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 100101, P. R. China; 2 University of Chinese Academy of Sciences, Beijing 100049, 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 100101, P. R. China. Telephone: +86-10-64807185. E-mail: daijy@ioz.ac.cn Competing financial interests: The authors declare no conflicts of interest. S1
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
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
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
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 (100 1000 m/z) and MS/MS (50 1000 m/z) throught information dependent acquisition (IDA) with a resolution of 30000. 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
Table S1. Physical and chemical properties of HFPO-TA Property Value CAS No. 13252-14-7 Chemical formula C 9 HF 17 O 4 Molecular weight Color/Physical State 496.08 g/mol Waxy white solid Solubility in water a 100-200 g/l at 25 ºC Melting point b Boiling point c Log Kow d 5.555 Log Koa d 7.520 pka e -0.07 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
Table S2. Information on the sampling sites No. Location Longitude ( E) Latitude ( N) Date and time S1 Xiaoqing River, upstream 117.08301 36.72504 29.11.2015 13:41 S2 Xiaoqing River, upstream 117.36152 36.91037 29.11.2015 15:04 S3 Xiaoqing River, upstream 117.70097 37.06833 30.11.2015 16:28 S4 Xiaoqing River, upstream 117.85625 37.06194 30.11.2015 8:05 S5 Xiaoqing River, upstream 117.92063 37.07227 30.11.2015 8:31 S6 Xiaoqing River, upstream 118.00153 37.08574 30.11.2015 9:09 S7 Dongzhulong, tributary 118.04091 36.97257 30.11.2015 9:40 S8 Dongzhulong, tributary 118.03743 37.00682 30.11.2015 9:52 S9 Dongzhulong, tributary 118.05121 37.04844 30.11.2015 10:16 S10 Dongzhulong, tributary 118.04803 37.09170 30.11.2015 10:25 S11 Xiaoqing River, downstream 118.09406 37.11093 30.11.2015 10:40 S12 Xiaoqing River, downstream 118.26309 37.12842 30.11.2015 11:22 S13 Xiaoqing River, downstream 118.30893 37.14063 30.11.2015 11:46 S14 Yubei River, tributary 118.31264 37.12505 30.11.2015 15:20 S15 Xiaoqing River, downstream 118.35409 37.14834 30.11.2015 15:55 S16 Xiaoqing River, downstream 118.39309 37.14123 30.11.2015 16:20 S17 Xiaoqing River, downstream 118.55746 37.18997 01.12.2015 9:32 S18 Xiaoqing River, downstream 118.83181 37.27456 01.12.2015 11:17 S7
Table S3. Information for common carp (Cyprinus carpio) collected from Xiaoqing River (n = 15) No. Gender Length (cm) Body weight (g) 1 male 30.6 435.19 2 male 25.5 354.05 3 female 20.3 169.23 4 male 17.5 124.57 5 female 18.4 105.84 6 female 14.9 77.86 7 male 28.4 253.85 8 male 27.3 357.76 9 female 24.6 290.81 10 male 26.5 287.60 11 female 22.3 228.84 12 female 20.9 183.40 13 female 30.4 587.21 14 female 23.5 187.30 15 male 20.8 140.49 S8
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 (%) 0.0 0.30 90 10 Gradient 1.0 0.30 80 20 4.0 0.30 10 90 6.0 0.30 10 90 6.1 0.30 90 10 9.0 0.30 90 10 Compound Ion transitions CV/DP (V) CE (V) Internal standard Multiple reaction monitoring (MRM) transitions PFBA 213 169 30 11 PFPeA 263 119 10 8 PFHxA 313 269 14 10 PFHpA 363 319 30 10 PFOA 413 369 30 10 PFNA 463 419 28 10 PFDA 513 469 12 10 PFUnDA 563 519 30 10 PFDoDA 613 569 2 10 PFTriDA 663 619 10 10 PFTeDA 713 669 10 15 PFBS 299 80 40 30 PFHxS 399 80 45 33 PFOS 499 80 30 39 4:2 Cl-PFESA 431 251 30 24 6:2 Cl-PFESA 531 351 30 24 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
8:2 Cl-PFESA 631 451 30 30 HFPO-DA 329 169 30 18 13 C 4 -PFOS 13 C 3 -HFPO-DA HFPO-TA 495 185 20 12 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
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 0.05 0.16 0.23 0.05 0.08 0.14 0.05 0.18 0.26 0.05 0.33 0.43 HFPO-TA 0.05 n.d. 0.05 0.10 n.d. 0.10 0.10 n.d. 0.10 0.10 n.d. 0.10 PFBA 0.05 0.37 0.50 0.10 n.d. 0.10 0.10 0.42 0.55 0.10 0.36 0.48 PFPeA 0.05 n.d. 0.05 0.05 n.d. 0.05 0.05 n.d. 0.05 0.05 n.d. 0.05 PFHxA 0.02 n.d. 0.02 0.05 n.d. 0.05 0.10 n.d. 0.10 0.05 n.d. 0.05 PFHpA 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 PFOA 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 PFNA 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 PFDA 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 PFUnDA 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 0.02 n.d. 0.02 PFDoDA 0.02 n.d. 0.02 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 PFTriDA 0.02 n.d. 0.02 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 PFTeDA 0.02 n.d. 0.02 0.01 n.d. 0.01 0.02 n.d. 0.02 0.02 n.d. 0.02 PFBS 0.01 n.d. 0.01 0.01 n.d. 0.01 0.02 n.d. 0.02 0.02 n.d. 0.02 PFHxS 0.05 n.d. 0.05 0.01 n.d. 0.01 0.02 n.d. 0.02 0.02 n.d. 0.02 PFOS 0.02 n.d. 0.02 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 4:2 Cl-PFESA 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 6:2 Cl-PFESA 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 8:2 Cl-PFESA 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 0.01 n.d. 0.01 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
Table S6. Reported and measured values (ng/ml) of NIST SRM1957. Analyte NIST reported values Present study PFHpA 0.305 ± 0.051 0.270 ± 0.024 PFOA 5.00±0.44 4.963 ± 0.369 PFNA 0.878 ± 0.077 0.843 ± 0.040 PFDA 0.39 ± 0.12 0.262 ± 0.028 PFUnDA 0.172 ± 0.036 0.118 ± 0.014 PFHxS 4.00 ± 0.83 3.854 ± 0.279 PFOS 21.1 ± 1.3 18.66 ± 0.521 S12
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 102.4 ± 6.4 102.7 ± 8.3 92.5 ± 7.3 101.6 ± 8.1 109.3 ± 0.9 154.4 ± 0.9 95.3 ± 5.8 97.3 ± 1.8 HFPO-TA 100.3 ± 2.2 90.7 ± 1.6 84.0 ± 4.8 79.9 ± 4.8 98.6 ± 6.7 113.0 ± 10.5 97.7 ± 4.7 75.3 ± 6.9 PFBA 104.0 ± 3.1 96.1 ± 2.3 85.2 ± 4.8 89.2 ± 4.2 99.4 ± 2.1 104.9 ± 2.4 92.4 ± 6.3 92.2 ± 4.0 PFPeA 103.5 ± 3.3 96.1 ± 2.3 87.5 ± 5.6 93.9 ± 3.3 101.2 ± 0.6 107.3 ± 1.9 97.9 ± 3.5 113.0 ± 4.8 PFHxA 104.5 ± 1.6 106.7 ± 3.0 92.2 ± 0.2 99.2 ± 5.1 100.2 ± 0.8 100.8 ± 1.4 102.8 ± 2.8 89.0 ± 4.2 PFHpA 104.7 ± 2.7 92.2 ± 3.8 90.4 ± 3.7 98.5 ± 5.4 101.1 ± 1.3 104.7 ± 4.6 95.8 ± 3.5 107.9 ± 4.2 PFOA 103.1 ± 3.3 93.5 ± 2.9 92.7 ± 1.0 97.7 ± 5.7 98.1 ± 2.0 84.2 ± 0.8 92.2 ± 2.6 113.3 ± 5.3 PFNA 105.7 ± 1.3 97.6 ± 1.7 94.2 ± 0.6 93.2 ± 5.7 100.9 ± 2.0 107.5 ± 2.9 88.6 ± 0.5 99.1 ± 2.8 PFDA 105.4 ± 5.0 97.5 ± 1.2 96.6 ± 3.1 96.4 ± 4.2 102.3 ± 2.6 102.2 ± 1.1 92.4 ± 2.1 104.8 ± 1.7 PFUnDA 106.1 ± 2.4 95.4 ± 2.0 92.1 ± 3.2 92.8 ± 7.6 102.3 ± 3.4 109.7 ± 2.1 85.1 ± 1.3 106.7 ± 3.1 PFDoDA 99.0 ± 8.5 96.2 ± 3.3 90.7 ± 2.3 93.0 ± 5.5 97.1 ± 7.4 111.1 ± 1.5 90.0 ± 1.9 106.6 ± 2.5 PFTriDA 93.3 ± 1.6 109.5 ± 1.9 123.9 ± 3.8 125.5 ± 7.1 108.8 ± 4.0 108.2 ± 4.3 95.5 ± 4.7 102.2 ± 2.9 PFTeDA 104.5 ± 1.8 93.7 ± 3.2 93.9 ± 3.7 91.9 ± 5.2 120.1 ± 3.8 93.5 ± 6.1 97.1 ± 2.7 87.7 ± 2.6 PFBS 104.3 ± 3.0 77.1 ± 3.1 72.0 ± 4.6 103.2 ± 5.6 99.2 ± 1.3 100.2 ± 4.7 106.7 ± 4.6 108.7 ± 1.4 PFHxS 105.0 ± 3.5 95.8 ± 2.4 89.7 ± 1.6 92.0 ± 4.1 98.6 ± 0.9 105.0 ± 2.8 109.6 ± 3.4 112.7 ± 3.0 PFOS 102.3 ± 3.7 92.6 ± 1.3 88.1 ± 2.4 90.5 ± 6.2 99.3 ± 1.5 109.9 ± 4.4 108.5 ± 4.1 120.1 ± 3.2 4:2 Cl-PFESA 102.5 ± 3.3 97.4 ± 3.1 93.4 ± 2.2 96.7 ± 3.7 99.2 ± 0.9 109.7 ± 5.5 103.7 ± 0.6 110.5 ± 3.9 6:2 Cl-PFESA 104.9 ± 2.5 93.4 ± 2.6 81.6 ± 3.8 95.9 ± 6.2 98.0 ± 1.4 107.4 ± 2.4 105.9 ± 4.0 106.2 ± 5.9 8:2 Cl-PFESA 104.9 ± 4.3 94.2 ± 3.4 78.4 ± 2.1 87.4 ± 3.8 99.2 ± 1.3 105.8 ± 3.9 89.5 ± 4.6 102.5 ± 4.5 S13
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 3.64 1.61 1.95 1.99 2.30 2.20 2.34 1960 2060 1750 960 410 327 198 417 294 120 118 HFPO-TA 44.8 8.93 42.7 10.7 3.99 48.2 148 68500 40200 47800 15500 7950 8050 5200 8400 7550 6950 5650 PFBA 3.19 2.78 9.70 7.71 9.40 8.37 12.7 3970 3280 3190 1390 463 407 464 467 532 369 273 PFPeA 0.97 0.93 2.08 3.32 3.16 2.75 7.11 3370 2920 2750 1210 403 385 426 417 483 332 235 PFHxA 1.35 1.08 4.20 4.39 3.75 3.59 3.77 3690 3300 3140 1290 524 497 475 497 561 443 343 PFHpA 1.10 0.66 1.28 3.26 2.19 1.66 1.97 3470 3160 2600 1100 280 301 330 291 334 303 598 PFOA 17.1 15.4 30.4 75.2 55.4 78.9 171 197000 158000 136000 59500 23300 23000 20500 21100 25400 23100 25600 PFNA 0.59 0.53 1.01 4.20 2.84 1.43 1.46 124 97.9 115 36.2 15.0 15.7 13.3 15.2 18.2 17.3 15.9 PFDA 0.59 0.44 2.10 3.99 2.91 1.61 0.56 25. 7 17.3 22.4 7.05 4.65 5.21 4.57 5.06 5.31 5.71 4.89 PFUnDA 0.13 0.09 0.27 0.33 0.26 0.16 0.08 4.90 3.72 3.97 1.39 0.67 0.69 0.52 0.59 0.63 0.67 0.54 PFDoDA 0.11 0.05 0.20 0.12 0.10 0.04 0.04 1.99 1.49 1.91 0.62 0.29 0.33 0.20 0.19 0.31 0.39 0.20 PFTriDA n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.63 0.45 0.35 0.18 n.d. n.d. n.d. 0.14 0.15 0.29 n.d. PFTeDA n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.44 0.26 0.24 0.09 n.d. n.d. n.d. n.d. n.d. n.d. n.d. PFBS 0.99 0.88 0.98 0.73 0.79 0.71 0.42 1.12 2.47 1.76 1.01 1.08 0.94 1.51 1.08 1.20 1.05 1.14 PFHxS 1.32 0.45 0.84 0.46 0.46 0.31 0.16 0.27 0.40 0.26 0.14 0.29 0.57 0.31 0.31 0.51 0.30 0.39 PFOS 2.07 3.15 3.81 4.26 3.35 2.05 2.71 4.68 10.2 4.43 2.66 3.67 6.24 3.93 3.65 3.91 4.72 4.68 4:2 Cl-PFESA n.d. 0.02 0.02 0.02 0.01 0.01 0.03 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 4.32 11.3 15.6 8.86 5.41 3.69 2.59 2.55 1.52 9.59 2.065 5.95 2.31 5.29 9.22 6.13 5.67 5.97 8:2 Cl-PFESA 0.01 0.02 0.02 0.02 0.01 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.05 n.d. ΣPFASs 82.3 48.4 117 130 96.4 156 355 282000 213000 197000 81900 33400 33000 27700 31600 35200 31600 32800 n.d., not detected S14
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 236.88 0.153969 HFPO-TA 7137.50 4.639375 PFBA 410.20 0.266632 PFPeA 366.32 0.238108 PFHxA 461.20 0.299782 PFHpA 381.24 0.247804 PFOA 23771.50 15.451474 PFNA 16.62 0.010802 PFDA 5.24 0.003406 PFUnDA 0.61 6.5 10 8 0.000393 PFDoDA 0.27 0.000175 PFTriDA 0.15 0.000096 PFTeDA 0.01 0.000007 PFBS 1.12 0.000725 PFHxS 0.38 0.000244 PFOS 4.24 0.002754 4:2 Cl-PFESA 0.01 0.000003 6:2 Cl-PFESA 6.75 0.004385 8:2 Cl-PFESA 0.02 0.000011 ΣPFASs 32800.22 21.320145 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
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 100 2.09 (0.93, 15.6) 94.1 1.37 (0.55, 2.08) 94.1 1.53 (0.44, 6.46) HFPO-TA 100 1510 (360, 3560) 100 587 (137, 1220) 100 118 (27.8, 230) PFBA 100 4.09 (1.68, 8.70) 100 1.13 (0.24, 2.75) 47.1 0.81 (n.d., 1.53) PFPeA 100 1.48 (0.63, 2.87) 100 0.45 (0.07, 0.94) 17.6 n.d. (n.d., 0.72) PFHxA 100 1.53 (0.58, 3.91) 100 0.45 (0.10, 0.88) 23.5 n.d. (n.d., 0.21 ) PFHpA 100 3.41 (1.46, 10.3) 100 1.28 (0.51, 1.94) 100 0.16 (0.06, 0.34) PFOA 100 2190 (563, 5050) 100 449 (93.2, 935) 100 73.6 (16.7, 166) PFNA 100 17.1 (4.02, 46.3) 100 5.16 (1.34, 9.10) 100 0.85 (0.26, 1.98) PFDA 100 41.0 (21.5, 70.8) 100 12.4 (6.04, 19.3) 100 2.18 (1.09, 3.63) PFUnDA 100 18.0 (9.77, 34.5) 100 5.90 (2.72, 8.80) 100 1.01 (0.49, 1.69) PFDoDA 100 15.6 (10.7, 39.2) 100 7.37 (3.56, 9.84) 100 1.41 (0.75, 2.19) PFTriDA 100 4.16 (2.41, 18.8) 100 6.29 (2.16, 9.38) 100 0.95 (0.27, 2.46) PFTeDA 100 3.66 (1.23, 15.90) 100 2.68 (0.58, 6.69) 100 0.58 (0.13, 1.54) PFBS 82.4 0.02 (n.d., 0.05) 35.3 n.d. (n.d., 0.04) 0.0 n.d. PFHxS 100 0.17 (0.04, 0.39) 94.1 0.09 (0.03, 0.30) 11.8 n.d. (n.d., 0.08) PFOS 100 41.5 (19.4, 60.5) 100 22.1 (12.0, 32.5) 100 2.59 (1.28, 4.92) 4:2 Cl-PFESA 94.1 0.02 (n.d., 0.04) 94.1 0.02 (n.d., 0.03) 0.0 n.d. 6:2 Cl-PFESA 100 42.7 (28.1, 65.9) 100 26.1 (17.0, 43.3) 100 3.21 (1.57, 8.58) 8:2 Cl-PFESA 100 3.95 (2.43, 8.99) 100 2.02 (1.32, 3.98) 100 0.25 (0.13, 0.45) ΣPFASs 3960 (1400, 7950) 1140 (287, 2030) 196 (53.3, 441) S16
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] -0.9723-6.8061 Frag 15 -F [fluorine, aliphatic attach] -0.0031-0.0465 Frag 1 -COOH [acid, aliphatic attach] -0.6895-0.6895 Frag 5 -CF2(-CF2)(-CF2) (linear -CF2- core) -0.2970-1.4850 Const Equation Constant -0.2290 Log Kow = 4.8141 PFNA Frag 8 C [aliphatic carbon - No H, not tert] -0.9723-7.7784 Frag 17 -F [fluorine, aliphatic attach] -0.0031-0.0527 Frag 1 -COOH [acid, aliphatic attach] -0.6895-0.6895 Frag 6 -CF2(-CF2)(-CF2) (linear -CF2- core) -0.2970-1.7820 Const Equation Constant -0.2290 Log Kow = 5.4832 HFPO-TA Frag 8 C [aliphatic carbon - No H, not tert] -0.9723-7.7784 Frag 2 -O- [oxygen, aliphatic attach] -1.2566-2.5132 Frag 17 -F [fluorine, aliphatic attach] -0.0031-0.0527 Frag 1 -COOH [acid, aliphatic attach] -0.6895-0.6895 Frag 2 -O-C(F)F or -S-C(F)F correction -0.5500-1.1000 Frag 1 -CF2(-CF2)(-CF2) (linear -CF2- core) -0.2970-0.2970 Const Equation Constant -0.2290 Log Kow = 5.5550 S17
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 ± 0.42 0.50 ± 0.24 0.61 ± 0.44 HFPO-TA 2.18 ± 0.44 1.75 ± 0.42 1.05 ± 0.43 PFBA 0.95 ± 0.30 0.37 ± 0.35 n.c. PFPeA 0.56 ± 0.25 0.01 ± 0.36 n.c. PFHxA 0.49 ± 0.26-0.08 ± 0.31 n.c. PFHpA 1.10 ± 0.29 0.61 ± 0.21-0.27 ± 0.29 PFOA 1.93 ± 0.34 1.24 ± 0.34 0.46 ± 0.34 PFNA 3.01 ± 0.37 2.42 ± 0.36 1.68 ± 0.34 PFDA 3.90 ± 0.18 3.39 ± 0.15 2.64 ± 0.19 PFUnDA 4.43 ± 0.19 3.91 ± 0.15 3.17 ± 0.19 PFDoDA 4.77 ± 0.21 4.32 ± 0.16 3.62 ± 0.17 PFTriDA 5.42 ± 0.31 5.43 ± 0.22 4.66 ± 0.29 PFTeDA 5.32 ± 0.38 5.08 ± 0.32 4.38 ± 0.34 PFBS 1.19 ± 0.33 n.c. n.c. PFHxS 2.56 ± 0.34 2.30 ± 0.36 n.c. PFOS 3.86 ± 0.19 3.65 ± 0.15 2.73 ± 0.20 4:2 Cl-PFESA 3.28 ± 0.24 3.27 ± 0.22 n.c. 6:2 Cl-PFESA 4.03 ± 0.11 3.80 ± 0.16 2.91 ± 0.26 8:2 Cl-PFESA 5.93 ± 0.23 5.64 ± 0.17 4.69 ± 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
Figure S1. Molecular structures of target PFASs in this study S19
Figure S2. 1 H NMR of HFPO-TA (400 MHz, DMSO-d6) S20
Figure S3. 19 F NMR of HFPO-TA (376 MHz, DMSO-d6 S21