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Supplementary file to Collet et al. 2022: Changes in serum testosterone and adrenal androgen levels in transgender women with and without gonadectomy. This supplemenary file contains: 1) Method comparison between the endocrine laboratories of the UZ Gent and Amsterdam UMC for total testosterone, androstenedione and SHBG. 2) Supplementary analyses

This are supplementary files 1-4 as described in the paper: Early decline of androgen levels in healthy adult men: an effect of aging per se? A prospective cohort study.Supplementary file 1: Exclusion Criteria.Supplementary file 2: Reproducibility and stability of serum testosterone concentrations.Supplementary file 3: Statistical Analyses.Supplementary file 4: Baseline Associations.

Presence of endocrine disrupting or active chemicals in waterways is an ever growing global problem as evidenced by the WFD (2013/39/EU) who prioritized some model estrogenic chemicals into their watch list. Analyzing these substances typically involves separation chromatography coupled to mass detection at high cost per sample. However, performing a first screening of water samples using in vitro assays that detect endocrine selective ligands can help prioritize monitoring sites at lower cost. We therefore studied a highly impacted and urbanized river (Zenne River, Brussels, Belgium) that is composed of up to 50% in WWTP effluent downstream. We collected river water and influent and effluent of 2 major WWTPs (North and South) discharging to the river and a hospital effluent in order to gain more knowledge on their fate upon reaching this urban river. To accomplish this we collected water samples on a monthly basis during 1 year, performed SPE clean-up on the dissolved water phase, and analyzed these extracts using the BG1Luc4E2 in vitro transactivational bioassay to detect endocrine agonist activity. Concentrations of activity (BEQs) are reported as mass E2-equivalent concentrations. One of the most interesting features observed is that activity levels in the river tend to increase along the flow of the river, ranging from 1.34ng E2-eq./L to 3.80ng E2-eq./L at the most downstream site. Effluent levels of the two WWTPs (1.47 and 2.55ng E2-eq./L) displayed similar activity, but are able to drive activity variations depending on the river flow and their relative contribution there to. Influent values of the WWTPs and hospital ranged 1 to 2 orders of magnitude higher and pose real concern when untreated wastewaters are released into the river during combined sewer overflow events, which occur frequently in this water system and urban area. Furthermore, by implementing this monitoring program we obtained (annual averaged) in vitro activity results that surpass the limits proposed by the EU WFD (0.4ng/L for E2 and 0.035ng/L for EE2). Keeping in mind there is no clear-cut extrapolation or correlation between these effect-directed analyses and the chemically based limits, our results indicate this river system is highly impacted with endocrine activity and the responsible agents need characterization.

Endocrine disrupting chemicals (EDCs) are gaining in worldwide attention due to their omnipresence, wide range in chemical properties and possible effects on wildlife populations. Problems with these EDCs are their continuous release into the environment (industrial, domestic, inefficient removal). For this reason, the Water Framework Directive (amended in 2013/39/EU) established a priority list of 33 new and 8 old pollutants and also placed 15 compounds (including estrogens) onto a watch list. Rather than looking solely for concentrations of targeted and well-known EDCs, this project strives to combine bio-analytical and chemo-analytical data on the Zenne river crossing Brussels and hospital effluents (a potential major source for EDCs due to high consumption of pharmaceuticals and personal care products) for the first time. Bioassays allow scientists to use in vitro receptor models to assess endocrine activity by comparing the mixture or cocktail effect originating from the sample extract. Samples areextracted using Oasis HLB SPE columns and analyzed with human ovarian adenocarcinoma (BG1) cells transfected with a luciferase reporter gene (BG1Luc4E2 CALUX assay). Over a 12 month sampling period, bioassay values ranged from 0.68 to 16.53ng EEQ/L for the Zenne river water and from 67.40 to 231ng EEQ/L for hospital effluent. WWTP effluent values ranged from 0.96 - 6.62ng EEQ/L and influent levels from 17.6 - 88.8ng EEQ/L. Results indicate that effluent values are high post-discharge of human activities, EDCs are only partially removed by processes in WWTPs, and that effluents can contribute significantly to EDC loads in the Zenne river.

Endocrine disrupting chemicals (EDCs) are gaining in worldwide attention due to their omnipresence, wide range in chemical properties and possible effects on wildlife populations. Problems with these EDCs are their continuous release into the environment (industrial, domestic, inefficient removal). For this reason, the Water Framework Directive (WFD, amended in 2013/39/EU) established a priority list of 33 new and 8 previously regulated chemical pollutants and, in addition, 15 compounds were also placed onto a watch list containing estrogen compounds (estradiol, ethinylestradiol). Octyl-, nonylphenol, DEHP, among others have shown to exhibit endocrine disrupting potential. Rather than looking solely for concentrations of targeted and well-known EDCs, this project strives to combine bio-analytical and chemo-analytical data on the Zenne river crossing Brussels and hospital effluents (a potential major source for EDCs due to high consumption of pharmaceuticals and personal care products) for the first time. Bioassays allow scientists to use in vitro receptor models to assess endocrine activity by comparing the mixture or cocktail effect originating from the sample extract. Added value is that this technique picks up effects from compounds that are not routinely monitored in targeted chemo-analytical methods and can detect them at low amounts (pg/L EEQ, Estrogen EQuivalence). The bioassay used in this study is the CALUX (Chemically activated luciferase gene expression) bioassay using a luciferase reporter gene as part of BG1luc4E2 cells (human ovarian adenocarcinoma). Responses are time, dose, and ligand specific using 17β-estradiol (the natural female hormone) as reference compound. Water samples were taken as grab samples over various locations upstream and downstream of the Zenne River and in a hospital nearby; 24h composite samples were collected in Brussels WWTP South (in- and effluent). Samples were filtered and extracted using Oasis HLB SPE columns. Values ranged from 0.77 to 1.85ng EEQ/L for the Zenne river water, 98ng EEQ/L for hospital effluent and, respectively, 52.3 and 1.2ng EEQ/L for WWTP influent and effluent. For comparison reasons, commercial drinking waters amount to 0.004-0.070 ng EEQ/L. These results indicate that effluent values are high post-discharge of human activities, EDCs are only partially removed by processes in WWTPs, and that the effluents contribute significantly to EDC loads in the Zenne river.

Endocrine disrupting chemicals (EDCs) are gaining in worldwide attention due to their omnipresence, wide range in chemical properties and possible effects on wildlife populations. Problems surrounding these EDCs are their continuous release into the environment through industrial, domestic and hospital effluents and inefficient removal by wastewater treatment plants (WWTPs). For this reason, the Water Framework Directive (WFD, amended in 2013/39/EU) established a priority list of 33 new and 8 previously regulated chemical pollutants, some of which have shown to exhibit endocrine disrupting potential (octyl-, nonylphenol, DEHP, …). Additionally, 15 compounds were also placed onto a watch list containing estrogen compounds (estradiol, ethinylestradiol). Rather than looking solely for concentrations of targeted and well-known EDCs, this project strives to combine bio-analytical and chemo-analytical data on the Zenne river crossing Brussels and hospital effluents (a potential major source for EDCs due to highconsumption of pharmaceuticals and personal care products) for the first time. Bioassays allow scientists to use in vitro receptor models to assess endocrine activity by comparing the mixture or cocktail effect originating from the sample extract. Added value is that this technique picks up effects from compounds that are not routinely monitored in targeted chemo-analytical methods and can detect them at low amounts (pg/L EEQ, Estrogen EQuivalence). The bioassay used in this study is the CALUX (Chemically activated luciferase gene expression) bioassay using a luciferase reporter gene as part of BG1luc4E2 cells (human ovarian adenocarcinoma). Responses are time, dose, and ligand specific using 17β-estradiol (the natural female hormone) as reference compound. Water samples were taken as grab samples over various locations upstream and downstream of the Zenne River and in a hospital nearby; 24h composite samples were collected in Brussels WWTP South (in- and effluent). Samples were filtered and extracted using Oasis HLB SPE columns. Values ranged from 0.77 to 1.85ng EEQ/L for the Zenne river water, 98ng EEQ/L for hospital effluent and, respectively, 52.3 and 1.2ng EEQ/L for WWTP influent and effluent. For comparison reasons, commercial drinking waters amount to 0.004-0.070 ng EEQ/L. These results indicate that effluent values are high post-discharge of human activities, EDCs are only partially removed by processes in WWTPs, and that the effluents contribute significantly to EDC loads in the Zenne river. Acknowledgements INNOVIRIS in financing the Prospective Research For Brussels project. The CALUX bioassay BG1Luc4E2 cell line was developed with funding from the National Institute of Environmental Health Sciences Superfund Research grant (ES04699) to Prof. M.S. Denison (UC Davis).