Figure 1. Sampling sites of Northern pike in the St. Lawrence River (QC, Canada). Figure 2. Liver mRNA levels (relative normalized expression fold [± SEM]) of genes involved in lipid metabolism of male pike collected upstream (n = 8, except pparα with n = 5) and downstream (n = 8) of the Montreal’s MWWE (Mann-Whitney U Test and Student t-test, *p ≤ 0.05). Figure 3. Correlations between A) the ∑34PBDE concentrations (ng/g ww) and relative normalized expression of pparα (upstream, n = 5; downstream, n = 7) and B) relative abundance of ∑2steroid lipids and the relative normalized expression of pla2g4ab (upstream, n = 8; downstream, n = 8) in male pike liver collected in the St. Lawrence River of the Montreal’s MWWE discharge point. Grey field represents the 95% confidence interval and sites are identified with symbols for upstream (▲) and downstream (○). Impact of a primary wastewater effluent on liver lipid metabolism and oxidative stress in St. Lawrence River Northern pike GRAPHICAL ABSTRACT HIGHLIGHTS  First analysis of 18 new PFAS in fish from the St. Lawrence River  MWWE exposure led to sex-specific lipid metabolism disruption in Northern pike liver  Downregulation of pparα and pla2g4ab transcription in male pike exposed to MWWE  Higher contribution of membrane and steroid lipids in male pike downstream of MWWE  Negative correlation between PBDE levels and pparα transcription in male pike 1 Impact of primary wastewater effluent on liver lipid metabolism and oxidative stress in Northern pike Supporting Information 2 Table S1. List of flame retardants analyzed in the liver of Northern pike collected upstream and downstream of Montreal’s municipal wastewater effluent (MWWE) discharge point (QC, Canada). PBDEs BDE-7 2,4-Dibromodiphenyl ether BDE-10 2,6-Dibromodiphenyl ether BDE-15 4,4’-Dibromodiphenyl ether BDE-17 2,2',4-Tribromodiphenyl ether BDE-28 2,4,4'-Tribromodiphenyl ether BDE-47 2,2',4,4'-Tetrabromodiphenyl ether BDE-49 2,2',4,5'-Tetrabromodiphenyl ether BDE-66 2,3',4,4'-Tetrabromodiphenyl ether BDE-71 2,3',4',6-Tetrabromodiphenyl ether BDE-77 3,3’,4,4’-Tetrabromodiphenyl ether BDE-85 2,2',3,4,4'-Pentabromodiphenyl ether BDE-99 2,2',4,4',5-Pentabromodiphenyl ether BDE-100 2,2',4,4',6-Pentabromodiphenyl ether BDE-119 2,3',4,4',6-Pentabromodiphenyl ether BDE-126 3,3’,4,4’,5-Pentabromodiphenyl ether BDE-138 2,2',3,4,4',5'-Hexabromodiphenyl ether BDE-139 3,3’,4,4’,5-Pentabromodiphenyl ether BDE-140 2,2',3,4,4',6'-Hexabromodiphenyl ether BDE-153/BB- 153 2,2',4,4',5,5'-Hexabromodiphenyl Ether/2,2',4,4',5,5'- hexabromobiphenyl BDE-154 2,2',4,4',5,6'-Hexabromodiphenyl ether BDE-171 2,2',3,3',4,4',6-Heptabromodiphenyl ether BDE-180 2,2',3,4,4',5,5'-Heptabromodiphenyl ether BDE-183 2,2',3,4,4',5',6-Heptabromodiphenyl ether BDE-184 2,2',3,4,4',6,6'-Heptabromodiphenyl ether BDE-191 2,3,3',4,4',5',6-Heptabromodiphenyl ether BDE-196 2,2',3,3',4,4',5,6'-Octabromodiphenyl ether BDE-197 2,2',3,3',4,4',6,6'-Octabromodiphenyl ether BDE-201 2,2',3,3',4,5',6,6'-Octabromodiphenyl ether BDE-203 2,2',3,4,4',5,5',6-Octabromodiphenyl ether BDE-204 2,2’,3,4,4’,5,6,6’-Octabromodiphenyl ether BDE-205 2,3,3’,4,4’,5,5’,6-Octabromodiphenyl ether BDE-206 2,2',3,3',4,4',5,5',6-Nonabromodiphenyl ether BDE-207 2,2’,3,3’,4,4’,5,6,6’-Nonabromodiphenyl ether BDE-208 2,2’,3,3’,4,5,5’,6,6’-Nonabromodiphenyl ether BDE-209 Decabromodiphenyl ether Other flame retardants PBEB Pentabromoethylbenzene HBB Hexabromobenzene PBT Polybutylene Terephthalate 3 BEHTBP Bis(2-ethylhexyl)tetrabromophthalate DBDPE Decabromodiphenylethane OBIND Octabromotrimethylphenyllindane Dec-602 Dechlorane 602 Dec-603 Dechlorane 603 Dec-604 Dechlorane 604 Dec-604CB Dechlorane 604 component B syn-DP Syn-Dechlorane Plus anti-DP Anti-Dechlorane Plus Cplus Chlordene Plus 4 Table S2. List of per- and polyfluoroalkyl substances (PFAS) analyzed in the liver of Northern pike collected upstream and downstream of Montreal’s MWWE discharge point (QC, Canada). PFAS Acronym Name PFECHS Perfluoro-4-ethylcyclohexane sulfonate PFHxSAm Dimethylammoniopropyl perfluorohexane sulfonamide PFOSAm Perfluorooctane sulfonamidoalkyl amine 6:2 FTAB 6:2 fluorotelomer sulfonamidoalkyl betaine FBSA Perfluorobutane sulfonamide FHxSA Perfluorohexane sulfonamide FOSA Perfluorooctane sulfonamide FOSAA Perfluorooctane sulfonamidoacetic acid EtFOSAA N-ethyl-perfluorooctane sulfonamido acetic acid EtFOSA N-ethyl-perfluorooctane sulfonamide MeFOSAA N-methyl-perfluorooctane sulfonamido acetic acid MeFOSA N-methyl-perfluorooctane sulfonamide PFBS Perfluorobutane sulfonate PFDoS Perfluorododecane sulfonate PFDS Perfluorodecane sulfonate PFHpS Perfluoroheptane sulfonate PFHxS Perfluorohexane sulfonate PFNS Perfluorononane sulfonate PFOS Perfluorooctane sulfonate PFPeS Perfluorohexane sulfonate PFPrS Perfluoropropane sulfonate PFTrDS Perfluorotridecane sulfonate PFOANO Perfluorooctane amidopropyl amine oxide PFBA Perfluorobutanoic acid PFDA Perfluorodecanoic acid PFHpA Perfluoroheptanoic acid PFHxA Perfluorohexanoic acid PFHxDA Perfluorohexadecanoic acid PFNA Perfluorononanoic acid PFOA Perfluorooctanoic acid PFOcDA Perfluorooctadecanoic acid PFPeA Perfluoropentanoic acid PFPrA Perfluoropropionoic acid PFTeDA Perfluorotetradecanoic acid PFTrDA Perfluorotridecanoic acid 5 PFUnA Perfluoroundecanoic acid PFHxSAmS Trimethylammoniopropyl perfluorohexane sulfonamide PFOSAmS Trimethylammoniopropyl perfluorooctane sulfonamide 6:2 FTCA 6 :2 fluorotelomer carboxylate 8:2 FTCA 8 :2 fluorotelomer carboxylate 10:2 FTCA 10 :2 fluorotelomer carboxylate 6:2 FTUCA 6:2 fluorotelomer unsaturated carboxylic acids 8:2 FTUCA 8:2 fluorotelomer unsaturated carboxylic acids 10:2 FTUCA 10:2 fluorotelomer unsaturated carboxylic acids 3:3 Acid 3:3 fluorotelomer carboxylate 4:3 acid 4:3 fluorotelomer carboxylate 5:3 Acid 5:3 fluorotelomer carboxylate 7:3 Acid 7:3 fluorotelomer carboxylate 10:2 FTSA 10:2 fluorotelomer sulfonic acid 4:2 FTSA 4:2 fluorotelomer sulfonic acid 5:1:2 FtB 5:1:2 fluorotelomer betaine 5:3 FtB 5:3 fluorotelomer betaine 6:2 Cl-PFESA 6:2 chlorinated perfluoroalkylether sulfonate 8:2 Cl-PFESA 8:2 chlorinated polyfluorinated ether sulfonic acids 6:2 FTSA 6:2 fluorotelomer sulfonic acid 8:2 FTSA 8:2 fluorotelomer sulfonic acid 6:6 PFPiA 6:6 perfluoroalkylphosphinic acids 6:8 PFPiA 6:8 perfluoroalkylphosphinic acids 8:8 PFPiA 8:8 perfluoroalkylphosphinic acids ADONA Dodecafluoro-3H-4,8-dioxanonanoate Gen-X Hexafluoropropylene oxide dimer acid MeFBSA N-methyl-perfluorobutane sulfonamide PFEESA Perfluoro(2-ethoxyethane)sulphonic acid PFMBA Perfluoro(4-methoxybutanoic) acid 3,6-OPFHpA Perfluoro-3,6-dioxaheptanoic acid PFMPA Perfluoro-3-methoxypropanoic acid FDSA Perfluorodecanesulfonamide PFDoA Perfluorododecanoate PFEtS Perfluoroethanesulfonate FHpSA Perfluoroheptane sulfonamide PFHxPA Perfluorohexylphosphonic acid PFOAB Perfluorooctane amidoalkyl betaine PFOSNO Perfluorooctane sulfonamidoalkyl amine oxide PFOSB Perfluorooctane sulfonamidoalkyl betaine PFOPA Perfluorooctylphosphonic acid PFUdS Sodium perfluoro-1-undecanesulfonate 6 Table S3. Number of individual lipids by class, grouped by biological function, analyzed in the liver of Northern pike collected upstream and downstream of Montreal’s MWWE discharge point (QC, Canada). Lipids Biological function Number of individual lipids in this lipid class Lipid class acronym Lipid class name Diacylglycerol 41 DAG Diacylglycerol Fatty acids 20 FA Fatty acids Energetic reserve 340 TAG Triacylglycerol Membrane lipids 5 3-KDS 3-Ketosphinganine 28 Cer Ceramide 43 SM Shingomyelin 10 Dihydro-SM Dihydro shingomyéline 11 Hex Cer Hexosyl ceramide 10 Hex2 Cer Dihexosyl ceramide 8 Hex3 Cer Trihexosyl ceramide 4 Sa Shinganine 1 Sph Sphingosine 1 SPC Shingosylphosphorylcholine 121 CL Cardiolipin 8 G Ganglioside 22 LPA Lysophosphatidic acid 17 LPC Lysophosphatidylcholine 37 LPE Lysophosphatidylethanolamine 32 LPG Lysophosphatidylglycerol 28 LPI Lysophosphatidylinositol 34 LPS Lysophosphatidylserine 84 PA Phosphatidic acid 95 PC Phosphatidylcholine 88 PE Phosphatidylethanolamine 116 PG Phosphatidylglycerol 147 PI Phosphatidylinositol 144 PS Phosphatidylserine 9 ST Sulfatide Steroid lipids 8 BA Bile acid 16 CE Cholesterylester 7 Table S4. List of genes, acronyms, and primer information used for real-time quantitative polymerase chain reaction (RT-qPCR) and digital droplet polymerase chain reaction (ddPCR) analysis in Northern pike liver. Gene name Acronym Species Accession number Primer Sequence RT-qPCR target genes Peroxisome proliferator- activated receptor alpha pparα Esox lucius XM_010883898.2 Forward CTGAGCCCACGGATTTG Reverse GTTGAGCTACGGTGATAGTG Sterol regulatory element binding transcription factor 1 srebf1 Esox lucius XM_013135832.4 Forward ACCACACTGAACCGTGATCC Reverse GGCAGCTTGTCAGAGTCCAT Fatty acid desaturase 2 fads2 Esox lucius XM_029114990.2 Forward CCCCTTGTATGTTGTTGACGGT Reverse CTGATTCCGTCTGCTGTCCT ddPCR target genes Acyl-coenzyne A oxidase 1, palmitoyl acox1 Esox lucius XM_010889413.5 Forward GAGGAAGAGTGCCCAGATGAT Reverse CAGGCGGGTATGAAGAAGGTG Elongation of very long chain fatty acid elongase 5 elovl5 Esox lucius NM_001310986.1 Forward CGCATCACCCAGAGGTTAGA Reverse AGTTATCCAGCAGCAGCCAT Phospholipase A2 group IVAb pla2g4ab Esox lucius XM_013135127.4 Forward GGACCCCTCCACATTGACTG Reverse CTCCACATCTTGCCAGGGTT Phospholipase A2 group XIIA pla2g12a Esox lucius XM_010888398.5 Forward GCAGTTCGGGTGTTGGGTTA Reverse CTTCATAGAGCGGGGTCACG Phospholipase A2 group XIIB pla2g12b Esox lucius XM_010868108.5 Forward CCCAACGGAGTGTGTCAGTA Reverse TGCCCAGGTCAAACTGGAATC Reference genes β2 microglobulin precursor b2mg Esox lucius BT079123.1 Forward TGTTGCCCTTGTTTTCTGCGTGGT Reverse TGGCCGTATTTGCCAGGGTTGC Peptidyl-prolyl cis-trans isomerase ppia Esox lucius BT080015.1 Forward TGCTAAAACTGCTTGGCTGGATGG Reverse TCGCTTTCGTCTTGCCGCTG Tubulin α chain tba Esox lucius BT079618.1 Forward GGTCACTACACCATCGGCAAGGA Reverse ACAGGCGTTCCATCAGCAGGGA Ubiquitin-like protein 4A ubl4a Esox lucius BT079424.1 Forward CAGCAGGCGAAAGGAGTGGAGT Reverse TGCCAGGACGGTGGACAAAGT 8 Table S5. List of genes involved in lipid metabolism analyzed in pike liver collected upstream (n = 20) and downstream (n = 20) of Montreal’s MWWE discharge point (QC, Canada). Acronyms for genes and their function in fish are also indicated. Name Acronym Function Références Peroxisome proliferator- activated receptor alpha pparα Transcription factor that enables the transcription of several genes regulating β-oxidation (Varga et al., 201110; Xu et al., 199912) Sterol regulatory element binding transcription factor 1 srebf1 Transcription factor that activates genes involved in synthesis of triacylglycerol, cholesterol and fatty acid synthesis (Goh et al., 20204) Fatty acid desaturase 2 fads2 Introduces unsaturation (double bonds) to polyunsaturated fatty acids (Garrido et al., 20193; Xie et al., 202111) Acyl-coenzyne A oxidase 1, palmitoyl acox1 Regulated by PPARα involved in the first step of long-chain fatty acid ß-oxidation in peroxisome (Madureira et al., 20196; Tocher, 20039) Elongation of very long chain fatty acid elongase 5 elovl5 Regulated by SREBF; in Northern pike it elongates C18 or C20 polyunsaturated fatty acids through the addition of two carbons (Carmona-Antoñanzas et al., 20132; Sun et al., 20208) Phospholipase A2 group IVAb pla2g4ab Cytosolic form that hydrolyzes ester bonds at the sn-2 position to release stored arachidonic acid (Bao et al., 20231; Okamura et al., 20217) Phospholipase A2 group XIIA pla2g12a Secreted form that hydrolyzes ester bonds of a glycerophospholipid to release fatty acids (Leslie, 20155) Phospholipase A2 group XIIB pla2g12b Secreted form that hydrolyzes ester bonds of a glycerophospholipid to release fatty acids (Leslie, 20155) 1Bao, Y., Shen, Y., Wu, Z., Tao, S., Yang, B., Zhu, T., Zhao, W., Zhang, Y., Zhao, X., Jiao, L., Wang, Z., Zhou, Q., & Jin, M. (2023). High dietary arachidonic acid produces excess eicosanoids, and induces hepatic inflammatory responses, oxidative stress and apoptosis in juvenile Acanthopagrus schlegelii. Aquaculture Reports, 29, 101506. https://doi.org/10.1016/j.aqrep.2023.101506 2Carmona-Antoñanzas, G., Tocher, D. R., Taggart, J. B., & Leaver, M. J. (2013). An evolutionary perspective on Elovl5 fatty acid elongase : Comparison of Northern pike and duplicated paralogs from Atlantic salmon. BMC Evolutionary Biology, 13(1), 85. https://doi.org/10.1186/1471-2148-13-85 3Garrido, D., Kabeya, N., Betancor, M. B., Pérez, J. A., Acosta, N. G., Tocher, D. R., Rodríguez, C., & Monroig, Ó. (2019). Functional diversification of teleost Fads2 fatty acyl desaturases occurs independently of the trophic level. Scientific Reports, 9(1), Article 1. https://doi.org/10.1038/s41598-019-47709-0 9 4Goh, P.-T., Kuah, M.-K., Chew, Y.-S., Teh, H.-Y., & Shu-Chien, A. C. (2020). The requirements for sterol regulatory element-binding protein (Srebp) and stimulatory protein 1 (Sp1)-binding elements in the transcriptional activation of two freshwater fish Channa striata and Danio rerio elovl5 elongase. Fish Physiology and Biochemistry, 46(4), 1349‑1359. https://doi.org/10.1007/s10695-020-00793-w 5Leslie, C. C. (2015). Cytosolic phospholipase A₂ : Physiological function and role in disease. Journal of Lipid Research, 56(8), 1386‑1402. https://doi.org/10.1194/jlr.R057588 6Madureira, T. V., Pinheiro, I., Malhão, F., Castro, L. F. C., Rocha, E., & Urbatzka, R. (2019). Silencing of PPARαBb mRNA in brown trout primary hepatocytes : Effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 229, 1‑9. https://doi.org/10.1016/j.cbpb.2018.12.001 7Okamura, Y., Miyanishi, H., Kono, T., Sakai, M., & Hikima, J. (2021). Identification and expression of phospholipase A2 genes related to transcriptional control in the interleukin- 17A/F1 pathway in the intestines of Japanese medaka Oryzias latipes. Fish and Shellfish Immunology Reports, 2, 100028. https://doi.org/10.1016/j.fsirep.2021.100028 8Sun, S., Ren, T., Li, X., Cao, X., & Gao, J. (2020). Polyunsaturated fatty acids synthesized by freshwater fish : A new insight to the roles of elovl2 and elovl5 in vivo. Biochemical and Biophysical Research Communications, 532(3), 414‑419. https://doi.org/10.1016/j.bbrc.2020.08.074 9Tocher, D. R. (2003). Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish. Reviews in Fisheries Science, 11(2), 107‑184. https://doi.org/10.1080/713610925 10Varga, T., Czimmerer, Z., & Nagy, L. (2011). PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation. Biochimica Et Biophysica Acta, 1812(8), 1007‑1022. https://doi.org/10.1016/j.bbadis.2011.02.014 11Xie, D., Chen, C., Dong, Y., You, C., Wang, S., Monroig, Ó., Tocher, D. R., & Li, Y. (2021). Regulation of long-chain polyunsaturated fatty acid biosynthesis in teleost fish. Progress in Lipid Research, 82, 101095. https://doi.org/10.1016/j.plipres.2021.101095 12Xu, H. E., Lambert, M. H., Montana, V. G., Parks, D. J., Blanchard, S. G., Brown, P. J., Sternbach, D. D., Lehmann, J. M., Wisely, G. B., Willson, T. M., Kliewer, S. A., & Milburn, M. V. (1999). Molecular recognition of fatty acids by peroxisome proliferator- activated receptors. Molecular Cell, 3(3), 397‑403. https://doi.org/10.1016/s1097- 2765(00)80467-0 10 Table S6. Final cDNA reaction concentration in reaction mix for each gene for real-time quantitative polymerase chain reaction (RT-qPCR) and digital droplet polymerase chain reaction (ddPCR). Method Gene Final cDNA concentration for samples analysis (ng/µl) RT-qPCR b2mg 0.08 ppiα 0.08 tba 0.1 pparα 0.19 fads2 0.1 srebf1 0.1 ddPCR b2mg 0.13 tba 0.13 ubl4a 0.13 acox1 0.25 elovl5 1.25 pla2g12a 1.25 pla2g12b 1.25 pla2g4ab 1.25 β2 microglobulin precursor (b2mg); peptidyl-prolyl cis-trans isomerase (ppia); tubulin α chain (tba); peroxisome proliferator- activated receptor alpha (pparα); fatty acid desaturase 2 (fads2); sterol regulatory element binding transcription factor 1 (srebf1); ubiquitin-like protein 4A (ubl4a); acyl-coenzyne A oxidase 1, palmitoyl (acox1); elongation of very long chain fatty acid elongase 5 (elovl5); phospholipase A2 group XIIA (pla2g12a); phospholipase A2 group XIIB (pla2g12b) and phospholipase A2 group IVAb (pla2g4ab). 11 Table S7. Real-time quantitative polymerase chain reaction (RT-qPCR) steps for primer validation and sample analysis. Polymerase activation 30 sec, 95°C Amplification, 40 cycles Denaturation 15 sec, 95°C Annealing/extension 20 sec, 60°C Plate read 60°C Melt curve analysis 31 sec, 65°C 60 cycles Temperature augmentation 5 sec, 65°C + 0,5°C/cycle Plate read 65°C + 0,5°C/cycle 12 Table S8. Digital droplet polymerase chain reaction (ddPCR) steps for sample analysis. Denaturation 5 min, 95°C Amplification, 40 cycles Denaturation 30 sec, 95°C Annealing/extension 60 sec, 60°C Enzyme deactivation 5 min, 4°C 5 min, 90°C Cooling 10 min, 4°C 13 Table S9. Mean (± SEM, range) concentrations (ng/g ww) of polybrominated diphenyl ethers (PBDEs) in Northern pike liver collected upstream and downstream of Montreal’s MWWE in the St. Lawrence River (QC, Canada). Means were provided if at least 50% of the samples had concentrations of the compound greater than the method limits of quantification (MLOQ). PBDEs that are not listed were below the MLOQ in all samples. MLOQ Females Males Upstream Downstream Upstream Downstream % sample > MLOQ (n = 10) % sample > MLOQ (n =12) % sample > MLOQ (n = 8) % sample > MLOQ (n = 7) BDE-47 0.02 100 31 ± 4 (15-53) 100 53 ± 9 (28-147) 100 33 ± 5 (15-54) 100 44 ± 12 (10-111) BDE-99 0.04 100 13 ± 2 (4-28) 100 18 ± 3 (8-45) 100 15 ± 3 (6-30) 100 9 ± 2 (5-18) BDE-100 0.04 100 8 ± 1 (8-16) 100 11 ± 1 (5-17) 100 11 ± 3 (4-25) 100 16 ± 7 (3-55) BDE-49 0.02 100 4 ± 0.5 (2-6) 92 5 ± 1 ( LOD (n = 10) % sample > LOD (n =12) % sample > LOD (n = 8) % sample > LOD (n = 8) PFECHS 0.05 70 0.1 ± 0.03 ( LOD (n = 10) % sample > LOD (n =12) % sample > LOD (n = 8) % sample > LOD (n = 8) PFCA PFBA 0.1 0 Limit of detection: PFBA, PFHpA, PFHxA, PFOA, PFPeA, PFPrA, PFBS, PFDoS, PFNS, PFPeS, PFPrS, and PFTrDS. Compounds excluded for males only because there was less than 65% of samples > Limit of detection: PFHpS. 17 Table S13. Statistical results of Student T-test (* p ≤ 0.05) to test differences between sites for the relative contribution (%) of the sum of each lipid biological function (mean ± SEM, range) in Northern pike (liver) collected upstream (n = 18) and downstream (n = 20) of Montreal’s MWWE in the St. Lawrence River (QC, Canada). Biological function Females Males Upstream Downstream T-test results Upstream Downstream T-test results (n = 10) (n =12) (n = 8) (n = 8) Energetic reserve lipids 67 ± 2 (58-77) 69 ± 2 (60-76) t = -1, df = 18, p = 0.3 67 ± 2 (60-75) 59 ± 2 (48-69) t = 2, df = 12, p > 0.05 Membrane lipids 23 ± 2 (15-30) 20 ± 1 (16-27) t = 1, df = 17, p = 0.2 22 ± 1 (17-27) 29 ± 2 (21-37) t = -2, df = 12, p = 0.04* Fatty acids 5 ± 0.4 (4-9) 6 ± 0.2 (4-9) t = -0.3, df = 15, p = 0.8 5 ± 0.4 (3-7) 7 ± 0.4 (5-8) t = -2, df = 14, p = 0.1 Diacylglycerol 4 ± 0.2 (3-5) 4 ± 0.3 (3-6) t = -1, df = 18, p = 0.4 4 ± 0.2 (3-5) 4 ± 0.3 (3-5) t = 1, df = 13, p = 0.6 Steroid lipids 1 ± 0.2 (0.4-2) 1 ± 0.1 (0.5-2) t = 1, df = 17.87, p = 0.5 1 ± 0.1 (0.6-2) 2 ± 0.2 (1-3) t = -2, df = 12, p = 0.04* 18 Table S14. Statistical results of Student T-test or Mann-Whitney U test (* p ≤ 0.05) to test differences between sites for the relative contribution (%) of the sum of each lipid class in Northern pike (liver) collected upstream (n = 18) and downstream (n = 20) of the Montreal’s MWWE discharge point in the St. Lawrence River (QC, Canada). The biological functions are arranged in descending order of relative contribution (%) importance. Each lipid class is also ranked in descending order of relative contribution (%) importance within its biological function. Biological functions of lipid classes Number of lipid compounds per class T-test or Mann-Whitney results Females (n = 23) Males (n = 16) Energetic reserve lipids ∑340TAG †t = -0.98, df = 17.69, p = 0.34 †t = 2.55, df = 13.07, p = 0.02 * Membrane lipids ∑43SM †t = 1.49, df = 15.78, p = 0.16 †t = -2.36, df = 12.16, p = 0.04 * ∑88PE †t = 1.61, df = 19.88, p = 0.12 †t = -1.76, df = 12.02, p = 0.1 ∑144PS †t = 0.73, df = 16.75, p = 0.47 †t = -2.68, df = 10.54, p = 0.02 * ∑147PI †t = 1.79, df = 17.44, p = 0.09 †t = -1.99, df = 11.59, p = 0.07 ∑28Cer ‡W = 76, p = 0.31 †t = -1.80, df = 8.92, p = 0.11 ∑95PC †t = 1.45, df = 17.60, p = 0.16 †t = -2.44, df = 11.75, p = 0.03 * ∑10Dihydro-SM †t = 1.03, df = 19.82, p = 0.31 †t = -2.26, df = 14, p = 0.04 * ∑116PG †t = 1.95, df = 18.99, p = 0.07 †t = -2.02, df = 12.62, p = 0.07 ∑84PA †t = 1.33, df = 20, p = 0.20 †t = -3.33, df = 11.69, p < 0.01 * ∑37LPE †t = -0.36, df = 20, p = 0.72 †t = -1.47, df = 11.37, p = 0.17 ∑17LPC †t = 0.09, df = 19.96, p = 0.93 †t = -1.42, df = 10.46, p = 0.18 ∑121CL †t = 0.03, df = 19.96, p = 0.98 †t = -2.33, df = 9.93, p = 0.04 * ∑28LPI †t = 1.13, df = 19.99, p = 0.27 ‡W = 32, p = 1 ∑11Hex Cer ‡W = 79, p = 0.23 †t = -1.32, df = 13.94, p = 0.21 ∑22LPA †t = -0.12, df = 19.97, p = 0.90 †t = -1.59, df = 10.68, p = 0.14 ∑32LPG ‡W = 78, p = 0.25 ‡W = 36, p = 0.72 ∑53-KDS ‡W = 80, p = 0.2 ‡W = 21, p = 0.28 ∑4Sa †t = -0.01, df = 18.09, p = 0.99 †t = -2.68, df = 10.31, p = 0.02 * ∑34LPS ‡W = 43, p = 0.28 †t = -2.67, df = 11,09, p = 0.02 * ∑9ST ‡W = 48, p = 0.46 †t = -4.78, df = 13.66, p < 0.01 * ∑10Hex2 Cer ‡W = 55, p = 0.77 ‡W = 25, p = 0.51 ∑8G †t = -2.29, df = 19.94, p = 0.03 * ‡W = 12, p = 0.04 * ∑8Hex3 Cer ‡W = 62, p = 0.92 ‡W = 17, p = 0.13 SPC ‡W = 65, p = 0.77 †t = -0.31, df = 12.93, p = 0.76 Fatty acids ∑20FA †t = -0.32, df = 14.74, p = 0.76 †t = -2.19, df = 13.78, p = 0.05 * Diacylglycerol ∑41DAG †t = -0.85, df = 17.85, p = 0.41 †t = -0.25, df = 12.93, p = 0.81 Steroid lipids ∑16CE †t = 0.84, df = 17.70, p = 0.41 †t = -1.59, df = 11.43, p = 0.14 ∑8BA ‡W = 60, p = 1 ‡W = 17, p = 0.13 See Table S3 for abbreviations † Student T-test (* p ≤ 0.05) ‡ Mann-Whitney U test (* p ≤ 0.05) 19 Table S15. Statistical results of Student T-test or Mann-Whitney U test (* p ≤ 0.05) to test differences between sites for the levels of mARN as relative normalized expression fold of selected genes in the liver of Northern pike collected upstream and downstream of the Montreal’s MWWE in the St. Lawrence River (QC, Canada). † Student T-test (* p ≤ 0.05) ‡ Mann-Whitney U test (* p ≤ 0.05) Method Gene Statistical results Females Males RT-qPCR pparα †t = 0.3, df = 16, p = 0.8 ‡W = 36, p = 0.02 * fads2 ‡W = 54, p = 0.8 ‡W = 42, p = 0.3 srebf1 †t = 0.7, df = 17, p = 0.5 †t = 1, df = 14, p = 0.2 ddPCR acox1 †t = -0.06, df = 15, p = 1 †t = -0.8, df = 13, p = 0.4 elovl5 ‡W = 47, p = 0.9 ‡W = 31, p = 1 pla2g4ab ‡W = 57, p = 0.6 †t = 3, df = 13, p = 0.01 * pla2g12a †t = 0.03, df = 18, p = 1 ‡W = 31, p = 1 pla2g12b ‡W = 62, p = 0.4 ‡W = 21, p = 0.3 20 Table S16. Mean (± SEM, range) of malondialdehyde (MDA)/triacylglycerol (TAG) concentrations (mmol/mg) in Northern pike liver collected upstream (n = 18) and downstream (n = 20) of the Montreal’s MWWE in the St. Lawrence River (QC, Canada). Females Males Upstream Downstream Upstream Downstream (n = 10) (n =12) (n = 8) (n = 8) MDA/TAG (mmol/mg TAG) 20 ± 2 (10-32) 17 ± 2 (1-30) 16 ± 3 (3- 26) 16 ± 3 (4-34) 21 Table S17. List of contaminants under the method limits of quantification (MLOQ) or limits of detection (LOD) in the liver of Northern pike collected upstream and downstream of Montreal’s MWWE in the St. Lawrence River (QC, Canada). Flame retardants under limits of quantification MLOQ 0.03 BDE-15 0.01 BDE-71 0.02 BDE-77 0.05 BDE-138 0.03 BDE-139 0.03 BDE-140 0.05 BDE-171 0.02 BDE-180 0.03 BDE-183/Dec-604 0.04 BDE-184 0.01 BDE-191 0.03 BDE-196 0.03 BDE-201 0.06 BDE-203 0.13 BDE-205 0.76 BDE-206 0.1 BDE-207 0.14 BDE-208 0.05 Cplus 0.6 DBCD 1.21 DBDPE 0.08 Dec-601 0.08 Dec-602 0.47 Dec-603 0.01 Dec-604CB 0.19 OBIND LOD PFAS under limits of detection 0.1 3:3 Acid 0.2 5:3 Acid 0.2 6:2 Cl-PFESA 0.02 8:2 Cl-PFESA 0.05 EtFOSA 0.02 EtFOSAA 0.02 FOSAA 0.02 6:2 FTAB 0.1 5:1:2 FtB 0.05 5:3 FtB 0.01 6:2 FTCA 22 0.02 8:2 FTCA 0.05 4:2 FTSA 0.02 8:2 FTSA 0.05 10:2 FTSA 0.1 Gen-X 0.05 MeFBSA 0.02 MeFOSA 0,01 PFBS 0.05 PFDoS 0.05 PFHxPA 0.1 PFHxSAm 0.1 PFHxSAmS 0.2 PFNS 0.02 PFOAB 0.05 PFOANO 0.1 PFOPA 0.02 PFOSAm 0.05 PFOSAmS 0.05 PFOSB 0.05 PFOSNO 0.05 PFPeS 0.1 PFPrS MLOQ PFAS under limits of quantification 0.2 FDSA 0.2 FHpSA 0.01 6:2 FTUCA 0.02 8:2 FTUCA 0.2 10:2 FTUCA 0.2 6:6 PFPiA 0.01 6:8 PFPiA 0.02 8:8 PFPiA 0.05 PFEESA 0.1 PFEtS 0.1 PFMBA 0.1 PFMPA 0.2 PFPrA 0.01 PFTrDS 0.05 PFUdS See table S1 and S2 for abbreviations. 23 Table S18. List of correlations close or under the alpha-cutoff value (p < 0.05) for Pearson correlation and Spearman’s rank correlation coefficient in the liver of Northern pike collected upstream and downstream of the Montreal’s MWWE point of discharge in the St. Lawrence River (QC, Canada). Sex Biological variables Correlation coefficient p value Females ∑76PFAS and ∑1103membrane lipids †r = 0.417 0.054 pla2g4ab and pufaLPC/pufaPC ratio ‡Rho = 0.44 0.052 Males ∑1103membrane lipids and ∑340TAG †r = -0.98 < 0.01* pla2g4ab and CE †r = -0.49 0.057 pla2g4ab and ∑9SFA †r = 0.49 0.056 Per- and polyfluoroalkyl substances (PFAS); phospholipase A2 group IVAb (pla2g4ab); Polyunsaturated fatty acid lysophosphatidylcholine (pufaLPC); Polyunsaturated fatty acid phosphatidylcholine (pufaPC); Triacylglycerol (TAG); Cholesterylester (CE) and Saturated fatty acid (SFA). † Pearson correlation coefficient (* p ≤ 0.05) ‡ Spearman’s rank correlation coefficient (* p ≤ 0.05) 24 Figure S1. Examples of the one-color plot outputs demonstrating methodological robustness of the digital droplet polymerase chain reaction (ddPCR) analysis in Northern pike liver. The positives droplets are represented by blue dots and negative by grey dots. Each reaction contains approximately 16,000-20,000 copies of the cDNA template analyzed using the QX200 Droplet Reader (Bio-Rad, Mississauga, ON, Canada) with QX200™ ddPCR™ EvaGreen Supermix (Bio- Rad, Mississauga, ON, Canada). A) The effect of annealing temperature (gradient 63.3°C to 53°C from left to right) on the final fluorescence intensity of the positive and negative partitions of the gene acox1. The well with the least interface droplets is the best annealing temperature; here in the well D01 (59.8°C). B) The effect of annealing temperature (gradient 63.3°C to 53°C from left to right) on the final fluorescence intensity of the positive and negative partitions of the reference gene b2mg. C) The effect of cDNA concentration (left: 0.25 ng/µL; right: 1.25 ng/µL) in reaction mix on the final fluorescence intensity of the positive and negative partitions of the gene acox1. The final concentration of positive droplet (copies/µL) for each sample needed to be above 1 copy/µL; here the mean final concentration is 3.14 copies/µL (left) and 15.25 copies/µL (right). A) B) C) 25 Table S19. Sex, age and morphological information of Northern pike collected upstream and downstream of Montreal’s MWWE effluent discharge point in the St. Lawrence River (QC, Canada). Some individuals were excluded from the analysis as they were not sexually mature and are not listed here. ID Site Sex Age (year) Total length (cm) Mass (kg) Liver mass (g) IBO-B-1 Upstream F 6+ 68.50 1.84 20.00 IBO-B-2 Upstream M 6+ 58.00 1.03 12.00 IBO-B-3 Upstream M 2+ 55.00 1.10 18.00 IBO-B-4 Upstream F 6+ 64.00 1.38 13.00 IBO-B-5 Upstream F 5+ 63.00 1.38 10.00 IBO-B-6 Upstream M 4+ 62.00 1.33 16.00 IBO-B-7 Upstream M 7+ 64.00 1.22 14.00 IBO-B-9 Upstream M 3+ 57.50 1.03 9.00 IBO-B-10 Upstream F 7+ 73.00 1.96 31.00 IBO-B-11 Upstream F 7+ 73.00 2.10 30.00 IBO-B-12 Upstream F 4+ 59.50 0.40 10.00 IBO-B-13 Upstream F 5+ 63.00 1.30 20.00 IBO-B-14 Upstream M 2+ 55.50 0.98 11.00 IBO-B-15 Upstream M 4+ 60.00 1.12 12.00 IBO-B-16 Upstream F 3+ 60.00 1.05 13.00 IBO-B-17 Upstream F 4+ 60.50 1.23 15.00 IBO-B-18 Upstream M 8+ 69.00 1.65 27.00 IBO-B-19 Upstream F 6+ 70.50 1.59 36.00 IBO-B-20 Upstream Not determined 5+ 61.00 0.93 10.00 IVT-B-1 Downstream F 8+ 76.50 2.17 18.00 IVT-B-2 Downstream F 6+ 75.00 1.88 30.00 IVT-B-3 Downstream M 3+ 54.00 0.79 9.00 IVT-B-4 Downstream F 6+ 66.00 1.39 13.00 IVT-B-5 Downstream M 3+ 55.00 0.82 7.00 IVT-B-6 Downstream F 5+ 65.00 1.36 21.00 IVT-B-8 Downstream M 3+ 58.00 1.01 10.00 IVT-B-9 Downstream F 5+ 64.00 1.45 12.00 IVT-B-10 Downstream F 5+ 61.50 1.16 19.00 IVT-B-11 Downstream M 6+ 58.50 0.85 6.00 IVT-B-12 Downstream M 6+ 61.50 1.21 20.00 IVT-B-13 Downstream M 7+ 64.00 1.49 11.00 IVT-B-14 Downstream F 4+ 58.00 1.05 17.00 IVT-B-15 Downstream M 3+ 51.00 0.73 9.00 IVT-B-16 Downstream F 4+ 62.00 1.29 19.00 IVT-B-17 Downstream F 8+ 55.50 0.98 16.00 IVT-B-18 Downstream F 5+ 62.00 1.23 10.00 IVT-B-19 Downstream F 5+ 67.00 1.74 29.00 IVT-B-20 Downstream F 3+ 52.00 0.80 7.00 IVT-B-21 Downstream M 3+ 53.50 0.82 10.00 26 Table S20. Date, site name and capture coordinates of Northern pike collected upstream and downstream of Montreal’s WWTP effluent discharge point (QC, Canada). Some individuals were excluded from the analysis due to their lack of sexual maturity and are not listed here. Date Site Fish ID Site name Latitude Longitude (degrees, decimal minutes) 17/06/2022 Upstream IBO-B-1 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 17/06/2022 Upstream IBO-B-2 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 17/06/2022 Upstream IBO-B-3 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 21/06/2022 Upstream IBO-B-4 Île de la Baronnie 45°33'52.9"N 73°29'47.5"W 21/06/2022 Upstream IBO-B-5 Île de la Baronnie 45°33'52.9"N 73°29'47.5"W 21/06/2022 Upstream IBO-B-6 Île de la Baronnie 45°33'52.9"N 73°29'47.5"W 21/06/2022 Upstream IBO-B-7 Île de la Baronnie 45°33'52.9"N 73°29'47.5"W 28/06/2022 Upstream IBO-B-9 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 28/06/2022 Upstream IBO-B-10 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 28/06/2022 Upstream IBO-B-11 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 28/06/2022 Upstream IBO-B-12 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 28/06/2022 Upstream IBO-B-13 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 28/06/2022 Upstream IBO-B-14 Île de la Baronnie 45°34'18.8"N 73°29'28.1"W 08/07/2022 Upstream IBO-B-15 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 08/07/2022 Upstream IBO-B-16 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 08/07/2022 Upstream IBO-B-17 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 08/07/2022 Upstream IBO-B-18 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 08/07/2022 Upstream IBO-B-19 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 08/07/2022 Upstream IBO-B-20 Île de la Baronnie 45°34'02.6"N 73°29'41.0"W 14/06/2022 Downstream IVT-B-1 Îles Robinet 45°44'24.5"N 73°25'42.2"W 20/06/2022 Downstream IVT-B-2 Îles Robinet 45°44'24.5"N 73°25'42.2"W 20/06/2022 Downstream IVT-B-3 Île Beauregard 45°44'56.0"N 73°24'55.8"W 20/06/2022 Downstream IVT-B-4 Île Beauregard 45°44'56.0"N 73°24'55.8"W 20/06/2022 Downstream IVT-B-5 Île Beauregard 45°44'56.0"N 73°24'55.8"W 20/06/2022 Downstream IVT-B-6 Île Beauregard 45°44'56.0"N 73°24'55.8"W 22/06/2022 Downstream IVT-B-8 Îlet Vert 45°42'30.1"N 73°27'06.4"W 2022-06-26 Downstream IVT-B-9 Île Beauregard 45°44'58.8"N 73°24'53.1"W 05/07/2022 Downstream IVT-B-10 Île Beauregard 45°44'58.8"N 73°24'53.1"W 05/07/2022 Downstream IVT-B-11 Île Beauregard 45°44'58.8"N 73°24'53.1"W 05/07/2022 Downstream IVT-B-12 Îlet Vert 45°42'18.1"N 73°27'15.2"W 05/07/2022 Downstream IVT-B-13 Îlet Vert 45°42'18.1"N 73°27'15.2"W 14/07/2022 Downstream IVT-B-14 Îlet Vert 45°42'18.1"N 73°27'15.2"W 14/07/2022 Downstream IVT-B-15 Îlet Vert 45°42'18.1"N 73°27'15.2"W 14/07/2022 Downstream IVT-B-16 Île Beauregard 45°44'58.8"N 73°24'53.1"W 15/07/2022 Downstream IVT-B-17 Îlet Vert 45°42'18.1"N 73°27'15.2"W 27 15/07/2022 Downstream IVT-B-18 Îlet Vert 45°42'18.1"N 73°27'15.2"W 15/07/2022 Downstream IVT-B-19 Îlet Vert 45°42'18.1"N 73°27'15.2"W 15/07/2022 Downstream IVT-B-20 Îlet Vert 45°42'30.1"N 73°27'06.4"W 15/07/2022 Downstream IVT-B-21 Îlet Vert 45°42'30.1"N 73°27'06.4"W MeunierM - Figures MeunierM - Graphical abstract GRAPHICAL ABSTRACT MeunierM - Highlights Highlights MeunierM - SI