Document em1BnpdaVp89gq1ELEG2wGY9g

BIODEGRADATION (ABS/LAS Shake CultureTest) TEST SUBSTANCE Identity:Perfluorooctanoaiccid,ammonium saltm;ay alsobe referretdo as PFOA ammonium saltA,mmonium perfluorooctanoatPeF,O, FC-116,FC-126,FC-169,orFC-143. (Octanoicacid, pentadecafluoro-a,mmonium saltC,AS # 3825-26-1) Remarks: The testsubstanceisa whitepowder. The 3M productionlot number was 83.The testsample isreferretdoby thetesting laboratorays FC-143. The puritoyfthesample was not sufficientclhyaracterizeda,lthoughcurrentinformatioinndicates itisa mixture-o9f6.5- 100% testsubstanceand 0 - 3.5% C6, C7, and C9 perfluoraonaloguecompounds. METHOD Methods: Shake Culturestudymodeled aftertheSoap and Detergent Association'psresumptivetestforthe determinatioonfABS/LAS biodegradability. Type: Aerobic GLP: No Year completed: 1978 Contact time (units):2.5months lnnoculum:Activatesdludgecollecteadtthe 3M ChemoliteFacility, Cottage Grove, MN, 3M DecaturFacilitDye,caturAL, and Metro Wastewater TreatmentPlant,St Paul,MN RESULTS No biodegradatiownas observedinthe2.5month shake culture biodegradatiosntudy. DATA QUALITY ReliabilitKyl:imischranking= 2. Thisstudymeets allcriterifaorquality testingb,uttheanalyticamlethodology isquestionable.The purityofthe sample was notsufficientclhyaracterized. REFERENCES Fate ofFluorochemicalsintheEnvironment,Projectnumber 9970612613, E.A.Reiner,July19,1978, 3M Company, EnvironmentalLaboratory. OTHER Submitter: 3M Company, EnvironmentalLaboratoryP,.O. Box 33331, St.Paul,Minnesota,55133 Last changed: 5/25/00 3mForm 6747-1 I-A TO: TECHNICAL TECHNICAL REPORT COMMUNICATIONS CENTEft - 201-2CN SUMMARY r@7/19/7@8 tl'nPOnwt - IfMPOrt iSPrintedon bo6 $;do$of~, land two coi*s to TCC.) Divlwon Environmental fto)oct Laboratory (EE & PC) Fwpon Tiiw Fate of Fluorochemicals-in the Environment Biodegradation Studies of Fluorocarbons TO D. L. Bacon C4& 4@.E. A. Reiner W*ub-00k",Bforome 44703, p. 6-14,- 2-1. 25-27, 29, 45727t P- 32-35; 49400, p. 11-12. BECURITYIO- Ooon lffCIOND 3?ACHEMICAL REGISTRY KEYWORDS: (Selewcatn from3m Thowrus.Sugpn other awksme (Biodegradation) CURRENT OBJECTIVE: To evaluate the susceptibilities to microbial decomposition. EE & PC-Div. Envir. Assess. Fluorochemical Degradation Usp-t. Numbw 0535 PraiecNtumaw 9970612613 ReportNumwr- 46; FmPlOVesNumboRs7- 47816 r4a. in Pqps incaucunrgove;wee-t 17 Now ChemkoisAmoorted 13 ya jo No of FC-95 and FC-143 REPORTABSTRACT1:2DO-2wSoOrdsT)hiasbsftivnaforffwitscifiogntribubtYetd heTechniccawlnmunicgtCigonntsgtro &Wt3M'wtoConv" R&C). A biodegradation study is described which allows the evaluation of the susceptibility of FC-95 and FC-143 to aerobic microbial degradation. The culturing pro- cedures used in this study are modeled after the Soap and Detergent Association's presumptive (shake culture) test for the determination of ABS/LAS biodegradability. Microbial inocula were obtained from activated sludge collected at Chemolite, Decatur and Metro waste treat"nt plants. Analytical procedures included GLC, TLC, C-scintillation counting and analysis for released fluoride. Degradation of reference compounds demonstrated the suitability of the biodegradation test conditions. informaLtiiaoinson initiolo: -2- SUMMARY Fluorochemicals FC-95 and FC-143 were shown to be completely resistant to biodegradation in a 21-month shake culture biodegrada- tion study. The mixed microbial test cultures used in this study were derived from activated sludge inocula obtained from three waste treat.%--ntsystems (Chemolite, Decatur, & the Twin Cities Metro plant). The cultures were maintained in dilute yeast extract-basal salts media supplemented with the hydrogen analog of the respective fluorochemicals. Test cultures also contained FC-95 or FC-143. Phenol and 1-dodecene-derived linear alkyl sulfonate (LAS) were used as reference compounds. Their degradation demonstrated that biodegradation could occur under the test conditions. All cultures were transferred 15 t@mes over the 2*-month period, and temperature was controlled at 25 C.-during the latter half of the experiment. In the final growth period, degradation products of 14 C-labeled fluorochemicals were assayed for by thin-layer chromatography (TLC) and gas liquid chromatography (GLC). Chemicals separated by TLC were visualized by TLC-autoradiograph. Metbylated and nonmethylated culture extracts separated by OLC were detected by electron capture. No degradation products were detected. Scintillation counting showed that all radioactivity associated with the labeled fluorochemicals remained in the culture medium. In all but the final growth period, fluorocarbon biodegradation was monitored simply by measuring the initial and final fluoride concentration in the media. No increase in fluoride concentration was observed indicating that if biodegradation did occur, it did not result in the release of fluoride. Control cultures supplemented with fluoride showed that fluoride is not lost from the media under the experimental conditions used. While this study cannot rule out the possibility that conditions could be found that would allow the biodegradation of these compounds, the results of this study suggest that these chemicals are likely to persist in the environment for extended periods unaltered by microbial catabolism. -3- INTRODUCTION The fluorochemicals selected for this study, FC-143 and FC-95, have perfluorinated carbon chains and are chemically stable. The perfluorinated portion of fluorocarbons have not been found to be susceptible to biological degradation (1). Therefore, biodegradation studies were conducted on these compounds primarily for the sake of completeness. Without such testing, it could not be said with certainty that these compounds would resist microbial modification. Since biodegradation was unlikely, the best feasible test conditions for biodegradation were selected. Inocula were obtained from areas considered likely to contain acclimated microorganisms. Long acclimation periods-were used in an attempt to select and develop populations of microbes capable of degrading these compounds, and hydrogen analogs of the fluorocarbons were added to try to select organisms that might gratuitously "cometabolize" the fIuorocarbons . -4- METHODS AND MATERIALS Chemicals FC-95, FC-143, the hydrogen analog of FC-95, ammonium octanoate (the hydrogen analog of FC-143), carbon-14 labeled FC-143, and carbon-14 labeled FC-95 were obtained'from Commercial Chemicals Division. These chemicals were used as received unless designated otherwise (Arthur Mendel-Report in Progress). Standard linear alkylate sulfonate prepared for use as a reference compound for biodegradation studies was obtained from the US/EPA Laboratory in Cincinnati, Ohio. Except where noted, all other compounds were reagent grade. Culture Media The control medium used in these studies had the composition shown in TABLE 1. TABLE I CONTROL MEDIUM COMPOSITION 1) Basal salts solutions: 1.0 g/l - NH ci 4 2.0 g/l - K HPO 24 0.25 g/l MGSO 4* 7H 2 0 0.002 g/l FESO 7H 0 4' 2 2) Well water 25 ml/l 3) Yeast extract - 0.3 g/l 4) Hydrogen analogs of either FC-95 or FC-143 - 20 mg/l Media were prepared from stock solutions which were combined and brought to volume just prior to each culture transfer. A fresh solution of FESO 7H 2 0 was prepared and dry yeast extract was used in media preparati*on at each transfer. The pH of all media was adjusted to 7.5 with 1.0 N HC1 and if overshot adjusted back with 1.0 N NAOH. The well water was added to insure an adequate supply of trace elements. Analyses of the well water made during the 12-month period prior to the initiation of this study showed its calcium hardness to range from 92 to 144 mg/l expressed CACO Any precip- itate resulting from the addition of well water was i;moved by filtration through a #54 Whatman filter. -5- The purified hydrogen analogs of FC-95 and FC-143 were used in biodegradation test media and controls. These compounds were included in an attempt to select a microbial population likely to degrade the fluorocarbons. Enzymes capable of catalyzing defluorination reactions are frequently identical to enzymes involved in carbon-hydrogen bond cleavage (1). Additional components of other specific media are listed in TABLE 2. GROWTH TABLE 2 MEDIA FORUMATIONS Media FC-95 FC-143 Phenol Test Controls LAS Controls Fluoride Controls 14 C-FC-95 Test 14 C-FC-143 Test FC-95 + LAS FC-143 + LAS Culturin_g Procedures Components FC-95 Control Medium + 50 mg/l FC-95 FC-143 Control Medium + 50 mg/l FC-143 FC-95 or FC-143 Control Medium + 30 mg/i Phenol FC-95 or FC-143 Control Medium + 30 mg/l Standard Linear Alkylbenzenesulfonate (LAS) FC-95 or FC-143 Control Medium + 33.2 mg/l NaF (15.0 mg/l F-) FC-95 Control Medium + 50 mg/l 14 C-FC-95 FC-143 Control Medium + 50 mg/l 14 C-FC-143 FC-95 Control Medium + 30 mg/l LAS + 50 mg/l FC-95 FC-143 Control Medium + 30 mg/l LAS + 50 mg/l FC-143 The initial growth period was started by inoculating 49 ml of each medium with 1 ml of activated sludge supernatant. The activated sludge used was a mixture of two sludges collected on the day of inoculation. The sludge was obtained from the Metropolitan Waste Control Conmission's Metro plant in Saint Paul, Minnesota, and the Chemolite Waste Treatment Plant in Cottage Grove, Minnesota. -6- Following inoculation, the cultures in polypropylene Erlenmeyer flasks were shaken at 200 rpm on rotary shakers at room temperature (4). At the end of each growth period, each culture was transferred to identical fresh media using a 1% inoculum from the preceding culture (i.e., 0.5 ml of existing culture to 49.5 ml of identical new medium). The growth period between transfers varied as is noted in TABLE 3. A 10 ml sample was taken from each culture at 10 minutes after inoculation or culture transfer and at the end of each growth period. Samples were centrifuged for 10 min. at 17,000 x g prior to analysis of the centrifugate. Deviations from this culturing procedure are noted in TABLE 3. The final growth per-iod differed from preceding periods. Media were prepared with Carbon-14- labeled FC-95 and FC-143. One hundred ml cultures were grswn in flasks on a rotary shaker in a growth chamber controlled at 25 C. + 1. Twenty ml samples were taken at 10 min., 2 days and at 7 days.- Chemical Analysis Fluoride ion concentrations were measured using a fluoride ion electrode (Orion ion analyzer fluoride electrode model 96-09), and a standard curve drawn from the results of measurements of accurately prepared fluoride standards. The concentrations of these fluoride standards bracketed the concentrations present in the experimental samples. Fluoride curves were set up at each sampling period, except for transfer 1. For the analyses following this transfer, a 1.0 ppm fluoride standard was used to calibrate the instrument with the assumption that the slope of the previous fluoride curve remained constant. Phenol analysis was done according to Standard Methods for the Examination of Water and Wastewater, 14th Edition, 1975. Linear alkyl nzenesulfonate (LAS) was analyzed for by the methylene blue, chloroform extraction method described in the 14th edition of Standard Methods (3), except in transfers 8-14, LAS was analyzed by a modification of this method. In this modified method, the samples was diluted to 100 ml in a separatory funnel. Also added to the separatory funnel were 25 ml of Standard Methods methylene blue solution and 100 ml of chloroform. This mixture was shaken for 30 seconds, allowed to settle, swirled, and the chloroform drawn off through glass wool into a 2.5 em diameter, spec 20 curvette. Percent transmittance was read at 652 nm and compared to a standard curve prepared with surfactant samples of known concentration treated in the same manner. -7- TABL,E 3 SUMMARY OF CULTURING PROCEDURES IN THE SHAKE FLASK BIODEGRADATION FC-95 AND FC-143 USED STUDY OF Transfer 0 1 2 Culture Growth Period (days) 3 3 4- Notes Used activated sludge inoculum from Metro and Chemolite. FC-143-hydrogen analog added to 143 cultures and controls. 3 3 At the time of culture transfer, I ml of Decatur sludge supernatant added to cultures. 4 3 LAS replaced phenol as a reference compound. LAS media was inoculated with a mixture of control culture and Chemolite and Decatur sludge supernatant. 5 3 6 3 The use of fluoride control was discontinued. 7 6 Shaker was inadvertently turned off,- possibly for 5 days, during this growth period. 8 3 ml of Metro sludge supernatant was added to all cultures. 9 6 10 4 11 4 In this and subsequent growth periods, cultures were grown in a reciprocating shaker-wateg bath at 100 strokes per min. and 25 C. 12 6 13 6 14 8 - +6 (2) 15 7 78 days Total Enrichment Period Carbon 14 Counting Techniques Scintillation counting was pe rmed on 1 ml samples of culture centrifugate added to Aquasol . and Counted with an internal standard quench correction. The radioac 14vity of thelt samples was compared to known weight samples of C-FC-95 or C-FC-143 added directly to Aquasol. Solid samples were collected directly 04to millipore HA 0.45 pm filters composed of cellulose acetate and cellulose nitrate. The filters were then washed with deioniz water and placed into paper combustion con wet with Combustaid zg) , and ombusttd in Agri- chem's Packard fl)combustion equipment. The CO resulting from combustion was trapped in a scintillation fluid egntaining an organic amine and countgd in Agrichem's Packard scintillation .counter. Samples were recounted with an internal standard for quench correction. Thin-Layer Chromatography (TLC) Thin-layer chrotqtography was,Rerformed to detect radioactive metabolites of C-FC-95 and C-FC-143. Ten ml culture samples were collected and immediately frozen. These samples were stored frozen for about 1 month. The samples were extracted immediately after thawing with 10 ml of ethyl acetate. The samples were then centrifuged at 17 ,000 x g to ensure the separation of the ethyl acetate, water, and solids phases. The water phase and portions of the ethyl acetate phase were evaporated to dryness under N The dried samples were resuspended in a 9:1 hexane:ethyl ethei mixture. (Some samples which evaporated to dryness in air before spotting were resuspended in methanol.) The resuspended samples were spotted on E. Merck silica gel GF Small spots of solids residue were also applied directly to,JRSLe plates. ill samples were referenced against a mixture of C-FC-143 and C-FC-95. The Plates were developed with 10% ethanol in ethyl acetate and visualized by exposing Kodak no-screen x-ray film on the plates for one week. TLC was repeated on the remaining portion of the solvent samples. The solvent was allowed to evaporate to dryness in air, and the residue resuspended in methanol. These plates were spotted more heavily, developed as before, and visualized with x-ray film for 2 weeks. -9- Gas-Liquid Chromatography (GLC) Ethyl acetate extracts were prepared as described in the thinlayer chromllography meth2ts. Control solutions were made by dissolving C-FC-95 and C-FC-143 in ethyl acetate. Portions of the ethyl acetate extract samples and the ethyl acetate control solutionswere also methylated. Aliquots of the methylated and nonmethylated ethyl acetate extracts and controls were injected onto the 5713 Hewlett Packard gas chromatograph with electron capture detector. Methylated samples were injected within 3 hrs. of their methylat.ion. The chromatographic column was 12 ft. x 1/811 O.D. stainless steel packed with 20% DC 200 (12,500 CB) on 10% Bentone 34 and ZO% SO/90 mesh Anakrom P.A. The injection port temperature was 250 C., and the detector temperature 300u S. The column temperasure was Brogrammed to hold for 4 min. at 35 C., to rise to 180 C. at 8 C. per min., and to hold at 180 C. The flow rate was adjusted to 35 ml/min. of Argon/methane, 95/5. Methylations were performed by adding a 20 ml aliquot of a I ug/ml C F19COOH solution, as a reference compound to each sample. DYazomethane was then added until a yellow color persisted. The samples were then loosely capped, swirled and allowed to stand for 15 minutes. Nitrogen was blown over the samples until the yellow color disappeared, and the sample was returned to its original volume with ethyl acetate. RESULTS AND DISCUSSION Fluoride Release In all but the final growth period, degradation of FC-95 and FC-143 was monitored only by analysis of fluoride concentration at the beginning and end of each culture period. It was assumed that if the fluorochemical portions of these molecules were degraded, fluoride ion would accumulate in the media. To ensure that fluoride was not lost from the culture by absorption, precipitation or volatilization, control cultures were grown with 15 mg/l of fluoride. This fluoride concentration is approximately what would result if FC-95 or FC-143 underwent degradation with 50 percent fluoride release. The results of the fluoride analyses conducted on different days showed considerable variation. This was due to the variable and very sluggish response of the fluoride electrode. TABLE 4a shows the results obtained at each transfer TABLE 4b shows the results obtained when the same samples, which had been stored in polyethylene containers, were analyzed together after the termination of the experiment. Despite the variability due to the analytical technique, the results indicate that fluoride, if released to the media through biodegradation, would not be lost from the media. The results of the fluoride analysis on fluorocarbon-containing cultures and controls are shown in TABLE 5. The results show that no biodegradation with fluoride release occurred. -10- Transfer 0 1 2 3 4 TABLE 4a INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLUORIDE SUPPLEMENTED CONTROLS MEASURED BY SPECIFIC ION ELECTRODE AT THE TIME OF TRANSFER FC-95 Fluoride Control initi-al Fing@l- FC-143 Fluoride Control Initial ----Final 21 23 -20 2616.5 23 22 22 17.5 19.2 20 2.1 21 20 18 21 25 16.5 16 17.3 TABLE 4b INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLU* ORIDE SUPPLEMENTED CONTROLS MEASURED BY SPECIFIC ION ELECTRODE MEASURED COLLECTIVELY AT END OF STUDY Transfer 0 1 2 3 4 5 FC-95 Fluoride Control Initial Final 16'.4 15.6 15.6 16.2 15.6 19.3 @16.2 16.2 16.2 16.2 15.7 17.0 FC-143 Fluoride Control Initial --7'@l-na-1 15.7 15.7 14.5 16.4 15.7 15.0 17.0 15.0 16.4 15.6 17.0 16.4 TABLE 5 INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FC-143 AND FC-95-CONTAINING CULTURES AND OF NONSUPPLEMENTED 143 CONTROL CULTURES 95 AND Transfer # 0 1 FC-95 TestInit. Final 0.46, 0 .51 0.50 0.46- 2 0.42 3 (5) 1.75 0.66 1.6 4 0.73 0.71 5 0.72 0.78 6 0.73 0.8 7 0.14 0.17 a 0.90 0.84 9 0.84 0.73 10 0.72 0.81 11 0.81 0.80 12 0.74 0.73 13 0.73 0.84 14 0.81 0.78 95 Control Init. Final FC-143 Test Init. Final 143 ControlInit. Final 0.31 0.33 <0.1 <0.1 <0.1 <0.1 0.36 0.36 <0.1 <0.1 <0.1 <0.1 0.34 0.56. <0.1 .<O.l <0.1 <0.1 1.75 1.5 .83 1 .81 1 0.68 0.60 <O.l <0.1 <0.1 <0.1 0.61 0.68 <0.1 <0.1 <0.1 0.56 0.63 0.70 eO.I <0. 1 <0. I <0. 1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.66 0.66 <0.1 <0.1 <0.1 <0.1 0.72 0.60 <0.-l <0.1 <0.1 <0.1 0.60 0.68 <0.1 <0.1 <0.1 <0.1 0.69 0.62 <0.1 <0.1 <0.1 <0.1 0.66 0.64 0.62 0.66 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.66 0.64 <0.1 <0.1 <0.1 <0.1 -12- Reference Compounds Reference compounds were used to demonstrate that the biodegradation test conditions used were suitable to degrade compounds known to be somewhat resistant to degradation. In the first four growth periods, 30 mg/l phenol was added to two cultures which were identical to the test cultures, except that they lacked fluorocarbons. Analytical problems prevented the measurement of phenol concentration during the first three growth periods. In the fourth growth period, phenol was found to degrade to less than 1.3 mg/l, the limit of sensitivity of the method as applied. This demonstrated that the test conditions were suitable for the biodegradation of phenol. In the fifth through final growth periods, reference linear alkyl sulfonate (LAS) was used as the reference compound. This compound is a standard reference material used in the Soap and Detergent Association's biodegradation test method for anionic surfactants(6). This material is considered to be relatively easily degraded. In the Soap and Detergent Association's shake flask biodegradation test, the results are considered invalid if the removal of 1-dodecenederived LAS is not nearly complete. The data showing the extent of degradation of LAS in surfactant supplemented controls are depicted in TABLE 6. The data showing the equivalent amount of methylene blue active substances in the controls not supplemented with LAS are depicted in TABLE 7. Little LAS degradation occurred during the first few adaptive transfers. Three transfers were required before the majority of the LAS began to degrade in the surfactant supplemented control for FC-95. Five transfers were required for LAS degradation in the 143 control. Therefore, it appeared that organisms capable of degrading 1-dodecene- derived LAS were not initially present in sufficient numbers for LAS degradation. The test condition allowed for enrichment of these organisms, but enrichment occurred at a slower rate than had been anticipated. Consequently, changes were made in the procedure to increase the rate and likelihood of acclimating organisms capable of degrading the fluorochemicals. Growth periods were extended from 3 to0 4-6 dags, and temperature was raised from room temperature (<20 to 22 C) to a constant temperature of 250C. Results of LAS degradation in the final growth period are shown in TABLE S. In the growth periods following transfers 11 and 12, an experiment was done to determine if 50 mg/l of FC-95 or FC-143 inhibited the degradation of LAS. These results are shown in TABLE 9. FC-95 appears to have an inhibiting effect on the microbial degradation of LAS. However, its presence was not completely inhibitory. Comparison with TABLES 8 and 6 shows-that the presence of 50 mg/l of FC-95 inhibited LAS degradation by 18% and 23% during these two test periods. On the other hand, within the limits of the precision of our method, FC-143 did not appear to have a significant effect on LAS degradation. -13- In the final growth period, 50 mg/l of carbon 14-labeled FC-95 and FC-143 were used as test substrates in place of the nonlabeled fluorochemicals. Both FC-95 and FC-143 cultures were prepared in triplicate. The concentrations of the radioactive fluorocarbons present in the aqueous phase as determined by scintillation counting are shown in TABLE 10. The initial FC-95 concentration is much lower than expected. This low value could have rectulted from a systematic error in the collection of the initial FC-95 samples. It is also possible that FC-95 had not completely dissolved in the cultures when the first sample was taken, but this seems unlikely, since the initial values for FC-95 concentration from all 3 parallel cultures were almost identical (30.3, 29,8 and 30.4 mg/1). Nevertheless, the remaining data show that the radioactivity associated with FC-95 and FC-143 remained in solution during the entire 7-day degradation test-period.' Anil@dfi;'df. th6 biological solids showed some binding of radioactive material, but the -vast majority remained in the liquid phase. TABLE 6 CONCENTRATION OF LAS (mg/1) IN SUPPLE)MTED CONTROLS AND % LAS REWVED Transfer 4 5 6 7 8 9 10 11 12 13 14 95 - Surfactant Control 143 - Surfactant Control 5@s@ Init. Final % Removal(7) Init. Final LAS Removal 31.5 26.8 18.4 35.5 29.5 19.4 28.3 27.5 0.1 32.8 25.8 21.2 29.8 25.0 '25.5 12.0 15.5 91.1 27.0 25.0 24.0 30.6 7.0 -2.0 31.2 33.0 3.75 3.17 89.8 95.1 37.0 38.9 35.8 13.7 11.1 94.6 32.7 2.33 95.9 42.7 12.8 89.7 31.0 2.0 95.0 39 13.7 93.8 31.3 2.33 96.5 41.3 19.7 77.9 31.7 2.5 31.3 3.0 93.5 92.8 41.3 40.3 18.0 13.7 88.3 90.7 -14- TABLE 7 CONCENTRATION OF METHYLENE BLUE ACTIVE SUBSTANCE (mg/1) IN NONSUPPLEMENTED CONTROLS Transfer 4 5 6 7 8 9 10 11 12 13 14 95 - Control Init, Final 1.0 1.9 1.15 .38 0.50 .75 5.2@@ 10.2 3.0 0.88 5.75 1.83 4.17 1.17 4.33 0.67 i.o 1.33 3.67 0.67 3.67 1.0 143 - Control Init. Final 4.5 4.5 4.5 3.5 7.1 5.50 5.0 10.2 9.0 10.9 10.9 12.2 12.3 9.67 11.5 12.0 12.3 13.3 11.3 14.5 14.5 11.3 TABLE 8 CONCENTRATION OF MBAS (mg/1) IN SURFACTANT SUPPLEMENTED AND NONSUPPLEMENTED CONTROLS DURING FINAL GROWTH PERIOD Time FC-95 Controls #1 LAS #2 LAS Non- Suppl., Suppl. Suppl. Initial 28.7 29.3 1.0 Day 2 13.0 26.0 1.0 Day 7 1.67 2.0 .7 % LAS 96.5 95.4 - Removal (7) - FC-143-Controls #1 LAS #2 LAS Suppl. Suppl. 34.0 8.0 6.3 36.7 22.3 6.3 Nonsuppl. 6.5 5.3 4.0 91.6 92.3 - -15- TABLE 9 EFFECT OF FC-95 AND FC-143 ON THE BIODEGRADATION OF LAS ANALYZED FOR AS MBAS Transfer 11 12 -FC-95 Init. + LAS Culture ck EAS- Final Removal( 58.0 -34.7 64'.3 40ka-3 73.6 78.9 FC-143 + LAS Culture % LAS Init. Final Remoyal(8) 53.7 53.7 27.3 34.0 97.8 71.4 TABLE .10 CONCENTRATION THE CENTRIFUGATE OF 14 C-FC-95 OR 14 C-FC-143 IN OF TEST CULTURE DURING THE FINAL GROWTH PERIOD Init. Day 2 Day 7 14 C-FC-95 Cultures Concentration Standard Deviation 30.1 mg/l 52.8 53.5 0.3 mg/l 0.5 3.1 14 C-FC-143 Cultures Concentration StandardDeviation 46.2 mg/l 48.0 49.7 0.9 mgll 0.3 0.4 Thin-layer chromatography did not reveal the presence of radioactive metabolic products of either FC-143 or FC-95. Likewise, gas liquid chromatography of the same culture extracts, both before and after methylation, showed no products that were not initially present or not also present in controls. From the combination of these results, it can be concluded that no biodegradation of these fluorochemicals occurred. REFERENCES AND FOOTNOTES (1)Goldman, Peter, Enzymology of Carbon-Halogen Bonds. Degradation of Synthetic Organic Molecules in the Biosphere, Nat. Acad. of Sci., Washington, DC (1972). (2)There was a six-da7yperiod before the onset of the final growth period during which the test cultures were shaken at 250 C. in the presence of FC-95 or FC-143. (3)Standard Methods for the Examination of Water and Wastewater, 14th Edition, Imerican IgublicHealth Associati n (:L975). (4)Dagtimetemperaturweesreobservetdo rangebetween20 and 22 F. Nighttemperaturweesrenotmeasuredduringthatpart of thestudyin whichculturewsereshakenat ambientemperature (see TABLE 3). However, measurement made near the termination of this 2*-month study, in Januagy showed that nighttime temperature frequently drops to 17 (5)At this transfer, Decatur sludge was added which contained a high fluoride concentration. (6)Subcommittee on Biodegradation Test Methods of the Soap and Detergent Association, A Procedure and Standards for the Determination of the Biodegradability of Alkyl Benzene Sulfonate and Linear Alkylate Sulfonate. J. of the American Oil Chemists' Society, 42:986 (1966). (7)Percent LAS removal was calculated as: Removal (MBAS si - MBASCI) - (MBASSF - MBASCF) m x 100 MBAS si - MBASCI Where: MBAS si MBAS ci The initial methylene blue active substances (MBAS) concentration of the surfactant supplemented culture. The initial MBAS concentration of the nonsupplemented control (TABLE 7). MBASSF '.The final MBAS concentration of the surfactant supplemented culture. MBAS ci @ The final MBAS concentration of the nonsupplemented control (TABLE 7). -17- (7) The percent LAS Removal was calculated as: IioRemoval (MBABSTI - MBASCI) - (MBABSTF (MBASSI - MBASCI) MBASCF) x 100 Where: MBABSTI The initial methylene b-lue active substances (--MBAS) concentration of the culture supplemented by both LAS surfactant and either FC-95 or FC-143. UBASCI The initial MBAS concentration of the nonsupplemental-control (TABLE 7). UBAS STF The final MBAS concentration of cultures supplemented with surfactant and fluorocarbon. MBAS cp The final MBAS concentration of the nonsupplemental control (TABLE 7). MBAS si The initial MBAS concentration of the surfactant supplemental culture (TABLE 6). It was assumed that MBAS concentration due to FC-95 or FC-143 was not reduced by the biodegradation or other loss of these compounds. BAR/cen