Effects of diclofenac on sentinel species and aquatic communities in semi-natural conditions.

Due to the potential hazard of diclofenac on aquatic organisms and the lack of higher-tier ecotoxicological studies, a long-term freshwater mesocosm experiment was set up to study the effects of this substance on primary producers and consumers at environmentally realistic nominal concentrations 0.1, 1 and 10 µg/L (average effective concentrations 0.041, 0.44 and 3.82 µg/L). During the six-month exposure period, the biovolume of two macrophyte species (Nasturtium officinale and Callitriche platycarpa) significantly decreased at the highest treatment level. Subsequently, a decrease in dissolved oxygen levels was observed. High mortality rates, effects on immunity, and high genotoxicity were found for encaged zebra mussels (Dreissena polymorpha) in all treatments. In the highest treatment level, one month after the beginning of the exposure, mortality of adult fish (Gasterosteus aculeatus) caused effects on the final population structure. Total abundance of fish and the percentage of juveniles decreased whereas the percentage of adults increased. This led to an overall shift in the length frequency distribution of the F1 generation compared to the control. Consequently, indirect effects on the community structure of zooplankton and macroinvertebrates were observed in the highest treatment level. The No Observed Effect Concentration (NOEC) value at the individual level was < 0.1 µg/L and 1 µg/L at the population and community levels. Our study showed that in more natural conditions, diclofenac could cause more severe effects compared to those observed in laboratory conditions. The use of our results for regulatory matters is also discussed.

Parental exposure to methyl methane sulfonate of three-spined stickleback: contribution of DNA damage in male and female germ cells to further development impairment in progeny.

Data regarding the link between DNA integrity of germ cells and the quality of progeny in fish exposed to genotoxicant are scarce although such information is of value to understand genotoxic effects of contaminants in aquatic fauna. This work aimed at studying the consequences of a parental exposure during the breeding season on offspring quality in three-spined stickleback. After in vivo exposure of adult fish to methyl methane sulfonate, a model alkylating compound, a clear increase in DNA damage was observed in erythrocytes of both genders, here used as a biomarker of exposure. MMS exposure significantly affected sperm DNA integrity but neither female fecundity nor fertilization success. In order to understand the contribution of each sex to potential deleterious effects in progeny due to parental exposure, mating of males and females exposed or not to MMS, was carried out. Exposure of both males and females or of males alone led to a significant increase in both mortality during embryo-larval stages and abnormality rate at hatching that appeared to be sensitive stages. Thus, in accordance with recent studies carried out in other freshwater fish species, such development defects in progeny were clearly driven by male genome, known to be devoid of DNA repair capacity in spermatozoa. The next step will be to investigate the link between DNA damage in stickleback sperm and reproductive impairment in natural populations exposed to complex mixture of genotoxicants.

Monitoring of dioxin-like, estrogenic and anti-androgenic activities in sediments of the Bizerta lagoon (Tunisia) by means of in vitro cell-based bioassays: contribution of low concentrations of polynuclear aromatic hydrocarbons (PAHs).

We used an array of in vitro cell-based bioassays to assess dioxin-like, estrogenic and (anti-)androgenic activities in organic extracts of sediments from the Bizerta lagoon, one of the largest Tunisian lagoons subjected to various anthropogenic and industrial pressures. The sediments were sampled both in winter and summer 2006 in 6 stations differently impacted and in one reference station located in the seawards entrance of Ghar el Melh lagoon. Chemical analyses of the 16 priority PAHs showed that the sediments were low to moderately contaminated (2-537 ng/g dry weight). By using the estrogen- (MELN) and androgen-responsive (MDA-kb2) reporter cell lines, significant estrogenic and anti-androgenic activities were detected only in the Menzel Bourguiba (MB) site, the most contaminated site, both in winter and summer. By using 7-ethoxyresorufin-O-deethylase (EROD) induction in the fish PLHC-1 cell line after both 4 and 24 h of cell exposure, dioxin-like activities were detected in all analysed samples. Dioxin-like activities were higher after 4 h exposure, and varied according to the sites and the sampling season. While highly significant correlation was observed between bioassay- and chemical analyses-derived toxic equivalents (TEQs), PAHs accounted for only a small part (up to 4%) of the detected biological activities, suggesting that other readily metabolised EROD-inducing compounds were present. This study argues for the use of short time exposure to assess biological TEQs in low contaminated samples and provides new induction equivalent factors (IEF(4h)) for 16 PAHs in the PLHC-1 cell line. Finally, our results stress the need to further characterise the nature of organic chemical contamination as well as its long-term impacts on aquatic wildlife in the Bizerta lagoon.

Decision trees versus support vector machine for classification of androgen receptor ligands.

With the current concern of limiting experimental assays, increased interest now focuses on in silico models able to predict toxicity of chemicals. Endocrine disruptors cover a large number of environmental and industrial chemicals which may affect the functions of natural hormones in humans and wildlife. Structure-activity models are now increasingly used for predicting the endocrine disruption potential of chemicals. In this study, a large set of about 200 chemicals covering a broad range of structural classes was considered in order to categorize their relative binding affinity (RBA) to the androgen receptor. Classification of chemicals into four activity groups, with respect to their log RBA value, was carried out in a cascade of recursive partitioning trees, with descriptors calculated from CODESSA software and encoding topological, geometrical and quantum chemical properties. The hydrophobicity parameter (log P), Balaban index, and descriptors relying on charge distribution (maximum partial charge, nucleophilic index on oxygen atoms, charged surface area, etc.) appear to play a major role in the chemical partitioning. Separation of strongly active compounds was rather straightforward. Similarly, about 90% of the inactive compounds were identified. More intricate was the separation of active compounds into subsets of moderate and weak binders, the task requiring a more complex tree. A comparison was made with support vector machine yielding similar results.

Endocrine disruption profile analysis of 11,416 chemicals from chemometrical tools.

A number of chemicals released into the environment have the potential to disturb the normal functioning of the endocrine system. These chemicals termed endocrine disruptors (EDs) act by mimicking or antagonizing the normal functions of natural hormones and may pose serious threats to the reproductive capability and development of living species. Batteries of laboratory bioassays exist for detecting these chemicals. However, due to time and cost limitations, they cannot be used for all the chemicals which can be found in the ecosystems. SAR and QSAR models are particularly suited to overcome this problem but they only deal with specific targets/endpoints. The interest to account for profiles of endocrine activities instead of unique endpoints to better gauge the complexity of endocrine disruption is discussed through a SAR study performed on 11,416 chemicals retrieved from the US-NCI database and for which 13 different PASS (Prediction of Activity Spectra for Substances) endocrine activities were available. Various multivariate analyses and graphical displays were used for deriving structure-activity relationships based on specific structural features.

Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.).

This study examined the response of 7-ethoxyresorufine-O-deethylase, glutathione-S-transferase, glutathione peroxidase, glutathione content, level of thiobarbituric acid reactive compounds and circulating vitellogenin, in three-spined sticklebacks after 21 days of exposure to Diquat herbicide, commercial nonylphenol polyethoxylate adjuvant and mixture between Diquat and adjuvant. The results showed that adjuvant exerted more important oxidative effects than Diquat and that mixture effects were unlike to single additivity. This study argues for ecotoxicological risk assessment of adjuvants and mixtures of adjuvants and pesticides.

SAR and QSAR modeling of endocrine disruptors.

A number of xenobiotics by mimicking natural hormones can disrupt crucial functions in wildlife and humans. These chemicals termed endocrine disruptors are able to exert adverse effects through a variety of mechanisms. Fortunately, there is a growing interest in the study of these structurally diverse chemicals mainly through research programs based on in vitro and in vivo experimentations but also by means of SAR and QSAR models. The goal of our study was to retrieve from the literature all the papers dealing with structure-activity models on endocrine disruptor xenobiotics. A critical analysis of these models was made focusing our attention on the quality of the biological data, the significance of the molecular descriptors and the validity of the statistical tools used for deriving the models. The predictive power and domain of application of these models were also discussed.

Homology model of the rainbow trout estrogen receptor (rtERalpha) and docking of endocrine disrupting chemicals (EDCs).

A model for rainbow trout (Oncorhynchus mykiss) estrogen receptor (rtERa) was built by homology with the human estrogen receptor (hERa). A high level of sequence conservation between the two receptors was found with 64% and 80% of identity and similarity, respectively. Selected endocrine disrupting chemicals were docked into the ligand binding domain (LBD) of rtERa and the corresponding free binding energies Delta(DeltaG(bind)) values were calculated. A Quantitative Structure-Activity Relationship (QSAR) model between the relative binding affinity data and the Delta(DeltaG(bind)) values was derived in order to predict which further organic pollutants are likely to bind to rtERa.

BFCOD activity in fish cell lines and zebrafish embryos and its modulation by chemical ligands of human aryl hydrocarbon and nuclear receptors.

Assessment of exposure and effect of fish to pharmaceuticals that contaminate aquatic environment is a current major issue in ecotoxicology and there is a need to develop specific biological marker to achieve this goal. Benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD) enzymatic activity has been commonly used to monitor CYP3A activity in fish. In this study, we assessed the capacity of a panel of toxicologically relevant chemicals to modulate BFCOD activity in fish, by using in vitro and in vivo bioassays based on fish liver cell lines (PLHC-1, ZFL, RTL-W1) and zebrafish embryos, respectively. Basal BFCOD activity was detectable in all biological models and was differently modulated by chemicals. Ligands of human androgens, glucocorticoids, or pregnanes X receptors (i.e., dexamethasone, RU486, rifampicin, SR12813, T0901317, clotrimazole, ketoconazole, testosterone, and dihydrotestosterone) moderately increased or inhibited BFCOD activity, with some variations between the models. No common feature could be drawn by regards to their capacity to bind to these receptors, which contrasts with their known effect on mammalian CYP3A. In contrast, dioxins and polycyclic aromatic hydrocarbons (PAHs) strongly induced BFCOD activity (up to 30-fold) in a time- and concentration-dependent manner, both in vitro in all cell lines and in vivo in zebrafish embryos. These effects were AhR dependent as indicated by suppression of induced BFCOD by the AhR pathway inhibitors 8-methoxypsoralen and α-naphthoflavone. Altogether our result further question the relevance of using liver BFCOD activity as a biomarker of fish exposure to CYP3A-active compounds such as pharmaceuticals.

Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes.

Pharmaceuticals are found in the aquatic environment but their potential effects on non-target species like fish remain unknown. This in vitro study is a first approach in the toxicity assessment of human drugs on fish. Nine pharmaceuticals were tested on two fish hepatocyte models: primary cultures of rainbow trout hepatocytes (PRTH) and PLHC-1 fish cell line. Cell viability, interaction with cytochrome P450 1A (CYP1A) enzyme and oxidative stress were assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide tetrazolium (MTT), 7-ethoxyresorufin-o-deethylase (EROD) and dichlorofluorescein (DCFH-DA) assays, respectively. The tested drugs were clofibrate (CF), fenofibrate (FF), carbamazepine (CBZ), fluoxetine (FX), diclofenac (DiCF), propranolol (POH), sulfamethoxazole (SFX), amoxicillin (AMX) and gadolinium chloride (GdCl(3)). All substances were cytotoxic, except AMX at concentration up to 500 microM. The calculated MTT EC(50) values ranged from 2 microM (CF) to 651 microM (CBZ) in PLHC-1, and from 53 microM (FF) to 962 microM (GdCl(3)) in PRTH. CF, FF, and FX were the most cytotoxic drugs and induced oxidative stress before being cytotoxic. Compared to hepatocytes from human and dog, fish hepatocytes seemed to be more susceptible to the peroxisome proliferators (PPs) CF and FF. In PLHC-1 cells none of the tested drugs induced the EROD activity whereas POH appeared as a weak EROD inducer in PRTH. Moreover, in PRTH, SFX, DiCF, CBZ and to a lesser extend, FF and CF inhibited the basal EROD activity at clearly sublethal concentrations which may be of concern at the biological and chemical levels in a multipollution context.

In vivo experimental model for silicosis.

The selection of an experimental model for silicosis requires a thorough understanding of many pulmonary parameters specific to the experimental animal (e.g., clearance time, penetration curves, anatomical differences), as well as the strain's sensitivity to different conditions. The pulmonary response of three rat strains (i.e., Fischer 344, Sprague-Dawley, and Wistar) to silica dust was compared using two methods of exposure: intratracheal injection and inhalation. The test period lasted 3 months for injection and 6 and 12 months for inhalation. The histological study of the lung revealed a distinct nodular reaction in Sprague-Dawley and Wistar strains. Intratracheal injections led to the development of fibrotic nodules in Wistar rats, whereas such silicotic nodules were infrequent in injected Fischer 344 rats, and almost absent when exposure was by inhalation. Sprague-Dawley and Fischer 344 rats showed frequent thickening and metaplasia of the alveolar walls near the terminal bronchioles. This tendency was particularly pronounced in rats exposed by inhalation (especially Fischer 344 rats). Macroscopic examination (wet lung weight) and biochemical dosing (lung silica, hydroxyproline, and lipid content) revealed an increase in the various parameters examined in Sprague-Dawley rats relative to the other two strains, regardless of the type of exposure. The histological examination, however, leads us to conclude that the Wistar rats offer the best experimental model for silicosis, because their pulmonary reaction is more characteristic of the human condition than that of the other two strains.

Induction of the hsp70 Gene Promoter by Various Anticancer Drugs.

HeLa cells containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter have been exposed in vitro to various anticancer drugs. Cisplatin induced CAT production with a dose-effect relationship at a non-cytotoxic dose, whereas no induction was detected with carboplatin. Etoposid induced a significant response at a cytotoxic concentration. The limited positive response with doxorubicin, daunomycin and mitoxantrone was not statistically significant. These chemicals are known to produce reactive oxygen species and induce apoptosis. No induction of the hsp70 promoter could be detected with the other cytostatic compounds that have been tested such as base analogues (5-fluorouracil, cytosine arabinoside 3'-MP), inhibitors of DNA synthesis (amethopterin, aminopterin), antimitotics (vinblastine, colchicine), and alkylating (streptozotocine, carboplatin, melphalan) or intercalating agents (bleomycin). In addition, the role of the transcription inhibitory activity of doxorubicin in this model is evidenced and the consequent question of the suitability of the reporter gene system is discussed. Our results suggest that specific genotoxic compounds are not able to induce the hsp70 promoter, and are in agreement with the concept that stimulation of HSP70 synthesis occurs through a biochemical process involving proteotoxicity.

Use of transepithelial electrical resistance in the study of pentachlorophenol toxicity.

The toxicity of pentachlorophenol (PCP), a polluting substance believed to exert a narcotic effect, was assayed using the Caco-2 cell line as a model. In order to assess this toxicity as fully as possible, several viability tests, each examining different endpoints, have been used. Neutral red uptake was found to be more sensitive to PCP than MTT and Alamar Blue tests. Transepithelial electrical resistance (TEER) was shown to be the most sensitive to PCP at concentrations and exposure times where the Alamar Blue, LDH leakage and Blue Dextran passage did not evidence any effect. Blue Dextran passage and optical microscopy revealed cellular detachment at concentrations where LDH and Alamar Blue showed little or no cytotoxicity. Thus, PCP seems to affect the integrity of the intestinal barrier at levels where no cytotoxicity is seen. Our results support the notion that TEER can be used as a very sensitive method for evaluating membrane-perturbing toxicants.

Use of the caco-2 model in the screening of polluting substance toxicity.

The aim of this work was to investigate the oral toxicity of representative chemicals chosen from each class of the list of 132 substances present in industrial effluents after the EEC Directive 76-464. Owing to its characterization as a model of the intestinal epithelium, the CaCo-2 cell line model was chosen. Cytotoxicity was assayed using the tetrazolium blue (MTT) test. For most of the substances, a linear correlation was observed between the octanol/water partition coefficient (log Kw) and the median inhibition concentration (IC(50)). This relationship between lipophilicity and toxicity is the hallmark of a narcotic mechanism of action. However, diethylamine appeared more toxic than the correlation would predict. Other amines were then tested (tert-butylamine, n-butylamine and benzylamine). All of these did not fit into the baseline correlation. The IC(50) were corrected by taking into account only the non-ionized, lipid insoluble, concentration at pH7.3. The amines still did not fit into the correlation, reinforcing the idea of a non-narcotic mechanism. The toxicity of a large number of substances can thus be predicted from their physico-chemical properties only when the substances exert a direct and non-specific effect. The amines appeared more toxic than substances with the same partition coefficient, showing that knowledge of the only lipophilicity is too restrictive to predict toxicity.

Uptake and metabolism of T-2 toxin in relation to its cytotoxicity in lymphoid cells.

The sensitivity of lymphoid cells to the cytotoxic effects of T-2 toxin (T-2) varies according to their degree of differentiation. To understand the mechanisms of these variations, the uptake and the metabolism of T-2 in susceptible (human lymphoma Daudi and phytohaemagglutinin-stimulated murine lymphocytes) and resistant (human leukaemia KE37 and REH) cells were studied in culture. When cells were incubated with [3H]T-2 a significant increase in the quantity of T-2 associated with the cell occurred during the first 30 min, this increased further from 10-16 hr, and decreased after 24 hr. Daudi and REH cells took up 20 and 3% of the T-2 present in the medium, respectively. Metabolites, extracted from the culture medium and from cells, were analysed by the thin-layer chromatography. The products were identified by comparison with standards for T-2 tetraol, T-2 triol, HT-2 toxin, neosolaniol and T-2. Qualitatively, similar metabolic pathways were found in all cells examined. The presence of these metabolites demonstrated that T-2 was taken up by these cells. A correlation existed between the relative sensitivities of the cells toward T-2 and the amount of intracellular T-2 and/or metabolites. It is thought that differences in the kinetics of uptake and processing of T-2 account for the known differences in cellular sensitivities to the toxin.

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Detection of immunotoxic effects of estrogenic and androgenic endocrine disrupting compounds using splenic immune cells of the female three-spined stickleback, Gasterosteus aculeatus (L.).

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100nM over 12h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.

Effects of aluminium and bacterial lipopolysaccharide on oxidative stress and immune parameters in roach, Rutilus rutilus L.

Aluminium is used in diverse anthropogenic processes at the origin of pollution events in aquatic ecosystems. In the Champagne region (France), high concentrations of aluminium (Al) are detected due to vine-growing practices. In fish, little is known about the possible immune-related effects at relevant environmental concentrations. The present study analyzes the simultaneous effects of aluminium and bacterial lipopolysaccharide (LPS), alone and in combination, on toxicological biomarkers in the freshwater fish species Rutilus rutilus. For this purpose, roach treated or not with LPS were exposed to environmental concentrations of aluminium (100 µg/L) under laboratory-controlled conditions for 2, 7, 14 and 21 days. After each exposure time, we assessed hepatic lipoperoxidation, catalase activity, glutathione reductase activity and total glutathione content. We also analyzed cellular components related to the LPS-induced inflammatory response in possible target tissues, i.e. head kidney and spleen. Our results revealed a significant prooxidant effect in the liver cells and head kidney leukocytes of roach exposed to 100 µg of Al/L for 2 days. In liver, we observed more lipoperoxidation products and lower endogenous antioxidant activity levels such as glutathione reductase activity and total glutathione content. These prooxidant effects were associated with a higher oxidative burst in head kidney leukocytes, and they were all the more important in fish stimulated by LPS injection. These findings demonstrate that environmental concentrations of Al induce oxidative and immunotoxic effects in fish and are associated to an immunomodulatory process related to the inflammatory response.

Relationship between DNA damage in sperm after ex vivo exposure and abnormal embryo development in the progeny of the three-spined stickleback.

Many xenobiotics released in the aquatic environment exhibit a genotoxic potential toward organisms. Long term exposure to such compounds is expected to lead to multigenerational reproductive defects, further influencing the recruitment rate and hence, the population dynamics. Paternal exposure to genotoxicants was previously shown to increase abnormal development in the progeny of mammalian or aquatic species. The aim of this study was to evaluate the relationship between DNA damage in sperm of the fish three-spined stickleback and progeny developmental defects. Spermatozoa were exposed ex vivo to an alkylating agent (methyl methanesulfonate) before in vitro fertilization and DNA damage was assessed by the alkaline comet assay. A significant relationship between abnormal development and sperm DNA damage was underlined. This study illustrates the interest to use germ cell DNA damage after ex vivo exposure to evaluate the impact of genotoxic compounds on progeny fitness in aquatic organisms.

Anti-androgenic activities of environmental pesticides in the MDA-kb2 reporter cell line.

Pesticides have been suspected to act as endocrine disruptive compounds (EDCs) through several mechanisms of action, however data are still needed for a number of currently used pesticides. In the present study, 30 environmental pesticides selected from different chemical classes (azole, carbamate, dicarboximide, organochlorine, organophosphorus, oxadiazole, phenylureas, pyrazole, pyrimidine, pyrethroid and sulfonylureas) were tested for their ability to alter in vitro the transcriptional activity of the androgen receptor in the MDA-kb2 reporter cell line. The responsiveness of the system was checked by using a panel of reference ligands of androgen and glucocorticoid receptors. When tested alone at concentrations up to 10 µM, none of the studied pesticides were able to induce the reporter gene after a 18 h exposure. Conversely, co-exposure experiments with 0.1 nM dihydrotestosterone (DHT) allowed identifying 15 active pesticides with IC(50) ranging from 0.2 µM for vinclozolin to 12 µM for fenarimol. Fipronil and bupirimate were here newly described for their AR antagonistic activity.