Document peEaaDjEa9BMDz9OJ4NGnMea6

AR226 _ 2800 ' `DuPontCompanyConfidentialInformation -For DuPontUseOn[ ly -- ( | | I | | || | | | | | 1 ||{ (--AE-- ROBIC-- BIODEG-- RADATI-- ON STUDY OF AMMONIUM! JORO-OCTANOATE (C8) E.L DuPonFltuDoreopNreomdouuctrssSanBdUCompany SoomineTod: FVrincoi M.nFeliwxrk JCaRm&eDs/GMl.aOsdgoomw SUBMITTED BY: ENVIROGEN, INC. P4r1in0c0eQtuoankReersberairdcgheCReonatedr Lawrenceville, N.J. 08648 Witten By: Mery F. DeFlaun, Ph.D. Applications Manager, Bioremediation Technologies Envirogen Job No. 57202 June 1996 Company Sanilzed. Does not contin Tes mor DuPont Company Confidential Information - For Pupont Use Only Executive Summary The purpose of this study was to evaluate the ability of aerobic bacteria, previously shown to have a broad range of degradative capabilities, to degradeHBR--------^H^JJB and ammonium perfluoro-octanoate (C8). Nine different bacterial strains, known to metabolize compounds similar to|HJand C8, were tested in bottle assays .for their effectiveness against these compound^. Experiments conducted with bothQK for dehalogenation_ analysis, andlffff^o test for production of ^COz, failed to show degradation oifffyy all nine bacterial strains. Analysis for C8 by existing methods lacked the sensitivity needed to analyze degradation of this compound. Due to the high degree of fluorination of bothUjand C8, however, it is more likely that these compounds would be degraded by anaerobic dehalogenation pathways. It is our recommendation that a more sensitive method for C8 analysis be developed, and that anaerobic consortia be tested for their ability to degrade botlfBjnd C8. Introduction of|HHBHBH^^I|Bi ENVIROGEN has experience in the biodegradation compounds. With a grant from the National Science Foundation. ENVIROGENhas hypothesized that the bacterial strains that were competent in degrading these compounds m^UU^----und would also be able to degrade ammonium perfluoro- octanoate (C8). This research was conducted under a joint development agreement between DuPont and ENVIROGEN (see attached agreement). Scope of Work ENVIROGEN proposed to investigate the degradation of two different fluorinated or perfluorinated compounds using three different bacterial strains previously shown to Materials and Methods I B H Strains and Media. A total of nine different strains were tested for their ability to degrade Pseudomonas mendocina KR1 (T4MO; Whited and Gibson, 1991) and Pseudomonas putida Fl (TDO; Wackett and Gibson, 1988) were both grown on a combination of toluene vapors and 0.04% glutamic acid in basal salts media (BSM; Hareland et al., 1975). The methanotroph Methylosinus trichosporium OB3b (OB3b; Stirling and Dalton, 1979) was grown on BSM with methane as the sole carbon and B N V O B energy source, and the propanotrophs Mycobacterium vaccae JOBS (JOBS; Wackett et were grown on propane. issay, the cells were collected by ^ ,, . --- ^ centrifugation, ^ - - washed - --- ----- - and suspended in an equal volume of fresh BSM medium to an 00550 of 2.0. Cultures prepared for ion chromatography analysis were washed and 2 Cnmnanv Sanitized. Does nit contain TSCA CBl DuPont Company Confidential Information - For Dupont Use Only suspended in phosphate buffer and those for HPLC analysis were prepared in MOPS buffer. Activity Assays. Oxygenase activity in certain strains {^P. mendocina, P. putida, M. trichosporium) was monitored by performing a ^I^Jdegradation assay to insure production of the degradative enzymes. The cultures were harvested by centrifugation and suspended in.BSM to an optical density of 2, as measured at 550 nm. Five milliliters of the cultures werethen aliquoted into 15-ml serum vials, in triplicate, and sealed with Teflon lined septa. fBHiat was diluted in methanol was then injected through the septa S"to a final concentration of 20 pM. The vials were then allowed to incubate at 25-30C, ' " ii t 100 rpm in a horizontal position. A standard curve was generated hymaking iitions to serum vials that contained only BSM. The concentration of^H|was rr mined by injecting 10 pJ ofheadspace gas from the sample vials onto a GCthat was equipped with an ECD. Activity in the propanotrpph JOB5 was monitored by degradation of BrMe in a bottle assay similar to the^^Rissay. The rest of the propanotrophs were grown on propane which induces the appropriate enzyme system, however, these strains were not monitored for activity prior to testing their activity againsfH^ vttfupe gradation Assays. Culture preparation was performed as above and 5 p.1 of '''C- ||H45 [id] was added with a micro-pipette prior to sealing the vials with Teflon-lined crimp caps. The cultures were then allowed to incubate at room temperature on a 100 rpm shaker until they were sacrificed at 0, 2, 7, 24, and 48 hours. Longer incubations with the propanotrophs JOB5 and ENVOB (68 hours, 5 days, and 13 days) were performed with a constant propane feed. The incubation was terminated by the addition of 1.0 ml of 1 N HC1. Acid addition kills the cells to prevent any further activity, and forces carbon dioxide out of solution and into the headspace of the serum vials. The vials were then returned to the shaker until all of the incubations were terminated. The vials were subsequently purged with nitrogen gas to strip the COz into an NaOH trap. For analysis, 0.1 to 1.0 ml of the NaOH solution was transferred to a scintillation vial containing 5 ml of scintillation cocktail. The assay vial was then centrifuged to obtain the cell-free supernatant which was removed from the vial and retained. The cells were suspended in fresh BSM, washed and centrifuged. The supernatant from this wash was added to the original supernatant. An aliquot of [he washed cells and the supernatant were both added to scintillation vials containing 5 ml of cocktail. The amount of radioactivity present was determined by scintillation counting. For analysis of degradation by ion chromatography, cells were washed twice and suspended in phosphate buffer to an 00550 of ~ 2.0. Ten milliliters of washed cells in phosphate buffer were added to 50 ml serum vials. Autoclaved cells were used as killed controls for this assay. 10 p.1 ofttjktock (2 mg/ml) were added to each vial for a final concentration of 10 ppm. The vials were sealed and placed horizontally on a shaker at room temperature (JOB5) or 30C (OB3b, ENV2C, ENV2R. and ENV2W). For analysis, the caps were removed from the vials and the contents were filtered (0.2 \im) into 10 ml 3 c>-~,,~^,, Cor;-Q< rr>oc nnt r-nntain ^K{*& CR1 DuPont Company Confidential Information - For Dupont Use Only serum vials to remove the cells. chromatography. The vials were placed at 4"C until analysis by ion ff^Anulysis by Ion Chromatography. HPLC grade^|HHBHHllCwas "sed to prepare a 254 ppm working standard in reagent grade deionized water. Combined with the fl|Bworking standard, fluoride was also added to ensure validity in mixture and concentration upon analysis. Ion chromatographic parameters were developed using 1.0 mM sodium carbonate/1.7 mM sodium bicarbonate as the mobile phase and a Dionex AS4A anions column as the stationary phase. Flow and pressure were all set to normal parameters for standard anions applications. Ion chromatographic standards were prepared forjjBand analyzed at the following concentrations: 0.8, 1.5, 3.1, 7.7, and 15.4 mg/1. The chromatographic retention time fo^Jys 1.75 minutes and the fluoride check is at 0.87 minutes. Both compounds calibrated to a 0.995 or better correlation coefficient encompassing all calibration-levels. The quality control measures for z|Rnalysis series is: ^^^^^r Reagent Blank A continuing calibration check standard (1-2 check standards) Matrix duplicates Matrix Spikes Final continuing calibration check standards A typical number of samples would range from 20 to 50 samples and be analyzed within 48 hours of receipt (usually upon receipt). Spike recoveries averaged 85 to 115% recovery foqBBspikes as well as additional anions spiked into the matrix. For each set, all samples were pre-filtered then placed directly into auto sampler vials. Each sequence was reviewed and presented in a simple data summary format as requested by the submitter. No significant peculiarities or matrix interference were noted on the analysis ofp^m samples. Hm^Ktandards were also prepared and analyzed by ion chromatography. JThese standards were compared to the 'cold'sSBgtandards with a 113% recovery of the IUHmaterial. No abnormalities were noted in the ion chromatographs. ^fiw\.nalysis. A commercially purchased stock offlBH^^HU^^ftAwas used to prepare a 5000 ppm working standard in reagent grade deionized water. Ion chromatographic parameters were developed using 1.8 mM sodium carbonate/I .7 mm sodium bicarbonate as the mobile phase and a Dionex AS4A anions column as the stationary phase. Flow and pressure were all set to normal parameters, for standard anions applications. Ion chromatographic standards were prepared foi^Uind analyzed at the following concentrations: 1.0, 2.0, 5.0, and 10 mg/1. The chromatographic retention time for^^ftis 0.97 minutes. C8 Degradation Assay. Culture preparation was performed as above except that C8 was added to a final concentration of 5 ppm prior to incubation, and the cultures were 4C..P--------^.-ncoalITSCAC61 DuPont Company Confidential Information - For Dupont Use Only suspended in MOPS buffer rather that BSM in an attempt to eliminate any potential interference with the analysis. Following a 12 hour incubation the vials were centrifuged and the supernatant was analyzed. C8 Analysis. Three different methods were attempted for derivitization and analysis of the compound C8 on various GC-detector configurations: I. DuPont Method 1. 1 gram (1 ml) C8 solution into a 60 ml serum vial. * 2. Add 20 ml 20% phosphoric acid solution and 20 ml methylene chloride. 3. Crimp seal vial and shake 1 hr. 4'. 'Remove'raelnylene enloffde and dry over sodiuin' sulfate anhydrous**. 5. Evaporate methylene chloride with a nitrogen stream at 40C. 6. To dried sample add 3N methanolic HC1 (10 ml). 7. Seal crimp vial. 8. Heat solution at 50 to for 2 hr. Cool, insert 25 gauge needle to vent. 9. Add 10 ml ofhexane with a syringe and mix. 10. Add 20 ml NaCI solution via syringe - remove vent needle and shake. 11. Analyze on GC. * this solution was prepared with 40 pi of C8 solution (provided by DuPont) in 79.96 ml methane!. Concentration of this solution is 500 ppm. * *this step was also performed using dry ice and a vacuum. n. A. BF-^ - Methanol Esterfication Method (Metcaife and Schmitz. 1961) 1. 25 ui of 20% solution (provided by DuPont) diluted in 5 ml ether in a 10ml serum vial. 2. 2 ml of ether solution placed in 10 ml serum vial and 2 ml of BF3 reagent (Supeico cat# 3-3021) added. 3. Bottle placed in a beaker of water and boiled for 3-5 minutes. 4. 1 ml of water is added to stop reaction. Allow layers to separate. 5. Remove top layer and analyze. n. B. BFt - Methanol Esterfication Method fSW 846: Method 6640 EPAt 1. Stock Solution prepared in ethyl ether. Final concentration of 1 mg/mL. 2. A serial dilution of stock prepared a. 1/100 (lOpLin ImL) lOng/pL b. 1/1000 (1 pL in 1 mL) (Ing/uL) c. 1/10000 (10 pL of 1/100 in 1 mL) 5 Company Sanilizsd. Does not contain TSCA CBl DuPont Company Confidential Information - For Duoont Use Only 3. Esterify 0.5 mL of each solution and a 0.5 mL hexane blank a. add 0.5 mL of solution to serum vial. b. add0.5mLBF3 c. heat at 50C in a sealed serum vial for 30 min in a water bath. d. cool, add NaS04 solution, shake 1 minute e. quantitatively transfer to flask and concentrate to 0.5 mL. f. analyze via GC/MS in. Pentafluorobenzvl Bromide Derivitization Method fFlanagan and May. 1993) 1. Extract 25 ml of 5000 ppm C8 solution with 25 ml ether. 2. Remove 1 ml of ether extract and place in 25 ml acetone 3. Add 0.5 gram of K2C03 (dispersing agent). 4. Add 250 ulpentaflurobenzyl bromide 5. Reflux for 3 hours 6. Analyze GC/MS High Performance Liquid Chromatographv fHPLC) Method 1. Standards were prepared in accordance with method guidelines provided by DuPont. 2. Methods were reviewed: The following was steps were taken: a. Standards were prepared in deionized water in sterile plastic containers. b. C8 stock was provided by DuPont c. Zorbax column was purchased d. Standards were analyzed via HPLC e. Standards were analyzed via Mass Spec f. Standards were analyzed via GC-ECD g. Standards were prepared a second time and analyzed methods listed above. again using the 6 Company Sanitized. Does not contain TSC& CB3 DuPont Company Confidential Information - For Dupont Use Only Results ___degradation. The results of activity assays on the bacterial strains tested indicated degradef labeled^j|to that the appropriate enzyme systems had been induced (Figure 1). Nine strains^ were tested for their ability to Assays were conducted with ^C determine if these strains would produce '"COi and with HPLC grade^^"^" dehalogenation analysis. ^^^^h ^^degradation or^^^^^^&lehalogenation were not detected in incubations with any of ^^^^^~ strains tested, even ^^w^i^th^^ij^^^B^B^Hkfil^nkcubations of up tothirteen days (JOB5 and --- ENVOB with propane feed), or two days incubation withjRBfor fluorine analysis (Figure 2). Both JOBJLand OB3b were assayed at several different densities (OD=1, 2, and 3) with negative |llHILresults (Figure^ 3). The levels of ^C carbon dioxide detected in the traps from the incubations'with-^ttEailedto exceed the jgv&ls that were observed in the controls which contained no bacteria, and/or no labeledflHR In some experiments, the vials containing live cells produced slightly higher levels of C trapped in the NaOH, however, the total was never more than 0.2% higher than the controls. Soluble and total non-volatile (including cells) ^C was never significantly different in the experimental and killed controls (Figures 4 and 5). ^^^ ^^C^^_yjialysis. The objective of the^Hf analysis was to detennmewhether it co-eluted with any currency calibrated compound (F, Cl, NOz. N03, fC^.^^^Bind804); and JJ^ whether thefafttock was contaminated with The analysis o^BHlemonstrated Jhat the elution time did not interfere or co-elute with any other compounds, and that the ^k|R^^BB^----^------^Hjwas not contaminated with thjscompound. The cioses^lutingcon^oiindwasTluonde which was 0.87 min. BothQBjand fluoride were easily distinguished and quantified. No contamination withj----ftvasobserved in samples or standards. C8 Degradation. JOB5 was incubated with C8 and analyzed by HPLC analysis and a variety of GC methods. Failure to detect C8 at sufficiently low concentrations by any of these methods terminated this work. C8 Analysis. A standard curve analysis of C8 by HPLC showed a good response at 50 ppm, but no quantification at 10 ppm. The lack of sensitivity of this method combined with the interference with the buffers in which the bacteria were suspended made it inappropriate for this application. The derivatization reactions for GC detection appeared to randomly fragment the C8 into smaller compounds. Repeated analysis of these compounds had a negative impact on both the instrument and the column. C8 was not detected by any of the methods described above. Company Sanifesd. Does not contain TSCA CE, DuPont Company Confidential Information - For Dupont Use Only Discussion A suitable method for the detection of C8 at low concentrations that is compatible with the buffer solutions necessary for bacterial degradation experiments needs to be developed before further work on the degradation of this compound can proceed. Due to the high degree of fluorination of this compound, however, it is unlikely that the oxygenase enzymes of aerobic bacteria will be effective in the degradation of this compound. In the work that ENVIROGEN did on the degradation of HCFCs and HFCs, compounds with a high degree of fluorination were generally not degraded. It is more likely that this compound would be degraded by an anaerobic dehalogenation pathway. None of the cultures, representing a range of bacterial oxygenase systems was able to degrade ----^----fl^H_|As noted above, in our previous work with fluorinated compounds, thelugnertneaegree of fluorination, tne" less likely IT "was to" Be'degraded. This compound is also more likely to be degraded by anaerobic bacteria. Recommendations/or Future Research C8 Degradation. It seems unlikely that an HPLC method will be sensitive enough to measure degradation at low concentrations, therefore, a suitable method for detection by GC/MS should be developed. Methyl perfluorooctonoate is available commercially as a standard. This standard could be used to develop the method for detection of the derivatized compound. Once this standard can be detected reproducibly at a range of concentrations, a reliable method for the derivatization of developed. Once a suitable analysis system has been recommendation that anaerobic consortia be tested for their the compound can be established it is our ability to degrade this compound. ^Iffpe gradation. Although a considerable amount of work has already been done, it is ENVIROGEN's recommendation that the anaerobic work on pjLdegradation be continued. This work is more likely to be productive than the further investigation of aerobic pathways for this degradation. 8 com^y Sanitized. Does not contain TSCA CB( DuPont Company Confidential Information - For Dupont Use Only References Cited EPA Test Methods for Evaluating Solid Wastes. Third Edition, November, 1986. US Government Printing Office, Washington, D.C. Flanagan, W.P. and May, RJ. 1993. Metabolite Detection as evidence for Naturally Occurring Aerobic PCB Biodegradation in Hudson River Sediments. Environ. Sci. Technol. 27:2207-2212. Hareland, W., Crawford, R.L. Chapman, PJ. and Dagley, S. 1975. Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacterial. 121:272-285. Metcalfe,L.D.andSchnfftz,AA. 1961. Anal. Chem. 33:363-364. Stirling, D.I. and Dalton, H. 1979. Properties of the methane mono-oxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzyme from Methyloccus capsulatus (Bath). Eur. J. Biochem. 96:205-212. Wackett, L.P. and Gibson, D.T. 1988. dioxygenase in whole-cell studies with Microbiol. 54:1703-1708. Degradation of trichloroethylene by toluene Pseudomonas putida Fl. Appl. Environ. Wackett, L.P., Brusseau, G.A., Householder, S.R. and Hanson, R.S. 1989. Survey of Microbial Oxygenases; Trichloroethylene Degradation by Propane-Oxidizing Bacteria. Appl. Environ. Microbiol. 55:2960-2964. Whited, G.M. and Gibson, D.T. 1991. Separation and partial characterization of the enzymes of the toluene-4-monooxygenase catabolic pathway in Pseudomonas mendocina KR1. J. Bacterial. 173:3017-3020. 9 Comsany Sanitized; Doss not contain TSCACBf PuPont Company Confidential Information - For Dupnnt Ifag Only 500000 CTIVITY ASSAY 11^7/95 400000 5 0 300000 U < ^ 200000 L-/ 100000 fi-- JOB-5 JOB-5 katod OB3b OBSbkflted BSM control TIME (hours) Fignre 1 Ezaaple of results from an activity assay for M, vaccae JOB5 and M. trichoeporiua OB3b. 10 Comoastv RRn!f!fait' rt- CONCENTRATION ( 0 01 0 PuPopt Company Confidential Information - For Dupont Use Only stippled ban = experimental (o=3) open bm = lolled control % = ujppedftoeal U S 3 OD=1 OD=2 OD=3 BSM stippled ban = experimental (n=3) open ban = killed control % = fss^csS/SsssI OD=1 OD=2 OD=3 BSM Figure 3 Two examples of representative resulCsobtalned by 14c in NaOH traps after incubation of^B^B&wich cell densities. Results for JOBS andOB3bare for and killed controls. 12 measuring three different both experimental CoiRT-i^^^'f .SarufSyorE rt*%i->e" n"-^5 -*--c*'-- ^<%< DuPont Company Confidential information - For Diipnnt \]se OnJ^ JOB-5 UUassay 11/27/95 ttippled bos = experimeotal (n=3) open bm = killed control OD=1 OD=2 OD=3 BSM stippled bars = experimental (n=3) open ban = killed control OD=I OD=2 OD=3 BSM Figure 4 ^Solublftl^C measured in che supernatant fraction of the [esperiments with JOBS and OB3b. 13 f^fyi->f" O^wSK-v-i,/^ 'n<- DuPont Company Confidential Information - For Dupnnt Use Only 2.0' U Totaf non-volatile 14-C stippled ban = experimental (o=3) open bars = killed control 1.0 0.0 OD=1 OD=2 OD=3 BSM OB3b 11/27/95 Total non-volatile 14-C stippled bars = experimental (n=3) open bars = lolled control 0.0 OD=1 OD=2 OD=3 BSM Figure 5 Total Non Vola washed cells ii ^UC measured in the supernatant and the ixperiments with JOBS and OB36. 14 Company Sanit;z6d. Does not contain TSCA CBt