Document 4QyMNNEmMgQGd9zq0EYQ8yY9a

mm TECHNICAL REPORT SUMMARY TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN *"/ 7 ^ 4 /im portant- a| i f report is printed on both sides o f paper, send two copies to T C C J ^ Data 7/19/78 f r]|jf naiTiAi '` ; : !'Llv !ML *&hrMon ; Environmental Laboratory (EE & PC) Project . Fate of Fluorochemi cals in the Environment '.TUportViaa ; Biodegradation Studies of Fluorocarbons - III TT. bept.Numbar 0535 ProtectNumbar 9970612613 RaportNumbar - '5 Authorial ,- EmployaaNumbarta) .- WotabookRafaranea 44703, p. 6-14, 21, 25-27, 2 , 35, 39-43, 46; No.ofPagetIncludingCovanhaet . 45727, p. 32-35; 49400. p. 11-12. 17 Stfe^cHu Br IiTt Vy ^ ^ OpBfl BO vlOMQ (Company Confidential) (Spada) Authorization) 3M CH EM ICA L R EG ISTR Y ^ . New Chamioal* Raportad Yaa ] No KEYW ORDS: (Sa)act tarmt from 3M Thesauru*. Suggatt othar applicabla tarmt.) (Biodegradation) EE & PC-Div. Envir. Assess. Fluorochemical CU RREN T O B JECTIV E: To evaluate the susceptibilities to microbial decomposition. of i FC-143 Degradation REPO R T A BSTRA CT: (200-260 words) This abstract information is distributad by tha Tachnical Communications Cantar alart 3M'ari to Company R&D. It it Company confidantial matarial. A biodegradation study is described which allows the evaluation of the susceptibility of 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 treatment 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. Information Liaiton . Initial: s 2 :6 m 6 n n r OtviN iciaQ ii'-*A 1 -- 3M CONFIDENTIAL b d im m or o'utO>o cooois SUMMARY Fluorochemicals FC-143 were shown to be conpletely resistant to biodegradation in a 2-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 ment systems (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 fluorochem icals. Test cultures also contained or FC-143. Phenol and 1-dodecene-derived linear alkyl sulfonate (LAS) were used as reference compounds. Their degradation demonstrated that biode gradation could occur under the test conditions. All cultures were transferred 15 times over the 2J-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. Methylated and nonmethylated culture extracts separated by GLC were detected by electron capture. No degradation products were detected. Scintillation counting showed that all radioactivity associated with the labeled fluoro chemicals 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. EID108681 rO ooo* uu>> c00019 * ft -3 ^ f ;|:, U U iv> INTRODUCTION The fluorochemicals selected for this study, FC-143 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 condi tions 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 fluorocarbons. GLK000934 EID108682 C00020 METHODS AND MATERIALS Chemicals FC-143, , ammonium octanoate (the hydrogen analog of FC-143), carbon--14 labeled FC-- 143, and 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 1 CONTROL MEDIUM COMPOSITION 1) Basal salts solutions: 1.0 g/1 - n h 4c i 2.0 g/1 - K 2HP04 0.25 g/1 - MgS04 .7H20 0.002 g/1 - FeS04 *7H20 2) Well water - 25 ml/1 3) Yeast extract - 0.3 g/1 4) Hydrogen analogs of either or FC-143 - 20 mg/1 GLK000935 Media were prepared from stock solutions which were combined and brought to volume just prior to each culture transfer. A fresh solution of FeS04 7H20 was prepared and dry yeast extract was used in media preparation^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-montb period prior to the initiation of this study showed its calcium hardness to range from 92 to 144 mg/1 expressed CaCO. Any precip itate resulting from the addition of well water was removed by filtration through a #54 Whatman filter. EID108683 000021 The purified hydrogen analogs of 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 defluor ination reactions are frequently identical to enzymes involved in carbon-hydrogen bond cleavage (1). Additional components of other specific media are listed in TABLE 2. Media TABLE 2 GROWTH MEDIA FORMULATIONS FC-143 Test Phenol Controls LAS Controls Fluoride Controls FC-143 Control Medium + 50 mg/1 FC-143 FC-143 Control Medium + 30 mg/1 Phenol c FC-143 Control Medium + 30 mg/1 Standard Linear Alkylbenzenesulfonate (LAS) FC-143 Control Medium + 33.2 mg/1 NaF (15.0 mg/1 F" ) 14C-FC-143 Test FC-143 Control Medium + 50 mg/1 14C-FC-143 FC-143 + LAS FC-143 Control Medium + 30 mg/1 LAS + 50 mg/1 FC-143 Culturing Procedures 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 Commission's Metro plant in Saint Paul. Minnesota, and the Chemolite Waste Treatment Plant in Cottage Grove, Minnesota. EID108684 GLK000936 000022 - 6 - A llowing inoculation, the cultures in polypropylene Erlentneyer asks were shaken at 200 rpm on rotary shakers at room temperature (4 the end of each growth period, each culture was transferred to entical fresh media using a 1% inoculum from the preceding culture .e., 0.5 ml of existing culture to 49.5 ml of identical new medium). e growth period between transfers varied as is noted in TABLE 3. 10 ml sample was taken from each culture at 10 minutes after oculation or culture transfer and at the end of each growth period, mples were centrifuged for 10 min. at 17,000 x g prior to analysis the centrifugate. Deviations from this culturing procedure are ted in TABLE 3. U e final growth period differed from preceding periods. Media were : epared with Carbon-14 FC-143. One hundred ml ltures were grown in flasks on a rotary shaker in a growth chamber . strolled at 25 C. + 1. Twenty ml samples were taken at 10 min., ; tdays and at 7 days. epical Analysis ..uoride ion concentrations were measured using a fluoride ion .ectrode (Orion ion analyzer fluoride electrode model 96-09), ;d a standard curve drawn from the results of measurements of itsaxately prepared fluoride standards. The concentrations of lesse fluoride standards bracketed the concentrations present in e experimental samples. Fluoride curves were set up at each yspling period, except for transfer 1. For the analyses following ii transfer, a 1.0 ppm fluoride standard was used to calibrate *e instrument with the assumption that the slope of the previous Inertde curve remained constant. ` : ieol analysis was done according to Standard Methods for the anipation of Water and Wastewater. 14th Edition, 1975. Linear i ikflbenzenesulfonate (LAS) was analyzed for by the methylene blue, chloroform extraction method described in the 14th edition of S aadard Methods (3), except in transfers 8-14, LAS was analyzed by a modification pf this method. In this modified method, the staples 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 cm 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. E ID 108685 o r1 oo * SuO> -4 C00023 7 r':r t\'T A rt TABLE 3 SUMMARY OF CULTURING PROCEDURES USED IN THE SHAKE FLASK BIODEGRADATION STUDY OF FC-143 Culture Growth Transfer # Period (days) Notes 0 3 Used activated sludge inoculum from Metro and Chemolite. . 1 3 FC-143-hydrogen analog added to 143 cultures and controls. . 24 3 3 At the time of culture transfer, 1 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. 53 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 i ml of Metro sludge supernatant was added to all cultures. 96 10 4 ' 11 4 In this and subsequent growth periods, cultures were grown in a reciprocating shaker~water bath at 100 strokes per min. and 25 C. 12 1134 15 6 68 +6 (2 ) 7 78 EID108686 days Total EnrichmentPeriod GLK000938 00024 -8- U W m iA L Carbon 14 Counting Techniques Scintillation counting was performed on 1 ml samples of culture centrifugate added to AquasolVIW, and counted with an internal standard quench correction. The radioactivity of these samples was compared to known weight samples of C-FC-- 143 added directly to Aquasol. Solid samples were collected directly onto millipore HA 0.45 ym filters conposed of cellulose acetate and cellulose nitrate. The filters were then washed with deionized.water and placed into paper combustion cones, wet with Combustaidv and.combusted in Agrichem's Packard^11' combustion equipment. The CO resulting from combustion was trapped in a scintillation fluid containing an organic amine and counted in Agrichem's Packard scintillation counter. Samples were recounted with an internal standard for quench correction. Thin-Layer Chromatography (TLC) Thin-layer chromotography was.gerformed to detect radioactive metabolites of *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 ether mixture. (Some samples which evaporated to dryness in air before spotting were resuspended in methanol.) The resuspended samples were spotted on . Merck silica gel GF25 4 * Small spots of solids residue were also applied directly to.fnese plates. -,11 samples were referenced against a mixture of C-FC-143 ; 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. GLK000939 EID108687 C00025 -9 - Lr!- *' i^.' GLK000940 Gas-Liquid Chromatography (GLC) Ethyl acetate extracts were prepared as described in the thin- layer chromatography methods. Control solutions were made by dissolving 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. Uethylated samples were injected within 3 hrs. of their metbylatlon. The chromatographic column was 12 ft. x 1/8" O.D. stainless steel packed with 20% DC 200 (12,500 CS) on 10% Bentone 34 and 70% 80/90 mesh Anakrom P . A . . The injection port temperature was 250 C., and the detector temperature 300C. The column temperature was programmed to hold for 4 min. at 75 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 pi aliquot of a 1 yg/ml C9Fi9C00H solution, as a reference compound to each sample. Diazomethane 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 EID108688 Fluoride Release In all but the final growth period, degradation 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 fluorocbemical portions o/ these molecules were degraded, fluoride ion would accumulate in the media. To ensure that fluoride was not lost from the culture by absorption, precip itation or volatilization, control cultures were grown with 15 mg/1 of fluoride. This fluoride concentration is approximately what would result if 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 o f '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. C 00026 -1 0 - r . i r : TABLE 4a INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLUORIDE SUPPLEMENTED CONTROLS MEASURED BY SPECIFIC ION ELECTRODE AT THE TIME OF TRANSFER FC-143 Transfer # Fluoride Control Initial Final 0 20 21 1 21 20 2 18 21 3 25 16.5 4 16 17.3 TABLE 4b . INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FLUORIDE SUPPLEMENTED CONTROLS MEASURED BY SPECIFIC ION ELECTRODE MEASURED COLLECTIVELY AT END OF STUDY Transfer # 0 1 2 3 4 5 - FC- 143 Fluoride Control Initial Final 15.7 15.7 14.5 16.4 15.7 15.0 17.0 15.0 16.4 15.6 17.0 16.4 GLK000941 EID108689 G 00Q 27 I -1 1 - UU TABLE 5 INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FC-143 -CONTAINING CULTURES AND OF NONSUPPLEMENTED 143 CONTROL CULTURES Transfer # 0 1 2 3 (5) 4 5 6 7 8 9 10 11 12 13 14 . FC-143 Test Init. Final 143 Control Init. Final <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 .83 1 .81 1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.56 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 HOV <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 EID 108690 000028 -1 2 - 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/1 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/1, 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. . _. . 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 to 4-6 days, and temperature was raised from room temperature (<20 to 22C) to a constant temperature of 25C. Results of LAS degradation in the final growth period are shown in TABLE 8. In the growth periods following transfers 11 and 12, an experiment was done to determine if 50 mg/1 *~ or FC-143 inhibited the degradation of LAS. These results are shown in TABLE 9. GLK000943 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. EID108691 000029 -1 3 - u i. In the final growth period, 50 mg/1 of carbon 14-labeled _ and FC-143 were used as test substrates in place of the nonlabeled fluorochemicals. 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. Never theless, the remaining data show that the radioactivity associated with FC-143 remained in solution during the entire 7-day degradation test period.' Analysis of the 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 SUPPLEMENTED CONTROLS AND % LAS REMOVED Transfer # ' i 143 - Surfactant Control LAS Init. Final % Removal 4 35.5 29.5 19.4 5 32.8 25.8 21.2 6 27.0 24.0 7.0 7 25.0 30.6 -2.0 8 37.0 35.8 11.1 9 38.9 13.7 94.6 10 42.7 12.8 89.7 11 39 13.7 93.8 12 41.3 19.7 77.9 13 41.3 18.0 88.3 14 40.3 13.7 90.7 EID108692 GLK000944 GOO03Q -1 4 - TABLE 7 CONCENTRATION OF METHYLENE BLUE ACTIVE SUBSTANCE (mg/1) IN NONSUPPLEMENTED CONTROLS Transfer # 4 5 6 7 8 9 10 11 12 13 14 143 - 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 Initial Day 2 Day 7 % LAS Removal EID108693 GLK000945 FC-143 Controls #1 LAS #2 LAS Non- Suppl. S u p p l. SuPPl 34.0 8.0 6.3 36.7 22.3 6.3 6.5 5.3 4.0 9 1 *6 92.3 - C00031 15- TABLE 9 EFFECT OF FC-143 ON THE BIODEGRADATION OF LAS ANALYZED FOR AS NBAS lM'. Transfer # 11 12 FC-143 + LAS Culture . las Init. Final Removalf8 *1 53.7 27.3 53.7 34.0 97.8 71.4 TABLE 10 CONCENTRATION OF 14C-FC-143 IN THE CENTRIFUGATE OF TEST CULTURE DURING THE FINAL GROWTH PERIOD Init. Day 2 Day 7 14C-FC-143 Cultures Standard"- Concentration Deviation 46.2 mg/1 48.0 49.7 0.9 mg/1 0.3 0.4 GLK000946 EID108694 CO0032 16 Thin-layer chromatography did not reveal the presence of radio active metabolic products of either FC-143 Likewise, gas liquid chromatography of the same culture extracts, both before and after mthylation, 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 bio degradation of these fluorochemicals occurred. REFERENCES AND FOOTNOTES (1) Goldman, Peter, Enzymology of Carbon-Halogen Bonds. Degradation of Synthetic Organic Molecules in the Biosphere. Nat. Aca(i. of Sci'.,' Washington, DC (1972). . (2) There was a six-day period before the onset of the final growth period during which the test cultures were shaken at 25 C. in the presence of FC-143. (3) Standard Methods for the Examination of Water and Wastewater, 14th Edition, American Public health Association (9t5$. (4) Daytime temperatures were observed to range between 20 and 22 F. Night temperatures were not measured during that part of the study in which cultures were shaken at ambient temp erature .(see TABLE 3). However, measurement made near the termination of this 2-month study, in January, showed that nighttime temperature frequently drops to 17 C. (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 Chemists1 Society. 42:986 (1966). "' 31 (7) Percent LAS removal was calculated as: (MBASgj - MBASCI) - (MBASgp - MBAScp) Where : EID108695 MBASSI * The initial methylene blue active substances (MBAS) &A concentration of the surfactant supplemented culture. MBAS- = The initial MBAS concentration of the nonsupplemented UA control (TABLE 7). MBASs f * The final MBAS concentration of the surfactant supplemented culture. MBASrT * The final MBAS concentration of the nonsupplemented CA control (TABLE 7). 000033 GLK000947 -1 7 - U (7) The percent LAS Removal was calculated as: (MBASgipj -- MBAS^j) - (MBASg^p -- MBAS^p) % Removal (MBASgj - MBASCI) X 100 Where: MBASo TT - The initial methylene blue active substances (MBAS) 55,11 concentration of the culture supplemented by both LAS surfactant and FC-143. MBASm The initial MBAS concentration of the nonsupC1 piemental control (TABLE 7). MBAS,,TP * The final MBAS concentration of cultures bl* supplemented with surfactant and fluorocarbon. MBAS-,., The final MBAS concentration of the nonsupplemental w control (TABLE 7). MBASer The initial MBAS concentration of the surfactant supplemental culture (TABLE 6). It was assumed that MBAS concentration due to FC-143 was not reduced by the biodegradation or other loss of these compounds. EAR/cen GLK000948 EID108696 e c o o a i.