04-09-2018 ENDOCRINE DISRUPTING CHEMICALS and ENDOCRINE POTENCY of WASTEWATER measured by IN VIVO and IN VITRO ASSAYS K. Ole Kusk1, Hanne Frederiksen2, Eva C. Bonefeld-Jørgensen4, Anna-Maria Andersson2, Anne E. Lykkesfeldt5, Christine Nellemann3, Kamilla M.S. Hansen1, Tanja Krüger4, Manhai Long4, Camilla Taxvig3, Henrik R. Andersen1 1Dept. Environmental Engineering, Technical University of Denmark 2Dept. Growth and Reproduction, Copenhagen University Hospital, Denmark 3National Food Institute, Dept. of Toxicology and Risk Assessment, Technical University of Denmark 4Cellular & Molecular Toxicology, Centre of Arctic Environmental Medicine, Institute of Public Health, Aarhus University, Denmark 5Dept. Tumor Endocrinology, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Corresponding author: kok@env.dtu.dk We have a lot of authors since each is only measuring with a few methods and everybody has a boss who need to be on the paper too. I am making this presentation on behaf of Ole who has been principial investigator but had to stay home and take care of the teaching while the group is enjoying the sun here with you. DanEd

04-09-2018 Aim of the project The question: Does wastewater cause other hormonal disrupting effects we need to consider in treatment evaluation besides estrogen agonists (– probably) Evaluate presence of different type of hormonal disrupting effects - 2x Influent to STPs - 2x Effluent from STPs Wastewater inlet Wastewater outlet We participate in a project about endocrine disruptors. Our task is to

Content Sampling and pre-treatment Chemical analyzes 04-09-2018 Content Sampling and pre-treatment Chemical analyzes Ecotoxicological test Bioassays Conclusions

1- Sampling and pre-treatment Influent and effluents from two sewage treatment plants (STPs) were sampled Lynetten STP (530 000 PE) Usserød STP (25 000 PE) Source Domestic, small industries Domestic, hospital Recipient Sea Small stream Total Nitrogen <15 ppm <8 ppm COD 25-75 ppm 10-30 ppm

1- Sampling and pre-treatment 04-09-2018 1- Sampling and pre-treatment Sample preparation Grab samples Influent Effluent pH → 3 pH → 3 Filter 500 ml subsamples 1000 ml subsamples SPE Dry. Freeze -18 °C

Treatment of extracts 0.5 or 1.0 L Chemical analysis Bioassay 04-09-2018 1- Sampling and pre-treatment SPE are distributed to laboratories Organic solvent Organic solvent 0.5 or 1.0 L Bioassay Chemical analysis 50-250 µL (concentrated samples)

Chemical analyses (Agilent triplequad) (Varian GC Iontrap) 04-09-2018 2- Chemical analyses Chemical analyses LC-MS/MS GC MS-MS (Agilent triplequad) (Varian GC Iontrap) 13 phthalate metabolites 3 industrial phenols 5 parabens 6 sunscreen chemicals 3 steroid estrogens

04-09-2018 2- Chemical analyses Selected estrogenic compounds in effluent from two Danish sewage treatment plants, Usserød and Lynetten, quantified by GC-MS-MS Concentrations in ng/L LOQ* Usserød effluent Lynetten Industrial phenols Octylphenol 2.5 16 50 isoNonylphenol 14 27 Bisphenol A 60 172 Sunscreen chemicals Homosalate 25 N.D** Benzophenone-3 158 81 Benzophenone-7 4 2 3-(4-Methylbenzyliden)camphor 7.5 N.D. Octyl methoxycinnamate 23 12 Octyl dimethylaminobenzoate Steroid estrogens Estrone 1 < 1 17-β-Estradiol Ethinyl estradiol *LOQ Limit of quantification; ** N.D. not detected;

04-09-2018 2- Chemical analyses Phthalate metabolites and parabens in influent and effluent from two Danish sewage treatment plants, Usserød and Lynetten, quantified by LC-MS/MS LOD* Usserød STP Lynetten STP Concentrations in ng/L influent effluent Mono-ethyl phthalate 1.6 751 2.5 1814 < LOD Mono-butyl phthalate 8.4 1970 46 2996 129 Mono-3-carboxypropyl phthalate 3.7 134 7.5 110 63 Mono-benzyl phthalate 2.3 1101 404 Mono(2-ethylhexyl) phthalate 3.0 383 37 891 52 Mono(2-ethyl-5-hydroxyhexyl) phthalate 83 245 Mono(2-ethyl-5-oxyhexyl) phthalate 1.5 53 126 Mono(2-ethyl-5-carboxyhexyl) phthalate 1.0 94 251 Mono-octyl phthalate 3.4 9.1 Mono-iso-nonyl phthalate 4.0 28 Mono-iso-nonyl phthalate (one hydroxy group) 30 58 Mono-iso-nonyl phthalate (one keto group) 16 34 Mono-iso-nonyl phthalate (one carboxy group) 2.4 57 119 Methyl paraben 2.8 17462 16322 262 Ethyl paraben 1.9 9193 10519 290 Propyl paraben 12105 50 18631 231 Butyl paraben 1.2 4483 4.6 5641 550 Benzyl paraben 0.9 14 12

Bioassays Steroid synthesis (H295R assay ) ER activation (YES assay) 04-09-2018 3- Bioassays Bioassays Steroid synthesis (H295R assay ) ER activation (YES assay) Cell proliferation (MCF-7 cell assay) Expression of the estrogen inducible progesterone receptor (MCF-7 cell assay) Transactivation of the ER (MCF-7 stably transfected MVLN cells) Androgenic or anti-androgenic (AR) transactivation assay (Chinese Hamster Ovary CHO cells) Thyroid hormone (TH) cell proliferation (GH3 cell proliferation (T-screen) Dioxin-like compounds assayed with transfected mouse heptoma cells (Hepa1.12cR carrying the AhR-luciferase reporter gene)

H295R Assay Steroid synthesis 04-09-2018 3- Bioassays H295R Assay Steroid synthesis Testosterone 500 1000 1500 2000 2500 3000 3500 Usserød Lynetten ng hormone/L SC 10 000 20 000 mL wastewater/L Influent Effluent * SC: Solvent Control

H295R Assay Steroid synthesis 04-09-2018 3- Bioassays H295R Assay Steroid synthesis Estradiol 300 * Usserød Lynetten * 250 200 ng hormone/L 150 100 * 50 SC 10 000 20 000 mL wastewater/L Influent Effluent SC: Solvent Control

YES assay ER activation 04-09-2018 3- Bioassays YES assay ER activation

Wastewater concentration (ml sample/L) 04-09-2018 MCF-7 cell assay (increased growth up to 100/2000 mL/L then cytotoxicity) 3- Bioassays Tox Wastewater concentration (ml sample/L)

GH3 cell proliferation Thyroid hormone effects 04-09-2018 3- Bioassays GH3 cell proliferation Thyroid hormone effects T - Screen Lynetten Usserød Lynetten Usserød Influent Influent Effluent Effluent 6 000 * * 5 000 *# # 4 000 GH3 cell proliferation/L # # * * 3 000 2 000 1 000 SC 625 1 250 2 500 5 000 10 000 SC 625 1 250 2 500 5 000 10 000 SC 1 250 2 500 5 000 10 000 20 000 SC 1 250 2 500 5 000 10 000 20 000 SC SC SC SC SC=Solvent Control Concentrations in: mL wastewater/L Values represent the mean ± SD of at least two independent assays performed in triple. * indicates a significant difference compared to the solvent control. # indicates cytotoxicity.

AhR-transactivity Dioxine like effects 04-09-2018 3- Bioassays AhR-transactivity Dioxine like effects SC 260 640 1 600 4 000 10 000 130 320 800 2 000 5 000 SC=Solvent Control Concentrations in: mL wastewater/L Effluent Usserød Lynetten Influent

Bioequivalents of hormones in assays 04-09-2018 3- Bioassays Bioequivalents of hormones in assays Assay Lynetten influent effluent Usserød Influent Bio-EEQ (ng E2/L water) YES  29 3.0 90% 26 2.7 ER transactivation 32 ± 0.3 2.5 ± 0.3 92% 31 ± 2.8 1.4 ± 0.2 96% Bio-TEQ (ng TCDD/L water) AhR transactivation 7.6± 1.6 3.0± 0.6 61% 7.0± 1.4 1.7± 0.4 76% Bio-T3EQ (ng T3/L water) GH3 proliferation 284 5.1 98% 5.8

04-09-2018 4- Ecotoxicity test Ecotoxicity test Early life-stage development test on a marine copepod Acartia tonsa

Early life-stage test with copepod 04-09-2018 4- Ecotoxicity test Early life-stage test with copepod Lynetten influent Lynetten effluent Usserød influent Usserød effluent EC10 110 (89-140) 770 (470-1250) 320 (200-530) > 2000 EC50 230 (210-260) > 1000 510 (450-590) Effect concentrations (EC) (mL wastewater/L) causing 10 and 50 % inhibition of the development of early life stages of Acartia tonsa

CONCLUSIONS Effects of wastewater extracts 04-09-2018 5- Conclusions CONCLUSIONS Effects of wastewater extracts All EDC assays responded to in- and effluents from both STPs Steroid synthesis and metabolism (Dose-response not good) E2 receptor activation E2 sensitive growth stimulation Androgen receptor activation AhR transactivation stimulated (presence of dioxine-like substances) Thyroid hormone like activity was increased during treatment (Agonist-antagonist mixture) Concentrations and effects of EDCs reduced but not eliminated in effluents. The ecological relevance of these findings is unknown – but active substances (EDCs) are discharged and potentially they may affect wildlife. Weak ecotoxic effect was observed in the effluent from one STP.

CONCLUSIONS Most relevant parameters for WW treatment Chemical analysis ? Industrial phenols (Bisphenol A, Nonylphenol). ? Steroid estrogen. + Sunscreen (BP-3, OMC). -/+ Paraben (butylparaben). - Phatalate ester metabolites. Cell based bioassays ? Testosterone & estradiol biosynthesis. (+ with fractionation). + Estrogen assays. YES or E-screen. + AhR (dioxin). Low treatment effectiveness. -/+ Thyroid hormone assay. Not so relevant with well treated WW. Ecotoxicity -- Copepod development. Low sensitivity. No effect in effluents. €.