Document pmz90yw3yn44zy1K4KekanOJB

A r n 6 -o w BIODEGRADATION (ABS/LAS Shake Culture Test) TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The test substance is a white powder. The 3M production lot number was 83. The test sample is referred to by the testing laboratory as FC-143. The purity of the sample was not sufficiently characterized, although current information indicates it is a mixture-of 96.5 -100% test substance and 0 - 3.5% C6, C 7, and C9 perfluoro analogue compounds. METHOD Methods: Shake Culture study modeled after the Soap and Detergent Association's presumptive test for the determination of ABS/LAS biodegradability. Type: Aerobic GLP: No Year completed: 1978 Contact time (units): 2.5 months Innoculum: Activated sludge collected at the 3M Chemolite Facility, Cottage Grove, MN, 3M Decatur Facility, Decatur AL, and Metro Wastewater Treatment Plant, St Paul, MN RESULTS No biodegradation was observed in the 2.5 month shake culture biodegradation study. DATA QUALITY Reliability: Klimisch ranking = 2. This study meets all criteria for quality testing, but the analytical methodology is questionable. The purity of the sample was not sufficiently characterized. C03S33 REFERENCES Fate of Fluorochemicals in the Environment, Project number 9970612613, E.A. Reiner, July 19,1978, 3M Company, Environmental Laboratory. OTHER Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/25/00 003634  Form #747-1 T A TECHNICAL REPORT SUMMARY ow 7/19/78 TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN (Im p o rta n t -- I f re p o rt is p rin te d on both sides o f paper, send w o copies to TCC.I Division E nvironm ental L ab o rato ry (EE & PC) Pro) a c t F ate o f F lu o ro ch em icals in th e Environm ent Report Title Biodegradation Stu d ies o f Fluorocarbons - I I I To Author(() D . ^ L . B[ac-on E . A. R einer TSwS rtat^nc. 4 4 7 0 3 , p . 6 - 1 4 , 2 1 , 2 5 - 2 7 , ' 2 , 3 5 , 3 9 - 3 , 45727, p . 32-35; 49400, p . 11-12. SSEECCUURRITITYY* ^ Ooon S Cloi-od I| 3MreCgHiEsMtrICyAL ^* 48; Bapt. Numbar 0535 ProJact lumbar 9970612613 Report Number 5 Employa* Numbar)) 47816 No. of Paga*Including Covarahaat 17 QNowYCeahemicalsRepJorNteod (KSEalYaWctOtaRrmDaS:from3M Tha.aurut. Suggaat other pplicsbla tarma.) (B iodegradation) EE & P C -D iv . E n v ir. A ssess. Fluorochem i c a l D egradation CURRENT OBJECTIVE: To e v a lu a te th e s u s c e p t ib ilit ie s o f FC-95 and FC-143 to m icro b ia l decom position. REPORT ABSTRACT: (200-250 word) Thi* abitract information ii di*tributtd by tha Technical Communications Cantar to start 3M'art to Company RAD. A b io d e g ra d a tio n stud y is d e scrib e d w hich allo w s th e e v a lu a tio n o f th e s u s c e p t ib ilit y o f FC-95 and FC-143 to aero b ic m icro b ia l d e g ra d a tio n . The c u ltu r in g p ro cedures used in t h is stud y are m odeled a f t e r th e Soap and D etergen t A s s o c ia tio n 's presum ptive (shake c u ltu r e ) te s t fo r th e d eterm in atio n o f ABS/LAS b io d e g r a d a b ility . M icro b ia l in o c u la were o b tain ed from a c tiv a te d slu d g e c o lle c te d at C h em o lite, D ecatu r and M etro w aste treatm ent p la n ts . A n a ly tic a l procedures in clu d ed GLC, TLC, X - s c i n t i l l a t i o n co u n tin g and a n a ly s is fo r re le a se d f lu o r id e . D e g ra d a tio n o f r e fe r e n c e compounds dem on strated the s u it a b ilit y o f the biodegradation te s t c o n d itio n s. Information Liaison . Initial.: 003635 2- - SUMlARY F lu o ro c h e m ic a ls FC-95 and FC-143 were shown to be c o m p le te ly r e s is ta n t to b iod egrad ation in a 2i-m onth shake c u ltu r e b io d egrad a tio n stu d y . The m ixed m ic ro b ia l t e s t c u ltu r e s used in t h is stu d y were d e riv e d irom a c tiv a te d slu d ge in o c u la o b ta in e d from th re e w aste t r e a t nent s y s te m s ( C h e m o lit e , D e c a t u r , & t h e Twin C i t i e s M e tro p l a n t ) . The c u ltu r e s were m ain tain ed in d ilu t e y e a s t e x t r a c t -b a s a l s a l t s m edia supplem ented w ith the hydrogen an alo g o f th e r e s p e c tiv e flu oroch em i c a ls . T est c u ltu re s a ls o co n tain ed FC-95 o r FC-143. Phenol and 1-dodecene-derived lin e a r a lk y l s u lfo n a te (LAS) were used as referen ce compounds. T h eir d egrad ation dem onstrated th a t b io d e grad atio n could occur under the te s t c o n d itio n s . A ll c u ltu r e s were t r a n s f e r r e d 15 tim e s o v e r th e 2 -m o n th p e r io d , and te m p e ra tu re was c o n tr o lle d a t 25 C._ d u rin g th e l a t t e r h a l f o f th e e x p e rim e n t. In th e f i n a l growth p e r io d , d egrad atio n p ro d u cts o f ^ C - la b e le d flu o ro ch e m ica ls were assayed fo r by t h in - la y e r chrom atography (TLC) and gas liq u id chrom atography (G L C ). C hem icals se p a ra te d by TLC were v is u a liz e d by T L C -au torad io grap h . M eth ylated and nonm ethylated c u lt u r e e x t r a c t s s e p a ra te d by OLC were d e te c te d by e le c t r o n c a p tu r e . No d e g ra d a tio n p ro d u cts were d e t e c t e d . S c i n t i l l a t i o n c o u n tin g showed th a t a l l r a d io a c t iv it y a s s o c ia te d w ith th e la b e le d flu o r o c h e m ic a ls rem ained in th e c u lt u r e medium. In a l l b u t th e f i n a l grow th p e r io d , flu o ro c a rb o n b io d e g r a d a tio n was m onitored sim ply by m easuring th e i n i t i a l and f in a l flu o r id e c o n c e n tr a tio n in th e m ed ia. No in c r e a s e in f lu o r id e c o n c e n tr a tio n was observed in d ic a tin g th a t i f b io d e g ra d a tio n d id o c c u r , i t d id not r e s u lt in the r e le a s e o f flu o r id e . C o n tro l c u ltu r e s supplem ented w ith flu o r id e showed th a t flu o r id e i s not lo s t from th e m edia under the experim ental co n d itio n s used. W hile th is stud y cannot r u le out th e p o s s ib ilit y th a t c o n d itio n s co u ld be found th a t would a llo w th e b io d e g ra d a tio n o f th e s e compounds, the r e s u lts o f th is study suggest th a t these chem icals are lik e ly to p e r s is t in th e environm ent fo r extended p e rio d s u n a lte re d by m icrobial catab olism . 003636  m : * l k * ' h 4 <4M 3- - INTROPUCTION The flu o ro ch e m ica ls s e le c te d fo r th is s tu d y , FC-143 and F C -9 5 , have p e rflu o rin a te d carbon chain s and are ch e m ica lly s t a b le . The p e rflu o rin a te d p o rtio n o f flu o ro carb o n s have not been found to be su sce p tib le to b io lo g ic a l degradation (1 ). T h erefo re, b io d e g r a d a tio n s t u d ie s were con ducted on th e s e compounds p r im a r ily fo r th e sake o f co m p leten ess. W ithout such t e s t in g , i t co u ld not be s a id w ith c e r t a in t y th a t th e se compounds would r e s i s t m ic r o b ia l m o d ificatio n . S in ce b io d e g ra d a tio n was u n lik e ly , th e b e st fe a s ib le t e s t co n d i tio n s fo r b io d egrad atio n were s e le c te d . In o cu la were o b tain ed from areas co n sid ered lik e ly to co n tain a cclim a te d m icroorgan ism s. Long a c c lim a tio n p e rio d s-w ere used in an attem pt to s e le c t and develop p o p u la tio n s o f m icrobes cap ab le o f d egrad in g th e se compounds, and hydrogen analogs o f th e flu o rocarb o n s were added to tr y to s e le c t organism s th a t m ight g r a tu ito u s ly "co m e ta b o lize " th e flu o rocarb o n s. 03637 4- - METHODS AND MATERIALS Chem icals F C -9 5 , F C -1 4 3 , th e h y d ro g en a n a lo g o f F C -9 5 , anunonium o c t a n o a t e (th e hydrogen analog o f FC-143), carbon-14 la b e le d FC-143, and carbon-14 la b e le d FC-95 were o b tain ed from Com m ercial C h em icals D iv is io n . These chem icals were used as re c e iv e d u n le ss d e sig n a te d otherw ise (A rthur M endel-Ueport in P ro g re s s ). Standard lin e a r a lk y la te su lfo n ate prepared fo r use as a refere n ce compound fo r b io d e g ra d a tio n s tu d ie s was o b ta in e d from th e US/EPA Laboratory in C in c in n a ti, O hio. Except where n o te d , a l l o th e r compounds were rea ge n t g ra d e . C u ltu re Media ~ The c o n t r o l medium used in th e s e s t u d ie s had th e c o m p o s itio n shown in TABLE 1 . TABLE 1 CONTROL MEDIUM COMPOSITION 1) Basal s a lts so lu tio n s: 1 .0 g/1 - nh4c i 2 .0 g/1 - K2HP04 0 .2 5 g/1 - MgS04 -7H20 0 .0 0 2 g/1 - F eS 0 4 ' 7H20 2) W ell w a te r - 25 ml/1 3) Y east e x tr a c t - 0 .3 g/1 4) Hydrogen an alogs o f e ith e r FC-95 o r F C -1 4 3 - 20 mg/1 Media were prepared from s to c k s o lu tio n s w hich were com bined and b ro u gh t to volume j u s t p r io r to each c u lt u r e t r a n s f e r . A fr e s h s o lu tio n o f FeS04 *7H_0 was p rep ared and dry y e a s t e x t r a c t was used in m edia p r e p a r a t io n ^ a t e a c h t r a n s f e r . The pH o f a l l m ed ia was a d ju s t e d t o 7 .5 w ith 1 .0 N HC1 and i f o v e r s h o t a d j u s t e d b a c k w it h 1 .0 N NaOH. The w e ll w ater was added to in s u r e an a d e q u ate s u p p ly o f tr a c e e le m e n ts . A n a ly se s o f th e w e ll w a te r made d u r in g th e 12-m onth p erio d p r io r to the in it ia t io n o f th is stud y showed i t s ca lciu m h a r d n e s s to ra n g e from 92 t o 144 mg/1 e x p r e s s e d C a C O g . Any p r e c i p i t a t e r e s u ltin g from th e a d d itio n o f w e ll w ater was removed by f i l t r a t i o n th ro u g h a #54 Whatman f i l t e r . 003638 5- - The p u r ifie d hydrogen an alo gs o f FC-95 and FC-143 were used in b io d e g ra d a tio n t e s t m edia and c o n t r o ls . These compounds were in c lu d e d in an attem p t to s e le c t a m ic r o b ia l p o p u la tio n l i k e l y to degrade the flu o ro c a rb o n s. Enzymes cap ab le o f c a ta ly z in g d e flu o r in a tio n re a c tio n s are fre q u e n tly id e n t ic a l to enzymes in v o lv e d in carbon-hydrogen bond c le a v a g e ( 1 ) . A d d itio n a l components o f o th e r s p e c i f i c m edia a r e l i s t e d in TABLE 2 . TABLE 2 GROWTH MEDIA FORMULATIONS M edia Com ponents FC-95 FC-143 T est Phenol C on trols LAS C o n tro ls F lu o rid e C on trols FC-95 C o n t r o l Medium + 50 mg/1 FC-95 FC-143 C o n t r o l Medium + 50 mg/1 FC -1 43 FC-95 o r FC-143 C o n tr o l Medium + 30 mg/1 P h e n o l FC-95 o r FC-143 C o n tr o l Medium + 30 mg/1 S ta n d a r d L in e a r A l k y l b en zen esu lfon ate (LAS) FC-95 o r FC-143 C o n tr o l Medium + 3 3 .2 mg/1 NaF ( 1 5 .0 mg/1 F~) 14C -F C -9 5 T e s t FC-95 C o n t r o l Medium + 50 mg/1 14C -F C -9 5 14C-FC-143 T e st . FC-95 + LAS FC-143 + LAS FC-143 C o n t r o l Medium + 50 mg/1 14C - F C - 143 FC-95 C o n t r o l Medium + 30 mg/1 LAS + 50 mg/1 FC-95 FC-143 C o n tr o l Medium + 30 mg/1 LAS + 50 mg/1 FC -1 4 3 C u ltu rin g Procedures The i n i t i a l grow th p e rio d was s ta r t e d by in o c u la t in g 49 ml o f each medium w ith 1 ml o f a c t iv a t e d s lu d g e s u p e r n a ta n t. The a c t iv a t e d s lu d g e u sed was a m ix tu re o f two s lu d g e s c o lle c t e d on th e day o f in o c u la t io n . The s lu d g e was o b tain ed from th e M e tro p o lita n W aste C o n tro l Com m ission's M etro p la n t in S a in t P a u l, M inn esota, and the C h e m o lite Waste Treatm ent P la n t in C o tta g e G ro v e , M in n e so ta . 003639 6- - F o llo w in g in o c u la tio n , the cu ltu re s in polypropylen e Erlenm eyer f l a s k s w ere sh ak en a t 200 rpm on r o t a r y s h a k e r s a t room te m p e r a tu r e ( 4 ) . At th e end o f each growth p e r io d , each c u ltu r e was tr a n s fe r r e d to i d e n t i c a l f r e s h m edia u s in g a 1% in o c u lu m from th e p r e c e d in g c u l t u r e ( i . e . , 0 .5 ml o f e x i s t i n g c u lt u r e to 4 9 .5 ml o f i d e n t i c a l new m edium ). The growth p e rio d between tr a n s fe r s v a rie d as i s n o ted in TABLE 3 . A 10 ml sam p le was ta k e n from e a ch c u l t u r e a t 10 m in u te s a f t e r in o c u la tio n or c u ltu r e tr a n s fe r and at th e end o f each grow th p e r io d . Sam p les w ere c e n t r i f u g e d f o r 10 m in . a t 1 7 ,0 0 0 x g p r i o r t o a n a l y s i s o f th e c e n tr ifu g a te . D e v ia tio n s from th is c u ltu r in g p roced u re are n o ted in TABLE 3. The f i n a l growth p e rio d d iffe r e d from p reced in g p e r io d s . M edia were prepared w ith C arb o n -1 4 -lab e le d FC-95 and FC-143. One hundred ml c u lt u r e s were grgwn in f la s k s on a r o ta r y sh a k e r in a grow th cham ber c o n t r o l l e d a t 2 5 C . + 1 . Twenty ml s a m p le s w ere ta k e n a t 10 m i n ., 2 days and a t 7 d ay s. Chem ical A n aly sis F lu o rid e io n co n ce n tra tio n s were measured u s in g a flu o r id e io n e le c tro d e (O rion io n an aly zer flu o r id e e le ctro d e model 9 6 -0 9 ), and a s ta n d a rd cu rv e drawn from th e r e s u lt s o f m easurem ents o f a c c u ra te ly prepared flu o r id e sta n d a rd s. The c o n c e n tra tio n s o f these flu o rid e standards bracketed the con cen tration s present in th e e x p e rim e n ta l sa m p le s. F lu o r id e cu rves were s e t up a t each sam pling p e rio d , except fo r tr a n s fe r 1. For the an alyses fo llo w in g t h i s t r a n s f e r , a 1 .0 ppm f l u o r i d e s ta n d a r d was u se d t o c a l i b r a t e th e instrum ent w ith th e assum ption th a t the slo p e o f th e p re v io u s flu o r id e curve rem ained c o n sta n t. P henol a n a ly s is was done a cco rd in g to Stan d ard M ethods f o r th e Exam ination o f Water and W astew ater, 14th E d itio n , 1975. L in e a r a lk y lb e n z e n e s u lfo n a te (LAS) was a n a ly ze d fo r by th e m e th y len e b lu e , ch lo ro fo rm e x tr a c tio n method d e scrib e d in th e 14th e d it io n o f Stan d ard Methods ( 3 ) , excep t in tr a n s fe r s 8 -1 4 , LAS was a n a ly ze d by a m o d ific a tio n o f t h is method. In th is m o d ified m ethod, th e sam p les was d ilu t e d t o 100 ml in a s e p a r a to r y fu n n e l. A ls o added t o th e s e p a r a to r y fu n n e l were 25 ml o f S ta n d a rd M ethods m e th y le n e b lu e s o lu t io n and 100 ml o f c h lo r o fo r m . T h is m ix tu r e was sh ak en f o r 30 s e c o n d s , a llo w e d t o s e t t l e , s w i r l e d , and t h e c h lo r o fo r m drawn o f f th ro u g h g l a s s w ool i n t o a 2 .5 cm d ia m e t e r , s p e c 20 c u r v e t t e . P e r c e n t t r a n s m it t a n c e was r e a d a t 652 nm and com pared t o a s t a n d a r d cu rv e p re p a re d w ith s u r f a c t a n t sam ples o f known c o n c e n t r a t io n tr e a te d in th e same m anner. 003640 7- - TABLE 3 SUMMARY OF CULTURING PROCEDURES USED IN THE SHAKE FLASK BIODEGRADATION STUDY OF F C -9 5 AND F C -1 4 3 C u ltu re Growth T ran sfer # P eriod (days) Notes 0 3 Used a c tiv a te d slu d ge inoculum from M etro and C h em o lite. 1 3 F C -143-hydrogen a n a lo g added to 143 c u ltu r e s and c o n tr o ls . 24 3 3 At th e tim e o f c u ltu r e tr a n s fe r , 1 ml o f D ecatu r slu d g e su p e rn a ta n t added to c u ltu r e s . 4 3 LAS re p la c e d phenol as a re fe re n c e compound. LAS m edia was in o c u la te d w ith a m ixture o f c o n tro l c u ltu re and C h em o lite and D ecatu r slu d ge su p e rn a ta n t. 53 6 3 The use o f flu o r id e c o n tr o l was discon tin ued . 7 6 Shaker was in a d v e r te n tly turned o f f , p o ssib ly fo r 5 d ays, during th is growth p e r io d . 8 3 i ml o f M etro slu d g e su p ern atan t was added to a l l c u lt u r e s . 96 10 4 11 4 In t h i s and s u b s e q u e n t grow th p e r io d s , c u lt u r e s were grown in a r e c ip r o c a t in g sh a k e r-w a te g b ath a t 100 s tr o k e s per m in. and 25 C . 12 6 13 6 14 8 - +6 (2) 15 7 78 d ay s = T o t a l E n rich m en t P e r io d 003641 8- - Carbon 14 C o u n tin g T e c h n iq u e s S c i n t i l l a t i o n c o u n tin g was p erfo rm ed on 1 ml sam p les o f c u lt u r e c e n tr ifu g a te added to A qu asol' and counted w ith an in te r n a l stan d a rd quench c o r r e c t io n . The r a d i o a c t iv it y o f thesji| sam ples was com pared t o known w e ig h t sa m p le s o f C -F C -9 5 o r C -F C -1 4 3 added d ir e c tly to A quasol. S o l i d sam p le s were c o l l e c t e d d i r e c t l y o q to m i l l i p o r e HA 0 .4 5 pm f i l t e r s composed o f c e llu lo s e a c e ta te and c e llu lo s e n i t r a t e . The f i l t e r s were then washed w ith d e io n iz e g .w a te r and p la c e d in to paper com bustion cones wet w ith Com bustaid'' , and*com busted in A g r ichem 's P ackard '' * com bustion equipm ent. The C0,, r e s u lt in g from com bustion was trap p ed in a s c i n t i l l a t i o n f l u i d c o n ta in in g an o rg a n ic amine and counted in A g rich e m 's P ack ard s c i n t i l l a t i o n c o u n te r. Sam ples were reco u n ted w ith an in t e r n a l sta n d a rd fo r quench c o r r e c tio n . T h in -Layer Chrom atography (TLC) T h in -la y e r chrom otography w as-perform ed to d e te c t r a d io a c tiv e m e ta b o lite s o f C -FC-95 and ' C -F C -1 4 3 . Ten ml c u ltu r e sam ples were c o lle c te d and im m ediately fro z e n . These sam ples were sto re d frozen fo r about 1 month. The sam ples were e x tr a c te d im m ediately a f t e r th aw in g w ith 10 ml o f e t h y l a c e t a t e . The sam p les w ere th e n c e n tr ifu g e d a t 17,000 x g to ensure th e s e p a r a tio n o f th e e th y l a c e ta te , w ater, and s o lid s p h ases. The w ater phase and p o r tio n s o f the e th y l a c e ta te phase were evap o rated to d ryn ess under N ,,. The d rie d sam ples were resuspended in a 9 :1 h e x a n e :e th y l e th e r m ix tu r e . (Some sam ples w hich e v a p o ra te d to d ry n e ss in a i r b e fo r e s p o ttin g were resuspended in m eth an o l.) The resuspended sam ples were s p o tte d on E . Merck s i l i c a g e l GF254. S m a ll s p o ts o f s o l id s resid u e were a lso a p p lie d d ir e c t ly to -th e s e p la t e s . -.A ll sam ples were re fe re n ce d a g a in s t a m ixture o f C -FC-143 and a*C -F C -9 5 . The p l a t e s w ere d e v e lo p e d w ith 10% e t h a n o l in e t h y l a c e t a t e and v is u a liz e d by exp o sin g Kodak n o-screen x -ra y film on the p la te s fo r one week. TLC was re p e a te d on th e rem ain in g p o r tio n o f th e S o lv e n t sa m p le s. The s o lv e n t was allo w ed to ev ap o rate to d ryn ess in a i r , and th e re sid u e resuspended in m ethanol. These p la t e s were s p o tte d more h e a v ily , developed as b e fo r e , and v is u a liz e d w ith x -ra y film fo r 2 weeks. CQ3642 9- - G a s-L iq u ld Chrom atography (GLC) E th y l a c e ta te e x tr a c ts were prepared as d escrib e d in th e th in la y e r chrom atography m ethods. C o n tr o l s o lu t io n s w ere made by d is s o lv in g *C-FC-95 and *C -FC -143 in e th y l a c e t a t e . P o r tio n s o f the e th y l a c e ta te e x tr a c t sam ples and th e e th y l a c e ta te c o n tro l solu tion sw ere a ls o m eth ylated . A liq u o ts o f th e m ethylated and nonm ethylated e th y l a c e ta te e x tr a c ts and c o n tr o ls were in je c te d onto th e 5713 H ew lett Packard gas chrom atograph w ith e le c tr o n cap tu re d e te c to r. M eth ylated sam ples were in je c te d w ith in 3 h r s . o f t h e i r m e t h y la t io n . The c h r o m a to g r a p h ic colum n was 12 f t . x 1/8" O .D . s t a i n l e s s s t e e l p a c k e d w it h 20% DC 200 ( 1 2 ,5 0 0 C S ) on 10% B e n to n e 34 and 70% 80/90 mesh Anakrom P .A . The i n j e c t i o n p o r t tem perature was 250 C ., and th e d e te c to r tem perature 300C. The column tem p eratu re was programmed to h o ld fo r 4 m in . a t 75 C . , to r is e to 180 C . a t 8 C . per m in. , and to h o ld a t 180 C . The flo w r a t e was a d ju s te d t o 35 m l/m in . o f Argon/m ethane, 9 5 /5 . U e th y la tio n s were perform ed by ad d in g a 20 p i a liq u o t o f a 1 ug/ml Cq F^ q COOH s o l u t i o n , as a r e f e r e n c e compound t o e a c h s a m p le . Diazom ethane was then added u n t il a y e llo w c o lo r p e r s is t e d . The sam ples were then lo o s e ly capped, s w irle d and allow ed to stan d fo r 15 m in u te s . N itr o g e n was blow n o v e r t h e sam p les u n t i l th e y e llo w c o lo r d isap p ea red , and th e sam ple was retu rn ed to i t s o r ig in a l volume w ith e th y l a c e t a t e . RESULTS AND D ISCUSSIO N F lu o rid e R elease In a l l but th e f i n a l growth p e rio d , d e g ra d a tio n o f FC-95 and FC-143 was m onitored o n ly by a n a ly s is o f flu o r id e c o n c e n tr a tio n a t the b egin n in g and end o f each c u ltu r e p e r io d . I t was assumed th a t i f th e flu o ro ch e m ica l p o rtio n s o/ th e se m o lecu les were d eg rad e d , flu o r id e ion w ould accu m u la te in th e m ed ia. To en su re th a t flu o r id e was n o t lo s t from th e c u ltu r e by a b s o r p tio n , p r e c ip i t a t i o n o r v o l a t i l i z a t i o n , c o n t r o l c u l t u r e s w ere grown w it h 15 mg/1 o f flu o r id e . T h is flu o r id e co n cen tra tio n is ap proxim ately what w ould r e s u l t i f F C -9 5 o r FC-143 underw ent d e g r a d a tio n w ith 50 p ercen t flu o r id e r e le a s e . The r e s u lts o f th e flu o r id e an a ly ses con ducted on d if f e r e n t days showed c o n s id e r a b le v a r ia t io n . T h is was due to th e v a r ia b le and very s lu g g is h resp on se o f th e flu o r id e e l e c t r o d e . TABLE 4a shows th e r e s u l t s o b t a in e d a t e a ch t r a n s f e r . TABLE 4b shows th e r e s u l t s o b ta in e d when th e same s a m p le s , w hich had been sto red in p o ly eth y len e c o n ta in e r s , were an alyzed to g e th e r a fte r the term ination o f the experim ent. D esp ite the v a r ia b ilit y due to the a n a ly tic a l te ch n iq u e, the r e s u lts in d ic a te th a t flu o r id e , i f r e le a s e d to th e m edia through b io d e g r a d a tio n , would n o t be lo s t from th e m edia. The r e s u lt s o f th e f lu o r id e a n a ly s is on flu o r o c a r b o n -c o n t a in in g c u lt u r e s and c o n t r o ls a re shown in TABLE 5 . The r e s u l t s show th a t no b io d e g ra d a tio n w ith flu o r id e r e le a s e o c c u rre d . C03643 - 10- TABLE 4a IN IT IA L AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLUORIDE SUPPLEMENTED CONTROLS MEASURED BY S P E C IF IC ION ELECTRODE AT THE TIME OF TRANSFER FC-95 FC-143 Fluoride Control F lu orid e C ontrol T ransfer # In itia l F in al In itia l F in al 0 21 23 20 21 1 23 22 21 20 2 20 22 18 21 3 26 1 7 .5 25 1 6 .5 4 1 6 .5 1 9 .2 16 1 7 .3 TABLE 4b IN IT IA L AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLUORIDE SUPPLEMENTED CONTROLS MEASURED BY S P E C IF IC ION ELECTRODE MEASURED COLLECTIVELY AT END OF STUDY Transfer # 0 1 2 3 4 5 F C - 95 Fluoride Control In itia l F in al 1 6 .4 1 5.6 1 5 .6 1 6 .2 1 5 .6 19 .3 1 6 .2 1 6 .2 1 6 .2 1 6 .2 1 5 .7 1 7 .0 F C - 143 F luorid e C ontrol In itia l F in al 1 5 .7 15 .7 1 4 .5 1 6 .4 1 5 .7 15 .0 17 .0 1 5.0 16 .4 1 5.6 1 7 .0 1 6 .4 03644 - 11- TABLE 5 IN IT IA L AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FC -1 4 3 AND FC-95-CO N TA IN IN G CULTURES AND OF NONSUPPLEMENTED 95 AND 143 CONTROL CULTURES FC-95 T e st T ransfer # In it . F in al 0 0 .4 6 0 .5 1 1 0 .5 0 0 .4 6 " 2 0.4 2 0 .6 6 3 (5) 1 .75 1 .6 4 0 .7 3 0 .7 1 5 0 .72 0 .7 8 6 0 .7 3 0 .8 7 0.1 4 0 .1 7 8 0.9 0 0 .8 4 9 0 .8 4 0 .7 3 10 0 .7 2 0 .8 1 11 0 .8 1 0 .8 0 12 0 .7 4 0 .7 3 13 0 .7 3 0 .8 4 14 0 .8 1 0 .7 8 95 C o n t r o l In it . F in al 0 .3 1 0 .3 3 0.3 6 0 .3 6 0.3 4 0 .5 6 1.75 1 .5 0 .6 8 0 .6 0 0.6 1 0 .6 8 0 .6 3 0 .7 0 <0.1 <0.1 0 .66 0 .6 6 0.72 0 .6 0 0.6 0 0 .6 8 0.6 9 0.6 2 0 .66 0.62 0 .64 0 .6 6 0 .66 0 .6 4 FC-143 T est In it . F in al <0.1 < 0 .1 < 0.1 < 0.1 <0.1 < 0 .1 .83 1 <0.1 < 0.1 <0.1 < 0 .1 <0.1 <0.1 <0.1 <0.1 <0.1 < 0 .1 <0.1 <0 .1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0 .1 143 C o n tro l In it . F in al < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 <0.1 .8 1 1 < 0 .1 <0.1 < 0 .1 0 .5 6 < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 <0.1 < 0 .1 < 0 .1 < 0 .1 <0.1 < 0 .1 <0.1 003645 - 12- R e fe re n ce Compounds R eferen ce compounds were used to dem onstrate th a t th e b io d e g r a d a tio n t e s t c o n d itio n s used were s u it a b le to degrade compounds known to be somewhat r e s is t a n t to d e g ra d a tio n . In th e f i r s t f o u r grow th p e r i o d s , 30 mg/1 p h e n o l was added t o two c u ltu r e s which were id e n t ic a l to th e t e s t c u lt u r e s , e x ce p t th a t they la ck e d flu o ro c a rb o n s . A n a ly tic a l problem s p re v en ted th e measurement o f phenol co n cen tra tio n d u ring th e f i r s t th re e grow th p e r io d s . In the fo u rth growth p e r io d , phenol was found to degrade to le s s than 1 .3 mg/1, th e lim it o f s e n s it iv it y o f th e method as a p p lie d . T his dem onstrated th at the te s t co n d itio n s were s u ita b le fo r the biod egrad ation o f phenol. In th e f i f t h through f i n a l growth p e r io d s , r e fe re n c e lin e a r a lk y l s u lfo n a te (LAS) was used as the r e fe r e n c e compound. T h is compound is a standard referen ce m aterial used in the Soap and D etergen t A s s o c ia t io n 's b io d e g ra d a tio n t e s t method fo r a n io n ic s u r f a c t a n t s ( 6 ) . T his m ateria l is considered to be r e la tiv e ly e a s ily degraded. In the Soap and D etergent A s s o c ia tio n 's shake fla s k b io d egrad atio n t e s t , the r e s u lts are considered in v a lid i f the rem oval o f 1-dodecene d e riv e d LAS i s not n e a rly com plete. The d ata show ing the e x te n t o f d egrad atio n o f LAS in s u r fa c ta n t su p p lem en ted c o n t r o ls a re d e p ic te d in TABLE 6 . The d a ta sh o w in g th e e q u iv a le n t amount o f m ethylene b lu e a c t iv e s u b s ta n c e s in th e c o n tr o ls n o t supplem ented w ith LAS a re d e p ic te d in TABLE 7 . L i t t l e LAS d e g ra d a tio n o ccu rred d u rin g th e f i r s t few a d a p tiv e t r a n s f e r s . Three tr a n s fe r s were req u ired b e fo re th e m a jo rity o f th e LAS began to degrade in th e s u r fa c ta n t supplem ented c o n tr o l fo r F C -9 5 . F iv e tr a n s fe r s were re q u ire d fo r LAS d e g ra d a tio n in th e 143 c o n t r o l. T h e re fo re , i t appeared th a t organism s cap ab le o f d egrad in g 1-dodecene d e riv e d LAS were n ot i n i t i a l l y p re se n t in s u f f i c i e n t numbers fo r LAS d e g ra d a tio n . The te s t c o n d itio n allow ed fo r en richm en t o f th ese organ ism s, but enrichm ent occurred a t a slow er r a te than had been a n tic ip a te d . C o n s e q u e n tly , ch a n g e s w ere made in t h e p r o c e d u r e to in cre a se the ra te and lik e lih o o d o f a c c lim a tin g organism s cap ab le o f d egrad in g th e flu o r o c h e m ic a ls . Growth p e rio d s were exten d ed from 3 to 4 -6 d a v s, and tem perature was r a is e d from room te m p era tu re (<20 to 22C) to a co n stan t tem perature o f 2 5 C . R e s u lts o f LAS d e g r a d a tio n in th e f i n a l grow th p e r io d are shown in TABLE 8 . In th e grow th p e r io d s fo llo w in g t r a n s f e r s 11 and 12, an e x p e r im e n t was done t o d e te rm in e i f 50 mg/1 o f F C -9 5 o r F C -1 4 3 i n h i b i t e d th e d e g ra d a tio n o f LA S. These r e s u l t s a re shown in TABLE 9 . F C -9 5 ap p ears to have an i n h i b i t i n g e f f e c t on th e m ic r o b ia l d e g r a d a tio n o f LAS. However, i t s p resence was not co m p letely in h ib it o r y . Com parison w ith TABLES 8 and 6 shows t h a t th e p r e s e n c e o f 50 mg/1 o f F C -9 5 i n h i b i t e d LAS d e g r a d a tio n by 18% and 23% d u r in g t h e s e two t e s t p e r i o d s . On th e o t h e r h a n d , w it h in th e l i m i t s o f t h e p r e c i s i o n o f our method, FC-143 d id not appear to have a s ig n if ic a n t e f f e c t on LAS d e g ra d a tio n . 03646 - 13- In th e f i n a l grow th p e r io d , 50 mg/1 o f carb o n 1 4 - la b e le d FC-95 and FC-143 were used as t e s t s u b s tr a te s in p la c e o f th e n o n la b e le d flu o ro c h e m ic a ls. Both FC-95 and FC-143 c u ltu r e s were prepared in t r i p l i c a t e . The c o n c e n tra tio n s o f th e r a d io a c tiv e flu o ro c a rb o n s p resen t in the aqueous phase as determ ined by s c i n t i l la t i o n co u n tin g a re shown in TABLE 1 0 . The i n i t i a l FC -9 5 c o n c e n t r a t io n i s much low er than e x p e c te d . T h is low v a lu e c o u ld have r e s u lt e d from a sy ste m atic erro r in the c o lle c tio n o f the i n i t i a l FC-95 sam ples. I t is a ls o p o s s ib le th a t FC-95 had not co m p letely d is s o lv e d in the c u lt u r e s when th e f i r s t sam ple was t a k e n , b u t t h i s seem s u n l i k e l y , s in c e the i n i t i a l v a lu e s fo r FC-95 co n ce n tra tio n from a l l 3 p a r a lle l c u ltu r e s were alm ost id e n t ic a l ( 3 0 .3 , 2 9 ,8 and 30 .4 m g/1). N ever t h e le s s , th e rem ain in g d a ta show th a t th e r a d i o a c t iv it y a s s o c ia te d w ith FC-95 and FC-143 rem ained in s o lu tio n d u rin g th e e n t ir e 7-day degradation te s t_p e r io d . 'A n alysis o f th e b io lo g ic a l s o lid s showed some b in d in g o f r a d io a c t iv e m a t e r ia l, b u t th e v a s t m a jo r ity rem ained in the liq u id ph ase. TABLE 6 CONCENTRATION OF LAS (mg/1) IN SUPPLEMENTED CONTROLS AND % LAS REMOVED T ransfer # 95 - S u r fa c t a n t C o n tr o l 143 - S u r fa c ta n t; C o n tr o l LAS LAS I n i t . F in a l % Rem oval(7) I n i t . F in a l % Removal 4 31.5 2 6 .8 1 8 .4 35.5 2 9 .5 1 9 .4 5 2 8 .3 2 7 .5 0 .1 3 2.8 2 5 .8 2 1 .2 6 2 9 .8 2 5 .5 1 5 .5 27 .0 2 4 .0 7 .0 7 25 .0 12 .0 9 1 .1 25 .0 3 0 .6 -2 .0 8 3 1 .2 3 .75 8 9.8 37.0 3 5 .8 1 1 .1 9 3 3 .0 3 .1 7 9 5 .1 38.9 1 3 .7 9 4 .6 10 3 2 .7 2 .3 3 9 5 .9 4 2 .7 1 2 .8 8 9 .7 11 3 1 .0 2 .0 9 5 .0 39 1 3 .7 9 3 .8 12 3 1 .3 2 .3 3 9 6 .5 4 1 .3 1 9 .7 7 7 .9 13 3 1 .7 2 .5 93 .5 4 1 .3 1 8 .0 8 8 .3 14 3 1.3 3 .0 9 2 .8 4 0 .3 1 3 .7 9 0 .7 003647 - 14- TABLE 7 CONCENTRATION OF METHYLENE BLUE ACTIVE SUBSTANCE (mg/1) IN NONSUPPLEMENTED CONTROLS T ransfer # 4 5 6 7 8 9 10 11 12 13 14 95 - C o n tro l In it . Fin al 1 .0 1.9 1 .15 .3 8 0 .5 0 .7 5 5 .2 5 10.2 3 .0 0 .88 5 .7 5 1 .8 3 4 .17 1 .1 7 4 .3 3 0 .6 7 3 .0 1.33 3 .6 7 0 .6 7 3 .6 7 1 .0 143 - C o n tro l In it . F in al 4 .5 4 .5 4 .5 3 .5 7 .1 5 .5 0 5 .0 10.2 9 .0 1 0 .9 10 .9 1 2.2 12 .3 9 .6 7 11 .5 1 2 .0 12 .3 1 3 .3 11. 3 14 .5 1 4.5 1 1 .3 TABLE 8 CONCENTRATION OF MBAS (mg/1) IN SURFACTANT SUPPLEMENTED AND NONSUPPLEMENTED CONTROLS DURING FIN AL GROWTH PERIOD Time In itia l Day 2 Day 7 EC-95 Con tr o ls #1 LAS #2 LAS N onS u P P l. S ^ g g l, SHEE-. 2 8 .7 1 3 .0 1 .6 7 2 9 .3 2 6 .0 2 .0 1 .0 1 .0 .7 % LAS 9 6 .5 Removal (7) 9 5 .4 - FC-143 C o n tro ls #1 LAS #2 LAS Non- S u p p l. Su P P l- SugP.l 3 4 .0 8 .0 6 .3 36 .7 2 2 .3 6 .3 6 .5 5 .3 4 .0 9 1 .6 9 2 .3 - 003648 - 15- TABLE 9 EFFECT OF F C -9 5 AND F C -1 4 3 ON THE BIODEGRADATION OF LAS ANALYZED FOR AS MBAS Transfer # 11 12 F C -9 5 + LAS C u^lt-uLAr eS FC-143 + LAS C u ltu re *nLA" I n i t , F in a l Rem oval(8) I n i t . F in a l R em oval(8i 58 .0 64 .3 3 4 .7 40-1-3 7 3 .6 78 .9 5 3 .7 5 3 .7 2 7 .3 3 4 .0 9 7 .8 7 1 .4 TABLE 10 CONCENTRATION OF 14C -F C -9 5 OR 14C -F C -1 4 3 IN THE CENTRIFUGATE OF TEST CULTURE DURING THE FINAL GROWTH PERIOD In it. Day 2 Day 7 4C-FC-95 C u ltu res Standard Concentration D eviation 3 0 .1 mg/1 5 2 .8 5 3 .5 0 .3 mg/1 0 .5 3 .1 14C-FC -14 3 C u ltu r e s Standard C oncentration D eviatio n 4 6 .2 mg/1 4 8 .0 4 9 .7 0 .9 mg/1 0 .3 0 .4 G03649 - 16- T h in -la y e r chrom atography did not re v e a l the presence o f ra d io a c tiv e m etab o lic products o f e ith e r FC-143 o r F C -9 5 . L ik e w ise , gas l i q u i d chrom atography o f th e same c u lt u r e e x t r a c t s , b oth b e fo re and a ft e r m th y la tio n , showed no p ro d u cts th a t were n ot i n i t i a l l y p resen t o r not a ls o p re se n t in c o n t r o ls . From the com bination o f these r e s u lt s , i t can be concluded th a t no b io degradation o f these flu oroch em icals o ccu rred . REFERENCES AND FOOTNOTES (1) Goldm an, P e t e r , Enzymology o f Carbon-H alogen B onds. D egradation o f S y n th e tic O rganic M olecu les in the B io sp h e re , N a t. A cad, o f S c i . , W ashington, DC (1 9 7 2 ). (2) There was a s ix -d a y p e rio d b e fo re th e o n se t o f th e f i n a l growth p erio d d u rin g w hich th e t e s t c u ltu r e s were shaken at 25 C . in the presence o f FC-95 o r FC-143. (3) Stan d ard Methods fo r th e E xam ination o f W ater and W astew ater, 14th E d itio n , Am erican P u b lic Health A s s o c ia tio n ( 19Y 5). (4) Davtim e tem peratures were observed to range betw een 20 and 22 F . N igh t tem peratures were n ot measured d u rin g th a t p a rt o f th e stud y in w hich c u ltu r e s were shaken a t am bient temp e r a tu r e (s e e TABLE 3 ) . How ever, m easurem ent made n e a r th e te rm in a tio n o f t h is 2^-month s tu d y , in Ja n u a r y , showed th a t n igh ttim e tem perature freq u en tly drops to 17 C . (5) At t h is t r a n s fe r , D ecatu r slu d g e was added w hich co n ta in e d a high flu o rid e co n cen tratio n . (6) Subcom m ittee on B io d e g ra d a tio n T e st Methods o f th e Soap and D etergen t A s s o c ia tio n , A Procedure and Stan d ard s fo r the D eterm ination o f the B io d e g ra d a b ility o f A lk y l Benzene S u lfo n a te and L in e a r A lk y la te S u lfo n a te . J . o f th e Am erican O il C h em ists' S o c ie t y , 42:986 (1966). (7) P e rce n t LAS rem oval was c a lc u la te d a s : (M BAS,,. - M B A S -.) - (MBASqi;, - M B A S-.,) % Rem oval --------- ^ ------------- -- X 100 MBASS I - MBASC I W here: M BAS-. * The i n i t i a l m ethylene b lu e a c t iv e s u b s ta n c e s (MBAS) co n ce n tra tio n o f the s u r fa c ta n t supplem ented c u lt u r e . M B A S-. = The i n i t i a l MBAS c o n c e n t r a t io n o f th e n o n su p p le m e n te d u c o n tr o l (TABLE 7 ). M BAS-j, The f i n a l MBAS c o n c e n t r a t io n o f th e s u r f a c t a n t supplem ented c u ltu r e . M B A S-. = The f i n a l MBAS c o n c e n t r a t io n o f th e n o n su p p le m e n te d c o n tr o l (TABLE 7 ). / C03650 F / j . O SO O - 12> 7i - 17- (S fO O O O O O O Ilf-) (7) The p e rc e n t LAS Removal was c a lc u la t e d a s : tm i'd % Removal (MBASgTI - MBASCJ) - (MBASstf (MBASgj - MBASCI) mbasc f ) ------------- X 100 W here: MBASgmjr * The I n i t i a l m e th y le n e b lu e a c t i v e s u b s t a n c e s (MBAS) co n ce n tra tio n o f th e c u ltu r e supplem ented by both LAS s u r fa c ta n t and e it h e r FC-95 or F C -1 4 3 . MBASC I = The i n i t i a l MBAS c o n c e n t r a t io n o f th e n o n su p plem ent a l c o n t r o l (TABLE 7 ) . MBASg_F * The f i n a l MBAS c o n c e n t r a t io n o f c u l t u r e s supplem ented w ith s u r fa c ta n t and flu o ro c a rb o n . MBASr -p = The f i n a l MBAS c o n c e n t r a t io n o f th e n o n s u p p le m e n ta l ^ c o n t r o l (TABLE 7 ) . M BASg. The i n i t i a l MBAS c o n c e n t r a t io n o f th e s u r f a c t a n t su p p le m e n ta l c u lt u r e (TABLE 6 ) . I t w as assum ed t h a t MBAS c o n c e n t r a t io n due t o F C -9 5 o r FC-143 was n ot reduced by the b io d e g ra d a tio n o r o th e r lo s s o f th ese compounds. r EAR/cen G03651