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Enantiomer Fractions of Chiral Perfluorooctanesulfonate (PFOS) in Human Sera Yuan Wang,† Sanjay Beesoon,† Jonathan P. Benskin,† Amila O. De Silva,‡ Stephen J. Genuis,§ and Jonathan W. Martin*,† †
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3 ‡ Aquatic Ecosystem Protection Research Division, Water Science and Technology Directorate, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, Canada, L7R 4A6 § Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Alberta, Canada, T6G 2R7
bS Supporting Information ABSTRACT: Perfluorooctane sulfonate (PFOS) is the most prominent perfluoroalkyl contaminant in humans and wildlife, but there is great uncertainty in exposure pathways, particularly with respect to the importance of PFOS-precursors (PreFOS). We explored the hypothesis that nonracemic proportions of chiral PFOS in serum are qualitative and semiquantitative biomarkers of human PreFOS exposure. A new chiral HPLC-MS/MS method was developed for alpha-perfluoromethyl branched PFOS (1m-PFOS, typically 23% of total PFOS) and applied to enantiomer fraction (EF) analysis in biological samples. In blood and tissues of rodents exposed subchronically to electrochemical PFOS, 1m-PFOS was racemic (EF = 0.4850.511) and no evidence for enantioselective excretion was found in this model mammal. 1m-PFOS in serum of pregnant women, from Edmonton, was significantly nonracemic, with a mean EF ((standard deviation) of 0.432 ( 0.009, similar to pooled North American serum. In a highly exposed Edmonton family (mother, father, and 5 children) living in a house where ScotchGard had been applied repeatedly to carpet and upholstery, EFs ranged from 0.35 to 0.43, significantly more nonracemic than in pregnant women. Semiquantitative estimates of % serum 1mPFOS coming from 1m-PreFOS biotransformation in both subpopulations were in reasonable agreement with model predictions of human exposure to PFOS from PreFOS. The data were overall suggestive that the measured nonracemic EFs were influenced by the relative extent of exposure to PreFOS. The possibility of using 1m-PFOS EFs for assessing the relative contribution of 1m-PreFOS (or PreFOS in general) in biological samples requires further application before being fully validated, but could be a powerful tool for probing general sources of PFOS in environments where the importance of PreFOS is unknown.
’ INTRODUCTION Perfluoroalkyl substances are widely used in industrial, commercial, and consumer products, and are globally distributed in wildlife, humans, and the environment. A major perfluoroalkyl substance in biological samples is perfluorooctane sulfonate (PFOS, C8F17SO3). It is generally the most prominent perfluorinated contaminant in biological samples from around the world14 and is regarded as among the most concentrated xenobiotics in human serum in some geographical areas.5 Like other perfluorinated acids, PFOS is not metabolized in the human body, it has a long elimination half-life in human serum (arithmetic mean of 5.4 years),6 it bioaccumulates in foodwebs,79 and it is extremely persistent in the environment. PFOS is a developmental toxicant in animal models10,11 and was found to disrupt the endocrine system.12 Due to its potential for adverse effects on biota and the environment, PFOS was listed as a “Persistent Organic Pollutant” under the Stockholm Convention.13 Nonetheless, its classification under Annex B of this agreement permits continued r 2011 American Chemical Society
production and widescale use of PFOS and its precursors. For example, in China, total production was greater than 200 t in 2006, and by 2009 there were 66 distinct PFOS-precursors registered with the Inventory of Existing Chemical Substances.14 PFOS-precursors (referred to hereafter as PreFOS) are a vast array of manufactured substances that have the potential to degrade to PFOS. These have diverse structures but are generally substituted perfluorooctanesulfonamides (C8F17SO2NRR0 ). These PreFOS compounds have been used as coatings on textiles, paper, carpeting, and food packaging to achieve oil, stain, and water repellence. It is estimated that the historic environmental emissions of PreFOS were higher than direct emission of PFOS,15,16 but most PreFOS molecules that were known to have been manufactured have never been analyzed in any sample.14 Received: July 7, 2011 Accepted: September 1, 2011 Revised: August 30, 2011 Published: September 01, 2011 8907
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Environmental Science & Technology It is notable that in human blood samples from China, 3070% of total organic fluorine remained unaccounted for by the perfluorinated analytes routinely monitored.17 Nonetheless, various forms of PreFOS are indeed detectable in wildlife, human samples, house dust, indoor and outdoor air,1820 and even in the oceans and ambient atmosphere, including at remote locations.21,22 Some evidence exists to show that PreFOS may degrade to PFOS through abiotic hydrolysis23 and atmospheric oxidation,24,25 but the current weight of evidence points to biodegradation as the dominant environmental degradation pathway that converts PreFOS to PFOS.2628 It is proposed that direct exposure to PFOS may be the dominant route of PFOS uptake under some scenarios, but the relative importance of PreFOS might increase and surpass direct exposure under others. This may help to explain the sometimes divergent temporal trends of PFOS observed in biota. For example, after the voluntary phase out of PFOS and PreFOS by the major manufacturer in North America between 2000 and 2002,29 PFOS concentrations declined in wildlife from the Western Arctic and Canadian Archipelago.30,31 On the contrary, in Greenland the concentrations in ringed seals32 and polar bears33 have been rising since 2000. Armitage et al.16 proposed that marine foodwebs around Greenland are being increasingly exposed directly to PFOS through ocean water that has been slowly transported north from legacy source regions in North America and Europe, while further west, the dominant sources to the Arctic marine foodweb must still be from atmospherically transported PreFOS in equilibrium with seawater. The PFOS anion is thought to be persistent in the marine water column, thus it is difficult to explain how PFOS can rapidly decline in foodwebs from the Western Arctic and Canadian Archipelago unless exposure to PFOS included semivolatile forms of PreFOS that rapidly partitioned out of seawater as global atmospheric concentrations of these declined. For humans, although serum concentrations of PFOS and PreFOS have declined in the U.S. since the phase out,34 PFOS concentrations in some Chinese populations have been increasing,35,36 and the relative role of PFOS and PreFOS is unknown. Knowing the human exposure sources in all areas of the world would more effectively facilitate exposure mitigation steps. Potential pathways of human exposure to PFOS and PreFOS are many and include food, food packaging, indoor air and dust, outdoor air, water, and contact with commercial materials. In two human exposure modeling studies, the relative contributions of PFOS and PreFOS to human body burdens from various pathways were estimated.37,38 Both models predicted that PreFOS contributes significantly to the PFOS body burden of the general population, but the accuracy of these models is questionable because the yields of PFOS from various forms of PreFOS are not known in humans. Furthermore, many commercially relevant PreFOS molecules have never been examined in the human environment, thus the total human dose of PFOS from PreFOS metabolism cannot be calculated with certainty, and all existing predictions may be underestimations.14 To date, there is only limited evidence to prove that human, wildlife, or environmental burdens of PFOS are affected by PreFOS, despite that PreFOS emissions are estimated to have been greater than PFOS emissions.14 One major obstacle has been the availability of analytical tools that can differentiate directly absorbed PFOS from PFOS formed through the metabolism of PreFOS. Historical emissions of PFOS and PreFOS
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have always contained a mixture of linear and branched isomers, owing to the electrochemical fluorination (ECF) manufacturing method. We demonstrated a general trend whereby branched PreFOS was metabolized more rapidly than linear PreFOS in vitro,39 thus highly branched PFOS isomer profiles in human blood40,41 may be a biomarker of PreFOS exposure. However, this biomarker has not yet been validated in vivo, and the wide applicability of the principle may be limited because fish were shown to rapidly excrete branched PFOS isomers in one study.42 A more recent consideration for PFOS source tracking is enantiomer fraction (EF) analysis. As noted by Arsenault et al.,43 and later by Rayne et al.,44 some of the major branched PFOS and PreFOS isomers contain a chiral carbon center, and thus each may exist as two nonsuperimposable mirror-image molecules, termed enantiomers. Manufactured or abiotically generated PFOS (i.e., from oxidized, photolyzed, or hydrolyzed PreFOS) should contain a racemic (i.e., 50:50) mixture of enantiomers. Conversely, if PreFOS is degraded to PFOS by enzymes, which are always chiral, it is likely there will be preferential metabolism of one PreFOS enantiomer, leading to a nonracemic mixture of the PFOS metabolite enantiomers. Thus, if nonracemic signatures of any PFOS isomer are observed in biological samples, this may be a useful biomarker of exposure to PreFOS. In a proof-ofprinciple in vitro study,45 an enantioselective chromatographic method was developed for a model 1m-PreFOS molecule (C6F13CF(CF3)SO2N(H)CH2(C6H4)OCH3), and its in vitro biotransformation was indeed enantioselective in human liver microsomes, based on disappearance of the parent compound. However, to date there has been no analytical method capable of resolving the enantiomers of the ultimate metabolite, alphaperfluoromethyl branched PFOS (1m-PFOS). Here we developed a novel chiral HPLC-MS/MS method for enantioseparation of 1m-PFOS which normally composes 23% of total PFOS in real samples. This was applied to measure EFs of 1m-PFOS in individual human serum samples for the first time. To aid with the interpretation the human EFs, the EFs of 1mPFOS were also examined in rodents exposed directly to 3M ECF PFOS.46,47
’ MATERIALS AND METHODS Chemicals. Optima-grade methanol, water, and tetrahydrofuran were purchased from Fisher Scientific (Ottawa, ON, Canada). HPLC-grade formic acid (50%) and triethylamine (g99.5%) were from Fluka (Oakville, ON, Canada). An ECF PFOS standard was provided by 3M Co. (St. Paul, MN). The composition of 1m-PFOS in ECF PFOS is 1.6%,48 and the purity of the ECF PFOS was 86.9%. The racemic 1m-PFOS standard in this study was synthesized at Wellington Laboratories Inc. (Guelph, ON, Canada) and was supplied in methanol at a concentration 4.59 mg/mL. Dilutions of the 1m-PFOS standard were prepared in methanol, or 50% methanol in water. Rat Disposition of 1m-PFOS Enantiomers. Archived samples from two previous PFOS isomer pharmacokinetic studies were analyzed here enantioselectively to confirm if direct exposure to 1m-PFOS would result in racemic EFs in exposed animals. Direct exposure to 1m-PFOS that results in racemic EFs in tissues or biological fluids would support the hypothesis that disposition (uptake, distribution, or excretion) of the 1m-PFOS anion is not enantioselective. Such preliminary evidence is essential if we are to claim that nonracemic 1m-PFOS signatures 8908
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Environmental Science & Technology in other organisms are a reflection of enantioselective PreFOS metabolism. From a single-dose oral gavage study (ECF PFOS, 400 μg/kg),46 samples from three individual male SpragueDawley rats, following 38 days of depuration, were randomly chosen for analysis. Available samples included urine, blood, liver, and kidney. From a subchronic (84 d) ad libitum dietary exposure study (425 ng/g ECF PFOS spiked in food),47 samples from 2 female and 1 male SpragueDawley rats were randomly selected for analysis. One of the female rats was sacrificed on day 84, while the other 2 rats were sacrificed after a further 60 days of depuration while consuming a PFOS-free diet. Serum, liver, and kidney samples (collected at the time of sacrifice) were analyzed. PFOS was extracted from all samples with an ion-paring method.2 For details of animal husbandry, PFOS administration, sample collection and treatment, please see refs 46 and 47. Sera of Individual Pregnant Women. Eight samples were randomly selected from a cohort of 271 pregnant women’s serum samples, collected during the 15th16th weeks of pregnancy between December 15, 2005 and June 22, 2006 in Edmonton, AB, Canada.49 All women were g18 years of age and delivered at g22 weeks gestation to live singletons. The samples were extracted by a quantitative solid-phase extraction technique.50 Ethics approval was obtained from the University of Alberta Health Research Ethics Board. Human Sera Collected from “High-Exposure” Family. The high-exposure family consisted of 7 members: father (52 yrs), mother (48 yrs), 4 sons (1523 yrs), and 1 daughter (18 yrs). All family members were healthy and were included in the analysis because they had previously been identified to have high exposure to PFOS. Their house carpet had been treated with ScotchGard at least 8 times over a 17-year period between 1991 and 2007. Serum samples were collected in November 2008. The same solid phase extraction method as mentioned above was used for extraction.50 Ethics approval was obtained from the University of Alberta Health Research Ethics Board. Enantioseparation of 1m-PFOS by HPLC-MS/MS. Two Chiralpak QN-AX HPLC columns (2.1 mm I.D. 150 mm each, 5 μm particles, Chiral technologies Inc. PA) in tandem with a C18 guard column (4.0 mm I.D. 3 mm, 5 μm particles, Phenomenex, Torrance, CA) were used for enantioseparation of 1m-PFOS. Chromatographic conditions were optimized in reversed-phase mode on an HPLC system consisting of an Agilent 1100 binary gradient pump, autosampler, and column oven (Agilent Technologies, Palo Alto, CA). Isocratic elution was applied and the mobile phase consisted of tetrahydrofuran, 0.2 M formic acid, triethylamine, and water in the ratio 70:20:0.05:10, by volume. Flow rate was 0.12 mL/min and column temperature was kept at 15 °C. Injection volume was 2 to 20 μL, depending on the concentration of 1m-PFOS in the samples. Detection of 1m-PFOS enantiomers was performed on an API 5000 triple quadrupole mass spectrometer equipped with a Turbo V ion spray source (Applied Biosystems/MDS SCIEX, Concord, ON, Canada) operating in negative ion mode using multiple reaction monitoring (MRM) for data collection. The precursor ion ([M H], m/z 499) and the unique product ion for 1m-PFOS 51 ([C8F17], m/z 419) were monitored (dwell time 150 ms). This MS/MS transition is free from interference by other PFOS isomers.51 The declustering potential was 64 V, the collision energy was 38 eV, the collision cell exit potential was 9 V, the entrance potential was 11 V, the ion spray voltage was 4000 V, and the source temperature was 500 °C.
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Nitrogen was used as nebulizer, auxiliary, curtain, and collision gas. Mass spectral data were collected and processed by Analyst 1.5 software. Peak Fitting and Calculation of EF. To accurately determine the EF, the partially resolved chromatogram of 1m-PFOS enantiomers was deconvoluted and integrated with Peakfit software (v4.06, Aspire Software International, Ashburn, VA), as previously described.52 As the elution order of the 1m-PFOS enantiomers was unknown, EF was defined here as the quotient of the first-eluting enantiomer (E1) peak area divided by the sum peak area of both enantiomers (E1 + E2). Statistical Approach and Definitions of Racemic and Nonracemic. Statistical differences between EFs in various groups of samples were tested by the Student’s t test (α = 0.05). Characterization of any group of samples as “non-racemic” was based on satisfying two criteria. First, the EF for any group of samples had to be significantly different from the racemic ECF standard. Second, the EFs in any group of samples should not overlap with the range of subtle matrix effect(s) measured for racemic 1m-PFOS spiked into various biological matrices. If these two criteria were not met, the group of samples was defined as racemic.
’ RESULTS AND DISCUSSION Enantioseparation of 1m-PFOS. As previously discussed, 1m-PFOS is the only PFOS isomer that produces a detectable m/z 419 product ion over the calibration range on our system.53 Thus, other PFOS isomers should not interfere in the enantioselective analysis of 1m-PFOS, and no special sample preparation or in-line isomer separation was needed. Unfortunately, PFOS lacks common moieties known to interact with enantioselectors of most chiral stationary phases, thus the enantioseparation of any PFOS isomer was predicted to be difficult. It has been acknowledged that chiral recognition requires a minimum of three simultaneous interactions between the stationary phase and one of the enantiomers, with at least one of these interactions being stereochemically dependent.54 Normally these interactions can include hydrogen bonding, dipoledipole interaction, ππ interaction, inclusion, or steric interaction.55 Because PFOS is anionic, a Chiralpak QN-AX column was chosen for the separation because it contains a quinine-based weak anion exchange site situated in a chiral environment.56,57 The column was previously shown capable of enantioseparating chiral acids in reversed-phase with polar organic solvents,58,59 thus also making it appropriate for mass spectrometry detection. For the selected analyte, 1m-PFOS, the chiral carbon is adjacent to the sulfonate moiety, thus theoretically increasing the chances of accomplishing an enantioseparation (i.e., of getting 3 simultaneous interactions on the stationary phase) on the chosen chiral column. Chiralpak QN-AX columns may be used in polar organic mode or reversed-phase mode. In this study, no separation in polar organic mode was achieved, but enantioseparation could be achieved under reversed-phase conditions using tetrahydrofuran as the organic modifier (methanol, acetronitrile, or dioxane were not effective). In practice, however, insufficient separation was accomplished with a single column, thus two QN-AX columns were connected in series (300 mm total length). Organic modifier types and concentrations, acid and alkaline additives, and separation temperature were further optimized. The acid additive was essential (20% of 0.2 M of formic acid), while small 8909
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Figure 2. Mean enantiomer fraction (EF ( 1 SD) of 1m-PFOS over a range of ECF PFOS standard injections (n = 9), in blood and tissues of rats subchronically exposed to ECF PFOS (n = 5 for blood, n = 3 for liver and kidney), and in human sera collected in Edmonton, Alberta, from pregnant women (n = 8) and a family (n = 7) with high exposure to PFOS, likely because of repeated carpet treatment applications of ScotchGard in their home. Dashed gray line shows the racemic reference point, EF = 0.5, while the dashed black lines show the boundaries of the subtle matrix effect (Table S1).
Figure 1. Deconvoluted and smoothed chromatograms (m/z 499/419) of 1m-PFOS on Chiralpak QN-AX showing (a) a racemic 1m-PFOS standard (calculated EF = 0.501), (b) an ECF PFOS standard (calculated EF = 0.500), and (c) human serum from a “high-exposure” individual living in a house with repeated ScotchGard applications (calculated EF = 0.350). The inset in (a) shows the original chromatogram, before deconvolution.
amounts of alkaline additive improved enantioseparation (0.05% triethylamine). Although baseline separation was not achieved, peak shapes were symmetric and resolution of the enantiomers was up to 0.85 (Figure 1). Although such separation was not optimal, it was nonetheless adequate for quantitative applications using peak deconvolution and integration software. When using this peak deconvolution method it was shown that the analytical bias in the EF is less than 1%, even when resolution approaches 0.5, and even when the EF is very nonracemic.52 It is germane to note that the other chiral isomers of PFOS did not enantioseparate under our conditions, and were therefore not monitored in real samples. It is notable that column stability was limited by this method. After approximately 100 injections, the retention time of 1mPFOS decreased from 160 to 115 min, and resolution of the enantiomers decreased from 0.85 to 0.55 for all samples presented in the current study. To assess the possible analytical bias resulting from variation in the performance of the method, two human serum extracts were repeatedly analyzed on the column over the usage period. Calculated EFs for each sample only shifted by 0.001 (maximum), thus the effect of any analytical bias was deemed negligible.
Method Sensitivity and Quality Control. The main objectives in this study were to evaluate the EF of 1m-PFOS in biofluids and tissues (humans and experimental rats); concentrations were not calculated. Nonetheless, the limit of detection (LOD) for the racemic 1m-PFOS standard was 0.45 pg oncolumn, and the limit of quantitation (LOQ) was 1.6 pg oncolumn, based on signal-to-noise ratios (S/N = 3 for LOD and S/N = 10 for LOQ). No carry-over was observed immediately after a high concentration 1m-PFOS standard was injected (i.e., up to 3.7 ng on column). There was also no shift in the baseline within the retention window of the enantiomers when blank matrix (liver extract in which 1m-PFOS concentration was nondetectable by this method) was injected, indicating an absence of nonspecific biological interferences. To avoid false positive detection of nonracemic EFs, possible matrix effects that may affect the electrospray ionization efficiency of the enantiomers were investigated. For example, if a matrix component coeluted with only one enantiomer of a racemic mixture, the signal for that one enantiomer may be artificially suppressed or enhanced, and the resulting EF would seem nonracemic. In this study, matrix effects were determined by spiking standard racemic 1m-PFOS into real sample extracts, including rat liver, kidney, urine, serum, and human sera; unfortunately there are no isotope-labeled internal standards for branched PFOS isomers. In the standard addition experiments, the additional peak area for each enantiomer was calculated by subtracting the prespike peak area from the postspike peak area, and in all types of samples the EF of the added spike was in the range of 0.48 to 0.52, indicating only subtle matrix effects (Table S1 in the Supporting Information). Overall, the developed HPLC-MS/MS analytical and peak integration method were regarded to be reliable and sensitive enough for EF determination of 1m-PFOS in real biological samples. The method was previously applied to two samples of pooled human 8910
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Environmental Science & Technology serum,14 but the analytical method is presented for the first time here. Absorption, Distribution, and Excretion of 1m-PFOS in Rats. We previously showed that a model chiral 1m-PreFOS molecule could be metabolized enantioselectively,45 thus it was hypothesized that nonracemic PFOS signatures may be a biomarker of PreFOS exposure. Nonetheless, it could not be ruled out that absorption, distribution, or excretion of PFOS are also enantioselective processes, as has been shown for some chiral drugs.60 Therefore, to aid in the interpretation of 1m-PFOS enantiomer profiles in humans, we examined the disposition of 1m-PFOS enantiomers in rats exposed to an ECF PFOS mixture.46,47 If 1m-PFOS disposition in rats is not enantioselective (i.e., EF = 0.5 in all tissues and biofluid), then finding EF 6¼ 0.5 in human samples would support the possibility of PreFOS exposure. The EF ((standard deviation) of 1m-PFOS in the ECF PFOS mixture was 0.498 ( 0.004 based on repeated measurements of the standard diluted in methanol (see example chromatogram in Figure 1b, and measure of the variation in Figure 2). This EF was not significantly different from 0.5, thus 1m-PFOS in ECF PFOS was confirmed to be racemic, as anticipated. The EFs of 1mPFOS in tissues of SpragueDawley rat exposed subchronically to ECF PFOS are shown in Figure 2. Mean EFs in blood, liver, and kidney were 0.485, 0.504, and 0.511, respectively, for all sample time points combined. Although the effect was subtle, blood (p = 0.04) and liver (p = 0.01), but not kidney (p = 0.14), were statistically different from the ECF standard, however these mean values were within the boundaries of the subtle matrix effect (Figure 2, Table S1). Furthermore, in kidney and urine of SpragueDawley rats exposed to a single dose of PFOS by gavage, the 1m-PFOS EFs in kidney (0.497 ( 0.015) and urine samples (0.503 ( 0.023) were racemic. Urine is the primary excretion route of 1m-PFOS in these animals,46 thus there is no evidence that rats can enantioselectively excrete 1m-PFOS. In subsequent sections, these combined results for a model mammal were used to assume that nonracemic EFs are only attributable to biotransformation of PreFOS; but it is acknowledged that these data are not proof of such. Other potential confounders of the EF are also discussed in the final section. Enantiomer Fraction of 1m-PFOS in Human Sera. The average EF of pregnant women (n = 8) in the current work was 0.432 ( 0.009 (Figure 2), ranging from 0.42 to 0.45. The EF for these samples was nonracemic based on statistical difference from the ECF standard (p < 0.001), and no overlap with the matrix effect range. The EF in these samples was also significantly lower than EFs measured in blood of the rats exposed subchronically to ECF PFOS (p < 0.001). Furthermore, these women had similar EFs compared to what we previously reported for human standard reference serum,61 NIST SRM 1589a (EF = 0.41), collected in 1996 from pooled blood samples of donors who consumed fish caught around the Great Lakes, and NIST SRM 1957 (EF = 0.44), collected in 2004 and pooled from serum of Americans. It has been estimated that PreFOS contributes 10% of PFOS to the body burden of most individuals,37 based on a presumed 20% metabolic conversion rate of PreFOS to PFOS. If this is taken to be true for 1m-PFOS, and using a very conservative assumption that metabolism of 1m-PreFOS is entirely enantiospecific (i.e., one enantiomer totally biodegraded while the other was entirely recalcitrant), an EF as low as 0.45, or as high as 0.55, is the expected outcome from a simple modeling exercise
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(see Table S2, and Figure S1 for model calculations and full set of assumptions). Using the same set of assumptions, the current result for pregnant women (i.e., EF = 0.432) implies that the biotransformation of 1m-PreFOS contributes at least 13% to the body burden of 1m-PFOS in this background population. This is comparable to the 10% figure estimated by Fromme et al. for total PFOS.37 Based on exposure modeling,38,62 it was furthermore predicted that a subgroup of humans in a “high-exposure” scenario (i.e., individuals with very high serum PFOS) might receive up to 6080% of PFOS from biodegradation of PreFOS. To test this hypothesis, serum from a “high-exposure” family (n = 7) was analyzed by the new method. Total PFOS concentrations in serum samples of these family members were 215 times higher than those in background Alberta populations.5 The family’s home had high coverage by carpeting which had been treated with ScotchGard at least 8 times between 1991 and 2007. The household furniture, mainly the family room couch, was also treated at least 5 times. Many ScotchGard formulations were known to contain various PreFOS compounds as residual material or active ingredient.14 The EFs in individual serum samples from this family ranged from 0.350 to 0.430 (mean = 0.392 ( 0.027) (Figure 2), strongly nonracemic, and the EF was significantly lower (i.e., more nonracemic) compared to the pregnant woman population (p = 0.003). The lowest EF (EF = 0.35) measured in the high-exposure family suggests that at least 30% of serum 1m-PFOS comes from 1m-PreFOS (Table S2, Figure S1), however the true contribution from 1m-PreFOS could be higher because of the conservative assumption for the model, discussed above. Overall, the current results for the background population and high exposure family corroborate model predictions38 that the relative importance of PreFOS (in this case 1m-PreFOS) exposure may be greater in highexposure circumstances. For all these human EF measurements, it is important to keep in mind that because exposure to PreFOS can come from the diet,37 it is possible that some biotransformation of PreFOS (to PFOS, or to some intermediate) occurs in the foodweb from which dietary items originate (i.e., from microbes through to vertebrates). Barring any enantiospecific absorption or elimination processes (none were observed in the rat study here for 1m-PFOS), the racemic or nonracemic EF signature of 1m-PFOS in a dietary item should therefore be transferred directly to humans following ingestion. Thus, 1m-PFOS EFs measured in humans likely represent an integration of both direct and indirect PreFOS exposure. Direct PreFOS exposure includes the in vivo human metabolism of PreFOS (i.e., absorbed through food, air, dust, or contact with commercial products), whereas indirect PreFOS exposure would include human exposure to PFOS that is resultant from the ex situ metabolism of PreFOS (e.g., PFOS in food that is a metabolite from PreFOS metabolism in the food chain). However, because all organisms in a foodweb are likely to have different relative enantiospecific biotransformation rates of 1m-PreFOS, it is never advisible to use the human serum 1mPFOS EF as a highly quantitative measure of PreFOS exposure. Rather, the strength of 1m-PFOS EF monitoring is as a qualitative biomarker of PreFOS exposure, and calculation of the minimum contribution of 1m-PreFOS to 1m-PFOS is, therefore, the only quantitative information that might be extrapolated from this technique. Nonetheless, it is important to consider the current limitations and potential confounders for both quantitative and qualitative interpretation of the 1m-PFOS EF. 8911
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Environmental Science & Technology Future Application, Limitations, and Potential EF Confounders. Significant PFOS and PreFOS manufacturing con-
tinues today, and further applications of enantiospecific PFOS analysis are warranted to examine hypotheses on the pathways of exposure to other human populations and wildlife. However, limitations of the method must be acknowledged in the current data set, and also in future applications. First, PFOS is very persistent in human blood,6 thus it should be noted that the measured 1m-PFOS EFs do not necessarily reflect a present day exposure scenario. Rather, it is most likely that the EFs represent an integrated historical exposure scenario, and long-term temporal trend studies of 1m-PFOS EFs would therefore be most valuable for assessing how the human exposure scenario has changed over the preceding decades. Second, the current estimates of % 1m-PreFOS contribution to body-burdens are highly conservative minima, but they are also specific to 1m-PFOS, which is a relatively minor PFOS isomer in human serum (i.e., 23% of total PFOS41). Exposure to PreFOS will occur as a mixture of linear and branched isomers (likely ∼70% linear PreFOS),14 but the relative metabolic yield of each PFOS isomer from its corresponding PreFOS isomer is unknown for humans, as are the elimination pharmacokinetics of each PFOS isomer once they are formed as metabolites. Therefore, we cannot quantitatively extrapolate the current result for 1m-PFOS to body burdens of total PFOS (i.e., branched and linear). However, given the highly conservative nature of the model used here to estimate % 1m-PFOS coming from 1mPreFOS, we argue that the same % values might be meaningful as a reasonable lower bound estimate for the body burden of total PFOS. Nonetheless, this will require further validation through continued application of the technique. Third, although it is assumed that the extent of biotransformation of PreFOS to PFOS is the major influence on the measured EFs, it remains a possibility that other biological phenomena might confound the interpretation of measured EFs. As examined here, evidence for the enantiospecific elimination of PFOS was not found in SpragueDawley rats, but it is possible that in humans or other animals that this could occur. Age, sex, and physiological status (including pregnancy) may also be important confounders that would make it difficult to interpret 1mPFOS EF data. Many of the above unknowns will only be understood or addressed through further application of this 1m-PFOS EF monitoring method to environmental samples, foodwebs, human studies, and temporal trend analysis in various matrices. Such studies are underway in our lab. Although still underutilized, years of cumulative EF data for other chiral persistent organic pollutants, such as certain PCBs, have proven to be powerful for understanding the processes controlling their environmental fate.63 Further method development aimed at extending the applicability of the current method to other chiral PFOS isomers is advisible to validate the current approach for PFOS. Improvements of the current analytical method, to increase sample throughput or improve resolution and sensitivity, would also be beneficial to future studies on 1m-PFOS.
’ ASSOCIATED CONTENT
bS
Supporting Information. Matrix effect calculations and a quantitative model of minimum relative contribution of 1mPreFOS to total 1m-PFOS in serum. This material is available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION Corresponding Author
*Phone: 1-780-492-1190; fax: 1-780-492-7800; e-mail: jon.martin@ ualberta.ca; mail: 10-102 Clinical Sciences Bldg., University of Alberta Edmonton, Alberta, Canada T6G 2G3.
’ ACKNOWLEDGMENT J.W.M. acknowledges Alberta Ingenuity (New Faculty Grant) and NSERC (Discovery Grant) for supporting the salary of Y.W. S.B. and J.P.B. acknowledge support from Alberta Heritage Foundation for Medical Research and Alberta Ingenuity, respectively. Dr. Scott Mabury (University of Toronto) is acknowledged for his significant shared contributions (study conception and design) to the single-dose and subchronic rodent ECF PFOS exposure studies. Ms. Emily Chan (University of Alberta) is acknowledged for extraction of pregnant women’s serum samples. Alberta Health and Wellness is thanked for support of daily laboratory operations. ’ REFERENCES (1) Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35, 1339–1342. (2) 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 (4), 766–770. (3) 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 (2), 373–380. (4) Smithwick, M.; Muir, D. C. G.; Mabury, S. A.; Solomon, K. R.; Martin, J. W.; Sonne, C.; Born, E. W.; Letcher, R. J.; Dietz, R. Perfluoroalkyl contaminants in liver tissue from east greenland polar bears (Ursus maritimus). Environ. Toxicol. Chem. 2005, 24 (4), 981–986. (5) Gabos, S.; Zemanek, M.; Cheperdak, L.; Kinniburgh, D.; Lee, B.; Hrudey, S.; Le, C.; Li, X. F.; Mandal, R.; Martin, J. W.; Schopflocher, D. Chemical biomonitoring in serum of pregnant women in Alberta (2005). The Alberta Biomonitoring Program 2008, 71–77. (6) Olsen, G. W.; Burris, J. M.; Ehresman, D. J.; Froelich, J. W.; Seacat, A. M.; Butenhoff, J. L.; Zobel, L. R. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ. Health Perspect. 2007, 115 (9), 1298–1305. (7) Martin, J. W.; Whittle, D. M.; Muir, D. C. G.; Mabury, S. A. Perfluoroalkyl contaminants in a food web from Lake Ontario. Environ. Sci. Technol. 2004, 38 (20), 5379–5385. (8) Houde, M.; Czub, G.; Small, J. M.; Backus, S.; Wang, X.; Alaee, M.; Muir, D. C. G. Fractionation and bioaccumulation of perfluorooctane sulfonate (PFOS) isomers in a Lake Ontario food web. Environ. Sci. Technol. 2008, 42, 9397–9403. (9) 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 (4), 664–672. (10) Lau, C.; Butenhoff, J. L.; Rogers, J. M. The developmental toxicity of perfluoroalkyl acids and their derivatives. Toxicol. Appl. Pharmacol. 2004, 198 (2), 231–241. (11) Shi, X. J.; Du, Y. B.; Lam, P. K. S.; Wu, R. S. S.; Zhou, B. S. Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS. Toxicol. Appl. Pharmacol. 2008, 230 (1), 23–32. (12) Jensen, A. A.; Leffers, H. Emerging endocrine disrupters: Perfluoroalkylated substances. Int. J. Androl. 2008, 31 (2), 161–169. (13) Wang, T.; Wang, Y. W.; Liao, C. Y.; Cai, Y. Q.; Jiang, G. B. Perspectives on the inclusion of perfluorooctane sulfonate into the 8912
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