and Perfluorinated Acid Formation in Rainbow Trout - American

Jun 2, 2010 - 867 Lakeshore Road, Burlington, Ontario L7R 4A6, Canada. Received January 27, 2010. Revised manuscript received. May 9, 2010. Accepted ...
0 downloads 0 Views 998KB Size
Download PDF
Environ. Sci. Technol. 2010, 44, 4973–4980

Elucidating the Pathways of Polyand Perfluorinated Acid Formation in Rainbow Trout CRAIG M. BUTT,† DEREK C.G. MUIR,‡ A N D S C O T T A . M A B U R Y * ,† Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada, and Environment Canada, Water Science & Technology Directorate, 867 Lakeshore Road, Burlington, Ontario L7R 4A6, Canada

Received January 27, 2010. Revised manuscript received May 9, 2010. Accepted May 19, 2010.

Several studies have shown that fluorotelomer-based compounds can be metabolized to poly- and perfluorinated carboxylates, such as perfluorooctanoate (PFOA). Research has predominately focused on the 8:2 fluorotelomer alcohol (8:2 FTOH), however, the biotransformation pathway is not well understood. Specifically, there is uncertainty regarding the biological fate of the 8:2 fluorotelomer unsaturated carboxylate (FTUCA) and 7:3 fluorotelomer saturated carboxylate (FTCA). The objective of this study was to further elucidate the pathway for 8:2 FTOH biotransformation through dosing rainbow trout with three 8:2 FTOH metabolism intermediates: the 7:3 FTCA (CF3(CF2)6CH2CH2COO-), 8:2 FTCA (CF3(CF2)7CH2COO-), and 8:2 FTUCA (CF3(CF2)6CFdCHCOO-). This study represents the first investigation of these three labile intermediate metabolites in an in vivo system. The parent compounds were dosed via the diet and the parent compounds and intermediates were monitored in the blood and liver during the 7-day uptake phase and 10-day elimination phase. Exposure to the 7:3 FTCA did not result in the formation and accumulation of PFOA, but resulted in low levels of the 7:3 FTUCA and perfluoroheptanoate, a novel finding. PFOA was formed in the 8:2 FTCA and 8:2 FTUCA dosing. In addition, the 7:3 FTCA was formed during exposure to both the 8:2 FTCA and 8:2 FTUCA. Elimination half-lives were 5.1 d (95% confidence interval: 3.1-14 d) for 7:3 FTCA, 1.2 d (1.1-1.3 d) for 8:2 FTCA, and 0.39 d (0.31-0.53 d) for 8:2 FTUCA. The observed differences in the elimination half-life may be the result of differences in either the depuration or metabolism rate. Based on the findings of this study, and reported analogous literature pathways, we proposed a “betalike-oxidation” pathway for PFOA formation proceeding from the 8:2 FTUCA > 7:3 β-keto acid > 7:2 ketone > PFOA. Alternatively PFOA could be formed directly through the β-oxidation of the 7:3 β-keto acid.

Introduction In 2001, Giesy and Kannan (1) reported the worldwide dissemination of perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), and perfluorooctane sulfonamide (PFOSA) in wildlife. Additional research (2) demonstrated that wildlife * Corresponding author e-mail: [email protected]; phone: 416-978-1780. † University of Toronto. ‡ Environment Canada. 10.1021/es100702a

 2010 American Chemical Society

Published on Web 06/02/2010

were contaminated with a much broader suite of perfluoroalkyl compounds (PFCs), including long-chain perfluorinated carboxylates (PFCAs). However, since their initial identification in human (3) and wildlife tissues, the source of PFCs has remained elusive. The role of precursor compounds as potential PFC sources has been extensively studied. One mechanism is through precursor atmospheric oxidation (4). Laboratory studies have shown that fluorotelomer-based compounds, including alcohols (FTOHs, CF3(CF2)xCH2CH2OH) (5, 6), olefins (CF3(CF2)xCHdCH2) (7), iodides (CF3(CF2)xCH2CH2I) (8), and acrylates (CF3(CF2)xCH2CH2OC(O)CHdCH2) (9), as well as fluorinated sulfonamide alcohols (10, 11), form PFCAs (CF3(CF2)xCOO-). An additional mechanism is through the biotransformation of precursors. The original work by Hagen et al. (12) showed that the 8:2 FTOH was metabolized in rats to form the intermediate metabolites, fluorotelomer saturated (FTCAs, CF3(CF2)xCH2COO-) and unsaturated (FTUCAs, CF3(CF2)xCFdCHCOO-) carboxylates, as well as PFOA. In recent years, several additional studies have shown the formation of PFCAs and intermediate metabolites from 8:2 FTOH biotransformation, including microbes (13-17), in vivo studies with rats (18, 19) and mice (20, 21), and in vitro experiments using rat, mouse, trout, and human hepatocytes and microsomes (22). As well, Liu et al. recently investigated the fate of the 6:2 FTOH in soil and a mixed bacterial culture (23). Further, fluorotelomer-based compounds that are metabolized to FTOHs also have been shown to form PFCAs, such as the FTOH-based phosphates (PAPs) in rats (24) and the 8:2 FTOH acrylate in rainbow trout (25). There is considerable variability in the literature regarding the proposed FTOH biotransformation pathway. It is commonly reported that the first step is the oxidation of the alcohol group to the fluorotelomer aldehyde (FTAL), followed by FTAL oxidation to the FTCA. It is at this step that most biotransformation pathways deviate. In particular, there is uncertainty regarding the fate of the 7:3 FTCA. This metabolite has been observed in several 8:2 FTOH biotransformation studies (14, 15, 17-19, 22). Martin et al. (18), Fasano et al. (19), and earlier work by Wang et al. (14, 15) suggested that the 7:3 FTCA will form PFOA through β-oxidation. However, Nabb et al. (22) and the more recent work of Wang et al. (17) have suggested that PFOA is not formed when 7:3 FTCA is used as the substrate in hepatocytes and soils, respectively. The objective of this study was to further elucidate the biotransformation pathway of 8:2 FTOH in rainbow trout by means of individual dosing with three labile intermediate metabolites identified in previous 8:2 FTOH biotransformation studies: 8:2 FTCA, 8:2 FTUCA, and 7:3 FTCA. The test compounds were identified in a recent rainbow trout biotransformation study (25) using the 8:2 FTOH acrylate as the parent compound. They represent important branching points in the 8:2 FTOH degradation pathway in which there exists uncertainty in the literature. This is the first investigation of these compounds in an in vivo system.

Materials and Methods Chemicals Used. The 7:3 FTCA (97%) was obtained from Synquest Laboratories Inc. (Alachua, FL). The 8:2 FTCA and 8:2 FTUCA were synthesized using methods described by Achilefu et al. (26) (full details in the Supporting Information (SI)). MS-222 was purchased from Sigma-Aldrich (Oakville, Ontario). Analytical standards for PFHxA, PFHpA, PFOA, PFNA, 8:2 FTCA, and 8:2 FTUCA, and the stable isotope VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4973

standards (13C4-PFOA, 13C5-PFNA, 13C2-8:2 FTUCA, 13C2-8:2 FTCA) were provided by Wellington Laboratories (Guelph, ON). Food Preparation. Separate batches of 8:2 FTCA, 8:2 FTUCA, and 7:3 FTCA spiked food (target concentration: 100 µg/g) were prepared by spiking commercial fish food (full details presented in the SI). The mean 7:3 FTCA concentration in the 7:3 FTCA spiked food was 71.5 ( 10.0 µg/g (arithmetic mean ( standard error) and no other analytes were detected. The 8:2 FTCA spiked food had a mean 8:2 FTCA concentration of 103.2 ( 6.9 µg/g with the 8:2 FTUCA detected as an impurity at a mean level of 1.5 µg/g ( 0.14 µg/g. In the 8:2 FTUCA spiked food, the mean 8:2 FTUCA concentration was 69.1 ( 1.2 µg/g with trace quantities of unreacted 8:2 FTCA (0.7 µg/g, ∼1% of the 8:2 FTUCA concentration) detected. Perfluoroheptanoate, PFOA, and PFNA were not detected in any of the food preparations. Fish Care and Sampling. Juvenile rainbow trout (initial weight ∼60 g) were purchased from a local hatchery and were maintained in fiberglass tanks under flow-through conditions at a water temperature of 18 °C. During the 168-h uptake phase, fish were fed dosed or control food once per day at 1.5% of the average initial body weight. Fish were fed clean food during the 240-h elimination phase. During all time points (n ) 7 for uptake phase, n ) 5 or 6 for elimination phase), 3 dosed and 1 control fish were collected. Fish always ate voraciously, consuming the introduced food within 1-2 s of offering. The 8:2 FTUCA dosing experiment was conducted on a separate occasion and has a unique set of control samples (n ) 5) and method detection limits. Blood was collected immediately after euthanization by overdose exposure to MS-222 and stored in polypropylene microcentrifuge tubes. Liver was removed by dissection within 30 min. Blood and liver samples were kept frozen (-20 °C) until analysis. Tank water was not analyzed at any point during the experiment. Given that only blood and liver tissues were analyzed, and that tank water concentrations were not monitored during the experiment, it was not possible to calculate an overall mass balance for the parent compounds. Full details on fish care and sampling are provided in the SI. Extraction and Cleanup Methods. Liver (∼0.5 g) and blood (300 µL) were subsampled and placed in 15-mL polypropylene centrifuge tubes. The suite of stable isotope internal standards (13C4-PFOA, 13C5-PFNA, 13C2-8:2 FTUCA, 13 C-8:2 FTCA) was added to each sample prior to extraction. Liver was homogenized for 1 min in 8 mL of ethyl acetate by using a mechanical mixer. The extracts were centrifuged and the solvent was decanted into a clean tube. The blood samples were extracted by gently shaking for 5 min with 4 mL of ethyl acetate. The mixture was centrifuged, the solvent was decanted into a clean tube, the extraction was repeated, and fractions were combined. The liver and blood extracts were blown down to dryness under N2 gas and reconstituted in 1 mL of methanol for LC-MS/MS analysis. Instrumental Analysis. Instrumental analysis was performed by liquid chromatography with negative electrospraytandem mass spectrometry under conditions described previously (27) (SI). Sample extracts were analyzed using an API 4000 Q Trap (Applied Biosystems/MDS Sciex, Concord, ON, Canada) coupled to an Agilent 1100 pump. Analyte responses were normalized to internal standard responses. Paired extractions using the current method and a wellestablished liquid-liquid extraction with MTBE showed similar values (within 20% for all analytes detected). Statistical Analyses and Data Treatment. Details on the determination of the instrumental detection limits and method detection limits (MDLs), blank correction of tissue concentrations, calculations of the liver somatic index (LSI), fish growth rate and tissue correction, elimination rate, and assessment of steady-state are presented in the SI. 4974

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

Results and Discussion Physical Indices. No mortalities were observed in either the dosed or control treatments. The overall mean LSI was not statistically different (ANOVA, Tukey’s HSD posthoc test) between the control and 7:3 FTCA (p ) 0.35), 8:2 FTCA (p ) 0.69), and 8:2 FTUCA (p ) 0.73) treatments (Table S1 in SI), suggesting that the fish were not under metabolic stress due to the dose exposure. However, given the small sample size per time point (n ) 1 for control, n ) 3 for each dose), there may not have been sufficient statistical power to discern differences in the LSI between treatments. The overall growth rate was significant for the 7:3 FTCA, 8:2 FTCA, and 8:2 FTUCA treatments with fish weight increasing at ∼0.9 g/day. Growth rate for the control treatment was ∼1.0 g/day, but this growth rate was not statistically significant (p ) 0.44). This observed discrepancy is most probably due to the much larger sample size of the dosed groups. 7:3 FTCA Exposure. The parent 7:3 FTCA compound was rapidly accumulated from the dosed food with very high levels detected in the blood and liver within 12 h after initial dosing (Figure S1 and mean tissue concentrations are presented in the SI). The 7:3 FTCA blood levels increased throughout the uptake phase, but reached steady-state by 72 h at ∼2050 ng/g ww. In contrast, liver concentrations achieved steady-state within 12 h of dosing at ∼1500 ng/g ww. The only metabolites detected were the unsaturated analogue, 7:3 FTUCA, and PFHpA. The 7:3 FTUCA was formed in very low yields (blood only), with steady-state concentrations reaching ∼0.01-0.02% of the 7:3 FTCA. The 7:3 FTUCA levels were only above the MDL (0.01 ng/g ww) in the latter stage of the uptake phase and early during the elimination phase. Similarly, PFHpA was formed in low yields, with mean steady-state levels of 3.0 and 2.4 ng/g ww in the blood and liver, respectively, representing ∼0.1-0.2% of the parent 7:3 FTCA steady-state levels. The observation of PFHpA formation from 7:3 FTCA exposure is a novel finding and this pathway may represent the source of PFHpA observed in several 8:2 FTOH exposure studies (19, 22, 28). PFOA formation was not observed in the blood or liver. These results do not support the earlier suggestions by Martin et al. (18), Fasano et al. (19), and Wang et al. (14, 15) that postulated the 7:3 FTCA may form PFOA through β-oxidation. These results are surprising since it was expected that one round of β-oxidation would yield PFOA. However, the present findings are consistent with those of Nabb et al. (22) who did not observe PFOA formation during rat, mouse, human, and trout hepatocyte incubations with 7:3 FTCA and 7:3 FTUCA. Further, Nabb et al. (22) did not detect the formation of PFHpA from 7:3 FTCA incubation. Instead, it has been postulated that the exclusive fate of the 7:3 FTCA is conjugation with taurine (22, 28). The 7:3 taurine conjugate was not observed in the present study, although an authentic standard was not available and was monitored using the expected MS > MS transition. Further, it is unknown if the 7:3 taurine conjugate is stable in the extraction and analysis procedure. The mechanism for PFHpA formation is not known and warrants further study. The growth-corrected 7:3 FTCA elimination half-life was 5.1 days (95% confidence intervals: 3.1-14 days) for blood and 10.3 days (6.4-26 days) for liver (Table S2). Elimination half-lives could not be calculated for PFHpA and 7:3 FTUCA since these metabolites were below MDL almost immediately after elimination began. The relatively long half-life of the 7:3 FTCA, as compared to the 8:2 FTCA and 8:2 FTUCA, may suggest that this compound is a suitable biomarker for 8:2 FTOH exposure. Powley et al. (29) measured low ng/g ww levels of the 7:3 FTCA in ringed seal and bearded seal liver from the western Canadian arctic, but did not detect the 8:2 FTCA or FTUCA. Our study showed that the 7:3 FTCA blood

FIGURE 1. Mean whole blood and liver concentrations (ng/g ww) of 8:2 FTCA, 7:3 FTCA, 8:2 FTUCA, PFOA, and PFNA resulting from 8:2 FTCA dietary exposure. The y-axis scale is equivalent for both graphs. Error bars represent 1 standard error. Error bars are not shown for PFOA blood concentrations 1 ng/g ww and thus cannot be properly displayed on the log y-axis. concentrations did not exceed those of the 8:2 FTCA or 8:2 FTCA when these compounds are used as the dosing substrates, which may potentially suggest a shorter half-life of the 7:3 FTCA in blood. However, it was shown that the liver 7:3 FTCA concentrations were approximately 10-fold higher than the 8:2 FTUCA when the 8:2 FTUCA was dosed. Considering that the liver is the tissue most commonly monitored for poly- and perfluorinated carboxylates, it suggests that the 7:3 FTCA may be a relevant biomarker. In addition, exposure to fluorotelomer products could imply several classes of precursors (i.e., alcohols, olefins, acrylates, and olefins) and presumably these parent compounds would have different pharmacokinetics and product yields as compared to the parent FTCAs and FTUCAs themselves. 8:2 FTCA Exposure. The parent 8:2 FTCA was rapidly accumulated with very high concentrations measured in blood and liver within 12 h after initial dosing (Figure 1). The 8:2 FTCA concentrations increased slightly during the experiment with apparent steady-state concentrations reached within 12 h, at ∼4400 and ∼2100 ng/g ww for blood and liver, respectively. In addition, the 8:2 FTCA was rapidly metabolized with levels of 8:2 FTUCA, 7:3 FTCA, PFOA, and PFHpA observed within 12 h of initial dosing. Levels of 7:3 FTUCA and PFNA were below the MDL at the 12-h time point, but were quantifiable at 24 h and throughout the rest of the experiment. The predominant metabolite was the 7:3 FTCA, reaching 940 and 870 ng/g ww in the blood and liver, respectively, by the end of the uptake phase. The 7:3 FTCA concentrations continued to increase throughout the uptake phase but the final three time-points were not statistically different from each other, implying steady-state had been reached by 72 h. The 8:2 FTUCA was also formed and accumulated in high levels, reaching steady-state concentrations of ∼100 and ∼200 ng/g ww in blood and liver, respectively.

Trace levels of the 8:2 FTUCA (1.5 µg/g) were detected in the 8:2 FTCA spiked food, and may be responsible for some of the 8:2 FTUCA observed. The average liver mass was ∼1 g, and the average fish mass was ∼75 g equating to a blood mass of ∼6.6 g, assuming the blood represents 7.4% of total fish mass (30). At 12 h after initial dosing, the mean blood and liver 8:2 FTUCA concentrations were 56 and 120 ng/g ww, respectively. These concentrations equate to a 8:2 FTUCA mass of ∼370 and 120 ng in the blood and liver, respectively. Since each fish was fed ∼0.75 g of food per day, the daily dose of 8:2 FTUCA from the 8:2 FTCA food was 1.1 µg. Therefore, the total 8:2 FTUCA mass measured in the blood and liver was approximately 0.5-fold lower than the dose from the food impurity. However, these conditions assume that the 8:2 FTUCA is completely accumulated by the fish, with no depuration or biotransformation occurring, and the 8:2 FTUCA is partitioned only and equally into blood and liver. These conditions are highly conservative and not representative of actual pharmacokinetics. Thus, it is doubtful that the 8:2 FTUCA contamination in the food contributed significantly to the 8:2 FTUCA body burden. The terminal metabolites, PFOA and PFNA, were formed in low yields from the 8:2 FTCA biotransformation. Tissue PFOA concentrations increased throughout the uptake phase and reached levels of 8.8 and 56 ng/g ww in the blood and liver, respectively. Further, PFOA blood concentrations continued to increase for 5 days after the beginning of elimination. These trends are suggestive of formation from PFOA precursors (i.e., FTCA and FTUCA) that were still present in the body. PFNA concentrations in the blood were predominately below the MDL during the uptake phase, but were above MDL during the elimination phase and showed mainly steady levels. Finally, the 7:3 FTUCA and PFHpA were formed and accumulated in low concentrations (∼1-2 ng/g ww). Drawing from the results of the 7:3 FTCA dosing, it is VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4975

FIGURE 2. Mean whole blood and liver concentrations (ng/g ww) of 8:2 FTUCA, 7:3 FTCA, 8:2 FTCA, and PFOA resulting from 8:2 FTUCA dietary exposure. The y-axis scale is equivalent for both graphs. Error bars represent 1 standard error. assumed that these metabolites were formed from the biotransformation of the 7:3 FTCA that was produced in high concentrations from the 8:2 FTCA metabolism. The parent 8:2 FTCA and the metabolite 8:2 FTUCA were rapidly eliminated, presumably through a combination of depuration and biotransformation (Table S2). Growthcorrected elimination half-lives in blood were 1.2 days (1.1-1.3 days) for 8:2 FTCA and 1.3 days (1.1-1.5 days) for 8:2 FTUCA, and in liver were 1.3 days (1.1-1.4 days) for 8:2 FTCA and 1.8 days (1.0-8.6 days) for 8:2 FTUCA. The 7:3 FTCA showed an immediate decrease in the blood, whereas 7:3 FTCA liver concentrations remained steady during the initial 5 days of elimination. The PFOA blood levels increased for the initial 5 days of elimination. As such, the slope of the PFOA versus time relationship was not statistically different (p ) 0.41) from zero during elimination and thus the half-life could not be calculated. However, PFOA liver levels showed an immediate decrease and liver elimination half-life was 4.1 days (2.8-7.7 days) which is within the range reported for PFOA when dosed as the parent compound (31, 32). As mentioned, blood PFNA concentrations were predominately steady during the elimination phase and thus the concentration versus time slope was not statistically significant from zero. These results are consistent with the relatively long half-life of PFNA in rainbow trout of 16 days in blood and 6 days in liver (32). The liver PFNA elimination half-life could not be calculated because most data points were below the MDL. 8:2 FTUCA Exposure. The 8:2 FTUCA was rapidly accumulated within the blood and liver tissues (Figure 2). The 8:2 FTUCA reached steady-state within 12 h of dosing at ∼1400 and ∼70 ng/g ww in blood and liver, respectively. Interestingly, liver concentrations were ∼70-fold lower compared to the blood, suggestive of rapid biotransformation within the liver. These results are in contrast to the 7:3 FTCA and 8:2 FTCA dosing experiments in which levels of the 4976

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

parent compounds were within ∼2-fold between blood and liver. The 7:3 FTCA was formed and accumulated from the 8:2 FTUCA exposure within 12 h of dosing with levels initially increasing and reaching steady-state near the end of the uptake phase at 150 and ∼270 ng/g ww in blood and liver, respectively. The 7:3 FTUCA was also formed (data not shown), reaching concentrations of ∼1 ng/g ww. Interpretation of PFOA trends was constrained by the relatively high PFOA blank levels resulting in MDL values of 5.4 and 9.6 ng/g ww in blood and liver, respectively. However, PFOA was formed and accumulated with levels increasing throughout the uptake phase, reaching concentrations of ∼10 and 15 ng/g ww in blood and liver, respectively. In addition, the 8:2 FTUCA-GSH was detected in the liver using the expected MS-MS transition, as suggested by other studies (18, 19, 22, 28). A standard was not available and thus was quantified using arbitrary units. Detection of the 8:2 FTUCA-GSH conjugate in liver is consistent with several in vitro and in vivo studies examining 8:2 FTOH exposure (18, 19, 22, 28). Trace quantities of 8:2 FTCA (0.7 µg/g, equivalent to 1% of the 8:2 FTUCA concentration) were detected in the 8:2 FTUCA dosed food, presumably as the result of unreacted starting material from the 8:2 FTUCA synthesis. The biotransformation of the residual 8:2 FTCA likely contributed to some of the 8:2 FTUCA body burden in this experiment. The 8:2 FTUCA was eliminated very quickly with an elimination half-life in blood of 0.39 days (0.31-0.53 days) (Table S2). Liver concentrations were below the MDL (1.2 ng/g ww) within 24 h of commencing elimination and thus an elimination half-life could not be calculated. The elimination half-life of PFOA in blood was 0.54 days (0.3-6.1), whereas liver PFOA concentrations were below the MDL (9.6 ng/g ww) in all of the elimination samples. Interestingly, the PFOA concentrations did not show an initial delay in elimination as observed in the 8:2 FTCA treatment. Presum-

ably, this was due to the much faster elimination half-life of the 8:2 FTUCA as compared to the 8:2 FTCA. Elimination Half-Lives: Comparison of Intermediate and Terminal Metabolites. The elimination half-lives for the intermediate metabolites (i.e., 8:2 FTCA, 8:2 FTUCA, and 7:3 FTCA) investigated were generally much shorter compared to those of the terminal metabolites (i.e., PFOA and PFNA) that had been directly dosed (31, 32) (Table S2). Presumably this trend is due to the fact that the intermediate metabolites are metabolized, increasing their overall elimination rate. The overall aim of this study was to elucidate the biotransformation pathway for the polyfluorinated intermediate metabolites, rather than quantification of the elimination kinetics. However, future studies investigating the in vitro biotransformation of these and other intermediates, such as using S9 or microsomal fractions, would greatly assist in understanding the overall pharmacokinetics of PFCA precursors. Environmental Implications. Formation of 7:3 FTCA. The present study showed that 7:3 FTCA is formed from the biotransformation of 8:2 FTCA and 8:2 FTUCA. Further, in separate dosing experiments it was empirically shown that 8:2 FTCA is metabolized to 8:2 FTUCA, and that 8:2 FTUCA is not metabolized to 8:2 FTCA. Therefore, 7:3 FTCA appears to be formed from the direct biotransformation of 8:2 FTUCA, which is formed via 8:2 FTCA biotransformation. These results are consistent with those of Nabb et al. (22) who observed the formation of 7:3 FTCA and 7:3 FTUCA from hepatocyte incubations with both the 8:2 FTCA and 8:2 FTUCA. Nabb et al. (22) proposed the formation of the 7:3 FTCA from the 7:3 FTUCA, originating from the 8:2 FTCA (8:2 FTCA f 8:2 FTUCA f 7:3 FTUCA f 7:3 FTCA). In the hepatocyte incubations performed by Nabb et al. (22), 7:3 FTCA formation was observed when 7:3 FTUCA was used as the substrate. However, 7:3 FTUCA was also formed when 7:3 FTCA was the substrate. Therefore, it appears that 7:3 FTCA can be both the source and the metabolic product of 7:3 FTUCA. A similar reaction involving 8:2 FTUCA formation from 8:2 FTCA is not feasible since this would involve the formation of a carbon-fluorine bond. Formation of PFCAs. The formation of PFNA was observed in the 8:2 FTCA exposure, but not during 8:2 FTUCA or 7:3 FTCA exposure. These results are consistent with the suggested PFNA formation mechanism, through the direct R-oxidation of 8:2 FTCA (18, 19, 22). Several studies, investigating 8:2 FTOH exposure in mammalian systems, have shown that PFNA is formed in lower yields as compared to PFOA (18, 20-22). In the present study, approximately equal concentrations of PFNA and PFOA were accumulated in blood samples of the 8:2 FTCA exposure, however, liver PFOA levels were much higher than PFNA. There is considerable variability in the literature regarding the hypothesized pathways of PFOA formation from 8:2 FTOH metabolism. Consistent with a recent study involving rainbow trout hepatic fractions (22), the study showed that 7:3 FTCA is not a precursor to PFOA. Rather, it was shown that PFHpA is formed from 7:3 FTCA biotransformation, a novel finding. The results from the present study demonstrate that PFOA is formed from the biotransformation of 8:2 FTCA and 8:2 FTUCA. Hagen et al. (12) originally postulated that β-oxidation was involved in the biotransformation of 8:2 FTOH in rats, while Dinglasan et al. (13) initially proposed the formation of PFOA from the β-oxidation of 8:2 FTCA in a microbial system. Further, Martin et al. (18) postulated PFOA formation from the “β-like-oxidation” of 8:2 FTUCA, which originates from either the 8:2 FTCA or 8:2 FTUAL. This pathway was supported by observations of Nabb et al. (22), although these authors proposed a unique pathway for the predominant formation of PFOA. Wang et al. (14) suggested that β-oxidation could not proceed through

either the 8:2 FTCA or FTUCA since these compounds do not contain sufficient hydrogen atoms required to reduce nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD). However, the empirical observation of PFOA formation from the direct exposure of 8:2 FTCA and 8:2 FTUCA (18, 22) does suggest a β-like oxidation pathway. Martin et al. (18) noted that literature precedent does exist, specifically the dehydrofluorination of 2,2,difluorosuccinate (CO2-CF2CH2CO2-) to monofluorofumarate (CO2-CFdCHCO2-) (33), which is nonoxidative and thus does not require the simultaneous reduction of FAD (34). The second biotransformation step, the hydroxylation of monofluorofumarate to 2-fluoromalate (CO2-CF(OH)CH2CO2-) is also NAD-independent. The 2-fluoromalate intermediate is unstable (35) and is rapidly dehydrofluorinated nonenzymatically to yield oxaloacetate (CO2-C(O)CH2CO2-). In addition, the defluorination of methoxyflurane (CHCl2CF2OCH3) has been shown in humans (36). The ultimate step preceding the defluorination is the cleavage of the methyl ether (-OCH3) yielding the 2,2dichloro-1,1-difluoroethanol (CHCl2CF2OH). Similar to 2-fluoromalate, this hydroxylated compound is chemically unstable and spontaneously decomposes, releasing HF, to yield 2,2-dichloroacetyl fluoride (CHCl2C(O)F) which is hydrolyzed to the dichloroacetic acid (CHCl2C(O)OH). Therefore, literature precedent demonstrates that hydroxylation of the carbon containing fluorine will result in spontaneous nonenzymatic defluorination, resulting in either a carboxylic acid or ketone. The reactions described are analogous to the β-oxidation pathway and demonstrate the formation of a β-ketoacyl compound, from a 2,2difluorinated carboxylic acid, that does not require NAD. As noted by Martin et al. (18), the β-ketoacyl analogue of 8:2 FTUCA would yield PFOA-S-CoA and acetyl CoA via thiolase. As noted above, Nabb et al. (22) observed the formation of PFOA from the 8:2 FTCA and FTUCA incubations, however, they postulated that the primary pathway was from the 8:2 FTAL through the 8:2 FTUAL > 7:3 β-hydroxy unsaturated aldehyde >7:3 β-keto aldehyde > PFOA. Recently, Fasano et al. (28) proposed a combination of that described by Nabb et al. (22) and a unique pathway, analogous to the 8:2 FTUAL biotransformation previously proposed (22) but originating with the 8:2 FTUCA. In this pathway, the 8:2 FTUCA is hydroxylated to yield the 7:3 β-hydroxyl carboxylate > 7:3 β-keto carboxylate > 7:2 ketone. The 7:2 ketone is subsequently reduced to the 7:2 sFTOH which then forms PFOA. Nabb et al. (22) did not propose the formation of PFOA from the 7:2 sFTOH. The direct formation of PFOA (or PFHpA) from the 7:2 (or 5:2) sFTOH was also suggested by Wang et al. (17) and Liu et al. (23) in soil and mixed bacterial cultures. Although the Wang et al. (17) and Liu et al. (23) studies did not investigate animal systems, the proposed biotransformation pathways were similar to those presented by Fasano et al. (28) and Nabb et al. (22). The formation of the 7:3 β-keto aldehyde and 7:3 β-keto carboxylate from the 8:2 FTUAL and 8:2 FTUCA are essentially analogous to the “βlike-oxidation” biotransformation pathways described for the fluorinated succinate and methoxyflurane and thus appear reasonable. However, formation of the β-OH intermediates seems unlikely given the instability of the fluorohydroxy compound. Beta-keto acids are susceptible to decarboxylation, forming the methyl ketone. Thus, the decarboxylation of 7:3 β-keto acid to 7:2 ketone via decarboxylase is possible. It has been shown that methyl ketones undergo R-hydroxylation and subsequent oxidation to yield ketocarboxylic acids (37), ultimately forming aliphatic carboxylic acid through oxidative decarboxylation. Therefore, the formation of PFOA from the biotransformation of 7:2 ketone appears to be reasonable. Aldehyde reductase enzymes, capable of meVOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4977

FIGURE 3. Proposed biotransformation pathway for the 8:2 FTOH. tabolizing xenobiotic compounds, are found in mammals (38) and fish (39). Therefore, literature precedent exists for the reduction of the 7:2 ketone to the 7:2 sFTOH. However, we could not find any precedent for carboxylic acid formation from a secondary alcohol and thus formation of PFOA from the 7:2 sFTOH, as postulated by Fasano et al. (28), is speculative. It is likely that the predominant biological fate of the 7:2 sFTOH, like the 8:2 FTOH, is glucuronidation. Regarding the 7:3 β-keto aldehyde, we could not find any literature precedent for the formation of carboxylic acids from β-keto aldehydes. Rather, we propose that this compound is oxidized to the 7:3 β-keto acid > 7:2 ketone > PFOA. Based on the findings of this study and reported analogous literature pathways, we expand on the “beta-like-oxidation” pathway (18) for PFOA formation as proceeding from the 8:2 FTUCA > 7:3 β-keto acid > 7:2 ketone > PFOA (Figure 3). Alternatively, the 7:3 β-keto aldehyde could enter the betaoxidation cycle to form PFOA. The overall formation and accumulation of PFOA was very low, consistent with other studies examining 8:2 FTOH biotransformation (22). To compare the PFOA yield between the 8:2 FTCA and 8:2 FTUCA doses, we calculated the formation efficiency, defined as the PFOA concentration in the tissues at the end of the exposure phase divided by the parent concentration in the blood (i.e., FE (%) ) tissuePFOA * 100/food8:2 FTCA or 8:2 FTUCA). The FE for the 8:2 FTCA dosing was 0.009% and 0.054% in the blood and liver, respectively. 4978

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

The FE values for the 8:2 FTUCA were similar at 0.014% and 0.022% in the blood and liver, respectively. The FE values do not represent actual biotransformation yield, rather they are a function of yield and pharmacokinetics. Therefore, higher FE values are expected for the biotransformation of longer chain fluorotelomer compounds which would yield longerchain PFCAs with comparably slower elimination half-lives. The current study has built upon previous metabolic investigations of telomer-based polyfluorinated compounds and has provided additional insights into the biotransformation pathway. Although this study specifically examined the 8:2 fluorotelomer-based compounds, it is expected that these results are broadly applicable for all telomer-based compounds, of various chain lengths, which degrade to the FTOH, such as polyfluorinated phosphates (24) and polyfluorinated acrylates (25). However, as recently noted by Liu et al. (23), slight differences may occur for differing chain lengths due to the steric hindrance of some enzymes.

Acknowledgments Norman White and staff at the Aquatic Facility in the Department of Cell & Systems Biology (University of Toronto) are thanked for fish care and husbandry. Clara Chan, Helen Sun, and Alex Tevlin provided assistance with sample preparation. We are grateful to Wellington Laboratories for donation of mass-labeled standards. Project funding was provided by the Natural Sciences & Engineering Research

Council of Canada (NSERC) (S.A.M.) and Environment Canada’s Chemical Management Plan (D.C.G.M.). C.M.B. also appreciates the support of NSERC through a PostGraduate Scholarship.

(15)

Supporting Information Available

(16)

Detailed information on the synthesis of 8:2 FTCA and 8:2 FTUCA, food preparation and analysis, fish care and sampling, instrumental analysis, and statistical analyses and data treatment; figures of blood and liver trends from the 7:3 FTCA treatment; tables of liver somatic index values, elimination half-lives, and mean and standard error values for blood and liver from the 7:3 FTCA, 8:2 FTCA, and 8:2 FTUCA treatments. This information is available free of charge via the Internet at http://pubs.acs.org/.

(17)

(18) (19)

Literature Cited (1) Giesy, J. P.; Kannan, K. Distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35, 1339–1342. (2) Martin, J. W.; Smithwick, M. M.; Braune, B. M.; Hoekstra, P. F.; Muir, D. C. G.; Mabury, S. A. Identification of long-chain perfluorinated acids in biota from the Canadian Arctic. Environ. Sci. Technol. 2004, 38, 373–380. (3) Hansen, K. J.; Clemen, L. A.; Ellefson, M. E.; Johnson, H. O. Compound-specific, quantitative characterization of organic fluorochemicals in biological matrices. Environ. Sci. Technol. 2001, 35, 766–770. (4) Wallington, T. J.; Hurley, M. D.; Xia, J.; Wuebbles, D. J.; Sillman, S.; Ito, A.; Penner, J. E.; Ellis, D. A.; Martin, J.; Mabury, S. A.; Nielsen, O. J.; Sulbaek Andersen, M. P. Formation of C7F15COOH (PFOA) and other perfluorocarboxylic acids during the atmospheric oxidation of 8:2 fluorotelomer alcohol. Environ. Sci. Technol. 2006, 40, 924–930. (5) Ellis, D. A.; Martin, J. W.; De Silva, A. O.; Mabury, S. A.; Hurley, M. D.; Sulbaek Andersen, M. P.; Wallington, T. J. Degradation of fluorotelomer alcohols: A likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. 2004, 38, 3316–3321. (6) Hurley, M. D.; Wallington, T. J.; Sulbaek Andersen, M. P.; Ellis, D. A.; Martin, J. W.; Mabury, S. A. Atmospheric chemistry of fluorinated alcohols: reaction with Cl atoms and OH radicals and atmospheric lifetimes. J. Phys. Chem. A 2004, 108, 1973–1979. (7) Nakayama, T.; Takahashi, K.; Matsumi, Y.; Toft, A.; Sulbaek Andersen, M. P.; Nielsen, O. J.; Waterland, R. L.; Buck, R. C.; Hurley, M. D.; Wallington, T. J. Atmospheric chemistry of CF3CH)CH2 and C4F9CH)CH2: Products of the gas-phase reactions with Cl atoms and OH radicals. J. Phys. Chem. A 2007, 111, 909–915. (8) Young, C. J.; Hurley, M. D.; Wallington, T. J.; Mabury, S. A. Atmospheric chemistry of 4:2 fluorotelomer iodide (nC4F9CH2CH2I): Kinetics and products of photolysis and reaction with OH radicals and Cl atoms. J. Phys. Chem. A 2008, 112, 13542–13548. (9) Butt, C. M.; Young, C. J.; Mabury, S. A.; Hurley, M. D.; Wallington, T. J. Atmospheric chemistry of 4:2 fluorotelomer acrylate [C4F9CH2CH2OC(O)CH)CH2]: Kinetics, mechanisms, and products of chlorine-atom- and OH-radical-initiated oxidation. J. Phys. Chem. A 2009, 113, 3155–3161. (10) Martin, J. W.; Ellis, D. A.; Mabury, S. A.; Hurley, M. D.; Wallington, T. J. Atmospheric chemistry of perfluoroalkanesulfonamides: Kinetic and product studies of the OH radical and Cl atom initiated oxidation of N-ethyl perfluorobutanesulfonamide. Environ. Sci. Technol. 2006, 40, 846–872. (11) D’eon, J. C.; Hurley, M. D.; Wallington, T. J.; Mabury, S. A. Atmospheric chemistry of N-methyl perfluorobutane sulfonamidoethanol, C4F9SO2N(CH3)CH2CH2OH: Kinetics and mechanism of reaction with OH. Environ. Sci. Technol. 2006, 40, 1862–1868. (12) Hagen, D. F.; Belisle, J.; Johnson, J. D.; Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector - the biotransformation of 1H,1H,2H,2H-perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981, 118, 336–343. (13) Dinglasan, M. J. A.; Ye, Y.; Edwards, E. A.; Mabury, S. A. Fluorotelomer alcohol biodegradation yields poly- and perfluorinated acids. Environ. Sci. Technol. 2004, 38, 2857–2864. (14) Wang, N.; Szostek, B.; Folsom, P. W.; Sulecki, L. M.; Capka, V.; Buck, R. C.; Berti, W. R.; Gannon, J. T. Aerobic biotransformation of 14C-labeled 8-2 telomer B alcohol by activated sludge from

(20)

(21)

(22)

(23)

(24)

(25) (26) (27) (28)

(29)

(30)

(31)

(32) (33) (34)

(35)

a domestic sewage treatment plant. Environ. Sci. Technol. 2005, 39, 531–538. Wang, N.; Szostek, B.; Buck, R. C.; Folsom, P. W.; Sulecki, L. M.; Capka, V.; Berti, W. R.; Gannon, J. T. Fluorotelomer alcohol biodegradation: Direct evidence that perfluorinated carbon chains breakdown. Environ. Sci. Technol. 2005, 39, 7516–7528. Liu, J.; Lee, L. S.; Nies, L. F.; Nakatsu, C. H.; Turco, R. F. Biotransformation of 8:2 fluorotelomer alcohol in soil and by soil bacteria isolates. Environ. Sci. Technol. 2007, 41, 8024– 8030. Wang, N.; Szostek, B.; Buck, R. C.; Folsom, P. W.; Sulecki, L. M.; Gannon, J. T. 8-2 Fluorotelomer alcohol aerobic soil biodegradation: Pathways, metabolites, and metabolite yields. Chemosphere 2009, 75, 1089–1096. Martin, J. W.; Mabury, S. A.; O’Brien, P. J. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interact. 2005, 155, 165–180. Fasano, W. J.; Carpenter, S. C.; Gannon, S. A.; Snow, T. A.; Stadler, J. C.; Kennedy, G. L.; Buck, R. C.; Korzeniowski, S. H.; Hinderliter, P. M.; Kemper, R. A. Absorption, distribution, metabolism, and elimination of 8-2 fluorotelomer alcohol in the rat. Toxicol. Sci. 2006, 91, 341–355. Henderson, W. M.; Smith, M. A. Perfluorooctanoic acid and perfluorononanoic acid in fetal and neonatal mice following in utero exposure to 8-2 fluorotelomer alcohol. Toxicol. Sci. 2007, 95, 452–461. Kudo, N.; Iwase, Y.; Okayachi, H.; Yamakawa, Y.; Kawashima, Y. Induction of hepatic peroxisome proliferation by 8-2 telomer alcohol feeding in mice: Formation of perfluorooctanoic acid in the liver. Toxicol. Sci. 2005, 86, 231–238. Nabb, D. L.; Szostek, B.; Himmelstein, M. W.; Mawn, M. P.; Gargas, M. L.; Sweeney, L. M.; Stadler, J. C.; Buck, R. C.; Fasano, W. J. In vitro metabolism of 8-2 fluorotelomer alcohol: Interspecies comparisons and metabolic pathway refinement. Toxicol. Sci. 2007, 100, 333–344. Liu, J.; Wang, N.; Szostek, B.; Buck, R. C.; Panciroli, P. K.; Folsom, P. W.; Sulecki, L. M.; Bellin, C. A. 6-2 fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture. Chemosphere 2010, 78, 437–444. D’eon, J. C.; Mabury, S. A. Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPs): Exploring routes of human contamination. Environ. Sci. Technol. 2007, 41, 4799– 4805. Butt, C. M.; Muir, D. C. G.; Mabury, S. A. Biotransformation of the 8:2 FTOH acrylate in rainbow trout. I. In vivo dietary exposure. Environ. Toxicol. Chem., in press. Achilefu, S.; Mansuy, L.; Selve, C.; Thiebaut, S. Synthesis of 2H,2H-perfluoroalkyl and 2H-perfluoroalkenyl carboxylic acid and amines. J. Fluor. Chem. 1995, 70, 19–26. Butt, C. M.; Muir, D. C. G.; Stirling, I.; Kwan, M.; Mabury, S. A. Rapid response of Arctic ringed seals to changes in perfluoroalkyl production. Environ. Sci. Technol. 2007, 41, 42–49. Fasano, W. J.; Sweeney, L. M.; Mawn, M. P.; Nabb, D. L.; Szostek, B.; Buck, R. C.; Gargas, M. L. Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats. Chem. Biol. Interact. 2009, 180, 281–295. Powley, C. R.; George, S. W.; Russell, M. H.; Hoke, R. A.; Buck, R. C. Polyfluorinated chemicals in a spatially and temporally integrated food web in the Western Arctic. Chemosphere 2008, 70, 664–672. Brown, R. P.; Delp, M. D.; Lindstedt, S. L.; Rhomberg, L. R.; Beliles, R. P. Physiological parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health 1997, 13, 407–484. Martin, J. W.; Mabury, S. A.; Solomon, K. R.; Muir, D. C. G. Dietary accumulation of perfluorinated acids in juvenile rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 2003, 22, 189–195. De Silva, A. O.; Tseng, P. J.; Mabury, S. A. Toxicokinetics of perfluorocarboxylate isomers in rainbow trout. Environ. Toxicol. Chem. 2009, 28, 330–337. Brodie, J. D.; Nicholls, P. Metabolsim and enzymology of fluorosuccinic acids. I. Interactions with the succinate oxidase system. Biochim. Biophys. Acta 1970, 198, 423–437. Tober, C. L.; Nicholls, P.; Brodie, J. D. Metabolism and enzymology of fluorosuccinic acids. II. Substrate and inhibitor effects with soluble succinate dehydrogenase. Arch. Biochem. Biophys. 1970, 138, 506–514. Walsh, C., Fluorinated substrate analogs: Routes of metabolism and selective toxicity. In Advances in Enzymology; Meister,

VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4979

A., Ed.; John Wiley & Sons: Toronto, 1983; Vol. 55, pp 197285. (36) Mazze, R. I.; Trudell, J. R.; Cousins, M. J. Methoxyflurane metabolism and renal dysfunction: Clinical correlation in man. Anesthesiology 1971, 35, 247–252. (37) Gabriel, M. A.; Ilbawi, M.; Al-Khalidi, U. A. S. The oxidation of acetoin to CO2 in intact animals and in liver mince preparation. Comp. Biochem. Physiol. 1972, 41B, 493–502.

4980

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

(38) Bachur, N. R. Cytoplasmic aldo keto reductases: a class of drug metabolizing enzymes. Science 1976, 193, 4253. (39) Tulayakul, P.; Sakuda, S.; Dong, K. S.; Kumagai, S. Comparative activities of glutathione-S-transferase and dialdehyde reductase toward aflatoxin B1 in livers of experimental and farm animals. Toxicon 2005, 46, 204–209.

ES100702A