Understanding target-specific effects of antidepressant drug pollution on molluscs: A systematic review protocol.

BACKGROUND: The environmental prevalence of widely prescribed human pharmaceuticals that target key evolutionary conserved biomolecules present across phyla is concerning. Antidepressants, one of the most widely consumed pharmaceuticals globally, have been developed to target biomolecules modulating monoaminergic neurotransmission, thus interfering with the endogenous regulation of multiple key neurophysiological processes. Furthermore, rising prescription and consumption rates of antidepressants caused by the burgeoning incidence of depression is consistent with increasing reports of antidepressant detection in aquatic environments worldwide. Consequently, there are growing concerns that long-term exposure to environmental levels of antidepressants may cause adverse drug target-specific effects on non-target aquatic organisms. While these concerns have resulted in a considerable body of research addressing a range of toxicological endpoints, drug target-specific effects of environmental levels of different classes of antidepressants in non-target aquatic organisms remain to be understood. Interestingly, evidence suggests that molluscs may be more vulnerable to the effects of antidepressants than any other animal phylum, making them invaluable in understanding the effects of antidepressants on wildlife. Here, a protocol for the systematic review of literature to understand drug target-specific effects of environmental levels of different classes of antidepressants on aquatic molluscs is described. The study will provide critical insight needed to understand and characterize effects of antidepressants relevant to regulatory risk assessment decision-making, and/or direct future research efforts. METHODS: The systematic review will be conducted in line with the guidelines by the Collaboration for Environmental Evidence (CEE). A literature search on Scopus, Web of Science, PubMed, as well as grey literature databases, will be carried out. Using predefined criteria, study selection, critical appraisal and data extraction will be done by multiple reviewers with a web-based evidence synthesis platform. A narrative synthesis of outcomes of selected studies will be presented. The protocol has been registered in the Open Science Framework (OSF) registry with the registration DOI: 10.17605/OSF.IO/P4H8W.

Common aquatic pollutants modify hemocyte immune responses in Biomphalaria glabrata.

Disruptions to reproductive health in wildlife species inhabiting polluted environments is often found to occur alongside compromised immunity. However, research on impacts of aquatic pollution on freshwater mollusc immune responses is limited despite their importance as vectors of disease (Schistosomiasis) in humans, cattle and wild mammals. We developed an in vitro 'tool-kit' of well-characterized quantitative immune tests using Biomphalaria glabrata hemocytes. We exposed hemocytes to environmentally-relevant concentrations of common aquatic pollutants (17ß-estradiol, Bisphenol-A and p,p'-DDE) and measured key innate immune responses including motility, phagocytosis and encapsulation. Additionally, we tested an extract of a typical domestic tertiary treated effluent as representative of a 'real-world' mixture of chemicals. Encapsulation responses were stimulated by p,p'-DDE at low doses but were suppressed at higher doses. Concentrations of BPA (above 200 ng/L) and p,p'-DDE (above 500 ng/L) significantly inhibited phagocytosis compared to controls, whilst hemocyte motility was reduced by all test chemicals and the effluent extract in a dose-dependent manner. All responses occurred at chemical concentrations considered to be below the cytotoxic thresholds of hemocytes. This is the first time a suite of in vitro tests has been developed specifically in B. glabrata with the purpose of investigating the impacts of chemical pollutants and an effluent extract on immunity. Our findings indicate that common aquatic pollutants alter innate immune responses in B. glabrata, suggesting that pollutants may be a critical, yet overlooked, factor impacting disease by modulating the dynamics of parasite transmission between molluscs and humans.

Uptake, Elimination and Effects of Cosmetic Microbeads on the Freshwater Gastropod Biomphalaria glabrata.

The presence of plastic cosmetic microbeads in the environment due to their extensive use in society and inevitable dispersal into wastewater is concerning. Therefore, it is vital to understand the processes of microplastic uptake and elimination by aquatic organisms, and to further assess their potential to cause harmful effects and wider impacts. We therefore investigated the short-term (48-h) and long-term (21-d) uptake, elimination, and effects of exposure to polyethylene microbeads (a mixture of fragments and spheres extracted from commercially available facial scrubs) on the freshwater snail, Biomphalaria glabrata. We found fast uptake in the short-term (75 µg/g/h) and the long-term (6.94 µg/g/h) in B. glabrata exposed to 800 particles/200-mL and 80 particles/200-mL, respectively. Irregular fragments were more easily ingested and egested compared to spheres (ANOVA, p < 0.05) in both 48-h and 21-d exposures. The mean size of the fragments in B. glabrata tissues (413 ± 16 µm) after 48-h exposure was significantly larger than that of the standard sample (369 ± 26 µm) (ANOVA, F3,20 = 3.339, p = 0.033), suggesting that aggregation in the gut may occur. Floating feces containing microbeads were observed in the long-term exposure, which could alter the fate, behavior, and bioavailability of egested microbeads. No significant effects on survival and growth were shown within 48-h or 21-d exposure periods. Thus, further studies on the specific features of microplastics (e.g., their shape and size) influencing uptake and elimination, as well as toxic molecular mechanisms, should be explored in future ecotoxicological studies.

A Comparison of Different Approaches for Characterizing Microplastics in Selected Personal Care Products.

Any uncertainty in determining numbers of microplastics in the environment may be a barrier to assessing their impact and may stem from various aspects of methodologies used to quantify them. We undertook a comparison of approaches to quantify and characterize microplastics in 4 personal care products. The aim was not only to determine how many particles were present but to assess any differences due to the methods used. Counting of extracted microplastics was undertaken using particle size analysis, light microscopy, and imaging flow cytometry. Micro-Fourier transform infrared spectroscopy (µ-FTIR) was used to characterize the particles in each product. The mean size distribution of microplastics differed depending on the method employed, and it was apparent that imaging flow cytometry was affected by high background noise that may require staining of plastics to overcome. The application of µ-FTIR confirmed polyethylene as the microplastic in each product. Methodological challenges encountered in the study and the literature have highlighted the need for standardization of methods for determining microplastics. Environ Toxicol Chem 2022;41:880-887. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Early embryonic exposure of freshwater gastropods to pharmaceutical 5-alpha-reductase inhibitors results in a surprising open-coiled "banana-shaped" shell.

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated "banana-shaped" shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals' efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

Steroid Androgen Exposure during Development Has No Effect on Reproductive Physiology of Biomphalaria glabrata.

Gastropod mollusks have been proposed as alternative models for male reproductive toxicity testing, due to similarities in their reproductive anatomy compared to mammals, together with evidence that endocrine disrupting chemicals can cause effects in some mollusks analogous to those seen in mammals. To test this hypothesis, we used the freshwater pulmonate snail, Biomphalaria glabrata, for which various genetic tools and a draft genome have recently become available, to investigate the effects of two steroid androgens on the development of mollusk secondary sexual organs. Here we present the results of exposures to two potent androgens, the vertebrate steroid; 5α-dihydrotestosterone (DHT) and the pharmaceutical anabolic steroid; 17α-methyltestosterone (MT), under continuous flow-through conditions throughout embryonic development and up to sexual maturity. Secondary sexual gland morphology, histopathology and differential gene expression analysis were used to determine whether steroid androgens stimulated or inhibited organ development. No significant differences between tissues from control and exposed snails were identified, suggesting that these androgens elicited no biologically detectable response normally associated with exposure to androgens in vertebrate model systems. Identifying no effect of androgens in this mollusk is significant, not only in the context of the suitability of mollusks as alternative model organisms for testing vertebrate androgen receptor agonists but also, if applicable to other similar mollusks, in terms of the likely impacts of androgens and anti-androgenic pollutants present in the aquatic environment.

The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: implications for developing new model organisms.

Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals.

Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17ß and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p>0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17ß and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10(-6)M for Gen and >10(-5)M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17ß or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.

17ß-Oestradiol may prolong reproduction in seasonally breeding freshwater gastropod molluscs.

Whilst the effects of oestrogenic contaminants in the aquatic environment are well documented in fish, effects in invertebrate species has been subject to debate, possibly due to differences in experimental conditions (temperature, timing and duration of exposure) between studies. It has been suggested that molluscs are only susceptible to oestrogens in periods either following the main spawning or leading up to the maturation of gametes. To investigate this possibility, two temperate, seasonally reproducing gastropods (Planorbarius corneus and Viviparus viviparus) were exposed to two concentrations of 17ß-oestradiol (E2; 10ng/l and 100ng/l nominal) in an outdoor mesocosm (subject to natural seasons). In addition, P. corneus was also exposed to E2 (1, 10 and 100ng/l) in the laboratory at temperatures and photoperiods to simulate summer and autumn. In the mesocosm, both snail species produced similar numbers of eggs/embryos as reference groups in the summer, but the groups exposed to 10ngE2/l (nominal) had significantly higher productivities after the onset of autumn, when entering their quiescent phase, whilst the snails exposed to a higher concentration (100ng/l, nominal) had an increased rate of mortality, and did not experience increased reproduction. In the laboratory, the rate of egg laying in P. corneus was unaffected in simulated summer (20°C, 16h photoperiod), but snails exposed to 10 and 100ng/l (nominal) in simulated autumn (15°C, 12h photoperiod) showed a concentration-dependent inhibition of the natural decline in egg laying observed in the control snails. Overall, rather than an increase in reproductive rate, the response of this species was a perpetuation of summer reproductive rates into autumn. We conclude that exposure to E2 can affect reproduction in the freshwater gastropods studied, but in P. corneus at least, this is dependent on the seasonal conditions (temperature and photoperiod) at which exposures are made.

Exposure to treated sewage effluent disrupts reproduction and development in the seasonally breeding Ramshorn snail (subclass: Pulmonata, Planorbarius corneus).

Experiments were conducted to assess the impacts of exposure to sewage treatment works (STW) effluent upon the growth, reproductive function, and sexual development of the European mollusc, Planorbarius corneus under seasonally varying temperatures and photoperiodic conditions. In river water, a clear seasonal change in the number and weight of egg masses (during both 2003 and 2004), and in the number of eggs produced, was found, providing evidence for profound effects of both changing temperature and photoperiod on reproduction. Exposure to STW effluent caused disturbances in this seasonal reproductive cycle at all concentrations tested. The effects included significant dose-dependent increases in fecundity and in the overall length of the reproductive cycle in adult snails exposed to both 50% and 100% effluent relative to river water for a period of up to 14 weeks. Disturbances in the development of both the male and female gametes of the both the adult snails and their developmentally exposed offspring were also seen. These effects were more evident in the offspring than in the adults.

Endocrine disrupting effects of herbicides and pentachlorophenol: in vitro and in vivo evidence.

The potential for agricultural chemicals to cause endocrine disruption (ED) in humans and wildlife is an increasing concern; however, the effects of commonly used pesticides at environmentally relevant concentrations are largely unknown. Therefore, 12 environmentally relevant pesticides (11 herbicides and pentachlorophenol (PCP)) were tested for their endocrine disrupting potential in two in vitro assays. A recombinant yeast screen was used to detect receptor mediated (anti-) estrogenic and (anti-) androgenic activity (concentration range: 0.01-1000 microM), and cultured Xenopus oocytes were used to measure effects on the ovulatory response and ovarian steroidogenesis (concentration range: 0.00625-62.5 microM). Eleven pesticides were active in at least one assay (isoproturon, diuron, linuron, 4-chloro-2-methylphenoxy acetic acid (MCPA), mecoprop, atrazine, simazine, PCP, trifluralin, chlorpropham, bentazone), and one had no effect (2,4-dichlorophenoxy acetic acid (2,4,-D)). The most common effects were antiestrogenic/ antiandrogenic activity in the yeast screen, and inhibition of ovulation in vitro, accompanied by decreased testosterone production. Estrogenic activity was never observed. In addition, the most potent compound identified in vitro (PCP) was tested for ED activity in vivo. A short-term exposure (6 days) of adult female Xenopus to low concentrations (0.1 or 1 microg/L; 0.375 or 3.75 nM) resulted in minor alterations in plasma hormone levels and toxic effects on the ovary. Changes in in vitro human chorionic gonadotropin (hCG) stimulated hormone production in ovarian follicles from exposed individuals was also observed. In conclusion, novel effects of herbicides and PCP at environmentally relevant concentrations were found, and the effects of these compounds on humans and/or wildlife warrant further investigation.

Plants used in Chinese medicine for the treatment of male infertility possess antioxidant and anti-oestrogenic activity.

In this study Chinese herbs commonly used in the treatment of male infertility were investigated for relevant biochemical activity. Male factor infertility predominantly arises via barriers to, or defects in, spermatogenesis. The process of spermatogenesis is under strict endocrine control; in addition oxidative stress has been implicated in male infertility with significant levels of reactive oxygen species detected in 25% of infertile males. A total of 37 individual herbs and seven herb decoctions used in the treatment of male factor infertility were therefore tested for endocrine activity using a recombinant yeast based assay and antioxidant activity using the FRAP (ferric reducing antioxidant potential) assay. Individual herbs tested did not show androgenic properties, 20 showed strong and 10 weak anti-oestrogenic activity (per g of dried herb tamoxifen equivalents ranged from 1.18-1280.66 mg and 0.06-0.98 mg, respectively). Oestrogenic responses were elicited for two herbs (85.30-550 microg oestradiol equivalents/g dried herb), with seven and three herbs exhibiting a strong or weak anti-androgenic response (per g of dried herb DHT equivalents ranged from 1.54-66.78 mg and 0.17-0.32 mg), respectively. Of these 37 herbs, strong (15 herbs), intermediate (7 herbs) and weak/no (15 herbs) antioxidant activity was detected (ranging from 0.912-1.26; 0.6-0.88 and 0-0.468 microg ascorbate equivalent/mg dried herb, respectively). The seven decoctions (previously used to treat patients) tested elicited strong (5 herbs) and weak (2 herbs) anti-oestrogenic responses (per g of dried herb tamoxifen equivalents ranged from 1.14-13.23 mg and 0.22-0.26 mg, respectively), but not oestrogenic, androgenic nor anti-androgenic, consistent with their individual composition. With regard to antioxidant activity the following responses were recorded: three strong, three intermediate and one weak (ranging from 1.02-1.2; 0.72-0.76 and 0.44 microg ascorbate equivalent/mg dried herb, respectively). The prospects for introducing Chinese herbal treatments into the Western-based medicine are discussed.

Isomer-specific degradation and endocrine disrupting activity of nonylphenols.

Degradation of technical nonylphenol by Sphingobium xenophagum Bayram led to a significant shift in the isomers composition of the mixture. By means of gas chromatography-mass spectrometry, we could observe a strong correlation between transformation of individual isomers and their alpha-substitution pattern, as expressed by their assignment to one of six mass spectrometric groups. As a rule, isomers with less bulkiness at the alpha-carbon and those with an optimally sized main alkyl chain (4-6 carbon atoms) were degraded more efficiently. By mass spectrometric analysis, we identified the two most recalcitrant main isomers of the technical mixture (Group 4) as 4-(1,2-dimethyl-1-propylbutyl)phenols (NP193a and NP193b), which are diastereomers with a bulky alpha-CH3, alpha-CH(CH3)C2H5 substitution. Our experiments with strain Bayram show that the selective enrichment of isomers with bulky alpha-substitutions observed in nonylphenol fingerprints of natural systems can be caused by microbial ipso-hydroxylation. Based on the yeast estrogen assay (YES), we established an estrogenicity ranking with a variety of single isomers and compared it to rankings obtained with different reporter cell systems. Structure-activity relationships derived from these data suggest that Group 4 isomers have a high estrogenic potency. This indicates a substantial risk that enrichment of highly estrogenic isomers during microbial degradation by ipso-substitution will increase the specific estrogenicity of aging material.

Benzotriazole is antiestrogenic in vitro but not in vivo.

Benzotriazole (BT) is an anticorrosive agent well known for its use in aircraft deicing and antifreeze fluids but also used in dishwasher detergents. It is highly persistent in the environment; therefore, BT is frequently found in runoff emanating from large airports as well as in the surrounding groundwater. In addition, BT has recently been found to be ubiquitous in Swiss wastewater treatment plant effluents and their receiving waters; however, very little chronic toxicity data is available on which to base a sound ecological risk assessment of this chemical. In vitro assays conducted using a recombinant yeast (anti-) estrogen assay indicated that BT possessed clear antiestrogenic properties. This chemical was approximately 100-fold less potent than Tamoxifen, which was used as a positive control. A subsequent in vivo study, however, involving analysis of vitellogenin induction and somatic indices in adult fathead minnows (Pimephales promelas) exposed to BT at concentrations of 10, 100, and 1,000 mug/L for two weeks showed no evidence of antiestrogenic activity by this compound. The possibility exists that higher concentrations of BT may yet induce the type of activity observed in vitro, although the concentrations used here already far exceed those reported in surface-water samples. Furthermore, adverse effects may be observed in fish or other organisms exposed to BT for a longer period than employed here, although such studies are costly and unlikely to be included in standard risk assessment procedures. A rigorous investigation of the chronic toxicity of BT is imperative.

Novel estrogen receptor-related Transcripts in Marisa cornuarietis; a freshwater snail with reported sensitivity to estrogenic chemicals.

We have isolated novel molluskan steroid receptor transcripts orthologous to vertebrate estrogen receptors (ERs) and estrogen receptor-related receptors (ERRs) from the freshwater snail Marisa cornuarietis. Radiolabeled ligand binding analyses showed that neither recombinant receptor protein specifically bound 17beta-estradiol over the range applied (0.3-9.6 nM). These novel receptor transcripts have thus been designated mcER-like and mcERR respectively. Quantitative PCR revealed mcER-like to be expressed ubiquitously throughout a range of male and female structures studied, including neural and reproductive tissues. Highest absolute levels were seen in the male penis-sheath complex. The mcERR mRNA was also expressed ubiquitously throughout all male and female tissues analyzed here, with very low absolute transcript numbers in female accessory sex structures compared to other tissues.

Estrogenic activity measured in a sewage treatment works treating industrial inputs containing high concentrations of alkylphenolic compounds--a case study.

Chemical analyses were combined with a biological assay to investigate the main estrogenic chemicals as they passed through a sewage treatment works (STW) and entered a river. The STW studied was unusual in that it received wastewater from the textile trade. This wastewater was shown to contain high concentrations of alkylphenol polyethoxylates and their degradation products, such as nonylphenol. High-performance liquid chromatography fractionation, combined with biological assay, showed that the majority of the estrogenic activity was contributed by the alkylphenolic chemicals and the natural estrogens 17beta-estradiol and estrone. Despite removal of a high proportion of the alkylphenolic chemicals by the various treatment processes within the STW, concentrations in the final effluent were still high compared to most other STW effluents in the United Kingdom. The effluent was very estrogenic to caged fish, as was the river water 2 and 5 km downstream of the STW, even though less so. Using various approaches, attempts were made to determine which group of chemicals contributed most to the estrogenic activity of the effluent. The analysis suggested that, in this unusual situation, the alkylphenolic chemicals may contribute the majority of the estrogenic activity of the effluent. However, this conclusion was based on a number of uncertainties that are presently unresolved and hence can be considered only tentative.

Reduction in the estrogenic activity of a treated sewage effluent discharge to an English river as a result of a decrease in the concentration of industrially derived surfactants.

As a result of the introduction of tighter discharge limits and effluent treatment processes at source, the concentration of alkylphenol ethoxylates and nonylphenol present in the final effluent discharge from a sewage treatment works that treats trade effluent from the textiles industry was reduced. The estrogenic effects of the final effluent discharge to the Aire River were compared over a four-year period during which various treatment measures were introduced. Male rainbow trout exposed to the effluent on four occasions in consecutive years (1994-1997) showed a reduction in the level of induced vitellogenesis between 1994 and 1997. A marked decrease in gonadosomatic index (GSI) and increase in heptaosomatic index (HSI) was measured in fish exposed to the effluent in 1994. In successive years, these differences diminished, and in the case of the GSI no measurable difference was observed between fish exposed to the final effluent or those in the control group in 1997. However, an increase in HSI was still measurable in 1997 in fish exposed to the final effluent and at sites farther downstream. The reduction in the effects of the effluent paralleled the reduction in the concentration of nonylphenol as well as its mono- and diethoxylates, which have been demonstrated to produce estrogenic effects in trout exposed to these compounds in the laboratory. This study demonstrates that the setting of more restricted discharge limits for known estrogenic chemicals of industrial origin can lead to significant reductions in the estrogenic activity of the watercourses into which the effluents are discharged.