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

1 2 Supporting Information (32 Pages) 3 4 5 Bioconcentration of aqueous film-forming foam (AFFF) in juvenile rainbow trout (Oncorhyncus mykiss) 6 7 8 Leo W.Y. Yeung and Scott A. Mabury * 9 10 11 Department of Chemistry, University of Toronto, 80 St George Street, Toronto, M5S 3H6, ON, Canada 12 13 14 15 16 17 18 19 *Corresponding author: SAM Phone: +1 (416) 978 1780; Fax: +1 (416) 978 1631 e-mail: smabury@chem.utoronto.ca S1

20 Table of Content 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 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

49 50 51 52 53 54 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

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 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

72 73 74 75 76 77 78 79 80 81 82 83 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

84 85 86 87 88 89 90 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, 2000. The ingredients (w/w) include water (69-71%), diethylene glycol butyl ether (20%), amphoteric fluoroalkylamide derivatives (1-5%), perfluoroalkyl sulfonate salts (0.5-1.5%), alkyl sulfate salts (1-5%), triethanolamine (0.5-1.5%), and residual organic fluorochemicals (% not known). 91 92 93 94 95 96 97 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

98 99 100 101 102 103 104 105 PFAS extraction Fish Approximately 0.2-0.4 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. 106 107 108 109 110 111 112 113 114 Water The PFASs in water samples collected coincident with fish sampling were extracted using an Oasis WAX cartridge following the ISO 25101 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. 115 116 117 118 1. ISO 25101 Water quality -- Determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) -- Method for unfiltered samples using solid phase extraction and liquid chromatography/mass spectrometry. 2009. S7

119 120 121 122 123 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. 124 125 126 127 128 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

129 MS parameters 130 131 132 133 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

Acronym Parent Daughter Cone voltage Collision energy Type of transition Perfluoroalkane sulfonate (PFSA) (m/z) (m/z) (V) (ev) Perfluorobutane sulfonate (PFBuS) 299.0 80.0 40 30 Qualifying 299.0 99.0 40 31 Quantifying Perflurohexane sulfonate (PFHxS) 399.0 80.0 45 33 Qualifying 399.0 99.0 45 31 Quantifying Perfluorooctane sulfonate (PFOS) 499.0 80.0 60 39 Qualifying 499.0 99.0 60 38 Quantifying Perfluorodecane sulfonate (PFDS) 598.9 80.0 50 56 Qualifying 598.9 98.9 50 38 Quantifying PFOS precursor N-Methyl perfluorosulfonamidoacetate (MeFOSAA) 570.0 419.0 40 18 Quantifying 570.0 483.0 40 14 Qualifying Perfluorosulfonamidoacetate (FOSAA) 556.0 418.9 34 24 Quantifying 556.0 498.0 34 24 Qualifying N-Ethyl perfluorosulfonamidoacetate (EtFOSAA) 584.0 419.0 32 20 Quantifying 584.0 526.0 32 20 Qualifying Perfluorosulfonamide (FOSA) 498.0 78.0 66 28 Perfluoroalkyl carboxylate (PFCA) Perfluorobutanoate (PFBA) 212.9 168.9 30 11 Quantifying Perfluoropentanoate (PFPeA) 263.0 219.0 24 8 Quantifying 263.0 243.1 24 18 Qualifying Perfluorohexanoate (PFHxA) 313.2 128.0 26 20 Qualifying 313.2 269.0 26 6 Quantifying Perfluoroheptanoate (PFHpA) 363.0 169.0 30 19 Qualifying 363.0 319.0 30 10 Quantifying Perfluorooctanoate (PFOA) 413.0 169.0 30 19 Qualifying 413.0 369.0 30 10 Quantifying Perfluorononanoate (PFNA) 463.0 169.0 28 20 Qualifying 463.0 419.1 28 10 Quantifying Perfluorodecanoate (PFDA) 513.0 219.0 12 18 Qualifying 513.0 469.1 12 10 Quantifying Perfluoroundecanoate (PFUnDA) 563.0 319.0 30 17 Qualifying 563.0 519.0 30 10 Quantifying S10

Perfluorododecanoate (PFDoDA) 613.0 169.0 24 22 Qualifying 613.0 569.0 24 10 Quantifying Perfluorotridecanoate (PFTriDA) 663.0 168.9 30 26 Qualifying 663.0 619.0 30 10 Quantifying Perfluorotetradecanoate (PFTeDA) 712.9 169.0 30 22 Qualifying 712.9 669.0 30 10 Quantifying Perfluorohexanedecanoate (PFHxDA) 813.1 169.1 38 32 Qualifying 813.1 769.1 38 10 Quantifying Perfluorooctanedecanoate (PFOcDA) 913.1 169.0 24 32 Qualifying 913.1 869.2 24 18 Quantifying S11

Acronym Type of Calibration LOQ Perfluoroalkane sulfonate (PFSA) Liver (ng/g) Carcass (ng/g) Water (ng/l) Perfluorobutane sulfonate (PFBuS) External 0.2 0.4 10 Perflurohexane sulfonate (PFHxS) Internal 0.2 0.4 10 Perfluorooctane sulfonate (PFOS) Internal 0.2 0.4 10 Perfluorodecane sulfonate (PFDS) External 0.2 0.4 10 PFOS precursor N-Methyl perfluorosulfonamidoacetate (MeFOSAA) Internal 0.2 0.4 10 Perfluorosulfonamidoacetate (FOSAA) External 0.2 0.4 10 N-Ethyl perfluorosulfonamidoacetate (EtFOSAA) Internal 0.2 0.4 10 Perfluorosulfonamide (FOSA) External 0.2 0.4 10 Perfluoroalkyl carboxylate (PFCA) Perfluorobutanoate (PFBA) Internal 0.2 0.4 10 Perfluoropentanoate (PFPeA) External 0.2 0.4 10 Perfluorohexanoate (PFHxA) Internal 0.2 0.4 10 Perfluoroheptanoate (PFHpA) External 0.2 0.4 10 Perfluorooctanoate (PFOA) Internal 0.2 0.4 10 Perfluorononanoate (PFNA) Internal 0.2 0.4 10 Perfluorodecanoate (PFDA) Internal 0.2 0.4 10 Perfluoroundecanoate (PFUnDA) Internal 0.2 0.4 10 S12

Perfluorododecanoate (PFDoDA) Internal 0.2 0.4 10 Perfluorotridecanoate (PFTriDA) External 0.2 0.4 10 Perfluorotetradecanoate (PFTeDA) External 0.2 0.4 10 Perfluorohexanedecanoate (PFHxDA) External 0.2 0.4 10 Perfluorooctanedecanoate (PFOcDA) External 0.2 0.4 10 S13

Fluorotelomer sulfonate (FTSA) Parent Daughter Cone voltage Collision energy Type of transition 4:2 FTSA 327.0 80.8 60 24 Quantifying 327.0 307.1 60 20 Qualifying 6:2 FTSA 427.1 80.8 38 28 Quantifying 427.1 407.1 38 22 Qualifying 8:2 FTSA 527.1 80.8 68 32 Quantifying 527.1 507.1 68 26 Qualifying 6:2 FTSAS 586.2 135.0 88 42 Quantifying 206.0 88 36 Qualifying 8:2 FTSAS 786.0 135.0 88 42 Quantifying 203.0 88 36 Qualifying Fluorotelomer saturated/unsaturated carboxylate (FT(U)CA) 5:3 FTCA 341.0 237.0 15 10 7:3 FTCA 441.0 337.0 15 10 6:2 FTUCA 357.0 293.0 10 10 Quantifying 243.0 10 34 Qualifying 8:2 FTUCA 457.0 393.0 10 10 Quantifying 343.0 10 40 Qualifying Mass-labeled standards PFOS-13C 503.0 99.0 60 39 18O2 PFHxS 403.0 103.0 45 30 d3 MeFOSAA 573.0 419.0 40 18 d5 EtFOSAA 589.0 419.0 32 20 13C4 PFBA 216.9 171.9 30 11 PFOA-13C 417.0 372.0 30 10 PFNA-13C 468.0 423.0 30 10 13C2-PFHxA 315.0 270.0 30 10 13C2 PFDA 515.0 470.0 12 10 13C2 PFUnDA 565.0 520.0 30 10 13C2 PFDoDA 615.0 570.0 24 10 13C2 6:2 FTUCA 359.0 294.0 10 10 13C2 8:2 FTUCA 459.0 394.0 10 10 S14

Fluorotelomer sulfonate (FTSA) LOQ LOQ LOQ Liver (ng/g) Liver (ng/g) Liver (ng/g) 4:2 FTSA Matrix-matched 0.2 0.4 10 6:2 FTSA Matrix-matched 0.2 0.4 10 8:2 FTSA Matrix-matched 0.2 0.4 10 6:2 FTSAS Matrix-matched 0.2 0.4 10 8:2 FTSAS Extrapolated matrixmatched using 8:2 FTSA standard 0.2 0.4 10 Fluorotelomer saturated/unsaturated carboxylate (FT(U)CA) 5:3 FTCA Matrix-matched 0.2 0.4 10 7:3 FTCA Matrix-matched 0.2 0.4 10 6:2 FTUCA Internal 0.2 0.4 10 8:2 FTUCA Internal 0.2 0.4 10 S15

134 135 136 137 138 139 140 141 142 143 144 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 0.99. 145 146 147 148 149 150 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. 151 152 153 154 155 156 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

157 158 159 160 161 162 163 164 165 166 167 168 169 Quick Furnace (AQF-100), Mitsubishi Chemical Analytech, Japan) maintained at 900-1000 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 >0.9999. 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 0.0655 ng F - /ml. 170 171 172 173 1. Rand, A. A.; Mabury, S. A. In vitro interactions of biological nucleophiles with fluorotelomer unsaturated acids and aldehydes: fate and consequences. Environ. Sci. Technol. 2012. 46, 7398-7406. S17

174 TOF-CIC combustion efficiency test 175 176 Combustion efficiencies for TF and EOF were evaluated by spiking different amounts of PFOS and 6:2 FTSAS in control fish samples as follows: 177 178 179 180 181 182 183 184 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. 185 186 187 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

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

191 192 193 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

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 100 80 60 40 20 0 196 S21

197 Details on the construction of the 8:2 FTSAS calibration curve 198 199 200 201 202 203 204 205 206 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

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 4 3.5 3 2.5 2 y = 3E-06x + 3.26 R² = 0.0215 208 1.5 1 0.5 0 0 5000 10000 15000 20000 25000 Concentration pg/ml S23

Peak area 209 Figure S5. Extrapolated 8:2 FTSAS calibration curve using liver extract as matrix 300000 250000 Extrapolated 8:2 FTSAS calibration curve y = 13.515x + 71.147 R² = 0.9985 200000 150000 100000 50000 210 0 0 5000 10000 15000 20000 25000 Concentration pg/ml 211 S24

Day Day Day Day Day Day 212 213 214 215 216 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) 1 3 6 11 14 18 25 36 1 3 6 11 14 18 25 36 Liver 0.01% 0.10% 1.00% 10.00% 100.00% 1 3 6 11 14 18 25 36 1 3 6 11 14 18 25 36 36 0.01% 0.10% 1.00% 10.00% 100.00% Carcass Homogenate Control 0.01% 0.10% 1.00% 10.00% 100.00% 1 3 6 11 14 18 25 36 9990 13300 15000 17700 16700 13400 14350 10800 1 3 6 11 14 18 25 36 1 3 6 11 14 18 25 36 4160 9540 8810 8490 10720 9711 7211 6800 0.01 0.1 1 10 100 0.01 0.1 1 10 100 % % TF TF 0.01% 0.10% 1.00% 10.00% 100.00% 0.01% 0.10% 1.00% 10.00% 100.00% Angus Niagara Fire Angus Niagara Fire ng-f/g ng-f/g 1 3 6 11 14 18 25 36 Control TF ng-f/g 6300 6560 6620 7130 7820 8630 10300 10600 0.01 0.1 known PFAS 1 EOF TF 10 100 % TF 0.01% 0.10% 3M 1.00% 10.00% 100.00% ng-f/g 1 3 6 11 14 18 25 13800 30000 30100 42600 21800 14300 12600 14000 0.01 0.1 1 10 100 % 1 3 6 11 14 18 25 36 PFAS EOF IF + NEOF 0.01 0.1 1 10 100 % 1 3 6 11 14 18 25 36 1 3 6 11 14 18 25 36 TF ng-f/g 2790 2700 2680 4540 3590 4010 4280 3260 0.01 0.1 1 10 100 % TF 0.01% 0.10% 3M 1.00% 10.00% 100.00% ng-f/g 1 3 7300 26100 37800 35400 24700 22500 11900 14300 6 11 S25 14 18 25

217 218 219 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

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 116 1.9 112 4.9 9.5 0.2 13.8 0.9 1.81 0.10 0 (0.84) (0.79) 3M 120 2.0 124 3.9 8.5 0.3 14.4 0.5 1.85 0.1 0 (0.79) (0.74) Angus Fire 147 2.1 * 169 9.3 * 8.7 0.5 14.8 0.8 1.82 0.1 0 (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

Day Day Day 224 225 226 227 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. 1 5.53 0.56 185 19.1 1.64 0.25 1.65 0.84 125 12.6 708 116 9990 104 3 6.42 0.44 411 46.3 6.25 1.24 2.49 0.31 276 30.9 817 48.2 13300 1070 6 12.2 0.46 902 86.8 13.8 1.35 2.56 0.13 602 57.3 1500 52.6 15000 1070 11 18.1 0.93 1440 128 32.4 2.44 2.44 0.28 965 81.8 1600 46.2 17700 1150 14 18.0 1.54 1050 59.4 27.1 5.36 2.85 1.48 603 94.7 1140 92.4 16700 849 18 16.3 1.29 886 144 26.0 1.52 3.84 0.22 711 41.2 1050 28.9 13400 466 25 14.4 3.35 819 177 18.9 2.27 3.17 1.84 510 154 866 242 14400 1230 36 5.23 1.26 282 47.9 5.40 0.43 <0.2 189 32.0 448 29.4 10800 460 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. 1 4.93 1.64 6.24 1.88 14.1 9.43 87.0 44.6 0.040 0.017 52.0 15.6 2100 759 13800 3320 3 6.30 1.92 9.05 1.32 35.8 5.70 234 52.5 0.466 0.047 130 23.4 2820 290 30000 3050 6 5.66 0.72 13.4 3.31 87.6 29.6 48.3 12.8 0.885 0.177 85.3 22.4 2400 662 30100 2900 11 6.94 0.56 10.5 1.12 108 23.0 197 95.2 2.270 0.464 159 47.5 3350 276 42600 3870 14 6.93 3.87 <0.2 187 24.2 7.3 7.34 2.200 0.534 122 17.9 944 31.2 21800 2700 18 10.8 4.92 <0.2 116 27.8 13.0 13.0 0.924 0.366 82.9 22.4 842 247 14300 4730 25 10.8 1.14 <0.2 97.6 4.80 <0.2 0.335 0.276 66.4 2.94 708 119 12600 1210 36 12.5 0.77 <0.2 4.5 2.05 <0.2 <0.2 10.4 1.26 384 53.8 14000 4300 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 6.65 0.03 <0.2 <0.2 4.30 0.02 189 17.2 6300 128 3 <0.2 7.09 1.35 <0.2 <0.2 4.59 0.88 153 4.37 6560 371 6 <0.2 7.69 0.47 <0.2 <0.2 4.98 0.30 170 53.3 6620 157 11 <0.2 10.9 0.40 <0.2 <0.2 7.08 0.26 119 20.2 7130 199 14 <0.2 12.4 1.56 <0.2 <0.2 8.04 1.01 141 30.0 7820 54.6 18 <0.2 11.8 0.83 <0.2 <0.2 7.66 0.54 141 50.5 8630 114 25 <0.2 10.7 2.64 <0.2 <0.2 6.93 1.71 214 8.80 10300 374 36 <0.2 19.6 5.73 <0.2 <0.2 12.7 3.71 185 42.0 10600 56.6

Day Day Day 229 230 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. 1 1.22 0.02 61.1 11.5 0.56 0.11 <0.4 36.3 6.72 145 6.94 4160 744 3 2.86 0.20 239 17.0 3.48 0.20 <0.4 142 9.99 326 23.8 9540 1110 6 4.57 0.10 343 2.68 4.80 0.06 <0.4 204 1.58 439 109 8810 2020 11 5.68 0.39 407 39.9 8.34 0.70 <0.4 244 22.9 391 21.7 8490 615 14 5.48 0.64 288 35.2 7.36 1.11 <0.4 148 26.8 330 31.1 10720 1160 18 4.37 0.40 245 45.1 6.03 0.43 <0.4 173 20.6 366 33.3 9710 865 25 2.44 0.32 111 24.9 2.98 0.36 <0.4 67.5 14.55 188 31.9 7210 1100 36 1.31 0.27 66.4 14.6 1.52 0.22 <0.4 40.0 8.63 99.0 20.0 6840 404 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. 1 2.07 0.69 5.16 1.01 5.4 1.82 46.3 31.8 0.020 0.017 27.1 12.9 476 87.5 7300 669 3 3.06 0.55 11.9 1.09 25.7 4.48 55.6 13.2 0.130 0.012 48.1 7.32 1090 78.8 26100 3700 6 2.68 0.17 15.9 3.17 92.4 1.47 42.6 6.85 0.160 0.024 85.5 4.81 1210 166 37800 6340 11 2.25 0.37 9.08 0.83 80.4 14.5 69.8 35.8 0.378 0.067 85.4 24.1 1180 170 35400 8670 14 2.45 0.23 <0.4 72.1 8.82 0.55 0.55 0.269 0.035 46.1 5.49 461 89.7 24700 6400 18 2.72 1.04 <0.4 34.2 6.31 5.02 5.02 0.407 0.044 24.9 5.89 299 33.6 22500 3350 25 2.63 0.17 <0.4 36.6 4.79 <0.4 0.074 0.019 24.1 2.94 144 21.0 11900 2000 36 5.25 0.27 <0.4 2.4 1.15 <0.4 <0.4 4.87 0.71 117 18.5 14300 2730 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 2.86 0.19 <0.4 <0.4 1.65 0.11 33.4 3.65 2790 106 3 <0.4 4.62 0.86 <0.4 <0.4 2.67 0.50 25.1 0.25 2700 256 6 <0.4 2.59 0.27 <0.4 <0.4 1.50 0.16 29.0 0.73 2680 517 11 <0.4 3.11 0.15 <0.4 <0.4 1.80 0.08 36.7 2.60 4540 474 14 <0.4 2.23 0.15 <0.4 <0.4 1.29 0.08 36.0 3.27 3590 180 18 <0.4 3.54 0.16 <0.4 <0.4 2.05 0.10 33.5 2.04 4010 97.3 25 <0.4 2.39 0.10 <0.4 <0.4 1.38 0.06 35.5 0.41 4280 283 36 <0.4 4.25 0.12 <0.4 <0.4 2.46 0.20 36.5 2.03 3260 115 S29

232 233 234 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. 2003 Present study Martin et al. 2003 Present study Martin et al. 2003 Present study Martin et al. 2003 SE r 2 L/kg/d SE r 2 SE r 2 SE r 2 SE SE SE SE Liver PFDS 83.0 17.6 0.89 N/A 0.0513 0.0124 0.64 N/A 13.5 3.3 N/A 1620 343 N/A PFOS 98.8 8.59 0.94 260 17 0.88 0.0598 0.0131 0.64 0.05 0.00740.66 11.6 2.5 14.0 2.0 1650 144 5400 860 PFHxS 8.84 3.03 0.93 5.8 0.31 0.91 0.0666 0.0140 0.74 0.06 0.00700.75 10.4 2.2 12.0 1.5 133 46 100 13 8:2 FTSA N/A N/A 0.0870 0.0218 0.79 N/A 8.0 2.0 N/A N/A N/A Carcass Homogenate PFDS 29.1 1.9 0.97 N/A 0.0685 0.0110 0.87 N/A 10.1 1.6 N/A 425 27.5 N/A PFOS 32.1 1.2 0.93 53 1.3 0.98 0.0737 0.0170 0.79 0.05 0.00650.70 9.4 2.2 15.0 2.0 436 16.4 1100 150 PFHxS 7.04 3.0 0.94 0.62 0.02 0.96 0.0751 0.0133 0.86 0.07 0.00620.82 9.2 1.6 11.0 1.0 93.7 39.4 9.6 1.0 235 8:2 FTSA N/A N/A 0.0949 0.0254 0.76 N/A 7.3 2.0 N/A N/A N/A S30

236 237 238 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 1 2.3 97.7 1 4.9 95.1 3 3.0 97.0 3 10.7 89.3 6 2.9 97.1 6 5.2 94.8 11 6.0 94.0 11 4.9 95.1 14 5.7 94.3 14 3.6 96.4 18 5.4 94.6 18 6.1 93.9 25 3.2 96.8 25 3.9 96.1 36 6.9 93.1 36 6.7 93.3 3M Foam Day known PFAS Unidentified PFAS Day known PFAS Unidentified PFAS 1 17.7 82.3 1 25.0 75.0 3 33.7 66.3 3 43.5 56.5 6 40.2 59.8 6 46.3 53.7 11 60.3 39.7 11 62.4 37.6 14 52.8 47.2 14 45.0 55.0 18 67.7 32.3 18 47.3 52.7 25 58.9 41.1 25 35.9 64.1 36 42.2 57.8 36 40.5 59.5 Angus Fire Day known PFAS Unidentified PFAS Day known PFAS Unidentified PFAS 1 2.3 97.7 1 5.4 94.6 3 4.5 95.5 3 4.2 95.8 6 3.4 96.6 6 6.9 93.1 11 4.6 95.4 11 7.1 92.9 14 12.5 87.5 14 9.6 90.4 18 9.1 90.9 18 7.7 92.3 25 8.4 91.6 25 15.5 84.5 36 2.7 97.3 36 4.1 95.9 S31

b) Liver Tissue Control Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF 1 0.1 3.0 96.9 1 0.1 1.2 98.7 3 0.1 2.3 97.6 3 0.1 0.9 99.0 6 0.1 2.6 97.4 6 0.1 1.1 98.9 11 0.1 1.7 98.2 11 0.0 0.8 99.2 14 0.1 1.8 98.1 14 0.0 1.0 99.0 18 0.1 1.6 98.3 18 0.1 0.8 99.1 25 0.1 2.1 97.9 25 0.0 0.8 99.1 36 0.1 1.7 98.1 36 0.1 1.1 98.8 3M Foam Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF 1 1.3 7.1 91.7 1 0.9 3.5 95.6 3 2.1 6.3 91.6 3 1.5 3.4 95.1 6 4.0 10.0 86.0 6 2.3 5.0 92.7 11 5.4 9.0 85.5 11 2.9 4.6 92.5 14 3.6 6.9 89.5 14 1.4 3.1 95.5 18 5.3 7.8 86.8 18 1.8 3.8 94.4 25 3.6 6.0 90.4 25 0.9 2.6 96.5 36 2.2 5.1 92.7 36 0.6 1.4 98.0 Angus Fire Day known PFAS EOF IF+NEOF Day known PFAS EOF IF+NEOF 1 0.4 15.2 84.4 1 0.4 6.5 93.1 3 0.4 9.5 90.0 3 0.2 4.2 95.6 6 0.3 8.0 91.7 6 0.2 3.2 96.6 11 0.4 7.9 91.8 11 0.2 3.3 96.4 14 0.5 4.3 95.1 14 0.2 1.9 97.9 18 0.5 5.9 93.6 18 0.1 1.3 98.6 25 0.5 5.6 93.9 25 0.2 1.2 98.6 36 0.1 2.7 97.2 36 0.0 0.8 99.1 239 S32