SUPPLEMENTARY INFORMATION. Towards streamlined identification of dioxin-like compounds in

SUPPLEMENTARY INFORMATION Towards streamlined identification of dioxin-like compounds in environmental samples through integration of suspension bioassays Xiao, Hongxia # ; Brinkmann, Markus # *; Thalmann, Beat; Schiwy, Andreas; Große Brinkhaus, Sigrid; Achten, Christine; Eichbaum, Kathrin; Gembé, Carolin; Seiler, Thomas-Benjamin; Hollert, Henner * Total page number: 27, including 7 tables and 7 figures. # These authors contributed equally and share first authorship. *Corresponding authors: Prof. Dr. Henner Hollert Worringerweg 1 52074 Aachen, Germany Phone: +49 (0)241 80 / 26669, Fax: +49 (0)241 80 / 22182 henner.hollert@bio5.rwth-aachen.de Dr. Markus Brinkmann 44 Campus Drive Saskatoon SK S7N 5B3 Phone: (306) 966-1204 Email: markus.brinkmann@usask.ca S1

Sampling Location Sediment sample of CNG-U were collected using a Van-Veen sampler in September 2011 at the upstream of the Yangtze River close to Chongqing. Sediment samples of HAN-C and HAN-D were collected in May 2013 at the artificial Hanfeng Lake. Figure S1: Overview map of sampling locations in the Three Gorges Reservoir area, China. The Three Gorges Reservoir covers the Yangtze River section between Jiangjin district (Chongqing) and the Three Gorges Dam. CNG-U: Upstream of Chongqing municipality, HAN-C/D: Hanfeng Lake. Detailed sampling information are available in Floehr et al. 1 S2

List of Standards for GC-APLI-MS Analysis For quantification of polycyclic aromatic compounds, a custom-made calibration standard and additional single compounds resulting in 73 PACs were used: naphthalene, 2- methylnaphthalene, acenaphthylene, acenaphthene, fluorene, 1-methylfluorene, phenanthrene, anthracene, 9-methylphenanthrene, 9-methylanthracene, 9,10-dimethylanthracene, fluoranthene, pyrene, 1-methylpyrene, 7H-benzo[c]fluorene, benzo[c]phenanthrene, benzo[a]anthracene, chrysene, 5-methylchrysene, cyclopenta[cd]pyrene, benzo[ghi]fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene, 6-methylbenzo[a]anthracene, 7,12-dimethylbenzo[a]anthracene, benzo[e]pyrene, benzo[a]pyrene, 6-methylbenzo[a]pyrene, perylene, 3-methylcholanthrene, dibenzo[a,h]anthracene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene, anthanthrene, dibenzo[a,l]anthracene, dibenzo[a,e]anthracene, dibenzo[a,i]anthracene, and dibenzo[a,h]anthracene (all custom-made PAH-mixture, Chiron, Norway), 2-methylchrysene, 4-methylchrysene, 6-methylchrysene, dibenzo[a,c]anthracene, dibenzo[a,j]anthracene, triphenylene (all single standards, Neochema, Germany), retene (single standard, Dr. Ehrenstorfer, Germany) coronene (single substance, Sigma Aldrich, Germany), 5- methylbenzo[a]anthracene, naphtho[2,3-k]fluoranthene, naphtho[2,3-b]fluoranthene, naphtho[2,3-j]fluoranthene, naphtho[1,2-k]fluoranthene, naphtho[1,2-b]fluoranthene, naphtho[2,3-e]pyrene, naphtho[2,3-a]pyrene (all single standards, Chiron, Norway), benzo[b]naphtho[1,2-d]thiophene, 4-methyldibenzo[bd]thiophene, naphtho[2,3-b]thiophene, 2,3-dimehtyldibenzo[bd]thiophene, 1-methylphenanthrene, 1,8-dimethylnaphthalene, 2,6-dimethylnaphthlene (all single standards, kindly provided by Prof. Jan T. Andersson from University of Münster), carbazole, 2-methylbenzofuran, acridine, benzo[b]furan, benzothiophene, quinoline, dibenzofuran, dibenzothiophene, indole, 2-methylquinoline (all PAH-Mix 229, Dr. Ehrenstorfer, Germany). S3

Fractionation Method Table S1: List of single compounds used for establishing the relationship between log K ow and retention time (RT). Octanol-water partitioning coefficients (log K ow ) were obtained using US-EPA EPI Suite. Experimental data were preferred over predicted values. No. Comp. CAS No. Supplier (purity) MW (g mol -1 ) log K ow (-) RT (min) 1 Quinoline 91-22-5 abcr chemicals, Karlsruhe, Germany (>98%) 129.16 2.03 5.72 2 Indole 120-72-9 abcr chemicals, Karlsruhe, Germany (>99%) 117.15 2.14 4.98 3 6-Methylquinoline 91-62-3 abcr chemicals, Karlsruhe, Germany (>98%) 143.19 2.57 6.96 4 Benzofuran 271-89-6 Sigma-Aldrich, Steinheim, Germany (>99%) 118.14 2.67 6.75 5 Benzothiophene 95-15-8 abcr chemicals, Karlsruhe, Germany (>98%) 134.20 3.12 8.29 6 2-Methylbenzofuran 4265-25-2 Sigma-Aldrich, Steinheim, Germany ( 96%) 132.16 3.22 8.75 7 Carbazole 86-74-8 abcr chemicals, Karlsruhe, Germany (~95%) 167.21 3.29 6.96 8 Naphthalene 91-20-3 LGC Standards, Wesel, Germany (analytical standard) 128.17 3.30 9.61 9 Acridine 260-94-6 Merck, Darmstadt, Germany (>98%)S 179.22 3.40 8.46 10 2,3-Dimethylbenzofuran 3782-00-1 Sigma-Aldrich, Steinheim, Germany ( 97%) 146.19 3.63 11.40 11 Acenaphthene 83-32-9 LGC Standards, Wesel, Germany(analytical standard) 154.21 3.92 13.55 12 Acenaphthylene 208-96-8 LGC Standards, Wesel, Germany (analytical standard) 152.20 3.94 10.83 13 Biphenyl 92-52-4 LGC Standards, Wesel, Germany (analytical standard) 154.21 3.98 12.29 14 Dibenzofuran 132-64-9 Sigma-Aldrich, Steinheim, Germany (~98%) 168.19 4.05 12.77 15 Fluorene 86-73-7 LGC Standards, Wesel, Germany (analytical standard) 166.22 4.18 13.47 16 Xanthene 92-83-1 Sigma-Aldrich, Steinheim, Germany (>99%) 182.22 4.23 13.59 S4

17 Dibenzothiophene 132-65-0 abcr chemicals, Karlsruhe, Germany (>98%) 184.26 4.29 13.80 18 Anthracene 120-12-7 LGC Standards, Wesel, Germany (analytical standard) 178.23 4.45 14.16 19 Phenanthrene 85-01-8 LGC Standards, Wesel, Germany (analytical standard) 178.23 4.46 13.84 20 β-naphthoflavone 6051-87-2 Sigma-Aldrich, Steinheim, Germany ( 98%) 272.30 4.68 14.50 21 Pyrene 129-00-0 LGC Standards, Wesel, Germany(analytical standard) 202.25 4.88 15.48 22 Fluoranthene 206-44-0 LGC Standards, Wesel, Germany (analytical standard) 202.26 5.16 14.96 23 Hexachlorobenzene 118-74-1 Sigma-Aldrich, Steinheim, Germany (analytical standard) 284.78 5.73 18.71 24 Benzo[a]anthracene 56-55-3 LGC Standards, Wesel, Germany (analytical standard) 228.29 5.76 16.16 25 Benzo[b]fluoranthene 205-99-2 LGC Standards, Wesel, Germany (analytical standard) 252.31 5.78 17.39 26 Chrysene 218-01-9 LGC Standards, Wesel, Germany (analytical standard) 228.29 5.81 16.17 27 Benzo[a]pyrene 50-32-8 LGC Standards, Wesel, Germany (analytical standard) 252.32 5.99 18.15 28 Benzo[k]fluoranthene 207-08-9 LGC Standards, Wesel, Germany (analytical standard) 252.32 6.11 17.48 29 Benzo[ghi]perylene 191-24-2 LGC Standards, Wesel, Germany (analytical standard) 276.33 6.70 20.59 30 Dibenzo[a,h]anthracene 53-70-3 LGC Standards, Wesel, Germany (analytical standard) 278.35 6.75 18.32 31 Indeno[1,2,3-cd]pyrene 193-39-5 LGC Standards, Wesel, Germany (analytical standard) 276.33 6.76 19.79 32 2,3,7,8-TCDD 1746-01-6 Sigma-Aldrich, Steinheim, Germany (analytical standard) 321.97 6.80 20.68 33 PCB 126 57465-28-8 Dr. Ehrenstorfer, Augsburg, Germany (analytical standard) 326.43 6.98 19.42 S5

Development and Validation of the Fractionation Method Figure S2: Linear regression of chromatographic retention time versus the n-octanol/water partitioning coefficient (log K ow ) for a total number of 33 chemicals. The straight line is the regression line; the shaded area indicates the 95 % prediction band. S6

Fractionation of Environmental Samples Table S2: Concentrations of the 16 EPA-PAHs in sediment samples from the Yangtze River, China; CNG-U, HAN-C, HAN-D (Figures S5 and S6). Limit of detection (LOD): 10 µg kg -1. From Floehr et al. 1 Concentration (µg kg -1 ) Compound CNG-U HAN-D HAN-C Naphthalene 220 63 86 Acenaphthylene 12 16 21 Acenaphthene 33 n.d. n.d. Fluorene 64 37 42 Phenanthrene 340 68 73 Anthracene 41 17 19 Fluoranthene 170 34 40 Pyrene 150 30 34 Benzo[a]anthracene 73 21 24 Chrysene 120 47 53 Benzo[b]fluoranthene 170 87 116 Benzo[k]fluoranthene 38 47 59 Benzo[a]pyrene 76 n.d. n.d. Indeno[1,2,3-cd]pyrene 57 n.d. n.d. Dibenzo[a,h]anthracene 12 n.d. n.d. Benzo[ghi]perylene 76 20 26 Σ 16 EPA-PAHs 1,653 484 590 n.d.: below detection limits S7

Table S3: Characteristics and concentrations of 17 PCDD/Fs and 12 dl-pcbs in sediments from EBR and ZE. Modified from Eichbaum at al. 2016. 2 Ehrenbreitstein harbor Zollelbe Acronym EBR ZE River system Unit km Rhine main stream 591.4 Elbe cut-off meander 0.1 Longitude 7.60792 11.65087 Latitude 50.35400 52.13256 Sampling date 12.04.2012 10.04.2012 Grab (Max. sampling depth) Van-Veen grab (15 cm) Van-Veen grab (15 cm) TOC (g kg -1 ) 49.6 64.3 Loss on ignition (%) 10.6 ± 0.4 14.1 ± 0.3 Concentration of 12 dl-pcb 4.38 ng g -1 dw 9.72 ng g -1 dw Concentration of 17 PCDD/F 1.06 ng g -1 dw 3.70 ng g -1 dw Micro-EROD EC25 TEQ of PCB fraction (pg g -1 dw) 16.7 ± 7.1 76.4 ± 18.8 Micro-EROD EC25 TEQ of PCDD/F fraction (pg g -1 dw) 63.7 ± 8.8 159.0 ± 32.1 S8

Recovery of Analytes During Fractionation Table S4: Recovery of the 16 EPA-PAHs after fractionation of 10 µl of a 625 µg L -1 standard solution was determined by means of GC-MS in three replicates. No. Compound Mean recovery (%) Standard deviation (%) 1 Naphthalene 3 2 2 Acenaphthylene 7 8 3 Acenaphthene 5 6 4 Fluorene 11 11 5 Phenanthrene 27 8 6 Anthracene 26 7 7 Fluoranthene 63 3 8 Pyrene 53 3 9 Benzo[a]anthracene 112 25 10 Chrysene 112 29 11 Benzo[b]fluoranthene 103 28 12 Benzo[k]fluoranthene 113 36 13 Benzo[a]pyrene 109 23 14 Indeno[1,2,3-cd]pyrene 103 35 15 Dibenzo[a,h]anthracene 142 44 16 Benzo[ghi]perylene 159 45 S9

Fractionation of Environmental Samples Figure S3: Micro-EROD results of HPLC-fractionated sediment extract HAN-D (Yangtze River, China); 10 µl of a 1 g SEQ ml -1 extract were fractionated. Asterisks denote significant differences between fractions and control (one-way ANOVA with Holm-Sidak s post-hoc test, p 0.05). Shaded areas indicate the typical retention time ranges of the substance classes screened as single substances. Figure S4: Micro-EROD results of HPLC-fractionated sediment extract HAN-C (Yangtze River, China); 10 µl of a 1 g SEQ ml -1 extract were fractionated. Asterisks denote significant differences between fractions and control (one-way ANOVA with Holm-Sidak s post-hoc test, p 0.05). Shaded areas indicate the typical retention time ranges of the substance classes screened as single substances. S10

Table S5 Concentrations of various polycyclic aromatic compounds (PACs) in fractions F1 to F5 of an extract of sediment sample CNG-U, Yangtze River, China. Concentration (µg kg -1 ) Compound F1 F2 F3 F4 F5 Naphthalene n.d. n.d. n.d. n.d. n.d. 2-Methylnaphthalene n.d. n.d. n.d. n.d. n.d. 1,8-Dimethylnaphthalene n.d. n.d. n.d. n.d. n.d. 2,6-Dimethylnaphthalene n.d. n.d. n.d. n.d. n.d. Acenaphthene n.d. n.d. n.d. n.d. n.d. Fluorene 0.01 0.05 n.d. 0.14 n.d. 1-Methylfluorene n.d. n.d. n.d. n.d. n.d. Phenanthrene 0.01 0.22 0.00 n.d. n.d. 9-Methylphenanthrene 0.01 0.05 0.04 0.06 0.01 1-Methylphenanthrene 0.00 0.03 0.03 0.05 0.00 Retene 0.02 0.31 0.03 0.04 0.02 Anthracene n.d. 0.05 n.d. n.d. n.d. 9-Methylanthracene n.d. n.d. n.d. n.d. n.d. 9,10-Dimethylanthracene n.d. n.d. n.d. n.d. n.d. Fluoranthene n.d. 0.14 n.d. 0.10 n.d. Pyrene n.d. 0.05 n.d. n.d. n.d. 1-Methylpyrene 0.05 1.62 0.03 0.07 0.05 7H-Benzo[c]fluorene 0.05 0.27 0.02 0.03 0.02 Benzo[ghi]fluoranthene 0.56 0.45 n.d. n.d. n.d. Benzo[c]phenanthrene 0.70 0.08 0.04 n.d. n.d. Benzo[a]anthracene 9.78 1.22 n.d. n.d. n.d. 5-Methylbenzo[a]anthracene 0.05 1.24 0.07 0.05 n.d. 6-Methylbenzo[a]anthracene 0.05 11.81 n.d. 0.06 0.05 7,12-Dimethylbenzo[a]anthracene 0.03 16.94 0.15 0.09 0.02 Chrysene 11.17 2.10 n.d. n.d. n.d. Triphenylene 2.09 0.18 0.02 0.03 0.03 S11

2-Methylchrysene n.d. 6.99 n.d. n.d. n.d. 4-Methylchrysene n.d. 0.27 0.11 n.d. n.d. 6-Methylchrysene 0.04 3.02 0.04 0.04 n.d. 5-Methylchrysene 0.04 1.05 n.d. 0.04 0.04 Benzo[b]fluoranthene n.d. 39.60 0.03 0.06 0.00 Benzo[k]fluoranthene n.d. 11.73 n.d. n.d. n.d. Benzo[a]pyrene n.d. 15.34 0.03 0.03 n.d. 6-Methylbenzo[a]pyrene n.d. 0.01 0.05 n.d. n.d. Benzo[e]pyrene 0.04 34.68 0.09 0.10 0.05 Perylene n.d. 12.36 0.01 0.00 n.d. 3-Methylcholanthrene n.d. n.d. n.d. n.d. n.d. Indeno[1,2,3-cd]-pyrene n.d. n.d. 30.06 2.34 n.d. Dibenzo[a,h]anthracene n.d. 6.28 n.d. n.d. n.d. Dibenzo[a,c]anthracene 0.03 4.76 0.04 0.03 0.03 Dibenzo[a,j]anthracene 0.02 7.66 0.04 0.13 0.03 Anthanthrene 0.03 0.03 0.03 0.48 0.04 Benzo[ghi]perylene n.d. n.d. 2.47 11.15 n.d. Coronene n.d. n.d. n.d. n.d. 0.01 Naphtho[1,2-b]-fluoranthene + Naphtho[2,3-a]-pyrene Naphtho[2,3-j]-fluoranthene + Naphtho[1,2-k]-fluoranthene 0.03 0.03 1.26 0.66 n.d. 0.03 0.03 1.40 1.38 0.04 Naphtho[2,3-b]-fluoranthene 0.05 0.04 n.d. 5.27 0.05 Naphtho[2,3-e]-pyrene 0.03 0.04 n.d. 1.31 0.04 Naphtho[2,3-k]-fluoranthene 0.05 0.05 0.05 0.23 0.04 Dibenzo[a,e]fluoranthene 0.03 n.d. n.d. 0.41 n.d. Dibenzo[a,e]pyrene n.d. 0.01 n.d. 20.89 0.24 Dibenzo[a,h]pyrene 0.05 n.d. 0.05 n.d. 0.06 Dibenzo[a,i]pyrene n.d. n.d. n.d. 0.52 0.32 Dibenzo[a,l]pyrene n.d. n.d. n.d. 4.54 n.d. n.d.: not detectable S12

Solvent control Fraction F2 S13

Fraction F4 Figure S5: A solvent control, as well as the factions F2 and F4 of extract of CNG-U sediment were analyzed by means of gas chromatographyatmospheric pressure laser ionization-time-of-flight-mass spectrometry (GC-APLI-MS). Labelled peaks are listed in Tables S6 and S7. Peaks present in the solvent control were excluded from further analysis. S14

Table S6: Detected and confirmed (bold) PACs in fraction F2 of extracts of CNG-U sediment as detected by means of gas chromatographyatmospheric pressure laser ionization-time-of-flight-mass spectrometry (GC-APLI-MS). Peak numbers refer to the peaks labeled in Figure S5. No. RT (min) Max. m/z Compound (possible) sum formula Area Intensity (cps) S/N ratio 1 37.5 220.1249 C3-Phenanthrene/Anthracene C 17 H 16 1504126 374216 35.3 2 39.28 234.1403 C4- Phenanthrene/Anthracene C 18 H 18 2492948 583007 42.9 3 40.43 248.1562 C5- Phenanthrene/Anthracene C 19 H 20 1886678 506281 52.1 4 40.7 230.1093 C 18 H 14 5025221 667387 54.2 5 40.85 230.1092 C2-Pyrene/Fluoranthene C 18 H 14 1922604 485532 51.1 6 41.05 230.1093 C2-Pyrene/Fluoranthene C 18 H 14 10920470 2010393 159.6 7 41.1 246.104 C 18 H 14 O 1001087 286585 39 8 41.62 230.1092 C2-Pyrene/Fluoranthene C 18 H 14 7076355 1638978 117.1 9 41.85 230.1093 C2-Pyrene/Fluoranthene C 18 H 14 7341201 1906936 118.8 10 42.04 230.1093 C2-Pyrene/Fluoranthene C 18 H 14 7236000 1509256 113.2 11 42.16 234.1405 C4- Phenanthrene/Anthracene C 18 H 18 5603097 1434820 97.4 12 42.35 230.1093 C2-Pyrene/Fluoranthene C 18 H 14 4863036 1234021 101.5 13 42.69 230.1094 C2-Pyrene/Fluoranthene C 18 H 14 5490919 1271725 102.7 14 42.85 232.1249 C4-Phenanthrene/Anthracene, one double bond C 18 H 16 5042549 904423 69.1 15 43.33 246.1404 C4-Phenanthrene/Anthracene, one double bond C 19 H 18 3740523 838588 61.4 16 43.45 230.1091 C2-Pyrene/Fluoranthene C 18 H 14 2790664 691664 72.8 17 43.85 244.1247 C3-Pyrene/Fluoranthene C 19 H 16 12425247 1828533 138.1 18 44.22 244.1247 C3-Pyrene/Fluoranthene C 19 H 16 10269512 1828806 150.3 19 44.74 244.1247 C3-Pyrene/Fluoranthene C 19 H 16 3413902 486689 86.4 S15

20 44.98 244.1248 C3-Pyrene/Fluoranthene C 19 H 16 12129466 2184668 203.7 21 45.13 228.0936 Benzo[a]anthracene 4831269 1107616 77.1 22 45.74 228.0936 Chrysene 9268673 1810228 145.9 23 46.02 246.1403 C5-Phenanthrene/Anthracene, one double bond C 19 H 18 3546529 638448 36.6 24 46.19 228.0935 Chrysene isomers (hypothetic) 3957840 925184 63.2 25 48.61 242.1093 Methylchrysene/Methylbenzo[a]anthracene 3587524 814440 100.3 26 48.79 242.1104 Methylchrysene/Methylbenzo[a]anthracene 22446558 4014567 144.8 27 49.01 242.1098 Methylchrysene/Methylbenzo[a]anthracene 16012979 2842546 296.8 28 49.18 242.1095 Methylchrysene/Methylbenzo[a]anthracene 1808789 413664 41.9 29 49.57 242.1101 Methylchrysene/Methylbenzo[a]anthracene 26296038 3738601 315.6 30 49.83 242.1095 Methylchrysene/Methylbenzo[a]anthracene 2074286 420295 51.4 31 50 242.1096 Methylchrysene/Methylbenzo[a]anthracene 9161719 1767170 166.3 32 50.18 242.1094 Methylchrysene/Methylbenzo[a]anthracene 1758807 365402 43.9 33 50.23 254.1094 C2-226 PAH C 20 H 14 12827453 2226509 193.4 34 50.52 240.0937 C1-226 PAH C 19 H 12 2341744 484410 54.2 35 50.73 242.1095 Methylchrysene/Methylbenzo[a]anthracene 26226396 4773642 240.3 36 51.16 254.1092 C2-226 PAH C 20 H 14 2628743 537318 49.6 37 51.19 240.0937 C1-226 PAH C 19 H 12 10152894 1930459 160 38 51.47 256.1249 C2-Chrysen/Benzo[a]anthracene C 20 H 16 3097397 593084 51.4 39 51.85 254.1095 C2-226 PAH C 20 H 14 18397622 3260454 259.3 40 52.31 256.1251 C2-Chrysen/Benzo[a]anthracene C 20 H 16 21511598 2607466 202.8 41 52.65 256.125 C2-Chrysen/Benzo[a]anthracene C 20 H 16 1877610 365794 43.7 S16

42 52.89 256.1251 C2-Chrysen/Benzo[a]anthracene C 20 H 16 7109362 1126322 102.8 43 53.19 256.1251 C2-Chrysen/Benzo[a]anthracene C 20 H 16 6030214 1001585 88.2 44 53.41 256.1252 C2-Chrysen/Benzo[a]anthracene C 20 H 16 3937216 606882 87 45 53.63 268.1251 C3-226 PAH C 21 H 16 2499502 498285 46.4 46 53.69 256.1252 C2-Chrysen/Benzo[a]anthracene C 20 H 16 4301335 747143 81.5 47 54.1 256.1251 C2-Chrysen/Benzo[a]anthracene C 20 H 16 5427942 843399 109.6 48 54.31 254.1095 C2-226 PAH C 20 H 14 4291103 557148 47.8 49 54.82 254.1095 C2-226 PAH C 20 H 14 4195685 817452 58.2 50 54.89 256.1252 C2-Chrysen/Benzo[a]anthracene C 20 H 16 8181162 1430636 102.9 51 55.16 256.1251 C2-Chrysen/Benzo[a]anthracene C 20 H 16 1871760 357018 47 52 55.43 268.1252 C3-226 PAH C 21 H 16 8023985 1030871 77.8 53 56.71 252.0947 Benzo[b]fluoranthene 31410450 5081941 168.1 54 57.03 252.0943 Benzo[k]fluoranthene 25619508 3484799 299.9 55 57.12 268.0891 8863195 1329341 70.1 56 59.14 268.0888 3841226 493827 47.2 57 60.77 268.0886 3533547 503915 48 58 61.24 252.0945 Benzo[e]pyrene 40375216 4094493 338.3 59 61.94 252.0943 Benzo[a]pyrene 34418476 4074781 320.9 60 63.47 252.0938 Perylen 7182816 1178747 93.1 61 63.93 266.1095 C1-228 PAH C 21 H 14 6538039 1044518 86.8 62 64.73 266.1095 C1-228 PAH C 21 H 14 5844461 956731 78.6 63 65.47 266.1096 C1-228 PAH C 21 H 14 6077388 882196 82.5 S17

64 72.13 278.1094 Dibenzoanthracene 5661685 1102572 90 65 73.17 278.1094 Dibenzoanthracene 3497836 730921 56.9 66 73.48 278.1093 Dibenzoanthracene 3330069 456114 43.7 67 74.43 278.1095 Dibenzoanthracene 4010815 799486 63.4 68 74.88 278.1095 Dibenzoanthracene 8382295 1644140 135.9 Table S7: Detected and confirmed (bold) PACs in fraction F4 of extracts of CNG-U sediment as detected by means of gas chromatographyatmospheric pressure laser ionization-time-of-flight-mass spectrometry (GC-APLI-MS). Peak numbers refer to the peaks labeled in Figure S5. No. RT (min) Max. m/z Compound (possible) sum formula Area Intensity (cps) S/N ratio 1 7.56 209.1892 241101 63456 282.1 2 21.5 172.1332 C 9 H 18 NO 2 303318 61764 336.1 3 27.38 192.0928 C1-Phenanthrene/Anthracene C 15 H 12 257866 62050 616.4 4 30.39 206.1085 C2-Phenanthrene/Anthracene C 16 H 14 511928 83452 644.6 5 30.72 206.1084 C2-Phenanthrene/Anthracene C 16 H 14 305649 67225 564.4 6 33.04 148.0236 243806 62444 251.6 7 40.39 262.1726 C6-Phenanthrene/Anthracene C 20 H 22 419093 90061 712 8 40.71 262.1725 C6-Phenanthrene/Anthracene C 20 H 22 452569 75822 486.5 9 42.15 262.1723 C6-Phenanthrene/Anthracene C 20 H 22 403562 93557 696.7 10 42.29 149.0233 C 8 H 5 O 3 636763 132382 1073.6 11 42.33 262.1722 C6-Phenanthrene/Anthracene C 20 H 22 387161 95361 75.8 12 42.45 262.1722 C6-Phenanthrene/Anthracene C 20 H 22 221598 63512 953.8 S18

13 42.78 276.1875 C7-Phenanthrene/Anthracene C 21 H 24 707083 63745 596.8 14 42.9 260.1199 C 19 H 16 O 322572 72685 65.8 15 42.95 262.1718 C6-Phenanthrene/Anthracene C 20 H22 503869 80268 432.5 16 43.02 260.12 C 19 H 16 1053407 176985 959.7 17 43.29 234.9953 C 12 H 7 C l2 N 346885 86842 188.8 18 43.53 260.1198 C 19 H 16 O 783251 151926 67.4 19 43.75 262.1714 C 20 H 22 594574 144794 638 20 43.77 260.1196 C 19 H 16 O 757560 118820 60 21 43.83 274.1714 276936 65751 132.2 22 43.94 231.1167 C 18 H 15 242368 62104 276.3 23 44.05 260.1195 C 19 H 16 O 256107 62904 83 24 44.13 260.1558 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 496332 60923 182 25 44.19 276.1872 C 21 H 24 395181 85309 96.4 26 44.26 260.1195 C 19 H 16 O 282313 79146 178 27 44.41 231.1169 C 18 H 15 2472696 601980 3945.9 28 44.57 260.156 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 1249581 272581 630.8 29 44.66 274.1715 708979 117928 1045.2 30 44.78 260.1194 315087 63348 49.2 31 44.91 240.1689 Benzo[a]anthracene-D10 3767221 875936 6569.1 32 44.95 239.1624 585769 122953 319.8 33 45.02 245.1325 1669492 378109 2940.4 34 45.12 260.156 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 1497613 290760 2480.9 S19

35 45.25 245.1323 363412 75823 586.4 36 45.35 260.1558 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 452614 116296 1205.8 37 45.47 240.1688 Chrysene D10 6166041 1492204 10899.5 38 45.56 239.1621 275612 64484 125.7 39 45.72 228.0933 Chrysene 221980 63256 563.2 40 45.8 260.1557 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 717133 140879 73.4 41 45.89 245.1326 3409909 764634 5822.5 42 46.02 260.1559 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 365917 79240 994.9 43 46.05 274.1716 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 397609 89154 341.6 44 46.14 231.1168 3164758 675300 5033 45 46.18 274.1714 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 631492 154768 459.7 46 46.34 260.1557 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 400853 69941 143.4 47 46.52 272.1558 C5-Fluoranthene/Pyrene C 21 H 20 402472 65321 295.8 48 46.65 260.1556 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 392942 79185 546.9 49 46.67 274.1715 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 321923 70436 1349.4 50 46.79 274.1717 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 321174 83825 1555.5 51 46.82 231.1168 837620 169833 1060.6 52 46.92 258.1404 C4-Fluoranthene/Pyrene C 20 H 18 1458179 265138 1748.9 53 47.05 260.156 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 872814 112676 127.1 54 47.16 245.1324 553412 105912 203.8 55 47.31 231.1168 987227 201745 1212.2 56 47.35 258.1404 C4-Fluoranthene/Pyrene C 20 H 18 1378098 203342 298.8 S20

57 47.65 274.172 11082270 2190489 17202 58 47.72 245.1326 1531999 288411 1642.7 59 47.81 258.1404 C4-Fluoranthene/Pyrene C 20 H 18 956941 147200 743.6 60 48.01 245.1325 363191 70765 291.6 61 48.11 274.1724 22412352 3647467 30571.4 62 48.23 260.1563 1687817 328317 2700.1 63 48.38 231.117 456571 86121 397.9 64 48.71 258.1408 C4-Fluoranthene/Pyrene C 20 H 18 2376955 361138 2757.2 65 48.8 245.1328 2822428 545252 3290.7 66 48.94 274.1718 399379 86021 1410.1 67 49.1 258.1406 C4-Fluoranthene/Pyrene C 20 H 18 729243 124983 530.1 68 49.33 262.0811 597927 105427 476.6 69 49.68 274.1718 411877 67878 152.2 70 49.75 276.1873 C7-Phenanthrene/Anthracene C 21 H 24 411868 70817 435.6 71 49.81 258.1405 C4-Fluoranthene/Pyrene C 20 H 18 930139 145281 967.5 72 49.88 260.1561 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 953027 155764 1037.2 73 50.06 260.1561 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 453977 81372 704.5 74 50.13 258.1406 C4-Fluoranthene/Pyrene C 20 H 18 1153110 200829 1195.8 75 50.18 272.1561 C5-Fluoranthene/Pyrene C 21 H 20 380506 61498 181.4 76 50.23 274.1718 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 299430 61579 261.8 77 50.48 258.1406 C4-Fluoranthene/Pyrene C 20 H 18 905103 145869 814.1 78 50.6 274.1719 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 673797 116972 1279.9 S21

79 51.1 260.156 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 615258 83502 222.5 80 51.38 274.1718 C7-Phenanthrene/Anthracene, one double bond C 21 H 22 1123744 177953 877.8 81 51.56 259.1484 1150238 214978 1385.3 82 51.63 272.1563 C5-Fluoranthene/Pyrene C 21 H 20 496031 79988 127.9 83 51.77 258.1406 C4-Fluoranthene/Pyrene C 20 H 18 706945 120954 398.4 84 51.86 258.1407 C4-Fluoranthene/Pyrene C 20 H 18 437966 91408 449.2 85 51.97 274.1719 C7-Phenanthrene/Anthracene, one double bond C21H22 414420 78394 838.6 86 52.03 260.1561 C6-Phenanthrene/Anthracene, one double bond C 20 H 20 366157 66887 350.6 87 52.79 258.1405 C4-Fluoranthene/Pyrene C 20 H 18 632513 110325 110.4 88 52.87 258.1405 C4-Fluoranthene/Pyrene C 20 H 18 659927 103709 57.5 89 53.23 258.1405 C4-Fluoranthene/Pyrene C 20 H 18 406078 70682 64.8 90 53.53 272.1562 C5-Fluoranthene/Pyrene C 21 H 20 643641 84310 784.2 91 53.6 276.0968 C 19 H 16 S 375395 60642 397.3 92 53.86 256.1249 C2-228 PAH C 20 H 16 470237 86843 638.3 93 53.95 276.0968 C 19 H 16 S 321241 64849 186.1 94 54.28 272.1562 C5-Fluoranthene/Pyrene C 21 H 20 887001 147542 329.1 95 54.48 256.1248 C2-228 PAH C 20 H 16 590310 78133 60.6 96 54.84 256.1249 C2-228 PAH C 20 H 16 799754 130374 727.4 97 55.48 272.1559 C5-Fluoranthene/Pyrene C 21 H 20 951977 69244 355.1 98 55.99 270.1401 C3-228 PAH C 21 H 18 535042 65511 165.4 99 57.85 256.1245 C2-228 PAH C 20 H 16 582363 71395 83.8 100 60.2 270.141 C3-228 PAH C 21 H 18 519834 60362 90.6 S22

101 61.24 282.105 827793 110639 90.6 102 62.37 270.1417 C3-228 PAH C 21 H 18 936422 84322 438.6 103 62.47 270.1417 C3-228 PAH C 21 H 18 619023 87408 618 104 63.62 282.1055 C 21 H 14 O 606790 61427 331.7 105 63.89 270.1421 C3-228 PAH C 21 H 18 775932 126541 961.9 106 64.44 282.1056 C 21 H 14 O 773522 94543 619.5 107 65.77 280.1261 C2-252 PAH C 22 H 16 439931 63583 400.6 108 66.48 266.1101 C1-252 PAH C 21 H 14 539709 76304 476.1 109 66.84 284.1569 C4-228 PAH C 22 H 20 347292 63066 293.8 110 67.17 280.1255 C2-252 PAH C 22 H 16 468651 62692 207.1 111 67.59 266.1097 475073 63483 177.1 112 68.44 280.1254 C2-252 PAH C 22 H 16 665959 133505 1086.7 113 68.62 280.1253 C2-252 PAH C 22 H 16 522210 95020 82.7 114 68.73 280.1253 C2-252 PAH C 22 H 16 1191008 143687 71.4 115 69.65 284.0657 C 20 H 12 S 345101 60049 365.6 116 69.7 280.125 C2-252 PAH C 22 H 16 698175 131616 1079.8 117 70.14 280.1251 C2-252 PAH C 22 H 16 526239 101406 1165.3 118 70.22 284.1562 C4-228 PAH C 22 H 20 609236 113273 755.6 119 70.55 280.1251 C2-252 PAH C 22 H 16 820985 140183 155.9 120 70.73 280.1249 C2-252 PAH C 22 H 16 591105 72608 290.6 121 71.04 278.109 Dibenzoanthracene C 22 H 14 336583 69718 219.1 122 71.57 310.1718 C 24 H 22 771720 135023 556.7 S23

123 71.66 280.1248 C2-252 PAH C 22 H 16 348959 64714 497 124 71.85 294.1405 C3-252 PAH C 23 H 18 337662 66214 202 125 73.79 292.0882 C 22 H 12 O 346061 70673 444.6 126 75.49 276.0937 Benzo[g,h,i]perylene 7195340 1433750 11349.8 127 76.37 292.0883 C 22 H 12 O 781866 134158 1046.8 128 76.59 276.0933 C 22 H 12 367186 70485 497.4 129 76.7 292.1245 C1-Dibenzoanthracene C 23 H 16 762494 62034 66.3 130 77.52 292.1248 C1-Dibenzoanthracene C 23 H 16 786943 111180 888.3 131 77.99 292.1247 C1-Dibenzoanthracene C 23 H 16 989103 183362 912.9 132 78.91 292.1246 C1-Dibenzoanthracene C 23 H 16 572822 78003 545.8 133 79.02 290.109 C1-Benzo[ghi]perylene C 23 H 14 578133 104292 835.1 134 80.81 304.1244 C2-Benzo[ghi]perylene C 24 H 16 268746 63186 148 135 81.17 306.14 C2-Dibenzoanthracene C 24 H 18 474420 66085 1340.3 136 81.31 306.1402 C2-Dibenzoanthracene C 24 H 18 747909 128685 1015.5 137 82.29 304.1239 C2-Benzo[ghi]perylene C 24 H 16 429995 61187 79.7 138 82.37 302.1088 Dibenzopyrene 661510 118433 903.6 139 82.55 302.1088 Dibenzopyrene 379185 64606 493.8 140 82.99 302.1092 Dibenzopyrene 3751711 592233 4630.7 141 84.21 318.1039 567889 63091 58.1 142 84.65 302.1093 Dibenzopyrene 715421 107220 809 143 85.31 302.1098 Dibenzopyrene 1449532 233051 1754.7 144 85.77 302.11 Dibenzopyrene 2390314 307887 2347.4 S24

145 87.15 316.1257 C1-Dibenzopyrene C 25 H 16 696968 82519 244 146 92.52 328.1263 Hexacene isomers C 26 H 16 677615 61479 105.6 147 92.86 328.1264 Hexacene isomers C 26 H 16 603556 75129 64.9 148 93.76 328.1263 Hexacene isomers C 26 H 16 425553 82449 789.7 S25

Figure S6: Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Ehrenbreitstein. Rhine. Germany); 10 µl of a 1 g SEQ ml -1 extract were fractionated. No significant differences between fractions and control were found (one-way ANOVA with Holm-Sidak s post-hoc test. p > 0.05). Shaded areas indicate typical retention time ranges of the indicated substance classes. Figure S7: Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Zollelbe. Elbe. Germany); 10 µl of a 1 g SEQ ml -1 extract were fractionated. Asterisks denote significant differences between fractions and control (one-way ANOVA with Holm-Sidak s post-hoc test. p 0.05). Shaded areas indicate typical retention time ranges of the indicated substance classes. S26

References 1. Floehr, T.; Scholz-Starke, B.; Xiao, H.; Koch, J.; Wu, L.; Hou, J.; Wolf, A.; Bergmann, A.; Bluhm, K.; Yuan, X.; Roß-Nickoll, M.; Schäffer, A.; Hollert, H., Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish. Journal of Environmental Sciences 2015, 38, 63-82. 2. Eichbaum, K.; Brinkmann, M.; Nuesser, L.; Buchinger, S.; Reifferscheid, G.; Codling, G.; Jones, P.; Giesy, J. P.; Hecker, M.; Hollert, H., Bioanalytical and instrumental screening of the uptake of sediment-borne, dioxin-like compounds in roach (Rutilus rutilus). Environmental Science and Pollution Research 2016, 1-15. S27