Complementary Nontargeted and Targeted Mass Spectrometry

Nov 3, 2014 - (46) Yunker and Cretney identified higher BSAFs for PCDDs relative to PCDFs in Dungeness crabs.(68) In the present study, PCDDs were not...
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Complementary Nontargeted and Targeted Mass Spectrometry Techniques to Determine Bioaccumulation of Halogenated Contaminants in Freshwater Species Anne L. Myers,*,† Trudy Watson-Leung,‡ Karl J. Jobst,‡ Li Shen,‡ Sladjana Besevic,‡ Kari Organtini,§ Frank L. Dorman,§ Scott A. Mabury,† and Eric J. Reiner†,‡ †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario Canada M5S 3H6 Ontario Ministry of the Environment and Climate Change, 125 Resources Road, Toronto, Ontario Canada M9P 3V6 § Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 107 Whitmore Laboratories, University Park, Pennsylvania 16802, United States

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‡

S Supporting Information *

ABSTRACT: Assessing the toxicological significance of complex environmental mixtures is challenging due to the large number of unidentified contaminants. Nontargeted analytical techniques may serve to identify bioaccumulative contaminants within complex contaminant mixtures without the use of analytical standards. This study exposed three freshwater organisms (Lumbriculus variegatus, Hexagenia spp., and Pimephales promelas) to a highly contaminated soil collected from a recycling plant fire site. Biota extracts were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and mass defect filtering to identify bioaccumulative halogenated contaminants. Specific bioaccumulative isomers were identified by comprehensive two-dimensional gas chromatography high-resolution time-of-flight mass spectrometry (GCxGC-HRToF). Targeted analysis of mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans (PXDD/PXDFs, X = Br and Cl) was performed by atmospheric pressure gas chromatography tandem mass spectrometry (APGC-MS/MS). Relative sediment and biota instrument responses were used to estimate biota-sediment accumulation factors (BSAFs). Bioaccumulating contaminants varied among species and included polychlorinated naphthalenes (PCNs), polychlorinated dibenzofurans (PCDFs), chlorinated and mixed brominated/chlorinated anthracenes/phenanthrenes, and pyrenes/fluoranthenes (Cl-PAHs and X-PAHs, X = Br and Cl), as well as PXDD/PXDFs. Bioaccumulation potential among isomers also varied. This study demonstrates how complementary high-resolution mass spectrometry techniques identify persistent and bioaccumulative contaminants (and specific isomers) of environmental concern.

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INTRODUCTION Nontargeted analytical techniques are unconstrained by preconceived notions of contaminants of concern and are becoming increasingly important in the analysis of complex environmental mixtures. Recently, nontargeted techniques, such as comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-ToF), have been used to identify unknown halogenated contaminants in environmental samples.1−4 The use of high-resolution mass spectrometry further facilitates unknown identification by determining elemental compositions of accurate mass measurements. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), an ultrahigh resolution technique, and mass defect filtering have been used to identify chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) in vegetation exposed to an industrial fire5 and Lake Ontario lake trout.6 Comprehensive two-dimensional gas chromatography highresolution time-of-flight mass spectrometry (GCxGC-HRToF) © 2014 American Chemical Society

has been used to identify Cl-PAHs and mixed brominated/ chlorinated polycyclic aromatic hydrocarbons consisting of three or more aromatic rings (X-PAHs, X = Br and Cl) in several environmental matrices.7−9 These approaches may serve as complementary techniques in identifying previously unknown halogenated contaminants. First, the mass defect filtering of high-resolution mass spectral data reveals compound classes within complex data sets and provides initial screening information to direct further analysis. Second, the highresolution chromatographic separation and mass spectral data achieved via GCxGC-HRToF allow examination of specific isomers within congener groups identified in initial screening. Finally, analytes of interest discovered through these nonReceived: Revised: Accepted: Published: 13844

June 25, 2014 October 29, 2014 November 3, 2014 November 3, 2014 dx.doi.org/10.1021/es503090s | Environ. Sci. Technol. 2014, 48, 13844−13854

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trophic levels: Lumbriculus variegatus (oligochaete), Hexagenia spp. (mayfly nymph), and Pimephales promelas (juvenile fathead minnow). L. variegatus and P. promelas were raised from inhouse cultures, while Hexagenia spp. eggs were collected in the field (J. Ciborowski, University of Windsor) and raised in the laboratory. Bioaccumulation studies were performed using clean field collected control sediment (Long Point, Lake Erie, ON, CAN) spiked with soil collected from the 1997 Plastimet Inc. recycling plant fire site in Hamilton, Ontario. This fire produced a multitude of halogenated organic contaminants.5,10,47 To ensure sediment contained high levels of Plastimet ash without being acutely toxic, initial four-day toxicity screening tests on all three species determined a mix of 20:80 Plastimet soil to control sediment (dry weight) to be most appropriate. Spiked sediment was equilibrated for two months prior to testing. Bioaccumulation test conditions were optimized for organism physiological needs and to reduce variability in bioaccumulation response due to extraneous variables. Tests were performed in 2 or 4 L glass jars with sediment volume corrected to a 27:1 ratio of sediment organic carbon to organism dry weight and a sediment/water ratio of 1:4. For each treatment, clean control replicate test vessels were used to assess accumulation and survival in control sediment and to confirm organism health and test system integrity. Test jars were equilibrated for 24 h prior to organism exposure and were gently aerated via a Pasteur pipet during the exposure. Exposures were conducted at 23 ± 2 °C with 16 h of light and 8 h of dark each day (∼500 lux). Test organisms were exposed to sediments for a 28-day period. Upon initial exposure, organism sizes were approximately 5 mg, 29 ± 19 mg, and 241 ± 60 mg for L. variegatus, Hexagenia spp., and P. promelas, respectively. To each test jar, approximately 3 g (Hexagenia spp. and P. promelas) or 3.5 g (L. variegatus) wet weight of test species was added. Throughout the exposure, invertebrates did not receive food; however P. promelas were fed ground Nutrafin fish flake food at 1% wet body weight per day. After 28 days, organisms were sieved from sediment and transferred to clean dechlorinated water for a 24h depuration period. Following euthanasia, they were weighed and frozen until extraction. Lipid and Total Organic Carbon (TOC) Analysis. Details of lipid and TOC analysis have been reported previously48 and are available in the SI. Sample Extraction. Details of sediment and biota extraction procedures have been reported previously49,50 and are available in the SI. FTICR-MS Analysis. Details of FTICR-MS analysis are available in the SI. Briefly, a Varian 920 FTICR-MS, positioned in a Varian 9.4 T superconducting magnet, was used to obtain ultrahigh-resolution mass spectra using electron ionization (EI).5 The Hexagenia spp. sample extracts were injected (1 μL) on a Varian CP-3800 GC equipped with a DB-5HT GC column (15 m × 0.25 mm ID × 0.10 μm, J&W Scientific, USA). The FTICR-MS was operated at a resolution of 40 000 at m/z 400 full width at half-maximum (fwhm). Elemental compositions from accurate mass measurements were determined using the Varian Elemental Composition Calculator. Mass spectra were interpreted using mass defect filtering on an H/Cl mass scale. Details of this approach have been reported previously5,6 and are available in the SI. Briefly, mass spectral data were converted from the International Union of

targeted approaches may direct development of targeted analytical methods, such as gas chromatography tandem mass spectrometry (GC-MS/MS) analysis of mixed brominated/ chlorinated dibenzo-p-dioxins and dibenzofurans (PXDD/ PXDFs, X = Br and Cl).10 This complementary analytical approach provides the highresolution chromatographic separation and accurate mass information necessary to characterize complex mixtures of halogenated aromatic contaminants. Recently, a similar approach was used to characterize soil collected from the 1997 Hamilton, Ontario Plastimet Inc. recycling plant fire site, in which a variety of halogenated contaminants were identified.11 Combustion-derived Cl-PAHs, X-PAHs, and PXDD/PXDFs have received little attention relative to other combustion products, such as polychlorinated naphthalenes (PCNs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs), despite toxicological concerns.12−16 While there has been widespread environmental detection of Cl-PAHs,17,18 there are fewer reports of X-PAHs7,19−21 and PXDD/PXDFs.10,20−34 Although Cl-PAHs have been measured in air and airborne particulates,15,19,35−40 physicochemical properties suggest that, once deposited to water, they will partition into sediments and may be more bioaccumulative than corresponding parent PAHs.18 Similar environmental fates are expected for X-PAHs and PXDD/PXDFs. As a result, exposure of freshwater organisms to sediments contaminated with ClPAHs, X-PAHs, and PXDD/PXDFs is of concern. Biota-sediment accumulation factors (BSAFs) estimate the bioaccumulation of contaminants that cannot be effectively metabolized and eliminated. Bioaccumulation describes absorption of contaminants by an organism through all routes of exposure in a natural environment, including dietary uptake.41 Since Cl-PAHs, X-PAHs, and PXDD/PXDFs are comprised of thousands of congeners of which toxicological significance is specific to halogen substitution and position,13,42 BSAFs for specific isomers may be more informative than the corresponding congener group BSAF. For example, 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibits increased toxicological effects over other tetrachlorodibenzo-p-dioxin isomers because it can bind with the aryl hydrocarbon receptor as a result of its particular stereochemistry.43,44 In addition, identifying bioaccumulative isomers can direct future studies to compounds that pose a greater toxicological risk. However, assessing BSAFs of particular isomers without corresponding standards presents a complex analytical challenge. To date, the study of Cl-PAHs, XPAHs, and PXDD/PXDFs has relied on limited commercially available or in-house synthesized analytical standards. Nontargeted analytical techniques incorporating high-resolution chromatography and mass spectrometry help to narrow the focus of study to congeners and particular isomers of interest without a corresponding analytical standard. In the present study, complementary nontargeted and targeted analytical techniques are used to determine BSAFs for halogenated organic contaminants in three freshwater organisms exposed to soil collected from the Plastimet Inc. recycling plant fire site.

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EXPERIMENTAL SECTION Chemicals. Analytical standards used in this study are listed in the Supporting Information (SI). Bioaccumulation Study. Details of the bioaccumulation study methods have been reported previously.45,46 The study assessed three freshwater species of varying physiologies and 13845

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transitions and samples. To account for variation in sample dilutions, PXDD/PXDF concentrations were adjusted in accordance to the response of 13C12-2,3,7,8-Cl4DF, an internal standard added to samples prior to extraction. BSAF Calculations. BSAFs were estimated using either peak intensities or concentrations (C). All BSAFs were calculated using analyte responses from a single Plastimet sediment sample collected following P. promelas exposure. BSAFs were normalized to fractions of lipid in target organisms and sediment TOC. Approximated lipid fractions (f lipid) were 0.0300 (P. promelas), 0.0125 (Hexagenia spp.), and 0.0150 (L. variegatus). The TOC fraction ( f OC) was 0.24. BSAFs were calculated using eq 1.

Pure and Applied Chemistry (IUPAC) mass scale (C = 12.0000 Da) to an H/Cl mass scale (−H/+Cl = 34.0000 Da). For the converted data set, the mass defects were calculated by subtracting the nominal mass (rounded down) from the exact mass. A mass defect plot was constructed by plotting the H/Cl mass defect values (y-axis) against the IUPAC mass scale m/z values (x-axis). GCxGC-HRToF Analysis. Details of GCxGC-HRToF analysis are available in the SI. Briefly, PCN, Cl-PAH, and XPAH analyses were performed on an Agilent HP 6890 gas chromatograph (Agilent Technologies, Mississauga, CAN) coupled to a Waters GCT Premier time-of-flight mass spectrometer with EI (Waters, Milford, USA). The GCxGC system consisted of a single jet loop modulator (Zoex Corporation, Houston, USA), an Rtx-5MS first dimension column (30 m × 0.25 mm ID × 0.25 μm), and an Rtx-50 second dimension column (2.3 m × 0.18 mm ID × 0.2 μm; Restek Corporation, Bellefonte, USA). The modulation time was 4 s, and a mass range of m/z 150 to 800 was scanned at an acquisition rate of 10 Hz. The mass resolution was approximately 7000 fwhm. MassLynx software (Waters, Milford, USA) data files were converted for data processing of two-dimensional chromatograms in GC Image (GC Image, LLC, Lincoln, USA). Two-dimensional selected ion chromatograms (SICs) with a mass window of 0.04 Da were examined for analytes identified as bioaccumulative via FTICR-MS analysis. For each isomer peak of interest, the intensity and mass of the molecular ion were recorded for the most prominent peak slice. Retention time coordinates of the peak were recorded and corresponding mass spectra were confirmed to contain appropriate isotope ratios and fragments. Peak intensities were normalized to peak intensities of the internal standard (13C12-2,3,7,8-Cl4DF). APGC-MS/MS Analysis. Analyses were performed on an Agilent 7890A gas chromatograph (Agilent Technologies, Mississauga, ON, CAN) coupled to a Waters Xevo TQ-S tandem quadrupole mass spectrometer with an atmospheric pressure ionization source (APGC-MS/MS; Waters, Milford, USA). Analytes were separated on an Rtx-Dioxin2 column (60 m × 0.18 mm ID × 0.10 μm, Restek Corporation, Bellefonte, USA). A deactivated fused silica column (2 m, 0.18 mm ID column, Agilent Technologies, Mississauga, CAN) connected the column and ion source through the transfer line at 360 °C. The injection volume was 0.5 μL with an injector temperature of 270 °C. The GC oven program was held at 120 °C for 1 min, increased to 200 °C at 35 °C/min, then to 280 °C at 4.5 °C/ min, and was held at 280 °C for 8 min before increasing to 330 °C at 20 °C/min and held at 330 °C for 10 min. The mass spectrometer was operated in multiple reaction monitoring mode (MRM) with a source temperature of 150 °C. Dwell times for MRM transitions ranged from 0.005−0.03 s. Method parameters are available in Table S1. Sample analysis was based on a previously reported method.10 Quantification was performed via internal calibration using a linear calibration range of 0.5 pg/μL to 100 pg/μL and 13 C12-2,3-Br2-7,8-Cl2DD as an internal standard added prior to analysis. As a result of multiple PXDD/PXDF isomer peaks within each MRM transition, the sum of peak areas in one or two transitions was used for quantification, as indicated in Table S1. Corresponding MRM transition peak area ratios were monitored for each analyte to ensure sample response was within 40% of standard peak area ratios. Isomer groupings were confirmed by visual peak pattern matching between MRM

⎛ ⎞ ⎛ ⎞ C C BSAF = ⎜⎜ biota ⎟⎟ /⎜⎜ sediment ⎟⎟ ⎝ flipid ⎠ ⎝ foc ⎠

(1)

The Student’s t test was used to determine whether mean BSAFs were statistically greater than or less than 1 (p ≤ 0.05). Physical Property Estimates. Estimated log KOW and water solubility values at 25 °C were generated by the United States Environmental Protection Agency’s EPISuite51 and obtained through www.chemspider.com. Quality Assurance/Quality Control (QA/QC). For the bioaccumulation study, QA/QC was assessed through analysis of pre-exposed organisms, control sediment test jars (n = 3 per species), and replicate Plastimet sediment mix test jars (n = 5, 4, and 3 for L. variegatus, Hexagenia spp., and P. promelas, respectively). Throughout the study, organisms met method survival and growth criteria indicating no impairment from exposure. An internal standard (13C12-2,3,7,8-Cl4DF) was added to samples prior to extraction for comparison purposes. No specific method recovery studies were performed due to the nontargeted nature of the analysis; however extraction procedures have previously demonstrated recoveries for 2,3,7,8-tetrachlorodibenzo-p-dioxin in biota and sediment of 98 ± 2% and 102 ± 1%, respectively.49,50 No contaminants were identified in biota extracts from control test jars. Sample analyses by GCxGC-HRToF and APGC-MS/MS included solvent blanks and replicate injections to assess instrumental performance and precision. The APGC-MS/MS instrumental quantification limit (LOQ) was defined by a peak signal-to-noise (S/N) ratio ≥ 10. The LOQ for GCxGCHRToF analysis was defined by a monoisotopic peak intensity ≥ 10 cps. Peak identification in GCxGC-HRToF analysis required monoisotopic accurate masses to be within 20 ppm of theoretical masses, isotopic peak ratios to be within 20% of theoretical ratios, the presence of corresponding mass spectral fragments, and the peak to be present in every replicate biota extract.

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RESULTS FTICR-MS Analysis. Initial screening of Hexagenia spp. sample extracts via FTICR-MS analysis and mass defect filtering identified bioaccumulative analytes of interest. A mass defect plot identifying PCNs, Cl-PAHs, X-PAHs, and PCDFs in Hexagenia spp. exposed to the Plastimet sediment mix is presented in Figure 1. Congener classes were identified by their common mass defect and characteristic compound spacing of 33.9610 Da, representing the exchange of hydrogen for chlorine. For example, PCNs (C10H8−nCln+, n = 4−6) are represented by three clusters of orange squares with an average 13846

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Figure 1. Mass defect plot, based on the H/Cl mass scale, generated from FTICR-MS analysis of a Hexagenia spp. extract. Highlighted peaks represent PCNs, Cl-PAHs, X-PAHs, and PCDFs and proposed chemical structures.

mass defect value of 0.2074. Moving from left to right across the plot, PCN chlorine substitution increases from 4 to 6. Each cluster represents the relevant chlorine isotopic peaks of that particular PCN congener with characteristic isotope spacing of 1.9970 Da. The congener classes identified helped direct GCxGC-HRToF analysis. GCxGC-HRToF Analysis. Analysis by GCxGC-HRToF demonstrated the complex nature of the Plastimet sediment mix and variation in isomer bioaccumulation. Estimated BSAFs (and standard deviations) for PCN, Cl-PAH, X-PAH, and PCDF isomers that met peak identification criteria are presented in Table 1. BSAFs ranged from 0.34 (±0.10) to 2.9 (±0.6) for PCNs, 0.43 (±0.40) to 21 (±20) for Clanthracenes/phenanthrenes, and 1.1 (±0.4) to 2.5 (±2.0) for Cl-pyrenes/fluoranthenes. BSAFs ranged from 0.10 (±0.04) to 2.2 (±1.7) for X-anthracenes/phenanthrenes and the BSAF for the X-pyrene/fluoranthene isomer (C16H8BrCl) was 3.5 (±1.7). The BSAFs for PCDFs ranged from 0.040 (±0.030) to 1.2 (±0.7). Variation in C10H3Cl5 isomer bioaccumulation (circled peaks) among species relative to the Plastimet sediment mix to which they were exposed is shown in Figure 2. As a result of mismatched peak retention time coordinates between the Plastimet sediment mix and biota extracts, other isomers (uncircled) could not be confirmed as bioaccumulative. Similarly, several compounds identified in the mass defect plot of the Hexagenia spp. extract (Figure 1) do not have corresponding BSAFs in Table 1 because positive identification of bioaccumulative isomers was restricted to peaks observed in every biota extract replicate. Despite these analytical limitations, Figure 2 illustrates that the number and type of bioaccumulative isomers varies among species. From available standard solutions, it was possible to confirm the identity of six isomers presented in Table 2. Isomers a and c of C10H3Cl5 were identified as 1,2,3,5,7-pentachloronaphthalene and 1,2,3,4,6-pentachloronaphthalene, respectively. Isomer a of C10H2Cl6 was identified as 1,2,3,4,6,7-hexachloronaphthalene and/or 1,2,3,5,6,7-hexachloronaphthalene (coeluting isomers). Isomer a of C14H8Cl2 corresponded to 9,10dichlorophenanthrene. The isomer of C12OH4Cl4 was identified as 2,3,7,8-tetrachlorodibenzofuran, and isomer b of

Figure 2. Two-dimensional selected ion chromatograms for mass range 299.8400−299.8800 highlighting bioaccumulative isomers of C10H3Cl5. Circled isomer peaks were detected in all sample replicates for the species indicated and peak intensities were used to determine BSAFs relative to those corresponding peaks in the Plastimet sediment mix (Table 1). Isomers a and c were confirmed with a PCN standard solution as 1,2,3,5,7-pentachloronaphthalene and 1,2,3,4,6-pentachloronaphthalene, respectively. The two remaining unlabeled peaks in the PCN standard chromatogram correspond to 1,2,3,6,7-pentachloronaphthalene and 1,2,3,5,8-pentachloronaphthalene.

C12OH3Cl5 corresponded to 1,2,3,7,8-pentachlorodibenzofuran. APGC-MS/MS Analysis. Due to low concentrations, PXDD/PXDFs were not observed in FTICR-MS initial screening, and a more sensitive technique was required. Analysis by APGC-MS/MS provided chromatograms with isomer peak patterns that were unique to the PXDD/PXDFs examined (Table S1). Due to the large number of isomers (e.g., monobromo-trichlorodibenzofuran has 140 possible isomers), significant coelution was expected. Peak pattern retention time regions were determined using PXDD/PXDF standards and the Plastiment sediment mix extract. Relative isomer peak patterns observed for PXDD/PXDF congener groups in Plastimet sediment mix and L. variegatus extracts are shown in Figure 3. For PXDFs (C12OH5BrCl2, C12OH4BrCl3, C12OH3BrCl4, and C12OH4Br2Cl2), BSAFs correspond to the congener group rather than individual isomers because entire 13847

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Table 1. Mean BSAFs for Halogenated Compounds Identified in Biota Extracts with Associated Standard Deviations (SD)a

a

BSAFs for PCNs, Cl-PAHs, X-PAHs, and PCDFs analyzed by GCxGC-HRToF represent isomers. BSAFs for PXDD/PXDFs analyzed by APGCMS/MS represent congener groups or multiple isomers. Shaded cells indicate BSAFs were statistically greater than (in bold) or less than 1 (p ≤ 0.05, Student’s t test). ND indicates analyte was not detected. LD indicates analyte was detected but did not meet peak identification requirements. *BSAFs based on analyte instrumental responses < LOQ. **BSAFs based on extrapolated sediment concentrations. †n = 4.

Table 2. Six Isomers identified by GCxGC-HRToF in Table 1 Confirmed through Comparison of Retention Time Coordinates with Analytical Standardsa BSAF congener groups and isomers C10H3Cl5 C10H2Cl6 C14H8Cl2 C12OH4Cl4 C12OH3Cl5

a c a a a b

P. promelas

Hexagenia spp

L. variegatus

confirmed isomer

log KOW

solubility (25 °C, mg/L)

1.0 ND 0.62 LD LD ND

LD 1.6 LD 21 LD ND

1.1 LD 1.0 LD 0.18 0.040

1,2,3,5,7- pentachloronaphthalene 1,2,3,4,6- pentachloronaphthalene 1,2,3,4,6,7- or 1,2,3,5,6,7- hexachloronaphthalene 9,10- dichlorophenanthrene 2,3,7,8- tetrachlorodibenzofuran 1,2,3,7,8- pentachlorodibenzofuran

6.4 7.0 7.7 or 7.0 5.6 6.6 7.3

0.043 0.013 0.0020 or 0.0075 0.029 0.0019 0.00034

a

ND indicates analyte was not detected. LD indicates analyte was detected but did not meet peak identification requirements. Corresponding estimated values for log KOW and water solubility were generated by the United States Environmental Protection Agency’s EPISuite.

isomer peak patterns in the Plastimet sediment mix matched those in the biota extracts. In contrast, for other PXDD/PXDFs (C12OH3Br2Cl3, C12O2H5BrCl2, and C12O2H4BrCl3), BSAFs correspond to selected isomer peaks that were more prominent in biota extracts than other isomer peaks in the Plastimet sediment mix, suggesting particular isomers may be more bioaccumulative than others. These peaks were not considered interferences, as they were not observed in solvent blanks,

extraction method blanks, pre-exposed biota extracts, or biota extracts from control sediment test jars. Of the PXDD/PXDFs examined, C 1 2 OH 3 BrCl 4 , C12OH4Br2Cl2, C12OH3Br2Cl3, and C12O2H4BrCl3 were identified in L. variegatus extracts, while C12OH5BrCl2, C12OH4BrCl3, and C12O2H5BrCl2 were identified in both L. variegatus and Hexagenia spp. extracts. Estimated BSAFs (and standard deviations) for PXDFs and PXDDs ranged from 0.14 13848

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Figure 3. APGC-MS/MS chromatograms corresponding to MRM transitions of bioaccumulative PXDD/PXDFs: (A) C12OH5BrCl2 (313.8 > 206.8), (B) C12OH4BrCl3 (347.7 > 240.8), (C) C12OH3BrCl4 (381.8 > 274.5), (D) C12OH4Br2Cl2 (393.6 > 286.9), (E) C12OH3Br2Cl3 (427.6 > 320.9), (F) C12O2H5BrCl2 (329.6 > 159.8), and (G) C12O2H4BrCl3 (363.7 > 256.7). Top and bottom chromatograms correspond to Plastimet sediment mix and L. variegatus extracts, respectively. Peaks shaded in black correspond to peak areas used in BSAF calculations.

both the Plastimet sediment mix and biota extracts; however isomer peak patterns were too weak to meet quantitative method requirements. Method Performance. Precision of the GCxGC-HRToF was demonstrated by a triplicate injection of a P. promelas biota extract which generated BSAFs with percent relative standard

(±0.05) to 1.3 (±0.2) and 0.89 (±0.13) to 3.0 (±0.3), respectively (Table 1). PXDDs with six to eight halogen substituents (C12O2H2BrCl5, C12O2HBrCl6, and C12O2BrCl7) were observed in the Plastimet sediment mix, but not in biota extracts. PXDDs, C12O2H3BrCl4 and C12O2H4Br2Cl2, were observed in 13849

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rates in P. promelas may influence time required for the system to reach a steady state.56 BSAFs of PCNs, Cl-PAHs, X-PAHs, PCDFs, and PXDD/ PXDFs. On the basis of the equilibrium partitioning model, the theoretical range for organic contaminant BSAFs is between 1 and 2.59 The BSAFs presented in Table 1 represent a simplified model for examining the relative bioaccumulation potential of nontargeted contaminant isomers and congener groups in three species. The BSAF model used in the present study makes several assumptions that may lead to BSAFs outside the theoretical range of 1−2. It was assumed that at 28 days test jars were at a steady state, sediment was the only contaminant source, no metabolic degradation occurred, and all types of lipid and organic carbon were equal.56,60 In addition, it was assumed that contaminant concentration in the organism is a linear uptake function of sediment concentration,61 and concentrations of neutral organic contaminants are a function of organism lipid and sediment organic carbon content.62 Congener groups of PCNs (C10H3Cl5 and C10H2Cl6) were identified in all three organisms with BSAFs ranging from 0.34 to 2.9. A study of PCNs in a Baltic Sea benthic food chain identified a similar BSAF range of 0.69−1.4,63 and a Lake Ontario study observed C10H3Cl5 and C10H2Cl6 to be prevalent PCNs in benthic species.64 PCNs are known contaminants of concern, and their environmental occurrence and toxicity has been reviewed by Falandysz65 and Domingo.66 In the present study, GCxGC-HRToF allowed identification of specific bioaccumulative isomers without the use of analytical standards. As shown in Table 1, most PCN isomers were identified in L. variegatus, whereas only certain PCN isomers exhibited bioaccumulation potential in Hexagenia spp. and P. promelas. Of the analytes examined, only PCNs were identified to have bioaccumulation potential in P. promelas. This may be a result of increased water solubility and associated bioavailability in the water phase of smaller PCN molecules. This is evident from the confirmed isomers presented in Table 2 substituted with four or more chlorine atoms for which estimated PCN solubilities (0.0020−0.043 mg/L) are greater than those of PCDFs (0.00034−0.0019 mg/L).51 The BSAFs estimated for Cl-PAHs and X-PAHs varied widely among isomers (Table 1). The Cl-anthracenes/ phenanthrenes exhibited the largest range of BSAFs from 0.43 to 21. Isomer a of C14H8Cl2 identified as 9,10dichlorophenanthrene had the highest BSAF of 21 in Hexagenia spp.; however this value was not found to be statistically greater than 1. A total of five X-anthracene/phenanthrene isomers corresponding to C14H8BrCl and C14H7BrCl2 were identified in L. variegatus with BSAFs ranging from 0.10 to 2.2. Among Xanthracenes/phenanthrenes, isomer c of C14H8BrCl had the highest bioaccumulation potential with a BSAF of 2.2; however this value was not found to be statistically greater than 1. Relative to other contaminants, the Cl-pyrenes demonstrated higher bioaccumulation potential in both Hexagenia spp. and L. variegatus extracts with BSAFs ranging 1.1−2.5. The only Xpyrene identified was C16H8BrCl with a BSAF of 3.5. To date, only two studies have reported Cl-PAHs in environmental biota samples,6,67 despite increasing evidence of the toxicological risks they pose.13,15,16 The present study provides further evidence of the bioaccumulation potential of Cl-PAHs and new evidence of the bioaccumulation potential of X-PAHs. The BSAFs estimated for PCDFs were not found to be statistically greater than 1. Similarly, Van Geest et al. observed BSAFs less than 1 for PCDD/PCDFs in L. variegatus,

deviations (%RSD) for C10H2Cl6 isomers a and b of 46% and 40%, respectively. All experimental masses were within 20 ppm of corresponding theoretical masses. Isotopic peak ratios were generally within 20% of theoretical values. A three-point internal standard calibration curve constructed from 10, 40, and 200 pg/μL C12-2,3,7,8-Cl4DF (with 100 pg/μL 13C12-2,3,7,8Cl4DF) standard solutions gave an R2 value of 0.9943. Good precision was shown for APGC-MS/MS analysis with %RSDs for multiple PXDD/PXDF standard injections ranging from 0.55% for 20 pg/μL 8-Br-2,3,4-Cl3DF (n = 4) to 23% for 1 pg/μL 1,3-Br2-2,7,8-Cl3DF (n = 3). A triplicate injection of an L. variegatus biota extract generated BSAFs with %RSDs ranging from 1.5% for C12O2H4BrCl3 to 7% for C12OH3Br2Cl3. For BSAFs that were statistically greater than or less than 1 (Table 1), %RSDs ranged 8−93% (mean = 37%, n = 31). The range in error associated with BSAFs is likely a cumulative result of GCxGC-HRToF instrumental precision error and variation among bioaccumulation study test jars.

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DISCUSSION Variation in Contaminant Uptake among Species. Several factors were considered when comparing contaminant uptake by different organisms. First, species behavior influences contaminant exposure in aquatic systems. L. variegatus and Hexagenia spp. are invertebrates that burrow into and ingest sediments and therefore have direct interactions with contaminants that are sorbed to sediment and dissolved in pore water.52 P. promelas are exposed to contaminants through ingestion and resuspension of sediments, as well as watersoluble contaminants via absorption through gills and skin.52 Second, lipid content influences uptake of halogenated organic contaminants. In the present study, approximate lipid contents were 1−2%, 0.5−2%, and 3% for L. variegatus, Hexagenia spp., and P. promelas, respectively. Third, the organism’s ability to metabolize and eliminate the contaminant reduces bioaccumulation. Finally, as a result of these factors, the time required for test jars to reach a steady state varies among species. Of the 36 PCN, Cl-PAH, X-PAH, and PCDF isomers and PXDD/PXDF congener groups listed in Table 1, 94% were identified in L. variegatus extracts, while 25% and 11% were identified in Hexagenia spp. and P. promelas extracts, respectively. The high bioaccumulation potential of these contaminants exhibited in L. variegatus may correspond to its direct interactions with sediment and slow metabolism of PAHs.53−55 Previous studies have shown similar bioaccumulation trends for polychlorinated aromatic contaminants in L. variegatus and Hexagenia spp.;46,56 however in the present study, fewer contaminants were identified in Hexagenia spp. tissue extracts relative to L. variegatus. This may be due to variation in contaminant uptake and elimination among individuals, although mechanisms of metabolism in Hexagenia spp. are unknown. Despite P. promelas having the highest lipid content among the three species tested, only four PCN isomers were identified in P. promelas tissue extracts. This may correspond to previous studies that demonstrated contaminant concentrations in fish do not always reflect exposure57 due to metabolism of organic contaminants, such as PAHs,58 or due to limited direct interactions with sediment. An exposure study with polychlorinated biphenyls (PCBs) observed higher uptake and elimination rates for Hexagenia spp. and L. variegatus relative to P. promelas.56 The authors attributed the findings to differences in exposure routes and suggested slower uptake 13850

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Hexagenia spp., and P. promelas exposed to various sediments.46 Yunker and Cretney identified higher BSAFs for PCDDs relative to PCDFs in Dungeness crabs.68 In the present study, PCDDs were not observed, which was due to higher instrumental detection limits. Yunker and Cretney also identified higher BSAFs for 2,3,7,8-substituted PCDD/PCDFs compared to other isomers.68 Similarly, in the present study, the only tetrachlorodibenzofuran isomer identified corresponded to the retention time coordinates of 13C12-2,3,7,8Cl4DF, confirming this isomer as 2,3,7,8-tetrachlorodibenzofuran. While only one or two isomers were identified for each PCDF congener group by GCxGC-HRToF analysis, isomer peak patterns of PXDFs (C12OH5BrCl2, C12OH4BrCl3, C12OH3BrCl4, and C12OH4Br2Cl2) observed in APGC-MS/ MS analysis indicated bioaccumulation potential for nearly all isomers in the congener group (Figure 3). In contrast, chromatograms for one PXDF (C12OH3Br2Cl3) and PXDDs (C12O2H5BrCl2 and C12O2H4BrCl3) indicated selected isomer peaks were more prominent in biota extracts than others observed in the Plastimet sediment mix (Figure 3). Higher bioaccumulation potential was shown for C12OH5BrCl2 and C12O2H5BrCl2 with BSAFs statistically greater than 1. To date, only three studies have reported PXDD/PXDFs in environmental biota samples,32−34 despite known toxicological risks.12,14 The present study provides evidence of the bioaccumulation potential of PXDD/PXDFs in freshwater invertebrates. Considerations for Combustion-Derived Contaminant Bioaccumulation. In some instances, the bioaccumulation potential of a contaminant may be predicted by its octanol− water coefficient (KOW) or hydrophobicity. The estimated log KOW values for confirmed isomers in Table 2 range from 5.6 to 7.7,51 suggesting bioaccumulation will be influenced by both organism uptake rates and contaminant desorption from sediment.55 For contaminants with log KOW values greater than 6, the time required for a test system to reach equilibrium, as well as measurement and extraction techniques, may also influence (and possibly underestimate) bioaccumulation potential.69 A correlation between bioavailability of planar molecules and black carbon content of sediment has been reported previously.70−74 It is expected that strong π−π interactions exist between soot particles and planar contaminants, which decreases their desorption rate from sediments, thereby affecting their bioavailability and bioaccumulation.72 In the present study, the extraction method isolated planar compounds through a carbon column cleanup, and the soil collected from the Plastimet fire site undoubtedly contained significant amounts of black carbon as a result of incomplete combustion. Since PCNs, Cl-PAHs, X-PAHs, PCDFs, and PXDD/PXDFs are strongly associated with combustion processes, their association with black carbon may influence their bioaccumulation potential. Potential and Limitations in Nontargeted Analysis of Environmental Contaminants. The nontargeted approaches applied here are accessible to a modern laboratory. Mass defect filtering may be performed with any high-resolution mass spectral data set and serves as a valuable initial step in screening sample extracts for analytes of interest. It has been applied in many fields of study,75 including petroleomics,76−78 but has only recently been used to identify halogenated contaminants in environmental samples.5,6,11

Analysis by GCxGC-HRToF provides detailed information about isomer specific bioaccumulation. The advantage of chromatographic peak separation and high-resolution mass spectral information for distinguishing between a Cl-PAH and X-PAH with a common unit mass is demonstrated in Figure S1 and has been described previously.11 While the peaks are clearly chromatographically separated in this example, corresponding high-resolution mass spectra provide additional confirmation of their identity through accurate mass, isotopic peak ratios, and mass fragments. Chromatographic separation is most valuable in the present study for its ability to distinguish between isomers in an unknown mixture that would not be resolved by high-resolution mass spectrometry alone. Further characterization of bioaccumulative isomers would entail comparison of chromatographic peaks of interest with analytical standards of the suspected isomer. Isomeric structure may also be deduced using relative retention time coordinates of other isomers in the congener group. An important limitation in the present study was the slow acquisition rate of the HRToF. Typically, GCxGC-MS analysis is performed at an acquisition rate greater than 100 Hz with unit mass resolution; however low mass resolution is limiting in the identification of unknown contaminants in complex mixtures.7 In the present study, the acquisition rate of 10 Hz produced peak slices with fewer than 10 points over the peak, which did not meet typical quantitative requirements. Since the intensity of the most prominent peak slice was used for analysis, the analytical accuracy and precision relied on a single mass spectrum. This likely contributed to error in replicate injections and BSAFs, despite accurate masses and isotopic peak ratios meeting other method requirements. Coupling GCxGC to an HRToF capable of higher acquisition rates, as several recent studies have done,7,9,79 serves as a powerful enhancement in nontargeted analysis of complex environmental samples.

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ASSOCIATED CONTENT

S Supporting Information *

Further method details are described. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Wellington Laboratories for PXDD/PXDF solutions, as well as Vince Taguchi for use of the FTICR-MS. Thanks to Liad Haimovici, Christie Hartley, Leo Yeung, Miren Pena, Bert van Bavel, Frank Wania, and Derek Muir for useful discussions and feedback. Thanks also to Kurunthachalam Kannan for ClPAH standards and the MOECC Aquatic Toxicology Unit.

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REFERENCES

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