Supporting Information. (32 Pages) Bioconcentration of aqueous film-forming foam (AFFF) in juvenile rainbow. trout (Oncorhyncus mykiss)

Transcription

1 1 2 Supporting Information (32 Pages) Bioconcentration of aqueous film-forming foam (AFFF) in juvenile rainbow trout (Oncorhyncus mykiss) Leo W.Y. Yeung and Scott A. Mabury * Department of Chemistry, University of Toronto, 80 St George Street, Toronto, M5S 3H6, ON, Canada *Corresponding author: SAM Phone: +1 (416) ; Fax: +1 (416) smabury@chem.utoronto.ca S1

2 20 Table of Content Pg S4: Standards and reagent Pg S5: Different forms of fluorine in a sample Pg S6: Detailed information of the AFFFs Pg S7: PFAS extraction Pg S8: LC and MS parameters Pg S16: Detailed instrumental analyses for PFAS and TF/EOF Pg S18: TOF-CIC combustion efficiency test Pg S19: Figure S1. Different forms of fluorine in a sample Pg S20: Figure S2. Schematic diagram showing total fluorine, extractable organic fluorine, and known PFAS analyses for fish samples Pg S21: Figure S3. Matrix recoveries of known PFASs in water, fish liver, and carcass homogenate (n=3) Pg S22: Details on the construction of the 8:2 FTSAS calibration curve Pg S23: Figure S4 Response factor between 6:2 FTSA and 6:2 FTSAS at different concentrations (pg/ml) in liver extract as matrix Pg S24: Figure S5. Extrapolated 8:2 FTSAS calibration curve using liver extract as matrix Pg S25: Figure S6. Percent composition profiles of fluorine in the liver and the carcass homogenate under different treatments Pg S26: Table S1: Combustion recoveries/efficiencies (%) of PFOS and 6:2 FTSAS in different tissues Pg S27: Table S2. Whole body and liver growth parameters (mean ± standard error) for juvenile rainbow trout under different treatments Pg S28: Table S3. Left: PFAS concentrations (ng/g); right: known PFASs (ng-f/g), EOF (ng- F/g), and TF (ng-f/g) in juvenile rainbow trout under different treatments for a) the liver and b) the carcass homogenate (n=3 in each treatment; SE: standard error) Pg S30: Table S4. Rate constants for uptake (k u ) and depuration (k d ), depuration half-lives, and steady-state bioconcentration factors (BCFs) estimated by k u /k d. (SE: standard error; n = S2

3 ; r 2 represents the coefficient of determination for the corresponding regression analysis; N/A: not analyzed Pg S31: Table S5. Percent composition profiles (%) of fluorine for (a) EOF (b) TF in the liver and the carcass homogenate under different treatments (EOF: extractable organic fluorine; IF: inorganic fluorine; NEOF: non-extractable organic fluorine) S3

4 Standards and reagents Perfluoropentanoate (PFPeA); perfluorohexanoate (PFHxA); perfluoroheptanoate (PFHpA); PFOA; perfluorononanoate (PFNA); perfluorodecanoate (PFDA); perfluoroundecanoate (PFUnDA); perfluorododecanoate (PFDoDA); 13 C 2 PFHxA; 13 C 4 PFOA; 13 C 5 PFNA; 13 C 2 PFDA; 13 C 2 PFUnDA; 13 C 2 PFDoDA; potassium salts of perfluorobutane sulfonate (PFBS), PFOS, and 13 C 4 PFOS; sodium salts of perfluorodecane sulfonate (PFDS), perfluorohexane sulfonate (PFHxS), and 18 O 2 PFHxS; N-methyl perfluorooctanesulfonamidoacetate (MeFOSAA); N-ethyl perfluorooctanesulfonamidoacetate (EtFOSAA); perfluorooctanesulfonamidoacetate (FOSAA); d 3 MeFOSAA; d 5 EtFOSAA; 6:2 and 8:2 FTUCA; 5:3 and 7:3 FTCAs; and 4:2, 6:2, and 8:2 FTSAs were obtained from the Wellington Laboratories (Guelph, ON). The purity of all standards was above 98%. In addition, 6:2 FTSAS was synthesized in-house as part of another project 13. Tetrabutylammonium hydrogen sulfate (TBAS, 99%), sodium fluoride (99%), methanesulfonic acid, ammonium acetate (>99%), and ammonia (NH 3, 30%) were obtained from Sigma-Aldrich. Methanol (MeOH, LCMS grade) and methyl-tert-butyl ether (MTBE, Omnisolv, >99%) were acquired from EMD Chemicals Inc. (Mississauga, ON). An Oasis WAX SPE cartridge (6cc, 150mg sorbent, 30 m particle size) was purchased from Waters Corporation (Milford, MA). S4

5 Different forms of fluorine in a sample Different forms of fluorines in a sample are given in Figure S1. The concentration of total fluorine (TF) in any sample, using the concept of mass balance, is equal to the sum of inorganic fluorine (IF) and organic fluorine (OF) concentrations in that sample. TF can be determined in the bulk sample using total organofluorine-combusion ion chromatography (TOF-CIC), and it should stay the same value. However, OF varies dependent on the extraction method being used. Only a proportion of the OF in a sample can be extracted, extractable organic fluorine (EOF); PFAS falls into this fraction. The difference between EOF and PFAS gives rise to unknown organofluorine (UOF). We believe that there is some OF that cannot be extracted from the samples, and it is regarded as non-extractable organic fluorine (NEOF). In the present study, the amount of TF in the liver and carcass homogenate samples were measured without any fractionation. The EOF fraction refers to the extract after ion-pair. S5

6 AFFF Samples of two commercially available AFFFs were used in the exposure experiment. The FC- 203CF light water AFFF 3%, manufactured, collected, and provided by 3M on October 11, The ingredients (w/w) include water (69-71%), diethylene glycol butyl ether (20%), amphoteric fluoroalkylamide derivatives (1-5%), perfluoroalkyl sulfonate salts ( %), alkyl sulfate salts (1-5%), triethanolamine ( %), and residual organic fluorochemicals (% not known) Niagara 1-3, batch number 80-15/19/F, manufactured on September 15, 2009 by Angus Fire, collected by Environment Canada July 11, 2011, is an alcohol resistant film-forming fluoroprotein firefighting foam concentrate for use at 1% dilution on hydrocarbon-based fires and at 3% dilution on polar solvent liquid-based fires. The composition of the foam is as follows: hexylene glycol (<10%), sodium chloride (5-10%), hydrolyzed protein (20-40%), fluorosurfactants (<5%), bactericide (<2%), and water (balance). S6

7 PFAS extraction Fish Approximately g of fish liver and of carcass homogenate samples were mixed with 2 ml of a 0.5 M TBAS solution and then vortexed; MTBE (2 x 5 ml) was added to the mixture, which was shaken on a horizontal shaker for 10 min at 250 rpm. The organic and aqueous layers were separated by centrifuging at 6000 rpm for 10 min, and the two supernatant fractions were transferred to a new tube and evaporated to dryness under a gentle stream of nitrogen. The sample extracts were reconstituted into 1 ml MeOH for PFAS or EOF analyses Water The PFASs in water samples collected coincident with fish sampling were extracted using an Oasis WAX cartridge following the ISO method. 1 In brief, the WAX cartridge was conditioned with 4 ml 0.1% NH 4 OH/MeOH, 4 ml MeOH, and 4 ml 18 m Milli-Q water. After conditioning, 10 ml of the sample was loaded onto the cartridge for extraction. Buffer solution (4 ml) was added, and the cartridge was dried by passing high purity nitrogen through it for approximately 2 min. The sample was first eluted with 4 ml MeOH and then 4 ml 0.1%NH 4 OH/MeOH ISO Water quality -- Determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) -- Method for unfiltered samples using solid phase extraction and liquid chromatography/mass spectrometry S7

8 LC method Samples were analyzed using an LC mobile phase composed of water and methanol each with 10 mm ammonium acetate. All separations were performed using a Acquity BEH C18 column (2.1 x 50 mm, 1.7 µm, 100Å) kept at 50 o C. Sample extracts were analyzed using 10 μl injection with 50:50 water/meoh composition PFASs were analyzed using the following linear methanol:water gradient at a flow-rate of 500 μl/min: from initial conditions of 65:35 methanol:water (t=0 min) proceed to 95:5 over 2 min (t=3 min), hold for 0.5 min (t=3.5 min), revert to initial conditions of 65:35 over 0.5s (t=4.0 min) and re-equilibrate at 65:35 for 2 min (t=6 min). S8

9 129 MS parameters The capillary was held at 1 kv. Cone- and desolvation-gas flows were kept at 150 and 1000 L/h, respectively. Source and desolvation temperatures were kept at 150 and 450 C, respectively. MS/MS was operated under multiple reaction monitoring (MRM) mode, and the parameters were optimized for transmission of the [M K] or [M H] ions, as shown below. S9

10 Acronym Parent Daughter Cone voltage Collision energy Type of transition Perfluoroalkane sulfonate (PFSA) (m/z) (m/z) (V) (ev) Perfluorobutane sulfonate (PFBuS) Qualifying Quantifying Perflurohexane sulfonate (PFHxS) Qualifying Quantifying Perfluorooctane sulfonate (PFOS) Qualifying Quantifying Perfluorodecane sulfonate (PFDS) Qualifying Quantifying PFOS precursor N-Methyl perfluorosulfonamidoacetate (MeFOSAA) Quantifying Qualifying Perfluorosulfonamidoacetate (FOSAA) Quantifying Qualifying N-Ethyl perfluorosulfonamidoacetate (EtFOSAA) Quantifying Qualifying Perfluorosulfonamide (FOSA) Perfluoroalkyl carboxylate (PFCA) Perfluorobutanoate (PFBA) Quantifying Perfluoropentanoate (PFPeA) Quantifying Qualifying Perfluorohexanoate (PFHxA) Qualifying Quantifying Perfluoroheptanoate (PFHpA) Qualifying Quantifying Perfluorooctanoate (PFOA) Qualifying Quantifying Perfluorononanoate (PFNA) Qualifying Quantifying Perfluorodecanoate (PFDA) Qualifying Quantifying Perfluoroundecanoate (PFUnDA) Qualifying Quantifying S10

11 Perfluorododecanoate (PFDoDA) Qualifying Quantifying Perfluorotridecanoate (PFTriDA) Qualifying Quantifying Perfluorotetradecanoate (PFTeDA) Qualifying Quantifying Perfluorohexanedecanoate (PFHxDA) Qualifying Quantifying Perfluorooctanedecanoate (PFOcDA) Qualifying Quantifying S11

12 Acronym Type of Calibration LOQ Perfluoroalkane sulfonate (PFSA) Liver (ng/g) Carcass (ng/g) Water (ng/l) Perfluorobutane sulfonate (PFBuS) External Perflurohexane sulfonate (PFHxS) Internal Perfluorooctane sulfonate (PFOS) Internal Perfluorodecane sulfonate (PFDS) External PFOS precursor N-Methyl perfluorosulfonamidoacetate (MeFOSAA) Internal Perfluorosulfonamidoacetate (FOSAA) External N-Ethyl perfluorosulfonamidoacetate (EtFOSAA) Internal Perfluorosulfonamide (FOSA) External Perfluoroalkyl carboxylate (PFCA) Perfluorobutanoate (PFBA) Internal Perfluoropentanoate (PFPeA) External Perfluorohexanoate (PFHxA) Internal Perfluoroheptanoate (PFHpA) External Perfluorooctanoate (PFOA) Internal Perfluorononanoate (PFNA) Internal Perfluorodecanoate (PFDA) Internal Perfluoroundecanoate (PFUnDA) Internal S12

13 Perfluorododecanoate (PFDoDA) Internal Perfluorotridecanoate (PFTriDA) External Perfluorotetradecanoate (PFTeDA) External Perfluorohexanedecanoate (PFHxDA) External Perfluorooctanedecanoate (PFOcDA) External S13

14 Fluorotelomer sulfonate (FTSA) Parent Daughter Cone voltage Collision energy Type of transition 4:2 FTSA Quantifying Qualifying 6:2 FTSA Quantifying Qualifying 8:2 FTSA Quantifying Qualifying 6:2 FTSAS Quantifying Qualifying 8:2 FTSAS Quantifying Qualifying Fluorotelomer saturated/unsaturated carboxylate (FT(U)CA) 5:3 FTCA :3 FTCA :2 FTUCA Quantifying Qualifying 8:2 FTUCA Quantifying Qualifying Mass-labeled standards PFOS-13C O2 PFHxS d3 MeFOSAA d5 EtFOSAA C4 PFBA PFOA-13C PFNA-13C C2-PFHxA C2 PFDA C2 PFUnDA C2 PFDoDA C2 6:2 FTUCA C2 8:2 FTUCA S14

15 Fluorotelomer sulfonate (FTSA) LOQ LOQ LOQ Liver (ng/g) Liver (ng/g) Liver (ng/g) 4:2 FTSA Matrix-matched :2 FTSA Matrix-matched :2 FTSA Matrix-matched :2 FTSAS Matrix-matched :2 FTSAS Extrapolated matrixmatched using 8:2 FTSA standard Fluorotelomer saturated/unsaturated carboxylate (FT(U)CA) 5:3 FTCA Matrix-matched :3 FTCA Matrix-matched :2 FTUCA Internal :2 FTUCA Internal S15

16 Instrumental analysis PFASs Calibration Internal calibration was used to quantify PFASs based on the corresponding mass-labeled standards. Since no internal standards were available for some PFASs (PFDS, FTCAs (5:3 and 7:3), FTSAs (4:2, 6:2, and 8:2) and 6:2 FTSAS) these compounds were quantified using a matrix-matched external calibration curve using the control fish extract as a matrix. The curve was prepared by spiking different amounts of standards into extracts of the control samples. The internal and matrix-matched calibration curves were prepared using a concentration series from 10-20,000 pg/ml for each analyte, with no point deviating by >20% from the calculated standard curve. The linearity of the calibration curve (r 2 ) was over Limits of quantification (LOQ) The limits of quantification (LOQs) were calculated based on four criteria: i) the lowest concentration of the standard on the calibration curve that could be accurately measured within ±20% of its theoretical value; ii) a signal-to-noise ratio equal to or greater than 10; iii) the concentration factor; and iv) the sample volume EOF and TF Separation and quantification of fluoride were performed using ion chromatography (ICS-2100, Dionex Co. Ltd., Sunnyvale, CA). An oxidative combustion process was required to analyze the fluorine content in the EOF (the MeOH extract) and TF (liver or carcass homogenate) fractions. In brief, the sample was placed on a ceramic boat and then combusted in a furnace (Automatic S16

17 Quick Furnace (AQF-100), Mitsubishi Chemical Analytech, Japan) maintained at C, with argon and oxygen as the carrier and the combustion gas, respectively. All the organic and inorganic fluorine in the sample was combusted into hydrogen fluoride (HF) in the furnace, which is equipped with a water supply. The HF was transferred into an absorption unit (GA-100), in which it dissolved into H + and F - in the absorption solution. A detailed description of the combustion furnace and IC parameters can be found elsewhere. 1 The external calibration curve included a series of calibration standards at 0.2, 2, 20, 100, 200 ng F - /ml, and the injection volume was 1.0 ml. The calibration curve exhibited good linearity with r 2 > Quantification was based on the response of the external standards that bracketed the concentrations found in the samples. Methanesulfonic acid (CH 3 SO 3 H) was added to the absorption solution as an internal standard to correct any changes in its volume during the combustion process. All solutions for the CIC were prepared in Milli-Q water (18 mω), and the fluoride concentration was found to be ng F - /ml Rand, A. A.; Mabury, S. A. In vitro interactions of biological nucleophiles with fluorotelomer unsaturated acids and aldehydes: fate and consequences. Environ. Sci. Technol , S17

18 174 TOF-CIC combustion efficiency test Combustion efficiencies for TF and EOF were evaluated by spiking different amounts of PFOS and 6:2 FTSAS in control fish samples as follows: For TF, liver or carcass homogenate (50 mg) of control fish were set onto the ceramic boat and 2000 ng (50µL) of PFOS or 6:2 FTSAS was spiked into the sample, and the spiked sample was combusted using TOF-CIC. Liver or carcass homogenate of the control fish without spiking any standard was also combusted. The contents of fluorine were determined for the spiked and nonspike control samples. After subtracting the background fluorine content from the non-spike control fish, the recovery was evaluated by the fluorine content in the spiked fish sample with the theoretical fluorine content of the compound Similar procedures were used for EOF, except that 50 µl of the sample extract (not sample homogenate) and 200 ng (50 µl) of PFOS or 6:2 FTSAS were used. 188 S18

19 TF TOF EOF 189 Figure S1. Different forms of fluorine in a sample Total Fluorine Analys TF TF = IF OF EOF PFCs IF - Inorganic Fluo OOF - Other Organ NEOF - Non Extrac 190 NEOF UOF PFAS IF NEOF OOF PFCs NEOF: Non-extractable organic fluorine IF UOF: Unknown organofluorine PFAS: poly-/perfluoroalkyl substance IF: Inorganic fluorine S19

20 Figure S2. Schematic diagram showing total fluorine, extractable organic fluorine, and known PFAS analyses for fish samples Fish liver/homogenate Ion pair extraction MeOH extract Known PFAS Extractable organic fluorine (EOF) Total fluorine (TF) 194 LC-MS/MS TOF-CIC TOF-CIC S20

21 PFDS PFOS PFHxS PFBS PFDoDA PFUnDA PFDA PFNA PFOA PFHpA PFHxA PFPeA EtFOSAA MeFOSAA FOSAA 4:2 FTSA 6:2 FTSA 8:2 FTSA 6:2 FTSAS 6:2 FTUCA 8:2 FTUCA 5:3 FTCA 7:3 FTCA % 195 Figure S3. Matrix recoveries of known PFASs in water, fish liver, and carcass homogenate (n=3) Matrix recovery (n=3) 120 Water Liver Homogenate S21

22 197 Details on the construction of the 8:2 FTSAS calibration curve Since there was no standard for 8:2 FTSAS, an extrapolated calibration curve using 8:2 FTSA and the response factor between 6:2 FTSA and 6:2 FTSAS was constructed to estimate the 8:2 FTSAS concentration in the sample. The response factors between 6:2 FTSA and 6:2 FTSAS were calculated by plotting the peak areas of 6:2 FTSA and 6:2 FTSAS at different concentrations using corresponding matrices (Figure S4). The mean response factor was found to be 3.27 (S.D.: 0.15, RSD: 4.50). The peak area of 8:2 FTSAS was calculated by dividing the peak area of 8:2 FTSA with the response factor at corresponding concentration (Figure S5) to construct the extrapolated 8:2 FTSAS calibration curve. The concentration of 8:2 FTSAS in a sample was estimated by the 8:2 FTSAS calibration curve corresponding to different matrices. S22

23 Response factor 207 Figure S4 Response factor between 6:2 FTSA and 6:2 FTSAS at different concentrations (pg/ml) in liver extract as matrix y = 3E-06x R² = Concentration pg/ml S23

24 Peak area 209 Figure S5. Extrapolated 8:2 FTSAS calibration curve using liver extract as matrix Extrapolated 8:2 FTSAS calibration curve y = x R² = Concentration pg/ml 211 S24

25 Day Day Day Day Day Day Figure S6. Percent composition profiles of fluorine in the liver and the carcass homogenate under different treatments (EOF: extractable organic fluorine; IF: inorganic fluorine; NEOF: nonextractable organic fluorine) Liver 0.01% 0.10% 1.00% 10.00% % % 0.10% 1.00% 10.00% % Carcass Homogenate Control 0.01% 0.10% 1.00% 10.00% % % % TF TF 0.01% 0.10% 1.00% 10.00% % 0.01% 0.10% 1.00% 10.00% % Angus Niagara Fire Angus Niagara Fire ng-f/g ng-f/g Control TF ng-f/g known PFAS 1 EOF TF % TF 0.01% 0.10% 3M 1.00% 10.00% % ng-f/g % PFAS EOF IF + NEOF % TF ng-f/g % TF 0.01% 0.10% 3M 1.00% 10.00% % ng-f/g S

26 Table S1. Combustion recoveries/efficiencies (%) of PFOS and 6:2 FTSAS in different tissues (Spike levels: Total fluorine (TF): 2ug; extractable organic fluorine (EOF): 0.2 ug, standard deviation of triplicate injection were given in parentheses) 220 TF PFOS 6:2 FTSAS Liver homogenate 89 (7) 85 (12) Carcass homogenate 81 (13) 81 (10) 221 EOF PFOS 6:2 FTSAS Liver extract 92 (5) 96 (6) Carcass extract 90 (8) 92 (7) S26

27 222 Table S2. Whole body and liver growth parameters (mean ± standard error) for juvenile rainbow trout under different treatments Growth rate (mg/day), r 2a Fish mass (g) Liver somatic index (%) b Mortality (%) Whole body Liver Predose Day 36 Control (0.84) (0.79) 3M (0.79) (0.74) Angus Fire * * (0.83) (0.7) a The growth rates were calculated by fitting all whole body and liver mass data to an exponential model ln(mass, g) = a + bt, where a is a constant, b is the growth rate (mg/day), and t is the time (day). The coefficient of correlation r for the model is shown in parentheses. b The liver somatic index (LSI) is the ratio of the fish liver mass to the whole body mass. The LSIs shown here are the overall means of the LSI calculated at each time point for each population. * significantly different from the control group (Mann-Whitney U test, p<0.05) 223 S27

28 Day Day Day Table S3. Left: PFAS concentrations (ng/g); right: known PFASs (ng-f/g), EOF (ng-f/g), and TF (ng-f/g) in juvenile rainbow trout under different treatments for a) the liver and b) the carcass homogenate (n=3 in each treatment; SE: standard error; 8:2 FTSAS concentration was an estimated value using 8:2 FTSA standard) a) 228 Liver PFAS concentration ng/g Fluorine content ng-f/g 3M PFDS S.E. PFOS S.E. PFHxS S.E. EtFOSAA S.E. Known PFAS S.E. EOF S.E. TF S.E < PFAS concentration ng/g Angus Fire PFOS S.E. 6:2 FTSA S.E. 8:2 FTSA S.E. 6:2 FTSAS S.E. 8:2 FTSAS S.E. S28 Fluorine content ng-f/g Known PFAS S.E. EOF S.E. TF S.E < < < < < <0.2 < PFAS concentration ng/g Fluorine content ng-f/g Control PFDS S.E. PFOS S.E. PFHxS S.E. EtFOSAA S.E. Known PFAS S.E. EOF S.E. TF S.E. 1 < <0.2 < < <0.2 < < <0.2 < < <0.2 < < <0.2 < < <0.2 < < <0.2 < < <0.2 <

29 Day Day Day b) 231 Carcass homogenate PFAS concentration ng/g Fluorine content ng-f/g 3M PFDS S.E. PFOS S.E. PFHxS S.E. EtFOSAA S.E. Known PFAS S.E. EOF S.E. TF S.E < < < < < < < < PFAS concentration ng/g Fluorine content ng-f/g Angus Fire PFOS S.E. 6:2 FTSA S.E. 8:2 FTSA S.E. 6:2 FTSAS S.E. 8:2 FTSAS S.E. Known PFAS S.E. EOF S.E. TF S.E < < < < < <0.4 < PFAS concentration ng/g Fluorine content ng-f/g Control PFDS S.E. PFOS S.E. PFHxS S.E. EtFOSAA S.E. Known PFAS S.E. EOF S.E. TF S.E. 1 < <0.4 < < <0.4 < < <0.4 < < <0.4 < < <0.4 < < <0.4 < < <0.4 < < <0.4 < S29

30 Table S4. Rate constants for uptake (k u ) and depuration (k d ), depuration half-lives, and steady-state bioconcentration factors (BCFs) estimated by k u /k d. (SE: standard error; n = 3; r 2 represents the coefficient of determination for the corresponding regression analysis; N/A: not analyzed k u (L/kg/d) k d (L/d) Half-life (d) BCF (L/kg) Present study Martin et al Present study Martin et al Present study Martin et al Present study Martin et al SE r 2 L/kg/d SE r 2 SE r 2 SE r 2 SE SE SE SE Liver PFDS N/A N/A N/A N/A PFOS PFHxS :2 FTSA N/A N/A N/A N/A N/A N/A Carcass Homogenate PFDS N/A N/A N/A N/A PFOS PFHxS :2 FTSA N/A N/A N/A N/A N/A N/A S30

31 Table S5. Percent composition profiles (%) of fluorine for (a) EOF (b) TF in the liver and the carcass homogenate under different treatments (EOF: extractable organic fluorine; IF: inorganic fluorine; NEOF: non-extractable organic fluorine a) Liver Tissue Control Day known PFAS Unidentified PFAS Day known PFAS Unidentified PFAS M Foam Day known PFAS Unidentified PFAS Day known PFAS Unidentified PFAS Angus Fire Day known PFAS Unidentified PFAS Day known PFAS Unidentified PFAS S31

32 b) Liver Tissue Control Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF M Foam Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF Angus Fire Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF S32