Document 5xMY2V9QN70kLvZJYY9LG3jJ

AR226-2710 AR226-2710 Aerobic Biotransformation o f 14C-labeled 8-2 Telomer B Alcohol by Activated Sludge from a Dom estic Sewage Treatment Plant Ning Wang*f, Bogdan Szostekf, Patrick W Folsomf, Lisa M. Suleckif, Vladimir CapkapJ., Robert C. Buck, William R Bertif, and John T. G arnonf * Corresponding author phone: (302)366-6665; fax: (302)366-6602; email: ning.wang@usa.dupont.com o t DuPont Central Research and Development, Glasgow Business Community 301, PO Box 6101, Newark, DE 19714 $ DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE 19714 ^ DuPont Chemical Solutions Enterprise, Wilmington, DE 19805 !! Present Address: Tandem Labs, A Division o f NWT Inc., Salt Lake City, UT 84124 This study investigated the biodegradation potential o f 3-14C, 1H, 1H, 2H, 2H-perfluorodecanol [CF3(CF2)614CF2CH2CH2OH, 14C-labeled 8-2 Telomer B Alcohol or 14C-labeled 8-2 TBAJ by diluted activated sludge from a domestic waste water treatment plant under aerobic conditions. After sample extraction with acetonitrile, biotransformation products were separated and quantified by LC/ARC (on line liquid chromatography/aceurate radioisotope counting) with a limit o f quantification (LOQ) about 0.5% o f the 14C counts applied to the test systems. Identification o f biotransformation products was performed by quadrupole time o f flight (Q-TOF) mass spectrometry. Three transformation products have been identified: CF3(CF2)614CF2CH2COOH (8-2 saturated acid), CF3(CF2)6l4CF=CHCOOH (8-2 1 Subject to Copyright. Do not reference. Not for further reproduction unsaturated acid), and CF3(CF2)6,4COOH (perfluorooctanoie acid, PFOA), representing 27,- 6.0, and 2.1% o f the initial 14C mass (14C counts applied) after 28 days, respectively. A transformation product, not yet reported in the literature, has also been observed and tentatively identified as CF3(CF2)614CH2CH2COOH (2H, 2H, 3H, 3H-perfluorodecanoic acid); it accounted for 2.3% o f the mass balance after 28 days. The 2H, 2H, 3H, 3H-perfluorodecanoic acid is likely a substrate for 6-oxidation, which represents one o f the possible pathways for 8-2 Telomer B Alcohol degradation. The 8-2 saturated acid and 8-2 unsaturated acid cannot be directly used as substrates for B-oxidation due to the proton deficiency in their B-carbon (C3 carbon) and their further catabolism may be catalyzed by some * other still unknown mechanisms. The 2H, 2H, 3H, 3H-perfluorodecanoic acid may originate either from the major transformation product, CF3(CF2)6I4CF2CH2COOH or from other unidentified transformation products via multiple steps. Approximately 57% o f the starting material remained < unchanged after 28 days, likely due to its strong adsorption to the PTFE (polytetrafluoroethylene) septa o f the test vessels. No CF3(CF2)614CF2COOH (perfluorononanoic acid) was observed, indicating that a- . oxidation o f CF3(CF2)614CF2CH2COOH did not occur under the. study conditions. Several 14C-labeled ' .ni . . transformation products that have not yet been identified (each less than 1% o f the mass balance) were . -* -> also observed and together accounted for 7% o f the total 14C mass balance after 28 days. It is not clear whether these unidentified transformation products was resulting from further metabolism o f 8-2 saturated acid or 8-2 unsaturated acid. The results suggest that perfluorinated acid metabolites such as perfluorooctanoie acid account for only a very small portion o f the transformation products observed. Also, the observed volatility and bioavailability o f I4C-labeled 8-2 TBA for microbial degradation was markedly decreased as a result o f the presence o f a strongly adsorbing matrix such as PTFE in the experimental systems. It is apparent that the biological fete o f 8-2 Telomer B alcohol is determined by multiple degradation pathways, with neither B-oxidation nor any other enzyme-catalyzed reactions as a single dominant (principal) mechanism under the study conditions. 2 Subject to Copyright. Do not reference. Not for further reproduction introduction | There is growing interest in the environmental fate and effects o f fluormated chemicals in the environment. As a result o f the identification and ubiquitous global presence o f perfluorooctane sulfonate pF(CF2)gS03"l in the environment and humans and to a lesser extent perfluorooctanoic acid [F(CF2)7COOH], the environmental fate and distribution o f perfluormated chemicals (PFCs) is o f growing interest in order to understand the sources o f these chemicals in the environment (1-6). Carbon-fluorine bonds me extremely strong (7). As a result, it is generally accepted that nm perfluorocarbon chains do not readily biodegrade and any biodegradation may be limited to hydrocarbon functionality to which a perfluorocarbon moeity is attached. Interest extends beyond perfluorooctane sulfonate and perfluorooctanoic acid to the broader class o f perfluormated chemicals which includes fluorotelomer alcohols [FTQHs], 8-2 Telomer B Alcohol [8-2 TBA, F(CF2)8CH2CH20H, CAS# 678-39-7] is one o f the raw material intermediates used in the manufacture o f fluorotelomer-based products. Fluorotelomer: alcohols are comprised o f an even number o f fluorinated carbons appended to an ethanol moiety.* The fluorotelomer functionality in \ j products delivers unique surface modification properties including water and oil repellency (polymeric products) and wetting and leveling (surfactants) (8- 10). h The environmental fate and routes into the environment o f fluorotelomer-based substances is still largely unknown. Biodegradation is one transformation route that will determine the fate o f 8-2 TBA in the environment. A limited number o f experimental investigations have been conducted to investigate microbial biodegradation o f FTOHs and telomer-based substances (11-16). The same is true regarding metabolism in living systems and pharmacokinetics (17, 18). Potential transformation pathways have been reported from predictive models based upon known abiotic and biotic functional group transformations and their probabilities (5). A fter submission o f the initial draft o f this study, a new paper was published (19), which reports the biodegradation o f non-radioisotope labeled 8-2 Telomer B Alcohol by microbial enrichment culture. In this study, the authors quantified three known transformation products, 8-2 saturated acid, 8-2 unsaturated acid, and PFOA mid found that 8-2 3 Subject to Copyright. Do not reference. Not for further reproduction unsaturated acid is the major metabolite and PFOA accounts for about 3% o f total mass at day-81. The authors conclude that the B-oxidation is the principal pathway that determines the fate o f 8-2 Telomer B Alcohol. In this study, PFOA concentration also decreased significantly from about 6% o f total mass at day 51 to 3% at day 81. Because the above studies in general were limited to identification and quantification o f transformation products already known from prior literature or predictive models, potential unknown transformation products were not identified or quantified. Hence, the transformation pathway(s) and stable transformation products remain unclear. In addition, while widely believed to be the major transformation product from microbiological transformation o f 8-2 TBA, perfluorooctanoic acid, F(CF2)7COOH, was only identified in small quantities. 8-2 TBA is highly surface active, sorptive (Unpublished results by Telomer Research Program), and both hydro- and oleophobic (20). Additionally, 8-2 TBA has exhibited unique physical-chemical properties which make it a challenging test substance. As a result, test substance bioavailability and partitioning in the test system presents real challenges. Headspace migration, strong, potentially(irreversible sorption, and rapid migration to test system surfaces have been reported (20, 21). Finaliy, feesisolation, identification and accurate quantitation o f the transformation product(s) as well as obtainingHauthentic standards o f them has been limited thus far by testing o f non-radiolabeled material. We report here the first experimental biodegradation study to achieve mass balance which more clearly defines the biological fete o f a fluorotelomer alcohol. To achieve mass balance and overcome identification and quantitation problems, a custom synthesized 14C-labeled 8-2 TBA F(CF2)714CF2CH2CH2QH], was used to investigated the biodegradation potential and transformation products after incubation wife activated sludge inoculums from a domestic waste water treatment plant The use o f 14C-labeled material allowed us to quantify and identify unequivocally potential transformation products down to a LOQ o f 0.5% o f initial 14C applied to the experimental system. The objective o f this study is to assess whether 8-2 TBA can be readily transformed aerobically and if so, discern fee likely biotransfoimation pathway(s) and products. 4 Subject to Copyright. Do not reference. Not for further reproduction Experimental Section M aterials and M ethods. Non-radioisotope labeled 8-2 TBA (CF3(CF2)6CF2CH2CH20H, 8-2 TBA) was from TRP (Telomer Research Program) and had a 99.9% purity. The 14C-labe! 8-2 TBA (CF3(CF2)614CF2CH2CH2OH) was custom synthesized with a radiochemical purity o f virtually 100%. No 14C impurity was detected with a LOQ o f ~ 0.02% as analyzed by HPLC coupled with a ,4C flow through detector. The specific activity was 54.3 mCi mmol'1based on 14C counts on a mass basis and was confirmed by gas chromatography-mass spectrometry (GC/MS) analysis to be within 2% difference. The chemical purity was 93% with CF3(CF2)6CH2CH20H as the major impurity. The R e label 8-2 TBA was dissolved in absolute ethanol (purity = 99.97%, Aldrich Chemical Co., Milwaukee, Wisconsin, USA) to about 2.2 mCi mL'1ethanol as the stock solution and was stored at -- 10C. The positive control reference chemical aniline-HCl (C6H7N-HCI) was from Sigma Chemical Co. (St. Louis, USA) with a purity o f 99.6% and uniformly labeled aniline-HCl (^ C ^ N -H C l or l4C(U)-aniline-HCl) was from Moravek Biochemicals (Brea, USA) with a specific activity o f 78 mOis;mmol' 1 and radiochemical purity o f 99.1%, Three milliliters o f 1 g aniline-HCl L"1 aqueous solution wa&addeditdif>' 0.28 mCi 14C(U)-aniline-HCl solids to give a final concentration o f approx. 1:2 g L"1 as the stock solution. All other solvents used were purity > 99% and all other chemicals were reagent grade or higher. The water used throughout die experiment was deionized water (~17.6 M2-cm) from Bamstead E-pure system. Activated sludge (2 L in a 4-L container) was collected the same day the experiment was initiated from the City o f Wilmington (Delaware, USA) Municipal Waste Water Treatment Facility - Aeration Basin #2. After arriving at the lab, the sludge was mixed by shaking to suspend die microorganisms. After settling for approx. 14 min, the upper aqueous phase o f the sludge was used as the inoculums or was autoclaved and then used as abiotic control. Experim ental System. Because o f the semi-volatile nature o f the starting material (20), a sealed test system was used to prevent loss o f 14C-labeled 8-2 TBA due to volatilization. Glass serum bottles (120-mL volume) were used as the test vessels. The mineral medium used in this study was composed o f 8.5 mg L' 1 o f KH2P 04, 21.8 mg L' 1 o f K2H P04, 33.4 mg L' 1 o f Na2H P04-2H20 , 0.5 mg L' 1 5 Subject to Copyright. Do not reference. Not for further reproduction o f NH4CL, 36.4 mg L'1 o f CaCl2-2H20 , 22.5 mg L' 1 o f MgSQ4-7H20 , and 0.25 mg L"1 o f FeGl3-6H20 , and the pH was adjusted to 7.0 (22). The test and appropriate control media were prepared in 0.5 to 1 L polypropylene containers and then dispersed with glass pipettes into individual serum bottles. Five different treatments were included in the test design to assure study integrity. 1) Biodegradation test vessels (4 replicates at each sampling time point): contain 30 mL test medium composed o f activated sludge (0.5%), 0.48 pL 14C-label 8-2 TBA stock solution, and about % saturated (~ 80 pg L*1) 8-2 TBA solution made in mineral medium. 2) Abiotic control vessels (4 replicates at each sampling time point): contain autoclaved sludge (0.5%) plus 0.5 mM NaCN were substituted the live sludge and the rest o f the components were identical as in biodegradation test vessels. 3) Spike recovery vessels (4 replicates at each sampling time point): contain 30-ml test medium with activated sludge (0.5%) in mineral medium, 5 mL o f which was withdrawn for fluoride analysis before being spiked (dosed) at each sampling time point (days 0, 7, 14, and 28) with 50 pL o f 226 mg L' 1 14C-labeled 8-2 TBA and 0.5 mL o f 4 mg L"1 ; > freshly prepared standard fluorinated acids, CF3(CF2)6CF2CH2CC)OH (8-2 saturated acid), 1 .-. KR jdfsdif'C F3(CF2)6CFK:HCOOH (8-2 unsaturated acid), CF3(CF2)6COOH (PFOA), and CF3(CF2)4CO0H (PFHA). The 14C-labeled spike solution (226 mg L '1) was prepared by mixing 2 mL o f 150 mg 8-2 TBA L '1 in ethanol and 8 pL original 14C-label 8-2 TBA stock solution (specific activity --54.3 mCi mmol"1). 4) Sample matrix vessels (2 replicates at each sampling time point): contain 30-ml test medium with activated sludge (0.5%) in mineral medium to be used as a blank solution for LC/MS/MS analysis o f spiked fluorinated acids. 5) 14C(U)-anilme-HCl positive control vessels (2 replicates at each sampling time point) to assess the inoculum microbial activity over time: contain 30-ml test medium with activated sludge (0.5%) in mineral medium plus 30 pL o f 1.2 g 14C(U)-aniline-HCl L"1 stock solution. Test Conditions and Sampling. After each test vessel was filled with appropriate test medium, the bottle was crimp-sealed with a PTFE septum (pre-washed with methanol and sterile water)/aluminum cap. It should be noted that the biodegradation system in this study contained only about 13 mg L "1 organic carbon, which included the microbial inoculum and the ethanol as a co-solvent 6 Subject to Copyright. Do not reference. Not for further reproduction to disperse the starting material. This low amount o f organic carbon plus enough headspace (90 mL air) assured aerobic conditions in sealed serum bottles. The diluted sludge (0.5%, 200-fold dilution) contributed to about 1 mg organic carbon Lf1. The inoculum level was also low (estimated to be approx. 105 bacterial cells per bottle), so that the test conditions were comparable to the "Ready Biodegradation" test according to the study guide provided by Organization for Economic Co-Operation and Development (22). All the glass serum bottles were shaken at 250 rpm in an environmental incubator in the dark and at room temperature (ranged 25 to 27.7 C monitored by a Dickson chart recorder during 28 days). The 90-mL air space inside a vessel ensured an aerobic environment. The test vessels from different experimental treatments were sampled at days 0, 7, 14, and 28 for extraction and analysis. Sample E xtraction and Analysis. To quantify the initial concentration o f the starting material, triplicate 30 mL medium from the 1 L polypropylene bottles for treatments l and 2 were extracted with i 50 mL MTBE-0.1 M H2SO4 for 1.5 h and the MTBE phase (top phase o f the extract) was used for >H quantification o f 8-2 TBA and 14C-labeled 8-2 TBA by GC/MS ias described (23). On days 0, 7, 14, >and 28, 5 mL o f the test medium from treatments 1, 2, 3, and>4 above was withdrawn from each o f the serum bottles with a syringe through a 26-gauge needle and was treated with 50 pL o f 5N NaOH first and then neutralized with 42 pL o f 6N H2SO4 for fluoride analysis. The remaining 25 mL medium in each bottle was extracted with 45 mL o f acetonitrile (injected into the sealed bottle) for approx. 1 h at room temperature (first extraction). Due to possible injection wound (piercing damage) to the septa, some o f the bottles were then re-sealed with fresh PFTE septa/aluminum cap to assure an air-tight seal during long-term storage at -- 10 C. The acetonitrile extract from each sample bottle was used for liquid scintillation counting o f 14C, for quantification and identification o f 14C-labeled transformation products using LC/ARC, and for LC/MS/MS analysis o f fluorinated acids. To measure the mineralization o f l4C(U)-aniline-HCl by the inoculum (Treatment 5), 0.1 mL aliquot from the serum bottles was mixed with 0.2 mL o f 0.1 M HCl to remove 14C(>2 remaining in the medium and 5 mL o f 7 Subject to Copyright. Do not reference. Not for further reproduction scintillation cocktail was added to the 0.3 mL mixed solution for 14C counting by a liquid scintillation counter. It was found later that initial 14C counts o f the acetonitrile extract (from first extraction) did not account for all 14C applied as 14C-labeled 8-2 TBA at day 0. For this reason, a second sequential extraction was carried out for the serum bottles and septa separately. Each sample bottle was re extracted with 10 mL acetonitrile for 3 to 4 h at room temperature after rinsing 2 times with deionized water, and each PTFE septum from individual sample serum bottles, if saved, was re-extracted with 10 mL acetonitrile 4 to 5 times at 50C (each extraction lasted 4 to 7 days) and 14C activity was measured for mass balance calculation. Also, some o f the acetonitrile solution was pooled from each sample for LC/ARC analysis to confirm whether extended extraction at 50C caused any degradation o f ,4C-labeled 8-2 TBA. As mentioned above, some o f die original septa (piercing damaged) were not saved for the re-extraction. Fluoride ion was measured by fluoride-selective electrode after mixing the sample.with equal volume (5 mL) o f Thermo Orion TISABII buffer (15) using 5 to 500 pg NaF L"1 as standard calibration. Fluorinated acids in the spiked samples were quantified by iLC/MS/MS in negative electrospray ionization mode by a Waters 2795 HPLC/Micromass Quattro Micro system. The 10-50 pg L 1calibration standards were made in sample matrix blank (from Treatment 4) and the injection volume was 10 pL. The HPLC column used is a reverse phase Zorbax Rx-Cs (150 x 2.1 mm, 5 pm particle size. Mobile phase was 0.15% acetic acid/acetonitrile in a gradient with a flow rate o f 0.4 mL m in'1. Multiple reaction monitoring (313 > 269 for CF3(CF2)4COOH, 413 > 369 for CF3(CF2)6COOH, 457 > 393 for CF3(CF2)6CF=CHCOOH, and 477 > 393 for CF3(CF2)6CF2CH2COOH) was used for compound quantification. Analysis o f 14C-labeled 8-2 TBA and 14C-labeled Transform ation Products by LC/ARC. Original acetonitrile-extracted samples from Treatment 1 and 2 in sealed glass GC vials were diluted 1:1 with 2 mM ammonium acetate in water. The diluted samples were analyzed by LC/ARC to quantify 14C-labeled 8-2 TBA and 14C-labeled transformation products by integrating radioactivity o f apparent 8 Subject to Copyright. Do not reference. Not for further reproduction (baseline-resolved) chromatographic peaks during the separation. The LC/ARC system (AIM Research Company, Newark, USA) utilizes advanced stop flow counting technologies to accurately detect and quantify radioisotope with a sensitivity o f several CPM (counts per minute) above background level (24). The initial analysis (LC/ARC Method 1) used a Zprbax Rx-Ci8 column (4.6 mm x 150 mm, 5 pm particle size) and mobile phase was 2 mM ammonium acetate/acetonitrile in a gradient with a flow rate o f 1.0 mL min-1. The injection volume was 0.5 mL, the fraction size was 10 sec, and the 14C counting time was 120 sec for each fraction. The initial analysis (LC/ARC Method 1) was unable to separate CF3(CF2)614CF2CH2COOH from CF3(CF2)614C F^H C O O H to give individual transformation product peaks. A second chromatographic method (LC/ARC Method 2) was used, with 0,15% acetic acid/acetonitrile as mobile phase, to further separate and quantify the two 14C-labeled transfoimation products. Identification of 14C-IabeIed Transform ation Products by LC/MS Analysis. A HPLC (Alliance HT, Model 2795, Waters)/Q-TOF (Micromass) system in negative.electrospray ionization mode was used to identify the transformation products observed with the LC/ARC system. The chromatographic conditions o f the LC/MS analysis were kept essentially the .same as for LC/ARC analysis in order to match the retention times o f the observed signals in the LC/MS and LC/ARC analysis as closely as possible. The LC/MS analysis was done off-line from the LC/ARC system. A Zorbax Rx-Cis (2.1 mm x 150 mm, 5 pm particle size) column was used with a gradient mobile phase ( 0,15% acetic acid/acetonitrile) and a flow rate o f 0.25 mL m in'1; the lower flow rate was used to compensate for column diameter change from 4.6 mm to 2.1 mm and was necessary for proper electrospray operation. Other MS parameters are as follows: capillary voltage o f 2.5 kV, cone voltage o f 10 V, source temperature o f 120C, and desolvation temperature o f 350C. The transformation product identification was based on matching the retention time, observed molecular ion, daughter ion spectrum, and accurate mass measurement o f the observed transformation product with that o f respective standards. The retention time regions o f the LC/MS chromatograms where the LC/ARC 9 Subject to Copyright. Do not reference. Not for further reproduction peaks were observed were carefully examined to identify transformation products that could not be matched with available standards. I4C R adioactivity M easurem ent By L iquid Scintillation C ounting. Throughout the study, 5 mL aliquots o f liquid scintillation cocktail (Packard BioScience Ultima Gold XR) were mixed with processed samples for 14C radioactivity counting by a liquid scintillation counter (Beckman LS 5000TD). The counting time was normally 10 min to reduce counting error to within 2% except for samples with only background or close to a background level o f 14C radioactivity. Typical counting efficiency was >95% and the final radioactivity was automatically calculated and reported as DPM based on: (CPM/eounting efficiency) --DPM. Results and Discussion The measured starting concentration o f the parent material [CF3(CF2)6l4CF2CH2CH2OH plus non labeled CF3(CF2)6CF2CH2CH2QH3 was 337 13 pg L"1 for the test vessels and 306 9 jig L '1 for abiotic control vessels, which is about 2.3-fold above 8-2 TBA solubility limit (20). The purpose o f using the concentration above the solubility o f the test chemical is to increase the sensitivitysdortv m < detecting the 14C-labeled transformation products. Based on the amount o f 14C counts available to each < >w test vessel after the sample extraction, the LOQ for l4C-labeled transformation products was approx. 0.5% o f the initial 14C counts applied. The recoveries o f the starting material and fluorinated acid standards that had been spiked (dosed) into the test medium were acceptable. The recoveries for spiked test medium averaged 92 7% for 14C-labeled 8-2 TBA, 100 20% for CF3(CF2)6CF2CH2COOH, 81 21% for CF3(CF2)6CF=CHCOOH, 104 14% for CF3(CF2)6COOH, and 100 20% for CF3(CF2)4COOH (n = 16 for day 0 ,7 ,1 4 , and 28 samples). The reference compound, 14C-labeled aniline-HCl with a starting concentration o f approx. 1.2 mg L '1, degraded over time, with 45% o f total l4C counts remaining at day 7 compared with that o f day 0,41% at day 14, and 29% at day 28 ( = 2 samples), indicating that more than 70% o f the 14C-labeled aniline-HCl had been mineralized. CO2 evolution in excess o f 50% o f theoretical is interpreted as complete or near-complete biodegradation o f a test chemical because a substantial portion o f 14C 10 Subject to Copyright. Do not reference. Not for further reproduction applied was incorporated into the structural components o f the microbes (25). This suggests that the sludge inoculums used in this study were metabolically active during the test Form ation of B iotransform ation Products. Under the test conditions with live microbial inoculums, the starting material (parent) was readily transformed to various transformation products (Figure 1). The LC/ARC Method 1 (Figure l A, left column) was initially used to separate and quantify the 14C-labeled transformation products. As shown in Figure 1A, at day 28, one major peak with a retention time (tn) o f 21.6 min emerged in addition to the parent (peak 5, tn --33,6 min) and two additional smaller peaks (peaks 1 and 4) with retention times o f approx. 20 and 23 min are clearly visible. For the abiotic control, only the parent was observed at day 28. These results demonstrate that the formation o f transformation products was due to microbial activity. An LC/MS analysis using analogous chromatographic conditions revealed that the major peak (t,, = 21.6 in Figure 1A) contained two 14C-labeled transformation products. A second LC/ARC method, Method 2, was developed that allowed efficient separation and quantification o f all observed transformation products (Figure IB, right column). Identification of B iotransform ation Products. The identification o f biotransfoEmation products was conducted using negative ion electrospray ionization on an LC/Q-TOF MS system and applying chromatographic conditions matching those used on the LC-ARC system. Figure 2 shows the daughter ion spectra o f major transformation products (LC/ARC peaks 1 ,2 ,3 , and 4 o f figure IB). The identity o f transformation products (metabolites) 1, 2, 3 was elucidated by matching the retention times and daughter ion spectra o f the respective authentic standards [CF3(CF2)6COOH, CF3(CF2)6CF=CHCOOH, and CF3(CF2)6CF2CH2COCiH] with those o f the 14C-labeled transformation products. The peaks 1, 2, and 3 were identified as CF3(CF2)6,4COOH, CF3(CF2)6l4CF2CH2COOH, and CF3(CF2)614CFK:HCOOH, respectively. The daughter ion spectra o f identified transformation products are presented in Figure 2A-C. The positive identification was based on that die daughter ion spectrum o f a metabolite from the sample extract is essentially identical to that o f a corresponding standard. The match o f the spectrum o f an unknown metabolite and a standard is evaluated by the 11 Subject to Copyright. Do not reference. Not for further reproduction comparison o f observed masses o f fragment ions in the daughter ion spectrum and their relative intensities in the spectrum. Taking into account that observed metabolites contain one 14C atom and therefore, some o f the resulting fragment ions would be shifted two mass units. Full agreement was observed between the ions in the daughter ion spectrum o f metabolites and non-laheled standards as well as their relative intensities. Further conformation o f the observed transformation products structures was obtained by accurate mass measurements using LC/Q-TOF MS (Table 1). The measured masses agreed well with the calculated masses for the proposed elemental composition o f transformation products, especially for the two transformation products (LC/ARC peaks 2 and 3 o f Figure IB) observed at higher concentration. The accurate mass measurement for CF3(CF2)6COOH exhibited highest error, outside o f the typically accepted level o f 5 ppm. The Signal for this transformation product was very weak, which in turn affected the accuracy o f the mass measurement. The above three transformation products had also been reported in blood plasma o f male rats after a single oral dose (11), in mixed bacterial cultures (15) and in enrichment culture dosed with 8-2 TBA (19), and are also predicted by the CATABOL software (5). This suggests that common biodegradation pathways for 8-2 TBA may be shared between animals and microorganisms. Accurate mass measurement for the ion m/z 443 (LC/ARC peak 4 o f Figure IB) pointed to an elemental composition o f the deprotonated molecular ion containing four hydrogens (Table 1). Further evidence o f the presence o f four hydrogens in the molecule was obtained from the daughter ion spectrum o f ion m/z 443 (Figure 2D). The ions observed in the daughter ion spectrum o f m/z 443 can be rationalized as follows: m/z 339 represents loss o f CO2 (molecular weight = 44) and o f 3 HF (3 * 20 - 60) from die deprotonated molecular ion (m/z 443) and m/z 319 represents additional loss o f HF from ion m/z 339. The fragmentation pattern o f m/Z 443 closely resembles the fragmentation patterns observed for standards CF3(CF2)6CF2CH2COOH and CF3(CF2)6CFK:HC00H that would predominantly form ions resulting from losses o f CO2 and HF from the deprotonated molecular ion. However, it is not possible from the daughter ion spectrum o f m/z 443 alone to assign the placement o f 12 Subject to Copyright. Do not reference. Not for further reproduction the four hydrogens in the carbon chain o f the molecule. The structure for peak 4 was postulated as: CF3(CF2)614CH2CH2COOH, 2H, 2H, 3H, 3H-perfluorodecanoic acid, a transformation product that has not been reported in the literature previously. A standard for the postulated structure is not commercially available. Further support o f the proposed structure was obtained from studies o f a commercially available analog: 2H, 2H, 3H, 3Hnonanoic acid (CF3(CF2)5CH2CH2COOH). Detailed comparison o f 19F-MMR spectra, LC/MS daughter ion spectra, and electron impact (El) GC/MS spectra o f methyl and trimethyl silyl (IM S) derivatives o f the transformation product (peak 4) derived from biodegradation studies conducted with non-labeled 8 2 TEA and the analog 2H, 2H, 3H, 3H-nonanoic acid clearly supports the proposed structure for the unknown peak 4 (unpublished results). The quantities o f the unknown compound (peak 4) generated in this study were not sufficient to obtain the above data. However, a very good match was obtained for the retention time and the daughter ion spectrum o f the metabolite obtained from a non-labeled 8-2 TBA biodegradation study and the peak 4 data from this study. However, matching all the spectral data obtained for die peak 4 metabolite with that o f an authentic standard (CFslCE^eGHzCHjGGOH) is still required for a final structural confirmation o f this new transformation product:; n. Among the unidentified 14C-Iabeled transformation products, no base-line resolved LC/ARC peak corresponding to 14C-labeled perfluorononanoic acid (CF3(CF2)614CF2COOH) was visible, suggesting that a-oxidation (26) o f CF3(CF2)614CF2CH2COOH, the major transformation product, did not occur. This is consistent with the result o f Dinglasan et al. (19), in which CF3(CF2)6CF2COOH and CF3(CF2)4CF2COOH were not detected in the experiment system. This conveys the notion that potential biodegradation o f fluorotelomers in the environment may not lead to the formation o f significant amount o f odd-numbered perfluorinated carboxylic acids such as perfluorononanoic acid due to microbial a-oxidation. M ass Balance. Compared with day 0, the total 14C radioactivity in the acetonitrile extract (from the first extraction) decreased continuously over 28 days in both the biodegradation test vessels and abiotic controls (Table 2). It was initially thought that the decrease was due to both adsorption to the 13 Subject to Copyright. Do not reference. Not for further reproduction test vessel surface and volatilization o f the parent. However, after analyzing the 14C counts recovered from the glass surface and PTFE septa, it was shown that the decrease was actually caused by the strong adsorption o f the parent to the PTFE septa o f the sealed vessels. The l4C recovered from the septa was 8-2 TBA. After taking into account the 14C counts recovered from the septa, the mass balance o f the parent plus all transformation products was nearly 100% (Table 2). This observation demonstrates that the potential volatilization o f 8-2 TBA from the experimental systems was minimized at the presence o f a strongly adsorbing matrix such as PTFE; otherwise, it is impossible to achieve a near 100% mass balance. The adsorption o f 14C-labeled 8-2 TBA to the glass was minimal or at least was reversible as it can be easily recovered by acetonitrile at room temperature (Table 2). In this regard, although some o f the original septa (days 14 and 28 for the biodegradation test vessels and day 28 for the abiotic control) were not saved after the first acetonitrile extraction for subsequent re-extraction and I4C counting, it is reasonable to assume that most o f die 14C counts not recovered from the first extraction also reside in these septa. Also, available septum I4C counts data (Table 2) indicate that the degree o f septum adsorption o f 8-2 TBA increased over time. The adsorption to the PTFE septa was so strong (that it took about 4 weeks at 50 C to recover most o f the 14C counts from the septa using acetonitrile. The parent, CF3(CF2)614CF2CH2CH20H, accounted for virtually all 14C counts recovered from the septa as revealed by LC/ARC analysis, indicating 8-2 TBA was remarkably stable abiotically in aqueous solution under moderately high temperature. Based on the 14C counts o f individual transformation products versus the total 14C applied at day 0, CF3(CF2)614CF2CH2COOH is clearly the most abundant transformation product accounting for about 27% o f the mass balance at day 28 (Table 2). The CF3(CF2)614CF=CHCOOH was the second major transformation product accounting for about 6.0% o f the mass balance at day 28. In contrast, CF3(CF2)6CF=CHCOOH was found to be the predominant metabolite in mixed bacterial cultures (15) and in microbial enrichment culture (19), in which a much higher bacterial density (microbial loading) in the test media and more favorable conditions may accelerate the conversion o f F 3(CF2)6CF2CH2COOH to CF3(CF2)6CF=CHCOOH, in comparison with the very low bacterial loading and organic carbon supply used in this study. The newly identified 14 Subject to Copyright. Do not reference. Not for further reproduction transformation product, CF3(CF2)614CH2CH2COOH accounted for about 2.3%, and CF3(CF2)6-14COOH accounted for about 2.1% o f the mass balance at day 28. The parent still contribute about 57% o f the mass balance at day 28, about 41% o f which resulted from adsorption to the septa. It appears that the strong adsorption o f die parent to the PTFE septa during the test reduced its bioavailability for microbial biodegradation. D efluorination o f 14C-Iabeled 8-2 TBA during the Biotransform ation. Under the test conditions, the increase o f fluoride ion due to defluorination o f the parent was not discernible from the background level o f fluoride present in the test vessels. The level o f calculated fluoride increase (0.9 pg L '1 at day 7, 1.4 jig L"1 at day 14, and 2.0 jig L '1 at day 28 compared with day 0) based on the biotransformation products formed is low compared with that o f the background level (day 0 level) o f fluoride in the test vessels (13.7 1.0 pg L '1 for n = 4 samples), and, thus, made the increase indistinguishable from the background. This observation was consistent with the result that in' CF3(CF2)6I4CF2CH2COOH is the most abundant ,transformation product, whose formation does not ; mat v Kvr involve defluorination. On the other hand, a significant increase o f fluoride ion concentration was observed when CF3(CF2)eCF=CHCOOH became a major transformation product, which involved defluorination o f the non-labeled 8-2 TBA (15). B iotransform ation Pathw ays of 14C labeled 8-2 TBA. Several pathways may be available to convert the parent, CF3(CF2)614CF2CH2CH2OH, to various observed transformation products (Figure 3). The first pathway for CF3(CF2)6I4CF2CH2CH20H transformation is conversion to CF3(CF2)614COOH most likely via HF elimination (19) and monooxygenase-mediated reactions. First, the parent can be oxidized to the major transformation product, CF3(CF2)614CF2CH2COOH (I), via alcohol and aldehyde dehydrogenase reactions. No 14C-labeled fluoroaldehyde (CF3(CF2)614CF2CH2CHO) was detected with a LOQ o f 0.5% o f total mass, indicating that the fluoroaldehyde is either unstable or may be quickly oxidized by an aldehyde dehydrogenase before day 7 sampling. The fluoroaldehyde was detected as a transient intermediate (19) and its level cannot be quantified, indicating that the observed level may be low. After forming CF3(CF2)614CF2CH2COOH, this acid may be further converted in multiple steps or 15 Subject to Copyright. Do not reference. Not for further reproduction via HF elimination to form CF3(CF2)6t4CF=CHCOOH (II) and then potentially to CF3(CF2)eJ4COOH perhaps catalyzed by monooxygenase-mediated reactions, as also predicted by CATABOL (5). Both I and II are the principal stable transformation products and do not rapidly degrade under the test conditions. A second transformation pathway may involve the 6-oxidation o f the newly identified transformation product, CF3(CF2)6l4CH2CH2COOH (HI). First, the 14C-labeled 8-2 TBA parent may be converted in multiple enzymatic reactions to form CF3(CF2)614CH2CH2COOH, although the mechanisms o f these reactions are currently unknown. Incubation o f 2 mg L '1 o f CF3(CF2)6CF2CH2COOH in mixed bacterial cultures resulted in the formation o f only trace amount o f CF3(CF2)6CH2CH2COOH (N Wang and B Szostek, unpublished results) , suggesting that some other transformation products may also contribute the formation o f CF3(CF2)6l4CH2CH2COOH via multiple reactions. Because CF3(CF2)614CH2CH2COOH is a likely fatty acid analog, it can be further oxidized via B-oxidation reactions. However, direct experimental evidence is still needed to further confirm this hypothesis. .. Although long-chain fluorinated carboxylic acids can induce the proliferation o f peroxisomal fl- oxidation in rats and mice (27), it is not clear whether such acids can be directly used as substrates for the B-oxidation reactions (28, 29). It has been proposed that 8-2 saturated acid and 8-2 unsaturated acid can be used as substrates for B-oxidation to form PFOA and the B-oxidation pathway may be a principal fete o f fluorotelomers such as 8-2 Telomer B Alcohol (19). However, based on current knowledge regarding the enzymology o f this pathway, a direct B-oxidation o f 8-2 saturated acid and 8-2 unsaturated acid cannot occur. The B-oxidation pathway for fatty acids is well understood and is illustrated by Nelson and Cox (29). As Nelson and Cox have stated, in one pass through the B-oxidation sequence, one molecule o f acetyl-CoA, two pairs o f electrons, and four protons (from C2 and C3 carbons) are removed from the long-chain aeyl-CoA, shortening it by two carbon atoms. Because 8-2 saturated acid contains no H atoms in its B-carbon (C3 carbon) and 8-2 unsaturated acid contains only one H atom, 6- oxidation o f these two acids cannot occur because the proton deficiency prevents the proton/electron 16 Subject to Copyright. Do not reference. Not for further reproduction shuffling that is essential for the completion o f the reactions. Also, since the 8-carbon o f 8-2 saturated acid is already highly oxidized with two fluorine atoms attached, it is unlikely that any direct oxidation o f this carbon can occur under aerobic conditions. On the other hand, the B-oxidation may occur via the newly discovered metabolite, 2H, 2H, 3H, 3H-perfluorodecanoic acid. The third possible transformation pathway may be related to the formation o f unidentified transformation products. Since each o f the unidentified transformation products in general contributed less than 1% o f the mass balance, it is extremely difficult to identify their molecular structures. However, formation o f these transformation products may implicate some new pathways not yet understood. Compared with perfluorinated acids, the ethanol spacer (-CH2CH2OH) o f CF3(CF2)614CF2CH2CH2OH makes this molecule more flexible than a perfluorinated acid and could enhance its molecular accessibility to an active site o f a given enzyme. This, in turn, could lead to the formation o f certain unique metabolites that would not normally occur when a folly fluorinated acid is being used as. a starting material for microbial biodegradation. ..In mixed;bacterial cultures, CF3(CF2)6CF2CH2CH2OH can be converted to perfluorohexanoic acid [CF3(CF2)4COOH] with about 0.4% o f the mass balance (15), implying a mechanism for defluorination from the perfluorinated portion o f C-F bonds. When CF3(CF2)614CF2CH2CH20H was incubated with a high concentration (330mL sludge L"1medium) o f activated sludge from the same source as for this study, 14CC>2release contributed to about 0.4% o f the mass balance, indicating a decarboxylation mechanism from the fluorinated 13carbon. In microbial enrichment culture (19), all the metabolites (8-2 saturated and 8-2 unsaturated acids plus PFOA) together accounted for 55% o f total mass balance at day 81. The portion o f the missing part (45%) may include the metabolites that resulted from the degradation o f 8-2 unsaturated acid by some other unknown mechanisms. Also, the PFOA level reduced significantly at day 81 (3% o f total mass) compared with at day 52 (approx. 6% o f total mass) (19). Does this indicate that PFOA may have been further transformed by the microbial culture? All above evidences so far raise the possibility that alternative pathways may be available for degradation o f 8-2 TEA beyond PFOA. Therefore, multiple pathways and enzymes, such as B-oxidation o f 2H, 2H, 3H, 3H-perfluorodecanoic acid, HF 17 Subject to Copyright. Do not reference. Not for further reproduction elimination and monooxygenase-catalyzed oxidation o f 8-2 saturated and 8-2 unsaturated adds, and other unknown mechanisms, may determine the biological fete o f 8-2 Telomer B Alcohol under the study conditions. This report indicates that additional work needs to be done to clarify the prevalence and availability o fthe pathways identified and their products in a range o f microbial cultures, conditions and time frames to truly define the ultimate, stable, microbial biodegradation products. Acknowledgements We thank Drs. Theodore H. Carski, Robert A. Hoke, Mary A. Kaiser, S. Mark Kennedy, and Watze de W olf for consultation and manuscript preparation and Kim Brebner, Keith Prickett, Richard Rossi, and Scott Swain for quality assurance and technical assistance. This research was partially funded by Telomer Research Program, with member companies include Asahi Glass Co., Ltd. (Japan), Clariant GmbH (Germany), Daikin Industries, Ltd. (Japan), and DuPont (USA). 18 Subject to Copyright. Do not reference. Not for further reproduction TABLE 1 Accurate Mass Measurements o f Observed Transformation Products determined 14C-labeled transformation product mass ion 478.9803 CF3(CF2)6I4CF2CH2COO- (Peak 2) 458.9764 CF3(CF2)614CF=CHCOO- (Peak 3) 414.9771 CF3(CF2)614COO- (Peak 1) 443.0037 CF3(CF2)614CH2CH2COO_ (Peak 4) elemental composition 12C9l4C H 20 2Fi7 i2C914C H 0 2Fi6 12C714C 0 2F,5 ,2C914C H 402Fl5 calculated mass 478.9816 458.9753 414.9691 443.0004 error in ppm -2.6 +2.3 +19.3 +7.4 19 Subject to Copyright. Do not reference. Not for further reproduction TABLE 2 Mass Balance o f 14C-labeled 8-2 TB and Quantified Transformation Products Compound L Test vessels First Acetonitrile Extraction CF3(CF2)614CF2CH2COCr (Peak 2) CF3(CF2)6I4CF=CHCOO~ (Peak 3) CF3(CF2)614COCr (Peak 1) CP3(CF2)6I4CH2CH2COO- (Peak 4) Unidentified transformation products CF3(CF2)614CF2CH2CH2OH (Parent; Peak 5) Second Acetonitrile Extraction CF3(CF2)614CF2CH2CH2OH (Adsorbed to septa) CF3(CF2)614CF2CH2CH2OH (Adsorbed to glass) Sum (Total label) Sum o f CF3(CF2)614CF2CH2CH2OH (Parent) Day 0 NDb NDb NDb NDb NDb 99 0.3 0.3 0.2 0.7 0.3 100 100 Day 7 Day 14 Day 28 % of total initial mass a 20.7 2.4 3.0 1.1 0.8 0.2 0.8 0.1 3.9 0.8 47.4 4.9 24.8 1.7 4.6 0 .7 1.1 0.1 1.6 0.1 6.0 0,4 27.0 1.8 26.5 3.6 5.9 0.2 2.1 0 .4 2.3 0.2 6,9 0.6 16.0 2.6 23.5 3.1 0.8 0.3 101 72 34,3C 0.6 0.1 100c 62 40.8C 0.5 0.0 100c 57 27. Abiotic control First Acetonitrile Extraction CF3(CF2)6l4CF2CH2CH2OH Second Acetonitrile Extraction CF3(CF2)6l4CF2CH2CH2OH (Adsorbed to septa) CF3(CF2)6l4CF2CH2CH2OH (Adsorbed to glass) Sum (Total Label) 99 0.3 0.1 0.0 0.5 0.3 100 74.7 1.9 27.0 1.9 0.5 0.2 102 61.1 4 .0 49.2 3.6 37.5 3.3 0.3 0.1 99 51.4C 0.4 0.1 100c a The mass balance was calculated based on the 14C counts for the parent or individual transformation products at each sampling time points versus the I4C counts applied initially at day 0. All values represent mean SD (standard deviation) for n --4 samples unless otherwise stated. bNot detected. c Because the original septa after the first acetonitrile extraction were not saved for re-extraction (second extraction), no direct l4C counts data are available. However, available 14C counts data indicated a virtually 100% mass balance for day 7 test vessels and abiotic samples and day 14 abiotic samples. Thus a 100% mass balance was reasonably assumed for the rest o f samples and l4C counts in these septa were calculated based on a 100% mass balance. 20 Subject to Copyright. Do not reference. Not for further reproduction Figure Captions ' FIGURE 1. LC/ARC chromatograms o f the parent, CF3(CF2)614CF2CH2CH2OH (peak 5) and 14Clabeled transformation products (peak 1-4; peak 1: CF3(CF2)614COO`", peak 2: CF3(CF2)614CF2CH2COO", peak 3: CF3(CF2)6,4CF=CHCOO", and peak 4: CF3(CF2)6I4CH2CH 2C001 at different sampling time points using LC/ARC method 1 (A, left column) and method 2 (B, right column). FIGURE 2. Daughter ion spectra o f deprotonated molecular ions: m/z 415 (A), 479 (B), 459 (C), and 443 (D) obtained for transformation products observed in a day 28 sample. FIGURE 3. Proposed biotransformation pathways o f 14C-labeled 8-2 Telomer B Alcohol. The solid arrows indicate proposed transformation steps. The dotted arrows indicate potential transformation steps which may or may not be occurring. 21 Subject to Copyright. Do not reference. Not for further reproduction A . LC/ARC M ethod 1 B . LC/ARC M ethod 2 Figure 1 22 Subject to Copyright. Do not reference. Not for further reproduction 100 394.99 (LC/ARC Peak 1 - 415 m/z; Parent: CF3(CF2)414COO" ) (LC/ARC Peak 3 - 459 m/z; Parent: CF2(CF2)614C F =C H C 001 100 D m/z LC/ARC Peak 4 - 443 m/z; Parent: CF3(CF2)614CH2CH2C O ) Figure 2 23 Subject to Copyright. Do not reference. Not for further reproduction CF3(CF2)614CF2C H 2CH20 H --------- - - '----------------- -, i CF3(CF2)614CF2CH2COO"(I) , icf3(Cf2)614cf=chcoo"(n) - i CF3(CF2)6,4CH2CH2COO" (UI) -oxidation cf3(cf2>614coo" CF3(CF2)614CF2COCr(not observed) %. Figure 3 24 Subject to Copyright. Do not reference. Not for further reproduction Literature Cited (1) Moody, C. A., Field, J. A. 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