o f a 4.3 hour plasm a T /2, f u rth e r i l l u s t r a t i n g th e c o n c e n tra tin g c a p a c ity o f th e k id n e y . At 100 mg/kg th e plasm a T /2 was 23.1 h o u rs f o r th e f i r s t 36 h o u rs , th e n was com parable to th e an im als g iv e n a low er d o s e . 16453 30 o O In humans (G ehring e t a l . (1973) found t h a t a S mg/kg d ose was ex c re te d w ith a T/2 o f 23 hours f o r b o th plasm a and whole body c le a ra n c e . E s s e n tia lly a l l o f th e ad m in istered m a te ria l emerged in u rin e unchanged. The phenoxy a c id s a re r e la tiv e ly stro n g o rg a n ic a c id s , which i s one o f th e reaso n s th ey a re ex cre ted in laTge p a r t as th e p a re n t m olecule. The p o s s i b i l i t y o f h y d ro ly s is by rumen f l o r a o r by t i s s u e en ty m a tic p ro c e s s e s was f i r s t c o n s id e r e d by W rig h t e t a l . (1 9 7 0 ) who fo u n d t h a t a f t e r feeding of 2(2,4,S -trichlorophenoxy) e th y l-2, 2-d ich lo ro p ro p io n ate to sheep, 2 ,4 ,5 -tric h lo ro p h e n o l resid u es could be d etected in tis s u e s . C lark e t a l. (1975) m easured phenoxy a c id and phenol re s id u e s in m uscle, f a t , l i v e r , and k id n e y o f sh eep f e d 2000 ppm 2 , 4 , 5-T f o r 28 d a y s , and fo u n d 1 .0 0 , 0 .2 7 , 2 .2 9 , and 2 7 .2 ppm 2 , 4 , 5-T and 0 .1 3 , < 0 .0 5 , 6 . 1 , and 0 .9 ppm 2 , 4 , 5 - t r i ch lo ro p h en o l, r e s p e c tiv e ly . Seven days a f te r w ith d raw al, 2 ,4 ,5-T re s id u e s w ere w e ll below 0 .1 ppm o r below t h e d e t e c t i o n l i m i t o f 0 .0 S ppm. The phenol rem ained a t s i g n i f i c a n t l e v e ls in b o th l i v e r (4 .4 ppm) and k id n e y (0.81 ppm). A ccording to K oschier and B em dt (1976c) th e r a te o f e x c re tio n r is e s a lm o st im m e d ia te ly t o a b o u t 80% o f in p u t in a n im a ls t r e a t e d d a i l y , th e n continues to r is e fo r 7-9 days u n til ex cretio n approxim ately equals in p u t, where i t rem ains th ro u g h th e d u ra tio n o f tre a tm e n t. The r a te o f adm inis t r a t i o n was 20 ra g /k g /d a y . As d o s e r a t e i n c r e a s e d t h e i n i t i a l p e r c e n ta g e e x c re te d was l e s s , b u t 7-9 days was s t i l l r e q u ir e d to re a c h a r a t e e q u a l to i n p u t . An a p p a r e n t o v e r - s h o o t th e n to o k p l a c e f o r 5 -6 d a y s . The m arkedly d if f e r e n t r a te s o f 2 ,4 ,5-T e x c re tio n by r a ts and dogs was s t u d i e d in v i t r o b y Hook e t a l . (1 9 7 4 ). They fo u n d t h a t k id n e y o f both sp e c ie s a c tiv e ly accum ulated 2 ,4 ,5-T by a s a tu r a b le , oxygen dependent p ro c e ss w hich was d e p re s s e d by o th e r o rg a n ic a n io n s . Rat k id n e y had a g re a te r dependence on p o tassiu m , and dog kidney in c re a s e d accum ulation o f 2 ,4 ,5-T in th e p re se n c e o f a c e ta te . The c a p a c ity o f kidney to tr a n s p o r t th e s ta n d a rd t e s t a n io n , p ara-am in o h ip p u r ic a c id (PAH), can be com p e titiv e ly in h ib ite d by 2,4,S -T , in d ic a tin g th a t both are tra n sp o rte d by th e same m echanism . The a u t h o r s c o n c lu d e d t h a t t h e g r e a t e r c a p a c i t y f o r PAH tra n s p o rt by r a t tis s u e s accounts fo r th e d iffe re n c e in r e te n tio n betw een r a ts and dogs. A pparently low er re n a l ex cre tio n is a lso re sp o n sib le fo r th e extended h a lf-tim e o f 2 ,4 ,5-T in new-born r a t s observed by Fang e t a l. (1 9 7 3 ); Hook e t a l . (1974) hav e found t h a t k id n e y s o f 10 day o ld r a t s a r e much l e s s e f f i c i e n t th a n a d u l t s . S u b seq u en t work by th e same group (Hook e t a l . , 1976) in d ic a te d th a t plasm a b in d in g in dogs was more te n a c io u s th an in r a ts . The high s p e c if ic ity o f 2 ,4 ,5-T in causing c l e f t p a la te has been in v e s tig a te d by Dencker (1976) as a d is tr ib u tio n phenomenon. The s e n s itiv e p e rio d h as been shown to be l a t e in f e t a l d ev elo p m en t, d u rin g days 12-13 (N 'eubert and D illm an , 1972). P a la ta l c lo s u re o c c u rs v e ry l a t e in o rg an o g en esis and is com plete a t about day 15. D encker (1976) p o in ts out th a t no unusual u p ta k e o f 2 ,4 ,5-T in th e se stru c tu re s occurs, but o v e ra ll uptake in the fe tu s is g re a tly in creased b etw een d ay s 11 and 18. I t may w e ll be t h a t t h i s i n c r e a s e , a t a tim e when form ation o f most o th e r s tru c tu re s have p ro g ressed f a r enough th a t they are not se n sitiv e , is responsible fo r c le ft p alate. C - h r 'O? " *\J 31 8.10.1100 I t is p o s s ib le f o r 2 ,4 ,5-T to reach m ilk i f a s u f f i c i e n t le v e l is in c lu d e d in th e d i e t . At 30 ppm in t h e d i e t f o r two w eek s, fo llo w e d by one week on u n tr e a te d fe e d , no 2 , 4 , 5-T o r tr ic h lo r o p h e n o l was found a t a d e te c tio n le v e l o f 0 .0 5 ppm; a t 100 ppm t r i c h lo r o p h e n o l was b a r e l y d e t e c t a b l e . At 1000 ppm 0 .3 1 - 0 .5 4 ppm 2 , 4 , 5-T and 0 .1 6 - 0 .2 7 ppm t r i c h l o r o p h e n o l was found i n m ilk , and somewhat l e s s was fo u n d in cream (B je rk e e t a l . , 1972). These fig u re s can be put in th e p e rs p e c tiv e o f forage consum ption. R esidues on g ra s s a re on th e o rd e r o f 100-150 p p m /lb /a c re (M orton e t a l . 1967) im m ediately a f t e r a p p lic a tio n . T hese am ounts drop s h a rp ly to about 0 20% o f i n i t i a l l e v e l s d u r in g th e i n i t i a l tw o w eeks p o s t - a p p l i c a t i o n . T h is f a c to r , w ith th e r a p id e x c r e tio n c h a r a c t e r i s t i c o f 2 , 4 , 5-T make even lim ite d human e x p o s u re th r o u g h d a i r y p r o d u c ts o r m eat h i g h l y u n l i k e l y u n le s s p r o d ucts are obtained im m ediately a f te r heavy sp ra y in g . B ehavior o f 2 , 4 , 5-T in th e E nvironm ent and E f f e c ts on Submammalian S p e c ie s One f a c t o r i n th e h a z a r d p o t e n t i a l o f an h e r b i c i d e i s d e te rm in e d by it s p e rs is te n c e a f te r a p p lic a tio n and i t s movement and re a c tio n s in s o i l , w ater, or in p la n ts . A ppearance o f 2 ,4 ,5-T in f o r e s t stream s has been shown to r e s u l t o n ly i f th e h e r b ic id e was in tro d u c e d d i r e c t l y to th e s tre a m , and d im in ish e d from a b o u t 0 .1 ppm to l e s s th a n 0.01 ppm w i t h i n one d ay a f t e r tr e a tm e n t (N o rris, 1967). N o rris (1968) a lso found th a t heavy ra in s s ix months a f t e r tre a tm e n t d id n o t move d e te c ta b le 2 , 4 , 5-T in to s tre a m s . The h e rb ic id e is u s u a lly degraded on th e f o r e s t flo o r q u ite ra p id ly . N o rris (1969) found t h a t n e a r l y 90% d is a p p e a r e d i n two m o n th s, a lth o u g h c o ld w e a th e r, s t e r i l e s o i l , and la c k o f m o is tu r e may e x te n d th e p e r io d o f d e g ra d a tio n o v er as much as 9 m onths. The tr i c h lo r o p h e n o l h y d r o ly s is p r o duct o f 2 ,4 ,5-T degrades f a s te r than th e p a re n t compound (A lexander and Aleem, 1961). E v a lu a tio n o f s e v e r a l s o i l s from S outh V ietnam in d i c a te s th a t c o n c e n tra tio n s on th e o rd e r o f th o se ex p ected a f t e r a ty p ic a l f o r e s t a p p lic a tio n should be v ir tu a lly gone in seven weeks (B yast and Hance, 1975). More th a n 20% o f th e r a d i o a c t i v e c a rb o n o f l a b e l e d 2 , 4 , 5-T h a d becom e i n corporated in th e s o il, but a c tu a l re sid u e s o f h e rb ic id e were u s u a lly ab o u t 1%. 2 , 4 , 5-T h a s a l s o b een fo u n d s h o r t l i v e d i n v a r i o u s T exas s o i l by Bovey and Baur (1 9 7 2 ). Altom and S t r i t z k e (1973) s tu d ie d 2 , 4 , 5-T in th r e e Oklahoma s o i l s and fo u n d h a l f - t i m e s o f 2 4 , 14, and 21 d a y s . The s h o r te r tim e was on g r a s s la n d s , th e o th e r s w ere f o r e s t s . Leaching t e s t s show t h a t 2 , 4 , 5-T bound in s o i l rem ained in th e u p p e r 6 inches o f t e s t columns even a f te r a p p lic a tio n o f 4 .5 in ch es o f w ater (Wiese and D av is, 1964). The v ario u s e s te r s o f 2 ,4 ,5-T a re o fte n i n t r i n s i c a l l y more to x ic than th e p a re n t a c id , p ro b ab ly b ecause th e y a re more f a t s o lu b le . In an aqueous system , how ever, th ey h y d ro ly ze r a p id ly to th e a c id , and th e r a te is enhanced by s o il and m icroorganism s (T easley and W illiam s, 1970, as quoted by Kenaga, 1974). I t seems r e a s o n a b le to e x p e c t a f a i r l y r a p id h y d ro ly s is in any system w ith some w a te r p r e s e n t. 32 QOW In essence, 2 ,4 ,5 -T disappears r e la tiv e ly ra p id ly a f te r a p p lic a tio n , p ro b ab ly w ith in two months in a f o r e s t e n v iro n m en t. The ev id e n c e a ls o seems firm th a t 2 ,4 ,5 -T d e p o s ite d on th e ground w ill n o t move in t o a w a te r course, and w ill n o t leach in to deeper la y e rs . I t is d i f f i c u l t to se p a ra te p h y s ic a l b eh av io r a f te r a p p lic a tio n from e ffe c ts on low er organism s, because th e re is a v ery c lo se in te r f a c e be tween an ag en t bound to s o il o r in w a te r, and in s e c ts o r f i s h . Lower organism s are o f d ir e c t im portance in a sse ssin g environm ental im pact o f any in t r o d u c e d c h e m ic a l._ Food c h a in e f f e c t s may p r e d i c t r e p e r c u s s i o n s a t' h i g h e r l e v e l s , and s p e c i f i c a l l y s e n s i t i v e o rg a n is m s may be u s e f u l i n d ic a to rs o f p o llu tio n . E cological displacem ent o f any kind must be ac cepted only a f te r c a re fu l exam ination, w hether secondary to th e in ten d ed im pact o f th e chem ical on p la n t sp e c ie s o r as a r e s u lt o f d ir e c t to x ic e ffe c t o f the ap p lied chem ical. The v ario u s d e riv a tiv e s o f 2 ,4 ,5 -T d if f e r in to x ic ity to f is h . The p aren t h e rb ic id e , th e amine s a l t s and is o o c ty l e s te r s a re r e l a t i v e l y lim ite d in to x ic ity , but th e propylene g ly c o l b u ty l e th e r and butoxy eth an o l e s te r s a re q u ite to x ic to some s p e c ie s . T here i s a la rg e number o f g ro ss e f f e c t stu d ie s on v ario u s aq u atic sp e c ie s; th e se g e n e ra lly use an end p o in t o f death o r im m o b ilizatio n . S h e llfis h s tu d ie s m easure s h e ll grow th as an index o f e f f e c t. S ta n d a r d iz a tio n o f m ethods h as n o t r e c e iv e d as much a t t e n t i o n as seems j u s t i f i e d . Study p e rio d s v ary from 24-96 h o u rs, w a te r te m p e ra tu re s sometimes ap p ear o u ts id e th e norm al a c c e p ta b le range fo r th e s p e c ie s , and most e v a lu a tio n s seem to be c a rrie d on under s t a t i c c o n d itio n s . N onethe le ss, th e re la tiv e gross e ffe c ts can be u sefu l in referen c e to a given a p p l i c a t i o n i n th e f i e l d . As a v e r y ro u g h a p p r o x im a tio n , a 2 k g / h e c t a r e a p p lic a tio n o v er a w a te r c o u rse would p roduce a c o n c e n tra tio n o f a b o u t 2 mg/L o r 2 ppm, i f an a s s u m p tio n o f d i l u t i o n i n t h e u p p e r 10 cm o f w a te r is a c c e p te d . Flow ing w ater w ill d im in ish th e c o n c e n tra tio n below d e te c ta b le lim its in a few hundred y ard s (Evans and D u seja, 197 3 ), and a p p lic a tio n over deeper w ater w ill d ilu te acco rd in g ly . C o n c e n tra tio n s o f 2 ,4 ,S -T o r i t s d e r iv a tiv e s found to have no e f fe c t a re li s t e d below as acid e q u iv a le n ts: Na s a l t S p ecies k illifish m ullet se a lam prey b lu e g ill No E f f e c t Exposure Acid E q u iv alen t P e rio d C o n c e n t r a ti o n ppm R eference 48 h 50 B u tle r , 1963 48 h 50 B u t l e r , 1963 72 h 2 A pplegate e t a l . , 1957 12 d 4 6 H iltib r a n , 1967 16458 33 DOW" 0011 820 2 ,4 ,5 -T butyl e s te r tro u t b lu eg ill lam prey 24 h 24 h r 24 h 2 ,4 ,5 -T is o o c ty l e s te r b lu eg ill green sunfish b lu eg ill 8d 8d 12 d 4,1 A pplegate e t *a l . , 1957 4.1 A pplegate et a l . , 1957 4.1 A pplegate et a l . , 1957 7 H iltib r a n , 1967 7 H iltib r a n , 1967 0 .7 H iltib r a n , 1967 Median le th a l c o n c e n tra tio n s o f 2 ,4 ,5 -T d e r iv a tiv e s a re s im ila r ly ta b u la te d : S p e c ie s Exposure LCso P eriod Acid E q u iv alen t (h o u rs ) C o n c e n tr a tio n ppm R eferen ce 2 ,4 ,5 -T DMA s a l t b lu eg ill 48 144 Hughes and Davis (1963) 2 ,4 ,5 -T b lu eg ill 24 54 Davis and Hughes (1963) TEA s a l t 2 ,4 ,5 -T 0 1 e ic -l,3propylene diam ine s a lt b lu eg ill 48 2.9 Davis and Hughes (1963) 2 ,4 ,5 -T is o p ro p y l e s te r b lu eg ill 48 1.7 Davis and Hughes (1965) 2 ,4 ,5 -T b lu eg ill 48 31 Hughes and Davis (1963) iso o cty l e s te r 2 ,4 ,S-T b u to x y e th a n o l e s te r b lu eg ill 48 1.4 Hughes and Davis 1963 2 ,4 ,5 -T PGBE e s t e r s b lu eg ill spot h a rle q u in 48 24 48 17 0.21 1.0 Hughes and Davis , 1963 Cope, 1965 A la b a s te r , 1969 W hile th e more complex e s te r s o f 2 ,4 ,5 -T ten d to g r e a te r t o x i c i t y in w a te r a t pH 6 .5 , th e y h y d r o ly z e t o th e p a r e n t a c i d i n a b o u t a day ( T e a s le y and W illiam s, 1970, as quoted by Kenaga, 1974). 1645 34 E ffe c ts o f 2 ,4 ,5 -T on o th e r a q u a tic organism s have a ls o been m easured. In f lo w in g s a l t w a te r 1 ppm 2 , 4 , 5 - T c a u s e d no m o r t a l i t y i n brown s h rim p and 2 ppm was w i t h o u t e f f e c t on o y s t e r s h e l l g ro w th ( B u t l e r , 1 9 6 3 ). The PGBE e s t e r o f 2 , 4 , 5 - T a t 0 .1 4 ppm (0 .0 9 ppm a c i d e q u i v a l e n t ) c a u s e d a 50% d e c r e a s e i n o y s t e r s h e l l grow th a f t e r 96 h o u r e x p o s u re ( B u t l e r , 1 9 6 3 ). S tu d ie s o f t e r r e s t r i a l non-mammals seem to have been lim ite d to honey b e e s. Honey bees a re an im p o rtan t component o f th e crop cy c le and in many a re a s a r e s y s t e m a t i c a l l y managed f o r p o l l i n a t i o n . Phenoxy h e r b i c i d e s , i n c l u d i n g 2 , 4 , 5 - T , f e d i n 60% s u c r o s e - w a t e r s o l u t i o n a t 10 ppm d id n o t a f f e c t h a t c h i n g , b u t r e d u c e d b r o o d d e v e lo p m e n t when f e d a t 100 ppm (Morton and M o f f e t t , 1 9 7 2 ). When s p r a y e d i n a w a t e r c a r r i e r a t f i e l d a p p lic a tio n r a t e s , 2 ,4 ,5 -T was n o n -to x ic . (P etroleum s o lv e n ts alone caused high m o rtality during the f i r s t day a f te r spraying.) A erial spraying did not re s u lt in h erb icid e accum ulation in honey sacs o r c o lo n ie s (M o ffett and M orton, 1972). The p o i n t was made t h a t th e prim ary damage would be f a il u r e o f flow ers and lo ss o f n e c ta r , r a th e r th a n d i r e c t t o x i c i t y . When f e d d i r e c t l y t o n ew ly em erged b e e s , t h e phenoxy h e rb ic id e s were e s s e n t i a l l y n o n -to x ic a t c o n c e n tra tio n s up to 1000 ppm i n t h e d i e t (Morton e t a l . , 1 9 7 2 ). 2.4- D T o x i c i t y t o Humans For some re a s o n t h e r e have been r e p o r te d s e v e r a l i n c i d e n t s o f 2,4-D p o iso n in g in humans, but v i r t u a l l y no r e p o r t s o f human i n t o x i c a t i o n by 2 .4 - T. P ro b a b ly t h e b e s t known was a s u i c i d e by a s i n g l e do se o f 2,4-D DMA s a l t o f which more t h a n 90 rag/kg was e s t i m a t e d t o h a v e b e e n r e t a i n e d by t h e v i c t i m , a f t e r e x t e n s i v e v o m i tin g ( N i e l s e n e t a l . , 1 9 6 5 ) . An a c c i d e n t a l p o i s o n i n g w i t h a f o r m u l a t i o n c o n t a i n i n g 49% S - e t h y l d i p r o p y l t h i o c a r b a m a t e , 9% k e r o s e n e , and 36.5% 2 ,4 -D i s o o c t y l e s t e r c a u s e d symp toms and l a b o r a t o r y f i n d i n g s a t t r i b u t a b l e t o 2 ,4 -D i n t o x i c a t i o n . Among these were tw itc h in g and p a ra ly s is of in te r c o s ta l m uscles, hem oglobinuria, and m y o g l o b i n u r i a . No e v i d e n c e o f p e r i p h e r a l n e u r o l o g i c a l damage was e v id e n t d u r in g 36 months o f fo llo w - u p o b s e r v a t i o n s (B erw ick , 1 9 7 0 ). G oldstein e t a l . (1959) re p o rte d on th re e cases in which 2,4-D e s te r s were absorbed through th e s k in , r e s u lt in g in p e rip h e ra l n e u ro lo g ic a l c h a n g e s . One p a t i e n t s p i l l e d 60 ml o f a 10% s o l u t i o n o f 2 ,4 -D e s t e r on th e forearm s and d id n o t wash. Unusual f a ti g u e developed in a few houTS, and o v e r 10 days a f t e r e x p o su re t h e r e was e x te n d e d n a u s e a and v o m i tin g , with co n sid erab le weight lo s s . A second exposure caused s im ila r symptoms, then p a in and numbness in a l l d i g i t s , lo s s o f sk in from th e palm s, and w ith in s ix weeks th e re was s u b s t a n ti a l g e n e ra l n e u ra l damage. A nother p a tie n t ev en tu ally developed m etacarpal pain and sw elling in both hands, and. l a t e r became p a r t i a l l y p a r a l y z e d . The t h i r d e x p e r i e n c e d g a s t r o i n t e s ti n a l d istu rb a n c e and v e rtig o , then p a r e s th e s ia in th e arms and le g s and p e r s i s t e n t g e n e ra l muscle f a s c i c u l a t i o n s . . In two o f th e c a s e s , th e r e were second exposures which appeared to cause g r e a te r im pact th a n th e i n i t i a l e v e n t. S im ila r cases have been d e s c rib e d by Todd (1962), B erkeley and Magee (1963), and W ilson (1 9 5 6 ). 35 DOW QQii' 822 Seabury (1963) attem pted use o f 2,4-D in treatm en t of a term inal case o f d issem in ated co cc id o ia o m y co sis, on th e r a t i o n a l e t h a t th e 2,4-D as a s y n th e tic p la n t hormone might a l t e r the co urse o f th e fungus i n f e c t i o n . At th e tim e o f th e tr e a tm e n t (1949) t h e r e was no t h e r a p e u t i c a g e n t f o r th e d i s e a s e . In 24 tr e a tm e n ts o v er a p e r io d o f more th a n a month th e dosage was r a i s e d t o a f i n a l t r e a t m e n t o f 3600 mg. No p r i o r d o s e s , up to 2000 mg c a u s e d any r e s p o n s e , b u t t h e f i n a l a d m i n i s t r a t i o n c a u s e d extreme q u iescen ce, and f i b r i l l a t i o n o f m uscles in th e fa c e and hands, followed by deep stu p o r and r e f le x f a i l u r e . The p a t i e n t reco v ered from th e 2 ,4 -D w i t h i n 48 h o u r s and d i e d o f t h e f u n g u s d i s e a s e a b o u t two weeks later. General T o x icity to Laboratory Animals One o f t h e e a r l i e s t p u b l i s h e d s t u d i e s o f 2 ,4 -D t o x i c i t y was by Eucher i n 1948. She fo u n d an a c u t e LD50 o f 280 mg/kg i n m ic e , and fo u n d th a t s in g le doses o f 150-200 mg/kg would produce a m yotonia p e r s i s t i n g s e v e r a l h o u rs . The an im a ls rem ained awake and a l e r t , b u t when moved e x h ib ite d gross in c o o rd in a tio n . They were capable of working out of the syndrome w ith c o n tin u e d e x e r c i s e , b u t i f a llo w e d t o rem a in q u i e t t h e myo to n ia would r e c u r . The chem ical a lso caused d ia rr h e a in mice a t th e higher doses, and g a s tr o in te s tin a l and upper r e s p ir a to r y i r r i t a t i o n in do g s. Mice w ere a b l e t o t o l e r a t e d a i l y d o s e s o f 1 /2 LD50 f o r t h r e e m onths. H i l l and C a r l i s l e (1947) e s t a b l i s h e d LD50 v a l u e s f o r s e v e r a l s p e c i e s : m ic e , 375 m g/kg; r a t s , 666 m g/kg; r a b b i t s 800 m g/kg; and g u i n e a p ig s , 1000 mg/kg. They a ls o t r e a t e d monkeys and were a b le to g iv e up to 214 mg/kg w ith o u t s e v e r e r e s i d u a l e f f e c t . Twice t h a t dose caused v o m itin g , i n c o o r d i n a t i o n , and l o s s o f m uscle t o n e . Rowe and Hymas (1954) sum m arized l e t h a l i t y d a ta on 2,4-D and f i v e s a l t s o r e s t e r s ; in g e n e ra l th e l e t h a l doses were very high, w ith th e exception of a few dogs given 2,4-D i t s e l f , f o r w hich t h e LD50 was e s t i m a t e d a t 100 m g/kg. The l a t t e r d a t a was a p p a r e n t l y d e r i v e d from t h e o b s e r v a t i o n s o f D r i l l and H i r a t z k a ( 1 9 5 3 ) . Hansen e t a l . (1971) conducted a tw o-year feed in g s tu d y on r a t s , a t dosages o f 5 t o 1250 ppm 2,4-D i n t h e d i e t , w i t h o u t a p p r e c i a b l e c h a n g e i n g r o w th , hem atologic v a lu e s, o r organ w eight. In the same s tu d y dogs were main t a i n e d f o r two y e a r s w i t h l i t t l e e f f e c t a t d o s e s up t o 500 ppm 2 ,4 - D . D r i l l and H ir a tz k a (1953) found t h a t 20 mg/kg d a i l y was f a t a l i n 3 o f 4 dogs w ith in 49 days o f a 90-day s tu d y . Grazing species o f anim als might be expected to s u f f e r exposures in the f i e l d from eatin g tr e a te d v e g e ta tio n . Palmer and R ad eleff (1964) evaluated a s e rie s of h erb ic id es a t high doses in lim ite d numbers of r u m i n a n ts . One sh eep t o l e r a t e d 481 d a i l y d o s e s o f 100 mg 2 ,4 -D a l k a n o l a mine s a l t , an o th er was u n a ffe c te d by th e same tre a tm e n t w ith 2,4-D propy le n e g ly c o l b u ty l e th y l e s t e r . The f i r s t compound was l e t h a l a f t e r 7-500 mg/kg d o s e s ; t h e s e c o n d was l e t h a l a f t e r 9 -2 5 0 mg d o s e s . A b o v in e t o l e r a t e d 112 d o s e s o f 50 mg o f t h e a l k a n o la m in e s a l t , b u t a n o t h e r d e veloped d ig e s t iv e problem s a f t e r 80 doses o f 100 mg/kg. Palm er (1963) a d m i n i s t e r e d d a i l y d o s e s o f 5 0 -250 mg 2 ,4 - D /k g as t h e a l k a n o l a m i n e s a l t to a group o f s t e e r s . The s t e e r given 100 mg/kg dev elo p ed ru m in al atony a f t e r 86 d a y s , p re s u m a b ly as a r e s u l t o f t h e h e r b i c i d e . A nim als g iv e n 200 and 250 mg/kg became i n t o x i c a t e d i n 34 and 15 d a y s , r e s p e c t i v e l y , w ith d e s s i c a t e d mucous membranes and ten d en cy to n o s e b le e d when r e s t r a i n e d . 36 DOW 0 0 11;. 8 2 3 Rum inants on s p r a y e d p a s t u r e o r cows g iv e n S .5 gm d a i l y d i d n o t show c lin ic a l evidence o f to x ic ity , nor did production d eclin e. 2,4-D appeared in serum o f a cow f e d S.S gm/day f o r 106 d a y s , b u t none was p a s s e d in milk (M itchell et a l . , 1946). Chickens and o th e r fowl also appear r e l a t i v e l y in s e n s itiv e . Bjorn and N o rth e n (1948) fo u n d no im p a irm e n t i n w e ig h t g a i n a t d o s e s up t o 28 mg 2,4-D alkanolam ine/kg th re e tim es weekly f o r fo u r weeks; a t 280 mg/kg t h e r e was a marked d e c r e a s e i n g a i n . S i n g l e d o se s o f 765 mg/kg w ere l e t h a l , 380 mg/kg was n o t. 2,4-D d id n o t cause d e c re a se in growth r a t e a t dose r a t e s up t o 1000 ppm i n t h e d i e t , b u t a t 7500 ppm g ro w th e s s e n t i a l l y s to p p e d (W h iteh ead and P e t t i g e w , 1 9 7 2 ) . The a c u t e LD50 was estim ated a t 900 mg/kg. Whitehead (1973) te s t e d d i e ta r y 2,4-D at concen t r a t i o n s up t o 100 mg/kg d i e t , and fo u n d t h a t a t d i e t a r y l e v e l s o f 10 mg/kg d i e t o r g r e a t e r , growth r a t e was d e p r e s s e d . Because food c o n v e r sio n e f f i c i e n c y was n o t a f f e c t e d , i t was s u g g e s te d t h a t p a l a t a b i l i t y was affected re s u ltin g in decreased consumption. Chickens w ill d iscrim in ate ag ain st 2,4-D co n tam inated food (Whitehead and P e ttig re w , 1972). The U.S. F ish and W ild lif e s e rv ic e te s t e d a number o f chem icals by fe e d in g to v a r io u s game b i r d s p e c ie s in th e e a r l y 1960s. 2,4-D a ce tam id e g iv e n to young q u a i l a t 2500 ppm c a u s e d 72% m o r t a l i t y i n 12 d a y s , t h e b u t o x y e th a n o l e s t e r a t 5000 ppm c a u s e d 28% m o r t a l i t y i n 155 d a y s , and 2S00 ppm d im e th y la m in e s a l t c a u s e d 12% d e a t h i n 158 d a y s . I n o l d e r b i r d s 2500 ppm was a l m o s t w i t h o u t e f f e c t a f t e r 50 d a y s , and a f t e r 111 d a y s o f 1000 ppm f e e d i n g . T e s t s on C o t u m i x i n d i c a t e d a s i m i l a r t o l e r a n c e ( S t i c k e l , 1 9 6 4 ). More r e c e n t l y , H i l l e t a l . (1975) h a v e shown t h e LC50 f o r 2 ,4 -D acetamide in th e d i e t o f bob w hite, c o tu m ix , p h easan t, and m allard to be i n e x c e s s o f 5000 ppm. The b u to x y e t h a n o l e s t e r and DMA s a l t h a v e a s im ila r low t o x i c i t y . A number of o th e r b io lo g ic a l e ffe c ts o f 2,4-D have been found ex p erim en ta lly , u s u a lly a t very high doses. Dybing and Kolberg (1967) sug gested th a t 2,4-D was a c tiv e ly reabsorbed, and i f so would c o m p etitiv ely decrease the clearance of p-aminohippurate w ithout in te rfe rin g with crea t i n i n e c l e a r a n c e . S t u d i e s w i t h r a b b i t s g iv e n 100 mg p r im in g d o s e s , th e n 2 mg 2 ,4 -D /m in p r o d u c e d d e c r e a s e d PAH c l e a r a n c e a t d o s e s o f 6 2 -1 1 5 m g/kg. Chang e t a l . (1974) s tu d ie d a number o f e f f e c t s on r a t l i v e r fo llo w in g tre a tm e n t w ith 2-5 gm/kg 2,4-D o v e r 4-7 w eeks. Glycogen c o n te n t was 50100% h i g h e r , and l i v e r n u c l e i s y n t h e s i z e d RN'A more a c t i v e l y i n v i t r o t h a n d id c o n t r o l s . DNA c o n t e n t p e r l i v e r was d e c r e a s e d . I n t h i s w ork, 2 ,4 -D was compared w i t h 2 , 4 , 5 - T ; t h e l a t t e r compound c a u s e d i n c r e a s e d l i v e r weight and p ro te in accum ulation, w hile 2,4-D decreased li v e r w eight and le s s e n e d RNA c o n t e n t , w ith no p r o t e i n a c c u m u l a t i o n . S p e c u l a t i o n i s j u s t i f i e d t h a t TCDD i n 2 , 4 , 5 - T was r e s p o n s i b l e f o r t h e d i f f e r e n c e . A d i e t a r y supplem ent o f 60 ppm 2,4-D to lambs had no e f f e c t on rumen l i q u o r o r sh eep or serum p r o t e i n s . Weight g a in was s l i g h t l y d e c re a s e d o v er a 12 week feeding p e rio d (Abou-Akkada e t a l . , 1975). The same au th o rs (1973) had p rev io u sly found th a t 2,4-D did not a l t e r rum inal m icrobial fu n c tio n . At f a i r l y h ig h d o s e s (80 m g /k g /d ay , f o r s e v e n d a y s) 2,4-D i n c r e a s e s 131l in tak e by th e th y ro id . The e f f e c t only occurs in a norm ally fu n c tio n in g th y ro id g lan d ; i t was not seen a f t e r hypophysectoray o r io d in e d e p le tio n (Florsheim and V elco ff, 1962) . The e f f e c t is a p p are n tly due to lowered thyroxine binding to serum p ro te in (Florsheim e t a l ., 1963) b u t w hether th is is secondary to 2,4-D binding or is a s p e c ific pharm acologic e ffe c t is not known. 37 16460 cv GO T h e r e h a s b een l i m i t e d i n v i t r o s t u d y o f 2 ,4 - D . Weiss and B e c k e r t found stim u lated m ito tic a c t iv it y and in c re a se d chrom atin a f t e r c u ltu re d monkey k id n e y c e l l s , G i r a r d i h e a r t c e l l s , and t r o u t gonad c e l l s w ere exposed t o 10 o r 50 ppm 2,4-D f o r 72 h o u r s . 2 ,4-D i n h i b i t s m e v a lo n a te incorporation into non-saponifiable lip id s o f liv e r, but only at concen t r a t i o n s in e x c e s s o f 1 mM (O lson e t a l . , 1 9 7 4 ) . O x i d a t i v e p h o s p h o r y la tio n in r a t m itochondria appears to be s e n s itiv e to co n ce n tra tio n s as low as 10- ^ M; a t 10- 3 m r e s p i r a t i o n was norm al b u t P /0 d e c r e a s e d to 5: 20% o f norm al (B rody, 1 9 5 2 ). I n L929 c e l l s i n m o n o la y e r c u l t u r e , 2 ,4 -D O causes t r i g l y c e r i d e accum ulation when p r e s e n t a t a c o n c e n tr a tio n o f 500 a Ug/ml (K olberg e t a l . , 1972) and i n h i b i t s a l l grow th a t 50 yg/m l (K o lb e rg et a l ., 1971). 2,4-D at high doses is used also as a chemical inducer o f e x p e ri mental myotonia, as a model o f the co n g en ital d is e a s e (Iy e r e t a l . , 1977; E yzaguirre e t a l . , 1948). The a c tio n i s a p p a re n tly due to an in c r e a s e in membrane r e s i s t a n c e and reduced c h l o r i d e t r a n s p o r t . E l e c t r i c a l a c t i v i t y o f t h e b r a i n was fo u n d t o be r e v e r s i b l y i n h i b i t e d by d o s e s o f 200 mg 2,4-D /kg, in r a t s (Desi e t a l . , 1962). The chem ical a ls o produces a primary myopathy th a t is usefu l in stu d y of th e fam ily o f d is e a s e s which includes w hite muscle d ise a se in lambs (Heene, 1969). These e f f e c t s a re not of toxicologic sig n ifican ce except in cases of massive acute in tak e r e s u ltin g in myoneural symptoms. E ffe c t o f 2,4-D on R eproductive F unction As w i t h 2 , 4 , 5 - T , t h e p o s s i b i l i t y t h a t 2 ,4 -D h a s t e r a t o g e n i c p o t e n t i a l was f i r s t s tu d ie d by th e B io n e tic s R esearch L a b o ra to rie s stu d y (3 io n e tic s Research L a b o ra to rie s , I n c ., 1970). The d a ta were s u g g e s tiv e t h a t i n c id e n c e o f f a i l e d low er jaw f o rm a tio n was somewhat g r e a t e r th a n t h a t r e s u l t i n g from t h e DMSO c a r r i e r . S chw etz e t a l . (1971) m e a s u r e d te r a to g e n ic and f e t o t o x i c e f f e c t s o f 2,4-D and two e s t e r s on r a t s . The h ig h e r d a i l y d o s e s (75 mg 2 , 4 - D /k g ; 75 mg p r o p y l e n e g l y c o l b u t y l e s t e r o f 2 ,4 - D /k g ; 8 7 .5 mg i s o o c t y l e s t e r o f 2 , 4 - D / k g , e a c h j u s t below t h e maternal to x ic dose) caused decreased f e t a l w eight, subcutaneous edema, delayed bone o s s i f i c a t i o n and wavy r i b s . Most o f th e s e changes a r e fe to to x ic r a th e r th a n te r a to g e n ic . The l a s t two a re developm ental e f f e c t s b u t h av e no e f f e c t on s u r v i v a b i l i t y . No t e r a t o g e n i c r e s p o n s e s w ere found a t any dose. T h ere i s some d i f f e r e n c e in d e f i n i t i o n o f t e r a t o g e n i c r e s p o n s e among authors in th e f i e l d . A v a r ie ty o f s k e l e t a l d e f e c ts t h a t do n o t i n t e r f e r e with p o s tn a ta l s u rv iv a l were found by Khera and McKinley (1972) ; most e f fe c ts observed were wavy r ib s or fu sed sternum . The in c re a s e d in c id e n c e of t h e s e changes was e v i d e n t a t d o ses a s low as 25 m g /k g /d a y . 2 ,4 -D t e r a t o g e n e s i s was s t u d i e d by Bage e t a l . (1975) b u t o n ly in p r e s e n c e o f 2 ,4 ,5 -T . The m ix tu re caused some t e r a t o g e n e s i s , b u t was le s s e f f e c t i v e th a n 2 , 4 , 5 - T a l o n e , so t h e im pact o f 2 ,4 -D i n t h e s y ste m was d i f f i c u l t to evaluate. Hamsters are su b ject to a te ra to g e n ic e ffe c t o f high doses o f 2,4-D. C ollins and W illiams (1971) found th a t 2,4-D from th re e d if f e r e n t sources 38 c C c a u s e d a low i n c i d e n c e o f a n o m a l i e s , u s u a l l y f u s e d r i b s , a t d o s e s o f 100 rog/kg/day th ro u g h days 6-10 o f g e s t a t i o n . T here was no s a t i s f a c t o r y dose re s p o n s e r e l a t i o n s h i p . D i e t a r y 2 ,4 -D a t 500 and 1000 ppm d id n o t a lte r reproductive function in a three-generation, 6 l i t t e r study with r a t s . P ercentage o f pups s u rv iv in g to weaning and w eanling w eight was d e c re a se d a t 1500 ppm, however (Hansen e t a l . , 1971). Sheep are ap parently not su b je c t to 2,4-D induced te ra to g e n e s is . Binns and J o h n s o n (1970) a d m i n i s t e r e d 2 grams d a i l y f o r 30, 6 0 , and 90 days follow ing breeding and caused no m alform ation. There were presum a b ly 6 sheep p e r group b u t th e number i n t h i s s p e c i f i c e x p e rim e n t was not stated. Eggs are p e c u l ia r l y v u ln e ra b le to exposure to h e r b ic id e s and s e v e ra l studies have been d ire c te d toward defin in g the hazard of 2,4-D to chicken o r game b i r d e g g s . An i n j e c t e d d o s e o f 10 mg 2 ,4 - D /e g g c a u s e d 50% mor t a l i t y , 5 mg/egg r e s u l t e d i n 30% l o s s , and a t 0 . 5 mg/egg 90% o f t h e eggs h a t c h e d . No d e f o r m i t i e s o c c u r r e d a t any d o s e (D u n ach ie and F l e t c h e r , 1967). A l a t e r stu d y by Dunachie and F le tc h e r (1970) confirm ed th e se findings fo r 2,4-D and 2,4-DB (2 ,4 -d ich lo ro p h en o x y b u ty ric a c id ). In c o n tra s t, L utz-O stertag and Lutz (1970) sprayed pheasant and grouse eggs w ith 2,4-D a t r a te s commonly used in th e f i e l d and caused embryonic m o rta lity and t e r a t a . Somers e t a l . (1973, 1974) were n o t ab le to support these fin d in g s a f te r sp ray in g m ixtures of 2,4-D and picloram a t usual f i e l d r a te s (2.8 kg/ha) and 2,4-D and 2 ,4 ,5 -T on hen eggs (1973) and p h e a s a n t eggs (1974) a t 10 t i m e s f i e l d c o n c e n t r a t i o n s ( 1 1 .2 k g / h a ) . They found no "ad v erse e f f e c t on h atc h in g s u c c e s s , in c id e n c e o f malformed embryo o r subseq u en t c h ic k m o r t a l i t y " . Kopischke (1972) a l s o found no e f f e c t on pheasant eggs sprayed a t f i e l d c o n c e n tr a tio n s , b ut found t h a t d ie sel fuel as a c a r r ie r blocked hatching com pletely. Dipping hen eggs i n 1% 2 ,4 -D f o r 10 s e c o n d s , t h e n c o n t i n u i n g i n c u b a t i o n was s i m i l a r l y in e ffe c tiv e (Gyrd-Hansen and D algaard-M ikkelsen, 1974). An i n t e r e s t i n g o b s e r v a t i o n o f 2 ,4 -D d i s t r i b u t i o n i n mouse f e t u s e s has been made by L in d q u is t and U llb e rg (1 9 7 1 ). L ab eled 2,4-D g iv e n l a t e in g e s t a t i o n accu m u lated e a r l y in th e y o lk s a c , p a s s e d on to th e f e t u s and was a lm o s t c o m p le te ly e l im i n a te d by 24 h o u rs a f t e r a d m i n i s t r a t i o n . D i s t r i b u t i o n among t i s s u e s was n o n - s e l e c t i v e , and c o n c e n t r a t i o n s te n d e d to p a r a l l e l th o s e o f th e dam, p e rh a p s e x p l a in in g in p a r t th e la c k o f teratogenic effect. "O 'O GOiO. c/r Carcinogenic and Mutagenic P o te n tia l o f 2,4-D Innes e t a l . (1969) screen ed 120 compounds f o r tu m o rig en ic p r o p e r ti e s in mice. 2,4-D and se v e ra l o f i t s e s te r s were in clu d ed ; none caused in creased tumor in cid en ce. Hansen e t a l. (1971) c a r r ie d out a c a rc in o genesis study in r a ts and concluded th a t although tumors were found, the observed incidence did not support a finding th a t 2,4-D is carcin o g e n ic . T here seemed t o be some i n c o n s i s t e n c i e s in i n t e r p r e t a t i o n t h a t may h av e c o n f u s e d t h e i s s u e . A p p a r e n t l y ho o t h e r e v a l u a t i o n s o f c a n c e r p o te n tia l o f 2,4-D have been made. A number of m utagenic scre e n s have included 2,4-D, however. Jenssen and Renberg (1976) found th a t 2,4-D 16462 39 would n o t induce in c re a s e d m icro n u clei in mouse bone marrow e ry th ro c y te , but th e compound d id s l i g h t l y depress m ito tic a c t i v i t y . S ex -lin k ed l e t h a l i t y assay o f 2,4-D in m ale-D rosophila was a ls o n e g a tiv e f o r m utagenic a c t i v i t y (Vogel and C handler, 1974). S ty le s (1973) t r e a t e d r a t s w ith 2,4-D , then used serum from the anim als in a h o st m ediated assay w ith h is tid in e - r e q u ir in g S. Typhimurium m u t a n t s . No e f f e c t o f 2,4-D was e v i d e n t . In a s c r e e n o f 110 compounds w i t h t h e "Ames t e s t , " u s i n g e i g h t h i s t i d i n e - r e q u i r i n g m u ta n t s t r a i n s , Anderson e t a l . (1972) was unable to d e t e c t m utagenic a c t i v i t y by 2,4-D . Absorption, Metabolism, Tissue D istrib u tio n , and E xcretion o f 2,4-D 2,4-D was absorbed r a p id l y from th e lung o f r a t s i n which the h e r b i c i d e was i n j e c t e d i n t r a t r a c h e a l l y as 0 .1 ml o f 0 .0 1 - 1 0 mM s o l u t i o n . The anim als were s a c r i f i c e d from one h a l f to 120 m inutes l a t e r . The h a l f time f o r 2,4-D a b s o r p ti o n was 1.4 m in u te s , and c o n c e n t r a t i o n had l i t t l e i n f l u e n c e on t h e r a t e (B u rto n e t a l . , 1 9 7 4 ). When i n g e s t e d , 2 ,4 -D i s absorbed p rim a rily by th e p o r ta l c ir c u la tio n ; th e lym phatic d rain ag e accounts fo r very l i t t l e 2,4-D absorption, as might be expected from the lim ited f a t s o lu b il ity of the h e rb ic id e (S ieb er, 1976). Blood concen tration of from rad io lab eled 2,4-D adm inistered o r a lly to sheep has been shown to r i s e v e ry r a p i d l y (C la rk e t a l . , 1964). The r a t e o f ab so rp tio n o f 2,4-D from th e rumen i s not known; th e immediate e le v a tio n in b lo o d 1 4C s u g g e s ts t h a t a t l e a s t some m a t e r i a l was removed d i r e c t l y from th e rumen. However, Gutenmann e t a l . (1963) f e d 5 ppm 2 ,4 -D t o c a t t l e and found t h a t th e c o n c e n tra tio n in th e rumen c o n te n ts d ecreased from 3 .5 t o 0 .5 ppm o v e r a 24 h o u r p e r i o d , w hich does n o t s u g g e s t r a p i d a b so rp tio n , but r a t h e r d il u tio n w ith p assag e o f rumen c o n te n t. The chemical did not disappear in an a r t i f i c i a l rumen. K o h li e t a l . (1974) g av e 5 mg o r a l l y t o s i x human v o l u n t e e r s and found peak plasma co n cen tratio n s by seven hours; the average plasma c o n c e n tr a tio n one week l a t e r was about 10% o f th e peak c o n c e n t r a t i o n . H a lf tim e f o r e x c r e t i o n was c a l c u l a t e d a t 33 h o u r s . C l i n i c a l o b s e r v a t i o n s o f human p a t i e n t s who had e x t e n s i v e s k i n c o n tact w ith 2,4-D showed sy stem ic to x ic resp o n ses w ith in a few hours (Goldstein et a l . , 1959; Todd, 1962), in d ic a tin g ready absorption through the skin. A p p l i c a t i o n o f 2 ,4 -D a s t h e DMA s a l t , i s o o c t y l e s t e r and b u t y l e s t e r t o th e body s u r f a c e o f r a b b i t s was r e l a t i v e l y i n e f f e c t i v e (Kay e t a l . , 1 9 6 5 ). T r e a tm e n t was w i t h 15 ml o f aqueous o r o i l s o l u t i o n a p p l i e d on a 4 x 3 gauze p a t c h , c o v e re d t i g h t l y w ith p l a s t i c f i l m . C o n ta c t was seven h ours d a i l y , f iv e days a week f o r th r e e weeks; c o n c e n tr a tio n s o f 2,4-D w ere 0 .6 2 6 and 3.13% a c i d e q u i v a l e n t . Some a n i m a ls w ere t r e a t e d on ab rad e d s k i n . A few a n im als d ie d d u r in g t h e e x p e rim e n t b u t th e pathology and symptoms were not ty p ic a l o f 2,4-D p o iso n in g ; most o f th e f a t a l i t i e s were among t h e a n im a ls w ith s k i n damage. No n e u r o l o g i c a l lesions were found, nor were any o th er observed param eters changed. The o n ly le s io n s o b se rv e d were a t th e s i t e o f a p p l i c a t i o n . 16463 40 Z9,,JTOO/Aa e. There seems to be g e n e ra l ag reem en t, o th e r th a n th e r e p o r t by K ohli et a l . (1974), th a t s i g n i f i c a n t amounts o f 2,4-D do not rem ain in an animal f o r much more th a n one o r two d a y s . Z e i l i n s k i and F i s h b e i n (1967) com pared whole body re s id e n c e tim es o f two e s t e r s o f 2,4-D and th e 2,4-D acid a f t e r su b cu tan e o u s i n j e c t i o n o f 100 mg/kg t o m ice. Of th e b u ty l e s t e r , 95% was gone a f t e r 6 h o u r s , and i n a n i m a ls t h a t had b e e n p r e t r e a t e d w ith f i v e s i m i l a r d a i l y d o se s, th e r a t e o f d is a p p e a ra n c e was f u r th e r enhanced. The is o o c ty l e s t e r was c o n s id e ra b ly slow er to d is a p p e a r, with a h a lf tim e o f somewhat le s s than fo u r h o u rs, and the ac id i t s e l f was r e t a i n e d s l i g h t l y l o n g e r . The a u t h o r s d i d n o t s p e c i f i c a l l y n o t e w hether h y d r o ly s is o f e s t e r s to 2,4-D a c id was i d e n t i f i e d as p a r t o f th e m etabolic p ro c e ss, but th e method appeared to account fo r th e d is a p p e a r ance o f a l l form s o f t h e compound. As a c o m p a r is o n , t h e d i s a p p e a r a n c e h a lf- tim e o f 2 ,4 ,5 - T a c id was on th e o r d e r o f 20 h o u rs . Khanna and Fang (1966) ad m in iste re d 2 ,4 -D -* 4C to male and fem ale r a t s at very high (80 m g /rat) and r e l a t i v e l y low (1 m g /ra t) doses and found tiss u e re sid u e s to be g re a te s t at about 6 h o u rs, and alm ost u n d etecta b le by 50 h o u r s a f t e r t r e a t m e n t . E x tr e m e ly h ig h c o n c e n t r a t i o n s w ere fo u n d in th e stomach b ut th e r e was no in fo rm a tio n about s e p a r a tio n o f stomach c o n t e n t from t h e t i s s u e p r o p e r . No l a b e l was fo u n d i n r e s p i r a t o r y g a s e s . A very sm all f r a c t i o n o f u r in a ry la b e l was found to be an u n id e n tif ie d m etabolite, but the d ata in d icate th a t almost a l l 2,4-D is excreted w ith out change by r a t s . Although 2,4-D binds re v e rsib ly to bovine serum albumin (Kolberg et a l ., 1973) and th e re fo re probably to o th e r plasm a p r o te i n s , th e re seem to be no s p e c i f i c t i s s u e binding s i t e s as a r e found in p l a n t s . Morre (1974) has examined f i s h muscle and r a t l i v e r a f t e r exposure to 2,4-D , seeking s p e c ific binding s ite s and has concluded th a t none e x is t. 2,4-D a p p a r e n t l y does n o t move i n t o m ilk o f l a c t a t i n g a n im a ls i n s ig n ific a n t amounts, w hether tre a te d by d ir e c t a d m in istra tio n of th e h e r b ic id e o r by g ra z in g on t r e a t e d land (B jerke e t a l . , 1972; Gutenmann et a l . , 1963; Bache e t a l . , 1964; S t. John e t a l . , 1964; Klingman e t a l . , 1966). Under f o r c i n g c o n d i t i o n s , h o w e v e r, (1000 ppm 2 ,4 -D i n d i e t ) , t h e le v e l o f 2,4-D r e s i d u e in cows m ilk was f o r c e d up to 0 .0 6 ppm, and w ere s t i l l barely d ete c ta b le seven days la te r . A su b stan tial capacity fo r conjugation of 2,4,5-T has been des c r i b e d e l s e w h e r e i n t h i s r e p o r t (Nony e t a l . , 1 9 7 6 ) . Grunow and Bohme (1974) have found th a t th e a b i l i t y to c o n ju g a te 2,4-D w ith g ly c in e and tau rin e e x ists but is considerably less than th a t fo r 2,4,5-T . In dog fis h and flo u n d e r, however, a major f r a c tio n of u rin a ry 2,4-D is ex cre ted as the ta u r in e co njugate (James and Bend, 1976), and in th re e o f th e fo u r d o g fis h s t u d i e d 10-20% o f t h e l a b e l was found i n b i l e a f t e r 48 h o u r s . In s p i t e o f th e ra p id lo s s o f 2,4-D by f i s h , p e r s i s t e n c e o f th e h e r b i c i d e o r i t s p r o d u c t s may be p r o l o n g e d . T r e a tm e n t o f pond weeds w ith up to 9 k g /h a u s u a l l y l e f t no d e t e c t a b l e r e s i d u e s in f i s h by 28 days p o s t-a p p lic a tio n , even a t the h ig h e st r a te s (S chultz and Harman, 1974). S c h u l t z (1973) h a s f o u n d , h o w ev er, t h a t some u n i d e n t i f i e d p r o d u c t s may be p re s e n t in f i s h 2-3 months a f t e r tr e a tm e n t. . 16464 41 DOW' ' O O li The r a p id appearance o f i n t a c t 2,4-D in u rin e is a p p a re n tly m ediated by a presum ably a c t i v e and s a t u r a b l e t u b u l a r e x c r e t i o n mechanism (Erne and S p erb er, 1974). The e x te n siv e e x c re tio n o f 2,4-D in u rin e has been noted in ste e rs (Lisk e t a l . , 1965), sheep (Clark e t a l ., 1964), ra ts (S ie b e r, 1976), and humans (K ohli e t a l . , 1974), and a p p a r e n tly i s r a p id enough to remove a l l but m assive exposures b e fo re s i g n i f i c a n t damage can occur. Behavior o f 2,4-D in th e Environment and E f f e c ts on Submammalian S p ecies T his r e p o r t i s p r im a r ily concerned w ith e f f e c t s o f h e r b ic id e s on n o n -p lan t s p e c ie s, b u t a c u rso ry q u a l i t a t i v e survey o f l i t e r a t u r e on f i e l d p e r s i s t e n c e i s u s e f u l i n ju d g in g how lo n g a g iv e n amount o f h e r b i c id e may rem ain a v a i l a b l e . Water concentrations are a major concern, in re la tio n both to aqua t i c s p e c ie s and to w a te r s u p p lie s f o r th e human p o p u la tio n . 2,4-D ap plied in a watershed area appears in streams to a very lim ited extent (White e t a l . , 1976; Krammes and W i l l e t t s , 1 9 6 4 ). Movement i n t o s u b surface w ater flows also appears n e g lig ib le (White et a l . , 1976). In a e r a t e d la k e w a te r 2 ,4 -D p e r s i s t s up t o 120 d a y s , b u t i n l a k e mud 2 ,4 -D hydrolyzes very ra p id ly , because o f m icro b ial a c t i v i t y (Aly and F aust, 1964). H alf-tim e o f 2,4-D d isap p earan ce seems o v e r a ll to be much le s s than two weeks in aqueous system s. F or some a p p l i c a t i o n s on w a te r , r e l a t i v e l y m assive a p p l i c a t i o n s o f up t o 40 l b s . p e r a c r e a r e n e c e s s a r y . Two r e s e r v o i r s on t h e T e n n e s s e e R iver were t r e a t e d a t 20 and 40 l b s / a c r e depending on th e n a t u r e o f th e water m i l f o i l i n f e s t a t i o n . The tre a tm e n ts did n o t a f f e c t f i s h o r o th e r fauna, and had l i t t l e adverse e f f e c t on most o th e r p la n ts . Plankton did re ta in s ig n ific a n t, amounts of h e rb ic id e over extended p e rio d s , and 2,4-D was d e t e c t a b l e on o c c a s i o n i n f i n i s h e d d o m e s tic w a t e r from t h e r e s e r voirs (W ojtalik et a l ., 1971). In s o ils , 2,4-D has been found to have a h a lf-tim e of 4-5 days by Altora and S t r i t z k e ( 1 9 7 3 ) . W hite e t a l . (1976) fo u n d t h a t 95% o f 2 ,4 -D in t h e t o p 0 .5 cm o f s o i l would d i s a p p e a r i n 7 d a y s . However, an i n v itr o s tu d y by A lexander and Aleem (1961) in d ic a te d th a t 2,4-D would be d e t e c t a b l e f o r as much as 94 days i n one t y p e o f s o i l and o n l y 23 days in a n o t h e r . The p r e p a r a t i o n i n c l u d e d 100 ml o f n u t r i e n t medium and 4 mis o f s o i l as an inoculum ; w hether such a system r e p r e s e n ts a c tu a l b re a k down c o n d i t i o n s i n s o i l i s p o s s i b l y q u e s t i o n a b l e . S u f f i c i e n t m a t e r i a l to cause d etecta b le p h y to to x ic ity p e r s is ts fo r about a month, according to M ullison (1972). In areas where 2,4-D is used annually, the break down may be more r a p i d i n t h e l a t e r y e a r s t h a n f o l l o w i n g t h e f i r s t treatm en t (Hurle and Rademacher, 1970). Wiersma e t a l . (1972) re p o rte d a t o t a l o f 28 w hich h ad b e e n t r e a t e d w i t h 2 ,4 -D w ere found t o h av e from 0.01 t o 0 . 0 3 ppm 2 ,4 -D p r e s e n t . I t seems u n l i k e l y t h a t 2 ,4 -D would p e r s i s t in e i t h e r w ater o r s o i l f o r more th an a month. 2,4-D has been used ex ten siv ely in co n tro l of aquatic weeds, p a r tic u la r ly water h y acin th and w ater m ilf o il, and has probably been sub je c te d to more stu d y o f a q u a tic t o x i c i t y than any o th e r h e r b ic id e . A 16405 42 DOW OOll 829 number o f sh o rt term screens have been conducted w ith f is h . A la b a ste r (1969) r e p o r te d on 164 compounds, i n c l u d i n g 2 , 4-D .sodium s a l t , which was fo u n d t o have a 24 h o u r LC50 o f 1160 ppra f o r h a r l e q u i n f i s h , and t h e b u t o x y e t h y l e s t e r o f 2 , 4-D which was much more t o x i c w i t h a 24 h o u r LD50 o f 1 ppm, f o r th e same s p e c ie s . The 24-96 h r LC5Q o f PGBE e s t e r s o f 2 , 4-D f o r ra in b o w t r o u t w ere s l i g h t l y above 1 ppm (Cope, 1 9 6 4 ). F or m u l l e t and k i l l i f i s h t h e LC50 (24 h r ) was 5 ppm f o r b o th t h e PGBE and b u t o x y e t h a n o l e s t e r s . The p a r e n t a c i d was i n e f f e c t i v e a t SO ppm ( B u t l e r , 1963) . O y s t e r s h e l l grow th was 50% i n h i b i t e d by 3 .7 5 ppm b u t o x y e t h a n o l e s t e r , b u t th e a c i d and d im eth y lam in e s a l t were n o t e f f e c t i v e a t 2 ppm ( B u t l e r , 1 9 6 3 ). E x p o su re t o t h e DMA s a l t f o r 24 h r s a t 2 ppm c a u s e d no mor t a l i t y i n s h r im p ; 48 h o u r e x p o s u r e c a u s e d 10% l e t h a l i t y . The b u t o x y e t h a n o l and PGBE e s t e r s c a u s e d no m o r t a l i t y t o s h r im p a t 1 ppm x 48 h o u r . The e t h y l h e x y l e s t e r o f 2 , 4-D c a u s e d 38% d e c r e a s e i n o y s t e r s h e l l g ro w th a t 5 ppm o v e r 96 h r s ( B u t l e r , 1 9 6 5 ) . The c o n s i d e r a b l e d i f f e r e n c e s in 2 . 4 - D e f f e c t among v a r io u s s p e c ie s were i l l u s t r a t e d by S a n d e rs (1970a) 2 . 4 - D a c i d h a s a 48 h o u r m edian r e s p o n s e c o n c e n t r a t i o n (TL50) o f 100 mg/L (100 ppm) f o r D aphnia and 3 .2 mg/L f o r s c u d . (C ro sb y and T u c k e r (1966) fo u n d th e same v a l u e f o r D a p h n ia .) The PGBE e s t e r TL50 v a r i e s from 0 . 1 f o r D ap h n ia t o 2 .6 f o r scu d t o more t h a n 100 ppm f o r c r a y f i s h . The DMA s a l t TL50 i s more th a n 100 ppm i n b l u e g i l l b u t TL50 f o r t h e b u ty l e t h e r e s t e r i n b l u e g i l l i s 1 . 1 ppm, and 100 ppm f o r c r a y f i s h . S t o n e f l y n a i a d s a r e l e s s s e n s i t i v e t o 2 , 4-D i t s e l f (LC50 x 96 h r = 15 ppra) th a n t h e b u to x y e t h a n o l e s t e r (LC50 x 96 h r = 1.6 ppm) ( S a n d e r s and C ope, 1 9 6 8 ). S a n d e r s (1970b) h a s a l s o r e p o r t e d a 96 h r TL50 f o r 2 , 4-D amine o f 100 ppm f o r t a d p o l e s . The l e t h a l e f f e c t on ta d p o le s i s a p p a re n tly lim ite d , b u t B uslovich and Borushko (1976) found t h a t 2 , 4-D sodium s a l t would i n h i b i t metamor phosis o f Rana teraporaria ta d p o le s , and blocked th y ro id in s tim u la tio n o f th e p r o c e s s . The DMA s a l t was e f f e c t i v e a t 2 ppm. The a u t h o r s s p e c u l a t e t h a t 2 , 4-D a n ta g o n iz e s t h y r o i d hormone. Few s t u d i e s o f t r u l y c h r o n i c e x p o s u r e o f f i s h t o 2 , 4-D h a v e been made. Mount and S te p h a n (1967) com pared t h e 96 h o u r LC50 f o r 2 , 4-D b u t o x y e t h a n o l e s t e r ( 5 .6 ppm) w i t h v a r i o u s 10 month e x p o s u r e s , and fo u n d t h a t l / 1 9 t h o f t h e LC5Q, o r 0 . 3 ppm c o u ld be t o l e r a t e d by f a t h e a d minnows w ith o u t e f f e c t on grow th o r r e p r o d u c t i o n . Eggs w ere much more s e n s i t i v e th a n a d u l t s o v er 48 h o u r e x p o s u r e s . S c h u ltz (1973) p la c e d b l u e g i l l , c h a n n e l c a t f i s h , and la rg e m o u th b a s s i n s o l u t i o n o f 2 , 4-D DMA s a l t , l a b e l e d w ith l^C a t c o n c e n tra tio n s o f 0 .5 , 1 .0 , o r 2 .0 mg/L (ppm). F ish and w ater sam ples were removed a t i n t e r v a l s up to 84 days f o r a n a l y s i s o f 14C and 2 , 4-D c o n te n t. Considerable ra d io a c tiv ity remained in fish tis s u e s but ap p aren tly none was a s s o c ia t e d w ith 2 , 4-D, s u g g e s tin g e x t e n s i v e m e ta b o lism . The f i r s t t i s s u e in which r a d i o a c t i v i t y ap p eared was th e g a l l b l a d d e r o f c a t f is h and b l u e g i l l s , and e v e n tu a lly ^4C appeared in ev ery t i s s u e an a ly z e d . 16466 43 In both c a t f i s h and b l u e g i l l , th e amount o f r a d i o a c t i v i t y in muscle te n d e d t o i n c r e a s e t h r o u g h t h e c o l l e c t i o n p e r i o d . No t o x i c i t y was e v i dent in any of the exposed f is h . To r e l a t e c o n c e n t r a t i o n s o f one ppm to f i e l d c o n d i t i o n , an a p p l i c a t i o n o f 2 k g / h e c t a r e (10000 M^) t o w a t e r w i l l g i v e a c o n c e n t r a t i o n o f 2 mg/L o r 2 ppm i f t h e w a t e r i s 10 cm d e e p . D eep er w a t e r w i l l r e s u l t i n f u r th e r d i l u tio n . I f a p p lie d to flow ing w a te r, w a te r movement w ill ? d i l u t e th e h e r b ic id e q u i t e r a p i d l y , and t h a t m a t e r i a l d i s t r i b u t e d on O s o il and vegetatio n w ill tend to remain in p la c e . Obviously a s p i l l Q or other accident p resen ts d iffe re n t problems. Among i n s e c t s , t h e e f f e c t s o f 2 ,4 -D on h o n ey b e e s has r e c e i v e d t h e most a tte n tio n . Palm er-Jones (1964) has l i s t e d a number o f in v e s tig a t o r s who c o n c lu d e d t h a t 2 ,4 -D i s s a f e f o r b e e s , a s w e l l as o t h e r s who have shown t o x i c e f f e c t s , some o f w hich may dep en d on t h e s p e c i e s o f p la n t on which the h e rb ic id e is d ep o sited . In a f i e l d in v e s tig a tio n o f a t h r e e l b / a c r e a p p l i c a t i o n P a lm e r - J o n e s found t h a t a 22% m o r t a l i t y o c c u rre d i n 48 h o u rs a f t e r d u s t i n g . However, when b e e s a t th e h i v e w ere h e a v ily d u sted d i r e c t l y , th e r e was no m o r t a l i t y , le a d in g to th e c o n c lu sio n th a t th e bees in th e f i e l d were a c q u irin g in to x ic a n t by some mechanism other than su rface c o n ta c t. M offett e t a l . (1972) sprayed caged bees d ire c tly with various phenoxy h erb icid es at a one pound/acre r a te w ith v e ry l i t t l e e f f e c t . When i n c l u d e d i n t h e d i e t a t c o n c e n t r a t i o n s up t o 100 ppm, 2,4-D d id n o t d e c r e a s e l i f e t i m e o f b e e s and t h e e s t e r used was i n e f f e c t i v e a t 1000 ppm (M orton e t a l . , 1 9 7 2 ) . The h e r b i c i d e d o es c a u s e d e c r e a s e d brood d e v e lo p m e n t when f e d a t 100 ppm b u t h a s no r e p r o d u c t i v e e f f e c t a t 10 ppm (M orton and M o f f e t t , 1 9 7 2 ). Treatment of C occinellid b eetle larvae with 2,4-D at a ra te equal to 8 oz. acid eq u iv alen t/acre caused g reatly increased m o rta lity , r e g ard le ss o f th e age o f th e la rv a e through day 12 a t tim e o f sp ra y in g (Adams, 1 9 6 0 ). SILVEX T h e re a p p e a rs t o be a much more l i m i t e d a c c u m u la tio n o f d a t a on b i o lo g ic a l e f f e c t s o f s ilv e x and r e l a t e d compounds th a n i s a v a i la b l e f o r o th er phenoxy h e rb ic id e s . The d e s c rip tio n s o f b io lo g ic a l e f f e c t s o f s ilv e x w i l l be c a te g o riz e d in two b ro a d e r s e c tio n s r a t h e r th an th e more detailed en tries prepared fo r the other agents. B iological E ffects of S ilvex and I t s D eriv ativ es The a c u te median l e t h a l dose o f s ilv e x and i t s e s t e r s i s q u it e h ig h , and f a l l s in a r e l a t i v e l y narrow ra n g e among s p e c i e s and among th e d e r i v a t i v e s . Rowe and Hymas (1954) sum m arized d a t a w h ich had b een e s t a b l i s h e d ' at th a t tim e. S ilv ex and i t s mono-, d i - , and tr i- p ro p y le n e g ly c o l e th e r e s t e r s w ere o f a lm o s t i d e n t i c a l LD50 i n g u i n e a p i g o f 1200 and 1550 m g/kg. The l e t h a l i t i e s t o r a t s were a l s o s i m i l a r a t a b o u t 650 m g/kg. The r a t appears to be most s e n s i t i v e , and chicks and mice r e q u ir e doses s im ila r to the guinea pig. 44 S u b c h r o n ic (90 day) s t u d i e s o f r a t s fed 10-600 m g /k g /d a y o f t h e PGBE e s t e r r e s u l t e d i n more t h a n 50% l e t h a l i t y a t t h e h i g h e s t d o s e . The tim e o f d e a th ran g ed from 15-85 d a y s. Growth was d e p re s s e d in r a t s fed 500 mg/kg. There is somewhat o f a paradox in th e fin d in g s , because pathology in the dead animals in dicated m alnutrition ra th e r than h erb icid e to x ic ity . A p a ire d fe e d in g s tu d y a t 300 and 600 m g/kg/day in d i c a t e s t h a t d e p ressed growth was n o t e n t i r e l y due to in a d e q u a te food consum ption. Dose r a t e s o f 50 and 100 m g /k g /d a y c a u s e d an i n c r e a s e i n l i v e r w e ig h t b u t 10 m g /k g /d ay caused no ch an g e. (M u lliso n , 19 6 6 ). The s tu d y was fo llo w e d by a 90 day f e e d i n g o f up t o 10000 ppm s i l v e x (sodium s a l t ) i n t h e d i e t . 10000 ppm i s 1% o f t h e d i e t , and i f t h e a n im a ls consumed 10 gm d a i l y and w eigh 300 gra, the dose r a t e would be on th e o rd e r o f 300 m g/kg/day. That dose r a t e was highly l e t h a l and was abandoned, but d id produce s w e llin g and g ra n u la r deg en eratio n o f h ep ato cy te s and u ltim a te c e l l u l a r n e c r o s is . Renal tu b u le c e lls were sw elled and v acu o lated , and sem iniferous tu b u les were deg en era t e d . A t l e v e l s above 10 ppm (a b o u t 3 m g /k g /d a y ) some g row th d e p r e s s i o n occurred. M ullison (1966) also fed Kurosol (potassium s a l t o f s ilv e x , c o n ta in in g 53.3% s i l v e x a c id ) f o r two y e a r s a t c o n c e n t r a t i o n s up to 300 ppm. The h i g h e s t d o s e c a u s e d a s l i g h t l y i n c r e a s e d k id n e y / b o d y w e ig h t r a t i o i n m a le s , b u t 100 ppm and lo w e r d o s e r a t e s c a u s e d no ch an g e i n fo o d consumption, growth, gross and m icroscopic morphology hematology or bone marrow. 100 ppm was s t a t e d by t h e a u t h o r t o be e q u i v a l e n t t o 2.6 mg/ kg/day of s ilv e x . K u ro so l was w i t h o u t e f f e c t on b e a g l e s a t a f e e d i n g r a t e o f 56 ppm (a b o u t 19 m g /k g /d a y ) o v e r 2 y e a r s . Females f e d 190 ppm s u f f e r e d some h ep atic d e g en e ratio n and n e c ro sis a f t e r a y e a r o f fe e d in g , but a t th e end o f two y e a r s no damage c o u ld be fo u n d . At t h e 56 ppm i n t a k e , t h e r e w ere no ch an g e s o v e r a v e r y b r o a d s p e c tr u m o f h e m a t o l o g i c , c l i n i c a l c h e m i s t r y and m o rp h o lo g ic a n a l y s e s . The l i v e r damage a t h ig h e r le v e ls in c lu d e d h e p a to c y te n e c r o s i s , b i l e duct p r o l i f e r a t i o n , and b i l e pigment d e p o s itio n th ro u g h o u t the l i v e r and in the epithelium of kidney tubules. In a subchronic stu d y in c a t t l e by Palmer e t a l . (1964), one y e a r lin g B rah m a-cro ss f e d 100 mg s i l v e x / k g / d a i l y d i e d a f t e r 29 d a y s . Two o t h e r anim als g iv e n 25 and 50 mg/kg f o r 73 days showed no e v id e n c e o f t o x i c i t y . A 90 day ex p erim en t w ith 50 m g/kg/day, e i t h e r by d ren ch o r by i n j e c t i o n into a rumen f i s t u l a r e s u lte d in death of one of th e l a t t e r group of t h r e e a n i m a l s . Two o f t h o s e t r e a t e d o r a l l y d e v e l o p e d s e v e r e i n f l a m m a t i o n s in th e p a r o tid a re a , ap p aren tly due to lo c a l i r r i t a t i o n . The s u rv e y by In n es e t a l . (1961) s c re e n e d s i l v e x a g a i n s t two s t r a i n s o f m ice f o r 18 m o n th s. The c o n c e n t r a t i o n o f s i l v e x was 121 ppm, which was th e maximum t o l e r a b l e d o s e r a t e , and t h e r e was no i n c r e a s e i n tu m o rs i n e i th e r s t r a i n . The m utagenic screen by Anderson e t a l . (1972) d id n o t d e t e c t p o i n t m u ta tio n s r e s u l t i n g from s i l v e x tre a tm e n t in e i t h e r T4 phage or S. typhimurium (h is tid in e re q u irin g ). S ilv ex is te ra to g e n ic a t high doses. Courtney (1975) found th a t 398 m g /k g /d a y , on d a y s 12-15 o f g e s t a t i o n , c a u s e d 3% c l e f t p a l a t e i n t h e a O -;o o kV 'CO' H* 1 45 group g iv e n s i l v e x i n DMSO s u b c u t a n e o u s l y , and 7% w here g iv e n o r a l l y in c o m o i l . F e t a l m o r t a l i t y i n c r e a s e d t o 25% i n t h e group t r e a t e d s u b c u t a neously. The Dow Chem ical Co. h a s a l s o c o n d u c te d a s e r i e s o f t e r a t o l o g y s t u d i e s w ith s i l v e x . Dose r a t e s o f 75, 100 and 150 m g /k g /d ay from d ay 6 t o day 15 caused s e v e r a l c a r d i o v a s c u la r a n o m a lie s; 50 m g/kg/day caused r e t a r d e d o s s i f i c a t i o n i n t h e s te rn u m and s k u l l (Dow C hem ical C o ., 1 9 7 2 ) . The no a d v e r s e e f f e c t l e v e l was c o n s i d e r e d t o be 25 m g /k g /d a y . The PGBE e s t e r o caused s k e l e t a l changes a t an in ta k e o f 50 m g /k g /d a ily but no changes a were found a f t e r 35 m g/kg/day (23 mg/kg s i l v e x a c i d e q u i v a l e n t ) . B irds seem p e c u lia r ly r e s i s t a n t to th e h e r b ic id e s . For exam ple, DeWitt e t a l . (1963) f e d 5000 ppm BEE e s t e r o f s i l v e x t o young bob w h i t e q u a il, p h easan t and m allard ducks. The average l e t h a l in ta k e s were 9350, 9240 and 21,000 mg/kg, and tim e o f d e a th v a r i e d from 10 t o 100 d a y s . M a lla r d s w ere a b l e t o consume 100 ppm f o r 100 days w i t h no l e t h a l i t y , b u t re p ro d u c tio n was im p a ire d . S i l v e x a c i d a t 5000 ppm i n t h e d i e t was t o l e r a t e d f o r 5 d a y s w i t h no m o r t a l i t y by C o t u m i x . The 5 day LD5Q f o r p h e a s a n t s was 4500 ppm o f s i l v e x BEE e s t e r , f o r b o b w h ite q u a i l t h e LD50 was 30 ppm o v e r 5 d a y s , f o r C o t u m i x i t was i n e x c e s s o f 5000 ppm, f o r p h e a s a n t s LD50 was l e s s than 3000, and no m allard s d ie d a f t e r 5 days on 5000 ppm. A ll b i r d s were two weeks o l d a t t h e i n i t i a t i o n o f t h e s t u d y (H eath e t a l . , 1 9 7 2 ) . S t i c k e l (1964) r e p o r t e d US F i s h and W i l d l i f e s t u d i e s i n w h ich b o b v h i t e w ere f e d 1000 ppm o f an u n s p e c i f i e d s i l v e x e s t e r ; 50% w e re d e a d by day 54 w ith a v e ra g e t o t a l dose o f 17000 m g/kg. Of a group f e d 5000 ppm, the a v e ra g e d a i l y dose was 2300 mg/kg. H a lf o f th e b i r d s s u r v i v e d more th a n 4 d a y s , 40% s u r v i v e d more t h a n 10 d a y s , and an a d d i t i o n a l 4% d i e d d u r in g t h e r e m a in in g 14 d a y s . The a v e r a g e dose t o b i r d s t h a t s u r v i v e d 24 days was 54,500 mg/kg. These fin d in g s in d ic a te th a t w ild fowl should not be ad v e rse ly a f fected by any f ie ld a p p lic a tio n of s ilv e x , or fo r th a t m atter even a gross over-app1ic a tio n . E ffe c t o f S ilv e x on A quatic and I n v e r te b r a te S p ecies S ilv e x has become an h e r b ic id e o f c h o ic e f o r c o n t r o l o f s u r f a c e and u n d e r w a te r w a t e r n e e d s . As a c o n s e q u e n c e , t h e r e h a s p e r h a p s b e e n more a t te n ti o n paid to i t s im pact on a q u a tic s p e c ie s th an any o f th e o th e r phenoxy h erb icid es. There is an u n fortunate d iv e rs ity of experim ental methods and conventions f o r ex p ressin g c o n c e n tra tio n and o th e r f a c to r s which sometimes makes com parison o f d a ta d i f f i c u l t . Table 1 has been constructed to sim plify co n sid eratio n of the various reports of toxicity to aquatic species. Among f a c t o r s t h a t i n f l u e n c e t o x i c i t y t o f i s h a r e t h e c h e m i c a l form of the agent, whether the form ulation is granular or liq u id , and w ater hardness. G enerally th e e s te r s a re more to x ic , and liq u id fo rm u la tio n s i-b L b 46 r I Table 1. Gross T o x ic ity of S ilvex and I ts D eriv ativ es to Fish Species Form o f S ilv e x C o n cen tratio n (Acid Equiv.) PPm Effect B lu eg ill (young) (fry) (eggs) (fry) S to n e ro lle r (eggs) Chorus frog (tadpole) Fowler's toad (tadpole) BEE e s t e r BEE e s t e r Isooctyl ester K sa lt liquid K sa lt granular PGBE e s t e r K s a lt, liquid K s a lt, granular PGBE e s t e r PGBE e s t e r PGBE e s t e r PGBE e s t e r K salt K salt K salt PGBE e s t e r BEE BEE BEE 0.36 1.7 1.4 83 100 0.3 100 150 10 5 1 3 10 75 25 5 20 10 22 LC50 LC50 LCS0 LC50 lC50 tolerated tolerated tolerated no e f f e c t on hatch 100% l e t h a l no e f f e c t liv e r degen no e f f e c t 35% l e t h a l 95% h a t c h 45% h a t c h l c 50 LCSO l c 50 Period 48 h 48 h 48 h 48 h 48 h 96 h 96 h 96 h 36 h 80 days 2 weeks 2 1 /2 mo 2 1/2 mo 72 h 72 h 24 h 96 h 24 h Reference Cope, 1964 Hughes and D avis, 1966 Hughes and D av is, 1965 Hughes and D a v is, 1965 J o n e s , 1962 Jo n e s, 1962 J o n e s , 1962 W ilber and W hitney, 1973 Wilber and Whitney, Cope, 1964 Cope, 1964 Cope, 1964 Cope, 1964 H i l t i b r a n , 1967 H ilt ib r a n , 1967 S anders, 1970b S anders, 1970b S anders, 1970b 1973 M O L`C8 tiooMoa r DO\4f`, 00li^ 8 3 4 axe more to x ic . Data on w ater h ard n ess i s c o n tra d ic to r y ; but th e te n dency to g re a te r to x i c it y in s o ft w ater seems to be asso ciated w ith the s a lt r a th e r than the e s te rs (Surber and P ick e rin g , 1S62). D i s t r i b u t i o n r a t e s f o r s i l v e x t r e a t m e n t o f a q u a t i c weeds may be as h ig h a s 40 l b s / a c r e . An a s s u m p tio n o f d i l u t i o n th r o u g h o u t an 8 f o o t d e p t h is a p p a r e n t l y c o n v e n t i o n a l , w hich would g i v e a c o n c e n t r a t i o n o f 1 . 8- 1 .9 ppm. I f t h i s c o n c e n t r a t i o n o f an e s t e r f o r m u l a t i o n w ere t o h o ld i n a given a r e a , i t would c l e a r l y be l e t h a l t o many f i s h . The s a l t s h o u ld have up t o a 100 f o l d s a f e t y f a c t o r f o r f i s h , d e p e n d in g on m i n e r a l c o n t e n t o f the w ater. Such a p p lic a tio n is not a co n sid e ra tio n in fo re s t o p era tio n , but accid en tal overwater d ilu tio n could occur. T e rre s tria l ap p licatio n is a t r a t e s a t l e a s t 10 f o l d l e s s th a n 40 l b / a c r e b u t many w a te r c o u r s e s a r e much s h a l l o w e r t h a n 8 f e e t , t h e r e b y l e a v i n g a s i m i l a r h a z a r d . I f f l o w i n g , d ilu tio n w ill be ra p id and p erio d s o f exposure should be lim ite d . In s t a t i c w a te r th e ag en t w ill d if f u s e away from th e a p p lic a tio n s i t e w ith time ex ce p t in sm all sh allo w ponds, where some damage might p o s s i b ly o c c u r. Lower a q u a t ic organism s have a ls o been w ell s tu d ie d because o f silv e x use on w ater weeds. R e p re s e n ta tiv e though n o t com prehensive d a ta is compiled in Table 2. Again, i t appears th a t the s a lts of s ilv e x are much l e s s t o x i c t h a n t h e more com plex e s t e r s . The most v i v i d c o n t r a s t i s i n t h e e f f e c t on D aphnia magna, w hich i s 50% im m o b iliz e d by 100 ppm s i l v e r p o t a s s i u m s a l t , and by o n ly 0 .1 8 ppm s i l v e x PGBE e s t e r . Some s p e c i e s , h o w e v e r, a r e p a r t i c u l a r l y r e s i s t a n t t o even t h e PGBE e s t e r o f s i l v e x . The c r a y f i s h LC50 i s i n e x c e s s o f 100 ppm; f o r a number o f o t h e r organism s t h i s f a c t o r i s le s s th a n 1 ppm. I t i s n o t n e c e ssa ry to c a talo g u e th e e n t i r e l i t e r a t u r e on s ilv e x e ffe c ts on e i th e r f is h o r o th e r a q u a tic s p e c ie s . The range of e f f e c tiv e concentrations is well illu s tr a te d as well as the ex ten t, i f not d e ta ils , of species differences. E ffe c ts o f s ilv e x on t e r r e s t r i a l in s e c t l i f e have been e v alu ate d in honey b e e s by USDA w o rk e rs a t T u c s o n . A l l o f t h e phenoxy h e r b i c i d e s in c lu d in g s i l v e x were found to be r e l a t i v e l y n o n - to x ic to bees when applied in w ater a t f ie l d co n ce n tratio n (Morton e t a l . , 1972; M offett e t a l . , 1 9 7 2 ). When f e d a t 100 o r 1000 ppm, s i l v e x r e d u c e d b r o o d p r o d u c tio n , b u t a t 10 ppm, no a d v e rs e e f f e c t was fo u n d . In each c a s e , when the t o x i c a n t was removed, th e c o lo n ie s r e g a i n e d t h e i r o r i g i n a l re p r o d u c tiv e e ffic ie n c y (Morton and M o ffett, 1972). M e ta b o lic F a t e o f S i l v e x i n Mammals There have been few s tu d ie s o f th e d is p o s iti o n o f s ilv e x a f t e r ab s o r p t i o n by mammals. B je rk e e t a l . (1972) fed s i l v e x a t 1000 ppm i n fe e d and fo u n d 0 .1 2 ppm 2 , 4 , 5 - t r i c h l o r o p h e n o l (TCP) in m ilk and 0 .1 6 ppm in cream . In one week o f f co n tam in ated fe e d th e c o n c e n tr a tio n s d e c re a se d below 0 .0 5 ppm. Leng (1972) in a re v ie w , r e p o r te d work in which 2,4,5-TCP was n o t d e t e c t e d ; h o w ev er, r e s i d u e s o f s i l v e x w ere s u b s t a n t i a l i n t h e l i v e r . A f t e r f e e d i n g 300 ppm s i l v e x 28 d a y s , 4 ppm w ere fo u n d i n l i v e r , 18 i n k i d n e y , 0 . 6 i n m u scle and 0 . 9 i n f a t . A f t e r 2000 ppm i n t h e d i e t , 1 2 , 3 0 , 2 and 4 ppm w ere fo u n d i n t h e r e s p e c t i v e t i s s u e s . I n a 16471 48 Table 2. Gross T o x ic ity o f S ilv e x and I t s D e riv a tiv e s Species Form o f Silvex Concentration (Acid Equiv.) ppm Daphnia magna Scud Seed Shrimp Glass Shrimp Sowbug Crayfish adult Daphnia pu lex Simocephalus S e rru la tu s Stonefly naiad Drown shrim p adult PGBIi e s t e r BEE e s t e r K salt PGBE e s t e r PGBE e s t e r BEE e s t e r PGBE e s t e r BEE e s t e r PGBE e s t e r BEE e s t e r PGBE e s t e r BEE e s t e r PGBE e s t e r PGBE e s t e r PGBE e s t e r acid PGBE e s t e r PGBE e s t e r 0.18 2.1 100 1.8 0.8 1.2 0.2 4.9 3.2 8.0 0.5 40 100 210 2.4 5.2 .34 5.6 .34 0.28 LCS0 m edian l e t h a l c o n c e n t r a t i o n ICj-q m edian c o n c e n t r a t i o n t o c a u s e i m m o b i l i z a t i o n *all f i r s t in s ta r unless otherw ise noted ot> -si tO to In v erteb rate Aquatic Species* Effect LLLLLLLLICCCCCCCCC555S55555O00000000 LLLLCCCC55550000 IC 50 ICSO IC 50 IC 50 IC SO LLCC5500 Period 48 h 48 h 26 h 24 h 96 h 24 h 48 h 48 h 48 h 48 h 48 h 48 h 48 h 48 h 48 h 24 h 96 h 24 h 96 h 24 h Reference Sanders, 1970a Sanders, 1970a Crosby and T u ck er, 1966 S anders, 1970a S anders, 1970a Sanders, 1970a S anders, 1970a Sanders, 1970a S an d ers, 1970a S anders, 1970a S anders, 1970a S anders, 1970a S anders, 1970a Sanders and Cope, 1966 Sanders and Cope, 1966 Sanders and Cope, Sanders and Cope, Cope, 1965 Cope, 1965 B u t l e r , 196S 1968 1968 ec8,iTtoo/Aoa CO CD 'GO M O P sim ilar experiment Clark et a l . (1975) found roughly s im ila r v a lu e s, with t r a c e s o f 2,4,5-TC P in each t i s s u e . In a s i n g l e cow e x p e r im e n t, 5 ppm kuron was f e d f o r f o u r days a t 5 ppm ( S t . John e t a l . , 1 9 6 4 ) . The k u ro n h y d ro ly ze d t o s i l v e x and was e x c r e te d as a s a l t in t h e u r i n e . U rin a ry c o n c e n t r a t i o n s peaked a t 4 .4 ppm on day 4 and d e c l i n e d t o 0 .3 2 on day 6 . About 67% o f t h e t o t a l f e d was found i n u r i n e ; f e c e s w ere n o t a n a l y z e d . No d e t e c t a b l e r e s i d u e s o f Kuron o r s i l v e x w ere fo u n d i n m ilk and no ku ro n as such e n te re d th e u r i n e . The pharm aco-kinetics o f s ilv e x have been stu d ie d in r a t s given 5 or 50 m g / s i l v e x / k g . The compound was r i n g - l a b e l e d w i t h ^ C . The h i g h e r d o se appeared t o be s a t u r a t i n g b eca u se plasm a c l e a r a n c e was n o t l i n e a r , as was th e c a s e a t 5 mg/kg. H a lf - ti m e f o r plasm a c l e a r a n c e was 9 -1 0 h o u rs a t b o th d o s e s . At t h e lo w e r d o se 76% o f t h e l a b e l e d m a t e r i a l em erged i n b i l e in 72 h o u r s and 90% o f t h e h i g h d o s e . A lm ost a l l o f t h e b i l i a r y e x c r e t i o n was i n t h e f i r s t 24 h o u r s . Most o f t h e m a t e r i a l was s u b j e c t t o e n t e r o hepatic r e c ir c u la tio n and ev en tu ally l e f t the body in u rin e . T o tal s ilv e x e x c r e t e d i n 192 h o u r s i n u r i n e was 78%, and 16% a p p e a r e d i n f e c e s ( S a u e r h o f f et a l ., 1977a). S a u e rh o ff e t a l . (1977b) have a l s o examined s i l v e x d i s p o s i t i o n by humans. One mg/kg was g iv e n o r a l l y t o se v e n men and one woman, and p la s m a u rin e and fe c e s were an aly zed f o r 168 h o u rs . C le a ra n c e from plasm a and appearance in u rine were b i-p h a s ic , each stag e follow ing apparent f i r s t o r d e r k i n e t i c s , maximum p la s m a c o n c e n t r a t i o n s o c c u r r e d i n 2-4 h o u r s , 65% o f th e dose was e x c r e te d in u r i n e in 24 h o u r s . H a lf tim e s f o r th e two phases i n plasm a were 4 and 16 .5 h o u rs and i n u r i n e w ere 5 and 26 h o u r s . The l a r g e s t amount r e c o v e r e d i n f e c e s was 3.2%. 50 DOW 0011 837 REFERENCES Abou Akkada, A .R ., M.A. H assa n , and M.A. Naga ( 1 9 7 3 ) . E f f e c t o f some p e s ti c id e s and h e r b ic id e s on th e rumen m icro-organism s o f sheep. J . Agr. Sci. 81:231-236. Abou A kkada, A .R ., M.A. H a s sa n , and M. Naga ( 1 9 7 5 ) . A n o t e on t h e e f f e c t o f h e r b ic id e s on th e m etabolism and perform ance o f lambs. Anim. P rod. 2 0 :311-313. Adams, J .B . (1 9 6 0 ). E f f e c t s o f s p r a y in g 2,4-D am ine on c o c c i n e l l i d la rv a e . Can. Jo u r. Zool. 38:285-288. A la b a s te r , J . S . (1 9 6 9 ). S u rv iv a l o f f i s h in 164 h e r b i c i d e s , i n s e c t i c i d e s , fu n g icid es, w ettin g agents, and m iscellaneous substan ces. In te m a t. P est. Control 11:29-35. A lexander, M., and M .I.H . Aleem (1961). E ffe c t o f chem ical s t r u c t u r e on m ic ro b ia l d e c o m p o sitio n o f aro m a tic h e r b i c i d e s . J . A gr. Food Chem. 9 :4 4 - 4 7 . A lle n , J . R . , J . P . Van M i l l e r , and D.H. Norback (1 9 7 5 ). T i s s u e d i s t r i b u t i o n , e x c r e t i o n and b i o l o g i c a l e f f e c t s o f [^4C ] t e t r a c h l o r o d i b e n z o p -d io x in in r a t s . Fd. Cosmet. T oxicol. 13:501-505. A lle n , J . R . , D.A. B a r s o t t i , and J . P . Van M i l l e r ( 1 9 7 7 a ) . R e p r o d u c t iv e dysfunction in non-human prim ates exposed to d io x in s. P resented a t 16th Annual M eeting, Soc. T o x ico l., Toronto, 1977. A b stract 111. A lle n , J . R . , D.A. B a r s o t t , J . P . Van M i l l e r , L . J . Abrahamson, and J . J . L alich (1977b). M orphological changes in monkeys consuming a d ie t c o n ta in in g low l e v e l s o f 2 ,3 ,7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n . Food and Cosmetics T o x ico l, (in p r e s s ) . Altom, J .D ., and J . F . S t r i t z k e (1973). D eg rad atio n o f dicam ba, p ic lo ra m , and f o u r phen o x y h e r b i c i d e s i n s o i l s . Weed S c i e n c e 2 1 :5 5 6 - 5 6 0 . Aly, O.M., and S.D. F au st (1964). S tu d ie s on th e f a t e o f 2,4-D and e s t e r d e r i v a t i v e s i n n a t u r a l s u r f a c e w a t e r s . J . A gr. Food Chem. 12:541-545. Anderson, K . J . , E.G. L e ig h ty , and M.T. T akahashi (1 9 7 2 ). E v a lu a tio n o f h e r b i c i d e s f o r p o s s i b l e m u tag en ic p r o p e r t i e s . J . A gr. Food Chem. 20:649-656. A pplegate, V.C., J.H . Howell, and A.E. H all (1957). T o x ic ity o f 4,346 chem icals to l a r v a l lampreys and f is h e s . U.S. F ish and W ild life Service Spec. S c ie n tific Report--Fish. 207. A t l a s , S . A . , E .S . V e s e l l , and D.W. N e b e r t ( 1 9 7 6 ) . G e n e t i c c o n t r o l o f in te rin d iv id u a l v aria tio n s in the in d u c ib ility of aryl hydrocarbon h y d ro x y la se in c u l t u r e d human lym phocytes. C ancer Res. 36:46194630. 51 A xelson, 0 . , and L. S u n d ell (1974). H e rb ic id e ex p o su re , m o r t a l i t y and tum or i n c i d e n c e . An e p i d e m i o l o g i c a l i n v e s t i g a t i o n on S w edish r a i l road workers. Work-Environ. H ealth 11:21-28. Bache, C .A ., D.D. Hardee, F.R. H olland, and D .J. Lisk (1964). Absence o f phenoxyacid h e r b ic id e r e s id u e s in th e m ilk o f d a iry cows a t h ig h feeding le v e ls. J . Dairy S ci. 47:298-299. BAge, G ., E. C ekanova, and K.S. L a r s s o n ( 1 9 7 3 ) . T e r a t o g e n i c and em bryoto x ic e ffe c ts of the h erb ic id es d i- and trich lo ro p h en o x y acetic a c id s (2,4-D and 2 ,4 ,5 -T ). A cta Pharm acol, e t T o x ico l. 32:408-416. B eatty, P.W., and R.A. Neal (1975). E f f e c t o f a l t e r a t i o n o f h e p a tic ` mixed f u n c t i o n o x id a s e a c t i v i t y on t h e t o x i c i t y o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o d i o x i n (TCDD) t o r a t s . T o x i c o l . A p p l. P h a r m a c o l. 33: 151. B eatty, P.W., and R.A. Neal (1976). In d u c tio n o f D T-diaphorase by 2 , 3 ,7 , 8 t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD). Biochem. B io p h y s . R e s . Comm. 68:197-204. B e a t ty , P .W ., M.A. H o l s c h e r , and R.A. N eal ( 1 9 7 6 ) . T o x i c i t y o f 2 , 3 , 7 , 8 tetrach lo ro d ib en zo -p -d io x in in la rv a l and a d u lt forms o f rana c a te s b e ia n a . B ull. Environ. Contain. T o x ico l. 16:578-581. Becker, B.A. (1975). T erato g en s. In : T oxicology. The B asic S cien ce o f P o i s o n s . L . J . C a s a r e t t and J . D o u ll ( e d s ) . M a c M illa n , New York. pp. 331-332. B e rk e le y , M .C ., and K.R. Magee (1 9 6 3 ). N eu ro p a th y f o l l o w i n g e x p o s u r e t o a dim ethylam ine s a l t o f 2 ,4 -D . Arch. I n t . Med. 1 1 1 :3 5 1 -3 5 2 . B em dt, W.O., and F. Koschier (1973). In v it r o uptake o f 2 ,4 -d ic h lo ro phenoxyacetic acid (2,4-D) and 2 ,4 ,5 -tric h lo ro p n e n o x y a c e tic acid (2,4,5-T ) by ren al c o rtic a l tis s u e o f r a b b its and r a t s . T oxicol. Appl. Pharmacol. 26:559-570. feerry, D .L ., P.K. Z a c h a r ia h , M .J. Namkung, and M.R. J u c h a u (1 9 7 6 ) . T r a n s p lacen tal induction of carcinogen-hydroxylating systems with 2 ,3 ,7 ,8 tetrach lorodibenzo-p-dioxin. T oxicol. Appl. Pharmacol. 36:569-584. Berwick, P. (1970). 2 ,4 -D ich lo ro p h en o x y acetic a c id p o iso n in g in man. J . Am. Med. A s s o c . 2 1 4 :1 1 1 4 -1 1 1 7 . Binns, W., and A.E. Johnson (1970). C hronic and te r a to g e n ic e f f e c t s o f 2,4-D (2,4-dichlorophenoxyacetic acid) and a tra z in e (2 -c h lo ro -4 e t h y l a m i n o - 6- i s o p r o p y l a m i n o - s - t r i a z i n e ) t o s h e e p . N o r th C e n t r a l Weed C o n tr o l C o n f e r e n c e P r o c . 1 9 7 0 :1 0 0 . Binns, W., and L. B a l l s ( 1 9 7 1 ) . N o n t e r a t o g e n i c e f f e c t s o f 2 , 4 , S - t r i chlorophenoxyacetic acid and 2 ,4 ,5 -T propylene glycol b u ty l e s te rs h erb icid es in sheep. Teratology 4:24S. 16475 52 DOW B ionetics Research L aboratories, Inc. (1970). Evaluation of the c a r cinogenic, te ra to g e n ic , and mutagenic a c t iv it y o f s e le c te d p e s tic id e s V and in d u s tria l chem icals. Vol. I I I . E valuation of the terato g en ic a c t i v i t y o f s e le c te d p e s t i c i d e s and i n d u s t r i a l chem icals in mice and r a t s . (R e p o r t t o t h e N a t i o n a l C a n c e r I n s t i t u t e on c o n t r a c t s PH 4 3 - 6 4 - 5 7 and PH 4 3 - 6 7 - 7 3 5 ) . B je rk e , E .L ., J . L . Herman, P.W. M i l l e r , and J .H . W e tte rs (1 9 7 2 ). R esidue s t u d y o f phenoxy h e r b i c i d e s i n m ilk and c re a m . J . A gr. Food Chera. 20:963-967. B j o r k l u n d , N - E ., and K. E rn e ( 1 9 7 1 ) . Phenoxy a c i d i n d u c e d r e n a l ch an g e s in the chicken. Acta Vet. Scand. 12:243-256. B l e i b e r g , J . , M. W a l le n , R. B r o d k in , and I . L . A pplebaum ( 1 9 6 4 ) . I n d u s t r i a l l y acquired porphyria. Arch. Dermatol. 89:793-797. Bovey, R.W., and J .R . Baur (1972). P e r s is te n c e o f 2 ,4 ,5 - T in g ra s s la n d s of Texas. B u ll. Environ. Contam. T o x ico l. 8:229-233. Brody, T.M. (1 9 5 2 ). E f f e c t s o f c e r t a i n p l a n t grow th s u b s ta n c e s on oxidative phosphorylation in ra t liv e r m itochondria. Proc. Soc. Exp. B io l. Med. 80:533-536. Bucher, N.L.R. (1946). E f f e c ts o f 2 , 4 -d ic h lo ro p h e n o x y a c e tic a c id on e x p e rim e n ta l a n im a ls . P ro c . Soc. Exp. B io l. Med. 6 3 :2 0 4 -2 0 5 . Burton, J .A ., T.H. G ardiner, and L.S. Schanker (1974). A bsorption o f h erb icid es from the r a t lung. Arch. Environ. H ealth 29:31-33. B u s e lm a ie r , W., G. R o h r b o m , and P. P ro p p in g ( 1 9 7 3 ) . C o m p a r a tiv e i n v e s tig a tio n s on th e m u tag en icity o f p e s tic id e s in mammalian t e s t system s. M utation Res. 21:25-26. B u slo v ic h , S . , and N. Boruschko (1 9 7 6 ). C h lo rin e d e r i v a t i v e s o f phenoxya c e tic acid as antagonists of thyroid hormones. Farmakol. Toksikol. 39:481-483. B u tler, P.A. (1963). P e s tic id e - w ild lif e e f f e c ts . U.S. Fish and W ild life S ervice C irc u lar 167. pp. 11-26. \ B u tle r, P.A. (1965). E f f e c ts o f p e s ti c id e s on f i s h and w i l d l i f e . U.S. Fish and W ild life C irc u la r 226. pp. 65-77. B yast, T .H ., and R .J . Hance (1975). D egradation o f 2 ,4 ,5 - T by South Vietnamese s o ils incubated in the la b o ra to ry . B u ll. Environ. Contam. T o x ico l. 14:71-76. C a r t e r , C . D . , R.D. Kimbrough, J . A . L i d d l e , R.E. C l i n e , M.M. Z a c k , J r . , W.R. B a r t h e l , R .E . K o e h le r , and P .E ., P h i l l i p s ( 1 9 7 5 ) . T e t r a c h l o r o d i b e n z o d i o x i n : An a c c i d e n t a l p o i s o n i n g e p i s o d e i n h o r s e a r e n a s . Science 118:738-740. Chang, H-C., J.W. Rip, and J.H . Cherry (1974). E ffe c ts o f phenoxyacetic a c i d s on r a t l i v e r t i s s u e s . J . Agr. Food Chem. 2 2 :6 2 -6 5 . 55 <jL'O' r " CQ P P 8 TTq o m o o C hhabra, R . S . , J .M . T r e d g e r , R.M. P h i l p o t , and J . R . F o u ts ( 1 9 7 4 ) . S e l e c t i o n o f i n d u c e r s : An im p o r t a n t f a c t o r i n c h a r a c t e r i z i n g g e n e t i c differen ces to induction of aryl hydrocarbon hydroxylase in s tra in s of mice. L ife S ci. 15:123-130. C la r k , D .E . , J . E . Young, R.L. Y ounger, L.M. H u n t, and J . K . McLaran (1 9 6 4 ) . The f a t e o f 2 ,4 -d ic h lo ro p h e n o x y a c e tic a c id in sh eep . J . A gr. Food Chem. 1 2 :4 3 - 4 5 . Q a rk , D .E ., J . S . Palm er, and C.H. Ayala (1970). R esidual and to x ic o lo g ic a l asp ects o f 2,4,5-T and an e s te r in sheep and c a t t l e . Presented a t m e e tin g o f Am. Chem. S o c. P e s t i c i d e D i v i s i o n , and C hem ical I n s t , o f Canada. T oronto, 1970. C lark, D .E ., and J . S . Palmer (1971). R esidual a s p e c ts o f 2 ,4 ,5 -T and an e s t e r in sheep and c a t t l e w ith o b s e rv a tio n s on concom itant to x ic o lo g i c a l e f f e c t s . J . A gr. Food Chem. 1 9 :7 6 1 -7 6 4 . C lark , D .E ., J . S . Palm er, R.D. R a d e le f f , H.R. Crookshank, and F.M. F a rr (1975). R esidues o f chlorophenoxy acid h e r b ic id e s and t h e i r p h e n o lic m e t a b o l i t e s i n t i s s u e s o f sheep and c a t t l e . J . Agr. Food Chem. 23: 573-578. C o llin s, T .F .X ., and C.H. W illiams (1971). T erato g en ic stu d ie s w ith 2 ,4 ,5 -T and 2,4-D in the ham ster. B u ll. E nviron. Contam. T o x ico l. 6:559-567. Cope, O.B. (1964). P e s t i c i d e - w i l d l i f e s t u d i e s . U.S. Fish and W ild lif e S ervice C irc u la r 199. pp. 29-43. Cope, O.B. (1965). E ffe c ts o f p e s ti c id e s on f i s h and w i l d l i f e . U.S. Fish and W ild life Service C irc u la r 226. pp. 51-77. C ourtney, K .D ., and J .A . Moore (1 9 7 1 ). T e r a to lo g y s t u d i e s w ith 2 , 4 , 5 tric h lo ro p h e n o x y a c e tic acid and 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n z o -p d io x in . T oxicol. Appl. Pharmacol. 20:396-403. C o u rtn e y , K .D ., D.W. G a y lo r , M.D. Hogan, H.L. F a l k , R.R. B a t e s , and I . M itc h ell (1970). T eratogenic ev alu atio n o f 2 ,4 ,5 -T . Science 168: 864-866. C o u rtn e y , K.D. ( 1 9 7 6 ) . Mouse t e r a t o l o g y s t u d i e s w i t h c h l o r o d i b e n z o - p d io x in s . B u ll. E nviron. Contam. T o x ic o l. 16:674-681. Crosby, D .G ., and R.K. Tucker (1966). T o x ic ity o f a q u a tic h e r b ic id e s to Daphnia magna. Science 154:289-290. C rosby, D .G ., and A .S . Wong ( 1 9 7 7 ) . E n v ir o n m e n ta l d e g r a d a t i o n o f 2 , 3 , 7 , 8 t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD). S c ie n c e 1 9 5 :1 3 3 7 -1 3 3 8 . C rosby, D .G ., K.W. M o ila n e n , and A .S . Wong ( 1 9 7 3 ) . E n v ir o n m e n ta l g e n e r a tio n and d eg rad a tio n of dibenzodioxins and dib en zo fu ran s. Env. H ealth. Persp. 5:259-266. 18477 54 O O Cunningham, H.M., and D.T. W illiam s (1 9 7 2 ). E f f e c t o f t e t r a c h l o r o d ib e n z o - p - d io x in on growth r a t e and th e s y n t h e s i s o f l i p i d s and p ro te in s in r a t s . B u ll. Environ. Contain. T oxicol. 4 :4 5 -5 1 . Davis, J . T . , and J .S . Hughes (1963). F u rth e r o b s e rv a tio n s on th e to x ic ity of commercial h erb icid es to b lu e g ill su n fish . Proc. S o u th e r n Weed C o n fe re n c e 1 6 :3 5 7 - 3 4 0 . Davring, L. (19 7 5 ). E f f e c ts o f a 2 ,4 ,5 - T e s t e r on e a r l y o o g e n e s is , f e r t i l i t y and development in D ro so p h ila m e lan o g aste r. H e re d ita s 80:255-262. D av rin g , L . , and M. S u n n er ( 1 9 7 1 ) . C y t o g e n e t i c e f f e c t s o f 2 , 4 , 5 tric h lo ro p h e n o x y a c e tic a c id on oog en esis and e a r ly em bryogenesis in Drosophila m elanogaster. H ereditas 68:115-122. Dencker, L. (1976). The h e r b ic id e 2 ,4 ,5 - T : E a r ly p l a c e n t a l b a r r i e r and accelerated f e ta l uptake with advancing g e sta tio n . In: T issue L o c a l i z a t i o n o f Some T e r a t o g e n s a t E a r l y and L a te G e s t a t i o n R e l a t e d to F eta l E ffe c ts . Acta Pharm. T o x ico l, [su p p l.] 39:59-72. D esi, I . , J . S os, J . O la s z , F. B u ie , and V. Markus (1 9 6 2 ). N ervous system e ffe c ts of a chemical h e rb ic id e . Arch. Environ. Health 4:95-102. DeW itt, J . B . , W.H. S t i c k e l , and P . F . S p r i n g e r ( 1 9 6 3 ) . P e s t i c i d e - w i l d l i f e s tu d ie s . U.S. Fish and W ild life S erv ice C irc u la r 167. pp. 74-105. D ougherty, W . J . , M. H e r b s t , and F . C o u l s t o n ( 1 9 7 5 ) . The n o n - t e r a t o g e n i c i t y o f 2 ,4 ,5 - tr ic h lo r o p h e n o x y a c e ti c a c id in th e rh e s u s monkey (Macaca m ulatta) . B u ll. Environ. Contain. T o x ic o l. 13:477-482. Dow C nem ical Company, I n t e r n a l C om m unication ( 1 9 6 1 ) . R e s u l t s o f 9 0 - d a y d i e t a r y fe e d in g s tu d ie s on Dowanol 9713 e s t e r 2 ,4 ,5 - T in r a t s . D r i l l , V.A., and T. H ira tz k a (1953). T o x ic ity o f 2 ,4 -d ic h lo ro p h e n o x y a c e tic acid and 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic a c id . Ind. Hyg. Occup. Med. 7 :6 1 - 6 7 . IXigois, P . , and L. Columb (19 5 7 ). C h lo r ic a c n e : Case r e p o r t s a r i s i n g from t h e p r e p a r a t i o n o f 2 , 4 , 5 - t r i c h l o r o p h e n o l . J . Med. Lyon 38: 899-903. D unachie, J . F . , and W.W. F l e t c h e r ( 1 9 6 7 ) . E f f e c t o f some h e r b i c i d e s on the hatching ra te of hen 's eggs. Nature 215:1406-1407. D unachie, J . F . , and W.W. F l e t c h e r ( 1 9 7 0 ) . The t o x i c i t y o f c e r t a i n h e r b i cides to h en 's eggs assessed by th e e g g -in je c tio n te ch n iq u e. Ann. Appl. B iol. 66:515-520. D /b in g , F . , and A. K o lb erg (1 9 6 7 ) . I n h i b i t i o n o f t h e r e n a l t u b u l a r tra n sp o rt of p-aminohippurate-(Tm-PAH) in the ra b b it caused by sub to x ic doses of dichlorophenoxyacetate (2,4-D ). Acta Pharm. e t T oxicol. 25: Suppl. 4:51. 16478 55 DOW Emerson, J . L . , D .J. Thompson, R .J . S tr e b in g , C.G. G erbig, and V.B. Robinson (1971). T era to g en ic s tu d ie s on 2 ,4 ,5 - tr ic h lo r o p h e n o x y a c e tic acid in the r a t and r a b b it. Fd. Cosmet. T oxicol. 9:395-404. E m e , K. (1966a) . D i s t r i b u t i o n and e l i m i n a t i o n o f c h l o r i n a t e d p h e n o x y a c e tic acids in anim als. Acta Vet. Scand. 7:264-271. E rne, K. (1 9 6 6 b ). S t u d i e s on t h e a n im a l m e ta b o lis m o f p h e n o x y a c e t i c h e rb ic id e s. Acta Vet. Scand. 7:264-271. E rne, K. ( 1 9 7 5 ) . Phenoxy h e r b i c i d e r e s i d u e s i n S w edish f i s h and w i l d l i f e . Environ. Q uality Safety (Suppl.) 3:192-195. Erne, K., and I . Sperber (1974). Renal tu b u la r tr a n s f e r o f phenoxy a c e tic acids in the chicken. Acta Pharmacol. T oxicol. 35:233-241. Evans, J .O ., and O.R. D useja (1973). H e rb ic id e co n tam in a tio n o f s u rf a c e r u n o f f w a t e r s . USEPA R e p o r t 2 - 7 3 - 2 6 6 . Eyzaguirre, C ., B.P. Folk, K.L. Z i e r le r , and J .L . L i lie n th a l, J r . (1948). E xperim ental myotonia and r e p e t i t i v e phenomena: The v e r a t r i n i c e f f e c t s o f 2,4-D i n t h e r a t . Am. J o u r . P h y s i o l . 1 5 5 :6 9 - 7 7 . Fang, S .C ., E. F a ll in , M.L. Montgomery, and V.H. Freed (1973). The metabolism and d is tr ib u tio n o f 2, 4 ,5 -tric h lo ro p h e n o x y a c e tic acid in female r a ts . Toxicol. Appl. Pharmacol. 24:555-563. F lo r s h e im , W.H., and S.M. V e l c o f f ( 1 9 6 2 ) . Some e f f e c t s o f 2 , 4 - d i c h l o r o phenoxy a c e t i c a c id on th y ro id fu n c tio n in th e r a t : E f f e c ts o f io d in e accum ulation. Endo. 71:1-6. F lo r s h e im , W.H., S.M. V e l c o f f , and A.D. W illia m s ( 1 9 6 3 ) . Some e f f e c t s o f 2 ,4 -d ich lo ro p h en o x y a c e tic a c id on th y ro id f u n c tio n in th e r a t : E ff e c ts on p e r ip h e r a l th y ro x in e . Endo. 72:327-333. F o w ler, B .A ., G.W. L u c i e r , H.W. Brown, and O .S . M cD aniel ( 1 9 7 3 ) . U l t r a s tru c tu ra l changes in ra t liv e r c e lls follow ing a sin g le o ral dose o f TCDD. E n v i r o n . H e a l t h P e r s p e c . 5 : 1 4 1 - 1 4 8 . F o w ler, B .A ., G .E .R . Hook, and G.W. L u c i e r ( 1 9 7 7 ) . T e t r a c h l o r o d i b e n z o p -d io x in in d u c tio n o f r e n a l microsom al enzyme sy stem s: u l t r a s t r u c t u r a l e f f e c t s on p a r s r e c t a (S3) p ro x im a l t u b u l e c e l l s . J o u r . Pharmacol. Exp. Therap. (in p re s s ) . F rie s , G .F ., and G.S. Marrow (1975). R e te n tio n and e x c r e tio n o f 2 , 3 ,7 , 8 t e t r a c h l o r o d i b e n z o - p - d i o x i n by r a t s . J . A g r. Food Chem. 2 3 :2 6 5 - 2 6 9 . Gaines, T .B ., J .F . Holson, J r . , C .J. Nelson, and H .J. Schumacher (1975). A nalysis of s tr a in d iffe re n c e s in s e n s i t i v i t y and r e p r o d u c ib ility of re s u lts in assessing 2,4,5-T te ra to g e n ic ity in mice. T oxicol. Appl. Pharmacol. 33:174-175. 1647-9 56 G e h rin g , P . J . , C.G. Kramer, B.A. S c h w e iz , J . Q . R o se, and V.K. Rowe (1973) The f a t e o f 2 , 4 , 5 - tr ic h lo r o p h e n o x y a c e tic a c id (2 ,4 ,5 -T ) fo llo w in g o ra l a d m in is tr a tio n to man. Toxicol A ppl. Pharm acol. 26:352-361. G o ld s te i n , N .P ., P .H . J o n e s , and J . R . Brown ( 1 9 5 9 ) . P e r i p h e r a l n e u r o pathy a f te r exposure to an e s te r of dichlorophenoxyacetic acid . J . Am. Med. A s s o c . 1 7 1 :1 3 0 6 -1 3 0 9 . G o ld s te i n , J . A . , P. Hickman, H. Bergman, and J .G . Vos ( 1 9 7 3 ). H e p a ti c p o r p h y r i a in d u c e d by 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n i n t h e mouse. R es. Comm. Chem. P a t h o l . P h a r m a c o l. 6 : 9 1 9 - 9 2 8 . G re ig , J . B . , G. J o n e s , W.H. B u t l e r , and J .M . B arnes ( 1 9 7 3 ) . T o x ic e f f e c t s o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n . Fd. Cosm et. T o x i c o l . 11:585-595. Grunow, W., C. Bhme, and B. B ud czies ( 1 9 7 1 ) . R en ale a u s s c h e id u n g von 2,4,5-T b ei r a tt e n . Fd. Cosmet. T o x ico l. 9:667-670. Grunow, W., and C. Bhme ( 1 9 7 4 ) . Uber den S t o f f w e c h s e l von 2 , 4 , 5 - T und 2,4-D b e i r a t t e n und mausen. Arch. T o x ic o l. 23:217-225. Gupta, B.N., J.G . Vos, J.A . Moore, J.G . Z in k l, and B.C. Bullock (1973). P a t h o l o g i e e f f e c t s o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n i n l a b o rato ry anim als. Environ. Health Perspect. 5:125-140. Gutenmann, W.H., D.D. H ardee, R.F. H ardee, and D .J. Lisk (1963). Residue stu d ies with 2,4-dichlorophenoxy acetic acid h erb icid e in the dairy cow and i n a n a t u r a l and a r t i f i c i a l rum en. J . D a iry S e i . 4 6 :1 2 8 7 1288. Gyrd-Hansen, N ., and S. D algaard-M ikkelsen (1974). The e f f e c t o f phenoxyh e rb ic id e s on th e h a tc h a b ility of eggs and th e v i a b i l i t y o f the chicks. Acta Pharmacol. Toxicol. 35:300-308. H all, S.M. (1972). E f f e c ts on p reg n an t r a t s and t h e i r progeny o f ade q u ate o r low p r o te i n d i e t s c o n ta in in g 2 ,4 ,5 -T ric h lo ro p h e n o x y a c e tic acid (2,4,5-T) o rp ,p '-D D T . Fed. Proc. 31:726. Hamada, N ., and R .E . P e te r s o n (1 9 7 7 ). R e v e rs a l o f 2 , 3 , 7 , 8 - t e t r a c h l o r o dibenzo-p-dioxin-induced depression in b ilia r y excretion of ouabain by p re g n e n o lo n e - 1 6 a - c a r b o n itr ile and s p iro n o la c to n e in r a t s . I n t. Cong. T o x ic o l., Toronto. A b stracts p . 25. Hansen, W.H., M.L. Q u a ife , R.T. Haberman, and O.G. F itz h u g h (1971). Chronic to x ic ity o f 2 ,4-dichlorophenoxyacetic acid in r a ts and dogs. T oxicol. Appl. Pharmacol. 20:122-129. Haugen, D .A ., M .J. Coon, and D.W. N e b e r t ( 1 9 7 6 ) . I n d u c t i o n o f m u l t i p l e forms o f mouse l i v e r cytochrom e P-450. E vidence f o r g e n e t i c a l l y controlled p ro tein synthesis in response to treatm ent with 6 naphth a f l a v o n e o r p h e n o b a r b i t a l . J o u r . B i o l . Chem. 2 5 1 :1 8 1 7 -1 8 2 7 . S7 til t* -.00 C: VH H "O -:0 o a Hay, A. (1976). Toxic cloud over Seveso ( E d i t o r i a l , no r e f e r e n c e s ) . Nature 262:636-638. Heene, R. (1969). Redeutung e x p e r i m e n t e ll- h i s t o c h e m is c h e r Befunde f u r d ie pathogenese von m uskeldystrophien. K lin. Wschr. 47:389-390. Heath, R.G., J.W. Spann, E.F. H ill, and J .F . K re itz e r (1972). Compara tiv e d ie ta ry to x ic itie s of p e s tic id e s to b ird s . Bur. Spt. F ish erie s and W ild life, Spec. S c ie n tific Report (W ildlife) 152. Highman, B ., T.B. G aines, and H .J . Schumacher (1 9 7 6 a). S e q u e n tia l h isto p a th o lo g ic , hem atologic, and blood chem istry changed induced in mice by a te c h n ic a l and a p u r if i e d p r e p a r a tio n o f 2 , 4 , 5 - t r i chlorophenoxyacetic acid. J . Toxicol. Environ. Health 1:469-484. Highman, B ., T.B. G aines, H .J. Schumacher, and T .J . Haley (1976b). S tr a i n d iffere n ces in h istopathologic, hem atologic, and blood chem istry changes induced in mice by a te c h n ic a l and a p u r if i e d p re p a ra tio n of 2,4,5-trichlorophenoxyacetic acid. J . Toxicol. Environ. Health 1:1041-1054. H i l l , E . V . , and H. C a r l i s l e (1 9 4 7 ). T o x i c i t y o f 2 , 4 - d i c h l o r o p h e n o x y a c e tic acid fo r experim ental anim als. J . In d u st. Hyg. T o x ico l. 29:85-95. H i l l , E . F ., R.G. H eath, J.W. Spann, and J .D . W illiam s (1 9 7 5 ). L e th a l d ie ta r y to x ic itie s of environm ental p o llu ta n ts to b ird s . U.S. Fish and W i l d l i f e S e r v i c e S p ec. S c i e n t i f i c R e p o r t ( W i l d l i f e ) 191. 61 p p . H i l t i b r a n , R.D. (1 9 6 7 ). E f f e c t s o f some h e r b i c i d e s on f e r t i l i z e d f i s h eggs and f r y . T r a n s . Am. F i s h . S o c . 9 6 :4 1 4 - 4 1 6 . Hook, G .E . R . , T .C . O r to n , J . A . Moore, and G.W. L u c i e r ( 1 9 7 5 a ) . 2 , 3 , 7 , 8 Tetrachlorodibenzo-p-dioxin-induced changes in the hydroxylation o f biphenyl by r a t liv e r microsomes. Biochem. Pharm acol. 24:335340. Hook, G . E . R . , J . K . Haseman, and G.W. L u c i e r ( 1 9 7 5 b ) . I n d u c t i o n and su p p ressio n o f h ep atic and e x tra h e p a tic microsomal foreign-com poundm e ta b o liz in g enzyme systems by 2 ,3 ,7 ,8 - te tr a c h lo r o d ib e n z o - p - d io x in . Chem.-Biol. In teractio n s 10:199-214. Hook, J . B . , M.D. B a i l i e , and J . T . J o h n s o n ( 1 9 7 4 ) . I n v i t r o a n a l y s i s o f tr a n s p o r t o f 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic acid by r a t and dog kidney. Fd. Cosraet. T oxicol. 12:209-218. Hook, J . B . , R. C ard o n a, J . L . O sborn, and M.D. B a i l i e ( 1 9 7 6 ) . The r e n a l handling of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in the dog. Fd. Cosmet. Toxicol. 14:19-23. Hughes, J . S . , and J .T . Davis (1963). V a ria tio n s in t o x i c i t y to b l u e g i l l s u n f i s h o f phenoxy h e r b i c i d e s . Weeds 1 1 :5 0 -5 3 . 164 58 / Hughes, J . S . , and J . T . Davis Cl965 (1962 c o n f e r e n c e ) ) . C om parative t o x i c ity to b lu e g ill su n fish o f g ran u la r and liq u id h e rb ic id e s . Proc. C onf. S o u t h e a s t A sso c , o f Game and F i s h Comm. 1 6 :3 1 9 - 3 2 2 . tiighes, J .S . , and J .T . Davis (1966). T o x ic ity of p e s tic id e s to b lu e g ill su n fish te s te d during 1961-1966. L ouisiana W ild life and F ish e rie s Comm. 8 p p . H u r l e , K . , and B. Radem acher ( 1 9 7 0 ) . S t u d i e s on t h e e f f e c t o f l o n g - t e r m a p p l i c a t i o n o f DMOC and 2 ,4 -D on t h e i r breakdow n i n t h e s o i l . Weed Research 10:159-164. H i s s a i n , S . , L. E h r e n b e r g , G. L o f r o t h , and T. G e j v a l l ( 1 9 7 2 ) . M u ta g e n ic e f f e c t s o f TCDD on b a c t e r i a l s y s t e m s . Ambio 1 : 3 2 - 3 3 . I n n e s , J . R . M . , B.M. U l l a n d , M.G. V a l e r i o , L. P e t r u c e l l i , L. F i s h b e i n , E .R . H a r t , A . J . P a l l o t t a , R.R. B a t e s , H .L . F a l k , J . J . G a r t , M. K lein, I . M itc h ell, and J . P ete rs (1969). Bioassay o f p e s tic id e s and in d u s tr ia l chem icals f o r tu m o rig e n ic ity in mice: A p re lim in a ry note. J . Nat. Cancer In s t. 42:1101-1114. I s e n s e e , A .R ., and G.E. J o n e s ( 1 9 7 5 ) . D i s t r i b u t i o n o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD) i n a q u a t i c model e c o s y s te m . E n v i r o n . S ci. Technol. 9:668-672. I y e r , V ., M. W h i tin g , and G. F e n i c h e l ( 1 9 7 7 ) . N e u r a l i n f l u e n c e on e x perim ental myotonia. Neurology 27:73-76. J a c k s o n , W.T. (1 9 7 2 ). R e g u la tio n o f m i t o s i s . I I I . C y t o l o g i c a l e f f e c t s of 2 ,4 ,S -tric h lo ro p h en o x y a c e tic acid and of d io x in contam inants in 2,4,5-T form ulations. Jour. Cell Sci. 10:15-25. James, M.O., and J . R . Bend (1 9 7 6 ). T a u rin e c o n ju g a tio n o f 2 , 4 - d i c h l o r o p h en o x y ac etic a c id and p h e n y la c e tic a c id in two m arine s p e c ie s . Xenobiotica 6:393-398. Jenssen, D., and L. Renberg (1976). D is tr ib u tio n and c y to g e n e tic t e s t o f 2,4-D and 2 ,4 ,5 -T p h en o x y ac etic a c id s in mouse blood t i s s u e s . Chem.-Biol. In te ra c t. 14:291-299. J i r a z e k , L . , J . K a le n s k y , K. Kubek, J . P a z d e r o v a , and E. Lukas ( 1 9 7 4 ) . Acne c h lo r in a , p o rp h y ria c u ta n e a , and o th e r m a n if e s ta tio n s o f g e n e ra l in to x icatio n during manufacture of h erb ic id e s. Czech. Dermatol. 49:145-157. Johnson, J .E . (1971). The p u b lic h e a l th im p lic a tio n s o f w idespread use o f the phenoxy h erb ic id es and piclo ram . B ioscience 21:899-905. J o n e s , G ., and W.H. B u t l e r ( 1 9 7 4 ) . A m o r p h o l o g i c a l s t u d y o f t h e l i v e r l e s i o n in d u c e d by 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n i n r a t s . J. Path. 112:93-97. o O o t ,,-O .'o 'GO- cn 16482 59 J o n e s , R.O. (1965 (1962 c o n f e r e n c e ) ) . T o l e r a n c e o f t h e f r y o f common warm-water f is h e s to some chem icals employed in f i s h c u l t u r e . P ro c. C onf. S o u t h e a s t A s s o c . Game and F i s h . Comm. 1 6 :4 3 6 - 4 4 5 . Kay, J . H . , R . J . P a l a z z o l o , and J . C . C a l a n d r a ( 1 9 6 5 ) . S u b a c u te derm al to x ic ity of 2,4-D. Arch. Environ. H ealth 11:648-651. Kearney, P .C ., E.A. Woolson, and C.P. E llin g to n , J r . (1972). P e rsiste n c e and metabolism of ch lo rod io x in s in s o i l s . Environ. Science Technol. o 6:1017-1019. Q K ellerm ann, G ., M. L u y te n - K e lle rm a n n , and C.R. Shaw ( 1 9 7 3 ) . G e n e ti c v a r i a t i o n o f a r y l hydrocarbon h y d ro x y la se i n human lym phocytes. Amer. J o u r . Human G e n e t. 2 5 :3 2 7 - 3 3 1 . Kenaga, E.E. (1974). 2 ,4 ,5 -T and d e r iv a tiv e s : T o x ic ity and s t a b i l i t y i n t h e a q u a t i c e n v i r o n m e n t. Down t o E a r t h 3 0 ( 3 ) : 1 9 - 2 S . Kenaga, E.E. (1975). The e v a lu a tio n o f th e s a f e t y o f 2 ,4 ,5 -T to b ird s in areas tr e a te d f o r v e g e ta tio n c o n tro l. Residue Rev. 99:1-19. Khanna, S ., and S .C . Fang (1966). M etabolism o f C ^ - l a b e l e d 2 ,4 - d i c h l o r o p h e n o x y a c e t i c a c i d i n r a t s . J . A gr. Food Chem. 1 4 :5 0 0 - 5 0 3 . Khera, K .S ., and J .A . Ruddick (1973). P o ly (c h lo r o )d ib e n z o - p - d io x in s . P e rin a ta l e f f e c ts and the dominant le th a l t e s t in W istar r a ts . ACS Adv. Chem. S e r i e s 1 2 0 :7 0 - 8 4 . Khera, K .S ., and W.P. McKinley (1972). P re - and p o s t n a t a l s tu d ie s on 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and t h e i r d e r iv a tiv e s in r a t s . T o x ic o l. A ppl. Pharm acol. 2 2 :14-28. K il i a n , D . J . , M.C. Benge, R.V. J o h n s t o n , and E.B. W horton, J r . (1 9 7 7 ). (Personal communication, unpublished m an u scrip t). C ytogenetic s t u d i e s o f p e r s o n n e l who m a n u f a c tu r e 2 , 4 , 5 - T . Kimbrough, R.D. ( 1 9 7 4 ) . The t o x i c i t y o f p o l y c h l o r i n a t e d p o l y c y c l i c com pounds and r e l a t e d c h e m i c a l s . CRC C r i t . Rev. i n T o x i c o l . 2 :4 4 5 498. KimSrough, R.D., C.D. C a r te r , J .A . L id d le, R.E. C lin e , and P .E . P h i l l i p s (1977). Epidemiology and pathology of a te trach lo ro d ib en zo -d io x in poisoning episode. Arch. Environ. H ealth 32:77-85. Kimmig, J . , and K.H. S c h u lz (1 9 5 7 ). B e r u f l i c h e Akne (s o g e n a n n te n C h lo ra k n e ) durch c h lo rie rte arom atische zyclische ther. Dermatologica 115: 540-546. Klingman, D .L ., C.H. G ordon, G. Y ip , and H .P . B u r c h f i e l d ( 1 9 6 6 ) . R e s id u e s in t h e f o r a g e and i n m ilk from cows g r a z i n g f o r a g e t r e a t e d w ith e s t e r s o f 2 ,4-D . Weeds 1 4 :1 6 4 -1 6 7 . Kocher, C.W., N.H. M ahle, R.A. Hummel, and L.A. S h a d o f f (1 9 7 7 ). A s e a r c h f o r t h e p r e s e n c e o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n i n b e e f f a t . B u ll. Environ. Contam. T oxicol, (in p r e s s ) . 60 / I t Kbciba, R .J ., P.A. K eeler, C.N. Park, and P .J . Gehring (1976). 2 ,3 ,7 ,3 t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD): R e s u l t s o f a 13-week o r a l to x ic ity study in r a ts . Toxicol. Appl. Pharmacol. 35:553-574. K ohli, J . D . , R.N. Khanna, and B.N. Gupta (1 9 7 4 ). A b so rp tio n and e x c r e t i o n o f 2 ,4 -d ic h lo ro p h e n o x y a c e tic a c id in man. Arch. I n t . Pharmacodyn. Therap. 210:250-255. K o lberg, J . , K. H e l g e l a n d , J . J o n s e n , and 0 . T j e l v e i t ( 1 9 7 1 ) . The h e r b ic id e 2 ,4 -d ic h lo ro p h e n o x y a c e tic a c i d . I . E f f e c ts on L c e l l s . Acta Pharmacol, e t Toxicol. 29:81-86. K o lb erg , J . , K. H e l g e l a n d , and J . J o n s e n ( 1 9 7 2 ) . The h e r b i c i d e 2 , 4 dichlorophenoxyacetic acid. I I . T rig ly cerid e accumulation in L c e lls . Acta Pharmacol. 31:481-487. K o lb erg , J . , K. H e l g e l a n d , and J . J o n s e n ( 1 9 7 3 ) . B in d in g o f 2 , 4 - d i c h l o r o and 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic acid to bovine serum album in. Acta Pharmacol. T oxicol. 53:470-475, Konstantinova, T.K. (1974a). E ffec t of a h e rb ic id e , the butyl e s te r of 2 ,4 ,5 - T on th e progeny o f ex p erim en tal a n im a ls. Gig. T r. P ro f. Z a b o l. 9 :2 9 - 3 2 (Chera. A b s t r . 8 2 :2 6 8 1 6 p ) . Konstantinova, T.K. (1974b). E ffect o f the h e rb ic id e 2 ,4 ,5 -T b u ty l e s t e r on th e m atern al body and on th e f e tu s under e x p erim en tal c o n d i t i o n s . G ig. S a n i t . 8:101-102 (Chem. A b st. 82:26851w ). Kopischke, E.D. (1972). The e f f e c t o f 2,4-D and d ie s e l fu e l on egg h a t c h a b i l i t y . J . W ild life Management 36:1353-1355. K b s c h ie r , F . J . , and W.O. B ern d t ( 1 9 7 6 a ) . S t u d i e s on t h e m echanism o f re n a l tra n sp o rt depression caused by p retreatm en t w ith 2 , 4 ,5 - t r i chlorophenoxyacetate. J . Toxicol. Environ. Health 2:323-334. K b s c h ie r , F . J . , and W.O. B ern d t ( 1 9 7 6 b ) . S p e c i f i c i t y o f 2 , 4 , 5 - t r i c h l o r o p h en o x y ac etate on tetraethylam m onium t r a n s p o r t . T o x ico l. A ppl. Pharmacol. 38:297-306. K b s c h ie r , F . J . , and W.O. B e rn d t ( 1 9 7 6 c ) . I n v i t r o u p t a k e o f o r g a n i c io n s by r e n a l c o r t i c a l t i s s u e o f r a t s t r e a t e d a c u te ly w ith 2 ,4 ,5 trich lo rophenoxyacetic acid. Toxicol. Appl. Pharmacol. 35:355-364. K buri, R . E . , H. R a t r i e I I I , S .A . A t l a s , A. Niwa, and D.W. N e b e r t ( 1 9 7 4 ) . A ryl h y d ro c a rb o n h y d ro x y la se i n d u c tio n in human lym phocyte c u l t u r e s by 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n z o -p -d io x in . L ife Sciences 15:1585-1595. Krammes, J .W . , and D.B. W i l l e t s (1 9 6 4 ). E f f e c t o f 2 ,4 -D and 2 , 4 , 5 - T on w ater q u a lity a f t e r a spraying tre a tm e n t. U.S. F o rest S e rv ic e Res. N ote PSW-52, P a c i f i c SW F o r e s t and Range E x p e r im e n t S t a t i o n , B e r k e l e y . 4 p. a o 61 K utches, A . J . , D.C., Q iu rch , and F .L . Duryee (1 9 7 0 ). T o x i c o l o g i c a l e f f e c t s o f p e s t i c i d e s on rumen f u n c t i o n in v i t r o . J . A gr. Food Chem. 18: 450-433. Leng, M.L. (1972). R esidues in m ilk and meat and s a f e t y to l i v e s t o c k from t h e u s e o f phenoxy h e r b i c i d e s in p a s t u r e and r a n g e l a n d . Down to Earth 28(1):12-20. 5 : Leng, M.L. (1977). Com parative m etabolism o f phenoxy h e r b ic id e s in a n i O m a ls . I n : F a te o f P e s t i c i d e s i n L a rg e A n im a ls . G.W. I v i e and H.W. Q Dorough ( e d s . ) . Academic P r e s s , New Y ork. L in d q u ist, N.G ., and S. U llb erg (1971). D is tr ib u tio n o f th e h e r b ic id e s 2 ,4 ,5-T and 2,4-D in pregnant mice. Accumulation in th e yolk sac epithelium . E xperientia 27:1439-1441. L isk , D . J . , W.H. Gutenmann, C.A. B ache, R.G. W arner, and D.G. Wagner (1963). Elim ination of 2,4-D in the u rin e of s te e rs fed 4-(2,4-DB) or 2,4-D. J . Dairy S ci. 46:1435-1437. L ucier, G.W., O.S. McDaniel, G.E.R. Hook, B.A. Fow ler, B.R. Sonawane, and E. Faeder (1973). TCDD-induced changes in r a t l i v e r microsomal enzymes. Environ. Health Persp. 5:199-209. L u c i e r , G.W., B.R. Sonawane, O .S. M cD aniel, and G .E .R . Hook ( 1 9 7 5 a ) . P o s tn a ta l s tim u la tio n o f h e p a tic microsomal enzymes fo llo w in g a d m i n i s t r a t i o n o f TCDD t o p r e g n a n t r a t s . Chem. B i o l . I n t e r a c t i o n s 11:13-20. L u c ie r, G.W., O .S. M cDaniel, and G .E.R. Hook (1 9 7 5 b ). N a tu re o f th e enhancement of hepatic urid in e diphosphate g lu cu ro n y ltran sferase a c t i v i t y by 2 , 3 ,7 ,8 -te tra c h lo ro d ib e n z o -p -d io x in in r a t s . Biochem. Pharmacol. 24:325-334. L u t z - O s t e r t a g e t M.H. Lutz ( 1 9 7 0 ) . TOXICOLOGIE-- A c t i o n n f a s t e d e l ' h e r b ic id e , 2,4-D su r le dveloppement embryonnaire e t la fco n d it du g ib ie r plumes. C.R. Acad. Sc. P a ris S e rie s D 271:2418-2421. MacKenzie, C . J . G . , W.K. Oldham, and W.D. P ow rie ( 1 9 7 5 ) . R e l a t i n g t o the co n tro l o f the use of p e s tic id e s and h e rb ic id e s and use o f animal and p la n t p e s t-c o n tro l mechanisms. F in al Report o f the Comm issioners, Royal Commission o f In q u iry in to th e Use o f P e s t i cid es and H erbicides. Mahle, N .H ., H.S. H ig g in s, and M.E. G etzendaner (1 9 7 7 ). S earch f o r th e p r e s e n c e o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n i n b o v i n e m i l k . B u ll. E nviron. Contain. T o x ic o l, (in p r e s s ) . Majumdar, S .K ., and R.C. H a ll (1 9 7 3 ). C y to g e n e tic e f f e c t s o f 2 , 4 , 5-T on in v i t r o bone marrow c e l l s o f Mongolian g e r b i l s . The J o u r n a l o f H eredity 64:213-216. 62 tejum dar, S.K ., and J.K . Golia (1974). Mutation te s t of 2 ,4 ,5 -tric h lo ro phen o x y acetic a c id on D ro so p h ila m e la n o g a s te r. Can. J . G enet. C y to l. 16:465-456. Nhtsumura, F . , and H .J. Benezet (1973). S tu d ie s on th e b io accu m u latio n and m i c r o b i a l d e g r a d a t i o n o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n . Environ. Health Persp. 5:253-258. May, G. ( 1 9 7 3 ) . C h lo r a c n e from t h e a c c i d e n t a l p r o d u c t i o n o f t e t r a c h l o r o d i b e n z o d i o x i n . B r i t . J o u r . I n d u s t . Med. 3 0 :2 7 6 -2 8 3 . M:Cann, J . , E. C h o i, E. Y am asaki, and B.N. Ames ( 1 9 7 5 ) . D e t e c t i o n o f carcinogens as mutagens in the Salmonella/microsome t e s t : Assay o f 300 ch em icals. P ro c. N a tl. Acad. S c i. 72:5135-5139. M cC ollister, S .B ., and F .J . Kociba (1970). R esults o f 90-day d ie ta ry fe e d in g s tu d y on 2 ,4 ,5 - tr i c h lo r o p h e n o x y a c e ti c a c id ( 2 ,4 ,5 -T ) in r a t s . Dow C hem ical Co. I n t e r n a l R e p o r t . M zC onnell, E . E . , J . A . Moore, J . K . Haseman, and M.W. H a r r i s ( 1 9 7 7 a ) . The com parative to x i c it y o f c h lo rin a te d dib en zo -p -d io x in s in mice and guinea p ig s. T oxicol. Appl. Pharmacol, (in p re s s ). KfcConnell, E . E . , J . A . M oore, and D.W. D a lg a r d (1 9 7 7 b ) . T o x i c i t y o f 2 , 3 , 7 , 8 t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD) i n r h e s u s monkeys (m acaca i m l a t t a ) follow ing a sin g le o ral dose. Toxicol. Appl. Pharmacol, (in p re s s ). MzNulty, W.D. ( 1 9 7 7 ) . T o x i c i t y o f 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n f o r rhesus monkeys: b r i e f r e p o r t. B u ll. Environ. Contam. T o x ico l. 18:18-19. MzQueen, E .G ., A.M.O. V e a le , W.S. A l e x a n d e r , and M.N. B a te s ( 1 9 7 7 ) . 2 ,4 ,5 -T and human b i r t h d e f e c ts . R eport o f th e D iv is io n o f P u b lic H e a l t h , New Z e a la n d D ep a rtm e n t o f H e a l t h . M e r c ie r , M .J. ( 1 9 7 6 ) . 2 , 3 , 7 , 8- t e t r a c h l o r o d i b e n z o - p - d i o x i n : An o v e r view . P roceedings o f th e e x p e rt m eeting on th e problem s r a i s e d by TCDD p o l l u t i o n . Comm ission o f t h e E u ro p ea n C o m m u n ities. M i l l e r , R .A ., L.A. N o r r i s , and C.L. Hawkes (1 9 7 3 ). T o x i c i t y o f 2 , 3 , 7 , 8 t e t r a c h l o r o d i b e n z o - p - d i o x i n (TCDD) i n a q u a t i c o r g a n i s m s . E n v i r o n . Health Persp. 5:177-189. M itch ell, J.W ., R.E. Hodgson, and C.F. G aetjens (1946). T olerance o f fa rm a n im a ls t o f e e d c o n t a i n i n g 2 , 4- d i c h l o r o p h e n o x y a c e t i c a c i d . J o u r . Animal S c i. 5:226-232. M o f f e tt, J . O . , H.L. M orton, and R.H. MacDonald (1 9 7 2 ). T o x i c i t y o f some h e rb ic id a l sprays to honey b ees. J o u r. Ec. Entom. 65:32-36. Morre, D .J. (1974). Brush co n tro l along a g ric u ltu ra l drainage d itc h e s . Environmental sa fe ty and effic acy of h erb icid e fo rm u latio n s. Tech. R eport, Purdue U n iv ersity Water Resource Research C enter. 1648 2 .0 OD i. 1 T-f O O o Q Morton, H .L ., E.D. Robison, and R.E. Meyer (1967). P e r s is te n c e o f 2,4-D , 2 , 4 , 5 - T , and dicam ba i n r a n g e f o r a g e g r a s s e s . Weeds 1 5 :7 6 8 - 7 7 1 . Morton, H .L ., and J.O . M o ffett (1972). O v ic id a l and l a r v i c i d a l e f f e c t s o f c e r t a i n h e r b ic id e s on honey b ees. E nviron. Entom. 1:611-614. Morton, H .L ., J .O . M o ffe tt, and R.H. MacDonald (1972). T o x ic ity o f h e r b ic id e s to newly emerged honey bees. Environ. Entom. 1:102-104. Mount, D . I . , and C.E. Stephan (1967). A method f o r e s t a b l i s h i n g a c c e p ta b le to x ican t lim its fo r f is h --m alathion and the butoxyethanol e s te r of 2 ,4 - D . T r a n s . Am. F i s h e r i e s S o c . 9 6 :1 8 5 - 1 9 3 . M i l l i s o n , W.R. ( 1 9 7 2 ) . E c o l o g i c a l e f f e c t s o f h e r b i c i d e s . Down t o E a r t h 28(2):21-24. M u lli s o n , W.R. ( 1 9 6 6 ) . Some t o x i c o l o g i c a l a s p e c t s o f s i l v e x . P r e s e n t e d a t t h e S o u th e r n Weed C o n f e r e n c e , J a c k s o n v i l l e , F l o r i d a , 18 J a n 1966. M jra n y i-K o v a c s, I . , G. R u d a l i , and J . Im b e r t ( 1 9 7 6 ) . B i o a s s a y o f 2 , 4 , 5 trichlorophenoxyacetic acid fo r carcin o g en icity in mice. B rit. Jo u r. Cancer 33:626-633. Murray, F . J . , F.A. Sm ith, K.D. N its c h k e , C.G. H um iston, R .J . Kociba, and B.A. Schwetz (1977). T h re e -g e n e ra tio n re p ro d u c tio n s tu d y o f r a t s in g estin g 2 ,3 ,7 ,8 -tetrach lo ro d ib en zo -p -d io x in . Presented at 16th Annual M eeting, Soc. o f T o x ico l., Toronto. A bstract 166. N atio n al Academy o f S cie n c e s and N a tio n a l R esearch C ouncil (1 9 7 7 ). D rin k ing w ater and h e a lth . (In p r e s s ) . N e b e r t, D.W., J . R . R o b in s o n , A. Niwa, K. Kumaki, and A .P . P o la n d ( 1 9 7 5 ) . Genetic expression of aryl hydrocarbon hydroxylase a c tiv ity in the mouse. J . C ell Physiology 85:393-414. Neubert, D., and I . Dillmann (1972). Embryotoxic e f f e c ts in mice tr e a te d w ith 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic acid and 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n z o p -d io x in . Naunyn-Schmiedeberg's Arch. Pharmacol. 272:243-264. N eubert, D ., P. Zens, A. R o th e n w alln er, and H -J. Merker (1 9 7 3 ). A su rv e y o f t h e e m b ry o to x ic e f f e c t s o f TCDD i n mammalian s p e c i e s . E n v i r o n . Health Persp. 5:67-79. Newton, M., and L.A. N o rris (1968). H erb icid e re s id u e s in b l a c k t a i l d eer from f o r e s t s tr e a te d w ith 2 ,4 ,5 -T and a t r a z in e . P ro c. West. S o c . Weed S c i . 3 2 - 3 4 . N i e l s e n , K ., B. Kaempe, and J . J en sen -H o lm ( 1 9 6 5 ) . F a t a l p o i s o n i n g i n man by 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d ( 2 , 4 - D ) : D e t e r m i n a t i o n o f the agent in fo ren sic m a te ria ls. Acta Pharmacol, et T oxicol. 22: 224-234. 64 s Niwa, A . , K. Kumaki, and D.W. N e b e r t ( 1 9 7 5 ) . I n d u c t i o n o f a r y l h y d r o carbon hydroxylase a c t i v i t y in v a rio u s c e l l c u ltu re s by 2 ,3 ,7 ,8 tetrach lo ro d ib en zo -p -d io x in . Molec. Pharmacol. 11:399-408. >iony, R . , M.C. Bowman, C.L. H o ld e r , J . F . Young, and W.L. O i l e r ( 1 9 7 6 ) . Trace analysis of 2,4,5-trichlorophenoxyacetic acid, its glycineamide, and t h e i r a lk a lin e h y d ro ly zab le co n ju g ates in mouse b lood, u r in e , and f e c e s . J o u r . Pharm. S c i. 1810-1816. Nsrback, D.H., and J .R . A llen (1969). M orphogenesis o f to x ic f a t induced c o n c e n tric membrane a r r a y s in r a t h e p a to c y te s . Lab. I n v e s t. 20(4) -.338-346. Norback, D.H., and J .R . A llen (1973). B io lo g ic a l responses of th e non human p rim a te , ch ick en , and r a t to c h lo r in a te d d ib e n z o -p -d io x in in g estio n . Environ. Health Persp. 5:233-240. N orris, L.A. (1967). Chemical brush c o n tro l and h e rb ic id e re s id u e s in th e f o r e s t environm ent. In : H e rb ic id e s and V eg eta tio n Management Symposium, Oregon S ta te U n iv e rs ity , pp. 103-123. N o rris, L.A. (1968). Stream co n tam in a tio n by h e rb ic id e s a f t e r f a l l r a in s on f o r e s t l a n d s . W este rn S o c i e t y f o r Weed S c i e n c e Res. P r o g . R e p t . , pp. 33-34. N orris, L.A. (1969). Degradation o f sev e ra l h e rb ic id e s in red a ld e r f o r e s t f l o o r m a t e r i a l . W est. S o c . f o r Weed S c i . R e s . , P r o g . R e p t . , pp. 21- 22. N o r r i s , L .A ., H. G r a tk o w s k i, C. Graham, and W.E. C u r r i e r ( 1 9 7 2 ) . R e p o r t on background in fo rm a tio n f o r th e phenoxy h e r b ic id e s 2,4-D , 2 ,4 ,5 -T , and 2,4,5-TP. In te rn a l Report, U.S. F orest S erv ice. O liv e r , R.M. (1 9 7 5 ). T o x ic e f f e c t s o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - l , 4 d i o x i n i n l a b o r a t o r y w o rk e rs . B r i t . J o u r , o f I n d u s t r . Med. 3 2 :4 9 - 5 3 . O lson, R . J . , T .E . T ru m b le , and W. Gamble ( 1 9 7 4 ) . A l t e r a t i o n s i n c h o l e s t e r o l and f a t t y a c id b io s y n th e s is in r a t l i v e r homogenates by a r y l oxy a c id s . Biochem. J o u r. 142:445-448. Palmer, J .S . (1963). Chronic to x ic ity o f 2,4-D alkanolam ine s a l t s to c a t t l e . J o u r . Am. V e t. Med. A s s o c . 1 4 3 :3 9 8 - 3 9 9 . Palmer, J . S . , and R.D. R a d e le ff (1964). The to x ic o lo g ic e f f e c t s o f c e r t a i n f u n g ic id e s and h e r b ic id e s on sheep and c a t t l e . Ann. N.Y. Acad. S ci. 111:729-736. P alm er, J . S . , D.E. C l a r k , and L.M. H unt ( 1 9 6 4 ) . T o x i c o l o g i c e f f e c t s o f s i l v e x on y e a r l i n g c a t t l e . J . Amer. V et. Med. A ssoc. 1 4 4 :7 5 0 -7 5 5 . P alm er-Jones, T. (1964). E f f e c t on honey bees o f 2 ,4 -D . N.Z. A g ric . Res. 7:339-342. 65 l Z $ Q T I O O vV\OC VO Pegg, D.G., W.R. Hewitt, K.M. McCormack, and J.B. Hook (1976). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on renal function in the rat. Toxicol. Environ. Health 2:55-65. Piper, W.N., J.Q. Rose, and P.J. Gehring (1973). Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Environ. Health Persp. 5:241-244. Piper, W.N., J.Q. Rose, M.L. Leng, and P.J. Gehring (1973). The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,S-T) following oral adminis tration to rats and dogs. Toxicol. Appl. Pharmacol. 26:339-351. Plimmer, J.R., V.I. Klingebiel, D.G. Crosby, and A.S. Wong (1973). Photo chemistry of dibenzo-p-dioxins. ACS Advances in Chemistry Series 120:44-54. Poland, A.P., D. Smith, G. Metter, and P. Possick (1971). A health sur vey of workers in a 2,4-D and 2,4,5-T plant. Arch. Environ. Health 22:316-327. Poland, A., and E. Glover (1973a). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ. Health Persp. 5:245251. Poland, A., and E. Glover (1973b). Chlorinated dibenzo-p-dioxins: Potent inducers of 5-aminolevulinic acid synthetase and aryl hydrocarbon hydroxylase. II. A study of the structure-activity relationships. Molec. Pharmacol. 9:756-747. Poland, A., and E. Glover (1973c). 2,3,7,8-tetrachlorodibenzo-p-dioxin: A potent inducer of 5-aminolevulinic acid synthetase. Science 179: 476-477. Poland, A.P., E. Glover, J.R. Robinson, and D.W. Nebert (1974). Genetic expression of aryl hydrocarbon hydroxylase activity. Jour. Biol. Chem. 249(17):5599-5606. Poland, A., and E. Glover (1974). Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Molec. Pharmacol. 10:349-359. Poland, A., and E. Glover (1975). Genetic expression of aryl hydrocar bon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: Evidence for a receptor mutation in genetically non-responsive mice. Molec. Pharmacol. 11:389-398. Poland, A., E. Glover, and A. Kende (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Jour. Biol. Chem. 251:4936-4946. Rip, J.W., and J.H. Cherry (1976). Liver enlargement induced by the herbicide 2,4,5-trichlorophenoxyacetic acid. Jour. Agr. Food Chem. 24:245-250. 66 16489 Roberts, R.E., and B.J. Rogers (1957). The effect of 2,4,5-T brush spray on turkeys. Poultry Sci. 36:703-705. Roll, R. (1971). Untersuchungen uber die teratogene. Wirkung von 2,4,5-T bei Mausen. Food Cosmetic Toxicol. 9:671-676. Rose, J.Q., J.C. Ramsey, T.H. Wentzler, R.A. Hummel, and P.J. Gehring (1976). The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36:209-226. Rowe, V.K., and T.A. Hymas (1954). Summary of toxicological information on 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Amer. Jour. Vet. Res. 15: 622-629. Sanders, H.O. (1970a). Toxicities of some herbicides to six species of freshwater crustaceans. J. Water Pollut. Cont. Fed. 42:1544-1550. Sanders, H.O. (1970b). Pesticide toxicities to tadpoles of the western chorus frog Pseudacris triseriata and Fowler*s toad Bufo woodhousii fowleri. Copeia 1970:246-2S1. Sanders, H.O., and O.B. Cope (1966). Toxicities of several pesticides to two species of Cladocerans. Trans. Amer. Fisheries Soc. 95:165-169. Sanders, H.O., and O.B. Cope (1968). The relative toxicities of several pesticides to naiads of three species of stoneflies. Limnol. Oceanogr. 13:112-117. Sauerhoff, M.W., W.H. Braun, G.E. Blau, and P.J. Gehring (1976). The dosedependent pharmacokinetic profile of 2,4,5-trichlorophenoxy acetic acid following intravenous administration to rats. Toxicol. Appl. Pharmacol. 36:491-501. Sauerhoff, M.W., W.H. Braun, and J.E. LeBeau (1977a). Dose-dependent pharmacokinetic profile of silvex following intravenous administration in rats. Jour. Toxicol. Environ. Health 2:605-618. Sauerhoff, M.W., M.B. Chenoweth, H.L. Gordon, W.H. Braun, G.E. Blau, and P.J. Gehring (1977b). The fate of silvex following oral administra tion to humans. Presented at 16th Annual Meeting of the Society of Toxicology, Toronto. Abstract 75. Schreiweis, D.O., and G.J. Murray (1976). Cardiovascular malformations in Oryzias latipes embryos treated with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Teratology 14:287-290. Schultz, D.P., and P.D. Harman (1974). Residues of 2,4-D in pond waters, mud, and fish, 1971. Pestic. Monit. Jour. 8:173-179. Schultz, D.P. (1973). Dynamics of a salt of (2,4-dichlorophenoxy) acetic acid in fish, water, and hydrosol. J. Agr. Food Chem. 21:186-192. 67 r* in oo T*4' O O O a Schweiz, B.A., G.L. Sparschu, and P.J. Gehring (1971). The effect of 2,4dichlorophenoxyacetic acid (2,4-D) and esters of 2,4-D on rat embryo nal, foetal and neonatal growth and development. Fd. Cosmet. Toxicol. 9:801-817. Schwetz, B.A., J.M. Norris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson, and C.G. Gerbig (1973). Toxicology of chlorinated dibenzop-dioxins. Environ. Health Persp. 5:87-99. Seabury, J.H. (1963). Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. Archives of Environ. Health 7:86-93. Shadoff, L.A., R.A. Hummel, L. Lamparski, and J.H. Davidson (1977). A search for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an environ ment exposed annually to 2,4,5-trichlorophenoxyacetic acid ester (2,4,5-T) herbicides. Bull. Environ. Contam. Toxicol, (in press). Seiler, J.P. (1973). A survey of the mutagenicity of various pesticides. Experientia 29:622-623. Sieber, S.M. (1976). The lymphatic absorption of p,p'-DDT and some struc turally-related compounds in the rat. Pharmacology 14:443-454. Sjoden, P-0., and U. Soderberg (1972). Sex dependent effects of prenatal 2,4,5-trichlorophenoxy acetic acid on rats open-field behavior. Physiology and Behavior 9:357-360. Smith, F.A., B.A. Schwetz, and K.D. Nitschke (1976). Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in CF-1 mice. Toxicol. Appl. Pharmacol. 38:517-523. Somers, J., E.T. Moran, Jr., B.S. Reinhart, and G.R. Stephenson (1973). Effect of external application of pesticides to the fertile egg on hatching success and early chick performance. 1. Pre-incubation spraying with DDT and commercial mixtures of 2,4-D: Picloram and 2.4- D:2,4,5-T. Bull. Environ. Contam. Toxicol. 11:33-38. Somers, J., E.T. Moran, Jr., and B.S. Reinhart (1974). Effect of exter nal application of pesticides to the fertile egg on hatching success and early chick performance. 2. Commercial-herbicide mixtures of 2.4- D with picloram or 2,4,5-T using the pheasant. Bull. Environ. Contam. Toxicol. 11:339-342. Sparschu, G.L., F.L. Dunn, R.W. Lisowe, and V.K. Rowe (1971). Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid on fetal development in the rat. Food Cosmet. Toxicol. 9:527-530. Sjparschu, G.L., F.L. Dunn, and V.K. Rowe (1970). Study of the terato genicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Food Cosmet. Toxicol. 9:405-412. 1649; 68 Stehl, R.H., and L.L. Lamparski (1977). Combustion of several 2,4,5trichlorophenoxy compounds: Formation of 2,3,7,8-tetrachlorodibenzop-dioxin. Science 197:1008-1009. St. John, L.E., Jr., D.G. Wagner, and D.J. Lisk (1964). Fate of atrazine, kuron, silvex, and 2,4,5-T in the dairy cow. J. Dairy Sci. 47:12671270. Stickel, L. (1964). Pesticide-wildlife studies. U.S. Fish and Wildlife Service Circular 199. pp. 77-115. Strange, J.R., P.M. Allred, and W.E. Kerr (1976). Teratogenic and toxi cological effects of 2,4,5-trichlorophenoxyacetic acid in developing chick embryos. Bull. Environ. Contam. Toxicol. 15:682-688. Styles, J.A. (1973). Cytotoxic effects of various pesticides in vivo and in vitro. Mutation Res. 21:50-51. Surber, E.W., and Q.H. Pickering (1962). Acute toxicity of endothal, diquat, hyamine, dalapon, and silvex to fish. Progressive Fish Culture 24:164-171. Thigpen, J.E., R.E. Faith, E.E. McConnell, and J.A. Moore (1975). In creased susceptibility to bacterial infection as a sequela of expo sure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infection and Immunity 12:1319-1324. Todd, R.L. (1962). A case of 2,4-D intoxication. Jour. Iowa Med. Soc. 52:663-664. Van Miller, J.P., R.J. Marlar, and J.R. Allen (1976). Tissue distribu tion and excretion of initiated tetrachlorodibenzo-p-dioxin in non-human primates and rats. Fd. Cosmet. Toxicol. 14:31-34. Va.n Miller, J.P., and J.R. Allen (1977). Chronic toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin in rats. Fed. Proc. 36:396. Vinopal, J.H., and J.E. Casida (1973). Metabolic stability of 2,3,7,8tetrachlorodibenzo-p-dioxin in mammalian liver microsomal systems and in living mice. Arch. Environ. Contam. Toxicol. 1:122-132. Vogel, E., and J.L.R. Chandler (1974). Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogaster. Experientia 30:621-623. Vos, G.J., J.A. Moore, and J.G. Zinkl (1973). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health Persp. 5:149-162. Vos, G.J., J.A. Moore, and J.G. Zinkl (1974). Toxicity of 2,3,7,8-tetrachloridibenzo-p-dioxin (TCDD) in CS7B1/6 mice. Toxicol. Appl. Pharmacol. 29:229-241. 69 D O W 0011 855 18 DOVy 0011 856 \i Vos, J.G., and J.A. Moore (1974). Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorodibenzop-dioxin. Int. Arch. Allergy Appi. Immunol. 47:777-794. Ward, C.T. (1976). Studies on the degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in lake water and sediments. M.S. Thesis, University of Wisconsin, Madison, Wisconsin. Weiss, S.V., and W.H. Beckert (1975). Herbicide effects on cultured animal cells. Jour. Cell. Biol. 67:451a. White, A.W., Jr., L.E. Asmussen, E.W. Hauser, and J.W. Turnbull (1976). Loss of 2,4-D in runoff from plots receiving simulated rainfall and from a small agricultural watershed. J. Environ. Qual. 5:487-490. Whitehead, C.C., and R.J. Pettigrew (1972). The subacute toxicity of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid to chicks. Toxicol. Appi. Pharmacol. 21:548-354. Whitehead, C.C. (1973). Growth depression of broilers fed on low levels of 2,4-dichlorophenoxyacetic acid. Br. Poult. Sci. 14:425-427. WLersma, G.B., H. Tai, and P.F. Sand (1972). Pesticide residue levels in sails, FY 1969. National soils monitoring program. Pesticide Monit. Jour. 6:194-228. Wiese, A.F., and R.G. Davis (1964). Herbicides movement in soil with various amounts of water. Weeds 12:101-103. Wilbur, R.L., and E.W. Whitney (1973). Toxicity of the herbicide kuron (silvex) to bluegill eggs and fry. Trans. Amer. Fish Soc. 3:631633. Wilson, W.E. (1956). Toxicity of 2,4-dichlorophenoxy acetic acid (weed spray). Jour. Am. Med. Assoc. 162:1269. Wbjtalik, T.A., T.F. Hall, and L.O. Hill (1971). Monitoring ecological conditions associated with wide-scale applications of DMA 2,4-D to aquatic environments. Pesticides Monit. Jour. 4:184-203. Woods, James S. (1973). Studies of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on mammalian hepatic c-aminolevulinic acid synthetase. Environ. Health Persp. 5:221-225. Wright, F.C., J.C. Riner, J.S. Palmer, and J.C. Schlinke (1970). Metabolic and residue studies with 2(2,4,S-trichlorophenoxy)ethyl 2-dichloropropronate (Erbon) in sheep. J. Agr. Food Chera. 18:845-847. Yang, K.H., P.D. Guiney, J.L. Seymour, and R.E. Peterson (1977). 2,3,7,8TetrachlOTodibenzo-p-dioxin (TCDD)-induced depression in biliary ex cretion of polychlorinated biphenyls (PCBs) in rats. Presented at 16th Annual Meeting. Soc. of Toxicol., Toronto 1977. Abstract 103. 16433 70 M //0 0 Yoder, J., M. Watson, and W. Benson (1973). Lymphocyte chromosome analy sis of agricultural workers during extensive occupational exposure to pesticides. Mut. Res. 21:335-340. Young, A.L. (1974). Ecological studies on a herbicide-equipment test area. Technical Report AFATL-TR-74-12 USAF Systems Command, Eglin AF3, Florida. Zielinski, W.L., Jr., and L. Fishbein (1967). Gas chromatographic measure ment of disappearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Agr. Food Chem. 15:841-844. Zinkl, J.G., J.G. Vos, J.A. Moore, and B.N. Gupta (1973). Hematologic and clinical chemistry effects of 2,3,7,8-cetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Persp. 5:111-118. I V 71 S.;ii.D./AI.4-47S;DOY/;^ A k c ^ - ' DOW CHEM ICAL U.S.A. 18.338 Juno 3, 1974 M ID L A N D . M IC H IG A N /OC-IO George? H. Lynn 10622 El C a p ! tan Circle Sun City, Arizona 85 351 cc: J. Davidson, 9008 " M.` J. Traynor, 20 50 M. R. Wessel, Washington,D.C. E. H. Blair, 2020 W. B. C r u m m e t t , 5 74 P. J. Gchring, 1803 TOXICOLOGY OF POSSIBLE CONTAMINANTS IN 2,4,5-T On Ma.y 5, Lefty asked that the toxicity of possible c o n taminants of 2,4,5-T be made available should EPA or EDF people ask for such information. I have searched our files for our information with the following results. Note that the possible contaminant list was supplied by Andy Watson. I found no tox.. data on the following: Amounts Likely in wt. percent 0.4 <0.02 3.2 4.1 <0.05 0.03 0.02 0.26 0.03 bis-(2,4,5-trichlorophenocy)acetic acid 2,3,6-trichlorophenoxyacetic acid 4,5-dichloro-2-methocyphenoxyacctic acid 2,5-dichloro-4-methocyphenoxyacetic acid 2 .4- dichloro-5-met.hoxyphenoxyacetic acid 2,3,6-trichlorophenol 2.5- dichloroquinone monomethyl ether 4.6- dichlororesorcinal monomethyl ether 4 , 5 -dichlorocatechol nionomethyl ether 2 ,6-dichlorophenocyacetic acid 2,5-dichlorophenoxyacetic acid 2,4,6-trichlorophenoxyacetic acid AN OPF.nATINCi UNIT O r THE DOW CHEM ICAL C O M PA N Y $ r.r.Af.j-f.,Y* ; e- \\ . I. .'-. V '*s0111Y.'\ "CARE V ( ( -2 - The following contain! nnnts had some data: 0.07 ])jchlorophonol 2,3-dich 'loronlieno 1 Acute oval guinea pig in range of 0.3 to 2.6 g/kg Acnegcnic activity - none 2,4 -d1ch1cropheno1 A c u t e oral guinea pig in range of 0.3 tO 2.0 g/kg Skin - old sample - caused skin burn in IS s e c . - new sample - non-corrosive by DOT test .Acnegcnic activity - negative Odor Threshold Dow 0.02 ppm Taste Threshold Dow - Fish 0.005 ppm Lit. - va ter - 0.00 2 mg/1 Fish Tox. LC100 5 to 100 ppm in water Lit. values Acutc oral Rat LD50 o-r 4.5 g/leg o-5* 3.6 7 g/kg Mouse LD50 1.6 g/kg Chronic oral 6 m o . - mice No effect 100 mg/kg/day Adverse effect 200 mg/kg/day Liver w t . - slight depression SGPT - slight depression 2 ,5-dichlorophenol Acute oral guinea pi.g in range of 0.1 to 2.8 g/kg 2 ,6-dichlorophenol Acute oral guinea pig in range of 0.8 to 2.0 g/kg Acute oral rat >2.0 g/kg Eye - moderate superficial burn which should heal in a week or so. Skin - a burn upon 24 hr. contact a superficial burn in l h hr. heals in 2 4 -4S h r s . Lit. Ref. says 2 ,6-dichlorophenol may be a 'sex hormone in a tick species. t -3- c < 0.02 2-Chlorophenoxyacotic acid (t Acute ora] - Rat in range of 1 to 3 g/kg Skin - minor irritation Skin absorption - no indication in skin tests y ^ t <0.09 4-Chlorophenoxy acetic aci d Acute oral - Rat o> LD50 1.54 g/kg Acute oral - Chicks LD50 1.60 g/kg Skin - dry powder - no irritation 0.06 2,4-Dichlorophciioxyacetic acid .Acute oral LD50 Rat Mouse G. Pig Dog Chicks 375 mg/kg 36S mg/kg 469 mg/kg 100 mg/kg 541 mg/kg Lit. Ref. Chronic oral Rats fed 114 day in diet ( 0.03% - no effect level 0.1% - adverse effects - minor liver and kidney path, and growth depression Skin - R.abbit Powder (100%) dry - no irritation slight irritation Skin - Human 1 application for 4 to 5 days, then rest 3 w k s . followed by challenge 24 hr. exposure "not a sensitizer" no to slight irritation depending on sample used Chloracne - negative ( v 1 (' <0.02 0.2 2 DO\ -4 - Teratology and Fetal Toxicity S7.5 mg/kg/day to dam during 6-15 day fetal toxicity - positive teratology - negative Odor Thresh.old 60 ppm + Metabolism 72 hrs. - Sheep - 96$ excreted - urine unchanged 1.4$ excreted - feces unchanged 3,4-Dlchlorophenoxyacetic acid Acute oral - Rat 250-500 mg/kg Eye - a transient burn lasting 2-3 days, completely- healed in t days. Skin - 100$ dry - no irritation - 10$ sol. - slight irritation Skin absorption - no indication in skin tests run. 2 ,4,5-Trichlorophenol Acute oral. - Rat - male - LD50- 2830 mg/kg body xt. Lethality - Rat - female - LD50-2460 mg/kg body v:t. Chronic oral - Rat Purified 2,4,5-trichlorophenol was fed in food at 100, 300, 1000, 3000 and 10,000 ppm for 98 days. No adverse effects were detected at levels of 1000 ppm or less; 3000 and 10,000 ppm caused mild diuresis and slight pathological changes in the liver and kidney. Eye Irritation - Rabbit - The product caused conjunc tival membrane burns, severe conjunctival chemosis, slight to moderate chemosis, and slight to moderate corneal injury. Skin irritation - Rabbit - A single short exposure resulted in slight erythema (minutes) , slight edema, and a slight to moderate burn (1-1% hr.). Rabbit - Twenty daily applications of a 10$ c h l o r o form solution of tlie product to the ears of rabbits on a bioassay test for chloracaegenic activity resulted in no activity. OTT rnoo DC -5- f <0.0 2 2 ,_i ,6 -Tri chi orophenol Acute oral - Rat - male - LDS0-3250 mg/kg body wt. v. Lethality - Rat - female - LD50-2830 mg/kg body w t . liyc Irritation - Rabbit - The product caused moderate to severe conjunctival redness and swelling, slight to moderate iritis, and slight to moderate corneal inj u r y . Skin irritation - Rabbit - A single short exposure of the product, on intact skin resulted in slight erythema (1 hr), and moderate erythema, slight . edema and slight necrosis (4-5 hr). Prolonged exposure (24 hr) of the product on intact and abraded skin resulted in moderate to severe erythema, very slight to slight edema, and a moderate necrosis (random abraded skin areas). Rabbit - Twenty daily applications of a lOS chl o r o form solution of the product to the ears of rabbits on a bioassay test for chloracnegenic activity r e sulted in no activity. Inhalation - Human - Experience has shown that the dust is irritating to the nose and throat. If any of you know of further data, please supply me with copies of it so I can update this. The data given is a quick summary. Should more detail be needed, please let me kno\. . Mark A Wolf Information Services Toxicology Research Laboratory 1803 Building f 164S9 Ov-O, {) bcc: S. B. Sachs G. E. Lynn-- Action File-_______________ KURON December 30, 1964 Mr. George M. Downard, Head (2) Registration Section Pesticides Regulation Division Agricultural Research Service U. S. Department of Agriculture Washington, D. C. 20250 Dear Mr. Downard: Subject: Proposed use of KUR0N<8> on Sugar Cane in Florida (KUAQN - USDA Registration No. 464-162) ^ O ^ ro 00 CCDD By copy of the attached communication from J. R. Orsenigo, Associate Horticulturist, University of Florida, Everglades Experiment Station, dated December 23, 19S4, it will be noted that certain weeds of sugar cane plantings in Florida are not now controlled with 2,4-D and 2,4,5-T herbicides. In some cases the long continued use of 2,4-D has apparently resulted in development of resistance of the weeds and in others the weeds have never been susceptible to the use of 2,4-D and/or 2,4,5-T. Several of these weeds, such as the nightshades, fennels, ground cherry, purslane and wild lettuce do respond to treat ment with silvex, the name of the active ingredient in HURON. Since KURON is registered for use in sugar cane plantings in Louisiana and Hawaii, and since it is effective against the weeds in question in Florida when used at the same rates and under similar application conditions, it seems appropriate to apply for use registration in Florida, as indicated by the attached proposed addition to the KURON label. We therefore respectfully apply for registration as indicated and will appreciate the earliest practicable action by the Pesticides Regulation Division pertaining thereto. Very truly yours, 0. II. Hammer Registration Section Bioproducts Department cc: W. W. Sunderland, Dow Washington Office I 16500 D O W 331106 ACD FILE y 254G AQUATIC VEGETATION CONTROL IN RECREATIONAL AREAS by Mark G Wiltse 1 Introduction Aquatic weed growth has become an increasing problem in recreational areas of the eastern half of the United States. Nature does abhor a vacuum in the plant world, and in aquatic areas of shallow water this especially true. A succession of biological organisms and algal growth followed by the invasion of aquatic weeds, emergent aquatic growth, and higher forms of emerged aquatic vegetation and terrestrial plants, is nature's way of converting aquatic areas to dry land. Aquatic vegetation has restricted utilization of water for such purposes as hunting, fishing, bathing, water skiing, boating, and in some instances has destroyed the normal beauty of aquatic areas. Fish pro duction can be limited by excessive growth of water weeds. Aquatic vegetation is often a problem in water areas with limited water movement. Man has been trying.to interrupt this evolution of aquatic areas for some time, in order to maintain the aquatic or water areas so they can be of maximum use to man. Aquatic vegetation control measures have been used for many years and haveconsisted primarily of mechanical techniques. These contrivances have ranged from underwater cutting bars to hand rakes. Although these mechanical methods are often effective for a short time, regrowth from roots or rooting of stem fragments often increases the problem. The control of water levels, or draining and reflooding water areas, has also been used to some degree. These methods have generally been laborious and provide only temporary relief. In recent years, chemical control methods have offered a new opportunity in aquatic vegetation control. In discussions on aquatic vegetation, it is desirable to consider the various aquatic weeds, depending upon their habitat and type of growth, since the control measures for the various groups differ considerably. For this discussion, let us consider control measures for the following broad classification of aquatic vegetation-- submerged, floating, and emergent aquatic weeds. Control of Submerged Aquatic Weeds Submerged weeds are probably the major weed problem in recreational waters or at least causes the greatest concern where the water is used for bathing, boating, fishing, water skiing, etc. Submerged aquatic weeds, such as milfoil, coontail, and pondweeds, can completely fill ponds that are 7 to 8 feet deep with aquatic growth, so thax is impossible to even row a boat through the water. Bathers have become.entangled in this type TThe Dow Chemical Company, Midland, Michigan (Paper presented at 1961 Michigan Forestry and park Association Annual Conference, East Lansing, Michigan.) -2- DOW 3 3H 07 of, growth and drown because of their inability to free themselves. Sub merged aquatic weeds also tend to create a fish imbalance by providing protection for the small fingerlings, s o .that the large fish cannot consume them. This gives rise to a large population of small fish which consume much of the food supply. Fishermen often have difficulty in getting their hook and line through these weed masses. The New Jersey Fisheries Laboratory at Milltown, New Jersey, conducted extensive tests on this problem and in 1956 demonstrated the activity of silvex for the control of submerged aquatic weeds (6). in their test, they found that silvex was effective on a broad spectrum of aquatic weeds using 2 ppm of the active ingredient. Since 1956, silvex has been tested and used commercially for submerged aquatic weed control through out the United States. In 1958, Kuron* herbicide, Dow's propylene glycol butyl ether ester formulation of silvex, was registered for this use by the Pesticides Regulation Branch of the United States Department of Agriculture. Kuron, when used at the rate of 5 quarts per acre foot, will control certain submerged aquatic weeds such as water milfoil, fanwort, bladderwort, coontail and waterweed. Kuron should be applied in early summer when the water temperature is 65-70F. and weeds are approaching the water surface. To apply properly, first dilute the Kuron with enough water for uniform distribution over the water surface with the available equipment. Kuron has been especially effective when applied to water areas that have a minimum amount of water movement. Ponds with some water movement can be successfully treated if the water level can be lowered so there will be no overflow for at least three days after treatment. This rate of application of Kuron will also give control of some of the floating and emergent aquatic weeds, such as water lilies and spatterdock. Applications of Kuron ordinarily have not been harmful to fish; however, in certain situations where complete pond treatments have been made and where concentrations higher than the 5 quarts per acre foot have been used, some fish kill has been observed. Recent work in Massachusetts (1) has indicated that lower rates of Kuron have been effective on such weeds as water milfoil and bladderwort, and they are using 1-2 quarts per acre foot of water in some of their lakes. Kuron has a low mammalian toxicity, and normal precautions used in handling agricultural chemicals is sufficient for the use of this material. The Kuron formulation of silvex is an ester and an emulsion is formed when it is sprayed into the water. Our company has been actively evaluating other formulations of silvex for aquatic vegetation control and has developed a salt formulation which forms a true solution with water. This has given very encouraging results; Silvex salt formulations will possibly be available in test market areas in Michigan this season as'Kurosal* G" and "Kurosal*SL". Kurosal G contains 20% silvex on a clay granule, while Kurosal SL contains 6 pounds of silvex per gallon. Kurosal G should be applied at 100 to 150 pounds per surface acre, depending upon the depth of `the -water. Kurosal SL is designed to be applied os a direct application .n-to the water surface by a boom or by injecting a stream of the chemical in the wash of an outboard motor. Kurosal formulations appear _____________________panli-cularly safe to use in waters inhabited by fish. T r a d e m a r k o f The Dow C h e m ic a l Company 16502 DOW331X08 -3 - Silvex is the most promising development in the aquatic herbicide field and offers the potential of solving many of the submerged and emergent aquatic weed control problems without the necessity of using materials which are toxic to animals. 2 . 4 - B is available both as an ester and an amine salt and has been used to some extent for submerged aquatic vegetation control. In 1956, Dr. Buford Grigsby, from Michigan State University, reported on his results using 2,4-D.on a granular carrier and there has been considerable interest in the use of granular 2,4-D for submerged aquatic vegetation control since that time. Esteron* 99 Granules herbicide, which contains 20% 2,4-D as the propylene glycol butyl ether ester on a clay granule, is one of several available commercial formulations of granular 2,4 - D . 2 . 4- D granules applied to the surface of the water by hand or mechanical devices sink rapidly to the bottom of the water releasing 2, 4-D. It was originally thought that 2,4-D adhered to the granule for long periods, releasing slowly and thus localizing the active ingredient around the roots of the submerged aquatic weeds. Some recent work has indicated that 2,4-D actually is removed from the granule fairly rapidly, and that it is dissolved into the water. However, the use of the granule carriers for distribution purposes is convenient for small scale applications around boat docks and in front of cottages, etc. Although some workers have indicated that granular 2,4--D was effective, regardless of water ^epth, it has become evident from later work that water depth is quite important to obtain satisfactory control. To insure effectiveness, 2 . 4- D should be applied at about 5 pounds acid equivalent per acre foot of water treated. Water movement may also decrease the effectiveness of 2,4-D granules, since the active ingredient can be removed from the site of application before it is active on the plant. Esteron 99 Granules or other 2,4-D granules containing 20% 2,4-D should be applied at 100 pounds per surface acre in water that is 4 feet deep with an additional 25 pounds per surface acre for each foot of depth in excess of 4 feet. 2 , 4-D granules must be applied early in the growing season to be effective. Later season treatments on weeds not actively growing have not been consistently effective. 2,4-D granules will control such weeds as water milfoil, coontail, and bladderwort, but is considerably less effective on pondweed (Potamogetn spp.). 2 , 4-D granules are not as effective on as broad a spectrum o weeds as sodium arsenite or silvex. In the granular form, 2 , 4-D is easy for the boat owner or cottage owner to use in small a r e a s . Control of Floating Weeds Water lilies are a problem in recreational waters since they often . i n t e r f e r e with boating, water skiing, and swimming. Water lilies can 2Come so dense that it is nearly impossible to get through the area with a boat. Water lilies can be most effectively controlled by surface sprays with silvex. We recommend the use of Kuron at 2 quarts per 100 gallons ....... 16503 T radem ark o f The Dow C h e m ic a l Company DOW -4- of water to be sprayed as a wetting spray on the water lilies when they are in an active growth period, and the leaves have fully developed. Late season applications when the water lilies have completed their vegetative growth have not been consistently effective. 2,4-D esters CO have also been effective for the control of water lilies; however, the CO results have not been as consistent as with applications of Kuron. Duckweed (Lemna s p p .) is a free-floating aquatic weed and is often a nuisance in sheltered areas and may completely cover the water surface. 5? Duckweed has been very troublesome to control, since it can reinfest areas very rapidly. It has been reported that several materials will give immediate kill of the duckweed but reinfestation within three or four weeks is common. Control of Emergent Aquatic Weeds Emergent aquatic weeds have been a severe problem in waterfowl marshes and have decreased the waterfowl populations in some areas. Phragmites, cattails, and other weeds and grasses can cover an entire marsh, and thus open areas are not available for geese or ducks to land, and nesting and feeding areas are not attractive to them. Of course, emergent aquatic weeds are also a problem along the shoreline of recreational waters, cottages, etc. Cattails and other narrow-leafed plants can be controlled effectively by using.sprays of Dowpon* or R a d a p o n * . Radapon at 10 pounds per 100 gallons of water should be applied as a wetxing spray when the weeds are in an active stage of growth. Aerial applications of Radapon at 10-15 pounds per acre in 5-10 gallons of water have been very effec tive for the control of cattails for opening up marsh areas. Radapon is particularly effective on narrow-leafed plants and has little effect on many broad-leafed plants such as smartweed, pokeweed, wild buckwheat, etc., which are desirable for waterfowl food. Radapon is less toxic than common table salt and has been used in water reservoirs for aquatic vegetation control without causing any deleterious effect on water quality. Broadleaf emerged weeds often grow along the shoreline and may be a problem in some recreational areas. Some of these problem weeds are pickerelweed, arrowhead, water plantain, etc. Spray applications of Kuron using 2 quarts per 100 gallons of water have been effective in control of these weeds. Applications should be applied when the plants are in an active stage of growth. Emergent aquatic vegetation consist ing of both narrow-leafed plants and broad-leafed plants can be effec tively controlled by a mixture of silvex and dalapon which is sold commercially under the trademark, Garlon*. Garlon, used at 3 gallons per 100 gallons of water, has given effective control of cattails, burreed, arrowhead, pickerelweed, etc. Applications should be applied when the plants are in an active growing stage to be most effective. * Trademark of The Dow Chemical Company -5- Summary O O S e v e r a l h e r b i c i d e s a r e now a v a i l a b l e f o r t h e c o n t r o l o f a q u a t i c weeds ^ and a l g a e i n r e c r e a t i o n a l a r e a s . New h e r b i c i d e s h a v e shown some fo r the c o n tro l of submerged a q u a tic weeds and a d d itio n a l re s e a rc h wi p ro b a b ly develop new c h e m ic a ls t o be u s e f u l in t h i s f i e l d . T here a r e ^ se v era l chem icals p re s e n tly a v a ila b le th a t can do a very e ff e c tiv e jo b o in the c o n tro l of a q u a tic weeds and should be very u s e fu l in the management of r e c r e a t io n a l w aters and m arshes. 16505 T.ITTCf *- i -6- Literature Reviewed oO 1. C o rtell, J. M. ? Re-evaluation of the Concentration Required f Effective Aquatic Weed Control with Silvex. PROCEEDINGS OF NORTHEASTERN WEED CONTROL CONFERENCE 14:478-482, 1960. 2. Heath, G. and L. ,C. Ruch. Aerial Control of Cattail with Radapon. DOWN TO EARTH, Winter 1957 Issue. 3. Mackenthun, K. M. The Chemical Control of Aquatic Nuisances. Committee on Water Pollution, Madison, Wisconsin, January 1958. 4. Palmer, C. M. Algae in Water Supplies. U. S. Department of Health, Education and Welfare. Public Health Service Publication No. 657, 1959. 5. Wiltse, M. G. Aquatic Vegetation Control with K u r o n . (Presented at I960 Meeting of Weed Society of America, Denver, Colorado.) 6. Younger, R. R. A Preliminary Report on Controlling Aquatic Vegetation in New Jersey with Kuron. DGWN TO EARTH, Spring 1958 Issue. 16506 X DOW 33110fl ACD FILE NO. 1IA-23 k\ 2547`i AQUATIC VEGETATION CONTROL WITH KURON* by Mark G W iltse** A q u a tic weed g ro w th ha3 become a n i n c r e a s i n g problem i n r e c r e a t i o n a l w a te r of the e a ste rn h a lf of the United S ta te s . V egetation has r e s tr ic te d u tiliz a tio n of waters fo r such purposes as bathing, water sk iin g , boating, and in some i n s t a n c e s has d e s t r o y e d th e norm al b e a u ty of a q u a t i c a r e a s . F ish p ro d u ctio n has o fte n been lim ite d by e x c e ssiv e growth of w ater weeds. A number of c o n tro l methods have been employed w ith varying degrees of success. Mechanical co n triv an ces have ranged from under w ater c u ttin g b a rs to hand rakes. Although these m echanical methods a re o ften e ffe c tiv e fo r a sh o rt tim e, regrowth from ro ots or ro o tin g of stem fragm ents o ften in creases the problem . The method i s g e n e r a l l y la b o rio u s and p ro v id e s only tem porary relief. Sodium a r s e n l t e has. been used a s a h e r b i c i d e f o r s e v e r a l y e a r s . In some a re a s, p a rtic u la rly s t a ti c w aters, th is well-known chemical has given o u t standing r e s u lts . P rin cip al lim ita tio n s to i t s use have been i t s in herent tc 'c it y , re sista n c e to .decomposition and f a ilu r e to co n tro l a broad range oi .e g e t a t l o n . S everal workers have been a c tiv e ly e v a lu a tin g products which would c o n tro l submerged a q u a tic weeds and overcome the l im i ta t io n s of p re v io u s ly e x is tin g m e t h o d s . T h e New J e r s e y D i v i s i o n o f F i s h a n d Game i n a u g u r a t e d a f i v e - y e a r re s e a rc h program in 1951 fin a n c e d by D ln g e ll-Jo h n so n funds to fin d an iq u a tic h e r b i c i d e (8 ). I n th e c o u rs e o f t h i s work, Kuron h e r b i c i d e was .'ound a d a p t a b l e t o t h i s p r o b le m and seemed t o o f f e r c o n t r o l c o m p a r a b le t o jodlum a r s e n i t e (8 ). Kuron c o n ta in s th e a c t i v e in g r e d ie n t s l l v e x -(2,4,5 -trlchlorophenoxy) propionic acid as the propylene glycol butyl :th er e s t e r and a c ts as a p la n t grow th r e g u la to r . A quatic Weeds C o n tr o lle d Early t e s t s in d ic a te d t h a t Kuron h e rb ic id e a t 5 q u a rts per a c re fo o t would C o n tro l many of t h e tr o u b le s o m e subm erged a q u a t i c weeds i n t h e n o r t h e a s t e r n United S ta te s ( 1 , 2 , 3 , 4 , 8 ). E x p erim en tal t e s t s In 1957 and 1958 in s e v e ra l sections of the country evaluated the effectiv en ess of th is concentration on s e v e r a l a q u a t i c w e e d s . Some t e s t 3 w e r e c o n d u c t e d a t r e d u c e d c o n c e n tr a tio n s by c e r t a i n w orkers ( 2 ) . Commercial a p p l i c a t i o n s in 1958 and 1959 contributed ad d itio n al performance inform ation fo r co n tro llin g certain * r a d e m a r k o f The Dow C h e m i c a l Company The Dow C h e m i c a l C om pany, 9 1 6 S h o r e h a m B u i l d i n g , W a s h i n g t o n 5 , D. C. P a p e r p r e s e n t e d a t I 9 6 0 M e e t i n g o f t h e Weed S o c i e t y o f A m e r i c a , Denver, C olorado.) 65G7 -2- p c c ie 3 . The fo llo w in g l i s t sum m arizes some of th e weeds c o n tr o l le d by Kuron. ' q A q u a tic Weeds C o n t r o ll e d by Kuron a t 1 . 2 5 , 2 .5 and 5 Q u a rts p e r Acre F oot CO 1 . 2 5 q u a r t s / a c r e f o o t o r l e s s ( 0 . 5 PPm) CO White water I lly P ickcrelw eed (Nymphaea spp.) (Pontederla cordata L .) 2 . 5 q u a r t s / a c r e f o o t o r l e s s ( l . O ppm) Yellow water I lly Mud p l a n t a i n Water m ilfo il 'N u p h a r s p p . ) Heteranthera sp p .) ,MyrlophyHum hetero p h y llu m ) 5 q u a r t s / a c r e f o o t o r l e s s (2 ppm) Bladderwort W atershleld C o o n tall Panwort Waterweed V a r ia b le pondweed N' \d Sp-ice ru sh Tapegrass W aterstarwort Soft rush B u lru sh Burreed < (Utrlcularla spp.) (Brasenla s c h r e b e r l ) fCeratophyHum demersum) Cabomba carollniana) Anadiarla canadensis) SPotamogetn dlverslfollus) Potamogetn amoilf oliua) Na.jar. flexills ) Eleocharls app.) Valllsnerla americana) (Callltrlche spo.) 1Juncus e f f u s u s ) Sclrpus amerleanus) Sparganlum spp. ) I n some t r e a t m e n t s , t h e s e weeds were c o n t r o l l e d a t lo w e r c o n c e n t r a t i o n s , b u t th e re were not s u f f i c i e n t t e s t s to in d ic a te c o n s is te n t e ff e c tiv e n e s s a t the lower r a t e s . T reated a re a s observed two y ears a f t e r treatm e n t Indicated a continued co n tro l over th is period of tim e. In to ta l t r e a t ments on ponds and la k e s w ith Kuron a t 5 q u a r t s /a c r e fo o t two y e a rs c o n tro l has been o b tain ed in some i n s t a n c e s . A pplication Technique Emerged s p e c i e s were c o n t r o l l e d i n many i n s t a n c e s by u s in g Kuron a t one g a llo n per 100 g a llo n s of spray s o lu tio n ap p lied to the emerged fo lia g e as a thorough w ettin g sp ra y . The a p p lic a tio n s f o r th e c o n tro l of the sub merged a q u a tic weeds were g e n e r a l l y a p p lie d by d i l u t i n g the Kuron a t one G a l l o n i n 10 t o AO g a l l o n s o f w a t e r a n d d i r e c t i n g t h i s s o l u t i o n on o r i n t o t!**> w a t e r . P r o p o r t i o n i n g pumps w e r e o f t e n u s e d t o mix K u r o n w i t h l a k e w ' r and pump i t t o t h e boom w h i c h w ould d i s t r i b u t e t h e s o l u t i o n o v e r a wide sw ath. Uniform d i s t r i b u t i o n appeared to be d e s ira b le to allow a l e t h a l c o n c e n t r a t i o n o f t h e Kuron t o come i n im m ediate c o n t a c t w ith a l l the vegetative growth. 1 I65G8 -3- * Injection of the undiluted formulation of Kuron wa3 used in some equipment, but.on a limited basis. i* dovv a a iio 2 Environmental Effects Applications of Kuron appeared to be most effective if treatments were iiade during periods of active growth. Treatments applied when water temperatures have been 65 F or higher have been most effective. The wate temperature may not Influence the reaction of the chemical to a signlflca degree,, but the aquatic weeds may be in their most active growth at these higher temperatures and thus more susceptible. Undoubtedly, the most active growth periods vary for the various aquatic weed species. Applications with Kuron have been most effective on submerged aquatic plants In lakes and ponds where there is no or limited water movement. Less satisfactory control of some plants has been observed where water cur rents replace the treated water soon after application. Translocation has seen demonstrated in water lilies where applications to the leaf surface las caused a herbicidal effect on the rootstock. However, some aquatic )lant3 have only primitive vascular systems which may not be functional m d translocation to root areas probably is not important. In these plants, i lethal concentration of Kuron may be required to contact all the vegeta tive portions of the plant which are capable of''growth or reproduction, vlthough the exact minimum contact or exposure time required is not clearly leflned, ^8 to 7 2 hours exposure to #treated water has given control. .'re slut and Williams (7) compared the effectiveness of Kuron for control ling coontail (Ceratophyllum demersum). in greenhouse tank experiments at three pH levels and three concentrations of calcium ion3. Kuron was most :ffective on coontail growing in slightly acid water. It was also more ictive on coontail growing in water wi t h 25 0 and 5 0 0 ppm of calcium ion than in water with no calcium. In these tests, the effect of calcium was lot differentiated from a possible effect of chloride ion which was added n equivalent amounts. Field plot treatments and commercial applications iave been only slightly better under acid water conditions compared with reatments to neutral or slightly alkaline water. Perhaps the water pH is nly important In water situations with a low level of calcium ions or ilmilar dissolved ions. 1 afety to Fish and Other Aquatic O r g a n i s m s . he experimental treatments to plots and complete ponds have caused no fish njury at concentrations of Kuron of 1.25 to 5 quarts per acre foot, ommurclal applications at concentrations of 5 quarts per acre foot or less avc given no appi'eciable fish kill in numerous treatments throughout the ountry. Slight fi3h kill has occurred in some instances where applications ere made at higher concentrations. The higher concentrations sometimes ccurred in shallow areas where the spraying equipment could not operate roperly and thus higher concentrations were developed. There has been no la)5" 'njury in over 99 of the treatments applied. Kapid incorporation of he pray solution particularly those associated with rates in excess of quarts per acre foot of Kuron should be avoided where fish kill cannot be * derated. 16509 DOW 331103 Pierce (Ji,5,6 ) found that applications of Kuron had no Influence on oxygen content In treated ploto. Aquatic weeds treated with Kuron normally dr 'rnpose slowly, in most situations 1 0 to 2 1 days are required for d e c o m position. T h i 3 3 low decomposition apparently does not lower the oxygen content of the water.. This is a problem with some aquatic herbicides which give rapid kill of weeds. Pierce also reported that Kuron treatments showed no effect on population of benthic organisms represented by the following groups: Annelida, mostly leeches; Mollusca, including seven species of Gastropoda and one specie of Pelecypoda (S p h a e r l u m ); Amphipoda; Insecta, larvae of mayflies, damsel flies, dragon flie3, midges and beetles. Applications of Kuron temporarily decreased populations of some plankton, but within two weeks after treatment the populations were equal to untreated areas. These plankton consisted of fifty identified species in the following large groups; Myxophyceae, Chloro p h y c e a e , Desmldaceae, Diatomaceae, Flagellata, other Protozoa, Nematoda, Rotifera, Annelida, Crustacea. There was no deleterious effect on large aquatic vertebrates such as frogs and turtles in the treated areas. Prolific algae growth is often a problem soon after treating aquatic weeds with some aquatic herbicides. Algae growth has not normally occurred until three to four weeks after treatment with Kuron. The delay in appearance of algae apparently is associated wit h the slow kill of the aquatic weeds m d thus the delay in release of nutrients into .the water f r o m the decaying /egetatlon. 3af~ty to User Curon herbicide is low-in toxicity and presents a low' degree of hazard in handling and U3e. /hen Kuron herbicide was fed by intubation to groups of various animal ipecies, the LD^q values were found to be as follows: rat, 1 0 7 0 mg/kg; guinea pig, 8 5 0 "mg/kg; rabbit, 8 5 0 mg/kg; mouse, 2140 mg/kg and chicks, OOO mg/kg. From these data, it may be concluded that there should be no >roblem from Ingestion Incidental to the handling and use of this product. _f large quantities are swallowed, accidentally or willfully, some injury light result. H o w e v e r , the likelihood of serious injury is r e m o t e , [evertheless, Kuron herbicide should not be left where children or live stock have access to it. 'atlle, sheep, swine, ducks and chickens have consumed water containing uron herbicide at 5 0 ppm over a four week period with no adverse effects s determined by water consumption, body weight gains, and gross observalons, including gestation and parturition in sheep. These data indicate lit foliage or water treated with Kuron herbicide, as recommended, should ot'present a hazard from ingestion. kin Irritation tests conducted upon rabbits have indicated that Kuron Crrbiclde and its dilutions are slightly irritating, upon repeated pro- oned contact. In these studies, there was no evidence of absorption hr 3 I1 the skin in acutely toxic amounts. The observation of, reasonable ire and personal cleanliness practices should be adequate to avoid skin Ifficulties. 16510 331104 Kuron herbicide, when diluted to 1% with water, caused no primary Irrita tion nor allergenic responses when applied to the skin of fifty human volunteers using a repeated Insult technique. One subject did develop a Id fatiguing response. In addition, the product was "patch tested" on a bocond group of fifty human subjects at concentrations up to $ 0 % without cutaneous reaction. Even when applied undiluted to the skin for two h o u r s , 0 Oit caused no skin response. Thus, it may be concluded with a high degree of assurance that when used as directed, this product should cause no skin ^ irritation or 3kin sensitization responses. When tested on the eyes of rabbits, Kuron herbicide and its solutions produced moderate pain but only slight irritation. Thus, this product presents no unusual hazards from eye contact. However, suitable eye pro tection, such a3 safety glasses, may be worn simply to avoid the discomfo that might result from contact. Swimmers should not experience any discom fort from water treated at recommended concentrations. Effect on Water Use Kuron may remain in water for three to four months after treatment where no dilution occurs. An 88-acre lake was treated in New York after the 'water level was lowered two feet. No flow occurred through the outlet until three months after treatment. In analysis of the lake water thirty days after treatment, 2.4 ppm of sllvex from Kuron was present, essentially the same amount that was applied. Analysis of the lake water seventy days after treatment showed 2.1 ppm of silvex present. Samples of water analyzed 100 days after treatment and thereafter showed no Kuron present. Ch leal analysis of the bottom mud Indicated no build-up of Kuron. Since Ku.jn herbicide Is an o r g a n i c .compound, the components would be expected to be bacterlologlcally decomposed principally to the elements carbon, hydrogen and oxygen. Sllvex acta as a growth regulating compound on terrestrial broadleafed plants and Is used as a.stop-drop treatment prior to harvest of apples at concentrations of 10 to 20 ppm. Although 2 ppm of silvex as used for aquatic weed control might not cause Injury if sprayed on some broadleafed plants, some susceptible species may be affected. Accordingly, it Is suggested that water treated with Kuron should not be used for spray pur poses or for Irrigation during the season of treatment until more informa tion is available on the possible hazards to crops. Summary Curon aquatic herbicide is effective on several submerged and emerged .quatlc weeds. Applications to still water at 5 quarts per acre foot are uggested for general aquatic weed control. Lower rates are effective on ortaln species. Conditions for growth, pH and dissolved ion content of atcr may influence the effectiveness of Kuron. The low mammalian toxicity, low action, and safety on fish food organisms are desirable properties of uron aquatic herbicide. 16511 -6- Litcrature Cited DOW 331105 1 . B o s c h e t t i , M. M . , " F i e l d T e s t i n g o f K u r o n a s a n A q u a t i c H e r b i c i d e i n M a s s a c h u s e t t s , 11 P r o c . NEWCC, 1 3 :315"321. 1959 2. C o r t e l l , J . M., "R e-E valuation of the C o n c en tratio n s Required f o r E f f e c t i v e A q u a t i c Weed C o n t r o l w i t h S l l v e x " , P r o c . NEWCC 14:478-482. I 960 3 . H a l l , WT C . , " C o n t r o l o f V a r i o u s A q u a t i c Weeds w i t h S l l v e x " , P r o c . NEWCC 1 4 : 4 7 6 - 4 7 7 . i 9 6 0 4 . P i e r c e , M. E . , "The E f f e c t o f t h e W e e d i c i d e K u r o n u p o n t h e F l o r a 'a n d F a u n a o f Two E x p e r i m e n t a l A r e a s o f Long P o n d , D u t c h e s s . C o u n t y , New Y o r k " , P r o c . NEWCC 1 2 : 3 3 8 - 3 4 3 . 1 9 5 8 5 __________________ " F u r t h e r S t u d i e s o f t h e E f f e c t o f K uron u p o n t h e F l o r a a n d F a u n a o f Long P o n d , D u t c h e s s C o u n t y , New Y o r k " , P r c c . NEWCC 1 3 : 3 1 O -3 1 4 , 1 9 5 9 6 "P ro g re ss R eport of th e E f f e c t of Kuron upon the B i o t a o f Long P o n d , D u t c h e s s C o u n t y , New Y o r k " , P r o c . NEWCC 14:472-475. i 960 7. T r u c h e l u t , G. B . , a n d W i l l i a m s , R . C . , "Some O b s e r v a t i o n s o n t h e I n flu e n c e of D isso lv e d Ions on t h e ' f f e c tlv e n e s s of Phenoxy-Type H erb icid es on Submersed Aquatic Weeds", Proc. swcc 13:221-227. i960 8 . Y o u n g e r , R. R . , "A P r e l i m i n a r y R e p o r t on C o n t r o l l i n g A q u a t i c V e g e t a t i o n i n New J e r s e y w i t h K u r o n " , DOWN TO EARTH, S p r i n g '1958. 9; Y o u n g e r , R. R . , " P r o g r e s s R e p o r t o n t h e Use o f K u r o n , 2 , 4 - D a n d 2 , 4 , 5 - T P G r a n u l e s o n A q u a t i c H e r b i c i d e s " , P r o c . NEWCC 13:322-329. 1959 / t 16512  ' .i :s 254 KURON silvex herbicide continues to gain acceptance By DLaowwreCnhceemSicoaulthUw.Sic.Ak. O O CO CO h* O CO i\3 For more than two decades, three chemicals in the phenoxy group of systemic herbicides, i.e. 2.4-D. 2.4.5-T and MCPA. have more or less dominated the attention of researchers, advisors, marketers and users. Generally speaking. 2.4-D and MCPA are still leaders in broadleaf herbaceous weed control, and 2.4.5-T. alone and in combination with 2.4-D. is widely used in wcody plant control. The purpose of this brief paper is to review som e of the herbicidal possibilities of silvex. This report does not and is not intended to encom pass an exhaustive survey of the con siderable literature generated over the past 20 years relative to this herbicide. Selected published papers are referenced to provide support for the thesis that silvex is indeed a very effective herbicide with a broad use spectrum. Historical Background Shortly following the introduction of 2.4-D in 1945. researchers began to characterize its herbicidal utility in several use areas. It rapidly found acceptance for controlling herbaceous broadleaf w eeds in turf, small grains, corn and in non-cropland. It also showed promise for controlling woody species. However, as early as 1946. it be came obvious that 2.4-D was not adequately effective on certain weeds, for example, chickweed in turf, and on many woody plants in non-cropland brush control. By 1947. Dow research workers in Michi gan. workers in Hawaii, and probably others reported significant differential species re sponse to 2.4-D and 2.4.5-T. The latter soon found a place in the control of woody plants and also in selective weed control in rice. The idea of combining these two herbicides to broaden the spectrum of woody plant 26 OOWN TO EARTH. Voi. 28. No 4. Sprinti 1973 control was put into practice. Comme spraying of right-of-ways with mixtun 2.4-D and 2.4.5-T became a general pra and such combinations are in wide us the present time. Early Dow studies in Michigan (1952 dicated that another related but chemi specific herbicide. 2(2.4.5-tnchlorophen propionic W3S v?ry on assortm ent of herbaceous w eeds and w< plants2. Silvex w a s coined as the com name. KURON herbicide, a low voi ester formulation of silvex. was first i keted in 1954. Beginning around 1954. many rep have been presented at the regional v. control conferences on the herbicidal c acteristics of silvex. For example, in Mi gan trials, it w as reported very effective four species of oaks, and on m aple3. In tensive te s ts in Pennsylvania, res checked three years after application sho silvex effective " --on mixed oaks and a. ciated sp ec ie s" 1. In Oklahoma, silvex excellent on "all oaks" which included p white, blackjack, black and chinquap Based on many field trials, silvex w as fo to be an excellent overall turf herbicide controlling a wide assortm ent of weeds eluding many that are tolerant to 2.4-D4 an aquatic herbicide, silvex was reporte- be superior to others in Oklahoma8. n o rth ea st9, and elsewhere. An early report pn basal treatment v KURON herbicide was made in 1956 on key oak in Florida17. Summer applicate were rated excellent while winter treatrr was moderately effective. Work in Ore proved the effectiveness of KURON in oi basal control of big leaf m ap le11. Curr 16513 LITERATURE CITED |1) |2) 13) (4) (5) 16) (7) i3l cQai2pFwSSaCv4CANRpEstBhstN2SS1noiii9ntloisoarh.8loorxeradayugtouatea4poe.idasyn.rripkpronraEremmtpeptd.evpvntir.uioeee7dk5roi1snam.iscewesodetsrfs.r1m-s7BsnerUt,josliisT.n.reoa.tnm.5t.JruihliasR.r..pnSnocynj-F.eJSNl.eLKetn.7ApY1lt.--dgHesU.o.eeincA....Ao8N9Notogr.DrRtsth.ttt3.v.rAL6einruoSirhuoLnctKo.eeiAa.i6shwrfcAeamcro..DSnewnt.tDe1nu5nihakhC1sbdctFt9Vdii5lA.FahtEina1tn9teToAal6er1h2ouLltodtz9o7jxirre7Ji9Cnno.ensl:roamr61.pl.F.6catTS2eoct1gern7F.r6er6hc4do:.lsatdo9Nn.Sw..o-ro.no..rtMCal7ir12hi.toeulmrdiriCnSnJSR0FN04ctaWhsaRSmismho..tdn.ocae.-rooeoelrtepa2nFdecieolnf.rimpeCasRarlP7kewostl-HseFPonee1e1urva.ilstiertceosrccoaoL:0aceeteaSptntsahreleo1phmarrmV2sorfe.pwEtcle.fnd.eikoatr1aeaelhis1rxmeiriaranbt1-teryRb.cgt91ipecm2ciron9ditN1lnS966ieuex.uhei7tn3nAtv9jr6E29vtorstels.Jeu1ie.uel1aigP7ia.ish:tmx.dnerc.eer!.t.r.eaapM7UeewFtieTSia9cpcef1.sUoswCIoNc.f.nehufoecre5SpnhorntDpeeitCoihrnlm-echaeut.senpFni7SaouDot-vtNsconks.riter9ti2wrnredtetrrroebdc.iCadn4roa0solaAgrdeydllynsi.-ttf:.lll (((((((1111111(046539.12))))))))__wfch2emow(jwFo_iWpDo2SSSWSoSNNNeceaua3a_a_4eaaf_ohhomotaiooaocicboitae:kkm:tkrrr__a_riruTwhltttrrhtslibpsesutiesrieiystt__ne22_tstonotruhnh.mi,hsshnovrcte_i_9o5_rnrgfnlra.eet..teenaTitodsean80t__r_rtdarcaatyportthVoolretn.e--H_._s_Jeentrihssi32ocoeo)nien.doe.__.o6_asltt.AeEh05eJrfn-eeals_-6_s_lt.a.Ca2ahn6VuWL.irrtR.re:m__t_npfnnmrr.te.e.2woJbo.Ptrdo__Le_9jphasr.0oaneSl.reo1wafboe_W_We1.lpp.lfsiaDcn9nduoi_e0_oo1lcuVuneeNce.5dtftsiu_d-_9noiePses1lhdeoeond99iot6ltCsidn9adeddlvad.ne1eh.L1oa.7e6unRionn1e129tfnrenc.1TCCcre8rat.1nnb9a96.r2oido.r:oCco.encp9sfs7oo6.in9IfJ.eln.icn2enuslyV0e49..nnau(br3hiP3jtlrrt.4e.doWettvs.onWnratoMirr-e5)aciinolaPcepo2oftsnwn.n:SEaenk-iecnelptoa7lesln.jToos2alo9deees.iekflll..aCCiotm.lr0.nflh.trcneedaefo.rJywio-otrTtaratnebNtre2PPraoie.nnr.Vtrr-ltejlCP3doehlpereddgwiirfrfvoossseeo..1o1eeaarcwroaahhlintorra99.ctntct4nrneevaomehfmc6d6ieAee:odsannfrsoPde442eaeseede.rreccbode1..lk0tnsernwdaeednrli6dcea:eedlkewEltnt..siCt.iohiirlnnvnsniseqna.teflhVVoeoaleesf1gcgguraieeinamdnmrocaoiro9etsJssicnsac'edhfdglslsns0......,.tl * DOWN TO EARTH. Vol 28. No A. Sixin.i 1073 25 6 o ie rMfV D w 332094 COOH CH-CHU iJ 0 1 Cl ^C Cl l 2 (2,4,5-trichlorophenoxy) propionic acid T Oregon recommendations include silvex for l this species as well as for vine maple '*. Dor1 mant treatm ents on salt cedar in New 4 Mexico were moderately effective'0. Comrl mercial use of basal application of silvex has j corroborated effectiveness on many species j of woody plants. An effect commonly noted i is delayed maximum response. Final control ' j has been satisfactory. Extensive aerial trials in Hawaii showed } KURON herbicide to be the m ost promising { of those tested to reclaim wetland brush j jungle for cattle ra n g e 13. Weedy species inj eluded melastoma, false staghorn fern. ohia. j and lantana. Work in Oregon proved the j superiority of silvex in air or ground applied I foliage sprays on dalmation toadflax and j this is the present state recommendation in i Oregon7 as well as in Montana. Control of j prickly pear cactu s with silvex is recomI mended in Montana. Nebraska. Wyoming i and elsewhere. Data in the Research Prog! ress Reports of the Western Weed Control j Conference, even prior to 1962. indicated j good control with KURON on several diffi:j cult species, including cholla cactus, comj mon tansy, dalmation toadflax, lantana. ! larkspur, mesquite, milkweed, perennial j peppergrass. poison oak. prickly pear cac! tus, Russian knapweed, salt cedar, snow Jberry, sorrel, turbinella oak. whitethorn, j wild rose, yucca and others. For many years. . i silvex has been used extensively in turf and ^ sugar cane and for aquatic weed control. j Weed and Brush Control with KURON ^ Two excellent references which ch arac terize comparative effectiveness of phenoxy herbicides are: USDA Farmers' Bulletin No. j 2183 (Jan. 1971 revision) "Using Phenoxy Herbicides Effectively"'1, and USDA Agricul ture Handbook No. 269 (March 1965) "Her bicide Manual for Non-Cropland Weeds" 5. Tables 1 to 8 were developed largely from these information sources. Since the effectiveness of silvex has not been adequately reported on many species, the listings in the tables cannot be con sidered "com plete". In Tables 1 through 4. effectiveness is given in terms of excellent, good, or fair which are characterized as follows: excellent --one application at a normal rate (1 Ib/acre) kills the plant in m ost cases. g o o d --more than one application at the normal rate or an application at a higher rate (2 to 4 Ib/acre for weeds and 4 to 8 Ib/acre for woody plants) needed for control. fair--repeated applications at the nor mal or a higher rate often required for adequate control. It is interesting to note in these tables the wide range of annual, biennial and peren nial weeds, woody plants and aquatic weeds that are controlled with silvex. This relatively old. established herbicide obviously has at tributes that may not have been fully eval uated and certainly were not fully developed due. in part, to the diversion of interest and research effort to the great influx of new families of herbicides which emanated from the research laboratories coincident with and shortly following the initial development of silvex. Yet the limited research studies with KURON herbicide and other silvex formula tions as well as successful commercial usage have delineated specific use' areas wherein the special attributes of silvex her bicide have become recognized. The infor- * 1 6 5 1 5DOWN TO EARTH. Vol 28. No 4. Simrai 1973 27  TABLE 1. C o n tr o l of A n n u a l a n d B ie n n ia l W e e d s w i t h S ilv e x EXCELLENT amaranth bedstraw beggarticks bitterweed bloemustard burdover burdock barhtad . buttercups chickweed. coo. coileebean corton dogfennei ducksalad evemngprinuw liddleneck lleabanes galinsoga groundcheny htdoimustard hemlock, poison knaweL annual kochie lambsquarters marshelder morningglory mustards partridgepai passionflower pennycress. field pepperweeds pigweeds plantains poisonhemlock radish, wild ragweeds rape, bird redstera sesbania (coffeebean) sneezeweed spanishneedles sunflower thistle, bull. Russian tievine vetch, wild witchweed woodsorrel. yellow wormseedmustard GOOD broomweed coffaeweed lieldpeppergrase geranium. Caroline goosefoot oakleaf groundsel, cressleaf henbit horseweed (marestail) knapweed, spotted knotweed, japonesa medic, black meiicantea mesicanweed mudplantaia nightshade, black pokewaed purslane rocket London rocket yellow shepherd's purse smartweeds 28 OOWN TO EARTH. Vot 28. No 4. Sdmixi 1073 o O FAir. buckwheat u CO buckwheat w CO carrot wild curlyindiga O jointvetch CO knotweed. prr cn mayweed (chr. mulleia poorjoe puncturevine spurry. cora 16516 TABLE 4. C ontrol of a q u a tic w e e d s w ith S ilvex anowhtad bladder*ort cocotail olodoo fanwort loosestrife Iotas Modlonisli panotfeathtr pickeralweed primrosawillow lush EXCELLENT spattordock swamploosestrife watarcrowfoot weterhyacintb w tto rlily watermilfoil waterplantain watershield waterstargrsss wataistarwort waterwoed, Canada wildcslary GOOD w Lr--~ . alligatorwood . -C bulrush bunted cattails ^ pondwtods .o 'CO mation in the tables is simply indicative of the acknowledged wide spectrum of species response to silvex. An exhaustive survey of literature and progress reports, and es pecially a compilation of user experiences undoubtedly would reveal additional sus ceptible species. Comparison with 2,4 ,5-T In Tables 5 and 6 . tw o lists of w eed and woody plant species are given to illustrate differential foliar responses to silvex and to 2.4.5-T. These ratings are taken from the information provided in the two perform ance references referred to above3- " with assists from other data including California Bulletin 812.'2 In general, silvex often is considered more effective on the species listed thereunder in the tables and 2.4 ,5-T for th o se listed under it. However, this does not mean that control of some species listed under silvex is not possible with 2 .4.5-T or vice versa. It simply m eans that reported field results on these particular species give the edge to one or the other herbicide. The differences m effectivity vary from moderate to very significant. However, it should be em phasized that other field 30 OOWN TO EARTH. Vul 28. No 4. Siiniuj 1973 data may not be in total agreement w that reported here, and likewise many c sired comparisons have not been reportt Considering the proven utility of combir tion treatm ents of 2.4.5-T with 2.4-D with picloram for broad spectrum and i proved woody plant control, it is likely th silvex plus 2.4-D or picloram will be efft tive com binations and commercial trie 'indicate that this is so. Comparison with 2,4-D In Tables 7 and 8. similar weed and w o o l plant lists are show n relative to silvex ve sus 2.4-D. again using information from tf same published sources. Here again it obvious that there is a place for both herb cides since they do have certain different; spectra of activity. It is com m on knowledge that many herb ceous weeds and woody plants respor quite similarly to m ost of the phenoxy he bicides. including silvex. 2 .4.5-T and 2.4-1 On the other hand, it likewise is true th the re sp o n se of many species to these th re chemicals varies significantly, which emph sizes the need for each of them in the herb cide arsenal. It also em phasizes the poss 16517 TABLE 2 . C o n t r o l o f P e r e n n i a l W e e d s w i t h S i l v c x EXCELLENT badstraw blackcytdsusaa broomwaed bursaga buttarcup tlU U f chickweed. mouse-ear caneilowtr coyotillo dandelion dasertparsloy firewead lupina, tilvary milkvetch mulesears norealbaan pennywart lawn ponyfoot (dichondra) arch watarhemlock. spotted whitadovor woodsorral * i i i TABLE 3. Control of woody plants with Silvex i EXCELLENT aider aspen chittam chollacactus maples oak. post oak, shinnery palmatto. saw persimmon. Tasas J poisonivy poisonoak pricklypearcactus r redbud lose, cherokee ulmonberry trumpetcroeper willowwaed I GOOD chicory cutgrass docks groundiay kudio larkspur, tall loeowsed, whits milkwasds pokawaad saga, creeping shaapsonal snakawaed. threadiest speedwell, purslana violet whitatop GOOD boselder buttonbusb dogwood honeysuckle locust black mosquito oak,turkey persimmon, common raspberry, wild rosa, McCartney sagabrush, sand salteador sumac traa-oi-haaven willows t FAIR bindweed, (ield cinqueioil . hoarycrass f larkspur, duncecapf^^ lactuca, blue f* * i skunk cabbage sorrel sourdoefc sowthistle spikerush spurges strawberry, wild tansyragwort thistle, Canada yarrow, common yellawrocket FAIR blackberry, wild * cherry, wild chokecherry aeosgtebush elm grouncherry, clammy gum, black & sweet hickory manzanita mulberry oak, blackjack oak. bluejack oak. scrub oak, white osageoranga tost, muitillora sagebrush, big tarb'ush (oyon whitethorn ysrba-sania yucca (soapweed) 16518DOWN TO EARTH. Vol 28. No 4. Sih'hhi l`J /3 29 uow 331097 CL bility of herbicide rotation to forestall the . development of weed spectra tolerant to a single phenoxy weed killer. It is obvious from the plant response tables that these herbi cides are not equivalent and cannot com pletely r e p l a c e o n e another. Considering silvex in particular, it see m s abundantly clear that this interesting herbi cide. which has been around for many years, probably merits increased consideration not ! only on the part of those now advising on its i present registered uses but also on the part ; of researchers to ferret out new possibili' ties, including combinations with other herbicides. SUMMARY KURON herbicide is a low volatile ester 1 formulation of silvex. having wide utility in s controlling broadleaf weeds and woody plants. Over the years, the product has j gained acceptance for use mainly in aquatic and turf areas, but published literature af- ifirms a broad range of utility for this herbi cide and suggests possibilities for further | use development. Many plant species show i excellent response and can be controlled at ' moderate ictec. The specie! utility, of silvex in aquatic and turf weed control is noted, j Concerning the possible environmental - impact of silvex use. it is known that break down and loss in soil occur via microbiologi- ) cal degradation. Silvex is degraded more i slowly than 2.4-D but buildup in soil does \ not occur from annual increments. Used as i recommended on the label. KURON herbi- i cide presents no health hazard from inges- j tion or skin contact to humans, farm animals ] or wildlife. A toxicological summary w as published in 1966IU|. IIij S1E.Lc2BEi.ya4rCt.ne5Ted-sTE.sADpWea.cnRiRedEs. .AeFtTEPaARrto. Eco1enN9e5mdC7iniE.xgeSAsdt1eo1sattkhsofNa2on.rd4th,5ae-saTssPot.-j ern Weed Control Conference: 188-195. jj|1jjI 432...rCPefChCCoroseoooornsrcuuubtterllluitrttoeceeeerlidrfrrpd...iCeonwLL.groL.e.ts.neDLLfo.deLO1f.a1raWecntnf1nhoidNed9cnl5KAedtJr4.Tn:.o9.OCnelW0..uxS-.EpaB9DieAllG1avOrRN.eriibrWexToosn.HnrNoctsnhea.9.TsC.nO11e9ewN95nw5Eoi6tt4.Arh.a.sR4KleAs:T1WUlieHl8pvRce-ertOe2o1ixvd0g2N.e..I. 5.NnbDooou.onn2kch:ra2oNm4po-l.2a5n2R.d6.9wSeed1s9.65U.SHDeArbAicgirdieculmtuarneuHalanfodr TABLE 5. Comparative control of certain herbaceous weeds with silvex and 2.4,5-T. __________ __________ _____ bedstraw (d inars) blackeyodsusan chickwnds evening primrose fiddlcneck (ireweid OFTEN BETTER CONTROL WITH SILVEX ground hry hemlock, poison htnbit knapweed, spotted kniwel knotweed. Japanese v ( marsbelder medic, black milkweed, showy nightshade, black spurges spurry. corn e c t & buckwheat wild cofieeweed daisy, exeye goosefoot indigo, curly ironweed. western OFTEN BETTER CONTROL WITH 2,4,5-T mayweed mosquito. velvet mesicantei mustards pennycress. field pepperwaed. field pootjoe purslane, common sag*, creeping shepherdsputse yellowrocket TABLE 6. Comparative control of certain woody species with silvex and 2,4,5-T. OFTEN BETTER CONTROL WITH SILVEX cactus, cholla & prickly pen honeysuckle maples mulbeiry oaks (liva. blue. turkey, and others) palmetto persimmon. Texas poisonoak redbud ross. Cheroket sagebrush, fringed saltcedar tnimpetvine yucca buckbrush chemise hawthorue OFTEN BETTER CONTROL WITH 2,4,5-T mesquite oak. whita osagt orange radbay sagebrush, big snowbrush , swealgum yirbasanta 16519OOWN TO EARTH. Voi 28. No 4. Siirjp'i 1'J73 31 11111342109867........jTMpMMmcctpssKLHewucyNfGHGhtEr1aOreueepehaoeonle1ueeouoeoailaalaclrorenwsalngtgerr.tletlalyettru.nllEshobngtcrelt.ihstcCcdleyes3snr'iraooOoimAoslhnecosar'oloA:rrWckx..Bnn2Rtgsirwd.lalnorndnyaeePt-.rweu.H.DTnfrfJ.4eieEnSig.e^tWornal.H.0aii.hlssCuEoorx!ohtlWePltlm.eshM.ueagmnpa.D-ue.tO2ioan.9ieolAlrler..entrvtSEW3ee5ndce1hkdbsfsr.NRse.e..L.senil9rieimaea.tNlxwicoesoHv:6atCar1Newhn.saiorrnaeae01.nodl92datdotool.Rkn.xi.9nhydoef6t3/m.Wn1PC.od.h6tW2esd17-C9taAiC6ra2r11npero91.yS.P7oWpoo..l48e:eo59ncsfe2A1cptrnlr.D3Odn.4r6ipaotSo.efDel8.ltrt.v.fti7l.CtCnorcokeodCr-eeaHOiReo.2aoeAlrt.ldmocdWnaorxmlaa2ClWHneHoetnitShnenrih.nsWdeovaoorlfavoNhfg.feegBiiesefuennoatnmnoaesrerotrulystCtdlrebvgfweeyeTlarx.elabaaniso.dralnsO..ciberann11corcplalc.ioiCitdq1d99oePo1ofpoUeli1ooWtnE9u6et:olk4uclfr:hwoS9irm7o5Aas:cnetsH4ne64g8he1Dc..1taRdt-e2i2i1a7irae.ea2AcCtacaDAdo0T.2wi6Nedp6oaUqnrhl-HOT.-A.dapC-4pnluno4Ces1FchWiil3glsRuaonar8imiia2orao1aent5reatgns2eNh7nn8rnndogiii.ecls...tf 15. 16. 17. 18. 4tPOOt1PmNN1hu7:4oirweeeaer2etnk2pwwhre-gdce0el4nttoee.y-oofN7sf2.nnne5.woo4D..Mc.arW.iCtttukMahh.M.etodeciecDBefihSacdh.lO'esheKalvtsCWaneU1eeseel9orxR.Nln5n.O1E6CtT.D9Wr.NOo1o7O1e9luC29EueW6n.Hh6dp2AtHeN0ya.oRmC..ennTTdrCoiPbcHYObtnorahi.ocotYnl1eroiEgdo.1tekrrAe.bloe.rRPsiNaCslorTsdooitonaoH.inccrf4ffeaeooe:1pte2bfrri8eeood3i.gLsnnri-nolt2tcNergn4eyoaoosg:..f.ff TABLE 7. Comparative control of certain herbaceous weeds with silvex and 2,4-0. bedstraw buttercups blackeyedsusan duckweeds dogfannel fiddlemck litewaed fleabanas bluewaed. T in s carpetwaed cinquefoil croton deathcamas garlic, wild OFTEN BETTER CONTROL WITH SILVEX groundchany grounditry hemlock. poison hanbit horcaweed knapweed, spotted knawal lupines OFTEN BETTER CONTROL WITH 2.4-0 heal- all ironweed. Ww'sIpm knapweed, oift'-ise meilcar.tea mudplantain mustards pennycress. iiaid medic, black milkweed, cam^w^ nightshade, blyk-\ purslane spurgas sputry. cornCO wnodsorrel C O yarroft 1 o CO CO pineappleweed poorjoa puncturevine sage, creeping smartweed speedwell yellowrocfcat T A B L E 8 . C o m p a ra tiv e co n tro l ot c e rt a in w o o u y s p a d e s w ith s ilu c x ar.d 2 ,4-D . aider blackberry cactus cherry. './Id cbokacherry iliu Hickory buckeye buckbrush chemise OFTEN BETTER CONTROL W lfH SILVEX honeysuckle locust black maples mesquitl mulberry oaks osage orange palmetto persimmon, common poisonivy poisonoak raspberry redbud saltcedar tree ol heawen trumpetcraeper OFTEN BETTER CONTROL WITH 2.4-0 cottonwood manzanita rabbilbrush snowfarush sagebrush, big toyon 32 DOWN TO EARTH. Vol 28. No 4. Sfirmu 1973 I 16520 . \ -/ , \ Program Your A quatic W eed Control N eeds By CHARLES T. LICHY P la n t S cien ce R esea rch a n d D e v e lo p m e n t T h e D o w C hem ical C o m p a n y Winter Park, Florida 2549 D o 3 331083 li.w title of this talk is PROGRAM YOUR AQUATIC VEED CONTROL NEEDS. What docs it-mean to program? )o wc realize what the word program can imply? Webster lefincs program as a "plan of future procedures." An apt lefinition but one which does not completely describe all of heside cfleets of programming. Another delinilion listed for >rogramis "a doctrine, theory or system whose validity can )e tested only in practical application." This, perhaps, is ven a better definition for us working in weed control. Basially, however, the word programming means "what arc we tying to accomplish" and "how can wc go about with the ask at hand." One way to do this is to list what the problems light be in regard to the type of aquatic weed "problem" ou have. "Problem" would include not only the species that irepresent, but which species you would like to have present, ir perhaps what is the purpose of the weed control program, >cit drainage ditch, irrigation canal, farm pond, water fowl efuge or what have you. Other points that need to be listed; vhat arc the ramifications of your control program, is spray irift a problem, is it a water shed.area, how about fish popuation, is the area used for swimming, irrigation or what :!sc?One factor that must beconsidered is that you obviously :annot useadillerent material for cadi species present. Tlicrcorc, you must use a material which has a broad enough pectmm so that the primary weed populations will be emoved. Perhaps the next year, then, the material will have o be changed to pick up the resistant weeds that were not killed "by the first year's application. Another factor which may be important is ecology. After you spray one weed population, what changes can be expected in the population? And, of course, last but not least, are the economics involved. Of course, they are always important'but sometimes these are relatively important, sometimes they are relatively unim portant when looking at the total program. In short, pro gramming your aquatic weed control needs actually becomes very complicated when all factors of control arc considered. Let us examine some of these factors in detail so that wc can arrive at principles where these factors might be taken into consideration in programming our control needs. Per haps the most obvious place to start is which weeds do wc have to control or which weeds arc our problem. It is not necessary to go into any discussion on identification of the various aquatic weeds since these have been discussed in some detail before the society on several occasions. One point, however, that must be considered is the considerable variation in the time when the aquatic weeds are most suscep tible to chemical treatment. For some species like the Arrow- tahreumflo(wPecrliunmgdsrtaages .p pF.o) rtohethemropslat nstussscuecphtibalsethpeerrioosde is .during mallows, hibiscus species susceptibility is daunrilincgattthaeilsla{iTcylplohwa csrpinpg.) the and time of greatest the early stages of seed production. (Jiaiitcutgrass can be killed most easily during the period of maximum runner growth, which usually 16521 . -- \ DOW 331089 `xltiids about a monlli after llie initial flowering period. home, lawns, gardens, etc. Some of these problems can be ,Villi the phragmiles the most susceptible period is at the avoided by using well trained and well equipped spray crews. ini* of flowering or pollination, two to three weeks after the Spray crews should he instructed or shown how to exercise tassels. Cattails are most easily killed when in the late care in proper chemical application. Materials should be used lowering or early fruiting stage. In the south the tropical which have, a non-volatile nature. It is important to use low rallail often flowers about two months after the narrow leaf pressure to provide largest sized droplets that arc compatible .atlail. For this reason multiple treatments sometimes have with the typo of application and control necessary. The end x:cn required to control both species. Further north, however, use of the water should be considered. That is, whether the he period between flowering of the two species is much water is used for swimming, irrigation, drinking, fishing; hortcr. In Maryland, for example, tropical cattail flowers whether it is a water shed area, and some attention must be lbout one month after narrow leaf cattail and one treatment paid to this when selecting herbicides for weed control use. las given satisfactory control of both species. The best time Practical considerations must also be included in your or treatment of ncedlcrush occurs during the flowering stage choice of weed control materials. You obviously cannot use md lasts for three to four weeks. Regardless of all this, a different cct*..".ontr'l material for each species you may lowevcr, spraying can and should be done over awide range find along a ditch bank or canal. You must, therefore, use if growth periods since satisfactory results can be obtained a material which will control the primary species involved. md frequently less chemical is required because of the This may require over a period of time that two or more mailer size of the plants. Whether the plant sites arc flooded herbicides be used if eradication is desired. It may even be >rnon-floodcd can also play an important role in determining desirable to make combinations of herbicides so that all the :flective control from a herbicide. species arc properly controlled. In this regard attention must Aquatic plant control is also complicated by the need to be paid to the ecology, that is, the changing population after nsidcr the role of plants in different situations. Weeds arc a herbicide application. In the case of waterfowl refuges, lcscribed as plants being out of place. Thus, plants that arc where certain plants arc desirable, this can be made to work jndcsirablc in one location may be beneficial in another. for you. Table III shows the effect of two chemicals on the To the waterfowl manager a weed is a plant that does not ecology of two plots before and after chemical application. irovidc enough food or cover to justify the space it occupies. Note that before application the plant composition is approxi ror example, buttonbush is responsible for 70% or more of mately the same, yet after application, the pldnt composition he coppice growth which is a problem in the marshes and is radically different. .wamps of the lower Mississippi valley area. Yet in Mary- A farm pond presents a somewhat different situation in and, Virginia and the Carolinas buttonbush contributes to aquatic weed control since you have a substantially stable /aluablc waterfowl habitat. Phragmitcs also can have a place water environment as contrasted to a ditch or canal. This n waterfowl management. The root growth will protect a means that chemicals applied to the water remain in contact na luring siuuns. /msu, Uie piiragniites lurnishes excellent for along period cf time. This has advantages in that it.'.ends :ovei for both the hunter and the hunted. For example, in to make the chemical more effective in weed control but has last years it has helped to make the Thousand Acre Marsh in adisadvantage in that it continues the water pollution problem Delaware one of the best duck shooting areas along the for along period of time unless after a period of weed control Atlantic Coast. But solid stands are undesirable since landing the pond can be flushed with fresh water. Ponds have an ireas arc not available. additional advantage in that construction of pond edges that Since water is intimately associated with aquatic weed drop off quite rapidly and fertilization which shades the ontrol many problems arc unique to the water weed control aquatic weed serves as a reasonable means of weed control eld. Aquatic plants differ in physiology and habitat so sig- without chemicals. However, once weeds get into a pond, ificantly from terrestrial plants that one cannot confidently then chemicals must be used to eliminate them. Weeds may pply to aquatic plant control the information gained from get.into the pond from seeding upstream or birds scattering spcricncc solely with terrestrial plants. Water quality may seeds or a number of other ways. Water weeds often prow : an extremely important factor. For example, work done in water stored for irrigation. They clog outlet and inlet y The Dow Chemical Company shows the effect of pH on pipes, spray nozzles and irrigation structures. They also xmtail as shown in Table I. Similar results were also harbor mosquitoes and interfere with fishing. Many irrigation btained with potamogeton and alligatorwecd. Table II shows reservoirs arc shallow and have fiat bottoms and arc subject heeffect of calcium as well as the effect of pH. Notice that hepresence of calcium in the water, particularly at a pH of to frequent draw-down and refilling. In this case neither deep edges nor fertilization control weeds in such impound or above, improved kill of coontail. Water temperature lay not influence the reaction of a chemical to a significant cgrcc but the aquatic weed may be in their most active mreesnetrsvoibrsecaoursethoosfethien fwluhcitcuhatidnrgaww-daotewrnslevaerlsc. inInfredqeueepnet,r fertilization and deep edges may be practical. However, in rowth at higher temperatures and tiius would be more usCcocnpttaibmlci.nation of water by hcrbicidal chemicals isof prime nportancc. Naturally, all due caution should be exercised henusing herbicides. It is necessary to use those materials 'Well are relatively non-toxic to humans and Fish as well as ossiblc desirable plant species along the edges of the aquatic rca.^lL js thus very important to control spray drift; spray rift his instance being that physical drift, which through igh pressure spray equipment or otherwise negligent use of iray equipment, in which particles of the spray material 16522iovc onto desirable areas such as vegetables, citrus trees, irrigation pond:; extreme care must be taken so that waters which have heen contaminated with chemicals arc not used for irrigating sensitive crops. Lakes have similar problems and normally not being equipped with deep edges and not being practical in most cases to fertilize, the problems are considerably magnified. Let's discuss some of the chemicals that arc available for aquatic weed control taking into account some of the prin ciples and problems wc have discussed above. I'm sure all of you have seen recommendations put out hy Hob Blackburn and Lylv Weldon from Ft. Lauderdale. One of the chemicals' you will jv.iticc r^entioi- >i in almost all of the aquatic weed , The Dow Chemical Company effects as determined by water consumption, body weight ' :i.mi of silvex as the propylene gains and growth observations including gestation and par l is not a new material, h has turition in sheep. These data indicate that foliage or water ;-flies for aquatic weed control. treated with Kuron herbicide as recommended should not i'-v Kuron herbicide at 5 qls/acrc \ a: the troublesome submerged States. Experimental tests in . lions of the country evaluated .Mceniration on several aquatic ill 1958 andsubsequent years ::;:.tncc information. Kuron con- ..n in Table IV. > ..catiKonurocnanatbeI used. Emergent gal./100 gal. of : eemerged foliage as a thorough . for the control of submerged made by diluting Kuron at 1gal. - .lr.J directing this solution on or ;g pumps arc also often used to anJ pump it to the boom which on over a wide swath. Uniform .- desirable to allow a lethal con- mc into immediate contact with present a hazard from ingestion. Skin irritation tests con ducted upon rabbits have indicated that Kuron herbicide and its dilutions arc slightly irritating upon repeated prolonged o o contact. through In these the skin studies there was no evidence of absorption in acutely toxic amounts. The observation j %^ of reasonable care and personal cleanliness practices should"*" be adequate to avoid skin dillicultics. Kuron herbicide wlien"*^ dpirliumteadrytoirr1it%atiownithorwaatlelerrgoerni1c graels.popnesres1 0 0whgeanl. caused applied to ^ the skin of nay human volunteers using a repeated insultCO/ technique. In addition, the product was patch tested on aC3 second group of fifty human subjects at concentrations up to 50% without cutaneous reaction. Even when applied undiluted to the skin for nvo hours it caused no skin response. Thus it may be concluded with a high degree of assurance that when used as directed, this product should cause no skin irritation or skin sensitization responses. Kuron may remain in water for three to four months after treatment where no dilution occurs. Accordingly it is sug .ectionsof undiluted formulations : on a limited basis with varying gested that water treated with Kuron should not be used for spray purposes or for irrigation. 't completely safe to fish. Experis in several ponds have caused Thus Kuron appears to present a possible solution to a widevariety of aquatic weedproblems. It has awide spectrum of activity on both submerged and emerged weeds, is rela areas where high concentrations 'lications at concentrations of 5 tively low in toxicity to fish and other aquatic animals and it has been exhaustively tested throughout the years. The omc fish kill in a limited number -ep. no fish injury in over 99% 2 apid incorporation of the spray associated with rates in excess scientists at Ft. Lauderdale and other places around the United States have had Kuron in test in the laboratory and finieltdhetefsietldfo.rTthheeCcoornptsrool toEfnaglilnigeaetrosrwheaesdp.uSt ioncuet aKhuurogeti sdcoaeles vc avoided where fish kill cannot Jersey found that applications on oxygen content in treated have a wide spectrum of activity it is very useful where mixed stands of aquatic weeds occur. For example, in areas where alligatorweed and water hyacinth are intermingled, vith Kuron normally decompose Kuron offers a solution for control of both species. Many :n to twenty days arc required chemicals suchas2,4-Dandothers will control water hyacinth decomposition apparently docs but will not appreciably alfcct the alligatorweed. Kuron ' of the water. This is a problem offers economic and efficient control of both. vhich give arapid kill of weeds, For the control of aquatic and ditch bank grasses amaterial "tmenis with Kuron showed no generally recommended is dalaoon or the Dow trademarked enthic organisms. Applications product Dowpon or Radapen. This material, like Kuron, dpopulations of someplankton 'catmcnt the populations were ' plankton studied consisted of 'crious effect on large aquatic irtlcs in treated areas. Prolific ti soon after treating aquatic 'ides. Algae growth does not four weeks after treatment has been extensively field tested in a wide variety of locations and species. It is particularly useful in drainage ditches since grass and cattails can seriously reduce the How of water. Cattails arc particularly troublesome. They interfere with the proper utilization and maintenance of drainage and iaigation canals by reducing the velocity and volume of How, and by causing deposition of sill and debris. Safety can also be an important consideration. In weed clogged drainage ditches iranee of algae apparently is ' the aquatic weeds and thus water, is.held for a longer period of time after storms, in creasing die chance that small children may tumble in and Uothe water from decaying drown. In reservoirs, farm ponds, marshes and lake margins toxieitv and presents a low cattails waste large quantities of water, crowd out plants 1 use. The Kuron herbicide which provide food for wildlife and often iixcrferc with >1iiuspweiciieisnaTnadbtlhee following V. From that theie should be no fishing, boating and other uses of these bodies of water. An excellent bulletin on this has been issued by the U. S. Dcpailmenl of Agriculture, July 1963, entitled "Siutlics on the to die handling and use tide should he left where it. Cattle, sheep, swine, ' tiler containing Kuron ivriod with no adverse Control of Common Cattail in Drainage Channels and Ditches." These studies indicated that Dowpon herbicide was extremely effective in controlling cattail. Additional wwlourcklihsahsobwesentiireepeoffrctecdiivciniaM-ssichoifgaDno,wOpiuengoinn acnodntorothlleinrgstactaels- 16523 tr.iJs. Ted Hall of tin* U. S. Fish ami Wildlife Service reported TABLE IV the results applying Dowpon by aircraft in the southeast. In Aquatic Weeds Controlled by Kuron at 1.25, 2.5 and this lest Dowpon was applied to control cattail, maidenenne 5 Qts./acro foot. --d giantcutgrass. All showed good response. In addition, .vpon is non-to.xic to fish and wildlife species as well as 'domestic animals. Brush control can also be important in certain eases of aquatic weeds, particularly in ponds and ditch banks. Brush control has been done by 2,4-D, 2,4,5-T or combinations of both. All of you are familiar with these results. Recently The Dow Chemical Company has discovered a new brush control material trademarked Tordon. Although this material is not yet in the marketing stage for aquatic weed control, exccllcn brus control results have been obtained on utility rights-of-way. The material is also currently under test throughout the United States with aquatic weed workers. In summary--remember that aquatic weed control is perhaps more complicated and lias more ramifications than 1.25 quarts/acrc foot or less (0.5 ppm) While waterlily --........................... Pickcrclwecd 2.5 .q...u..a...r.t.s.../.a..c..r..c....f.o...o..t....o..r...le(sVso (n1tc.0d c pr ipamc)o r ( d Ny a ta m ph L .) a c a s p p . DOW 331Q91 Yellow water Mud plantain Water milfoil 5 .l.i..l.q..y...u.....a_...._r...t._..s.._../.._a...._.c.._..r.._.c..._..._.(f.o_M.._o.._y.t_.rdoi_o._r:_p_l_h_e__sy_sl_l u_((_m2f i e( N uphar spp tcra n th era phpcmt c )r o p h y l l .) spp. um ) ) FWBWClaoaaantdotecwdnrrwctsoraehwreitil_ocd__lr_dt________________________________________________:_________________________________________(____C______c____r___a(___At__(o_nCp_(a/_aDtcyb(r/lUtoalaumstrernibnisinai ai dt accesaramcirhnaoeralrcidssnbpetincnaprnsn.i))i)us ) ) Variable pondweed____________( P o ta m o g e t n d iv e r s ij o li u s ) terrestrial weed control. Be sure to take into consideration all the problems and possibilities of problems in controlling aawquaareticowf eyeodsurbycomchpelmicaictaelds. pTrhoeblecmhsemaicnadl caorempcaonniesstanatrlcy striving in research and development to oiler better solutions to those problems. It is up to each of you to be aware not only of the problems of aquatic weed control but also the latest solutions to these problems. Naiad__________________________(_P__o _t a__m_o__g _e_t ( n N a aj m as p lifu liu s fle x H is ) ) STpaipkeegrruasssh_________________________________________________(V__u_l_li_s n( E e l r eo ia c h a ari me s r i spp. cana ) ) BSBWouuafrltrtrcuerruscsshdtah___n_____v___o___r___i________________________________________________________________________________________(___S___c_(_i_Sr_pp(anC( rJsagl ilaanl inmet rineuiscrmihcccafsjnpusapspsu.p))s. ) ) TABLE I Effect of pH of W ater on Silvex Effectiveness Sifrex Kill pH ppm rating 5.0 0 0 7, 0 0 9.0 0 0 5.0 3.5 10 7.0 3.5 8 9.0 3.5 3 TABLE V . Animal Species Rat Ciiiinr.n pig R ab b it Mnnsr: Chicks Ltl V alues 50 mg/kg 1070 . . 850 850 7140 2000 TABLE II Effect of Ca an d pH on Response of Coontail to Silvex at 2.5 ppm Co Kill ppm pH rating 0 5 10 0 77 0 92 250 5 10 250 7 9 250 9 8 500 5 10 500 7 9 500 9 8 TABLE III Plant Composition as Affected by Chemical Treatm ent rordgrass libiscus )ock ' -'yp'- . 'lunrlwvvd Plot 1 Before After 96% 3% 1% Trace 1% 98% 1% Before After Plot 2 95% 3% 2% Trace Trace 20% 70% 2% 5% B ib lio g ra p h y 1. WBualtl,erEfodwwlinA, r"eRaessuolftsthine ASpouptlyhienagstD,"aDloOpoWnNbyTOAirEcAraRfTt Hto, SPuemstmPelra, n1ts95i8n. 2. DDaOyW, N8. TE.OanEdARSTwHe,zcSyp, riAn.g W19.,56"C. ontrol of Phragmites with Dalapon," .3. CGuotogdrarusms , inPhEilaDst.eranndTeGxraasy,," CDhOarWlesN ET,,O"ExEpAeRrTimHe, nWtalinCteornt1ro9l58o.f Giant 4 coFGeofribngthrtsrueobalyrMy,oicfB1h.9Cig5Ha5a.t.,ntaRiAlc,gimTriyecpru,hltaCur.5apHl p.E.,xapnbedyrimCCheuentlmterSic,taaWltio.SnpA,r.aV,yo"sOl,."b3Qs7eur(av3ra)tteiporplnys4B0o0un-ll4et0thi6ne, 5. HDeOaWthN, RT. OG. EanAdRTRHu,chW, Lin.tCer., "1A9e57ri.al Control of C attail W ith Radapon," 6. YFfiaeourrcnkea,",OPMfr.oTcwE. o.,NE".TExh.pWee.rCimE.Cfefen. ctat1l2o:Afr3et3ah8se-3oW1f3c,Lcod1ni9gc5id8P.conKdu, roDnuchoenssthCeouFnlotyra, Naenwd 7. oPfierLcoen.gMP.onEd,,, "PDruocghreessss CRoeupnotyrt, oNf. thYe.,"EfPferocct. oNf .EK.uWro.Cn.CU.po1n4: th4e72-B3io7t5a, 1960. 8. SAtucgcunsist, J3.0 Ha,,nd"Aq3u1,atiIc9,6W0,eeTdheCoPnetnrnosl,y"lvParnoicaeeSdtiangtes FUonrievsetrrsyityS.ymposium, 9. TDirmaminaognes, CFh.anL.neclst aanl d"SDtuidtciehses,o"nUt5hDeACToenctrhonl icoafl CBoumllemtoinn IC2Sa6tt,aiJl uliyn 1963. 10. aWnedldiotsn,CLo.nWtro.,l,""AUSSDuAm,mCaorrypsReovlieEwngoinf eIenrvse, sCtiKga3ti3o-n6s0 oSnepAtellmigbnetor rw19c<ri0d. 11. OWtill1s9c6,0 Mm.eeGti.n. g"Aonlutnhleic WVeeegdetaStoiocnietyCoonf trAoml eWricitah. Kuron, Presented 16524 r* ; . t. } ' i . i^' ^_.. . > c?\. J# 'Mj-:*.- i 2 5O : o 03 03 J 1 I i U.ik*jU*t.lu O ff fK 4 CONTROL FOR UNW ANTED VEGETATION IN LA K E S A N D P O N D S Th e d efin itio n -o f-thumb says a weed is " a plant out of place." A weed can be out of place in a garden, in a lawn, in a farm field . . . or in a lake. Finding suitable methods of controlling unwanted aquatic vegetation has been a goal of agricultural chemists, conservationists and water sports enthu siasts for years. The nature of aquatic weed problems depends largely on one's aquatic interests. Odors that ac company excessive aquatic weed infestations are offensive to sightseers. Vegetation can jam boat propellers, trip water skiers or ruin swimming spots. Anglers know that excessive vegetation can destroy fish spawning beds or unbalance fish populations. And to industry, excess aquatic growth in cooling ponds can interfere with circulation of cooling water. T o qualify for aquatic use any herbicide chemical mu*' meet rigid specifications. First, it must do a got ,ob of controlling aquatic planta. Secondly, >> the chemical must have a wide margin of safety to birds, animals, fish and humans. The recent acceptance of K u ro rt as an aquatic weed control agent marks a big step forward in this field. K u ro rt has been known for years as a brush killer, especially effective on hard-to-kill species such as oak and maple. The new use registration has been granted by government regulatory agencies on the basis of performance coupled with substantial safety in use. The story of Truesdale Lake shows the possibili ties for K u ro rt in this new field. Truesdale Lake is an attractive body of water in New York State. The lake is ringed with summer homes and was a popular watering spot until it became choked with weeds which virtually ruled out swimming, boating and fishing. Members of the Truesdale Lake Property Owners Association faced decreasing fur. and declining property values and f Kepnnfed from A pril 1959 Oow Diamond . 152! TW 331Q86. of Trwesdole LpUe in New York before treatment nov vegetation. The lake ha an area of 88 and iff'ihores are populated with summer homes, i t pout several years weeds hod interfered / a vn g ly with swimming, boating and fishing Treating the lake with Kuron to remove vegelotion. A mixture of the chemical and wafer w o i applied as o coarse spray over the surface o f the lake. Karon form ed a white emul sion in the w ater, persisting fa r about 20 minutes and elim inating the need fo r m arker buoys. Kuron is e ffe ctive in co ntrolling same o f the most obnoxious oquofic weeds when as little os two ports per m illion o re a p p lie d in lake water. The treatm ent was made by Robert Sheridan knew that something had to be done. " Something" proved to be a big order. Various materials were tried with discouraging results. When association members heard of successful tests in New Jersey using K u r o n to control aquatic weeds, they deter mined to try the method on Truesdale Lake. Necessary arrangements were made with the City and State of New York to make the application last summer. Since the lake is a part of the watershed serving New York City, special arrangements were made to measure chemical concentrations in the water. Th e program was conducted under the general supervision of Dr. E. C. Raney, professor of fisheries biology and zoology, Cornell University, with assist ance from R oy R. Younger of the N ew Jersey Department of Conservation. Robert Sheridan, a custom spray operator from Dover, New Jersey, was engaged to make the treatment. The entire operation came off with no adverse effects, save a minor kill of fish. This seems almost inevitable in aquatic vegetation control work since decaying vegetation ties up oxygen in the water. However, K u r o n kills aquatic plants gradually, hold ing this problem to a minimum. A t Truesdale Lake, cottage owners were glad to overlook the slight fish kill in favor of greatly improved fishing conditions. And this past summer, for the first time in five years, boating, swimming and fishing were possible and pleasant in July. Four weeks a fte r application the lake had become 10 dea r that the bottom could be seen in many areas. O bservers wotching the operation re ported a groduoi kill o f w a te r weeds. In a b o u t a week a fte r application plants be gan turning dow n, with most weed going to the bottom tw o weeks or so a fte r a p p li cation 1652 f[Il ! I - [ i S ____ / Tc <L d ' 71 -'-w \171 Kurze Origin- 'mittcilnngcn y A .i/iu.vj vi<*it ie iaten iiunr> . even w hen duxes f.ir s u p e r io r In the l . l ) ^ Arc a d of the C o in m itte e on ( nuinu n.e (W.S. Selnlatlio'). April 7 a n d l 5. ministered). mid high potencv ns :in e n z v m e inilucer 4|, li)70. 11.S. G o v t. I'r in tin g IMluo. W as h in g to n . I > .. . 1 ,)7ii, / suggest th a t this eiuilil l>e n( a c a s c a d e - t y p e involving a series p. 1 cji 1. -- | j | I ligginlKilhain, (i. K.. rl at.. N a tu r e 220, 7112 of biochem ical lesions. W'c ha v e th erefore investiga te d the (1968). -- [4) Iiiu-IIo. N. I'.. at.: (..It. Acad. Sci. (I'aris). effect on the enzym e e q u ip m e n t of r a t s i n to x ic a te d w ith (I), Sr. D 272. 1447 ( 1'>71 ). -- [5] H eilm an. S., Krankel. S.: Amcr. so as to define the targets of this biological damage. J. Clin. I'a th . 28. 56 {1` 57)- -- Id] S h a p ira. G.. ft al.: Scm. A d u lt W is ta r r a ts ( i n j a n d lO V) weighing 2 t o ;h 20 R were ogiven a single dose of lit mg of (l)/k g in olive oil l>y intra - H 6p. Paris 29. 1 0 1 7 ( 1 953). -- [7l W rohlcwski. et a l.: Proc. Soc. l'.xper. Biot. Med. 90, 21 0 1195)). -- [8 ] Kosalski. S. H.. peritoneal injection: the controls ( I 0 J a n d ID?) received the W olk insnn. J. H . : N a t u r e 188. l i m ( i 960). -- [9] U o da nsky. solvent alone. B oth t r e a te d rats a n d c o n tro ls received a norm al O .: J. Biol. Chcm. 99. 197 (19)2). -- [ 1 0 ] V incent. I).. et at.: KiVsi,lift a n d w a ter m l libitum . After in d a y s, blood sam ples were Ann. Biol. Clin. 18. 4 X9 ( 1960). taken liy cardiac puncture and the levels of the following scrum enzymes were measured: glutamic oxaloacetic trans aminase (G.O.T.) [$j. glutamic pyruvic transam inase (G.P.T.) {5. aldolase (Aid.) [6 ], lactic d e h y d r o g e n a s e (L .D H .) [ 7 ], h y d r o x y b u t y r ic deh y d ro g en a se (H b.O M .) [K], alkaline phos-. ^ Organs as Targets of " Dioxin" (2,3, p h a ta sc (A.P.) [9 ], arvlestcrase (A.K.) [ 1 0 ], an d cholinesterase c_h_ lorodjbenzo-ji-dioxin) Intoxication - 1 etra- (C.h.K.) [ i n ] . T h e results, a n a ly z e d s ta tis tic a lly b y S t u d e n t 's / " T r _ ,, i7'VN ,, e t-test. are gwen in Table 1. Pham -Huu C h an h (M ,ssM ; . Scsquo. (Mrs.) M. C. A z um -G ela dc, and G. b a in t - K u f O CO "< CO Centre National de la Recherche Scientifique, Paris, France Table 1 . Effect of (1) on serum enzyme levels Enzyme Sex* Controls Treated S.G.O.T. S.G.P.T. Aid. L.DH. Hb.DH. A.P.b A.E.C Ch.E.c O* -i- ? 65-21 4.19 0+9 32.SS X 1-55 + 9 2 7 . 4 S 1.74 0 + 9 473-60 31.73 + 9 250.44 2S.01 + 9 ' 17-S6 1.22 333-20 19 05 i ? 3 1 6 . SO 14.50 U . 16.88 2.13 18.48 1.61 344.3S 40.089 43-64 3 879 29.30 2 .S 5e 716.33 76.S49 339 92 41.02 1 17-14 t . 2 6 e 1 65 .60 i 2 S.1S 1 9 2 1 3 .60 6 .73a 4.40 O.649 3*22 O.359 In previous papers [ 1 . 2 ], we showed by m eans of enzym e studies th a t the liver is a main target of intoxication by the extraordinarily potent toxic and teratogenic agent "d io x in " (2. 3. 7.S-tctrachiorodibcnzo-/>-diuxin) (1). T h e p r e s e n t p a p e r supplies anatomo- and histopathologic evidence of damage to this organ, ami to other organs which we have also found to be profoundly affected within the short duration (10 days) of our experiment. W is ta r r a t s (10<J a n d 10?) w eighing 2 l 0 2 0 g w ere given a single dose (lOm g/kg) of (1) by intraperitoneal injection in olive oil; t h e c o n tro ls ( 1 0 c? a n d 1 0 ?) received the s o lv e n t only. AH'the animals received a normal diet and water ad libitum. The treated animals lost considerable weight as from the Expressed in nilL'/ml. b Expressed in Bodansky units. 0 Eor A.E., results expressed in nig of phenyl acetate hydrolyzed in 1 h b y 1 ml ol; se rum ; for Ch.E .. results e xpressed in mg of a cetylcholine hvd ro lv ze d in 1 h l>v 1 nil of serum . d Sta tistically highly significant. * Non-significant. ( P < 0.02. * S e p a rate fig ures for 0 a n d ? given only when there arc statistically significant differences. T ab le 2. E ffects o (I) on blood sc ru m c o m p o n e n t s ir, r a t s (<J a n d ?) Component Unit Controls Treated Glucose Cholesterol L'rca Total lipids I*ruU*in Ihlirulmi Sodium l 'oiasbjum Chloride K/l I . 1 7 0.04 K/l 2.37 0 . 1 R/l O.34 0 . 0 2 k/l 3-79 0 .1 5 K/l 69-79 l.SS i n ^ / lo o nil 3- l o ti-3 J"l,liK:>qi//1i *146.11 m 2.4 1 4.S 7 U.30 102.4 O.71 0.68 0.03* 2.65 0 . 1 5 " 0.4 3 0.03c 6.25 0 . 4 J9 57.1 1 . 9 5 23.57 4.899 139 25 5.42 U3.-4311 c 996 j;l.5 b P < 0.001. `' N on-sig nificant. c / ' < 0.02. 11 H ig h ly signif icant. *i-/. V V N j-A -v..VtdfY.-.-eh- v''i-:'-'I-"a ^V.. \ V .,,____ - ~ -?. 'v*\ a *<-( . .1 I,'"..,? Q* f ' ^ v V .>V : It is evident from T a b l e ! t h a t " d i o x i n " produces deep perturbations m several enzymatic systems in rats; the dis turbance in.iv be a decrease (particularly remarkable in the case of cholinesterase) or an increase (particularly considerable in the case of S.G.O.T.). T able 2 . which records the effect of (1) on the m ain c o m p o n e n t s of blood serum, also shows a profound disturbance in the h o m eo s ta sis indicating, tnter lo'oi. t h a t the liver is one of the main targets of " d i o x i n " intoxication. 1 1 istopatholoeic evidence of this damage to the liver ami to other target organs (notably the th y m u s and heart) will be picser.ted in a separate paper. Keccived J a n u a r y 17, 1972 [ 1 ] B u u - l lm . N. 1'.. et a t. : C.K. Acad. Sci. (Paris). Sdr. 17 273. 70S (1971). - - 1 2, !-!>,,, ru. 22.$ p g / k g in the ra t. see V errett. J.. in: Effects of 2,4,5 T o n Man and the E nvironm ent. Hearings f e w ? A: ;STy v \,- /.r:I*4. . \! .Yav- * :* 4. < . N'; X* , I:;g. 1 a a n d b E ffec t of " d i o x i n " in to x i c a t i o n on liver and tliynu.s. a) Hep.ilucvte deterioration: steatosis, k.iryocl.isi.i, voluminous pkismodial (nrms ( x 25u). b) Inverted picture of th y n u ja - ^ t r u c t u r e ( X (i2. 5) ZU'J g ., lie ft 4. 1071 nuchlftcsprcch'i'tKcn 175 2n<l il.iv of tin* <*\|HTimMit. ami lv tin* lutli day thev writhed u> n*i ^ U">> ill.m a I tli' Mart, ulifrv.is llir rnntrnls had ";nnr<| (41- 7 -6 I iarm.iltijn^ic r\amiiMli<>n >*Iim\t*tl a lii^lilv signif icant increase (Stiulciu's /-test) m the haematnent (controls: 3*>.5< .i. <>*>; treateti: 4*i.2$ }2 37). siynificnnt increase in the leukocyte count (controls: 5iS; treated: 73 1 5 :^ 4 5 1 ) with a decrease of lymphocytes (controls: 79% 1.64; treated: {!%; 11) and augmentation of polynuclear neutro phils (controls: i.S; treated: 37-3x11). However, no significant effi*ct was made on the lionc marrow within this hrief period. All the trea te d anim als had hepatic lesions, the females being more severely affected. The lobules were reshaped by im portant centrihdnilar stasis ilis.viciatm^ the Kcmockc trabe culae (pronounced amsokaryosis, frequent binucleation. cyto plasm leaded with chromopholx' vacuoles and hvnhne inclu sions). and by a focal hyperplasia of the Kupffer cells; along side the hinucleated iiepatocvics. voluminous plasmodes were often present. These findings were already prominent by the Ath flay (Fig. 1 a). Apart from these hepatic lesions, which confirm the biochem ical findings rejxrted ; i. 2 two o th e r organs manifested con siderable involvement. The modifications sustained by the t k \ t i : u i ranged from dhfused pvknosis of the thym ocytes to an inverted picture of the structure (migration of thvmocytcs towards the medulla) or total involution: these lesions were manifest as early as the $th d:iv (Fig. I b). This thym us involve ment explains the particularlv pronounced toxicity of (1) in young animals. The third organ to undergo considerable change was the heart. the anim als presenting a valvulitis with erosion of the cnditciirdiinu on th e v e n tricu la r f.tce <f t h e valves, fibrinoid throm bi, and oedema of the vn!\es u iu ili were thickened by fibroblastic proliferation. Focal leMons located deep in the myocardium were also observed, with a dis appearance of myofibrillae and interstitial polynuclear in filtration. At the same tune, intra-arterial fibrino-Ieukocytithrombi, pcriartciiolar fibroblastic reactions, and foci of alveolitis were seen in the lungs. These target organs suffered qualitatively the same damage when a considerably smaller dose, i.c., i mg/kg, of (1 ) was administered. The pathological deterioration observed in the liver and heart, and. to a lesser degree, the lungs, of rats, is similar to th a t reported by Allen and Carstens [3] in an experim ent with monkeys on a long-term diet which included a toxic fat con tam inated with a "cluck edema factor" which was later found to contain chloro derivatives of dibcnzo-p-dir.xm including (1) itself f4); the th ymus was not included in their study. th a n k Dr. S. S. E pstein, Cleveland, Ohio, for helpful dis- ycussions and advice. __ _____ Received J a n u a r y |S, 1 972[l]*4 [ l ] B uu-H ot. N. P.. et air. C.R. Acad. Sei (P ,, n s), S*'*r 0 272, 1447 (197U- -- [2] B uu-H oi. N. P . pcl ai. : Natr-rw rsscnschaften 59. 1 73 ( 1 972). -- [3] Allen, J. K.,, Cars*..ms, L. A.: Amor. J. G. ( S);Vet. Kcs. 23, 1513 (iyo7); i-ab. In v e st 15, 970 ( i 960). -- [4] Higginbotham. K.. e`. at.: N a t u r e 221. 702 19 6 Cantrell, J . S., el c l.: A c ta Cryst. 25 B. \ '0 (1969). Buchbesprechungen O tto H a h n -- Eine Bilddokumcntation. Von Ernst Berninger. M n c h e n : H einz .Moos 1909. l o S . . 1 37 Abb.. DM IS.-- . Das Buch ist ein dokum entarischer Bericht ber einen auerge whnlichen .Menschen und sehr orluigreichen Forscher. Die bebilderte Biographie ist in sich vollstndig und umfassend, wenn cs auch zu beklagen ist. d a historische, wissenschaftlich interessante D okum ente und ein Teil des Schriftwechsels im Arbeitszimmer Otto Hahns durch Kriegseinwirkung ver nichtet worden sind. Hahns Charakter, sein Charme, seine Klugkeit -- Weisheit -- sprechen aus dem Buch, wie wir. seine Schler, ihn k a n n te n . Ein W issenschaftlcr hohen Kav.gcs, sicher im Leben und seiner Umwelt stehend, kontaktfreudig und heiter erscheint er uns als eine f'crsnlichkeit. die in ihrer Art einzigartig war u n d lilcii>en wird. Ein interessantes B uc h l>cr einen D eutschen, aber* zugleicn auch ber eine vergangene und vielseitige :-.|iochc der hohen Zeit der Wissen schaft in Deutschland. W. Scelmann-Eggcbcri (Karlsruhe) Elastische, piezoelektrische, plczooptischc, elektro optische K onstanten und nichtlinearc dielektrische S u s z e p t i b i l i t t e n v o n K r i s t a l l e n . Von K. Ucchm ann. H. IC S. lle .irn u m u n d S. K. K u rtz (I-andnlt-B ornstcin, Neue Serie. O r u p p c l l l . Bil. 2. H rsg. K .-H . Hellw egc und A. .M. Heilwege). Beriin-1 icidellicrg-Xcw Y ork: Springer )9y. 232 S.. 196 Abb.. DM 136.-- , S 37 -t". N ur 3 Jahre nach dem Erscheinen des ). Bandes der O ruppc III ..Kristall- und J-Ysikur|x r|ihvsik" (Neue Serie) h a t sich ein Ergnztingsband als notwendig erwiesen. Er bringt Nculicsliinmuugeii und Erg.iii/iingen zu den elastischen, piezo elektrischen. piezo'iplis, heu und elektrooptischen Koiisl.iiiten. sowie zu deren 'I e m p e r .itu r - und D rut kkorlli/.ienten. Die Literatur ist bis etw a Milte lot.S erfat. Der vorliegende . Band ist aiillerilem stofflich erweitert um Angalien ber die Klastizitalskonstanten dritter ( Inlnuiig, ober die Erzeugung optischer f tlji-rweileu von l.iclil in Kristalli n (unter Einschlu einzelner sehr genauer Messungen von rctluingsindi/rs) und liier elektrooptische Konstanten zweiter Ordnung. Die Kapitel werden zugnglich gemacht durch klar abgeiate ausfhrliche Einfhrungen, die aus der ersten Ausgabo (gestrafft) bernommen sind. Der Zusammenhang zwischen den kristallocranhischen Eigenschaften wird durch vielfache Querverweise hergestellt. Ein gemeinsames, naeli Br jttoform cln alphabetisch geordnetes Subs tan Verzeichnis erschliet und ver bindet den Inhalt beider Bnde. Leider sind hierin keine Hin weise auf die o r t der nacligc wicsencn Daten enthalten. -- Beide Bnde gehren zusammen und sollten in keinem Institut fehlen, das sich mit Festkrperphysik oder Kristallographie beschftigt, sei es grundlagcmig. sei es hinsichtlich der Entw icklung neuer physikalischer Bauaggregate. Gerade die neu zugefgten Kapitel sind in diesem Arbeitsbereich von besonderer Aktualitt. A. N euhaus (Bonn) T heorie und praktische A nw endung von Komple:cblIdn e r n . (M ethoden der Analyse m der C hemie. Ild. 9). Von B. U m land. A. J a n s s e n . D. H u e r i g und G. W n sc h . F r a n k f u r t / Main: Akadem ische Verlagsgcsclisci.aft 1971. 760 S., DM 1 50. T h is trea tis e h a s 2-P>7 rcievcnccs and offers an e x te n siv e discussion of complexes, mainly with iiiultidentatc organic ligands, in gravimetric, volumetric, photom etric and fluuromctric quantitative analysis of virtually all elements. The theoretical in troduction is ra th e r weak a n d relies on the a r chaic h y b r id iz a tio n t h e o r y : thus. 011 page 1 3 . the possibility is left open that square-planar (tms redundant worn ought to b e " q u a d r a t i c " ) c o p p e r (II) c o m p le x es c o n ta i n one -t p e lec tron. On the other hand, the general description of what ligating atoms arc preferentially bound to a given central a to m is quite instructive. The tex t contains detailed prescrip tions for a large n u m b e r of s(>cci(ic m eth o d s, a n d highly useful tables. The sources of errors and interference are carefully discussed. This book, representing a tremendous am ount of critical stu d y of tlie literature, can tie recom mended to everybody interested in inorganic analytical chemistry. A specific asjicct is th e discussion of d ip h cn y llin ra lc derivatives. C. K. Jorgensen (Genf) C o l o u r C h e m i s t r y . B y R. L. M. Allen. L o n d o n : Nelson 1971336 S.. 5- Ungeachtet der lawinenartig zunehmenden Bedeutung der Kunststoffe bleiben die technischen F'arbstoffo ein wichtiges, iu me ratio ol "ca on tnc i u m j c 01 yrc pit sac tne number ofjumps made was counted M. F. Holick. J. E. Frommcr. S. C. McNeRl. N. M. Richland. J. W. Henley. J. T. Polls, It.. Biorhcm. Biophvs. Res, Commun. 76. 10/11977). 2. M.>F. Holick. N. M. Richland. S. C.hlcNeiU. S. A: Holick. J. E. Frommer. J. W. Henley. J. T . . Polls. Jr.. Biochemistry 1*. 1003 16979). J. M. F. Holick. J. A. MacLauphlin. M.B. Clark. J. T. Pons. Jr.. R. R. Andersop/ I. H. Blank. J. A. Parrish. P. Elias. Science 210. 203 (1980). 4. T. Okano. K. Mizuno. Hr Matsuyama. N. Nobuhara. T. Kobayashi./RrW. Trav. Chim. Pavs-Bas. 98. 2J3 (1979)./ 3. J. W. Mclski. L. Tanenbaum. J. A. Parrish. T. B. Fitzpatrick. H- L. Blrich.7. Invest. Derma tol. 68. 328 (1977). `T he criteria for patients with skin types I. I l /l I I . and IV. generally of European cxtraction/are based on the history of the usual reaction t / t h e first hour of full sun ex posure in early summer. Skin type V generally includes patients/of Asiatic. American Indian. Mexican, and Puerto Rican extraction. Skin type V] generally includes patients of African extraction." / 6. I. H. Blank. R. O. Griesemer. E. Gould. J. In vest. Derma/ol. 29. 292 (1957). 7. R. Belsey. M. B. Clark. M. Bernal. J. Glowacki. M. F. Holick. H. F. DeLuca. J. T. Potts. Jr.. Am . J. Med. 57 . 50 (1974). 8. D. Schacter. O. V. Kimber. H. Schenker. Am . J. Physiol. 200. 1263 (1961). Male weanling Holliman rats were fed a diet deficient in vitamin/D but containing 0.47 percent calcium and 0.3 percent phosphorus for 4 weeks. SIM refers . (serou! side) to the incubation medium on the outside of the gut sac (mucosal ^ide). There were six animals in each group; the values are reported as the mean - standard error. 9. W. F. Loomis. Science 157. 503 (1967). 10. F. Daniels. Jr.. P. W. P o s t . / . E. Johnson, in Pigmentation: Its Genesis and Biological Con trol. V. Riley. Ed. (Appleton-Century-Crofts. New York. 1972). p. 13. / It. At the time that Loomis proposed this theory, it was not known that during exposure to sunlight, vitamin D, is not directly formed in the skin (/- V) and that vitamin D/must undergo a series of regulated metabolic / hydroxylations to I-25- dihydroxyvitamin D/before it is biologically ac tive U2). / 12. E. Havinga. Espenentia 29. 1181 (1973). 13. M. F. Holick and7!. T. Potts. Jr., in Harrison's Principles o f Internal Medicine. K. J. Issel- bacher. R. D./Adams. E. Braunwald. R. G. Petersdorf. J./D. Wilson. Eds. (McGraw-Hill. New York. 1980). 14. S. A. Leviniohn. Am. J. Dis. Child. 34. 955 (1927). / 15. lniematiodal Commission on Illumination. CIE Publication No. 20ITC-2.2). 1972 (Paris. 1972). 16. We thank R. R. Anderson. J. A. Parrish, and J. T. P o t/. Jr., for helpful advice and assistance, and L /B . Fred. J. S. Adams, and S. A. Holick for editorial assistance. Supported in pan by a NASA grant (NAS9-15205) and N1H grants (Aty25395-01 and R01-AM27334-01). 8 July 1980; revised I October 1980 by eye. We scored ambulat^iyjfor.spme groups by dividing the floqC,c2)tti)t`:box into quadrants and counting the number of times the chick crossed into a new quadrant. We tested visual discrimina tionlearningonday 9oflifeby usingthe " pebblefloor" test (4 ), whichconsistsof asearchfor grainsoffoodscatteredover abackground of small pebbles adhering to the floor. Within a 60-peck trial, a measure of visual learning is generated z by recording thenumber of incorrect re- sponses (pecks at pebbles) within con- ^ secutive 20-peck intervals. ' ^ General activity, jumping, and visual learning rate were altered by 2,4,5-T ^ treatment (Fig. 1). Sincethe samedoses < wereadministeredonday8 andday 15of - incubation, these data could be exam ined by a two-way analysis of variance (that is, dose, age, andtheir interaction). Aone-wayanalysis ofvariancewasused DOH 2123634 for the data from day 2 after hatching. General activity scores conformed to a 2,4,5-Trichlorophenoxyacetic Add Causes Behavioral parametric distribution, butjumpingand learning scores required logarithmic Effects in Chickens at Environmentally Relevant Doses transformation. Jumping behavior proved to be the Abstract. A d m in is tr a tio n o f th e h e r b ic id e 2 .4 ,5 -tr ic h lo r o p h e n o x y a c e tic a c id to in most sensitive parameter affected by c u b a tin g c h ic k e n e g g s a lte r s b e h a v io r a fte r h a tc h in g . S in g le d o s e s , w ith n o m o r p h o 2.4.5- T. For those treated on days 8 and lo g ic a l e ffe c ts, re ta rd learning (lo w e st d o se , 7 m illig ra m s p e r kilogram o f b o d y w eig h t) a n d in crea se general a ctivity (27 m illig ra m s p e r kilo g ra m ) a n d ju m p in g (13 m illig ra m s p e r kilo g ra m ). D a y 15 o f in cu b a tio n is th e m o st su sc e p tib le sta g e o f d e v elopm ent. Asfar asweknow, therehasbeenon The median lethal dose (LDM) on day 15of incubation,jumping showedboth a significant dose effect (F = 2.78: d.f. = 3, 90;F < .05) andsignificant interaction between age and dose (F = 3.33; d.f. = 3, 90: F < .05). Comparisons of the groups given the two highest doses ly one earlier study reporting teratologi- 2 after hatching was 200 mg/kg. and on with their control groups (/-test) showed cal effects of 2,4.5-trichlorophenoxyace- day 15 of incubation it was 53 mg/kg. a significant increase in jumping in the tic acid(2,4,5-T) onbehavior(/). Admin There was 70 percent hatching in eggs groups treated with 13 mg/kg (F < .05) istration of 2,4,5-T (100 mg/kg) to- given 2,4,5-Ton day 15of incubation at and 27mg/kg (F < .01) on day 15of in pregnant rats produced no morphologi dosesup to27mg/kg. Some5to 10per cubation, but there were no significant cal deformities in (heoffspring, but it did cent of the hatched chicks, irrespective differences inthosetreatedonday8 (see cause themto have learningdeficits and of dose, showed abnormal leg develop Fig. 1). There wasalsoasignificant dose to exhibit increased activity in the open ment: theyeither draggedonelegor held effect for jumping in the groups treated field. We have shown that even lower it off the ground. Approximately 5 per on day 2 after hatching (F = 4.06; doses can produce behavioral abnormal cent of thetreated chicks showed depig d.f. = 2, 20: F < .05), and a /-test of ities in the chicken. A single dose of mentation of feathers and down. Both datafor the control group and the group 2.4.5-T (Sigma; containing 0.03 part per of these effects have been reported (5). receivingadoseof 150mg/kgrevealed a million of 2.3,7.8-tetrachlorodibenzo-p- No differences in body weight gain were significant increase in jumping after dioxin (TCDD). accordingtotheAustra observedinchicks receiving2.4.5-Tdur 2.4.5- T treatment (F < .025), this dose lian Government.Analytical Laboratory] ing incubation or at doses less than 150 beingtenfoldhigher thanthelowest dose was administered to the chicken egg on mg/kg on day 2 after hatching. found to be effective on day 15 of in either day 8 or day 15of incubation (2). A third group received 2.4,5-Tby intra- The chicks without apparent morpho logical deformities (after doses shown in cubation. General activity, which includes mea peritoneal injection onday 2 after hatch Fig. 1) were given behavioral tests in sures ofjumping, ambulation, and other ing. The 2.4,5-T was suspended in a0.5 week2after hatching. Thetester wasun movements, shows asimilar tendency to percent solution of gumtragacanth made aware of the treatment that each chick increase after 2.4,5-T treatment on day with sterilized, distilled water. Control had received. There were 8 to 20chicks 15of incubation andday 2after hatching animals (matched for each separate in each group tested. We measured the (Fig. 1). Analyses of variancejust failed hatching) received the vehicle alone. Doses were calculated according to the weight of the chicken or eggat the time of injection and ranged from7to 53mg/ kg for eggs and 75 to 225 mg/kg for chicks after hatching. activity in anunfamiliar environment ori day 7 of life by placing the chick in a gray-walled box(30cmby jfl cmuith25cm walls). Cumulative activity for a 3minute interval ("general activity") was scored by anAnimex activity meter, and toreveal significant effects, but /-tests of data for the control groups and those croups that had received the highest doses at each age revealed asignificant elevation of general activity in the groups treated with 27 mg/kg on day 15 SCIENCE. VOL. 211. 6 FEBRUARY 1981 0036-807? 8 0306-0?93?00 ?0 0 Copyright 1981 AAAS 1652S  E 9 E Z I 7 . 140 Q kiwaiiuii \r ^ .uii anamosereceiv cant effects werepresent after treatment which is 2 .S liler/ha of commercial 80 ing <50 mg/kg on day 2 after hatching at the other two ages. percent 2.4,5-T(weight tovolume). 1 m1 (P < .025). There were nosignificant ef Day 15of incubation appears to be a would receive 224 mg of 2.4.5-T. Since fects after treatment onday 8 . stage of maximum sensitivity to the be the surface area of an egg is approxi 'Ambulation, the other component of havioral effects of 2,4,5-T. Neuronal cell mately 50 cm2, the dose of 2.4.5-T re genera] activity that we measured, did division is at a peak on day 8 of in ceived by one eggafter spraying would not show any dependence on dose and cubationandislargely completedbyday be 1.1 mgor 22 mg/kg, which is greater io significant differenceswerefoundasa 15when synaptic proliferation is occur than thelowest dosefound to bebehav- result of examination of the data by t- ring (5). Synaptic proliferation is com iorally teratogenic. As 2,4.5-T has been testsi even though there was atendency pleted before day 2 after hatching (4). foundtopassthrough theeggshell (5), it for ambulation to increase with increas-. The brain therefore seems to be most may therefore present areal risk to bird ing doseafter treatment onday 15of in sensitive to 2.4.5-T at a time of maxi species. It isquestionablewhether there cubation and day 2 after hatching (Fig. mum synaptic formation. The fact that isadosebelowwhich2,4,5-Tcanbesaid 1 ). Thus, as found in rats, 2,4,5-T has 2.4.5- T caused adverse effects at lower to haveno effect since some individuals long-lasting effects on activity, but in doses than reported previously may in appear to be highly susceptible. At all chicks themost sensitiveparametercon dicate an extreme susceptibility of the the doses weused, morphological defor tributing to the increased activity is developing nervous system and hence mations wereseeninsomeofthetreated jumping rather than ambulation, which behavior. This isconsistent with onere chicks, and, asthe rawdataforjumping may indicate increased fear responsive port of the possible adverse effects of in Fig. 2illustrate, someindividuals with ness to novelty. 2.4.5- T in the human species (6 ). no morphological deformations were There was asignificant doseeffect for The extent to which our results are highly susceptible to the behavioral ef visual discrimination learning in those due to 2,4,5-T or to very small amounts fects of 2,4,5-T, even at thelowest dose groups treated on days 8 and 15 of in of its highly toxic contaminant, TCDD, (7mg/kg) that we administered. cubation (F = 4.19; d.f. = 3, 86; .005 remains unresolved. However, it is im Thereisconsiderablespeciesvariation < P < .01) but not for those treated portant to remember that in this study insensitivity tothetoxic effectsof2,4,5- on day 2 after hatching. Comparison of weused2,4,5-Tofgreater purity, partic T. At present one of the best indicators treated animals with their controls' at ularly with respect to TCDD content, of interspecies toxicity is the rate at eachagerevealedasignificant slowingof than that available commercially and which 2,4,5-T is metabolized and ex learning inthosetreated onday 15of in sprayed in the environment. Based on creted (9). Based on such measures, cubation with 27mg/kg(P < .001) and7 the concentration recommended inAus chicks and rats appear to be of roughly mg/kg (.01 < P < .025), but no signifi tralia for spraying blackberries (7), similar sensitivity whereashumans have General activity too - Ju m p in g A m b u latio n Visual learning 52 48- 32 28 - Jumping oay 15 incubation 200 20 - I 'H Z 100 12 7 13 O ose (mg/kg) 27 to- S|i . iBL.ia._i 150 0 75 150 Oose (mg/kg) Fig. I (left). Plots of means and standard er rors for four behavioral measures after the ad ministration of 2.4.5-T at the doses indicated on day S or 15 of incubation or day 2 after hatching. Behavior has been tested in week 2 of life. The ordinate scales are as follows; for general activity, counts on the electronic ac tivity meter in the total 3 minutes of scoring: for jumping, the number ofjurr.ps in 3 minutes: for ambulation, the number of times :tie chick o "ej with-both feet into a new quadrant of the box: and for visual learning, (he number of errors (pecks at pebbles) in the last 20 pecks of the j. .e-floor learning task. In the latter test, control groups as determined by one-tailed a/-theisgthse: r' e,rPro<r s.c0o5r:e" i`n.dPica<te.s01s:loawnder'*le*a.rnPin<g. The .001. asterisks Fig. indicate 2 (right!. significant differences from the The raw data for jumping scores obtained during 3 minutes in a novel environment after treatment with 2.4.5-T on day 15 of incubation. La. h point represents the score for a single 1 individual. The fact that some individuals are highly susceptible to the behavioral teratolocical effects of 2.4.5-T is demonstrated by the 'J Q that, even after treatment with the lowest dose (7 mg/kg), some individuals show a marked increase in jumping. 5W SCIENCE. VOL. 211 DOIf 2 l 2 3 6 3 5 a least a inreetold greater sensitivity W. Reicher. Nature iLondnnl 215. |auu (|ytw). the nerve endings, whereas the mean di (75). Our findings therefore imply arisk tothe human species. 4. L. J. Rogers. H. D. Drennen, R. F. Mark. Brain Res. 7. 213 (1974). 5. B. M. Freeman and M. A. Vince. Development ameter of the osmotically sensitive NSG resembles that of the granules/in the o f the Avian Embryo (Chapman and Hall, Lon- C. A. swellings.1 Sanderson don, 1974); J. Sedlacek, in Advances in Psycho / L. J. R o g e r s "P harm acology D epartm ent, biology, G. Newton and A. Riesen. Eds. (Wiley, New York. 1972), vol. 1. pp. 129-170. 6. B. Field and C. Kerr. U n c e t 1979-1. 1341 Inthisreport wepresent evidencethat the osmotically insensitive granules are M onash U niversity, C layton, 3168, V ictoria, A u stra lia (1979). 7. Vermin and Noxious Weed; Destruction Board. Keith Turnbull Reseat ch Institute Bulletin JE newlyformed NSG(NF-NSG) that have recently arrived at the neural lobe and , References end Notes (Department of Crown Lands and Survey. Vic toria. 1977). 8. N. Gyrd-Tlansen and S. V. Dalgaard-Mikkelsen, that the osmotically sensitjvc NSG are probably derived from th^former. Fur 1. P.-O. Sjodfcn and U. Soderberg, Physiol. Psy chol. 3, 175 (1972); in Chlorinated Phenoxy Acids and Their Dioxins. C. Ramel. Ed. (Swed ish Natural Science Research Council. Stockholm. 1978). p. 149. 2. J. McLaughlin et at.. Toxicol. Appl. Pharmacol. S. 760 (1963). 3. H. Lutz and Y. Lutz-Osterug. Adi-. Exp. Med. Biol. 27. 127 (1972); J. F. Dunachie and W. Acta Pharmacol. Toxicol. 35, 300 (1974). 9. F. J. Koschier and M. Acara. J. Pharmacol. Exp. Ther. 208, 287 (1978). 10. W. N. Piper. J. Q. Rose, M. L. Leng, P. J. Geb- ring, Toxicol. Appl. Pharmacol. 26, 339 (1973); K. Eme, Acta Vet. Scand. 7, 240 (i960). 11. We thank Drs. G. Bentley and M. Culen for valuable advice. 23 July 1980; revised 29 September 1980 thermore, weshowthat theNF-NSGare located in the nerve callings, whereas most of the aged granules(A-NSG) can not immediately releasi their contents and are thus likely to/be found in the swellings. Protein spediesof amolecular weight lower than thit of neurophysins canbedemonstrated/intheA-NSG. This suggests possible/ postmaturaiional Neurosecretory Granules: Evidence for an Aging Process cleavage of intragr^nular proteins. Albino(Wistar) maleratsweighing250 Within the Neurohypophysis to 300gwere anesthetized with ketamine chlorohydraie (10 mg per 100 gof body Abstract. W h en c y s te in e la b e le d w ith su lfu r-3 5 is in je c te d in to th e th ir d v e n tr ic le o f weight) and given intracistcmal injec th e r a t b ra in , it is f i r s t in c o r p o r a te d in to o n ly o n e d f th e tw o p o p u la tio n s o f n e u r o tions of [" Sjcysleine mat was obtained s e c r e to r y g r a n u le s th a t c a n b e is o la te d on a n is o s m o tic g r a d ie n t. T h e s e c o n d p o p u from the supplier (New England Nucle la tio n o f g r a n u le s is la b e le d m u c h la te r . S tim u la tio n o f h o r m o n e r e le a s e fr o m is o la t ar, 500 Ci/mm^le) or by reduction of e d la b e le d n e u r a l lo b e s a n d s u b s e q u e n t is o la t io n o f n e u r o s e c r e to r y g r a n u le s a t d if [35S)cysiine (470 Ci/mntolc) with 10m id fe r e n t tim e s a fte r th e in je c tio n o f la b e le d c y s te in e s h o w s th a t th e ra d io a c tiv ity dithiotbreitol in O.lAf phosphate buffer d e c r e a s e s in o n ly o n e p o p u la tio n o f g r a n u le s . O n e o f th e f r a c t i o n s o f th e g r a d ie n t (p H 7.0). Each]rat was injected with 10 r e p r e s e n ts th e g r a n u le s f o u n d n e a r th e r e le a s e s ite ; th e s e c o n d p o p u la tio n is p r o b p \ of the final solution (50 p C \ of radio a b ly lo c a te d d e e p e r in th e n e rv e e n d in g s o j i n th e n e n -e s w e llin g s . W h e re a s n e u active cysteini). The rats were decapi r o p h y s in s a re f o u n d in b o th p o p u la tio n s , sin a lle r p r o te in s c a n o n ly b e d e te c te d in tated at different times after the injection o n e . T h u s it a p p e a r s th a t n e u r o s e c r e to r y g r a n u le s u n d e r g o a n a g in g p r o c e s s a n d th e : ana their neural lobes weredissected out isosm otic gradients can separate the aged granules fro m th o se new ly fo rm ed . in less than 1 minute. Immediately after their isolation, ten of the radioactive Neurosecretory granules (NSG) in tl(e motic gradient yields two populations of lutes were/homogenized in 1.0 tnl of hypothalamo-neurohypophysial tractAre NSG. each of which contains ncurophy- 0 3/./ sucrose buffered atpH 6.8 with 10 formed in the magnoceilular neuron of sins, oxytocin, and vasopressin. One mM Hep^s, and mixed with 20 non- thesupraopticandparaventricularXuclei population has an isopycnic density of radioactive neural lobes homogenized (/). Labelingwith [35S]cysteineindicates' 1.13 g/cm3 and is insensitive to osmotic under tHe same conditions. The NSG that the magnoceilular neuron/ mainly changes in the surrounding medium, wereisolatedasdescribedby Nordmann contain a large precursor protein (~ whereas theother sediments at adensity et a l. w). The gradient fractions were 20.000 daltons), which pre/umably is of 1 . 1 1 g/cm3 andismuch affectedbythe collected with a Buchler device, and a packaged into granules. Thar contents of osmotic pressure of the surrounding me portion of each fraction was kept for a the granules mature during/transport to dium. Furthermore, the mean diameter determination of density. Scintillation ward the neurosecretory/ nerve termi of osmotically insensitive NSGafter fix fluid wasadded tothefractionsandtheir nals. Whenthegranulesreachtheneural ationisverysimilar tothat oftheNSGin total radioactivity was measured with a lobe, almost all the pr^ursor has been convened into proteins of 12.000 i 2.000 daltons, neurophysins, and pep tides that may include the hormones Fig. I. Time course oxytocin and facopressin (2 ). of the appearance of The neurosecretory axons in the [34S]cysteine in NSG neurohypophysis can besubdivided into threecompartments, all of whichcontain fractions isolated on a sucrose-metrizamide isosmotic gradient at NSG(3)'- undilated axons containingfew different times after NSG. nerve endings characterized by the injection of the thepresenceof microvesicles, andnerve swellings containing secretory granules but few if any microvesicles. We pre isotope. The radioac tivity in NF-NSG () and A-NSG (O) was measured and the re viously showed that the mean diameter sults are given as the jf NSGintheendingsdiffersfromthat of NSG in the swellings (4). Furthermore, we showed that fractionation of NSG percentage of the total radioactivity found in both fractions. from the neural lobe of rats on an isos- Day* T im e a l t e r i n j e c t i o n of &J s ] c y s l e i n e 16531 SCIENCE. VOL. 211. 6 FEBRUARY 1981 0036-807.V8I/0206-0595S00.50 0 Copyright S 1981 AAAS '595 N e w S c ie n tis t 12 F e b r u a r y 1981 O .K . 25o0 4 Herbicide is `real-risk* to birds and people -UOt_ Vrt V* TtmHtctecwepwem2ttmfdoSaUcsotebt2rtistrwhrhonehhhhuaxmhox,oaoocmi,rguafeiie4enaco4atpcretoeepreensPresTvvcmTgrnmhibn,h,ttrriouaeagetmtgteu55evehsrhhdeeeurhrteflhssebalccchAe-soreyreesrTeelelrvn8rie--Tyeeithsvhcnaaotdcsergsdrsaruteeato--diioirasslhittilsmesouetahwoicdtrtwseo(ei1fhnhohipfbislyocu0dkflstt,retlrnwswphthnehcaiyereohdnoossoots0sgetwrottaahieackrhlniewodurieohsenmwvuynhisersltniineteasrieegiotifnieenrlstogtttblenehadtohmarceensrsrhyitnef--toosevebiaihhntigo7isecitddegchshnixneshrbninailfhamrpAfcdacthalmthstaaeeynitaewetizsoienantoiskogLhu.rmpuSarrbtdofomeortmxbec.seimcg.oledprchsraren5eueote-irai5.hn1rcyieTrehcgoe3ticpceteteaeHJts3ceh5rao0omsssevanoeshehneaa.hna2arrttes-rmuemevrsfyethcetsnmnhr0scetmls,liui:relamihRac4mdsorrtoieaoi3itargsnkgiotgcdhat5e-bunfsponfhatlsdhorad/ooisghuostle-l/eo(ypisaltrekkginagacrkTelecvaipsfvsiureipaeodtdegnysiwnigetirro,serlmhde2mvnddfdiyresxribagegttg.erlca,tesaenodr.)haoia4vosiyiabxlosnsimhTcinsbn.toe2stenc,ernworacmoaet)e5iihoiiae1huopieTdcnhcnelhnltdutomns-ooaS1inmffgeeohdockehtTegcn.ddyaftsc,pidlrtothsprarstraaeeuieortoriMhhoidkmeat'plyoiwertpssmbteisee2iepcicsevolttonol(nefC.xpeay,oiee,damdhhgs25edco3li1bnfesgpn.rytoluicaoii9o,h,5nlhigooagcviet4d7tstitoboskcwsr3Ahohhibkgdhesaxe,Aciertn,otntienaooi)oo8seoeeg5yonsonssihyntislk.ldsrt.:l-.fflfff tswoctaibb2nhhohn,neee4eeniytrcr,awy5eklnteo-aeehsTgnnffe'efulsdeoneit--nbacrtiamefftfRfrsaooihenenbbmoocatstegvdhtaveihieleionitodarahtguosouverrhstriahtoyle:htyeieeunrrdntbsrjrsehduiv.tisece1aemicra0idmiodTrnrpminpeahmd;tmeggeehimreeaynsaeednsmancanueedtyctfaetrr;onhoealluthhistl.chnaodpkSveadvwiesosiaecsbcfnntuboitwtduhvahhltveheiamelaaetslreydyltrt.l T he h erb icid e 2,4,5-T hits ch ick em bryos hardest w h e n th eir n erve colls are beginning to lin k up wnt82oicpbtsctsehi,otnoelyhvr4hereHanTlneea,ltr5sisneaonhttnad1.ho-tedwpeTpata5getcotteyhittiSethdhicosaivseoanecln1n2oetyfndniiir5cdo,ecfrcesad4t,htnarhh--feyfi,minoto5tlec,rehlitcssyrrTksahdfetemotuf8yhesateenbtsniiiyoahcmssnnaiaagttenepgst2badnedpi"osr,ornmam4vira.eyffnode,iioysos5ctic,nst--tis-aeinTomttcRnsthocrwfehnestobeeulacieigelebbtniseehtrsecxheaoc^firhefvauttefasenoihermivoLcrcettedeeimhnkaotoacsy.d--etseutaraihemltorOylryxdlemgsninanotsm.ihcdbvoeek"heoouirntirntnnnBhashyvmsmsloegdegeoeeyyit AdkHcw02wmssutesfawtds9rroSmoKt--hmitShEhohnoeniheiomou,gupnneohusoufieouo4anerr0eusnolaomBagTsdrsbftwsidkoemxnuamu1,ntdaeceocrtrcwsntet5hho2aieAudtclredltrhiyaychngealhRtdanctheTeprbaepih,eghtesvoeturnpenon4aohnegysposirceldeshrowciarsei2ttusea,ffenscsisbmghatirdhaW5nr.rbtod,sshesnsooed,beScr.n4saneTidBlenTepayaenttnnraehreitByo,nbhifroWavehssnthepr5rtll2tttilsldhyoefiseeehcaaiatioericaeteaota,Tedwttmebtneiehstt4shkcyieombidhrntrye,dhnvrssulaesa,,.o2aBaietarlandies5setefhyehoiyatb2ntic2htnctafnac"h-lohcnrharholryxiTAlco,dieeRetrkioa.reorerbe4tUtnemsehirml.bmrwehtsisbfrnuailunf,yoeeilochrsleoc5--anare(smaeed.lgltsgenihaxehtphdB-)lprrwaihdteudrlfeT/vetraimiaeorleitiaarocrnhkcriusmc2iSim,oireteupriarcmrsftbekhasuiegekotue,rtfbefervhrtpkcbpFk4spxmoehuausnsuh--sa,eulidnistoterpilca,sluoiramtshdoriielsyg5eeaetocecetrsnotiedttuehahtdwyasogyrhTylihaaaptssntwdosds.nweteotignamic"ee2engttoruatBn,ndwpb.iseTcai,iaerfmaosthbnhoitgii,foehrcialnyhnhsrtdmtddrnrtelceririhbniueinhsoidtgtoicmeiircaAmnmm,onscSiriossaeardg'emsohsmatewSmtmghruakbhfatatioe/shtneelcsdenhsskahyitosinaeyeisrbpmCtecdpshavn--eisaepgtarthdgusg2e22ltptsiermeoehenthuttedeo.nete,gtbpaoa,dh,4ocecrsltras4er4rraseeswrNhl"na,eotewiibcsirtiirB5e,natoth,sgtotahaveaitr5lrohesto5iriihhi-eneeecvlvhodaneteatudeuea1nandvh'w1neeTh:sCdiee:siesleslsti.t,tr A plant cell th at can score electricity iwBhbaOtsRbtapccfacpatpicepsneahrpaaaheeocnnalhrrilioahagdoiataaasTocEvlelpdntsoehivmlshcneeottceenimhtetflsifiethoiofdwnftntprssczestdeyfireiorhbmcidcciegatcytstofc.cboimeheemooiaorlansbttriatrrlleaerocWeleseimnsknslltetalivklomwsnsh.osuyttnteecbshmotchsBeestedtwosshsfloihhefyueyNoanthoumerlhilinaeoppGor(ceursesscnaooacaaaNrraapdmteoloerorwohenlbpidmlowrdteoopoflaixrcpgeaducipeugswoh"prnactioiltrttprsltcuhGaalscreoiei.yOcsoniatoaosrvrhnsotes.rthratosaohjnoCatrneoiautetlddlofcyntninsuiofsst,rs-siocihmhe,reishcnsod--dtgtyr.slvecoaeipttolipeonm,oiae"seeoenscvmoOtpehdthStnnnmsogslreahhtslcecoRndishaatl2eceietasiemnetafrvwectashomu8aeuslittenleietnanlee9slooalldtiechisassloseg,gcrcreom,aatckeoeegdtphrhpclttmdtoenrldlleUfhhabiwleluetrinuPardomcrctsw3eoernaaitcnhhosnlatrvlaihti9luekephtgotooiyeinitbnenyrdevc6e-clcgouaenpuoolseib,emehe)hhonhefllisnf.waadssrwclllesanicf,oosossacocslehatntlmeooeierarrihgenhiatbltlatraeotvaowsnlpwchdyeesdeeelilsttl,--, wptmstimtmliisonshinhheglhufaateeeutaTnepohdoskmmttshnptitheafsireblaftteoyeptiihetrrcvchiohcsoeanelceueaeeefenwntoiylosbpaeacretuaeeotwrttroornubcerttcuihoafrhstoceitiregcetesuellwtmmlideurthtpaawscoprleoephdlceo3'oolacsrecetisnocmw-,psoaectvadpuhvpradepupeoluetreiecrtlhreorroepcc;rrudrtaeereehfrolsotetadcddlatrhs.otrxhetthsnmoeeirabaoniecaotlnrcyuattnipestpftntvllahtitncyoaooocahcocahtsfeonlnheifthtsltto.ora.tlysototatrsrgh.hmBwarsFnoeuoeeTeepeisprfwcamolmaoseaelattkahusodtouvsuua,iusistnntetcscxaatsrlidngehihsyseit,ll J^=j tecpadsseagmsttthhhhhtohafrfiorrfoefvfueoaiteGtmeiisuecrcicdnadmtrreeisihrhueowec.tcusyeiacshcnnceaaCanrvsoeltclmcalyaolbeleonyylrtroorlel,ihvaaglotngwscuralsenrmaepeseioteegysdbnntltcenashatneeathiotaiittscnatcomrmehrtabstihaaittmncebsialuvopnuoeflaicurlnirsutsoynoeaylteacen--sruintmconootthpiisaowspmnnunteehsllnhtryhhlsstnhtopoeortoae.ottetliafddmhersbontupeg--uatecuSuhoruicraesist'bcselewskeei,lurlnhoiuclcesosetmgolltehimailrflhmgnotlleo-mforuwleciodghoonmrmoehiuitrsfacpcwntnueinl--icaherateenvechteodhenlhlnaeuhalrobdogeedrstla.osteitayreiaacohhntbignlohnenfisanegnulyilinddneyolystr,l.,l C ells based on w hole chloroplasts h a ve b een u sed to convert th e Su n 's energy into electricity. P art of X72a c h lo r o p la s t, 000, is show n. T he p la n t's use o f mlig h t e n e r g y b e g in s the th in *-* ""$ 6 3 3 2 lOOlN UlLiO -UO 12 March 1982 Volume 215, No. 4538 LETTERS NucleDHHAas..irgsSLheWaawsyesnaraigneypbg;:oeTnIAhmshegepirMmaicCca;utrtDleatourayniram-a1lIsa:ln:gdRWiTinea..gsnRBesS.a.:trSeDcCmrho.e:icoCthWht.yr;.paPSneW.,:yG.nBMKtr.h.ooeHsMntsikamc.lneaVH:snaeo;TcnerXcna.i.-inn.gr.es.a,.as.y.:mW...H.a..ozM..ol.o.n.g.A.r.a.r..pk.i.h.n.y.-.: ed ito r ial Energy and Chemicals fromTrees. as 1344 o VJ1 V* CM oU1 1349 CD CD AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE t^o f'O reports Recent Volcanic Activity at Glacier Peak: J . E . B e g e t................................................ Kinetics of Delignification: A Molecular Approach: J . F. Y a n ................................... Dating M of aFault . Ikeya , T. bMyiEkil,ecKtr.oTnaSnpaink aR..e..s..o..n..a..n..c..e....o..n....I.n..t.r..a..f.a..u...lt...M....a..t.e..r.i.a..l.s..:....................... o^-AdIrnetnraocceelplutolarr-MSetuddiaietsedinHvyivpoe:rpGo.laKri.zaAtgiohna joa nf iLaoncuasndCCoe.rPu.leVusa nNdeeurMroanesl:en . OxytoInciitniaRtioecneoptfoLrsabaonrd: Human A .-R . F uPcahrstuerittiaoln..:...A....D...u..a..l. Role for O-- RapidDEimleecntsroionnicalAGuetolsf:luJo.roBg. rDaphy' of 1 HumawnithLivMer` M' AC .................... p ~ ,u ch ,,iainedctR . oLn. LWifea-lSfopradn............ -wu Ketinotectal Projection During Chick ............................................................................................. i-. M .S ^,eroid chw artz RanedguJl.atiWon. TorfuMmuasnc.l.e....D...e..g..e..n..e..r.a...t.io...n...i.n....a..n...I.n...s.e...c..t.:......................... T e c h n ic a l M. L Comm . Leng e n ts : : L. J Jumping . Rogers CanhdickCe.nAs.: SRaenledvearnscoent.o....H...a..z..a..r.d....in....H...u..m...a..n...s.:.............. CO CO 1309 -- 1390 1392 ,`f11 1413 1415 1 41S 1420 1421 products and m aterials PersoHnaulmCaonmTpuCteelrl:GCreollwCthulFtuarcetoCr;haInmfrbaerer:dRSepfreigcetrroamtoerste:rS; aLmitpelreaPturerep.a..r.a..t.i.o..n...:....... 1424 SCIENCE. VOL. 215, 12 MARCH 1982 Jumping Chickens: Relevance to Hazard in Humans Sanderson and Rogers have reported evaluation of the data because (i) the the results of a study on the behavioral doseswerenot environmentally relevant effects in chickens given "environmen tally relevant doses" of 2,4.5-trichloro- for bird eggs, muchlessfor humans, and (ii) chick embryo data arenot asuitable phenoxyacetic acid (2,4,5-T), and they postulated that humans would be three model for safety assessment in humans and are not used by any government times more sensitive lo this herbicide agencies for this purpose. than chicks (J). The conclusions drawn Recent studies by Lavy et ul. (2) have from this study do not reflect a valid shown that the maximum exposure to 0036-8075/82/0312-1421SO1.00/0 Copyright 1982 AAAS . 16&33 2.4.5- T likely to be encountered in hu nearly lethal dose of 2.4,5-T into newly mpns. i'Sabout 0.1 mg per kilogram.of hatched chicks to the potential effects body weight, which is orders of magni from low-level exposure in adult hu tudes less than in the chicken studies. mans. Unfortunately, reports of such Furthermore, such exposure would be studies done under highly exaggerated likely to occur only in applicators who conditions continue toadd to theconfu areusingknapsacksprayersandwearing sion surrounding the use of beneficial contaminated clothing or in mixers or agricultural chemicals such as2,4,5-T. loaders who are not careful to avoid M a r g u er ite L . L eng contact with the concentrate and spray H ea lth a n d E n v iro n m e n ta l S c ie n c e s , solutions. Measurableexposureto2,4,5- D o w C h em ica l C o m p a n y , T isextremely unlikely in bystanders or M id la n d , M ic h ig a n 48640 thegeneral population (3). Inthestudyof SandersonandRogers, References end Notes the doses ranged from 7 to 53 mg of 1. C. A. Sanderson and L. J. Rogers. Science 211. 2.4.5- T per kilogramof body weight in 393 (19811. 2. T. L. Lavy. J. S. Shepard. J. D. Maltice. J. jectedintotheyolk sacoffertile chicken eggs on day 8 or day 15of incubation, Agric. Food Chem. 28. 626 (1980): T. L. Lavy. J. S. Shepard. O. C. Bouchard. Bull. Environ. Contam. Toxicol. 24. 90 (1980): T. L. Lavy. andfrom75to225mg/kg injected intraperitoneally into chicks on day 2 after paper presented at the meeting of the Weed Science Society of America. Toronto. Canada. February 1980. hatching. The 2,4,5-T contained 0.03 part per million of the highly toxic con 3. M. L. Leng. T. L. Lavy. i. C. Ramsey. W. H. Braun, in Pesticide Residues and Exposure (Symposium Series 182. American Chemical So taminant 2,3,7.8-teirachIorodibenzo-p- ciety, Washington. D.C.. 1982). pp. 133-156. 4. J. D. Somers. E. T. Moran. Jr.. B. S. Reinhart. dioxin (TCDD), which is representative of the level in 2,4,5-T produced world Bull. Environ. Contam. Toxicol. 11. 511 (1974). 5. W. N. Piper. J. Q. Rose, M. L. Leng. P. J. Gehring. Toxicol. Appl. Pharmacol. 26. 339 wide today. " Environmental relevance" was (1973). 6. K. Erne. Acta Vet. Scand. 7. 240 (1966). 7. P. J. Gehring. C. G. Kramer. B. A. Schweiz. J. basedon anestimated dose of 22 mg/kg Q. Rose. V. K. Rowe. Toxicol. Appl. Pharma col. 26. 352 (1973). for an egg contaminated with 1 . 1 mg of 30 March 1981: revised I June 1981 2.4.5- T over the entire shell surface (area, 50cm2), presumed to be equiva Several aspects of Leng's technical lent to spraying an 80 percent 2,4,5-T comment require correction. We found product (derivative not specified) at a that, forchickeneggs, themost sensitive rate of 2.8liter/ha. The latter is equiva- period to 2,4,5-trichloroplienoxyacetic 'ent to 224mgof 2,4,5-T (or derivative) acid (2,4,5-T) was day 15of incuOaiion, per square meter of horizontal surface whentheinjectionofaslittle as7mgper area, not the entire egg surface, and kilogramof body weight caused subse 2.4.5- T is generally applied as a 2 to 5 quent behavioral abnormalities (/). This percent solution in oil, oil-water, or wa amount does not causemarked morpho teremulsion. Injectionof2,4,5-Tintothe logical abnormalities and is well below eggwasalsoassumedtobeequivalent to the LD50 of 53mg/kg (the dose lethal to contaminationof theshell surface. How 50 percent of the animals iestedi [see ever, only asmall fraction of the herbi (/)]. cide applied to the surface of an egg Thequestionof the amount of 2.4.5-T penetrates the shell and reaches the absorbed through the eggshell is. of chick embryo (4). course, controversial. If we accepi that Sanderson and Rogers concluded that only 30to 50percent of an egg may be humans would beat least three limes as covered by the spray and if we use the sensitive to 2,4.5-T exposure as rats or concentrationrecommended by thegov chickens. This conclusion was basedon ernment of Victoria, Australia, for studies by Piper e t a l. (5) and Erne (6 ), although neither study implied such a spraying blackberries, an egg would re ceive7to 11mg/kg. Evenif only someof relationship. What happensasaresult of this isabsorbed, it is still uncomfortably theinjectionofalargedoseofachemical closetotheamounts that caused behav intoanisolated eggis not representative ioral abnormalities in our sample. Leng of what might happen in ahuman fetus connectedtoitsmother'sefficient detox ification system, particularlyfor achem ical such as 2,4,5-T which is rapidly excreted in the urine of humans (7). does not allow for a safety factor. At tempts to extrapolate from these results tothecalculationofthe" no-effect" dose inhumansarecomplicated by individual variations in sensitivity to 2.4.5-T [see Evenmorequestionableistheir extrapo- ourjumping data in (/)]. ' ;on of effects from the injection of a We used the acid form of 2.4,5-T, whichislesstoxic thanitsesters, usually used for spraying. Moreover, we gave only one dose to each egg. Repeated dosing may well be toxic at lower daily rates of administration. Leng quotes a maximumexposureto2.4.5-Tof0.1 mg/' kgfor knapsack sprayers but fails tosay that this value u'as calculated for one operation lasting 180 minutes. Most sprayersarerepeatedly exposedandfor longer periods. The maximumexposure measuredby Lavy et al. 12) wasactually 1.85 mg/kg per spraying operation, and this did not include spray inhalation, which is probably a more important route of exposure, particularly in the vicinity of aerial spraying. We agree that other species must be tested with 2,4.5-T. We have in fact shown that the oiai dosing of pregnant rats with aslittle as 3or 6 mg/kgonday 8 of pregnancy causes behavioral ab normalities in the pups tested several months later (J). This dose is one-hun dredth of the LD50 in this species and one-tenthof thelowest dosereported to causephysical deformities(4). Phenoxyaceticacids themselves appear to beable to cause these effects; we have found similar effects with 2,4-dichlorophenoxyacetic acid (2.4-D), which does not con tain 2,3.7,8-tetrachlorooibep.zo-p-dioxi.i (TCDD) (J). Our statement that humans are likely to bemore sensitive than rats and chicks is based ondeduction., fromthe plasma half-lif,.. w-hichtor ar, oral doseof 2.4.5Tof5mg/kgis23.1 hoursinhumansand 4.7 hours in rats 15). The results for chicks appear to be similar to those for rats (5). We therefore maintain that 2.4,5-T(and2.4-D) may present risks to brain development and function in hu mans andother species. L. J. Rogers C. A. Sanderson P harm acology D epartm ent, M onasit U niversity, C layton, Victoria 3168, A u stra lia References 1. C. A. Sanderson and L. J. Rogers. Science 211. 2. 593 (1981). T. L. Lavy. J. S. Shepard. D. C. Bouchard. Bull. Environ. Contam. Toxicoi. 24. 90 (1980 3. M. A. Crampion and L. J. Rogers, in prepura- lion; P. J. Booth and L. J. Rogers, in prepara tion 4. K. S. Khera and W. P. McKinley, Toxicoi. Appl. Pharmacol. 22, 14 (1972J. 5. P. i. Gehring. C. G. Kramer. B. A. Schwetz. J. Q. Rose. V. K. Rowe. ibid. 26. 352 (1973): W. N. Piper, J. Q. Rose. M. L. Leng, P. J. Gehring. ibid., p. 339; M. W. Saueroff, W. H. Braun. G. EE.rnBe.laAu.c tJa. E. LeBeau, Vet. Scand. ibid. 37. )36 (1976); 7. 240 (1966). K. 23 November 1981 16534 DOH 21/3632 I I I i i i SCIENCE. VOL. 215 B. W 3 25dl ^gAoXU. 'TW r L itid ^ So^T' TxrS">rcoVA EPIDEMIOLOGY OF SOFT-TISSUE SARCOMA AND RELATED HUMAN RESEARCH (as related to herbicide exposure) z Noi O0s0 O' .e* LAWRENCE B. HOBSON, M.D., P h .D . Deputy Director Agent Orange Projects Office Veterans Administration March 1983 DOM 2120649 16535 DOW 2 1 2 0 6 5 0 EPIDEMIOLOGY OF SOFT-TISSUE SARCOMA AND RELATED HUMAN RESEARCH Initiation of Swedish Studies of Herbicides and Cancer In 1972 Swedish newspapers published rumors that rail road workers were dying from lung cancer as a result of exposure to herbicides used in their work. The National Board of Occupational Safety, as a result, requested Professor Olav Axelson, a specialist in occupational medi cine, to undertake an epidemiological investigation of the matter. The results of this investigation have been reported in a series of four papers, Another series, J5-7_/ was prompted by criticism of the epidemiological and statistical methods employed in this and related studies. Attention in the United States has focussed on the pre-publication manu script of the 1980 paper by Axelson, Sundell, Andersson, Edling, Hogstedt, and Kling, 4_. This paper dealt with two aspects of the study of rail road workers. The initial phase was a cohort study of 348 men who had been exposed, individually rather than as a group, to herbicides for more than 45 days during 1957 to 1972 and who were followed through October 1978. Exposure information was incomplete but the workers were divided into subcohorts with exposure to phenoxy acids (which include the ingredients of Agent Orange), amitrol, or to both herbicides. The mortality rates for these exposed subcohorts were com pared to., the age-specific national death rates for Swedish men, the latter serving as the control cohort. Overall 49 deaths were expected in the exposed cohort; 45 occurred, a result attributed to the "healthy worker effect." There were, however, 17 tumors found where 11.85 were expected. Among the deaths occurring at least ten years after the first exposure, 6 cancers were found although only 1.78 were expected. Dr. Axelson, _8, later increased this to 7 tumor cases. Each subcohort had an excessive number of tumor deaths, the greatest being in the group exposed to both phenoxy herbicides and amitrol. Although initially, _2, amitrol was associated with an increased tumor mortality, somewhat different results were found in a second phase of the examination, described as a case-referent study (identical to a case-control study). The data indicated a "statistical association" of phenoxy herbicides and excess tumor mortality, 3 . Suspicion was increased by finding that workers exposed to phenoxy acids 16536 DOW 2 1 2 0 6 5 1 /> alone had a "statistically significant excess of stomach cancer", specifically 2 cases compared to 0.33 expected when this type of herbicide was used alone and 3 cases as compared to 5.1 expected .(increased from 4.1, _8) for all workers exposed to phenoxy herbicides, alone or with amitrol. The series of papers on railroad workers has been criti cized on several methodological grounds, _9, and Axelson has replied to these criticisms, 6. Richard D. Remington, Dean of the School of Public HealtF, University of Michigan, re viewed this and other Swedish studies for the Office of Tech nology Assessment. His evaluation, J_0_, was that the Axelson investigations had been "carefully conducted" and "well re ported." He pointed out the limitations of the statistical methods used and found that "the numbers available ... are inadequate to permit definite conclusions" although "the results ... are suggestive." Of interest in connection with the question of softtissue sarcomas and phenoxy herbicides is the type of tumors found by Axelson's group, 4_. One case of Hodgkin's lymphoma occurred among the eight tumors in men exposed to phenoxy herbicides alone and no soft-tissue sarcomas or non-Hodgkin's lymphomas was diagnosed among the eight tumors appearing in workers exposed to both amitrol and phenoxy herbicides. In other words, no sarcomas were reported for the total of 207 men exposed to phenoxy compounds, 2_. A reticulum cell sar coma and a Hodgkin's lymphoma were found among the 7 tumors of workers exposed to amitrol alone. Thus, there was one soft-tissue sarcoma reported for 152 men exposed to amitrol, 2 . Another 28 persons described as exposed to "other herbi cides and combinations" cannot be identified as to exposure to specific herbicides but apparently none developed a tumor. Axelson's work is directly related to the later work on soft-tissue sarcomas and lymphomas by Lennart Hardell. In deed, Axelson suggested to Hardell in 1976, that he conduct a case-control study of soft-tissue sarcomas and has actively assisted in Hardell's work since then. Swedish Investigation of Soft-Tissue Sarcoma That work began when Hardell admitted for treatment 3 patients in the autumn of 1976, each with a soft-tissue sa r coma, and a history of exposure to phenoxy herbicides. He then found a total of 7 patients with "malignant mesenchymal tumors" (soft-tissue sarcomas) who gave a history of having worked with phenoxy herbicides 10 to 20 years earlier. The cases were among 87 patients with soft-tissue sarcomas, 55 of 2 16537 3 whom were men. Of these men, 9 were forestry workers, 6 worked in forestry and on farms, and 6 were employed in saw . mills or pulp plants. Another two tumors appeared in men whose connection with forestry was less direct. The malig nancies found were two leiomyosarcomas, two rhabdomyosar comas, two neurofibrosarcomas, with one each of fibroid liposarcoma, myxofibrosarcoma, and polymorphocellular sarcoma, 12. Following Axelson's advice, Hardell began a case-control study that was published in two journals, _1_3_, J_4_, a common practice of reporting in Swedish with an almost identical paper in English. The second paper in English aroused much interest in the United States. Hardell and Sandstrom found 21 living and 31 dead men who were diagnosed as having soft-tissue sarcomas in Hardell's oncology department in northern Sweden. They were matched for age and place of residence, as well as date of death for the deceased, with other men selected from the Swedish National Population Registry or from the National Registry for Causes of Death. Each living patient had 4 living controls? each dead man had 4 deceased controls. Ex posure information was sought by the use of a mailed que s tionnaire that has never been published. It contained 130 questions, including 16 about the use of organic solvents, 4 about plastics, 3 about glues, 4 about drugs, "several" about smoking habits and an unstated number about exposure to phenoxy herbicides and chlorophenol as used in the lumber and paper mills. This questionnaire was mailed to the patient or to his next of kin if he were dead, 15. When the answers to the questionnaire were less than clear, a supplementary interview was obtained, usually by telephone, with the interviewer unaware of the health status of the person in question. Employers, neighbors, and others were consulted "if necessary to verify and monitor the accu racy of the exposure information," 15. Using the criteria for exposure established for the study, 36.5% of the 52 patients and 9.2% of the 208 controls had been exposed to phenoxy herbicides and/or chlorophenols. The "relative risk" of developing soft-tissue sarcomas was calculated as 5.7, i.e. men exposed to the chemicals had almost six times as great a change of developing a sarcoma as did those who were not exposed. The relative risk was 5.3 for the 46 men exposed to phenoxy herbicides alone, and 6.6 for the 40 men exposed only to chlorophenols. It was thought that confounding factors had an insignificant effect. 16538 7 DON 2 1 2 0 6 5 2 don 2120653 The authors concluded that "the investigation showed an increased risk for soft-tissue sarcomas" but "a specific evaluation of the effect of separate chemical substances was not possible," _V4_. The study's methods have been criticized and doubts have been expressed about the 100% response rate to the question naire approach, 9_. (Actually, 2 of 208 controls did not answer, J_4_. ) The statistical approach was described as slightly misrepresented and a major criticism was leveled because of the possibility of "selective recall," the greater tendency for an ill person to remember a supposed "cause" for the illness than a well person would have to remember the same "causal" event. The criticisms evoked several replies. Axelson defended the case-control design, the objectivity of obtaining expo sure data retrospectively, and the statistical techniques, 6_. He concluded that the use of interviews for determining ex posure is justified, 1_, and defended in principle the treat ment of confounding factors, 16. Hardell recalculated the 1979 results and his subsequent papers to substantiate his earlier findings and performed a separate investigation in support of his confidence that "no substantial observational bias could exist in the studies," 15 Remington, J_0_, expressed the view that "the findings of this particular investigation are suggestive" and that "a relative risk of 5.3 for exposure to phenoxyacetic acids must be taken seriously." However, "case-control studies are uniquely susceptible to hidden sources of bias" even when the investigators are "unusually careful" as they are in this "excellent investigation." Hardell's group also undertook a second case-control study of identical design in southern Sweden which is more devoted to agriculture than to forestry, J_7_, _1j8_. In this investigation each of 72 living and 38 dead patients was matched with two controls. Among the 110 cases, 22.7% reported exposure to phenoxy herbicides or chlorophenols and, among the 219 controls, 5.9% were so exposed. This gave a relative risk of 5.1 with matching and 4.7 when the matching was dissolved, i.e. when sorting by age was ignored during statistical calculations. The relative risk from exposure to phenoxy herbicides was calculated to be 6.8, and that from chlorophenols to be 3.3. Exposure to more than a dozen other noxious materials, e.q. asbestos, smoking, DDT, and lindane, were considered as possible confounding factors although none was found to be clearly associated with an increased risk by itself. 4 16539 The reports list the diagnoses of all 110 cases of softtissue sarcoma as: leiomyosarcoma, 33; malignant fibrous histiocytoma, 19; liposarcoma, 15; neurogenic sarcoma, 11; angiosarcoma, 9; myxofibrosarcoma, 7; fibrosarcoma, 5; dermatofibrosarcoma, 3; atypical fibroxanthoma, synovial sarcoma, sarcoma NOS, 2 e a c h ;Ewings 1s sarcoma (extraskeletal) and rhabdomyosarcoma, 1 each. No statement is made as to which of these tumors was found in the 25 cases with identified exposures and no histological diagnoses are reported for the northern Swedish series, 13, 14 . ' The authors of the southern Swedish study conclude that "exposure to phenoxy acids and chlorophenols might constitute a risk factor in the development of soft tissue sarcomas," 18. The investigation has been the subject of the same criticisms and refutations as the earlier study. Remington concludes that "the results are consistent with the hypothesis that phenoxy acid exposure increases the risk of tumors of this type" but adds that "case-control methodology is intrinsically susceptible to subtle and un measurable biases." UUH 2 12 0 6 51 Swedish Investigation of Lymphoma In May, 1978, Hardell was prompted to a new study by a patient with a malignant histiocytic lymphoma and a history of "massive exposure to phenoxyacetic acids." All men admit ted to the oncology department with this type of tumor during the first nine months of 1978 were asked about their occupa tion and possible chemical exposure. Of 17 patients, 14 re ported an occupation consistent with exposure and 11 of them had had contact with phenoxy herbicides or chlorophenols ten or more years earlier, 19. These observations led to a case-control study, the report of which in 1981, _2J_, differs considerably from that in 1980, _20_. The earlier report was commented upon in manu script form by various experts but the later version will be used here. The investigation, in collaboration with Axelson, 2 0 , included both Hodgkin's disease and non-Hodgkin lymphomas. The 169 cases consisted of 60 Hodgkin's disease patients (lymphocyte predominance, 20; nodular sclerosis, 3; mixed cellularity, 27; lymphocyte depletion, 10), 105 men with nonHodgkin's lymphomas (follicular center cell (FCC) type, 53; non-FCC type, 52), and 4 individuals with unci assifiable lymphomas. Each case had two matched controls, 338 in all. Of the cases, 62 had died as had 124 of the controls. 5 16540 Questionnaires and interviews were used to determine , exposure to phenoxy herbicides, chlorcphenols, organic solvents, or medicines and to characterize jobs, hobbies, and smoking as they were determined in the soft-tissue sarcoma* investigations, 20 . All cases and controls were from northern Sweden. Cases in which exposure was reported to chlorophenol, or to "mutagenic" solvents (benzene, trichloroethylene, perchloroethylene and styrene) were divided into high-grade and lowgrade exposure groups. Continuous exposure for"a week or l e s s o r repeated exposures totaling less than a month were considered low-grade. Analyses also divided cases into two groups depending on whether 5 years had elapsed as a latency period between the first exposure to the chemical and the tumor diagnosis. Of the cases, 36.1% had been exposed to phenoxy herbi cides or chlorophenols; 9.6% of the controls had been so exposed. The relative risk for these exposures was 6.0 with matching and 5.3 without it. Phenoxy herbicides gave a rela tive risk of 4.8 although it was greater if exposure was for 90 days or more. Chlorophenols gave relative risks of 8.4 for high-grade exposure, 2.9 for low-crade. High- and lowgrade exposure to organic solvents gave relative risks of 2.8 and 1.2 respectively. On the other hand the few cases with both phenoxy herbicide and high-grade organic solvent expo sure was calculated to have a relative risk of 11.2 and some other combinations also gave large relative risks. The length of the latency period, however, seemed to have no effect. The authors conclude that "this investigation suggests that exposure to organic solvents, chlorophenols, and/or phenoxy acids constitutes a risk factor for malignant lym phoma," 2 1 . Dr. Remington commented that "a substantial and statistically significant relative risk is found for this group of tumors. And again, pnenoxy acid exposure is specifically incriminated." He continues, however, that the limitations of case-control methods have to be considered as well. Swedish Investigation of Carcinoma of the Colon Hardell undertook to answer doubts that his question naire and interview methods allowed observational bias in assessing exposure by conducting a case-control study of "colon cancer." The condition is not suspected of having any association with phenoxy herbicides or chlorophenols. In consequence, if the previously used exposure determination DOM 2 1 2 0 6 5 5 16541 DOW 2 1 2 0 6 5 8 i resulted in a relative risk of 1.0 or near it, there had been no observational bias in the questionnaire-interview procedure used in the earlier studies of soft-tissue sarcomas and lymphomas. Of the 157 men with colon cancer all but 3 answered the questionnaire. The controls consisted of the control groups from the soft-tissue sarcoma study (206 men) and the malig nant lymphoma study (335 men). In all, 41% of the cases and 45% of the controls were dead. Of the cases and controls, 11.0 and 10.4% respectively had been exposed to phenoxy herbicides or chlorophenols. For phenoxy herbicides, the relative risk was calculated to be 1.3 and for chlorophenol it was 1.8. Neither was significantly above 1.0. The con clusion was that "the previously reported associations be tween exposure to phenoxy acids or chlorophenols and softtissue sarcoma and malignant lymphoma cannot to any essential degree be explained by observational bias," 15 Later Criticism of Swedish Studies There remain, however, doubts about the practical significance of the Swedish epidemiological studies stem ming from several of their characteristics. The main criticism is the reliance on recall of the men or their relatives, employers and associates for undramatic events years earlier as well as the possibility of unconscious bias on the part of the interviewer, the "observational bias" discussed above. Coggan and Acheson point out that the positive association between exposure to phenoxy herbicides and the development of several or many softtissue sarcomas, Hodgkin's disease and non-Hodgkin's lymphoma may indicate "a serious undetected bias" even though the explanation has been offered that all these tumors are embryologically related, 22. These authors conclude that "it is as yet impossible to estimate with any precision the risk of soft-tissue sarcoma due to p h en ox y.herbicides" but add that "there is suggestive evidence of a biological association between phenoxy herbicides (or their contaminants) and soft-tissue sar comas." They feel that there is weaker evidence for an association between herbicides and lymphomas. Hardell and Axelson reject the idea of observational bias, citing the colon cancer study as evidence, 23. They also defend the aggregation of tumors because of the "so-called addition theorum for chi-square and Poisson distributions" as well as the embryological relationship of the neoplastic tissues. 16543 2 S 9 0 Z I Z non American Support for Swedish Conclusions Support for the connection between soft-tissue sarcomas and exposure to phenoxy compounds has been reported in several papers from outside Sweden. The data most often cited as favoring the relationship are derived from observa tions in the American chemical industry.* The first was a note by Honchar and Halperin in which they pointed out that of 105 deaths in four exposed industrial "cohorts" 3 (2.9%) were due to soft-tissue sarcoma, whereas only 0.07% of deaths , among adult American men are so caused. The three cases were malignant fibrous histiocytoma, fibrosarcoma, and liposarco ma. The authors felt that these "suggest a common pat tern," 2A_. Cook added a fourth case, another malignant fibrous histiocytoma and noted that all four were smokers and two had chloracne, 25 . Moses and Selikoff reported a fifth case, a non-smoker, with neurogenic sarcoma (malignant schwannoma). They give the total annual incidence of soft-tissue sarcomas as 4500 (less than 1% of newly diagnosed cancers) in the U.S. and quote 4.9% of soft-tissue sarcomas as malignant schwannoma, 26. Johnson and his co-workers briefly described a young man who died of fibrosarcomatous mesothelioma some four years after first being exposed to phenol. His father had a liposarcoma after "prolonged exposure" in a plant manufacturing chlorinated phenols among other chemicals, 27. Hardell and Ericksson accepted the two additional cases to total 7 deaths from soft-tissue sarcoma among 105 deaths among American industrial workers, the expected number being 0.07%. This would "fit in with" the Swedish investigations, they believe, 28. To date no critical review has been made of the cases and the industrial population in which they were detected. The reports have been brief "Letters to the Editor" and each discusses one to three cases. The total of 105 deaths used as the number of dead workers has not been kept current as new soft-tissue sarcoma cases were added and the total number *Data given by Honchar and Halperin, Cook, Moses and Selikoff, and Johnson et al pertain to workers at Monsanto Company and Dow Chmical Company. For details of studies of these workers see 24a and 25a. 8 16543 DON 2 1 2 0 6 5 8 of exposed workers has not been given. No use has been made of controls, even in the form of a retrospective cohort comparison. A case report without statistical data briefly describee three soft-tissue sarcomas among Vietnam veterans who re ported exposure to phenoxy herbicides in that country. One man had an inflammatory histiocytoma, another suffered from a fibrosarcoma, and the third had a leiomyosarcoma, 2 9 . European Support for Swedish Conclusions Barthel determined the frequency of malignant neoplasms among 1791 pesticide sprayers and agricultural technicians ir. East Germany during 1976 to 1979. He states the ret ros pec tive cohort study used police as controls but gives no data for them. After eliminating "on statistical grounds" 133 cases who died before 1970, he compared the mortality rate and cancer incidence with corresponding figures from the death statistics and the cancer registry of the Health and Social Welfare. The "case" group had multiple exposures over the years to fungicides, insecticides, and herbicides in cluding phenocyacetic acids. Among 169 malignant neoplasms in 1658 exposed men were 1 lymphosarcoma, 3 plasmacytomas, 1 described as a malignancy of lymphoid tissue, and 1 of softtissue, not otherwise characterized. Bronchogenic carcinoma was the most common malignancy with 59 cases, double the expected occurrence, although the cases had smoking habits like those of the general population, 30. A brief report describes a case of non-Hodgkin's lymphoma and a second of malignant lymphoma among 158 workers with pen tac hlo rop he nol . This tyoe of neoplasm would have an expected occurance of 0.28, 31. Studies Not Supporting Swedish Conclusions In contrast to the reports of an association between phenoxy herbicides or related compounds, and soft-tissue sar comas and malignant lymphomas, some investigators have found no association. Some of these investigated a possible rela tion, others were "follow-up" studies of industrial workers in whom no' sarcomas or lymphomas were found. Dr. Riikimaki and his collaborators have completed nine years of mortality study following 1,926 persons who worked with phenoxy herbicides in Finland during the 1955-1971 period. All had at least two weeks of exposure and a quarter of the men totalled eight weeks or more as of 1971. The mortality rates among the workers were compared with the national death rates. As of 1980, there had been 82 deaths 16544 9 DOM 2 1 2 0 6 5 9 { of exposed men as compared to 91 expected and, of these, 17 were cancer deaths with 18.4 expected. There were no cases ,of soft-tissue sarcoma nor of lymphomas although 0.1 and 0.8 would have been expected. The authors believe that "the investigation cannot be regarded as a conclusive negative study" but point out that the "results do not confirm the ... association between mixed herbicide exposures" and cancer risk, 32. Hogstedt and Westerlund compared the mortality rate of Swedish supervisors and workers in forestry. The supervisors were fewer in number (142) than the workers (244) but the former were judged to have been more heavily exposed. The relative risk of death was about the expected but, after a 10-year latent period, the relative risk for cancer was about 4 for the supervisors and only about 0.4 for the workers. The fatal tumors were of various types but there was no softtissue sarcoma or lymphoma, 33. Two case-control studies in N e w Zealand have been initi ated by Smith et al to examine the association suggested by Swedish studies of phenoxy herbicides with soft-tissue sar comas and malignant lymphomas. In the first investigation, 102 cases of soft-tissue sarcoma have been identified in men from the New Zealand Cancer Registry between 1976 anmd 1980. An equal number of matched controls with other forms of cancer were selected for comparison. The sarcomas are fibro sarcomas, 25; 1 iposarcomas, 20; rhabdomyosarcomas, 9; leio myosarcomas, 7; malignant histiocytomas, 6; other types, 22; and unspecified, 13. The preliminary report compares cases and controls as to the occupation shown on the Registry en rollment..' There was no significant difference betwen the groups as to the number of men working in agriculture, fores try, and fishing, the occupations with the greatest likeli hood of exposure to phenoxy herbicides and chlorophenols. The only occupations associated with soft-tissue sarcomas exclusively are blacksmiths, machine tool operators, electri cal fitters, and electrical workers. The investigators are now obtaining work histories for cases and controls by tele phone interviews and warn that later results may change their conclusions. The data at present "do not give evidence for a relationship (of soft-tissue sarcoma) with occupational expo sure to phenoxy herbicides and chlorophenols" but "should not be taken as substantive evidence against the hypothesis", 34. A second report by Smith et al includes the results of the telephone interviews regarding 80 cases and 92 controls already completed. Probable or definite exposure to phenoxy herbicides for more than one day earlier than five years before cancer registration was found in 17 cases and .13 con- 16545J 00M. 2120660 trols, giving an odds ratio of 1.6. This would be expected to increase when the exposure criteria were more stringent but, when exposure was at least five days and more than ten years before registration, there were 13 cases and 12 controls included reducing odds ratio of 1.3. Neither ratio is statistically significant and there have been no soft-tissue sarcomas reported among the most highly exposed group of 2000 aerial and ground sprayers. The results, the authors believe, "do not generally support the hypothesis that exposure to phenoxy acid herbicides cause soft-tissue sarcoma," 35. A brief initial report by Edling and Granstam compared the causes of death for 375 Swedish forestry workers, aged 25 to 69 years, who died during 1968 to 1977, with the mortality figures from the Swedish national statistics. There were 75 deaths from all malignant tumors, as compared to 86 expected. Renal tumors killed 8 with 3.84 expected and "tumors of lym phatic and hematopoetic systems" were responsible for 14 deaths with 7.5 expected. No deaths were attributed to softtissue sarcoma, 36. In addition to these studies, several small industrial groups have been followed well into the latent period for solid tumors. None has been reported to include cases of soft-tissue sarcoma or malignant lymphoma. May examined 41 of 79 workers who developed chloracne following accidental exposure to trichlorophenol in 1968 at the Coalite Company in Great Britain. Another 54 employees were possibly exposed. None of the workers had significant changes ten years after the accident and neither death from nor evidence of neoplasm was found, 2 2 -' Jirasek's group has closely followed 55 men who were intensely exposed during the manufacture of 2,4,5trichlorophenoxyacetate from 1965 to 1968 in Spolana, Czecho slovakia, and who developed evidence of acute intoxication. Two workers died of bronchogenic carcinoma 5 to 5.5 years after the first exposure. There was no other evidence of malignant neoplasms during a ten-year follow-up, 38. In 1963 an explosion at Phi lip s-D uph ar , Amsterdam, ex posed 106 workers involved in manufacturing 2,4,5-tetrachlorophenyoxy-acewtate. Among the 93 workers followed to 1977, only one death 14 months after the accident was due to cancer and the pancreatic carcinoma involved was apparently symtomatic before the explosion. No case of soft-tissue sarcoma or malignant lymphoma was reported, 39. One study is often cited with the Swedish studies al though it did not deal with soft-tissue sarcomas and mali g nant lymphomas, 40. A more recent review by Thiess et al reports that all- T4 exposed persons are still being followed 1654S DON 2120661 after 26 years. There have been 21 deaths, about equal to the 18 to 20 deaths expected from major comparative popula tions and 18 and 19 deaths expected among matched unexposed controls. Cancer was responsible for 7 deaths as compared, to 4.1 expected from the comparative populations and 5 in each internal control group. Gastric carcinoma in 3 exposed persons exceeds the expected 0.61 to 0.70 expected cases. There were, however, no soft-tissue sarcomas or malignant lymphomas among these chemical workers at BASF, 4 1 A number of other industrial exposures to phenoxy herbi cides, their precursors or contaminants were reported before 1973, 4 2 . The populations were small but generally heavily exp osed. Unfortunately it has not been possible to locate late reports on the exposed populations although ten years or more have elapsed since exposure. The accident at the ICMESA factory in Seveso, Italy, in July 1976 exposed many people to trichlorphenol; more than 5400 adults and children of.both sexes are known to have been in contact with the chemicals for several days, 4_3_. Although only about six years have elapsed since the exposure, the population has been under surveillance and the rate and causes of death are being followed. To date no soft-tissue sarcomas or malignant lymphomas have been reported. Another less systematic observation bears on the situa tion. The phenoxy herbicides have been used frequently and extensively in agriculture and forestry in the United States since the late 1940's. They were used on lawns in cities, as well, for. most of that period. If the relative risk of d e veloping so distinctive a group of tumors as the soft-tissue sarcomas and the malignant lymphomas had increased by 5 of 6 fold over that before 1945 as the Swedish studies would pre dict, it almost certainly would have been evident to clini cians and pathologists, especially in the rural areas, even without systematic studies. No such increase was noted. Critical Evaluations' The Swedish investigators have been cautious in inter preting their results. In his medical dissertation based on his epidemiological studies, Hardell judges that the similar results in the two case-control investigations (J_2, 13, 14, 16) "seem to increase the confidence that the observed asso ciation of exposure to phenoxy acids and soft-tissue sarcoma was not spurious" and did not believe that confounding fac tors "could account for the observed relation." In summary, he concluded that "it is suggested that exposure to phenoxy 12 16547 DOW 2120662 acids should be looked upon as an occupational cancer hazard," 44 . Other reviewers have been more skeptical as to the sig nificance of the work. Remington's overall opinion was that "in to t o , the Swedish work is credible if not fully conclu sive. Certainly this work would seem to justify further investigation," 10. Coggan and Acheson, after reviewing other work as well as the Swedish studies, state that "on the present evidence it seems possible that soft-tissue sar comas have arisen in association with exposure to phenoxy herbicides" but continue that "it is as yet impossible to estimate with any precision the risk of soft-tissue sarcoma due to phenoxy herbicides." They conclude that "there is suggestive evidence of a biological association between phenoxy herbicides (or their contaminants) and soft-tissue sarcoma. The evidence relating these products to the occurance of lymphoma is weaker," 22_. An unsigned editorial in Lancet commenting on the opinions of Coggan and Acheson seems to agree with their conclusions with regard to soft-tissue s a r c o m a s , 45 . Hardell and Axelson disagreed with both the Coggan and Acheson's opinions and the Lancet editorial, 23. They have been at some pains to counter charges oT- "ob servational bias," 15, but have not convinced everyone that faulty memories do not result in significant errors in evaluating exposure, 45. The causal connection between phenoxy herbicides and soft-tissue `sarcomas would be much more likely if there were a unique preponderance of one type or even of a few types in the exposed men. The Swedish reports never compare the morphological types or location of the malig nant tumors in cases with those in controls, 45. Their only justification for aggregating the types, and presum ably for omitting the data from their reports is "the uncertainty of relations between the various histological groups in terms of causal mechanisms" and "the so-called addition theorem for chi-square and Poisson distribu tion," 23_. The uncertainty of causal relations is precisely the reason for reporting the groups and the addition theorem cannot justify the aggregation of unlike *2,3 ,7,8 -tet rachlorod ibenzo-p-d iox in has be en suggested as the ;prin cipal carcinogen in th e phenoxy herbicide 2,4 ,5-T and trichlorophenols but this has been disputed, See 22, 23, 45, 46 . The controve rsy is no t considered in this dis cussion. ( 13 16548 W 2120663 entities unless significant common factors have been demonstrated. Scientific results are strengthened greatly when independent investigators substantiate them. The Swedish studies have been said to be independent and confirma tory. The two soft-tissue sarcoma investigations do support one another (_1_2, 13, _1_4, _1_6) but they are the work of the same group of investigators. The investiga tion of malignant histiocytic lymphoma was also conducted by the same group but was a case-control study of a separate entity, (19-21 ). Axelson's work on herbicide exposure and cancer _4) was not truly independent from Hardell's efforts since Hardell has recognized his in debtedness to Axelson for his assistance in the first case-control study, (13, 14). More important Axelson did not associate phenoxy herbicides or chlorophenolic com pounds with soft-tissue sarcomas nor with malignant lym phomas among railroad workers, J_-4_. The reports of soft-tissue sarcomas among chlorphenol workers in the United States (24-27 ) have been cited as supporting Hardell's conclusions, _2_4, _28_, _4, 4_6. The data have been reported piecemeal without a clearly enumerated total population from which they were drawn. The comparison was made to mortality data for the general population of the appropriate age and sex. The type of soft-tissue sarcoma is known for each case; among the 7 men were 2 malignant fibrous histiocytomas, 2 1 iposarcomas, as well as one each of fibro sarcoma, malignant schwannoma, and fibrosarcomatous mesothe lioma. As before, the tumors are not of a uniform type. Coggan and Acheson comment that the Swedish studies anc the American reports taken separately do not "provide con vincing evidence that the incidence of soft-tissue sarcomas is increased after exposure to phenoxy acids and chlorophenols, -- Considered together the whole becomes more persuasive." They add that "it is surprising that the asso ciation should apply to tumors of such a variety of tissues," 2 2 . The Lancet editorial finds only that "the number of deaths due to soft-tissue sarcomas [in the American data] is disturbing;" 45. In addition to the American experience, the British (37), European (30, 32, 33 , 36, 38-4 1) and New Zealand (3 4 , 3~5T medical and scientific writers "Have studied populations TTve years or longer after exposure to phenoxy herbicides and/or chlorophenols in a variety of situations, some intense and acute, others prolonged. Only one observer (30 ) reported a case described as a soft-tissue malignant neoplasm without 16549 14 further characterization. The same report included a lymphosarcoma, a malignant neoplasm of lymphoid tissue and 3 plasmocytomas. No other study found a soft-tissue tumor. In summary, the Swedish studies of soft-tissue sarcomas cannot be considered to have proved that exposure to phenoxy herbicides is the cause of one or more types of this varied group of malignant tumors. There are no fully reported systematic studies to confirm what the Swedish investigators describe as an association. There are an epidemiological study (_3_2_) and observations of exposed populations that do not support the finding as opposed to uncorrelated American observations and an East German study (3 0 ) that do strengthen the case for such an association. At best, the Scottish v erdict of "Not proven" seems most realistic at this time. The Advisory Panel on Toxic Substances of the American Medi cal Association says that "while 2,4,5-T and 2,4-D pesticides (phenoxy herbicides in Agent Orange) have been used in ag riculture, forest management and residential landscaping for over 30 years, there is still no conclusive evidence that the y and/or TCDD (a contaminant of Agent Orange) are mutagenic, carcinogenic, or teratogenic in man, nor that they have caus ed reproductive difficulties in the human," 47. L. B. HOBSON 3/5/83 moziz'Hoa 16550 1 =; BIBLIOGRAPHY 1. Sundell, L . , Rehn, M., and Axelson, 0. Exposure to herbicides - mortality and tumor incidence. An epidemiological investigation in Swedish railway workers. Lakartidningen 71:2466-2470. 1974 2. Axelson, 0. and Sundell, L. Herbicide exposure, mortality, and tumor incidence. An epidemiological investigation on Swedish railroad workers. Work Environ. Health 11:21-28. 1974 3. Axelson, O. and Sundell, L. Phenoxy acids and cancer. Lakartidningen 74:2887-88. 1977 4. Axelson, 0., Sundell, L . , Andersson, K., Edling, C., Hogstedt, C. , and Kling, H. Herbicide exposure and tumor mortality. An updated epidemiological investigation on Swedish railroad workers. Scand. G Work Envirn. & Health 6:73-79. 1980 5. Axelson, 0. Aspects on confounding in occupational health epidemiology (Letter.) Scand. J. Work Environ. & Health 4:85-89. 1978 6. Axelson, 0. Views on criticism of pesticide studies (Reply to note by Jannerfeldt, E.). Lakartidningen 77( 12) :1096-1 099. 1980 7. Axelson, 0. A note on observational bias in case-referent studies in occupational health epidemiology. Scand. J. Work Environ. & Health 6:80-82. 1980 8. Axelson, 0. Testimony before EPA Hearing, EPA Exhibit No. 587. 1980 9. Jannerfeldt, E. Epidemiologic methodology and pesticide studies. Lakartidningen 77(12): 1096 . 1980 10. Remington, R. D. Specific summary critique of five investigations related to concerns about Agent Orange. Congressional Record, p p . S- 10911, S 10912, August 6, 1980. 11. Hardell, L. Verbal testimony at EPA Hearing. September 29, 1980. a O O 5E 16551 S 990 1 /1 12. Hardell, L. Malignant mesenchymal tumors and exposure to phenoxy acids - A clinical observation. Lakartidningen 7 4 ( 3 3 ) :2753-2754. 1977 13. Hardell, L. and Sandstrom, A. Malignant mesenchymal soft tissue tumors and exposure to phenoxy acids or chloramphenacol. Lakartidningen 75:3535-3536. 1978 14. Hardell, L. and Sandstrom, A. Case-control study: Soft tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. Brit. G. Cancer 39:711. 1979 15. Hardell, L. Relation of soft-tissue sarcoma, malignant lymphoma anc colon cancer to phenoxy acids, chlorophenols and other agents. Scand. G. Work Environ. & Health 7(119-130). 1981 16. Axelson, 0. Aspects cf confounding and effect modification in the assessment of occupational cancer risk. J. Toxicol. & Environ. Health 6(5-- 6):1127-- 1131. 1980 17. Eriksson, M. , Hardell, L., Berg, N. 0., Mller, T. and Alexson, 0. Case-control study of malignant mesenchymal tumors of the soft tissue and exposure to chemical substances. Lakartidningen 76:3872-3875. 1979 18. Eriksson, M . , Hardell, L., Berg, N. 0., Mller, T . , and Axelson, O. Soft-rissue sarcomas and exposure to chemical substances: a case-referant study. Brit. J. Industr. Med. 38:27-32. 1981 19. Hardell, L. Malignant lymphoma of histiocytic type and exposure to phenoxvacetic acids or chlorophenols. Lancet 1(8106):55-56. 1979 20. Hardell, L . , Erikson, M. , Lenner, P. Malignant lymphoma and exposure to chemical substances, especially organic solvents, chlorophenols and phenoxy acids. Lakaricningen 77( 4 ) :208-21 0. 1980 21. Hardell, L . , Erikson, M., Lenner P., and Lundgren, E. Malignant lymphoma and exposure to chemicals, especially organic solvents, chlorophenols and phenoxy acids: a case-control study. Brit. J, Cancer 43:169. 1981 b 9930ZIZ MOO 16552 V M00 22. Coggon, D. and Acheson, E. D. Do phenoxy herbicides cause cancer in man? Lancet 1(8278)1057-1059. 1982 23. Hardell, L. and Axelson, 0. Soft--tissue sarcoma, malignant lymphoma, and exposure to phenoxy acids on chlorophenols..Lancet 1 (8286 ): 1408-1409. 1982 24. Honchar, P. A. and Halperin, W. E. 2,4,5-T, trichlorophenol, and soft-tissue sarcoma. Lancet 1(8214) :268-269. 1981 24a. Ott, M. G . , Holder, B. B . , and Olson, R. D. A mortality analysis of employees engaged in the manufacture of 2,4,5-trichlorophenoxyacetic acid. J. Occup. Med. 22:47-50. 1980 24b. Cook, kR. R . , Townsend, J. C . , Ott, M. G . , and Silverstein, L. G. Mortality experience of employees exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (T C D D ). J. Occup. Med. 22:530-532. 1980 25. Cook, R. R. Dioxin, chloracne, and soft-tissue sarcoma. Lancet 1(8220)618-619. 1981 25a. Zack, J. A. and Suskind, R. R. The mortality experience of workers exposed to tetrachlorodibenzodioxin in a trichlorophenol process accident. J. Occup. Med. 22:11-44. 1980 25b. Zack, J. A. A mortality study of workers employed at the Monsant Plant in Nitro, W.Va. Unpublished. 26. Moses, M. and Selikoff, I. J. Soft-tissue sarcomas, phenoxy herbicides, and chlorinated phenols. Lancet 1(8234) :1370. 1981 27. Johnson, F. E . , Kugler, M. A., and Brown, S. M. Soft tissue sarcomas and chlorinated phenols. Lancet 2(8236):4 0. 1981 28. Hardell, L. and Erikson, M. Soft-tissue sarcomas, phenoxy herbicides and chlorinated phenols. Lancet 2(8240):250. 1981 29. Sarma, P. R. and Jacobs, G. Thoracic soft-tissue sarcoma in Vietnam veterans exposed to Agent Orange. New Engl. J. Med. 306(1 8):1 1O S . 1981 c DON 2 1206G8 .. J 30. Barthel, E. Cancer risk in agricultural workers exposed to pesticides. Arch. Geschwulstforsch. 51 (7):5 79-58 5. 1981 31. Bishop, C. M. and Jones, A. H. Non-Hodgkin's lymphoma of the scalp in workers exposed to dioxin. Lancet 2( 8242):369. 1981 32. Riikimaki, V. , Asp, S., and Hernberg, S. Mortality of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid herbicide applicators in Finland. Scand. J. Work Environ. & Health 8:37-42. 1982 33. Hogstedt, C. and Westerlund, B. Cohort study of mortality rates of forestry workers with and without exposure to phenoxy compounds. (No translation available) Lakartidningen 77(19):1828-1830. 1980 34. Smith, A. H., Fisher, D. 0., Pearce, N. and Teague, C. A. Do agricultural chemicals cause soft-tissue sarcoma? Initial findings of a case-control study in New Zealand. Unpublished report at ANZSEARCH Annual Conference, May, 1982 35. Smith, A. H . , Fisher, D. 0., Giles, H. J . , and Pearce, N. The New Zealand soft-tissue sarcoma case-control study: interview findings concerning phenoxyacetic acid exposure. Unpublished report at Third International Symposium on Chlorinated Dioxins and Related Compounds. October 1982 36. Edling, C. and Granstam, S. Pattern of causes of death among forestry workers, a pilot study. Hygiea 8 8 (3):7 4 . 1979 37. May, G. Tetrachlorodibenzodioxin: a survey cf subjects ten years after exposure. Erit. J. Industr. Med. 39:128-135. 1982 38. Pazderova-Vejlupkova, J . , Nemcova, N., Pickova, J., Lukas, E., and Jirasek, L. The development and prognosis of chronic intoxication by tetrachlorod ibenzo-d iox in in rr.an. Arch. Environ. Health 36( 1): 5-1 1. 198 1 d 16554 DOM 2 1 2 0 6 6 9 39. Cited in Huff, J. E., Moore, J. A., Saracci, R. and Tomatis, L. Long-term hazards of polychlorinated dibenzodioxins and polychlorinated dibenzofurans. Environ. Health Prespectives 36:221-240. 1980; and Hay, A. Accidents in trichlorcphenol plants: a n^-ecl for realistic surveys to ascertain risks to health. Ann. N.Y. Acad. Sci. 320:324. 1579 40. Thiess, A. M. and Frentzel-Beyne, R. Mortality study of persons exposed to dioxin following an accident-, which occurred in the BASF on November 13, 1953. Presented at the Fifth International Conference on Medichem. September 1977 41. Thiess, A. M . , Frentzel-Beyme, R. , and Link, R. Mortality study of persons exposed to dioxins in a trichlorophenol process accident that occurred in the BASF AG on November 17, 1953. Am. J. Industr. Med. 3:179-189. 1982 42. Young, A. L . , Calcagni, J. A., T h alk en , C. E., and Tremblay, J. W. The toxicology, environmental fate, and human risk of Herbicide Orange and its associated dioxin. USAF OEHL Technical Reoort (Report OEHL TR 78-92) pp. V-7, V-8 43. Caramaschi, F., del C o r n a , G., Favaretti, C., Giambelluca, S. E., Montesarchio, E., and Fara, G. M. Chloracne following environmental contamination by TCDD in Seveso, Italy. Intern. J. Epidemiol. 10(2):135-143. 1981 44. Hardell, L. Epidemiological studies on soft-tissue sarcoma and malignant lymphoma and their relation to phenoxy acid or chlorophenol exposure. Umea University Medical Dissertations, New Series No. 65. 1981 45. Anonymous. Phenoxy herbicides, trichlorophenols, and soft-tissue sarcomas (Editorial). Lancet 1(8278):1051-1052. 1982 46. Hardell, L. and Axelson, O. Phenoxy acids, chlorophenols, and cancer. Lakartidningen 78( 34 ):2862-2863. 1981 47. Council on Scientific Affairs' Advisory Panel on Toxic Substances (American Medical Association). The health effects of "Agent Orange" and polychlorinated dioxin contaminants. October ', 1981 e 16555 j 2614 7 DIOXIN AND HUMAN HEALTH During the past decade, considerable controversy has developed over the human health effects of exposure to TCDD, an unintentional contaminant from the production of some herbicides and antibacterial agents and a substance created by the process of combustion. This controversy has been fueled by reports on the effects of Agent Orange exposure on returning Vietnam veterans, the Love Canal situation, the explosion of a 2,4,5-trichlorphenol plant in Seveso, Italy, and, most recently, the buy-out by the U.S. government of Times Beach, Missouri, a town contaminated with dioxin, (i.e., 2,3,7,8-tetrachl orodibenzo-p-dioxin [TCDD]). During this time period, much speculation and misinformation regarding this issue have been generated, with resulting confusion and apprehen sion in the public mind. It is the purpose of this paper to state the facts regarding TCDD and human health and to place the issue in its proper perspective. This paper will document the following key points: Certainly 2,3,7,8-TCDD is a potent and toxic compound that can cause a variety of ill health effects at relatively low doses in laboratory animals. We should be concerned with reducing any unreasonable risk of human TCDD exposure in our environ ment. Based on the preponderance of scientific studies, the trace environmental presence of TCDD does not pose a health hazard to people. There is a wealth of epidemiological data supporting the position that humans are far less susceptible to TCDD than several of the animal species which have been studied. The mere presence of TCDD in the environment (soil , water, fish, etc.) is not proof of contamination from industrial chemical sources. The scientific evidence does not demonstrate that TCDD exposure causes cancer in humans. There is no valid epidemiologic evidence linking TCDD exposure in humans with a higher-than-normal rate of birth defects. 2,3,7,8-tetrachl orodibenzo-p-dioxin D0 2 1!389 2 -la- TABLE OF CONTENTS What is D i o x i n ? .................................................... The American Medical Association Report ........................... Seveso.............................................................. Epidemiological Studies ............................................ Soft Tissue S a r c o m a ................................................ The Dow Mortality S t u d y ............................................ Birth D e f e c t s ...................................................... The Toxicity of TCDD in Animal Species............................. Environmental Hazard Evaluation (Soil/Fish) ....................... References.................................................. . . . . 2 4 7 9 10 17 19 21 24 28 DOH 2113893 16557 -2- What is Dioxin? The term "dioxins" (or more scientifically, "chlorinated dibenzo-pdioxins") refers to 75 similar, but different substances.1 Each of the 75 dioxins that has been studied has its own unique identity and toxicological properties. On the basis of animal studies, 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the most toxic of the dioxins, and it has been comprehensively examined for its toxicological effects. This report will generally address the health impact of TCDD exposure. 1 6 8 E I 1 1 KOQ TCDD is a highly toxic compound that can cause a variety of ill health effects at relatively low doses in laboratory animals. We must be concerned with establishing safe levels of human TCDD exposure in our envi ronment. TCDD is formed as a trace unwanted contaminant in the production of 2,4,5-trichlorophenol from tetrachlorobenzene. The herbicide 2,4,5trichlorophenoxyacetic acid (2,4,5-T), other trichlorophenoxyacids, and the germicide hexachlorophene are synthesized from 2,4,5-trichlorophenol and may contain trace amounts of TCDD and other polychlorinated dioxins. 2 Dow has not produced the herbicide 2,4,5-T in the United States since 1979 due to reduced market demand from an Environmental Protection 16558 DOH 1 1 13 8 9 5 - 3- Agency (EPA) emergency suspension which restricted some applications of the product. There are no manufacturers currently producing 2,4,5-T in the United States. A Dow joint venture company produces 2,4,5-T in New Zealand. Dow currently supplies 2,4,5-T from inventories for the rice and rangeland applications presently approved by EPA. The trace amount of TCDD present in Dow-inventoried 2,4,5-T is less than 10 parts per billion. TCDD can also be created by combustion. Data indicate that chlorinated dioxins may arise from the combustion of most types of organic material, and suggest that chlorinated dioxins result from trace chemical reac tions occurring in fire.3 Many chemical reactions occur during combustion at very low concentra tions -- parts per billion and lower. With more advanced analytical capabilities, scientists have discovered numerous sources where various dioxins have been formed, including TCDD. Some of these sources include refuse incinerators, wood-burning stoves, gasoline- and diesel-powered automobiles and trucks, fireplaces, char coal grills and cigarettes. TCDD and the other dioxin compounds have been entering the atmosphere via airborne particulates from these various sources and settling on soil and in bodies of water. There have been a number of industrial incidents throughout the world where people have been exposed to TCDD. Epidemiological studies have 16559 DO^ 2 1 13096 - 4- evaluated these incidents and ail of the evidence so far tends to reduce the initial concern about the health effects of TCDO on humans. An overall look at the mounting data has been undertaken by the American Medical Association. These findings and conclusions are of interest and bear mention. The American Medical Association Report In 1981 the American Medical Association (AMA) published a technical report reviewing the medical evidence regarding the toxicity and long term health effects of the TCDO contaminant in Agent Orange*. The report's preface stated: "In spite of the voluminous data...there is still very little substantive evidence for many of the alleged claims that have been made against these compounds (dioxins). The most serious of these allegations assert that Agent Orange, or compounds of a like nature, have caused malignant tumors, spontaneous abor tions and birth defects. Although data from studies on experi mental animals tend to support some of these claims, it is not certain that the animal data are extrapolatable to man. No laboratory animal can fully substitute for man; we must, therefore, depend on the results of ongoing epidemiological studies on persons who are known to have been exposed." *(Agent Orange was a 50/50 herbicide mixture of 2,4,5-T and 2,4-D pro duced according to specifications set by the U.S. government for use as a defoliant in Vietnam to protect American troops from enemy ambush.) 16560 - 5- A close look at the summary of the report gives a clearer insight into what the AMA's Advisory Panel on Toxic Substances found. The AMA Panel reported that, in addition to the marked variations in the sensitivity and susceptibility of animal species to all toxic substances, there are significant differences between some of the toxic effects of TCDD in experimental animals and the human experience. People appear to be less sensitive to the health effects of TCDD than some of the animal species studied. DON 2 1 1 3 8 9 7 One of the more pronounced biological effects of heavy exposure to TCDD, as well as a number of other chlorinated aromatic compounds, is a tendency to cause chloracne in certain animals and humans, the AMA Panel reported. This particular skin condition is regarded as the clinical marker for TCDD over-exposure in people. The Panel noted that human systemic dis orders from exposures to TCDD are unlikely to occur in the absence of chloracne. Such findings as impaired liver and kidney function, gastro intestinal irritation, muscle and nerve disorders, and depression and irritation of the central nervous system have been reported after expo sure to relatively large amounts of TCDD. However, these disorders have not been progressive, and they have disappeared with time, the AMA report stated. In short, if there is no chloracne, there are unlikely to be other persistent toxic effects from exposure to TCDD. The AMA Panel also reported that other toxic effects of TCDD, when experimentally produced in test animals, appear as pathological changes in the liver, lymphoid and epithelial tissue. 16581 -6 - TCDD is a powerful enzyme inducer. In addition to altering normal enzyme activity, it may potentiate or inhibit the action of other toxins. The AMA report stated that TCDD may act as a promoter of carcinogenesis in some strains of rats and mice. For cancer to start, it must go through several distinct steps. The first stage, called initiation, is produced by a carcinogen (an initiator) and probably involves irrevers ible mutational changes. Subsequent growth and development of an actual cancer may require promotion, which may be the result of additional applications of a carcinogen, or of other, non-carcinogenic materials. Promotion may involve additional unknown processes which could aid the growth of the cancer or reduce resistance to the developing cancer. D0';i 2 113 8 9 8 TCDD is among the group of chemicals that may secondarily influence the formation of cancer by promotion. The carcinogenicity associated with TCDD in laboratory animals is typically accompanied by other signs of systemic toxicity in contrast to some other chemical carcinogens for which cancer is the only observable toxic effect. TCDD has reportedly induced genetic changes in some forms of bacteria, but evaluations in rodents and humans do not indicate that these mutational changes will occur in higher animals, let alone people. The AMA Panel made clear in its summary that the extensive information collected for more than 30 years provides no conclusive evidence that 1656? - 7- 2,4,5-T and/or TCDD are mutagenic, carcinogenic or teratogenic in humans, nor that they have caused reproductive difficulties in people. Furthermore, the AMA report stated that 2,4,5-T undergoes relatively rapid decomposition in the soil. However, TCDD does persist in soil longer than 2,4,5-T, but, in general, the half-life of TCDD in soil may be no longer than one year. TCDD exposed to ultraviolet light is broken down rapidly when present as a thin film on plants, water and the surface of soil.4 Seveso The most well-known and widely studied incident of community exposure to TCDD followed the July 10, 1976, explosion at the ICMESA trichlorophenol plant at Seveso, Italy. As a result of that accident, approximately seven square miles of the countryside were contaminated by chemicals, including TCDD, exposing as many as 30,000 p e o p l e . T h e heaviest con tamination involved an area of 180 acres (Zone A), occupied by 736 people. TCDD was reportedly detected on soil at levels in excess of 5.5 parts per million ( p p m ) . 7,5 in general, little effect was noted on plant life, but wild animals did die, particularly in Zone A (nearest the chemical plant). About four percent of the domestic animals in contaminated zones died, and virtually all of these were small animals.5 Some of these deaths may have been due to other chemicals released at the same time. DOS 2113899 1S5S3 DOW 2113900 -8- Chloracne, which has generally been regarded as the hallmark or sentinel sign of TCDD exposure^, was observed in this population, although at generally low rates and primarily in children.9.10,5,6 j^e highest incidence of chloracne reported was about 13-14 percent among elementary school children in Seveso, and this was generally mild and rapidly resolved in most affected peopl e.5,6,11 other than chloracne (of which there were ultimately 100-200 cases), nausea, vomiting, itching and head ache were the most commonly observed symptoms^ readily attributable to the exposure which may have involved chemicals other than TCDD. Head ache, stomach and intestinal upset were more commonly noted in individuals with chioracne.9,6 Bisanti'12 concluded that no "major event" had taken place with respect to birth defects, miscarriages (spontaneous abortions), births and deaths. While this seems to be the consensus, changes in birth defect reporting and in the reporting of "spontaneous" (sometimes "thera peutic") abortions have obscured any small changes which might have occurred.13,5,6,14,11 Data on reproductive outcome are limited in number, but Reggiam'H reports that examination of fetuses in 34 cases of medically-induced abortion revealed no evidence of injury attribu table to TCDD. Pregnancy outcome in the years following 1976 appears to be comparable with Western experience. 6 Of particular interest is the fact that no birth defects were observed during 1977 among the 70 births in Zones A and B. Only those pregnan cies coming to term after January 1977 would have been relevant, i.e., DON 21(3901 - 9- those in which July 1976 maternal exposure would have taken place during the critical period for the developing fetus. Many factors are known to cause birth defects in humans, which occur in three to six percent of live births: viral infections, genetic predis position, smoking, alcohol, and even medications, including excess quantities of certain vitamins. Epidemiological Studies In recent years a number of investigations have been conducted concern ing the human health effects associated with exposures to TCDD. The following brief review of the human exposure experience with TCDD and the epidemiological studies of those people are taken from a recent report by the Advisory Committee on Pesticides, which is charged with making recommendations concerning the safety and safe use of pesticides in the United Kingdom. Monsanto's plant at Nitro, West Virginia: 121 workers were followed up for 30 years after exposure to TCDD. No apparent excess in total mortality or in deaths from cancer or diseases of the circulatory system was observed. 15 Coalite plant (1968) in the United Kingdom: Chloracne was initially seen in 79 workers and some had initial abnormali ties in the liver but these returned to normal limits within 10 days. No clinically recognizable disease has been demon strated. There is no evidence of liver or cardiovascular di sease.15 16535 DOW 2113902 -10- Dow Chemical Company workers exposed to 2,4,5-T: No adverse effects have been noted in an epidemiological study in these workers (mortality was actually less than e x p e c t e d ) . 15 Vietnam: Exposure to Agent Orange is blamed for a host of illnesses. The U.S. Veterans Administration found that no cause-effect relationship was established between TCDD exposure and alleged medical complications.15 Finnish railroad workers using 2,4,5-T and other phenoxyacetic acid herbicides in forestry and on roads and railways: 1,960 men were studied and no increased mortality from cancer was demonstrated. There were no deaths from cancer in forestry workers although six deaths would have been statistically predicted.15 Soft Tissue Sarcoma The most persistent hypothesis deals with the possible causal associa tion between TCOD and soft tissue sarcomas. This hypothesis is derived from the epidemiological work done by Dr. Hardell and his associates in Sweden.15 Soft tissue sarcoma is a generic term for a group of lesions that include more than 100 different types of tumors.1? These tumors are relatively rare; thus few pathologists have been able to achieve a high degree of consistency in their diagnosis.18 Most experts believe that if TCDD were acting as a carcinogen it would cause an increase in one type of soft tissue sarcoma, but would be unlikely to raise the risk all across the board. One of the reasons vinyl chloride is accepted as a human carcinogen is the specific way it acts. At high levels, it produces a single type of cancer: angiosarcoma of the liver. 16536 DOW 2113903 -11- The question which arises is: If there is a common exposure link between various reports of soft tissue sarcomas in occupational groups, is TCDD the link? The exposure documentation differs from study to study. In some, based on detailed technical knowledge of the chemical process, analyses of reaction contaminants, and clinical evidence of excessive group exposure (such as chloracne), there is evidence that TCDD is at least one of the common exposure links. In other reports, the evidence is more presump tive, and in some cases it is totally lacking. Epidemiologists from Dow Chemical and Monsanto have reported on the soft tissue sarcoma cases occurring in their occupational groups presumably exposed to high levels of 2,3,7,8-TCDD.19,20,21 In 1964, a group of Dow trichlorophenol production workers experienced an outbreak of chloracne. Sixty-one workers were identified as being involved with the trichlorophenol process. Forty-nine of these workers developed a rash diagnosed as chloracne. Epidemiologists followed up on the status of all 61 men through 1978, 14 years after the incident. Statistically, 7.8 deaths would have been expected in this group, but only four had occurred. Among the causes of death, one was from cardio vascular disease when 3.8 would have been expected. Cancer was idehtified as the cause of three deaths when 1.6 were expected. None of these findings represent a statistically significant deviation from the expected experience. 20 One of the cancer deaths in the Dow group was -12- due to a type of soft tissue sarcoma called fibrosarcoma. In 1981 another employee in the Dow trichlorophenol worker group developed another type of soft tissue sarcoma, malignant fibrous histiocytoma, one of a multitude of tumors which comprise the soft tissue sarcoma category. The employee died in 1983. In the 1978 Monsanto study, no link between exposure to TCDD and excess deaths due to cancer or other causes was found among 121 current and former workers employed at a plant in Nitro, West Virginia. The employees who participated in the study included workers who developed chloracne as a result of over-exposure to TCDD in a manufacturing accident in 1949. E i i z MOO Among the Monsanto workers studied, 32 deaths were reported versus 46 which would be expected. Cancer accounted for nine deaths among the Monsanto group compared with slightly more than nine deaths expected. One of these cancer deaths was attributed to.soft tissue sarcoma, a malignant fibrous histiocytoma. * 06 A second Monsanto study evaluated the mortality experience of male hourly workers employed at the Nitro plant during or after 1955 and followed through 1977.22 when adjustments were made for prior exposures to a known bladder carcinogen, the observed deaths due to all causes and due to cancer were less than expected. Among the subset of employees who had worked in 2,4,5-T production, the proportion of deaths due to cancer was lower than that found in the rest of the plant population. In this subset one individual died as a result of a soft tissue sarcoma, a liposarcoma. XGSS'S DOH 2 1 1 3 9 0 5 -13- These four soft tissue sarcoma cases were summarized in the literature in 1981.23,24 /\11 four had occupational exposures to trichlorophenol. Only one, the second Monsanto case, had also been exposed to 2,4,5-T. Three had frank chloracne and the fourth, a Dow employee, a facial rash during his assignment in the trichlorophenol plant. All four had a history of cigarette smoking. It was suggested that the hypothesis of soft tissue sarcomas being caused by heavy TCDD exposure among cigarette smokers needed to be tested. Three other cases of soft tissue sarcoma were also reported in 1981.25,26 Exposure to 2,4,5-T, trichlorophenol or TCDD was not well documented. In one case, the individual was considered unexposed until his disease was discovered. The other two occurred in a father and son. In a group of Dow 2 , 4 , 5-T employees (none of whom had chloracne)27 there have been no cases of soft tissue sarcoma. At Dow, the mortality experience of 204 persons exposed to 2,4,5-T during its manufacture from 1950 to 1971 was studied through 1978. No adverse effects were observed with respect to occupational exposure to either 2,4,5-T or its feedstock, 2,4,5-trichlorophenol. Mortality of the studied workers was favorable compared to that of U.S. white males. There have been no cases of soft tissue sarcoma reported in a BASF worker group, many of whom experienced severe chloracne. Seventy-four DOH 2113906 - 14- members of the BASF group were exposed to TCDD during a 1953 accident in a trichiorophenol production unit or during cleanup and repair following the accident. Exposures were heavy. Twenty-seven years after the accident a mortality study of those exposed to TCDD in the accident was undertaken. Overall mortality did not differ in this group from the rate expected in three external reference populations or from that observed in two internal comparison groups.28 Dow is cooperating with the National Institute for Occupational Safety and Health (NIOSH) to prepare a study of more than 4,000 industrial workers potentially exposed to dioxins. Swedish Epidemiological Studies Dr. Hardell's work, which has lead to the question about soft tissue sarcomas, uses a different methodology than in the foregoing studies -- the case control approach -- and reports on different patterns and levels of exposure.16.29 in his first study, he reported a five- to six-fold increased risk of soft tissue sarcomas for those presumably exposed to phenoxyacetic acids, predominantly 2,4,5-T, and chiorophenols ("pre sumably" because the exposure information was subjectively reported by the study participants and/or next of kin). Objective records were incomplete and were difficult to interpret. In the second study, he also estimated the risk associated with exposure to phenoxyacetic acids not contaminated with TCDD and found the same magnitude of risk. D09 2113907 -15- Some epidemiologists reviewing the information find this confusing. The justification for combining chiorophenols and 2,4,5-T was the presumption of a common contaminant, TCDD.29 if that were the case, increased risk would not be expected for the phenoxyacetic acids without this contaminant. In both of his studies, Dr. Hardell first administered mail questionnaires and then followed up with telephone interviews on a selected subset of subjects. While the questionnaire was designed with the idea of masking the intent of the investigation, the telephone inquiry specifically focused on the exposures of pertinent interest: phenoxyacetic acids and chlorophenol s. Interestingly, in his second study, Dr. Hardell reported the frequency of the various exposures among both cases and controls and most of the risk estimates were elevated. The risk from only one out of 15 chemi cals, sodium chlorate, was not elevated. Nicotine had a high risk. Unfortunately, the risk estimates derived from the questionnaire alone are not known, nor is it known what the risk estimates would have been for other agents, if they had been probed in the same depth as were the phenoxyacetic acids and chiorophenols. There are a number of other concerns relating to Hardell's work. The accuracy with which exposure to the chemicals is determined is of vital importance, and the memory recall techniques Or. Hardell used are subject to error. Workers are unlikely to remember with accuracy the chemicals they used some years in the past. It is also extremely difficult to estimate the extent or duration of the exposures. Furthermore, the DOW 2 1 1 3 9 0 8 - 16- identification and classification of the various soft tissue sarcomas have not been consistent. The diversity of tumors is particularly difficult to explain. If all the tumors had been of a single type, the data would be much more convincing. Furthermore, research currently underway does not support Harden* s hypothesis concerning soft tissue sarcoma: The U.K. Government Advisory Committee on Pesticides noted in February 1983: "It is our view that the procedures used for establishing the Swedish study groups, and some aspects of the collection of the exposure data, were not reliable." Smith and associates^ are conducting a case-control study in New Zealand. Herbicide application is a registered profession in New Zealand, and phenoxyacetic acids have been used in bulk since the late 1940s. In a preliminary report presented at the 1982 Inter national Symposium on Chlorinated Dioxins and Related Compounds, Smith noted he was unable to establish an association between the use of phenoxyacetic acids and soft tissue sarcoma. In fact, not one of the soft tissue sarcoma patients had ever worked as a licensed phenoxyacetic acid herbicide applicator. Milham31 explored this issue in the state of Washington using death certificates. While there has been considerable exposure over the years to phenoxyacetic acids and chlorophenols in Washington state, occupations related to their use did not show consistent patterns of death due to soft tissue sarcomas. Surprisingly, the two occu pations with the highest proportional mortality ratios were marine engineers and bankers. Among the deaths in the U.S. Air Force "Ranch Hand" Study, none were due to soft tissue sarcomas.32 Riihimaki studied 1,960 Finnish herbicide applicators and found no difference in death rate from any natural cause, including cancer, compared with the total Finnish male population. There were no soft tissue sarcomas reported in this group of workers.33 The Michigan Department of Public Health (MDPH) is investigating the possible relationship between dioxin and a small number of soft and connective tissue cancer deaths among women in Midland County 16572(where Dow has facilities).^ In a recent report (May 1983) MDPH DON 2113909 - 17- did not identify a specific association between any environmental factor and the reported increase of soft tissue cancer in the county. While overall age-adjusted cancer mortality rates in Midland County were below the state of Michigan average, soft and connective tissue cancers among white females in the county were elevated. As part of the initial study, MDPH reviewed data for 28 other U.S. counties in which TCDD or other dioxins were likely produced as a chemical manufacturing contaminant. They found no increase of soft tissue cancer in these counties versus counties that did not have industrial manufacturing sources suspected of generating such dioxins. Various animal species did not develop soft tissue sarcomas when fed TCDD during laboratory experiments. Additional research is under way in the United States and elsewhere. The results of these studies should offer a better perspective on this issue. But right now, the preponderance of evidence does not demon strate a link between TCDD exposure and soft tissue sarcomas. The Dow Mortality Study In 1976 a research study was published that summarized the mortality experience of over 8,000 men employed by The Dow Chemical Company. The population of interest was defined based on a March 1, 1954, employee census of the Midland, Michigan, manufacturing location of the Company.35 The study originally developed background mortality data on the Dow worker population so that findings obtained from smaller groups directed at more specific questions, (e.g. questions regarding particular chemi cal exposures or processes), could be placed in perspective. The observation period for mortality follow-up was 1954 through 1972, and 16573 0 I 6 1 IZ ftOQ - 18- comparisons were made to the mortality experience of the corresponding United States white male population. At that time, mortality among Dow employees was found to compare favorably with that of the general U.S. population. The objectives of the most recent update of this group of 8,181 employees were to extend the observation period through 1978, and to include comparisons with a community-based group of employed men not 0 engaged in chemical manufacturing activities. In addition, the effects of cigarette smoking on mortality were investigated. The Dow group consisted of personnel from research departments, the corporate headquarters, and a major manufacturing division of the Company. It has been estimated that as many as 500 distinct chemical processes ranging from small batch operations to large continuous and highly-automated production units have been onstream at the Midland manufacturing site, including production of trichlorophenol and related products.36 For the most part, the men in the study represent long-term Company employees. Some were hired prior to the 1940s. Cause of death was obtained from death certificates for all but nine o.f the 1,932 deceased members of the study population. - 19- Overall mortality in the Dow group was 19 percent less than that expected for the corresponding United States population. Expressed in another way, a Dow worker of age 25 in 1972 could expect to live 2.2 years longer than a non-Dow worker of the same age. There were no statistical findings of excess mortality among Dow employees for any cause of death category. For total malignant neoplasms, there were five percent fewer deaths observed than expected based on the United States white male population. Significantly fewer deaths were observed than were expected for all categories. Presumably, if low-level TCDD exposure caused a variety of increased illnesses, the opposite from what is stated above would have been found. DOH 2113911 Comparing the Dow workers with a comparable employee population in Michigan, the overall mortality risk was about 10 percent lower in the Dow group than the corresponding non-Dow group.37 Birth Defects Concerns that TCDD may cause birth defects are not substantiated by epidemiological science. A brief review of some areas of concern and the corresponding results, and a more specific look at the Dow situation in Midland, Michigan, are presented: i Aerial spraying of 2,4,5-T in Oregon: Claims of a link between incidence of miscarriages and 2,4,5-T herbicide could not be established when all the data were available.15 isS''5 DOK ZI 13 912 -20- A six-year study of agriculture and forestry workers in Hungary exposed to 2,4,5-T: Data do not suggest that there has been any significant increase of birth defects.^ New Zealand workers exposed to 2,4,5-T: An alleged abnormally high incidence of neural tube defects (such as spina bifida) were chance occurrences, and there is "a very high assurance of safety in normal use" of 2 , 4 , 5 - T . 15 Australian worker exposure to 2,4-D and 2,4,5-T: Claims of high incidence of birth defects were investigated, and available information revealed no evidence that the birth defects were caused by exposure to these two compounds.15 Facial cleft defects in Arkansas: Study of exposure to 2,4,5-T from 1948-74 reveals these defects to have no association with 2,4,5-T.15 About five years ago it was reported that the birth defect rates in Midland County (Michigan) were three times above those for the state of Michigan. At that time, the Michigan Department of Public Health (MDPH) examined this issue. It found an increase had occurred between 1971 and 1974 due to additional reporting of minor congenital malformations. Before 1971 and after 1974, the birth defect rates in Midland County were either at or below state rates. If widespread chemical contamina tion of the environment was responsible for birth defects, the rates should have gone up and stayed up. They did not. The changes may have been due to reporting techniques, not additional cases of birth defects. It is very important to note that the numbers of county births and birth defects were small, and thus, rates calculated from them tended to be more variable from year to year than the comparable rates for the entire state. The MDPH recently announced (May 1983) an updated report which concludes that the birth defect elevations noted in the early 1970s were not unique and that no additional studies are warranted at this time.58 -21- In 1982 Dow scientists published a report of a survey of reproductive events among 370 wives of Dow Michigan Division employees exposed to dioxins.39 They analyzed the associations between exposure and spon taneous abortion (miscarriage), stillbirths, infant deaths, and several categories of congenital malformations before and after controlling for multiple confounders (i.e., sources of error or confusion) independently and in various combinations. They did trend analysis to determine if duration of paternal exposure was related to adverse pregnancy outcome. There were no associations between any exposure and adverse pregnancy outcomes. These findings have recently been confirmed in another study conducted by Australian scientists^ who examined the impact on birth rates involving Vietnam veterans potentially exposed to Agent Orange. The Toxicity of TCDD in Animal Species 2113913 There are wide variations in how different animal species are affected by TCDD. For example, the single oral dose LD50 (the amount that it would take to kill 50 percent of the test animals) in guinea pigs is 0.6-2.0 ug/kg (micrograms per kilogram or every 2.2 pounds of body weight [a microgram is one millionth of a gram; a kilogram is 1,000 grams, or 2.2 pounds]).41j42 jn hamsters, however, TCDD is much less toxic, with an oral LD50 of 1157-5051 ug/kg.43,44 Therefore, the guinea pig is approximately 5,000 times more sensitive than the hamster, although both species are closely related biologi cally. The direct relevance of any single animal experiment to humans must be evaluated with respect to all other available data. 16577 DON 2113914 -22- In acute and subchronic studies, liver toxicity is a prominent component of TCDO toxicity in rats, mice and rabbits, but not in monkeys, where effects on the bone marrow and epithelial tissue are more prominent. Thymic atrophy in early toxicity studies suggested that TCDO might decrease the immune response. In laboratory animals, scientists can induce immunotoxic effects with high doses of TCDD. However, as the Seveso incident shows, these effects have not been demonstrated in exposed humans.45,46 Teratogenicity/Animal Species Of the various toxic responses of animals to TCDO, the potential for teratogenicity (birth defects) has perhaps received the most attention. Teratogenic effects resulting from TCDD have been realized only in mice, which show an increased frequency of cleft palate, along with an abnor mality of the central collection system of the kidney. It is well known that many factors can cause birth defects in mice, including the stress of being transported by air during pregnancy, or being deprived of drinking water overnight. In both rats and mice, dose levels of TCDD have been identified at which these effects do not occur. In rats, TCDD does not cause a teratogenic effect but sufficiently high doses can cause embryo- and fetotoxicity. 16578 - 23- Many of the studies with TCDD in monkeys have been conducted at the University of Wisconsin. In experiments where the high dose level of about 0.011 ug/kg/day TCDD in the diet was given to the monkeys for up to 9.3 months, there were substantial toxic effects.47,48 A preliminary abstract of a follow-up study49 showed that monkeys given diets contain ing approximately 0.0017 ug/kg/day of TCDD had only slight toxicity. There are studies currently in progress at the University of Wisconsin at lower doses. DOW 2113915 In a three-generation reproduction study^O of rats at incremental dose levels of TCDD in the diet, high doses caused decreased fertility and neonatal survival. The intermediate dose level caused decreased fertil ity and other effects in the first two generations, but not in the third. At the low-dose level , there was no impairment of reproductive capacity through the three consecutive generations, indicating that 0.001 ug/kg/day was a no-adverse-effect level over multiple generations. Mutagenicity/Animal Species A number of mutagenic studies have been conducted with TCDD. The major ity of the tests have been negative or uninterpretable. Only two of the tests were positive in one strain of Salmonella bacteria, showing that there is low possibility for mutagenesis with TCDD in bacteria. In studies46,51,52,53,54,55,56,57 with rats or human cells, there is little indication that TCDD elicits a mutagenic response. 165T3 DON 2 1 I39 I6 - 24- Thus, while a few positive or questionable mutagenic responses have been observed in certain plant or microbial test systems, there appear to be no definitive correlates of mutagenicity in higher animals or humans. Carcinogenicity/Animal Species Carcinogenic studies following ingestion of TCDD have been conducted in rats and mice.58,59,60 Review of these data indicates good correlation of the results in rats and mice, with a carcinogenic response associated only with lifetime ingestion of higher dose levels that also induce other toxicity. The liver was the primary target for cancer in both rats and mice. No cancer response occurred at dose levels of 0.0010.0014 ug/kg/day in the rats and 0.001-0.03 ug/kg/day in mice. TCDD does cause cancer in animals. However, it is only at very high dose levels -- dose levels that are higher than those that elicit other kinds of toxicity. Cancer induced by exposure to TCDD would be preceded by substantial signs of toxicity. This toxicity (which is reversible when exposure stops) would act as a sentinel, or warning at dose levels below those that might cause cancer. Environmental Hazard Evaluation Soi 1 Potential human exposure to TCDD from environmental contamination concerns many people. The following factors should be considered when evaluating potential human health hazards due to TCDD in soil: DOW 2 I I39 17 - 25- TCOD is tenaciously bound by soils and other materials (carbon, charcoal), and studies have shown that plants grown in soils containing TCDD do not accumulate or translocate the material. The amount of material absorbed depends on contact with the skin, or ingestion. Soil binding of TCDD reduces the amount that can be absorbed through the skin or internally from ingestion. Animal studies show that only a small amount of TCDD is absorbed from contaminated soil. In animal studies, when such soil con tained about 500 parts per billion (ppb) ppb TCDD and when the soil was held in contact with skin for 24 hours (covered with aluminum foil), about 1/1000 of the TCDD in soil was absorbed. Estimates of absorption from the Seveso incident suggest that people would be expected to absorb about 1/2000 of the average amount of material present in 1 square meter (approximately 1.2 square yards) of their environment each day. If one uses the laboratory or Seveso data, one can estimate that 100-1000 ppb TCDD in soil could result in human absorption of as much as a dose equivalent to that which produced no effect when fed to laboratory rats for a lifetime. If one assumes that soil is eaten in signifi cant amounts by children, the permissible level is less, perhaps 10 ppb. DOH 2 II39 I8 - 26- Based on the assumptions and calculations presented here, (particularly those based on the work of Dr. Kingsley Stevens),61 it appears that general environmental soil contamination in the range of low parts per billion would not pose a health hazard to the general population, including the unborn, children, pregnant women or the old and infirm. For the general population, excluding children with intimate soil con tact or those who ingest soil, a level of 100 ppb would still pose no hazard. In occupational settings, where workers are protected by standard industrial hygiene practices, higher levels of contamination would not be accompanied by greater exposure or hazard. Dioxin and Fish Rats consuming a diet containing 22 parts per trillion (ppt) ppt TCDD (1 nanogram TCDD/kg of body weight per day) for a lifetime showed no adverse health effects due to TCDD. Rats consume about 10-20 percent of their body weight in food each day, while people daily consume about 2-3 percent of their body weight. Thus, for a given TCDD concentration, rats will ingest more of the compound than people. A person eating about 2-3 pounds of food per day uniformly contaminated with 25 ppt TCDD would ingest about 0.5 nanogram/kg body weight/day or about one-half the dose (weight of TCDD per unit of body weight) shown to produce no adverse health effects when fed to rats for a lifetime. Again, this is true because people eat less food in proportion to their body weight compared to laboratory rats. - 27- The dose received by eating contaminated fish is much less. Most people in the United States (99 percent according to U.S. government figures) eat less than 0.6 pound of fish per week. Nine out of ten people in the United States eat less than 0.3 pound of fish per week, while the ''average" person eats still less. Since fish is such a small part of the Anerican diet, 99 percent of the Anerican public would receive less than 1/70 of the amount of TCDD shown to produce no effect in laboratory rats fed TCDD daily for their lifetimes, if those people ate fish con taining 25 ppt of TCDD. Or said another way, a person could eat nearly one ton of fish per year containing 25 ppt of TCDD and not exceed the no-effect level established by laboratory animal experiments. The fish consumption guideline of 25 ppt established by the Food and Drug Administration assures an adequate margin of safety to protect people from overexposure to TCDD.62 ** DOii 21 139 19 August/1983 (6) 1658J DOH 2 1139 20 - 28- REFERENCES 1. Kociba, R. J. and Schwetz, B. A., (1982): Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Drug Metabolism Reviews, 13(3):387-406. 2. Garattini, S., (1982): TCDD Toxicology with Particular Reference to Seveso: Introductory Remarks. Drug Metabolism Reviews, 13(3):345-353. 3. Bumb, R. R., Crummett, W. B., et al., (1980): Trace Chemistries of Fire: A Source of Chlorinated Dioxins. Science, 210, pp. 385-390. October 24, 1980. 4. Beljan, J. R., MD, et al., (1981): The Health Effects of "Agent Orange" and Polychlorinated Dioxin Contaminants, Technical Report, Prepared by the Council of Scientific Affairs' Advisory Panel on Toxic Substances, Dept, of Environmental, Public and Occupational Health, American Medical Association, Chicago, IL, October 1, 1981. 5. Homberger, E., et al., (1979): The Seveso Accident: Its Nature, Extent and Consequences. Ann. Occup. Hyg., 22:327-370. 6. Pocchiari, F., et al., (1979): Human Health Effects from Accidental Release of Tetrachl orodibenzo-p-dioxin (TCDD) at Seveso, Italy. Ann. N.Y. Acad. Sci., vv:311-320. 7. Fanelli, R., et al., (1982): TCDD Contamination in the Seveso Incident. Drug Metab. Rev. 13(3)407-422. 8 . Crow, K., (1978): The Chemical Disease. New Scientist. April 13, 1978. 9. Caramaschi, F., et al., (1981): Chloracne Following Environmental Contamination by TCDD in Seveso, Italy. International J. Epidemiology, 10(2): 135-143. 10. Fara, G. M., et al., (1980): Chloracne After Release of TCDD at Seveso, Italy. In: Chlorinated Dioxins and Related Compounds: Impact on the Environment, Oxford: Pergamon Press, pp. 545-559. 11. Reggiani, G., (1979): Estimation of the TCDD Toxic Potential in the Light of the Seveso Accident. Arch. Toxicol. Suppl., 2:291-302. 12. Bisanti, L., et al., (1979): Experience of the Accident of Seveso. Proceedings of the 5th European Teratology Society Conference, September 4-7, 1978, Budapest, Hungary, 1979. 13. Abate, L., et al., (1980): Mortality and Birth Defects From 1976 to 1979 in the Population Living in the TCDD Polluted Area of Seveso. In: Chlorinated Dioxins and Related Compounds: Impact on the Environment, pp. 571-587, Pergamon Press, Oxford. DOW 2113921 -29- 14. Reggiani, G. , (1977): Toxic Effects of TCDD in Man. NATO Workshop on Ecotoxicology, Guilford, England, July-August, 1977. 15. Kilpatrick, R., et a l . , (1980): Further Review of the Safety for Use in the U.K. of the Herbicide 2 ,4 ,5 -T , Report of the Advisory Committee on P e s t i c i d e s , Ministry of Agriculture, Fisheries and Food, United Kingdom, December, 1980. 16. Hardell, L. and Sandstrom, A., (1979): Case-Control Study: Soft Tissue Sarcomas and Exposure to Phenoxyacetic Acids or Chlorophenol. British Journal of Cancer, 39:711-717. 17. Hajdu, S. I . , (1981): Soft Tissue Sarcomas: C l a s s i f i c a t i o n and Natural History. CA-A Cancer Journal for C l i n i c i a n s , 31(5):271-280. 18. Rubin, P. , Ed., and Bakemeier, R. F ., Assoc, e d . , (1978): Soft Tissue Sarcoma, W. 8. Patterson, In: Clinical Oncology for Medical Students and Phy sicia ns . Chapter XIX, American Cancer So c ie ty , pp. 210-217. 19. Zack, J. A. and Suskind, R. D . , (1980): The Mortality Experience of Workers Exposed to Tetrachlorodibenzodioxin in a Trichlorophenol Process Accident. Journal of Occupational Medicine, 22:11-20. 20. Cook, R., et a l . , (1980): Mortality Experience of Employees Exposed to 2,3, 7,8 -T etra ch lo ro d ib en zo -p -d io xi n (TCDD). Journal of Occupa tional Medicine, 22:530-32. 21. Zack, J. A., Gaffey, W., (1980): A Mortality Study of Workers Employed at the Monsanto Chemical Plant in Nitro, West V ir gin ia, Unpubli shed. 22. Zack, J. A. Gaffey, W. R . , (1983): A Mortality Study of Workers Employed at the Monsanto Company Plant in Nitro, West V ir g in ia . In: Human and Environmental Risks of Dioxin and Related Compounds, R. E. Tucker, Ed.; Plenum Publishing, pp. 575-591. 23. Cook, R. R . , (1981): Dioxin, Chloracne, and Soft Tissue Sarcoma. Letter to the e d i t o r . The Lancet, March 14, 1981, p. 618. 24. Cook, R. R., (1983): Soft Tissue Sarcoma: Clues and Cautions. In: Human and Environmental Risks o f Dioxin and Related Compounds, R. E. Tucker, Ed.; Plenum Publishing, pp. 613-618. 25. Moses, M., and S e l i k o f f , E. J . , (1981): Soft Tissue Sarcoma, Phenoxy Herbicides, and Chlorinated Phenols. Letter to the e d i t o r . The Lancet, June 20, 1981, p. 1370. 26. Johnson, F. E., Kugler, M. A., and Brown, S. M., (1981): Letter to the e d i t o r . The Lancet, July 4, 1981, p. 0. 165b5 D0H 21 1 3922 -30- 27. Ott, M. G., et a l . , (1980): A Mortality Analysis of Employees Engaged in the Manufacture of 2,4,5-Trichlorophenoxyacetic Acid. Journal of Occupational Medicine, 22:47-50. 28. Thiess, A. M., et a l . , (1982): Mortality Study of Persons Exposed to Dioxin in a Trichlorophenol-Process Accident That Occurred in the BASF on 13 November 1953. American Journal o f Industrial Medicine, 3:179-189. 29. Eriksson, M., e t a l . , (1979): Case-Control Study on Malignant Mesenchymal Tumors and Exposure to Chemical Substances. Lakartidningen 76:3872-75, (EPA Translation). 30. Smith, A. H., et a l . , (1982): The New Zealand Soft Tissue Sarcoma Case-Control Study: Interview Findings Concerning Phenoxyacetic Acid Exposure, Third International Symposium on Chlorinated Dioxins and Related Compounds, Salzburg, Austria, October 12-14, 1982. 31. Milham, S . , (1982): Herbicides, Occupation and Cancer. Lancet, i :1464-1465, 1982. 32. Chesney, M., (1982): Status of the Ranch Hand Study Concerning Agent Orange, Presentation to the House Committee on Veteran's Affairs Subcommittee on Oversight and I n v e s t i g a t i o n s . 33. Riihimaki, V., et a l . , (1982): Mortality of 2,4-Dichlorophenoxya c e t ic Acid and 2,4,5-Trichlorophenoxyacetic Acid Herbicide Applicators in Finland. Scand. J . Work. Environ. Health, 8:37-42. 34. "Evaluation of Soft and Connective Tissue Cancer Mortality Rates for Midland and Other Selected Michigan Counties Compared Na tionally and Statewide," Michigan Department of Public Health, May 4, 1983. 35. Ott, M. G., e t al . , (1976): Determinants of Mortality in an Industrial Population. J. Occup. Med., 18:171-77. 36. Ott, M. G., e t a l . , (1975): Linking Industrial Hygiene and Health Records. Am. Ind. Hyg. Assoc. J . , 36:760-766. 37. Ott, M. G., (1982): E ffe cts o f Selection and Confounding on Mortality in an Occupational Cohort (Doctoral D is s e r t a t io n ) (Mortality Among Men In A Chemical Manufacturing Company -- Executive Summary), University Microfilms, Ann Arbor (In P r e s s ) . 38. "Evaluation o f Congenital Malformation Rates for Midland and Other Selected Michigan Counties Compared Nationally and Statewide", 1S7S-19Si, Michigan Department of Public Health, May 4, 1983. 39. Townsend, J. C., et a l . , (1982): Survey of Reproductive Events of Wives of Employees Exposed to Chlorinated Dioxins. Am. J . Epidem., 115:695-713. 16586 DOH 2 1 1 3923 -31- 40. Armstrong, B ., (1983): Australia Reports No Link Between Service in Vietnam and Birth Defects Among Offspring. The Epidm. Monitor, 4(3):1- 41. Schwetz, B. A., et a l . , (1973): Environ. Health Perspect. Exp.. Issue No. 5:87. 42. McConnel, E. E . , e t a l . , (1978): T oxi co l. Appl. Pharmacol., 44:335. 43. Olson, J. R., et a l . , (1980): Toxicol. Appl. Pharmacol., 55:67. 44. Henck, J. W., e t a l . , (1981): T oxic ol. Appl. Pharmacol., 59:405. 45. Homberger, E., et a l . , (1979): The Seveso Accident: Its Nature, Extent and Consequences, Report of the Givaudan Research Company. 46. Reggiani, G., (1980): J. Toxicol. Environ. Health, 6:27. 47. Al l en, J. R. , e t al . , (1977): T oxic ol. Appl. Pharmacol., 41:177. 48. Al l en, J. R. , et a l . , (1977): Food Cosmet. T o x i c o l . , 15:401. 49. Schantz, S. L., (1979): T o x ic o l. Appl. Pharmacol., 48:A180. 50. Murray, F. J . , e t a l . , (1979): T oxic ol. Appl. Pharmacol., 50:241. 51. Green, S . , Moreland, F. S . , (1975): T oxic ol. Appl. Pharmacol., 33:161. 52. Green, S . , e t al . , (1977): DA By-Lines, 6:292. 53. Wassom, J. S . , e t a l . , (1977-78): Mut. R e s . , 47:141. 54. Khera, K. S . , Ruddick, J. A., (1973): "Polych loro dib enzo -p -dic xins : Perinatal E ff e c ts and the Dominant Lethal Test in Wistar Rats" , in Chiorodioxins - Origin and Fate. Advances in Chemistry S e r i e s , No. 120 (Etcyl H. B l a i r , e d . ) , American Chemistry S o c ie t y , Washington, DC. 55. Beatty, P. W., e t a l . , (1975): T oxic ol. Appl. Pharmacol. , 31:309. 56. Tenchini, M. L., et a l . , (1977): Approaches to Examination of Gene t i c Damage After a Major Hazard in Chemical Industry: Preliminary Cytogenic Findings on TCDD-Exposed Subjects After Seveso Accident, Special Project o f I n v e st i g a ti o n s on TCDD-Exposed Pregnancies (Prof. G. B. Candiani and Prof. L. DeCarli), University of Milan, I t a l y . 57. Rehder, H., et a l . , (1978): Schweiz. Med. Woc.henschr.. 108:1617. 58. Kociba, R. J. , e t a l . , (1978): T oxic ol. Appl. Pharmacol., 46:279. 59. National Cancer I n s t i t u t e , (1980): DHHS Pu blic. No. NIH80-1765. 16587 * Z 6 e i h o qn 60. Toth, K ., et a l . , (1979): Nature, 278:548. 61. Stevens, K. M., (1981): Agent Orange Toxicity: The Quantitative Perspective. Human Toxicology, 1, pp. 31-39. 62. Cordle, F . , (1981): The Use of Epidemiology in the Regulation of Dioxins in the Food Supply. Regulatory Toxicology and Pharmacology 1, Academic Press, pp. 379-387. August/1983 (6) MN n7 ? 283 X r> H ealth s Environmental Information 2615 For Further Information Contact: R. W. (Bob) Charlton (517) 636-9303 DIOXIN AND HUMAN HEALTH Executive Summary The issu e of dioxin in the environment and the p o s s ib le human health e f f e c t s of th is compound has been the subject of much speculation over the past decade. This paper dis cusses s c i e n t i f i c research and makes the following major points: The word "dioxin" refers to a family of 75 compounds of which the one c al le d TCDD* i s the most t o x ic based on animal t e s t s . Dioxins can be produced as an unwanted contaminant of some manufacturing processes and through some combustion sources. An American Medical Association review of a v a il a b le research has concluded that there i s no convincing support for a l l e g a t i o n s that TCDD causes cancer, birth de fe c ts or other reproductive d i f f i c u l t i e s in humans. Studies of some 30,000 people exposed to TCDD a f t e r an indus trial accident in Seveso, I t a l y , found a chemically-induced acne that occurred in approximately 200 people and no p e r s i s t e n t e f f e c t s which could be v e r i f i e d . There was no excess of birth d e f e c t s , abortions or deaths in the population. A number of s tu d ie s of workers a c c i d e n t a l l y exposed to large amounts of TCDD as long as 30 years ago have i d e n t i f i e d cases of chloracne in some of those workers. However, no long-term health e f f e c t s have been demonstrated among t h i s worker group. The st ud ies have looked for cancer, heart d i s e a s e , l i v e r damage and other ailments. no CO CO CO CO The work of a Swedish s c i e n t i s t who purports to find a l i nk between TCDD exposure and s o f t t i s s u e sarcoma (a r e l a t i v e l y rare cancer) is not consistent with other s tu d ies . This research is not supported by the bulk of st u d ie s of exposed ind ustrial employees, herbicide applicators and others which show no excess of t h i s type of cancer. A study of 8,000 Dow employees in Midland, Michigan, where herbi cides were produced, found t h e i r death rate to be 19 percent l e s s than the general U.S. population. There was f i v e percent l e s s cancer than predicted. *2,3,7,8-tetrachlorodibenzo-p-dioxin i THE DOW C HEM ICAL COMPANY MIDLAND, MICHIGAN 48640 DOH 2 1 1 389 I I n ve st i ga ti on s of herbicide a p p l i c a t o r s , fo r e st workers and indus t r i a l workers and t h e i r wives found no l i nk between TCDD exposure and birth d e f e c t s . Studies o f animals exposed to TCDD show a var ie ty of adverse health e f f e c t s that vary widely from s pe cie s to s p e c i e s . In most s t u d i e s , s a f e l e v e l s of dioxin exposure have been i d e n t i f i e d . Cancer occurred only at l e v e l s where animals were su ffering from obvious TCDD poisoning. A review of the s c i e n t i f i c l i t e r a t u r e suggests that humans are l e s s s u s c e p t i b le to TCDD than various animal s p e c i e s . As i s the case with other t o x i n s , TCDD exposure should be c a r e f u l l y con t r o l l e d . The s c i e n t i f i c evidence to date does not warrant undue concern about individual exposure to trace amounts of TCDD presently measured in the environment. Government environmental gui del i ne s currently provide fo r more than adequate margins of s a f e t y . 11 165S0 c c 2616 AfEonnravtilhryoetincUma.lSeSn. ttauldiPersotection Agency PVDinOreLotUchtMieesEcioltEiainonnvMirAaogkniemnngecnytal CASE STUDIES Prepared by R obert E. B u rt, N athan J. K arch, R aphael G. Kasper, R ic h a rd W. L o w erre , L a w re n ce E. M c C ra y , and J. R o x a n n e A r n o ld for the Committee on Environmental Decision Making Commission on Natural Resources National Research Council DO II204 4 507 i t NATIONAL A CAD EM Y OF SCIENCES Washington, D.C. 1977 165S1 cc EXPLICIT CRITERIA AMD PRINCIPLES FOR IDENTIFYING CARCINOGENS: A FOCUS OF CONTROVERSY AT THE ENVIRONMENTAL PROTECTION AGENCY Nathan J. Karch^ 00112 0 4 4 508 CONTENTS I. DESCRIPTION OF THE DECISIONS II. INTRODUCTION A. The Nature of the Disease B. Cost and Extent of Cancer C. Environmental Origins of CancerD. Strategy for Control: New Emphasis on Prevention III. CANCER PRINCIPLES A. Identification of Carcinogenic Substances B. Early Development of Principles C. Statutory Framework D. Use of Cancer Principles in the DDT Cancellation Proceeding E. Use of Cancer Principles in the Aldrin/Djeldrin Suspension Proceeding F. Use of Cancer Principles in the Heptachlor/ Chlordane Cancellation and Suspension Proceedings 1. Magnitude of the Cancer Problem 2. Uncertainty in the Mechanism of Cancer Induction 3. Establishment of Carcinogenicity and Assessment of Risk 4. Problems with Experiments in Animals G. Use of Cancer Principles in the Mirex FactFinding Proceeding H. Use of Cancer Principles in Rule.Making I. Origins of the Cancer Principles 1. Extrapolation from Rodents to Humans 2. Can All Chemicals Cause Cancer? 121- 121 122 123 124 124 126 126 128 130 131 133 137 142 142 142 143 1 5 3 ___ 159 151 162 164 113 165S2 . 3. Do Benign Tumors Characterize a Carcinogenic Response? 4. Can "No-Effect" Levels be Set for Chemical Carcinogens? J. Organized Opposition K. Attempt at Peer Review IV. CONCLUSIONS A. Nature of Controversy B. How the Controversy Might Have Been Averted V. NOTES VI. APPENDICES 165 168 172" 176 184 184 186 188 204 CD CD r"o co C,~i CO CO 120 0 G S /DZ/JOCI many scientists in OPP had extensive toxicological experience in industry, USDA, or FDA with substances that induce reversible effects below a certain "threshold" of action. Thus, they believed "no-effect" levels should be set for carcinogens as they are for substances posing acute and certain other chronic hazards. Furthermore, many in OPP believed rodents (mice especially) were inappropriate animal models for cancer testing. Because cancer is such a poorly understood disease, there are no unequivocal answers to a great many questions. The principles were an attempt to summarize reasonable presumptions concerning the design and interpretation of studies to detect carcinogens. They sought to present in simple terms the thinking of expert committees that had addressed the problems of carcinogenicity testing for over 25 years. Not surprisingly, many scientists in OPP opposed the use of the principles, and OGC took the lead in preparing the scientific and technical basis for the Agency's position on carcinogenicity in cancellation actions- Under other circumstances, OPP would have had a large hand in the cancer issue during cancellation actions; OPP and other Agency scientists testified on many issues other than cancer. I. Origins of the Cancer Principles *09 Although it is not the intention in this case study to evaluate the scientific validity of the cancer principles, it seems appropriate to examine in detail some of the underlying documentation. For this reason, seme of the reports and articles that were entered into the record of various fact-finding, cancellation, and suspension proceedings, and that form the basis for the principles will be discussed. The following discussion is irtended to illustrate the surprising consensus-- short of unanimity-- on the concepts embodied in the cancer principles and is limited to four such concepts: -- It is necessary to extrapolate from animal studies, particularly in rodent species, to humans in order to evaluate the risk of cancer. -- Not all chemicals are capable of causing cancer, even at maximum doses of administration. -- Although a distinction can frequently be made between benign and malignant tumors, for the purposes of . establishing the carcinogenicity of a substance, they should be considered synonymous. 161 165 D 0 1i20 59 I c U c. -- There is r.c practical nrethod for determining a "noeffec*-" or. chreshold exposure level in humans for carcinogens. 1. Extrapolation from Podents to Humans The first issue of reliance on studies in rodents has already been discussed earlier to some extent. ` It should be recalled that the various expert committees agree that reliance on animal studies is necessary because of the practical and ethical limitations in reliance on epidemiological studies to identify ca rcinogeni-c agents and because of the evidence of the validity of studies in experimental animals as the basis for establishing the carcinogenicity of a substance. Discussions by expert committees of the optimal conditions for carcinogenicity testing has included consideration of appropriate mammalian species for testing. After a thorough review of carcinogenicity testing, the Mrak Commission reported in 1969: The species most practical for testing are rats, mice, and--as more recently shown-- hamsters. Strains and colonies should be selected to provide adequate sensitivity to tumor induction, as revealed by positive control tests with kncwn carcinoqens.... The use of nonrodent species, recommended in the earlier reports, has now been substantially dropped. A suitable, practical nonrodent species would be useful but it is not available at this time..... While dogs have taen employed for tests of carcinogenicity, with noteworthy success in selected cases (bladder carcinogenicity of aromatic amines), the requirement of lifetime feeding makes this species too expensive, in terms of time and funds, to be employed routinely.10 Again in 1975, an MRC committee confirmed the views expressed in many of the earlier reports: Rodents are the animal of choice for carcinogenesis tests because of their convenience, comparatively short life span and proven susceptibility tc a broad range of carcinogenic agents.111 Another MRC report published in 1975 concluded: ...the carcinogenic risks, and other health hazards, of pesticides require continuing evaluation by testing with laboratory mammals; and... despite the problems involved in translating 162 c 103 Ibid. c 104 Federal Register, Vol. 40, (July 3, 1975), pp. 2824228296. 105 Ibid. . p. 28256., Z G E H OZ/IO 106 Ibid.. . p. 28257. 107 Ibid.. p. 28263. 108 Bob Wyrick, "EPA Officials Devised Cancer Tests on People,' Washington Post. Thursday, June 23, 1977, p. A1; "EPA Proposed Carcinogen Test on Mexicans," Ibid. Wednesday, May 11, 1977, p. A1. 10 [ wish to acknowledge the contribution made to the writing of this section by John E. Bonine, Office of General Counsel, EPA. Bonine gave me a preliminary draft of a paper tracing the term "principles" and reviewing some of the reports of expert committees. 110 Mrak Commission (1969), supra note 37, p. 465, text and footnote 2. 111 National Research Council (1975) Evaluating Chemicals, supra note 37, p. 150. 112 National Research Council (1975) Pest Contro l , supra note 34, p. 66. 113 Mrak Commission (1969), supra note 37, p. 11. 114 National Cancer Advisory Board (1977), supra note 31, p. 463. 115 Tomatis, supra note 37, p. 1. 116 U.S. Department of Health, Education and Welfare, Survey of Compounds Which Have been Tested for Carcinogenic Activity. Volumes I-III, (1974-1976), (Washington, D.C.: Government Printing Office). 117 The study was performed by the Litton-Bionetics Research Laboratories under contracts PH 43-64-57 and PH 43-67735; see U.S. Department of Health, Education, and Welfare (1969), supra note 31, pp. 461-506; J.R.M. Innes, B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A-J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell, and J. Peters, "Eioassay of Pesticides and Industrial Chemicals for Tumorigenicity in Mice: A Preliminary Note," J. National Cancer Institute, Vol. 42 (1969), pp. 1101-1114. 198 16536 / 4/^4 A/JT7 2 6 2 6 hoq e n e z iz SUMMARY OF DOW'S RESPONSE TO EPA'S RISK ANALYSIS ON THE USE OF 2,4,5-T HERBICIDE compiled by Marguerite L. Leng Product Registrations Health $ Environmental Sciences THE DOW CHEMICAL COMPANY Midland, Michigan USA 2 O \j\ V*l VM O ui February 1979 Excerpts from Volume I of Dow risk rebuttal and from Dow legal brief bmitted to U.S. Environmental Protection Agency on August 4, 1978 I fibok Mo. A? j Jut ^s**TM* Cj^yx / 5 J ^ C 1/ cmyu ^ f /1 ^y, ji-f-n+ASl, t* APflfZ. ( / <Mu*u ^vyufvti^ r^J^U ^G , ' 1. </'s- r APPENDIX II /<du-rr\ 4 *v>- / uUsX z j n r - t ^ . J * / a&x L u t t u r s l i u l w n l t t e d l o y:I*A I n H u a p o n i i u i n 2 , 4 , 5 - T HPAUi Hu 111 E f f e c t s f r o m H o y l s t e r o d D n u a . EI`A | 5 6 7 LJ 10 2(1 31 37 43 77U 0G yuA ai yj 1 02 Name o f !>ubm l i t e r James Erwin Mack 1.. L I t L l a C. I'.ddlo Mucks D o n a l d H. C u u t u l l n w Howard G. HcDowul1 .1. F . Howe 1 1 .John W. I l l n r i e l i a F r u d o r l c k DruinmonJ Dm. J . W a l d r l | > l l l c l i a r d A. MeHouijal Ja m es L. C o lo llm ry F. Maine Janion l(. F lo y d P a t r i c k J . Konny liona Id A. M i c l i l e l l Town S t n Lo Occupation 'Years of usu Kosebnrij Oil Sucrutary, S tate Sprayers 400 man y e a r s Lufkin rx r u n c h e r / s p r a y u r s l n c a 1950 Thomas ton GA C o u n t y a e j u n t c o o p o x t u n u i o n service Moscow 10 p o u t c o n t r o l c o n s u l t a n t 20 y e a r s Ml it n on I n C l ** ltuition MT K x c o . V . P . I n l a n d F o r e s t llcsourco Council T>' r n n c l i a r yearn Grant County MM County oxtenuion ayent yuiira I' a wl i i i n k t t OK s p r a y e r / r n n o b e r your n Lubbock TX re s e a rc h and ramjo nu.nagor 30 y e u rs HuLlonal C a t t l e m a n 's A ssoc. . (200,000 jtrof oils lonft 1aj Leavenworth IN s m a l l r e n c h and woods o v e r 25 yuaru It 1n e o n GA m a n a g e r , w o o d l u n d i : r e n o a r c l i y o a i b 1,1 b o r n 1 KS a e r i a l n p p l l c n L o r 200.000 acres Woutborouyh MA Now E n g l a n d P o w e r S o r v i c u C o . 20 y e a r s Wnulilnijton D.C . Exec. D lroctor Public Lands C o u u c 11 m CJ? U i E Z I Z KOQ & L .J ^ /V o./* TRANSLATION GERNAN PARLIA M EN T 9th Election Period ; r / L r .L e / t h mi Publication 9/379 April 29, 19 8 1 cr Ev r-o REPLY of the Federal Government to O CjO wn ^ CjO ^ on O U the inquiry of members of the parliament, Frau Dr. Hartenstein, Sch fer (Offenburg), Catenhusen, Duve, Jansen, Kiehm, Dr. Kbler, Marschall, Frau Dr. Martiny-Glot2 , Reuter, Dr. Schwenk (Stade), Frau Vi'eyel, Witek, Frau Zutt, Ibrgger, B e r g e r o w s k i , E n g e l hard, Dr.Hirsch, Kleinert, Dr. Viendig, Wol fg ra nm (Gttinger.) and The Secretary of the Dept, of Food, A gri cu ltu re and Forestry - 913-3320-2/1 - has- answered the inquiry on beh alf of the Federal Government by letter of April 14, 19^1, as follows: . The Biologische Bundesanstalt fr Land- und Forstwirtschaft (BBA) and the Bundesgesitodheitsa-mt (BOA) v.Tiich are responsibl.e for the registration of pesticides, have repeatedly expressed their expert opinion on the herbicide 2,4,5-T. It was established in a joint statement of the year 1975 that a hazard does not exist if it is used properly and according to the regulations. Necessary restrictiens for the use are ruled by the dec ee about prohibitions and restrictions of the use of pesticides, dated December I9 , I980 BG31. I p- 2355) or by requirements. 2,4,5-t containing herbicides as registered in the F e dera l Republic of Germany today, cannot be put in a relation to the "agent o r a n g e ' which was used as a defoliant for forests in the V i etn am v;ar. These products were contaminated very much with the toxic TCDD (Dicxin), which also reached the environment during the accident, of.Seveso. The defoliant used in Vietnam contained up to 5000-times more TCDD than the formulations registered in the Feder al Republic of Germanj today. The BBA has required from the m a n u f a c t u r e r s in, the registra: that a TCDD content of 0.01 mg/ kg 2,4 ,5- T is n o t exceeded, i.e. 100 g/t a.i.. In contrast to this, the g u i d e l i n e level of the World Health Organization amounts to 0.5 mg/kg, i.e. 5 0 - times highc than the German guideline level. IS59,,9_ 2 A\O OUI/OC I \ S n i \ i Ioo 2632 A symposium sponsored primarily by the American Chemical Society's Division of Pesticide Chemistry, with the cooperation of the Committee on Chemistry and Public Affairs, and the Divisions of Agricultural andFood Chemistry, Chemical Health and Safety, and Professional . Relations. Held o n fficRefcT^6, 1980, in Las Vegas, Nevada, during the' 180th national meeting of the Society. American Chemical Society 1155 16th St., N.W. Washington, D.C. 20036 ; % 2644 APFENDIJ B PROPOSALS FOR EPIDEMIOLOGICAL INVESTIGATIONS OF MORBIDITY ASSOCIATED WITH HERBICIDES Cc r- t'' The assessment of Che available daca concerning a possible biological association between exposure Co phenoxy acid herbicides and sofc tissue sarcoma has led co Che conclusion Chat there is a need for further research co permit Che confirmation or refutation of chis suggestion. Ic has also been noted thac a number of studies are already in progress or are about to be initiated iu several councries around the world using data which are available on boch a national and international basis. These include systematic epidemiological studies of both a retrospective and a prospective nature which constitute the only appropriate methods available for this purpose. In view of the known ceraporal and geographical variations in the incidence of a number of malignant conditions and of the importance of the phenoxy acid herbicides to agriculture and forestry in this country it is considered advisable to investigate che situation as it exists in the United Kingdom. Appropriate informacin is available to enable epidemio logical studies of boch a retrospective and a prospective nature to be carried out. In addition to being the only way of obtaining information of direct relevance to the UK workforce it should be possible, from the data available, to obtain evidence which is less open to the criticisms thac have been levelled against some of the reports thac have been published so far. There would also be an opportunity to extend che scope of che investigations to include aspeecs ocher than the occurrence of sofc tissue sarcoma and the possible involvement of ocher chemicals such as the chlorophenols. Retrospective Study 2 This would examine whether patients suffering from sofc tissue sarcomas (or ocher specified malignant conditions) had an unusual history of exposure Co phenoxy acids during their manufacture or use as herbicides. The firsc step would be to establish a group of sarcoma patients and an appropriate control group. Since comprehensive cancer registration data are available covering the whole country, basic information eg age, sex, type of malignancy, when and where registered, for all identified cases in specified categories of soft tissue sarcoma is readily accessible. However, these are relatively rare tumours, and a decision would have co be taker, whecher co scudy all of them In relevant age groups, or only chose in parts of the country where herbicide use is likely co hu more common. The control group, which would have co be matched at least by age and sex, could be cancer registrations for body sites where there are no reasons to believe thac tumour occurrence could be associated with herbicides. 3 The major problem with retrospective studies is In obtaining the history of herbicide exposure. For mosc patients the occupational exposure will not have been documented precisely, if ac all. Memories are notoriously fallible, even In the short term and recall concerning events many years 1 # a p p e n d ix b previously will probably be uncertain. For patients who have died recourse aighc be had to their families to obtain the history, as in some of the published reports, but this would be even.more unreliable. Furthermore, whether obtained from the patient or n relative, there is the possibility of a bias in the histories of herbicide use from cases compared with controls because of the publicity on possible hazards. A retrospective study would have to be conducted in the expectation that only in a small proportion of cases and controls could the histories of herbicide use be validated. In such an investigation particular attention would have to be paid to ethical considerations. Information in cancer registries is confidential, and a proper procedure would have to be established before any approach could be made to consultants, general practitioners, to the patients and their families, or to any other source of data. CD Cj Despite these drawbacks a retrospective study has.- certain advantages. It ' can be mounted fairly quickly and is relatively cheap. It does noc take long, the duration being governed largely by the time required to obtain occupational histories. cn Prospective Study In this study a cohort with known exposure to herbicides would be Identified and monitored to determine whether they subsequently suffered any excess of soft tissue sarcomas or morbidity of other kinds. There would be a need for a control cohort comprised of individuals not exposed to herbicides. The subsequent morbidity of this group would be recorded in exactly the same way as for the herbicide user cohort. Since reliable information concerning exposure is of paramount importance in such a study an essential pre-requisite is that there should be good documentation available concerning herbicide use. Organist lens such as the Forestry Commission or commercial sprayers who keep routine records should therefore be selected in preference to farmers whose occasional use of herbicides may go unrecorded. The documentation system would have co be reviewed to assess the need for possible changes to ensure that complete, uniform and appropriate data are obtained. ' The essence of the prospective study would be the collection of morbidity information in cohorts defined as either using or not using herbicides. Leaving aside the choice of the contrul cohort, the non-users, there are different problems depending on what aspect of morbidity is selected for study. The extreme event, death, could be readily ascertained, even if members of the cohort moved or changed their occupation, by tagging their entries in the MHS Central Register. Cancer registrations, as well as deaths from any cause, are reported to this Register so that the cancer morbidity as well as mortality can be obtained for the defined cohort. Other expressions of morbidity, leading to consultation with a general practitioner, out-patient referral or admission to hospital, are not obtainable from a central register. If there was a case fur seeking evidence on anything besides general mortality and cancer morbidity, 2 16802 t APPENDIX B 9 * i z z i z tioa7 t g on liver disease, this would need a separate organisation such as a questionnaire to the patient or his doctor to determine whether there was any episode of illness worth further enquiry. It would add considerab to the cost and complexity of the study to extend the .range of outcomes V in this way, and there would need to be a strong case for such addition. 10 It could be difficult in a prospective study to establish a satisfactory control cohort. Ideally it should comprise individuals of similar age, sex, and social class distribution, and probably from the same areas. In occupational studies of this kind it is sometimes possible to find groups within tne same organisations, such as administrative and clerical staff, who are not in contact with the process, but their numbers may well be too small in this instance. It is always possible to use national or Regional data on mortality and cancer registration as a standard of comparison for the cohort of users, and this compromise may be a reasonable one to adopt. / 11 i The main disadvantage of a prospective study, is the length of time before sufficient events have occurred to warrant comparisons betu user and non-users particularly where rare events such as the occurrence of soft tissue sarcomas is concerned. Furthermore, the cohorts defined initially will not remain 'pure' in the sense that additional exposures to similar or different agents may occur. Nevertheless this type of study is more likely to lead eventually to a valid measure of che risk, if any, of exposure to specific herbicides and should, therefore, be given serloud consideration, although the difficulties which have been outlined above should not be underestimated. 12 A cohort, proepecclve, study set up now could provide evidence only on pnenoxy a d d herbicides currently produced. However, chere may be scope for conducting a cohort study retrospectively. If in selected organisations, such as the Forestry Commission, it was possible to prepare nominal lists of all employees in some period lr. the past, say die decades 1945-54, 1955-64, end Co categorise them by pesticide use, t-heir subsequent experience up to the present day of mortality and cancer registration mighc be retrievable. The mortality picture could be virtually complete, but cancer rcgiscradon was not fully established in the earlier years and would be reliable only for che last 10-15 years. The study would not be easy, but might be valuable because it would provide information on exposure to herbicides with a relatively high' dioxin contamination. Unlike a new cohort study established now, some measure of the outcome 20-30 years after exposure could be obtained relatively quickly. 2645 APPENDIX A SUMMARY OP EPIDEMIOLOGICAL AND CLINICAL STUDIES CONTRIBUTING ORICINAL OBSERVATIONS CONCERNING A POSSIBLE ASSOCIATION BETWEEN CHEMICAL ' EXPOSURE AND SOFT TISSUE SARCOMAS Country of Origin Study Population Summary of findings O o n: CASE CONTROL STUDIES Sweden 52 coats of soft tissue sarcoma and 208 matched controls from Northern Sweden co CO Exposure to phenoxy acids reported by 252 of cases and 7Z of controls. Exposure to chlcrophcnols reported by 13Z of cases and 3Z of controls. Reladve risk* of developing a safe tissue sarcoma associated with exposure to phenoxy acids - 5.3. (Reladve risk associated with exposure to chlo 'henols - 6.6). Chlorinated dibenzodioxiu.; and dlbenzofurans possibly Involved. Relative risk Is the statistically derived expression of die ratio between the lnclden of exposure reported by cancer patients and that reported by the control group. Sweden Sweden Sweden 110 cases of soft tissue sarcoma and 219 macched controls from Southern Sweden. Exposure to phenoxy acids reported by 13Z of cases and 3.2Z of control:. Exposure to chlorophenols reported by 10Z of cases and 3.7Z of controls Relative risk of developing a soft tissue sarcoma associated with exposure to phenoxy acids h.8. (Relative risk associated with exposure to chlorophenols 3.3). 169 cases of nallgnant lymphoma (60 Hodgkin's disease and 109 non-Kodgkin's lymphoma) and 338 matched controls. Exposure to phenoxy acids reported by 24Z of cases and 7Z of controls. Exposure to chlorophenols reported by 30Z of cases and 10Z of controls. Relative risk of developing a lymphoma associated with exposure to phenoxy acids - 4.8. (Relative risk associated with exposure to chlorophenols - 4.3). 156 esses of colon cancer and 541 unmatched controls. (Supplementary study to 1, 2 and 3 abovu). A re-examlnation of the data presented In 1, 2 and 3 above confirms relative risk factors of 4.2, 5.6 and 4.1 respectively for an association between the develop ment of soft tissue sarcoma and malignant lymphoma and exposure to phenoxy acids following a S year latency period. No association was established between exposure to such chemicals and the development of colon cancer, relative risk - 1.3. Countrv of Ref ' Origin-- _ Study Population ArFENDIjC n\ Summary of findings 5 Hew Zeeland 102 caeca of soft tissue sarcoma. Preliminary report. On the basis first diagnosed between 1976 of Cancer Registry Records there end 1980 and 306 ocher cancer is no evidence of an excess of soft patienCs as matched controls. tissue sarcomas among the occupational group Involving agriculture and forestry. 6 New Zealand 80 cases of soft tissue sarcoma and 92 ocher cancer patients as controls. (Further data from Che study reported at 3 above). COHORT STUDIES 7 West Germany 75 chemical workers involved in a trichlorophenol plant accident in 1953 Further preliminary report. Exposure to phenoxy acids reported by 26Z of soft tissue sarcoma cases and 21Z of controls. Estimt, for the relative risk of developing a soft tissue sarcoma following exposure to phenoxy acids varied from 1.3 for potential exposure of more chan 1 day at least 5 years before cancer regis tration to 1.6 for probable exposifgNo soft tissue sarcoma patient had-- : worked full time cn phenoxy acid ^ herbicide spraying. rvj r"0 "vl 42 cases of severe chloractie associated with exposure during the accident or subsequent cleanlSf^ work.' By 1973 17 deachs had occurred (II to 25 expected), 6 due to cancer (4 expecced). The Incidence of stomach cancer (3 cases was higher than expected. There were no cases of soft tissue sarcoma 8 Finland 1,971 herbicide sprayers (Railway, forestry, highway and electric company workers) Retrospective study 1955-1971, 49 de occurred (87.8 expected), 13 dee to cancer (16.4 expected). Prospective follow-up, 1972-1976, 62 deaths occu (92.9 expected) 9 due to cancer ( 13. expected). Prospective follow-up, 1977-1980, 82 deaths occurred, (91.1 expected), 17 due to cancer, 13.4 expected. No cases of chlur.icne. Nc cases of soft tissue sarcoma. 9 USA 6 1 `chemical workers involved in trichlorophenol production and exposed to 2,3,7,8-TCDD in 1*64 49 casim of chlorncrie ut-rc rc;:or ed ranging from mild to severe. By 1978 4 deaths had occurred (7.8 expected), 3 due to cancer (1.6 expected). One case was a soft tissue sarcoma (f Ibrusarcuma). 2 Ot I LI I l liOQ Country of lef Orlgln~ Study Population APPENDIX A Summary of findings 10 USA 11 USA i2 Sweden 13 Sweden 14 Czecho slovakia 15 USA 16 USA 204 chemical workers Involved In 2 ,4,5-T production between 1950 and 1971 No case of chloracnc. By 1976 11 deaths had occurred (20.3 expected) only 1 due to cancer (3.6 expecced). This was noc a case of soft tissue sarcoma. 121 chemical workers Involved in a crichlorophenol plant accident in 1949 All members of the selected cohort exhibited chloracne attributable to exposure during the accident. By 1978 32 deachs had occurred (46.6 expected), 9 due to cancer (9 expected). One case was a soft tissue sarcoma (malignant fibrous histiocytoma). 142 forestry workers exposed to phenoxyacetic acid herbicides between 1954 and 1978 and 244 forestry workers not so exposed The pattern of-deaths among both exposed and unexposed cohorts is in accord with Swedish national mortality statistics. However among foremen, the cost extensively exposed workers, there is an excess of mortality (8 observed/4.1 expected) due to an excess of bath cancer (3 observed/1.0 expected) anu cardiovascular disease (4 observed/ 1.9 expected). There were no soft tissue sarcomas. 348 railway workers (herbicide sprayers between 1957 and 1972) No cases of chloracne. By 1978 45 deaths had occurred (49 expected), 17 due to cancer (11.9 expected). No specific type of tumour predominates and no soft tissue sarcoma was reported. 55 chemical workers exposed to 2,3,7,8-TCDD over a 10 year period during the production of 2,4,5-T from trlchlorophenol 52 cases of chloracne. By 1980 6 deAths had occurred, 2 due to cancer. There were no cases of soft tissue sarcoma. Review of 9, 10 and 11 above plus additional data on chemical workers Involved in tha production of 2,4,5-T from trlchlorophenol An additional case of soft tissue .sarcoma (generalised llposarcoma) is reported in a worker Involved in the production of 2,4,5-T from trlchlorophenol. There was no prior history of chloracne. Supplementary data concerning the cohort studied at 9 above An additional case of sofc tissue sarcoma (malignant fibrous histiocytoma) in a trlchlorophenol production worker is reported. There was a previous history of chloracne. 1660' Oouncry of Origin Study Population APPENDIX A Summary of findings 17 United 126 chemical workers involved Kingdom in a trlchlorophenol plant accident in 1968 18 USA Retrospective analysis of mortality data - Washington Scat (1950-1979) 19 USA Retrospective analysis of mortality data - butchers end slaughterhouse workers (1965-1980) 79 cases of chloracne were reported after the accident, ily 1981 no deaths had occurred nor had any case of cancer been identified. Mo abnormal biochemical or clinical effects attributable to exposure. In occupations with exposure to phenoxy acid herbicides or chlorophenols 49 soft tissue sarcomas were observed (41 expected). In those occupations with the greatest excess of deaths from soft tissue sarcoma there was no obvious association with exposure to phenoxy acid herbicides or chlorophenols. An analysis of cancer mortality among butchers and slaughterhouse workers reveasled an Increased risk relative for soft tissue sarcoma of 5.6 (1 observed case/ 0.18 expected). m h iz non CLINICAL REPORTS 20 Sweden / cases of soft tissue sarcoma (included in the case concrol study reported at 1 above) 21 Sweden 22 USA 17 cases of malignant lymphoma (included in the case concrol study reported ac 3 above) 1 case of soft tissue sarcoma In a chemical worker 23 USA 2 cases of soft tissue sarcoma in chemical workors (facher and son) Exposure to phenoxy acids is reported in all 7 cases. In 5 cases the exposure was dln-.cc and heavy. Exposure to phenoxy acids, is reported in 8 cases. A case of retroperitoneal neurogenic sarcoma is reported in a truck driver and maintenance worker in a chemical plane producing trlchlorophenol or 2,4,5-T. Ho history of prior chloracne. Two cases of soft tissue sarcor.n are reported (a fibro-sarcoraatous mesothelioma and a liposarcoma). Both individuals had been employed at a chemical plant producing chlorophenols. No history of prior chloracne. z i i 11 iioon REFERENCES 1. Hardell, L., SandstrUm, A., (1979), British Journal of Cancer _39_ 711-717 "Caac-concrol study: soft-tissue sarcomas and exposure to phcnoxy acetic acids or chlorophenols". 2. Eriksson, M., et 1., (1981), British Journal of Industrial Medicine 38 27-33, "Soft-tissue sarcomas and exposure to cheaical substances: a case-referent study". 3. Hardell, L., et al., (1981), British Journal of Cancer _43 169-176, "Halignant lymphoma and exposure to chemicals, especially organic solvents, chlorophenols and phenexy acids: a case-control study". A, Hardell, L., (1981) Scandinavian Journal>of Vork Environment and Health _7 119-130 "Relation of soft-tissue sarcoma, malignant lymphoma and colon cancer to phenoxy acids, chlorophenols and other agents". 3. Smith, A.H., et al., (1982) Coasninity Health Studies 114-119, "Do agricultural chemical cause soft tissue sarcoma? Initial findings of a case-control study in New Zealand". 6. Smith, A. H. , et al., (1982) Proceedings of the 3rd International Symposium on Chlorinated Dioxins and Related Compounds. In Press. "The New Zealand soft tissue sarcoma case-control study: Interview findings concerning phenoxyacetic acid exposure". 7. Thiess, A.M., Frentzel-Beyme, R., (1978) Working paper, quoted in Incernation Agency for Research on Cancer, (1978). "Long-term hazards of polychlorinated dlbenzodioxlns and polychlorinated dihnnzofurar.s" Internal Technical Report No 78/001, Lyon. "Mortality study of persons exposed to dioxin after an accident which occurred in Che BASF on 13th November 1933". 8. Rllhlmaki, V., et al., (1982) Scandinavian Journal of Work Environment and Health J3 37-42 "Mortality of 2,4-trlchlorophenoxyacetlc acid herbicide applicators in Finland". 12_9. Cook, R., et al., (1980), Journal of Occupational Medicine 530-532 "Mortality experience of employees exposed Co 2,3,7,8-tetrachlorodibenzo-p-dioxiu (TC1)D)". 22^10. Ott, MG., et al., (1980) Journal of Occupational Medicine 47-50 "A mortality analysis of employees engaged in Che manufacture of 2,4,3-Crlchlorphenoxyacedc acid". 11. Zack, J.A., Suskind, R.R., (1980) Journal of Occupational Medicine 22 11-14 "The mortality experience of workers exposed to letrachLurodibenzodioxln in a trichlorophenol process accident". 1 12. Hogstedt, C . , Westerland, B., (1980) Lilkartldnlngcn 19 1829-1831 "Cohort study of causes of deach of forestry workers with sad without exposure to phenoxy a d d preparations". 13. Axelson, 0., et al., (1980) Scandinavian Journal of Work Environment and Health 6^ 73-79 "Herbicide exposure and tumour mortality. An up-dated epidemiological investigation on Swedish railroad workers". 14. Pazderova-Vejlapkova, J., et al., (1980) Pracovnl Lekanstri "Chronic poisoning by 2,3,7,8-tctrachlorodlbenzo-p-dloxln". 204-209 15. Honchar, P.A., Halperin, W.E., (1981) Lancet 1 268-269 "2,4,5-T, trlchlorophenol, and soft-tissue sarcoma". 16. Cook, R.R., (1981) Lancet 1 618-619 "Dioxin, chloracne, and sof t-tlssuq-- t sarcoma" . > 22.17. May, C., (1982) British Journal of Industrial Medicine 128-135 "Tetrachlorodlbenzodloxin: a survey of subjects ten years after exposure". 18. Milham, S., (1982) Lancet 1 1464-1645 "Herbicides, occupation, and cancer". i'o r--s j 19. Johnson, E.S., Flschcan, H.R., (1982) Lancet 1 913 "Cancer mortality c- among bucchers and slaughterhouse workers". 20. Hardell, L., (1977) LUkartldningen ^ 2 2753-2754 "Malignant mesenchymal tumours and exposure to phenoxy acids - a clinical observation". 21. Hardell, L., (1979) Lancet i 55-56 "Malignant lymphoma of histiocytic type and exposure to phenoxyacetic acids or chlorphenols. 22. Moses, M., Selikoff, I.J., (1981) Lancet i 1370 "Soft tissue sarcomas, phenoxy herbicides and chlorinated phenols". 23. Johnson, F.E., et al., (1981) Lancet ii 340 "Soft tissue sarcomas and chlorinated phenols". RK/JAK The Rt Hon Peter Walker, MBE, MP, Minister of Agriculture, Fisheries and Pood, Whitehall Place, London SW1A 2HH. 13th December, 1982 % 2646 DON 2 1 2 7 1 3 4 Dear Minister, 1 . In your letter of 3 May 1982, you sought the Advisory Comnittee on Pesticides' observations on new evidence concerning the safety of 2,4,5-T herbicides, with particular reference to a review article published in the Lancet on that same date. We have new reached a point in our enquiries at which we think it would be helpful to report to you and the other Departmental Ministers with an interest in the safety of these herbicides. Background to Study 2. The Ccnmittee decided that a panel of independent members should be set up under my chairmanship to consider the latest evidence, and the advice in this letter reflects their detailed examination of all the relevant studies known to us which are surmarised in Appendix A to this letter. The panel included all areas of expertise needed for a study of a postulated association between use of a herbicide and a particular type .of cancer, and consisted of Professor C.L. Berry Dr R.L. Carter Dr R. Goulding Professor J. Knavelden Professor R.I. McCollum Professor G.R. Sagar 3. Before giving you our conclusions, I think that a brief review of the evidence we have considered since publication of our December 1980 report might.help you and your colleagues to assess our advice. You will recall chat much of our 1980 enquiry was concerned with the question of whether the use of 2,4,5-T herbicides might be associated with miscarriages and birth deformities among humans and animals, and the evidence included a number of specific cases in which such an association was claimed. Each of these was investigated in detail by the appropriate Government Departments and the Health and Safety Executive in consultation with the general practitioners, hospital consultants and veterinary surgeons concerned, before the Ccnmittee reached its conclusion. Our examination revealed no evidence linking any of the cases with use of 2,4,5-T herbicides; and we could find no other evidence which established any association between use of these products and harmful effects on humans, other living creatures or the environment. We were thus able to conclude that; "2,4,5-T herbicides can safely be used in the UK in the recaimended way and for the reccnmended purposes". 4. Our present enquiry arose out of a separate and specific question that there might be an association between exposure to phenoxy acid herbicides, chlorophenols or the contaminants that may be present in these products and substances, and an increased incidence of a group of tuoours classified as soft tissue sarcomas. The term soft tissue sarcomas includes seme 50 varieties of tunour. As a group they are rare, in that they represent approximately one in every 1500 individuals who die each year, and three iron every 1,000 people who die from cancer each year. Obviously such a low overall incidence makes assessment of changes related to possible causal agents a most difficult exercise in statistical significance. 16610 DOH 2 1 7 . 7 1 3 5 5. Hie evidence we have reviewed is based on studies of two quite distinct types of populations. The first is of users of various herbicides and wood preservative products, and the second is of factory workers engaged in the production of chemicals. The ACP is concerned with advising on all risks that may arise from the use of pesticides, as distinct from any risks which may arise in the course of manufacture, and which are strictly speaking outside our remit. V.'e, therefore, concentrated on the first type of population - pesticide users - and our advice is based on the weight of evidence emerging for this type. Nevertheless, we also examined factory studies to ensure that no evidence which might have a bearing on our task had been overlooked. Discussion of Evidence - Herbicides and Wood Preservatives 6. Studies of pesticide user groups have been undertaken in various countries and consist both of case control and mortality investigations. The case control studies involved a comparison of individuals with soft tissue sarcomas whose exposure to herbicides and wood preservatives had been assessed mainly by memory recall, with control groups who either had no cancer or other types of cancer. In the mortality studies the incidence of death among groups of workers largely identified on the basis of occupational histories was compared with national or regional mortality statistics. 7. For users of herbicides and wood preservatives, a positive statistical association between reported exposure and soft tissue sarcomas had been suggested from two studies carried out in Sweden. Successive reports on a similar case control study have also become available from New Zealand. These showed no correlation between exposure to phenoxy acid herbicides and soft tissue sarcomas. It is our view that the procedures used for establishing the Swedish study groups, and seme aspects of the collection of the exposure data, were not reliable. In that country, separate case control studies-were undertaken in Northern and Southern Sweden following publication of a previous clinical report in which 7 individuals with soft tissue sarcomas recalled being exposed occupationally to phenoxy acid herbicides at some time during the 1950's and 1960's. I should explain that whenever clinical evidence of that kind becomes available in any area, it is normal epidemiological practice to follow up the findings by means of investigations amng totally different groups, thus preventing the possibility of bias. In the follow up study in Northern Sweden, however, the 13 cases in which exposure to phenoxy acid herbicides was claimed (amongst a total of 52 cases of soft tissue sarccmas) included the 7 cases identified during the earlier clinical investigations. We consider this to be unorthodox epidemiological practice; if these 7 cases are emitted from the study, the relative risk is reduced to within error limits. This was not the. only defect in the study. For example, patients and controls who reported exposure only to chlorophenols were excluded from the calculation of the relative risk factors associated with phenoxy acid herbicides while the control group excluded patients with any malignancies. The relative risk factor for chlorophenols was similarly artificially raised by excluding patients and controls who reported exposure only to phenoxy acid herbicides. ISoreover, that study, and the further one in Southern Sweden, were conducted at least one year after a publication drawing attention to the possibility of the suggested association from clinical experience. Because public awareness of alleged risks from 2,4,5-T herbicides had been acute for some years, there is a probability that a bias frem exposure recall occurred in both studies. 2 1 8. Although, they may be valuable, case controlstudies are not a precise tool for establishing a causal relationship. They can be very unreliable and misleading unless supported by sound documented information an exposure. Such information was not available in either of the Swedish studies, where it was largely based on the memory recall of individual patients or their relatives. We have, therefore, looked for corroborative evidence iron mortality studies of cohorts of herbicide users. We have found none. Tito such cohorts have been studied in Sweden and one in Finland. A total of 2,461 individuals were included in them. There was no overall increase in deaths iron all causes or from cancer. No case of soft tissue sarcomas was identified. Similarly, no correlation between soft tissue sarcomas and occupations involving the use of phenoxy acid herbicides emerged from a study of deaths in Washington State between 1950-1979. We cannent further on this study in paragraph 13 belcw. 9. Che feature of our 1980 enquiry again became evident during the current one and has already been referred to above. This is that nuch of the evidence depends on personal recollection of what took place well before the association was claimed, and that recollection may have been influenced by later publicity. In our 1980 report we argued, in relation to miscarriages and biith deformities, that it can be relatively easy to select seme products, or other factor, and without enquiring very closely, to assume sane connection with family misfortunes of that kind. This tendency has again been illustrated by a recent publication in this country ("Portrait of a Poison: The 2,4,5-T Story") in which two deaths were attributed to soft tissue sarcomas developing after alleged exposure to 2,4,5-T herbicides. We have enquired carefully into these cases, which have been given sane publicity. In addition to the lack of reliable exposure data, we have established beyond doubt that in neither case was the tumour involved a soft tissue sarcoma. Conclusions - Herbicides and Wood Preservatives 10. In stannary, a rnanber of factors have emerged from our analysis of studies of user groups that are relevant to the assessment of the safety of 2,4,5-T herbicides in this country. Firstly, none of the reports we have examined is concerned only with use of formulated 2,4,5-T herbicides, or indeed, only with production of 2,4,5-T. These studies relate to the phenoxy acid group of herbicides as a whole, os well as to wood preservatives. In this country, use of 2,4,5-T herbicides constitutes only a snail fraction of the total use of all these products. Secondly, deficiencies in those studies that gave positive results are such as artificially to raise the calculated relative risk factor. Anu thirdly, we are not aware of any instance in this country where an individual has used 2,4,5-T herbicides and developed a soft tissue sarcana. We conclude that there continue to be no grounds for changing our previous advice that femulations of 2,4,5-T herbicides as presently cleared, or, for that matter, other phenoxy acid herbicides and related wood preservatives, do not pose a safety hazard,'whether used in agriculture, forestry, the bane and garden, or elsewhere. 11. However, it has always been our normal practice to keep the safety of any pesticide under continuous review, and to take account of any additional relevant evidence that may become available. In the present case, we believe it would be practicable and desirable to pursue some further enquiries, based on available UK cancer mortality data, in order to establish whether there has been any change in the incidence of soft tissue sarcomas over the past 20 years, and whether there has been any correlation between such sarcomas and particular occupations. This will take a little more time, but without such an analysis the examination we havemade of all known relevant studies would not be complete. We will, therefore, be reporting further. i 5 r ` c ^ DOU 212/ 137 12. Separately freon this we suggest that the Government should consider arranging for systematic and complementary retrospective and prospective case control investigations to be undertaken as a contribution to the work being done internationally in this area. To this end we set out, in Appendix B, details of the kind of enquiries which would be appropriate and cannent on the problems involved in setting them up, and on their potential value. ' Discussion of Evidence - Production of Chemicals 13. The second type of evidence we have reviewed (paragraph 5 above) ccmes from mortality and disease data of cohorts of chemical workers in the USA, West Germany, Czechoslovakia and the UK. Whereas in the last three of these countries the findings were negative, positive evidence has emerged only frem the USA, where 7 individual cases of soft tissue sarcomas have been identified in various studies. Three of these cases were the subject of individual clinical reports where neither the size of the exposed population nor the nature and extent of exposure, if any, of the patients was established. Another case occurred among an undefined group of chemical production workers. The remaining three cases, each of which had a prior history of chloracne testifying to the fact and intensity of toxicologically significant exposure to dioxins, were drawn fran two cohorts comprising a total of 182 chemical production workers. Exposure to chlorcpfaenols was cannon to the latter 4 cases. Other cohort studies in the USA and elsewhere among workers similarly exposed to chlorophenols have revealed no cases of soft tissue sarcomas among a total of 460 workers. Two further American studies, involving the retrospective analysis of mortality data, did not establish any association betwen the incidence of soft tissue sarcomas and likely occupational exposure to either phenoxy acid herbicides or chlorophenols. 14. We do not consider these studies to be relevant to any assesanent of the safety in use of the phenoxy acid group of herbicides, because the circumstances, nature and extent of possible exposure differ greatly between the factory and the field, the forest or the garden. The studies of cohorts of chemical production workers which we have examined, and which identified the occurrence of chloracne among sans of the individual cases, indicated that exposure was to both a wider range cf substances and to higher levels than was the case with users of formulated products, and that it included exposure to contaminants which have now been virtually eliminated iron foimulated products. There was no such evidence among the populations who had merely used the forimlated products. Conclusion - Production of Chemicals 15. In this country, chlorophenols are produced and used in many situations outside agriculture and wood preservation. A few have limited use in their own right, but the majority are used, or have been used, in the production of a wide range of products such as adhesives, dyes, paints, inks, disinfectants, wax emulsions, cutting oils, fire extinguishers, ropes, tents, pharmaceuticals, toothpastes and cosmetics, as well as in the leather, textile and paper and board industries. The evidence -we have examined concerning chemical production workers leads us to the conclusion that it would be prudent for the Government to undertake further relevant investigations in factory situations in this country. Neither the factory processing of chlorophenols nor the safety of the products and practices described above is within the remit of the Advisory fiamittee on Pesticides; but we would .wish to be involved in any such wider investigations in so far as these might relate to the specific area of the production of herbicides and wood preservatives. Yours sincerely, Robert Kilpatrick Chairman Advisory Conmittee on Pesticides 4 16613 30115* U" 2124046 2649 i DOW C H EM IC AL U.S.A. August 28, 1980 ROUTE 1. BOX 1313 davis, caufo rnia m i 916 753-6608 J. B. SWEELEY MASONITE CORPORATION WESTERN WOODLANDS DIV. P.O. BOX 97 CALPELLA CA 95*18 bcc: W. E. Allison 5. H. Davidson D. D. Hunt M. G. Norris A. 3. Watson Dear Jack, Regarding your letter of August 12, I just returned from vacation --hence the belated reply. Based on our knowledge of 2,*-D and 2,*,5-T, the claim or insinuation that these materials could cause any adverse effect on humans or animals at the low exposure levels resulting from applications several years previously is spurious. Hundreds of water samples have shown that the levels im mediately below the treated area are less than 10-15 ppb. Data from Norris and others indicates that the levels are reduced to non-detectable ones in Yi mile or so downstream. There is a tolerance for 2,*-D in potable water for 100 ppb. At the rate 2,*,5-T was applied, there would have been very little in the water immediately after application and only if run-off .occurred in the spring after treatment. If there was some TCDD in the 2,*,5-T it would have been decomposed mostly by sunlight and if it did get to the soil or duff it is sc insoluble that it would be extremely unlikely to get to the water by run-off. It is interesting that workers in the Dow 2,*,5-T plant have not reported chloracne during the 25 years of operation. Chloracne is the earliest ex pression of reaction to TCDD. Nor have any adverse effects been reported by workers in the 2,*-D plants.- These groups have been observed very care fully. You may want some references to support the above statements; a copy of the CAST report on Phenoxy Herbicides and several others concerned with phenoxy herbicides that may be of interest are enclosed. Since it Is very unlikely that the "itchy back" resulted from exposure to the forest herbicides, we can look for other causes. If Mr. Johnson "swims" in the creek that is too shallow to swim in, he may be exposed to poison oak or a number of other plants that can cause skin problems. Of course, many other things, like soap, cleaners, oils, etc., are known irritants. AN OPERATING UNIT OP THE DOW CHEMICAL COMPANY M O U I Z tnn J. B. Sweeley 2- - August 28, 1980 It seems odd that the "itchy back" has only now become serious enough to cause a complaint -- 7 years after the last application. You may recall the Seveso incident where 2-4 lbs. of TCDD was distributed over 200 to 400 acres in Italy. Children and adults were not moved out for 2 weeks. Many cases of serious chloracne were treated, but after 12-18 months all cases had healed upj there have been no other observed adverse effects above those in the surrounding populations. The 2,4-D may last for 2-3 months and the 2,4,5-T about 4 to 6 months. It's quite impossible for these materials to last more than a year or 2 in the trees and it disappears in 24 weeks from soil. The spring treatments would have allowed these or faster rates of decomposition because of warming conditions and good soil mois ture. The previous treatments should have enriched the microorganisms and enhanced 2,4-D breakdown. I will pass this on to other members of our staff who may have added comments of value. I hope this analysis and the reports will help refute the allusion of Mr. Johnson. Yours very truly, L. c. warren Agricultural Products Department Technical Service and Development Ehe. cc: J. Jones, DOW CHEMICAL U.S.A., Sacramento. Matt Anderson, CFPA, Sacramento Norman Parker, Western Helicopters, Newberg, OR. kirn DOH 2 0 6 M I I 26Q Dow Chemical Co'. EPA Hearing Submissions Do : Cancellation - Volume 2 16. "Petition for the Establishment of Tolerances for the Pesticide Chemical, (2,^1,5-T) on Raw Agricultural Commodities", Sec. C: Full Reports of Investigations re: 2,l|,5-T, 12/8/67'. (1 ) Summary of 11 tests on safety of 2,il,5-T (A-K below) (2 ) Conclusion: -more data on 2,^,-D than 2,i1,5-T is available, but indi cations are that two materials are comparable; acute oral toxicity of both in lab animals is in same range, 3 0 0 1 0 0 0 mg/kg. -long history of usefulness not produced evidence of unusual toxicity. A. "Acute Oral Toxicity of 2,^,5-T to Rats, Mice, Guinea Pigs and Chicks", Rowe, McCollistcr and Spencer, (Dow, 1950). (1 ) Single oral doses (administered by stomach tube) of 2,^, 5-T as a 10^ suspension in olive oil, to mixed sex group of rats , mice and guinea pigs, 3% solution in olive oil given to chicks. (2 ) Symptons of poisoning = ataxia, myotonia; death at highest dosage levels; doses ranged from 0 . 1 - 1 . 0 grams/kg. (3) LD 50's: rats mice guinea pigs chicks = = = = 0.500 grams/kg. 0.389 O.3 8 I " " 0.310 " 0 0 2,Jj,5-T significantly more toxic to chicks than to rats. B. "Toxicity of 2.JI-D and 2,^,5-T - a report on their acute and chronic toxicity in dogs", Drill and Hiratzka, 1953("There have been no reports'concerning the toxic propertie of 2,^,5-T"). (1 ) Experimental Procedures : adult mongrel dogs of both sexes 1 16616 00li 206(J4I 2 used, fed "l-'riskios" ad^ 1 :i b , commercial 2,4-D and 2,4,5-T used.. -single oral dose administered in capsules. Survivors autops.ied after 14 days (acute studies). -oral administration in capsules imbedded in *1-5 gram piece of commercial conned dog food, 5 days/wk for 13 weeks (chronic studies). (2) Results of Acute Studies : (a) 2,4 -D : -400 mg/kg caused death in 2/2 'dogs in 2-3 days fol lowing administration. -250 mg/kg t death in 3/3 in 4-8 days -100 mg/kg - 2/4 died in 9 days, 2 survived to 14th da - 25 mg/kg - no deaths , all 3 survived to day 14 2 4 5r.T: -400 mg/kg - 1/1 died on day 2 -250 mg/kg - 1/1 died on day 3 -100 mg/kg - 1/4 died on day 7, 3 survived to day 14 mg/kg - no deaths , all survived to day 14 (c) Oral LD(-q for 2,4-D = approx, 100 mg/kg; for 2,4,5-T, 100 mg/kg or higher (d) Body weight - decrease with larger doses of both 2,4-D and 2,4,5-T; these animals developed anorexia, those that died refused food towards end. (e) Symptoms: ataxia, stiffness in hind legs, myotonia 2. 10617 r o LA 1 OOHZ 0 6 0 41 3 (f) Patholo g y : gastrointestinal chances - redness in small intestine, some necrosis and inf lamina: hepatic necrosis, mild renal tubular degeneran deaths due to nonspecific changes - hepatic cor./ many cases o' death due to pneumonia. (3) Results of Chronic Studies: (a) Mortality - -2,5jl0 mg 2,4-D or 2,4,5-T/kg: all dogs surviv 90-day test period -20 mg/kg of compounds: all d6gs died during s `; (b) Body Height - loss in weight occurred signifies, only in those animals that died t (c) Symptoms - survivors were free of any symptoms; both 2,4-D and 2,4,5-T dead, weakness, leg stii: difficulty in swallowing food, bleeding from gu:\ (d) Blood Count - only change observed = fall in % c; lymphocytes in 3 animals that died, (e) Organ Weights - no change in survivors; in 2 of : 4-1) deaths, slight increase in heart and kidney similar change in 2 of 4 2,4,5-T deaths. (f) Gross Pathology - 2,4-D 12 dogs showed areas of ness in duodenum; 2,4,5-T one dog showed dir. reddened duodenum and jejunum, another demonstr;. a generalized icterus. (g) Microscopic Changes - 5 cases of focal necrosis liver (not related to dosages nor death, thus, ' significance). Some duodenal hyperemia, some infiltration of cells in the mucosa. Sub,:luco f-':. serosa were normal. Slight increase in // of c::. in kidney sections in some dogs (unrelated to d: and of doubtful significance). '3 16.618 OOH20604 I 4 ('0 Com mo nt : -effects of 2,*l ,5-T not as severe as those of 2,*I-D in acute studies. -delayed death in chronic studies with 2,*1-D may indicat a cumulative effect of 2,*I-D. r -signs observed in dying animals in acute study differs from those In chronic study. (5) Summary : 2,*I-D acute oral LE>^q in dogs = 100 mg/kg, doses r at this level or higher produced a definite myotonia with anorexia and weight loss. r 2,*I,5-T acute .oral LDj-0 in dogs = 100 mg/kg or higher; such doses produced only signs of a mild spasticity. r Death during the repeated administration of 2,*1-D or 2,^ 5-T was not related to pathological changes in the liver, kidney, or other organs examined. C "Summary of Toxicological Information on 2,"-D and 2 h y rJ - rirt Type Herbicides and an Evaluation of the Hazards to livestock associated with their use", (Rowe and H y m a s , 195*0. (1) Early in 1950, Grigsby and Farwell published results of experiments with 2,'I-D and 2 , 5-T involving domestic r livestock, concluded "it seems that the farm use of these materials for pasture weed control is a reasonably safe p ro ced ur e." r (2) Commercial Herbicides - formulations of active ingredients appearing as a sa'l'i. (V/ater-soluble) or an ester (soluble r in oil); most formulations also contain a dispersant, a solvent, a wotting, agent, perhaps a diluent (usually con sidered inert ingredients though they may affect herbicidal e f f e c t i v e n e s s ). f' (3) Oral Administrate i of S 1.::'d c Doses : (a) test material;: administered primarily by intubation. (b) aqueous, or olive oil or corn oil, solutions used r r 'i r O0H2O6O I 5 (c) rata mico guinea piga chiclea dogs 2 ,'1,5-T 500 mg/kg 309 mg/kg 3 0 l mg/kg 3 10 mg/kg 100 mg/kg 2 Jl-D 375 mg/kg 368 mg/kg *169 mg/kg 5;H mg/kg 10 0 mg/kg (d) toxic symptons of LD^q ,s include loss of appetite, weight loss, depression, roughness of coat, general tenseness, muscular weakness; post mortem findings stomach irritation, minor liver and kidney injury, lung congestion. (4) Oral Administration of Repeated Doses of 2,4 -D: (a) Rats - fed 2,^1-D 5 times/week by intubation, in olive oil solutions. Dosages were 0.0, 3.0, 10.0, 30.0, 1 0 0 .0 , 300 mg/kg. -- wj*inu ar>uvt cftiftftft cft cft ^u^cftCftU oft aMu Uu.ftU.ft.ifltU4. .if'tUJ.j aJ > TJLAVy AA mg/kg levels; 3.00 mg/kg group - gastrointestinal irritation, swelling in liver,- depressed growth rate; 3 0 0 mr/kg group failed .rapidly C-. died, severe gastro irritation observed. (b) Another Rat Study - fed 2,4-D for 113 days at levels of 0 , 1 0 0 , 3 0 0 , 1 0 0 0 , 3 0 0 0 and 1 0 , 0 0 0 ppm. Results - no adverse effects in control, 100 and 300 ppm groups; 10 0 0 ppm group suffered depressed growth rate, excessive mortality, slightly increased liver weights, liver swelling; 3000 and 1 0 , 0 0 0 ppm groups were destroyed after 1 2 days since they were not eating and wore losing weight; examination revealed increased liver and kidney weight, 1 . and k. patho logical changes. (c) Chicks - fed 2,^-D for 7 days at levels of 0,500, 1 ,0 0 U and 30 00 ppm. Results - only adverse effects in 3000 ppm group reduction in food intake and retarded growth.rate (no histopathological exams made). 16620 5 DOHZ0 6M I6 (5) Oral Administration to Stoops - (experiments performed to see if extrapolation of data on small animals to larger animals is justified). (a) "Esteron Brush Killer" used (formulation of esters ol 2,4-D and 2 , 11,5-T); administrations made by lntubatic -experiment i l l : 291 leg steer given single dose of 1000 mg/kg - no toxic symptoms. -experiment //2: 295 kg steer given 1000 mg/kg esterc for 3 days. results : general depression, decreased food and water intake, decreased rumen motility; animal died on 3rd day after last dose (death was undra matic). Necropsy revealed dry rumen contents with strong smell- of esteron, abomasum was im pacted, intestinal contents entirely fluid, con gested mesenteric vessels, spleen dark and shrunken. -experiment # 3 ` 2 9 5 kg steer given 500 mg/kg for 2 days. r e su lt s: on 3rd day, animal was off feed, rumen motility ceased. On 4th day, steer appeared perfectly normal, no discernible after-effects. -experiment //4: 336 kg steer given 500 mg/kg for 3 days. results: no toxic symptoms observed, animal remained on full feed. -experiment //5: 250 kg steer given 100 mg/kg for 15 days. results: outward appearance normal. Killed 4b brs after last dose for study. Duodenal hemorrhage, abomasum irritation, hemorrhagic necrosis of liver, fatty degeneration, slight edema- and congestion in kidneys were found. 6 16621!. DOH20604 I 7 I (b) "Huron11 used In another experiment: 250 kg steer given 100 mg/kg Huron for 15 days. r e s u l t s : outward appearance normal. Killed >i8 hours after last dose for examination. Very slight edema in kidney, but this is frequently observed in untreated animals; its significance is questionable. (6) Discussion: (a) Dogs more susceptible to 2,^-D and 2,^,5-T than- other r species studied; chicles appear tolerant. (b) "Inert" ingredients in commercial formulations have no r' effect on toxicity of active materials. (c) 2,*1-D and 2,iJ,5-T can be tolerated without adverse effects in doses only slightly smaller than those which r cause toxic effects when given only once - these mater ials have a low degree of chronicity, therefore. t (d) Ther-. is a similarity in susceptibility of cattle and small lab animals to these herbicides. (e) If esteron were applied at the normal rate of no more than *17 mg/sq. ft. (or 2 q t s . per acre) a 3 5 0 kg (770 l b . ) animal would have to consume a 11 forage I" on 7 ^ s q . ft. to acquire a 1.00 mg/kg dose of herbicide, a level at which ill effects would not be expected. r (f) " . . . there is little, if any, direct hazard to live stock or wild-life foraging areas treated with herbi cides of the type described herein. This has been con firmed by the extensive use of these materials over a r number of years without any proved cases of adverse effects." i (g) Spraying of certain plants with 2,^1-D can increase their nitrite content to hazardous levels, but some of these plants were sprayed accidentally, and others are not ordinarily eaten by livestock. Toxic amounts of these materials can be obtained if animals have access to spray tanks or other containers of the material. 7 1SS22 r DOH 2 0 6 CU I 8 D. "Results of Toxicological Tests on (2,^1,5-T) ", Dow, August 1950. (data submitted to FDA Hearing on residue tolerances). (1) Because of similarities between 2,J|-D and 2,1|,5-T, residue tolerances for both should be same - 20 ppin "would provide a wide margin of safety." (2) 10# solution of 2 , ^ 5 - T in propylene glycol introduced into rabbit eye caused pain and irritation immediately, severe conjunctivitis and moderate corneal damage apparent 5 hours later, continued for 2 days before healing began. (3) 10# solution of 2,^,5-T in butyl carbitol acetate applied to rabbit ear and abdomen caused slight irritation. (0 2,^1,5-T is moderate in acute oral toxicity for the four species, rat., mouse, cavie, and chick (rat more resistant, chic): most susceptible)-. (5) Handling hazards and precautions: -swallowing, although unlikely, could cause serious ill effects; induce vomiting. -avoid contact with eyes and skin, d o n 't wear clothing or shoes contaminated with material'. -no Inhalation studies to date, but doubtful that toxic concentrations would be inhaled due to irritation it will produce in upper respiratory passages. E. "Toxicity tests in NuAmine D / T " , Report to Diamond Alkali Company by Hazleton labs, February 1962. (1) NuAmine D/T = 1,3 - propylene diamine esters of 2,^-D and V>9-T. (2) Acute Oral Administration - NuAmine D/T given to SpragueDawiny rats (6 groups of 5 each) by stomach tube, cither in water emulsion or undiluted dosages of 10,31.6, 100, 316, 1000 or 3160 micrograms/kg body weight. Concentra tions of material varied from 1#, 10#, or 100#. Observa tion period of lh days following dosing. 8 16 523 DOH2060419 (a) ..Mor tal it y: After 2 1! hrs., 2 in 1000 microgram/kg group, arid all ( 5 / 5 ) in 3 1 6 0 microgram/kg group died. On day 12, another in 1000 microgram group died, and another on day 1*J. These were only deaths recorded. (b ) Other E f f c o h s : -1 0 ,3 1 -6 , and 1 0 0 micrograms/kg g r o u p s : normal appearance and behavior, increase in body weight, no pathological findings. - 3 1 6 m i cro ;./k g : b / 5 seemed normal, 1 was depre on days 12-.') 4. Slight increase in average body weigh 2 / 5 showed congestion and hemorrhage of lungs. - 1 0 0 0 mic ro g . /k g: b / 5 normal at first, 1 shov/ed depression, a t axi a, and labored respiration after h r s . 2 died after 2 ;l h r s ., survivors showed depressio and labored respiration. After 48 hrs., all survivor appeared normal; at 2 nd week, all showed unthriftines depression, labored respiration and ataxia. 2 more died on days 1 2<T;1 h . Autop sie s c*V^ 1.ori 1nn(* U ObJ.UJ1y JUUiJCJ and stomach hemorrhage, hematuria, intestinal inflame tion, discoloration and/or reduced size in kidneys spleen and liver, stomach inflammation, congestion of fat around tests. - 3 1 6 0 microgra:-.s/kg :appeared normal following intubation; depression and ataxia after 1 h r . ; pros tration, ptosis, salivation, labored respiration, poo reflexes after ') hours; 1 death at 5 hours, survivors showed hematuria. Total mortality by 2 b hours. Autopsies revealed congestion of lungs, adrenals liver and spl*.. on; kidney and stomach mucosa hemorrhag intestinal inflammation; bloody fluid in stomach and urinary bladder. (3) Acute Eye Application - NuAmine D/T applied to eyes of 3 albino rabbits Cleft eye only, right served as control) 9 00H2060420 Single application of 0.05 ml of undiluted tout jnatcrJ.nl made into conjunctival sac. Periodic observations for 1 week. Food (Turlna rabbit pellets) and water ad_ l i b . (a) R e su lts : Animals seemed normal throughout study, slight decrease in body weights in some. Eyes showed erythema, film over cornea, slight vas cularization of sclera and nictitating membrane, lacrimation, edema of lids (obscuring eye and preventing thoroug exam after a few hours), exudation, corneal opacity, and corneal lesion (1 animal). (*0 Acute Dermal Application - albino.-rabbits used; single application of 5 0 // volume/volume aqueous emulsion of NuAmine D/T at dose of 0.1 mg/kg body weight, and single application of undiluted test material at doses of 0.1, 0.5, 2.0 ml/kg.- Test material applied to abdominal skin. "Purina Rabbit Pellets." and water ad_ l i b . (a) Results : 3 deaths at 2 ml/kg level, no deaths at any other level. Estimated acute dermal LD5r0o of NuAmine D/T for albino rabbits = 1.2 ml/kg. 1. 50% e m uls io n: application(0.1 ml/kg) produced no signs of systemic toxicity, no pathological findin 2^ hours after application - moderate erythema and edema. At fourth and fifth days, erythema and ede subsided, but animals showed slight or moderate atonia and desquamation- By 12th day, almost all signs of irritation gone. 2. Undiluted: -0.1 ml/kg - normal appearance and behavior. Autopsy showed kidney congestion, mottled and blanched"livers. -0.5 ml/kg - normal appearance and behavior. Autopsy showed kidney congestion, and granular app ance of liver. 10 16625 DOH2O60 4 2 I -2.0 m l / h i ' - 3 deaths at this level, on *)th, 5th, and '/th day:-; of observation. 2 h lira, post application, 3 animale showed depression and laborc respiration, *lth acoined normal. Toxic signs gradu ally bocair.o more severe: depressed reflexes and coordination, labored respiration, ptosis, ataxia, coma phonation, salivation. Autopsies showed con gestion or hemorrhage of lungs and kidney; bloody urine in 1 bladder, bright yellow urine in another; liver damage (blanching and discoloration) and toughened liver surface; spongy brain tissue with excessive fluid in skull; extremely congested or hemorrhagic appearance of skull cap and bone marrow One survivor was sacrificed^ showed congestion of kidneys. Subcutaneous edema, damage to all layers of the skin. Dermal' effects - 2*1 hrs. after application, skin at all dosage levels showed edema and blanched areas surrounded by moderate erythema. In 3 animal that died at highest dosage, derma] irritation was relatively unchanged at time of death. Survivors in all levels - edema subsided after 3-5 day.'-, scab formations or marked desquamation, atonia, coriaceousness, and fissuring developed to end of observation. (5) Repeated Dermal Applications - albino rabbits given 1 ml/k NuAmine D/T in varying strengths (*1, 12, 20 oz./gal. aqueous emulsions) 5 days a week for *1 w e e k s . R e su lts : each group showed moderate degree of dermal irritation - erythema, edema, atonia, desquamation. Several in 20 oz./gal. group showed fissuring. One animal in each of the test groups died, all showed systemic toxicity - depression, labored respiration, sprawling of limbs, weakness, weight loss. Moribund animals also showed prostration, poor coordination, depressed reflexes, clonic convulsions. Abnormal blood and/or urine findings in each group, severe liver, kidney and skin damage in *1 and 20 oz./gal. groups . E-l "Microscopic examination of bone marrow sections" fro;1. Nu/.minc D/T study, Hazleton Labs report to Diamond Alkali Co., 12/16/65. - supplement to report of 2/23/62; tests on bone marrow taken from rabbits receiving repeated dermal applications of NuAm.i.ne-D/T. ( 11 16826 OOW2060422 .T Results : No distinctive effect on bone marrov/ at 2 concentrations (*1 and 12 oz./gal.). Higher con centration (20 oz./gal.) crouP showed somewhat atrophied adipose tissue. A possible effect - mega karyocytes (in 20 oz./gal. group) underwent degen erative changes, more so than in controls. F. "Results of 90-day Diet"vy Feeding Studies of Dowanol 97B (ester of 2,*i,5-T) in Rats", Dow, 11/27/61 (1) Dowanol 97B ester of 2,4,5-T is an agricultural herbicide Timber liquid) (2) experimental procedure: 5 groups of 20 m & f rats were maintained for 90 days on diets containing O.O(control), 0.3^ 0.1, 0.03, and 0.01# of the ester. Food (ground Famo Laboratory Chow) and water ad lib. . r (3) Results . i 0.03 and 0.01% levels: no evidence of adverse effects increase in average weight ,of spleen in males, increase in body weight of females - believed due merely to chance. 0.3% level: growth retardation in males, liver and kidney/body weight ratio increased in males. M and F livei were large and light in color,"some swelling and necrosis. r Some swelling in male kidneys; female kidneys appeared greenish, some swelling and necrosis. Increase in serum i alkaline-phosphatase determinations in males. r 0.1% level: livers (m and f) showed some swelling and necrosis, females shov/ed hypercellularity of glomerular tuft, swelling of renal tubular epithelium. Increase in average male kidney weight." (*0 Conclusions: the Dowanol 97B ester of 2,4,5-T is moderate r ' in repeated oral toxicity in 90 day feedings. Levels of Ii 0.03# and below were tolerated without evidence of adverse effect. Male and female rats showed some injurious effects (liver & kidney-pathology) at levels of 0.1# and above. Long-term dietary studies (2 years, with rats and dogs) may be required if ester is found in significant amounts as residue in food. G. "The Toxicologic Effects of Certain Fungicides and Herbicides on Sheep and Cattle", Palmer and Radeleff, 196*1 (1) "In contrast to insecticides, organic fungicides and herbi cides (with only a few exceptions) have generally been considered to provide comfortable margins of safety for grazing livestock when the compounds are used according (. to directions." 16627 DOH2060423 (2) Sheep and cattle maintained on grain concentrates witli hay supplements. (3) Commercially available formulations used, as fluid dilutions or in gelatin capsules; number of doses varies. Results : (1) Fungicides - a) "Captan11: sheep are regularly poisoned by doses of 250 mg./kg or higher; reduction to 50 or 100 mg/kg permits animals to eliminate most of material without being harmed. Cattle more tolerant of Captan than sheep. Depression and anorexia,., 'tendency to stay away from group were only toxic signs in sheep; in one heifer, diarrhea and abortion observed. At necropsy, hepatic and renal changes noted but not remarkable. b) "Zineb" : Single doses of 500 mg/lcg. tolerated by sheep and cattle. 90 daily doses of 100 or 2 5 0 mg./kg. did not poison sheep. In single poisoned sheep (15 doses of 500 mg./kg.), anorexia and bright yellow diarrhea observed. Necropsy showed congestion of the lungs., hepatitis, and nephritis. c) "Cere.san M " : Sheep fed 1, 1.5, or 2 lbs. of treated grain per day. Lethal to all, at doses of 1 lb./day x 33 doses, 1.5 lb./day x 22 doses, or 2 lb./day x 6 doses. Signs of poisoning included anorexia, diarrhea, weight loss, nasal discharge, stilted gait and lameness. Necropsy showed lesions characteristic for mercurial poisoning. (Ceresan contains mercury). (2) Herbicides - a) 2, *I-D: administered as alkanolamine salt and as propyler glycol butyl ether ester to sheep and cattle. Very little difference in toxicity of 2 derivatives. "Remark able tolerance" of ;l8l daily doses of 100 mg./kg. by sheep indicates little probability of acute poisoning in normal applications of the compounds. Toxic signs in sheep - anorexia, depression, weight loss; in cattle muscular weakness, ataxia, dry, cracked muzzle, ulcera tion of nasal mucous membrane. Necropsy - lesions varied usually liver and kidney degeneration;rumen stasis with bright, undigested ingesta; hemorrhages in heart and excess of pericardial fluid. 13 16628 OOH2QSOm b) 2 , _Jl, 5- T: admi nisl.i.red a;; trielhy lam.'uie salt and us propylene glycol butyl other eater to r.heejj and cattle. Salt appeared leant toxic (sheep tolerated *18.1 doses of 100 ing./kg.) while eater:.: were lethal at 36<J donee of 100 mg./kg. Mstern were lethal to sheep and cattle after 7 doses of 2 `j Q mg./kg. 2 , ^1, f3--'1` somewhat more toxic to sheep and cattle than 2, >I-D, "although poisoning under ordinary circumstances of use would be most unlikely. 11 c) 2 , 1I, 5-TP (2 - (2, *1, 5-trichlorophcnoxy) - propionic acid) administered as butyl ether esters of propionic acid derivative to sheep and cattle. Markedly more toxic than 2;/l-D or 2, , 5-T (sheep succumbed to 11 doses of 100 mg./l;g., cattle to 29 such doses). Signs of poisoning: anorexia co progressive weight loss. Necropsy showed degeneration of liver and kidneys, hemorrhages on heart surface, congestion of visceral blood vessels, swelling of lymph glands, rumen atony with large quantities of ingesta. ( 3 ) Benzoic Acid Compounds - dimethylamine salts of 2, 3, 6-trichloro- (Trysben) and 2-methoxy - 3,6-dichloro enzoic acids (Banvel D) tested. Both compounds slightly more toxic than phenoxyacetic acids studies. Trysben at dosage of 250 mg./kg. x 10 doses was lethal to the one sheep, but the cow was u p ;'ffee ted; at- 500 mg./kg. x 2 doses, the compound produced mild poisoning in one other cow. Banvel D in doses of 250 mg./kg. x 10 doses did not affect a sheep, but killed another sheep at 500 mg./kg. x 2 doses. A cow was not affected by 5 doses of 250mg/kg. Signs of poisoning: salivation, trembling, depression, tympanites. Necropsy showed congested lungs, hemorrhages on heart, pericardium and larger blood vessels, undigested feed in rumen, edema in respiratory system, inflamed kidr.e liver engorged with blood. (4) Triazincs - (Propazine, Simazinc, and Atr az in e) : admin istered to sheep and cattle; Atrazine was lethal at all doses tested (50 mg./kg. x 199 doses, 100 mg. x 16, 250 x 2) for sheep, and lethal to the cow tested at 250 mg./kg x 2 doses. At the 250 mg./leg. level in sheep, Si marine was more toxic than Propazi n e ; at 50 mg./kg. Simazinc was more toxic than Air:;::ine. Signs of poisoning (Propazi anorexia and depression. Signs of poisoning (Atrazine and Simazinc): muscular spasms and fasciculation, stiff gait, .increased respiratory rates. Necropsy in sheep and cattle given Atrazine showed degeneration and dis coloration of the adrenal glands; congestion of lungs, liver and kidneys; presence of undigested food in rumens. l'l 16629 DOH2060425(5) Mir,ccl 1;n coik; compounds : a ) Dalapon (2, 2-d.i.chloropropionlc acid) - sodium salt is of a low order of toxicity (1300 mg./kg. x 10 doses not toxic to' sheep or cattle, one sheep tolerated >l8l doses of 100 mg./kg.) No poisonous signs. b) MCP Am jno (2-mcthyl (^l-chloro-o-toloxyacetlc acid) alkanolamine salts poisoned one sheep lethally at 383 doses of 100 mg./kg.; a cow was poisoned by 8 doses of 500 nig./kg. but survived. Signs of poison ing in cattle: anorexia, chronic tympanites; in sheep, anorexia, depression and ataxia before death. c) Fenuron (3-phcno.l-l, 1-dimethyl, urea) - appeared morctoxi-~ than most herbicides discussed, but high dosages still required to produce poisoning (e.g., 250-500 mg./kg. x 2-10 doses). Toxic signs: de pression, anorexia, complete incoordination (heifer abortion); slow recovery. Necropsy: lung congestion, heart hemorrhages. d) Bandane (polychlorodicyclo pentadiene isomers) - sheet poisoned by 3 doses of 2 5 0 rng./kg. but survived, while same dosage was lethal for cattle. Toxic-signs i4,n11 s*- h ir -- f-- n :1 ts- a l i v a h i n n j. a--nl . m- -' -P-Y-T- a- 9 t - . v m--n--a- n i t . a a ~~\J -- t- and depression. Toxic signs in cattle: tense muscles muscular spasms. (Even when dosing stopped, signs progressed, duplicating tetanus). Death preceded by convulsions. Necropsy: hemorrhages of cerebrum and medulla oblongata, hemorrhage of right psoas muscle, skeletal fat replaced by gelatinous material, lung congestion. (6) Discussion: studies offer no "reason to alter the already generally accepted concept of the hazards pr e sented by these herbicides." Further study needed re. toxic signs. (7) Conclusions: feeding Ceresan M (13-38 mg./kg.) on treated grain by mistake can easily poison sheep; other materials studied are safe when used as recom mended . "Commercial Fishery Investigations", Philip A. Butler - Acute and Chronic Toxicity Studies, 196^1 (1) purpose of project: to learn how to protect and pre serve marine environment from possibly adverse effects of agricultural chemicals; discovery of pesticides that may be useful in improving fish harvests 15 16630 DOH2060426 (2) "Tlic need for this rcr.car'cli does not imply that the wJdespread use of pesticide formu]ations automatically constitutes a serious throat to marine life." (3) "Even the mo:;I, harmless of chemicals, including viator, Is toxic when present in sufficient quantity." (*0 different forms, of marine life may react quite dif ferently to the same chemical (!5) four bioassay groups (pbytoplankton metabolism, oyster shell growth, shrimp survival, fish survival) tested for sensitivity to four pesticides (dyrene, methyl parathion, phosdrin, phosphamidon). Phosdrin and phosphamidon produced the least reaction; dyrene had some effect on all *1 groups, especially fish survival: shrimp survival was very sensitive to methyl parathion. (6) phytoplankton - microscopic sea plants, essential link in conversion of solar energy into food for more complex marine animals. Using decrease in growth, or decrease in carbon fixation as a standard, tcxicitiec of pesticides may be compared. (7) Crustacea - shrimp is most valuable fishery; most pesticiues kill! terrestial members of shrimp's phylum (arthropod). Some pesticides paralyze shrimp and other crustaceans, rather than kill; toxic cri teria for this, group is effective concentration causing paralysis or death to half the samples within a stated period (EC,-0 ) (8) mollusks - clams and oysters store high concentrations of chemicals that exist in only trace amounts in the surrounding sea water. Mollusks, however, are able to close their valves and protect themselves from toxic substances in (lie environment. Thus, tests must be conducted at nihlethal. concentrations; growth is an objective index to measure, using EC,-0 measure of effect (9) f i s h .- younger specimens usually most quickly affected by pestici des , (10) 2, *1, 5-T (poly glycol butyl ether ester): - E C TM for oys lor shell growth = 0.1*1 mg./liter (96 h r s . 1 we^lc recovery period. 1. "Coiiiincrc:i al_Pi shier 1e:. fnvost 1gat,ions ," Philip A. Butler Laboratory Studies ana Toxicology ` 16 166 3 1 DOK 206 M 2 7 r (1) phy top.lankl-on - majority of chemic a la ter:ten1 canned significant deorcane in productivity at 1 ppm, al r` though at lower concentrations an increase in pro ductivity rate war, frequently observed (probably because pesticide was toxic to animal part of natural i* phytoplankton community). Herbicides and DDT very toxic to some plant species. (2) Crustacea - post-larvae of brown shrimp and blue r- crabs are quite sensitive to chlorinated hydrocarbon insecticides. All shrimp and most crab tests con ducted in sea water with acetone stock solution of c pesticide (constant flow systems, in which test solu tions are renewed continually, have advantage of in suring sufficient oxygen and desired concentration r~ of pesticide). After exposure to lethal or near lethal concentrations, crabs and shrimps often mori bund for h o urs. r~ (3) mollusks - oyster and clam tested, constant flow system with acetone-stock solutions of pesticides. Oyster closes its shell when sufficiently irritated by contaminated water - does not feed while closed and therefore does not grow; therefore relative chemical toxicity is growth). indicated by changes in t>XgvirW/-.ioUlw4U- tV1-4sh rate XCi. v^. (shell inated hydrocarbon insecticides were most toxic pesti ( cides to oysters - most inhibited shell growth at well below 1 ppm; other pesticides (crganoplr sphorus and carbamate insecticides) had little or no effect on oyster growth - they decomposed in water. Meta bolism increased at summer temperatures, therefore, at higher temperatures some chlorinated hydrocarbon insecticides killed oysters at concentrations which at lower winter temperatures only stopped growth. 1-4 week recovery period for reduced growth rate; data indicates that "a single field application which might temporarily affect oyster growth would probably not have a lasting effect." Chronic, rather than acute, exposure to pollutants is more likely. Chemicals tested included aldrin, dieldrin, DDT, toxaphene, malathion and acetone (acetone used as a solvent in program, so tested for possible toxicity). In general, no decrease in growth rate; treated groups sometimes grew slightly better than controls. Oysters did not acquire tolerance to pesticides. -ECr(, for decrease in shell growth: J 2,4,`j-T acid (temps, of 1 6 , 30 C) - no decrease at 2 ppm 2,4-D acid (temps: 9, 30C) - no decrease at 2 p; 17 1 6 6 3 2 ' OOK2060428 (4) fish - juvenile while mullet longnose killifish used ;io tost animals in running sea water aquaria. 24 arid 48 hr. median tolerated limit values (TLm) ol tained. Chronic low-level pollution studies .im portant, since many marine fish pass their early growth stages in estuaries. 3 groups of Spot (leiostomus xanthurus) exposed 3 m o s . to sublelhal concentrations of dieldrin (0 .1 , 0.01, 0.001 ppb), in running sea water. High mortalities, 30-37#> but also in controls. No dif ferences in mean lengths, though some experimental fish had axial skeletal distortions, not in controls. Survivors exposed to 2 ppb dieldrin to determine if earlier low-level exposure had created any resistance: 80# of experimental' fish survived 24-hr. exposure, while previously unexposed fish all died. -2,4,5-T acid tested on longnose killifish showed no effect at 50 ppm (24 and 48 h r s . TLm) with 19 C water temperature. -Esteron 99 (formulation): TLm for 50# mortality in mullet - 1.5 ppm (24 and 48 hrs.) at 20C; in killifish.= 3-5 ppm (24 hrs.) and 3.0 ppm (48 hrs. at 19C. (5) Future considerations : "... to determine which pesticid: now considered necessary may be expected to cause the least damage to marine resources. . . careful selection of the control agent and method of application can lessen the current pesticide hazards." J. "Commercial Fishery Investigations", P.A. Butler - Acute Toxicity, 19b5 (1 ) Crustacea - shrimp - most valuable commercial fishery resource, spend part of life span in estuaries sus ceptible to pesticide pollution. Since they are rather closely related to insects, insecticides are particularly toxic to them - either kill quickly or paralyze them so they lose sense of balance. Tests conducted 24, 48 hrs. in flowing seawater. Resul t s : 2~,4,5-T acid had no effect on brown shrimp mortality or equilibrium at 1 ppm after 24, 48 hrs. (aVg. water temp. = 28C) 2 _4.5-T pn ]y rju/c o 1 butyl ether ester tested on brown shrimp in 2buC water caused 10# mortality (or loss of equilibrium) at 1 ppm after 24 h r s ., and 20# mortality at 1 ppm after 48 hrs. * 18 1 water had no effect on mort Uin after 2;l hr. and >!0 hr. exposure at. I. ppm level. 6 Z!/ 090Z?iOO therefore juveniles used in teats. r,ua water used. Results : -2,jl,5-T polyp;.!ycol butyl e 111e r_ oster tested on Spot in l'(7C water ternp. iVad" 2^1 and h8 hr. EC,-q of 0.32 ppm for mortality. -Veon 2^ 5, also tested on Spot, in 2 7 0 , had no effect after 2^1 and ^8-hr, concentrations at 1 ppm. "Recommendations for Minimizing Danger's of Test Control and Pesticides to Fish and Wildlife", John L. George, June, 1963 (1) be sure there is real need for pesticide use (2) discuss possible hazards with biologists prior to u (3) avoid treatment of any significant portion of range of a rare species or one with specialised habits consult with conservation officials (1) treat minimum neces.sary area; avoid direct treatmen of streams, etc., consider danger of runoff contam ination (5) judge danger of chemical both by its toxicity and its disappearance rate (6) use method of application which will minimize contact with wildlife. Emulsions are more toxic to water environ ments than oil solutions or suspension;-.. Granules often concentrate the effect of the toxicant in one stratum. (7) no double-dosing (8) avoid main spring migration, nesting periods of birds, etc. when choosing time of treatment consider possi bility of using repoli.ants or scare devices to move mobile wildlife from area. (9) physical or biological control methods have advantageover chemical methods - more specific, reduce pests without loss of bnficia] population. (10) susceptibility to different pesticides varies greatly among animals 19 (11) DOT susceptlb11 1ty : 0.]. - 9 lbs./acre heavy kJ ].1 effect; on crustaceans and fish, moderate kill to amphibians and reptiles, some kill to birds, no effect on mammals until 5 lbs./acre level (12) table with toxiclties of other chemicals as compared to DDT: 2 },\}lj-T derivatives (DDT=1): rats LD 50 of 0 . 4 (single oral dose) bobwhites - 0.2 chr onic toxicity l d 50 (10- 0 0 1--1 0 m Q i-} pheasants - 0 . 1 n ii tf mallards 0.2 ii It L D 50 11 (13) bluegills - L D 50 of 0 . 1 (96 hr. test) iposal of pes ticid es always diffic ult proced ure unopened containers should be returned to wholesaler or sold; disposal of opened containers: - burned in a h o t flame (liquid organic chemicals) - addition of lime (wettable powders) - dumping into sanitary landfill-type dump, contain erized if soluble - empty containers - burned or dumped - do not clean equipment at creeks or waterways 0 v 0 9 0 Z00 . 20 16635 17 Dow 'Tiit* A n a l y s i s o f R i c e f o r Residue:. ; o f [ 2 , 5 , 5 - T ]" V 2 5 / 5 ` (1) ingreclients of Do'./, 2 , , 5-- P !\-iin<"> ivpii k j11 p i* (2) d.iroetioris for use - ;l-8 weeks after emergence of rice (7-9 weeks when flooded), treat with 1 - 2.5 pts. product in approx. 5-7 Gallons water per acre (= 0.5-1.25 lbs. 2, 1, 5-T acid) (3) with some weeds, up to 3 pts./acre may be necessary, but yield may be reduced (^1) treatment after flooding is safer than treatment before flooding (5) warning: do not apply to vegetables, flowers, grapes, fruit trees, ornamentals, cotton or other desirable plants which are sensitive to 2, 4, 5-T; do not permit spray mist to drift since even minute quantities of the spray may cause injury to them. (6) residues - methods for determination - extraction, chromatography, colorimetric determination, standard curve. Method capable of determining residues at level of 0.1 ppm.. (7) results indicate residues less than 0.1 ppm when applied at levels of 0.75 and 1.25 lbs./acre. (2 locations: Beaumont, Texas and South Dos Palos, Texas 21 1 6 6 3 S I t ' i O S O Z l ion 1 8 . Devi no , ,1.M ., Syracuse University Research Corporation Deport on 2,4 5-T Residues j.n Rough Rico and Rico Straw" A. P u r p o s e : to determine residues of 2,4,5-T in rough rice and rice straw Prom Arkansas, Louisiana and Texas (1) no detectable residues found in any rough rice sample (2) Louisiana straw samples contained 1.04 ppm residue after 0.75 and 1.5 lb./acre treatment;. 0.85 ppm found after a single 1-5 lb./acre treatment (3) Texas straw contained 0.18 ppin after double treatment (0.75 and 1.5 lb./acre), no detectable residue after single treatment (1.5 lb/acre) (4) Arkansas straw - detectable residues of 0.02 ppm 2,4,5-T only after single treatment (this level residue is close to limit of detection and may be due to background interferences) (5) no detectable residues of "bound" 2,4,5-T found (both double and single treatment) in rough rice from all 3 states (6) 1.5 lb 2,4,5-T/acre mistakenly applied over a Silvex treatment; analysis revealed 0.02 ppm 2,4,5-T. (7) average recovery of 2,4,5-T from rice and straw was 84+10.52 B. Supplements, e t c . (1) procedure for free chlorophenoxy acids (2) procedure for bound chlorophenoxy acids (3) appendix: Arkansas - rice seeded 5/6/69, treated 6/16 and 7 / H harvested 10/3-6 Louisiana - rice seeded 5/12/69, treated 6/19 and 7/ harvested 8/28 Texas - rice emerged 5/4/69, treated 5/28 and 6/23, harvested 8/15 C. Letter from Woodbury to Dunn (both Hercules, Inc.) 6/3/69 re cooperators in phonoxy rice residue project, samples they arc using and approximate harvest dates. 22 EE'/OOOZIIOO D. Sugme:: Led Protoco l. - Plu-nox y Herb 1c ido Res 1chirr,_- Rice: - object iv(;: to obtain ramp] e:; of rou^li rice, straw, mill rice, rice hull:.; arid rice polish and bran of adequate size for residue analyse.*:.; - data to be used in supplementing a petition for establishing tolerances for 2, ^l-D, 2,/l,5-'f, silvex and MCPA in or on rice and milled fractions thereof. - general considerati o n : conditions for growing should conform to those typical of the area - r c e variety: should be typical of those used in the state. - formulations: amine salts of 2,^-D, 2,iJ,5-'f, low volatile ester of silvex, and MCPA to be used - tills form is most widely used and recommended - ra tes and timing of application: highest registered rates of application and shortest interval between applications and harvest consistentwith registration limitations should be used, to obtain highest residues. Single treatment, unless two applications are required. - Suggested treatments: (1) ',k, 5-T - 0.7b, followed by 1.5 lb./acre, 3-5 weeks after emergence (2) 2, ^1, 5-T - 1.5 lb./acre, just before panicle initiation (when internode length is l/2 in. or slightly less) - size of pl o t : V x 2 0 1 minimum, replicated *1 times, is suggested - rice products for analyses: about 10 lbs. straw and 20 l b s . rough (unrnilled) rice from each treatment will be required. E* Rice - USDA registered u s e s : 2 , 4 , 5-T: 1.25 lb./acre - tiller to boot and before flooding 0l-8 weeks after rice emerges) 1.5 lb./acre - apply after flooding (2-3 weeks) or 7-10 weeks after planting *23 16638 F . Rico Produci:loti and Aorcago Arkansas = 430,061 acres (24.899 of US production) California = 323,630 acres (2*1.12 of US production) Louisiana = 512,884 (24.269 of US production) Texas = 464 ,732 (23.99% of US production) CD o C3 CD CD Average US yield/acre 3,963 lb (5 year average) co G. Letter from Woodbury (Hercules) to Baker (Louisiana State Univ.), 3/11/69 re : rice residue project, enclosing copy of suggested protocol. Letters also sent to : -Flinchum (Texas A & M Univ., Beaumont, Texas) -Smith (USDA Rice Branch Experiment Station, Stuttgart, Arkansas) H. Letter from Flinchum to D e v i n e , 11/14/69, notifying of shipment of samples from project and listing treatments. Treatment. D (1.5 lb/Acre Silvex) mistakenly applied over Treatment B (0.75 + 1.5 lb/Acre Silvex). Each treatment replicated *1 times. I Letter from Smit.h to Woodbury , 11/25/69 , notifying of shipment of samples from project and listing treatments. J Letter from Baker to Woodbury , 11/24/69, notifying of shipment of samples from project and listing treatments. K. Letter from Flinc.hnn to Woodbury , 2/5/70, describing pro ject at Beaumont. 24 1 0 6 3 9 '01;he i* pesticides used besides 2^1-D, 2,J|,5-T, Si .1vex' and M C PA : Arasan, >I2-S, Aldrin; propanil Yield Data : -2,4,5-T (0.75 -2^1,5-T (1.5) no herbicide + 1.5) = = = 2830 lb/acre (lowest in grou 3031 lb/acre 3579 lb/acre (highest in gro 001/2060 ^35 25 1 S 6 4 0 H 0 9 0 Z H 00 i f t 19. D o w , effect of irrudiut.i on upon TCDD - 5/25/70: -1.02 rnf- T C D D , in 100 m 1. chloroform saturated with water, exposed for 2-1/2 hrs to UV licht (GE sunlamp) at 35C. -50 to 100# degraded into other compounds (not identified), "a dramatic chance" 26 1 6 6 4 1 20. Pou .(12/8/6*1, *1/13/70) "Sol ub 1 1.U,y of [TCDD] In various solventi: at 2DC. -least soluble In water -most soluble in 0-cIichloroboir,viio ''0902/100 / 27 /H)fa 00 r OOH 2 0 6 C H 3 8 USDA. Notice, NRC Pesticide Residues Coinnii1;t'.ee "Statement for Implementation of Report; on No Residue and Zero Tolerance" (approved by Orville Freeman, Sec'y, k/l/CG). (l) 11 recommendations of NRC committee, from June 1965 study and subsequent report: (a) "No residue" and "zero tolerance" concepts are scientifically and administratively untenable and should be abandoned. (b) "Negligible residue" or "permissible residue" registration suggested. (c) Registration for residue-producing pesticides only if negligible residue in line with FDA regulations. (d) "Negligible residue" figure should be published, as well as analytical method for determining residues. (e) If pesticide is known to be too hazardous for o r> UUUil uo C should be refused. (f) Responsibility for registration properly rests with USDA. (g) Schedule for transition to new procedure to be decided by USDA and HEW. (h) Manuals, mutual assistance encouraged. (i) Formal program for education in residue analysis is needed - agencies should cooperatively sponsor program. (j ) Expanded research program needed in this area. (2) After considering report, Agricultural Research Service and FDA agreed on certain general principles and procedures. (3) Federal Food, Drug and Cosmetic Act: any pesticide in or on food shall bo deemed unsafe unless a "tolerance" lias been proscribed. (^1) Both agencies agree in establishing finite tolerances concept. 28 16643 (5 ) Re:; l.dui'--'omi.i ng pestle .tie:; shall not he regis lered under F'IFKA mile:;:; a l'.Ln.i le residue Level is provided for by ,D,l.C Act. 6M)90ZfJOQ (6) ' "Tolerances should bo established on Die bao jo of data in petltiono presented by proponents to establish that ouch uses will be safe". (7) "Agricultural uses of pesticides for which it can be concluded there is no reasonable expectation of any residues on the food will be considered as non food uses and can continue to be registered in the absence of a finite tolerance" (until or unless residues are discovered later on). (8) For tolerance purposes, chronic feeding studies in 2 species of animals and reproduction studies re quired (unless only negligible levels are involved, when 90 day feeding studies on 2 species may be sufficient to provisional tolerance). (9) "No procedure or formula is to be employed which will serve to override scientific judgement based on ade quate safety data". (10) "Prompt action will be taken on petitions for toler ances, for negligible residues of such pesticide's". (11) All petitions should supply an analytical method. (12) "It is reasonable to expect that uses of persistent pesticides on crops or in soil in which crops to be grown may result in residues on the crop at harvest". (13) "Zero tolerance" or "no tolerance" registrations should be discontinued as of 12/31/67 unless finite tolerances established or in process of being es tablished; in no case should former registrations extend past 12/31/70. (1*0 Both agencies (FDA and USDA) can receive ano process petitions. (15) Comparable problem with inadvertent and unavoidable residues in foods which needs resolution. (16) Public Health Service of HEW is available for consul tation and correlating human experience wish animal experience. 29 DOH2060U 0 22. Statement, by Julius E. Johnson (Dow) before Senate Subcommittee on Energy, National Resources, and the Environment, *1/15/70. (1) 10/29/69 announcement by Dr. Dubridge (Office of Science and Technology) referring to birth defects observed in Bionetics test. This pre ceded final report of Panel on Teratology to Mrak Commission (Commission on Pesticides and their Relationship to Environment and Health, appointed 5/69 by Secretary of H E W ). Commission members had not seen Bionetics report at that time. (2) 2,4,5-T sample in Bionetics tests came from Diamond Alkali Co. (no longer makes 2,*I,5-T); 2 > j5-tr/ichlorophenol came from Coleman Mathison Bell, via McKesson Robbins, via Dow. Quality of 2,*1,5-T is related to quality of its inter mediate 2, *1,5-trichlorophenol; possibility of a highly toxic impurity being formed in 2 ,*1,5trichlorophenol as a side reaction under con ditions of elevated processing temperatures^ Most sensitive toxic reaction observed in humans - chloracne, mostly prevalent on face, neck and back. Impurity not formed during manu facture of Dow 2,*1,5-T from the trichlorophenol, nor does it form on storage even at high tem peratures. Impurities concentrated in waste oils from 2, *1,5-trichlorophenol process. (3) Early control test - rabbit ear test for dermatitis. (*0 196*1 - Some Dow workmen developed chloracne; rabbit ear test shov/ed chloracne potential of waste oil from 2 ,*1,5-trichlorophenol process was building up to a danger point, caused by operating changes made to improve production capacity. Waste is routinely destroyed by incineration at high temperature. Exposure to waste oil caused acne in workmen. Plant shut down; it was discovered that principal offending impurity was TCDD (found by gas chromatography). Plant was redesigned to ensure minimal TCDD production. Since 30 16645 ' DOH208044I i 1966 Dow 2,4,5-T and 2, 4 ,5-trichloropheno.l have mot specificntion of less than 1 ppm (moat were less than 0.5 ppm) TCDD. (5) In Blonctics test, 2 ,4, 5-trichlorophehol showed r no significant increase of anomalies, but 2,4,5-! sample did display a significant increase of anomalies. Samples of Diamond Alkali 2,4,5-T contained TCDD up to levels of 16 ppm, according r to records. (6) Above information presented to Mr ale Commission on r 11/7/69. (7) Low wamr.r-lian toxicity of 2,4,5-T, and absence of reports of increased birth defects in cattle or r sheep grazing 2,4,5-T treated ranges caused Dov/ t o d o u b t Bionetics study's reliability. r (8) Tests performed at National Institute of Environ mental Health Science (NIEHS) labs at Research Triangle, North Carolina. Previously agreed to r' try to duplicate Bionetics study, using SpragueDowley rats (superior to mice tests) and using Dow 2,4,5-T of regular production grade. Dow i v/ould provide samples of 2,4,5-T and TCDD to NIEHS labs. (9) On 12/11/69, Dr. Courtney (NIEHS) provided sample r of 2,4,5-T used by Bionetics labs; examined at Dow and showed to cause positive rabbit ear re r action characteristic of the contaminant, and gas liquid chromatography indicated presence of 27+ 8 ppm TCDD. (10) 1/12/70' report to HEW showed that Dow 2,4,5-T of regular production grade did not cause birth defects in rats as determined by gross examina tion of fetuses. Dosage levels used were selected in consultation with Di*s. Falk, Courtney and Gaylo of NIEHS. Pilot study with pregnant rabbits also showed 2,4,5-T had not caused birth defects. Later 16646 31 DOH2 0 6 0 U 2 microncopie oxami nation of tissues and Skeltons confirmed preliminary findings of no causality. (11) TCDD teratology study in rats showed high level of maternal and fetal toxicity; concluded that presence of TCDD in Bionetics sample could well have accounted for observations reported and at tributed to 2,I,5-T. (12) Dow physicians have made in-depth evaluation of health of 130 male employees exposed to 2,),5-T in manufacturing operations, for from 6 months to approximately 20 years; 50 clinical parameters evaluated; control population = ^,600 individuals for whom similar data were available. Conclusion: no evidence of adverse effects. (13) Dow's belief : 2,4,5-T with less than 1 ppm TCDD presents no practical hazard when used in accordance with good agricultural practices. (10 Industry Task Force on Phenoxy Herbicide Toler ances established by National Agricultural Chemica Association, pooled information, submitted petitio for negligible residue tolerances of 2,(,5-T in Dec. *67; in April of 1963, FDA said more informa tion needed on metabolism and residues. (15) 2,^,5-T originally registered under-"no residue" tolerance; on 1/13/66 t Secy's of USDA and HEVJ changed policy to finite residue tolerances. Sine Task Force data v/ould not be ready before Jan. 1, 1970 deadline for new registration, the Task Force withdrew its petition without prejudice to future filing, ,and requested extension of 2,J,5-T regis trations since o imminent hazard from continued use was anticipated. In response, USDA extended registrations to 1/1/71; Industry Task Force to refile before 1/1/71. 32 16647 OOW2060443 B "The Response of Rabbit Skin to Compound:: Reported to Have Caused Acneform Hennatitis". Adams, Irish, Spencer and Rowe (Dow), from Jan. 19^1 issue of Industrial Medic:i ne . (1) Materials have been applied to inner surface of the ear of albino rabbits and to the shaven belly both undiluted materials and solutions of various concentrations in olive oil, paraffin oil U.3.P., propylene glycol, ethanol and water. Applications made once a day, 5 days a week, for 4 weeks or until a marked reaction resulted. (2) Some of strongest irritants produce a rapid des truction of the tissue (necrosis) without the skin having an opportunity to show an active response; milder irritants have some effect upon the tissues Most irritants resulted in responses which tend to develop rapidly and to subside in a short time; response may include hyperemia, congestion, inflar:. mation, exfoliation, edema, blistering, sloughing, exudation, crustation, necrosis, induration, hair loss. One type of response has a latent reaction and a much more prolonged course - epithelial hyperplasia, with its resultant thickening of the skin, follicle enlargement and sequellae (varying degrees possible, from slight to severe). (3) Indications of a relationship between reaction ob served in rabbit and acneform dermatitis of man: r action in rabbit produced by 5 types of substances known to cause an acneform dematitis in man (chlorinated diphenyls, chlorinated naphthalenes, chlorinated diphenyloxides, crude chlorinated phenols, and petroleum oils). (4) Other reports: epithelial hyperplasia from varies oils, fats, paraffin, dyes; eczema from aniline and coal tar exposure (also wai'ts and acneform dermatitis (chloracne)). i 33 1Jkv w DOH 2060U 4 (5) ChJoracrie - visible response of skin to irritant, talcing form of (1) epithelial changes, (2) inflam matory and degenerative changes, and (3) regenera tlve processes, (in order of appearance). Dow: "Determination of (TCDD) in (2,4,5-T) by gasliquid Chromatography" 6/22/65 - Analytical Method. (1) Dioxin can be detected at 1 ppm level with a lowc> limit of 0.5 ppm possible at optimum operation conditions. (2) Accuracy of this method = + 5% or less. Abstract from "Teratogenic Study of (2,4,5-T) in the Rat", Emerson, Thompson, Gerbig and Robinson, Dow (presented 3/17/70). (1) Study to determine embryotoxicity or teratogenicit of 2,^,5-T containing less than 1 ppm TCDD, tested on Sprague-Dawley rats. : (2) 5 treatments groups, 25 females each (3) Given 1,3,6,12 or 2k mg/kg/day of compound via gavage in 0.25% HETHOCEL on days 6-15 of gestation Control group (50 females) received vehicle alone. (0 No clinical or gross pathologic signs of adverse chemical effect were observed in treated dams; no teratogenic or embryotoxic effects. (5) Results of study failed to substantiate Bionetics Study. Abstract from "Teratogenic Study of (TCDD) in the Rat", Sparschu, Dun n , and Rowe, Dow (presented 3/17/70). (1) Study to determine if TCDD impurity could account for fetal abnormalities in test animals in Bionetic tests. 3'I 16649 OOW2060445.. I r (2) Dioxin Given by navaga in 9:1 corn oil . acetone solution in doses of 0(control), 0 .0 3 , 0.125, 0.9, 2.0, and 8.0 microgrums/kg/day to group:: of 2*1 (control) and 12 (treatment) preg nant Spraguc-Dnwley rats on days 6-15 of gestatio dams sacrificed on day 20. r (3) No differences in fetuses from control and 0.03 micrograms group. f* (*1) 0.125 microgram group showed slight decrease in average weight and intestinal hemorrhage (18/127) and subcutaneous edema (22/80) in fetuses. t"* (5) 0.5 microgram level - number of fetuses reduced, number of resorptions and fetal deaths increased; average 'weight of viable fetuses slightly decrease intestinal hemorrha^ : (36/99) and subcutaneous ede (31/65). (6) 2.0 microgram level - only 7 viable fetuses, from *1 of 11 litters, numerous resorptions, intestinal hemorrhage in ^ of the 7; subcutaneous edema in k ; 1 fetus had kinked tall and 2 misshapen feet. (7) 8.0 microgram level - t o x i c 'to darns; no viable fetuses; all resorptions were early and no fetal tissue found. (8) Delayed ossification of some sternebrae and skull bones occurred throughout various groups, includ ing controls - not considered to be of practical significance. (9) Results indicate high level of maternal and fetal toxicity to be associated with TCDD; could account for Bionetics observations attributed to 2,*I,5-T. f 35 DOH 2O 6O 44 6 23. USDA notice to Hnnufacturcrs, Formulators, Distributors, a n d Registrants of Economic Poisons re "extension of cert no residue' and 'zero tolerance' registrations beyond Dec. 31, 1969" (dated April 9, 1970). (1) Lists were extended until Jan. 1, 1971. (2) 2,4,5-T uses on McIntosh apples, low bush blue berries, grains (cereal-undesignated) , pastures (grasses), rangeland clearances, rice and sugar cane - extended by this notice. (3) Signed by Harold G. Alford, Ass't. Dir. for Registration. `36 1 6 6 5 1 OOW2Q6Q44 7 2k. Dow,.. "Product Specification for 2, *1,'-'j"'} 3/ 27/6 9 , released for use by USDA etc. by G. L, Lynn. (1) Lists TCDD to be at a non-detout.nblc level, whether produced by Dow itself or puroli;K-,cd by Dow for resale, etc. r r r r r r r t r f r 37 f C -.rf sv> r* 8V090ZMOQ 25. Dow, "Sales Specification for 2,'1,5-T", 2/13/70 (1) Shows TCDD to bo lees than 1 ppm. 30 DOM2060449 26. Dow y "Analytical Method for quality testing of 2, >1,5 - T " , giving procedures for determining assay and freezing poir 6/5/65. jo osmoztioo 2 7 . Dow, ."Analytical Molhod for determination of TCDD In 2> 4 ,5-T by car.-liquid Chromatography", 6/22/6j`. 5^ V* END OP VOLUME TWO .)(> I>r. IIv i :i : i .v . I understand. il is .simply ;i matter of (11<` care :nul sophist ir:ifion of IIn* method, llie time, reipiiml to got. the job done. AVo have built, Dr. Huyley has pointed out, in isolation laboratory-- 1 say built., I change that to equipped in isolation laboratory. We. Imvo. tho scientists. Wo. are, in fact, ready to proceed. Tho time, therefore., would ho the. time 0 0 retpiired to do the ana lyses and verify them. CO Air. Hick w i t . Tluml; you very much. CQ Senator J I a k t . (ionl lemon, thank you. It lias been an interesting mil in format ivo. morning. Congratulations again for the. effort that, yon, I am sure, put into developing and tho.ii persuading depart mental acceptance of tho suggested amendments. I hope improve ment- in that, basic, law soon will be written. Dr. H.wi.r.v. Thank yon, Mr. Chairman. Senator I I a u t. Wo. adjourn, to resume, tomorrow at II n.m. in tho morning in the heaving room of the Committee on Commerce, 5110. (Whereupon, at. 12 40 p.m., the hearing recessed to reconvene at 11 n.m. on Thursday, Juno 18,1070.) ; ncocci co co 7-- t EFFECTS OF 2,4,5-T ANI) RELATED HERBICIDES ON MAN ANI) THE ENVIRONMENT THURSDAY, JUNE 18, 1070 U.S. S knatk, ( ,'oMMI17KK OS (!(ISI MKIK'li, S t J I t C O M M ITIT.K O.V l'NKISOY, N a TIMI.M, Kl:S< lUIKT.S, ANI 'INK KnVIHKNMKNT, W a sh in g to n , D .C . The. hiiI init(*( 1111*1 , pursuant. to adjournment, ut 11 :'i> a.ni. in ninni lio, New Sennic. Olliro Umldiii", Hon. IMiilip A. Hurt, (chairman <>f lln* siihrummitlee) presiding. I 'resent : Senni o r 11u r i . Sennini- Il \iir. Tlie r n m m i l l c e will I0. in o l d e r . I.el un- :i11en| >1 lo |lulouize | o (ho wit nesses w h o have, lieeii i n runs eiiiele ed b \ tliis .".i i ni nnl e d e la y. A m e r l i n ^ w as r ai le d ycsleri|;i \ nf (In- I >111 ' u-III t i< r:i liens fur III a.III. fi lid I fell eum|i(-|]eil In |,iii ;. ;|i Mi-. I ssi h sse ruiild ini n:i*ri* t i l i ngs a Iit t If inure r es pons i bl y in ilei in i e. a . I.ii \ a ( 'ili_rl is ninnili:.' el e r y l m d y ri se's Inisiiie-.s. T .r |'|i ' ss :I*ii- luilas i-. I!-e di Iniqui lied --rienee advisor tu Ilit* I '| i eli-nl. 1*r, I mi Iii el I1'-. statement or dii. i.r.r. a. dhIiridoe. science advisor to the TREIWDENT and director, oi-fice of science and tech- NOI.Of V -. ACCOMl'ANIEI) DY DR. EDWARD J. BURGER, JR., TECHNICAL ASSISTANT inn lr. Iti limimi:. .Mr. ( `l ia i r m a n , I lune. ashed m y assuri al e, l>r. l i m b e r , nl' my udire, tu a r r u m p a n y me. Ile. is a n M. l ) . win lias lieeu followin'.' llie mail ers relaled to health and the oilier ell'eels of JX-- 1 r i d e s . Mr. ( 'li-.Mi-mnn. I have, testified he.fore l.liis eommiltee nn April l.r> on Ihe `J.l.n T subject, and I am not .sure there is very much to add In whai I said at Ihat-1ime, hut. there aro u few poinls I would lils-e lo review and emphasize. I reviewed Ihen sonici bin' nhont the history and development and value o f t he use o f Ihis lierhieide arid I used t h a t r evi e w as a P-xf f r o m whirli to d r a w w h a t T c o ns i d er ed to he s ome impnrl ni i l ji-iu-ralizalinns almut pesi ieides. and Illese are some of (he m a il e rs I would lik-e lo repeat . Eel. me, lieyin hv pointing out Mint `Jyl.fi-T is a pesi iridai chemical whieli has lienn ini rod need intent iomilly into man's surroundings beenuse of Ilie lit*IlffiIs presumed to follow. Tls purpose was In s e n e ns an adjunct to oilier means of weed and brush contr i land and (57) 5S waterway iiikI aprirull ural manapemenl. Over a period of 20 years il lias proved ifs utility so tlmt we, uru now in 1 position of relative dependence on this material. Ilowuvcr, especially in recent months, we have. bepun to ipiestion in preater and preater depth the. possible human health ellects of pesticides like. 2,l,f>-T and this lias required a preater so|)histieatiou iu research and lest up than was previously thouphl. adequate. The. example, of course, was the Bionetirs study for the National ('aneer Institute. Previous research oil 2.l..ri-T had concentrated on the acute, toxicity of that compound and had shown this to he of a low level. The Phonetics study represented n departure in that it invesfipaled the potential of the herbicide to provoke tumors, birth defects and penetie. alteration in appropriately exposed experimental animals. J.l.o-T cmerped from this study as a. possible, tcrafopenic apeut. As I have, said, this study was n departure in several ways. Whereas nearly all of the backpround toxieolopy on pesticides had been performed as part, of the development process by the devclopinp company or industry, this study was launched and paid for by the ( iovernment. I hinted that this miphl represent a precedent. If our society de mands a very liiph level of sophistication in this type of research, industry may not lie able to athird the increased cost, of develop ment. and further development, of valuable new products may be diseouraped or prevented, lienee I snppeslcd that, new ways of disIrilmlinp the costs of this work may have to he found. ICxpendittires of public funds and (iovernment participation in this research may he desirable. I emphasized that. at. any point, in lime, we liml it. dillicult. to pet complete, information about, the true hazards of any pesticide or any other chemical substance. 'I'llat. is, research in this area (as in any oilier) has no finite*, end points, ft may take lonp experiments wifii all kinds of levels and all kinds of circumstances to make any such assertion and one can never he sure. what, new research results will turn out. As one performs more research to investipato various hypotheses, one inevitably raises additional questions--as well as answers. Tt follows from this that any repula lory system for pesticides must be able to accommodate new and unexpected information. I pointed out that, our present arranpement for reputation is not sullieienlly flexible, to relied, new information ns it emanates from research. Apain, these, mints were dearly illustrated by the case of LU.r.-T. What T said in April was that there, does not exiut. a mechanism whereby the (Iovernment may exercise prudent and unequivocally elVcrtivo restraint temporarily on the receipt, of new, unexpected in formation and possibly preliminary results and while, awaitinp more definitive, conclusions. In many wavs the. Federal (Iovernment. did net with dispatch in the case of 2.t,.r>-T. After the October sit) announcement about restric tions imposed on 2,l.r>-T additional research studies were bepun in a m ?r of apencies. These studies were initiated both by the C!ov6. ..ment and by industry. The aim in every case was to confirm r** 5<J I D nr extend llm unexpected results obtained I'rnm >lit- Hinnrl>0- Nat iomil ( ':iiii'cr Insl it nil*- si udies. On of lliu now issues examined in lie new set of invest igal .ou.i was Ilie. imporlnnee. of impurit ies present, in many samples nl 2,4,.r>-T. If' bad been diseovered that over a period of years commer cial i,t,r>-rr contained varying amounts of a highly toxic impurity which was a me.mlier of a family of polychlorinated dioxins. It was of olivious imporlaiicu to ascertain llm relat ive coni rihul ions ol llm `2,l,ii-T and tlm dioxin impurity as potential teratogenic, agents. The dioxin was laiowu to he very toxic. Hence this (pieslion hecame part of llm experimental aim. Fortunately, tcrnlogenesis is a relatively acute allair and experi ments necessary to investigale this phenomenon are short-term experiments. Answers were expected in a fairly short period of lime. Some of these, confirmatory experiments were undertaken by one of tlm National Institutes of Health--the. National Institute of Environmental Health Sciences. The results of these experiments were reported to yon as fresh out, of t.lm laboratory at the lime of the. last, hearings. In brief, you may recall, these, results implicated both `2.l.f>-T and '2,."i,7.H-lelrachlorodibenzo-p-dioxin as potentially teratogenic in nature. In rats, over the same dose range, only llm dioxin appeared to produce birth defects. It. was principally on the basis of these results that Secretary Hardin, Secretary Finch and Secretary Ilickel jointly announced the. series of restrictions on llm use of `2,-l.fi-T. These, were related to you by the. Surgeon (icneral, Dr. Steinfeld. In brief, the philosophy behind these restrictions was Imped for protection of women of childbearing ape. Thus the Department of A i.'iicnll ure suspended the rcgM rat ion of liipiid formulations of the "icdkilli.r for uses around the home and of all formulations for use on l a k e . . ponds and ditch banks. In aI<Iition, resist rat ions were cancelled for uses of nonli<|iiid forinulai ions around the home and of all formulations for use on food crop- intended lor publii.... n- umpl ioii. Dl Ilie total amount-, of -J.|..iT n-I in this comil rv for all pur poses it was e-limalei| that these restrictions applied to about l2U percent -the `2U percent, of the cases where human exposure was possible. i (irmly believe, that, the issues raised by the ease, historv which I outlined in April continue, to be prominent. In a way, I suppose, wo can thank the existence of the. ipicsl inning about `2,-l..ri-T for bringing to our attention matters such as the ones I have described. This study has served ns a most, useful vehicle and we mav learn some, lessons for fuluro studies. However, as I warned, any attempt to answer the. research questions raised will inevitably raise some additional questions. I suggested that in some wavs we. were fairly lucky in our investigntinns of iM.ibT. The issues have appeared fairly straight forward and it was possible, to start confirmatory experiments fairly quickie and to pet confirmatory results quickly. Yet. while this appenrs to have boon a modest sure story, some may rightly ask: Shouldn't the kinds of experiments which were *"* ii nii (0 mobilized on (lie .spur of the. moment, for 2,1,5-T luive. been ace.omjdislied on :t more systematic lmsis mul without, (lie spirit of n crisis neeess.'iry to urge. tliem on '{ Further. one, might isle whether or tint it might bp, desirable to .support a J'airly sophisticated level of investigation for a large number of post ieidal chemicals--not, just. 2,l,r>-T. Now, I and others have, outlined the research work on 2,'l,f>-T and I have, termed it- relatively sophisticated. Yet. 1 will have to admit that there has hee.n almost, no work done to elucidate the metabolic handling of this herbicide in the animal organism. There is little known in biochemical terms of the mechanism oi its actions and there is essentially no knowledge of any possible interactions between this chemical and oilier nialerinls. In similar fashion we are poorly informed about the character istics of the dose-response relal.unship for very low dose levels. This, of course, is the, situation which wo lace, in real life in the ease of a variety of environmental agents-- including pesticide residues. Hero the problem is a statistical 011c. Jn order to derive meaningful answers with any useful level of confidence very large colonies of ex perimental animals must be tested, often over a long period of time.. T suggest these, comments to illustrate that there are, various levels of sophistical ion in research. Til the realm of pesticides the level of our research activities may not. have kept, up with tIn* slate of that art. nor with a corresponding level of ipicslinning to which policy makers ami the public are now seeking answers. The very excellent report on research needs compiled hy an advi sory task force to the National Insiitule of Knvironmcntal Health Sciences outlined these research areas very well and very explicitly. This report. I am informed, is just now being published. All of this --more sophisticated research, more expensive, research, research sponsored by the ( <n\eminent--will cost, money. Again. I repeal, if a really serious thrust is taken in this direction we may bo obligated to (ie.d new institutional avenues for accommodating this research since, as a part of the cost of development, the bill to industry may be higher tInin we might desire. All of this discussion brings me once again to a point, which I maile in my previous testimony and which I feel is worth emphasiz ing. While we as a society have recently begun to ask more jienel rat ing ipiesiions iihonl the possible adverse health ell'ccls of environ mental agents it is not clear that we know how penetrating this q i i r s l inning should lie or must. be. Wlial I said before was that we had set our sights higher. AVhat I should add is that wo are not sure, lmw high they should be set. For example, up to the present, time we have hern willing to live with a system under which the amount of toxicological research performed on a pesticide was to some, extent related to the. probabil ity of lnnnan exposure. With a low or seemingly negligible probabil ity of exposure, relatively little understanding was sought, and little research was underlaken. In fact, one could argue that, since the appearance of residues of `2.-l,ri-T have been rare events--it. is very rare to (ind measurable residues of 2,'l.o-T on food--therefore, ono > R0PQRR1 I 01 00 dhl mil. have lo know Inn much about Ilu*. Uiximlngy nf the In-ihi- ride. Now wo are more, particular. f l feel Unit wo should li morn explicit about assumptions such as CD these. J f limy mo valid they will stand on tlm.ir own merit.. If tlm.y arc not valid wo should change them. I am happy to say that my nIVico is examining questions such ns these at tho present time. Thank yon, Senator Hart. Senator ] Iaiit. Thank yon. You suggest, the possibility that the Government .m ay have to nssumo a greater role in any testing area but specifically in (ho matter of pesticides. Dr. DuHuinm-:. Yes. Senator ILurr. You indicate that, otherwise, the hill to industry might he higher than we. might, desire, which 1 suppose is another wav of saying an industry could not afford it. I low should we read that? Dr. Dtilhiioor,. "What J meant to snv is if industry is required to carry on years of very expensive research before anv new product ran he manufactured, ohviousiv industry will no longer he interested in manufacturing new products because, they could not. recover the loss. Thus the community, the society would he, robbed of many future very valuable chemicals which society might liml extremely important.'for health nml oilier reasons. To impose the harden on a particular company that heTorn it can market, a product, it must, undertake millions and millions of dollars more worth of research than it lias in the past would simply slop the development, of new products. Therefore it seems to me only fair, since, we. want to protect society as a whole hut. also to encourage benefits to society, (hat. soi ii-iy is a whole, ought to participate ill the cost of determining u!isi ihc ilamages may bo as well iih what the benefits may he. Si n i i o r 11 \ i:t . That then would he your basic, answer to the in.ii nr argument that research is just another element of the, - - f P I Mi l 11 ill. in-. In I-1'rI,, i . Vi 'I,in. li : i. I ! IlI'l||ir Willi Iil!,irill I r o f t h e product .'Imilld b e a r that cost. Dr. D rl 'ii:ii-<.i. V r -. I l.vacl | \ . I a m i m i s a y i n g ( h a i ( h e . i i i a u u f a e - t n r e r s l i n i l l ' l lint :i| n l.i-ar . l a M i n i i -m -I-.-. | J e s h n i i h l i i i a h r s i i l o t h a t t h e | n-i ii 11|i-| I h i t h i ' i i ].i | i l i ; ' I n i n a l i . i - l i s i m i d a l i g r r n l i s l V I m i Iiliull--, l i l i e s I mi | i :i \ e n h i n o n d \ i - r " i > )>11111:i 11 l i e a l l h e l l e e l s . 11ii(i I r v : h u i i l d h e n - < p i i I I n m i d i - r i a l , e a r e a ^ n i i a l i l e sol. o f e x p e r i - m i - i i l - . a m i l l i e y a l w . - n - - In. t u ir-' i u v l l i a t i b i s p r u i l i i i - l h a . s a e i a - l i ' r | v g o in i s a f e l y hn-lnr. l'.ul il m a v labi- y c a r s In tio d olii l o w li'vol a m i ea sily liidden d a n i/e rs w h ic h sninel iim-s m a v h ee n m e n ln i- oiis only w l i e n m a s s i r e is i m d e r t n k c i i . Tu l i e l p n v o i d Ihese. d a n g e r s T lliillk so m e l'YJeral pal i ieipal u m in Ilio resean-li p r o g r a m w m ild he desiralde. Senalor 1 .M.-r. Do voli know whothor th ndminisl rat ino inlends to einhark on hroad new researeli ]imgrams in ibis area? Dr. 7)trIif.'fif;i-;. Tliere aro sevorrei agcneies which aro develoiiing plans, pursninir rescarch in Ibis nron, and T helie.ve an advisorv commi (tee. of tho Department, of IIK W lieaded hy T IOuiil Mralc I M A Ne 'io*l in 02 is making some proposals to J i K W about substniil.iitlly extending ith research ami testing ncl.ivilies in this field of pesticides, nnd about, developin'' extensive facilities, hire; animal colonies and lnrgoscale lestin' equipment ami personnel, to carry out extended tests in this hold. These arc recommendations that aro bein'' formulated and nro bein',' proposed to lliCW. I do not know what the status of them is at the moment. Senator J I akt. It is likely you would not have the figure with you, hut. Jet me ask the question. Perhaps it could bo provided for the record arid I have no idea wliat it will show. Hut would it lie possible to identify llmso activities which am not undertaken by the Dopnrt.ment of Defense that are comparable to the line item research and dvelopiucnt that is done by the Department of Defense? Tbo total fipure for research by Hie Department of Defe.nse is in the range, of $7 billion a. year, flow much are we spending in other research? Dr. Dnl' amni:. The total Federal budget for research and develop ment in Ibis current year is close to $ 17 billion. Senalor IIaut. Including(lie. DOD? Dr. I)i;I Including Ilie DOD. I f you lake, out somewhat, over $7 billion of DOD funds, it is $!> billion lo $1(1 billion in all other branches of Ilie ( invcniiornl. Senalor I I .m it. We will lei olliers judge whether (lie allocation o f (lie resources is or is noi prudenl. I think il is good lo have it in the record. Yesterday we recriwil h- Iimoiiy from llie Department of Agri- cull lire. When we lliink ab'iiil chaiiL'fS in Ilie system of pesticide research and control ivc have In lliiok ahoni changes in the. basic, law of pesticide regulations. Included in Ilie tr-limniiv yesterday from the Department of Agriculture Mere a number of suggestions and changes that they recommend lie. made, in the. basic ad. Are you familiar with those suggest inns? Dr. D i;l>i:11><:i:. Dr. Jturge.r told me. about them this morning and T have a. copy of them here, and we. have, discussed them. Those, aro in line with some, of the. suggestions wo have bad with Agriculture and other agencies; namely, that, there is not suDieie.nt flexibility in Hie present, statute to lake, a suitable action in all cases wbo.ro now in formal ion becomes a va iIahie. Sometimes new in formation like Hie Hinnetics study is very sug gestive but cannot be. said to be finally conclusive because of the small number of animals and small number of circumstances in volved and Hie separation of an impurity may not, be. taken care, of. It will frequently happen that y o u will gel. preliminary sug gestive rosnlls not sufficient- to abolish the. use of a chemical but suliicient to take, some prudent action until more iinnl results have been ohlained. I lliink Hie Agriculture suggestions do movo in this direction to give, for example, wliat they call preliminary suspension authority. So if a danger sign is raised yon take, prudent temporary action and eonliimo further research. Tf the, further research confirms that safety is there after all you can remove the. suspension. I f further rosea1 proves the danger, then perm anent suspension can be aehipv 05 in (Vi Cjp CO Senator J I \l(T. I 11i(1 nol. <|ii:iI'l'cl yesterday with tin1 I) j>:i1 1111*-1.1 Agricnll lire's interpret ;t( ion of 1lio basic n d . Overnight. I haw 11 nd. ns wo always do, to justify wlmt wo did, namely, those of u- who wrote the low. 1 inn not convinced Mint. Mint statute prohibits the Department of Agriculture, wlicn, ns you sny, the signal goes up from suspending the marketing of tho produel.--I in not sure Mint ('ongress should lie. held to have said Mutt wo excluded the possibility of a lomporury suspension. .fust ns I wus not yesterday, 1 am not sure you are not equipped, nnd did not plan to debate wlmt limits there are. under tho exist ing law. J)r. Di/l'.iuiinr.. No, I nm not nn expert on tho question of interpretation of (lie. law, but it has simply been taken for g r a n t e d , maybe, not properly nnd mnybo under mi interpretation Mint should be further developed, but it has been taken for granted that pre liminary or temporary' suspension wns not provided for, explicitly at least, in tlio statute. I f the law wore interpreted to allow this, it would be fine. Senator IIaiit. I cannot imagine there would lie any different criteria for a temporary suspension than a suspension. 1 still have tho feeling that the suspension is nol. for a thousand years. If you suspend nnd then discover that your alarms were, groundless, surely you can unsnspend. That would argue that you can temporarily suspend. l)r. IJnlbimci'. Tf that is the. case l think' that is fine. Tt apparently needs to lie made more explicit to the people, who are. doing the sus pension because, (.hey do not feel that, they have this authority. Senator I Taut. Clearly they do nol. A re. you yet. in n position to advise, whether you would support or rci oniinend support, of the suggestions for law changes mndo yeslcrd iv I.v Agric.ull.iiro? Dr. In r.innoi'.. I think it is fair to say that, we would believe. 1 ' ! :"'e proper moves. Again, ns T say, we. are not, experts in 1j " 1 --i-.' rv iii-M nid the legal field. "What wo tried to do is to find 1 ......... "id liilinology that is applicable, nnd wo leave it to * "O :"' aoi I l lie legal authorities to determine wlmt specific l:i! 11mi :11111 i 11 hii*. :i|*( re11|\n*(J, l*ot I lliiid. i 'll ; :i-1<111i11n I llc\ 1tiI1 y docs in principle sound uu-v desu alile lo u,:, simple t.< 111 - ii'-ivirrli results, you know, are iic\er the final answer null- ' Ihev are cxhomelv conclusive, resells of extreme, danger. It is almost never possible. In . :,v ||,:li ,,11 the research lias been done and it, proves (hat the. thing is f.,re\er safe. Senator IbMrr. Conversely, forever unsafe. There wer0 several other suggestions (lint. Agriculture made. One regarded (ho ineffectiveness of handling pesticide regulation by Inlieling. They were going to recommend rcsl nirl uring^l fie. law to require a grading by degree of tho hazards oT a pesticide and to ensure Mint, extremely hazardous products would lie permitted to he handled only hy individuals or institutions licensed to do so I)o von liavo any comment on that one? Dr. DuBninnrc. I think I have no very expert o.< tent except, to noto tho experience within my own family that labels on packages it nn 04 iiv oftcm not, adequately read and flint the labeling problem is a ditlirult inic. Sena to r ll.wrr. As Agriculture, indicated, not iv very reliable hnndlo for protection ngainst injury lo health or environment. Well, wo, would hope that yon will loud yonr distinguished scicn- tilir. support lo the recommendations that Agriculture lias made. A couple of other questions, and this joes to tho different notions that have been taken with respect to pesticides or herbicides by dif ferent departments of the Government. Agricull nro suspended I think you said about 20 percent of tbo use of L\l.r>-T. Dr. Dulhunm:. T did not intend to imply that was all Agriculture. I said the total suspension amounted to 20 permit of the total use. Senator ]I.\kt. It is my understanding that, the Department of Defense has suspended it ent irely for use in Vietnam. 1 )r. 1)(11ii:11x;k. That is correct.. Senator II.wit. And the Department of Interior very recently suspended it, for use on public lands; lands in ils ownership. I>r. Didhunni;. Yes. Senator ll.wrr. Ifow do wo explain the dill'crcnt reactions from tho several departments with re>ped lo the same, product? Dr. Did iiunci-:. I do not know that I ran fully explain it, hut tho sit imIion in military operations is not necessarily the situation in normal peaceful operat ions in this roiiiil rv. In military operations, by necessity, these chemicals are distributed by aircraft, and it is not always easy to control where they go and bow far away they blow or even that the aiivrafi is hilling the. right, target. 'Therefore T think somewhat more prudence may be required. When you are doing it in a normal way. in agricultural practice or land managemcul, you can be much mocc careful and make sure that Ihe material does not get. on food crops or in your waters, which will contaminate the waters, or get. on grazing lands on which ani mals will he grazing ami therefore, get. into milk or meal. Fortimalcly 2,l.ii-T degrades quite rapidly. If is not like DDT. If Volt spray a past lire with 2.1 ..>T. essentially all (races of if are gone after about three months' exposure, to wind, rain, and sun. Therefore rattle can quite safely graze, on glazing hind treated with 2.1,o-T after this period. . Also, if it gets oil food it is likely |o degrade prelly rapidly, though it is desirable lo have no tolerance for food. ISul where, it is used in areas where there is no human population and where (hero is no ilanger of eoiilammal ing food or water, then it coni rolled use can he. extremely valuable, and sinco.no dangers would he resulting f think- if is perfectly proper to have coni rolled use in this country. Senator ll.wrr. 'What do you say, then, lo Inferior's prohibition for use on its lands? Dr. D i.ISiudoio. I guess I am not familiar with how oxfensivo Unit use is. Senator II,wit. I nm told that Interior's action was taken only yesterday. Dr. DirUmnr.n. I see. I am not familiar with the background for thut action or the e.xlcnt. of it; or maybe they found flint other mil- T T eafifii Icrials could 1x* used oh )mi1>Ii lands oilier limn i i , - l w h i c h would serve the purpose. I jnsl, do mil know. Smalm- II.m;t. I'.ut il. mnl lilmles lo (lie. uneasiness of t.lio pnlili.i wlieii we si'e these. srrmiii<dy ronllirl in" reactions. I)r. DoJbtmoi:. This is one of Ihe very ilelie.nlo thinps (lint (ho Government faces; Mint is, nol. lo nverreaet to situations which will do damage hy overrear.tion. lint. Ihern is an opposito injury of uniler reaction and not taking prompt, or adeonato ncf.ioii when dangers are evident. I would like to defer to t.lio De]uirtinont of tho Inferior in this ease, and with your permission, have (lint- Depnrtmonl. submit a statement Tor the record. ('I'he informal ion follows:) StATUMKNT t>N INTI'.'lllOll PliNTM'lltK 1'OUCY 'j*Iip 1>)>jin infill ftf* llm ftiiorlnr policy Ntulrinrut, ly Hrrrrl i\ry 111*l<<*l nn Jiif#* IN, tlll'/Vr DWirKwiliy frmii ilii' mu* llwil lui* bmi fullowril fnr HrViTHl yt'Mr*. Nrvcrl IiHcmn, ||. ||tIf|hHOIIH*rItrillIniN mIhmiI \\ hit'll wc ItfIvr 1`rrrl Veil uilillltiHinl 'lain wllhin Hit* hint, yrtir mill rrcm'iiP/.rH Hie cmitcrh cxprcNHctl by Coopui'ill Illtc I>i*|nrl mrtilmmi Huitir ulliri'H, rJ'lir Jiriilillil11oil Mf'nmst iiim.hI: of llir rlilnrl milnl livdrumrlmri lltvpri Irltlrs It.td h(`on In (MTccf for w'vrnil yrniN nml (hut mi IVI.fi-T kIikm! (Mnhcr JIM'!). Tim lui/.urds of mercury him* lirrn recuj'iilxi'tl fur smim Ihnr hill, writ* urrmitiiuted hy inonllorhur rrsiillM III llir l;>s( six or rl^lil ijhimIIin. Amlliol wiih included hr* Cinisr of llir ohjrrl Ions of llir Fond nml iH'iij' Adinlhisl nil loo (o (fir iihi* of CMl*rlMoi'rliM. The h;i:i 11mriit of Hip Inlrrior pulley Is H"| lolrndrd jim It Federal pesllridi! jmliry. II li:is liMitf Invlt (hr policy of IIiIm I>rprili mriil In m*| n standard III IlH use <d` prsljchlrs flint, is liryoud reproach from (hr fdomlpoiot of safely, Cmisr* ipHiilIy, our posINon may hr more sirirl Him moo*- iIi. i-; will wish lo m-i. If if-rarrli ilnnoiisl ni Irs llial sour* of Iin* pe*>iride-' II-led urty hr Ir-e: liaxardoiis ttinit \vr MliHprrl; Him lliry Jimy hr rrslurcd for Ur on Inhrior lutein. S e n a t o r JI.Mil'. Von r emi nded mi' of a <|ucOion I I,iehed m y s e l f yesterday for not having a-ked Ihe Department of Agrirulluro wit nesses. P e r h a p s you can help. A' <m liieiil ioneil l>l>T. A- I recall t h e l e si imo n y y e s i e n l a y , u n d e r llm K I K I t A Act. a M-ifin I* a d v i s o r y r o m n i i l l r r is iv l a bl ishe d when there is a raiuvllal ion proeedure aimed at a produel. Tliev exp la ine d t h a t in I lie rase of D D T t h a t some six month s have passed since, t he raiieellal imi proeedure. was i n i ti al ed a nd no e o m m i ll e es have hern f o r m e d : lienee th e p assa ge o f time Inis hcen e x t e n d e d a t l a st l>y t hi s a mo un t d u r i n g w hi ch , u n d e r t h e e.aneellal ion p r o c ed u r e s, continued marketing' o f the pro d u c t goes on. W h y the {-month d e lay ? Dr. Di lli.-inm:. T c a n n o t e x pl a i n t h a t . T do n o t k n o w w h v I hern should lie a long l ug between these, two events. T h e onl v t h i n g | r;ni t h i n k o f is since, (lie. Sccr i'i'tI'V s pesticide a d v i s o r y c om m i s s i o n ha s lieeu rout iumiusly at work on the pesticide problem, cspeeiallv with attention to DDT, they were depending on it to examine (his partieular prohlem. Senator H aiit. T repeat, I should have asked them yesterday and T did not. I' ut if they read so strictly Ihe sfatuto with respect to suspension, I would nssurno tho samo strict, rending would fell llmm that they ennnot substitute tho At rale Commission for t.h explicit statutory ro<|uiremont that Micro bo a fioioneo hoard estal ,ed for each of these products. GG Dr. DuHr.mim. I would fierce. I th in k maybe the law does not set itiii: a t which the soienco advisory commission .shall be established. Senator H a u t . N o ; it does not. I t was assumed th a t it would bo ablished a t least with nil deliberate speed, and G months seems to limine delay. :)r. Di/lliumm. Yes, I agree. W ith your permission, I would like ask the D epartm ent of A griculture to provido an explanation of s apparent delay. 'The inform ation follows:) T.i'i:A. Dulliiinni:, D kpartmknt or AonicoLTumi, Ol'TR'K o r t i i r Hkciiktaky, W a x h l n p t o n , 1). G, J u l y 7, rut Ir a S e c r e t a r y , C o u n c i l o n / E nvironm ental Q u a l i t y , E x e c u t i v e O ffic e o f the. 1'r c x h l c n t , I V a x I i l n y l o n , D .C . i:au J jh. JH jlliuiiar.: I n r e f e r e n c e l o 11m (pie.-dhiii r.'iiscd n t tlrn H a r t mil l l e e h e a r i n g n s t o w h y 1L 1ms t a k e n s o l u n g (o c s l u h l l x h n o a d v i s o r y m i l Leo o n I >1/1', w e s u b m i t llu* f o l l o w i n g ex pin mi I Ion ; () A fter the nmiounrcmciit. of ennei'llnlIon, the companies Involved did rapicst. nn ndvlsory eommlitee or public lien ring for the four uses of D PT e cii neclh'd uni II Ilie hi Ilev purl, of Ilie .'lii-iluy period provided for appeals lilt* Federal Jnseellehle, Fungicide, mid Uodeiitlelde Art. i) Meet lugs and discussions were held with represeulnliven of tho National deiii.v of Selenees on makeup of |ho committee. .) previously we lmd asked for lists of names of persons to serve on nd- ry eoiimilttees for oilier cnneellcd products, and they were already In the ess of compiling them. t) Ino to mifiivoruhln puhllelty related to connlet.-of-hilerest. ehnrces roIIiiu'niilhorllles Hint served ns consulImils lo Ilie Department In llu> punt, exports wre not willing loserve. ) Two ndditionnl recpiesls to NAS for candidates lo serve, besides tho lunl. were necessary In order to complete the enmiulllee. ') Two of three companies rciiuosllng ndvlsory coniiulllees withdrew, ing only one forn tracking powder use. i) (elLiiif; written posilion from DIIHW ns to whether trucking powder cotisldercd nn csscnllnl use from n public hcnllh standpoint, i) (liintnellng mid gelling approval of proposed (imlhlates to serve on tho inlllcc. ) Nidifying committee members that they were selected to serve on tho III 11I**. .) Cimlllcl-nf-lnlorost review nnd evaluation wllhln the TJfJDA. Sincerely, N u 1. llA vt.s.r, Director, Science anil Education. eiiulur il.Mir. T wmihl hnpn in cinmeelinn willi tin*. cancellation ....ling on ,l,`>-T an ailvisorv commit lee will he, formed with delay. his next, question bears directly on your broad background. Ono lie. diH'ienll.ies in Ibis `2,-l.ii-T slorv was Ihe dill'irnll.y experienced blaining information which might, have, he.cn of public health sig- anre. nder what conditions do you suggest scientific in formation ought ic kept secret when a question is raised as .to the. safety of the Inel ? m\ D i'l'itnicu. Tf yon nro talking about scie.nl.ilic information, i not. think it should ho kept secret. When yon are. talking about mint ion having to da with tho iniinu facture of commercial prod. ilnit is a very dilTercnt situation because tlio costs of dovolop- t, testing and g mg a commercial product into production are /s n o i l l i t M-fV high. Il is i|iiile proper 111:iI l In* 11 1111 f e Iin r or li < ...< ' r |,,. |i|iiiiTlri| so licit. Ini will h av e hi o|i| I mill y In ........i I'o.-ls. 111! should hall! 1 patent or IV |II-|.I.-.| II.II 11>r liH IIIi!it toll o|his product so licit. Ini can recover Ilie. very Isii'^r' eo.-.U to develop il. Whereas, if Ini inslanlly pulilislic.il nil llic. information about lcnv to in l,-ii llii.H product so that others woulil inslnnlly si ml. making il. without Ilie expense of development, this woulil obviously bo no unfair kind of competition. Our wholo system is bused upon tho fuct tlint inventors of new processes mid products have protection to refrain their investment and recover thoir costs. Therefore I think tho publication and tho dis tribution of information with regard to tho manufacture of products nml mnferinls is a proper trade secret. On tho other hand, when it is clear that human he.nllh is lit stnko I would assume there should ho mechanisms by which (Jnvoriimont agencies in proper authority could he told something nbout the com position of tho product so that they would he aide to determine whether or not l.hero might, bo materials in tho product which ought to bo investigated for their harm. I do not know exactly what the law is on this, but it would seem sensible* Senator ITart. Your suggestion is that when a ipicstion of health is raised Avit.lv respect to a product, data and information oiv tho product, should be. made available to the appropriate (lovernuu-nt agency for ils determination as to a question of health and safety? Dr. DuHmnnK. Yes, sir. Senator TIaut. That excludes, of necessity, tho judgment and tho cninmont of perhaps very gifted men nml Avomcn of science in ar riving at tho determination of whether public, health is or is not in j'"ip:udy. This is not to suggest that the appropriate agency lacks qualified anil competent peoplo hut surely they do not have a mon , |P"ly on that. I ihere sumo way, notwithstanding the obligation to protect, trade H''i'i ! and eucouriige invention and discovery, we ran do a belter jnli i.f pi-rmiiling the outsider, whether it. is the head of the chemi11iv lb |ri n 111**111. n|. ('i| Tech or someplace else, being brought, in and haiing an >.p| mil mi il y fo sharpen the judgment, nf evervbndv? Dr. Di l*.i:11.i:i. Well, I think that (lie various advisory mcehancans uvailahln in il,i> various fiovcrnmenl. agencies might in general to accomplish dial nlijccl ive. A science, advi.-'orv group can be called in to consult mi n |i.ail ini lar problem, on IIn*, possible dangers of tho particular chemiial.s iliat happen lo lie. in a particular commercial product. T nnv sure. tbal. Ibcv could t; [> Ilie rest, of the scientific, community to find out, Avhelher chemical A or chemical B is of a nature that it would likely ho harmful. They do not havo to reveal the wholo conposition of the product in order to say this product happens to con tain a certain amount of compound A, is there any evidence or nnv chance or any reason to believe that, this compound A is harm ful. T think tho knowledge of tho scientific community could bo ob tained. Senator TTaht. Should he obtained? Dr ^qBniDOB. And should bo, of course. O Hi Soimlor D.u.'r, Tlm sii^cslion Iiiih bruii jiikIo llmt niI of uh uh a pooido ivonld lu bol.fur oli' if ivo litui .some, conivalimi c.leurin/ihouso or dalli Iniuk iato ubid c.ouM lui fod till of (lui infonimtion not. uh- jot'f,fO friniti SflilvlH. Ulivo yt>ll /rivoli IIuy (IlOII/fld. I() flint. HII^/rOHlioli? Di'. IluKimitir,. I itmli'inlnini tlint, l)r. .Sluinfold ih /oiii/x lo dimmem (Imi. tjiifHloli wlion luiii|i|ioill'H. Soiinlor ll.wrr. Vonr mietimi ivonld not.stop liiin. Dr. DirMiiintir.. Ilo knoivs so moli inoro itimut it. I do not wnnt to nidioipufo il. II. is Ibis t|iiostion of Ilio dissoininntio of scioliiic inforinidio ivliirli is orni oT onr inosf. diU'indl. probloms. , In |>iis(. yonrs ivo uliviiys nssimiod tluit. if you piiblisliod in scion- tilio joiinmls nini Iioolvs llmt. unybody litui umiss to, niiyouo would just, /pi to flu Iilnirv nini look up wlmt lui iviinlod to knoiv. 'Mio volimi of soionliUo. infonimi io Iiiih Involuti /irmi, (lui iii/ituioy of liiIin/r piofos of soifiililio, iformili io tpiioklv Iiiih burmno /rout., nini (iis luts lod lo (lui ipio.st.io iis to ivhufbur olio cun or ouimot uso niotlorn soionl ilio oi|iiipioiil. to sloro miti ivirim o scionl ilio in- forin:dion moro o\podil iously. Tini only troulilo. is if, is vorv oxpoiisivo, miti Ilio doiolopinont of ft*t*liiitjiifs for putii il. in suil.'ililt' fori fur tisifu prooossin/r. tini tpiosfioll of litui' mmiv IIjcllcicH slioilltl 1)0. ilivolvotl 1 fottilill/r t.lio inforiii:i(io i unti lioiv lo off. 1 oii( filoso uro ooinplo.'i looli- imlijLM':tI proliloin.-t ivltioli inorilior nidi Ilio rxpoiiso Iiilvo not boon ivorkml otil. I iliink il isn vory ur^r-ciiI pniUrm. Tlioro uro pooplu in onr olfioo limi uro working mi (bis and I Jiopt' ivo. oun liuti ivavs lo liuvo. a sloraoo of iioodml loolmioul infor- liial itili, pai Iioniarlv in Ilio houli li loltl. Sonulor ll.urr. I.siluro in lliat./orlino. M y Itisi. t|oslio, uoain, is u /onorai tino. W o limi un o.\ulnui"o yoslortlay willi Ilio Dopurlmi'iit, of A/rrio.idluro wilnossos nn f.lio ulti probloni limi, is rroalod in Ilio inintls of sodio wlion you liuvo a tlo- purlmonl. limi, is olm rotai willi Ilio promol ion of un nulivity nuder- liikin/r liso lo ivoululo il- In Ibis o.aso, oxpandin/i a/rriciilllimi pro lliti'!imi is il l't'spousibilily of Ilio dopar)uuint, ami ut. tini smini limo ivo suy inuko suro (.Imi. borieidos uro not ponnillud llmt. do tlumu/ro. Do you liuvo any onorili rido us lo wlieilior promolionul mid l't*"uIti1u'_\' liiiirtioiissbolliti bo sopiiriiloti? Dr. DrP.miitn-.. I do noi. tbhik I xvoidd proposi il ooiirrid rido on (Imi. In il- pa riiridar raso of posiioitlos il is Irno limi A c'i'iciiIfure is involimi. I'.ni iiuder ilio i111<r;i^cncy aoirrinriil, 111:i!ili. falliraiio, nini Wrlfarr ami Inlorior ari joinllv tiif11 A /rimiliun on Iliesr. mioa- lions. If Iboro is imi a^rioiillm a Iprodiirl limi is i uso inni JIKW bus ifonimi io llmt llirro may In limllli rlIVrls fru Ibis, tlioy ran iiiiiiinlinlclv bri/: il lo ilio alloiilio of Aoriniliuro unti action un bo lakoii (boli hy jtiinf ii^ivvnioiil l i m i n o iboso liuvo tloparlinonts. I lliink (bis is mio purposo of litivili; ibis inlordepnrl monlal ar- raiiooinonl. I uni suro llmt. no Dopa rimoni, is unxions to promoto Fomolliinir llmt. Ims ilun/'or lo hmiiuii boallli, and iis suoli as human hoitllh nspot'ls uro bron/rht. onf. 11 !'AAr lms Ilio ohlioulion unti Ilio nu- fliorily lo brino Ibis lo flu uttenfion of any otlior Dopurfiiiont. In firn caso of postioitlos (boy nioot ami talee nelion jointly. "1 f f Ji 'rl ;. j j Sennini1 11 vut. Ah I melei.-l od :i!l Imi. mil^' orni viIc, nini l)i:it I><p it ; n.- 1.1 <' CO culi un' . ^ J^i*. |hiI iiiiimi:. Ni, I ilo uni. lliinU limi in ipiile <<>11 l. I 1 1>>>1. it,r~' Iiiih berli ngreril limi, (lui unuiiimuiis eomeiil. ni' llm lime ud Ini sminili; for specillo ucliou. 1 think 1liis is u privalo, agreement. I do noi. kimw whetlier ithas boon inailo pub)ic or noi. Sennlor H aiit. I t is my undersf unding if tlio.ru we.ru disagreement llm decisimi would he Agrioulturo's. Mr. Jiidcwit't Mr. IUckwit. You bave tolti uh tlmt Ilio Dopai!meni of Agrioul- turo doos not bclicvo thut Micy bave cerlain aulborily Hiut you would libo, lo neo them bave, in ornis of lemporary snspensions and (lui libo. Davo (boy commmiinilcd lo you, as llmy commimicaled lo u h ycKl.onlay, limi. I.bov would ne ver liavo aulborily lo Imo Ibi* use. of a luizurdmis pesticide oli food e.rops wlien il, was kimwn llial. (he ingostion of Food wliicli liad been trealed by limi, pesticide, was dangerous to man? Dr. DuHidiMu:. When there is clear evidence of danger to man they have, full authority, of course, to cancel or suspend Ilie use of it. The problem cornea when l.lio evidence, is not eleur or is not con- elusive or delinitive. Mr. biouwiT. That, was my impression us well. Dill (hey contend (bat Ihe. use. of a peslieide on food erops will never create, an im minent hazard to the public because it will lake several months for that food to arrive on the tables of those, people, who ingest it, nml therefore, that the. hazard ensiled is not in fai l, imminent. Dr. Dirlbimm:. I Veil, (.hero is a legal delcrmitml ion arid interpre tation of the word " imminent." Sometimes food reaches your (aide prompt ly, sometimes it does not. Mr. Dii.utwrr. Cun we impute to Congress the intent, to leave the public unprotected in nne.li a ease? As I understand it, there is no legislative, history on the use of Ihe terminology " imminent haz ard" in ihe. relevant act, and Congress has not defined that teriui- J l o l o g y 11 | ],,)(. ,1(.|,, l>r. In 1 ' 1:11 m:i-:. A s I understand kind nf m l imi Imt. hazard present it, imminent hazard leads hut. not imminent leads lo alo il0l 1111'0- civiii kind nf 11I ion. Oho can have, either suspension or .;nn't-!I:iI ion )n eilbej-1-,1 V. Suspension oeeiirs when there is iinmiiienl hazard, and kiis|ii'iisuin, (I !1 ii snoods more, temporary, really is not. Mr. Iiii'K wit. Il eei'lainlv is not, given Ibe euiirellnl ion prnre.durcs may take as limi/ as \ |.;i iv. Dr. Did i i u i m.i .. I bey lake Iimr. So. (here eau he a suspension ill flic case of iiiimiiienl. hazard, I l i n e ran he eaoeellal ion if there, is hazard hut; noI. iuimiuenl. I do not know hoc- you interpret, imminent, whether il. is a dav,a week or a niouih or wliul. Mr, I?imewit. They inlerpreied il so IIml. they would not he allowed to move in the case, of known hazards to human health when (ho hazards result, from the use. of a hazardous pesticide, on food erops. Dr. D itB iuduf.. Then possibly a clarification of Iho statute would bo desirable. I-- 70 L. \ Mr. BincwiT. I just, wonder whether it is needed in view of tho fact. Unit it seems perverse to mo to assume Unit Congress would in tend to exclude protective notion in coses such ns Mint. Dr. DuBnuxn:. I' lenso do not nslc me. to understand the, views of legal counsels for the various departments, or how (hey come to tlio various conclusions ns to wind, their departments enn or cannot do under Ilie Inw. Mr. Bicicwrr. I will not if yon do not. nslc me to understand it. I have jest, one other question which relates t.o a legal term, hut. I would like to hear your reaction to it from a scientific standpoint wit hoot legal context, attached to it. Do you have any reasonable doubt, about the safely of pesticides such as 2,1-D and Silvex? Dr. DwBimxm. I have reasonable doubt, about anything in which tlio research and testing have not. been adequately curried out, Tho qeustions of substantial dangers in tlmso eases I think hnvo not, been proved so I would he much more coin fort able about the use. of Ihoso than I would about 2,4,5-T whoro the toratogcnic c/Teet is now clearly established. For those others, I think imminent or serious hazards have not. been found. Mr. B ickwit. Thank you very much. Senator 11 art. Thunk you. Dr. Burger, is there, anything you want to add in light of our exchnngo? Dr. B ukof.r. N o, I do not believe so, Scnntor. Senator IIaut. I renew my apologies as T asic Dr. Sloinfeld to como up. I know there, are many things lie. would hope, to he. able to do this morning, which have been delayed. Dr. Jesse Steinfeld, the Surgeon ( eiierul. STATEMENT OF DR. JESSE STEINFK1.D, SURGEON GENERAL, D E P A R T M E N T OF HEALTH. EDUCATION. AND WELFARE; ACCOM PANIED BY DR. PAUL KOTIN, DIRECTOR, NATIONAL INSTITUTE OF E N V I R O N M E N T H E A L T H SCIENCE; A N D DR. WILLIAM M. UFIIOLT, ACTING STAFF DIRECTOR, SECRETARY'S PESTICIDE ADVISORY COMMITTEE Dr. S tkini-t.i.d. Thunk you. Senator Hurl. With me this morning are Dr. Paul Koiin. who is the Director of the National Institute of Kiivimumruial llcallh Sciences, lie. is on my left. And on my right, is Dr. William (iphull, who is the. execu tive secretary of the Secretary's Pesticide Advisory Committee. Before I begin, I would like to apologize, for a number of mis spelled words, run-on sentences and so forth in the statement, of which yon have a copy. Tt will ho somewhat different than it is be Toro you ns T rend it, if T may he permitted to rend it. Scnntor 1 I . \ i:t . You may ho almost certain 1 won't spot, the misspell ings. Dr. Sri:iNM i:r,n. I am pleased to appear before you today to discuss flic actions that have lwen taken to protect the public health by the Department of Health, Education, and Welfare regarding tho chloro"honoxynciil herbicides, particularly 2,4,5-T and 2,4-D. i'TFRRR t l :j l ._.. u.. '1 --J IJuI'inA? <>nr bist ippcarnnre before Ibis rnmmiuic. wo (*;!< nolo ,,f iviliiia needs to inuruii.su the Federal (overnmrui's o l i i ivmu" ^ in with (ptcstious of hazards (o Ilio pulii if. health )>n- t-nted by l!,,- pesticides nini illudami ourselves to action. Wo have m:id:i jini^i'uss, even in thu extremely short timo siimi our April 15, H't'h appearance. Wo uro now dolininf' how host to undertake to study llm moans for predictin'^, in laboratory animal systems, the potential li:ix.:iids posed for mini hy oliomioal iusieidos. It is certainly desirable. and may provo ossoutiul (.hut wo find some moans of extra pointing the re sults from reeding animals very lur^o douses of pesticides lo Ilio real life situation in which man is exposed for u Ion' period to very small nmoiinls of these chemicals. However, 1 must, emphasize (hat even wilh results based on studies in two species of mammals, un certainties remain as to tlm sif'iiiliennco of those, studies when ap plied to limn. Complete information outlie pesticides is essenti,al to the ollieioiit performance of all a^eneies concerned with the public, health aspects of pesticides, he they Federal, Slate., or loe.nl. A centralized clcarinfjliouso for information on all typos of pesticides is beimi estab lished jointly by the National Library of Medirine and the. Food and Drug Administration. It. is now being established. The l)ivismn of Toxicology of F D A and the National Library of .Medicine, are now sharing toxicological infonnation and are building on this base to form the clearinghouse. I inn very [lease.d with the progress on (iiis information center to date. The Food and Drug Administration has issued instructions that spedili attention to Hie extent of available resources is to be given to the annlyses .for residues of 2,1.5-T on food crops for which this herbicide was formerly registered for use. This step was (alien as an additional precaution to prevent accidental exposure to residues of 2.1.5- T even though our surveillance, activities liad not delected signilicant residues of 2, 1,5-T on these food crops. I In* seieiillie research on which the April 15 ammimeemenl was hie i d has continued. Ilio Naiiomd Institute of T-lnvimnmcnliil Ifeallli Sciences is eondiii-ting flirt tier research on 2,1.5-T, eerlain related lierbieide com pound-. and o.!'i.7,.S let rac.lilorodilicnzopa radioxin which is l!ic dioxin we reliTri d to in our previous test imonv. _Aijdij ama I ?fimIi(-s on the Iera I"logy of 2.1.5 T and Iet rarlilomdio.xin in tin- random hn-d ino-ie ban- eiinllrmi'il tbe earlier ; Indies that, 2,1,5-1 |inii|le a-; eli-li liliali- in Ibi- moii-e. ( he- slinlv wli'u-h utilized a combinat ion of Id r.irhloroilinxin and tin- purest. 2.1.5-T avili laide indicates (Imi tliere is no synergistic elicci, of these two compounds on the. production of elrll palate in (lie mouse. Preliminary studies have been nil ialed wilh three esters of 2.1.5-T, namely, the isobutyl-esler. the isont-tyl-oslcr, and (lie propvlenc-gl vcol-bulyl-ester. 'Die experiment design is the same ns that, uscii to study tlm acid form of 2 .1,5-T earlier. 'Lhc results that lire available to date are suggestive t h a t a t least some esters may be comparable, in teratogenic activity to that of 2.1.5- T. At this time more definitive studies on these esters are underway. 2631 SS062T 1 MOQ ! reason-- Jl .au pArosjseocct--ia- | j irays the | tiozin moleJbout how I about the |r precursor] laa or wood lfed by the 1 1say when i-'w, regards *at anybody | nlicated to <to an outf n it would nt dis1tests show ag/kg and i a the chick Indent," he ' *11 2,4,5-T however. ! -Vational :/Meets for |3e's wrong. V;aiits dolepi* of mal- ' to studies [' defects in l^ade avail- *ttcb data fi? Pbeips, [p Develop8SUres on ' Jnne 8 - n Ld 20 galfr-an Mww doctors fTJ duck, a U'dlt: two f ond one r f Tonto I,if 'copter l.;d convulr^ositosis, f'etys who ^foisoning . 14 *ouchCase. that i^d duPc"klsn. L bains of L toes, as 0 any 0f if411 cases TMnn and 107 I'm no toxicologist People here are emotional and each morning wake up with new nails pounded into their palms. What's needed is solid scientific investiga tion. All I hope is they don't leave ns hanging in the air for the next 20 years." Appendix 3 [From the New Yorker, Feb. 7, 1970] A R eporter at L arge: D efoliation By Thomas Whiteside Late in 1961, the United States Military Advisory Group in Vietnam began, as a minor test operation, the defoliation, by aerial spraying, of trees along the sides of roads and canals east of Saigon. The purpose of the operation was to increase visibility and thus safeguard against ambushes of allied troops and make more vulnerable any Vietcong who might be concealed under cover of the dense foliage. The number of acres sprayed does not appear to have been publicly recorded, but the test was adjudged a success militarily. In January, 1962 following a formal announcement by South Vietnamese and American officials that a program of such spraying was to be put into effect, and that it was intended "to improve the country's economy by permitting freer communi cation as well as to facilitate the Vietnamese Army's task of keeping these avenues free of Vietcong barassments," military defoliation operations really got under way. According to an article that month in the New York Times, "a high South Vietnamese official" announced that a seventy-mile stretch of road between Saigon and the coast was sprayed "to remove foliage hiding Commu nist guerrillas." The South Vietnamese spokesman also announced that defol iant chemicals would be sprayed on Vietcong plantations of manioc and sweet potatoes in the Highlands.. The program was gathering momentum. It was doing so in spite of certain private misgivings among American officials, partic ularly in the State Department, who feared, first, that the operations might open the United States to charges of engaging in chemical and biological war fare, and .second, that they were not all that militarily effective. Roger Hilsman, now a professor of government at Columbia University, and then Direc tor of Intelligence and Research for the State Department, reported, after a trip to Vietnam, that defoliation operations "had political disadvantages" and, furthermore, that they were of questionable military value, particularly in accomplishing their supposed purpose of reducing cover for ambushes. Hilsman later recalled in his book, "To Move a Nation," his visit to Vietnam, in March, 1962: "I had flown down a stretch of road that had been used for a test and found that the results were not very impressive. . . . Later, the senior Austra lian military representative in Saigon, Colonel Serong, also pointed out that defoliation actually aided the ambushers--if the vegetation was close to the road those who were ambushed could take cover quickly; when it was removed the guerrillas had a better field of fire." According to Hilsman, "The National Security Council spent tense sessions debating the matter." Nonetheless, the Joint Chiefs of Staff and their Chairman, General iMaxwell Taylor, agreed that chemical defoliation was a useful military weapon. In 1962, the American military "treated" 4,940 acres of the Vietnamese country side with herbicides. In 1963, the area sprayed increased five-fold to a total of 24,700 acres. In 1964, the defoliated area was more than tripled. In 1965, the 1964 figure was doubled, Increasing to 155,610 acres. In 1966, the sprayed area was again increased fivefold, to 741,247 acres, and in 1967 it was doubled once again over the.previous year, to 1,486,446 acres. Thus, the areas defoliated in Vietnam had increased approximately three hundredfold in five years, but bow adverse opinion among scientists and other people who were concerned about the effects of defoliation on the Vietnamese ecology at last began to have a braking effect on the program. In 1968, 1,267,110 acres were sprayed, and In 1969 perhaps a million acres. Since 1962, the defoliation operations have cov ered almost five million acres, an area equivalent to about twelve per cent of the entire territory of South Vietnam, and about the sire of the state of Mas sachusetts. Between 1962 and 1967, the deliberate destruction of plots of rice, manioc, beans, and other foodstuffs through herbicidal spraying--the word "deliberate" is used here to exclude the many reported Instances of accidental 45-302--70----- 8 C; . an estimated cropgrowing least half a -c defoliation 3ore effective ' lime of that a on a life of -tration from vealed jungle -an military Son of either 'Hat it is an "a? of ehemi- 1Hint it is an lisruption of ^lons simply 1; they were "^ent report, ' of crops guerrillas, '"d gave a * mild and ^ngerous to aring seven lor develop- 54 thirties) * b,logical Torld War, '"t of the 'Mention of of War, *advice on jo* of hor"J,n Plants ' ight be r. for the on Detriclc, r rdjog to ,, advisorv ^ls In aii ' or .^eloped and ,!nosrh.phTehne- 'd acid, affected :vUnitebde ' and the . "f both, applica_1'er. and to con- **<1 ^ "trr, to conten. 'd 1963, ry; 109 since 1963, their use lind risen two hundred and seventy-one iiercent--more tliau double the rate of increase in the use of pesticides, though pesticides are still far more extensively used. By 1960, an area equivalent to more than three tier cent of the entire United States was being sprayed each year with herbi cides. Considering the rapidly growing civilian use of these products, it is perhaps not surprising that the defoliation operations in Vietnam escaped any signifi cant comment in the press, and that the American public remained unaware of the extent to which these uses had their origin in planning for chemical and biological warfare. Nevertheless, between 1911 and the present, testing and experimentation in the use of 2,4-D, 2,1,S-T, and other herbicides as military weapons were going forward very actively at Fort Detrick. While homeowners were using herbicidal mixtures to keep their lawns free of weeds, the military were screening some twelve hundred compounds for their usefulness in biologi cal-warfare operations. The most promising of these compounds were testsprayed on tropical vegetation in Puerto Bico and Thniland, and by the time fullscale defoliation operations got under way in Vietnam the U.3. military had settled on the use of four herbicidal spray materials there. These went under the names Agent Orange, Agent Purple, Agent White, and Agent Blue-- designations derived from color-coded stripes girdling the shipping drums of each type of material. Of these materials, Agent Orange, the most widely used as a general defoliant, consists of a fifty-fifty mixture of n butyl esters and of 2,1-D and 2,1,5-T. Agent Purple, which is interchangeable with Agent Orange, consists of the same substances with slight molecular variations. Agent White, which is used mostly for forest defoliation, is a combination- of 2,4-D and Picloram, produced by the Dow Chemical Company. Unlike 2,1-D or 2,4,5-T, which, after application, is said to be decomposable by micro-organisms in soil over a period of weeks or months (one field test of 2,4,5-T in this country showed that significant quantities persisted in soil for ninety-three days after application), Picloram--whose use the Department of Agriculture has not authorized in the cultivation of any American crop--is one of the most persist ent herbicides known. Dr. Arthur W. Galston, professor of biology at Tale, has described Picloram as "a herbicidal analog of DDT," and an article in a Dow Chemical Cpmpany publication called "Down to Earth" reported that in field trials of Picloram in various California soils between eighty and ninety-six and a half per cent of the substance remained in the soils four hundred and sixty-seven days after application. (The rate at which Picloram decomposes in tropical soils may, however, be higher.) Agent Blue consists of a solution of cacodylic acid, a substance that contains fifty-four per cent arsenic, and it is used in Vietnam to destroy rice crops. According to the authoritative "Merck Index," a source hook on chemicals, this material is "poisonous." It can be used on agricultural crops in this country only under certain restrictions imposed by the Department of Agriculture. I t is being used berbicidally on Vietnamese rice fields at seven and a half times the concentration permitted for weed-killing purposes in this country, and so far in Vietnam sometlving like five thousand tons is estimated to have been sprayed on paddies and vegetable fields. Defoliation operations in Vietnam are carried out by a special flight of the 12th Air Commando Squadron of the United States Air Force, from a base at Bien Hoa, just outside Saigon, with specially equipped C-123 cargo planes. Each of these aircraft has been fitted out with tanks capable of holding a thousand gallons. On defoliation missions, the herbicide carried in these tanks is sprayed from an altitude of around a hundred and fifty feet, under pres sure, from thirty-six nozzles on the wings and tail of the plane, and usually several spray planes work in formation, laying down broad blankets of spray. The normal crew of a military herbicidal-spray plane consists of a pilot, a ccpilot, and a technician, who sits in the tail area and operates a console regu lating the spray. The equipment is calibrated to spray a thousand gallons of herbicidal mixture at a rate that works out, when all goes well, to'about three gallons per acre. Spraying a thousand-gallon tankload tnkes five minutes. In an emergency, the tank can be emptied in thirty seconds--a fact that has par ticular significance because of what has recently been learned about the nature of a t least one of the herbicidal substances. The official code name for the program Is Operation Hades, but a more friendly code name. Operation Banch Hand, is commonly used. In similar fash ion, military public-relations men refer to the herbicidal spraying of crops sup- tton,Ameritioutcareful of friendly tlittledoubt ets of the slightly jos fire from fieherbictwo parallel Vdistrict and p South VietV Slateschain, r-iradviser, a find in South 1ieAmerican 1 likely to VV-atwioorndsinwgittho I'dtheend of M turn down 1ionot have l-iestion. And 1a the words p 'ive one of r*ofdenying F>at deal of pas sent up pwe "enthuponders and Li!' are ti k ,'n acr l,u0t at n vJlustratic ' rte bottli President Johnson from twenty-two scientists, Including seven Nobel laureates. The petition pointed out that the "large-scale use of anticrop and `nonlethaP antipersonnel chemical weapons in Vietnam" constituted "dangerous precedent" in chemical and biological warfare, and it asked the President to order it stopped. Before the end of that year, Dr. Edsnll and Dr. Matthew S. Meselson, a Harvard professor of biology, obtained the signatures of five thousand scien tists to co-sponsor the petition. Despite these protests, the area covered by defoliation operations in Vietnam in 1967 was double that covered in 1966, and the acreage of crops destroyed was nearly doubled. These figures relate only to areas that were sprayed intentionally. There Is no known way of spraying an area with herbicides from the air in a really accurate manner, because the material used is so highly volatile, especially under tropical conditions, that even light wind drift can cause extensive damage to foliage and crops outside the deliberately sprayed area. Crops are so sensitive to the herbicidal spray that it can cause damage to fields and gar dens as much as fifteen miles away from the target zone. Particularly severe accidental damage is reported, from time to time, to so-called "friendly" crops In the 111 Corps area, which all but surrounds Saigon and extends in a rough square from the coastline to the Cambodian border. Most of the spraying in III Corps is now done in War Zones C and D, which are classified as free fire zones, where, as one American official has put It, "everything that moves in Zones C and D is considered Charlie." A press dispatch from Saigon in 1967 quoted another American official as saying that every Vietnamese farmer in tlmt- corps area knew of the defoliation program and disapproved of i t Dr. Galston, the Yale biologist, who is one of the most persistent .critics of Ameri can policy concerning herbicidal operations in Vietnam, recently said In an interview. "\Ve know that most of the truck crops grown along roads, canals, and trails and formerly brought into Saigon have been essentially abandoned because of the deliberate or inadvertent falling of these defoliant sprays: many crops in the Saigon area are simply not being harvested." He also cited reports that in some instances in which the inhabitants of Vietnamese villages have been susis-oted of being Vietcong sympathizers the destruction of food crops has brought about complete abandonment of the villages. In 1966, herbi cidal o|KT:iti.uis caused extensive Inadvertent damage, through wind drift, to a very large rubber plantation northwest of Saigon owned by the Micheliu rubber interests. As tbe result of claims made for this damage, the South Viet namese authorities paid the corporate owners, through the American military, nearly a million dollars. The extent of the known inadvertent damage to crops in Vietnam can be Inferred from the South Vietnamese budget--In reality, the American military budget--for settling such claims. In 1967, the budget for this comiiensarion was three million six hundred thousand dollars. This sum, however, probably reflects only the barest emergency claims of the people affected. According to Representative Richard D. McCarthy, a Democrat from upstate New York who has been a strong critic of the program, the policy of allowing applications for defoliation operations to flow, usually without question, from the level of the South Vietnamese provincial or district chiefs has mennt that these local functionaries would order repeated sprayings of areas thut they had not visited in months, or even years. The thought that a Vietnamese dis trict chief can initiate such wholesale spraying, In effect without much likeli hood of serious hindrance by American military advisers. Is a disquieting one to a number of biologists. Something that disquiets many of them even more is wlint they believe the long-range effects of nine years of defoliation operations will be on the ecology of South Vietnam. Dr. Galston, testifying recently before a congressional subcommittee on chemical and biological warfare, made these observations: "It has already been well documented that some kinds of plant associations . subject to spray, especially by Agent Orange, containing 2,4-D and 2,4^5-T, have, been irreversibly damaged. I refer specifically to the mangrove associations that line the estuaries, especially around the Saigon River. Up to a hundred thousand acres of these mangroves have been sprayed. . . . Some (mangrove areas) had been sprayed as early as 1961 and have shown no substantial signs of recovery. . . . Ecologists have known for a long time that the mangroves lining estuaries furnish one of the most important ecological niches for the completion of the life cycle of certain shellfish and migratory fish. If these plant communities are not in a healthy state, secondary effects on the whole D8WJ 129059 t] are ahead the ni anyhow, food in are the * Research ''sit to Viet- ? 'Wrt on the 4 ^ Dr. Gal- } towever, he ! "ithenst of i H llnd been ; '>*aa killed l ri-r- and he ruld take ; " that a `nof vir- ; 113 latex production in the affected plantations fell off b ; an average of between thirty-five and forty per cent According to a report by the two scientists, "A large variety of garden crops were devastated in the seemingly endless number of small villages scattered throughout the affected area. Virtually all of the . . . local Inhabitants . . . depend for their wellbeing upon their own local pro duce. These people saw their crops . . . literally wither before their eyes." The Cambodian claim is still pending. Until the end of last year, the criticism by biologists of the dangers Involved in the use of herbicides centered on their use in what were increasingly con strued as biological-warfare operations, and on the disruptive effects of these chemicals upon civilian populations and upon the ecology of the regions In which they were used. Last year, however, certain biologists began to raise serious questions on another score--possible direct hazards to life from 2,4,5-T. On October 20th, as a result of these questions, a statement was publicly issued by Dr. Lee DuBridge, President Nixon's science adviser. In summary, the statement said that because a laboratory study of mice and rats that had been given relatively high oral doses of 2,4,5-T in early stages of pregnancy "showed a higher than expected number of deformities" in the offspring, the government would, as a precautionary measure, undertake a series of coordi nated actions to restrict the use of 2,4,5-T in both domestic civilian applica tions and military herbicidal operations. The DuBridge statement identified the laboratory study as having been made by an organization called the Blonetics Research Laboratories, in Bethesda, Maryland, but gave no details of either the findings or the data on which they were based. This absence of specific information turned out to be characteristic of what has been made available to the public concerning this particular research project. From the beginning, it seems, there was an extraordinary reluctance to discuss details of the pur ported ill effects of 2,4,5-T on animals. Six weeks after the publication of the DuBridge statement, a journalist who was attempting to obtain a copy of the full report made by Bionetics and to discuss its details with some of the gov ernment officials concerned encountered hard going. At the Blonetics Laborato ries, an official said that he couldn't talk about the study, because "we're under wraps to the National Institutes of Health"--the government agency that commissioned the study. Then, having been asked what the specific doses of 2,4,5-T were that were said to have increased birth defects in the fetuses of experimental animals, the Blonetics official cut off discussion by saying, "You're asking sophisticated questions that as a layman you don't have the equipment to understand the answers to." At the National Institutes of Health, an official who was asked for details of or a copy of the study on 2,4,5-T replied, "The position I'm in is that I have been requested not to dis tribute this information." He did say, however, that a continuing evaluation of the study was under way at the National Institute of Environmental Health Sciences, at Research Triangle Park, North Carolina. A telephone call to an officer of this organization brought a response whose tone varied from wari ness of downright hostility and made it clear that the official had no intention of discussing details or results of the study with the press. The Blonetics study on 2,4,5-T was part of a series carried out under con tract to the National Cancer Institute, which is an arm of the National Insti tutes of Health, to investigate more than two hundred compounds, most of them pesticides, in order to determine whether they induced cancer-causing changes, fetus-deforming changes, or mutation-causing changes in experimental animals. The contract was a large one, involving more than two and a half million dollars' worth of research, and its primary purpose was to screen out suspicious-looking substances for further study. The first visible fruits of the Blonetics research were presented in March of last year before a convention of the American Association for the Advancement of Science, in the form of a study of possible carcinogenic properties of the fifty-three compounds; the find ings on 2,4,5-T were that it did not appear to cause carcinogenic changes in the animals studied. By the time the report on the carcinogenic properties of the substances was presented, the results of another part of the Blonetics studies, concerning the teratogenic, or fetus-deforming, properties of the substances, were being com piled, but these results were not immediately made available to biologists out side the government. The data remained--somewhat frustratingly, in the view of some scientists who had been most curious about the effects of herbicides-- out of sight, and a number of attempts by biologists who had heard about the le e e s D0W_1 129061 I Harvard. In early October, Miss Johnson's friend, In a conversation with Pro fessor Matthew Meselson, mentioned Miss Johnson's report on the preliminary Bionetics findings. This was the first that Dr. Meselson had beard of the exist d - ence of the Bionetics study. A few days previously, he had received a call from a scientist friend of his asking whether Dr. Meselson had heard of cer tain stories, originating with South Vietnamese Journalists and other South Vietnamese, of an unusual incidence of birth defects in South Vietnam, which were alleged to be connected with defoliation operations there. A few days later, after his friend sent him further information, Mr. Mesel son decided to obtain a copy of the Bionetics report, and he called up an acquaintance in a government agency and asked for i t He was told that the report was "confidential and classified," and inaccessible to outsiders. Actually, to in addition to the preliminary report there were now in existence the full Bio CD netics report and a statistical summary prepared by the National Institute of Environmental Health Sciences, and, by nagging various Washington friends. O Dr. Meselson obtained bootlegged copies of the two latest reports. What he 0 5 read seemed to him to have such serious implications that he got in touch with acquaintances in the White House and also with someone in the Army to alert them to the problems of 2,4,5-T, in the hope that some new restriction C would be placed on its use. According to Dr. Meselson, the White House people apparently didn't know until that moment that the reports on the adverse C' effects of 2,4,5-T even existed. (Around that .time, according to a member of Nader's Raiders, "a tremendous lid was put on this thing" within government i-O agencies, and on the subject of the Bionetics work and 2,4,5-T "people in gov ernment whom we'd been talking to freely for years Just shut up and wouldn't Hw say a word.") While Dr. Meselson awaited word on the matter, a colleague of hl3 Informed the press about the findings of the Bionetics report. Very shortly thereafter. Dr. DuBridge made his public announcement of the proposed restrictions on the use of 2,4,5-T. In certain respects, the DuBridge announcement is a curious document In its approach to the facts about 2,4,5-T that were set forth in the Bionetics report, it reflects considerable sensitivity to the political and international r. issues that lie behind the widespread use of this powerful herbicide for civil ian and military purposes, and the words in which it describes the reasons for restricting its use appear to have been very carefully chosen: "The actions to control the use of the chemical were taken as a result of findings from a laboratory study conducted by Bionetics Research Laboratories which indicated that offspring of mice and rats given relatively large oral doses of the herbicide during early stages of pregnancy showed a higher than expected number of deformities. "Although it seems improbable that any person could receive harmful amounts of this chemical from any of the existing uses of 2,4,5-T, and while the relationships of these effects in laboratory animals to effects in man are not entirely clear at this time, the actions taken will assure safety of the public while further evidence is being sought" These actions, according to the statement, Included decisions that the Department of Agriculture would cnncel manufacturers' registrations of 2,4,5-T for use on food crops, effective at the beginning of 1970, "unless by that time the Food and Drug Administration has found a basis for establishing a safe legal tolerance in and on foods," and that the Departments of Agriculture and the Interior, in their own programs, would stop the use of 2,4,5-T in populated areas and in all other areas where residues of the substance could reach man. As for military uses of 2,4,5-T, the statement said, "The chemical is effective in defoliating trees and shrubs and its use in South Vietnam has resulted In reducing greatly the number of ambushes, thus saving lives." However, the statement continued, "the Department of Defense will [henceforth] restrict the use of 2,4,5-T to areas remote from the population." All this sounds eminently fair and sensible, but whether it represents a candid exposition of the facts about 2,4,5-T and the Bionetics report is debata ble. The White House statement that the Bionetics findings "indicated that offspring of mice and rats given relatively large oral doses of the herbicide during early stages of pregnancy showed a higher than expected number of deformities" is, in the words of one eminent biologist who has studied the Bio netics data, "an understatement" He went on to say that "If the effects on experimental animals are applicable to people It's a very sad and serious situa- '16667 : sofficiently ipeirainmgeenrts- ag further (rery small | j increased b\ anluatleitsi,esanIdn rHyerimen- i additional : dosages oi >46.4 millir?bt] susI -tta through p*t SO per i the begin^nality was 16 tng/kg. i with the ' rats when ' of 2,4,5-T a mortality * herbicide F' an accur- [ `-ibles pre- - dosage of I ' of gesta-. T-ree times I >-tlon be l f 2L5 and r* Percent- [^y, or a f 1 Mgher [^probable ^ any of '* this is 2,4,5-T i in the M iaboraM'rtaken. y^mals to ,,? animal these J`a"tseddoeins , 1sed to L`r,r l by ment of li,y. y begin Com- l*tpes- 5 their ^ st two 'testing K* PestlL? cio?y *, Permis, sPecles , 'terage | * . eiehty l ^ g lit- and 117 their total number was, in fact, greater, not less, than this average Including controls but excluding litters, the total number of animals used In the 2,4,5-T studies was two hundred and twenty-five Analysis of the results by the National Institute of Environmental Health Sciences found them statistically "significant," and this Is the real purpose of such a study: it is meant to act ns a coarse screen to shake ont of the data the larger lumps of bad newe Such a study la usually Incapable of shaking out anything smaller; another kind of study is needed to do th at Thus, the DuBridge statement seems to give rise to this question; If the Bionetics study, based on the effects of 2,4,5-T on two hundred and twenty-five experimental animals of two species, appears to be less than conclusive, on the ground that "the study Involved relatively small numbers of laboratory rats and mice," what is one to think of the adequacy of the tests that the manufac turers of pesticides make? If, as the DuBridge statement says, "at best It Is difficult to extrapolate results obtained with laboratory animals to man," what is one to say of the protection that the government affords the consumer when the results <?f tests of pesticidal substances on perhaps a hundred and twenty rats are officially extrapolated to justify the use of the substances by a popu lation of two hundred million people--not to mention one to two million unborn babies being carried In their mothers' wombs? The very coarseness of the screen used in all these tests--that Is, the rela tively small number of animis involved--means that the bad news that shows up in the data has to be taken with particular seriousness, because lesser effects tend not to be demonstrable at all. The Inadequacy of the scale on which animal tests with, for instance, pesticides are currently being made In this country to gain F.D.A. approval is further Indicated by the fact that a fetus-deforming effect that might show up if a thousand test animals weTe used is almost never picked up, since the studies are not conducted on that scale; yet if the material being tested turned out to have the same effect, quantitatively, on human beings, this would mean that it would cause between three and four thousand malformed babies to be produced each year. The tera togenic effects of 2,4,5-T on experimental animals used by the Bionetics people, however, were not on the order of one in a thousand. Even in the case of the lowest oral dose given rats, they were on the order of one in three. Again, It is fair to say that what is applicable to rats in such tests may not be applicable to human beings. But It Is also fair to say that studies involving rats are conducted not for the welfare of the ra t kingdom but for the ultimate protection of human beings. In the opinion of Dr. Epstein, the fact that the 2,4,5-T used in the Bionetics study produced teratogenic effects in both mice and rats underlines the seriousness of the stndy's implications. In the opinion of Dr. McLaughlin, this is even further underlined by another circumstance-- that the rat, as a test animal, tends to be relatively resistant to teratogenic effects of chemicals. For example, in the late nineteen-fifties, when thalido mide, that disastrously teratogenic compound, was being tested on rats In oral dosages ranging from low to very high, no discernible fetus-deforming effects were produced. And Dr. McLaughlin says that as far as thalidomide tests on rabbits were concerned, "You could give thalidomide to rabbits in oral doses at between fifty and two hundred times the comparable human level to show any comparable teratogenic effects." In babies born to women who took thalido mide, whether in small or large dosages and whether in single or multiple dos ages, between the sixth and seventh weeks of pregnancy, the rate of deforma tion was estimated to be one in ten. Because of the relatively coarse testing screen through which compounds like pesticides--and food additives as well--are sifted before they are approved for general or s|>eciallzed use In this country, the Food and Drug Administration theoretically maintains a policy of stipulating, as a safety factor, that the maximum amount of such a substance allowable In the hjiinan diet range from one two-thousandth to one one-hundredth of the highest dosage level of the substance that produces no harmful effects in experimental animnls. (In the case of pesticides, the World Health Organizatl(TO takes a more conservative view, considering one two-thousandth of the "no^effect" levdl in animal studies to be a resaonsable safety level for human exposure.) According to the standards of safety established by F.D.A. policy, then, no human being the Bionetics anywhere study of rsahtosuledveervyerdohsaavgee been level exposed to 2,4,5-T, produced deformed because fetuses. In A "no-effect" level was never achieved. i. 16668 ,0 -S906ZT nwoo |:t5-T for human opriate populacwell-fed United 1 withstand the 1ambers of civllI numerable horf IIJ5-T on human >the amount of Lsit have been |* *>. a compnri- i and in Viet| V average recpptint control is ire about five s3lc.4a.l5-CTolminpeaniys of Esteron |*vir allegations | and clotb- "iag safe use the following "lc purposes 'arerning the whether in I"* warnings, " raries with ?* would be ler acre. In 'ed has been ["raged thir" States. The " *d to enter | " the areas ied from 'Sat, taking L , , Per acre ['.?*! is Quite 'ehty-eigtit/'r contamitodred and ? f 2.4.5-T . tfr kilo of IIn.iPe4r0cernatnasgee V * every i the quesL '*>concenTj'talttee to afe nl^t'Ote ago, K one >ltre i ? ? " 1 VietKi:;4'5-t in "es the t u ? I" not Orations. C Mon- IS 0r the Lq|,n load eans of 119 the thirty-second emergency-dumping procedure. Dr. Pfeiffer has recalled going along as an observer on a United States defoliation mission last March, over tim Plain of Heeds area of Vietnam, near the Cambodian border, during which the technician at the spray controls was unable to get the apparatus to work, and thereupon dumped his whole load. "This rained down a dose of 2,4,5-T that must have been fantastically concentrated," Dr. Pfeiffer has said. "I t was released on a very watery spot that looked like headwaters draining Into the .Mekong Elver, which hundreds of thousands of people use? In another instance, he has recalled, a pilot going over the area of the supposedly "friendly" Catholic refugee villages of Ho Nai, near Bien Hoa, had serious engine trouble and dumped his whole spray load of herbicide on or near the village. In such Instance, the concentration of 2,4,5-T dumped upon an Inhab ited area in Vietnam probably averaged abont a hundred and thirty times the concentration reccommended by 2,4,5-T manufacturers as both effective and safe for use in the United States. Theoretically, the dangers Inherent in the use of 2,4,5-T should have been removed by means of the steps promised in the White House announcement last October. A quick reading of the statement by Dr. DuBridge (who is also the executive secretary of the President's Environmental Quality Council) cer c-< tainly seemed to convey the impression that from that day onward there would be a change In Department of Defense policy on the use of 2,4,5-T in Vietnam, just as there would be a change In the policies of the Departments of Agriculture and the Interior on the domestic use of 2,4,5-T. But did the White House mean what it certainly seemed to be saying about the future mil itary use of 2,4,5-T in Vietnam? The White House statement was issued on October 29th. On October 30th, the Pentagon announced that no change would be made in because--so ththeepoWlicayshginogvteornninPgotshte military reported use on of 2,4,5-T in South October 31st--"the Vietnam, Defense tDiveep."arTtmheenPt ofsetealsrtiictsle present went o policy n: conforms to the new Presidential direc "A Pentagon spokesman's explanation of the policy, read at a morning press u .; * briefing, differed markedly from the written version given reporters later. "When the written statement was distributed, reporters were told not to use the spokesman's [previous] comment that the defoliant . . . is used against enemy `training and regroupment centers.' `The statement was expunged after a reporter asked how use against such centers conformed to the Defense Department's stated policy of prohibiting its use in `populated areas.' " But the statement wasn't so easily expunged. A short time later, it was made again, in essence, by Hear Admiral William E. Lemos, of the Policy Plans and National Security Council Affairs Office of the Department of Defense, in testimony before a subcommittee of the House Foreign Affairs Committee, the only difference being that the phrase "training and regroup ment centers" became "enemy base camps." And In testifying that the military was mounting herbicidal operations on alleged enemy base camps Bear Admi ral Lemos said: "We know . . . that the enemy will move from areas that have been sprayed. Therefore, enemy base camps or unit headquarters are sprayed in order to make him move to avoid exposing himself to aerial observation." If one adds to the words "enemy base camps" the expunged words "training and regroupment centers''---centers that are unlikely to operate without an accompanying civilian population--what the Defense Department seems : actually to be indicating is that the "areas remote from the population" against which the United States are "remote from the population" is at cloeansdtucintinpgarmt iblietcaaruyseheorfbitchiedsael operations operations. As for the Bionetics findings on the teratogenic effects of 2,4,5-T on experi mental animals, the Department of Defense Indicated th the dangers suggested by the report A reporter for the aYt ailte pDuta illiyttlNeetsotoscwk hIon telephoned the Pentagon during the first week in December to inquire about the Defense Department's attitude toward its use of 2,4,5-T in the light of the Bionetics report was assured that "there Is no cause for alarm about defol iants." A week or so later, be received a letter from the Directorate for Defense Information at the Pentagon which described the Bionetics resnlts as based on "evidence that 2,4,5-T, when fed in large amounts to highly lmbred and was susceptible normal for mice these and rats, animats." gave a higher Incidence After reading this letter, otfhebiYrathle dDefaeiclyts Htehvaons "16669 r methods -1 States J rations [-fare. The I - consider F^oritative J it official fWlshment. Jbcal war|a l prodf an, ani| "'(-growth ' it. and kdentists, ! ^cation of of bioJ '`Ctionary f'*' and the l^rprise to 1 ^ 1 state- 'anescent tenable to J r-4l>y that that u` It is in Kjnonnced. edltioa \ * Herbi /Sued as >ring or and goes wth of Srowth, 7ment of '-'rations fs and as ^ ln Part Jf-MW 'I 511!' Pro,->* of a Present K 'b a t the I study i C ^ "P'e te a [ * ' to the business of manufacturing 2,4,5-T. It appears that the presence of a dioxin con taminant in the process of manufacturing 2,4,5-T ia a constant problem among nil manufacturers. Three years ago, Dow was obliged to close down its 2,4,5-T plant in Midland, Michigan, for several months and partly rebuild it because of what Dow people variously described as "a problem" and ``an accident." The problem--or accident--was that workers exposed to the dioxin contami nant during the process ef manufacture came down with an acute skin irrita tion known as chlor-acne. The Dow people, who speak with considerable pride of their toxicological work ("We established our toxicology lab the year Balpb Nader was born," a Dow public-relations man said recently, showing, at any rate, that Dow Is keenly aware of Nader and his career), say that the chloracne problem has long since been cleared up, and that tbe current level of the dioxin contaminant in Dow's 2,4,5-T Is less than one part per million, as opposed to the dioxin level in the 2,4,5-T used in the Bionetics study, which is alleged to have been between fifteen and thirty parts per million. A scientist at the DuBridge office, which has become a coordinating agency for informa tion having to do with the 2,4,5-T question, says that the 2,4,5-T used by Bio netics was "probably representative" of 2,4,5-T being used in this country--and presumably in Vietnam--at the time it was obtained hut that considerably less of the contaminant is present in the 2,4,5-T now being produced. Evidently, the degree of dioxin contamination present in 2,4,5-T varies from manufacturer to manufacturer. What degree of contamination high or low, was present in the quantities of 2.4.5-T shipped to South Vietnam at various times this spokesman didn't seem to know. The point about tbe dioxin contamination of 2,4,5-T is an extremely Impor tant one, because if the suspicions of the Dow people are correct and the cause of the fetus deformities cited in the Bionetics study is not the 2,4,5-T but the dioxin contaminant, then this contaminant may be among the most teratogenicaliy powerful agents ever known. Dr. McLaughlin has calculated tjiat if the dioxin present in the Bionetics 2.4.5-T was indeed responsible for the terato genic effects on the experimental animals, it looks as though the contaminant would have to la- at least ten tnousaDd times more teratogenically active in rats than thalidomide was found to be ln rabbits. Furthermore, it raises alarming questions about tbe prevalence of the dioxin material in our environ ment It appears thnt under high hent the dioxin material can be produced in a whole clnss of chemical substances known as trichlorepbenols and pentachlorophenols. These substances Include components of certain fatty acids used in detergents and in animal feed. As a consequence of studies that have been made of the deaths of millions of young chicks in this country after the chicks had eaten certain kinds of chicken feed, government scientists are now seriously speculating on the possi bility that the deaths were at the end of a chain that began with the-spraying of corn crops with 2,4.5-T. The hypothesis is that residues of dioxin present in tlie 2,4,5-T remained in tlie harvested corn and were concentrated into certain byproducts that were then sold to manufacturers of chicken feed, and that the dioxin became absorbed into tbe system of the young chicks. One particularly disquieting sign of tbe potential of the dioxin material is the fact that bio assays made on cluck emoryos in another study reveuled tlial all the embryos were killed by one twenty-millionth of a gram of dioxin per egg. Perhaps an even more disquieting speculation about the dioxin is that 2,4.5-T may not be the only material in which it appears. Among the compounds that several experienced biologists and toxicologists suspect might contain or produce dioxin are the trichiorophenols and pentachlorophenols, which are rather widely present in die environment in various forms. For exafnple, a number of the trichiorophenols and pentachlorophenols are used as slime-kill ing agents in paper-pulp manufacture, and are present in a wide range of con sumer products, including adhesives, water-based and oil-based paints, var nishes and lacquers, and paper and paper coatings. They are used to prevent slime in pasteurizers and fungus on vats in breweries and are also used in hair shampoo. Along with the 2,4,5-T used in the Bionetics study, one trichlorophenol and one pentachlorophenol were tested without teratogenic results. But Dr. McLaughlin points out that since there are many such compounds put out by various companies, these particular samples might turn out to be --by the reasoning of the allegation that the 2,4,5-T used by Bionetics was unu sually dirty--unusually clean. DOW .1 129069 ' |J0W;i 129071 123 There have been a number of reports from Vietnam both of animal abortions and of malformed human babies that are thought to have resulted from spray ing operations in which 2,4,5-T was used. But such scattered reports, however 'ell founded, cannot really shed much more light on the situation. The fact is that even in this country, the best-fed, richest, and certainly most statisticsminded of all countries on earth, the standards for testing materials that are put into the environment, into drugs, and into the human diet are grossly inadequate. The screening system is so coarse that, as a teratology panel of the Mrah Commission warned recently, in connection with thalidomide, "the teratogenicity of thalidomide might have been missed had it not produced mal formations rarely encountered." In other words, had it not been for the fact that very unusual and particularly terrible malformations appeared in an ubrious pattern--for example, similarly malformed babies in the same hospital at about the same time--pregnant women might still be using thalidomide, and lesser deformations would, so to speak, disappear into the general statistical background. As for :more subtle effects, such as brain damage and damage to the central-nervous system, they would probably never show up as such a t all. If such risks existed under orderly, normal medical conditions in a highly developed country, bow is one ever to measure the harm that might be done to unborn children in rural Vietnam, in the midst of the malnutrition, tbe dis ease, tbe trauma, tbe poverty, and tbe general shambles of war? DEPABTJtEXXTeico rYAormkp, lMifaicrcahtio5,n ,1970. TThbee XEdniotoYros,rker Dear S ir s : In an article that appeared in The Xeic Y o rker on February 7th, I wrote that Dr. Lee DuBridge, the President's science adviser, issued a state ment last October a t the 'White House saying that because a laboratory study bad shown a "higher than expected number of deformities" in the fetuses of mice and rats exposed to the herbicide 2,4,5-T, agencies of the United States government would take action to restrict the use of that substance in this country and in Vietnam, where it was being used in extensive m ilitary defolia tion operations. Tbis action, Dr. DuBridge announced, would include tbe can cellation, by January 1st of this year, of Department of Agriculture permits for the use of 2,4,5-T on some American food crops unless the Food and Drug Administration had by then been able to determine a safe concentration of the herbicide in foods. Dr. DuBridge further announced th a t the Department of Defense would thenceforth "restrict the use of 2,4,5-T to areas remote from the population" in Vietnam. His statem ent added that these actions and others "will assure the safety of the public while further evidence [of the alleged harmful effects of 2,4,5-T] is being sought" Four months have passed, and 2,4,5-T is still being used as widely as ever. The Department of Agriculture has yet to cancel its permits for the use of the herbicide on food crops in this country, and the Department of Defense is con tinuing to use it in populated areas of Vietnam. In the meantime, officials of tlie Dow Chemical Company, which is one of the largest producers of 2,4,5-T, have been maintaining that the samples of 2,4,5-T used in the study cited by Dr. DuBridge, which was done by the Bionetics Research Laboratories, of Bethesda, Maryland, were uncharacteristic of the 2.4,5-T currently being pro duced, because the material tested by Bionetics--which did not come from Dow--was contaminated to an unosnal extent by a toxic substance identified as symmetrical 2,3,0.7-tetrachlorodibenzo-p-dioxin. This contaminant, usually called dioxin, was alleged by tbe Dow people to be present in the Bionetics samples a t a concentration of approximately twenty-seven parts per million, and they claim that the 2,4.5-T that Dow is currently producing contains tbe dioxin contaminant in concentrations of less than one part per million. The Dow people maintain that their currently produced 2,4,5-T does not appear to have the effect of deforming rat fetuses. In January, a Dow official told tbe Department of Health, Education, and Welfare, "We strongly urge th at action concerning the status of 2,4,5-T be held in abeyance until [Dow's] testing pro gram is completed [in] April." The United States government's failure so far to place tbe promised restrictions on tbe use of 2,4.5-T in this country may in Part be attributed to tbis plea. 45-362-- 70--- 9 16671 2693 Toxicology of Chlorinated Dibenzo-p-dioxins by B.A. Schwetz,* J.M. Norris,* G.L. Sparschu,* Y.K. Rowe,* P.J. Gebriy,* J.L. Emerson/ and C.6. Gerbiff w Severe toxicological responses have been associated with certain chlorodibenzodioxins. One of these responses is chloracne, a folliculosis first associated with skin contamina tion by chlorohydrocarbons in 1899 (1 ). Serious outbreaks of chloracne-Iike lesions associated with runaway reactions in the' production of 2,4,5-trichlorophenol occurred in Germany in the early 1950's (2). 2,4,5Trichlorophenol itself does not cause acne (3) , but the contaminants which may be formed in the uncontrolled production of 2,4,5-trichlorophenol are extremely potent acnegens (2 ). 2,3,7,8-Tetrachlorodibenzo-pdioxin and tri- and tetrachlorodibenzofuran were isolated from the contaminants formed in 2,4,5-trichlorophenol production and were demonstrated to be strongly positive acne gens when applied to rabbit ears (3). By using the rabbit ear test, the acnegenic potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was confirmed in 1962 (4 ) . In addition, 2,3,7,8-TCDD is extremely toxic in the chick embryo assay (5) and is highly embryotoxic in rats (6). Another chlorodibenzodioxin, hexachloro'dibenzo-pdioxin (HCDD), is known to be positive for the chick edema factor, a condition char acterized by hydropericardium, ascites, and anasarca (5, 7). Chemical Biology Research, The Dow Chemical Co., Midland, Michigan 48640. t Human Health Research and Development Cen ter, The Dow Chemical Co., Zionsville, Indiana 46077. Experimental . wW ro IV) )-> "Ni tO Materials The chlorodibenzodioxin samples used in these studies are identified and described in Table 1. Studies were limited in some cases by availability of pure samples. Acute Lethality Samples of 2,7-dichlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, hexachlorodibenzo-p-dioxin, and octachlorodibenzo-pdioxin were evaluated for acute oral lethality in several animals as summarized in Table 2. Test materials were administered as sus pensions in corn oil or as corn oil: acetone (9:1) solutions in single doses by gavage. The animals were deprived of feed for 16 hr before dosing. After dosing, they were ob served for signs of toxicity including body weight changes for two to eight weeks. Lethality of 2,3,7,8-TCDD via skin ab sorption was tested on rabbits of mixed sexes with doses of 31.6, 63, 126, 252, and 500 /ig/kg body weight. The compound was applied as a 0.01% solution in acetone to the abdominal skin which had been shorn. After the acetone evaporated, the trunk of each rabbit was wrapped in cotton to pre vent ingestion. The rabbits were housed in individual holding cages and were observed for signs of toxicity including body weight changes for three weeks. Parenteral lethality was determined by injecting rabbits of mixed sexes intraperi- September 1973 CORRECTION: t h e t e r m u g o r m g /k g - d a y ' on pp 89-97 sh o u ld read ug o r m g /k g /d a y . 87 16672 / /-> i toneally with 31.6, 63, 126, 252 and 500 fig/kg of 2,3,7,8-TCDD as a 0.01% corn oil suspension ; control rabbits were injected with corn oil. The rabbits were housed in individual holding cages and were observed for signs of toxicity for four weeks. The LD.-,o's were calculated by the Weil modi fication of the Thompson method (8, 9) or by the Litchfield and Wilcoxon method (10). The acute lethality studies were terminated when it was evident that the survivors were not showing signs of toxicity. Eye Irritation Rabbit eyes were examined prior to ex periments and found to be free from defects or irritation. Approximately 2 mg of 2,7DCDD, 2,3,7,8-TCDD, HCDD, or OCDD were instilled in the conjunctival sac of one eye; the contralateral eye served as a control. The eyes were examined at various times after treatment for conjunctival redness and chemosis, iritis, and corneal injury. Re sponses were categorized according to in tensity. Rabbit Ear Bioassay For Acnegenic Activity Acnegenic activity of 2,7-DCDD, 1,2,3,4tCDD, 2,3,7,8-TCDD, HCDD, and OCDD was tested by applying 0.1 ml of either a solvent solution or the supernatant of a solvent suspension of each compound to the inner surface of the rabbit's ears five days a week for four weeks. The ears were ex amined weekly for signs of chloracne, in flammation and hyperkeratosis. The re sponses were divided into five categories: (1) none, (2) very slight, (3) slight, (4) moderate, and (5) severe. Responses in the first three categories in clude no response to mild irritation, in creased ear thickness, slight enlargement of the follicular aperture, slight exfoliation and slight crust formation. These responses alone are not considered indicative of chloracnegenic activity. Categories 4 and 5 are indicative of acnegenic response and are characterized by comedo formation, in creased ear thickness and hyperkeratosis. September 1973 Teratology Pregnant adult Sprague-Dawley (Spartan strain) female rats weighing approximately 250 g were used to study teratogenicity of the chlorinated dibenzo-p-dioxins. The day sperm were first present in a vaginal smear was considered day zero of pregnancy. The animals were housed individually in wirebottom cages in a room controlled for tem perature, humidity, light cycle and noise. Commercial laboratory rat chow and water were provided with choice. Corn oil: acetone (9:1) solutions with varying amounts of test material were given, in 2.5 ml/kg dosages by gavage. Dosages were calculated, using daily body weights. Rats were treated with 100 mg of 2,7DCDD/kg-day, 0.1; 1.0, 10, or 100 .g HCDD/kg-day and 100 or 500 mg OCDD/ kg-day on days 6 through 15 of gestation. Control rats received 2.5 ml/kg of corn oil: acetone (9:1) orally. All rats were observed daily throughout pregnancy and were weighed on days 6, 13, and 21 of gestation. Pregnant females were sacrificed by carbon dioxide anesthesia on day 21 of gestation; the uterine horns were exteriorized through a midline incision in the abdominal wall, and the number and position of live, dead, and resorbed fetuses were noted. After be ing weighed and sexed, the fetuses were examined for external anomalies; the crownrump length was measured with a vernier caliper. Half of each litter was preserved in Bouin's solution and later examined for soft tissue anomalies (11) \ the other half was preserved in alcohol, cleared and stained with Alizarin Red-S, and examined for skeletal abnormalities (12). A 2 x 2 contingency table was used to evaluate the frequency of anomalies and resorptions within the fetal population and between litters. Body weight and body mea surements were statistically analyzed by an analysis of variance and Tukey's test (13). In all cases, the level of significance was P<0.05. Chick Bioassay for Chick Edema Factor The bioassay for chick edema factor was 89 i i 16673 Table 3. Lethality of 2,3,7,8-tetraehlorodibenzo-p-dioxin * Species and sex Rat, male Rat, female Guinea pig, male Guinea pig, male Rabbit, mixed Dogs, male Dogs, female Sample " c c c d c c c c c Route of adm inistration Oral Oral Oral Oral Oral Skin intraperitoneal Oral Oral Time of death, days postadminis tration 9-27 13-43 5-34 9-42 6-39 12-22 6-23 9-15 -- LD,, m g/kg 0.022 0.045 (0.030-0.066) 0.0006 (0.0004-0.0009) 0.0021 (0.0015-0.0030) 0.115 (0.038-0.345) 0.275 (0.142-0.531) -- Dose, m g /k g 0.008 0.016 0.032 0.063 0.032 0.063 0.126 0.252 0.500 0.30 3.00 0.03 0.10 Number deaths/ number treated 0/5 0/5 10/10 5/5 0/5 2/5 2/5 2/5 3/5 0/2 2/2 0/2 0/2 Responses to individual doses are given in thne cases in which an LD*> could not be calculated. The LDm for oral adm inistration to rabbits was calculated by using the method of Litchfield and Wilcoxon (9); the rem aining values were calculated by using the Weil modification of the method of Thompson (15, 16). " Letters refer to sample identification in Table 1. ient pain and conjunctival inflammation, initially. Treatment with 2,3,7,8-TCDD was associated with delayed conjunctival chemosis 13-22 days later. By day 27, the chemosis had subsided, but the rim of the eyelid was thickened and encrusted. In rabbits treated with HCDD, the rim of the eyelid was en crusted 27 days after treatment. Neither corneal injury nor iritis was observed in any of the animals following: instillation of the chlorodibenzodioxins in the conjunctival sac. Acnegenic Response Both 2,3,7,8-TCDD and HCDD produced acne in the rabbit ear bioassay as indicated by the formation of comedones. Solutions of 2,3,7,8-TCDD (sample c) in benzene rang ing in concentration from 0.04 to 400 ig/ml produced a positive response with severity increasing with concentration. A negative response was obtained with a solution of 0.004 Mg/ml. In contrast, a chloroform solu tion of 1,2,3,4-TCDD, 50 /*g/ml, did not produce a positive response. With HCDD (samples a, b, c, and d), a response was produced by solutions of 10 to 50 ig/ml in chloroform and dimethoxyethane. Chloro form extracts from 10% suspensions of 2,7DCDD or OCDD were negative, indicating that these have a low order or possibly no acnegenic activity. Teratogenicity The effects of chlorodibenzodioxins on maternal and fetal body measurements, in cidence of fetal resorptions and anomalies are given in Tables 4 and 5. 2,7-DCDD. Rats treated with 100 mg/kgday on days 6 through 15 of gestation gained slightly more weight during pregnancy than controls but showed no toxicity. There was no effect on fetal body measurements, or in cidence of resorptions, or gross, soft tissue or skeletal anomalies. HCDD. Administration of 0.1-100 ng HC DD kg-day was associated with a doserelated decrease in maternal weight-gain September 1973 91 September 1973 Table 4. Effect of treatm ent with chlorinated dibeuzo-p-dioxin on m aternal and fetal body measurem ents and the incidence of fetal resorption. Test compound (sample) * Maternal weight gain, g " Petal Fetal Fetal resorptions, % No. of ---------------------------------------------------------------------- body weight, cro w n -ru m p ' ---------------------------------------- litters Days 6-13 Days 13-21 Days 6-21 g* length, mm Population" Litter * Control 30 2,7-Dichlorodibenzo-p-dioxin (d) 100.0 m g/kg-day 7` Hexachlorodibenzo-p-dioxin (c) 0.1 ^g/kg-day 19 1.0 ^ g/kg-day 10.0 ^g/kg-day 100.0 fig/kg-day 19 18 19 36 2 31 1 28 2 27 3 22 3 ' 6 2' 101 6 122 4 102 5 99 5 97 6 13 7 ' 137 8 5.68 0.05 44.5 0.1 7 ( 22/337) 47 (14/30) 162 5 6.80 0.09 44.2 0.2 6 ( 5 / 86) 67 ( 4 / 7) 130 5 5.73 0.04 43.8 0.1 5 ( 10/217) 47 ( 9/19) 126 6 5.93 0.16 45.7 0.5 9 ( 20/218) 74 (14/19) 119 6 5.12 0.0 5 ' 42.6 0 .2 ' 25 ` ( 57/229) 9 4 ' (17/18) 19 9 r 3.65 0 .2 8 ' 36.2 0.7 r 8 6 ' (194/227) 1 0 0 ' (19/19) Octachlorodibenzo-p-dioxin (d) 100.0 m g/kg-day 500.0 m g/kg-day , 12 17 32 2 35 3 100 8 115 4 131 7 150 6 5.73 0.09 5.69 0.05 43.6 0.4 44.6 0.2 8 ( 11/131) 42 ( 5/12) 5 ( 9/199) 41 ( 7/17) ` Sample identified in Table 1; administered on days 6-16 of gestation as a corn oil: acetone (9:1) solution. b Mean S.E. for various gestation times. ` Mean of litter means S.E. *% (number resorptions/number implantations). * % (number litters with at least one resorption/number litters). ` Significantly different from control by an analysis of variance and Tukey's test (measurements) or the 2 x 2 tions) , P <0.05. contingency table (resorp .. C* M O Q 05 CJt er 1973 Table 6. Result* of chick edema bioassay: body weight, food consumption, and pericardial fluid volume calculations of chicks treated with chlorodiozins. Treatm ent (sample)* n Pericardial fluid volume Body weight, g 11 ----------------------------------- --------------------------------------- Food Mean log Day 0 Day 21 consumption, g ` ml S.E. (100 X ml) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (d)d o.oi0 MgAg iig/ke 0.10 /g /k g 1.0 MgAg * 10 46 1 210 44 1 10 46 1 j 42 1 10.0 M gA g' 9 42 1 Hexachlorodibenzo-p-dioxin (c)d 0 0.1 1.0 10.0 100.0 Mg/kg Mg/kg Mg/kg Mg/kg Mg/kg* 9 10 10 9 10 38 1 42 1 43 1 38 1 36 1 199 6 196 7 203 7 196 24 No survivors 6194 197 4 196 6 187 7 No survivors 17.4 16.7 17.2 33.7 11.2 ' 17.4 17.8 18.2 16.7 13.3 Octachlorodibenzo-p-dioxin (d )` 0% of diet 0.1% of diet 0.6% of diet 12 11 11 46 1 46 1 43 1 (Day 20) 141 8 124 6 196 10 13.3 9.5 10.9 * Sample identified in Table 1. ` Mean S.E. * G ram s/chick/day. 'A dm inistered orally as a corn oil: acetone solution. * Animals died on days 9, 11, 11, 14, 16, 17, 18, and 19 of treatm ent. ' Animals died on days 3, 4, 4, 4, 6, 8, 8, 9, 12 and 16 of treatm ent. * One animal died on day 19 of treatm ent. ` A nim als died on days 6, 6, 6, 7, 8, 10, 11, 11, 16 and 17 of treatm ent. 1Fed in the diet { 0 .1 % = 100 m g/kg, 0.6% -- 600 m g/kg). 0.16 0.02 0.14 0.02 0.19 0.01 2.34 0.08 1.29 0.62 0.16 0.01 0.11 .0.02 0.09 0.01 0.81 0.01 0.62 0.24 0.08 0.01 0.06 0.01 0.09 0.01 1.1717 1.1181 1.2688 2.3680 1.5661 1.1771 0.9978 0.9387 1.7294 1.5650 0.8053 0.7889 0.9002 Positive for chick edema factor based on Calculated ---------------------------------------t value Calculations Gross lesions -- -0.74 + 1.69 + 21.7 + 1.47 -- -1 .9 3 -4 .6 7 + 3.82 + 2.72 -- -0.21 +0.91 -- No No No No -- No No No No -- No No -- No No Yes Yes -- No No Yes Yes -- No No IN* .eIDn 05 05 T / ^ / M O a { 8 6 tC e ^ o a Microscopic examination of this organ re vealed a highly variable pattern and degree of hepatic necrosis with various degrees of degeneration and regeneration of the hepatocytes, depending upon the post-treatment in terval. Necrosis was observed both in the centrilobular and periportal areas. The de gree of necrosis of the liver was not sufficient to conclude that it was responsible for death. Hepatic lesions were observed in rats, mice, rabbits, and dogs. In addition to hepatic involvement, other changes observed sporad ically include fat necrosis, periarteritis, ser ous atrophy of fat, and ascites. DiscussionandSummary The studies reported here confirmed the high toxicity of 2,3,7,8-TCDD. In addition, some perspective of the relative toxicities of 2,7-DCDD, HCDD, and OCDD has been obtained. 2,7-DCDD and OCDD failed to cause death in female rats given oral doses of 1 g/kg; even larger doses were given to mice without causing death. Limited data suggest that oral doses of approximately 100 mg/kg of HCDD are needed to cause death in male rats. In the teratology study, no deaths occurred following administration of 100 /ig/kg of HCDD to female rats for 10 consecutive days. 2,3,7,8-TCDD is much more toxic than the other chlorodibenzodioxins studied; the LDso ranged from 0.6 /tg/kg in male guinea pigs to 115 /g/kg in rabbits. Dogs appear to be less sensitive than rabbits. Others have reported 100% mortality in rabbits treated with 10 /ig/kg (15) and chick embryos treated with 0.05 /*g/egg (5). Death following treatment with a lethal dose of 2,3,7,8-TCDD is often delayed for several weeks. Among the animals which died following treatment, approximately half the deaths occurred between 13 and 18 days after treatment, with one animal dying as late as 43 days after a single oral dose. In mice and rabbits, there is a marked in dividual difference in susceptibility to this compound which makes it difficult to con duct acute lethality studies. If the results of the rabbit eye irritation September 1973 test can be extrapolated to man, accidental contact of these chlorodibenzodioxins with the eyes should not present a serious threat to vision. However, repeated contact with the skin of small amounts of either 2,3,7,8TCDD or HCDD may be expected to produce chloracne. Sensitivity to 2,3,7,8-TCDD was recognized by industry years ago, and preTcautions have been taken to minimize itV* occurrence and prevent contamination o worker's skin. HCDD is apparently a less'^ potent acnegen than 2,3,7,8-TCDD. As previously reported, 2,3,7,8-TCDD is highly embryotoxic (6). The no-effect level for embryotoxicity was 0.03 ^g/kg-day of 2,3,7,8-TCDD. In contrast to the high em bryotoxicity of the symmetrical 2,3,7,8TCDD, 1,2,3,4-TCDD was not embryotoxic at doses as high as 800 /tg/kg-day (IS). By previously described definitions of ter atogenicity and embryotoxicity (17), HCDD is teratogenic in the rat at a 100 /xg/kg-day dose level, given orally on days 6 through 15 of gestation. Treatment of pregnant rats with HCDD caused embryotoxicity evid enced by a dose-related decrease in fetal body weight and crown-rump length and an increase in the incidence of fetal resorp tions (Table 4). Likewise, the incidence of certain soft tissue and skeletal anomalies increased in a manner related to the dose level of HCDD (Table 5). A 0.1 Mg/kg-day dosage of HCDD had no effect on embryonal or fetal development. OCDD caused embryotoxicity but was not teratogenic at 500 mg/kg-day. OCDD and 2,7-DCDD caused neither teratogenicity nor embryotoxicity at 100 mg/kg-day. Khera and Ruddick (16) reported that the ad ministration of 2 mg 2,7-DCDD/kg-day was associated with microscopic myocardial and pericardial lesions in rat fetuses. However, examination of sections of myocardium and pericardium from fetuses of dams treated with 100 mg doses in this study revealed no morphological differences from controls. Both 2,3,7,8-TCDD and HCDD give posi tive results in chick edema bioassays (Table 6). This HCDD result is consistent with a previous report that the HCDD isolated 97 16677 16. Khera, K. S., and Ruddick, J. A. Polychlorodibenzo-p-dioxins: P erinatal effects and domi nant lethal te st in W istar rats. Adv&n. Chem. Ser. 121, R. F. Gould, Ed., American Chemical Society, W ashington, D.C., in press. 17. Schwetz, B. A., Sparschu, G. L., and Gehring, P. J. The effect of 2,4-L and esters of 2,4-D on ra t embryonal, foetal and neonatal growth and development. Food Cosmet. Toxicol. 9: 801 (1971). DOW 331986 September 1973 r 99 166TS 2694 /* # DOW Formation of Dibenzodioxins and Other Condensation Products from Chlorinated Phenols and Derivatives kr H.G. Lanier,' T.P. Brady,' aid P.R. B rin s ' Introduction Chlorodioxins can be formed in a two-step condensation reaction from oriAo-substituted chlorophenoxy radicals or anions (1-3). The rst route is of significance only where strongly oxidizing conditions exist such as a reaction of chlorine with pentachlorophenol at elevated temperature. For the second route we have investigated the condensation of alkali metal salts of chlorinated phenols which occurs spontaneously when these metal salts are heated to temperatures above 300C. Since this reaction is strongly exother mic, it proceeds to completion in a very nar row temperature range once initiated. For bimolecular reactions involving ortho chlorines of both phenate molecules, dioxins would be formed according to . the scheme shown in eq. (1) as one of the possible reac tion pathways. If these phenates contain halogen substituents in meta or para posi tions, these also are involved in the conden sation reactions and the product distribu tion depends on a variety of factors such as (1) the total number of halogen substitu ents which determines the ease of removal of halogen ; (2) the arrangement of the mole cules within a crystal if this reaction takes place in the solid state, which in turn is in fluenced by the metal cation involved; (3) a ste rn Research Laboratory, Liow Chemical USA, W ayland, M assachusetts 01778. September 1973 steric effects which may facilitate the at tack by the second molecule at a particular carbon or may prevent a nucleophilic attack at a particular site; (4) electronic effects which in this case allows halogen atoms in meta positions to be removed more readily than those in the other ring positions (4). Experimental Thermal reactions were carried out in bulk as described below or in a differential ther mal analysis cell using a DuPont 900 Differ ential Thermal Analyzer. Residues and sublimates were analyzed by mass spectrometry on an A.E.I. MS-12 in strument coupled to a D.E.C-PDP-12 com puter using a program called MASH (5). Results The results of our extensive studies are summarized in Table 1, which contains all the pertinent information on this compli cated reaction system and with this the an swers to many questions regarding dioxin formation. From electronic considerations Cl atoms marked with an asterisk (*) and located in meta positions are more likely to be re moved than any of the others (4). The first two reactions occur in the solid state where the molecules--anions as well as alkali metal cations--are locked into positions which may have an important effect on the reaction 3 DOW-331898 oo Table 1. Pyrolysis products from Na chlorophenates by mass spectrometry. Compound Sodium pentachlorophenate (solid) m/e 456 684 912 Rela tive inten sity, % Species Dioxin 2 Trimer 0.3 T etram er (8 Cl) (12 Cl) (16 Cl) Approx, yield, % o- C1 i Cl Sodium 2,3,4,6-tetrachlorophenate (solid) 388 582 100 Dioxin 3 Trimer (6 Cl) (9 Cl) gO Sodium 2,4,5- 320 100 Dioxin trichlorophenate (at mp) 35-356 30 Dimers 480 4 Trim ers (4 Cl) (5 Cl) (6 Cl) o- C1 1 C\ Cl* Cl Sodium 2,4,6- 320 100 Dioxin trichlorophenate (a t mp) 480 33 T rim er (4 Cl) (6 Cl) 640 50 T etram er (8 Cl) 800 30 Pentam er (10 Cl) Sodium 2,4dichlorophenate (at mp) 252 100 Dioxin (2 Cl) 378 10 Trim er (3 Cl) 504 1 Tetramer (4 Cl) o0- Q1 Cl Cl % I Sodium Irgasan (molten) 252 100 Dioxin (2 Cl) 378 10 Trim er (3 Cl) 414 20 Trim er (4 Cl) 504 16 T etram er (4 Cl) 540 12 T etram er (5 Cl) September 1973 f 16680 Cl o:_m _____c i: OM Cl Cl Cl' Cl ooG T eeM oa etc. (2) REFERENCES 1. Kuika, M. Octahalogenodibenzc-p-dioxins. Can. J. Chem. 39: 1937 (1961). 2. Tomita, M., Ueda, S. I., and N arisada, M. Dibenzo-p-dioxin derivatives. XXVII. Synthesis ox polyhalcdibenzo-p-dionin. Yakugaku Zasshi 79: 186 (1959). 3. Pohland, A. E., and Yang, G. C. P reparation and characterization of chiorinated dibenzo-p-dioxins. J. Agr. Food Chem. 20: 1093 (1972). 4. W eingarten, H. Ullman condensation. J. Org. Chem. 29: 977 (1964). 5. Briggs, P., Dix, D. T., Glover, D., and Kleinman, R. A m u lti instrum ent d ata acquisition system for use with mass spectrom etry. Am. Lab. 4: 57 (Sept. 1972). 6. Buu-Hoi, N. P., et al., P rparation, proprits et identification de la "dioxine" (tetrachloro-2,3,7,8dibenzo-p-dioxine) dans les pyrolysats de dfoli ants a base d'acide trichloro-2,4,5-pihenoxy-acetique, et de ses esters et des vgtaux contaminns. C. R. Acad. Sci. (P aris) Ser. D 273: 708 (1971). 7. Boer, F. P., et al., The crystal and molecular structure of 2,3,7,8-tetrachloro-dibenzo-p-dioxin. Acta Cryst. B 28: 1023 (1972). September 1973 . .. ;7 1 16681 TESTIMONY OF A. T. TALCOTT .) -t-A \ SHOECRAFT ET AL. VS. DOW 2723 DOW 1469660 .J I am A. T. Talcott, administrator of Regulations and Labeling in the Quality Assurance Department of The Dow Chemical Company. I have a Bachelor of Science degree in chemistry from Alma College, Alma, Michigan, and have done graduate work at Michigan State University but have no advanced degree. I joined The Dow Chemical Company, Midland, Michigan, in 1957 as a chemist in their analytical laboratories and moved through a series of technical promotions in the analytical chemical field. In 1965 I moved to the Labeling/ Product Registration section of the Legal Department, becoming supervisor of that section in 1975. In 1976 I became manager of Product Safety Compliance in Dow's Quality Assurance Department, which included the functions of labeling, material safety data sheets, and product registration. In 1979 I assumed my present position where I am responsible for labeling policies on Dow products. I am a member of the American Conference on Chemical Labeling, the Labeling Standard Revision Committee of the Chemical Manufacturers Association, a member or chairman of several committees dealing with regulations and labeling within the Chemical Specialties Manufacturers Association, a former member of the Labeling and Precautionary Information Committee of the Chemical Manufacturers Association, and the Labeling Committee of the National Paint and Coatings Association. I was also one of two industry representatives participating with EPA to put together their National Symposium on Pesticide Labeling in 1974.' I will testify on the history and procedures used in the development of ; labels by The Dow Chemical Company and to the registration requirements for_ labeling used by the Pesticide Registration Division of EPA which was pa of the USDA in 1968 prior to the formation of EPA. I will also testify to J u 0 jL -- -------f w ic o o ^ u iu u s , L e c iin ic a i specialists, and legal and regulatory counsel in areas, pertinent to such a product. The labeling used on KURON weed and brush killer in-the~time"period"1967" through 1969 was in^part specified in*rgultions for pesticides which were called economic poisons under the statute in effeet, at'that -time-i Theselrequired the product to bear a label containing the name of the product, the name and address of the manufacturer or registrant, the net contents, an ingredient statement which was prescribed in a special manner, a warning or caution statement as appropriate to regulations prescribing same, and directions for use-- which if complied with, would be adequate for the protection of the public. These elements of a label were further prescribed in a certain manner by the regulations and interpretations promulgated by the Pesticide Registration Division and required a preclearance or acceptance by that Division before they could be used on the package of the pesticide. This labeling was also submitted to the state of Arizona and other states for review as part of the state registration of the product required prior to sale. The product name KURON, the Company name, and certain other elements such as the registration number required to appear on the label are for the most part self-explanatory. For precautionary labeling purposes,pesticides were placed in one of four dLiAM idi different categories based on certain toxicity data that would be animal tests TUX' ' provided to the Pesticide Registration Divisions These were aallad-categories one, two, three, and fourj[ vfpih category one being materials that were highly toxic and requiring the word "poison" and the skull and crossbones to be used on *Trademark of The Dow Chemical Company 1 ' 1469662 ___ _ LHJ.1.U LaLcgutiw were ten fold decreases In toxicity from the lower-numbered category. During this time precautionary statements used on pesticides were set forth in a document called "Interpretation 18, Revision 2" published in the Federal Register of March 9, 1962 as an interpretation of 7 CFR, Part 362.- There were two ways in which this interpretation was used. .One related to signal words and statements of hazard that were used on pesticidesycontaining specifically-listed active ingredients and the other set forth general procedures based on the toxicity categories previously mentioned which- was applicable to KURON weed and brush killer. Applying the principles stated in Interpretation 18, one comes up with the precautionary labeling which was accepted by the government as adequate to protect - -- ---v the public.and used by Dow on.its product KURON weed and brush killer.-^ Quoting from section 362.116(b)(2)(III), "Labels of products in the third category should carry the word "caution" in statements indicating the means of avoiding the principal hazards of use. Use of the skull and cross bones, the word "poison,'* and antidote statements are not necessary for these products.'/ Another part of the label which may need more explanation is the ingredient statement. Ingredient statements are required to be made on all pesticide products and must be stated in certain ways. The constituents of a product must be characterized as either active or inert ingredients. The active ingredients must be described by their chemical name. The two ways in which ingredient statements can be listed were specified 3 ? ,K j a J A i - 4 . in section 362.7 of the-regulations. -Thesesw?e to list each active ingredient and its percentage and the total of the inert ingredients or to list the total of the active ingredients and each individual active ingredient in descending order of percentage.without the actual percentage of each ingredient.plus the ijeSF-"iXiKis jr.rxr .1 this product. Such a listing would not have been acceptable from the government's standpoint since this is not an active pesticidal constituent; additionally, the initials TCDD do not represent a common chemical name acceptable to the federal government so such listing would again be prohibited. The other reason for not listing TCDD as an ingredient is that cue limits of analytical detection at the time of production of the KURON in question were such that TCDD was not found in this product. There are other sections of the label that are, of course, pertinent to the allegations made concerning the product KURON weed and brush killer. Among these are the instructions under a section titled "Warning" that "applications by airplane, ground rigs, and hand dispensers should be carried out only when there is no hazard from drift." Other instructions which are meaningful to a user of such products are the instructions that "coarse sprays are less likely to drift" as well as the instructions on application rates and times for specific applications of the product. The procedures followed for labeling pesticidal products are an application of the principles that are included for Labeling of chemicals set forth in the American National Standard for Precautionary Labeling ANSI Z129.1. '.This is a consensus standard which was not published until 1976, howevery~.does-have the benefit of review of such organizations as the American Society of Agricultural Engineers, the National Safety Council, the American Society of Safety Engineers, the Society of Toxicology, AFL-CIO, the American Conference of Governmental I in * --J -- u r / J a i 'l j ; ! ' 0 ;? \z Industrial Hygienists, and numerous other government, industry, and independent organizations with an interest in conveying precautionary information. These principles prescribe a signal word, statement of hazard, precautionary measures, and as appropriate, instructions in case of contact or exposure as procedures for providing precautionary labeling. The labeling used by Dow on KURON weed and brush killer demonstrates these principles. The procedures used within Dow to develop labeling in 1967-involved the initial labeling being developed from a use standpoint by a technical specialist from our Agricultural Products Department along with the individual responsible for submitting registration applications to the federal government, and precautionary information coming from a labeling specialist like myself. This draft copy then would be typed and sent for review by the numerous specialists within the Company with input on the health and safety aspects coming from toxicologists, industrial hygienists, medical doctors, individuals from our quality function in connection with our manufacturing people providing input on the composition of the product, trademark and patent comments coming from our attorneys, and the original three persons reviewing these inputs and those of our marketing personnel. Once these inputs had been composited,, the labeling was sent to the federal government for their acceptance or suggestions for change if they^-did-not accept it. upon return of a copy of the accepted labeling, the labeling specialist would initiate che actions responsible for getting the labels or the preprinted container copy to our production people for application on containers of the final product. It is important to remember when developing or reviewing labeling for a product that the labeling must be based on the product as a whole; consequently, the labeling must be viewed in its entirety, not just in parts-- since it is the. whole product that is used, not a part. 16686 DOW 1469665 I have compared the labeling used by other pesticide producers of similar products and find the- labeling used, which also was required to be submitted to the Pesticide Registration Division before being used, to be essentially the same where applicable as that used on the product KURON weed and brush killer. Based on my education, training, and experience, it is my opinion that the label used on the product KURON weed and brush killer in the time period 1967' through 1969 met or exceeded all regulations and standards of care pertaining to such products; and further the directions and precautions if followed were adequate to prevent accident or injury with the handling and application of the product. ; 16687  D o v y 758726 CM O MN073280 > CM i it iii r r c=* 0 VJ n l 16688 ii\ i i W. -.'m ' ; . ( j p . i t . l i - ? li b | i f ' i . m - . l l)m\ Cli. >-=- at USA )1 ,C r - ' - < pThheencouxrryehnet srbtaictuidseosf if /y DOW 758727 U-VCKCIU >UND In January of 1971 a review of "The Status of 2.4-D. 2.4.5-T. Silver and MCPA_Herbi cides" was reported in DOWN TO EARTH* (Vol 26. No 4). It now seems desirable and beneficial to repeat pertinent parts of that paper as well as to include additional data and information generated since then This new review concerns itself with 2.4-D (2.4,-dichlorophenoxyacetic acid). 2.4.5-T |2.4.5-trichlorophenoxyacetic acid), silvex 12-i2.4.5-trichlorophenoxy) propionic acicf| and MCPA 12-methyl-4-chlorophenoxy acetic acid) While these herbicides appear to be quite similar in structure, each com pound exhibits unique characteristics with respect to selectivity for broadleaf weed and/or brush control. The formulation ol these compounds into specific esters or amines further contributes to species se lectivity as v.ell as to particular techniques for spray preparation and application The phenoxy herbicides have been uti lized commercially lor twenty years or more without harm to humans or wildlife and were registered on the basis of safety tests considered adequate by USDA at the time In 1966 the USDA and FDA. acting on a Study conducted by the National Academy of Sciences, declared that the concepts ol "no-residuo" and 'zero tolerance" rcqistrabon of pesticides on food crops wen; no longer tenable and residue tolerances would have to be established to enable mntmued registration Linder these concept.-> In ordei tii accomplish ties, additional 1 ixicity ami analytical studies undated to meet i ui rent leguitemenls. have been i rguued by USO A and FDA Pesli. ii It- ri11 .lialii.i 11.a, now been transferred to the Environmental Protection Agency (EPA) Scientific studies subsequent to April 1966. when the USDA abolished the no residue status, have shown the phenoxy herbicides to represent no hazard to man and his environment when properly applied in accordance with label recommendations. The issue of 2.4.5-T on food cr ops is soil undergoing review hy EPA. primarily at the insistence of environ mental groups and as an outgrowth of ns previous usage in Viet Nam. It is extremely important to recognize that industrial, for estry or rangeland usage of 2.4.5 T are not affe cte d by the current controversy The 2.4.5-T issue has become one of principle rather than monetary value to the manu facturers and it has been in this context that Dow has continually pursued the sci entific route to resolve the potential hazard of 2.4.5-T to man and his environment I Ir.;*;..:i.:: i,| Events Development of the phenoxy hedmad- in the; mid-forties resulted in three nui|oi hei bicidos for right-of-way. forestry nun. other industrial markets These product-. (2.4-D. 2.4 5-T and silvexi ate not pinim ; by patemts Seven commercial compac.ie were manufacturing one or more of tl..... three products and oi MCPA at l1'.- b" whim USDA announced abolishne-nt u! ' no residue status on food and (mi . on April 13 1966 Tli^ai; worn D... Dow Heii.ules Monsanto Rbudci In son and Thompson Hayward Tlu-y to Ioiiii III..- Imlusliy T.r.n Fun im m ' Hi-iOk 1.1. Ti.11>i an. .m . ITf hi 11 . Ti tit.-1.:y V i r i .>. --i 1-, .a.'.. . .1 16689 v`- ' Task Fhim1 mfHlilics prlilions by rt-(|iK.'slin<| 300 ppm U!!,hImi: I H ) | f h ^ l l i l i t t i f M I I t y C r i t o n i w i ................. li.-.iiiii'j lo '.iftoril regi*.Iranis .io1iln- : il.'i tolei.Miu! for eiK.li |Jienoxy hcrhu:id<: iii/oii tilt.' nppothinily to present Hu: |/crlini:n: range or pastuie grosses economic and other data relating to the toil ...... .. > i / : Judgment entered l>y ance that the law requires to be struck be U S Circuit Court of A|j|xj;iIs. District of tween benefit and risk " Hearing to lie held ... aV. Columbia. Pa::. Petition for. review, of prefer . in the fall but no specific date set. Harrison Weilford. et al (fratiiionorsf wk*. ?..' ^Company ob|0C if H i^ r r v 7w ller> of Aiiqus 6 and requests EPA administrator to write " "..y' . .* : V ` 't iiii-i.f~e' - #y; ~*4V registrations I 1 All granular 2.4.5-T formulations for use around the home, recreation areas and similar sites. ...: .r 2. All 2.4.5-T uses on food crops intendod for human consumption. The case is remanded to EPA adminis trator "for further consideration and for a decision supported by a reasoned analysis of the relevant,factors." - . x y . : . . . Id. 1971 Ruckelshausannounces 60 day review beginning January 18. All interested groups and individuals ate in vT iittWeMd ti wo wsuwbh mm miti ,. iiin| .wt irii ti iimnmg an fy *andmdmitwioi ni hai l N o v i - t 977rJ1EPA states th'ug- ust 6 order should remain in effect in spite of Dow's objections to the order,, thereby continuing4he(cancellation of, 2.4.5-T on rice. Ruckelshaus states that the hearing should proceed "forthwith" October N oyeivbet 1971 Task Force sub mits amendments to petitions containing .,. ,, . additional data oh residues inmeat and meat b y < p r ^ c Y ^ ^ ^ f ) ^ ;1)hd 'X phenoxy compound and its corresponding'- ^ phenol as requested ,by EPA July ,12^19X1..^ ReceipPiackriovvIedged by EPA, and re < . ;.i' 1 " ' , m Cy V^,, ,'f 4.; ...V O data.or a r g u m e n t o n 2.4.5-T is or is not an imminent hazard to - r- -- ' : the public. - .: , ,0^ P ub^ February Scientific Advisory'Com- *" y word. : v*,. jnmiitttteee' hlds^first;.ImMeeettiinngg bopn canec?ejlll^atiobpn: wc'OSAifiiffJQ: me.no ---------je beep receivea! ' ii Of 2.4.5-T jf^^rpl^u^o a p f ^ J and Hercules (May 28. 1970). /Way 7. 797./.. 2,4.5-T Advisory Commit- .^ __ tee report suiifpitied to ERA a d m in is t r a t o ir .^ ^ ^ J ^ M g buJtunlyot 2r3e,tea1sQe7d.1a- t his time. ' - ; 2.4.5-T Advisory -' Com mittee report'released by EPA. Committee recommends^ by a 8:1 vote, restoring regis tration of 2.4,5-T lo status existing' prior to April 1970 with following conditions: (JJ0.1 ppm tolerance of 2.4.5-T on edjbje food crops . (2) limit of 0 5 ppm TCDD* on existing ' To(er^ c e s ^ several formulations of 2.4-D in apples. ;;V barley, grapefruit.'lemons, oats; orangesJ ; pears, rye a r^ ^ ^ a ^ A tplerance has afs^' been establi^^^or^Tfl-P in asparagus ` '' 'v V ii- from uM ofonry;1He!s0dium salt. Oata?;priginalfy yBfnttted with the peti tions in December, 1967 is expected to be sufficient fqr 2.4-D"1ri blueberries, crar.- r-j-it'.- J . -A- 7..S2,^.^-T..'|fwent.pries and 0.1 ppm on berries and grapes and for silvex in aiiples 'future,2,4,5-T. production with.certi-/; and piums.' Data qrf^r'e.sidues in grass in fied analysis being furnished by man- . eludes that" from previous work and from ;V- ufacturer:toiEPA;v^rrv.Vrf-v:^.<.:;-. " residuap'Sjamjifes analyzed by Dow in 1970. (3) formulations around home bear wartv Additional work' supported by the Task : . . ing of -possible .danger to pregnant women and exposurej o : (4) condual^p^^ffic ri (5) additional post registration monitor- - inq phenol in the anml tissues and milk mg for adverse affects Residue analyses indicate 0 1 ppm In, ,, ' 7- 7 EPA issues a purported a|| phenoxy acifJs in mC)bI food m o ;.s 1' Q A Determination and Order" on 2 4,5-T which tHlle of harvesl Neq!l(J(ljie continuos tHeMoceUmjon f y lJ,J'O>W` ^ ' ^r.v.v* ,r^, . -,il>n: levtrl cst Iu l i l i s h i :i III April 1`J67 (oi wlnai ..Iiiilu o by Bioiiclu s Ri.M'.iii.h LaPfir.i .-an,ill (|(.llllb lory implii.-d Ili.it 2 4 5 T a vmy high f P i .i(t Feuding studies in d.nry i.ows won; i.on- levels w as teiat uijt.-i ii'. (producing mat ducted for each plienoxy compound at levels formed fetusesi m mice and rais Subse- of 30. 100 and 300 ppm for two weeks and (|uent studies showed that a toxic contam i at 1000 ppm for three weeks in the total nant. 2.3.7.8 tetraohlorodiljoiuo-p -dioxin diet. Milk w as collected and analysed for (TCDD) w as responsible for som e. residuesitJhifrrdata iodicaieyteljtigffliir i M i P i y ' "- t 5resiBCTS S ^ fi^|Sfrv'gtt le ~fifi for 2.4.5-T, and that t w o 'w es"`sfimid contained z 27 ppm TCDD.-x? so ffic^C w ren t .restrictions^ *r q ir e f & six w eek^ffjg wi!t^2,4iS1v N restriction "" ?osos as^ i h l essary for'dairy cattle grazing^on' areas and in rabb'ts at doses as treated with 2.4-D. silvex or MCPA. day. The obvious concqrn is to produce The phenoxies were also fed to beef cattle 2.4.5-T without the contaminant. and sh ee p at levels up to 2000 ppni in J h f . i y '.,, TCDD can be formed in.th^(m an^fagture!.. total diet for four weeks. Animals !w'ere " of. 2.4.5-trichlorophenoi; t h e ^ V r e c t i r e ^ 'i ^ * : ^ .- ^ then slaughtered immediately or one week 2.4.5-T. The conditions required for its after withdrawal from a diet containing 2000 formation are high temperatures in th e :, pirin herbicide Based upon the 300 ppm presence of base, conditions which ' c a n . tolerance requested in/on g rass from appli- occur in th e alkaline hydrolysis of 1.2.4.5- ' for 2,4.5-T approved by EPA should suffice , cursor 2.4-dichlorophonol is made W S m S k S-i . for both silvex and 2.4.5-T. This states: "Da chiorinaticjn of phenlfand^ngCby ----------- vo. :> : r-> treated cation:- on/in a v' -wt; cHssmalteti'icrv; is similar'. _____ a location, vtr--------**-*- -- * phenoxy h Toxicplpg Negligible tained based on information, front. 90 day feeding studies in two species of rjtammals. However,.tolerances at higher (permissible) m u i. " '.y i t i residue""levels require twd^yeari feeding* studies in {airs and dogs, plus fertility and reprr^iicti^t studies in rat&^AOtM'.timej the jftencwy "herbicides were dei^ioped.* m m - ' " "';i L* -V f-!:z :* `It. _ uti # liti tvV.L 'f^.^&'rvwv d i Y~T-" y 1 these long , term feeding studies'were not S neceis^iyijsincB these compounds were 3 registere^l^^^h'-rsidue'rbsialf. -`, FDA "ital^onducted two & it &:*U. * - K 1 * j- ' ;/:>v U *S,- B..'/' i} t 'i . -.;v studies :an studies wi(ftHr2;4-D. Dow has^cohducfed , . - -Vg-- ^ two-year, f a d i n g . studies with .-.silvex but. fc,. not reproduction or fertility studies! Ninety^i day feed in g stu d ies have been^rud on ra js '* :'-V; andfftfo^s ii<5r?.2.4-D. , sruxiFte>U'srotf^bt;.4 S T-iv MCPA. Thd^no-ill effect i e v e i s ^ e 'I H ^ n l i d i t p Table 2 .^ -1^ 'U: ' t T -41 _ az Based on single oral doses in rats. 2.4-D. 2 .4.5-T. silvex and MCPA are classed as 'slightly toxic'' wnh LD-.o values caiujing from 300 to 700 mg'kg body weight /-!';; J'WEEKS AFTER APPLICATION 16691 2 2 . 7,8.7*eti(ichloioditu.-/uo p Dioxin. M o U 1 CiUpi aitJ M-.ijei s a n i c i for re;ra^e [ ) umici (he IlfPHT proijrm Vr i'A .1 S :-. rt'jne O.ix ; : ->; r. : ; r ..- . . x .. . , V : ' , ^ J .?>. V ; -'fevinW-:)? :-w. To date, analytical methods have been developed and validated to a sensitivity of 0.1 ppm for ;TCDD in 2.4.5-T acid. With {jersistenf^^ nof riB a d ily Je ^ n ^ ^ ^ ^ ^ ^ ^ ^ ^ s plants nor translocated to other plant pnaar/ttse';. *r.:;.-.,. : . and !itac_a_n_|_>_e_w_a_sh!.qX<|Jouf*'. . i <k *&*?**& ft' proper manufacturing controls. ?,4.5-T can ,'be.;Pfo<^ !jLil^yino..fl teqt0|^TQ O T^^;|JfcTCPDj^ j^ | ^ ^ j U teUel measured by net analytical met] '"; C^o--nsid'-*3--raub,l'-,''daia-ta---o--n- 'T tC'nDn0 ,hay -b--en t. except in tfi^m^jpi mm. regulations1? icaref T q u iii ,,rijtiHi. the Amertcahihstftutedf BiologicalSciences meeting in ^iQomington,. Indiana;i August r 26. 1970 iw.^TWa Public Health ^plications; : TH e^B JtypI of Widespread Use of the Phenoxy Herbi Rbcent^dey^ofweQ^piwpcalS; cides and Picloram." This paper was pub cloud surrounding trie; Z.i.'5-T controversy2*.:'1 lished in the September 1. 1971,. issue of is clearing! Trade ifiemdr^ndum R-.14 BioScience (Vol. 21. No. 17) and concludes ber 15.1971 from the Canada Departnv^-g^c.r.' with the st&ement that "Impurities! can be of an important factor--particulaily the chloro- "jrade Vnemaran^^ T-53 of May 1|,;S dibenzo-prdjgijfig^but these can be ,con and T-53 amentSwSTs of June 12 trolled by proper manufacturing techniques." gust 24. 1970defirilrth current registration: Photodecomposition of chlorinated dibenzo- status of 2.4.5-T^:.'products. Since,., this. p-dioxinsWas'shown by 0. G. Crosby at al memorandum was' issued, a great deal of/^T?t * (Scienra^g||73. pp. 748-74$. August 20. research" |faii'beri':iarried out. pd th^.tera^^ v.;^; 1971). Work ed'dioxins was culminated in a togedicity of 2.4.5-T and' of .full day sy(f|{iigsium in sessions of,the Pesti nant^ ^.3j5^-tetrachlorpdibenzo:paradiitin cide Chemistry Division at the American . Chemical,ocigty meeting held iryA/ashing- Jfcbb);4tf}s1Fecoghized that "purei "2;4.5-T- 1 il ` _ ton DC. Se^t^^gr^ 13-17. 1971^%eySDA Agrjcullural'DtaSearch Report of"bctober 1971 * " " the compoum The Agricultural Research Service studies se n js hp i^ndue risk tr^pumane.. antm als^y^r on TCDD in the chlorinated com pounds th''nvirnmerif wfieri used as outlined'in; v - with particular emphasis on 2.4 5-T were started in February 1970 at Beltsville Mr I . by n research team led by biochemist Philip '1^r -v *C. Kei memorandum T-53 X6BS3 F e n o p ro p is re co g n ize d j s being sub*:- t to the same TCDD contaminanoii as 2.4 5-T jitamif^cltirinyj i-i'i DOW 758732 lies The standards of acceptance for tech nical fenoprop. in terms of TCDD content, will be the same as for technical 2.4.5-T. It is now proposed that for registration purpose all 2.4.5-T and fenoprop products to be sold in Canada must have a declared and guaranteed TCOD content in the tech nical material of less than 0.5 ppm. Also, the source (i e. the manufacturer and place) of technical 2.4.5-T and fenoprop must be identified The quality control measures used are also to be described Finally, the analytical method used to determine the TCDD content must be fully described and shown to be sensitive to a level of 0.5 ppm TCDD 2.4.5-T and fenoprop products are not to be registered for sale for 1972 unless these conditions are met. These criteria will in future be a part of the protocol for regis tration and re-registration of 2.4.5-T and fenoprop products. Adherence to these standards will justify deletion of the following warnings from 2.4.5-T product labels: (1) Exposure of pregnant women by any route of contact to 2.4.5-T may be harmful to the offspring ' 1 2 ) Direct exposure of some women to this product may cause a health hazard " The council of the Society of Toxicology in a letter to the editor of Science (Vol 174. November 5. 1971) commented as follows on 2.4.5-T: "The 2.4.5-T controversy involves a funda mental issue in safety evaluation. The issue is simply whether or not demonstration of a teratogenic effect m some species of ani mal. at a dosage level far in excess of any possible human exposure constitutes scientific grounds for banning the cheinu al A sin.ill minority ol Ioxn.olu>|isls believe in the affirmative, but the overwhelming ma jority do not. The EPA scientific advisory committee on 2.4.5-T. which included sev eral toxicologists and teratologists. specif ically endorsed the majority opinion. To accuse the scientific advisory committee of "bias" for defending a commonly held sci entific principle is unwarranted. To select a committee of ten experts who might share the opposite "bias" would be extremely difficult if net impossible ' The USDA has issued two reports deal ing with the b ' lefits of phenoxy herbicides Restricting the Use of Phenoxy Herbi cides--Costs to Farmers Agricultural Economic Report No. 194 Restricting the Use of 2.4.5-T: Costs to Domestic Users Agricultural Economic Report No. 199 The USDA states that prohibiting the use of phenoxy herbicides "would cost the U S farmers an additional $290 million to main tain current agricultural prod1etion. In addi tion. farmers and their families would have to work 20 million more hours to control the weeds without these herbicides. For this extra labor, the farmers would obtain no additional income " Over a million dollars has been expended over the past two years to prove the safety of phenoxy herbicides to man and his en vironment. From a scientific base the phenoxy herbicides can contribute econom ically. efficiently, and safely in the luture for the control of broadleaved weeds and brush on food crops, pastime, rangeland, and noncropland areas as they have for over 20 years 11 166^ OAsri l MOO r>~ . :\r>oi A,, ~Mn. iG,risw--o-l--d-,----E-x--e--c-u--t-i--v--e---R-e--a-.- J . C. V an d e rW eele, P e n ta O f f i c e- ......................................................................................... - flows/Hoyle, Biochem. Rea. Lab. W. A.;Melching, Dowicidea ^ KJ .n aRp.pMA yleeirns e, Z urich Oty PBS Pile H ft," '/.TV' DOW C H IM IC a L INTERNATIONAL LIMITED 1714 BAfTMAN ROAD MIDCAND MICHIOAN IUA. February 2 3 , 1957 C. H. Boehrlnger Sohn 22b Ingelheim am Rhein Germany References Dr. Vey/B .. ., Gentlemens Subject tf Preparation of Trlchlorophenol to Avoid Chloraone Thanlca very much for your letter of February 11 and the attached description of the work you have done on the pre paration of Triohlorophenoxyacatic A d d to avoid the for mation of Chloraone exciter, and consequent dermatitis to your workmen. This work la of very gr^at Interest to u s ' and we .appreciate your thoughtfulness In remembering our , earliercorrespondence on, .this matter. ......... . ' / / ;i. "i l; : YoursVvOfcy1'truly, , : i,, : ~:Z ~F-.rf ..' (* 1 i J l - ... . I' Frank B> 'Smith Technical Director/ I i n .*Vi PBS/Wc r.B.s. 'S?l-'-'r-' t. :-v cos O t t Erahn ,.4.,-.-:*'` j: I-.; -: 1 NOTES ,Early in 1955 subject company communicated with L Givaudan & C i e . S . A In Geneva concerning skin Irritation from 2r4^5-Triohlophenol. which was referred to us by the Givaudan Corp.,Delawann&, New Jersey. On January 27, 1955 we wrote jto Boehrlnger enclosing a data sheet describing the hazards due to toxicity and precautions fra* safe handling usd use of 2 ,5-Triohlorophenol and answered seven specific questions: regarding experience in our own pluit. 7 ; -v: \: ** ; vf. :J;:'*i -T:r,;r r-*4:i > rfcesfri .t.pa.1.. ' vr.`'1"m u i n flOW l 627630 2718 STATEMENT OF LEWIS A . SHADOFF ON 2 , 3 , 7 , 8 -TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN THE ENVIRONMENT 8/4/76 X o ro *M O Ul 166S9 My name is Lewis Shadoff. My residence address is 3031 N. 11 Mile Road, Coleman, Michigan. I am an Analytical Specialist of the Analytical Laboratires of the Dow Chemical Company's Midland Division. I graduated from the Polytechnic Institute of Brooklyn in 1961 with the B.S. degree in chemistry. I then attended Kansas State University and was awarded a Ph.D. in chemistry in 1966 having done my doctoral research in mass spectrometry. Since then, I have been employed by Dow Chemical working in mass spectrometry. In my present capacity, I have responsibility for the operation and maintenance of three gas chromatograph-mass spectrometers including a high resolution unit. ANALYTICAL INVESTIGATION OF TCDDI I have been working on the analysis of TCDD since 1970 determining the purity of synthesized material (1) and the TCDD content of 2,4,5-T ester products. Since April, 1973, I have been involved in the development of gas chromatography-mass spectrometry methods for the determination of TCDD at the picogram level (corresponding to the part per trillion level in biological samples after sample preparation). Gas chromatography is an analytical technique used to separate a mixture into its component parts. The separated components may then be analyzed by a mass spectrometer which yields information about the atomic composition of these components. This combination has been used as an extremely sensitive and specific detection method for TCDD. The concept of parts per trillion is difficult to envision. If it were salt we were analyzing, it would correspond to the 627631 16700 .-2- a b i l i t y to d e te c t one g ra in o f s a l t in an Olympic size-sw im m ing pool of fre s h w a te r by a n a ly z in g one s h o t- g la s s f u l l o f w a te r. DOW 627632 We have searched for TCDD in the following samples by this technique: ENVIRONMENTAL SAMPLES ANALYZED FOR THE PRESENCE OF TCDD SAMPLE Human Milk (ref. 2) Fish (2) Mud (2) W a t e r (2) g w 1s M i1k (3 ) Beef F a t (4) Rice (5) Shrimp SOURCE Texas Michigan Texas Arkansas Arkansas Texas Arkansas Texas Oklahoma Missouri Arkansas Texas Oklahoma Missouri NUMBER SAMPLES 6 1 20 20 2 2 2 2 25 TCDD CONTENT* N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 24 21 spls N.D. 3 spls 3-4 ppt 19 N.D. (12 of them commercia 1) Japan . Vietnam 1 3 N.D. N.D. LIMIT OF DETECTION A v e . 3 p p t , range 1-6 Ave. 7 range 2-18 ppt 4 and 6 ppt 3 ppt A v e . 0.2 ppt 0.1 ppt 0-5 or 1 ppt Ave. 4, range 2-7 ppt Ave. 4, range 2-7 ppt 1 ppt *N.D. = not detected at the detection limits listed. The significance of low part per trillion TCDD content is not pre sently known. It is under study at the EPA, Dow Chemical, and IITRI. . The methodology and some of the results have been reported at j scientific meetings (2). Publication of the work is now pending in jrnals whose articles are reviewed by scientific peers. 1t 16701The cow's milk was collected from cow's grazing on grass known to h a v e ^een treated with 2,4,5-T. DOW 1 627633 t- 3 - The fish, human milk, mud, and water were taken from environments where 2,4,5-T had been used on rangeland and rice fields. Those samples from Texas were from the San Angelo, Texas Reservoir which is an impoundment of the North Concho River and has its water shed large acreages that have been sprayed with 2,4,5-T herbicides for the control of mesquite (0.5 lbs/acre 2,4,5-T acid equivalent) and brush (3-4 lbs/ acre 2,4,5-T acid equivalent). The samples from Arkansas were taken from a 125-acre pond in the heart of the Arkansas rice growing area near Grady, Arkansas. .. This water is used to flood the rice fields which have been sprayed with the equivalent of 1.25 lb/acre of 2,4,5-T acid four to eight weeks previously. Later, when the water is drawn off the fields it it pumped back into the pond for reuse. Water in the pond also collects run-off from surrounding rice fields during the rair.y season and is supplemented by wells. This cycle has been in use (including the proper use of 2,4,5-T herbicide) for 18 years up to the time of this study. Thus, these should represent likely locations for the accumulation of TCDD if it occurs. Twelve of the rice samples were collected from grocery Stores throughout the United States. The rest were taken from experimental plots. The shrimp was supplied by the Kien Hoa Shrimp Company. REFERENCES 1. 120, (1973). W. W. Muelder and L. A. Shadoff, Adv. in Chem. Ser. 2. R. A. Hummel and L. A. Shadoff, 170th National Meeting of the American Chemical Society, Chicago, Illinois (1975). -4-, 3. Mahle, N. H . , Higgins, H. S., and Gertzendaner, M. E., ."Search For The Presence of 2,3,7,8-Tetrachloro-p-dioxin in Bovine Milk," submitted to Bull, Environ. Cont. Toxic, for publication. 4. Kocher, C. W . , Mahle, N. H. , Hummel, R. A., Shadoff, L . , and Getzendaner, M. E . , "Search For The Presence of 2,3, 7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Beef Fat," submitted to Bull, Environ. Cont. and Toxic, for publication. 5. Jensen, D. , Huimnel, R. A., Madrid, Ester, and Turley, June, "Search For 2,3,7,8-Tetrachloro-p-dioxin in Rice," to be published. CD CO jr- 1670? Acl:i 'c l. xeaml. I *)!(>, `te 2080 j-nmi |In- Xntimint Yi tiiiiia i y Institute, .Slm-Midlm, Mwi-ilcn. D 1ST IU BUT I ON AND ELIMINATION OF CHLORINATED PM ENOXYACETIC ACIDS IN ANIMALS *> By K u r l Erne. Tlic distribution, elimination anil metabolism in plant tisanes of chlorinated plumoxyaliphalic acids have been the subject of numerous investigations (see o. g. Audits HKH). Tlu: fate, of phenoxy acids in tlie animal organism, however, lias not been so thoroughly investigated. According to early studies unsuhstilnlcd phcnoxyaeelic acid is excreted unchanged in urine by man, dogs, rats and rabbits ( W i l l i a m s Hla!)). The o- and /whloroderivalives also were, shown to be excreted renally, without conjugation, in the rabbit ( L e a r y A- Lewis 1SM7). On feeding oi-phenoxybulyric and -eajirnie acids to rabbits, some pheuoxyaeclie. acid was delected in the. urine, along with the unchanged acids, indicating partial (t-oxiduliou. Recently a group of workers at Cornell University, N.Y., in a series of feeding experiments with cattle anil using a gas chromatographic method of analysis, found Hint chlorinated phenoxyaeelic aeids (2,-1-JJ, M Q 'A and 2,'l,a-T) were completely eliminated with the urine in intact form, as was a chlorinated 2-phrnoxypmpionie. arid (2,J,f>-TL>) ( ( i n t c 'n m i i n n cl al. lOO.'i a, h; List; c l nl. 1iKi.'l;lla ch c. c r < il. Ii)Cl a, h ; 'S I . J o h n r t al. 1%-l). ('.hloriuated 4-jdienoxyhutyrie aeids (2,1-1)15 and MC.UR) were largely degraded in the rumen of cattle, 1ml small amounts of the e-oxidation produels, the corresponding phenoxyaeelic aeid-., were also iletecli'd in Ilie urine. A 2,-1,.'>-Tl' ester was laigety elimmalt-il in urine as the parent aeid. None of the pln noxy In i bii i.li s given ') Sii|i|ii.i leil ))\ ;i :;i:1111 fri.ni " Allu-rl ) lj.ii ivfmnlin " . f A o fO ro d r\oCO XIco Xll--s MCO t J I 2 lo colili' toltili In- ilflfHftl in Dio milk. Cimi; ri ni. (I!l(il), a|>plymg :1 lr.'ii'i-)' Ifrlmitjiif, rojtorloil Ilio rapiti renai crmiinnlhm of orally oilminislftfil 2,l-ilirhliirnjilienoxyarelit: acid il) :i t.lioop. !)C> <>CIlio aclivily was excreted willt Ilio urine in 12 hours, aliti lilootl lovols ilri)|iju`il lo below O.flf p.p.m. in 21 hours. Exjierimcnls with oUkt animai spcr.ics do noi seem lo have hoon rejiorlvd. The jrosoni paper presents results from studies of Die dislriIiuliou and elimination of some chlurophcnoxyaretie acids in rats, Jill's, calves and chickens, The experiments were jierformed in eimneelion with toxicity studies which will he. rejiorle.d separately (lijn rkh u u l cC Erne HKi(i). EXTEIUM EXTAL Male ruila 2.4- 1) limine. Aijneotis solution containing 20 mg of 2,-I-D jier ml, as Ihe triethanolamine sail, prepared hy diluting a crnumereial for mulation. 2.4- 1) K-Xa anil. Atjuenus .solution containing 20 mg of 2,l-l> per ml, jirejiareti hy dissolving (lie. pure, acid (m.p. 13S--M2'C',) in a slight excess of aqueous KOlJ-XaOll (1:1) and adjusting pH to about 7. 2,4,5-T aininc. Aqueous solution containing 20 mg of 2,1,5-T per ml, prepared hy dissolving the pure acid (m.p. 155--157'C) in a slight excess of aqueous triethanolamine tinti adjusting pii la about 7. 2.4- 1) caler. Emulsion containing 20 mg of 2,-I-D per ml, as the butyl ester, jireparetl by homogenizing a commercial formulation, in a petroleum solvent, with water. Animals Pala given for weight and age refer to conditions at start of expcrimenl. Albino rats, Autieimex strain, 1SO--22(1 g. bolli sexes. 1'igs, Swedish "lamIras" breed, 20 -25 kg, 1(1...12 weeks, castrated males, and females. Calves, Siili breed, 5a--G5 kg, G--S weeks, both sexes. Clucks, broiler, out: week, male. Chickens, While Leghorn breed, 0.ft --1.0 kg, S--10 weeks, hoih sexes, and .New llunipshice breed, 2.2 -2.0 kg, adult bens. Mil hails Adminislrnlitiii. i'.rfore dosing animals were starved tnermghl. Ibises were given orally, in short term espi-rimemis bv stomach tube, in long Iciin experiments in'feed or drill I. in;; water. .Vimipnm/. At selci led lime intervals bcloiT anil alter dosing, b lo o d samples were withdrawn in ht-pai mmol lu lu s , in rais al o O to cn GO CO If li (N'.vni,;tmi:iluiii f r i ni i :i n e r i ; a r l i - r y , in p i g s f r n m v e n a r:iv:i c r a u i r d i s , i n c a l v e s f r i nii :i j u g u l a r v e i n uni i i n r h i e k r n s f r i nii ;i w i n g - v r i u . J ' I a s m a w a s s r p n r a l e d Ity r i ,nlrifu;::ilimi. A il li 1111>*)' o f Un: c x p r i ' i m r n l a l a n i m a l s w e r e s a r r i f i r r d al x r l i c l i d l i mi : itili rvnl.s, b y N.s;ii u i 11: iIii11 a f l r . r a n c s l l u I i / i n g wi l l i r i l l n - r cliliiriifoi in (rais ami p o u ltry ) o r inebmnnl soiliiim (pigs). Tissues W e r e (xaiiiiiH-il f o r g r o s s p a t h o l o g i c a l a m i lii:.lo,.ii;, ical c h a n g e s ( H j i r l : I n t i i l X U r n e l'Ki(i) a m i s a m p l e : ; r o l l e c l c d f o r a n a l y s i s . S a m p l e s sveli: sloreil al -20'C unlil a n a le /e d . A n a ly tic a l n ie llim i. Tlie elilnrinaleil jilieno.syacelir. acids sverc isolaieil frinii b o d y flniii.s a m i issues liy snlvc ni e.slraelion, sejiarateil from e.\Irai-lives by lliin-laycr cliroinnlngraphy and ijuaulilalively ( l e l e r i n i m - d j 111tito n u- ( r i c n 11y a c c o r d i n g l o a l e c h n i i p i e d e s c r i b e d p r e v i o u s l y ( / i r n e 19(50 a ) . M' I i e n r c i p i i n a i , Un- p r o c e d u r e , w a s s c a l d i d o w n l o h a n d l e s a m p l e s a s s m a l l as ] g. T h e m e t h o d gi ves I he l oial e n n efiilralion of clilorophenosyaeelie acids. The limit of delcrminalion o f Ilio n i e l l i m i i s (1.1-- 0.2 p g n i 2,1-1) pi r g o f s a m p l e , wi l l ) t h e r e d u c e d s a m p l e a m o m t l s , h o w e v e r , a l m i i l 0.5--1 p g / g . HMSUr/IS A. Plasma lends 1. Siiu/lt: <lose a. 2 /i-D an iin i'. (Iruups of rals (male unci female), pigs, cal ves am! chickens (Leghorn ami New Hampshire) were given 2,-1-1) amine, sail, in (lie form of a technical funnnlalion, as single oral doses equivalent lo 50--200 mg of 2,1-1) jut kg laxly vcighl. On each dosage level 2a male ami 10 female rals, a- -10 pigs and chickens and 2 calves were used. I',local samples were taken (in rals aflo.r killing) nl 2--d-hour intervals during Ihe first 12 hours and Ihrrcafli-i: Jess frequciilly, and jilasma analyzed for 2,-1-1). Plasma eoneenlraliuu-lime curves are shown in Figs. 1--4. (In addition, a numlier of Ihe animals were killed al certain inlervals in order lo sludy (issue dislrilmlion, sec seeliou C, below.) The peak plasma eoncenlralion of 2,4-1) was usually allained wi IIt in 2 hours aflcr dosing in chickens and within 4 -7 hours in Ihe inammaliau species. In-flic sjieeivs examined a (lose of 100 nig/kg gave a peak level of about 100- 200 pg/ml. The plasma levels for female rats were lower Ilian lliose.for Ihe males. The. half-life values of 2,4-1) in plasma, ealeulaled from Ihe curves alter plotting on a semi-logaril hmic scale, are given in Table 1. 1). 2 , 1 - / ) A -A a . ' .n il. I h i 1 e x p e r i m i - n 1s w e r e r e p e a l e d w i t h I h e | i o l a - . s i u i n : ( n l i n m s a i l o l 2 , 1 1 ) . T e n m a l e t a l s a n d 2 r a i s e s w e r e J'.ivrn single du-.es o| |(nl m g /l;g . o ro cn GO CO ` 16708 I a 1 O re CR cc CO cn F i g u r e s 1-- 1. Plasma levels of 2,1-D in different species niter a a i n r j t e oral dose of 2 / t - l ) m u t i n ' . (50, 100 or 200 mg 2,1-1) A f i ) . l '11' <`tirves for pigs, calves and chickens were cdilained with 3- a animals on each dosage level ami (lit* curves for rals will 25 males and 10 females, 2-- -1 animals on each point of Hie curve. T h e c ur ve s o bt ai ne d and llte half- lif e values f ound ( Table 1) were, similar lo those ohluinrd willi 2,1-1) amine. e. 2,5,5-7' n i n t n c . Ten male rals and 2 pigs were given single oral d o s e s of a 2,-1,5-T a m i n e sail (1IM) in:;/1 ; i . Th e plasma cu i i e en l i al i o o - l i m , curves, as well as llie plasma ball' lile values, fo und, were similai lo ll m w oldaiueil willi !be 2,1-1) sails. 1670 <1. `1,'/-]) rslrr. A lorlinicnl f<>rm u!;iIi; t imlainiiig Mie- luilyl csh-r of 2.'1-1) w js given :is silicio orili dnses of Itili n>:;/kg lo M in.-ili' r a K -1 jii;;s n(1 2 ralvrs. Plasma Sniliplrs vane analyzed fin- lolnl 2,1-1) as in Hit' prevedili:; cxperimenls. Typiea! elirves ire sliown in l'io. fi. Curves for pigs woi'f intermediale helwecn Illuso for rais ;iiid ealvos. Tilt' plasma half-lile valut'S are given io Tallio 1. DI 3. 200- <N < 100-i s al -f 0~-- O RdIs O - --"O Ccilves --0 ------------- i--- r~-- y-- 10Time (ho.u20r's] -- 50 F i j li r e fi. Plasma k'vt'ls of 2,'I-D in rais ami calves after a s i n t / l c orai (Itisi* of 2, i - l ) b u h j l c s l c r (100 nifi 2,T-))/.kg). Tln* curves v e r e ubiamoti svilii 11 rais (2 un ca d i mini of tilt* curve) ami 2 calves. T a l l i i * 1. Tliisma half-life values, in different species, of phemisy acetic derivatives given ns a single oral dose of 100 mg/kg. Compound 2,1-1) stilline* *tall If >> ft it II J* f If ,* If K-Na >f >1 If If ester > 1* If H 2.1.a- T amine salt Species Hal, male Hat, female Pig Cair Chicken H:il, male Calf Hal, male P'g Cali Hal, male Plasma litilf-lifc hours 2.1) - 0.1 3-2 a: 0.5 12 2 7.5 O.S 7.7 0.7 3.5 0.5 3 0.0 0 I 10 0.8 10 at 1 3 0.0 2. l i c p c u l c i l i i d n i i i i i x l i u l i o u 2,1-1) inline anil 2,1-1 ) ester were given as repealed oral doses (ad Ul;; 2, l - ! ) / U ; ;/ (l u\ ' ) to t wo g r o u p s of and ! pigs, 1 esperi i \ el y. In htdh g r o u p s (he p l a s m a l e s t i td 2,1 1) w a s sitai (o d ecl ine .anil Hit- uri nary r \ e r r ! k u i lo inert-use as es em p l i ! it tl in rc ca or; CO 38318 j n :t\ ii 1. ti ; v a m i i . i . m i c i i i j n n :.J,;>('> " i p u r . n o i ii;;. l i l i e s ill m ilcuole Ilie eiinv.,.|,niiiliiii' urinary eoueetili'sliinis nf 2,l-l>. is Kuinlirr of doses Riven Pltt.sinu 2,1-D, (at 2 i hrs. liter fci.sl dose) Amine lister Pitf nt>. <1 A Pi|* no. ii A Pit; no. 0 A PiR no. 15 A 3 35 (200) 230 190 155 4-- 315 240 -- 7-- 335 24 295 88 520 (90) 25 -- 23 0 (550) -- 7 (250) -- Table 2 by pigs no. 4 A and 0 A. One animal in each group, how ever, did nol lolerale Die. repealed administration; the plasma lcve.ls conlinued rising during Hie experimental period, and signs of poisoning gradually developed (pigs no. S A and 15 A, Table 2). In an experiment wilh two hens, 2,4-1) amine. (300 m g /k g / day) was given, orally for 12 and 24 days, respeelively, and during the experimental period the plasma level (at 3 hours afler dosing) dropped from 150 lo 20 pg/ml, on the average. I).- Blood cells In in vilro experiments 2,4-D was added, at two concentration levels, to horse blood and the. samples agitated at room tem perature. for 5 hours and 24 hours, respeelively. Then the blood cells were separated by centrifuging for .`10 minutes at 2500 r.p.m., washed by suspending in two volumes of either fresh plasma or physiological saline for 5 minutes and centrifuging for another 5 minutes and then hacmolyzcd by diluting wilh two volumes of water. For comparison some samples were washed in physio logical saline three times. The 2,4-1) levels found in plasma and blood cells are presented in Table 2. A small proportion of the 2,4-1), about 10 r,o of the plasma level, was found in blood cells washed once. The proportion did nol change on prolonged contact between plasma and cells. W ashing with pl.-iMii.i removed more of the 2,1 I) Iroiu the blood cells Ilian did washing wilh saline. I'epeated' washing with .`.aline removed 2, I I) a lm o ! completely (tom the cells. 7 'J':i I) ) c .'I. I n v i t r o ili.slriliiilinn of 2,11) between liorsi* ]iI:imii:i :iiiI 1,1(10(1 n ils ;i| | wo ronrcntrulinn levels. before. analysis (In* blood rclix were washed cither once (;"> min.) v i ll i 1! volumes of fresh plasma (A) or of pin siologiral saline (II) nr 111rcc limes (15 min. each linn) villi 'J volumes of physiological saline ((!). Values veil arc means of re.siills from c111]>Iic-itIc experiments. 2,4-1). /.g'lnl Time. af coitlrul hours 1(1 /,({ 2.1-1) added per ml whole Wood Plasm:i Jllood cells A 1! C 100 //ft 2,-t-I) Gilded per .ml whole blood blasma lllood cells A U /. C r> 13 i.r> 2.1 < 0 . 1 135 11 17 <0.1 21 12 1.5 2.0 122 10 IS The results reported in Tnlilc 4 were obtained under jiliysiological conditions v illi rats receiving single oral doses of 2,1-1) inline and 2,4-D esle-. ' T a b ! e I. In vivo distribution of 2,4-D between rat plasma and blood cells after a single oral dose of either 2/1-1) amine or 2,1-1) butyl ester (100 mg 2,l-l)/lcg in eaeli ease). before analysis the blood cells were washed fur 5 min. with 2 volumes of physiological saline.. Values given arc the means of results from two animals. Time after" dosing hours 2,4-1), /'({/ml Ainiuc lister Plasma IJloori cells Plnsma blood cells 5 170 20 25 3 2i 2.5 0.S 8.5 1.2 C. Tissues G r o u p s of nils (male mid-female), pigs and chickens (Leghorn and New Hampshire) wen: given xinylc oral doses of 2/1-1) amine (100- 200 mg 2,1-])/kg), and al different lime inlervals 3 -1 aninoils from each group were taerifired and (issues and body fluids nnalyy.cd for 2/1-1). The experiments were performed in romierl ion with Ihe jilasnm level studies described above. The results me su m imiri/.ed in Tables 7. I he cllerl oil tissue dial rihtil ion of ;v i , i ; t ci! m ! m i l : i:;l r c l i . m ol I- 1) v a s s Ilulled in Ie ('din:; e \ pe imen Is v i l l i ral s ( m a l e and o O cr CO 16710 DOW 8 T ;i b I e 5. Tissur li-vels <>f 2,1-1) in male ;iii(l iemale n i l s :il differciil inlcrvals afler a s t i i j / t r orai dose of 2,1-1) amine (100 ni;,' 2,1-D/kg). Karli valor is llir oiran of resoli;; frmn i aiiimal.s, svilii Ilo- range Riveli in hrael.els. Tnmh* 2,1-1). (i hours Males Kcnmlrs (fresh sviagli!) 24 hours Males J'cinalts I'lasma I.ivrr Kidnev Lung .Spleen .Skeletal muscle Frees la!) iI "0 - - ! / ) 00 ( 05 - 12(1) 250 (1X2 --2X0) M0 ( 1 0 1 - 170) 80") 25 ( 12-- 20) 70 ( 55 - - 00) To (.7r>-- 7) ;15 ( 2 2 - - -12) 1 15 (00--IX5) 00 (15 -- 75) -- 1 1 ( X - 21 ) -- n) One animai. 2 ( 1.2-- 3.0) 5(3--9 ) 27 (10 -- 10 ) X ( G -- 11. ) G 2 ( 1.3-- 3.1) IX ( 1 1 -- 27 ) 1.5 ( OX - a.l) 3 ( 2.0 - 5.2) 15 (10 -- 23 ) G ( -1 -- 8 ) -- 0.G ( 0.2-- 1.3) -- ro c: \ co fO o T a b l e 0. Tissue levels of 2,1-1) in i/js al different inlervals afler a sinr/lc orai dose of 2,1-1) amine ( I00 mg 2,1 -D /k g ). Farli value is Hu mean of results from 2 --I animals, svilii Hie range given in brnelu-ts. Tissue Ghours 21 hours 48 hours 72 hours I 'l a s ma Liver Kidney Lung .Spleen Ilearl musele Skeletal muscle Hraii) Fat .Skin JljJe llrine Feces Thyroid A ilrcnnl Ovary 1`a n r r c u s Salivary gland Lymph gland ( iiit'M'iilrrir} 210 (1X5 -- 230 ) 115 ( XO -- 125 ) 100 (105 -- 225 ) 00 ( 7-1 -- 107 ) 5 ( 51 -- 78 ) 15 ( 38 -- 53 ) 21 ( 15 -- 28 ) 12 ( 8 -- IX ) 3 ( 1. 2 -- 3.3) 15 ( 30 -- 55 ) 20 ( J7 -- 30 ) 85 ( 55 ---125 ) 150 ( 77 -- 205 r 55 ( 12 -- 03 ) 05 ( XII --1II5 ) 105' ') (it) ( 50 - 71 ) (ill ( IS 00 ) 55 ( 13 -- 0! ) 27 ( IS -- 35 ) 30 ( 23 -- -IS ) 20 ( M -- 32 ) 17 ( 0 .... 2 0 ) -- 3( 3( 2( -- 2.5- 2-- 1.0 _ 1. 5) 3. ">) ) 1) 55 ( III -- 71 ) ISO (110 -300 ) 50 ( 10 -- 05 ) -- .... 7: ( 01 ;;:i ) ") Our animal. 10 ( 5 -- 10 ) 0 (3 -- 0 ) 10 ( 3 -- IX ) 1 ( 3 -- G ) 3 (2 -- A ) 3") 2 ( 1 . 0 - 2<7) 1.5 ( 1.2-- i. X) -- -- 30 (15 -- 38 ) 05 (00 -- 135 ) 15 (10 - - 25 ) ___ __ __ ___ _. X"). 3 ( L.5--- -1.5) 1 ( 2..5- - 7 ) 5 ( 2 --- X ) 3 (2 - 1) -- -- l.fi ( L.0-, . ' 2) -- -- -- 3 { 1..5--- 1.5) 25 (10 50 ) -- -- -- -- -- --- .... 16711 9 'J'a li I c 7. Tissue levels of 2,l-l> ill r l i i r l . r t i s al different ulcrvais afire a s i / i i / h - oral dnsr of -.1-1 > amine (200 in;! 2, l-D/l;;;) Larli value is Hie mean (if rcsnlls friim 3 ! animals, will! Ilic ran;;e given in hrarkcls. Tissue (j limit s 2,4 0, (fresh wci;;!il) 24 hours 48 hotirr Plasma Liver Kidnev Lung Spleen Skelelal muscle Drain ]'"al Kgg, yolk ,, while ] 0(1 (72 -- 131 ) 80 (50 -- 105 ) 120 (80 -- 170 ) (ill (10 -- 82 ) 80 (50 -- <1(1 ) 3.5 ( 1.5-- 1.2) 1.5 ( 1.2-- 2.1) 1.1 ( 0.0--- 1.5) <0.1 <0.1 15 (10 -- 20 ) 25 (20 -- 32 ) 80 (53 -- 120 ) 10 (28 -- 10 ) 20 (10 -- 28 ) i.5 ( 0.8-- 1.0) -- -- 0.2 ( 0.1 -- 0.3) <0.1 5 (2 -- 7 ) 3 (2 --1 ) 7 (1 -- 8 ) 1 (2 -- 5 ) -- '. 1.2 (0.5-- l.C) -- -- 0.2 (0.1-- 0.1) <0.1 a O ro cn qq T a )> 1e S. Tissue levels of 2,1-1') in ralx, pigs-, chicks and chickens afire r c j n ' i i l c J oral adminisl ralion for 2 moulds, Pigs were given 500 p.p.m. of 2,1-1) amine in slumlord feed Iwiec daily and Ihe oilier species 1000 ]).]).m. of 2,4-1) amine in their drinking waler. The pigs were killed 21 hours afler Ihe Iasi meal; for Hie oilier species Ihe interval between Iasi dose and killing was irregular. Kuril value given is Ihe mean of rcsnlls from 2 -I animals, with Ihe range given in hrackcls. T issu e Ma les Hals I'cm alcs 2,4-1), /ig/g (fresh weight) Pigs Chicks Chickens Plasma Liver Kidney Lung Skelelal muscle Skin Drain l-.it K;-.g. \nlk .. whi le 1(1 ( 1 -- 2(1) 25 (10 -- 30) 15 (22 -- 00) 30 (10 ---12) 5 (2- -10) 22 ( 8 35 ) 12 (3 - 2 5 ) 10 (3 -- 31 ) 3 (3 -18) 0 ( 3 -- 11 ) 8 (5 -- 10 ) 15 (5 -- 20 ) 15 (8- -20) 12 (10 ---Id ) 30 (7 - 7 1 ) 20 (8 - -50 ) <`i (3- - I D - 1 ( 2 .-- 0 ) 15 (0 --10 ) 12 (5 -- IS ) 7 ( 3 -Id) ln) -- 1.5 ( o. 5- - 2) 3 ( 2- - 5) -- -- ") fine animal. 2 ( 1.5 - 3 ) 1. 3 (0.3 - 1.0) 3 (2 -- 3..3) 3") ____ ___ 2 ( LI- ... 2 (!) 1.3 ( 0.0 . 2, 2) -- 1. 8") 1. 0 (0.5 1. 5 (11.5 1. 3) I 1) 0. 2 (0.1 (1. 1> IO female), pigs, chicks (broiler) ;iml chickens (Leghorn). Tin* pigs were foil ;i standard d id , with fiOO p.p.m. of 2,4-1) amine added, twice :i d:iy, and the oilier animals were given 2,4-1) aniiiic, at a level of 1(100 p.p.m., in llicir drinking wafer. Animals were sacrificed after feeding periods of 2 months lo 2 years. The m ajor clinical sinus, observed in all groups were anorexia and a reduced weight gain. Further dinie.al and morphological ohservalions of Ihe experimental animals will he reported else where (Bjorkluntl Or Erne. I00G). 'J'ahle S indicates the. tissue levels of 2,4-1) found after a feeding period of '.'.l least 2 monllis. T a h 1e 0. Tissue levels of 2,-J-D in pigs .showing signs of poisoning on repeated oral administration of 2,'t-D amine. 'j'ixsuc ---2,4-1), /'f/ (iresti w-e--t-"--I-i-l-)---------------------- . l'l'Uno. 3 A Pig no. fi ,\ ') Pifi no. 12Ac> Plasma 300 580 530 I.ver 230 '300 225 Kidney 200 370 185 Lung 220 340 190 Spleen 130 225 150 Skeletal illusele 105 -- 120 Drain 40 -- 05 Urine 100 90 -- ") 10 daily doses of 50 ing/kg. Killed 18 hours after last dose. ) 8 1 1 1* 1 1 ,, 90 -,, 1 > 1 c) 3 M too ,. G ,, * tr t* In another experiment, repealed oral doses of 2,1-D amine (50 and 100 mg 2,'1-D/kg/day) were given by stomach lube lo fi and 2 pigs, respectively. Two animals of (be first and one of the second group developed signs of poisoning (vomiting, iocoimdory disturbances, depression) and were killed. On analysis of tissues from Illese animals, Ihe insults given in 'fable 0 were ohlained. Further, four pigs were given repealed oral doses of l.-l-1) caler (;j0 mg 2,4-I)/kg/duy) for up lo ime moniti. In three of Illese animals no untoward effects were seen, :md in their tissues only low levels u| 2,4-1) were found (Table Id), (lutaci ester could noi he dclcvlcd in tissues from any of the animals.1, (hie animal, however, exhibited signs of poisoning ami was killed alter 1(1 days (pig im. la A, Table 2). On aiialNMS compara Ii\ely a O < ro CJT cc CO to to 16713 11 T it I) I c 1 (I. Tissue levels of 2,1-D in pigs aflcr repealed oral nilliiiiii- sl r:i Ii<>:i uf 2,1-1) h i i l i / l ester ( (.-(|i ii v;il fi 11 In fid mg 2 , 1-1)/k "/Iny) for 1 inondi. Till' :11 iJi):11s Were I. lied .`.'I hours aflcr I;is ( (lose. J'lm-li value "ven is Ilic mean of resol!:. frulli II :tni 111:Is, willi Hit- range gi\eu in hraelirls. Ti.vMIC 2,4-1), ! ffccslt wci|;lil) Plasma Liver Kidney Lung Skeletal illusele Fai 7 ( 5 -- 12 ) 10 ( 5 -- 17 ) 17 (12 -- 25 ) 4 (2 -- 7 ) 1.5 ( 0 . 5 - 3 ) 0.5 ( 0.2-- 1.1) high (issue levels of 2,4-D were foimcl (230, 100, 2fl.'i and SO |ig/g in liver, kidney, lung :md skeletal muscle, respectively. Finally, will) (lie main juirjiose of studying (lie. possible effee.is of phenoxy acids on reproduction, -u pregnant sow was given 2,4-D amine (500 p.p.m.) in (lie feed during the entire pregnancy (see BjijrkUuxd X Erne). Of fifleen piglels delivered on parturition one was Jjorn dead and nine died within 24 hours. On analysis of (issues from (lie dead piglels considerable amounts of 2,1-D were found; for liver, kidneys and lungs die mean values, and (he range, were .*15 (14-- 77), 27 ( I3 --5S) and 30 (13 -04) |ig/g, respectively. The placenta conlained 40 |ig/g of 2,4-D and (he plasma of the sow at parluriliou 28 |ig/ml. DISCUSSIOX A b s o r j i l i o n . 2,4-D, when a d mi ni sl ei ed orally as a (eehnieal formnl-ation of an amine sali, was readily ahsorhed in all (he animai species sludied, as indiealed hy Ihe peak plasma eoncentratinns afler a single dose heing allained in a few hoiirs (ahoul 2 liours for ehickens and 4---7 Iiours for rais, pigs and calves, l'igs. 1-- 4). Simi lar r csnll s were oldained wi lh an alleali salt of p ur e 2,1-D, (li il s indiealiiig Ihe ahsorpliuu l'ale of 2,'I-D lo he cs.senlially unaffee.led hy Ihe amine or olher ingredienl of Ihe tedili irai for mul al ion. 2/l,.ri-T ami ne hcliaVcd similarly. Orai ailuiiuislraliou of 2,4-D esler, ou Ihe olher hand, residled in low p l as ma an d li.ssue levels of 2,4-1) as comparili wilh Ihe soluhle sali:;, jirohahly a?. Ihe rcsull ni mrompl cl c ahsm p!imi (big. a, Jalile III). C.mu.idrriiig Ihe poni Mduliilily in walcr of Ihe esler, a low ahsoi pi imi l'ale is alio lo he esperirti. Noi al any a 0 $ to 0*4 oCcO ro r *. 16714 r~~ / 12 l i m e a f t e r d o s i ng coul d intact c sl cr In- ilrlc.rled in J)I:imii:i or ur ine of nils, pigs :md calves. Only t]ti* :iri(l was detectable. indicating lln- osier lo under!,fii hydrolysis during absorption (LY/ir I'.KTi li). D i s l r i b n l i o n . Tin*, absorbed piicnoxy acids were tlislrilniled r:i|ddly throughout lln* body (Tallies 5-- 7). hi :i11 species the highest tissue levels were found in Ilie excretory organs, liver and kidneys, and in (lie lungs and spleen. The levels in liver, kidney and lung al 0 hours approached, and al haler limes oflen exceeded, Hie plasma level. This was pnrtir.ularly line for nils and hens. A comparison between species m ay he based on Ilie kidney levels. If expressed relative lo (he fixer levels, these values at (i hours and 21 hours, respectively, were 1.0 and 1.5 for pigs, 2.1! and 5.4 for male rats, 4.1 and 5.0 for female rats and 1.5 and 5.2 for chickens. Thus, rats exhibited the highest values throughout, and in both rats and chickens the values increased during the. elimi nation phase. The difference in 0-hour values for male and female rats probably is not significant. Likewise, there seems to be no sex-difference in distribution pattern in rats, although the tissue levels found in the females consistently were inferior lo those found in the males. From the more detailed study performed with pigs (Table (!) is seen that comparatively high levels can be attained also in endocrine and other secretory organs. Concentrations of 2,4-1) comparable lo that in liver, or about 50 of the plasma level, were found in ovaries and .adrenals. In skin and heart muscle the levels were approximately 25 and in skeletal muscle about 10 /f-, relative lo plasma. The brain level was usually low, about 5 relative lo plasma, although after toxic doses higher concentrations could be attained (see Table In adipose lissue only traces of 2,4-1) were found, or 1--2 V<- relative to plasma. The. low brain level is consistent with current concepts of drug transfer across body membranes. For most drugs and foreign organic compounds the transfer can be explained in terms of a simple diffusion, of the unionized compound, across a lipoid membrane, llm rale-determining fac tors being IIn* ionization of the compound and the lipid-solubility of the unionized form (see for instance / H u n s I'.Mil). Now, plienoxyaeelie acids, as being rclnlivclv slrong acids (plv, values aboil) .11, will be hugely ionized ami llierefoie not readily diffusible al plasma Jill. Areoi iliugly. Ilicy Would nol be expected lo llaxcrsv Ihe blood luain harrier, as well as the placental bailier. icadilv. 16-; D O W 258321 j:i A;;nms( tills bnrkpmuml. IIn1 results ublaii'cd with the jireipiaiil sow, su:;"cslu>" :i ready jilaecnlal transfer of 2,t-D, seem inlereslin:.`>. Tlie results maV lie :i11rilmlable In (lilfeivnl factors, sueli ;is eli.ni:;e uf jhici'M1.11 jivrmrability tlin iu:; jircipiancy, lowcl'in" of Jilarcntal j11 .is eouijMl ed In j 11:is111:i, with ;i rniiscqurnl lowerill" of Ilie decree of ionization of I lie. jibenoxy ;icid, :md c.xlrapl:ieeut;d transfer. However, Hie jiresenl limited evidence does mil permit an assessment of Ilie relative imjiorlanee of Hie various factors. Tlie dislrilmtion pallern of 2,1-0, administered as Hie ester, did not differ appreeiaMy from Dial observed with 2,4-D amine, altlui(i"li tissue levels were eeii.sisleu!!y lower. The ajijiarenl dislrilmlion volume of (lie compounds under study can lie estimated by |ilolliii" tlie du>.c-c)iminalion curves (Fij's. I - -4) on a semi-lo"arilbii;ie scale (lot;. eoue. v. time.) and cxlrapolalin" Hie terminal, linear pari of (lie plot bark to zero lime. Iiy rclatin" Hie dose rjivcn to Ilie " zero lime plasma levels" thus obtained, apparent, distribution volumes of 25---50 % of Hie body weight were found for Ibe salts of 2,4-1) and 2,4,5-T, when administered orally as a single dose. These values are intermediate between Hie exlraeellul.ir body volume (about 20 r/c of Hie body wei"lil) and lolal body water (about 70 % of the body weight), thus sut;;;eslini; some {lenelratiun of Hie jibenoxy acids into Ilie cells. Direct evidence for Ibe permeability of blood cells to jibenoxy acids was obtained in tlie experiments sum marized in Tables 3 and 1. In Ibe blood cells 2,4-D regularly could lie delected in concentrations runc.iii" between 10 and 20 '/ of Ibe corres|iondin" jilasma levels. Moreover, Ibe. 2,4-1) eonlciil of tlie cells could be easily rciuovcd by rejiealed washing, wbieb indicates a ready jinssagr of jibenoxy acids across Ibe. erythrocyte wall. Tbe distribution equilibrium between jilasma and blood cells is obviously displaced towards plasma, a fa d which is also reflected in jilasma bein" sujierior to saline.in washing efficiency. One reason for Ibe equilibrium beiii" disjilaccd could conceivably be a jiarlial bindin" of Ibe jibenoxy acids lo jilasma jiroleins, (bus reducin" Ihe free (diffusible) fradiou of jibenoxy acids in Jilasma. Indications oI such a jirnleiii bindiii:; have been obtained ( J'lnir I'.If.li b ). . l-.liin imil inn. I-71i11 i11:11i,11 of Ibe jibcioi\y acids f*i mu jilasma. aflcr be in:; adiiiiiii'.li'i cd as amine oi all.ali sails, was r;iji.I in all sjuvics sludlcd. I lie sen',ilop,;; i ilb nin do-e v!iotin.d ion ciliscs o O ro ci CO CO ra ci 1671$ D O W 2583 I M were linear in their terminal courses, iiidiraling a firsl order fliiiiin:iltin rale. Tlic plasma half-life values calculated Icom these curves wrii- aboiil .`I hours for rals, ii hours for calves and chickens and 12 hours for pigs (Tallin 1). No sex difference. in respect lo idiniinal ion rale was observed in rals, although llie plasma levels wen' lower in llie females Ilian in llie. males. 'J'lie elimination rale from plasma of 2,1-1), given as Ihc ester, was slightly reduced as compared willi the soluble sails. The rale, of elimination of 2,1-1) from tissues was lower Ilian Dial from plasma (Tables ii-- 7). From the. tabulated data, halflife values of 2,1-1) ranging between a and III hours can he cal culated for rals anil between 10 and .'10 hours for the oilier species. Generally, in 72 hours or less, 1issue levels had dropped lo a few |ig/.". No retention of phenoxy acid was noted in Ihc tissues examined. The results arc in agreement willi (hose obtained in sheep using C"-labcl!cd 2,1-D (Clark cl al. 100-1). The distribution pattern of 2,1-D on repealed administration was essentially similar lo that obtained after a single dose, although individual variations were considerable, presumably owing lo variations in feeding habil of (he animals and in time, between last water or food intake and lime of killing (Tables (1 and 10). The results seem lo support (lie view that halogenalcd phenoxyac.clic. acids arc not likely to accumulate- in lissues of clinically healthy animals. Excretion. Judging from the. analytical results llie major excretory route is via llie. kidneys, at least in the mammalian species studied. Ilisluputhologicul examination suggests that Ibis is true for chickens as well (Ujiirklnnd & '.me. l%Ci). Only low levels of 2,1-13 were, found in feces from rals and pigs and in bile from pigs. Thai plienoxyaeelic acids are predominantly excreted rcnally in callle and sheep, was reported by LisI: d <il. (10011), Ilachc cl al. (lUtil ;l), St. John cl til. (ltl(il) and Clack cl al. Inlercslingly, hens were found lo be able lo excrete part of ingested 2,1-D with the eggs (Table (i), a finding, not previously reported. Most of Ihc 2.1-1) content of Ihc egg was found in Ihc yolk. A t l a p l n l i i n i . On p r o l o n g e d a d m i n i s t r a t i o n of 2,1-1), given as a m i n e or as c;.|cr In l os e, In-low llie a c u l r l v l o s i c level, sonic <\ J ><-j 1111<'111;:1 a n i m a l s d evel oped sign', of an adaptati on. In pigs, p l a sm a level1, ol 2, !-l > U-.ilallv slarlril lo uVrkio' oi l er aboul a cn 16717 D O W 25832 ir> week. Hie urinary exrrcl i<>11 cuiic.iirrenlly iicrt-:isi 11 (Table 2, pigs nu. -I A :ni(l !1A). A similar response was seen in chickens. ]| .should In- i*i-111:iil ;i-;1, however, III.-I I pigs seem lo In: less loh-ranl In prolonged exposure lo phciioxy herbicides lh;ui arc* llio ofln-r sjii-rics studied. lu several inslauees, 2,-1-1) given as daily doses of fid m g/l.g/dny eveuliially produced signs of poison ing in pigs with rising jdasina and (issue levels of 2,-i-D (ISjrkinn/ A'- / '.rnr). id -ff .kfa'cfs Andns, !.. J.: The Physiology and lliochrmislry of Jlcrhieidcs. Acadcinie Press, London A New York 10G-I. Raehe, A., I). J. Lisl:, I). G. Waynet- it- R. G. Warner: Klimiiialiou of 2-iiieMiyl--l-i-liloro|)!i<'iio.\yaei:l ie acid and -l-Ci-melliyl-J-cIiloroplicno.xy) butyric aeid in the urine from cows. J. Dairy Sri. I'.Hil a, 47, 93--9. Hache, C. A., D. D. lardee, R. Holland it I). J. Lisk: Absence of pheiioxyacid herbici-h- residues in (lie milk of dairy cows at high feeding levels. .). Dairy Sci. lilti-l b, .47,'298--2`J9. Rians, T. I!., ed.: Absorption and Distribution of Drugs. Livingstone, Fdinhiirgh it l.ondim 100-1- Rjorklnnd, X.-K. (t A". Kmc: Toxicological studies of phcnoxyacclic herbicides. Ada vel. seand. 19GG, 7. In press. Clark, I). K., J. Yonny, R. !.. Yonnyer, L. M. Rant it J. K. McLarini: `Flic fate of 2,-l-dichl(]rophcuo.\yarclic aeid in slieeji. .1. Agrie.. 1*00(1 Clu-in. J9G1, I'J, dfl --15. Kmc, A'..- Delerniinalio.-i of phciioxyaccth: herbicide residues in bio logical materials. Acta vet. seand. JDGtia, 7, 77--UG. Krnr, A'.: Studies on Ibe animal melabotisin of pbenoxyacelic lierbicides. Acta vet. seand. 19GG b, 7. In press. Gnlcnnmnn, W. 11., J). /J. Hardee, R. R. Holland .t H. J. Risk: Disappearauce of l-n.-I-dicliloroplu-noxy) luilyric acid herbicide in the dairy cow. J. Dairy Sci. 19G3a, -id, 991--092. Gnlcnniann, It'. //., I. 1). Hardee, R. /'. Holland it 1). J. Lisk: Residue studies wild 2,'t-dieldorophenoxyacelie aeid herbicide in the dairy cow and in a natural and artificial rumen. J. Dairy Sci. JUG:! b, ill, 12S7 12X.3. / . c r e y . A". A'- //. /!. L e n d s : The uielaholisui of plu-noxy-.icelie acid, ils homologues, and sonie iimnoelil.n opln-iiux \ aeclic acids. New examples I.f jj-ox ida Iion. .1. bint. Clu-in. P.II7, it':''., 2K) 221. I.isk. />. If. //. Itiilemn.i:m, C. A. Hache, It. It. Warner ,t It. It. If,ay n c r : 1'.!i :i i i::i! i n n of 7,1 |1 in I b e l i r i a , of -,l e e r s led ! (!!,-! D I o r 2,1 I), D a i r y .'iei. Pin.'.. /It, ] f ; .i I l.'i','. 1C, S I . J o h n . I .. II- (' ll'(,i j n c r A- I ) . ./. J ; i s k : Kale of alra/.inr, kmon, silvcx ;m(1 in llic dairy cow. .1. Dairy Sri. Iill! I, {/, 12(17-- 1270. W i l l i m n s , I \ . T - : I h l o x i ca l i nn Mechanisms, 2ml lid., (7)i:i| uii;in A' Mull, l.iiiiilon 1il.'i!), p. ."(31). DOW258328 .SUMMARY The distribution and elimination of (wo phennx vacclic acids, 2,1-L> and 2,1,3-T, were .sludied willi a clieinical nielliod in rals, pigs, ealees and chickens. When adiiiini.slei'ed orally as amine or'alkali salts, llic eoiii]oiind.s were readily absorbed and distributed over (lie organism in all species sludied. The ahsorplion of 2,-1-D in (lie form of an estep was incomplete, . however, the ensuing plasma and (issue levels of 2,1-D being only low. (Intact ester could not he detected in plasma). The highest tissue, levels of 2,1-1) and 2,1,0-T were found in liver, kidney, lung and spleen, the levels sometimes exceeding the plasma level. In blood cells 10-- 20 of the plasma level was found. Pene tration of 2,1-D 11i111 adipose tissue and into Iho central nervous system was restricted, whereas a ready placental transfer was deuionslralvd in swine. The dislrihulion pattern did not show any significant species or -- in rals -- sex differences. Isliminatioii of (lie compounds was' rapid, the plasma half-life being about 3 hours in rats, about ft hours in calves and chickens and about 12 hours in pigs. The tissue half-life, values ranged between 3 and 30 hours, the: l ower values being found in rals. No retention in tissues was noted, nor was aceiimnlalicin seen cm repealed admini stration. In pigs and rhiekeus an inervased elimination rale was observed after repealed administration. The major excretory mule seemed lo he via the kidneys in all species studied. Mens excreted sinaii amounts of 2,1-D wilt, tin- eggs. /. IiSAMMI.XI 'ASSI1X(1 YcrtciUiinj nod EUininnlioii non c.hloiicrtcn I'luiici.njcssi.-jXtiiii.n ini j'Uriinjaitismns. Die. Ycrlcihing mid Klunlnalinn run /.wei eldoricrlcn Phcnuxyessigsauren, 2,1-1) uud 2,1,3-1', wurdeii an Mailed, Sellweiiien, Kalbcm >md Jliilniern unhiMichl. Nacli oralcr Kingabe, in Korin drr Amin- oiler Alk.di.sab'.e, wiirdcu die Ycrldniliingrn lici alien iinlci sin hleii Ticr.irtcn selmell resorhierl, ond iiber den Orgaiiismus vei li ill. Orale Zufiihr vmi 2, I D In Korin clues IXlers rr,,ali jcdoili nnr iiieilrigr Plasma- mid (Irwr.V.grhallr von 2,1 M, aiisrlieinciid infolgr gr ringer 1`esorpl inns;;! noli wiisdi:.1.ciI. Nacli cln/h'i-r oralrr /.nliilii von 1'Iieioi\ \ (.sipsiiiii'c;i:iiio ..il.wa worileii die In'n lr.li o (Is wel -.1.mi a i.11 il l on e o, h i e i l c i i den I'l.isio.ispir;;il I'ihersli i;,i nil, in l.clier. Ni tre, I.tinge Ulnl Mil.. ;;i-1'11 [1111ii. In 17 den rolcn Hhilkiirpcrrlien wurden 1(I--20 des 111;i.s111: i i : i 11.-i nnrh;e'vii-.rn. 1', 1-1) ;;t-!;iii;;ti- nur in niedri;;er Konzenl ral ion ins ]-'cll;;ewchc und ins Z.i-nlralucrveiis\.-.lein. I)u:;c;;cn wurde ein leichtes Durchdriiii;rn der I*I:n-111:mh.uTiere lirim Schwein midier wiesen. Im Yerlcilimi'smii-.lrr waren keine eschlcrhllielicn (hei Hallen) mul mir inxsj;{e licrurf\j;chiiiidciie Variationen wuhrzunrhnicn. Dir Yerhindunuf-ii wurden selinell eliminierl; die llalbwerlzeil in Plasma, nach einziger oraler Kinjpihc, liclnig e;i. 3 Slundeu liei Hullen, e:t. K Slundeu hei Klhern mul Hhnern und ca. 12 Slunden hei Schweinen. Die 1Ialbwerlzeilcn in den Geweden waren hoi linden 5--10 Slundeu und hei den linderen Tierarten 10--30 Slundeu. Keine (iewehsrelenlinn von Phenoxysiiuren wurde fcst"cstclll, ehen.su keine Aekumuhdinn mich wiederhuller Zufuhr. liei Sehweinen und iilincen wurde in einigen Fllen mich wiederhuller Zufuhr eine {jesleijji-rle Ausscheidim;/ von Phenoxysumi nachi{ewirseii. Die Phcnoxysauren wurden lio.i ullen wnlrrsuehlen Arten haupt schlich durch die Nieren unsi'cscliicdcn. Hei Hennen wurde eine Aus.seheidunu von kleinen Mengen 2,-l-D mit den Eiern naclii-ewicscn. SAMMANFATTNIXG Dislrilmliun neh cliitincrtny au klorcradc. [ciw.ridlliksyrur Jtux tljitr. Tvii fcixixialliksyrcdcrivals, 2,-l-D och 2,-1,5-T, dislrihulion orli eliniineriuj' lio.s ritor, {risai-, kalvur och hiin.s Ime undersiikls. Tillfiirda orali som amili- ellcr alkalisaller resorherades foreninfjara kilt och fordclades suahhl ver 'iir;;unismen hos alla iindcrsiikla djurarler. lfler lillforscl av 2,-1-1) esler pvisndes c-ndasl hijja iilnsmahaller, synliarli"en heroende. ]) ofuMshindiu resoi-jilion. J-ever, njnre, lulipa eli mjiille visade de luosla viivnadshallerna, vilka ildand oversleg jilasmanivim. 1 erylrue.yler fdcrfann.s 0--20 '/ av plasmahallcn. Fellvav neh centrala nervsyslemel pcnctrorades eli dasi i riii};a {rail av 2,-l-D, Hiedan |ilaeeulaharriiren lili synes ohehindral lasserades hos svili. Dislribulionsmoiislrot visade cj miyol si{nfikanl ari- eller, fiir rfdlor, kousherocnde. llonrfillor visade dock n{oI lii),'re plasma- nell vavnadshallcr iin hanr/illor. Fiireninipirna t-limincradcs siiahld; lialverinifsliden i plasma vaieia 3 liminar fr ridine av hada koiiuen, e.:a S (humar fiir kalvur orli hiin.s orli e:a 12 limiuar fiir {risai-. Hai velinosi den i viivnaderna vai ai- slorlek.sordnin;en "1--30 limiuar; rfdlorna linde de liijjrc vrdena. Inveii relenlion i viiviiaderua ohserverades neh ej heller n;i"oii arkumulaliim efler upprepad lillfiirsel. lilis ;;rxar (>(.|, liiius iakllops en upplianad uisnudrin;; efler upprepad lillfrsel av 2,1-1). I Isiinili'injp-n skedile huviidsal. li;a'H ;;eiinn njuiarnn ho-, undersiikla arler. llons iiImmoIi .uh* soia maiimlrr 3,1 D nied ae.r.ru. ( Un rii'i d Muti li 2'.', IVI'ihK o O rc coni co to CO 69 i1 6`('>Jr > D. Toxic Effects of Herbicides and Conclusions As mentioned in the introduction of this chapter, we are listing here a variety of information which could not properly be listed in the other sections. The items specifically included here are the following: (l) the mutagenic activity of herbicides on plants, bacteriophage, and bacteria, (2 ) the effect of herbicides on the embryonic development of beetle, (3) the con centration of herbicides in the tissues of fish, (4) the importance of select ing a formulation which will minimize the.hazards of herbicides in aquatic environments, (5) an estimation of the hazard involved in the spraying of a man with herbicides,and (6 ) some general conclusions about the toxicity hazards relative to the use of herbicides. Mutagenic Effects Some of the persons who are critical of the use of herbicides feel that we are creating a monster which will eventually destroy us. One area of expressed concern deals with the possibility that herbicides will produce heritable changes in the genetic material of plants. The resulting plants, they fear, will no longer meet our need for the production of foods or they may be insidious weeds which do not respond to either chemical or biological control measures. Some studies have been conducted to determine the likelihood that herbicides may produce heritable changes in plants. Doxey and Rhodes (1949) conducted a study of a plant-growth-regulator >type of herbicide known as 4-chloro-2-methylphenoxyacetic acid (MCPA). In this study they exposed onion rootvtips to MCPA and observed the effects of this treatment on cell division and on the chromosomes. Chromosome "stickiness" and breakage were observed and the effects were judged to be similar in many respects to changes in mitosis produced by X-irradiation. In a series of three papers, Unrau and Larter (1952) and Unrau (1953 and 1954) discuss their findings on the effect of 2,4-D on the genetics of wheat and barley. The seed grain fields were sprayed with 8 to 16 oz of 2,4-D (EE) per acre 30 days prior t o cytological examination. The chromosomes were studied in detail in those cells that were dividing during the process of pollen formation. Regardless of the date of application or the concentration of treatment, the herbicide caused "highly significant" abnormalities of chromosome behavior and the changes were visible as much as 30 days after treatment. Unrau warns that indiscriminate use of herbicides could have grave and far-reaching consequences especially in relation to the prime seed stocks of seed growers. There is reason to suspect that the herbicide interferes with self-pollination and this could explain the increase in the number of off-types or crosses obtained in Unrau's experiments. 201 '16721 DOW 120471 Muhling et al. (i960) has studied the cytological effects of 2,4dichlorophenol, 2,4-dichlorophenoxyethanol, 2,4-D, and other herbicides on pea seedlings (Pisum sativum). 2,4-D was clearly shown to be a prophase poison and to produce spindle inhibition resulting in chromosome configura tions very similar to those produced by colchicine. Dr. Kenneth J. Andersen (1967), Senior Research Microbiologist at Battelle Manorial Institute, Columbus Laboratories, is engaged in a research program for the United States Department of Agriculture to determine the pos sible mutagenic and genetic effects of the herbicides. As a part of this study, he grew histidine-requiring mutants of Salmonella typhimurium on a histidine-deficient medium to screen 120 of the 130 herbicides listed in the journal, Weeds. These included, 2,4-D, 2,4,5-T, picloram and cacodylic acid and the other herbicides which are currently important for noncropland vegetation control (see Table II-2).Since t h e -6 . typhimurium used is histidinedependent it does not grow very well on the deficient medium unless there is a mutation which eliminates the histidine requirement. In his experiments he found that diethyl sulfate, beta-propiolactone, acridine mustard (ICR-91) and K-methyl-N'-nitro-N-nitrosoguanidine all produced the mutations required for the growth of the organism. On the other hand, none of the herbicides caused this mutation. A second approach to the problem involved the use of T^ bacteriophage to detect chemically induced mutations of the rll type. The number of mutant plaques, which characteristically were larger, with a clear center and a sharp edge, were compared to the normal T4 plaques which were small, with a clear center surrounded by a halo. In addition to this characteristic plaque morphology, rll mutants form plaques on Escherichia coli but not E. coli KB while wild type T^ bacteriophage forms plaques on both of the E. coli strains. In, this evaluation system noninhibiting levels of the compounds (approximately 20 p.g-1 mg/20 ml) were employed. The rate of spontaneous mutation (no test chemical present) by T 4 bacteriophage'to the rll-type mutant, was about 0 .11$. In the presence of the known mutagen, 5-bromouracil, the mutation frequency increased to about 2.5$. In the test system it was concluded that none of the herbicides were mutagenic. Dr. Andersen has also performed experiments using a 2-aminopurineinduced rll mutant of T 4 bacteriophage to detect revisions from the mutant to the wild type which might be caused by the herbicides. Herbicides, in this detection, were again observed to be nonmutagenic. McGahen and Hofflnan (1966) have also investigated the possible o mutagenic activity of herbicides. In their program they employed a screening O technique similar to that used by Andersen (described above). In their study. 1 1202 nz 120472- DQW they used both the 3- and also the 6-alkyl bromouracil herbicides (including bromacil) on a bacteriophage and concluded that it was not mutagenic by this test. Dr. Verne Comstock (1967), U. S. Department of Agriculture and Agronomy Department at the University of Minnesota, has conducted a seven-year study on the effect of herbicide applications on the growth, yield and quality of flaxseed. During the years of 1956 to 1963 paired lines of four varieties of flaxseed were grown on plots which had received no herbicide or had re ceived 15 to 20 oz/acre of MCPA per year (treatment was omitted in 1959). The performance of unsprayed subpopulations were compared with that of the sub populations which had been sprayed in the Fg, Fj, and F^ generations. "It s can be concluded that the application of high rates of MCPA to successive generations did not cause a significant shift in these flax populations to more resistant biotypes." Insect Development Several investigators have considered the possibility that pesticides may under certain situations cause a disturbance in insect population which will result in a stimulation of a secondary pest. The reason for the increase in aphids in the grain fields in which 2,4-D amine has been used for weed control has now been investigated by Adams (i960). In her study three species of coccinellid beetles (Coccinella transversoguttata Fald., Hippodamia tredecimpunctata (L.), and Coccinella perplexa Muls.) were grown in the > laboratory and sprayed with 2,4-D at various stages of their larval development (l to 12 days). It was observed that spraying on the 12th day of larval development produced the highest mortality (70$) and about a 75$ increase in time to maturity. Since this bettle is an important factor in the control of aphid populations on grain, weed control with herbicides should be timed so as to minimize the damage to the useful beetle. Concentration in Food Chains One of the big concerns of the ecologists is this,are the herbicides concentrated at seme point in the food chain so that some life form at or near the top of the food pyramid will receive a toxic dose? Fortunately there are few life forms which concentrate herbicides at all and in most species this concentration is a reversible process so that the concentration level remains small. Table VI-10 shows the amount of herbicide present in the tissues of fish after exposure to the concentrations shown. For comparison it may be noted that under these same conditions heptachlor accumulates in the bluegill 203 16723 120473 b O ty , 120474 Species Bluegill Redear Sunfish Rainbow Trout Green Sunfish Bluegil L Channel Catfish Bluegill Bluegill TABLE VI' 10 AMOUNTS OF SOME HERBICIDES MEASURED AS RESIDUES IN ANIMALS AFTER 30-DAY EXPOSURES IN PONDS Pesticide Locality Amount of Pesticide in Water (ppm) 2 ,4-D PGBE in oil Tishomingo, Okla. 10 Silvex,* PGBE in oil Paraquat emulsion Tishomingo, Okla. 1 i Denver, Colo. 1 Paraquat emulsion Denver, Colo. 1 Paraquat emulsion Denver, Colo. 1 Paraquat emulsion Sodium arsenite Denver, Colo. S LaCrosse, Wis. 1 0.23 Diquat LaCrosse, Wis. 1 Amount of Residue in Animal (ppm) None detected 0.5 0.11 0.05 1.21 0.37 0.40 0.09 * Fenoprop. Source: Cope, O.B., Contamination of the Freshwater Ecosystem by Pesticides , J. Appl. Ecology , 3 (Suppl.) 33-44 (1966). 16724 to the point where the tissue level of heptachlor is 300 times as great as the heptachlor level in water in which the fish is swimming (Cope, 1966). Selection of Herbicides At the present time there is no magic formula which will automatically select the herbicide which will control the vegetation best and provide the minimum damage to fish and wildlife. Instead herbicides must be carefully selected on the basis of the weed that needs to be controlled, the location of that weed, and the possible fish and wildlife which may be affected by its use. That selection of the proper formulation for the job is exceedingly s important in the control of aquatic weeds without unnecessary toxic effects on the fresh-water ecosystem. In Table VI-11, the acute toxicities of a series of herbicide formulations to lake Emerald Shiners (Notropis atherinoides) are presented for three different exposure periods. The importance of the formula tion employed is borne out by the observation that both the most toxic and the least toxic formulations are formulations of endothall and one is about 6,000 times as toxic as the other. Spraying of Herbicides on Man From an examination of unpublished results from the Patuxent'Fish and Wildlife Center at Laurel, Maryland (Robert G. Heath, 1967), we conclude that dieldrin is at least 30 times more toxic than amitrole, atrazine, 2,4-D(BEE*), 2,4-D(DMS*), dalapon, diquat, diuron, monuron, picloram, ^imazine, 2,4,5-T(BEE) and 2,4,5-T acid to the two-week old young of mallards, ring-necked pheasants, bobwhite and coturnix. 7 O One further comparison of the toxicity of herbicides and pesticides: the Federal Aviation Agency is much concerned about the toxicity of pesticides and their effect on the ability of the pilot to fly his plane safely. Dr. Paul Smith, Head of the Pharmacology Section at the Aeromedical Laboratories in Oklahoma City, said that the pilots spray insecticides during the early part of the year and then spray defoliants and herbicides in the summer for brush control. He said that the pilots themselves are always glad when they are through spraying the insecticides. He added that the flagman on the ground during the aerial spraying operation probably receives a higher dose of herbicides than the pilots. So far as he is aware there has not been a serious case of poisoning or toxicity as a result of the spraying of herbicides. * BEE = Butoxyethyl ester; EMS = Dimethylamine salt. 205 16725 TABLE VT-11 COMPARISON OF THE ACUTE TOXICITIES OF COMMERCIAL HERBICIDES TO LAKE EMERALD SHINERS (NOTROPIS ATHERINOIDES) IN MEDIUM HARD WATER. MEAN TEMPERATURE 69a' - 72F Product Mfr. Liq. Active Gr. Ingredient TLm , PP active 4 hr 24 hr 4e hr 96 hr AquatholSilvex V/eed Rhap 20 AmitroL T Pennsalt ReasorHill Attehem L. Gr. L. Kurosal G Dow Gr. Crop Rider Diam. Aik. Gr. KurosaL SL Dow L. Regione (DB) Regione (DC) Chipman Chipman L. L. Atlas A Chipman L. Esteron 99 Garln Dow Dow Gr. L. Urab Allied L. Kuron Hydrothol Dow . L. Pennsalt L. Endothall + > 1^000 780 Silvex 2,4-D, ethyl- > 1,000 620 hexyl ester Aminotriazole 910 455 + am. thio- cyanate Silvex, potas- 1,100 540 slum salt 2,4-D, ethyl- 500 280 hexyl ester Silvex, potas- 509 420 slum salt Diquat, di- > 180 > 180 bromide form Diquat, di- > 180 15.5 chloride form Sodium > 32 13.5 arsenite 2,4-D, 2,4,5-T > 10 4.3 butyl esters Dalapon + >10 4.2 Silvex (butyl) 3-phenyl-l, 5..6 4.7 1-dimethylurea trichloroacetate Silvex, butyl ester > 10 4.0 Endothall, coco- 0. 29 0.12 amine, salt 612 620 455 450 280 310 86.2 11.7 8.1 4.3 4.2 4.3 2.4 0.10 510 510 420 370 280 270 25.8 9.1 8.1 ' (As) 4.3 4.2 .3.8 2.0 0.08 Source: Y..H. Swabey and C.F,. Schenk, Proc. Third Annual Aquatic Weed Control Soc. 1963, pp. 20--28. 0^120476 206 ` 1672^ :J 3 A ~ . li ^' ;i-- 14.036 m DOW 1122i REVISED and UPDATED ' AFTER TWENTY YEARS * _ G r a n v il l e F. K n i g h t , M.D., F.A.C.A. President . PRICE-POTTENGER NUTRITION FOUNDATION'. Orcinally Published In M O D ERN N U T R IT IO N MAGAZINE ' ; April. September mid December, 1902 ' 'J ' " Copyright 1972 by A uthor .1 J I ' .. ;! . . i What Are Pesticides Doing to Human Beings? , B y G ranville F . ICnisht, M .D ., F .A .C .A . S anta Monica, California 90403 "Nothing!"--say the publicity agents of the insecticide manufacturers. . "Hys terical alarmists" is the quaint descrip tion applied to some of those who even suggest that the public is being harmed by their use. Groups urging that the newer pesticides not be released com mercially until proven harmless to hu mans by exhaustive testing, have been accused of obstructing research and scien- of new poisons added to the diet. This is particularly true in view of the fact that DDT is very stable and m ay be concen tra te d in body fat up to a t least 30 tim es the level in the food consumed. U n fo r tunately, scientific proof of toxicity to humans from low levels of intake is dif ficult. The potential hazard to health is tre.mcntlous. It must be recognized by all parties concerned and controlled until the /acts arc established beyond all doubt! The Delaney Committee In spite of the assurance th at th e re is nothing to fear, the public has become alarmed over the widespread use of poi sonous sprays and dusts on our crops. And rightly so. T his alarm was communicated to the Congress which appointed a Select Com mittee to Investigate the Use of Chemi cals in Food Products. T h e chairman is "Nothing to worry about!"-- repent m ost John J. Delaney, M.C. In addition to of o u r entomologists. ''P e stic ides arc p o i pesticides, this Committee is investigating sonous. Yes. But with proper precautions the use of chemicals in other fields, such those handling them arc safe. And with as drugs, cosmetics, food preservatives, the establishment of tolerances for resi and fertilizers. dues on food the public is protected. Moreover, farmers can not bring to h a r vest the large crops needed throughout the world without the newer prsf icides. Better the risk of some toxic residues on our fqod than widespread crop failure from insect attack." These conflicting statements remind one of the cheery whistle adopted by a small boy when passing a cemetery at night. "Danger!" Widespread illness from the use of DDT on crops and in homes. This is the belief of a few keen observers in the medical profession. A handful of agri For the past two years the Committee has been hearing testimony from prom i nent scientists, entomologists, nutrition ists, food growers, m anufacturers, physi cians and others interested in the prob lem. In the field of pesticides, testimony was overwhelmingly against the idea that the public health was being endangered by their use. However, a few physicians p re sented convincing clinical and laboratory evidence of widespread illness from con tact with DDT on foods or in sprays. culturalists, entomologists and others Louis Bromficld was not only con . agree. T here is no question at all in the vinced of the hazard b ut from his ow n minds of those who have personally ex experience told the Committee, that with perienced DDT poisoning. correct farming methods, few insecticides How may wc account for such marked were necessary or desirable. divergence of opinion? It is n o t difficult. Recently L. G. Cox. d ir e c to r of t e c h The manufacturing companies arc con nical projects for the Bcceh-Nut packing cerner! with a booming business. The company stated that h is `firm had spent entom ologists' responsibility lies m a in ly SGGU.OOO in the p a s t six y e a r s in a n a t in protecting our crops from insect pests. tempt to keep the newer pesticides out Both groups arc loath to accept evidence of baby foods and peanut butter. He also which m ight interfere with the p ro d u c stated that a fund, of $119,000 had been tion and use of the new insecticides. raised by the manufacturing chemists "to Knowing that some individuals exposer! counteract unfavorable publicity." Mr. to large amounts of DDT remain in a p Cox related that in a num ber of instances p a r e n t gootl health, they refuse to believe his com p an y had b een forced to r eje ct ~ that small, or even large, residues on food large orders of fruits and vegetables be might oc harmful to the general public. cause of contamination with DDT, Chlor- Physicians, on the other hand, are pri- danc and Benzene Hcxachloridc. ,, marily concerned with the health of the In a preliminary report to Congress in - people and arc quick to sense th e th re a t J a n u a r y , 1951, the C o m m ittee recognized Reprinted from The Modern Nutrition Afo^o^ine April, September en d December, lp }2 : . K b- t'C re 16725 \J Z iZ Z T T the existence of a definite hazard to pub that some docs. Spot check tests can dls- . lic health and continued hearings. These cover only a portion of it. arc still taking place. As a result of the evidence it is to be hoped that remedial legislation will be recommended to the Congress. (-1) Our knowledge of the long range toxic effects of most of the new com pounds is woefully inadequate. Therefore liow can tolerance limits be arbitrarily The purpose of this article is to give' set? Particularly when no tests have yet the members of the Academy the latest been perfected for even delecting the information on this highly controversial most recent ones. Logic dictates that until and extremely important subject. all the facts are known any residue on Extent of the Problem food is too much. For the past thirty years or more there Other Contacts has been a gradual increase in the amount As though poisonous residues on food of poisonous chemicals used on crops for were not enough, the public is exposed to the control of insects and fungous dis powerful chemicals in other ways. eases. During the same interval a corre sponding decrease in the protein content of our plants and the nitrogenous content For some unknown reason--and in spite of evidence to the contrary--we have been sold the idea, that DDT and chlordanc of our soil has been noted. arc harmful for all practical purposes only Since the introduction of DDT and to insects. The main basis for this impres similar compounds in 1!M6 the tonnage sion seems to be these facts: (1) A com of the new insecticides in use has tre paratively few, healthy young men, when mendously increased, while that of the exposed to considerably larger doses of cider chemicals has dropped. The marked DDT than would be encountered in or toxicity of these new compounds for most dinary usage, showed no ill effects. (2) insects and animals, together with their Animals exposed to DDT aerosols came unusual stability, has .exchanged new through unharmed except for local irri problems for olcl. tation. No allowance has been marie for It is no exaggeration to say that at the present time, with the exception, of fish, it is a rare article of food that, has not been dusted or sprayed with one or sev the fact that many humans are malnour ished. allergic and chronically ill; nor for the fact that the young are particu larly susceptible to toxins of all kinds. eral poisons before reaching the con As a result of this propaganda, few . sumer. Never before has the long-suffer households arc without DDT aerosols or ing public been made a guinea pig for sprays for fly control. And where ants, the mass testing of so many exceedingly arc a problem chlorckmc (even more toxic powerful chemicals. than DDT) is used for their destruction. Food Contamination Both of these substances may be encount ered in hotels and restaurants. Naturaily, Theoretically crops arc treated long enough before harvest so that weathering has reduced the residue below toxic levels. Practically this safeguard is of little com fort for the following reasons: contamination of food and utensils is to be feared. Most commercial tree sprayers tisc the chlorinated hydrocarbons. In addition to this source of exposure, pleasure drivers fl) Enforcement of regulations is not on the highways in agricultural areas may feasible except in the ease of large farms. be dusted by airplanes. It is physically and financially impossible to police all the small fanners. How often Is lho P-ublic Protected? has ignorant enthusiasm led to the appli At the present time the Food and Drug - cation of many times the recommended Administration offers some protection, amounts? Who knows? And how many but only has jurisdiction over interstate small producers, if faced with the destruc commerce. The great weakness of the tion of a crop by insect pests shortly be agency is this--while it has authority to fore harvest, would have the fortitude to set tolerances for pesticide residues on or refrain from spraying at that time? And in plant and animal foods, under existing why should they hesitate? The trade jour "law it can not prevent the use of a clicm- ~ - nals tell them any danger to the consumer ical before a tolerance has been set and - has been greatly exaggerated.j the relative safely of lho chemical dc- :- (2) When airplanes arc used for dusting iermined. fields, drift to adjacent areas is frequent. Most of the state laws are similar. This has been reported as leading to inno Therefore the stale agencies labor under cent, but heavy contamination of sonic similar handicaps. : crops. Potent new compounds arc being syn (3) While our state and federal inspec thesized at an amazing rate. Rough lethal tors are conscientious and hard-working, duscs for animal life can be determined they arc-limited in number. One wonders in a few months by acute toxicity studies how much food grossly contaminated .with in the rat and larger animals. However, poisons reaches the consumer. We know to learn the cumulative effects of repeated 2 16729 sj conl.ict with small amounts of a chemical requires careful observation over several generations. This may take two or three years. In addition to being time-consum ing. it is expensive. In the meantime the partially tested pesticide may be manufactured, adver tised. sold, and widely used.' Is this pro tection? It is only fair to state that some manu facturers insist on thorough investigation before releasing a new product. Others arc not so careful, partly because they lack the funds and facilities for such work, and partly because the law does not de mand it. Under the present laws any company wishing to use a new chemical in or on food is not required to first consult with the Food and Drug Administration rela tive to merits or potential harmfulncss. This agency may confiscate or condemn products only if they contain chemicals proven to be deleterious. The inadequacy of this law is obvious, but some illustra tions ar.c illuminating. Agcnc was used for artificially aging flour over a period of thirty years before it was discovered that dogs fed with bread made from such flour developed "canine hysteria" or "running fits." The use of this substance lias now been abandoned by the baking fraternity in favor of a less ? ? ? harmful substance. Denaturation of the proteins and damage to vita mins undoubtedly remains. Incidentally, the United Slates Supreme Court in ltiis) declared bleached flour to be unfit for human, consumption. If this judgment is still on the books, why has it never been enforced? When (he second of tho "sulfa" drugs, sulfanilamide, was introduced some years ago. in addition to tablets, a liquid preparation was sought. The drug was relatively insoluble in the ordinary sol vents, hut finally a chemical was found that would dissolve an adequate dose. This was one of the group now used as an anti-freeze in car radiators. Without adequate animal tests the product was marketed. It was condemned only after a number of deaths due to the solvent occurred. Other examples could be cited. IIow long must we wait before adequate pre testing may be counted on to prevent tragedies of this soil? The Older Insecticides Let us now consider tin- more impor tant chemicals used for the destruction of pests. First of ali we .shall discuss those in use for many yours--adequate enough to bring tremendous crops to maturity during liic recent world conflict. With the exception of selenium and arsenic, these chemicals do not transmi grate from the non-edible portions of a 3 plant to those parts used for food. Nor do they penetrate to any extent into the fruit or leaf. Therefore, most of the resi dues may be removed by scrubbing with soap and water and/or by dipping for one minute in dilute hydrochloric acid. The chronic toxicity of arsenic is well known. In small doses it may nave a tonic effect. "Arsenic caters" in the Tyrol feed it to their horses to increase their stamina and cat it themselves for the same reason. The custom is not highly recommended. As the chemical is usually metabolized and excreted in from one to six weeks, high chronic doses arc necessary to pvoducc damage. However, such high contamination may occasionally be found. Livestock grazing in orchards which have been heavily treat ed with arsenic compounds may show serious symptoms from such fodder. Some years ago the American Medical Associa tion condemned apples from one county in the State of Washington. These or chards had been so heavily treated with arsenic that dangerous amounts of this chemical had been taken up by the roots of the trees and translocated to the fruit. Arsenic is often applied to fruits in the form of lead arsenate. The limit for the compound on apples was set in 1040 ;it 3.0 ppm (parts per million). The lead, component is more dangerous in small amounts than the arsenic. Lead is stored ' in the bones at all dietary levels and may cause kidney damage. A high level of calcium in the diet tends to be protective.. Mercury is highly toxic and residual compounds are dangerous. As little as 0.5 ppin may induce storage in the kidney with resultant harm to that important organ. Mercury vapor used to preserve grain in storage has not been shown to contaminate such grain. Selenium in the soil is absorbed by the roots and transmigrates to all parts ofplants. The same process occurs following the application of sprays. This substance may not therefore be used ort food crops. It causes abdominal pains, anemia, weight loss anil liver damage. Catllc. cropping plants in sclcnifcrous areas such as parts of Wyoming, develop the "blind staggers." This is followed by paralysis and death. Selenium sprays arc used only on orna mental flowers. Fluorine compounds arc less toxic than lead arsenate. Their toxicity lies in their ability to precipitate calcium in tbc body. "Cryolite" is used as an apple spray. When so used tin: shipper is required to wash off the residue. Urmlung water containing from 0.1 to I ppiu lias sonic d i e d in lowering the incidence of dental crudes in young children, liven tlicso concentrations may produce mottled enamel and interfere with none metabolism. * 112219 4 16730 u- ' Nicotine is one of our most powerful, ' rapidly-acting poisons. One (train is the minimum lethal dose for man and even one-fifteenth of that may cause severe symptoms. It is a nerve poison, causing stim ulation which is rapidly followed by depression. T he alkaloid is volatile a n d therefore presents no chronic toxicity. Some of its sails may provide a residue hazard. For all practical purposes jt is no problem except to the applicator. ' responsible authorities, DDT was released for_com m ercial use in 19-15. F arm ers, on- "lomulogists and manufacturers had heard The pyrethrins. These were originally of the wonderful new insecticide and- obtained from the petals of -.member of clamored for supplies. T he pressure was the chrysanthem um family grown in the great. Early results justified all. expec Far East. Synthetic analogues are now tations. ' . available and have similar properties. Tiicsc compounds arc rapidly detoxified in anim al tissues and th e rlc th a l dose is very high. Therefore, they are comparalively safe. However, they may produce severe asthma or other allergic reactions in those sensitive to ragweed pollen. During the latter part of World War II, DDT had received extensive field triis * ,in.l 'typ h h c u s protection and mala of ria . our Th i a s rmed was forces a calcu from lated risk--and there .is no doubt that the d e struction of lice and mosquitoes radically reduced the mortality from these danger R otenonc from derris root is most p oi ous diseases. sonous to fish and not very toxic to man. In Southern Europe our troops were The thiocynates of which lethanc is an ` dusted with DDT powder for the elimina- example are absorbed through the skin. tln- of body lice. The same treatment They release hydrogen cyanide, a pow er vigorously applied to the population of ful enzyme poison, in the body. Caution Naples undoubtedly halted an epidemic should be exercised if they arc used as of typhus lever. garden for the sprays. control The solutions formulated of flies arc of a low order- In the tropics whole, islands were " c'd by airplanes. T he unsuspecting dustmos- - of toxicity for man. Nevertheless, inhala tion should be avoided. quitos and flies were decimated. O ther insets, bees and birds shared the same fate Since the introduction of the newer to a lesser extent. ' . pesticidesdescribed culiar pro the has pert use of rapidly ies of t the chemicals decreased. Th he new compo e u just" pe nds, ' . - An called tiu^Ia epidemic--! gas virus hepatitis nila at the- time troen affec this terit ted city is and so our troops was given together with their application to almost all foods, has created a potential hazard the same treatment. The cause was never definitely determ ined but. was attributed to health of unprecedented proportions. to unwashed vegetables acquired from Part-II the natives. .mopiMi * . For practical purposes, the newer pcsti- In the y e a r 1950 it is es tim ated th a t one cides may be divided into two main billion pounds of agricultural chemicals groups--the chlorinated- hinlrncnrhonx and w ere m anufactured. This gives an idea of th e ortjaiiic ihoxiihntr.x. W hile differing in ..the p roblem before us. mode of action' and oilier properties, both types of compounds in sullic-icnt quantity Physical and Chemical Properties arc lethal to all animal life and most DDT is the least w ater soluble organic insects. compound known to science. This fact The Chlorinated Hydrocarbons In 1939 Swiss chem ists discovered t h e insecticidal properties of DDT or diehlorodiphcnyltrichloroetlianc. Soon afterwards BllC.tjjg^acliloi-ocvclolicxanel. commonly known asoenzenc hexaciilondc, was conir ib u lc d b y F ra n c e a iu l' t h e 1' U nited Kingdom. Since that time American ingenuity has developed other compounds of similar type, namely toxaphcnc, TOE, chlordnn, mcthoxyehlor, lindane, aUlrin and dietdrin. New compounds arc constantly be ing synthesized. helps to explain why chemists assumed -- incorrectly as events have proved--that dairy cattle might be safely sprayed or dusted with that compound. It is however, quite soluble in m any fats. Its usefulness ns a pesticide and its danger, is to a con- ' siderablc extent dependent upon this jii-uperty. DDT is exceedingly stable. It is not normally decomposed by sunlight nor by cuoking temperatures. The leaching ef fect of ram is very slight. Hosidues on c i t rus fruits and foliage in California were found to be reduced by only 5091. in 40 days. Ten per cent still remained after A lth o u g h still undergoing investigation, 90 days, in soil the com pound is m uch nnd with, some reluctance on the part of more stable. One test plot still shows 4 to to to o 112221 I heavy contamination six years after one application to the soil. BIIC is a m ix tu re of optical isomers with a musty smell. Its application has been found to produce an oil-flavor in potatoes, walnuts and other crops. The Beech-Nut Packing Company has had to reject shipments of peanuts and vege tables purchased for its line of baby foods for this reason. With the exception of its gam m a isomer, lindane, the com pound is fairly stable. Lindane is the most active fraction and can now be purchased in a relatively pure form. Chlordan has properties intermediate between DDT and BHC, but on the whole is more toxic. Aldrin and dieldrin arc chlorinated naphthalenes of similar na tu r e --<jicldrin being the m ost toxic and rcsidualiy effective of the present in secticides. Toxanhene is a chlorinated cam phcnc of marked stability. Melhoxycnlor shares with lindane the fact that it is relatively unstable and therefore presents less resi dual hazard. However, one investigator has stated, that with repeated exposure, the toxicity of lindane may increase as m uch as 100 times! This 'm erits confirm a tion. Toxicology The chlorinated hydrocarbons are nerve poixnnx and th e ir action is associated p r i m arily v/ith their solubility in tissue fats or lipoids. The cuticle or chilinous cov ering of most insects is w ater-repellent and scents to protect them from dcssication or dcowning. The outer layer of this cuticle is of a w ax y n a tu re and has a definite affinity for DDT. This fortuitous property renders most insets as vulner able to contact with DDT as though ho cuticle were present. And the marked sta bility of this chemical presents prolonged opportunity for such contact, Flics readily absorb the compound through their feet. DDT has a similar action on the n e r vous system of insects and animals. Overstimulation' of tlte sensory and motor nerves >y lethal doses leads to tremors, convulsions and spastic paralysis. Death from exhaustion follows. . Toxicity for Man and Animals The acute toxic oral dose for animals is abo u t 250 m g /k g . For a 150 lb. m a n this would'be about ounce. The toxi city is enhanced w hen DDT is dissolved in dietary fat or in oils. Suspensions and powders applied to the skin arc believed to he non-toxic, but symptoms in m an have occurred follow ing contact with oily solutions. The ordinary household aerosol bomb used for flics and mosquitos is thought to be harmless. Animal toxicity only ap pears after exposure to a concentration 4000 times that found in this contraption. However, Biskind has reported severe reactions to these sprays, probably as a result of hypersensitivity. Recent experi ments suggest that repeated exposure may lead to respiratory infection in animals. Because DDT is poorly metabolized and excreted by animals and is fat soluble, it is stored in the body fat. It is therefore a cum ulative li&inon. T h e a m o u n t ingested in food may be concentrated as much as 30 limes in fatly tissue. W hen the diet is cnntainintacd, DDT is excreted in c o m paratively large quantities in bovine and hum an milk, being found primarily in the cream. Thus, cows fed through the w in te r on h ay containing (54-134 ppni of DDT, secreted enough of this chemical in their milk to cause loss of w eight in steers drinking it. Their ow n suckling calves accum ulated DDT to -'th e e x t e n t of 825 ppm in their body fat. When rats were fed for several years on a diet containing only 1 ppm, they stored up to 30 ppm in their adipose tissue. Chickens consuming contaminated ra- t tions magnify the DDT intake and excrete it in the yolks of their eggs. It is conceivable th at a rapid loss of weight in animals or humans storing large amounts of DDT in their body fat couid release enough in the blood stream to cause symptoms of acute poisoning. Clin ically this seems to occur. At this point it.should be noted that fat is a living tissue w hich is continuously being broken down and rebuilt. More over, all types of body ceiis are surrounded by semipermeable m em branes contain-, ing lipoids, which can theoretically hold DDT. This chemical is known to inhibit one of the oxidative enzymes, cytochrome oxidase and may interfere with the action of others. Therefore this type of storage in body fat is not like keeping b u tle r in a refrigerator. Toxic effects on each body coll even with high dilution are not too remote a possibility; Pathology Whereas rats do not show obvious signs or symptoms of poisoning on a ration co n taining less th a n 100 ppm of DDT, the i n clusion of as little as 5 ppm in the diet over an extended period of time produces minimal but definite liver damage. This is reversible, if all DDT is rem oved from the diet. Liver necrosis may be preceded or accompanied by fatty degeneration or enlargement. Fatty changes arc resistant to treatment with choline. While early stages of dam age m a y be r e p a ire d , A. V/. A. Drown states, "-- hut if fu rth e r chronic doses prevent its regeneration, the detoxi cation mechanism of the liver for DDT is impaired." The damage rrsulting from sc cere poiMining with tnc chlorinated carbons varies somewhat with the animal and the com pound. However, few organs escape. P ath ological changes reported include edema 16732 saying that the "Virus X" epidemic was the result of DDT poisoning. Oiskind is emphatic in remarking that he "did not, and could not from the evidence at hand, make such a statement." While a virus believed to be responsible for this cpi. dcmic has been isolated, similarity of symptoms is conducive to much thoughL Evidence of Danger of the brain and spinal cord, slight kid ney damage, necrosis of the muscles, stom ach and gall bladder, hemorrhages in the heart and gastrointestinal tract and degen eration of the endocrine glands. TDE has a unique tendency to cause atrophy of the adrenal gland. Symptomatology- At present the harmful effects of the chlorinated hydrocarbon pesticides on Mr. and Mrs. John Q. Public arc debatable. The great majority of entomologists be lieve that residues on crops, in milk and "eggs and meat are low enough to be harmless to humans, and that any pos sible toxicity is overbalanced by the ne cessity of these chemicals for the produc Acute DDT poisoning produces, in labo tion of adequate food supplies. Their con ratory animals, a period of nervousness clusions are open to question and I shall and hyporexcitabilily, with excessive blinking, cold skin, ruffled fur, lack of appetite and muscular weakness followed by the onset of tine tremors due to muscle fibrillation, particularly in the heart muscle, hind legs and back. Advanced poisoning lends to rapid weight loss, clonic convulsions, paralysis, and death. In ad dition to these, cattle and sheep develop staring eyes, diarrhea and staggering gait. Symptoms of mild chronic poisoning may cite some evidence to the contrary. Biskind, as one of the original physi cians suspecting toxic effects from the now insecticides, has reported numbers of cases illustrating definite reactions to DDT, chlordan and EHC suggesting both allergic and direct toxic reactions from these compounds. While some afflictions were short-lived, others persisted for many weeks or months. include only nervousness and failure to Pot'.cngcr noted that patients who re gain weight normally. ported gastrointestinal upsets, followed Through 1P5I a total of only 14 human fatalities due to DDT were reported. Most of these were the result of swallowing, accidentally or with suicidal intent, rela tively large amounts of DDT dissolved in oil. A few severe reactions probably of allergic nature'have been described. These included two deaths. Six cases of hemorrhagic purpura and one of agranulo cytosis were recently reported. Symptoms resulting from the accidental ingestion of unHisynh'vil DDT: include nau sea, vomiting, diarrhea, nervousness, sore throat, joint pains, muscular twitching, impairment of sight and insomnia. Recov ery often takes place in a few days, but one or two weeks later by respiratory symptoms, often showed mild jaundice and evidence of liver damage. Fat biopsies analysed for DDT varied in content from 0 to 33 ppm. lie fell the exhaustion and oilier associated symptoms could be at tributed to exposure to DDT in food or household sprays. There was no question in his mind but that exposure to DDT could cause serious illness including one type of pneumonia. . Pottcngcr strongly recommends that any foods sprayed in the process of grow ing, preparation for market, or the final handling, shouid carry labels to that effect. Let the public know what it is buying. . in one case took as long as twelve months. Krohn and Pottcngcr in a medical pa Biskind from his observations has added the following: overwhelming fatigue and - per awaiting publication, and included in their, testimony before the Select Com muscular weakness, peripheral neuritis, mittee of the House of Representatives shooting pains, areas of skin hyperesthe sia, sense of a lump in the throat, severe to Investigate the Use of Chemicals in Foods and Cosmetics, conclude as follows: emotional depression and vague fear, and (1) The widespread use of chlorinated decreased vibratory .sense. hydrocarbon insecticides has produced It will be noted that many of these many cases of toxicity. symptoms are typical of the so-called (2) These insecticides accumulate in fat virus infections that have been widespread and principally damage fat containing or in the past five or six-years. gans, as the liver and nervous tissue. Diskind has called attention to the fact (3) In the presence of characteristic that the symptoms described during an epidemic of "Virus X" in California sev eral years ago corresponded almost 100% with those tabulated by him in cases of DDT toxicity. As a result of these obser signs, symptoms and blood studies, de tection of chlorinated hydrocarbon insecti cides in a biopsy of the patient's fat con firms. the diagnosis of insecticide poison ing. vations some writers have quoted him as (4) Chronic toxicity from these insccli- fi I l ..l F.Z77JT cidcs has been observed more frequently Ironically enough, flics in most areas than acute toxicity. arc becoming resistant to DDT. And what The writer has observed a small number is more--once resistant to this chemical of eases with negative fat biopsies but they rapidly seem to acquire immunity to evidence of liver damage, that gave a sig others. Authorities therefore, arc once nificant history of exposure, lie has also again stressing the old methods of control seen several eases of indisputable allergic --cleanliness and eradication of breed reactions to these substances. ing places. And so we coinc full circle In other words, the presence of DDT once more. in body fat proves that absorption of this While translocation of DDT from the' chemical has taken place but docs not ground or foliage to the edible portion of necessarily indicate actual poisoning from plants seldom occurs, enough EHC and that substance. lindane may be so transported to give an However the presence of liver damage favors such a supposition--particularly if more than 5 ppm of DDT :s present in off-flavor to potatoes and other vegetables. Therefore the use of these chemicals is decreasing. adipose tissue. Exposure to DDT in the home is all too Contrariwise, an allergic or hypersen sitive individual may theoretically ex hibit violent reactions to minute amounts of DDT without absorbing measurable amounts. The only criterion necessary for diagnosis is the reproduction of symp toms upon repeated contact. common. Aerosol bombs for control of flies and fleas should be considered un safe--as should ciiiordan powder or sprays for the destruction of ants and other pests. A case of poisoning from the latter com pound was recently reported in the J.A.M.A. Tlve increasing use of lindane. DDT and Sources of Exposure similar compounds in restaurants and There is adequate evidence to prove that other public places by vaporization has many foods raised commercially contain been mentioned in a previous report and at least traces of DDT or similar com is.attended by definite hazards. pounds. These include staple vegetables Hvpcrsusccptiblc individuals may incur and fruits. damage from contact with outdoor sprays For all practical purposes DDT docs not used domestically or commercially. They penetrate through the skin of fruits. How should be .avoided. ever, it becomes incorporated in the skin Tentative Conclusions - or rind and can not be removed there-' from by any washing method known. In spite of the loss of nutrients present in this layer the only safe method of avoid ing possible high contamination is to tecl all fruit. Beef, chicken, lamb and pork may con tain relatively large amounts of DDT if the fodder of these animais was contami nated with the substance. The vise of corn husks, pea vines and other heavily tainted sources of livestock feed renders this pos sibility a real one. Fatty portions of meat, contain the largest quantities. The purpose of this article is not to create-alarm but to alert thoughtful per sons to the hazards inherent in the use of DDT and allied substances. At the present lime food production in the United States would be disrupted in a few weeks if the newer pesticides were discarded. This would be disastrous. However, he evidence herein presented points out inherent dangers which were not forscen, and if suspected were -- and still are -- ignored by many of those in responsible positions. ?-iorc evidence of human toxicity is needed.v ... j As has been mentioned before, milk, cream, butler and eggs may be serious offenders if contact with sprays or DDT containing food is'adequate. Nor is margarinc innocent. The vegetable fats in this Your..House Select Committee is doing a fine piece of investigation and deserves encouragement. PART III I product may be contaminated as a result The Organic Phosphates I - of spraying or dusting, These compounds were developed in j ' It took many months before it was rea lized that trol with dairy DDT coarttlfeilslCpray>nedlofwor fly con dilution would exen-te these substances in their milk fur ten days or inure. This even occurred when barns were fogged or whitewashed with DDT in spile of the fact that food bins were covered and the ' cattle removed. At the present lime these chemicals arc not recommended for use Germany during the past war for use as insecticides. Humor has it that they may be the so-callcil "nerve gases" whose polenlialily for harm hrmighl bail dreams to our inlellii:i-nee staff at that lime. He that as it may, these products are so toxic that they arc limited to applications in the field by trained operators. Their use on livestock and pets is not recommended. (Department of Understatement.) " in dairies or oil beef cattle. Instead, lin Physical and Chemical Properties dane and inethoxychlor are suggested as The organic phosphorus compounds arc substitutes.. among the most toxic chemicals used for 7 16734 pest control. Exposure to relatively small blood sample for cholinesterase levels may I amounts may result in severe illness or help in diagnosis. Sedatives and the inira- " death. venous adm inistration of atropine in doses [ T E P P Cor te tra clhylpyrophosphatc ) is the most potent of the group from the acute standpoint. It is several limes more toxic th a n nicotine. llfc'.TP (hclraetliyltelrnphosphnte) is on a p ar w ith nicotine amt owes most of its destructive potential to the percentage of TEPP present in the mixture. Both these compounds are ra of 1 to 2 mg. as often as every hour may be life saving in severe eases of poisoning. Death may occur within six to ten hours of the development of symptoms. Recovery from acute attack s is usually complete. Adequate protective clothing and special masks most he used by those handling these chemicals. '| pidly hydrolysed and therefore dangerous TE PP and 11ETP present no problem. 1 for only a few days after application. Fruit pickers have become ill after enter- ; They arc also rapidly detoxified in the ing an orchard treated with parathion > body and therefore not stored in the several days after its application. Food . tissues. crops are not supposed to be sprayed J P arathion is an ester of thiophosphoric acid and contains a su lfu r radical. It is more slowly decomposed and as a result presents more of a hazard. It is also fat soluble to some extent. Toxicology w ithin thirty days of harvest. A safe rcsi- due on ^ny one item of diet is considered to be 2 pp:n of parathion. In the ease of citrus fruit the peel will absorb and r e tain higher residues than arc allowable. This is also true of other fruits. As r e gards apples and pears, if parathion is ; f * There is little difference in suscepti applied--"strictly in accordance, w ith the ' bility among various animal species to recommendations of tiic Bureau of Ento the toxic effects of these compounds. mology & P la n t Q u aran tin e of th e U. S, Several hundred human eases of poisoning Dept, of Agriculture with particular refer have been reported with a num ber of ence to the time between the last spray fatalities--most of them due to careless ing and the harvesting of the fruit, norm al ness in handling the products. weathering should result in parathion The organic phosphates are toxic by absorption through the sltin, by inhalation residue no greater than a fraction of a part per million." and by ingestion. As little as-one drop of N evertheless toxic parathion residues concentrated solution in the eye has have been reported by Pottenger in bread caused death. W hereas T E P P and ITF.TP and canned fruit. Again the h u m a n e q u a have failed to show cumulative effects, tion enters the picture. animal experiments have shown that re peated contact with small amounts of Susceptibility to the organic phosphates ' is variable as one would expect. In cases ' parathion may produce poisoning. of suspected poisoning a physician should It has been estim ated th a t 25 mg. of alw ays be called. Blood tests m a y u n co v e r TEPP by mouth will produce moderately chronic eases. severe sym ptom s in m an. 100 mg. orally would probably be fatal as would 500 mg. applied to tltc skin. The alkyl phosphates inhibit the enzyme cholinesterase. This enzyme destroys ace tylcholine. one of the chemicals liberated at nerve junctions and endings to transmit the nerve impulse. Thus a lack of the Absorption and Translocation T E P P is rapidly absorbed into p la n t tissues as shown by growth inhibition or stimulation and other metabolic disturb ances. The rapid breakdown of these com pounds limits the importance of their absorption to the effect on plants. enzyme results in a marked prolongation Parathion-has been reported to kill a of each nerve impulse affecting prim arily' variety of insects on plants w hen applied the parasym pathetic p a rt of the nervous only to the soil. Biochemical studies and . system. Nicotino-likc eficcts are also pro bioassay have shown that absorption and duced. . translocation docs take place in insecti '1 On acute exposure symptoms develop rapidly once the cholinesterase level has been sufficiently lowered. They include loss of appetite, nausea, vomiting, abdom inal cram (is, excessive sw eating, pupillary constriction, diarrhea, respiratory diffi culty, headache, dizziness, muscle twitch cidal amounts. The relative stability of this chemical together with its toxicity makes these findings of great im portance to animals and humans. Chemically al tered parathion has been .found in some citrus fruits. . The toxicity has not yet been reported; 1 ing and weakness. Severe cases dcvclopl coma, convulsions and death. Systemic Insecticides These arc chemicals absorbed and Repealed exposure to small dos'-s may translocated by actively growing plants result in illness of severe degree. The e n in sufficient amounts to kill insects feed zyme system recovers fairly rapidly after ing at a site distant from the original a p one episode, but may take weeks after plication. The principal compounds of this n um erous subclinicat contacts. Testing a group arc "O.ViPA" (octam cthlypyrophos- 112224 ( ft t 112225 phoramidc). "Syslox'* (diethyl clhylmcrcaptocthylthiopliosphalc), triphosphoric acid penta dimclhylomidc and the bis fluorophosphme oxides. After absorption the chemicals may b? changed to less or more toxic compounds. Injury to the plant is possible. Transloca tion to edible portions may make them toxic or produce a tainted llavor. At the present lime the use of these svstemie pesticides on food crops is not permitted. They are still in the experimental stage and should always remain so as far as food for man or animal is concerned. It should be staled once more that in addition to the systemics just dcscrihcd, evidence of absorption and translocation exists for arsenical compounds, paratluon, benzene hcxacldoricle. DDT, loxnpucne. chlordane, dieidrin, selenium con-mounds, fluoroacetates, and fiuorohyririns. The last four groups.have been considered too toxic for use on food crops, although dieidrin has recently been released for use on peaches in California. Benzene hexachloride (as reported in Modern Nutrition for October) may no longer be applied in vegetables or fruits in this state. However, where it has been used in the past the soil may remain contaminated to a decreasing extent for live years or more. It has taken a long time for the au thorities to take such a step and now treated ground will not be free from traces of the chemical for eight or ten years more. In the meantime the popula tion has been exposed to the effects of BHC for a period of four or five years. This is but one example of the price the public is paying for the use of new chem icals in and on our food without adequate investigation of possible harmful effects from these poisons. Practical Aspects By this time those who have followed these articles should be considerably alarmed over the number of poisons hu man beings in this atomic age must en counter. And rightly so! It is a deadly and grim situation. Naturally the question now arises--so what? Where no we go from here? What can I do to protect niy family, myself and our wonderful Nation from what seems to be serious harm? Unfortunately there is no pat answer. However, I am going to discuss steps which seem logical at the present time. Legislative Action While your government representatives in Ideal, state and federal capacities real ize there is a problem and wish to pro tect the public, they are under, pressure from pesticide manufacturers and dis tributors whose chief interest naturally is in sales. Those groups refuse to behove that exposure to small amounts of insecti cides constitutes a great threat to public health. Entomologists being primarily respon sible for the production of large crops to feed a hungry world are loth to accept proof of chronic toxicity--and such proof in humans is difficult to obtain. (There is plenty of evidence of such putcntial haz ard from animal experiments.) Meanwhile Departments of Agriculture are busy con ducting investigations along lines of ioxicily (or crops, animals and man. You arc the guinea pigs and must write your slate and federal representatives asking them to take action. You must also contact your local departments of health along the same lines; The other side of the question must be heard from. Personal Precautions In an attempt to avoid contaminated food? one must not go off the deep end and develop malnutrition in the process. Many foods from large truck farms where spraying directions are cnrcfuily followed and supervised contain 1V0 detectable resi dues. However, a certain number of crops, because of ignorance, willful neglect of advice, drift from nearby elds or other factors show slight or moderate and oc casionally, heavy contamination. Surveys in the Los Angeles area in 1P50 showed detectable residues of DDT in more than half the samples examined. About four percent were heavy. Arsenic contamina tion was negligible This risk is unpre dictable and at the present lime cannot be prevented. The following suggestions may be helpful: 1. Whenever possible, purchase' foods that have been grown without the use of toxic insecticides. At present the supply of such foods is markedly limited. Encour age the development of organic farms in your area. Grow your own vegetables. 2. Do not use sprays or aerosols in your house that contain DDT. Chlordane, lin dane. BiiC or mcihoxychlor. Make sure that commercial exterminating companies do not use these chemicals in your house or on your trees and shrubs. Do not cal in restaurants using vaporizers for fly control. 3. Peel all fruits and vegetables that lend themselves to such treatment. Scrape the outside layer from celery. Washing will not remove residues. Purchase eecs from local ranches that arc not using DDT, BHC or chlordane in hen houses. (Motiinxyelilor and lindane are not cumulative and do not ordinarily build up in chicken tissues, hence in eggs.) Milk, cream and butter are much .infer lb.in formerly. Most dairies now use melhoxycldur and lindane for fly control and very little is excreted in the milk. Ciu-rk with your local dairy to find out what chemicals they use. Shift to another dairy if yours is still using DDT, UI ICC or chlordane or dieidrin by the "strip'* method. 16736 AA I Most vegetables growing in the ground Summary and Conclusions such as potatoes, carrots and beets are comparatively safe. Contamination by The various types of chemicals used for the contrul of insect pests have been de 13HC will produce an off flavor and thus scribed together with their potential tox give warning. Potato skins may be eaten. icity for man and animals. The fact that Fruits and vegetables that have at in tervals shown contamination with DDT some of the chlorinated hydrocarbons, even when absorbed in trace amounts, may be ill the Los Angeles area are celery, cab concentrated at least thirty fold in the bage, cauliflower, greens (spinach, par sley. endive, turnip, etc.) and lettuce. Apples and tomatoes arc included, but quite safe when peeled. In case of stom ach upsets it would be wise to avoid these body fat of animals and human beings, renders even slight contamination of our foods a public health problem, of first importance. Such contamination is pres ent and in susceptible individuals is pro vegetables for a few weeks. Dried fruits may be higher in DDT than fresh fruit and they cannot be peeled. Some olives and grapes arc suspect. Prunes show* very little residue as a result of the alkali treatment given them. ducing illness. This fact must be faced and steps taken to protect the public in terest. The Delaney Congressional Com mittee has been investigating the problem and will recommend legislation if this seems needed. The Committee needs the support of all. Moderate amounts of salads greens arc fairly safe and desirable food stufTs. Home sprouted soy beans, miing beans, alfalfa and the like from untreated seeds provide an excellent source of uncontaminutcd and Suggestions have been made by means of which the public may reduce the intake of insecticides to a minimum. It is impos sible to avoid ail contact. nutritious salads. If the use of insecticides were 'suddenly Lean meats are satisfactory sources of protein and sea foods are naturally free of any residues. 4. Attention to other factors' affecting the general health is important. Adequate rest, avoidance of worry and tension, mod eration in the use of coffee, alcohol and tobacco, sufficient exercise and a diet low in refined carbohydrates need emphasis. . Vitamin supplements such as yeast or stopped crop failure and hunger would be widespread. Perhaps the solution may lie in the following steps: (1) Fairly rapid abandonment of the more toxic pesticides in favor of less harm ful ones. (2) The development of widespread pest control by natural enemies. Excellent re search on this subject is being carried on at the Riverside Experimental Station. vegex, dcssicatcd liver, rice bran con (3) As Poltengcr-has suggested, foods centrates, fresh wheat germ, cod liver oil that have been sprayed or dusted should or its concentrates and kelp for trace min be so labelled including the insecticides erals are helpful. More potent prepara used. The public has the right to know tions arc often indicated but should pre what it is buying. ferably be taken only under medical super vision. (4) Testing of food crops before market ing. While costly it might oc cheaper m Allergic.slates and chronic infection of the end, to test for BIIC, DDT, arsenic, any sort interfere with adequate metabol lead and parathion, where any or all of ism and should be corrected if possible. Naturally other ailments should receive these have been used on food crops before tlic crop is sent to market. To allow ; the necessary care. for differences in concentration and other 5. Repeated attacks consisting of up per respiratory infections and/or gastro intestinal symptoms together with Joss of weight and marked fatigue suggest pcstLcidc toxicity or allergy. In any case of this sort see your physician. Other causes may be operative. If he finds nothing wrong suggest the possibility of insecticide factors, a sample from each acre might be indicated. Thus heavily contaminated crops would be discovered before reaching . the consumer. (5) The passage of State or National laws condemning tire manufacture of aerosols, sprays or vaporizers containing DDT, chlordanc ,lindane or mcthoxyehlor for use in homes or public places. These poisoning and ask for a blood-count and chemicals should be used only. outdoors liver function tests. Do not try to diag and then with extreme caution. nose and treat yourself. Yon are almost (C) The gradual spread of organic farm bound to be mistaken and to do more ing methods niclutiing the use of city harm than good. wastes for fertilizer purposes. The work of In' definite cases of DDT poisoning a strict diet In,"ether with other nutritional therapy will he necessary. Recovery is often discouragingly slow but to be expected once the cause has been recugnizcd. -K. Pfeiffer along the lines of special cul tures to produce a rapid breakdown of organic material shows much promise. In spile of a great deal of skepticism, an in creasing number of farmers and soil ex perts believe that healthy plants arc more 10. ! j t } J ; 1 i C5 16f t resistant to insect pests and that healthy plants can only grow in rich soil. It is probable that the future of America and, indeed of the world, depends upon the rebuilding of our impoverished soil through the addition of organic m atter, trace minerals and judiciously used com mercial fertilizer--not upon the develop ment of newer and more powerful insec ticides. 2901 Wilshire Dlvd., Suite 3-15 References BICISnKomF1NomnDidt.tePeMro.todSuI,ci:itvscSMtlUamtcencmt.ce1nT2t)he1b9Ue3f0so.ereoftChehemSeicleaclst BtPSsKyIcNhoDt.heMra.pyS.. 11a1n.dNoI.rv2in(AgprRili)ch1e9r4:9. Am. Jl. BRCtrO.oMmFoKmoIdiEttLePeDr,otdoLuoIcunt*ivse:(sMStitgaaaytt)eem1De95nie1t.Ubesefooref CthoeemSeteleactet BR71O7905W1.pN.., ANewW. YAo.r:kI:nsJeochtnCoWn.tiricoyl bcy SCohnem. iIcnacls... CAAaSngTdnOcATu.rila.Jn.CslhoEec.mata.i.nondVoTlo..f7C,in.NsoeAc.LtGiLciEidJNeus:mcAbiybs1o9p5r2lpa.tniotsn. COUNCIL o1n49:P.7H`i7ARiMMnAyCYZ\) AMNSDI. CHEMISTRY: CO9Pp)UhhoaN1rr5Cum5Is0La.irnooslenocctyiPcHiadnAedsR.MtoJAx.AiCrn.YMin.rAA*y-ND1o4fl:Ccl0eH4rE*tatMvin3ISipTMhR-opYst:. DEmFNoiotDtdeYe.PJrtooohdInun:cvtSsestat'iMtgeamatyee)ntth19eb3e1Uf.osree othfeCSheelmecitcaClsomin EDinITaOdRipIeAsLe tDisEsPuTe.. MJ.5A:.M73.5A-7.3:GIn(Mseactri.ciIdDe) s1t9o51r:age HiO4tni2cSs.ee9Ksc9It6Nii.cn.Si1d.9Ce5Ws0a.l.rifeNosrfu.n:ltiiaDn.egpJoforsuoirtmna.vnEadrcioroneusosimdcuiocentEorfonltroepmcreoanlc.t. KNCFIooGomdHmsTiat.ntedeGC.tooFsm.:IneTtvicce.ssit.tiigPm.auoronty2th,be1e9f5oU1rs.eetohfe CHhoeumseicaSleslecint KCRSheOleHemcNitc.alCsBoienmrnFmaonritddte:PerSotdtaoutcetmIs.nevPneatsrttibg2e.aft1oe9r5e1t.hteheUHseouosef LA2AON4.cGMGcu.(.AMrEr.a.eArn?.c)r..ech1F9oi.5vf1Me.s.DKDluTUdN. iZHnEygha.unmr&laCnO.cScla.. tPMRaeIndCd..K3Em:2Ti4lTk5-:. LEo1H0f5M0.iAnsNe.ctiAc.idJe.s:. PJh.aAr.mMa.Aco..log1i4c4a:l10c4o*1n0s0idetrSaetpiotn.) LEVFHoooMl.dAXaNInV.d,ADN. roJu..g:3BO(ufJfluliceiityaiinte7,o9o5ff0.tthhee AUsnsiotecdiatSiotnateosf. LE3sc0H7rMti(cAJidNucn.se.A).B1J9u.4:l9l.eTthine MN.ajYor. Atocxaidc. aMcteiodn..s2o5f:38in2-LEOeHrftfMfmicoAionaNdlsc. haAodef.mdJiUit.ci.:vaSeSlss.o.mVmoeBalyu.tlo1ol.x4r.iAcmNosalosoyn.g.3incFoa(otJloubrdleeya)apsnoe1drn9m5s0Di.wtrtheudye LEVABMounlMln.lueO3tai4Nin..,rNeADpooe.llrpet4tn.,fo1A3r9.5c.0trh.iCceuhclitae..lfe,nSdbtaautrreeyaeouafroC1fa9l5iC0f.ohreTnmiha..e-. MDIEn.TsCeCcA.t.Lic1Fi9d.-eiaKs... L.N: aTth.eRMeosdeaerochf ACctoiounncoilf. OWrcaasnhi.c. rOCI'lPhoTeumFse.iNcaSGlesKleRicnt. CFFon.wnMuls.nitatJncerd.,tCooTsIemnsvteietmsictoisg.naytPearbttheefo2r,\eJ1s*95th1oe.f ROJa.nIAIdW.MtEo.RAx.i..cSil1,v4A4o:.1f0a4cn-ed1r0t3aIIi.n(SLep.pntHo.s.vp9i1uleor1r:9u5Ps0h.ianrsmecatcicoildoegsy. SelCtiecivaocettnesgC.rioenUsmsn.Fmioo2niotntddeCeaPStlreoronvdIdnnuanvcrnets.sN:tioHgR.aoet1uep1so3et3rhteo(JfNUaonsR.e.e3o3p2)fr5e4C1s.9he5nr81it1.nas*t. SIpMMneMidroO.,nNsVSoa.ln.Sd3.1r.WeNl.a:ote.Hd4eas(ulJtbuhslytha)anz1ca9e5rs1d..sAomf. EJc.oniormopic. ADDENDUM In reprinting'this article, which first ap- Ceared 20 years ago, it seems logical to riefiy review what has happened since that time. Chemical roulaminalion of mir environ m ent has In-come obvious. D D T and other chlorinated hydrocarbon pesticides have un expectedly been spread by wind currents thouglumt (he world: They have penetrated our food chains and are now round in high concentrations in some fish and seals, boll) in the Arctic and Antarctic. Many of our rivers arc sewers and some of our lakes are riving as a result of changes caused by chemical effluents. Our coastal waters are so badly coniaiuiiialed llial in some areas many fisli have lu-en found with eaneernus growths. As a result nf DDT. the brown pelican may soon In- extinct am i oilier birds are seriously threatened. Human beings carry in their fallv tissues about 12 parts , per million of D I ) T or its degradation products. Many individuals have measurable residues nf oilier chlori nated hydrocarbons, including the piienoxy herbicides and the polychlorinated bi phenyls (PCBs). Degenerative diseases, in cluding cancer, arc on the increase. Studies hy the Dionclics Laboratories in 1905 -- but not released until 1909 -- showed th a t the piienoxy herbicides. 2.-1--D. 2.4.5-- T. 2.4-D P and Silvex not only pro duced birth defects but cancer in experi mental mice. So did D D T and related chlorinated hydrocarbons, as well as other herbicides such as the T ria zincs. P araquat ami /Cert ran. il is oj considerable concern that D D T and 2,4-D have been used on nnr land crops since about 1Via and th a t 2/i-U. 2.4.5-- 'T, Silvex and other herbicides have In-on available for use on lawns also during this periiwl. Since 2.1.5--T anrl Silvex arc contaminated hy the 2.3,7.3 telrnehlorudi- lii-nzo-para-<lixius. ii liirli, in anim al exp eri m ents, hurt: p ro em In be lip In n n r millinti lim es m are nelit'c than llinlidnm ide in pro ducing tlefnrmrd yuan;:, can lum inal ion hy these herbicides m ay be much m ore'serious than suspected, incidentally, the toxic effects ot ef ntshi ve e lIyH rdaocchuhmu neinhtlend- nb/y. o -Spt-. dJiaocxqi nuse wer line e ex Ver- retl, i'b.D., Division of Toxicology, F.D.A., 11 112227 ( i 112228 in April of 1370. Obviously, the results of animal experiments cannot bo necessarily applied to human beings. Nevertheless, the results arc of great importance since minute amounts of carcinogenic chemicals may, at a much later date, produce cancer in suscepti ble individuals or animals. According to the Delaney Anietidnieut to the Food, Drug and Cosmetic Act of l'J-13. auv chemicals which produce cancer in animals must ho banned from foods or cosmetics. I'henoxy herbicides .even though in trace amounts, now contaminate about thirty per cent of our water su p p lie s and a recent report indicates that the ubiquitous l'CUs have heen detected in fifteen to twenty per cent of our drinking water. Such contamina tion may occur from local applications, effluents of various types, drift from aerial spraying, transport by winds from other parts of the country, or by as yet unknown means. "Agent Orange," a mixture of equal parts of 2 / . - D and 2.4,5--T . was used for m any years as a defoliant in South Vietnam. This chemical weapon may have irreversibly de stroyed the balance of life in the estuaries, os well as ruining as much as thirty percent of their hardwood forests. Not many indi viduals are aware of. the fact that "Agent Orange," contaminated by the fantastically potent tctrachlorodihcn/o-p-dioxiii3, was used for many years in our ov.-n forests be fore it _>vas sprayed as a defoliant in Viet nam. Since it v.ns banned as too dangerous in Vietnam in 1370, why has its use not been forbidden anywhere in the United Slates? Mr. Ruckleshnus. Director of the Environ mental Protection Agency, is attempting to limit the use of dangerous chemicals, such as D D T , 2.4,5--T . Silvex and sim ilar chlori nated hydrocarbons, in order to protect our environment, lie needs support. Powerful in terests are opposing this move. Proof of definite damage is now a t hantl through the repeated pesticide spraying of Globe, Arizona, and the resulting extensive research of one of the victims -- Rillec Shoccraft -- whose hook "Sue the Dastards!" provides a cogent and moving account of her experi ences. In the Globe area, plants and crops were destroyed; domestic and wild animals died or disappeared human beings and ani mals became ill: birth deformities were noted in animals, as well is hum an licings. R epro ductive difficulties affected women and some of them long past the menopause -- much to their consternation -- tiled from the uterus. Recurrent attacks of "virus" were frequent. In 1371, the Dureau of Reclamation used Silvex on (ho ltiu Grande ltiver near San Acacia*, about sixty miles south of Albu querque, in an attempt to clear salt-hush from the river books. The Bureau felt that Ibis was using too uiueli water. Without prior warning, helicopters sprayed the area, con taminating ill llur process members of two families and two herds of cattle. Ruth the h um an beings and the cattle became ill and many of the latter died. T h e remainder of the herds, even though they hart been re moved from the are a and were eating vora- ** ciously, lost weight and had to he sold a t a tremendous loss. Post-mortem examinations indicated damage to the liver and intestines, as well as to other internal organs. T h u s ,' wo know that Silvex can not only cause se rious illness in cattle, hut death as welt, if the exposure is great enough, Fortunately, the human licings recovered. 2.4--D is contaminated by some of the d i oxins and. other noxious compounds. A re cent study, reported to the French Academy of Sciences, involving the use of pheasant ' eggs injected wifli 2.4--D , revealed startling findings: Matchahility was reduced by about 70 percent and numerous embryos were deformed. Most alarming was the finding that in fifty percent of the-em bryos, " the genital tract of the male embryo resembled that of a normal female." T he authors stale. " I t is important to note that even the vapors of 2. -1-D have an analogous action . . . W hen noil-treated eggs were p u t in treated nests, the embryos show the same sexual anom alies.'' Conclusion: " It is a question of fem inization or physiological castration." (Are human beings exempt from this influence?) Disturbing reports of cancer developing in S w e d ish workers .using 2,4--D and 2.4.5--T along railmtnf'rights-of-way and of deformed babies being IKirn to two women in A ustralia exposed to 2,4.5-T, have apparently had no inllucnco on those who a re using these com pounds in our forests an d on our ranges. Californians should know that for the past twenty years. 2,4-D, 2.4.5--T . 2.4--DU a n d / or Silvex have been, and still arc being used in various combinations in our national for ests up and down the const, in order to con trol weed growth along firebreaks and power lines, as well as to destroy brush in planned brush conversion areas.- W h eth er spraying is done by helicopter or ground crews, drift is inevitable and this m ay account for the fact that many Californians arc showing j the presence of these herbicides in their blond.- Whereas herbicides arc being used alo n g . state highways, th e application of 2.4.5--T and Silvex have n o t boon authorized by the Stale of California for three years. Compounds being used include Simazine and smaller amounts of A lr a z in e ind I'ara- ( qunt. I t is. therefore, p robable th a t most of the phenoxy hcrhicklo contamination is . the' result of nir-hornc residues: This needs - ! more study. _j The extrem ely high incidence of virus jm rum onitis in experimental animals ex posed to pesticides during the itinnetics Studies for teratogenicity an d carcinogenic ity is highly significant. In the |inst few monllis. a n increasing jm m hor of illnesses seem to lx: associnled with herbicide spraying in the Los Angeles area. T hese so-called "virii: infections" tend to recur in waves and arc extrem ely debili tating. Half a dozen patients have said they 12 112229 felt they were dying. These occurrences are extremely disturbing. ! Some specimens of beef contain residues of the synthetic estrogen. dicthylstiibcstrol; which has cancer-inducing properties. even in minute amounts. Meat, and particularly liver from these cronlurcs. nmv also contain residues uf tiie phonoxy herhieides and other ! chlorinated pesticides. Chickens supplied \ with food conlainin:: stilhcstrol. of course. I may also exhibit residues in their moat and : livers. Ju st recently, the use of slilbostrol I in animal feeds has been' haunt'd. However. 1 pellets may still bo implanted subcutaneous* 1 ly in animals, and whereas the residues will probably bo less, tiicre is no proof that some will not remain. Lindane vaporizers arc being phased out but arc still being advertised anti sold. "Mo . Pest" slrips m ay bo just as dnn::erous as tiio vaporizers. (Two monkeys who chewed on I their flea collars containing dichlorvns, the t . active ingredient of many "strips," were re- ! ported as suffering from repeater! attacks of virus pneumonia together with other com plications. One of them died and (he other recovered after a prolonged illness.) Hoxnchhirnphcni- sponge baths for infants were recently found to cause serious poison ing and ;i unrulier of death;;. A three percent , solution of hcxoehlorophcnc had been used I and it is now known diat the scrotal area , absorbs practically 100 percent of most compounds applied lo it. ilcx aciJorophcnc ` has also been shown to contain other toxic contaminant.;, socii as tiie dil>em;odin:<ins. Hexachlorophtme lias now been banned for use in drugs and cosmetics, except on pre scription. T h is is another example of long delay in recognizing toxic effects front Lite chlorinated hvdrocarhons. Last year, liquid formulations of 2.4,5-T Were banned, together with any applications j near water supplies, campgrounds, etc. On ' J u n e 14. 1972. with a few exceptions, the j use of D O T in tin; United Slates was ordered discontinued by the Environmental Prntec- ' tion Agency as of December 31, 1972. This I is as it should he. If it sticks! | . I suspect that recurrent, so-called "virus infections" which have appeared with in creasing frequency over the years and which ; produce symptoms such as nausea, vnniilI mg. diarrhea, prostration, severe headache, I hody pains, depression, sweat;;, low grade fever, sinus congestion, sore throat, laryn gitis, hoarseness, feeling of constriction u n der the breast hone, "virus pneumonia," and t . severe pains in various parts of the body | m ay all lie due 1 contact with pesticides in nur air. food and water, either orling as " virus perse, or lowering resistance lo viruses. . (Morion ltisl.ind, M.U., Westport, Cotiuectieul, speculated on this fuel 21) years ago.) T he suggest ions ill my original article are slid valid. However, since the subject of mercury has .surfaced, swordfish were found " lo lie higher ill m ercury eonlr.nl th in other fish. T hey were, therefore, banned tempo rarily and arc now carefully screened 60 that swordfish appearing on the shelves is fairly safe if one does not cat it more than once weekly. Examination of museum speci mens of various types of fish has hnwn the presence of mercury in those which were twenty, thirty or forty years old. This indi cates that the importance of mercury has not yet been completely evaluated. Lead contaminates the air of our cities, as do lluoridcs, nitrous oxides, etc. A good in take of Vitamin C helps to reduce lead toxicity. Vitamins A and E. together with C, help to minimize smog damage to the lungs. Liver, yeast and B-compiox in addition to these vitamins ami a good diet high in raw, uncoutaniinalcd foods grown, in fertile soil, const itulc logical nutritional insurance against infections and the development of degenerative diseases including cancer. Obviously, resistance to toxic chemicals is related to active enzyme chemistry which, in turn, depends upon good nutrition and avoidance, insofar as that is possible, of ail chemical compounds which are enzyme poisons. Fluorides arc now one of the most serious contaminants of our ecosystem. T h e y are producing allergic and toxic reactions. Therefore, the endless drive by the U.S. I'uoiic Health Service and the American Dental Association -- bockcd by our tax dollars -- to promote artificial fluoridation of water supplies, seems not only stupid and foolhardy but dictatorial. Unfortunately, other toxic herbicides, aside from tiie phenoxy herbicides arc stiil being used for weed control on vacant lots' and along highways and freeways in Los Angeles County, ns well as in agricultural areas. These include Diquat. Paraquat, Atrazino anti Sirnar.ine. (Paraquat possesses the unique quality of musing lung damage when it is inhaled; swallowed, or applied to the skin in sufficient quantity.) Research funds arc urgently needed to . monitor foods, water, plants, and people for tin; presence of organic phosphates, herbi cides and other chlorinated pesticides as well as lend, arsenic ami mercury. Tw'enty thou sand dollars is needed for ;m adequate in vestigation. Any donation will help to keep tiie work moving until larger gills are re ceived. Checks made payable to the PriccPotlenger Mutriliou Foundation, labeled for " research purposes and forwarded to me, will be most welcome. Such donations are deductible for income tax purposes and will, he acknowledged. We need your help! Once again. I would suggest that you look for sources of uncoulniiiinntcd foods; grow vegetables in your own backyard; in.ike viiur ['pinions felt by writing your legislators. If nur civilization is lo survive, tiie use of potent and persistent pesticides must he re duced to an absolute minimum: mineral and organic matter letiiniod lo our soils and food grown for quality" rath e r than "quantity." ns recommended m any years ago by \V. A. Albrecht. Ph D., Chairman. Dept, of Soils at the University of Missouri. tc i 1674 I f * s A l*I*r i . . X . Your future and that of your family de pend upon your actions. Whereas individual activity is important, croup action-- is more effective. Southern Californians should con tact and join an American Nutrition So ciety chapter. There are chapters in Pasa dena, San Fernando Valley. I.oa Ancclcs, Pomona and South Bay at Palos Verdes. Whereas special situations may require the use of pesticides and the judicious use of artificial fertilizers, the widespread, in discriminate use of these substances must be controlled if our fuod supply and health arc to ho preserved. ' "Vcrlmm sapienti salis est,'* ItE F & nK N C E S H A ltM E T l, Tl: M .: U nfit foe Itu m n n C onsum ption, .1V4 ,*>!.. HW NTKll. New U. Acts c r: l*rralce*(i.iU, Inc., V.: Con-mmcr Acu-nrc. 440 1071, pp., $N<Ve9w5 Ynr\" Sim on nml Shusicr^ 1^71. S8.PS M l U .Mil. M . W. & C . (l._U rc: Chem ical F ctteut, -- ji; ..,S o rin /:ic l(i:`*nina' C r'"l,honiiui,-ir>O>,- 512.25, SHOM Cif A l" l\ 1.: **Sur the tiantard!" <G0 po I'W m * : F'-tnklin 245 iC, ImiiAit School i<d 1971. *4.45. a O :io ro CO O' LV '