Characterization of vitellogenin concentration in male fathead minnow mucus compared to plasma, and liver mRNA.

The objective of this study was to characterize vitellogenin (VTG) protein in male fathead minnow (Pimephales promelas) mucus compared with more conventional measures in plasma and mRNA isolated from liver. To assess the intensity and duration of changes in mucus VTG concentrations, male fathead minnows were exposed to 17α-ethinylestradiol (EE2) for 7 days with a subsequent depuration period of 14 days. The experiment was conducted in a flow-through system to maintain a consistent concentration of EE2 at a nominal EC50 concentration of 2.5 ng/L and high concentration of 10 ng/L as a positive control. Mucus, plasma and liver were sampled at regular intervals throughout the study. Relative abundance of vtg mRNA increased after 2 days of exposure and returned to control levels after 4 days of depuration. VTG protein concentration displayed similar induction kinetics in both mucus and plasma, however, it was found to be significantly increased after 2 days of exposure using the mucus-based assays and 7 days with the plasma-based assay. Significantly elevated levels of VTG were detected by both assays throughout the 14-day depuration period. The elimination of the laborious plasma collection step in the mucus-based workflow allowed sampling of smaller organisms where blood volume is limiting. It also resulted in significant gains in workflow efficiency, decreasing sampling time without loss of performance.

Effects of two Bisphenol A replacement compounds, 1,7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane and Bisphenol AF, on development and mRNA expression in chicken embryos.

Concerns about the estrogenic properties of Bisphenol A (BPA) have led to increased efforts to find BPA replacements. 1,7-bis(4-Hydroxyphenylthio)-3,5-dioxaheptane (DD-70) and 4,4'-(hexafluoroisopropylidene) diphenol (bisphenol AF, BPAF) are two potential chemical substitutes for BPA; however, toxicity data for these chemicals in avian species are limited. To determine effects on avian embryonic viability, development, and hepatic mRNA expression at two distinct developmental periods (mid-incubation [day 11] and term [day 20]), two egg injection studies were performed. Test chemicals were injected into the air cell of unincubated, fertilized chicken eggs at concentrations ranging from 0-88.2 µg/g for DD-70 and 0-114 µg/g egg for BPAF. Embryonic concentrations of DD-70 and BPAF decreased at mid-incubation and term compared to injected concentrations suggesting embryonic metabolism. Exposure to DD-70 (40.9 and 88.2 µg/g) and BPAF (114 µg/g) significantly decreased embryonic viability at mid-incubation. Exposure to DD-70 (88.2 µg/g) decreased embryo mass and increased gallbladder mass, while 114 µg/g BPAF resulted in increased gallbladder mass in term embryos. Expression of hepatic genes related to xenobiotic metabolism, lipid homeostasis, and response to estrogen were altered at both developmental stages. Given the importance of identifying suitable BPA replacements, the present study provides novel, whole animal avian toxicological data for two replacement compounds, DD-70 and BPAF. DATA AVAILABILITY: Data, associated metadata, and calculation tools are available from the corresponding author (doug.crump@canada.ca).

Concentrations levels and effects of 17alpha-Ethinylestradiol in freshwater and marine waters and bivalves: A review.

Pharmaceutical drugs are contaminants of emerging concern and are amongst the most frequent in the aquatic environment. Even though a vast literature indicate that pharmaceuticals exert negative impacts towards aquatic organisms, mainly in vertebrates, there is still limited information regarding the effects of these drugs in freshwater and marine bivalves. Marine bivalves have a high ecological and socio-economic value and are considered good bioindicator species in ecotoxicology and risk assessment programs. Furthermore, another lacking point on these studies is the absence of bioconcentration data, with no clear relationship between the concentration of drugs on tissue and the biological effects. 17alpha-ethinylestradiol (EE2) is a synthetic hormone with high estrogenic potency that was added to the Watch List adopted by the European Commission stating the priority substances to be monitored. Thus, this review summarizes the current knowledge on the concentration levels and effects of EE2 on freshwater and marine bivalves. The inclusion in the Watch List, the presence in freshwater and marine systems, and the impact exerted on aquatic biota, even at trace concentrations, justify the review devoted to this pharmaceutical drug. Globally the available studies found that EE2 induces individual and sub-individual (e.g. tissue, cellular, biochemical and molecular levels of biological organization) impacts in bivalves. Essentially, this estrogenic compound, even in trace concentrations, was found to have accumulated in wild and laboratory exposed bivalves. The most common effects reported were changes on the reproductive function and energy metabolism. The studies used in this review support keeping the EE2 on the Watch List and highlight the need to increase the number of monitorization studies since clear negative effects were exerted on bivalves by this drug.

Astrocytes Mediate Protective Actions of Estrogenic Compounds after Traumatic Brain Injury.

Traumatic brain injury (TBI) is a serious public health problem. It may result in severe neurological disabilities and in a variety of cellular metabolic alterations for which available therapeutic strategies are limited. In the last decade, the use of estrogenic compounds, which activate protective mechanisms in astrocytes, has been explored as a potential experimental therapeutic approach. Previous works have suggested estradiol (E2) as a neuroprotective hormone that acts in the brain by binding to estrogen receptors (ERs). Several steroidal and nonsteroidal estrogenic compounds can imitate the effects of estradiol on ERs. These include hormonal estrogens, phytoestrogens and synthetic estrogens, such as selective ER modulators or tibolone. Current evidence of the role of astrocytes in mediating protective actions of estrogenic compounds after TBI is reviewed in this paper. We conclude that the use of estrogenic compounds to modulate astrocytic properties is a promising therapeutic approach for the treatment of TBI.

Sex-specific endocrine-disrupting effects of three halogenated chemicals in Japanese medaka.

Several halogenated chemicals are found in an array of products that can cause endocrine disruption. Human studies have shown that endocrine responses are sex specific, with females more likely to develop hypothyroidism and males more likely to have reproductive impairment. The objective of this study was to assess sex differences on thyroid and estrogenic effects after exposure of Japanese medaka (Oryzias latipes, SK2MC) to halogenated compounds. This strain is an excellent model for these studies as sex can be determined non-destructively a few hours postfertilization. Medaka embryos were exposed to sublethal concentrations of Tris(1,3-dichloro-2-propyl) phosphate (TDCPP, 0.019 mg/L), perfluorooctanoic acid (PFOA, 4.7 mg/L) and its next generation alternative, perfluorobutyric acid (PFBA, 137 mg/L). Methimazole (inhibits thyroid hormone synthesis) and the thyroid hormone triiodothyronine served as reference controls. Fish were exposed throughout embryo development until 10 days postfertilization. Females displayed significantly larger swim bladders (which are under thyroid hormone control) after exposure to all chemicals with the exception of triiodothyronine, which caused the opposite effect. Females exposed to TDCPP and PFOA had increased expression of vitellogenin and exposure to PFOA upregulated expression of multiple thyroid-related genes. Upregulation of estrogenic-regulated genes after exposure to TDCPP, PFOA and methimazole was only observed in males. Overall, our results suggest that females and males show an estrogenic response when exposed to these halogenated chemicals and that females appear more susceptible to thyroid-induced swim bladder dysfunction compared with males. These results further confirm the importance of considering sex effects when assessing the toxicity of endocrine-disrupting compounds.

Assessing wild fish exposure to ligands for sex steroid receptors from pulp and paper mill effluents in the Biobio River Basin, Central Chile.

Bioactive substances in the Biobio River Basin in Chile were examined by deploying Semi Permeable Membrane Devices (SPMDs) upstream and downstream of 4 pulp mill effluent discharges. Androgenic and estrogenic activity of SPMD extracts were then evaluated using in vitro fish sex steroid receptor binding assays. The results indicated the occurrence of estrogenic type compounds associated with one of the mill discharges. A significant correlation among the presence of these compounds, an increase in gonadosomatic index GSI and induction of hepatic EROD activity of two native fish species was observed. However, no significant presence of mature oocytes in female gonads was detected. Although EROD induction was observed in sites impacted by mill effluents, an increase of its activity occurred towards the downstream areas, suggesting other non-mill sources. More research is needed to understand the environmental changes in context of the new technological improvements in treatment systems to MBBR (Moving Bed Biofilm Reactor) recently implemented by the pulp mill industries.

Estrogenic effects associated with bisphenol a exposure in male zebrafish (Danio rerio) is associated with changes of endogenous 17ß-estradiol and gene specific DNA methylation levels.

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17ß-estradiol (E2), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500µg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1a's promoters were determined. Our results indicated that for the 500 and 1500µg/L treatment groups, VTG might be induced mainly by the elevated E2 levels; increases of E2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish.

Examining effects of a novel estrogenic perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the fathead minnow EcoToxChip.

In a previous in vivo study, adult male fathead minnows (Pimephales promelas) were exposed via water for 4 days to 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol). The present study expands on the evaluation of molecular responses to this perfluoro-alcohol by analyzing 26 male fathead minnow liver RNA samples from that study (five from each test concentration: 0, 0.018, 0.051, 0.171, and 0.463 mg FC8-diol/L) using fathead minnow EcoToxChips Ver. 1.0. EcoToxChips are a quantitative polymerase chain reaction array that allows for simultaneous measurement of >375 species-specific genes of toxicological interest. Data were analyzed with the online tool EcoToxXplorer. Among the genes analyzed, 62 and 96 were significantly up- and downregulated, respectively, by one or more FC8-diol treatments. Gene expression results from the previous study were validated, showing an upregulation of vitellogenin mRNA (vtg) and downregulation of insulin-like growth factor 1 mRNA (igf1). Additional genes related to estrogen receptor activation including esr2a (estrogen receptor 2a) and esrrb (estrogen related receptor beta) were also affected, providing further confirmation of the estrogenic nature of FC8-diol. Furthermore, genes involved in biological pathways related to lipid and carbohydrate metabolism, innate immune response, endocrine reproduction, and endocrine thyroid were significantly affected. These results both add confidence in the use of the EcoToxChip tool for inferring chemical mode(s) of action and provide further insights into the possible biological effects of FC8-diol. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol.

Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Occurrence and environmental risk assessment of 4 estrogenic compounds in surface water in Belgium in the frame of the EU Watch List.

The presence of natural estrogens estrone (E1), 17ß-estradiol (E2), estriol (E3) and synthetic estrogen 17α-ethynylestradiol (EE2) in the aquatic environment has raised concerns because of their high potency as endocrine disrupting chemicals. The European Commission (EC) established a Watch List of contaminants of emerging concerns including E1, E2 and EE2. The proposed environmental quality standards (EQSs) are 3.6, 0.4, 0.035 ng/L, for E1, E2, EE2, respectively. A thorough evaluation of analytical procedures developed by several studies aiming to perform sampling campaigns in different European countries highlighted that the required limits of quantification in surface water were not reached, especially for EE2 and to a lesser extent for E2. Moreover, data regarding the occurrence of these contaminants in Belgian surface water are very limited. A sampling campaign was therefore performed on a wide range of rivers in Belgium (accounting for a total of 63 samples). The detection frequencies of E1, E2, E3 and EE2 were 100, 98, 86 and 48%, respectively. E1 showed the highest mean concentration (= 4.433 ng/L). In contrast, the mean concentration of EE2 was 0.042 ng/L. The risk quotients (RQs) were calculated based on the respective EQS of each analyte. The frequency of exceedance of the EQS was 31.7% for E1, EE2, while it increased to 44.4% for E2. The extent of exceedance of the EQS, represented by the 95th percentile of the RQ dataset, was higher than 1 for E1, E2, EE2. The use of a confusion matrix was investigated to try to predict the risk posed by E2, EE2, based on the concentration of E1.

Estrogenic, Genotoxic, and Antibacterial Effects of Chemicals from Cryogenically Milled Tire Tread.

Tire and road wear particles (TRWP) contain complex mixtures of chemicals and release them to the environment, and potential toxic effects of these chemicals still need to be characterized. We used a standardized surrogate for TRWP, cryogenically milled tire tread (CMTT), to isolate and evaluate effects of tire-associated chemicals. We examined organic chemical mixtures extracted and leached from CMTT for the toxicity endpoints genotoxicity, estrogenicity, and inhibition of bacterial luminescence. The bioassays were performed after chromatographic separation on high-performance thin-layer chromatography (HPTLC) plates. Extracts of CMTT were active in all three HPTLC bioassays with two estrogenic zones, two genotoxic zones, and two zones inhibiting bacterial luminescence. Extracts of CMTT artificially aged with thermooxidation were equally bioactive in each HPTLC bioassay. Two types of aqueous leachates of unaged CMTT, simulating either digestion by fish or contact with sediment and water, contained estrogenic chemicals and inhibitors of bacterial luminescence with similar profiles to those of CMTT extracts. Of 11 tested tire-associated chemicals, two were estrogenic, three were genotoxic, and several inhibited bacterial luminescence. 1,3-Diphenylguanidine, transformation products of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, and benzothiazoles were especially implicated through comparison to HPTLC retention factors in the CMTT samples. Other bioactive bands in CMTT samples did not correspond to any target chemicals. Tire particles clearly contain and can leach complex mixtures of toxic chemicals to the environment. Although some known chemicals contribute to estrogenic, genotoxic, and antibacterial hazards, unidentified toxic chemicals are still present and deserve further investigation. Overall, our study expands the understanding of potential adverse effects from tire particles and helps improve the link between those effects and the responsible chemicals. Environ Toxicol Chem 2024;43:1962-1972. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The neuroprotective effects of estrogen and estrogenic compounds in spinal cord injury.

Spinal cord injury (SCI) occurs when the spinal cord is damaged from either a traumatic event or disease. SCI is characterised by multiple injury phases that affect the transmission of sensory and motor signals and lead to temporary or long-term functional deficits. There are few treatments for SCI. Estrogens and estrogenic compounds, however, may effectively mitigate the effects of SCI and therefore represent viable treatment options. This review systematically examines the pre-clinical literature on estrogen and estrogenic compound neuroprotection after SCI. Several estrogens were examined by the included studies: estrogen, estradiol benzoate, Premarin, isopsoralen, genistein, and selective estrogen receptor modulators. Across these pharmacotherapies, we find significant evidence that estrogens indeed offer protection against myriad pathophysiological effects of SCI and lead to improvements in functional outcomes, including locomotion. A STRING functional network analysis of proteins modulated by estrogen after SCI demonstrated that estrogen simultaneously upregulates known neuroprotective pathways, such as HIF-1, and downregulates pro-inflammatory pathways, including IL-17. These findings highlight the strong therapeutic potential of estrogen and estrogenic compounds after SCI.

Salmonid Pituitary Cells as a Test System for Identifying Endocrine-Disrupting Compounds.

The pituitary gland is a central regulator of reproduction, producing two gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which regulate gonadal development, sex steroid synthesis, and gamete maturation. The present study sought to optimize an in vitro test system using pituitary cells isolated from previtellogenic female coho salmon and rainbow trout, focusing on fshb and lhb subunit gene expression. Initially, we optimized culture conditions for duration and benefits of culturing with and without addition of endogenous sex steroids (17ß-estradiol [E2] or 11-ketotestosterone) or gonadotropin-releasing hormone (GnRH). The results suggest that culturing with and without E2 was valuable because it could mimic the (+) feedback effects on Lh that are observed from in vivo studies. After optimizing assay conditions, a suite of 12 contaminants and other hormones was evaluated for their effects on fshb and lhb gene expression. Each chemical was tested at four to five different concentrations up to solubility limitations in cell culture media. The results indicate that more chemicals alter lhb synthesis than fshb. The more potent chemicals were estrogens (E2 and 17α-ethynylestradiol) and the aromatizable androgen testosterone, which induced lhb. The estrogen antagonists 4-OH-tamoxifen and prochloraz decreased the E2-stimulated expression of lhb. Among several selective serotonin reuptake inhibitors tested, the sertraline metabolite norsertraline was notable for both increasing fshb synthesis and decreasing the E2 stimulation of lhb. These results indicate that diverse types of chemicals can alter gonadotropin production in fish. Furthermore, we have shown that pituitary cell culture is useful for screening chemicals with potential endocrine-disrupting activity and can support the development of quantitative adverse outcome pathways in fish. Environ Toxicol Chem 2023;42:1730-1742. © 2023 SETAC.

Comparison of Two Estrogen Chemically Activated Luciferase Expression Cell Bioassays to Liquid Chromatography-Mass Spectrometry for Quantifying Estrone in Water Samples.

Chemically activated luciferase expression (CALUX) cell bioassays are popular tools for assessing endocrine activity of chemicals such as certain environmental contaminants. Although activity equivalents can be obtained from CALUX analysis, directly comparing these equivalents to those obtained from analytical chemistry methods can be problematic because of the complexity of endocrine active pathways. We explored the suitability of two estrogen CALUX bioassays (the Organisation for Economic Co-operation and Development-approved VM7Luc4E2 cell bioassay and the VM7LucERßc9 cell bioassay) for quantitation of estrogen. Quadrupole-time of flight ultraperformance liquid chromatography-mass spectrometry (LC/MS) was selected as a comparative method. Regression analysis of measured estrone (E1) calibration samples showed all three methods to be highly predictive of nominal concentrations (p ≤ 7.5 × 10-51 ). Extracts of water sampled from laboratory dilutor tanks containing E1 at 0, 20, and 200 ng/L alone and in combination with atrazine were selected to test the quantitative capabilities of the CALUX assays. Process controls (0 and 100 ng E1/L) and a separate E1 standard (10 ng/ml, used to prepare the E1 process control) were also tested. Levels of E1 determined by LC/MS analysis and bioanalytical equivalents (ng E1/L) determined by CALUX analyses were comparable except in certain instances where the samples required dilution prior to CALUX analyses (e.g., the E1 process control and E1 standard). In those instances, measurements by CALUX were slightly but significantly decreased relative to LC/MS. Atrazine had no effect on the ability of either LC/MS or the CALUX bioassays to quantify E1. The present study illustrates the CALUX bioassays as successful in quantifying an estrogen in simple water samples and further characterizes their utility for screening. Environ Toxicol Chem 2023;42:333-339. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Gonadal Development in Smallmouth Bass (Micropterus Dolomieu) Reared in the Absence and Presence of 17-α-Ethinylestradiol.

Testicular oocytes in wild adult bass (Micropterus spp.) are considered a potential indication of exposure to estrogenic compounds in municipal, agricultural, or industrial wastewater. However, our ability to interpret links between testicular oocyte occurrence in wild fish species and environmental pollutants is limited by our understanding of normal and abnormal gonadal development. We previously reported low-to-moderate testicular oocyte prevalence (7%-38%) among adult male bass collected from Minnesota waters with no known sources of estrogenic compounds. In the present study, two experiments were conducted in which smallmouth bass (Micropterus dolomieu) fry were exposed to control water or 17-α-ethinylestradiol (EE2) during gonadal differentiation, then reared in clean water for an additional period. Histological samples were evaluated at several time points during the exposure and grow-out periods, and the sequence and timing of gonadal development in the presence of estrogen were compared with that of control fish. Testicular oocytes were not observed in any control or EE2-exposed fish. Among groups exposed to 1.2 or 5.1 ng/L EE2 in Experiment 1 or 3.0 ng/L EE2 in Experiment 2, ovaries were observed in 100% of fish up to 90 days after exposure ceased, and approximately half of those ovaries had abnormal characteristics, suggesting that they likely developed in sex-reversed males. Groups exposed to 0.1, 0.4, or 1.0 ng/L in Experiment 2 developed histologically normal ovaries and testes in proportions not significantly different from 1:1. These findings suggest that, while presumably able to cause sex reversal, juvenile exposure to EE2 may not be a unique cause of testicular oocytes in wild bass, although the long-term outcomes of exposure are unknown. Environ Toxicol Chem 2022;41:1416-1428. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Evaluation of Three ISO Estrogen Receptor Transactivation Assays Applied to 52 Domestic Effluent Samples.

Estrogens are released to the aquatic environment by wastewater treatment plant (WWTP) effluents and can affect wildlife. In the last three decades, many in vitro assay platforms have been developed to detect and quantify estrogenicity in water. In 2018, the International Organization for Standardization (ISO) standardized protocols became available for three types of in vitro estrogen receptor transactivation assays (ERTAs) detecting estrogenicity in 96-well plates (ISO19040 1-3). Two ERTAs-lyticase Yeast Estrogen Screen (L-YES) and Arxula YES (A-YES)-use genetically modified yeast strains, whereas the third utilizes stably transfected human cells. One human cell based assay is ERα-CALUX, which is based on a genetically modified human bone osteosarcoma cell line. In the present study, we characterized the performance, comparability, and effectiveness of these three ERTAs, including an evaluation involving proposed water quality thresholds (effect-based trigger values [EBTs]). For a robust evaluation, we collected 52 effluent samples over three sampling campaigns at 15 different WWTPs in Switzerland. Estrogen receptor transactivation assay results were correlated and compared with results from chemical analysis targeting known estrogens. The three ERTAs showed comparable data over all campaigns. However, the selection of EBTs plays a significant role in the interpretation and comparison of bioassay results to distinguish between acceptable and unacceptable water quality. Applying a fixed cross-assay EBT for effluent of 4 ng L-1 resulted in varying numbers of threshold exceedances ranging between zero and four samples depending on the ERTA used. Using assay-specific EBTs showed exceedances in eight samples (ERα-CALUX) and in one sample (A-YES), respectively. Thus, proposed EBTs do not produce similar risk profiles across samples and further refinement of assay-specific EBTs is needed to account for assay-specific differences and to enable the application of ERTAs as effect-based methods in environmental monitoring. Environ Toxicol Chem 2022;41:2512-2526. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Pioneering studies of the "morning-after" pill.

Yale School of Medicine produced the first proof-of-concept study on the viability of a "morning-after" pill for human use. This study was a result of a fruitful collaboration between a pair of Yale scientists, Drs. John M. Morris and Gertrude van Wagenen, who sought a non-abortion, post-coital contraceptive. They tested a variety of hormones, hormone-based synthetic drugs, and other compounds in monkeys in an effort to uncover a compound that was non-toxic but highly effective. Unfortunately, although they were unable to identify such a drug, their initial studies inspired other scientists to further pursue the concept of a "morning-after" pill, leading to the development of Food and Drug Administration (FDA)-approved emergency contraceptives.

Multiple organochlorine pesticide exposures and measures of sex steroid hormones in adult males: Cross-sectional findings from the 1999-2004 National Health and Nutrition Examination Survey.

BACKGROUND: Organochlorine pesticides are detectable in serum from most adults. Animal studies provide evidence of pesticide effects on sex hormones, suggesting that exposures may impact human reproductive function. Mounting evidence of sex differences in chronic diseases suggest that perturbations in endogenous sex hormones may influence disease risk. However, the association between organochlorine pesticide exposure and sex hormone levels in males across the lifespan is not well understood. METHODS: We evaluated cross-sectional associations of lipid-adjusted serum concentrations of ß-hexachlorocyclohexane, hexachlorobenzene, heptachlor epoxide, oxychlordane, dichlorodiphenyldichloroethylene (DDE), p,p'-dichlorodiphenyltrichloroethane (DDT), trans-nonachlor, and mirex in relation to sex steroid hormone levels [testosterone (ng/dL), sex hormone binding globulin (SHBG; nmol/L), estradiol (pg/mL), and androstanediol glucuronide (ng/dL)] in a sample of 748 males aged 20 years and older from the 1999-2004 cycles of the National Health and Nutrition Examination Survey (NHANES). Survey-weighted linear regression models were performed to estimate geometric means (GM) and their 95% confidence intervals (CIs) for quartiles of lipid-adjusted pesticide concentrations, adjusting for age, race, body mass index, serum lipids, smoking, education, and survey cycle. RESULTS: Hexachlorobenzene concentration was positively associated with total estradiol (GM Q4 = 43.2 pg/mL (95% CI 36.5-51.1) vs. Q1 GM = 25.6 pg/mL (24.1-27.3), p-trend <0.0001) and free estradiol (GM Q4 = 0.77 pg/mL (95% CI 0.64-0.93) vs. Q1 GM = 0.47 pg/mL (0.44-0.51), p-trend = 0.002). Serum DDT concentration was positively associated with total estradiol (GM Q4 = 31.6 pg/mL (95% CI 25.9-38.5) vs. Q1 GM = 27.3 pg/mL (25.9-28.7), p-trend = 0.05) and free estradiol (GM Q4 = 0.60 pg/mL (95% CI 0.48-0.76) vs. Q1 GM = 0.50 pg/mL (0.47-0.53), p-trend 0.02). There was a suggestive inverse association of DDT and SHBG (GM Q4 = 29.2 nmol/L (95% CI 23.8-35.9) vs. Q1 GM = 33.9 nmol/L (32.3-35.5), p-trend 0.07). A positive association of ß-hexachlorocyclohexane with total estradiol (GM Q4 = 30.3 pg/mL (95% CI 26.5-34.6) vs. Q1 GM = 26.7 pg/mL (24.5-29.0), p-trend = 0.09) was also suggestive but did not reach statistical significance. No distinct associations were observed for other hormone levels or other organochlorine pesticides. CONCLUSIONS: Our findings suggest that select organochlorine pesticides may alter male estradiol levels. The positive associations with estradiol may implicate sex hormones as a possible mechanism for disease risk among those with organochlorine pesticide exposure.

Removal of 17α-ethinylestradiol and caffeine from wastewater by UASB-Fenton coupled system.

In aquatic systems, some substances considered as endocrine disruptors have been detected, which can be due to their incomplete elimination in wastewater treatment plants (WWTPs) and inadequate disposal of pharmaceuticals. Among these contaminants are 17α-ethinylestradiol (EE2) and caffeine (CAF). Moreover, it has been reported that this kind of contaminants may provoke different adverse effects in many aquatic organisms. Because of that, in the present study, up-flow anaerobic sludge blanket reactors (UASB) coupled with the Fenton process was evaluated for EE2 and CAF removal spiked in wastewater samples. First, the best reaction conditions were established in each process. For UASB reactor, two hydraulic retention times (HRT 8 and 24 h) were evaluated, achieving the highest chemical organic demand (COD) removal (70 %) and drug elimination (84 %-86 %) with HRT 24 h. Subsequently, Fenton process was conducted at pH 3 with different levels of Fe2+ (0.05-0.5 mmol/L) and molar ratios Fe2+:H2O2 (1:1-1:10). Better results were obtained with 0.5 mmol Fe2+/L, and 1:10 ratio molar Fe2+:H2O2. Finally, UASB-Fenton coupled system allowed 80 % of COD decrease, almost complete removal of drugs and the toxicity of samples on Vibrio fischeri was reduced from 73 % to 30 %, demonstrating that this coupled system is a promising and efficient system for pharmaceutical compounds removal from wastewater.

Secondary treatment phase of tertiary wastewater treatment works significantly reduces estrogenic load.

Estrogenic compounds enter waterways via effluents from wastewater treatment works (WWTW), thereby indicating a potential risk to organisms inhabiting adjacent receiving waters. However, little is known about the loads or concentrations of estrogenic compounds that enter Australian WWTWs, the efficiency of removing estrogenic compounds throughout the various stages of tertiary WWTW processes (which are common in Australia), nor the concentrations released into estuarine or marine receiving waters, and the associated risk for aquatic taxa residing in these environments. Therefore, seven estrogenic compounds, comprising the natural estrogens estrone (E1), 17ß-estradiol (E2) and estriol (E3), the synthetic estrogen (EE2), and the industrial chemicals bisphenol A (BPA), 4-t-octyl phenol (4-t-OP) and 4-nonyl phenol (4-NP), in wastewater samples were quantified via liquid chromatographic-mass spectrometry (LC-MS) after solid-phase extraction at different stages of wastewater treatment and associated receiving waters. The concentrations of the target compounds in wastewater ranged from < LOQ (limit of quantification) to 158 ng/L for Tanilba Bay WWTW and < LOQ to 162 ng/L for Belmont WWTW. Most target compounds significantly declined after the secondary treatment phase. Appreciable removal efficiency throughout the treatment process was observed with removal from 39.21 to 99.98% of influent values at both WWTWs. The reduction of the natural estrogens (E1, E2 and E3) and 4-t-OP were significantly greater than EE2, BPA, and 4-NP in both WWTWs. Risk quotients (RQs) were calculated to assess potential ecological risks from individual estrogenic compounds. In predicted diluted effluents, no targeted compounds showed any ecological risk (RQ ≤1.65 × 10-2) at both WWTWs. Similarly, all RQs for shore samples at both WWTWs were below 1. Finally, the hazard index (HI), which represents combined estrogenic contaminants' ecological risk, indicated no mentionable risk for predicted diluted effluents (HI = 0.0097 to 0.0218) as well as shoreline samples (HI = 0.393 to 0.522) in the receiving estuarine or marine waters.