Document bBjXaO12544MV3zw44Y54pxpO

Dec: Kollcix PaDKtsaaey 27 H&R7 Ox. M. i7 = TM!ioi,vs JtancKjrcii Division llti lei-liitf 1-0. 3'3 tiaticisai Ca:\H uacis-ter Dytc> 'Jf Ohio . . ' y( ' . . Dear Dr-. %'tecanaat Attached is a. photDBttnt tiio orrii^inol paper o 2>r- 'Jeer stem Xn Sweden ,, 5rl-a fcinfj to tioiychlornt'ad .. bipl>o^rl3 X vi.ll bo happy to huvo youe ideas after yow rem-, .it. As.'fur ad tSia 'seetion en . tetiedlccr/ is ooncornofi, it-..... ; is fenws t-liSt :b;vloracid>; ntfl liver trouolo-. can result frh-a ?. r-r.Tr"dlcac'r> < ' w;'.othoi-"or hot this is at oil iclavant: -to treall cyvftati ti.n5? n:<i.ot-irr iu Ituiann ; Sat is ,' of .course ,, nj entirely diffuirimf qoantlop. At' any raw,' X 'ooliova before v* yoacry about, tiro toxicoloqioal. part of the protliad, vfc should sottlo *tho analytical parti . .Gineesely, jKi.-.u/in aUt. P.. Kmtrvot Kcilly, n,, D. Kcsdiojrvl. Director 0631149 PHGNCR 2000087 MONSFOXOOOOOQ88 WATER PCB-00044033 O' . Vr Chixiroan, L&dloe and goatlenvnt 0U ' < \ ' ' V.:-' la honor %o our Brittiah Je&t X will try to hold this lecture in Engliffho Aa the title of thlfi locturs statesp X aa today going to toll about tho discovery of eotae hlthorto unobserved Chlorinated hydrooarbc&D having up to oight chlorine in tli nolocule and round'in residue ana-- lycico fha ohonioal nano of polyohlorlnatod bifenyle ( In the following collofi ICE)o So get ficailiar with JOB X will start with the chemistry and oxloologla ` .. phpttqetry ' .: . S'n She main'characteristic of PCB lo U Sheir'vory high stability0 Is on ,| oxcnple they can he boilodwith nitrlu acid without being tiostro?ft<U r to they ore hardly metabolised Sn living organism* 3* If more than 4 chlorine arelpre^sent they ere non inflsonbloa It is clear that these three charap^iriatiofl floeo it eaoy to understand that when they havo * entorod the ^Living organisms the will have a low peroiotance * But it is difficult "to explain bow they find their way into the living organism* : One thing opens to bo clear, they don't oose fro* agricultural ubq, ; but from a technical one end most probable it donas to the nature vie . wastes that ore tried to bo burnt up, because then >ro have then, at .onoo ' in the *irv-baoause of their non inflemability a , ; .ToXlQOlOKl ho FOB wero Introduced la 1929 and an early as 1936 7Ones.and Alien j reported tint ?J ant of 24 nan employed In manufacturing of FOB ouiforod Iron an acne tors' eruption of th* skim. Acne did not appear until 6 to 8 months after the material was first'mood. In 1957 Drinker reported J-J that rats exposed to chlorinated 'biphenyls In concentration of epproxl- 2 nately 1 ne/t? for 16 hours a day for 6 nooks chowdi damage of the liver. g After that tine the allowed concentration of'FOB in air' ie.0,5 ng/a^. , (For DDt the same value 1 0.5 - 1 ag/ffl?). Ihe same authors finished their experiments in 1939, and related that those compoun&d have on injourlouo effect, manifested solely in the liyor. Cblorinotud blpehnylo apponred to. be'.the^most imjourtouo ohlorlnated. compounds of 'all tested. PHGNCR 2000088 MONSFOXOOOOQ089 WATER PCB-00044034 Greenburg, Moyer end Smith 1939 reported' that K and polychlorinated . naphtsloaee are blamed tor the death fit three young workers,~7 and thaiT* pregnant women and pereono who have at any tine had any liver disease* are particularly s>uspectitle, , Wedolj Bailer end Benton gave 1942 animals FOB ineluding adniniotration by inhalation, ingestion and shin absorbtion. Histological esoalnotlon S the viscera showed important toxio effect only in the skin and liver, and the degeneration effects in the liver are essentially the oone what ever was'the method for the adaini etration, Faribok (1955/ found ao an occupational poison in the eleotrical induBtry, mixed totra and posts ohlorobiphecyl eauoos folllculitle, oomodo, pyoderala and other olio affections, ond that its principal toaio offset la fatty degeneration of the liver, ' Miller (1944) injected 69 mg FCB (4 end 5 chlorine) euboonteneouoly in _ 32 guinea pigs. Bight to ton days after Insertion, fat droplets wero noted In. the liver oello, and after 16 days tbey were present in modorato Dr very large numbers. Babbits and rats were also tested in thle investi gation, ao well as the FC5B was adninistated both oontinouely, ouboontin- ously or ingested in the food. In the feeding experiment 0 guinea pigs received 2 donee of 69 mg of the chlorinated biphenyl 1 week apart. Death occurred In 11 to 29 days. Finally Uo Daughlin 1964 reported a method to toot the chemical toxicity and teratogenlo effect by in)eotion into the yolk sao of tortile eggs prior to incubation. PCD wan found between the eight compounds among 100 tooted having tho highest order of toxicity. Uo hatoh was found at a level of 25 mg pa egg. it a level of JO mg per egg, one chi ok hatched out of 20 Injected eggs, but died 2 days later. Some embryos which were examined after they died, Bhovred weak deformities (often a short upper brek) | and growth retardation. Dead acetate resulted as an example In no hatch at a level of 1 g per egg. Autopsy of the dead eobyoe have showed exton sivo brain damage. Mercuric chloride shoved no hatoh evon at a level of , 0,5 mg per egg. As the analytical ohemistry is a pronounced service science X have boen in centnot with many ooientiots from other fiolds during tbs work with residue analysis, and I have always found this contact very stimulating for my own work. Ihie oo-oporatioh often demands that wo era talking the same solontlfio language. Booauso of thle need I will today try to give a lecture in low level analytical chemistry for biologists, illustrated by the rooidue analysis of polyohlorinatod biphenyln. ?he looturo will bo divided in the following three sub-divisionei . PHGNCR 2000089 MONSFOX00000090 o0 o . 1# Chemistry of PCB end -tbofr tocloology, 2, Analytical methede for residue analysis toad proof of structuree, 3o Behaviour of PCB la naturet differences in metabolising *ato of the PCS compononto, patoasation in an soological oerio, con centration levels and examples of samples which hare boon proved to con tain PCOo A residue analysis can be divided ins '. ' ' 1,, Extraction of the pestloidea from the biologloal material, , , followed by a careful oleaning-up to tako away interferring substances, meet often fats. oe 2. Identification analysis by aeon of gas ohromstography. Thin- . r layer chromatography and mass spectrometry, 3. Quantitative analysis.. '. ' At as ecological laboratory in Blksnuedet is Stockholm 1-2 g of a sample is cut out of the biological material and transferred into a weighed and o oerefully cleaned test tube, and stored at -20 until analysis. Smaller campion have boon used, min. 3 ag of body fat, and with dry materials such as hair, feathers, pise needlee 100 mg ere sufficient to reach the desired 10 e/s level in residue analysis. In oases of water proofs 1 1. is used for reaching the 10 pg/g. level. B.l(hooog) la order to facilitate complete extraction of the fatty moteriale from the biological sample, the doulbe amount of finely powdered, anhydrous ' mag&eeiun sulphate is added to the sampling tube, and the wholo In-homo genised with an lnecrtable homogenisor'. fhe rasuiting powder Is transferred into a special Soxhlot extractor. After d hours of extraction the solvent ie evaporated, leaving the fat in a small weighed toot tube at the bottom 'sox.-tube) of the extractor. Ihls fat is dissolved in methylene chloride in such a way that 100 ul (o,l ml) contain 20 g of fat. She 100 ul solution is now transferred te a little object glass, 5 J 1 cm, covered with a sllioagel layer 1 mm thick, in order to form a line 0,7 cm from one end of tha slide. Inserting this thin-layer plate into a vessel . the bottom of which ie covered by a few mm of methylene chloride, the olvont will be euclced up in the dry layer of ailleagel, and at leaet 3 reach the upper end of the plate. She faot is that the fat has a greater affinity to. the powder on the plate than the chlorinated hydrocarbon have, -- and we get a Depuration. (The fat being more polar than the chlorinated hydrocarbons will nsver go longer than 2 om before the ' . .1 028**22 PHGNCR. 200009 MONSFOXQ0000091 WATER_PCB-00044036 0 2X02IOS tuba o solvent roaehos tbs uppop part of the gloss. Eh* front of the tat appears quite visible against a leap, and with the aid of a razor blade the sons above the fat is transferred to the olutlon tube and the chlorinated biooidoo absorbed on the powder can nos be oluted by ono ml of ethers The concentration is sufficient for detection of tho chlorinated hydrocarbons denn to the 10"^ g levels Eho next stop in tho analytical proeeduro conooxns tho separation of tho different chlorinated hydrocarbons that the cample may contain. Ac a mat-tor of foot,, thin 4e a troublesome task. It io easy to estimate what is not proaentp but acre difficult to say exactly one la present. We suffer from tho negative demonstration, as trill bo shown later. , At first a few words about tho separation of the components present In the sample. and their visualization. _ 2be> separation is ocoonpliohod by mean of a gas chromatograph fitted to a detector that transfers Its impulse to a recorder. ' 2?ho systom io shortly .described! A spirally formod glass tube with an inner diaaoter of 2 mm and about 2 B in length is filled up by a support ,, covorcd with on thin layer of on oil. The tube is heated In tho ohromatograph to about 00. Through tho tube n stream of nitrogen oontinously follows. When about 10 ul (1/100 of 1 l) of the purified 'sample is lnjoctod Into th* tubs, the eompononta of the sample will be ovaporltod and go forward through the column with the gas stream. As the constituents have different affinity to the column filling tboy will pass the column with different speed and.it will take different time for then'to reach the detoctor at tho other end of tho glass tube. If tho temperature and the nitrogen flow ore held constant thin time, tho retention tine. has a apocifioualuo for a contain compound. Ehio ie true, but unfortunately it la bIbo a fot that two oompenonto can have ths-samo retention time. Shis is one of the bigger problems in gas ebrematograpbie analyola of unknown samples, as will soon be obvious. So moke it possible to estimate tho rotontion time It is nocoBD&ry to visuallxe the chlorinated hydrocarbons. For that purpose mora or loss spoolflo dotootora aro used. She dotootor moot often.used in postlcldo analysis is the so celled elootxon capture detector, which can detect down to ono picograa (<* IO"1 g of linden). Onfortunatoly thio detector is not spoolflo for chlorine, bu gives answer aloo for orygenoontalning compoundsIhe rosponso her* la mucj: lower but can be oountorbalanood If tho conoon- trntlon of tho oxygon containing is much higher, 0281x23 Tho prlnciplo for tho electron capture detector is ohortly: At the end of the gas chromatographic tube io placed a little tuba cen tal nlnr m foil Dado of titanium trltido. This Is An-radlant. Eha o- PHGNCR 2000091 MONSFOXOOOOQ092 WATER PCB-00044037 . V* . . &-------------------------- ------- o . - <3 particle a arc reacting with the nitrogen aoleculse coning from the ooluan. ^hen wo get t Mg -- e o Hgo Over the Aeteotor we have a tendon of 90 volt and by mean of the electrons ere will get a oonetant elootrieal current over the detector, fhls standing current is transferred to a . one*-aV rooordor ae a conetant baseline, Wien now a chlorinated hydrocarbon leaveo the oolxmn thie oeapound has a high affinity to the ulootrons and this noene that tho amount of oloctrons will diminish,, and they will diminish proportionally to the amount of chlorine, The oleotrioal current will ilea diminish and this is noted as a peek on the recorder, $he crea of tho peak will be proportional to the amount of substance in the sample* By noon of a standard injection it is now possible to compare the reten tion time and the area of on unknown component with the retention time and area of tho known standard* As said before this detector la not ape ci vile far chlrlne but anyhow very useful^ because of Its high sensitivity ?he system described has, as we have Been, two disedvantagoo: to wo different compounds can have the name retention time and be detected as one peak* '. 2d A xegifitrated peak does not need to be chlorinated , because the detector la not specific* If tho sample la injected in two different columns with different chemical properties we have increased' the chance for a good ceparatlon. If two compounds have the same retention tine on one column they may not have it on anotherB When a result eeeme doubtful, - if the compound being responsible for a certain peak contains chlorine or not - it ia possible to concentrate the sample and analyse ,-lt od a less sensitive detector' such as the micro doume trie one, which is specific fox chlorine* the compound is burned in a furnace and teh generated chlorine titrated directly, , Is is seen from tho two last mentioned possibilities it la anyhow possible to get a rather high degree of certainty in rebiduo analysis, but it is t a rather fcime-oonsumeing work.When using this method Just described, we * very ofton found that many chromatograms from residue tuaalyois of most carefully purified samples still contain a large number of peaks* Ua*y of these have retention times that do not agree with any knovm chlorinated pesticides, or their metabolites* fhls chromatogram con serve ae an example* It was obtained by residue analysis of a oen~oafile found dead in the archipelago of Stockholm, In the range of the known peaks, ihore JJ 5 are do many unidentified thot there oloo ouct be an obvious riok of the . known peaks to be covered by unknown ones* If thlfl rwmarJr in fAxiYirt th< runnrtffl nf wnnv nv**virmnn*. PHGNCR 2O0OO2 MONSFOX00000093 WATER PCB-00044038 tltnbive anal^^ie must be brought Into Quest In. In the present invocti- gation it is shown that most of the unknown peak of chromatograms at rcoidue analysis of chlorin&ted. pesticides axe due to polychlorinated biphenyleo - I wi11 shot? <& chromatogram of human fat analyoed on a so called 8? 96 c0lwan.o the moat often need type in pesticide analyse. Early retention tines more in agreement with DEEff BE?op and PWpp, Host ollde show tho oaae sample analysed on a QP-t column. Hot? the former 2 pf pecks hay * divided Into 4 peaks,end two of them are allll in agreenont with BBSpp and op., tho two now were unknown0 Xogioally, those unknown component were at first thought to be metabolites of the inoectioideo* Against that spoke that noigthsr treatment nor conoontratod sulfurio sold in others This treatment made it rather cure that the compounds did not contain onygene In Sweden residues of organic me? cury have boon investigated rather intensively in the Swedish fauna. Ae these compounds give very high responses to the electron capture detec tor it was also investigated if the unknown peaks could havo a mercuric origin* It was found that the water-eoologioal series had high residues of both moroury (v/eotemark,Johnelo) and the unknown ones, when the same indivi duals were analysed. Anyhow,, the pheasant Buffering most from mercury poisoning only contained low levels of electron capturing compounds and these belonged * ' ' to the normal in&ectiaidco. Therefore the unknown oould hardly be mercu rials or.metabolites of'them. . . As the eagle sample giving the chromatogram shown In fig. 10. could be oetim&t&d to oontaln EDI' end PBB up td 15 gAg in extractable fat, the amount of unknown compounds oloo- were suggested to be in the same range, and tehn sufficiently high to do a run on the combined gae chromatograph mass spectrometer* If this could be done eucccoeSully it would be possible' to get very important information about the ohenio&l nature of the unknown, fqr ex. the molekular weight numbers of chlorine etc. Thin method is up to now the method giving the highest degree of oprtainty in the low level analytical chemistry, amounts of 100 ng substance being enough. 6 Z V IS Z 0 As this method for identification of totally unknown residues surely will bo very important in the future (when f.ex* a biologist has found that fishes in a river die) it may^possible by mean of this method to find out exaotly what compounds are responsible for the death* 7or this reason, X will go into come detail with thto method. In the actual oaoo we took the extract from 20 mg eagle and concentrated it as touch as possible and made Injection on the gaa chronntOgr&ph combined with the mass spectrometer* The result was the chromatogram shown on the next olldo. Every time the recorder shewed that a compound io leaving the column, the effluent is led to the mass Bpeotromotoro Now Just a few worde about the aaao spec* Tho molecule* leaving the column are bonded with electrons at B* ^ have Sow got the molocule positive oharg*dB but with the earn* maoo no before * This XI0* ieacooloroted in a vacuum and will then get a kinottc enargi . where is the speed* Next cones the magnetic field that . trios to bend the direction of the molecule* This will be big for. a email molecule and less for If wo have & ciovo In the other end we can directly read the molecular weight* Added to this parent molecule H+ we will also got addition in~ formations, because of the fact that II* may not be atbblo, a port of thorn will be broken down before they reach the sieve In the other end* " 2>Hf H HD2 - CCIj Maos Dpeatrogroas froa th# Siffarent unknown peaks In the ooglo enmpla ob shown* Tho mass numbers equal to the molecular weights of the unknowns could be read to 42d392B 358, >24* jfetoniBhingly, the molecular diffe rences were constantly >4 mase unite* This difference ehowa a familerity in origin of the unknown* Now the fact i that chlorine emcinto as a mixture of two isotopes with atom wolghto >5 end 57 in proportion 75*25# If the molecule has one chlorine, thie will give *two molecule pecks, one for C1^ and on* for (Jl^* If there are two chlorine wo hare the possibility , of one with only 01 one with hoth 01^ and >7 and one with 2 Cl^ and therefore 0281426 PHGNCR 2000090 MONSFOXOOOOD095 WATER_PCB-00044040 An explanation of the familiarity of the oompounds can be given If one substance is built from the former by substituting a hydrogen with ohlorins . ' ?hon it is posslb} 0 calculate tho molecular weight of the parent hydrocarbon yBC0 UjjHj E " s Bci x Sg 9 ??here H is the molecular weight of the cpnponont having x chlorine atoms. F.ex. for m t> 426 and 8 Cl w will got Upgg 426 - 280 + e 154 and Qual with tho other molekyle. Tho noet probable formula with carbon and hydrogen giving thlo moleoular volght ie C12 and `this oan only bo satisfied when -tho pnre&t-hydro- oarbon Is biphenylp end the unknown being polychlorinated biphenyls, fhic explanation was later fully verified by injection of a oynthotlo PBC on the mass spec. yurthermore'extensive gas chroaatographi0 Investigations provod that the PBO standard gave peaks with the case retention time as the unknown peaks from the sea eagle* ' * With the mothod Just deoorlbed X suppose thot we have a new possibility to study the residues In the air because the pine needles can allwsyB bo . 0e havo had great difficult; in quantifying the ?CB, but when getting a little more time it will be possible. We have done ft few calculations on a few' species, and X suppose they ore right within a factor 2. We have found the residue to bo frost . It has been my statement here to~day to present this method for studies of defiling of the nature, and with this method a new typo of defiling agents has been found to be present in nature, and a few experiment haro shown where they may bo found, How this method is going to bo used in the first hadn to estimate how the situation la in nature as a whole, and in the other hand to find tho leaks throug whloh they find its tray to nature* Soem neybe are prooont hero today to get news about the leaks? and to then I want to cay ooso book In a year* P0G2VCR 200009S MONSFOXOOOOOQ96 WATER PCB-00044041 So mueh X think X can Day again that the PCB herdly can come from agriculture. Be support for this suggestion I can say that wo havo found FCB In eagle feathers from Blkemueeet from 1944, whero hardly any chlorinated pesticides sere used la egrlcultrue. One more thing that X find Important to say Is that In contrast to the mercury problem this docs not oeom to ho a pure Swodiah problem. X have 3ust studied chroma! ogramo token from Xondoa air,, and they clearly contain FCB, sad dr. Moldoa hue tola mo- that ho also find them in his fiahBamples. But finally in waiting mt more results Xnshould like to point8&e morething. It is proved that FCB combo to nature0 wo dont fanervr non where they are usod, hut they are very poroiotant to chomicale and to fire. X think the poison Jury should try to state that a content of FCB shall always bo found in an open deolar{w tion. . ' Of81428 f PHGNCK. 20000% MONSFOXOOOOOQ97 WATER PCB-00044042 PHGNCR 2000097 MONSFQX00000098 WATER_PCB-00044043 Uiu/ tl f3 rz If k> o o O /V t't 13 /3 a /0 * 7 /2 fi-p'oor & S' fo O r*-- o fo u PHGNCH 2000098 MONSFOXOOOOOG99 WATER PCB-00044044 PHGNCR 2000099 MONSFOXOOOOO1O0 WATER_PCB-00044045 u fk r> r* n n - fk o IQ ro a n // O n /2 // /<? o <Mf*r* o Pp'-DOT p-p'-m. phgncr 2000100 MONSFOXOOOOOIOI WATER PCB-00044046 PHGNCft 2000101 MONSFOX0OOOO1O2 WATER PCB-00044047 -T 6 t & O. . V /i w O ft, n l wofive l*p.p'-DOT. pp. qoq X> DffLD/VlJ pp-ooe nt-D/iffs i WDiW PHGNCK 2000102 MONSFOXOOOOQ103 WATER PCB-00044048 0281435 o.frr PHGNCR 2090103 MQNSFOX0Q000104 WATER PCB-00044049 o . . o.. o 0281636 0 fe 0 6 ' PBGNCR 2000104 PONSFOXOOOOOI05 WATER_PCB-00044050 PHGNCJR 2000305 MONSFOXOOOOOIO6 WATER PCB-00044051 PBGNCK. 2000106 MONSFOX000Q0107 WATER_PCB-00044052 OJ8l<i39 raGNCJR 2000! 07 MONSFOXOQOOOI08 WATER PCB-00044053  '. ' 0 W ,. o PHGNCR 2000108 MONSFOX00000109 WATER PCB-00044054 /pr i? *..;Y,./ * ' * . <r a o '!> PHGNCR 2000109 MONSFOXOOOOOIIO WATER PCB-00044055 SO '' * v. 3 tf r* . ' 0K9 a PHGNCR 20001 SO MONSFOXOOOOO111 WATER PCB-00044056 /cyzyo >?/. //, 0281<i43 PHGNCR 2000111 \ I MONSFOXOOOOO112 WATER PCB-00044057 ,' ^ . . . <jTy 2> *o* PHGNCR 2000112 MONSFOXOOOOO113 WATER PCB-00044058 fo/sy* r? r. /3 r\ PHGNCR 2000113 MONSFOXOOOOO114 WATER PCB-00044059