Document yp1NdXzv5g0xGkG0YBEbeLze6

A R 30G -03: BIODEGRADATION TEST SUBSTANCE Identity: N-ethylperfluorooctane sulfonamidoethanol; may also be referred to as N-EtFOSE Alcohol or FM-3422. (1-Octanesulfonamide, N-ethyl1,1l2,2,3.3,4)4 I5,5,6,6,7,7,8,8.8-heptadecafluoro-N-(2-hydroxyethyl)-I CAS #1691-99-2) Remarks: Material is an off-white, waxy solid of uncharacterized purity. The following summary includes a history of studies. METHOD: Methods followed: Modification of OECD 302A - Inherent Biodegradability: Modified SCAS Test.; Warburg Determination (Standard Procedure included with study); COD analysis; Shake-Flask Die Away Study (Procedure described in study) Test Type: Aerobic GLP: No Years studies were performed: 1976 -1978 Contact time: 7 days to 6 months Inoculum: Activated sludge collected from an aeration basin at the municipal waste treatment plant in St. Paul, Minnesota was primarily used. Alternate inocula taken from the Decatur, Alabama manufacturing facility were also utilized with the understanding that these media were the most likely to have developed naturally enriched cultures capable of degrading fluorochemicals. Remarks: The initial study in 1976 was a Warburg oxygen uptake experiment. This testing resulted in oxygen uptake equivalent to approximately 3% of the theoretical oxygen demand for the hydrocarbon moiety. However, no degradation products were detected. Further experiments included testing on non-stabilized and surfactant-stabilized N-EtFOSE alcohol. In the initial semicontinuous activated sludge (SCAS) study, N-EtFOSE Alcohol was simply added to the media in an ethanol solution. The NEtFOSE Alcohol rapidly congealed and separated from the w a te r p h a s e . It w a s postulated that biodegradation may not have been observed because enzymes could not "attack" the coagulated N-EtFOSE Alcohol molecules. Since emulsions of water-insoluble molecules have been shown to be more susceptible to enzyme attack (Dickson, L. S. Liu, M.Sc., Ph.D.), a second SCAS degradation study of NEtFOSE Alcohol was conducted in 1978 using emulsified N-EtFOSE Alcohol. RESULTS___________________________________ __________________________ 006322 None of the testing conducted could definitely prove loss of N-EtFOSE alcohol through biodegradation process. Analytical capability at the time could not verify the presence of degradation products or give explanations for why the test substance became non-extractable during some of the experiments. C O N C L U S IO N S ________________________________________________________ It appears that N-EtFOSE Alcohol does not degrade, at least to detectable levels or by verifiable processes, under the conditions of these tests including with high cell densities and extended contact periods. These studies cannot rule out the possibility that conditions could be found that would allow for biodegradation, nor could they exclude the possibility that a microorganism could eventually be enriched that could metabolically alter this chemical. Testing did indicate that NEtFOSE alcohol was not inhibitory to bacteria. DATA QUALITY___________________________________________ Reliability: Klimisch ranking = 3. The analytical methodology is questionable. Sample purity was not sufficiently characterized. Procedural problems were present in several of the studies. Analytical methodology did not exist at this time to detect possible degradation products. REFERENCES_________________________________________________ _________ 3M Technical Report Summary, "Biodegradation Studies of Fluorocarbons", E. A. Reiner, 8/12/76 Request for Laboratory Work, Lab Request No. 3844, COD, Dale Bacon, July 1977. 3M Interoffice Correspondence, "Chronological Review of Biodegradation Studies on FM 3422", E. A. Reiner, August 12,1977 3M Technical Report Summary, "Biodegradation Studies of Fluorocarbons-II", Report number 4, E. A. Reiner, 1/9/78 O T H E R ________________________________________________________________ Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/18/00 006323 TO: T e c h n ie o l C orr/num cofiorti C e n te r, 20J-25 M ICR O FO R M COPIES: 310 T E C H N I C A L R E P O R T S U M M A R Y E n v iro n m e n tal_______ _ L A B O R A T O R Y . D E P T . N U M B E R T itle B iodegradation Studies o f Fluorocarbons P ro je c t: Fate o f Fluorochem icals in th e Environm ental To: R. L. Bohon By: E. A. R ein er S E C U R IT Y Company Conf identiol(Open) O b ia c tiv e : To d e term in e th e b io d e g r a d a b il ity o f s e l e c t e d 3M flu o ro c a rb o n compounds. Special Authorixatian(C lo.ed) August 12. 1976 0222 Project Number: i R ep o rt No ! (3 d ig it.) 75-6398-29 ! Employ Number: 47816 Notebook Reference: 40671 Pgs. 29-37, 41-50 IF SUMMARY R E PO R T Has inform ation in th is report been covered by other reports subm itted to T C C ? No. of pages Including coversheet A B S T R A C T and C onclusions. (System can accommodate 200*250 words! 12 X* P ortio lly : C om pletely P le a s e keyw ord inform ation not in clud ed in other reports and give page numbers o f new m oterial: 3M C H E M IC A L R E G IS T R Y chem icals reported? - [ ) No P i Yes B iodegradation s tu d ie s u sing a Warburg re sp iro m e ter were conducted on FC-95, FM 3422, FC-128, and hydrogen an alo g s o f FC-95 and FM 3422. No b io d e g r a d a b il ity was o b se rv e d on FC-95, a lth o u g h an approxim ate hydrogen analog o f FC-95 was r e a d ily d e g ra d a b le . FM 3422 and FC-128 b o th were d e m o n stra te d to undergo some b io d eg rad atio n . Attempts to is o la te degradation products o f C* M14 *X/A-*O*.O* . | AF.rAA.Vi/HnI tWhAoV. W a r i i i i r f f Wc tVm** *ii *D.^c., J n r* A f r n m Va c n K c o n n p n t a r t i v a t A / 1 sludge study were u n su c c e ssfu l. KEYW ORDS Select gen eral, s p ecific, and 3M product term s from 3M Th esaurus. E n c lo s e suggested term s in parenth eses. EE S PC - Div. Envron - Assess B io lo g y B acteria B ioscreening Fluorocheraical B io d eg rad ab le i, i S P E C IF IC P R O B L E M S rem ainina to roach objective. - V C ontinued a tte m p ts w i l l be made to i s o l a t e and i d e n t i f y th e b io d e g ra d a tio n p ro d u c ts o f FM 3422 and o th e r 3M flu o r o c a rb o n s . Work w ith r a d i o a c t i v i t y la b e le d FM 3422 i s b e in g c o n s id e re d . . rk ' N' ! , \ |,r,i o" ''*JTion JpC ip nt'S f 1006324 / BIODEGRADATION STUDIES OF FLUOROCARBONS SUMMARY AND RECOMMENDATION No b io d e g ra d a tio n was o b serv ed in Warburg s tu d ie s on FC 95. B io d e g ra d a tio n o f FC 95 i s im probable b e ca u se i t i s c o m p le te ly f lu o r i n a t e d . The r e s i s t a n c e o f t h is compound to b io d e g ra d a tio n by an a c c lim a te d m ic ro b ia l c u ltu r e , however, has n o t y et been dem onstrated. Warburg s tu d ie s on FM 3422 and FC 128 b o th in d ic a te d t h a t some b io d e g ra d a tio n o c cu rred . The p ro d u cts o f th is b io d e g ra d a tio n are n o t known. Sem icontinuous a c t i v a t e d slu d g e s tu d ie s on FM 3422 d id n o t co n firm o r d isp ro v e th e Warburg fin d in g s . F u tu re in v e s tig a tio n o f th e b io d e g r a d a b ility o f th e flu o ro carb o n compounds would be g re a tly f a c i l i t a t e d by th e developm ent o f an a n a ly tic a l procedure f o r FC 95. Warburg s tu d ie s u sin g p u r i f i e d FC 128 sh o u ld be made to co n firm the present findings. S tu d ie s on th e b i o d e g r a d a b il ity o f FM 3422 w ere h in d e re d by i t s low w a te r s o l u b i l i t y . T his problem co u ld be overcome u s in g FM 3422 r a d i o a c t i v i t y la b e le d on i t s hydrocarbon p o rtio n p rovided th is m a te ria l had a high s p e c ific a c t i v i t y (>5 mci/m mole) and p u r i t y . B io d e g ra d a tio n o f a s a tu r a te d s o lu tio n o f th e la b e le d compound c o u ld be m easured by d e te c tin g 14CC>2 e v o lu tio n . INTRODUCTION The s u s c e p tib ility to m ic ro b ia l m o d ific a tio n i s an im portant param eter in the study o f th e environm ental f a te o f any c la s s o f compounds. I t i s the most im portant form o f degradation fo r o rg an ic compounds. ,v A v a s t a rra y o f o rg a n ic compounds can be com pletely degraded by m icroorganism s. So v a s t in f a c t t h a t i t was once b e lie v e d by some t h a t given enough tim e and the p ro p er c o n d itio n s, m icroorganism s could degrade any organic m a te ria l. T h is d o c tr in e o f m ic ro b ia l i n f a l l i b i l i t y i s s t i l l a common m is c o n c e p tio n (1 ) . P e r f lu o r in a te d compounds a re extrem ely r e s i s t a n t to b io d e g ra d a tio n (2 ). A lthough compounds w ith s in g le f lu o r in e s have been shown to r e le a s e f lu o r id e io n s as a r e s u l t o f b io d e g ra d a tio n , p e rflu o r in a te d compounds have r a r e ly o r n ev er been shown to undergo n a tu r a l d e g ra d a tio n . For t h is re a so n , no m o d ific a tio n o f th e p e r f lu o r o components o f compounds in t h i s stu d y was a n tic ip a te d . However, m o d ificatio n o f i t s hydrocarbon components seemed p o s sib le . An u n d e rs ta n d in g o f th e p a r t i a l d e g ra d a tio n p ro d u c ts i s im p o rta n t s in c e th e environment w ill be exposed to th ese products in ad d itio n ' to the undegraded m aterials. METHODS AND MATERIALS Cheroicals The chem icals used in th e s e ex p erim en ts a re shown in Table I . 006325 -2N TABLE I . CHEMICALS USED IN BIODEGRADATION EXPERIMENTS FM 3422 Hydrogen Analog o f FM 3422 g2h5 C8F17S02NC2H40H c 2h 5 c 8h 17so2nc2h4oh FC 95 c8f 17S03 K S ip e x -o ls FC 128 C8^ 17OSO3Na f 2 HS CsF , 7S02NCH2C00IC They w ere o b ta in e d from Don R ic k e r o f th e Commercial Chem ical D iv is io n in S eptem ber, ly 7 5 . FM 3422 (N -e t Fose a lc o h o l) was i d e n t i f i e d as 788 CC 745-2. The FC 95 u s e d was from l o t 583. Lot numbers w ere n o t g iv e n f o r th e FM 3422 hydrogen a n a lo g o r th e s ip e x - o ls (RM 2 6 4 4 2 ). These c h e m ic a ls were s e l e c t e d f o r a number o f r e a s o n s . FC 95 i s e s s e n t i a l l y the,, flu o ro c a rb o n c o n s t i t u e n t o f a la r g e number o f 3M flu o ro c a rb o n compounds. FM 3422 i s an in te r m e d ia te in th e p ro d u c tio n o f 3M flu o ro c a rb o n s , and FC 128 i s a f i n i s h flu o ro c a rb o n p ro d u c t. S ip e x -o ls and th e Hydrogen Analog o f FM 3422 were s e le c te d fo r comparison to the flu o ro carb o n s. S ip ex -o ls is an approxim ate hydrogen a n a lo g o f FC 95. These hydrogen a n a lo g s were t e s t e d b e c a u se b i o l o g i c a l l y la b ile fluorocarbons have freq u en tly been found to be g ra tu ito u s ly d e flu o rin a te d by enzymes which norm ally remove a hydrogen. Thus, i t seemed p ro b ab le th a t m ic ro b ia l growth on hydrogen analogs c o u ld ^ s e le c t p o p u la tio n s o f organism s which co u ld more com pletely degrade flu o ro c a rb o n s . WARBURG DETERMINATION Warburg s tu d ie s were conducted according to th e a tta c h e d stan d a rd p ro ced u res. (A ttachm ent) M icroogranism s were c o lle c te d from th e mixed liq u o r o f the Pigs Eye tr e a tm e n t system ,w ash ed , and su sp en d ed in a b a s a l s a l t s medium and used a t a c o n c e n tr a tio n o f 2000 mg o f b i o l o g i c a l s o l i d s p e r l i t e r . W ater in so lu b le s u b s tra te s were e m u lsifie d in w ater p r io r to a d d itio n to th e Warburg f l a s k s . E m ulsions were made u s in g a B la c k sto n e model EP-2 u l t r a s o n i c p ro b e , b a se 1/2 in c h , a t 100% pow er. L o g a rith m ic d i l u t i o n s I in w a te r w ere made o f th e t e s t s u b s t r a t e s , and 1/2 ml was p la c e d in th e f i r s t s id e arm o f th e Warburg f l a s k s . C o n tro ls c o n ta in e d 1/2 ml o f 10 g /1 g lu c o se s o lu tio n o r d e io n iz e d w a ter in t h is s id e arm. The second s id e arm co n tain ed e ith e r glucose or deionized w ater. 006326 -3- Oxygen in ta k e was f i r s t o b serv ed in each f la s h f o r a p e rio d up to 1 .5 h r s . w ith readings a t 10-15 min. in te r v a ls to e s ta b lis h th e endogenous a c ti v ity . This was fo llo w ed by a d d itio n o f th e f i r s t s id e arm and c o n tin u e d oxygen m onitoring f o r approxim ately 2 h r s . A d d itio n o f th e second s id e arm containing glucose, a re a d ily degradable m a te ria l, allowed a fu rth e r evaluation o f the to x ic ity of the previously added m aterial. Semicontinuous A ctivated Sludge S tudies A w eek-long sem ico n tin u o u s a c t i v a t e d s lu d g e (SCAS) stu d y was co n d u cted on FM 3422. The m icroorganism s u sed w ere o b ta in e d , as b e f o r e , from P ig s Eye T reatm en t P la n t. One Hundred F i f t y ml o f a c t i v a t e d slu d g e was added to 3 SCAS r e a c to r s and ta p w a te r as a c o n tr o l t o a f o u r th . FM 3422 was added to 3 r e a c to r s below th e w a te r s u r f a c e in 1/2 ml o f a b s o lu te a lc o h o l. Each a d d itio n in c re a s e d th e FM 3422 c o n c e n tr a tio n by 33 m g/1. P ure e th a n o l was added to one s lu d g e -c o n ta in in g r e a c to r as a c o n tr o l. The o p e ra tio n o f th e sem icontinuous r e a c to r s i s shown in F ig u re 1, The SCAS r e a c to r s w ere a e r a te d f o r 23 h r s . w ith 500 m l/m in. o f a i r w h ile the contents o f each re a c to r were s t i r r e d w ith a m agnetic s t i r r e r to p rev en t s e t t l i n g . A fte r th e a e ra tio n p e rio d , th e slu d g e was s e t t l e d f o r an hour and one l i t e r o f s u p e rn a ta n t was re p la c e d w ith p rim ary e f f lu e n t from th e P igs Eye P la n t. FM 3422 was added a t th e b e g in n in g o f th e T e s t C y cles 1, 2 , and 4. Samples were taken a t the s t a r t and end o f each t e s t cy cles and from th e su p ern ata n t after settlin g . The a e r a tio n chambers u sed in th e SCAS s t u d i e s w ere p l e x i g l a s s c y lin d e r s 13" high w ith a 4" in te r n a l d iam eter. A s id e arm allow ed d rain ag e o f th e s u p e r n a ta n t le a v in g th e 500 ml w ith th e s e t t l e d slu d g e u n d is tu r b e d . A nalytical Samples ta k en a t th e te rm in a tio n o f th e f i r s t W arburg s tu d y on FM 3422 were e v a lu a te d by th in la y e r chrom atography (C e n tra l R esearch a n a ly t ic a l work re q . No. A59412). The sam ples w ere e x t r a c t e d i n t o d ic h lo ro m e th a n e , d r i e d to a sm all volum e, and s e p a r a te d on Woelm s i l i c a p l a t e s . The d e v e lo p in g so lv e n t system was 10:90 e th a n o l, chloroform (V :V ). The developed p la te s w ere v is u a liz e d by th e io d in e s ta r c h te c h n iq u e and compared to known s ta n d a rd s w ith a d e te c tio n l i m i t o f one yg o f FM 3422. Samples f o r th e SCAS s tu d y were e x t r a c t e d i n t o n - o c ta n o l and s e p a r a te d by gas chromatography w ith an e le c tro n cap tu re d e te c to r. E x tra c tio n s were perform ed in capped 50 ml p o ly p ro p y le n e c e n tr if u g e tu b e s and p h a se s s e p a r a te d by c e n tr if u g in g a t 26,700XG f o r 10 m in u te s . RESULTS AND DISCUSSION Warburg - FM 3422 R e s u lts from th e Warburg s tu d y on FM 3422 a re sum m arized in F ig u re 2 . T h is experim ent was p erfo rm ed by f i r s t s o n ic a tin g FM 3422 and i t s a n alo g in w a te r to make em ulsions o f ap p ro x . 24,000 mg/1 o f th e FM 3422 and 11,000 mg/1 o f th e FM 3422 a n a lo g . S in c e FM 3422 and i t s hydrogen a n alo g a r e n o t very s o lu b le in w a te r , i t was f e l t t h a t form ing an em u lsio n w ould p u t more o f th ese compounds in c o n ta c t w ith th e m icroorganism s in th e Warburg s tu d y . 006327 -4- STEP 1: Add t e s t compound, media, and m icroorganism s KsrtowmiitoLE STEP 2: A e ra te and mix fo r 23 hours 'H / STEP 5: Re-add ' t e s t compound/ and media. / Repeat cycle. / m agnetic stirrin g bar s' s u p e rn a ta n t, drain STEP 4: D rain s u p e r n a ta n t. sludge II 'I " i STEP 3: Stop a e r a t i o n and m ix in g . Let sludge s e ttle . FIGURE 1: T est c y c le f o r sem ico n tin u o u s a c ti v a te d slu d g e r e a c t o r . 006328 >ii moles 0o u p tak e c o rre c te o r ''T endogenous -5FM 3422 FM 3422 -6- W hile th e FM 3422 a n a lo g was r e l a t i v e l y e a s i l y e m u ls ifie d and s t a b l e once e m u ls if ie d , th e FM 3422 was n o t. A p p ro x im ately one h o u r was r e q u ir e d to p u t 75% o f th e FM 3422 i n t o e m u lsio n , and t h i s m a te r ia l p ro c e ed e d to slo w ly come back o u t o f e m u lsio n . In a b o u t two t o th r e e h o u rs , e x cess FM 3422 emulsionwhich had not been used in the experim ent, turned in to a sem i so lid gel. Complete ch em ical o x id a tio n o f th e h y d ro carb o n component o f th e FM 3422 a t th e h ig h e s t c o n c e n tra tio n (M 6 u m oles) would r e q u ir e 87 y moles o f O2 b a se d on th e fo llo w in g e q u a tio n : C8F17S02NCC2H5)C2H40H + 5 ` 52 ^ C8F17S2NH2 + 4C2 + 4H2 M ic ro b ia l o x id a tio n r a r e l y ex ceed s 60% o f th e ch em ical o x id a tio n . In t h i s ex p erim en t, only 2-3 m icro moles o f oxygen up tak e was o b serv ed . However, oxygen uptake was c o n tin u in g a t th e end o f t h is experim ent. A ddition o f g lu c o se to th e FM 3422 c u l t u r e a ls o p ro d u ced in c r e a s e d oxygen u p ta k e , c o n firm in g t h a t th e FM 3422 em u lsio n was n o t i n h i b i t o r y to th e m ic ro b ia l c u ltu re . On th e o th e r h a n d , th e hydrogen a n alo g o f FM 3422 showed s i g n i f i c a n t t o x i c i t y . Upon a d d itio n o f th e m ost c o n c e n tra te d em u lsio n o f th e a n a lo g , endogenous oxygen uptake ceased and was n o t r e s to r e d even a f t e r th e a d d itio n o f glucose to th e c u l t u r e . The n e g a tiv e s lo p e o f th e hydrogen a n a lo g 's oxygen uptake curve (F ig u re 2) i s due to th e endogenous c o r r e c t io n and n o t oxygen e v o lu tio n . S im ila r r e s u l t s were o b ta in e d i n a second Warburg e x p e rim e n t w ith FM 3422 and i t s hydrogen analog. A n a ly sis o f FM 3422 h a s shown i t t o be q u i t e p u re . The oxygen u p tak e ob serv ed was g r e a te r th a n would be e x p ected from im p u ritie s in th e compound. I t is conceivable th a t so n ic a tio n produced d eg rad atio n p roducts th a t were biodegradable, b u t not d e te c ta b le by th in la y e r chromatography. I t is also p o s s ib le t h a t some o f th e h y d ro carb o n components o f FM 3422 m o lecu le were d e g ra d ed . However, u s in g t h i n la y e r chrom atography we were u n a b le,-to d e te c t any m a te r ia ls form ed as a r e s u l t o f th e b io d e g ra d a tio n o f FM 3422. I t i s n o t known i f th e hydrogen a n a lo g o f FM 3422 i t s e l f i s t o x i c . Thin la y e r and gas chrom atography showed t h i s m a te r ia l to b e im p u re. Gas chrom atograph* showed th e a n a lo g to be 90% p u re w ith two m ajor c o n ta m in a n ts . The co n tam in an ts may have been the cause of the observed to x ic ity . SCAS - FM 3422 t The se m ico n tin u o u s a c t i v a t e slu d g e (SCAS) s tu d y was a second a tte m p t to i s o l a t e th e h y p o th e s iz e d d e g ra d a tio n p ro d u c ts o f FM 3422. T his s tu d y was conducted over a p e rio d o f 1 week w ith sam ples tak en a t th e i n i t i a t i o n and end o f each 2 4 -h r . c y c le . The FM 3422 sam ples added in an e th a n o l s o lu tio n r a p id ly s e p a r a te d from th e l i q u i d p h a s e , and as a r e s u l t may have had to o sm all a su rface area to allow s ig n ific a n t m icrobial degradation. n -o ctan o l e x tra c ts o f th e samples were analyzed by gas chromatography. No new le a k s w ere form ed as a r e s u l t o f e x p o su re o f th e FM 3422 to th e m icro o rg an ism s. I f some o f th e FM 3422 had been degraded to th e s u lf o n ic a c id , i t would n o t have been d e te c te d . The s u lfo n ic acid i s n o t s u f f ic ie n tly v o la tile to pass through th e gas chrom atography column. * perform ed by Commercial Chem icals D iv isio n 006330 -7- The n -o c ta n o l e x tr a c ts c o u ld n o t be s e p a ra te d by th in la y e r chrom atography because o f th e low v o l a t i l i t y o f t h i s s o lv e n t. Frozen n o n e x tra c te d sam ples s t i l l e x is t a t t h is d a te and co u ld be e x tra c te d in to a more v o l a t i l e s o lv e n t for thin layer analysis. T hree a d d itio n s o f FM 3422 i n 33 ppm in c re m e n ts were made d u rin g th e SCAS e x p e rim e n t. The FM 3422 s e t t l e d w ith th e s o l i d s and f o r th e m ost p a r t rem ained in th e r e a c to r when th e s u p e rn a ta n t was w ithdraw n. The f in a l c o n c e n tra tio n (a lth o u g h n o t in s o lu tio n ) was a p p ro x im a te ly 100 m g/1. This m a te r ia l was n o t homogeneously d is trib u te d and accum ulated on th e sid e s o f th e re a c to r s . WARBURG FC-9S The r e s u l t s o f Warburg s tu d ie s w ith FC-95 a re graphed in F ig u re 3 . No oxygen u p ta k e was o b se rv e d as a r e s u l t o f th e a d d itio n o f FC-95. T h is m a te ria l a lso caused no to x ic e f f e c t s . S ip e l- o l, an approxim ate hydrogen analog o f FC-95, was shown to be r e a d ily b io d e g ra d ab le and to have no to x ic effects. The S ip e l- o ls was s o lu b le a t a l l c o n c e n tra tio n s t e s t e d (as h ig h as 1700 m g/1). FC-95 was in c o m p le te ly s o lu b le a t 4000 m g/1, b u t was c o u p le te ly i n s o lu tio n a t 400 mg/1. The lack o f d e g ra d a tio n w ith FC-95 was ex p ected s in c e p e r f lu o r in a te compounds a re c h a r a c t e r i s t i c a l l y n o n b io d e g ra d ab le . WARBURG FC-128 Oxygen u p tak e curves from Warburg s tu d ie s on FC-128 as shown in F ig u re 4. These r e s u lts in d ic a te th a t FC-128 i s r e a d ily b io d e g ra d a b le . Assuming b io d e g ra d a tio n o c c u rs as i s shown b elo w , a p p ro x im a te ly 70% o f th e t h e o r e t i c a l maximum oxygen u p ta k e o c c u rre d w ith in th e 7 - h r . e x p e rim e n ta l p e r io d . ..This oxygen uptake is g re a te r than expected and appeared to be co n tinuing a t the end o f th e experim ent. These r e s u lts are somewhat in q u e stio n sin c e th is FC-128 i s known to be an im pure ch em ical. C2HS CgF17S02NCH2C00K + 4 .2 5 0 2 + H+ =* CgF17S02NH2 + 4C02 i 3H20 + K+ 006331 -8- 'S 7_. 6. cw3O -(oOoUc cV h <2 **<uOato1-*>3u> U Q> la +-> CL 3 (N / ton *--oi E 5- 2- mmvkmm glucose addition / ! / 1_ 0 -* s u b stra te addition ^ tim e hours glucose a d d itio n FIGURE 3: Warburg s tu d y o f FC 95 and S ip e x - o ls . -98 1 FC 128 UpUlKC L U l R 'C l t u lO l CiluugClluuij IllOiCb 7 <I I * * 1 I i t i ( Ii i Ii 12 3 4 tim e hours , FIGURE 4 : Warburg stu d y o f FC 128. i ,11 1i i 1 ' ii T' 5 6 006333 10REFERENCES: (1) A lexander, M.; B io d eg rad atio n : Problems o f M olecular R e c a lc itra n c e and M icrobial F a l l i b i l i t y . Adv. Appl, M icrobial 7: 35-80, 1965. (2) Chapman, P. J . ; D epartm ent o f B io ch em istry , U n iv e rsity o f M innesota, S t. P a u l, M innesota, P erso n al Communications 2 /2 4 /7 6 . ) v i . >) 006334 attachment I STANDARD PROCEDURE FOR WARBURG DETERMINATIONS 7/10/75 E. A. R ein er 1. D esign e x p erim en t and c a l c u l a t e c o n c e n tr a tio n s o f m a te r ia ls to add. 2. F i l l w a te r b a th (DI w a te r i f l e f t in b a t h ) . 5. A djust temp, o f bath (s e v e ra l h rs . o r o v e rn ig h t). 4. I'la c e manom eters in d e s ir e d o r d e r . 5. P re p a re th erm o b aro m eter. Add about 3ni o f H^O to 1 f l a s k . 6. S e t o u t g la ss w a re in d e s ir e d o rd e r ( t o match th e manometer w ith which they were c a lib ra te d ). 7. L ig h tly g rease c e n te r-w e ll top w ith stopcock g re a se th a t can be removed w ith s o lv e n t . Add 0 .2 ml 10% KOH. 8. P re p a re sam ples in Dl w ater (o r a c c o rd in g to r e q u e s t) to add to s id e arm s. Keep r e f r i g e r a t e d u n t i l u sed . 9. Prepare c e lls (keep c e lls cold a t a ll tim es b u t avoid fre e z in g ). A. C e n trifu g e 0 C. B. Wash w ith c o ld BSM - c e n tr if u g e . C. R esuspend in c o ld BSM. D. D eterm ine c o n c e n tra tio n o f an a l i q u o t w ith th e s p e c tr o n ic 20 a t 600 nm. A djust rem ainder to d e s ire d cone. C b a sic s a l t s medium. R e frig e ra te u n til use. F.. Take sam ple o f f i n a l a d ju s te d slu d g e fo r s ta n d a rd MLSS a n a l y s i s . <, 10. Add sam ples to s id e arm; ( u s u a l l y 1 ml i f one s id e arm, % ml to each s id e a r m - if 2 side arms). 11. Add 2 ml o f washed c e l l s to f la s k s . 12. Add f i l t e r p a p e r s t r i p to a l k a l i in c e n te r cup. Id. A ttach fla sk s to the c o rre c t manometer. 14. R etig h ten fla s k s a f t e r about 5 min. shaking in b a th . 15. Leave sto p co ck open to a tm o sp h e re , and l e t temp, a d ju s t f o r an a d d itio n a l 10 min. 10. A d ju st le v e l in manometer to 150 w ith sto p co c k open ( c lo s e s to p c o c k ). 17. Begin re a d in g s (alw ays a d ju s t c lo se d arm o f manometer to 150 mm b e fo re r e a d in g ) . 15. Add c o n te n ts o f s id e arms a c c o rd in g to e x p erim en t design requirem ents. 006335 19. Take read in g s p e rio d ic a lly (on open arms) th ro u g h o u t course o f experim ent. 20. Disconnect and clean fla s k s . A. R inse w ith w a te r. B. Wash o f f g re a se w ith a c e to n e . ('. Acid wash. D. R inse w ith DI W ater. v * 006336 V. BEST COPY AVAILABLE 006337 WARBURG DATA FORM 5^ P X t " Ti t l e : \^fk Ihi T j7>xic,(h^ v t 3 ^ 2 2- Run No. D ate: C e ll Cone. :3oa ^V^-Temp. ; 3 ( - S tro k e s/M in . : / / S C ontents o f Inner W ell: No F la sk No.Mt*v Time -*/I ! 0 7 ) i 5_ k 5. s 1. a i . s s s 71 'S 97 h ? in 21____ + Flask Contents + 1 ___Reading in mm. 11\ 1 aI>j-*or^ m -flwtsiHD L o-lntm d'* 73TJ Y'lf\ + ,,St-opP^r- ...2 //*> +-.....A . . '3 i*Sc7 2- Ctiucos' -h K.& J-'X-Jrf'u.'w *S4 IboH ^^ Iluoo-S^i f rU1i , U M l Z / # !$% t t f f l 7 191 ts s lit 3 ! t (h it l t (5 0 i # Hit / / / 1*7 m /0/ n 1 $ l i ? /&} 13\ /f M fa* 1* % Y f f io b l -fsf 15/ t 1 lit. l/e U o foi n % P i % L A i f / ~>?7 ' 11121098567JM>3M6JCS>527ii'o3>U7SOo33/f/b>"H7l^S?7>f?iii1lt?f 13 14 "3?9S*S<5??1Yt5 3 lvc o s *- F n i+ U l (SZ /35 IZ 1r3.fidl1uei0o*'se<-t-t- f2MP/4MiiXl3^3<li?/2/z2. /SI M73! i 75/ m ascose 1/50 1st /S3 r9j.Vjlj\/"v1,u.crcapt.SeDJ.+.S-+C/A+1U/u.^/an222frn.^W0tlP.i.o&/2/nlyit=-W.j4.iF/tr*F*^PlA\'0r/si6Pi4*nm-2?2L^27.///h55&5tCf i/2#5mmm%I/<oi ili /2$ 9 1 1(1 f0 l m in fa? m Ml Mi /ft IM !/Z m /an% f/t HQHi,l /{& ?>u f a 9 l ?( no f lz( m (ft loi ?7 V $1 1*I t 5^ los lo\ % 1 s 1H jiltl*S Mi M IQOU m Mt &7f> u f \ t n? y* S?11 s 11 p 3 n s/ 7Au Bipit M: 72- t l 51 i l 1 11? HS V PI 7/ Ifi 10 ?% i/ %2i /55& I__ i1 N otes: ytu-d-W CM ccf^J f-2 .7 -7 5 Lx^U<J *- fy'-hi t~> ^ on M i i i 'j .J x t ^ t s Q g j ~ /2 /v!,'h * C lu^ fiSd h . j k 11\ S i J i c M A / n $ l h i k ` (*/uQS.e O CXpr<\ :c u ls o u k W ) ode n i mi'A, " i m ."n -f- n i II L I I u> tA ^ r ir\Jo oX m - a.kat\. VOOJV'4.'' 1j * j \ c Q \]J . * S h n /ity . M q . 'ff - . f/jki/iL M et. U 5 <- ^ * 4 " o **> '*" C 3 Vv *\ *+6 6 ?/ tuJ~ 1 , i 1 1 i -n1 P ~n v/> -c .y . PRO'SE^T NO_____ - 3M TECHNICAL NOTEBOOK NO. PAGE 40671 28 Suiect: F M' 3 iv ^jeet: ^ Y Date: % Z ^ 7 1 -------- its 6'^dvy' < $/ e ^y y & * 1 t- d s i4 fl(U sC sL r*yd**l~ * b ./C i- t' FlUU-llLL- ^ . C ^~ ~ ^ \ *UM,^1 CZa +- /*C*- U<X-A r L & `,* s J U ~ 4 r c a ^ L * . W (jL o j\4 ^jL c^U * \ C o~c~^fc-> J'V-*-^ P'V OJV/.4,)' U iU -. /^1 J-T -J-IS fnOUC&L<s<s~ t y7 ivV ^ O V \^ (^ ) ^Stoiw)" (X X ^ y y ^ / t 7 / i t p s i CL^J jS/m *CEA& @ C& *<st <cu<> +*~ iP ^ jo fc d U r& t* -/ s*~L 4+^ A * " y ' ~" > ^0<y^- 2. / . lr~rtM C ^irF*^ & /b *& 'L&rry s ? jra & d & - At ja d ico < * 4 ~ M l .s J & 3 p kk> jA<**i CUinM <~ l* .> d te< J ^ ^ Q K 'Z s /<* i ytp '& d * }* - c^ a c -^ C U d& i^nus*x*- /f7 L j2 A ^A ^ ^ r - w & r * ^ d/\s< *Q -rr^ , ' > i V O rs f j ? &yiL: J ^y- t^-~ & & rr \ %?Z ttT ^ r C<4x a >< t<? h> b ? fr X.(rkrj 0 i w A : - SignatureRead and Understood- 006338 Date:_ Date: PROJECT NO....... ...... ^ bject: f rr\ - 3 4 ^ 3M TECH NICAL NO TEBO O K NO. 40671 29 Date: !I Reference: IO&A, ' WIMPYmilMLE -Ue. W B nr< Mfr**c las^ J T ^ \ ]/jU l\ * ' ! - -+--t-M / ^ ! "I 1 I ^nvdLo^<^at4-o ) / p .)J . ^ J o o o ^ i k v ^ ( k ) & JL $ s L M a- ) l/lM - C P ); -i / ' / ,, va oj\ Vptfts^ : i ( S / A 5* ^ r >V) H & 3 - F k\X L/ 3 ^ 7 ^ ) ^ o '* 3 ^ ^ Pm ^ h 5 5 y? / 9 *tA~. ; --- i-----r Solwit*' I X I ... l . _ j _ J / K - W -- s j / j c > ^ \ .. . ID . 1 "T"" --- _<Z-,----- --- ---------- - r -- 1----- ?-- 1-- r .......... ............ .V i..^ . . L /: : F JA 3 4 V*- , ; Q) ^ U ^ e 'Si W t i 57 . . =T=- : 57 r -j - i W ^: ; t Fa - y -ix x p > 3 4f ? ^ ' i -- -*- ? j> * M- >7 m _ .rt .: ? ? >1 KA ^ ^ 6 0 0 t ^ X 7> F tv \ 15 ^ (>0 x ti k u / J M x ^ f ^rv- d / f~ (f\~ 3 . y ^ . '` ~ X lt , ft r~ . SignatureRead and Undcrstood- 006339 Date:_ Date:_ *t PROJECT N0~ ^bjcct: Reference: P(uo^C -"Uv^ 41^ Cov4"<*v(i> / f f i ) / b . l f i IVJ/A. \ / . M <f / h'jdf'U*' 3M TECHNICAL NOTEBOOK NO. - f r x'AGfc 40671 30 1 . .. t -, S JU J^k ! ^ itC r " ; # - / _______ __ *- ^ l-IZ^ i ^ J S*li~h.<- J ^4*14Ua S cU-m^ i 4^ C e *h ~ 6>^ /p ^ ic o i (u ) f i U (/V ^\ ) 3^^ / ) Y. ^ >-- a f\r < if) /h^Xry. / F tu o 't^ /~ d j y tr - r> )) >> r> 1 : (i M- "-^ y^ , &*<*ky*- S ^ `*r 5) saFi-r 1----,,j/ 3) jkt,K-^- )l~l> ' iiXfyU r .UtjoJ SfysijtO''. 1 ' ; /LM- l j Uyp^t^i :f r.?>";! 1- >- t- - - -:- . -- ............... , ----- ,-------- -- * j # ~ t i O K? c^CH W k J F M * /2 2 (C jzfl/o Fil S t Vf 3^> - fh W H>/r*)tK C t ^7 fJ. 0>) *=- r t * 3 Y * * S U ^ f7 /2 9 f& yir (-Y Signature.------------------------------------------------------------------- ' Pr*`iH unH I!niLr**.fnnii ^) b"v ^ bvii . 006340 Date:______ Date:. y"lnta* 3 'y.i ssmuH V ;' t * i 1-- I S S '? tri** t 006344 1 r `-- -t JL>-i 1-- r*.....`i * o /<l$ jS t.lc^ 2*0 Chri.) .y  V^JW-vi' v f BEST COPY M I E 006346 A o> R. #V ' J. -v ^>V `- > fA .*rg'\ :;V.- " Ufr >7 vVj^'; ,; 1' .!%>: $*r; WARBURG 02 UPTAKE CALCULATION SHEET yz iU ltc o t>t< s . X <?*'*-< T itle: JAct'robiUA*"/\f*- FtA ^4.tuxfYi. ,i Date 7-75 -7 5 FlasX.No.: Time Elapsed Time o Reading T. B.1 My Reading Flask (ran) ISO Change^ in mm. (mm) 0 T.B. Correction^ d> A cS itS ts: er^dc^i^, t^-uX Actual C_ hange4 o2 Uptake^ EndogenousTCorrected Upt2ake I Uptake** (mm) (nmoles) (nmoles) liusol&). 0 r~ " - '5 i s * ISC, 1*4 +3 17 /..HI P-5 /52* M %l t s 74 i.w .25 n x + 3 3 1 a > 5? 45 ~55TM 15H 51 nr lTMU ""> 34 3 l +5 +? HI 3 . 2. 3.81 6 5 /5 * /<>7 H 3 + ! 5 3 43~v 7 5 51 />H H 4 + 10 5 4 </,<5 8 5 l 101 n . . . . r* 97 H>2> 1* 5 x +W ic 5- 5 5-*/r 101 /4 5 15 5 7 + iC. 7 3 o 7 U ? /6 4 A t 59 +n 74 131 n% 88- 47* ^19 ? / 73 . -.... % Footnotes over. & 0 * & : ,''''.. \ - -'>.At-7/j i,/ - v - ; \- v,. -i iVv ' t. l > f c V- r*i  ' I 1' V..YAA': ,,'.A ./T; i7-t*irT-" WARBURG 02 UPTAKE CALCULATION SHEET 3 *r " ir"* 'S r :r # / v : ' r (0 O o f':V; kA i.c.E* T ine E lapsed T ine R ead in g T .B .1 y^Y h' -..A -,.'; . ' - O a /s a 't ?$ ?i F. ;. *\ ` ?!5v - '' *. v: . 35 HS ss 5 75 Vi,*v A52. /5V /5^ /57 /5* _ /S l HI . ?? lii A; n s . -v>n ' t i t : JU u t tn Pte:9^5-75 F la s k N o .: >3r ; C o n te n ts : 9 ^ ^ CrJ^X / R ead in g F lask 2 Change ^t sp; A ctual in an . C o rrectio n Change* #2 5 U ptake "E ndogenous C o r r e c te d 2 U p tak e 1U p tak e6 C*1. . . . i*rt Iia o le s V ^luB SU gFl. /$o - o 6 0 00 13 9 - u f i r -lt ifio 7,9/ t3 77 2.17 /,95 ^. . . . //? ^ 3 iS />9 -Hi + S HC 3 . ^ ~3-z .U /0 / -H I S 7 9,77 3 `S/ 94 -SH 92 -SB - 43 7 7 -73 + IO f */ 43 Cr 74 S' 7 5,7./. / .3 r 7,3/ H'3'1, H.te ,9 ? 1 Ob. !.}S ).? i 7o - 6/ ; .W - 'I t . -/* V i .,,-'v/ii-l'-i.' i-' '. > /4 +i) M ) 94 /d * 7-1 .. . $ .0 6 < t.o7 /Q .tl " /.3 v t -73 1.99 7 75 3 ,3 A iV-MSSK* C vjT '. :. i ' s s a .it ttvr-ri*--Vrv r i-wa- A ' ' A ' - ,, '-if *.* / .* '' lr , " .' :y J;........ . .. . '''`V ` ' B E srcopv AVAILa h [ E r r :Wfs:;' . >. ', . ................................. ' V / ' . ' ' 'V / 'v . % . -. ' ,' * ' > . f 1 : * r i f " ' . :A "' : . 1 , . _ '. - > v Jr; 1 r'-*-ll m , jV*:;.'fSA'. : * !;,*,*if.-^W^/'^iv'i:fe'i?-i'V.i^iJ;^;...?:: v>vV',j.;V '../ S & A g ^ il8 ^ iS 6 ** .SKS*TM- V/`?r*- wAryyi^u WARBURG p 2 UPTAKE CALCULATION SHEET tos'AiV'>' i T itle : t c t v b v t r y * - 0 tv>iiDu'f> s o / _ :* ? S -? 5 FlMk.No.: V Contents: / f uo t Di Tine Elapsed Reading Tiae T.B. 1 1 iaal Reading Flask 0- 1 ._ .i .2 Change 1 in an. Correction C1 _ Actual C_ hange4 (SW_ --------------- |Endogenous Corrected Upt2akes Uptake 4Uptake6 (uaolesl . iMHUSF.U fUB9U?J . /Y ? i5 j D 0 O 0 O O il* I3 Y /7 f 3 ^LO l,U Ay/ ` .Z5- i f:-;: V'' " > 5 l$1~ I ; 7 5 t3 /.?? Ik ;- 3 5 / s v n r 3 y * 3 .2.9 f 7 5ir HI HS / 5 Y no *#5 H i 3.V1 3 ,a U i ;. SS isn or i o Sr ..3.-9Z .. .4/ A 65 IS* 9 5 5& ........... + ! 5 5>H0 13% fa * 7 5 iSl 9 0 + 10 7 / ~ t j o ~ H-iS . u ^ - . 55 u t ev 67 0 s./s />5 0 9 7 /*3 75 7 I0 r T F " S.H* ^ .6 0 \t o 'v . . . ' !'." 707 /4 5 5 ^. f t l o b S C.oi T t r //* 13/ /u . lit . ,<V l . ; : , ,5Y;:;;:'r' HI - , > y ,"; / fl7 _ + n / /o> - t , cf W t ' . " j. _ 1 M -- o h t.73 l.lt H.oQ r " - . . i / . v ' '. *. ..V ^ ' '* ' " ' ; X ; V ' ,'/j. (. ' v.- - ` ' .' , A ;. V ; : v ; iriTA Itto iC a a t; . ... -y\" :i'^ ' ;' '..:'' i',;' - - CO V PS co 9 Jv.'"vjj,' >$> ..; 1;'V :'' ft n. '#,.*:+.<i ..'** *y'!> > Kr , tJ f py & 1 S**WU * .*f V-;v ,- 0 :,.';SF^ri'f:'i^^i>k;ftj ..v" v,.'..' c r N & : v :lii'-T., *,- /. ' J,': '/`Ay ' f ;i^vvV;V*', v $&::;\ iV,-'"v'y>'t-' r- <\ ' . ,-V *r. . ; WARBURG 0 2 UPTAKE CALCULATION SHEET 0^ T itlet T in e 0 *fVT>Hiw6*n E la p s e d T in e R a id in g T. .1 Dt:-.?3-75 FImKHo.: U n d in g F la s k (m l _J 2 Change in n . *) ' T 5F F i Correction ,S~ c S tS t.: g ' 'rf V -* Fw A c tu a l 4 Change 2 5 Uptake Endogenous Corrected Uptake | Uptake6 tan) (m ol . O My !S ^ 0 -<J> 0O j *> t b i ' , M 7 -:,:.. 1 ^ 3 7 0 ~ T T T ~ h i! .> a r v7.* '- ... 151. 35 -JS > H S 15*1 IZ -i I'L l fJi >9 3/ 3? t3 %1 yo v 1 , 1 *) ^ 3 - S3 7 *7 2 .5 * +S H5 ~ 5T 5~ 3 .1 , 3-1 ML *" 5 S -- if .. 76 1S T IS * IS 1 107 . L O J ,i 97 is 51 Ss _ M O 53 o (o 5 H 31 5W 5 */? 3 *1 H .31 H .iS `" " T i ' ` ,75 .. .SrH ' 7 is AT !(> % . /i 9/ 11 9 71 S IS /. * 0 *3 101 S .H ? l . 52> 4.'?.-.>^.-%'. - . v-. v w 'V 4- > V 1 to l m f: Ul . 1U Ht 2/ 2> % n lo o ' 'TV. /6 +1 > to i l , l 3 IS / 0 .o 5 Lb 1 & 3 iC 73 ' / ,? r t 7 >C3 asii'V .'%>.: .- >v V # :'. :i ,: V ; ` 4 r ; T: > ., j ' -V 'iiv 'v - RES n n p v A | All API F fV ILIID LC - - ' ; '' SeW V ta* ** t o Q 0 '.';-t,.'-' -.yva '-V:'>' ::A . i* - ^ '.1 ^ i -j ':i ' i ..'"if.**?* * t ; Tu *fJ . ' -, > . : .* , ; ... ` .4 ' . "''' " V'vVi-:',' *;;:/ < *.;y .><< * T itle : T iae -"'*1 ' -1' '' ; ,v. * - V.". . ;;-r we*. J Jcnrhi**'\ ^ x.cfy & E lapsed T iae O /5 PS _ 3 5 5 65 75" 5 97 /P 7 //t /< 3 / R ead in g T .B .1 real /* i9 is * 151- IS ^ /** 151 16% ISI ui - Libus HI lit -'z'J' Y -Vr-'* v.; ' V*.: i.J-,> .:,& y '!jy, WARBURG 0 2 UPTAKE CALCULATION SHEET 0* ^/kwv*" 1 V^Nj tC<*\ c^t i __ i |h tWj ... o '. - > ... . ,, ,. ,/<?. F lask V . D a t e : 7 ^ 5 - 7 5 F l g K .H o .1 > ____ C o n t e n t s : Change2 in aa. CPP) T .B . i C orrection A ctual Change4 CaaT IEndogenous C orrected 2 s U p tak e 2 U ptake t u a o l e s ) i.. . 26 | Uptake IU K lg?J . IS / / 37 /2M in to1 /o 5 /o / 96 W **. 73 Y ; " Oo O --. V L / *o **3 t2> 11 3-3 7 ? #* 3 3 4 +5 . K? 5oHC > 9 + IO 3o 3* io 55 4o 55 w o 2 i -m y ..7 6 + U ^5... 79 -- + o IS 97 -A.: . m 0 t>HH ~TW ~ 7.51 * .* 3 0 LI.Vi 14, U so* sy L h 7> 7,310 %.01 z-n `, % > 1,41 / ,71 ^ .5 ir *f'tS S',is S.ili C.oi 31(?S ... X 0 +. o3 ~ o 'tj - >o i */ ..,s> .3 H ,Hb. .....*1 |.ii i o 1- V7 -^v W ; v 10 c? o O O ;;X i .v,' X ' - * J, t 'l >1 -V. ?-|*7^ . ~ ,. y k .* - <;V, ' :6 ' ' .:i .. ' ,v ,t.':-,. ~1*n ( . . UILABLE _ _ _ _ _ _ _ _ J::i z j T - .)' w' : . :.'';*'.'*/*.*'.-'.`r,-. 'A''.,*.,*i .w'; WARBURG 0, UPTAKE CALCULATION SHEET T itle : ii T ine E la p se d T iae R ead in g T .B . 1 M_ O /-? ? IS*- 5 1S t 3 5 l$Ts HS 5 5 51 75 5 - SS /5 S IS1 U>1 toi1 7 ..... - . n i - . . Ilf m - 131 Ut *'-r;-''' "'';.-V '. .... . ;.1. .- , '.* 1 ; F o atM tM OVIf >....;fc*.s; 1V=> .. M ' '''.'' .' to D a te :? ~ 7 5 - 7 5 F la s K .N o .:___ _ 2 _ ___ <*5? * t s : R ead in g F lask (M ) 2 Change in m i. tan) T.B . i C orrection A ctual Changed M T 5 U ptake ^ ^ 5V ; T' J Endogenous . C ogrected 2 U p tak e I Uptake6 t inaolesl 15 0 i,s r '3 3 it5 6 / l +*3 i 7 f*3 5 1 + 3 0 <*>' 6 15 ..>71 - .N 707> o 1 9 \ M r - `1 >* a .s ^ -,7.1 n r lib / oSr 1oS 10 o 32 3% iX H S ... so -MO He 5/ 55 c$ 5-i 3 3 9 ,3 / 9 .6 5 S .3 -1 >9/ Hhx Li>C$ 5 ,1 5 - .07 ^ i07. - . 6/ O +.7 f z : 63- U zx n 9 iU 1H h */ + i C + 1) C-13 5>H9 --M 5 *1 7,C9 ,0 7 ... h i %.Sl :>c 13 1 5 5 .,. f ,?j . a . v i ' . . k-V.'; '/ * . ... ---- ------ , 'y- ;<*. $ ,' 7 % J& ' :V*'. ;kkV-.k' 006351 '.i >i- 4 ii '"; . i < ' *' . y ^ v -i- \ *".' j :"' Ofipy Ai f A l l l P i r mm-* - . V*' rn u iP L C T - ..- .I " - ,`vV , , v ,. , yf'-K M S T COPY AVAILABLE 006352 Till.: & TttkTif Tine Elapsed Reading Tine t--T.AB.1 O Ml *S i s * PS IS'L 35 ..} k HS k s 151 iis IS* 76 iS l SS l i i ..Z _ (01 m 131 It2> Its litUK WARBURG 02 UPTAKE CALCULATION SHEET P a t e 5 - 7 5 F1m K.No. : 9 Contents: Reading Flask Oml . . 2 Change in aa. CM) _ T.B. Actual 1 Correction Change4 Cm ) . ISO O 0 0 /3* H . **3 - 1 7 .... 111 PI t 3 110 30_ #*3 ......... I 0* HP' +S i(o 5H + i 4.7_ <ol 7 0 . . . L I . . +10 It +151 H . . . u V H +N 77 1 *}9 9 3A. +It H9 l 0 ^ + 0 02 ni J s Uptake Cw p I m I 0 IH 5 1 .0 5 1.91* H'OP 6 .3 0 5% .58 7.*/i <U3 lb,il 5 % - ) / 2. +.'*- - H I + Endogenous Corrected 12 Uptake Uptake6 fuaoles) lim its J , . _> . . '( i j 0 OH .0 4 ; 3' V -/ .' .... - - , *3' 'i '/ -> -- ^ > .. l.iC . 7*9 3 2 -7V' 2 ,5 1,2S M.Hi < * BEST COPY AVAILABLE ,0 WARBURG 0, UPTAKE CALCULATION SHEET lt *: &a,c.ly_/r W J V ^ ^ L l Pate:7yS-75 FlasK.No.: If CFolnatseknts: Tine Elapsed Reading Tine T.B.1 Raading Flask Change2 in an. Correction1 Actual C_hange4 Upt*akes pa)_ Cm) _ Endogenous Corrected Upt2ake 1Uptake* aPlAAX.JliaaUsJ :vcV-ii11i O ISo 0 > 0 0 _ > ^ " ' 0 / 5 * n-H 2 ^ +*3 /. S5 1.9 i ry--' 5 tS'L /i 1 . 3 ? _ _ f*3 Dl . _ 3 .*S I. 91 3 5 IST* / o i 1 * * 3 5 1 3>7b .15 H6 1 * 9 0 CO cs 3i"*i r * * ,3 ^ mWi"'- i s 151 6* V s I S * 72. 7 ? 1C +9 ?7 H.Ol H,U . ia/* "*71L... 16 . 0*-f 7 6 iSl u 8 6 l(>7- 5 6 + 10 HO 9H lol S . o 1? 5 '3 '- , r^ *^->' 1.^#- .. ^ ,58 9 7 h $ .... m /or + i*4 n*> 69 5-ii' l,tl ton u s / 3-^ + l i 1 3 * 7 , 9 0 * / . J , 3 2 . tit HL - n 121 + n I S C 9, 34 b -5 i 2,09 131 H t * 12,1 IS? >'<* . J ? 7 . . _ 1 .H1 ;C 7 : %nc % Footnotes o w r. r\ & X b*3> -fo iS tt - : t- * * i * 1 Ji t ' - V - * if rV- sr^'i O063S3 006354 < ; BEST COPY AVAILABLE WARBURG 02 UPTAKE CALCULATION SHEET o^6 *>= T ine i y; ; ">'.r // E lapsed T ine o IS 5 35 HS s 65 75 55 97 /P ? lit 13/ + R eed in g T .B . 1 /" /? D ate 7 - 7 5 - 7 5 F le sk N o.: 7 F l e s k ' c C ontents: R eed in g F la sk 2 Chenge T .B . A ctual 1_ 4 i n an. C o r r e c tio n C hange * S U p tak e ( - __ ten) -tW - ISO 00 /^ t 1* 5 7 -5 #" 3 * Z KH5 t5l> III 39 t* 3 1% M ? IST* I * 1* lol H ..... 97 59 p 3 SO p.ssr 69 3 30 isn IS* !St U>1 Hi ns tu HZ */ 72 _ SH HI 27 *,&L nr 9 ..... 7? n loi 72*3 73 7 .._/_4L5 t- i ^ 7 7-9 * 7 + IO W3> 09 7-W 1*3 7* / C 139 +n ISi * ` 9 _ 179 3* 9 ? " M-fl hM(, 5. 3 6.3S 7 . lit iM /9-.6 0v'* 6r y '+ * ' Endogenous' C orrected 2 U ptake I U ptake6 r ,L w p.U i)r JllB ftigiJ . O' ;, H: -r. o*f 1.99 7 .5?" + ,o o 1- l o ..3 .5 ' r A t -- o 5 .0 7 . 17 .3/ 5Yk' 1 .7 i> O / ^ Ml ).43 .25 &&- ( *$5& X,- ` V; Jfc / < 4 uku .( f - / 5 0 6^+*' Y*f-elihy .> .. 3 s k # ; 006355 BEST COPY AVAILABLE . ..a T ine E lapsed Time O '5 >5 35 H5 5 65 76 SS 97 tPl Ut Ul R ead in g T .B . 1 A ari /*t<? tS * 52- 'ST* 1*1 151 /5 * iSl li>Z m li* IU ~ lit WARBURG 0 ,, UPTAKE CALCULATION SHEET P a tei?-?5-7S Flssfc N o .: "7 t O- F lasi C ontents: R ead in g F la sk Lml _ j Change2 in an. ta n ) A ctual 1^ 4 C o rrectio n lange _im) -- > S U p tak e Endogenous C orrected 2 U ptake 4U p 2 tak e 6 15 0 35 l* s 1 5 > f3 t*3 b 0 o -~"6 4' V __Lp '1 ... -',3 5 1,46 ],**) - >3* n* 3* * 3 I / O HO ^ 5 3.5 l.l ") 5 ? -,$ 45 1 .1 4 --i, z - *59 lOh 9* ,, .... t l 11 _ HI 55 U l % ..... . *9 + 10 *-J3> +W 55 CH h 93 3 .7 * . . -- 5*r>' u . 'IK L H ,*5 5 ,$ 0 j. > 5 `i f 4 & -5V -,H 5 3 + 7. ?z . HO _ T "1 / 10 *n /D9 //5 6 .3 0 7,d3 1 o7 * *? j j -* ,o * do e J 9SC900 T iae ' BEST COPY AVAILABLE WARBURG 02 UPTAKE CALCULATION SHEET 7? --- ,---1--- E lapsed T lae O IS >5 36 HS SS 65 76 SS 97 R eed in g iiiiL w /*f9 S* 15 2 Si* \sn IS* iS! K>2, J62> 101 i t s l i t 1U ~ 13/ l i t -- ----------- R eed in g F le sk ( 1 ... !*4% ihH IIS /* / /a i IZS izi . /?? m ISO 130 i2>0 F iM h.X o.: >1 c S S S t , : --r 2 Change t S P ' A ctual J 5 in n a. C o rre c tio n Change4 Uptake M_ M . O0 3 1 5 -3 M3 <"}/* Endogenous C orrected 2 U ptake 1 U *> ptak e 6 - h a o l t j - 1 - Mr -- .. o ; .u ! - , `x v 7 1 f*3 %7 I I.ttf -- .j o O 3 ) . |,5 " 3 +5 3* .Ol 2- 2 -1 . ^ 3 Z 2 . 6 ) . . 5,^' . - W O at 3 I 1, 9 5 -* XV- 1 X + I0 3X 34 ?-0' ;-/3 / -> - 2,6 4 <e* ^0 3 9 S iil -3 . 3s i i .... ii *n V ..... ..t. 35 _ 3C 2.26 3 r 2.3 i o ' ~ Hio k /- '?' 'St '*f. bS ---- - ~ i_ f orm 020-6-PWO m : ftZGiiEST BOOK - 42-8W Ref: ENVIR. ASSESS. INQ. PRODUCT: 1 PRODUCT ECOLOGICAL ASSESSMENT REQUEST FOR LABORATORY WORK DIVISION LAB REQUEST NO ^ Name: Reauest Date: Proiect No.: Code No.: Semole Date: _ REQUESTER "7/ 7 7 *1 f 3 0 12- 0~0 Phorte No. Prof. No. Date Needed: DescriDtion: PurDOse,of Tests. C -t p f T ) // c - '? i~ y //C PHYSICAL & CHEMICAL PROPERTIES Heatof Combustion Cal/G (BTU/Lb.) Density Ash Other . f e n U ja - T LABORATORY I Date Received Sample: 1 Analyst: I Date Completed: I Hours Spent: IY B P 1 Please Call Reauester If Delavs Are Encountered. 1/ f t a & za .T c T S _L * WATER POLLUTION pH COD bod5 BOD. Ultimate TOC ^ GC Oxygen Uptake Dehydrogenase Activity (TTC) Other T ^ 53 . 1 0 0 o' BIOASSAY Specify Organism and Method: O rJ Ti L LEACHATE FROM LEACHING TEST pH COD bod5 BOD. Ultimate TOC GC Bioasvtv Other OTHER Bioaccumulation Persistence Degradation Products ~T? ts : O' ,CTFO H O " o o is ' ^ .r ^ s ' i l ;A1 \b'iU-.>/ ^ * * Vr O'" * - 1 * \ % 1 i ___ !1 006357 - W eturn to W aauaeter C anary . E nvironm entI E nera. O ffice F II ^ lnte tn v ire n m e m e l nQ rj. 5 1 Interoffice Correspondence S u b je c t: C hronological Review o f --^ B iodegradation Studies ? on FM 3422 ? A ugust 12, 1977 TO: FROM: D. L. BACON E. A. REINER The fo llo w in g rev iew s th e p ro g re s s t h a t h as b e e n made i n th e b io d e g r a d a tio n stu d y o f FM 3422 o v e r th e c o u rse o f th e F a te o f F lu o ro c h e m ic a ls P r o je c t. The f i r s t s te p in o u r b io d e g ra d a tio n s tu d y o f FM 3422 was a Warburg oxygen u p tak e e x p e rim e n t. The W arburg work was co n d u cted u s in g an u n s t a b i l i z e d FM 3422 e m u lsio n in w a te r p ro d u ced by s o n ic a tio n . When FM 3422 was exposed to a c t i v a t e d slu d g e as th e s o le carbon s o u rc e , 0 u p ta k e e q u iv a le n t to ab o u t 3% o f th e t h e o r e t i c a l 0^ demand f o r th e h y d ro c a rb o n p o r ti o n was o b se rv e d w ith in a few h o u r s . No d e g ra d a tio n p ro d u c ts were d e te c te d by t h i n - l a y e r chrom atography (TLC). However, th e se r e s u lts were confirm ed by a second Warburg stu d y . T his work was follow ed by attem p ts to c o lle c t d e g ra d a tio n p ro d u cts in q u a n titie s s u ffic ie n t fo r id e n tific a tio n . A sem icontinuous a c tiv a te d sludge study using n o n e m u ls ifie d FM 3422 was conducted f o r a one-w eek p e r io d w ith d a i l y r e a d d itio n o f FM 3422. No d e g ra d a tio n p ro d u c ts were d e te c ta b le by gas l i q u i d chrom atography (GLC) in n - o c ta n o l e x t r a c t s o r by TLC in e th y l a c e t a t e e x t r a c t s . The FM 3422 added in e th a n o l r a p i d l y co n g ealed and s e p a r a te d from aqueous s u s p e n s io n . As v a r e s u l t , to o l i t t l e s u r f a c e a re a may have b een a v a i l a b l e f o r d e te c ta b le m ic ro b ia l d e g ra d a tio n . A m ethod was th e n d ev elo p ed to make n o n to x ic , s t a b l e FM 3422 em u lsio n s i n o r d e r t o in c r e a s e th e lik e lih o o d o f m ic ro b ia l m o d if ic a tio n o f FM 3422. L iu h a s shown t h i s approach to be e f f e c t i v e w ith p o ly c h lo r in a te d b ip h e n y ls (PCB) (1 ) . S t a b i l i z e d PCB em ulsions a r e b io d e g ra d a b le ; ( n o n e m u ls ifie d PCB's a re n o t. However, th e in c u b a tio n o f e m u ls ifie d FM 3422 i n SCAS r e a c to r s a g a in d id n o t y ie ld eth y l a ce ta te ex tractab le m aterial th a t could be id e n tifie d as biodegrada t i o n p ro d u c ts o f FM 3422 by e i t h e r TLC o r GLC. I t a ls o was im p o s sib le to q u a n tif y th e lo s s o f FM 3422 by t h i s approach s in c e FM 3422 d id n o t rem ain homogeneously d is trib u te d throughout th e system . D ie-aw ay e x p e rim e n ts w ere co nducted to q u a n t i t a t i v e l y m easure d e p le tio n o f FM 3422 by b i o l o g i c a l d e g ra d a tio n . M easured q u a n t i t i e s o f FM 3422 were shaken w ith a c tiv a te d sludge in Erlenm eyer fla s k s . Flasks were s a c rif ic e d a t 0, 1, 2, 4, and 8 d a y s, a c i d i f i e d , e x tr a c te d and a n a ly z e d f o r FM 3422 by GLC. No c o n s is te n t r e d u c tio n in GLC peak h e ig h t was o b se rv e d in t h i s 8-d a y e x p e rim e n t. A ne\i_GLC peak was o b serv ed t h a t was n o t p re s e n t in c o n tro l c u ltu r e s . 006358 D. fc. Bacon -2 - August 15, 1977 S u c c e s s iv e sev e n -d a y a d a p tiv e t r a n s f e r s o f FM 3422 a c c lim a te d c u l t u r e s were th e n made i n t o n u t r i e n t m edia c o n ta in in g e m u ls ifie d FM 3422. U n ex p ected ly , th e f i n a l c o n c e n tr a tio n o f e th y l a c e t a t e e x t r a c t a b l e FM 3422 was found to d e c r e a s e (s e e a tta c h e d b a r g r a p h s ) . The newly o b se rv e d u n i d e n t i f i e d GLC peak was p r e s e n t b o th in i n i t i a l and f i n a l FM 3422 c o n ta in in g sam p les. T his s u g g e s te d t h a t th e m a te r ia l c a u s in g t h i s peak was formed ch em ica lly (n o t b io lo g ic a lly ) from FM 3422 as a r e s u l t o f th e e x t r a c t i o n a n d /o r a n a l y t i c a l te c h n iq u e . T his m a te r ia l was found o n ly in sam ples a c i d i f i e d t o pH 1 p r i o r to e x t r a c t i o n . A ttem pts to i d e n t i f y t h i s m a te r ia l by TLC in o ur la b and by C e n tr a l R esearch f a i l e d . The a c id i f ic a tio n s te p in th e e x tra c tio n p ro cedure was found un n ecessary and dropped. M o d ific a tio n s i n th e m ethods o f making FM 3422 em u lsio n s and in th e e x tr a c ti o n tech n iq u e reduced th e v a r ia b ility in r e s u lts . Our a b ility to o b ta in re p e ata b le r e s u lts o ver th e same in c u b a tio n p e rio d is dem onstrated in th e a tta c h e d b a r graphs. A lte rn a te in o c u la , in a d d itio n to P ig 's Eye s lu d g e , were a lso t r i e d as sou rces o f m icroorganism s. A y e a r-o ld D ecatur s o il sample gave no lo ss o f e x tra c ta b le FM 3422 d u rin g th e f i r s t se v e n -d a y grow th p e r io d . However, lo s s was o b serv ed a f te r the second p erio d and increased in subsequent adaptive tra n s fe rs (see g ra p h 3 ). New D e c a tu r s o i l and D e ca tu r a c t i v a t e d slu d g e were found c a p a b le o f d e c r e a s in g th e amount o f e x t r a c t a b l e FM 3422 d u rin g th e f i r s t sev en -d ay grow th p e rio d as w e ll as in subsequent growth p e rio d s . However, i t must be noted th a t s t e r i l e tech n iq u e was n o t used in a l l o f th e se experim ents. Thus, i t is p o s sib le th a t a l l o f th e se c u ltu re s became contam inated by th e alread y developed acclim ated cu ltu re present in the laboratory. No m e ta b o lite s have y e t b een i d e n t i f i e d . An a tte m p t i s now b e in g made to i d e n t i f y a p o s s i b l e (b u t I f e e l im p ro b ab le) m e ta b o lite s e p a ra te d by TLC.* Very d i s t i n c t s p o ts o f t h i s m a te r ia l w ere p r e s e n t i n two 135 ppm FM 3422 c o n ta in in g c u l t u r e s , b u t a s p o t o f s im ila r i n t e n s i t y was a ls o observ ed in a c o n tro l c u ltu r e . The c o n tr o l c u l t u r e c o n ta in e d o n ly 2 .7 ppm o f FM 3422 t h a t was p r e s e n t i n i t s v in o cu lu m . Both TLC and GLC co n firm ed t h i s low FM 3422 le v e l in th e c o n tr o l. A num ber o f e x p e rim e n ts a r e now underway in th e la b o r a to r y and o th e r s a re b e in g w r i t t e n w ith th e o b je c t iv e o f d e te rm in in g what c a u se s FM 3422 to become non- e x t r a c t a b l e . An a tte m p t i s b e in g made t o d e te rm in e i f FM 3422 h a s been c o n ju g a te d , p o ly m e riz e d , o r m o d ifie d t o a form t h a t can be c o n v e rte d back to u n m o d ified FM 3422 by a c id o r b a s e h y d r o l y s i s . A lthough i t i s u n l i k e l y t h a t FM 3422 i s d eg rad ed to th e p o in t o f flu o rid e ion re le a s e to th e media, th is p o s s ib ility is being in v e s ti g a te d . An a tte m p t w i l l b e made u s in g s t e r i l e te c h n iq u e to c l a r i f y w h eth er v ia b le a c c lim a te d c u l t u r e s a r e n e c e s s a ry to e f f e c t th e change in FM 3422 which makes i t nonbiodegradable. A m a terials balance experim ent w ith la b e le d FM 3422 w i l l be c o n d u cte d t o d e te rm in e i f th e n o n d e te c ta b le p o r tio n o r p ro d u c t o f FM 3422 i s located in the eth y l a cetate phase, the w ater phase, or in the m icrobial so lid s. O ther fu tu re o b je c tiv e s a re to look fo r b io d e g ra d atio n pro d u cts o f FC-128. This conpound i s c h e m ic a lly v e ry s i m i l a r t o FM 3422. FC-128 h a s a c a rb o x y l s a l t in p la c e o f a h y droxyl s u b s titu te d carb o n , and FC-128 i s a p o s s ib le d e g ra d a tio n p ro d u c t o f FM 3422. FC-128 was found to b e r e a d i l y d e g ra d a b le in Warburg s t u d i e s . * I n f r a r e d , GLC and TLC a n a ly s is o f th e e x t r a c t now s u g g e s t t h a t t h i s m a te r ia l i s a com plexed form o f FM 3422 ( 8 /1 7 /7 7 ) . 006355 D. L. Bacon -3 - August 15, 1977 I d e n t i f i c a t i o n o f th e b io d e g r a d a tio n p ro d u c t o f FM 3422 has p ro v e n to b e a more e lu s i v e g o a l th a n o r i g i n a l l y e x p e c te d . However, b e c a u se o f FM 3422*s c e n t r a l p o s i t i o n in th e flu o r o c a rb o n l i n e , i t i s im p o rta n t t h a t we d e te rm in e w hat i s h a p p en in g to t h i s m o le c u le . I t i s l i k e l y t h a t th e n o n c o s tly e x p e rim e n ts we have p la n n e d w i l l g iv e us th e answ ers we n e e d . EAR/cel R eference: (1) D ickson L. S. L iu , M .S c ., Ph..D. ( B r i t i s h C o lu m b ia), " B io d e g r a d a tio n , An E n v iro n m en tal S o lu tio n to Some T oxic O rg an ic Compounds," E n v iro n m e n tal C o n s e rv a tio n , V ol. 3 , No. 2 , Summer 1976. * 006360 Concervtrc-fwn f/n o O N> Ou O o 0c* o <A CN V J Oo O O oo O 2; 'x . v <3> 006361 29C900 ai / /"/k9 A -/ k__ * - A- l x^jjyzhz uij ^ <*/ r~ VJ5 o- o !\ <S^ c o r'O <5o o Vr\ O r-* CO ^3- o ) 2 ^Z) n u Ay orscey\`^t-~(<)r\ Fm 3 ^ 2 _ C "vj ' V * O............. -o ......-- i............... oc d 1 ) o O M O 1 _____L_ O O ~vj .V.... -.... ,o -s -J O 0"\ "vi V? O N? 1 j I i I -vj i ,! 006364 __________________L 0 rtlp k? Lo I v lu wo 9i $0 $ 2> o Cor\ CCh F ft1 3 / z 7 (/Z3 006365 ( i:*s rr> n fiij'd rp ji o ^ ~ i 7 \--i-- ---- i-- r r mj --*!1 - pi c*H-< r 1 !y' i >&:. _L__L: <r> 1i"?u .; i-- __ _CC 1svf_L1___,_L Jl! ! . ? 11i ___!__ }. - ' id ! ; ! i ' : : J .i i ! ! ! i i ! !1 \fLf s i -1i' . ! .<=! 1 i! ___:___ _____ !__ Li_:__ ;__ ! i! ll~e_o ;1 > ! j (ppbO \ : i : ! 1 : : !: i i i - i , . >'<>'- ! - ^3 f i ; O. i 3 -1________ !__ __li___1__ L . L i i ! I ! .: - ' i '1. i- ' ! i ! i : : .i i ' j i i ' ;. ! I. i i i .'i ' ; . 1 ! ! - i :. ! 1 ! C> ! !I !; ; ; : ! !! !; :' i> i; i! i1 1; ' !! ;i i1 i; i; i! i1 1 ! ! ' 1, . 4 - i ! ! ' -i ' ; . 7 i J ; 1 i r i ; i 1 i i r I : ! :: |. 1 , !' i i ' i vl_ ti i - i M I i7 i i ` `! ! ' *i : ! i - i 1 : ! : ! i i ! i' i i1 i - i : ;' ii -, j. ; 1i . I .'i ; -| ! ... i , , i ' h; ii i i i i: i . !, M. ; !! i : ; i ; ]y ! i !. ! I " if<\ ii. ; 1 -s^,t ? I ! ! ; . i 1: ! : : ! T i .1. . i-- 1 i j i i i i ! ! ' ' ` ! i ` i 1 j- 1 i : i i ! ; ' i 1 *;' ' !! 1 7! 1, - i i i- ! ! ! i ! ! |. j i i - - * ! 1 i ' : ii! !j ! ! ;1 ! . ; : 1M ! i i iI : ; , 1 ! 1 - i ! - i i ! T f i ! ! i . i 7 : 1 i " i !T i i. ' I ! i -- rt - i i ; ' 1 --iTT^ i 1 : :1 - j 1 i;' i '; i : k !' ! - ; i . i i .1 ' 1 : f I: 1 i !; ! i. ! : i . : ' ;; I i " i? V - , i - i i j M : i ; : iii 1 i i l M e : ' i i !i i; . ;! ,. :: : . ` i i "i : :; ; !: ' i; -i, !i 1! ; ; . iiM r : i ! . M M :i : : i -M i' M ; 1 : i . ! - -;- ! ; 1 > ; 1I ! i. , ! - ! .J - -i! ' i- :1 _ 1i1' j *`*-* i1: i i i- i ; i !> r. i! 'i } i- ; ; ; ; ; !' i j; :: i ; - ili j j _ - i i . *f!- i ; I' " ' i -i : -I i- ... . \! !i M ! . ! i. s i : i -i ! ; : i ;iii ;.; i . - i i. . i n .; ; ; ; i ! .T 7 : : i 7! ! ! i! i1 i. .i! . i*l i ii i ! f ! i : .: i ; ! . .j., ' . Vs : 1. '! V- i ' i i l i .. : i ' 1 1. *11 i i 'i -i i ! [ M . i" ! ! !S- ii '* :.s.&'*-; \H 7*^ - 1 1 -1 i!iii1 .,;7 i ! . t : 1 ' ! 1 i . i i- i i " -V. ! i ; i . : ; -i : ; : : i ; : i ;J ! ! : ; :.: ! ! ! ; : ;1 i! ! - i i `i :1i 1 -1 . i , , .I ! !. i ' 'j; ".i , MI : ! ' !" : i r: !" ! !c . M - 11 ' J:tr ' J1' j~ ;1 1i .! ! . !i .l i l ! ! i ! - ii i .! i 1 7J V !i . ! \J1 ` !i U- l ;! il . ."!* - [:- I . i. - i !t ii .'!i . \i l \i \! ' i ! i . i; 1! V;rr 7 -; `: ii -4 V -iJ " s^v i V --'A ; . ' i i -i .1 . . : ' -1' / M 77 7 ..T h h-'7)s ' . * t 'ti , ' 77 7 *r' ! !' i " ! : 1 1 .' ! 1 ! ! i .[ i< i i .i i 1i, 1 : . __ '7 i7 -` -- d o UC *n F * h i v FM V l/\ 006367 V7)/,rMVy2 ''1/,L '^ * p '* ) <\ si 5orm 6747-11-A TECHNICAL REPORT SUMMARY Data 1/9/78 TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN (Im portant - I f report sprinted on both sides o f paper, send two copies to TCC.) Division Proiact Raport Title To E nvironm ental L ab o ra to ry (EE 8 PC) Fate o f Fluorochem icals in the Environment B iodegradation Studies o f Fluorocarbons - II Oept. Number 0222 Proiact Numbar Report9N9u7m0b6e1r 2613 4 D. L. Bacon____________________________________________________________________ Author!) Employas Numbar(s) E* A. RkGiiiioir 47816______________ Notebook Ratatines * 4 0 6 7 l, p . 54 -5 6 ; 45727, p . 1 -3 0 ; 46269, p . 7, 1 4 , 15, No. of Pages Including Covarthaat 18 2 1, 25, 29; 41947, p . 50, 51, 55; 44191, p . 27-32___________ 20 SECURITY G Open (Company Confidential) O Closed (Special Authorization) 3M CHEMICAL REGISTRY ^ New Chemicals Reported Yes 2 No KEYWORDS: (Select terms from 3M Thesaurus. Suggest other applicable terms.) (B io d eg rad atio n ) EE 8 PC-Div. Envir. Assess. Fluorochem ical (D egradation) CURRENT OBJECTIVE: To e v a lu a te th e s u s c e p t i b i l i t y o f FM 3422 to b i o l o g i c a l decom position. REPORT ABSTRACT: (200-250 words) This abstract information is distributed by the Technical Communications Center to alert 3M'ers to Company R&O. It is Company confidential material. B iodegradation stu d ie s are d escrib ed which allow th e e v a lu a tio n o f th e s u s c e p t i b i l i t y o f FM 3422 to a e ro b ic m ic ro b ia l d e g r a d a tio n . B io d e g rad a t i o n t e s t p ro c e d u re s used a re m o d if ic a tio n s o f common b io d e g r a d a tio n t e s t m ethods. They in c lu d e sem icontinuous a c tiv a te d slu d g and shake fla sk die-away stu d ies w ith seq u en tial adaptive tra n s fe rs . M icrobial * in n o c u la w ere o b ta in e d from s e v e r a l e n v iro n m e n ta l s o u r c e s . Some s o u rc e s were se le c te d fo r th e ir lik e lih o o d o f co n tain in g m icro b ial p o p u latio n s e n ric h e d in th e c a p a b i l i t y o f d e g ra d in g FM 3422 o r s i m i l a r f l u o r o - c h e m ic a ls. A n a ly tic a l p ro c e d u re s used in c lu d e d GLC, TLC, ^ C - s c i n t i l l a t i o n counting and analyses fo r re le a s e d .flu o rid e . In te rp re ta tio n o f b io d e g ra d atio n r e s u l t s from th e se s tu d ie s was h in d e re d by th e low s o l u b i l i t y o f FM 3422, i t s v o l a t i l i t y , and i t s a f f i n i t y f o r su sp en d ed o rg a n ic m a te r ia l. T his b in d in g made i t d i f f i c u l t to q u a n t i t a t i v e l y e x t r a c t FM 3422 from th e b io l o g i c a l s o lid s o f th e t e s t c u l t u r e s . N o n to x ic d e terg en t s ta b iliz e d em ulsions were used to circum vent problem s caused by low w a te r s o l u b i l i t y . Information Liaison Initials: 006368 -2- SUMMARY AND CONCLUSIONS FM 3422 was found to be co m p le tely r e s i s t a n t to b io d e g ra d a tio n u n d e r th e co n d itio n s described in th is re p o rt. These conditions are considered to be optimum f o r b i o l o g i c a l d e g ra d a tio n . FM 3422 was a ls o found to h av e a s tr o n g a ffin ity fo r organic so lid s. In a l l e x p e rim e n ts d e s c rib e d h e r e i n , FM 3422 was exposed a s a s u r f a c t a n t s t a b i l i z e d em ulsion, to heterogeneous m icrobial c u ltu re s , under aerobic c o n d itio n s, and n e ar n e u t r a l pH ( 7 .1 - 7 .5 ) . C o n c e n tra tio n s o f FM 3422 ran g ed from 100-500 m g/1. M icro o rg an ism s w ere o b ta in e d o r d eveloped from s o i l o r w a ste tre a tm e n t s lu d g e s . Some m icro b ial sources were s e le c te d because o f t h e ir previous o r continuous exposure to flu o ro c h e m ic a ls. I t was f e l t th a t th e se sources were lik e ly to have developed n a t u r a l l y e n ric h e d c u ltu r e s c a p a b le o f d e g ra d in g flu o ro c h e m ic a ls . FM 3422 was e x p o sed t o b o th h ig h (^2000 mg/1) and low (^lOO mg/1) c e l l d e n s i t i e s . Weekly t r a n s f e r s o f th e low c e l l d e n s ity c u ltu r e s a llo w ed a c c lim a tio n t o FM 3422 to p ro ceed fo r s ix months. This study cannot ru le out the p o s s ib ility th a t con d itio n s could be found th a t would allow b io d e g ra d atio n o f th is compound, n o r can i t exclude th e p o s s i b i l i t y th a t a m icroorganism could ev en tu ally be enriched th a t could m e ta b o lica lly a l t e r th is chemical under the conditions described in th is re p o rt. N evertheless, the r e s u l t s o f t h i s q u ite e x te n s iv e stu d y s tr o n g ly s u g g e s t t h a t FM 3422 i s l i k e l y to p e r s is t in th e environment fo r extended periods u n a lte red by m etabolic a tta c k . I t s o b se rv e d a f f i n i t y f o r o rg a n ic s s u g g e s ts t h a t FM 3422 r e le a s e d t o th e e n v iro n ment is lik e ly to s o rb to organics such as those p resen t in s o il o r in the sedim ents o f aq u atic ecosystem s. Table 1 summarizes the experim ents conducted in th e s tu d y o f FM 3422 b io d e g ra d a tio n . 006369 -3- TABLE 1 SUMMARY OF EXPERIMENTS PERFORMED IN FM 3422 BIODEGRADATION STUDY Experim ent 1) 10- day sem ico n tin u o u s activ ated sludge biodegrada tio n study o f su rfa c ta n t s t a b i l i z e d FM 3422. 2) 6-m onth sh ak e f l a s k d ie -a w ay study w ith weekly tr a n s f e r s . 3) M ate ria ls b alan ce experim ent. 4) F luoride re le ase 5) A ttem pts to re c o v e r n o n e th y l a c e t a t e e x t r a c t a b l e FM 3422 from c e ll c u ltu re s: A) H y d ro ly s is B) B in d in g to C o n ta in e r s C) V o l a t i l i z a t i o n from C e lls D) Hot e th y l a c e t a t e e x t r a c t s E) R eflux w ith Dioxane Findings No FM 3422 b io d e g ra d a tio n p ro d u c ts were d e te c te d in e th y l a c e t a t e e x t r a c t s by TLC o r GLC, Again no d eg rad atio n products were d e te c te d in e th y l a c e t a t e e x t r a c t s . However, >50% and som etim es >75% o f th e i n i t i a l l y e th y l a c e ta te e x t r a c t a b l e FM 3422 was m issin g from e x t r a c t s from 7 -d ay -o ld c u ltu re s . An a tte m p t to m easure th e f r a c t i o n s o f 14CFM 3422 p r e s e n t i n th e w a te r , e th y l a c e t a t e , and s o lid s phases o f 7-day-old c u ltu re s fa ile d ;'. T his a p p a r e n tly was due t o FM 3422 v o l a t i l i z a t i o n d u rin g s o l i d s d ry in g . When mixed w ith grown c e l l s , FM 3422 became n o n e x tr a c ta b le to o r a p id ly to be ex p lain ed by a m etabolic phenomenon. Fluoride concentration d id n o t increase during die-away stu d ie s w ith cu ltu re s acclim ated to FM 3422. T h is s u g g e s ts no m o d if ic a tio n s o c c u r to th e p e r f lu o r o p o r tio n o f FM 3422. V- N eith er acid o r base hydrolysis freed non e x t r a c t a b l e FM 3422. Irre v e rsib le binding-to cu ltu re container did n o t e x p la in n o n e x tr a c ta b le FM 3422. N o n e x tra c ta b le gM 3422 was 1 /3 re c o v e re d by v o la t i l i z i n g a t 100 C. from c u ltu r e s o lid s and condensing. E thyl a c e ta te e x tra c ts a t 60C. d id n o t r e c o v e r a d d i t i o n a l FM 3422. R efluxing c u ltu re so lid s w ith 1,4-dioxane re c o v e re d a l l n o n e x tr a c ta b le FM 3422 from th e so lid s o f 7-day-old cultures,C om plete recovery o f FM 3422 shows t h a t no b io d e g ra d a tio n occurred. 006370 -4- INTRODUCTION Our e a r ly work e v a lu a tin g th e s u s c e p t i b i l i t y o f FM 3422 to m ic ro b ia l d e g ra d a tio n has been d e sc rib e d in a p re v io u s re p o rt (1 ). T his work in clu d ed two s e p a ra te Warburg re s p iro m e tric experim ents w hich, c o n v in cin g ly , showed in c re a se d oxygen u tiliz a tio n by unacclim ated b io lo g ic a l sludge im m ediately follow ing ad d itio n of u n s ta b l iz e d em ulsio n s o f u n p u r if ie d FM 3422. In th e s e two e x p e rim e n ts , w ith FM 3422 as th e s o le exogenous carbon s o u rc e , oxygen u p ta k e above th e endogenous l e v e l a cc o u n te d f o r 3% o f th e oxygen r e q u ir e d to c o m p le te ly o x id iz e th e h y d ro carbon p o rtio n o f th e m o le c u le. The reaso n f o r t h i s rem ains u n e x p lain ed . One p o s s i b i l i t y i s t h a t b io d e g ra d a b le frag m en ts o f FM 3422 may have been form ed as a r e s u lt o f th e em ulsion form ation by so n ica tio n . A ttem p ts to i s o l a t e FM 3422 d e g ra d a tio n p ro d u c ts from th e Warburg f l a s k s fo llo w in g th e observed oxygen uptake were u n su cc e ssfu l, as were th o se from a subsequent sem ic o n tin u o u s a c t i v a t e d s lu d g e (SCAS) b io d e g ra d a tio n s tu d y . In th e SCAS s tu d y s o l u t i o n s o f FM 3422 i n e th a n o l were added d i r e c t l y to th e b i o l o g i c a l r e a c t o r s . Added in t h i s m anner, th e FM 3422 r a p i d l y c o n g e a le d and s e p a r a te d from th e w a te r phase accum ulating on th e re a c to r w alls and w ith th e b io lo g ic a l s o lid s . This s e p a r a tio n from th e w a te r p h a se may h av e made FM 3422 u n a v a ila b le to r e a c t w ith d egradative enzymes. In the p resen t rep o rt, fu rth e r attem pts to c h aracterize the s u s c e p tib ility o f FM 3422 to b io d e g r a d a tio n a re d e s c r ib e d . METHODS AND MATERIALS Most methods are d e sc rib e d in th e ex p erim en tal s e c tio n . The fo llo w in g sta n d a rd methods and m a te ria ls were used re p e a te d ly throughout th e experim ent. E xtraction Procedure 10.0 ml sam ples w ere e x t r a c t e d in p o ly p ro p y le n e c e n tr if u g e tu b e s by ad d in g 1 0 .0 ml o f e th y l a c e t a t e . The sam ples w ere th e n capped w ith p o ly p ro p y le n e caps . and in v e r te d 50 tim e s . P hase s e p a r a tio n was e n s u re d by c e n t r i f u g i n g f o r 10 m inutes a t about 12,000 x g r a v ity . A c o r r e c t io n h a s to be made f o r th e d e c re a se d volume o f th e s o lv e n t phase and th e in c re a s e d volume o f th e w a te r phase fo llo w in g e x tr a c tio n . A fte r sh ak in g th e volume o f th e aqueous phase in c re a s e s from 10.0 to 11.0 ml and th e volume o f th e e th y l a c e t a t e s o lv e n t p h ase d e c r e a s e s from 10.0 t o 9 .0 m l. T h e re fo re , a d e te r m in a tio n o f th e number o f ppm o f FM 3422 o r i g i n a l l y p r e s e n t in th e w a te r p h a se w hich i s e x t r a c t e d i n t o e th y l a c e t a t e i s made by m u ltip ly in g the co n cen tratio n found in the eth y l a c e ta te phase by 0.90. Media The b a s a l s a l t s media used in th e sem icontinuous a c tiv a te d slu d g e experim ent consisted of: 1.0 g/1 nh4c i 2.0 g/1 K2HP04 0 .2 5 g /1 MgS04*7H20 A d ju ste d to pH 7 .2 w ith s tr o n g a c id 006371 -5- The stan d ard media used in die-aw ay and subsequent s tu d ie s c o n tain ed : 133 ppm o f FM 3422 (e m u ls ifie d ) 2 g/1 K2HP04 2 .4 g/1 YM Broth 830 mg/1 Alconox ^6 g/1 E th y l a c e ta te pH ^ 7 .2 I t was p re p a re d by e m u ls ify in g 0 .2 g o f FM 3422 d is s o lv e d i n w a t e r - s a t u r a t e d e th y l a c e ta te in 100 ml o f 1 2 .5 g /1 A lconox s o l u t i o n t h a t h a d b een p re p a re d u sin g e th y l a c e ta te - s a tu ra te d w a ter. E m u lsific a tio n was accom plished by m ixing f o r 30 seconds w ith a model SDT T iss u m iz e r. The r e s u l t i n g e m u lsio n was th e n d i l u t e d in a ^HPO^ s o lu tio n w hich in t u r n was d i l u t e d i n a weak YM b r o t h m ed ia. The s u rfa c ta n t c o n tain in g media were dem onstrated to be n o n to x ic to m icroorganism s by in o c u la tin g th ese media and comparing th e d isso lv e d oxygen c o n c e n tra tio n d ep letio n ra te s from th ese c u ltu re s w ith th o se o f n o n su rfa c ta n t-c o n ta in in g c u ltu re s . These co n p ariso n s w ere made i n i t i a l l y , a f t e r one h o u r , and a f t e r one day o f exposure to th e su rfa c ta n t-c o n ta in in g media. Chemical Analyses TLC and GLC a n a ly se s a re d e s c rib e d in th e P ro g re s s R ep o rt by A. Mendel (2 ) . C hem icals The follow ing chem icals were used in th is stu d y : 1) S ublim ated FM 3422 (s e e r e p o r t o f A rt M endel - (2 )) 2) R a d io a c tiv e FM 3422 (s e e r e p o r t o f A rt M endel - ( 2 ) ) 3) E thyl a c e ta te - M a lle n c k ro d t;.A.R. 4) YM B roth - DIFCO - B acto D ehydrated B acto Y east E x tr a c t - 14.3% DIFCO M alt E x tr a c t - 14.3%- B acto -P ep to n e - 23.8% ' Bacto-D extrose - 47.6% 5) E ther - Anhydrous,J . T. B aker,"B aker A nalyzed." 6) S ip o n a te DS-10 - A p u r i f i e d a l k y l a r y l - s u lf o n a te s u p p lie d by A lcolac, Incorporated. 7) Alconox - A m ix tu re o f a l k y l a r y l s u l f o n a t e s , l a u r y l a lc o h o l s u l f a t e s , phosphates, carbonates and s y n e rg is tic agents - su p p lied by Alconox, In co rp o rated . 8) 1,4-D ioxane - R eagent g ra d e - re p a c k a g e d , s u p p l i e r unknown. 9) A quasol - X y len e-b ased s c i n t i l l a t i o n s o l u t i o n - s u p p lie d by New E ngland N uclear. 006372 EXPERIMENTAL -6- Sem icontinuous A ctivated Sludge Study In an e a r l i e r s tu d y , no d e g ra d a tio n p ro d u c ts o f FM 3422 were d e te c te d by TLC o r GLC in a sem ico n tin u o u s a c ti v a te d slu d g e (SCAS) s tu d y in which FM 3422 was sim p ly added t o th e media a s an e th a n o l s o l u t i o n , and FM 3422 r a p id ly co n g ealed and sep a ra te d from the w ater phase (1 ). I t was p o s tu la te d th a t b io d eg rad atio n may n o t have been o b serv ed b e ca u se enzymes c o u ld n o t " a tta c k " th e c o a g u la te d FM 3422 m o le c u le s. S in ce em u lsio n s o f w a te r - in s o lu b le m o lecu les hav e been shown to be more s u s c e p tib le to enzyme a t t a c k ( 3 ) , a seco n d SCAS d e g ra d a tio n s tu d y o f FM 3422 was c o n d u cted u s in g e m u ls if ie d FM 3422. P r io r to th e i n i t i a t i o n o f th is stu d y , s e v e ra l s u rfa c ta n ts were te s te d f o r t h e i r a b i l i t y to s t a b i l i z e FM 3422 e m u lsio n s. The two m ost s a t i s f a c t o r y were A lconox and S ip o n a te DS-10. These w ere b o th u se d in th e SCAS s tu d y . The em ulsions f o r t h i s s tu d y were made by d is s o lv i n g 3 g o f FM 3422 in 3 ml o f e th y l a c e t a t e . P o rtio n s o f t h i s s o l u t i o n w ere th e n added t o 5 g/1 s o lu tio n s o f A lconox o r S ip o n a te DS-10, and e m u ls ifie d by m ixing f o r 30 seconds w ith a tis s u m iz e r . The c o n c e n tra tio n s o f chem icals in th e em ulsions th u s formed were 5 g/1 Alconox or DS-10, 2 .7 g /1 e th y l a c e t a t e , and 3 g /1 FM 3422. Em ulsions w ith o n ly e th y l a c e t a t e and one o f th e two s u r f a c ta n ts were used as c o n tr o ls . S ix sem icontinuous a c tiv a te d sludge u n its were s e t up by adding 2 l i t e r s o f fre s h a c tiv a te d slu d g e to each u n it. T his slu d g e was c o lle c te d from an a e ra tio n b a sin a t th e m unicipal w aste tre a tm e n t p la n t in P ig 's Eye, M innesota. The sludge was s e t t l e d and th e bottom 500 ml, c o n ta in in g a l l o f th e s e t t l e d c e l l s , was r e t a i n e d . One l i t e r o f medium was th e n added t o th e s e t t l e d slu d g e in each r e a c to r , and i d e n tic a l medium readded a t th e b e g in n in g o f each t e s t c y c le . These m edia w ere made by d i l u t i n g p o r ti o n s o f th e a b o v e -d e s c rib e d em u lsio n s in th e b a s a l s a l t s medium d e s c rib e d in th e m ethods and m a te r ia ls s e c tio n . * Table 2 shows th e c o n c e n tra tio n s o f e th y l a c e ta te , Alconox, S iponate DS-10, and FM 3422 p r e s e n t i n 1 .5 L c o n te n ts o f each r e a c t o r a t th e b e g in n in g o f th e f i r s t te s t cycle. TABLE 2 CONCENTRATION OF ORGANIC COMPOUNDS IN SEMICONTINUOUS ACTIVATED SLUDGE REACTORS AT THE BEGINNING OF THE FIRST TEST CYCLE _________I n i t i a l C o n c e n tra tio n (ppm)________ SCAS R eacto r 1 2 3 4 5 6 Ethyl A cetate 560 560 460 460 90 90 A1conox 830 0 830 0 170 0 DS-10 0 830 0 830 0 170 FM 3422 0 0 510 510 100 100 006373 -7- The sem ico n tin u o u s a c tiv a te d slu d g e p ro ced u re was d e sc rib e d in a p re v io u s r e p o r t (1 ), and is b r ie f ly o u tlin e d in Figure 1. I M s e x p e rim e n t was conducted f o r 10 days w ith sam ples ta k en a t th e b e g in n in g and end o f th e f i r s t and la s t t e s t cy cle. A fter th e samples were ta k en , they were c e n trifu g e d . The s o lid s and c e n trifu g a te were e x tra c te d s e p a ra te ly . The c e n t r i f u g a t e was e x tr a c te d two tim e s , f i r s t a t th e pH o f th e sam ple (^pH7) and a g a in a f t e r a d ju s t in g to pH 1 w ith c o n c e n tra te d s u l f u r i c a c id . The c e l l s were e x t r a c t e d o n ly once a t pH 7. The e th y l a c e t a t e e x t r a c t s w ere th e n e v a p o ra te d , re s u sp e n d e d in m ethanol and s e p a r a te d by TLC. Problems w ith foaming developed in a l l re a c to rs during th e f i r s t t e s t c y c le . T h is foam ing c a u se d th e lo s s o f some suspended s o l i d s in r e a c t o r s 1 , 2 , 3, and 4 . L i t t l e o r no s o lid s were lo s t from r e a c to r 5, and most o f th e suspended s o lid s w ere l o s t from r e a c t o r 6. In a l l su b se q u e n t t e s t c y c le s , foam ing and lo s s o f s o l i d s w ere c o n tro lle d by blow ing a stream o f a i r on th e w ater su rfa c e o f each r e a c to r . This a lso in c re ase d th e ra te o f ev ap o ratio n , and re a c to rs were brought back to t h e i r o r ig in a l volume w ith d eio n ized w ater p r io r to d ra in in g o r s a i l i n g . T here w ere no new s p o ts t h a t d ev elo p ed on TLC p l a t e s t h a t would i n d i c a t e t h a t a d e g r a d a tio n p ro d u c t o f FM 3422 was a c c u m u la tin g . T his was t r u e b o th in th e e x tra c ts o f th e suspended s o lid s and in the e x tra c ts o f th e c e n trifu g a te s . There were in d ic a tio n s th a t biodegradation o f th e su rfa c ta n t took place during the course o f th e f in a l t e s t c y cle . Spots which could be a ttr ib u te d to Alconox o r DS-10 w ere p r e s e n t in th e e x tr a c ts o f th e i n i t i a l sam ples. These sp o ts w ere r e p la c e d by new s p o ts from l a t e r s a n p le s and th e s e new s p o ts w ere c o m p le te ly gone (e x c e p t in r e a c t o r 4) a t 24 h o u rs . I t a p p e a rs t h a t FM 3422 had n o t d e g ra d ed , a t l e a s t to a d e te c ta b le l e v e l , u n d er th e s e c o n d itio n s w ith h ig h c e l l d e n s i t i e s and a 10-d a y a c c lim a tio n p e r io d . These a r e c o n s id e re d to be optimum c o n d itio n s f o r b io d e g r a d a tio n . , v Shake F la s k Die-Away S tu d ie s w ith A daptive T ra n sfe rs Growth o f a hetero g en eo u s c e l l p o p u la tio n in n u tr i e n t media c o n ta in in g e m u ls ifie d FM 3422 w ith s e q u e n tia l a d a p tiv e t r a n s f e r s was i n i t i a t e d f o r two r e a s o n s . F i r s t , i t was v e ry d i f f i c u l t to q u a n t i t a t i v e l y m easure th e rem oval o f FM 3422 from SCAS s t u d i e s . T h is was due to FM 3 4 2 2 's v e ry low w a te r s o l u b i l i t y and i t s a f f i n i t y f o r c e l l u l a r m a t e r i a l . On th e o th e r h a n d , th e n t i r e c o n te n ts o f d ie-aw ay c u l t u r e s could e a s ily be e x tra c te d elim in a tin g th is problem o f nonhomogeneous sam pling. The second reason fo r s ta r tin g shake fla s k s tu d ie s is th a t th is a d ap tiv e tr a n s f e r pro ced u re i s an accepted method o f e n ric h in g organism s from n a tu re th a t a re capable o f growth on xen o b io tics o r ra re ly -o c c u rrin g o rganic m a te ria ls . In an attenqpt to o b ta in organism s c a p a b le o f grow th on FM 3422, in o c u la w ere o b tain ed from a number o f sources. These sources included th re e s o il samples from 3M's D e c a tu r, Alabama p l a n t , ta k en from a re a s t h a t were l i k e l y to have been exposed to flu o ro ch em icals, b io lo g ic a l sludge from th e D ecatur p la n t, and slu d g e from th e M e tro p o lita n Waste T reatm ent P la n t a t P ig 's Eye, M innesota. S ep arate shake fla s k c u ltu re s were s ta r te d from th ese vario u s in o c u la , b u t they w ere e v e n tu a lly combined in to a s in g le m aster c u ltu re . 006374 -- -4 -8- STEP 4: D rain s u p e r n a ta n t, STEP 3: S to p a e r a t i o n and m ix in g . Let sludge s e ttle . FIGURE 1 T e st c y c le f o r sem ic o n tin u o u s a c t i v a t e d slu d g e r e a c t o r . 006375 -9- I n i t i a l l y , the adaptive tra n s fe rs were conducted in g lass Erlenm eyer fla s k s c o n ta in in g 50 ml o f c u ltu r e medium. The t o t a l c o n te n ts o f th e f l a s k w ere a c i d i f i e d w ith s u l f u r i c a c id to pH 1, su p p lem en ted w ith a s a tu r a te d NaCl s o l u t i o n , e x tr a c te d two tim e s , and th e in s id e o f th e f l a s k washed w ith s o lv e n t two tim e s. T o ta l phase s e p a ra tio n was accom plished by c e n tr if u g a tio n . E x tra c ts and w ashings w ere combined p r i o r t o GLC a n a l y s i s . A c id if ic a t io n and s a l t a d d itio n w ere l a t e r elim in ated from th e e x tra c tio n p ro ced u re. S a lt a d d itio n proved to be o f no b e n e f i t and s u l f u r i c a c id a d d itio n c au se d th e fo rm a tio n o f a new u n i d e n t i f i e d peak in th e GLC a n a ly s is . Acid a d d itio n was a ls o o f no v a lu e in im p ro v in g th e e x t r a c t a b i l i t y o f FM 3422. T his new ly -fo rm ed peak a p p a r e n tly was a ch em ical m o d if ic a tio n o f FM 3422, and i t was c o n v e rte d back t o FM 3422 on t h i n - l a y e r p l a t e s . S in ce i t was p r e s e n t b o th in e x tr a c ts taken i n i t i a l l y and a t th e end o f th e growth p e rio d , i t was o b v io u sly n o t a m e ta b o lic p ro d u c t. The p r e p a r a tio n o f th e m edia u sed in e a r ly sh ak e f l a s k c u ltu r e s d i f f e r e d from th e l a t e r improved and sta n d a rd p ro ced u re d e sc rib e d in th e methods and m a te ria ls s e c tio n only in th a t w a te r-sa tu ra te d eth y l a c e ta te , and eth y l a c e ta te -sa tu ra te d w ater were n o t u sed in i t s p r e p a r a t io n . As a r e s u l t , how ever, th e FM 3422 was n o t a s c o m p le te ly e m u ls ifie d i n t h i s m edia. The n o n e m u ls ifie d FM 3422 was i n t e n t i o n a l l y s e p a r a te d and n o t in c lu d e d in th e c u l t u r e m edia. T hus, th e i n i t i a l FM 3422 c o n c e n tr a tio n was v a r ia b le and i t s c o n c e n tra tio n known o n ly to be som ething le s s th a n 133 ppm, which was th e c o n c e n tr a tio n t h a t would hav e been p r e s e n t had a l l th e FM 3422 b een emul s i f i e d . As w ith th e s ta n d a r d m edia, t h i s i n i t i a l l y - u s e d media was d e m o n stra te d to be nontoxic to m icroorganisms by th e fa c t th a t c e ll re s p ira tio n occurred norm ally in its presence. I n i t i a l l y , c u l t u r e s were shaken a t room te m p e ra tu re (2 0 -2 5 C .) and t r a n s f e r s made every 8 days. T ran sfer tim es were l a t e r changed to 7 days fo r convenience. C ultures were e x tra c te d bo th a t th e beginning and end o f each c u ltu re growth period. The r e s u l t s were q u ite v a r ia b le from t r a n s f e r to t r a n s f e r . T his can be seen in F ig u re 2 . In most c a s e s , th e amount o f FM 3422 t h a t c o u ld be e x t r a c t e d w ith , e th y l a c e ta te a t th e end o f th e in c u b a tio n p e rio d was le s s th an t h a t w hich was e x t r a c t a b l e im m ediately fo llo w in g c u l t u r e in o c u la tio n . U su a lly , 50% o r more o f th e i n i t i a l l y - e x t r a c t e d m a te ria l was n o t re c o v e ra b le from th e w eek-old grown c u l t u r e s . No d e g ra d a tio n p ro d u c ts w ere d is c o v e re d by e i t h e r TLC o r GLC o f th e e th y l a c e t a t e c u l t u r e e x t r a c t s . A t t h i s p o i n t , i t was n o t known w h e th er th e u n re c o v e re d FM 3422 were b e in g c o m p le te ly d e g ra d e d , m e ta b o lic a lly m o d ifie d to m a te ria ls which could n o t be e x tra c te d , o r w hether i t o r d t s m e ta b o lite s were bound to th e biomass o f th e c u ltu re c o n ta in e r. A number o f experim ents were subsequently conducted to determ ine what caused a p o r tio n o f th e FM 3422 t o become n o n e x tr a c ta b le by e th y l a c e t a t e . 006376 -10- F r a c tio n o f I n i t i a l FM 3422 C o n c e n tra tio n 23 4 8 12 Weekly T ra n s fe r # 16 20 ^ I n i t i a l c o n cen tratio n s were not measured in th e second and th ir d weekly adaptive tra n s fe r p erio d s. FIGURE 2. R a tio o f f i n a l to i n i t i a l FM 3422 c o n c e n tr a tio n in e q u a l volume e th y l a c e ta te e x tra c ts o f w eek-old shake fla sk c u ltu re s from adaptive tra n s fe r experim ent. 006377 -11- TLC and GLC A n a ly sis o f E x tr a c ts from Shake F la sk S tu d ie s In one TLG e x p e rim e n t, e th y l a c e t a t e e x t r a c t s o f 3 c u ltu r e s w ere found to c o n ta in a m a te r ia l which produced v ery d i s t i n c t s p o ts w ith a s m a lle r R f th a n FM 3422. Hie TLC v i s u a l i z i n g p ro c e d u re s u g g e s te d t h a t th e new sp o ts c o n ta in e d flu o ro c a rb o n s . Two o f th e c u ltu r e s p ro d u c in g th e s e s p o ts c o n ta in e d 135 ppm o f FM 3422 p r i o r to e x tra c tio n . The t h i r d c u l t u r e c o n ta in e d le s s th a n 3 ppm o f FM 3422 (in tro d u c e d w ith th e FM 3422 a c c lim a te d in o c u la ) . D e sp ite t h i s g r e a t d if f e r e n c e i n i n i t i a l c o n c e n tra tio n , the "new" sp o ts from th e e x tra c ts o f th e th re e c u ltu re s proved to be o f a p p ro x im a te ly e q u al i n t e n s i t y , b u t FM 3422 was d e te c te d by TLC o n ly i n th e e x t r a c t o f th e two 135 ppm c u l t u r e s . Hie e x tr a c ts o f th e s e th re e c u ltu r e s w ere combined. The m a te ria l c au sin g t h i s new s p o t i s o l a t e d by p r e p a r a t iv e TLC and a g a in r e p u r i f i e d by t h i n - l a y e r chrom atog ra p h y . A tte n p ts w ere made to c h a r a c t e r i z e t h i s m a t e r i a l . GLC o f t h i s m a te r ia l in o u r la b o n ly y ie ld e d a m a te r ia l w ith GLC c h a r a c t e r i s t i c s i d e n t i c a l to FM 3422. M easurem ents w ere n o t made o f th e amount o f m a te r ia l i n j e c t e d . IR s p e c tr a o f t h i s m a te r ia l showed i t t o have a c a rb o n y l a b s o r p tio n a t 'vbpm and an a b s o rp tio n f a i r l y c h a r a c t e r i s t i c o f a flu o ro c a rb o n conpound betw een 8 and 9pm. Bands s i m i l a r to FM 3422, b u t n o t o v e rla p p in g w ith FM 3422, w ere a l s o p r e s e n t . T his su g g e ste d a c o n p lex w ith a m a te ria l lik e u re a . Mass s p e c tr a o b ta in e d fo llo w in g d i r e c t i n s e r t i o n o f th e unknown in to th e mass s p e c tro p h o to m e te r showed ammonia, C0_, g ly c e r in , and FM 3422. The r e l a t i v e c o n c e n tr a tio n o f FM 3422 t o o th e r m a te ria ls ^ c o u ld n o t be d e te rm in e d . The p re se n c e o f u re a was n o t n o te d . FM 3422 a p p ea re d i n th e mass a p e c tr a work to v o l a t i l i z e a t a h ig h e r te m p e ra tu re th a n p u re FM 3422; t h i s may have been due t o i t s b e in g com plexed. GC-mass sp e c o f th e unknown m a te r ia l y ie ld e d m ainly g ly c e r in as i d e n t i f i e d by th e mass sp e c . (In f a c t , more th a n one peak was i d e n t i f i e d by mass spec as g l y c e r i n .) U n lik e th e work in o u r la b , FM 3422 was n o t d e te c te d , a lth o u g h th e same l i q u i d p h a s e , Carbowax 20M, was u se d a t a h ig h e r te m p e ra tu re . T his p ro d u c t h as n o t b een f u r t h e r c h a r a c te r iz e d . I t does n o t a p p ea r v. to be a major degradation product. Fluoride Release D eterm inations M easurem ents o f F c o n c e n tra tio n s , ta k en i n i t i a l l y and a f t e r 7 d ay s, have shown no change i n e i t h e r c o n tr o l c u l t u r e s o r c u l t u r e s t h a t showed *'50% d e c re a s e s in e th y l a c e t a t e e x tr a c ta b le FM 3422 c o n c e n tr a tio n . In t h i s experim ent, n in e c u ltu re s were p rep ared and in o c u la te d w ith a sm all amount o f a h e te ro g e n e o u s c u ltu r e t h a t h ad been m a in ta in e d f o r 6-8 t r a n s f e r s on FM 3422c o n ta in in g m edia. Three o f th e s e c u l t u r e s w ere grown in th e ab sen ce o f FM 3422 and e x t r a c t e d , th r e e were grown in th e p re s e n c e o f e m u ls ifie d FM 3422 and e x tr a c te d , and th r e e w ere e x tr a c te d p r i o r t o c e l l grow th and im m ed iately a f t e r FM 3422 a d d itio n . The e th y l a c e ta te phase was removed from a l l c u ltu r e s . F ifty -o n e , s i x ty - o n e , and f i f t y - s i x p e r c e n t o f th e i n i t i a l l y e x t r a c t a b l e FM 3422 co u ld n o t b e e x t r a c t e d from th e th r e e c u ltu r e s t h a t had b een grown in th e p re s e n c e o f FM 3422. Less th a n 0 .1 ppm o f F was p r e s e n t in any o f th e n in e c u l t u r e s . Had a l l o f th e n o n e x tr a c ta b le FM 3422 been d e g ra d ed in th e c u l t u r e grown w ith FM 3422 w ith com plete F io n r e l e a s e to th e m edia, ^40 ppm w ould have been found. These r e s u l t s in d ic a te t h a t , as e x p e c te d , th e p e r f l u o r i n a t e d p o r ti o n o f th e FM 3422 m o lecu les i s n o t m etabolically attacked. 006378 -12 - A ttem pts to F ree N o n e x tra c ta b le FM 3422 by H y d ro ly s is A p o s s ib le e sq jla n a tio n f o r th e o b s e r v a tio n t h a t 50% o r more o f th e i n i t i a l l y e x t r a c t a b l e FM 3422 f r e q u e n tly c a n n o t b e e x t r a c t e d i n t o e th y l a c e ta te a f t e r incubation w ith acclim ated m icrobial c u ltu re s is th a t i t has been conjugated o r polym erized form ing a n o n e x tra c ta b le m a te ria l. The o b je c tiv e o f th is e x p e ri ment was t o d e te rm in e i f a c id o r b a s e h y d r o ly s is c o u ld c o n v e rt FM 3422 b ack to an e x tra c ta b le form. In t h i s ex p erim en t, c u ltu re s which had been grown in th e p resen ce o f e m u lsifie d FM 3422 on th e s ta n d a r d m edia w ere e x t r a c t e d w ith e th y l a c e t a t e . The c o n c e n tra t i o n o f FM 3422 in th e e t h y l a c e t a t e e x tr a c ^ was d e te rm in e d . The c u ltu r e s were th e n e x t r a c t e d a g a in a f t e r h y d r o ly s is a t 80C. and a t pH 1 o r pH 13 f o r v a rio u s le n g th s o f tim e . C o n c e n tra te d H^PO. was u s e d f o r a c i d i f i c a t i o n and 20 m o lar NaOH was u s e d t o in c r e a s e t h e pH; F o llo w in g h y d r o l y s i s , a l l sam ples w ere n e u tr a liz e d and a d jlis te d to eq u al volumes b e fo re e x tra c tio n a t room te m p e ratu re. The r e s u l t s o f t h i s e x p e rim e n t a re shown in T a b le 3. An a v erag e o f 28.4% o f th e FM 3422 was e x t r a c t a b l e b e f o r e c e l l u l a r g ro w th . Very l i t t l e o f th e re m a in in g FM 3422 c o u ld be e x t r a c t e d even a f t e r h y d r o l y s i s . In f a c t , i t a p p ea rs t h a t lo n g e r e x p o su re t o th e s e h y d r o ly s is c o n d itio n s d e c re a s e d th e amount o f FM 3422 th a t could be recovered in a second e x tra c tio n . TABLE 3 RECOVERY OF "NONEXTRACTABLE" FM 3422 FOLLOWING ACID OR BASE HYDROLYSIS AT 80 C Time a t pH . 80C 70 10 1 0.25 h r. 1 2.5 h r. 1 25 h r . 13 0 h r . 13 2 .5 h r . 13 25 h r . 00 % o f "N onextractable" FM 3422 R ecovered 2 .7 12 3.9 1.5 8.2 3.2 0.4 006379 - 13- M aterials Balance Experiments 14 B io d e g ra d a tio n s tu d ie s w ere co n d u cted u s in g C-FM 3422. The p rim a ry o b j e c t i v e o f th e s e ex p erim en ts was to d e term in e th e r e l a t i v e p o r ti o n s o f FM 3422 p r e s e n t in the aqueous, so lv e n t, and soj^ds phases o f e x tra c te d c u ltu re s a f t e r growth in th e p re s e n c e o f e m u ls ifie d C-FM 3422. In t h i s e x p e rim e n t, th e s ta n d a rd s m edia was p re p a re d b o th w ith and w ith o u t 14CFM 3422 (100 ppm). I b i s m edia was in o c u la te d w ith a h e te ro g e n e o u s c u l t u r e t h a t had been m a in ta in e d f o r a p p ro x im a te ly 20 t r a n s f e r s a t w eekly i n t e r v a l s on th e s ta n d a rd FM 3422 m edia. Three o f th e new c u l t u r e s c o n ta in in g 4C-FM 3422 were e x tra c te d immediately a f te r in o c u la tio n , and th re e id e n tic a l c u ltu re s were e x tr a c te d a f t e r one week o f grow th. T h r e j .c u l t u r e s n o t c o n ta in in g FM 3422 w ere a ls o grown f o r one week. At t h i s p o i n t , AX-FM 3422 em ulsion was added t o th e s e c u ltu r e s , b rin g in g t h e i r c o n c e n tra tio n to 100 ppm. The c u ltu r e s w ere th e n shaken fo r an a d d itio n a l fif te e n m inutes and e x tra c te d . Three one-ml sam ples from th e aqueous and e th y l a c e ta te phases were taken from each o f th e n in e e x tra c te d c u ltu r e s , added to Aquas ol'- * in s c i n t i l l a t i o n v i a l s , and c o u n te d . The s o l i d s from th e grown c u ltu r e s ( e s s e n t i a l l y no s o l i d s w ere p r e s e n t i n i t i a l l y ) were tra n s fe rre d to combustion cups, d rie d in an oven a t 103 C. f p r two hours and com busted u s in g A grichem 's P ackard com bustion e q u ip m e n t. The 14C0- 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 hM ine and counted. Samples were counted w ith an in te r n a l stan d a rd .a u e n ch c o r r e c tio n , and t h e i r r a d i o a c t i v i t y compared to known volume sam ples o f i 4C-FM 3422 em u lsio n added d ire c tly to Aquasol. T his ex p erim en t was conducted two tim e s . In th e f i r s t a tte m p t, th e 14C-FM 3422 e m u lsij^ was in a d v e rte n tly n o t s ta b i liz e d w ith A lconox, and i t broke r a p id ly . A lso , C-FM 3422 em ulsion was n o t added to th e c u l t u r e s t h a t h a d b een grown b e f o r e FM 3422 a d d itio n b e c a u se th e w eek -o ld u n s t a b i l i z e d em u lsio n had a lr e a d y broken. The r e s u l t s o f th e f i r s t attem p t a t t h i s exp erim en t a r e shown in T ab les 4 and 5. Ethyl acetate W ater S o lid s T otal Recovered C ulture E x trac te d In itially 103%11% 0.04%0.008% 103%11% C ulture E xtracted A f te r 1 Week 102%6% 0. 02%0. 02% 0. 3%.4% 102%6.4 T able 4. P e rc e n t 1 S ta n d a rd D e v ia tio n o f i n i t i a l l y added 14C-FM 3422 re c o v e re d in f i r s t of 2 M aterials Balance experim ents. 0063S0 -14- C ulture E x trac te d In itially C ulture E xtracted A fter 1 Week Method o f D e term in in g FM 3422 C o n c e n tra tio n E le c tro n C a p tu re GLC S c in tilla tio n Counting E x trac t C o n cen tratio n % o f Added M aterial Accounted For. E x trac t C o n cen tratio n % o f Added M aterial Accounted For. 91 ppm4.2 ppm 82% 83 ppm+5.0 ppm 75% 115 ppm+12 ppm 103% 113 ppm7 ppm 102% T a b le 5. C o n c e n tra tio n o f FM 3422 i n e t h y l a c e t a t e e x t r a c t s + 1 s ta n d a rd s d e v ia tio n from the f i r s t M a te ria ls B alance experim ent as determ ined by s c i n t i l l a t i o n c o u n tin g and by e l e c t r o n c a p tu r e GLC, The r e s u l t s o f th e second run o f th e M a te ria ls B alance ex p erim en t a r e shown in T ab les 6 and T. C ulture E x tra c te d In itially C ulture E xtracted A fter 1 Week Grown C u ltu re s E xtracted A fter F ifte e n Minutes Ethyl acetate 89.7+3.6 39.9+9.8 33.6+3.0 W ater 4 .0 + 1 .3 0 .4 + 0 .3 1. 0+0.4 S o lid s -- 3 .9 + 6 .6 0 .1 + 0 .0 '5 ' T o tal Recovered 9 3 .7 + 4 .9 4 4 .2 + 1 6 .7 34.7+3.4 T ab le 6. P e rc e n t one s ta n d a r d d e v ia t io n o f i n i t i a l l y added 14C-FM 3422 reco v ered in second o f two M a te ria ls B alance ex p erim en ts. 4 Method o f D eterm in in g FM 3422 C o n c e n tra tio n E le c tro n C ap tu re GLC S c i n t i l l a t i o n C o unting C u ltu re E x tr a c te d I n i t i a l l y 9 9 .8 ppm+0.7 ppm C u ltu re E x tr a c te d A f te r 1 Wk. 4 4 .0 ppm+8.7 ppm 9 9 .6 ppm+4.0 ppm 4 4 .3 ppm10.8 ppm Grown C u ltu re E x tra c te d A f te r 15 M inutes 3 1 .1 ppm+2.7 ppm 3 7 .3 ppm3.3 ppm T a b le 7. C o n c e n tra tio n o f FM 3422 in e th y l a c e t a t e e x t r a c t s from th e second M aterials Balance experim ent as determ ined by s c in t illa tio n counting and by e le c tr o n c a p tu r e GLC. 006381 -15- The r e s u l t s o f th e se experim ents were v a lu a b le d e s p ite th e f a c t th a t com plete r e c o v e r ie s o f FM 3422 were n o t o b ta in e d . In th e f i r s t a tte m p t, i t was found t h a t when FM 3422 was added as an u n s ta b i liz e d em u lsio n , th e a b i l i t y to e x t r a c t t h i s m a te r ia l was n o t l o s t a f t e r 7 days o f sh ak in g . We have o b se rv e d t h a t u n s t a b i l i z e d em ulsions b re a k w ith in a few h o u rs on s i t t i n g w ith th e p r e c i p i t a t i o n o f c o a g u la te d clumps o f FM 3422. T his presum ably happened i n t h i s experim ent as w e ll, and in th is form, i t remains e x tra c ta b le d e sp ite co n tact w ith a viable b io lo g ical cu ltu re. I t is possible th a t su rfactan t f a c ilita te s th e u p ta k e <J.f FM 3422. However, i t i s more l i k e l y t h a t e m u ls if ic a tio n in c r e a s e s th e number o f FM 3422 m o le c u les c o n ta c tin g and, s u b s e q u e n tly , b in d in g w ith m icrobial so lid s. As n o te d ab o v e, a l l o f th e 14C-FM 3422 was n o t acco u n ted f o r in th e second a tte m p t a t t h i s e x p e rim e n t. As i s seen in T able 6, most o f t h i s m a te r ia l c o u ld be reco v ered i n i t i a l l y , b u t i t was n o t recovered a f t e r s ig n if ic a n t b io lo g ic a l s o lid s had accum ulated. The most p ro b ab le ex p lan atio n fo r th is lo ss o f ra d io a c t i v e FM 3422 i s t h a t i t was ab so rb ed by th e c e l l u l a r m a te r ia l and bound so s tro n g ly th a t i t could n o t be e x tra c te d by e th y l a c e ta te . However, i t a p p a re n tly was n o t so s tro n g ly bound J h a t i t could n o t be v o la tiliz e d from th e c e l l u l a r m a te r ia l by h e a tin g to 103 C. Loss t o v o l a t i l i z a t i o n i s th e most p ro b a b le e x p la n a tio n f o r th e low re c o v e rie s . T h is e x p e rim e n t a ls o s u g g e s ts t h a t a b s o r p tio n i s a p h y s ic a l phenomenon. FM 3422 was a ls o n o t e x tr a c ta b le from th e pregrown c u ltu re s th a t were shaken w ith e m u ls if ie d FM 3422 f o r o n ly 15 m in u te s. T his r a t e o f rem oval i s to o r a p id to be ex p lain ed by a m etabolic phenomenon, p a rtic u la rly sin ce the organisms had n o t b een a c c lim a te d to FM 3422. I t c a n ' t b e d eterm in ed from t h i s e x p erim en t w h e th e r o r n o t th e FM 3422 which a s presum ably bound to th e c e l l s i s e v e n tu a lly m etab o lized to an o th er chem ical form. This would re q u ire recovery o f th e bound FM 3422 and c o n firm a tio n by a method such as GLC t h a t th e re c o v e re d m a te r ia l i s s t i l l FM 3422. A dsorption on C ulture C ontainers v A p o s s i b l e e x p la n a tio n f o r th e d e c re a se i n e x tr a c ta b le FM 3422 in grown c u l t u r e s i s t h a t th e m a te ria l was b ein g s tro n g ly bound to th e polypropylene t e s t c u ltu r e c o n ta in e r s . I b i s p o s s i b i l i t y was co n sid ered rem ote s in c e s im ila r r e s u l t s were o b tain ed in both g la ss and polypropylene c o n ta in e rs. N e v erth ele ss, i t was in v e stig ate d in the follow ing procedures; A m edia was p re p a re d t h a t d i f f e r e d from th e s ta n d a rd 133 mg/1 FM 3422 em u lsio n m edia o n ly i n t h a t i t d id n o t c o n ta in th e n u t r i e n t YM b r o th n e c e s s a ry as a n itr o g e n s o u rc e f o r c e l l grow th. Ten ml o f t h i s media was added t o each o f n in e tu b e s . Three were e x tra c te d im m ediately, th re e were capped w ith polypropylene caps, and t h r e e w ere p lu g g ed w ith foam s to p p e r s . Tbe capped and s to p p e re d tu b e s w ere shaken f o r one week and e x tr a c te d and a n aly z e d by GLC u s in g th e s ta n d a rd procedures. 006382 -16- Sample I #1 I #2 I #3 P #1 P #2 P #3 F #1 F #2 F #3 Cap P o ly p ro p y le n e Polypropylene Polypropylene Polypropylene Polypropylene Polypropylene Foam Foam Foam Time in P o ly p ro p y len e Tube P rio r to E xtraction 15 min. 15 m in. 15 m in. 7 Days 7 Days 7 Days 7 Days 7 Days 7 Days % I n i t i a l l y Added FM 3422 R ecovered 79 79 78 75 78 84 92 93 92 T a b le 8. P e rc e n t o f i n i t i a l l y added FM 3422 e x t r a c t e d from p o ly p ro p y le n e tu b e s . The r e s u l t s o f t h i s e x p e rim e n t a r e shown in T a b le 8. The re a s o n s f o r th e low FM 3422 r e c o v e r ie s o b ta in e d i n t h i s ex p erim en t a r e n o t known. There a lso is no ex planation fo r the g re a te r reco v eries obtained w ith th e tu b es th a t were shaken w ith foam s to p p e rs. In th e w eek-long shaking p ro c e d u r e , th e c o n te n ts o f th e tu b e s n e v e r came i n c o n ta c t w ith e i t h e r th e p o ly p ro p y le n e cap o r th e foam s to p p e r. A lso, th e foam sto p p e rs had n o t been used b efo re in any experim ents. D e s p ite t h e in c o m p le te re c o v e ry o f FM 3422, th e s e r e s u l t s do n o t e x p la in th e phenomenon rep e ate d ly seen in e a r l i e r experim ents. In e a rly experim ents, high r e c o v e r i e s o f FM 3422 w ere o b ta in e d i n i t i a l l y b e f o r e th e c e l l c u l t u r e h a d d e v e lo p e d and low r e c o v e r ie s o f FM 3422 were o b ta in e d in e th y l a c e t a t e e x t r a c t s done a f t e r th e c u ltu re had grown f o r seven d ay s. In th is experim ent w ith n e g l i g i b l e m ic ro b ia l p o p u la tio n s , th e i n i t i a l 1FM 3422 r e c o v e r ie s were n o t s ig n if ic a n tly d if f e r e n t from th e re c o v e rie s a f te r 7 days. I t , th e re fo re , a p p e a rs t h a t th e in c r e a s in g lo s s o f a b i l i t y t o e x t r a c t FM 3422 from th e c u ltu re w ith tim e req u ires the presence of b io lo g ic a l s o lid s. R ecovery o f FM 3422 From C e lls By V o l a t i l i z a t i o n and C o n d en satio n As p r e v io u s ly n o te d , th e m ost p ro b a b le e x p la n a tio n f o r th e lo s s o f FM 3422 in th e M a te ria ls Balance experim ent is th a t i t v o la tiliz e d from th e s o lid s phase d u rin g th e two h o u rs d ry in g p e rio d . For t h i s re a so n , i t was b e lie v e d t h a t i t m ig h t b e p o s s i b l e to q u a n t i t a t i v e l y re c o v e r so rb e d FM 3422 by v o l a t i l i z i n g i t from c e l l s and c o l l e c t i n g i t by c o n d e n sa tio n on a " c o ld f i n g e r ." 006383 -17- F ig u re 3. A p p aratu s used t o r e c o v e r FM 3422 from c e l l s by v o l a t i l i z a t i o n and condensation. 006384 r -18-. The f e a s i b i l i t y o f t h i s approach was e v a lu a te d by th e fo llo w in g ex p erim en t which was run in d u p l i c a t e . FM 3422 (1 mg) was p la c e d in th e b a se o f a la r g e t e s t tu b e . A second s m a lle r t e s t tu b e was p la c e d in s id e th e f i r s t , and was e f f e c t i v e l y made in to a c o ld f i n g e r by c o n tin u o u s ly ru n n in g w a te r i n t o i t s base and a s p ir a tin g i t from th e to p . The b ase o f t h is ap p aratu s was then p la c e d in a steam b a th (F ig u re 3 ). A f te r 15 m in u te s , th e c o ld f i n g e r was removed and th e s u b lim a te d FM 3422 washed from i t s s u r f a c e i n t o a known volume o f e th y l a c e t a t e . GLC a n a ly s is in d i c a t e d t h a t 32% and 42% o f th e o r i g i n a l l y added FM 3422 was s u b lim a te d and c o l l e c t e d in two t r i a l s . The above su b lim a tio n p ro c e d u re was th e n re p e a te d in d u p lic a te u s in g w eek-old10 ml c e l l c u l t u r e s in th e s ta n d a r d m edia. The s m a ll inoculum f o r th e s e c u l t u r e s came from a h e te ro g e n e o u s FM 3422 a c c lim a te d c u l t u r e . These c u l t u r e s w ere e x tra c te d w ith equal volumes o f e th y l a c e ta te (10 m l), and th e liq u id phases removed. The u n d ried biom ass was tr a n s f e r r e d to th e o u te r tu b e o f th e aboved e s c rib e d a p p a ra tu s and exposed to th e steam b a th f o r 45 m in u te s. The e th y l a c e t a t e w ashings from th e c o ld f i n g e r w ere a n a ly z e d by GLC, b u t v e ry l i t t l e o f th e n o n e x tra c te d FM 3422 was re c o v e re d . The c e l l s w ere d r ie d u n d e r n itr o g e n and again exposed in th e steam b a th f o r an a d d itio n a l two h o u rs . The r e s u l t s o f t h i s e x p e rim e n t a re shown in T ab le 9 . The FM 3422 t h a t was n o t e x tra c ta b le in to e th y l a c e ta te and presum ably bound to th e c e l l s , was n o t q u a n tita tiv e ly recovered by sublim ation and condensation. Perhaps com plete recovery would have been p o s sib le had th e c e lls been exposed to th e steam b ath fo r a longer p e rio d . Although th is experim ent d id n o t ru le out th e p o s s ib ility t h a t some o f th e bound FM 3422 may have been c h e m ic a lly m o d ifie d by m e ta b o lism , i t d id in d i c a t e t h a t a la r g e f r a c t i o n o f th e bound FM 3422 was unchanged. C u ltu re #1 C u ltu re #2 E xtracted in to Ethyl A cetate 15.6% 2 1 .0% V o la tiliz e d from "Wet" C ells 0.5% 0.3% V o la tiliz e d from ^ D ried C ells 25.6% 2 1 .8% T o ta l FM 3422 R ecovered 41.7% 43.1% T ab le 9 . P e rc e n t o f i n i t i a l l y added e m u ls if ie d FM 3422 re c o v e re d from c e l l s by e th y l a c e ta te e x tra c tio n follow ed by v o la tiliz a tio n and c o n d e n s a tio n o f re m a in in g FM 3422. Hot Ethyl A cetate E xtracts In an a tte m p t to in c r e a s e th e re c o v e ry o f th e FM 3422 s o rb e d to th e c u l t u r e biom ass, e x tra c tio n s were done using h o t eth y l a c e ta te . This procedure involved a d d itio n o f 2 .5 ml o f FM 3422 em ulsion to w e e k -o ld , 10 ml c u l t u r e s t h a t had b een grown on n u t r i e n t m edia. FM 3422 em u lsio n was a l s o added t o tw o, 10 ml d e io n iz e d w a te r sam p les. The r e s u l t i n g 12 .5 ml sam p les, w hich c o n ta in e d 133 ppm FM 3422, w ere shaken f o r 15 m inutes on a r o t a r y s h a k e r to a llo w s o r p tio n o f th e FM 3422 by th e biom ass, and e x tra c te d w ith equal volumes o f e th y l a c e ta te a t room tem p eratu re o r a t 40 C. o r 60 C. Phase s e p a r a tio n was in s u r e d by c e n t r i f u g a t i o n a t 12,000 rpm and th e c o n c e n tr a tio n o f FM 3422 in th e e th y l a c e t a t e p h ase was d e te rm in e d by gas chrom atography. -19- The r e s u l t s o f th is e x tr a c tio n p rocedure a re shown in T able 10. Aqueous Phase Sample Composition E xtraction _ T em perature ( C) ppm FM 3422 in E th y l A cetate E x tract 1 D eionized Water 22 115 2 D eionized Water 22 133 3 M icrobial C ulture 22 52 4 22 55 5 40 35 6 40 31 7 60 51 8 60 56 Table 10. Hot ethyl a c e ta te e x tra c ts of w ater o r b io lo g ic a l c u ltu re s a f te r 15 m inutes o f sh ak in g w ith e m u ls ifie d FM 3422 (133 ppm). These r e s u lts in d ic a te th a t h o t e th y l a c e ta te e x tra c tio n does n o t improve th e e x t r a c t a b i l i t y o f FM 3422 from th e b io m ass. The amount o f FM 3422 e x t r a c t e d a t 60 C. i s n o -h ig h e r th a n t h a t e x tr a c te d a t 22 C. Hie re a s o n f o r th e low er c o n c e n tr a tio n o f FM 3422 in th e 40 C. e x t r a c t i s n o t known. The r e s u l t s a ls o c o n firm th e p re v io u s o b s e rv a tio n t h a t e m u ls ifie d FM 3422 i s a b so rb e d by c e l l s w ith in 15 m in u te s . T h is r a p id lo s s from a n o n a c c lim a te d c u l t u r e s u g g e s ts a physical/chem ical absorption process ra th e r than b io lo g ic a l degradation. D ioxane E x tr a c tio n o f FM 3422 from C u ltu re S o lid s In a p r e lim in a r y e x p erim en t, i t was found t h a t a l l o f th e FM 3422 n o t e x tr a c te d by e th y l a c e ta te could be recovered by re flu x in g th e c u ltu re so lid s w ith 1,4-dioxane. A ttem pts to re p e at th is observ atio n were i n i t i a l l y hindered by problem s w ith the e le c tro n capture d e te c to r o f our gas chrom atograph. These problems were ap p aren tly caused by th e presence o f dioxane. C en tral Research overcame th is a n a ly tic a l problem and th e experim ent was re p e a te d . The r e s u l t s (T able 11) d em o n strate q u ite c o n c lu s iv e ly t h a t FM 3422 i s n o t m e ta b o lic a lly a l t e r e d in sev en days b y sh ak e f l a s k c u l t u r e s t h a t have been m a in ta in e d w ith w eekly t r a n s f e r s on FM 3422- c o n ta in in g media fo r over s ix months. 006386 - -20- C ulture E x trac te d w ith Ethyl A cetate a t: 15 min. 15 m in. 15 m in. 7 days 7 days 7 days 7 days 7 days % o f FM 3422 E x trac te d By E th y l A cetate 99.3 99.3 101.3 25.8 27.1 31.1 30.4 29.6 Procedure fo r E xtracting C ulture S o lid s % FM 3422 Recovered From S o lid s No s o l i d s p r e s e n t -- No s o l i d s p r e s e n t -- No s o l i d s p r e s e n t -- (R efluxed in Dioxane 77.9 for 2 h rs.) If It 80.1 (D ried so lid s w ith Nj refluxed in Dioxane fo r 2 h r s .) 75.0 (R efluxed in Dioxane 73.5 fo r 1 m in.) (E xtracted w ith D ioxane a t Room T em perature) 68 Total % Recovery o f FM 3422 99.3 99.3 101.3 103.7 107.2 106.1 103.9 97.6 T ab le 11. R ecovery o f FM 3422 from c u l t u r e s o l i d s by e x t r a c t i n g o r r e f lu x in g w ith Dioxane. REFERENCES (1) R e in e r, E. A ., B io d e g ra d a tio n S tu d ie s o f F lu o ro c a rb o n s , 3M T e c h n ic a l R ep o rt, August 12, 1976. i (2) M endel, A ., A n a ly tic a l M ethodology on FM 3422, 3M T e c h n ic a l R e p o rt, November 15, 1977. (3) D ickson, L. S. L iu , M .S c ., Ph.D. ( B r i t i s h C o lu m b ia), " B io d e g ra d a tio n , An E n v iro n m en tal S o lu tio n to Some T oxic O rg an ic Compounds," E n v iro n m en tal C o n s e rv a tio n , V ol. 3, No. 2 , Summer 1976. EAR/cen 006387