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BiodegradationStudiesof Fluorocarbons(8112176)report and BiodegradationStudiesof Fluorocarbons- III (7/l9f78r)eport.(Note:bothreportsummarized vath one robustsummary) BIODEGRADATION TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredtoas PFOS or FC-95. (1-Octanesulfbniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsalt,CAS # 2795-39-3) Remarks field: The testsubstance isa white powder of uncharacterizedpurity. The followingisa reduced overview ofpreviouslycompleted biodegradationstudies.While these studieshave concluded thatIOctanesulfonicacid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-, potassium saltispersistentn,ew testingisunderway todetermine ifa mechanism existsforthe biologicadlegradationofthissubstance. METHOD Methods: Warburg Determinafion';Shake Culturestudy modeled the Soap and DetergentAssociabon'spresumptivetestforthe determinationofABS/LAS biodegradability2. Type: Aerobic GLP: No Year completed: 19761,1978 2 Contact time (units):3 hours',2.5 month S2 lnnoculum: Activated Sludge after RESULTS No biodegradationwas observed ineitherthe Warburg study orthe 2.5 month shake culturebiodegradationstudy. DATA QUALITY ReliabilityK:Jimischranking= 21. This study meets allcriterifaorquality testingb,ut analyticamlethodology isquestionable. lgimischranking= 22. This study meets criterifaorqualittyestingb,ut analyficamlethodology isquestonable. No referencecompounds orabiotc vesselswere includedineitherofthe studies. Footnotes: 'Warburg Determination 2Shake Culturestudy modeled afterthe Soap and DetergentAssociation's presumptivetestforthedeterminationofABS/LAS biodegradability REFERENCES 1. Fate of Fluorochemicalsinthe Environment,Projectnumber 75-639829, E.A. Reiner,August 12, 1976,3M Company, Environmental Laboratory. 2. Fate ofFluorochemicalsinthe Environment,Projectnumber 9970612613, E.A. Reiner,July19,1978, 3M Company, Environmental Laboratory. OTHER Submitter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 Last changed: 5/3/00 -F 4$.-! TO:, .T*chnicalCommunications Center,201-2S TECHNICAL REPORT SUMMARY DATE, Environmental Title LABORATORY,DEPT.NUMBER MICROFORM COPIES: Biodeeradation Studies of Fluoroc*aThons Project: Fate of Fluorochemicals in the Environmental Tor R. L. Bohon By% E. A. Reiner-. Objectivat SECURITY CompanyConfidential(Opin) To determine the biodegradability of selected 3M :Eluorocaxbon compounds. 'SpeciaAluthorizotion(Closed) Augmt 0222 12, 197m6 ProjectHumbor: 7S-6398-29 (3digits' Employ*@Number. 47816 Note1 ls-kReference40671 Pgs. 29-37, 41-SO IF SUMMARY REPORT Me.ofPages includincgovorsho. Has Informatioinnthisreport 12 bsouobnmictotveedtroedTbCyC?oth*r.reportsABSTRACT and Conclusions..(Sysctaonmaccommodate200-250w*rds) FX.No F-1,Partially El completely Please keyword inf@rmation not includedin otherreportsi and giv*page nurri6*rosf new material: 3M CHEMICAL REGISTRY law chemicalsreported? Biodegradation studies using a Warburg respirometer were conducted on FC-9S, FM 3422, FC-128, and hydrogen analogs of FC-95' and FM 3.422. No biodegradability was observed on FC-95, although.an approxima te hydrogen analog of FC-95 was readily'degradlable. FM 3422 and FC-128 both were demonstrated to underg-o some biodegradation. Attempts to isolate degradation products of FM 3422, from the Warburg studies, and from a subsequent activated sludge study were unsuccessful. NO 0 Yox KEYWORDS Selectgeneral,specifica,nd 3M productermsfrom3M rnesourus.Enclosesuggested termsinparentheses. .EE & PC Div. Envron Assess Biology Ba@6teTia Bioscreening Fluorochemical .Biodegradable SPECIFIC PROBLEMS remainintgoreachobjective. Continued attempts will be made to isolate and identify the biodegradation products of FM 3422 and other 3M fluorocathons. Work with radioactivity labeled FM 3422 is being considered. informatioSncientist Initials BIODEGRADATION STUDIES OF FLUOROCARBONS SUMMARY AND RECOMMENDATION No biodegradation was observed in Warburg studies on FC 95. Biodegradation of FC 9S 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. Semicontinuous activated sludge studies on FM 3422 did not confirm or disprove the Warburg findings. Future investigation'6f the biodegradability of the fluorocarbon compounds would be greatly facilitated by the development of an analytical procedure for FC 9S. 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 activi'ty(>s mci/m mole) and purity. Biodegradation of a saturated solution' of the labeled compound could be measured by.detecting 14C02 evolution.. INTRODUCTION The susciptibility 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 :factthat 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(l). PeTflWTinated 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 perflwro 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 Ch micals The chemicals used in these experiments are shown in Table I. -2-TABLE I. CHEMICALS USED IN BIODEGRADATION EXPERIMENTS FM 3422 Hydrogen Analog of FM 3422 C2HS C8Fl7SO2 NC2H40H C2HS C8Hl7SO2NC2H40H FC 9S CSF17SO3 x Sipex-ols FC 128 C0170S03Na C2 HS C8Fl7SO2NCli2COOK They were obtained from Don Ricker of the Commercial Chemical Division in. Sept ember, 1975. FM 3422 (N-et Fose alcohol) was identifiedas 788 CC 74-5-2. 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 essentia:llythe fluorocarbon constituent of a large number of 3M fluorocarbon compounds. FM 3422 is an intermediate in-the production of 3M :fluorocarbonsa,nd FC 128 is a finish fluorocarbon product. Sipex-ols and the Hydrogen Analog of FM 3422 were selected for comparison to the fluorocaxbons. Sipex-ols is an approximate 'hydrogen analog of FC 9S. 'These hydrogen analogs were tested because biologically labile fluorocarbonshave frequently been.found to be gratuitouslydefluorinated by enzymes which normally remove a hydrogen. Thus; it seemed probable that microbial growth on hydrogen analogs could select populations of organisms which cou]Ldmore coapletel:y*degradefluorocarbons. 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 concentrationof 2000 mg of biological solids per liter. Water insolublesubstrateswere 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. Logarithmicdilutions 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 gil glucose solution or deionized water in this side arm. The second side am contained either glucose or deionized water. -3- Oxygen uptake was first observed in each flash for a period up to 1.5 hrs. with readings at 10-lS 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-l*ongsemicontinuous activated sludge (SCAS) study was conducted on FM 3422. The microorganisms used were obtained, as before, from-Pigs Eye. Treatment Plant. One Hundred Fift)@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/l. Pure ethanol was added to one.sludg@7-containingreactor 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 cylinders1311high with a 411internal 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).,@T,he developed plates were visualized by the iodine'-starchtechnique and compared to known standards with a detection limit of one Ug-of FM 3422. Samples for the SCAS study were extracted into n-octanol and s6parated-by gas chromatography with an electron capture detector. Extractions were performed in capped SO ml polypropylene centrifuge tubes and phases separated by centrifuging at 26,70OXG for -LOminutes. 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/l of the FM 3422 and 11,000 mg/l 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. -4- STEP 1: Add test compound, media, and microorganisms STEP 2: Aerate and mix for 23 hours STEP S@ Re-add/ test compoiLmd,," and media. Repeat cycle. /* magnetic stirring bar air sparger supernatant drain STEP 4: Drain supernatant. its sludge,.., STEP 3: Stop aeration and mixing. Let sludge settle. FIGURE 1: Test cycle for somicontinuous ictivated ,Itidgerc;ictor. -sFM 3422 4 FM 3422 @-3000 mg/ (16 u mole. 3 2 0 t4 0 -i F) 0 FM 3422 n-300 mg/l (1.6.u mole 0 u 0 rpt3422@@dtioii cli Analog Addition 0 -2* glucose addition -3 .2S S .75 time hours 1.0 1.25 1. lo1g.,75 FIGURE 2: Warburg study of ni 3422 and its hydrogen analog. 2.0 llydrt)gcnAn:tlog mg/ I -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 semisolid gel. Complete chemical oxidation of the hydrocarbon component of the FM 3422 at the highest concentration (1@16'pmoles) would require 87 p moles of 02 based on the following equation: c a F 17S02" cc2H 5 )C2H 4OH + 5.50 2 C F SO NH + 4CO + 4H 0 8 17 2 2 2 2 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,.continuingat 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 34.22showed significant toxicity. Upon addition of the most concentrated emulsion of the analog, endogenousoxygen uptake ceased and was not restored even after the addition of glucose to' the culture. The negative slope of the.hydrogen analog's oxy en uptake curve (Figure @) is due to the endogenous correction anclnot 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. 7he oxygen uptake tAou;,'40observedwas 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. Ihin layer anCL-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 se6ond attempt to isolate the hypothesized degradation products of FM 3422. This study was conducted over a period of--l-weekwith samples taken at the initiation and end of each 24@4ir.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 extractsof 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. performed by Commercial Chemicals Division The n-octanol extracts could not be separated by thin layer chromatography because of thd low volatilityof this solvent. Frozen nonextracted samples still exist at this date and could be extractedinto a more volatilesolvent 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 supernatantwas withdrawn. The final concentration (althoughnot in solution) was approximately100 mg/l. This materialwas not homogeneously distributed and accumuliltedon the sides of the reactors. WARBURG FC-95 The results of WaThu:r@sgt,udies with FC-9S 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 approximatehydrogen analog of FC-95, was shown to be readily biodegradable and to have no toxic effects. The S3'.P'el-owlass s. oluble at all concentrations tested (as high as 1700 mg/1) FC-95 was incomple tely soluble at 4000 mg/l, but was completelyin solution ai 400 mg/l. The lack of degradation with FC-95 was expected since perfluorinate compounds are chaxacteristically nonbiodegtadable. 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 thiitheoretical maximilmoxygen 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 c 8F 17 SO 2NC2HHS2COOK + 4.250 2 H-+ C 8F 17S02 NH 2 + 4CO 2 3H 20 + 76 0 4 C) 2 172 mgll Sipex-ols glucose addition 000 mg/i FC 9S 400 mg/l FC 95 substrate 0 addition -7t time hours 2 3 glucose addition FIGURE 3: Warburg study of.FC 9S and Sipex-ols. A I 4 -98- FC 128 -70 6500,mg/l Soo mi so 4 -40 ,3 30 CD 20 2- fj c 100 mg/l 110 0 Substrate addi-L.ion It + 1 2 3 4 5 6 7 time hours FIGURE 4: Warburg study of FC 128. -10- REFERENCES: (1) Alexander, M.; Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility. Adv. Appl. Microbial.7: 35-80, 1965. (2 Chapman, P. J.; Depariment of Biochemistry, University of Minnesota, St. Paul, Minnesota, Personal Communications 2/24/76. STANDARD ATTAMIENT I PROCEDURE FOR WARBURG DETERMINATIONS 7/10/75 E. A. Reiner I. Design experiment and c:alculateconcentrations of materials to add. 2. Fill water bath (DI water if left in bath). 3.. Adjust temp. of bath (several hrs. or overnight). 4. Plac& manometers in desired order. S. Prepare the=obarometer. Add about 3idof If0 to I flask. 2 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. 8. Prepare samples in DI water (or according to reiuest) to add to side arms.. Keep refrigerated until used. 9. Prepare cells (keep cells cold at all times but avoid freezing). 0 A. Centrifuge 0 C. B. Wash with cold BSM - centrifuge. C: Resuspend in cold BS@t. D. Determine concentration of an aliquot with the spectronic 20 at 600 nm. Adjust remainder to desired conc. C ba'sic salts medium. Refrigerate until use. E. Take sample of final adjusted sludge for standard MISS analysis. 10. Adil-samples to side arms (usually 1 ml if one side arm, 2 side arms).. ml to each side.arm if. 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. -2- 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 WateT. lb Faft4674@- $3 A 1 TECHNICAL REPORT SUMMARY TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN (important-Itraport;sprintedon boM sidesofpaper,ondtwo copiesto TCC.) Divisjon . Project Environmental Laboratory Pate of Fluorochemicals (EE & 16 the PC) Environment Biodegradation Studies of Fluorocarbons - III Date 7/19/78 DGPL fqumbw 0535 ProloctNumbw 9970612613 t Repowt Number D. L. Bacon Author(%) E. A. Reiner r"OtOb*OkRetwence 44703, p. 6-14, @45727, p.'32-35; SECURITIV.10, 0 Open (Company Confidential) 21, !Z5-27; . 49400,_ P. 29, 31 1 -12. 39-43, Closed ..-PeciAaulthorization) 3PACHEMICAL REGISTRY 49',7- Employee Number($) 47816 N& of Paw Inc'udinsco"nhoa 7 Now ChemicalsRomrted [3Ya No KEYWORDS: (Sole=termsfrom 3M ,Thw*urus. othi* applicableS=)" (Biodegradation) EE & PC-Div. Envir. Assess. Fluorochemical gradation CURRENT OBJECTIVE: To evaluate the suseeptib@ilitieg to microbial decomposition. of FC-95 and FC-143 . I REPORT ABSTRACT: I200-250words)ThissbttraainfornutioInsdistributbeyd theTechnkalCommunicationsCenterto alert3M'ersto C@onVmw R&D. A biodegradation study is described which-allows the evaluation of the susceptibility of FC-95'-$LndFC-143 to aerobic microbial degradation. The culturing procedures used in this study are modeled after the Soap and Detergent Association's.presumptive (shake culture) test for the determination of ABS/LAS biodegradability. Microbial inocu:La were obtained from.activated sludge collected at Chemolite, Decatur and Metro.waste treat" nt plants. Analytical procedt-ies included GLC, TLC, . C-scintillation counting and analysis for released fluoride. Degradation of reference c6nipounds demonstrated the suitability of the biodegradation test conditions. InformatioLniaison initial$,, -2- SUMMARY Fluorochemicals FC-95 and FC-143 were shown to be completely 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- ,m--ntsystems (Chemolite, Decatur, & the Twin Cities Metro plant) - The' cultures were maintained in dilute yeast extract-basal salts m6dia supplemented with the hydrogen analog of the respective fluorochem- icals. Test cultures also contained FC-95 or FC-143. Phenol and 1-dodecene-derived linear alkyl sulfonate (LAS) were used a's reference compounds. Their degradation demonstrated that biode- gradation could occur under the test conditions. All cultures were transferred 15 t@mes 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 14 C-labeled fluoroch' emicals were assayed for by thin-layer chromatography (TLC) and'gas liquid chromatography (GLC). Chemicals separated by TLC were visualized by TLC-autoradiograph. Meihylated and nonmethylated culture extracts separated by GLC were detected by electron'captu're, 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 bbserved 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 cannbt 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- INTFTODUCTION The fluo'@rochemicalsselected 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 sak6 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 akdas'.considered likiely'to contain acclimated microorganisms. ]4on9 Acclimation pei;r:,iods were used in an attempt to select.and develop populati6n's of microbes capable of.degrading these compounds, and hydrogen analogs of the fluorocarbons were added to try to select'organisms that might gratuitously "cometabolize.1t1he fluorocarb.ons. -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 Co rcial 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 Cine innati, Ohio. Except where noted,.all other compounds were reag' ent 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 - K2HPO 4 0.25 g/l - *gSO 4. 7H 2 0 0.002 g/l FESO 4* 7H 2 0 2) Well water 25 ml/l 3) Yeast extract 0.3 g/l 4). Hydrogen analogs of either or FC-143 - 20 mg/l FC-95 Media were prepared from stock solutions which were combined and brought to volume just prior to each culture transfer. A-fresh solution of FeSOi47H 2 0 was prepared and dry yeast extract was used in media prepara ion 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-inonth 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 P;moved by filtration through a #54 Whatman filter. The purified hydrogen analogs of FC-95 and FC-143 were used in biodegrtdation 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 sjpecific,mediaare listed in TABLE 2. GROWTH TABLE 2 MEDIA FORMULATIONS Media FC-95 FC-143 Test Phenol Controls LAS Controls Fluoride Controls 14 C-FC-95 Test 14 C-FC-143 Test FC-95 + LAS FC-143 + LAS 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/l Phenol FC-95 or FC-143 Control Medium + 30 mg/l Standard Linear Alkylbenzenesulfonate (LAS) FC-95 or FC-143 Control Medium + 33.2 @ag/lNaF (15.0 mg/l F-) FC-95 Control Medium + 50 mg/l 14C-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 ControiMedium + 30 i6g/lLAS + 50 mg/l FC-143 Culturing Procedures The initial growth period was started by inoculating 49 ml of etch 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. -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 mediu'm). 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 CarVdn-14 labeled FC-95 and FC-143. One hundred ml, cultures were gr,gwn 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 t-ransfer, a 1.0 ppm fluoride standard was used to calibrate the instrument with the assumption that the slope a.fthe previous fluoride curve remained constant. Phenol analysis was done according to Standard Methods for the Examination of Water and Wastewater, 14th Edition, 1975. Linger 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 modificati.on 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 cm diameter, spee 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- TABLE 3 SUMMARY OF CULTURING PROCEDURES USED IN THE SHAKE FLKSK BIODEGRADATION STUDY OF FC-95 AND FC-143 Transfer Culture Growth 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. 2 4 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 controi culture and Chemolite and Decatur*sludge supernatant. 5 3 6 3 The use of fluoride control was discontinued. 7 6 Shaker was inadverten-tlyturned 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 ii 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 TAI , and counted with an internal standard quench correction. The radioact4vity of theit 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 Um filters composed of cellulose acetate and cellulose nitrate. The filters were then washed with de'lonizpi.[)water@and placed into paper pombust;ion conipft)wet with Combustaid' , andliombusted in Agrichem's Packard' combustion equipment. The CO resulting from combustion wag trapped in a.s'eintillation fluid egntaining an organic amine andcounted in Agrichem's Packard scintillation counter. Samples were recounted with an internal standard for quench correction. Thin-Layer Chromatography (TLC) Thin-layer chroTVtography wasrerformed to detect radioactive metabolites of C-FC-95 and. C-FC-143. Ten ml culture sample's 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 1.7,000x g to.ensure the separation of the ethyl acetate, water, and solids phases. The water phase and portion-s 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 be. fore. 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 se plates, were referenced against a mixture of 1 C-FC-143 and ill samples 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 remainin 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 weie@ks, -9- Gas-Liquid Chromatography (GLC) Ethyl acetate extracts were prepared as described in the tblinlayer chromilography methigs. 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 1/811O.D. stainless steel packed with 20% DC 200 (12,500 CS) on 101%Bentone 34 and ZO% 80/90 mesh Anakrom P.A. The injeesion port temperature was 250 C., and the detector temperature 300 S. The column tempera8ure was grogrammed to hold for 4 min.'at 35 C. to rise to 180 C. at 8 C. per min. , and to hold at 180 C. ihe flow rate was adjusted to 35 ml/min. of Argon/methane,95/5. Methylations were performed by adding a 20 ul aliquot of a 1 ug/mi C 9 F19COOH 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 dtand 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 fluprochemical 'portions of these.molecules were degraded, fluoride ion would 'accumulate in fliemedia. To ensurethat fluoride was not lost from the culture by absorption, pr6cipitation 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 du*e 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 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 Fluoride Control Fluoride Control Initial Final Initial Final 21 23 20 26 16.5 23 22 2217.5 19.2 20 21 21 20 18 21 25 16.5 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 16.216.2 16.2 15.7 17.0 FC-143 Fluoride Control Initial Final 15.7 .15.7 14.5 16.415.7 15.0 il.o 15.0 .16.4 15.6 il.o 16.4 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 Transfer FC-95 Test Init. Final 0 0.46,, 0.51 1 0.50 0.46 2 0.42 0.66 (5) 1.75 1.6 4 0.73 0.71 5 0.72 0.78 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 i2 0.74 0.73 13 0.73 0.84 .14 0.81 0.78 95 Control Init. Final 0.31 0'033 0.36 0.36 0.34 0.56. i.75 1.5 0.68 0.60 0.61 0.68 0.63 0.'70 <0. 1 <0. 1 0.66 0.66' 0.72 0.60 0.60 0.68 0.69 0.62 0.66 0.62 6.64 0.66 0.66 0.64 FC-143 Test Init. Final 143 Control Init Final -cO.l <0. 1 <0.1 <0.1 <0. I <0. I <0. 1 <0. 1 -<O.I -<Ool <0. I <0. 1 .83 1 .81 1 <0.1 <0.1 <0.I <0. 1 <0. 1 <0.1 <0. 1 0.56 ,@0.1 <0. I <0'.1 <0. 1 Q.1 <Oil <0. 1 <0. 1 <0. 1 -<O.I <0.1 -<O.l <0. .1 <0. .1 <0.I <0. 1 @0. 1 .;0.1 <0.I <O.i CO. 1 <0. I <0.I <0. 1 ejO1. <0.1 <0. 1 <0.1 <0'.1 <0.1 <0. 1 <0. 1 <0.1 <0.1 <o.i <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-pi the LAS began to degrade in the surfactant supplemented control for PC-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 acelitbating organisms capable of degrading the fluorochemicals. Growth periods were extended from 3 to0 4-6 dags, and temperature was raised from0 room temperature (<20 to 22 C) to a constant temperature of 25 C. 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/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 re,,,Iultefdrom 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 cultur' es 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.durijig'.theentire 7-day deg' radat @ion'test period.'" 'Analkiifg'df*.thd 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 2608 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 12.0 91.1 25.0 30.6 -2.0 8 31.2 3.75 89.8 37.0 35.8 1.1.1 9 33.0 3.17 95.1 38.9 13.7 94-06 10 32.7 2.33 95.9 42.7 12.8 .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 13.1 90.7 -14- TABLE 7 CONCENTRATION OF METHYLENE BLUE ACRIVE SUBSTANCE (mg/1) IN NONSUPPLEMMTED CONTROLS Trahsfer 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 5.25 10.2 3.6 0.88 5075 1.83 4.17 1.17 4.33 0.67 @.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 iO.2 9.0 10.9 10.9 i2.2 12.3 9..67 11.5 12.0 12.3 13.3 11.3 14.5 11.3 TABLE 8 CONOMTRATION OF.M.BAS (mg/1).IN SURFACTANT SUPPLEIMENTED AND NONSUPPT.VMENTED CONTROLS DURING FINAL GROWT]I PERIOD -Time' FC-95 Controls #1 LAS #2 LAS NonSuppl. Suppl. Su@pl. 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 Removal (7) 95.4 FC-143 #1 LAS supl@l. Controls #2 LAS suppl. Nonsuppl. 34.0. 36.7 6.5 8.0 22.3 5.3 .6.3 6.3 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 + LAS Culture FC-143 LAS Culturis LAS Init. Final RemovalCS)'Init. Final ReppyalCa) 58.0 64.@i 34.7 40.3 73.6 78.9 53.7 53.7 27.3 34.0 97.8 71.4 TABLE. 10 COliCENTRAT,ION OF 14 C-FC-95 OR 14C-FC-143 IN THE CENTRIFUGATE OF TEST CULTURE DTJRING THE FINAL GW%17H PERIOD init. Day 2 Day' :t4C@@.FC-95 Cultures Stand icoiieentration De'viat*ion 30.1 M'9/1 52.8 53.5 0.3 mg/l 0.5 3.1 14C-FC-143 Cultures StanXard doncentration Deviation 46.2 mg/l 48.0 49.7 0.9 mg/l 0.3 0.4 -16- Thin-layer chromatography did not reveal the presence of radioactive metabolic products of either FC-143 or PC-95. Likewise, gas liquid chromatography of the same culture extracts, both before and after methylatioii, showed no products that were not initially present or not also present in controls. From the c6mbination of these results, it can be concluded that no biodegradation of these fluorochemicals occurred.. REFERENCES MD FOOTNOTES (1) Gold.in=, Peter, Enzymology of Car. bon-Halogen Bonds@ Degradation'of Synthetic Orgabic Hole@du'les irfth*6 Biosohe're, Nat. Acad..of.Sci., Washington, DC (1972). (2)Th ere was a six-day period before the onset of the final gr'owtg period during which the test cultures were shaken iLt-95. C. in the presence of FC-95 or PC-143. (3)Standard Methods for the Exami'nation*of-water and Wastewater, 14th Edition, Imerican Public Health Association (1975). (4)Dagtime..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 temperature (see TABLE 3). However, measurement made near the. termination of this 2*-month study, in Januagy,.showed that. tiifittiwilt-emperature 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 big-iergentAssociation, A Procedure and Standards for the. Determination . of the Biodegradability-of Alkyl Benzene Sulfonate and Linear Alkylate Sulfonate. J. of the American Oil Chemists' Sociea, 42:986 (1966). (7)Percent LAS Removal removal was calculated as: (MBAS si - MBASCI) - (MBAS SF MBAS si - MBAS ci MBASCF) x 100 Where: MBAS si MBAS ci MBAS SF MBAS ci The initial methylene blue active substances (MBAS) concentration of the surfactant supplemented culture. The initial MBAS concentration of the nonsupp'lemented control (TABLE 7). The final MBAS concentration of the surfactant supplemented culture. The final MBAS concentration of the nonsupplemented control (TABLE 7). (7) The percent LAS Removal'was calculated as: Removal Where: (MBAS STI MBASCI) ()IBAS STF (MBAs si - MBAS CI) MBAS CF) x 100 MBAS STI The initial methylene blue active substances (.-MBAS).'. concentration of the culture supplemented by both LAS surfactant and either FC-95 or FC-143. MBIAS MBAS STF 'The*initial MBAS concentration of the nohsupplemental cdimtjrol(!rABIt 7)* The final- MBAS concentration of cultures ..supplemented with surfa'eta*ntand-fluorocarbon.- MBASCF The final IM@AS concentration of the jionsupplemental control (TABLE 7). MBAS s I. The: initial MBAS concentration of the surfacta'nt 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. EAR/c-en