Supporting Information

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1 Supporting Information (21 pages, 12 table, 3 figures) First Report on the Occurrence and Bioaccumulation of Hexafluoropropylene Oxide Trimer Acid (HFPO-TA): An Emerging Concern Yitao Pan, 1,2 Hongxia Zhang, 1 Qianqian Cui, 1 Nan Sheng, 1 Leo W.Y. Yeung, 3 Yong Guo, 4 Yan Sun, 4 and Jiayin Dai 1, * 1 Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing , P. R. China; 2 University of Chinese Academy of Sciences, Beijing , China; 3 Man-Technology-Environment Research Centre (MTM), School of Science and Technology, Örebro University, SE-70182, Örebro, Sweden; 4 Key Laboratory of Organofluorine Chemistry Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China *Correspondence author: Jiayin Dai, Institute of Zoology, Chinese Academy of Sciences, Beijing , P. R. China. Telephone: daijy@ioz.ac.cn Competing financial interests: The authors declare no conflicts of interest. S1

2 Table of Contents Pg S3: Standards and reagents Pg S4: Synthesis of HFPO-TA Pg S4: Sample extration Pg S5: Qualitative analysis of HFPO-TA Pg S6: Table S1. Physical and chemical properties of HFPO-TA Pg S7: Table S2. Information on the sampling sites Pg S8: Table S3. Information for common carp (Cyprinus carpio) collected from Xiaoqing River (n = 15) Pg S9: Table S4. LC-MS/MS instrument parameters for the quantification of the target analytes Pg S11: Table S5. Instrument limits of quantification (LOQ), average blank levels, and method detection limits (MDL) in different matrices Pg S12: Table S6. Reported and measured values (ng/ml) of NIST SRM1957. Pg S13: Table S7. Method validation for PFASs: matrix spike recoveries and matrix effects Pg S14: Table S8. PFAS concentrations (ng/l) in water samples from Xiaoqing River Pg S15: Table S9. Estimated total riverine discharge of PFASs from Xiaoqing River Pg S16: Table S10. PFAS concentrations in common carp (Cyprinus carpio) from Xiaoqing River (n = 15) Pg S17: Table S11. Details of the fragment constant methodology in the KOWWIN model. Pg S18: Table S12. Log BCF of PFASs in blood, liver, and muscle samples from common carp (Cyprinus carpio) (n = 15) Pg S19: Figure S1. Molecular structures of target PFASs in this study Pg S20: Figure S2. 1 H NMR of HFPO-TA (400 MHz, DMSO-d6) Pg S21: Figure S3. 19 F NMR of HFPO-TA (376 MHz, DMSO-d6) S2

3 Standards and reagents The 19 target PFASs included HFPO dimer acid (HFPO-DA), HFPO trimer acid (HFPO-TA), perfluorobutanoate (PFBA), perfluoropentanoate (PFPeA), perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnDA), perfluorododecanoate (PFDoA), perfluorotridecanoate (PFTriDA), perfluorotetradecanoate (PFTeDA), perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), and chlorinated polyfluorinated ether sulfonates (4:2, 6:2 and 8:2 Cl-PFESA). 6:2 Cl-PFESA is the major component of F-53B, a mist suppressant product in China, while 4:2 and 8:2 Cl-PFESAs are the impurity components of it. Except for HFPO-TA and Cl-PFESAs, all native and mass-labelled internal standards (purities exceeded 99%; listed in Table S4) were purchased from Wellington Laboratories (Guelph, ON, Canada). Native standards of HFPO-TA, 4:2, 6:2, and 8:2 Cl-PFESA (purity > 98%) were synthesized by Dr. Yong Guo at the Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences. Tetra-n-butyl ammonium hydrogen sulfate (TBAS), ammonium hydroxide, ammonium acetate, potassium hydroxide, sodium carbonate, and sodium bicarbonate were obtained from Sigma (St. Louis, MO, USA). LC-MS grade water and methanol and LC grade methyl tert-butyl ether (MTBE) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Solid phase extraction (SPE) cartridges (strata X-AW, 200 mg/6 ml) were purchased from Phenomenex (Torrance, CA, USA). Standard reference material (SRM1957, organic contaminants in non-fortified human serum) was purchased from the National Institute of Standards and Technology (NIST, USA). S3

4 Synthesis of HFPO-TA. The HFPO-TA was synthesized by its ester and was a gift from Sanming Hexafluo Chemicals Co. Ltd. The ester was first purified by distillation under reduced pressure. After purification, the ester was subjected to hydrolysis in the presence of potassium hydroxide in ethanol solution. The potassium salt was acidified by hydrochloride aqueous solution and neutralized by aqueous ammonium hydroxide solution to give HFPO-TA. The purity of HFPO-TA was identified by 1 H-NMR, 19 F-NMR, and elemental analysis, and the impurity was less than 2% (Figure S2 and S3). Sample extration. Water sample. 40 ml of water was spiked with 0.5 ng of internal mass-labelled standard and then loaded onto a solid phase extraction (SPE) cartridge (Phenomenex strata X-AW, 200 mg/6 ml) preconditioned with 8 ml of 0.5% ammonium hydroxide in methanol, 8 ml of methanol, and 4 ml of ultrapure water. The cartridges were washed with 4 ml of buffer solution (25 mm acetic acid/ammonium acetate, ph = 4) and centrifuged for 30 min at 4000 rpm to remove residual water. Target compounds were eluted into fractions separately by adding 4 ml of methanol (fraction 1) and then 4 ml of 0.5% ammonium hydroxide in methanol (fraction 2); fraction 2 was evaporated to dryness under nitrogen at 40 C and reconstituted with 200 µl of methanol for instrumental analysis. Biota sample. For the extraction of fish blood, fish liver, and human serum, 0.2 g of each sample was transferred and spiked with 0.5 ng of mass-labelled standard, 1 ml of tetra-n-butylammonium hydrogen sulfate solution (TBAS, 0.5 M), 2 ml of NaHCO 3 /Na 2 CO 3 buffer solution (ph = 10), and 4 ml of methyl tert-butyl ether (MTBE). After shaking and S4

5 centrifugation, the supernatant was collected by a Pasteur pipette, with the remaining residue extracted twice more with 4 ml of MTBE. All three extracts were combined and evaporated to dryness under nitrogen and reconstituted with 200 µl of methanol. Additional cleanup was performed for fish liver samples, with the extract further diluted to 10 ml with water, and then loaded onto the X-AW cartridge following the same procedure as that for the water samples. For fish muscle, 0.2 g of each sample was spiked with 0.5 ng of mass-labelled standard, then sonicated for 1 h in 10 ml of 10 mm KOH methanol solution, and shaken at 200 rpm overnight. The supernatant was concentrated to dryness and diluted to 10 ml with water for further SPE cleanup following the same procedure as that for the water samples. Qualitative analysis of HFPO-TA The confirmation of the occurrence of HFPO-TA in water and biota samples were conducted using a X500R Q-TOF System (AB SCIEX, Framingham, MA, USA) in ESI- mode. The instrument was calibrated in high sensitivity mode and the automated calibration device system (CDS) was set to perform an external calibration every five samples using calibrate solution. The Q-TOF was operated in full sacn MS ( m/z) and MS/MS ( m/z) throught information dependent acquisition (IDA) with a resolution of The sacn rate was 10 and 20 times per second in MS and MS/MS mode, respectively. The source parameters were optimized as follows: ion spray votage, 4500 V; curtain gas: 20 psi; collision gas: medium; temperature, 500 C; nebulizing gas (GS1), 50 psi; heater gas (GS2), 45 psi. In MS/MS mode, collision energy (CE) was set at 30 V, while the collision energy spread (CES) was 15 V, which meaned an interval from 15 to 45 V (30 ± 15 ev). S5

6 Table S1. Physical and chemical properties of HFPO-TA Property Value CAS No Chemical formula C 9 HF 17 O 4 Molecular weight Color/Physical State g/mol Waxy white solid Solubility in water a g/l at 25 ºC Melting point b Boiling point c Log Kow d Log Koa d pka e Not available Not available (decomposition starts above 149 ºC) a Empirical data from our laboratory. The solubility of HFPO-TA (ammonium salt) was lower than that of the ammonium salt of PFOA (above 500 g/l). b HFPO-TA is waxy solid hence unable to acquire the melting point c HFPO-TA is decomposed before the boiling point. The decomposition point was analyzed by differential scanning calorimetry (DSC) d The partition coefficient of octanol/water (Kow) and octanol/air (Koa) were predicted by EPI Suite v4.11 e The acid-dissociation constant (pka) was predicted by SPARC (ARChem). S6

7 Table S2. Information on the sampling sites No. Location Longitude ( E) Latitude ( N) Date and time S1 Xiaoqing River, upstream :41 S2 Xiaoqing River, upstream :04 S3 Xiaoqing River, upstream :28 S4 Xiaoqing River, upstream :05 S5 Xiaoqing River, upstream :31 S6 Xiaoqing River, upstream :09 S7 Dongzhulong, tributary :40 S8 Dongzhulong, tributary :52 S9 Dongzhulong, tributary :16 S10 Dongzhulong, tributary :25 S11 Xiaoqing River, downstream :40 S12 Xiaoqing River, downstream :22 S13 Xiaoqing River, downstream :46 S14 Yubei River, tributary :20 S15 Xiaoqing River, downstream :55 S16 Xiaoqing River, downstream :20 S17 Xiaoqing River, downstream :32 S18 Xiaoqing River, downstream :17 S7

8 Table S3. Information for common carp (Cyprinus carpio) collected from Xiaoqing River (n = 15) No. Gender Length (cm) Body weight (g) 1 male male female male female female male male female male female female female female male S8

9 Table S4. LC-MS/MS instrument parameters for the quantification of the target analytes. Instrument Analytical column Trap column Column temperature Injection volume Mobile phase Acquity UPLC coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA) or coupled to a API 5500 triple-quadrupole mass spectrometer (AB SCIEX Inc., Framingham, MA, USA) Acquity BEH C18 column (100 mm 2.1 mm, 1.7 µm, Waters, MA, USA) C18 column (50 mm 2.1 mm, 3.0 µm, Waters, MA, USA) 40 C 2 µl 2 mm ammonium acetate in water (A) and methanol (B) Time (min) Flow rate (ml/min) A (%) B (%) Gradient Compound Ion transitions CV/DP (V) CE (V) Internal standard Multiple reaction monitoring (MRM) transitions PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTriDA PFTeDA PFBS PFHxS PFOS :2 Cl-PFESA :2 Cl-PFESA S9 13 C 4 -PFBA 13 C 5 -PFPeA 13 C 2 -PFHxA 13 C 4 -PFHpA 13 C 4 -PFOA 13 C 5 -PFNA 13 C 2 -PFDA 13 C 2 -PFUnDA 13 C 2 -PFDoDA 13 C 2 - PFTeDA 13 C 2 - PFTeDA 13 C 4 -PFOS 18 O 2 -PFHxS 13 C 4 -PFOS 13 C 4 -PFOS 13 C 4 -PFOS

10 8:2 Cl-PFESA HFPO-DA C 4 -PFOS 13 C 3 -HFPO-DA HFPO-TA CV: cone voltage; DP: declustering potential; CE: collision energy 13 C 5 -PFNA Other mass parameters Xevo TQ-S, Waters Capillary voltage, -0.5 kv; Source temperature, 150 C; Desolvation temperature, 450 C; Desolvation gas flow, 850 L/h; Cone gas flow, 150 L/h; API 5500, AB Sciex Ion Spray Voltage: -4.5 kv; Curtain Gas: 20 psi; Collision Gas: Medium; Temperature: 500 C; Ion Source Gas 1: 50 psi; Ion Source Gas 2: 45 psi S10

11 Table S5. Instrument limits of quantification (LOQ), average blank levels, and method detection limits (MDL) in different matrices Water (ng/l) Serum (ng/ml) Muscle (ng/g w.w.) Liver (ng/g w.w.) LOQ Blank MDL LOQ Blank MDL LOQ Blank MDL LOQ Blank MDL HFPO-DA HFPO-TA 0.05 n.d n.d n.d n.d PFBA n.d PFPeA 0.05 n.d n.d n.d n.d PFHxA 0.02 n.d n.d n.d n.d PFHpA 0.02 n.d n.d n.d n.d PFOA 0.02 n.d n.d n.d n.d PFNA 0.02 n.d n.d n.d n.d PFDA 0.02 n.d n.d n.d n.d PFUnDA 0.02 n.d n.d n.d n.d PFDoDA 0.02 n.d n.d n.d n.d PFTriDA 0.02 n.d n.d n.d n.d PFTeDA 0.02 n.d n.d n.d n.d PFBS 0.01 n.d n.d n.d n.d PFHxS 0.05 n.d n.d n.d n.d PFOS 0.02 n.d n.d n.d n.d :2 Cl-PFESA 0.01 n.d n.d n.d n.d :2 Cl-PFESA 0.01 n.d n.d n.d n.d :2 Cl-PFESA 0.01 n.d n.d n.d n.d LOQ: Lowest concentration spiked in matrix blank resulting in S/N ratio above 10. MDL: Average plus three times the standard deviation of matrix blanks. Most PFASs were not detected in blank samples, MDLs were set as their LOQs. S11

12 Table S6. Reported and measured values (ng/ml) of NIST SRM1957. Analyte NIST reported values Present study PFHpA ± ± PFOA 5.00± ± PFNA ± ± PFDA 0.39 ± ± PFUnDA ± ± PFHxS 4.00 ± ± PFOS 21.1 ± ± S12

13 Table S7. Method validation for PFASs: matrix spike recoveries and matrix effects Spike recovery (2 ng) Matrix effect Water Serum Muscle Liver Water Serum Muscle Liver HFPO-DA ± ± ± ± ± ± ± ± 1.8 HFPO-TA ± ± ± ± ± ± ± ± 6.9 PFBA ± ± ± ± ± ± ± ± 4.0 PFPeA ± ± ± ± ± ± ± ± 4.8 PFHxA ± ± ± ± ± ± ± ± 4.2 PFHpA ± ± ± ± ± ± ± ± 4.2 PFOA ± ± ± ± ± ± ± ± 5.3 PFNA ± ± ± ± ± ± ± ± 2.8 PFDA ± ± ± ± ± ± ± ± 1.7 PFUnDA ± ± ± ± ± ± ± ± 3.1 PFDoDA 99.0 ± ± ± ± ± ± ± ± 2.5 PFTriDA 93.3 ± ± ± ± ± ± ± ± 2.9 PFTeDA ± ± ± ± ± ± ± ± 2.6 PFBS ± ± ± ± ± ± ± ± 1.4 PFHxS ± ± ± ± ± ± ± ± 3.0 PFOS ± ± ± ± ± ± ± ± 3.2 4:2 Cl-PFESA ± ± ± ± ± ± ± ± 3.9 6:2 Cl-PFESA ± ± ± ± ± ± ± ± 5.9 8:2 Cl-PFESA ± ± ± ± ± ± ± ± 4.5 S13

14 Table S8. PFAS concentrations (ng/l) in water samples from Xiaoqing River S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 HFPO-DA HFPO-TA PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTriDA n.d. n.d. n.d. n.d. n.d. n.d. n.d n.d. n.d. n.d n.d. PFTeDA n.d. n.d. n.d. n.d. n.d. n.d. n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d. PFBS PFHxS PFOS :2 Cl-PFESA n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 6:2 Cl-PFESA :2 Cl-PFESA n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d n.d. ΣPFASs n.d., not detected S14

15 Table S9. Estimated total riverine discharge of PFASs from Xiaoqing River Concentration (ng/l) Water flux (m 3 /yr) Riverine mass discharge (t/yr) HFPO-DA HFPO-TA PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTriDA PFTeDA PFBS PFHxS PFOS :2 Cl-PFESA :2 Cl-PFESA :2 Cl-PFESA ΣPFASs Measured PFAS concentration was derived from the mean level in water samples from S15 to S18. Annual water flux was acquired from the Ministry of Water Resources of the People s Republic of China: Annual Hydrological Report P. R. China, Hydrological Data of Huaihe River Basin, 2014 (in Chinese). S15

16 Table S10. PFAS concentrations in common carp (Cyprinus carpio) from Xiaoqing River (n = 15) Blood (ng/ml) Liver (ng/g w.w.) Muscle (ng/g w.w.) Detection rate (%) Median (5th, 95th) Detection rate (%) Median (5th, 95th) Detection rate (%) Median (5th, 95th) HFPO-DA (0.93, 15.6) (0.55, 2.08) (0.44, 6.46) HFPO-TA (360, 3560) (137, 1220) (27.8, 230) PFBA (1.68, 8.70) (0.24, 2.75) (n.d., 1.53) PFPeA (0.63, 2.87) (0.07, 0.94) 17.6 n.d. (n.d., 0.72) PFHxA (0.58, 3.91) (0.10, 0.88) 23.5 n.d. (n.d., 0.21 ) PFHpA (1.46, 10.3) (0.51, 1.94) (0.06, 0.34) PFOA (563, 5050) (93.2, 935) (16.7, 166) PFNA (4.02, 46.3) (1.34, 9.10) (0.26, 1.98) PFDA (21.5, 70.8) (6.04, 19.3) (1.09, 3.63) PFUnDA (9.77, 34.5) (2.72, 8.80) (0.49, 1.69) PFDoDA (10.7, 39.2) (3.56, 9.84) (0.75, 2.19) PFTriDA (2.41, 18.8) (2.16, 9.38) (0.27, 2.46) PFTeDA (1.23, 15.90) (0.58, 6.69) (0.13, 1.54) PFBS (n.d., 0.05) 35.3 n.d. (n.d., 0.04) 0.0 n.d. PFHxS (0.04, 0.39) (0.03, 0.30) 11.8 n.d. (n.d., 0.08) PFOS (19.4, 60.5) (12.0, 32.5) (1.28, 4.92) 4:2 Cl-PFESA (n.d., 0.04) (n.d., 0.03) 0.0 n.d. 6:2 Cl-PFESA (28.1, 65.9) (17.0, 43.3) (1.57, 8.58) 8:2 Cl-PFESA (2.43, 8.99) (1.32, 3.98) (0.13, 0.45) ΣPFASs 3960 (1400, 7950) 1140 (287, 2030) 196 (53.3, 441) S16

17 Table S11. Details of the fragment constant methodology in the KOWWIN model, which illustrates the contributions of the functional groups in the molecular structure to the total hydrophobic property of the target chemical (EPI Suite V4.11). Type Number LOGKOW fragment description Coefficient Value PFOA Frag 7 C [aliphatic carbon - No H, not tert] Frag 15 -F [fluorine, aliphatic attach] Frag 1 -COOH [acid, aliphatic attach] Frag 5 -CF2(-CF2)(-CF2) (linear -CF2- core) Const Equation Constant Log Kow = PFNA Frag 8 C [aliphatic carbon - No H, not tert] Frag 17 -F [fluorine, aliphatic attach] Frag 1 -COOH [acid, aliphatic attach] Frag 6 -CF2(-CF2)(-CF2) (linear -CF2- core) Const Equation Constant Log Kow = HFPO-TA Frag 8 C [aliphatic carbon - No H, not tert] Frag 2 -O- [oxygen, aliphatic attach] Frag 17 -F [fluorine, aliphatic attach] Frag 1 -COOH [acid, aliphatic attach] Frag 2 -O-C(F)F or -S-C(F)F correction Frag 1 -CF2(-CF2)(-CF2) (linear -CF2- core) Const Equation Constant Log Kow = S17

18 Table S12. Log BCF of PFASs in blood, liver, and muscle samples from common carp (Cyprinus carpio) (n = 15) Blood Liver Muscle Mean ± SD Mean ± SD Mean ± SD HFPO-DA 0.86 ± ± ± 0.44 HFPO-TA 2.18 ± ± ± 0.43 PFBA 0.95 ± ± 0.35 n.c. PFPeA 0.56 ± ± 0.36 n.c. PFHxA 0.49 ± ± 0.31 n.c. PFHpA 1.10 ± ± ± 0.29 PFOA 1.93 ± ± ± 0.34 PFNA 3.01 ± ± ± 0.34 PFDA 3.90 ± ± ± 0.19 PFUnDA 4.43 ± ± ± 0.19 PFDoDA 4.77 ± ± ± 0.17 PFTriDA 5.42 ± ± ± 0.29 PFTeDA 5.32 ± ± ± 0.34 PFBS 1.19 ± 0.33 n.c. n.c. PFHxS 2.56 ± ± 0.36 n.c. PFOS 3.86 ± ± ± :2 Cl-PFESA 3.28 ± ± 0.22 n.c. 6:2 Cl-PFESA 4.03 ± ± ± :2 Cl-PFESA 5.93 ± ± ± 0.19 n.c., not calculated because the compound was detected in less than 50% of samples. BCFs for PFTriDA, PFTeDA, 4:2 Cl-PFESA, and 8:2 Cl-PFESA should be higher than the estimated values because the concentrations in the corresponding water samples were below MDL. S18

19 Figure S1. Molecular structures of target PFASs in this study S19

20 Figure S2. 1 H NMR of HFPO-TA (400 MHz, DMSO-d6) S20

21 Figure S3. 19 F NMR of HFPO-TA (376 MHz, DMSO-d6 S21