Effect‐Directed Analysis of Progestogens and Glucocorticoids at Trace Concentrations in River Water

Abstract

Effect-based monitoring is increasingly applied to detect and-in conjunction with chemical analysis-to identify endocrine-disrupting compounds (EDCs) in the environment. Although this approach of effect-directed analysis has been successfully demonstrated for estrogenicity and androgenicity, data on progestogens and glucocorticoids driving endocrine disruption are quite limited. We investigated progestogenic and glucocorticoid activities in Danube River water receiving untreated wastewater from Novi Sad, Serbia. After a 2-step fractionation, all fractions were tested with reporter gene bioassays for agonistic and antagonistic hormonal responses at progestogenic and glucocorticoid hormone receptors as well as with target and nontarget analytical screening of active fractions by liquid chromatography-high-resolution mass spectrometry. Due to masking by cytotoxic mixture components, the effects could not be detected in the raw water extract but were unraveled only after fractionation. Target chemical screening of the fraction that was active in the progesterone receptor (PR) assay revealed that progesterone and megestrol acetate were predominant drivers of PR-mediated activity along with medroxyprogesterone, dihydrotestosterone, androsterone, and epiandrosterone. Hydrocortisone was detected at sub-ng/L concentration in the active fraction in the glucocorticoid receptor (GR) assay but could not explain a significant fraction of the observed GR activity. The present study indicates that effect-based monitoring is a powerful tool to detect EDCs in the aquatic environment but that fractionation may be required to avoid masking effects of mixture components. Future effect-directed analysis studies are required to better understand the occurrence of EDCs and masking compounds in different lipophilicity windows, to finally reduce fractionation requirements for monitoring to a smart clean-up. Environ

Fish biomarkers from a different perspective: evidence of adaptive strategy of Abramis brama (L.) to chemical stress

“…Levels of significance were *p < 0.05, **p < 0.01, and ***p < 0.001. Median values, interquartile ranges, minimum and maximum are given in Additional file 1: Table S5 pharmaceuticals, industrial chemicals, pesticides, and personal care products in the Danube River in this area, to activate toxicity pathways and disturb biochemical and physiological homeostasis in various cell lines, zebrafish embryos and fish [22,[42][43][44][45]. Still, the questions whether the biological effects observed in vitro can be regarded as an indication of probable ecological impact and weather the impact could be confirmed by adverse effects in resident biota, on individual and population/community level remain open.…”

Factors Affecting Sampling Strategies for Design of an Effects‐Directed Analysis for Endocrine‐Active Chemicals

“…Therefore, we felt it was important to assess these 2 types of bioassays in the EDA framework ( Figure 1). The 3 remaining studies employed fluorescence resonance energy transfer in cell-based Gene-BLAzer assays or vitellogenin messenger (m)RNA expression in primary rainbow trout hepatocytes (listed as "Other" bioassay in Table 1; Lavado et al 2009;Hashmi et al 2018Hashmi et al , 2020. Although the tg(CYP19a1b)-GFP zebrafish bioassay has been utilized in an EDA of EACs from sediment samples (Fetter et al 2014), we could not find any studies incorporating transgenic zebrafish in an EDA of EACs from water; nevertheless, we chose to include this bioassay in our EDA framework for comparative purposes against the other 2 selected bioassay types (yeast and LucTA).…”

“…Lastly, although the GeneBLAzer cell assays were not assessed in our framework, the 2 EDA studies that utilized these assays (listed as "Other" in Table 1) also identified several of the EACs listed in Table 3 as active components in fractionated and whole sample. For example, P4 was identified as one of the 2 primary progestogens responsible for activity in fractionated river water (Hashmi et al 2020). Similarly, E2 was identified as the main estrogen responsible for much of the activity in different fractions of the same river water, and testosterone partly explained androgenic activity in fractions in that same study (Hashmi et al 2018).…”

“…Effects-directed analysis (EDA) is a technique utilized to isolate and identify biologically active chemicals such as endocrine-active chemicals (EACs) from complex mixtures. The technique has been applied for EAC isolation from waters and sediments (Reineke et al 2002;Jenkins et al 2003;Furuichi et al 2004;Beck et al 2006;Fetter et al 2014;Jonker et al 2015;Hashmi et al 2018Hashmi et al , 2020Sonavane et al 2018;Zwart et al 2018). Not only can EDA guide the identification of the responsible EACs, but it can also help rule out candidate EACs not responsible for the observed biological activity or effect(s).…”

“…Not only can EDA guide the identification of the responsible EACs, but it can also help rule out candidate EACs not responsible for the observed biological activity or effect(s). The technique can be necessary when cytotoxic or interfering chemicals in the whole sample prevent accurate assessment of bioactivity (Hashmi et al 2020;Pochiraju et al 2020). This is accomplished through fractionation of the sample extract, using a bioassay to target "active" fractions (i.e., fractions that induce activity in a particular bioassay) for further subfractionation until the resulting active fractions are sufficiently clean to allow identification of the active components (Zwart et al 2018;Hashmi et al 2020).…”