Atmospherically Deposited PBDEs, Pesticides, PCBs, and PAHs in Western US National Park Fish: Concentrations and Consumption Guidelines

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1 Supporting Information For: Atmospherically Deposited PBDEs, Pesticides, PCBs, and PAHs in Western US National Park Fish: Concentrations and Consumption Guidelines Luke K. Ackerman, Adam R. Schwindt, Staci L. Simonich*, Dan C. Koch, Tamara F. Blett, Carl B. Schreck, Michael L. Kent, Dixon H. Landers Number of Pages: Number of Tables: Number of Figures: - 1 -

2 Experimental Chemicals All solvents were Optima grade from Fisher Scientific (Fairlawn, NJ). Anhydrous sodium sulfate was Mallinckrodt Baker (Phillipsburg, NJ) pesticide grade. Isotopically NONlabeled chemical standards (natives) in mixes and individually were purchased from, the EPA repository, Chem Services Inc. (West Chester, PA), Restek (Bellefonte, PA), Sigma- Aldrich Corp. (St. Louis, MO), AccuStandard (New Haven, CT), or Cambridge Isotope Labs (Andover, MA). Isotopically labeled standards were purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada) or Cambridge Isotope Labs (Andover, MA). The isotopically-labeled recovery surrogates were d 10 -fluorene, d 10 -phenanthrene, d 10 -pyrene, d 1 -triphenylene, d 1 -benzo[a]pyrene, d 1 -benzo[ghi]perylene, d 1 -EPTC, d -atrazine, d 10 - diazinon, d 7 -malathion, d 10 -parathion, d 8 -p,p -DDE, d 8 -p,p -DDT, d 6 -methyl parathion, d - alachlor, d 11 -acetochlor, C 1 -PCB 101 (,,,, -pentachlorobiphenyl), C 1 -PCB 180 (,,,,,, -heptachlorobiphenyl), d 10 - chlorpyrifos, C 6 -HCB, d 6 --HCH, d - endosulfan I, d -endosulfan II, d 1 -trifluralin, C 1 -BDE 8 (,, -tribromodiphenyl ether), C 1 -BDE 7 (,,, -tetrabromodiphenyl ether), C 1 -BDE 99 (,,,,- tetrabromodiphenyl ether), C 1 -BDE 100 (,,,,6-tetrabromodiphenyl ether), C 1 - BDE 118 (,,,,-tetrabromodiphenyl ether), C 1 -BDE 8 (,,,,, - tetrabromodiphenyl ether), C 1 -BDE 1 (,,,,, -tetrabromodiphenyl ether), and C 1 -BDE 18 (,,,,,,6-tetrabromodiphenyl ether). The isotopically-labeled internal standards were d 10 -acenaphthene, d 1 -benzo[k]fluoranthene, d 10 -fluoranthene, and C 1 -PCB 8 (,,,,, -hexachlorobiphenyl). All standards were stored at C and remade as needed, or at least once a year to insure stability. The organophosphate standards were stored separately from other chemical classes in ethyl acetate (EA) to minimize degradation. All lab ware was washed, rinsed, covered in foil, baked at C for 1hrs, and solvent-rinsed prior to use. Samples Standard Reference Material (SRM) #196 Lake Superior Lake Trout was purchased from the National Institute of standards and Technology (NIST) (Gaithersburg, MD). Representative fish samples for method detection limit and recovery studies were collected from remote lakes of US National Parks, specifically Wonder Lake, Denali NP, AK, Emerald Lake, Sequoia NP, CA, and Mills Lake, Rocky Mountain NP, CO. Most fish were caught with hook and line, stored in 8 gall buckets with lake water for less than 0 min prior to euthanasia and field dissection. Gill-nets and set lines were employed for the collection of some fish from Denali National Park. Upon capture ~100uL of blood was removed from the caudal vein for hormone, biomarker, and hematological analysis, and the fish was euthanized, weighed, and fork length measured. A quick field dissection to obtain pathological slices of kidney, spleen, liver, gills, and gonads was performed on solvent rinsed and baked foil with solvent rinsed scalpels and forceps. Gut contents were evacuated, and the carcass was wrapped in foil or aluminized poly-bags, double-bagged, and shipped to the laboratory on dry ice. Field blanks of the collection procedure without a fish were also obtained. When received at the lab the samples were logged into a - C freezer until analysis. - -

3 Sample Preparation Whole fish carcasses were homogenized in liquid nitrogen inside a Blixer BX-6 HP stainless steel food processor with polycarbonate lid until a fine, frozen slurry of nitrogen and fish homogenate was obtained. Personal protective equipment included face shields and cold gloves. Frozen homogenate was transferred to cleaned jars, sealed, weighed, and stored at - C, until extraction. Method blanks were obtained by grinding 10 g clean sodium sulfate with the liquid nitrogen and processing samples through the method. A 1- g subsample of the fish homogenate was gravimetrically analyzed for moisture content by baking for hrs at 10 C and re-weighing (until stable). Sample Extraction Samples and method blanks (~0g fish, 1.8g SRM) were weighed out onto a clean tarred glass mortar with a bed of clean sodium sulfate, sodium sulfate was added and the mixture ground until free flowing (<10min). The fish sodium sulfate mixture was transferred into 100mL stainless steel Accelerated Solvent Extractor (ASE) cells (Dionex, Sunnyvale, CA) with each cell containing ~10 g of the fish/sodium sulfate mixture. Once in the ASE cells, 1.-10ng of isotopically labeled recovery surrogates in ng/ul solutions were distributed equally among the tops of the cells containing sample or blank. The cells were capped. The sample or blank was extracted using dichloromethane (DCM) (100 C, 100 psi, cycles of minutes, 10 % flush volume). The extract was dried with clean sodium sulfate, decanted and filtered with glass wool into a 00mL volumetric flask, which was filled to the mark. Gravimetric lipid analysis was performed on % of the sample by heating the evaporated 10ml extract sub-sample at 10 C for one hour and weighing the extracted lipid that remained (until stable). The remainder of the extract was concentrated to 0. ml in the TurboVap II (Zymark, Hopkinton, MA) with nitrogen, and solvent exchanged to hexane. Polar matrix interferences were removed by loading the hexane extract on a 0 g silica solid phase extraction (SPE) cartridge (Mega Bond Elut Varian). Analytes were eluted from the silica SPE using ml hexane, ml 60:0 hexane:dcm, and 7 ml 70:0DCM:hexane. The combined eluate was then concentrated and solvent exchanged to DCM. Elemental sulfur and the high molecular weight interferences were removed using a Waters Gel Permeation Chromatography (GPC) Cleanup System (Milford, MA). The target fraction was concentrated to 0. ml with ethyl acetate rinses under a gentle stream of nitrogen and spiked with 1 µl of a 10 ng/µl isotopically labeled internal standard solution. Blank extracts were carried through the entire method together with batches of to samples. GC/MS Analysis In general extracts were analyzed by gas chromatographic mass spectrometry (GC/MS) on an Agilent 6890 GC coupled with an Agilent 97N MSD, using both electron impact (EI) ionization and electron capture negative ionization (ECNI) modes. The ECNI compounds monitored, their SIM windows, and characteristic ions are listed in Table S. Analysis was attempted for a larger list of compounds than that for which the method was subsequently validated. One microliter of the 0. ml extract was injected in pulsed splitless mode with a HP 768 auto-sampler. The inlet was a mm id glass gooseneck with wool, at 00 C (pulse of 0 psi until 0.6 min). Chromatographic separations were achieved on a DB-MS column (J&W Scientific, 0m x 0. mm id.; 0. Rm film thickness) with helium carrier gas flowing at 0.9 ml/min. The GC oven temperature program used for the GC/EI-MS was - -

4 reported previously and the program for the ECNI/MS was modified as follows to permit quantitation of the PBDEs: 10 C for 1 min, ramped at C/min to 7 C, ramp 6 C to 0, held 6.7 min for a total runtime of minutes. QA/QC Internal standard calibration curves were prepared from standard solutions, ranging. fg/ul to ng/ul. A minimum of calibration points (S per order of magnitude) were included in every curve and the curve was required to yield an r >0.98, or less than 1% deviation at every point. Calibration accuracy was checked with a low level calibration solution at a frequency of at least 18%, and the instrument re-calibrated as needed. Calibration solutions were re-prepared at least once a year or as needed. Duplicate injections were performed at a frequency of 10% and duplicates with > % relative deviation were flaged and re-run as needed. Standard reference material was analyzed at a frequency of 10%. Target compounds were identified only when the following criteria were met: a peak of greater than :1 S:N was observed for all three characteristic ions within 0.01 min or one scan of each other, the quantitation ion peak matched standard RT within 0.0 min, and the relative response of the three ions was within 0% of standards relative responses. Sample values were flagged if the lab blank was greater than % of the sample analyte mass, the field blank greater than 0%, the calibration check >0% deviation, or if the recovery surrogate yielded >0% or <0% recovery. Quantitation values were later censored according to method detection and quantitation limits. The method quantitation limit was set as the mass of the lowest concentration calibration solution used for each curve, normalized to each sample s mass extracted. If the mass in any blank was greater than % of the corresponding sample s mass, the sample value was deleted and flaged. All reported values have been blank subtracted and recovery corrected. Results The following compounds were detected in less than 0% of the samples and less than 0% of the samples from any single lake: b-hch, d-hch, heptachlor, aldrin, endrin, endrinaldehyde, PCB 7, BDEs #10, 7, 8, 1,, 1, 0,, 17,,, 7, 7, 71, 66, 77, 119, 116, 16, 118, 8, 166, 181, 190, triallate, chlorpyrifos oxon, etridiazole, parathion, ethion, and all PAHs. - -

5 Table S1. Average Fish Sample and Site Characteristics Lake US National Park Latitude (dd) Longitude (dd) Elevation (m) Fish Species Length (mm) mass (g) Age (yr) Growth Rate (g/yr) Condition Factor (cg/cm ) Sex Ratio (M:F) Date Sampled 1) Emerald Sequoia-Kings Canyon brooktrout //0 10 ) Pear Sequoia-Kings Canyon brooktrout //0 10 ) LonePine Rocky Mountain brooktrout //0 10 ) Mills Rocky Mountain rainbow trout /08/0 10 ) LP19 Mt. Rainier brooktrout /08/0 10 6) Golden Mt. Rainier brooktrout //0 10 7) Hoh Olympic brooktrout /08/0 10 8) PJ Olympic brooktrout //0 10 9) Oldman Glacier cutthroat trout /19/ ) Snyder Glacier cutthroat trout // ) McLeod Denali burbot, whitefish /10/0, 1) Wonder Denali lake trout /1/0 10 ) Matcharak Gates of the Arctic lake trout /01/0 10 1) Burial Noatak National Preserve lake trout /0/0 10 Total 6 n

6 Figure S1 Map of National Parks/Preserves Sampled 1

7 Table S. ECNI Target Analytes, Surrogates, Internal Standards, & SIM Windows Analytes, Surrogate for Internal Standard Retention Time Quantiation Confirmation nd Confirmation & Surrogates Quantitation for Quantitation (min) Ion m/z Ion m/z Ion m/z SIM Window 1 d 1 Trifluralin C 1 PCB Trifluralin d 1 Trifluralin SIM Window HCH, alpha d 6 gamma-hch C 6 -HCB HCB C 1 PCB C 6 HCB SIM Window HCH, beta d 6 gamma-hch d 6 gamma-hch C 1 PCB HCH, gamma d 6 gamma-hch SIM Window HCH, delta d 6 gamma-hch Triallate d 6 gamma-hch SIM Window PBDE #10 C 1 PBDE Metribuzin d 6 gamma-hch Heptachlor d 6 gamma-hch PBDE 7 C 1 PBDE SIM Window 6 Chlorpyrifos oxon d 10 Chlorpyrifos d 10 Chlorpyrifos C 1 PCB Aldrin d 6 gamma-hch PBDE #8 C 1 PBDE Chlorpyrifos d 10 Chlorpyrifos SIM Window 7 Dacthal d 6 gamma-hch PBDE 1 C 1 PBDE PBDE C 1 PBDE PBDE 1 C 1 PBDE SIM Window 8 Chlordane, oxy d Endosulfan I Heptachlor epoxide d Endosulfan I PCB 7 C 1 PCB SIM Window 9 Chlordane, trans d Endosulfan I PBDE 0 C 1 PCB 101 SIM Window 10 C 1 PBDE C 1 PCB PCB 101 C 1 PCB d Endosulfan I C 1 PCB Endosulfan I d Endosulfan I Chlordane, cis d Endosulfan I SIM Window 11 Nonachlor, trans d Endosulfan I SIM Window 1 Dieldrin d Endosulfan I PBDE C 1 PBDE SIM Window Endrin d Endosulfan II PBDE 17 C 1 PBDE PBDE C 1 PBDE

8 Table S. ECNI SIM Windows continued. SIM Window 1 d Endosulfan II C 1 PCB PCB 118 C 1 PCB Endosulfan II d Endosulfan II SIM Window 1 Nonachlor, cis d Endosulfan II C 1 PBDE 8 C 1 PCB PBDE 8 C 1 PBDE SIM Window 16 Endrin aldehyde d Endosulfan II PBDE C 1 PBDE # PCB 1 C 1 PCB PBDE 7 C 1 PBDE SIM Window 17 Endosulfan sulfate d Endosulfan II PCB 8 C 1 PCB C 1 PCB 8 Internal Standard SIM Window 18 PCB 187 C 1 PCB PCB 18 C 1 PCB PBDE 7 C 1 PBDE PBDE 9 C 1 PCB 180 SIM Window 19 C 1 PBDE C 1 PCB PBDE 7 C 1 PBDE 7 C 1 PBDE C 1 PCB SIM Window 0 PBDE 66 C 1 PBDE Mirex C 1 PCB PBDE 66 C 1 PBDE SIM Window 1 PBDE 100 L C 1 PBDE C 1 PBDE 100 C 1 PCB PBDE PBDE 99 L C 1 PBDE C 1 PBDE 99 C 1 PCB PBDE 116 PBDE 118 L C 1 PBDE 118 PBDE 8/1 PBDE 1 PBDE 16 PBDE 1 PBDE 1 L C 1 PBDE 1 PBDE 8 PBDE 166 PBDE 18 L C 1 PBDE 18 PBDE 181 PBDE 190 C 1 PBDE SIM Window C 1 PBDE C 1 PCB SIM Window C 1 PBDE C 1 PBDE C 1 PBDE SIM Window C 1 PBDE C 1 PBDE C 1 PCB C 1 PBDE C 1 PBDE SIM Window C 1 PBDE C 1 PCB C 1 PBDE C 1 PBDE

9 Figure S. Representative Final Fish Extract Selected Ion Chromatograms

10 Table S.Fish analytical method recovery, estimated method detection limits, accuracy, and precision Compounds Log K ow Method Recovery 1 (%) Estimated Method Detection Limit (pg/g ww) Determined Values for NIST SRM 196 (ng/g ww) Deviation from Certified Values (%) Avg. SD Avg. RSD Avg. RSD % Diff % % Method Averages Median Average Min Max Organochlorines Pesticides & Metabolites HCH, gamma HCH, alpha HCH, beta HCH, delta Methoxychlor Heptachlor epoxide Endrin Heptachlor Hexachlorobenzene o,p'-dde Chlordane, oxy Dieldrin Chlordane, cis p,p'-ddd Nonachlor, trans o,p'-ddd Chlordane, trans Nonachlor, cis Aldrin o,p'-ddt p,p'-dde Mirex p,p'-ddt OrganochlorineSulfide Pesticides & Metabolites Endosulfan sulfate Endosulfan I Endosulfan II Phosphorothioate Pesticides Parathion Ethion Chlorpyrifos Miscellaneous Pesticides Etridiazole Dacthal Triallate Trifluralin PolyChlorinated Biphenyls PCB PCB PCB PCB PCB PCB PCB spike, n=; - n=; - Values within NIST confidence intervals = 0% diff

11 Table S.Fish analytical method performance, continued Compounds Log K ow Method Recovery 1 (%) Estimated Method Detection Limit (pg/g ww) Determined Values for NIST SRM 196 (ng/g ww) Deviation from Certified Values (%) Avg. SD Avg. RSD Avg. RSD % Diff % % Polycyclic Aromatic Hydrocarbons Acenaphthylene Acenaphthene Fluorene Anthracene Phenanthrene Pyrene Fluoranthene Chrysene /Triphenylene Benzo(a)anthracene Retene Benzo(k)fluoranthene Benzo(a)pyrene Benzo(b)fluoranthene Indeno(1,,-cd)pyrene Dibenz(a,h)anthracene Benzo(e)pyrene Benzo(ghi)perylene PolyBrominated Diphenyl Ethers BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE n/a BDE BDE BDE BDE BDE BDE 8/1 7.7 / BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE spike, n=; - n=; - Values within NIST confidence intervals = 0% diff 6

12 Figure S - Concentrations of select current (A,B) and historic use contaminants (C,D) in average (bars) & individual fish (symbols) from western US national park lakes compared to estimated non-cancer (A,B) and cancer (C,D) health thresholds for recreational fishing. * - nd>0% of lake fish. 7

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