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BIODEGRADATION A R22-0057 FT--3 TEST SUBSTANCE_______________________________________________ Identity: Periluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) Rem arks field: The test substance is a white powder of uncharacterized purity. The following is a reduced overview of previously completed biodegradation studies. While these studies have concluded that 1Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-, potassium salt is persistent, new testing is underway to determine if a mechanism exists for the biological degradation of this substance. METHOD _______ _____________________________________________ Methods: Warburg Determination1; Shake Culture study modeled after the Soap and Detergent Association's presumptive test for the determination of ABS/LAS biodegradability2. Type: Aerobic GLP: No Year com pleted: 19761, 19782 C ontact tim e (units): 3 hours1, 2.5 months2 Innoculum : Activated Sludge RESULTS_____________________ __________________________________ No biodegradation was observed in either the Warburg study or the 2.5 month shake culture biodegradation study. DATA QUALITY___________________________________________________ Reliability: Klimisch ranking = 2 1. This study meets all criteria for quality testing, but analytical methodology is questionable. Klimisch ranking = 22. This study meets criteria for quality testing, but analytical methodology is questionable. No reference compounds or abiotic vessels were included in either of the studies. Footnotes: 1Warburg Determination 2Shake Culture study modeled after the Soap and Detergent Association's presumptive test for the determination of ABS/LAS biodegradability 000228 REFERENCES 1. Fate of Fluorochemicals in the Environment, Project number 75-639829, E.A. Reiner, August 12,1976, 3M Company, Environmental Laboratory. 2. Fate of Fluorochemicals in the Environment, Project number 9970612613, E.A. Reiner, July 19,1978, 3M Company, Environmental Laboratory. OTHER _________ __________________________________________ Subm itter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/3/00 000223 Far-67&-J T O :. T ech nical Communications Center, 207-25 310 TECHNICAL REPORT SUMMARY August 12. 197 Environmental________ LABORATORY, DEPT. NUMBER 0222 MICROFORM COPIES: T itle Biodegradation Studies of Fluorocarbons____________ ____ P raia : P ro je ct Number: R epart 1 Fate of Fluorochemicals in the Environmental To: R. L. Bohon .! (3 d ig it 75-6398-29 1 B y : Employee Number: E. A. Reiner 47816 . SE C U R ITY ' Company C onfid entia l(O po n) O bja ctlva: To determine the biodegradability of selected 3M fluorocarbon compounds. Notebook Reference: 40671 Pgs. 29-37, 41-50 JS pecial A uthorlzatlon(C losed) IF SUMMARY REPORT Has Inform ation In th is report been covered by other reports *0submitted to T C C ? E I j Partially | | Com pletely P le a s e keyword Information not included in other reports and give page numbers of new m aterial: 3M C H E M IC A L R E G IS TR Y 'ew chem icals reported? [UNo DYt. No. o f poges Including covorshi ABSTRACT and Conclusions. (System can accommodate 200-250 wards) 12 Biodegradation studies using a Warburg respirometer were conducted on FC-95, FM 3422, FC-128, and hydrogen analogs of FC-95 and FM 3422. No biodegradability was observed on FCr95, although an approximate hydrogen analog of FC-95 was readily'degradable. FM 3422 and FC-128 both were demonstrated to undergo some biodegradation. Attempts to isolate degradation products of FM 3422, from the Warburg studies, and from a subsequent activated sludge study were unsuccessful. KEYWORDS Select general, specific, and 3M product term s from 3M Thesaurus. Enclose suggested terms in parentheses. EE PC - Div. Envron - Assess Biology Bacteria Bioscreening Fluorochemical Biodegradable SP E CIFIC PROBLEMS remaining to reach objective. Continued attempts will be made to isolate and identify the biodegradation products of FM 3422 and other 3M fluoroeaxbons. Work with radioactivity labeled FM 3422 is being considered. In fo rm a tio n S c ie n tis t In iti al s 000230 BIODEGRADATION STUDIES OF FLUOROCARBONS SUMMARY AND RECOMMENDATION No biodegradation was observed in Warburg studies on FC 95. Biodegradation of FC 95 is improbable because it is completely fluorinated. The resistance of this compound to biodegradation by an acclimated microbial culture, however has not yet been demonstrated. * Warburg studies on FM 3422 and FC 128 both indicated that some biodegradation occurred. The products of this biodegradation are not known. Semi continuous activated sludge studies on FM 3422 did not confirm or disprove the Warburg findings. Future investigation"^ the biodegradability of the fluorocarbon compounds would be greatly facilitated by the development of an analytical procedure for FC 95. Warburg studies using purified FC 128 should be made to confirm the present findings. Studies on the biodegradability of FM 3422 were hihdered by its low water solubility. This problem could be overcome using FM 3422 radioactivity labeled on its hydrocarbon portion provided this material had a high specific activity 0 5 mci/m mole) and purity. Biodegradation of a saturated solution of the labeled compound could be measured by detecting 14C02 evolution. INTRODUCTION The susceptibility to microbial modification is an important parameter in the study of the environmental fate of any class of compounds. It is the most important form of degradation for organic compounds. A vast array of organic compounds can be completely degraded by microorganisms. So vast in fact that it was once believed by some that given enough time and the proper conditions, microorganisms could degrade any organic material. This doctrine of microbial infallibility is still a common misconception(1). PexfluoTinated compounds are extremely resistant to biodegradation (2) Although compounds with single fluorines have been shown to release fluoride ions as a result of biodegradation, perfluorinated compounds have rarely or never been shown to undergo natural degradation. For this reason, no modification of the perfluoro components of compounds in this study was anticipated. However, modification of its hydrocarbon components seemed possible. An understanding of the partial degradation products is important since the environment will be exposed to these products in addition' to the undegraded materials. METHODS AND MATERIALS Chemicals The chemicals used in these experiments are shown in Table I. 000231 -2 - ' TABLE I . CHEMICALS USED IN BIODEGRADATION EXPERIMENTS FM 3422 Hydrogen Analog of FM 3422 2H5 C8F17S02NC2H40H C2H5 c 8h 17s o 2n c 2h 4o h FC 95 C8f17S03K Sipex-ols FC 128 CgH170S03Na ?2H5 CgF17S02NCH2C00K They were obtained from Don Ricker of the Commercial Chemical Division in September, 1975. FM 3422 (N-et Fose alcohol) was identified as 788 CC 745-2. The FC.95 used was from lot 583. Lot numbers were not given for the FM 3422 hydrogen analog or the sipex-ols (RM 26442). These chemicals were selected for a number of reasons. FC 95 is essentially the fluorocarbon constituent of a large number of 3M fluorocarbon compounds. FM 3422 is an intermediate in the production of 3M fluorocarbons, and FC 128 is a finish fluorocarbon product. Sipex-ols and the Hydrogen Analog of FM 3422 were selected for comparison to the fluorocarbons. Sipex-ols is an approximate hydrogen analog of FC 95. ' These hydrogen analogs were tested because biologically labile fluorocarbons have frequently been found to be gratuitously defluorinated by enzymes which normally remove a hydrogen. Thus,- it seemed probable that microbial growth on hydrogen analogs could select populations of organisms which could more completely degrade fluorocarbons. WARBURG DETERMINATION Warburg studies were conducted according to the attached standard procedures. (Attachment) Microogranisms were collected from the mixed liquor of the Pigs Eye treatment system,washed, and suspended in a basal salts medium and used at a concentration of 2000 mg of biological solids per liter. Water insoluble substrates were emulsified in water prior to addition to the Warburg flasks. Emulsions were made using a Blackstone model EP-2 ultrasonic probe, base 1/2 inch, at 100% power. Logarithmic dilutions in water were made of the test substrates, and 1/2 ml was placed in the first side arm of the Warburg flasks. Controls contained 1/2 ml of 10 g/1 glucose solution or deionized water in this side arm. The second side arm contained either glucose or deionized water. 000232 -3 - Oxygen uptake was first observed in each flash for a period up to 1.5 hrs. with readings at 10-15 min. intervals to establish the endogenous activity. This was followed by addition of the first side arm and continued oxygen monitoring for approximately 2 hrs. Addition of the second side arm containing glucose, a readily degradable material, allowed a further evaluation of the toxicity of the previously added material. Semicontinuous Activated Sludge Studies A week-long semi continuous activated sludge (SCAS) study was conducted on FM 3422. The microorganisms used were obtained, as before, from,Pigs Eye Treatment Plant. One Hundred Fifty ml of activated sludge was added to 3 SCAS reactors and tap water as a control to a fourth. FM 3422 was added to 3 reactors below the water surface in 1/2 ml of absolute alcohol. Each addition increased the FM 3422 concentration by 33 mg/1. Pure ethanol was added to one sludge-containing reac.tor as a control. The operation of the semicontinuous reactors is shown in Figure 1. The SCAS reactors were aerated for 23 hrs. with 500 ml/min. of air while the contents of each reactor were stirred with a magnetic stirrer to prevent settling. After the aeration period, the sludge was settled for an hour and one liter of supernatant was replaced with primary effluent from the Pigs Eye Plant. FM 3422 was added at the beginning of the Test Cycles 1, 2, and 4. Samples were taken at the start and end of each test cycles and from the supernatant after settling. The aeration chambers used in the SCAS studies were plexiglass cylinders 13" high with a 4" internal diameter. A side arm allowed drainage of the supernatant leaving the 500 ml with the settled sludge undisturbed. Analytical Samples taken at the termination of the first Warburg study on FM 3422 were evaluated by thin layer chromatography (Central Research analytical work req. No. A59412). The samples were extracted into dichloromethane, dried to a small volume, and separated on Woelm silica plates. The developing solvent system was 10:90 ethanol, chloroform (V:V)v The developed plates were visualized by the iodine starch technique and compared to known standards with a detection limit of one yg of FM 3422. Samples for the SCAS study were extracted into n-octanol and separated by gas chromatography with an electron capture detector. Extractions were performed in capped 50 ml polypropylene centrifuge tubes and phases separated by centrifuging at 26,700XG for 10 minutes. RESULTS AND DISCUSSION Warburg - FM 3422 Results from the Warburg study on FM 3422 are summarized in Figure 2. This experiment was performed by first sonicating FM 3422 and its analog in water to make emulsions of approx. 24,000 mg/1 of the FM 3422 and 11,000 mg/1 of the FM 3422 analog. Since FM 3422 and its hydrogen analog are not very soluble in water, it was felt that forming an emulsion would put more of these compounds in contact with the microorganisms in the Warburg study. 000233 4- FIGURE 1: Test cycle for scmlcontlnuous activated sludge reactor. 000234 5 3_ 1- -5 FM 3422 yy s0 FM 3422 ^3000 mg, (16 ju mol< 0 j* 0 &* / / FM 3422 '''SOO mg/1 (1.6 m mol 0/ / f y --- f r f y F d 3422 Addition moles 0- uptake corrected for endogenous j -1 * : -2 _ Analog Addition \ V X glucose '`^addition 1. 0- 3 i....... ......... _ _ .4 1 '.i ' - d J ?t x r -j-- r L - - v .25 .5 .75 1.25 1/5^ 1.75 2.0 time hours FIGURE 2 : Warburg study of FM 3422 and its hydrogen analog: 000235 Hydrogen Analog v1#(>0 mg/1 - (,M u molosl -6 - While the FM 3422 analog was relatively easily emulsified and stable once emulsified, the FM 3422 was not. Approximately one hour was required to put 75% of the FM 3422 into emulsion, and this material proceeded, to slowly come back out of emulsion. In about two to three hours, excess FM 3422 emulsion,which had not been used in the experiment, turned into a semi solid gel. Complete chemical oxidation of the hydrocarbon component of the FM 3422 at the highest concentration (^16 ]x moles) would require 87 y moles of 02 based on the following equation: C 8F17S02NCC2H 5)C2H4OH + 5 `502 ^ C8F17S02NH2 + 4C02 + 4H2 Microbial oxidation rarely exceeds 60% of the chemical oxidation. In this experiment, only 2-3 micro moles of oxygen uptake was observed. However, oxygen uptake was^continuing at the end of this experiment. Addition of glucose to the FM 3422 culture also produced increased oxygen uptake, confirming that the FM 3422 emulsion was not inhibitory to the microbial culture. On the other hand, the hydrogen analog of FM 3422 showed significant toxicity. Upon addition of the most concentrated emulsion of the analog, endogenous oxygen uptake ceased and was not restored even after the addition of glucose to' the culture. The negative slope of the hydrogen analog's oxygen uptake curve (Figure '2) is due to the endogenous correction ana not oxygen evolution. Similar results were obtained in a second Warburg experiment with FM 3422 and its hydrogen analog. -Analysis of FM 3422 has shown it to be quite pure. The oxygen uptake ' observed was greater than would be expected from impurities in the compound. It is conceivable that sonication produced degradation products that were biodegradable, but not detectable by thin layer chromatography. It is also possible that some of the hydrocarbon components of FM 3422 molecule were degraded. However, using thin layer chromatography we were unable to detect any materials formed as a result of the biodegradation of FM 3422. It is not known if the hydrogen analog of FM 3422 itself is toxic. Thin layer and; gas chromatography showed this material to be impure. Gas chromatograph* showed the analog to be 90% pure with two major contaminants. The contaminants may have been the cause of the observed toxicity. SCAS - FM 3422 The semicontinuous activate sludge (SCAS) study was a second attenpt to isolate the hypothesized degradation products of FM 3422. This study was conducted over a period of 1 "week with samples taken at the initiation and end of each 24-hr. cycle. The FM 3422 samples added in an ethanol solution rapidly separated from the liquid phase, and as a result may have had too small a surface area to allow significant microbial degradation. n-octanol extracts of the samples were analyzed by gas chromatography. No new leaks were formed as a result of exposure of the FM 3422 to the microorganisms. If some of the FM 3422 had been degraded to the sulfonic acid, it would not have been detected. The sulfonic acid is not sufficiently volatile to pass through the gas chromatography column. dr performed by Commercial Chemicals Division 000236 -7 - The n-octanol extracts could not be separated by thin layer chromatography because of the low volatility of this solvent. Frozen nonextracted samples still exist at this date and could be extracted into a more volatile solvent for thin layer analysis. Three additions of FM 3422 in 33 ppm increments were made during the SCAS experiment. The FM 3422 settled with the solids and for the most part remained in the reactor when the supernatant was withdrawn. The final concentration (although not in solution) was approximately 100 mg/1. This material was not homogeneously distributed and accumulated on the sides of the reactors. WARBURG FC-95 The results of Warburg studies with FC-95 are graphed in Figure 3. No oxygen uptake was observed as a result of the addition of FC-95. This material also caused no toxic effects. Sipel-ol, an approximate hydrogen analog of FC-95, was shown to be readily biodegradable and to have no toxic effects. The Sipel-ols was soluble at all concentrations tested (as high as 1700 mg/1). FC-95 was incompletely soluble at 4000 mg/1, but was completely in solution at 400 mg/1. Hie lack of degradation with FC-95 was expected since perfluorinate compounds are characteristically nonbiodegradable. WARBURG FC-128 Oxygen uptake curves from Warburg studies on FC-128 as shown in Figure 4. These results indicate that FC-128 is readily biodegradable. Assuming biodegradation occurs as is shown below, approximately 70% of the theoretical maximum oxygen uptake occurred within the 7-hr. experimental period. This oxygen uptake is greater than expected and appeared to be continuing at the end of the experiment. These results are somewhat in question since this FC-128 is known to be an impure chemical. c 2h 5 CgF17S02NCH2C00K + 4.2502 + H -* CgF17S02NH2 + 4C02 + 3H20 + K 000237 -8 - 172 mg/l Sipex-ols ja. moles O2 uptake corrected for endogenous. FIGURE 3: Warburg study of FC 95 and Sipex-ols. 000238 '8 - -9 FC 128 ^r\ T T i n T i a i o a U l t-\t IIIU J .L J time hours FIGURE 4: Warburg study of FC 128. 000239 -10REFERENCES: (1) Alexander M . ; Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility. Adv. Appl. Microbial 7: 35-80, 1965. (2) Chapman, 1'. J.; Department of Biochemistry, University of Minnesota, St. Paul, Minnesota, Personal Communications 2/24/76. 000240 ATTACHMENT I STANDARD PROCEDURE FOR WARBURG DETERMINATIONS 7/10/75 E. A. Reiner 1. Design experiment and calculate concentrations of materials to add. 2. Fill water bath (DI water if left in bath). 3. Adjust temp, of bath (several hrs. or overnight). 4. Place manometers in desired order. 5. Prepare thermobarometer. Add about 3ml of 1^0 to 1 flask. 6. Set out glassware -in desired order (to match the manometer with which they were calibrated). 7. Lightly grease center-well top with stopcock grease that can be removed with solvent. Add 0.2 ml 10% KOH. B. Prepare samples in DI water (or according to request) to add to side arms. Keep refrigerated until used. 9. Prepare cells (keep cells cold at all times but avoid freezing). A. Centrifuge 0 C. B. Wash with cold BSM - centrifuge. Cl Resuspend in cold BSM. D. Determine concentration of an aliquot with the spectronic 20 at 600 nm. Adjust remainder to desired cone. C basic salts medium. Refrigerate until use. * E. Take sample of final adjusted sludge for standard MLSS analysis. 10. Add samples to side arms (usually 1 ml if one side arm, h ml to each side arm if. 2 side arms). 11. Add 2 ml of washed cells to flasks. 12. Add filter paper strip to alkali in center cup. 13. Attach flasks to the correct manometer. 14. Retighten flasks after about 5 min. shaking in bath. 15. Leave stopcock open to atmosphere, and let temp, adjust for an additional 10 min. 16. Adjust level in manometer to 150 with stopcock open (close stopcock). 17. Begin readings (always adjust closed arm of manometer to 150 mm before reading). 18. Add contents of side arms according to experiment design requirements. 000241 >' V. 19. Take readings periodically (on open arms) throughout course of experiment. 20. Disconnect and clean flasks. A. Rinse with water. B. Wash off grease with acetone. C. Acid wash. D. Rinse with DI Water. CC0242 3$0T3y1a technical report summary D a ta 7/19/78 TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN ( im p o rtan t - I f rep o rt sp rin ted on both sides o f paper, send two copies to TC C.I D ivision Environmental Laboratory (EE & PC) P roiact Fate of Fluorochemicals in the Environment R e p o rt Title Biodegradation Studies of Fluorocarbons - I I I To D. L. Bacon A uthor(s) j/f E. A. Reiner N ote book Reference 44703, p. 6-14, 21, 25-27, 2S , 35, 39-43, 46; 45727, p. 32-35; 49400, p. 13l- 1 2 . apt Number 0535 Project Number 9970612613 ! Report Number 5 E m ployee N um berls) 47816 No. of Pages In c lu d in g C oversheet 17 S EC U R IT Y ^ ^ ^ Open (Company Confidential) Closed special Authorization) 3M CHEMICAL ^ REGISTRY * New Chemicals Reported Yes No (Select terms from 3M -Thesaurus. Suggest othet applicable terms.) (Biodegradation) EE & PC-Div. Envir. Assess. Fluorochemical ^gradati on C U R R E N T OBJECTIVE: To evaluate the susceptibilities-oi FC-95 and FC-143 to microbial decomposition. R EP O R T ABS TRACT: (200-250 words) This abstract information is distributed by the Technical Communications Center to alert 3M'ers to Company R&O. 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 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. J Information Liaison . Initials: 000243 SUMMARY Fluorochemicals FC-95 and FC-143 resistant to biodegradation in a w2er-emonshtohwnshatkoebeculcotmuprleetbeiloydegrada 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 1i-cadlosd.eceTnees-tderciulvteudrelsineaalrsoalckoynltaisunlefdonFaCt-e95 or FC-143. (LAS) were Phenpl used as and reference compounds. Their degradation demonstrated that biode gradation could occur under the test conditions. All cultures were transferred 15 times over the 2i-month period, and temperature was controlled at 25 C. during the latter half of the experiment. In the final growth period, degradation products of 14C-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. 000244 INTRODUCTION The fluorochemicals selected for this study, PC-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 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 brganisms that might gratuitously "cometabolize" the fluorocarbons. 000245 -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 1 CONTROL MEDIUM COMPOSITION 1 ) Basal salts solutions: ) 1 . 0 g/ 1 - n h 4c i 2 .0 g/1 - K2HP04 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 FC-95 or FC-143 - 20 mg/1 Media were prepared from stock solutions which were combined and brought solution toofvFoelSum0e4 just '7H_0 prior to each culture transfer. A was prepared and dry yeast extract fresh 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-month 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 J filtration through a #54 Whatman filter. 000246 -5- Tbe 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 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. TABLE 2 GROWTH MEDIA FORMULATIONS Media Components FC-95 FC-143 Test FC-95 Control Medium + 50 mg/1 FC-95 FC-143 Control Medium + 50 mg/ 1 FC-143 Phenol Controls FC-95 or FC-143 Control Medium + 30 mg/1 Phenol LAS Controls FC-95 or FC-143 Control Medium + 30 mg/1 Standard Linear Alkyl- benzenesulfonate (LAS) ) Fluoride Controls FC-95 or FC-143 Control Medium + 33.2 mg/1 NaF (15.0 mg/1 F~) 14C-FC-95 Test FC-95 Control Medium + 50 mg/1 14C-FC-95 14C-FC-143 Test FC-95 + LAS FC-143 + LAS FC-143 Control Medium + 50 mg/1 14C-FC-143 FC-95 Control Medium + 30 mg/1 LAS + 50 mg/1 FC-95 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. J 000247 -6- Following inoculation, flasks were shaken at 2 0th0 e rpcmulotnurersotairny poslhyapkreorpsylaetneroEormletnemmepyeerrature (4 ) At the end identical forfesheacmhedigarowutshinpgeraio1d%, each culture inoculum from was the transferred to 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 period differed from preceding periods. Media were prepared with Carbon-14 labeled FC-95 and FC-143. One hundred ml cultures were grown 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, this transfer, a e1x. 0cepptpm for transfer 1. For the analyses following 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 alkylbenzenesulfonate (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 pf 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 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. 000248 -7- TABLE 3 SUMMARY OF CULTURING PROCEDURES USED IN THE SHAKE FLASK BIODEGRADATION STUDY OF FC-95 AND FC-143 Transfer # 0 1 2 3 Culture Growth Period (days) 3 3 4 s/ 3 Notes Used activated sludge inoculum from Metro and Chemolite. . FC-143-hydrogen analog added to 143 cultures and controls. 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. )5 '6 7 8 9 10 11 3 3 The use of fluoride control was discontinued. 6 Shaker was inadvertently turned off, possibly for 5 days, during this growth period. 3 ml of Metro sludge supernatant was added to all cultures. 6 4 4 In this and subsequent growth periods, csuhlatkuerre-swawteerge bagtrhownat in1 0 0a reciprocating strokes per min. and 25 C. 12 13 J 14 15 6 6 8 +6 ( 2 ) 7 78 days Total Enrichment Period 000249 Carbon 14 Counting Techniques Scintillation centrifugate acdoduedntitnogAwqausaspoelr'1formeadndoncou1nmtledswaimtphlesanofinctuelrtnualre standard quench correction. The radioactivity of these samples was compared to known weight samples of C-FC-95 or *C-FC-143 added directly to Aquasol. Solid samples were collected directly onto millipore HA 0.45 pm filters composed of cellulose acetate and cellulose nitrate. The filters were then washed with deionized.water and placed into paper combustion cones, wet with Combustaid' , andjcombusted in Agrichem's PackardtR? combustion equipment. The CO resulting from combustion was trapped in a scintillation fluid containing ah 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 chromatography was-gerformed 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 N9 . 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 E. Merck silica gel ^ 0 5 4 * Small spots of solids residue were also applied directly to-fnise plates. -.All samples were referenced against a mixture of A C-FC-143 and A*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 h2eawveieklsy., developed as before, and visualized with x-ray film for 000250 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-95 and C-FC-143 in ethyl acetate. Portions of the ethyl acetate extract samples and the ethyl acetate control solution^were 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 methylation. 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 to rise to 180 C. was at 8prCo.grpamemredmitno.,hoalndd for 4 min. to hold at at 75 C . , 180 C. The flow rate was adjusted to 35 ml/min. of Argon/methane, 95/5. MCe9tFh1y9lCaOtOiHonssolwuteiroen,perafsoramerdefbeyrenacdedincgompao2u0ndyltoaelaicqhuotsamopflea. 1 yg/ml Diazoraethane 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 voiume 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, precip itation or volatilization, control cultures were grown with 15 mg/1 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. 000251 .7. ' -10- ) 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 FC-143 Transfer # Fluoride Control Initial Final Fluoride Control Initial Final 0 21 23 1 23 22 2 20 22 20 21 21 20 18 21 3 26 17.5 25 16.5 4 16.5 19.2 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- 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 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 J 000252 TABLE 5 INITIAL AND FINAL FLUORIDE CONCENTRATION (mg/1) OF FC-143 AND FC-95-CONTAINING CULTURES AND OF NONSUPPLEMENTED 95 AND 143 CONTROL CULTURES FC-95 Test Transfer # Init. Final 1 2 .3 (5) 0.46v 0.51 0.50 0.42 1.75 0.46 0.66 1.6 4 0.73 0.71 5 0.72 0.78 6 0.73 0 . 8 7 0.14 0.17 8 0.90 0.84 9 0.84 0.73 10 0.72 0.81 11 0.81 0.80 2 0.74 0.73 13 0.73 0.84 .14 0.81 0.78 95 Control Init. Final 0.31 0.33 0.36 0.36 0.34 0.56 1.75 0.68 0.61 1.5 0.60 0.68 0.63 <0 . 1 0.66 0.70 <0 . 1 0.66 0.72 0.60 0.60 0.68 0.69 0.66 0.64 0.66 0.62 0.62 0.66 0.64 FC-143; Test Init. Final <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 .83 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 .1 <0 . 1 <0 . 1 <0 . 1 <0 .1 <0 . 1 <0 . 1 <0.1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 <0 . 1 143 Control Init. Final <0 . 1 <0 .1 <6 .1 <0 .1 <0 . 1 <0 . 1 .81 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 000253 -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/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 Association's reference material used in biodegradation test method the for SaonaiponiacndsDuertfearcgteanntts(6 ). This material is considered to be relatively easily degraded. In the Soap and Detergent test, the results are Association's shake considered invalid if fltahesk rbeimoodveaglraodfat1i-odnodecene- derived LAS is not nearly complete. The data showing the supplemented controls exatreentdeopfictdeedgraindatTiAoBnLEof6 LAS in surfactant . 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 to 4-6 days, and temperature was raised from room temperature (<20 to 22C) degradation in to a constant temperature of 25C. the final growth period are shown Results in TABLE 8 of . LAS In the growth periods following transfers 11 and 12, an experiment was done to determine if 50 mg/1 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 CofompLAaSr.isonHowweivtehr,TABiLtEsSpr8esaenndce6 was not completely inhibitory. shows that the presence of 50 mg/1 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. 000254 -13- In the final growth period, 50 mg/1 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 resulted 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). Never theless, the remaining data show that the radioactivity associated w7-idtahy FC-95 and FC-143 degradation test remained period. in solution during the entire Analysis o'f 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 # 95 - Surfactant Control 143 - Surfactant; Control LAS LAS Init. Final % Removal (7) Init. Final % Removal 4 31.5 26.8 18.4 35.5 29.5 19.4 5 28.3 27.5 0.1 32.8 25.8 21.2 6 29.8 25.5 15.5 27.0 24.0 7.0 7 25.0 1 2 .0 91.1 25.0 30.6 -2 .0 8 31.2 3.75 89.8 37.0 35.8 11.1 9 33.0 3.17 95.1 38.9 13.7 94.6 10 32.7 2.33 95.9 42.7 1 2 . 8 89.7 11 31.0 2 . 0 95.0 39 13.7 93.8 12 31.3 2.33 96.5 41.3 19.7 77.9 13 31.7 2.5 93.5 41.3 18.0 88.3 14 31.3 3.0 92.8 40.3 13.7 90.7 000255 -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 5.25 3.0 .75 10.2 0.88 5.75 1.83 4.17 1.17 4.33 0.67 3.0 1.33 3.67 3.67 0.67 1.0 143 - Control Init. Final 4.5 4.5 4.5 3.5 7.1 5.50 5.0 1 0 .2 9.0 10.9 10.9 12.2 12.3 11.5 9.67 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 FC-95 Controls FC-143 Controls______ #1 LAS #2 LAS Non- #1 LAS #2 LAS Non- Time Suppl. Suppl. Sugpl... glffiP.1 -. gyppJL. Suppl. Initial 28.7 29.3 1 . 0 34.0 36.7 6.5 J Day 2 Day 7 13.0 26.0 1.67 2 . 0 1.0 .7 8 . 0 22.3 5.3 6.3 6.3 4.0 % LAS 96.5 Removal (7) 95.4 - 91.6 92.3 00256 -15- TABLE 9 EFFECT OF FC-95 AND FC-143 ON THE BIODEGRADATION OF LAS ANALYZED FOR AS MBAS Transfer # 11 . 12 FC-95 + LAS Culture % LAS FC-143 + LAS Culture % LAS Init. Final Removal(8) Init. Final Removal i8") 58.0 64.'$ 34.7 40.3 73.6 78.9 53.7 27.3 53.7 34.0 97.8 71.4 TABLE 10 CONCENTRATION . OF 1144 C-FC-95 OR 14 14C-FC-143 IN THE CENTRIFUGATE OF TEST CULTURE DURING THE FINAL GROWTH PERIOD Init. Day 2 Day'7 ^C-FC-95 Cultures Standard" Concentration Deviation 30.1 mg/1 52.8 53.5 0.3 mg/1 0.5 3.1 14C-FC-143 Cultures -- ' 77 Standard Concentration Deviation 46.2 mg/1 0.9 mg/ 1 48.0 0.3 49.7 0.4 000257 -16- Thin-layer chromatography did not reveal the presence of radio active metabolic products of either FC-143 or FC-95. 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. Acad, 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-95 or FC-143. (3) Standard Methods for the Examination of. Water and Wastewater. l'4th Edition, American Public Health Association (1975). (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 2i-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 Chemists* Society, 42:986 (1966). (7) Percent LAS removal was calculated as: (MBASTM - MBAS ) _ (M B A S TM - MBASTM) % Removal - ----- --------- 2--------- 1------- SL- X 100 MBASgj -- MBAS^j Where : MBASgj =' The initial methylene blue active substances (MBAS) concentration of the surfactant supplemented culture. MBASrT = The initial MBAS concentration of the nonsupplemented ^ control (TABLE 7). MBASgF = The final MBAS concentration of the surfactant supplemented culture. MBASTM The final MBAS concentration of the nonsupplemented control (TABLE 7). 000258 -17- (7) The percent LAS Removal was calculated as: (MBASg^j - MBASCI) - (MBASgTF % Removal = (MBASgj - MBASCI) m b a s cf) --- --- X 100 Where: MBASSti = The initial methylene blue active substances (MBAS) c>ii concentration of the culture supplemented by both LAS surfactant and either FC-95 or FC-143. MBASrT = The initial MBAS concentration of the nonsupplemental control (TABLE 7). MBASgTF =* The final MBAS concentration of cultures & supplemented with surfactant and fluorocarbon. MBASrl7 = The final MBAS concentration .of the honsupplemental control (TABLE 7). MBASsi TshueppilneimteinatlalMBcAuSltucroence(nTtArBaLtEio6n).of the surfactant It was assumed that MBAS concentration due to FC-95 or FC-i43 was not reduced by the biodegradation or other loss of these compounds. EAR/cen 000259