Identification of Novel Hydrogen-Substituted Polyfluoroalkyl Ether. Sulfonates in Environmental Matrices near Metal-Plating Facilities

Transcription

1 Supporting Information Identification of Novel Hydrogen-Substituted Polyfluoroalkyl Ether Sulfonates in Environmental Matrices near Metal-Plating Facilities Yongfeng Lin 1,2, Ting Ruan 1,2 *, Aifeng Liu 1,3, Guibin Jiang 1,2 1 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, , China 2 University of Chinese Academy of Sciences, Beijing , China 3 CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao , China *Corresponding author Dr. Ting Ruan Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences Tel: Fax: tingruan@rcees.ac.cn Number of pages: 44 Number of Tables: 9 Number of Figures: 15 S1

2 Contents Preparation of super-reduced CCA assay...s5 Non-target screening workflow...s6 Laboratory purification of the 1H-6:2 PFESA and 1H-8:2 PFESA standards...s7 Spike recovery experiments in river water and sediment samples...s8 References...S10 Table S1. Detailed information on the 26 features generated in the non-target screening workflow with VIP threshold > 1...S11 Table S2. Homodimers of Cl-6:2 PFESA and 1H-6:2 PFESA in the 2-fold diluted supernatants in the super-reduced CCA assay...s13 Table S3. Detailed information (acronyms, chemical names, molecular structures, theoretical monoisotopic masses, retention behaviors and characteristic fragmentation ions) of per- and polyfluoroalkyl substances in the suspect screening procedure...s14 Table S4. Instrumental parameters on chromatographic separation and mass spectrometry...s16 Table S5. Method quantification limits (MQL) of all target analytes in river water and sediment samples...s19 Table S6. Spike recoveries of target analytes in river water and sediment samples..s20 Table S7. Recoveries of surrogate standards and matrix effects of injection standards in river water and sediment samples...s21 Table S8. Descriptive statistics of identified per- and polyfluoroalkyl substances in the sampling regions...s22 Table S9. Spearman's correlation analysis of quantified PFAS concentrations in river water samples from the Fenghuajuang River... S23 Figure S1. Negligible changes of Cl-6:2 PFESA and 1H-6:2 PFESA amount in an additional incubation period of up to 40 min when 3.6 ml of methanol was added in the super-reduced CCA incubation tubes at appointed incubation time, showing transformation reaction effectively retarded...s24 Figure S2. The sampling map showing spatial distribution of Cl-6:2 PFESA, 1H-6:2 PFESA, Cl-8:2 PFESA and 1H-8:2 PFESA in river water and sediment samples in Pan S2

3 River in Shandong Province. (Pink pentacle, metal-plating manufactory; black tetragon, sampling sites for river water and sediment samples)...s25 Figure S3. Non-target screening workflow for generation, filtering, ranking and structure confirmation of potential metabolites in Cl-6:2 PFESA reductive transformation process...s26 Figure S4. Results of principal component analysis (PCA, p < 0.05) show a clear separation of the metabolite-transformation and dosed-control dataset groups, which were illuminated by the red and blue dots, respectively. A. Kruskal s non-metric multidimensional scaling presents distinct distribution of samples in metabolite transformation group in association with incubation time; B. Score plots of PCA analysis; C. R 2 and Q 2 represent goodness of fit and prediction of the three primary factors in the statistical analysis procedures...s27 Figure S5. Results of orthogonal partial least-squares-discriminant analysis (OPLS-DA, p < 0.05) show a clear separation of the metabolite-transformation and dosed-control dataset groups, which were illuminated by the red and blue dots, respectively. A. R 2 (cum) and Q 2 (cum) represent goodness of fit and prediction in the statistical analysis procedures; B. Score Scatter Plot represents distinct distribution of samples in metabolite-transformation group in association with incubation time; C. Twenty-six features were identified with VIP values > 1; D. Distribution of the identified 26 features in the III and I quadrants of the S-Plot, illustrating the Cl-6:2 PFESA and potential metabolite candidates in elimination (DOWN) and generation (UP) trends with high constituent abundances, respectively...s29 Figure S6. MS 2 fragmentation patterns of the Cl-PFESA and H-PFESA standards (A. Cl-6:2 PFESA; B. 1H-6:2 PFESA; C. Cl-8:2 PFESA; D. 1H-8:2 PFESA). Solid-line arrows represent characteristic neutral loss of HF, C2F4SO3 and CF2O in the HCD fragmentation process...s31 Figure S7. Reconstructed total ion chromatography of deprotonated molecular ions of the dosed Cl-6:2 PFESA and proposed transformation products in both standard solution (10 ng/ml in methanol) and supernatant in the super-reduced CCA assay. Mass windows were set as theoretical monoisotopic mass ± 5 ppm. Instrumental responses (IRs) in the supernatant represented metabolite contents in samples at incubation time of 50s...S32 Figure S8. MS 2 fragmentation patterns of 2H-6:2 PFESA (A. HCF2(C5HF9)OC2F4SO3 - ) and 1H-6:2 PFUESA (B. HCF2(C5F8)OC2F4SO3 - ) in the HCD mode in the super- S3

4 reduced CCA assay at incubation time of 50s. Solid-line arrows represent characteristic neutral loss of HF, C2F4SO3 and CF2O in the HCD fragmentation process...s33 Figure S9. MS 3 fragmentation pattern of [C6HF12O] - generated from MS 2 of 1H-6:2 PFESA, and MS 2 spectra for homodimers of Cl-6:2 PFESA and 1H-6:2 PFESA in HCD mode. (A. [C6HF12O] - ; B. [2M-2H+Na] - homodimer of 1H-6:2 PFESA; C. [2M- 2H+NH4] - homodimer of Cl-6:2 PFESA) in the super-reduced CCA assay at incubation time of 50s. Solid-line arrows represent characteristic neutral loss of HF and CF2O in the HCD fragmentation process...s34 Figure S10. Tentative molecular structures of 2H-6:2 PFESA (CL: Level 2b) and 1H- 6:2 PFUESA (Cl: Level 3)...S35 Figure S11. Retention time of 1H-6:2 PFESA and 1H-8:2 PFESA in the LC column of Autopurification HPLC-MS system...s36 Figure S12. 1 H-NMR and 19 F-NMR spectrum of laboratory-purified 1H-6:2 PFESA and 1H-8:2 PFESA standards. (A. 1 H-NMR of 1H-6:2 PFESA; B. 19 F-NMR of 1H-6:2 PFESA; C. 1 H-NMR of 1H-8:2 PFESA; D. 19 F-NMR of 1H-8:2 PFESA)...S37 Figure S13. Detailed information on the purity of the 1H-6:2 PFESA and 1H-8:2 PFESA standards based on the LC-ELSD method. (A. 1H-6:2 PFESA; B. 1H-8:2 PFESA)...S41 Figure S14. Extraction efficiency of target analytes spiked in blank sediment samples. Each sample was extracted for three times and each extract was analyzed individually. Only about 2% of the target analytes were recovered in the third extraction, indicating duplicate extraction as performed in our experiment could reach sufficient extraction for all analytes....s43 Figure S15. Retention behaviors of 1H-6:2 PFESA, linear-pfos and Cl-6:2 PFESA on reversed C18 HPLC column. MS channels were generated by injection of standard solution (10 ng/ml in methanol)...s44 S4

5 Preparation of super-reduced CCA assay Preparation of super-reduced cyanocobalamin (CCA, Vitamin B12) assay was similar to procedures reported in previous literature with minor modifications. 1,2 In brief, all incubation solutions were produced under nitrogen gas condition in a Lab 2000 anaerobic chamber (Etelux Inc., Beijing, China). Water and oxygen contents were less than 0.1 and 274 part-per-million in volume (ppm, v/v), respectively. Solution of reductive agent titanium (III) citrate was firstly prepared by mixing 10 ml of 0.4 M sodium citrate and 3 ml of 20% w/v titanium chloride in 2 N HCl in a 50 ml polypropylene centrifugation tube (Corning Inc., Corning, NY), with ph value subsequently adjusted to 7.0 by adding 5.1 ml of 2 M sodium carbonate and 21.9 ml of ultrapure water. Approximately 1.0 mg of cyanocobalamin was added into the centrifugation tube, and super-reduced working solution (18.4 μm in case of CCA) was obtained with detectable endpoint of color change into dark black. Transformation process was performed by mixing 400 μl of Cl-6:2 PFESA methanol solution and 1 ml of super-reduced CCA solution in 15 ml polypropylene centrifugation tubes at room temperature (25 ± 2 C). Control samples containing the same amounts of dosed analytes, solvent and agents except for CCA (CCA-lacking assay) were also applied. An additional test was performed by adding 2 ng of Cl-6:2 PFESA and 1H-6:2 PFESA standards in CCA-lacking assay. Measured recoveries by external calibration curves ( ng/ml methanolic solution) were 95 ± 4% (n = 6) and 95 ± 4% (n = 6), respectively. The data were consistent with quantified results (98%, 97%, 94% and 97% for M3PFHxS, MPFOS, M8PFOS and M2-6:2 FTSA, respectively) of isotope-labeled injection standards, indicating negligible matrix effects of the PFESA compounds in the assay. S5

6 Non-target screening workflow A non-target screening workflow using multivariate statistic tools for mass detection and filtering was described in Figure S3. Raw data of MS 1 spectrum in both metabolitetransformation and dosed-control groups was introduced in the XCMS processing package, which resulted in recognition of 3389 features. A clear separation of the two dataset groups was observed by principal component analysis (PCA, Figure S4), in which the first two primary factors contributed to 60% of variations. Additionally, distinct distribution of samples in metabolite-transformation group was observed in association with incubation time, further indicating representativeness of the identified features for the transformation process. All information was subjected to an additional check by the 80% Rule, 3 and 610 features with missing values were further ruled out. CF2 Normalized AKMD plot was successfully applied to identify several novel fluorinated chemicals in water by visualizing instrumental signals in the form of homologue series. 4,5 Considering the normal AKMD of PFASs was generally ranged , CF2 normalized AKMD was then calculated and applied to exclude irrelevant mass information with 1412 features remained for further analysis. Furthermore, baseline noise of was set as instrumental response (IR) threshold, with another 103 features removed in the workflow. The remaining 1309 features were considered as the most informative masses, in which 679 features were found to be significantly different in relative abundance between both dataset groups (averaged IR change > 1.5 fold, p < 0.05, student s t-test). Moreover, orthogonal partial least-squares-discriminant analysis (OPLS-DA) was carried out to locate potential metabolites in the remaining features that contributed S6

7 primarily to the changes of chemical profiles. As shown in Figure S5, distinct distribution of samples (R 2 (cum) = 0.981, Q 2 (cum) = 0.963, p < 0.05) in association with incubation time was quite similar with that in the PCA analysis, in which 77% of variation represented differences between the metabolite-transformation and dosedcontrol groups. Twenty-six features with variable importance in the project (VIP) value > 1 (Table S1) were discerned in the III and I quadrants, illustrating Cl-6:2 PFESA and potential metabolite candidates in elimination (DOWN) and generation (UP) trends with high constituent abundances, respectively. Laboratory purification of the 1H-6:2 PFESA and 1H-8:2 PFESA standards Approximately 1.0 g of F-53B (solved in 10 ml methanol) was added into 40 ml of prepared super-reduced CCA solutions under anaerobic conditions. After an incubation time of 24 h, the solution was vaporized and remaining solid was resolved in 30 ml of ethyl acetate. Further washing was conducted by adding 30 ml of ultrapure water for 3 times to remove inorganic salts. After the ethyl acetate solvent vaporization, remaining solid was resolved in 10 ml of acetonitrile as product solution for further purification. An Waters Autopurification HPLC-MS System (Waters Inc., Milford, MA), including a System Fluids Organizer, a 2545 Binary Gradient Module, a 2767 Sample Manager, a 515 Makeup Pump and an ACQUITY SQD single-quadrupole MS detector, was used to generate the 1H-6:2 PFESA and 1H-8:2 PFESA standards. Analytes separation was achieved by an XBridge C18 column (Waters, 19 mm i.d. 150 mm length, 5 μm), using acetonitrile and ultrapure water (with 0.05% ammonium hydroxide additive) as mobile phases. Flow rate was set at 20 ml/min, and 500 μl of sample was injected in each separation cycle. The initial flow gradient comprised 35% of acetonitrile and then S7

8 linearly increased to 45% in 2.5 min. Fraction collection time was confirmed by the single-quadrupole MS detector. 1H-6:2 PFESA and 1H-8:2 PFESA were collected with retention time in the range of min and min, respectively (Figure S11). Both the collected fractions were lyophilized and remaining solids were obtained. The molecular structures and purities of collected 1H-6:2 PFESA and 1H-8:2 PFESA standards were validated by a nuclear magnetic resonance spectrometer ( 1 H-NMR and 19 F-NMR, Bruker AVANCE III 500WB, Billerica, MA) and HPLC coupled to evaporative light-scattering detector (Shimadzu LC20A-ELSD II, Kyoto, Japan). The detailed information was shown in Figure S12 and S13. Purities of the 1H-6:2 PFESA and 1H-8:2 PFESA standards were all > 95%. Spike recovery experiments in river water and sediment samples Blank river water samples (n = 3) were used to assess the spike recovery of target PFASs. Briefly, 1 ng of each native standard (C6 C12 PFCAs, C4 C10 PFSAs, 4:2/6:2/8:2 FTSA, Cl-6:2/8:2 PFESA and 1H-6:2/8:2 PFESA) in 20 μl of methanol was spiked in 200 ml of river water samples, reaching a final concentration of 5 ng/l for each analyte. The spiked samples were allowed to equilibrate for 24 h and then pretreated according to the procedure described in sampling and pretreatment procedures section in the Materials and Methods. Blank sediment samples (n = 3) were also used for spike recovery of target PFASs. In brief, 1.0 g of sediment was soaked with 600 μl of methanolic solution containing 1 ng of each native standard (C6 C12 PFCAs, C4 C10 PFSAs, 4:2/6:2/8:2 FTSA, Cl-6:2/8:2 PFESA and 1H-6:2/8:2 PFESA). The soaked sediment samples were homogenized and dried using nitrogen gas. After aging for 72 h in room temperature, an alkaline S8

9 extraction method was used as described in sampling and pretreatment procedures section in the Materials and Methods. An external calibration curve was applied to calculate the final concentrations of target PFASs in sample extracts. The spike recoveries for target PFASs in river water and sediment samples were summarized in Table S6. S9

10 References 1. Melcher, J.; Olbrich, D.; Marsh, G.; Nikiforov, V.; Gaus, C.; Gaul, S.; Vetter, W. Tetra- and tribromophenoxyanisoles in marine samples from Oceania. Environ. Sci. Technol. 2005, 39 (20), ; DOI /051090g. 2. Ruppe, S.; Neumann, A.; Diekert, G.; Vetter, W. Abiotic transformation of toxaphene by superreduced Vitamin B12 and dicyanocobinamide. Environ. Sci. Technol. 2004, 38 (11), ; DOI /es034994f. 3. Bijlsma, S.; Bobeldijk, I.; Verheij, E.R.; Ramaker, R.; Kochhar, S.; Macdonald, I.A.; van Ommen, B.; Smilde, A.K. Large-scale human metabolomics studies: A strategy for data (pre-) processing and validation. Anal. Chem. 2006, 78 (2), ; DOI /ac051495j. 4. Liu, Y.; Dos Santos Pereira, A.; Martin, J.W. Discovery of C5-C17 poly- and perfluoroalkyl substances in water by in-line SPE-HPLC-Orbitrap with in-source fragmentation flagging. Anal. Chem. 2015, 87 (8), ; DOI /acs.analchem.5b Myers, A.L.; Jobst, K.J.; Mabury, S.A.; Reiner, E.J. Using mass defect plots as a discovery tool to identify novel fluoropolymer thermal decomposition products. J. Mass Spectrum. 2014, 49 (4), ; DOI /jms S10

11 Table S1. Detailed information on the 26 features generated in the non-target screening workflow with VIP threshold > 1. Feature ID Fold of change p value Trend Measured RT b Molecular formula Averaged IR c Isotopes MIM a (minutes) ( [M-H] - ) M447T6_ UP [M] - C7HF14SO4 (1H-5:2 PFESA) M459T6_ UP [M] - C8HF14SO4 (1H-6:2 PFUESA) M481T7_ DOWN d [M] - ClC7F14SO4 (Cl-5:2 PFESA) M483T7_ DOWN [M+2] - M497T7_ UP [M] - M497T UP d [M] - C8HF16SO4 (1H-6:2 PFESA) M498T UP [M+1] - M499T6_ UP [M+2] - M500T6_ UP [M+3] - M531T7_ DOWN d [M] - ClC8F16SO4 (Cl-6:2 PFESA) M532T7_ DOWN [M+1] - M533T7_ DOWN [M+2] - M534T DOWN [M+3] - M535T7_ DOWN [M+4] - M531T DOWN [M] - S11

12 M597T UP [M] - C10HF20SO4 (1H-8:2 PFESA) M191T1_ DOWN [M] - C6H7O7 (citric acid) M1012T UP d [M] - C16H6F32S2O8N (NH4 + dimer of 1H-6:2 PFESA) M1013T UP [M+1] - M1014T UP [M+2] - M1017T UP [M] - C16H2F32S2O8Na (Na + dimer of 1H-6:2 PFESA) M1053T UP [M] - M191T1_ UP [M] - M479T6_ UP [M] - C8H2F15SO4 (2H-6:2 PFESA) M497T7_ UP [M] - M499T6_ UP [M] - a MIM: monoisotopic mass. b RT: retention time. c IR: instrumental response. d Features of the same retention behaviors were isotope clusters ([M] -, [M+1] -, [M+2] -, [M+3] - ) of the same compound. S12

13 Table S2. Homodimers of Cl-6:2 PFESA and 1H-6:2 PFESA in the 2-fold diluted supernatants in the super-reduced CCA assay. Molecular formula Theoretical RT Mass error Adduct type MIM ( [M-H] - ) MIM (min) (ppm) Homodimers of 1H-6:2 PFESA C 16 H 6 F 32 S 2 O 8 N [2M-2H+NH 4 ] C 16 H 2 F 32 S 2 O 8 Na [2M-2H+Na] C 16 H 3 F 32 S 2 O 8 [2M-H] Homodimers of Cl-6:2 PFESA Cl 2 C 16 H 4 F 32 S 2 O 8 N [2M( 35 Cl)-2H+NH 4 ] Cl 2 C 16 F 32 S 2 O 8 Na [2M( 35 Cl)-2H+Na] Cl 2 C 16 HF 32 S 2 O 8 [2M( 35 Cl)-H] S13

14 Table S3. Detailed information (acronyms, chemical names, molecular structures, theoretical monoisotopic masses, retention behaviors and characteristic fragmentation ions) of per- and polyfluoroalkyl substances in the suspect screening procedure. Acronym Chemical name Molecular structure Theoretical MIM RT MS 2 fragmentation ions Perfluoroalkyl carboxylates (PFCAs) PFHxA Perfluorohexanoate [F(CF2)5COO] [C5F11] -, [C2F5] - PFHpA Perfluoroheptanoate [F(CF2)6COO] [C6F13] -, [C3F7] - PFOA Perfluorooctanoate [F(CF2)7COO] [C7F15] -, [C3F7] - PFNA Perfluorononanoate [F(CF2)8COO] [C8F17] -, [C4F9] - PFDA Perfluorodecanoate [F(CF2)9COO] [C9F19] -, [C5F11] - PFUdA Perfluoroundecanoate [F(CF2)10COO] [C10F21] -, [C5F11] - PFDoA Perfluorododecanoate [F(CF2)11COO] [C11F23] -, [C6F13] - Perfluoroalkyl sulfonates (PFSAs) PFBS Perfluorobutane sulfonate [F(CF2)4SO3] [SO3] -, [FSO3] - PFHxS Perfluorohexane sulfonate [F(CF2)6SO3] [SO3] -, [FSO3] - PFOS Perfluorooctane sulfonate [F(CF2)8SO3] [SO3] -, [FSO3] - PFDS Perfluorodecane sulfonate [F(CF2)10SO3] [SO3] -, [FSO3] - Fluorotelomer sulfonates (FTSAs) S14

15 4:2 FTSA 4:2 Fluorotelomer sulfonate [F(CF2)4(CH2)2SO3] [C6F8H3SO3] -, [HSO3] - 6:2 FTSA 6:2 Fluorotelomer sulfonate [F(CF2)6(CH2)2SO3] [C8F12H3SO3] -, [HSO3] - 8:2 FTSA 8:2 Fluorotelomer sulfonate [F(CF2)8(CH2)2SO3] [C10F16H3SO3] -, [HSO3] - Chlorinated polyfluoroalkyl ether sulfonates (Cl-PFESAs) Cl-5:2 PFESA 5:2 Chlorinated polyfluoroalkyl ether sulfonate [Cl(CF2)5O(CF2)2SO3] [C5F10ClO] -, [FSO2] -, [FSO3] - Cl-6:2 PFESA 6:2 Chlorinated polyfluoroalkyl ether sulfonate [Cl(CF2)6O(CF2)2SO3] [C6F12ClO] -, [FSO2] -, [FSO3] - Cl-7:2 PFESA 7:2 Chlorinated polyfluoroalkyl ether sulfonate [Cl(CF2)7O(CF2)2SO3] [C7F14ClO] -, [FSO2] -, [FSO3] - Cl-8:2 PFESA 8:2 Chlorinated polyfluoroalkyl ether sulfonate [Cl(CF2)8O(CF2)2SO3] [C8F16ClO] -, [FSO2] -, [FSO3] - Cl-10:2 PFESA 10:2 Chlorinated polyfluoroalkyl ether sulfonate [Cl(CF2)10O(CF2)2SO3] [C10F20ClO] -, [FSO2] -, [FSO3] - Hydrogen-substituted polyfluoroalkyl ether sulfonates (H-PFESAs) 1H-5:2 PFESA 5:2 Hydrogen-substituted polyfluoroalkyl ether sulfonate [H(CF2)5O(CF2)2SO3] [C5HF10O] -, [C5F9O] -, [C4F7] -, [FSO2] - 1H-6:2 PFESA 6:2 Hydrogen-substituted polyfluoroalkyl ether sulfonate [H(CF2)6O(CF2)2SO3] [C6HF12O] -, [C6F11O] -, [C5F9] -, [FSO2] - 1H-7:2 PFESA 7:2 Hydrogen-substituted polyfluoroalkyl ether [H(CF2)7O(CF2)2SO3] sulfonate 1H-8:2 PFESA 8:2 Hydrogen-substituted polyfluoroalkyl ether sulfonate [H(CF2)8O(CF2)2SO3] [C8HF16O] -, [C8F15O] -, [C7F13] -, [FSO2] - S15

16 Table S4. Instrumental parameters on chromatographic separation and mass spectrometry. Instrument Analytical Column Ultimate 3000 ultrahigh performance liquid chromatography tandem Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) equipped with a heated electrospray source. The mass spectrometer was operated in negative mode. ACQUITY BEH UPLC C18 (2.1 mm i.d. 100 mm length, 1.7 um, Waters) Column Temperature 35 C Mobile Phases A: 1mM NH4Ac in H2O B: 1mM NH4Ac in methanol Gradient Profile Time (min) Flow rate (ml/min) Percentage A (%) Percentage B (%) Injection Volume 5μL S16

17 Orbitrap HRMS Parameters General instrumental parameters: Spray Voltage: V Ion Transfer Tube Temperature: 350 C Vaporizer Temperature: 200 C Sheath Gas: 35 Arb Aux Gas: 10 Arb Sweep Gas: 1 Arb Instrumental parameters for MS 1 scanning: Orbitrap Resolution: Mass Range: Normal Scan Range (m/z): RF Lens (%): 60 Automatic gain control (AGC) Target: Maximum Injection Time (ms): 100 Instrumental parameters for MS 2 scanning: Isolation Mode: Quadrupole Isolation Window (m/z): 1 Activation Type: HCD HCD Collision Energy (%): Detector Type: Orbitrap Orbitrap Resolution: Mass Range: Normal Scan Range: RF Lens (%): 60 AGC Target: Maximum Injection Time (ms): 100 Instrumental parameters for MS 3 scanning: MS Isolation Mode: Quadrupole MS Isolation Window (m/z): 2 S17

18 MS Activation Type: HCD MS HCD Collision Energy (%): 30 MS 2 Isolation Window (m/z): 2 MS 2 Activation Type: HCD MS 2 HCD Collision Energy (%): 30 Detector Type: Orbitrap Orbitrap Resolution: Mass Range: Normal Scan Range: RF Lens (%): 60 AGC Target: Maximum Injection Time (ms): 100 S18

19 Table S5. Method quantification limits (MQL) of all target analytes in river water and sediment samples. Analyte River water (pg/l) Sediment (pg/g) Surrogate standard PFHxA MPFHxA PFHpA M4PFHpA PFOA MPFOA PFNA MPFNA PFDA MPFDA PFUdA MPFUdA PFDoA N.D. a 15 MPFDoA PFBS MPFHxS PFHxS MPFHxS PFOS MPFOS PFDS N.D. N.D. MPFOS 4:2 FTSA N.D. N.D. M2-6:2 FTSA 6:2 FTSA M2-6:2 FTSA 8:2 FTSA M2-6:2 FTSA Cl-6:2 PFESA MPFOS Cl-8:2 PFESA MPFOS 1H-6:2 PFESA MPFOS 1H-8:2 PFESA 14 9 MPFOS a N.D.: not detected S19

20 Table S6. Spike recoveries of target analytes in river water and sediment samples. River water (mean ± SD a, %, n = 3) Sediment (mean ± SD a, %, n = 3) PFHxA 72 ± 2 77 ± 17 PFHpA 74 ± 5 71 ± 4 PFOA 86 ± ± 5 PFNA 86 ± 5 91 ± 6 PFDA 80 ± ± 6 PFUdA 72 ± ± 9 PFDoA 67 ± 4 79 ± 11 PFBS 70 ± 5 82 ± 6 PFHxS 78 ± 3 73 ± 2 PFOS 69 ± 8 71 ± 2 PFDS 69 ± ± 8 4:2 FTSA 95 ± ± 13 6:2 FTSA 95 ± ± 10 8:2 FTSA 105 ± ± 13 Cl-6:2 PFESA 68 ± 9 82 ± 1 Cl-8:2 PFESA 65 ± 6 57 ± 10 1H-6:2 PFESA 72 ± 5 70 ± 5 1H-8:2 PFESA 69 ± ± 5 a SD: standard deviation. S20

21 Table S7. Recoveries of surrogate standards and matrix effects of injection standards in river water and sediment samples. Recoveries (mean ± SD a, %, n = 30) Matrix effects (mean ± SD, %, n = 30) River water Sediment River water Sediment MPFHxA 71 ± 8 78 ± 8 M5PFHxA 93 ± 5 90 ± 5 MPFOA 82 ± 5 79 ± 7 M6PFDA 86 ± 8 84 ± 8 MPFNA 76 ± 6 70 ± 9 M3PFHxS 93 ± 2 90 ± 7 MPFDA 83 ± 6 75 ± 9 M8PFOS 95 ± 2 88 ± 9 MPFUdA 72 ± 5 70 ± 8 MPFDoA 74 ± 9 73 ± 13 MPFHxS 78 ± 3 74 ± 10 MPFOS 108 ± 4 77 ± 6 M2-6:2 FTSA 100 ± 5 86 ± 10 a SD: standard deviation. S21

22 Analyte Table S8. Descriptive statistics of identified per- and polyfluoroalkyl substances in the sampling regions. Range (pg/l) River water GM a (pg/l) Fenghuajiang River (n = 34) Pan River (n = 26) Sediment Range (ng/g, d.w. b ) GM (pg/g, d.w.) DF c (%) AC d (%) Range (pg/l) River water Sediment GM (pg/l) Range (pg/g, d.w.) GM (pg/g, d.w.) Perfluoroalkyl carboxylates (PFCAs) PFHxA ( ) N.D < MQL e N.D. < MQL N.D PFHpA N.D. N.D N.D. N.D PFOA ( ) N.D ( ) PFNA N.D N.D. N.D N.D PFDA N.D N.D. N.D N.D N.D PFUdA N.D. N.D. N.D. N.D. 0 0 N.D N.D PFDoA N.D. N.D. N.D. N.D. 0 0 N.D. N.D. N.D Perfluoroalkyl sulfonates (PFSAs) PFBS ( ) N.D N.D N.D. N.D PFHxS ( ) N.D N.D PFOS < MQL ( ) PFDS N.D. N.D. N.D. N.D. 0 0 N.D. N.D. N.D. N.D. 0 0 Fluorotelomer sulfonates (FTSAs) 4:2 FTSA N.D. N.D. N.D. N.D. 0 0 N.D. N.D. N.D. N.D :2 FTSA N.D N.D. N.D :2 FTSA N.D. N.D. N.D. N.D. 0 0 N.D N.D Chlorinated polyfluoroalkyl ether sulfonates (Cl-PFESAs) Cl-6:2 PFESA ( ) Cl-8:2 PFESA N.D. N.D. N.D N.D N.D Hydrogen-substituted polyfluoroalkyl ether sulfonates (H-PFESAs) 1H-6:2 PFESA N.D N.D H-8:2 PFESA N.D. N.D. N.D N.D N.D a GM: geometric mean concentration. b D.W.: dry weight. c DF: detection frequency. d AC: average composition. e MQL: method quantification limit DF (%) AC (%) S22

23 Table S9. Spearman s correlation analysis of quantified PFAS concentrations in river water samples from the Fenghuajiang River. PFHpA a PFOA PFNA PFBS PFHxS PFHpS PFOS 6:2 FTSA Cl-6:2 PFESA 1H-6:2 PFESA PFHxA PFHpA ** ** ** ** ** ** * ** ** PFOA ** ** ** ** ** ** ** ** PFNA ** * * ** * ** ** PFBS ** ** ** ** ** ** PFHxS ** ** ** ** ** PFHpS ** ** ** ** PFOS ** ** ** 6:2 FTSA ** ** Cl-6:2 PFESA ** a Analytes with detection frequency less than 70% in the river water samples were not included. * and ** Significant correlations at 0.05 and 0.01 levels, respectively. S23

24 Figure S1. Negligible changes of Cl-6:2 PFESA and 1H-6:2 PFESA amount in an additional incubation period of up to 40 min when 3.6 ml of methanol was added in the super-reduced CCA incubation tubes at appointed incubation time, showing transformation reaction effectively retarded. S24

25 Figure S2. The sampling map showing spatial distribution of Cl-6:2 PFESA, 1H-6:2 PFESA, Cl-8:2 PFESA and 1H-8:2 PFESA in river water and sediment samples in Pan River in Shandong Province. (Pink pentacle, metal-plating manufactory; black tetragon, sampling sites for river water and sediment samples). S25

26 Figure S3. Non-target screening workflow for generation, filtering, ranking and structure confirmation of potential metabolites in Cl-6:2 PFESA reductive transformation process. S26

27 (A) (B) S27

28 (C) Figure S4. Results of principal component analysis (PCA, p < 0.05) show a clear separation of the metabolite-transformation and dosed-control dataset groups, which were illuminated by the red and blue dots, respectively. A. Kruskal s non-metric multidimensional scaling presents distinct distribution of samples in metabolite transformation group in association with incubation time; B. Score plots of PCA analysis; C. R 2 and Q 2 represent goodness of fit and prediction of the three primary factors in the statistical analysis procedures. S28

29 (A) (B) S29

30 (C) (D) Figure S5. Results of orthogonal partial least-squares-discriminant analysis (OPLS-DA, p < 0.05) show a clear separation of the metabolite-transformation and dosed-control dataset groups, which were illuminated by the red and blue dots, respectively. A. R 2 (cum) and Q 2 (cum) represent goodness of fit and prediction in the statistical analysis procedures; B. Score Scatter Plot represents distinct distribution of samples in metabolite-transformation group in association with incubation time; C. Twenty-six features were identified with VIP values > 1; D. Distribution of the identified 26 features in the III and I quadrants of the S-Plot, illustrating the Cl-6:2 PFESA and potential metabolite candidates in elimination (DOWN) and generation (UP) trends with high constituent abundances, respectively. S30

31 Figure S6. MS 2 fragmentation patterns of the Cl-PFESA and H-PFESA standards (A. Cl-6:2 PFESA; B. 1H-6:2 PFESA; C. Cl-8:2 PFESA; D. 1H-8:2 PFESA). Solid-line arrows represent characteristic neutral loss of HF, C2F4SO3 and CF2O in the HCD fragmentation process. S31

32 Figure S7. Reconstructed total ion chromatography of deprotonated molecular ions of the dosed Cl-6:2 PFESA and proposed transformation products in both standard solution (10 ng/ml in methanol) and supernatant in the super-reduced CCA assay. Mass windows were set as theoretical monoisotopic mass ± 5 ppm. Instrumental responses (IRs) in the supernatant represented metabolite contents in samples with incubation time of 50s. S32

33 Figure S8. MS 2 fragmentation patterns of 2H-6:2 PFESA (A. HCF2(C5HF9)OC2F4SO3 - ) and 1H-6:2 PFUESA (B. HCF2(C5F8)OC2F4SO3 - ) in the HCD mode in the superreduced CCA assay with sample incubation time of 50s. Solid-line arrows represent characteristic neutral loss of HF, C2F4SO3 and CF2O in the HCD fragmentation process. S33

34 Figure S9. MS 3 fragmentation pattern of [C6HF12O] - generated from MS 2 of 1H-6:2 PFESA, and MS 2 spectra for homodimers of Cl-6:2 PFESA and 1H-6:2 PFESA in HCD mode. (A. [C6HF12O] - ; B. [2M-2H+Na] - homodimer of 1H-6:2 PFESA; C. [2M- 2H+NH4] - homodimer of Cl-6:2 PFESA) in the super-reduced CCA assay with sample incubation time of 50s. Solid-line arrows represent characteristic neutral loss of HF and CF2O in the HCD fragmentation process. S34

35 Figure S10. Tentative molecular structures of 2H-6:2 PFESA (CL: Level 2b) and 1H- 6:2 PFUESA (CL: Level 3). S35

36 Figure S11. Retention time of 1H-6:2 PFESA and 1H-8:2 PFESA in the LC column of Autopurification HPLC-MS system. S36

37 S37

38 S38

39 S39

40 Figure S12. 1 H-NMR and 19 F-NMR spectrum of laboratory-purified 1H-6:2 PFESA and 1H-8:2 PFESA standards. (A. 1 H-NMR of 1H-6:2 PFESA; B. 19 F-NMR of 1H-6:2 PFESA; C. 1 H-NMR of 1H-8:2 PFESA; D. 19 F-NMR of 1H-8:2 PFESA) S40

41 S41

42 Figure S13. Detailed information on the purity of the 1H-6:2 PFESA and 1H-8:2 PFESA standards based on the LC-ELSD method. (A. 1H-6:2 PFESA; B. 1H-8:2 PFESA) S42

43 Figure S14. Extraction efficiency of target analytes spiked in blank sediment samples. Each sample was extracted for three times and each extract was analyzed individually. Only about 2% of the target analytes were recovered in the third extraction, indicating duplicate extraction as performed in our experiment could reach sufficient extraction for all analytes. S43

44 Figure S15. Retention behaviors of 1H-6:2 PFESA, linear-pfos and Cl-6:2 PFESA on reversed C18 HPLC column. MS channels were generated by injection of standard solution (10 ng/ml in methanol). S44