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This is the first study on how a substance with anti-progestogenic activity affects amphibian reproduction. Mifepristone, a synthetic anti-progestin used in abortion pills, was chosen as model compound. African clawed frog (Xenopus laevis) females were exposed to four mifepristone concentrations (0.7, 9, 120, and 1380 ng∙L -1 ) for 30 days. A control group was also included. Egg-laying during the experiment was significantly less at the highest concentration. At the experiment's end, mifepristone-exposed and control females were randomly mated with sexually mature males. Breeding rate for females exposed to 1380 ng∙L -1 mifepristone was only 50 %. Histology revealed no significant changes in gonads, thyroid, or liver. Females exposed to 1380 ng∙L -1 mifepristone had lower estradiol levels in plasma, lower mRNA expression of lh and fsh in brain-pituitary complex, and p450scc in ovaries. In liver, mRNA level of npr was significantly increased in females exposed to 120 ng∙L -1 mifepristone. mRNA expression of mpr, erβ, dio2, and dio3 were upregulated in animals exposed to 9 ng∙L -1 and 120 ng∙L -1 mifepristone, whereas vtg expression was significantly downregulated in females exposed to 1380 ng∙L -1 mifepristone. All these findings show that exposure to compounds with anti-progestogenic activity affects the hypothalamus-pituitary-gonad axis and decreases reproductive success., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

One of the less studied in vitro biological activities in the aquatic environment are thyroid hormone receptor beta (TRβ)-mediated agonistic and antagonistic activities and transthyretin (TTR) binding activity. They were measured mostly using active sampling methods, but rarely found. It is unclear if these activities co-occur, and the drivers of the (anti-)TRβ activity are mostly unknown. Therefore, the main aim of the study was to determine (anti-)TRβ activities as well as transthyretin (TTR) binding activity in passive samplers from Czech surface waters in combination with the search for the effect drivers based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis by applying suspect screening. Passive samplers (polar organic chemical integrative samplers, POCIS) were deployed at twenty-one sites (all ends of watersheds and other important sites in Elbe River) in the Czech rivers. The (anti-)TRβ and TTR binding activity were measured using (anti-)TRβ-CALUX and TTR-TRβ-CALUX bioassays. Anti-TRβ activity was found at eight sites, and TTR binding activity co-occurred there at six of these sites. The co-occurrence of TRβ-mediated antagonistic activity and TTR binding indicate that they may have common effect drivers. No sample exhibited TRβ agonistic activity. The extract from the site Bílina River, the most burdened with anti-TRβ activity, was further successfully fractionated, and this activity was revealed in the fraction, where mid-polar compounds prevailed. However, the suspect LC-HRMS analysis did not reveal the chemical effect drivers. Our results showed that anti-TRβ activity can be found in surface waters by employing passive sampling and frequently co-occurs with TTR binding activity. Overall, the fractionation procedure and non-target data acquisition used in this study can serve as a basis for searching the effect drivers in future research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The occurrence, environmental risks and contribution of organic UV filters to detected (anti-)progestogenic activities were examined in samples of wastewater treatment plant influents and effluents, various surface waters and fish from the Czech Republic. Of the 20 targeted UV filters, 15 were detected in the WWTP influent samples, 11 in the effluents, and 13 in the surface water samples. Benzophenone-3, benzophenone-4, and phenyl benzimidazole sulfonic acid (PBSA) were found in all water samples. Octocrylene, UV-327 and 4-methylbenzylidene camphor exceeded the risk quotient of 1 at some sites. In the anti-progestogenic CALUX assay, 10 out of the 20 targeted UV filters were active. Anti-progestogenic activities reaching up to 7.7 ng/L, 3.8 ng/L, and 4.5 ng/L mifepristone equivalents were detected in influents, effluents, and surface waters, respectively. UV filters were responsible for up to 37 % of anti-progestogenic activities in influents. Anti-progestogenic activities were also measured in fish tissues from the control pond and Podroužek (pond with the highest number of detected UV filters) and ranged from 2.2 to 9.5 and 1.9 to 8.6 ng/g dw mifepristone equivalents, respectively. However, only benzophenone was found in fish, but it does not display anti-progestogenic activity and thus could not explain the observed activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Concerns are growing about adverse effects of progestins on biota, even at ultra-trace concentrations. The enrichment factor (EF) from extraction of analytes in environmental samples that is needed for sample pre-concentration can affect not only performance of the analytical method but also the matrix effect. Therefore, the present study aimed to assess the influence of high sample EF on performance of the high-performance liquid chromatography with atmospheric pressure chemical ionization and photoionization coupled with high-resolution mass spectrometry (HPLC-APCI/APPI-HRMS) method for analysis of progestins in waste water treatment plant (WWTP) effluents and surface waters and analysis of (anti-)progestogenic activities measured by (anti-)PR-CALUX bioassays. The results showed that HPLC-APCI/APPI-HRMS coupled with solid-phase extraction and a high EF (33,333 L water /L extract ) enabled the detection of more compounds compared to samples with lower sample EF (10,000 L water /L extract ). The matrix effect did not increase proportionally compared to lower EFs (10,000 and 16,666 L water /L extract ), and lower limits of quantification were achieved in WWTP effluents and surface waters. The results of bioassays have shown that relative EF of 25 L water /L bioassay appears high enough to detect progestogenic activity in treated waste water. Our study is one of the first to provide insights into sample pre-concentration in analysis of progestins and progestogenicity in aquatic environments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The objective of this study was to assess the effects of a model substance with anti-progestogenic activity on development of African clawed frog (Xenopus laevis) from tadpole to juvenile stage. Mifepristone, a synthetic progesterone receptor-blocking steroid hormone used in medicine as an abortifacient, was chosen as a model compound with anti-progestogenic activity. In the experiment, African clawed frog tadpoles were exposed to mifepristone at three concentrations (2, 21, and 215 ng L -1 ). A control group was exposed to dimethyl sulfoxide (DMSO; 0.001 %). The experiment started when tadpoles reached stages 47-48 according to Nieuwkoop and Faber (NF; 1994) and continued until stage NF 66, when metamorphosis was complete. Exposure to mifepristone had no significant effect on the rate of tadpole development, occurrence of morphological anomalies, weight, body length, or sex ratio. Mortality was within an acceptable range of 0-3.6 % throughout the test and did not differ among the groups. Histopathological examination of the gonads and thyroid gland revealed no significant changes. Therefore, we can conclude that mifepristone had no negative effect on development of the African clawed frog up to juvenile stage. Nevertheless, at the highest tested mifepristone concentration (215 ng L -1 ), gene expression analysis revealed up-regulation of mRNA expression of nuclear progesterone receptor (npr), membrane progesterone receptor (mpr), estrogen receptor beta (esrβ), and luteinizing hormone (lh) in the brain-pituitary complex of exposed frogs at stage NF 66. Higher mRNA expression of npr was also found in frogs exposed to 22 ng L -1 mifepristone compared to the solvent control. These findings confirmed the anti-progestogenic activity of mifepristone in frogs because the up-regulation of progesterone receptors occurs if progesterone availability in the body is reduced. All the observed changes in combination may have negative consequences for reproduction and reproductive behavior later in life., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20β-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hana Kocour Kroupova reports financial support was provided by Czech Science Foundation. Patrick Balaguer reports financial support was provided by National Agency for Food Environmental and Occupational Health and Safety., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Immunohistochemistry (IHC) is a laboratory method widely used to characterize tissue and cell origin, both in human and veterinary medicine. In fish, however, little is known about staining characteristics of most tissue types, and especially for less studied chondrostean fish. The aim of this study was to examine the specificity of various immunohistochemical markers in tissues of chondrostean and teleostean fish and to validate diagnostic tests. Sterlet (Acipenser ruthenus L.), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio L.) were examined. Markers were chosen as representatives of epithelial (cytokeratin AE1/AE3), mesenchymal (vimentin), neuroectodermal (S-100 protein), lymphoid (leukocyte common antigen, LCA) and endocrine (thyroglobulin, thyroxin) tissues and organs. Applied antibodies were of monoclonal or polyclonal mammalian origin and primarily intended for human medicine research or diagnostic application. No species differences were obvious while examining sterlet, shortnose sturgeon and carp. Cytokeratin AE1/AE3, vimentin, S-100 protein and thyroxin were positive on targeted tissues and structures. Leukocyte common antigen (LCA) and thyroglobulin were negative on targeted structures, however, and with clear cross-reactivity on non-targeted tissues (vascular wall, granulocytes). Conclusive results were obtained when using polyclonal antibodies with dilution adjusted to laboratory practice, while application of ready-to-use (RTU) kits with pre-diluted antibodies or monoclonal antibodies often showed conflicting or inconclusive results., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Bisphenols are increasingly recognized as environmental pollutants with endocrine-disrupting potential. Nonetheless, the study of environmental occurrence and some endocrine-disrupting activities of some bisphenols came widely into focus of research only recently. The aims of the present study were to: 1) determine the predominant bisphenols in Norwegian sewage sludge and sediment and in Czech surface waters, and 2) characterize the binding of bisphenols to a transport protein transthyretin (TTR) and their (anti-)thyroid, (anti-)progestagenic, and (anti-)androgenic activities. High-performance liquid chromatography with atmospheric pressure chemical ionization or photoionization coupled with high resolution mass spectrometry (HPLC-APCI/APPI-HRMS) and Chemically Activated LUciferase gene eXpression (CALUX) in vitro reporter gene bioassays were used to detect the target compounds and to determine endocrine-disrupting activities, respectively. Bisphenol A (BPA), 4,4'-bisphenol F (BPF), bisphenol S (BPS), and bisphenol E (BPE) were the most frequently found compounds in municipal sewage sludge. Furthermore, bisphenol TMC (BPTMC) and bisphenol AF (BPAF) frequently occurred in sediment and surface waters, respectively. BPA was the major contributor to Ʃ of bisphenols in Norwegian sewage sludge with exception of one sample where BPF predominated. We also monitored a few bisphenols in sediment but only BPTMC was found. BPA, BPAF and BPF were the dominant bisphenols in Czech surface waters. Some bisphenols have shown TTR binding potency (BPAF = BPF > BPA = BPE) and some have displayed the following endocrine-disrupting activities: anti-thyroid (BPAF), anti-progestagenic (BPTMC > BPA = BPAF), and anti-androgenic (BPAF > BPE > BPA > BPTMC > BPF > BPS). It is noteworthy that BPAF exhibited stronger or similarly potent endocrine-disrupting activities compared to BPA. Our results provide new insights into these less-studied endocrine-disrupting activities of environmentally relevant bisphenols and may be useful in prioritizing those compounds that deserve further attention in environmental monitoring and eco-toxicological research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Synthetic progestins are emerging contaminants of the aquatic environment with endocrine disrupting potential. The main aim of the present study was to investigate the effects of the synthetic progestins gestodene, and drospirenone on sex differentiation in common carp (Cyprinus carpio) by histological analysis. To gain insights into the mechanisms behind the observations from the in vivo experiment on sex differentiation, we analyzed expression of genes involved in hypothalamus-pituitary-gonad (HPG) and hypothalamus-pituitary-thyroid (HPT) axes, histology of hepatopancreas, and in vitro bioassays. Carp were continuously exposed to concentrations of 2 ng/L of single progestins (gestodene or drospirenone) or to their mixture at concentration 2 ng/L of each. The exposure started 24 h after fertilization of eggs and concluded 160 days post-hatching. Our results showed that exposure of common carp to a binary mixture of drospirenone and gestodene caused increased incidence of intersex (32%) when compared to clean water and solvent control groups (both 3%). Intersex most probably was induced by a combination of multiple modes of action of the studied substances, namely anti-gonadotropic activity, interference with androgen receptor, and potentially also with HPT axis or estrogen receptor., (Copyright © 2019 Elsevier Inc. All rights reserved.)

High rates of progestins consumption in the form of active ingredients in women's oral contraceptives and other hormonal preparations may lead to their increased concentrations in aquatic environments and subsequent harmful effect on fish reproduction. The objective of the present study was to assess the effect of etonogestrel, a third-generation synthetic progestin, on the reproductive behavior, fertility, gonads histology, and secondary sexual characteristics of male and female Endler's guppies (Poecilia wingei). Fish were subjected for 34 days to two concentrations of etonogestrel, including one possibly environmentally relevant (3.2 ng L -1 ) and one sublethal (320 ng L -1 ) concentration. A mating behavior study was subsequently conducted and revealed that the treatment with etonogestrel significantly reduced mating frequency in the exposed fish compared to controls. All the exposed females were unable to reproduce. In addition, female fish exposed to the highest level of etonogestrel were masculinized, as their anal fins and body coloration showed patterns similar to those of male fish. Etonogestrel-exposed females also had fewer developed oocytes. In conclusion, the low etonogestrel concentration (3.2 ng L -1 ) led to a reduction of mating activity in males without effect on their reproductive success, but it completely inhibited reproduction in females. Exposure to etonogestrel clearly has more severe consequences for females than males., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Synthetic musk compounds are extensively used in personal care and cosmetic products around the world. Because they are not completely removed in sewage treatment plants, they eventually end up in aquatic environments. The aim of this review was to summarize published information on effects of polycyclic musks on aquatic organisms and to discuss whether the experimental design of toxicological studies involving these substances could influence the results obtained. With the exception of one study run in a flow-through system, all published toxicological studies on synthetic polycyclic musks have been conducted in semi-static or even static systems. Based upon data in the literature and our own results, we conclude that in toxicological tests with semi-static set-ups, concentrations of polycyclic musks decrease with time between bath exchanges, and, as a result, tested organisms are not being exposed to stable concentrations but rather to concentration pulses. The duration and character of these pulses are influenced mainly by aeration of experimental baths, as polycyclic musks have a tendency to volatilize from water baths. Under semi-static conditions, tested organisms may be subjected to lower concentration of the tested substance for relatively long periods. Those levels may even fall below the limits of quantification. During these periods, some level of detoxification and/or elimination (depuration) of the toxicant may reduce toxic effect of the previous exposures. Consequently, toxicity of polycyclic musk substances for aquatic organisms obtained under these conditions may be underestimated. Based upon existing data in the literature, therefore, it is very difficult to correctly estimate risk of polycyclic musks to aquatic organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 μg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)