Dietary Bioaccumulation of Perfluorophosphonates and

Feb 15, 2012 - Amila O. De Silva, Christine Spencer, Ki Chung D. Ho, Mohammed Al Tarhuni, Christopher Go, Magali Houde, Shane R. de Solla, Raphael A...
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Dietary Bioaccumulation of Perfluorophosphonates and Perfluorophosphinates in Juvenile Rainbow Trout: Evidence of Metabolism of Perfluorophosphinates Holly Lee,† Amila O. De Silva,*,‡ and Scott A. Mabury*,† †

Department of Chemistry, University of Toronto, 80 St. George St., Toronto, Ontario, Canada, M5S 3H6 Water Science and Technology Directorate, Environment Canada, 867 Lakeshore Rd., Burlington, Ontario, Canada L7R 4A6



S Supporting Information *

ABSTRACT: The perfluorophosphonates (PFPAs) and perfluorophosphinates (PFPiAs) are high production volume chemicals that have been observed in Canadian surface waters and wastewater environments. To examine whether their occurrence would result in contamination of organisms in aquatic ecosystems, juvenile rainbow trout (Oncorhynchus mykiss) were separately exposed to a mixture of C6, C8, and C10 monoalkylated PFPAs and a mixture of C6/C6, C6/C8, and C8/C8 dialkylated PFPiAs in the diet for 31 days, followed by 32 days of depuration. Tissue distribution indicated preferential partitioning to blood and liver. Depuration half-lives ranged from 3 to 43 days and increased with the number of perfluorinated carbons present in the chemical. The assimilation efficiencies (α, 7−34%) and biomagnification factors (BMFs, 0.007−0.189) calculated here for PFPAs and PFPiAs were lower than those previously observed for the perfluorocarboxylates (PFCAs) and perfluorosulfonates (PFSAs) in the same test organism. Bioaccumulation was observed to decreased in the order of PFSAs > PFCAs > PFPAs of equal perfluorocarbon chain length and was dependent on the charge of the polar headgroup. Bioaccumulation of the PFPiAs was observed to be low due to their rapid elimination via metabolism to the corresponding PFPAs. Here, we report the first observation of an in vivo cleavage of the carbon−phosphorus bond in fish, as well as, the first in vivo biotransformation of a perfluoroalkyl acid (PFAA). As was previously observed for PFCAs and PFSAs, none of the BMFs determined here for the PFPAs and PFPiAs were greater than one, which suggests PFAAs do not biomagnify from dietary exposure in juvenile rainbow trout.



Total organofluorine analyses of freshwater20 and marine23 animals revealed that known PFSAs and PFCAs may not fully account for the total fluorochemical contamination observed in these samples, which implies the presence of other unidentified fluorinated chemicals. Perfluorophosphonates (PFPAs) and perfluorophosphinates (PFPiAs) are newly discovered PFAAs that structurally differ from the PFSAs and PFCAs in that their perfluorinated carbon tails are attached through a carbon− phosphorus (C−P) bond to either a phosphonate (RP(O)O2−; PFPA) or phosphinate (R2-P(O)O−; PFPiA) headgroup (Table 1). PFPAs and PFPiAs are commercial fluorinated surfactants marketed for use as leveling and wetting agents in household cleaning products24 and defoaming agents in pesticide formulations,25 although the latter application has been banned in the United States (U.S.) since 2008.26 Human exposure to the PFPiAs was recently confirmed in U.S. human

INTRODUCTION Historical and current use of fluorinated chemicals has led to the widespread occurrence of two classes of perfluoroalkyl acids (PFAAs), the perfluorocarboxylates (PFCAs) and perfluorosulfonates (PFSAs), in aquatic wildlife1 and their surrounding environments.2,3 Global contamination of PFCAs and PFSAs has been extensively reported in fish sampled from U.S. rivers, 4−6 the Great Lakes, 1,4,7,8 a German lake, 9 the Mediterranean and Baltic coasts,10,11 the Japanese coasts,12,13 the Chinese Yangtze river,14 Greenland and the Faroe Islands,15 and the Arctic.16−19 Analysis of wildlife species at different trophic levels consistently reports detection of PFOS and the longer chain PFCAs (≥7 perfluorinated carbons, CF’s),4,7,16−20 with significant concentrations (mid to high ng/g wet weight (ww)) observed in both benthic feeders and predatory fishes at the top of the aquatic food web. This is consistent with the bioaccumulation trend observed for PFAAs in which rainbow trout exposed through water21 and the diet22 exhibited increased accumulation of longer chain PFSAs (≥6 CF’s) and PFCAs (≥7 CF’s). © 2012 American Chemical Society

Received: Revised: Accepted: Published: 3489

December 16, 2011 February 13, 2012 February 15, 2012 February 15, 2012 dx.doi.org/10.1021/es204533m | Environ. Sci. Technol. 2012, 46, 3489−3497

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(>98%) were provided by Wellington Laboratories (Guelph, ON). Food Preparation. Three batches of commercial fish feed (Silver Cup 1.5 mm extruded floating feed, Martin Mills Inc., Elmira, ON) were prepared for the separate dosing of PFPAs and PFPiAs and the control feed, as described in detail in the SI. Determination of the PFPA and PFPiA concentrations in the dosed and control feed is described in the SI. The mean (±standard error) concentrations in the PFPA- and PFPiAdosed feed were 485 ± 28 ng/g C6 PFPA, 474 ± 37 ng/g C8 PFPA, and 533 ± 37 ng/g C10 PFPA; and 468 ± 12 ng/g C6/ C6 PFPiA, 510 ± 24 ng/g C6/C8 PFPiA, and 420 ± 12 ng/g C8/C8 PFPiA, respectively (Table 2 and SI Table S1). The PFPAs and PFPiAs were not detected in the control feed. Fish Care and Sampling. Juvenile rainbow trout (5−7 g) were purchased from Humber Springs Trout Hatchery (Orangeville, ON) and allowed to acclimate for two weeks prior to chemical exposure. The animals were housed in three 475 L fiberglass tanks under flow-through conditions (4−8 L/ min) using carbon-filtered and dechlorinated water, maintained at 18 °C, at the Aquatic Facility of the Department of Cell and Systems Biology at the University of Toronto. A 12 h daily photoperiod was used. One tank was designated for the control fish, whereas the remaining two tanks were designated for fish to be dosed separately with the PFPAs and PFPiAs. The initial fish loadings in the three tanks were ∼0.5 g/L. All animals in this research were treated and used under approval by the University of Toronto Animal Care Committee and in accordance with the guidelines of the Canadian Council on Animal care. Prior to chemical exposure, fish were deprived of food for 24 h to ensure an aggressive first feeding. During the exposure phase, fish received daily feeding of the dosed or control feed at 0.015 g feed (dry weight (dw))/g fish (ww), adjusted throughout the experiment for growth, followed by a depuration phase during which the fish were fed untreated feed at the same rate. Fish sampling always occurred before feeding. The fish were sampled on days −1 (predose), 1, 3, 6, 13, 20, and 31 of the exposure phase and days 1, 3, 8, 15, 25, and 32 of the depuration phase. The fish were sampled in triplicate (n = 3) at each time point until days 25 and 32 of the depuration phase during which the control fish were sampled in duplicate (n = 2) and the dosed fish were sampled in triplicate (n = 3) from their respective tanks. Each fish was euthanized by a lethal overdose of tricaine methanesulfonate (MS-222, 4 g/L solution buffered to pH 7 with sodium bicarbonate). After

Table 1. Structures, Full Names, and Acronyms of the Target Analytes Monitored

sera in which the C6/C6 and C6/C8 congeners were observed at 4−38 ng/L concentrations.27 Given their high annual production volumes (10 000−500 000 lbs) as reported in 1998 and 2002,28 PFPAs and PFPiAs are likely to be widely disseminated in the environment. The PFPAs are prevalent contaminants in Canadian surface waters, with concentrations ranging in the mid-to-high pg/L range.29 The presence of PFPAs in wastewater treatment plant (WWTP) effluents29 and PFPiAs in WWTP biosolids30 suggests the potential of these chemicals to partition from the aqueous phase into environmental solids like sediments, as was previously observed for PFSAs and PFCAs.31 Benthic feeders, such as Lumbriculus variegatus, have been observed to bioaccumulate PFAAs upon exposure to laboratory-spiked and contaminated freshwater sediments.32,33 Together with the significant PFSA and PFCA contamination observed in other benthic organisms, such as the freshwater invertebrate, Diporeia, and predatory fish, sculpin,7 these results suggest sediment may be an important source of fluorinated chemicals to these and possibly higher trophic organisms within the aquatic food web. The present research aims to evaluate the uptake and depuration of three PFPA (C6, C8, and C10) and three PFPiA (C6/C6, C6/C8, and C8/C8) congeners in juvenile rainbow trout (Oncorhynchus mykiss) upon dietary exposure for 31 days, followed by a 32-day depuration phase.



MATERIALS AND METHODS Chemicals. A list of all standards and reagents used in this study is provided in the Supporting Information (SI). Neat material (∼1 mg for each congener) of C6 n-PFPA (>98%), C8 n-PFPA (>98%), C10 n-PFPA (>98%), C6/C6 n-PFPiA (>98%), C6/C8 n-PFPiA (>98%), and C8/C8 n-PFPiA

Table 2. Concentration of Food (Cfood, in Dry Weight (dw)), Depuration Rate Constant (kd), Depuration Half-Life (t1/2), Assimilation Efficiency (α), Biomagnification Factor (BMF) of the Dosed PFPAs and PFPiAs, and Estimated Time to Achieve 90% Steady State (tss)a analyte

Cfood (ng/g dw)

kd (/day) (r)

C6 PFPA C8 PFPA C10 PFPA

485 ± 28 474 ± 37 533 ± 37

0.19 ± 0.03 (0.97) 0.16 ± 0.03 (0.96) 0.13 ± 0.02 (0.94)

C6/C6 PFPiA C6/C8 PFPiA C8/C8 PFPiA

468 ± 12 510 ± 24 420 ± 12

0.13 ± 0.01 (1.00) 0.03 ± 0.01 (0.88) 0.02 ± 0.01 (0.81)

t1/2 (day)

α (%)

Perfluorophosphonates (PFPAs) 0.13 ± 0.02 (0.94) 5.3 ± 4.4 ± 0.7 7± 5.3 ± 0.8 16 ± Perfluorophosphinates (PFPiAs) 5.5 ± 0.2 34 ± 20.4 ± 4.9 24 ± 52.7 ± 15.8 17 ±

BMF

logBMF

tss (day)

0.8 4 6

0.007 ± 0.003 0.007 ± 0.003 0.018 ± 0.006

−2.13 ± ± 0.17 −2.18 ± 0.21 −1.74 ± 0.14

12 14 18

6 9 15

0.041 ± 0.007 0.106 ± 0.033 0.189 ± 0.167

−1.39 ± 0.07 −0.97 ± 0.13 −0.72 ± 0.38

18 77 115

The coefficient of correlation (r) for the linear regression analysis to determine kd is shown in parentheses. The error is represented by ±1 standard error. a

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S3) was observed in PFPA- and PFPiA-dosed fish homogenate extracts, as discussed in the SI. Data Analysis. The whole-body and liver growth rates (SI Table S5) were calculated by fitting all fish and liver mass data to an exponential model: (ln(mass, g) = b·t + a; where b is the growth rate (g of fish/day), t is the time (day), and a is a constant. Whole-body concentrations in each treatment population were corrected for growth dilution by using the individual whole-body growth rates shown in SI Table S5. The liver somatic index (LSI) was calculated as LSI (%) = [liver mass (g)/whole fish mass (g)] × 100%. Mean LSIs calculated for each batch of fish sampled from the three treatment populations throughout the experiment are plotted as SI Figure S4. Depuration rate constants (kd) from the whole-body homogenates were calculated by fitting the growth-corrected concentration data in the depuration phase to the first-order decay model: ln(Cfish) = kd·t + a; where Cfish is the growthcorrected whole-body concentration, kd is the depuration rate constant (/day), t is the time (day), and a is a constant (StatsDirect, Version 2.7.8, 2010). Depuration half-lives (t1/2) were calculated as ln(2)/kd. Assimilation efficiency (α) was determined by using iterative nonlinear regression to fit the growth-corrected concentration data in the exposure phase to the integrated form of the kinetic rate equation for constant dietary exposure (SigmaPlot, Version 9.01, 2004): Cfish = (α·F·Cfood/kd)·[1 − exp(-kd·t)]; where Cfish is the growth-corrected whole-body concentration, F is the feeding rate (0.015 g food dry wt/g of fish ww/day), Cfood is the concentration in the food, and t is the time (day).36 Assimilation efficiency is expressed as the ratio of the amount of chemical absorbed to the amount fed. Biomagnification factors (BMFs) were calculated using the kinetic equation method since steady-state was not achieved for all the analytes during the exposure phase (BMF = α·F/kd). The estimated time to achieve 90% steady-state (tss, day) for each analyte was calculated by rearranging the above kinetic rate equation, as described in the SI. Statistical Analysis. Analyte concentrations observed below their corresponding LODs in the depuration phase were imputed as the LOD divided by square root of two, so that they can be fitted as nonzero values to the first-order decay model described above for calculating kd and t1/2. All tests were performed using StatsDirect (Version 2.7.8, 2010). An α-value of 0.05 was chosen as the criterion for statistical significance in all analyses. Further details describing the test results and the pvalues of the tests are provided in the SI.

weighing, the fish were dissected to remove the livers and subsequently minced into small carcass pieces. To minimize potential PFPA and PFPiA contamination of the carcass from undigested food, the digestive tract, containing the esophagus, stomach, pyloric ceca, and intestines, was discarded. The livers were weighed separately then returned to their corresponding carcasses for further homogenization. All samples were archived at −20 °C until further analysis. The masses of the whole fish and their corresponding livers are plotted as SI Figure S1. Tissue Distribution of PFPAs and PFPiAs. On the last day of the exposure phase (i.e., day 31), three additional fish (n = 3) from each of the control and dosed tanks were sampled to investigate tissue distribution. Fish were euthanized by a 1 g/L solution of MS-222 (buffered to pH 7 with sodium bicarbonate). Whole blood was collected through cardiac puncture with heparin-rinsed syringes and stored in heparinized vials (BD Vacutainer, Franklin Lakes, NJ). Fish were dissected to remove the heart, liver, kidneys, and gills. The remaining carcass was homogenized, as described above. All samples were archived at −20 °C until further analysis. Extractions and Instrumental Analysis. Whole-fish homogenates, livers, kidneys, hearts, gills, and whole blood samples were extracted using a modified version of the ionpairing method developed by Hansen et al.34 The livers, kidneys, and gills were homogenized in 1−2 mL of 1 M tetrabutylammonium hydrogen sulfate (TBAS) prior to extraction. Detailed extraction methods, chromatographic gradients, instrumental conditions (Table S2 and S3), and sample chromatograms (Figure S2) are provided in the SI. Quality Assurance of Data. The C5−C11 PFCAs were quantified using mass-labeled internal standards (SI Table S3). The C6, C8, and C10 PFPAs and C6/C6, C6/C8, and C8/C8 PFPiAs were quantified using matrix-matched calibration standards where control fish homogenate served as the matrix. This was necessary since isotopically labeled surrogates of the PFPAs and PFPiAs were not available at the time of analysis. Further details on preparation of the matrix-matched standards are provided in the SI. Spike and recoveries ranged from 73 to 126% for the PFPAs and PFPiAs in the different fish tissues (SI Table S4a) and 68− 123% for the C5−C11 PFCAs in whole-fish homogenates (SI Table S4b). All reported tissue concentrations were not corrected for recovery. Further details on the spike and recovery procedures are described in the SI. The limits of detection (LOD) and limits of quantiation (LOQ) were defined as the concentrations producing a signalto-noise (S/N) ratio of equal to or greater than 3 and 10, respectively. The method LOD and LOQ values for each analyte in the different fish tissues are listed in SI Table S4a,b. For the purposes of calculating means, concentration values below the LOD were assigned a value of zero and values greater than the LOD but below the LOQ were used unaltered. All reported concentrations are presented as arithmetic means with standard error. One procedural blank (high pressure liquid chromatography (HPLC) grade water, n = 1) was included in the extraction of each time point. No PFPA and PFPiA contamination was observed in the procedural blanks. The Canadian government recently listed the PFPAs and PFPiAs of varying perfluorocarbon chain length as potential precursors to long chain PFCAs (≥8 CF’s)35 and therefore PFCAs were monitored in fish, although they were not a component in the dosing. No production of PFCAs (SI Figure



RESULTS AND DISCUSSION Physical Effects Observed in Fish. No mortality occurred in either of the dosed and control populations. Significantly higher whole-body and liver growth rates (16.3 mg/day, wholebody; 16.1 mg/day, liver; p < 0.05, SI Tables S5 and S6b) were observed in the control population than in either of the dosed populations (5.9−9.6 mg/day). The fact that these growth rates were lower in the dosed populations than in the control contrasts a number of studies in which rainbow trout, exposed to PFCAs and PFSAs at concentrations similar to those used here, exhibited growth rates that were not significantly different than those for the control.21,22,37 LSI factors were calculated since this is a measure of liver enlargement and is used as an indicator of metabolic stress in an animal upon chemical exposure. No significant difference was observed in the mean 3491

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Figure 1. Growth-corrected whole-body homogenate concentrations (ng/g in wet weight, (ww)) of C6, C8, and C10 PFPAs and C6/C6, C6/C8, and C8/C8 PFPiAs in rainbow trout during exposure and depuration phase. The top panels represent the data collected from PFPAdosed fish and the bottom panels represent the data collected from PFPiA-dosed fish. Each data point represents the arithmetic mean concentration of the triplicate (n = 3) sampling at each time point. The error bar represents the standard error.

to steric constraints in crossing biological membranes during absorption into the gut. Although the pKA of PFPA is unknown, it is expected to be similar to the experimentally determined pKA’s of PFOA and PFOS ( 0.05, SI Table S8). Together with their estimated times of 700 amu) is consistent with the poor assimilation typically observed for chemicals with molecular weights greater than 600 amu,38 as was observed for the C16-chlorinated alkanes39 and other organochlorines with logKOW ≥ 7,40,41 and may in part be due 3492

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Figure 2. (A) Growth-corrected concentrations of PFPA metabolites (ng/g wet weight, (ww)) observed in fish dosed with a mixture of C6/C6, C6/ C8, and C8/C8 PFPiAs. (B) Percent PFPA yield with respect to accumulated parent PFPiAs (mol basis) in fish dosed with a mixture of C6/C6, C6/ C8, and C8/C8 PFPiAs. Each data point represents the arithmetic mean concentration of the triplicate (n = 3) sampling at each time point. The error bar represents the standard error.

profile previously observed for PFCAs and PFSAs in fish.14,21 Liver-to-blood (LBRs), liver-to-carcass (LCRs), and blood-tocarcass (BCRs) ratios (SI Figure S7, Table S10) were calculated and generally exceeded one, which together suggest PFPAs and PFPiAs are similar to other PFAAs in their tendency to predominate in proteinaceous compartments like the liver and blood in fish. The magnitudes and trends of these ratios are discussed in detail in the SI. Upon absorption into the bloodstream, some of the PFPAs and PFPiAs may exit enterohepatic recirculation and enter systemic circulation in the fish, as evidenced by their detection in the heart (nd−9 ng/ g ww, PFPA; 42−57 ng/g ww, PFPiA) and the gills (0.96−7 ng/g ww, PFPA; 34−57 ng/g ww, PFPiA) (SI Figure S6, Table S10). The gill contamination observed here suggests respiration may be an additional mode of depuration of the PFPAs and PFPiAs from the fish. Effect of Biotransformation on Bioaccumulation Parameters. Production of the C6 and C8 PFPAs was first observed in the PFPiA-dosed fish homogenates on day 6, with their concentrations increasing until the third day of the depuration phase (Figure 2). Metabolism of the parent C6/C6 and C6/C8 PFPiAs may yield the C6 PFPA either separately or synergistically, as with the C6/C8 and C8/C8 PFPiAs to C8 PFPA. Since the PFPA metabolite here may derive from two potential parent compounds, metabolite yields were estimated on a molar basis as the ratio of the amount of PFPA metabolite observed to the total amount of the two parent PFPiAs observed, as described in the SI. These yields should be treated conservatively as they are estimated by assuming equal contribution from the metabolism of both parent PFPiAs to the corresponding PFPA. On average, 12% of the C6/C6 and C6/C8 PFPiAs observed in the fish was metabolized to C6 PFPA, whereas 4% of the accumulated C6/C8 and C8/C8 PFPiAs was metabolized to C8 PFPA (Figure 2). No PFPA contamination was observed in the PFPiA neat material used to dose the fish feed, which further support the detection of C6 and C8 PFPAs in the PFPiA-dosed fish as biotransformation products. The lack of detection of C10 PFPA in the PFPiAdosed fish is also consistent with the congener profile in the dose in which none of the three PFPiA congeners contained a perfluorodecane (C10) linkage.

blood upon dietary exposure to a mixture of branched and linear isomers of PFOA and PFNA (3.7 days in liver and 5.6 days in blood, n-PFOA; 6.0 days in liver and 15.9 days in blood, n-PFNA).37 As was observed for water-borne21 and dietary22 exposure to PFCAs and PFSAs, the depuration half-lives observed here were positively correlated with the number of perfluorinated carbons present in PFPAs and PFPiAs (p < 0.05, r = 0.94, SI Table S9, Figure 3). This relationship is also consistent with the depuration trend observed in rats upon intraperitoneal injection of a mixed dose of PFPAs and PFPiAs.30 It is important to note that the difference between the headgroups of the doubly charged PFPAs and singly charged PFPiAs may also contribute to the differences observed in their clearance rates. Furthermore, geometry differences between the dialkylated PFPiAs and the monoalkylated PFPAs, PFCAs, and PFSAs limit direct comparison of the clearance rates between the PFPiAs and the three other classes of PFAAs. Assimilation of PFPAs and PFPiAs into Different Tissues. The highest concentrations of PFPAs and PFPiAs occurred in the liver (35−572 ng/g ww) and blood (33−116 ng/g) of rainbow trout collected on the last day of the exposure phase (SI Figure S6, Table S10), which suggest these chemicals primarily accumulate in the enterohepatic system. The high kidney concentrations also observed here (8−52 ng/g ww, PFPAs; 116−212 ng/g ww, PFPiAs; SI Figure S6, Table S10) are consistent with the demonstrated affinity between renal proteins and PFAAs.49,50 Although urine samples were not collected here, the potential of urinary excretion as a major route of elimination for PFPAs has been previously demonstrated by their observed high excretion efficiencies (up to 96% of the administered dose) in the urine of dosed rats.30 In that same study, the higher molecular-weight (MW) PFPiAs were not observed in any of the urine samples,30 the lack of which mirrored the low renal excretion (100 °C),53 but a biological degradation pathway has yet to be reported. Organic phosphonate and phosphinate biodegradation has been primarily studied in in vitro systems using cultured microbial enzymes (e.g., C−P lyase) that are known to be capable of cleaving the C−P bond.54,55 The mechanism by which this microbial bond cleavage occurs is widely debated, but various pathways, involving α-oxidation or free-radical dephosphorylation,55 have been proposed. To our knowledge, this is the first observation of an in vivo production of a phosphonate from a parent phosphinate in any organism, as well as, the first observation of an in vivo biotransformation of a PFAA. Literature on whether PFAAs biodegrade is limited to the observed disappearances of PFOA and PFOS in spiked WWTP sludge during anaerobic incubations without further confirmation by the detection of PFOA and PFOS metabolites and/or fluoride ions released from their mineralization.56 As was also observed for their parent PFPiAs, the C6 and C8 PFPA products were observed at the highest concentrations in the liver (27−36 ng/g ww), blood (10−13 ng/g ww), and kidneys (5−14 ng/g) (SI Figure S8). This tissue distribution suggests the liver and kidneys may be potential sites of biotransformation, although biotransformation in the gut cannot be precluded. Higher PFPA yields with respect to their corresponding parent PFPiAs were observed in the liver (17%, C6 PFPA; 12%, C8 PFPA) than in the kidneys (8%, C6 PFPA; 2%, C8 PFPA), which suggest potentially higher metabolic activity in the liver. Given that certain bacterial strains have been shown to cleave the C−P bond,54,55 the microbial flora present in the digestive tract and internal organs (e.g., liver and kidneys) of a fish57 may also be responsible for the biotransformation of PFPiAs observed here. A number of studies have demonstrated that biotransformation of a chemical can decrease its overall bioaccumulation potential in fish.58−60 The relatively low assimilation efficiencies observed here for the PFPiAs (Table 2) are consistent with previous reports of metabolizable compounds, like the shortchain polychlorinated alkanes58,59 and fipronil,60 having small assimilation efficiencies due to confounding of this parameter by the rapid metabolic depuration of these chemicals. The 3494

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bioaccumulation into rainbow trout despite their relatively large MWs (>700 amu). In vivo biotransformation of a PFAA is reported for the first time here, although it is unclear whether the fish or the bacterial flora present in the fish was responsible for this observed metabolism. In general, BMFs decreased in the order of sulfonate > carboxylate > phosphonate headgroups of PFAAs of equal perfluorocarbon chain length. Direct BMF comparisons cannot be made between the PFPiAs and other PFAAs, because no other PFAAs studied in rainbow trout22 had the same number of CF’s as the PFPiAs and PFPiA bioaccumulation was complicated by biotransformation, a problem that was not present with the other persistent PFAAs. As was observed for the PFCAs and PFSAs,22 the BMFs of PFPAs and PFPiAs were less than one, which suggest PFAAs, in general, do not biomagnify in juvenile rainbow trout from dietary exposure. However, there is considerable evidence that trophic magnification of PFOS and PFCAs (≥7 CF’s) does occur in aquatic and marine food webs4,7,15−20 and even in a remote terrestrial food web in northern Canada.63 Despite the relatively low bioaccumulation potential of PFAAs observed in fish, PFOS and PFCAs have been detected in higher trophic level animals, such as birds, minks, seals, foxes, caribou, and polar bears.4,7,10,12,15−20,63 This suggests the BMF data obtained here for the PFPAs, PFPiAs, and other PFAAs22 in rainbow trout cannot necessarily be extrapolated to predict the likelihood of their biomagnification in higher trophic level biota. The presence of PFPAs and PFPiAs in aquatic environments29,30 warrants their monitoring not only in aquatic biota, but also in terrestrial wildlife to evaluate the potential of these chemicals to undergo trophic magnification upon release into the environment.



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ASSOCIATED CONTENT

S Supporting Information *

A list of chemicals, extraction procedures, instrumental details, quality control data, statistical results, sample chromatograms, physical indices of growth data, and concentration data. This material is available free of charge via the Internet at http:// pubs.acs.org.



Article

AUTHOR INFORMATION

Corresponding Author

*Phone: (416) 978-1780 (S. A. M.); (905) 336-4407 (A. O. D. S.). Fax: (416) 978-3596 (S. A. M.); (905) 336-4699 (A. O. D. S.). E-mail: [email protected] (S. A. M.); amila. [email protected] (A. O. D. S.). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We gratefully acknowledge Nicole Riddell (Wellington Laboratories, Guelph, ON, Canada) for donating the PFPA and PPFiA neat material, native and mass-labeled standards, Norman White, the staff at the Aquatic Facility, Alicia Sales De Andrade, Leo Yeung, Derek Jackson (University of Toronto, ON, Canada), and Craig Butt (Duke University, NC, U.S.) for their assistance in this study. The present study is supported by the Environment Canada’s Chemicals Management Plan, and funded by Natural Science and Engineering Research Council of Canada (NSERC), and a NSERC Postgraduate Scholarship awarded to H.L. 3495

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