Evidence of estrogenic mixture effects on the reproductive performance of fish

Recent research into the effects of mixtures of estrogenic chemicals has revealed the capacity for similarly acting chemicals to act in combination, according to the principles of concentration addition. This means that, collectively, they may pose a significant environmental risk, even when each component is present at a low and individually ineffective concentration. The aim of this study was to investigate the ecological significance of mixture effects at low-effect concentrations by assessing the combined effect of estrogenic chemicals on the reproductive performance of fish. Pairs of fathead minnows were exposed to five estrogenic chemicals. Endpoints analyzed included fecundity, the expression of male secondary sexual characteristics, somatic indices, and vitellogenin induction. In the first phase of the study, a concentration-response analysis was performed to investigate the relative sensitivity of these endpoints. In the second phase, mixture effects at low-effect concentrations were explored by exposing fish to each of the mixture components, individually and in combination. Data from these experiments provide evidence of mixture effects on fitness and fecundity, demonstrating the capacity for chemicals to act together to affect reproductive performance, even when each component is present belowthe threshold of detectable effects. This has important implications for hazard assessment and contributes to our understanding of mixture effects at increasing levels of biological complexity.     

Aquatic hypoxia is an disrupter and impairs fish reproduction

There is increasing concern that certain chemicals in the aquatic environment can disrupt endocrine systems, leading to reproductive impairment and threatening survival of wild populations of invertebrates, fish, bird, reptiles, and wildlife. For the first time, we report that hypoxia is also an endocrine disruptor and poses a significant threat to the reproduction and hence sustainability of fish populations. Serum levels of testosterone, estradiol, and triiodothyronine significantly decreased in carp (Cyprinus carpio) upon chronic exposure to hypoxia. These hormonal changes were associated with retarded gonadal development in both male and female carp, reduced spawning success, sperm motility, fertilization success, hatching rate, and larval survival, indicating that adverse effects of hypoxia on reproductive performance resulted from endocrine disruption. Since aquatic hypoxia commonly occurs over thousands of square kilometers in aquatic systems worldwide, our results imply that endocrine disruption and reproductive impairment in fish may be a widespread environmental problem.     

Something from "nothing"--eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects

We tested whether multicomponent mixtures of xenoestrogens would produce significant effects when each component was combined at concentrations below its individual NOEC or EC01 level. The estrogenic effects of eight chemicals of environmental relevance, including hydroxylated PCBs, benzophenones, parabenes, bisphenol A, and genistein, were recorded using a recombinant yeast estrogen screen (YES). To ensure that no chemical contributed disproportionately to the overall combination effect, a mixture was prepared at a mixture ratio proportional to the potency of each individual component. The performance of four approaches for the calculation of additive combination effects (concentration addition, toxicity equivalency factors, effect summation, and independent action) was compared. Experimental testing of the predictions revealed that concentration addition and its application, the toxicity equivalency factor approach, were valid methods for the calculation of additive mixture effects. There was excellent agreement between prediction and observation. In contrast, independent action and effect summation led to clear underestimations of the experimentally observed responses. Crucially, there were substantial mixture effects even though each chemical was present at levels well below its NOEC and EC01. We conclude that estrogenic agents are able to act together to produce significant effects when combined at concentrations below their NOECs. Our results highlight the limitations of the traditional focus on the effects of single agents. Hazard assessments that ignore the possibility of joint action of estrogenic chemicals will almost certainly lead to significant underestimations of risk.     

Biological validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens

The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassaywere examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used accelerated solvent extraction (ASE) and gel permeation chromatography (GPC) and was validated with respect to recovery of biological response taking mixture effects into account. Four mixtures of three to six xenoestrogenic compounds (bisphenol A, 4-nonylphenol, (4,4'-dichlorodiphenyl)trichloroethane, (2,4'-dichlorodiphenyl)trichloroethane, dieldrin, 4-n-octylphenol, alpha-chlordane, dibutylphthalate, (4,4'-dichlorodiphenyl)dichloroethylene, and 2,4,5-trichlorobiphenyl) were prepared. Experimentally determined mixture effects were well described by the concept of concentration addition (CA), as expected for similarly acting compounds. Observed estradiol equivalence factors of the mixtures (on average 1.2 +/- 0.3) agreed very well with the value predicted according to CA. The sample preparation method was then applied to pure mixtures of standards and to sediment spiked with one of the mixtures. Recoveries of estrogenic compounds were estimated by determination of their mixture potencies in ER-CALUX and compared to the mixture effects predicted by CA. Recoveries of estrogenic activity were between 80 and 129%, indicating that the additive behavior of mixtures of xeno-estrogens is well conserved during sample preparation. Together with an average repeatability of 18.3%, low average limit of detection (2.6 +/- 1.8 pg of EEQ/ g), and coefficient of variance (3.5 +/- 3.3%),this demonstrated the suitability of the sample preparation method for the analysis of mixtures of (xeno-)estrogenic compounds in sediment with the ER-CALUX assay.     

Predictive environmental risk assessment of chemical mixtures: a conceptual framework

Environmental risks of chemicals are still often assessed substance-by-substance, neglecting mixture effects. This may result in risk underestimations, as the typical exposure is toward multicomponent chemical "cocktails". We use the two well established mixture toxicity concepts (Concentration Addition (CA) and Independent Action (IA)) for providing a tiered outline for environmental hazard and risk assessments of mixtures, focusing on general industrial chemicals and assuming that the "base set" of data (EC50s for algae, crustaceans, fish) is available. As mixture toxicities higher than predicted by CA are rare findings, we suggest applying CA as a precautious first tier, irrespective of the modes/mechanisms of action of the mixture components. In particular, we prove that summing up PEC/PNEC ratios might serve as a justifiable CA-approximation, in order to estimate in a first tier assessment whether there is a potential risk for an exposed ecosystem if only base-set data are available. This makes optimum use of existing single substance assessments as more demanding mixture investigations are requested only if there are first indications of an environmental risk. Finally we suggest to call for mode-of-action driven analyses only if error estimations indicate the possibility for substantial differences between CA- and IA-based assessments.     

Modulation of the hepatic CYP1A1 system in the marine fish Gobius niger, exposed to xenobiotic compounds

Anthropogenic chemicals in the aquatic environment are known to cause reproductive disturbances in vertebrate and invertebrate organisms by interfering with the endocrine systems. Large efforts have recently been devoted to dissect the mechanisms of action of xenobiotics in aquatic species, with the ultimate aim of detecting and controlling the effects of chemical exposure on the aquatic ecosystem and humans. In the present paper, males of a marine species, the black goby (Gobius niger), were treated with estrogenic and dioxin-like compounds commonly discharged into the environment from industry, agriculture, and urban waste such as nonylphenol (NP) and beta-naphthoflavone (beta-NF). Their effects were compared with those induced by estradiol (E2), analyzing the expression of biomarkers commonly used in ecotoxicological studies such as vitellogenin (VTG) and cytochrome P4501A1. The treatment with NP induced the synthesis of the female specific protein VTG in males, showing its estrogenic activity. NP and E2 lowered cytochrome P4501A1 basal levels while beta-NF determined a significant rise of its expression. The detoxification pathway was investigated, and the most relevant finding of this paper was the evidence that cytochrome P4501A1 inhibition by estrogen and estrogen-like compounds is mediated through the activation of the aryl hydrocarbon receptor repressor.     

Assessment of chemical mixtures and groundwater effects on Daphnia magna transcriptomics

Small organisms can be used as biomonitoring tools to assess chemicals in the environment. Chemical stressors are especially hard to assess and monitor when present as complex mixtures. Here, fifteen polymerase chain reaction assays targeting Daphnia magna genes were calibrated to responses elicited in D. magna exposed for 24 h to five different doses each of the munitions constituents 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, trinitrobenzene, dinitrobenzene, or 1,3,5-trinitro-1,3,5-triazacyclohexane. A piecewise-linear model for log-fold expression changes in gene assays was used to predict response to munitions mixtures and contaminated groundwater under the assumption that chemical effects were additive. The correlations of model predictions with actual expression changes ranged from 0.12 to 0.78 with an average of 0.5. To better understand possible mixture effects, gene expression changes from all treatments were compared using high-density microarrays. Whereas mixtures and groundwater exposures had genes and gene functions in common with single chemical exposures, unique functions were also affected, which was consistent with the nonadditivity of chemical effects in these mixtures. These results suggest that, while gene behavior in response to chemical exposure can be partially predicted based on chemical exposure, estimation of the composition of mixtures from chemical responses is difficult without further understanding of gene behavior in mixtures. Future work will need to examine additive and nonadditive mixture effects using a much greater range of different chemical classes in order to clarify the behavior and predictability of complex mixtures.     

Applications of contaminant fate and bioaccumulation models in assessing ecological risks of chemicals: a case study for gasoline hydrocarbons

Mass balance models of chemical fate and transport can be applied in ecological risk assessments for quantitative estimation of concentrations in air, water, soil, and sediment. These concentrations can, in turn, be used to estimate organism exposures and ultimately internal tissue concentrations that can be compared to mode-of-action-based critical body residues that induce toxic effects. From this comparison, risks to the exposed organism can be evaluated. To demonstrate the use of fate models in ecological risk assessment, we combine the EQuilibrium Criterion (EQC) environmental fate model with a simple screening level biouptake model for three representative organisms: a bird, a mammal, and a fish. This effort yields estimates of internal body concentrations that can be compared with levels known to elicit toxic effects. As an illustration, we present an analysis of 24 hydrocarbon components of gasoline that differ in properties but are assumed to elicit toxicity by a common narcotic mode of action. Results demonstrate that differences in chemical properties and mode of entry into the environment lead to profound differences in the efficiency of transport from emission to target biota. We discussthe implications of these results and draw attention to the insights gained about regional fate and ecological risks associated with gasoline. This approach is suitable for assessing single chemicals or mixtures that have similar modes of action. We conclude that the model-based methodologies presented are widely applicable for screening level ecological risk assessments that support effective chemicals management.     

In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish

The polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are used as fragrance ingredients in perfumes, soaps, and household cleaning products. They are known to be ubiquitously present in the aquatic environment, and because of their lipophilic nature, they tend to bioaccumulate in aquatic biota. In surface waters, concentrations between 1 ng/L and 5 microg/L have been found, depending mainly on the proportion of sewage effluents in the water. In fish, under normal environmental conditions, concentrations in the microgram per kilogram fresh weight (fw) range are found. In a previous study we showed that AHTN and HHCB exert mainly antiestrogenic effects on the human estrogen receptor alpha (ERalpha) and ERbeta in an in vitro reporter gene assay. In the current study, we assessed the in vitro antiestrogenic effects of both musks on zebrafish ERs. Antagonism was observed on zfERbeta, and more pronounced on the newly cloned zfERgamma. Using a transgenic zebrafish assay, we studied antiestrogenicity of the musks in vivo. Dose-dependent antagonistic effects were observed at concentrations of 0.1 and 1 microM AHTN and HHCB. GC-MS analysis showed that the musks bioaccumulated in the fish, with internal concentrations (15-150 mg/kg fw) which were roughly 600 times higher than the nominal test doses. To our knowledge, this is the first time that environmental contaminants are shown to be antiestrogenic in an in vivo fish assay that focuses solely on ER-mediated effects. This makes the transgenic zebrafish assay a promising tool for the rapid detection of both estrogenic and antiestrogenic effects of chemicals in fish.     

Identification of estrogenic compounds in fish bile using bioassay-directed fractionation

Conjugates of estrogenic chemicals, endogenous as well as xenobiotic, are mainly excreted via bile into the intestine. Therefore, measurement of estrogenic activity in bile yields useful information about an organism's internal exposure to (xeno-)estrogens. Although previous studies in The Netherlands have reported estrogenic activity in male fish bile, the contribution of natural hormones and xenobiotic substances to this activity is unknown. To identify compounds responsible for estrogenic activity in fish bile, we developed a bioassay-directed fractionation method for estrogenic chemicals. In this approach, the in vitro reporter gene assay ER-CALUX (Estrogen Responsive Chemical Activated Luciferase Gene Expression) was used to assess estrogenic activity in deconjugated bile samples and to direct RP-HPLC fractionation and chemical analysis (by GC-MS) of estrogenic compounds. The method was applied to bile from male breams (Abramis brama) collected at three locations in The Netherlands. At one of these locations, the River Dommel, extremely high levels of plasma vitellogenin and a high incidence of intersex gonads in these male breams have previously been observed, indicating the exposure to estrogens. In this study, the natural hormones 17beta-estradiol, estrone, and estriol accounted for the majority of estrogenic activity in male bream bile. At the River Dommel, the synthetic contraceptive pill component ethynylestradiol was found in effective concentrations as well. The detected natural and synthetic hormones may be responsible forthe estrogenic effects observed in wild bream from this location. Furthermore, a large number of xenobiotic chemicals was detected at relatively high levels in bile, including triclosan, chloroxylenol, and clorophene. Although chloroxylenol was shown for the first time to be weakly estrogenic, these compounds did not contribute significantly to the estrogenic activity observed.     

Pesticide residues and estrogenic activity in fruit and vegetables sampled from major fresh produce markets in South Africa

Food is likely to be one of the major pathways through which people are exposed to endocrine-disrupting chemicals. With the exception of residual effects, there are concerns that a number of naturally occurring and synthetic chemicals exert adverse effects upon endocrine systems in wildlife and humans. The current study reports selected pesticide concentrations and the total estrogenic activity of fruit and vegetables using the recombinant yeast oestrogen screen (YES) and T47D-KBluc reporter gene assays. A total of 53 food samples (27 fruit and 26 vegetables) from Johannesburg and Tshwane fresh produce markets (in South Africa) were analysed. Of these, 17 contained one to three different pesticide residues with concentrations ranging between 0.01 and 0.68 mg kg^–1, whereas in the rest of the samples no residues were detected. All pesticides detected except in one sample were below the maximum residue level (MRL), but others were unauthorised for use in specified fruit and vegetables. Estrogenic activity was detected in 26.4% (14 samples) of the samples tested, and the estradiol equivalents ranged from 0.007 to 2 pg g^–1. Although the estrogenic activity was low, it may contribute to adverse health effects. Continuous monitoring for pesticides in fruit and vegetables is important in view of the unauthorised pesticides detected in produce from South Africa and the endocrine-disrupting chemical activity found.     

Are oral contraceptives a significant contributor to the estrogenicity of drinking water?

Recent observed feminization of aquatic animals has raised concerns about estrogenic compounds in water supplies and the potential for these chemicals to reach drinking water. Public perception frequently attributes this feminization to oral contraceptives (OCs) in wastewater and raises concerns that exposure to OCs in drinking water may contribute to the recent rise in human reproductive problems. This paper reviews the literature regarding various sources of estrogens, in surface, source and drinking water, with an emphasis on the active molecule that comes from OCs. It includes discussion of the various agricultural, industrial, and municipal sources and outlines the contributions of estrogenic chemicals to the estrogenicity of waterways and estimates that the risk of exposure to synthetic estrogens in drinking water on human health is negligible. This paper also provides recommendations for strategies to better understand all the potential sources of estrogenic compounds in the environment and possibilities to reduce the levels of estrogenic chemicals in the water supply.     

Relative potencies and combination effects of steroidal estrogens in fish

The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.     

Deviation from additivity with estrogenic mixtures containing 4-nonylphenol and 4-tert-octylphenol detected in the E-SCREEN assay

An intriguing deviation from expected additivity is reported with mixtures containing 17beta-estradiol, 17alpha-ethinylestradiol, genistein, bisphenol A, 4-nonylphenol, and 4-tert-octylphenol. The effect of these chemicals on the proliferation of estrogen-dependent MCF-7 human breast cancer cells (the E-SCREEN) was measured. Data variance-component analyses, carried out to optimize the assay for mixture studies, showed that between-experiment variability was the dominant source of data variation. Adoption of a data-normalization procedure reduced the impact of this variability and allowed the pooling of historical E-SCREEN data. Concentration-response relationships for all six chemicals were recorded and utilized to calculate predictions of their joint effects by employing the model of concentration addition. Surprisingly, the observed combination effects of the mixture fell short of the additivity expectations, indicating weak antagonism. Experimental or prediction errors were ruled out as possible explanations for this deviation, which suggested that it might be the result of interactions between mixture components. With the aim of identifying the responsible components, mixtures were designed by excluding one or more of the chemicals from the original six-component mixture, and the resulting combination effects were assessed. These permutation studies allowed us to conclude thatthe presence of 4-nonylphenol and 4-tert-octylphenol is associated with the antagonisms observed with the six-component mixture and thus negatively affected the predictability of mixture effects. Future mixture studies utilizing the E-SCREEN with endocrine disrupters that also exhibit toxicity or growth-inhibitory effects will have to take account of the possibility that such interactions might compromise the predictability of estrogenic combination effects.     

Removal of estrogenic activity from municipal waste landfill leachate assessed with a bioassay based on reporter gene expression

The leachate of a municipal waste landfill was tested for estrogenic activity using a reporter-gene-based bioassay with a human breast-cancer-derived cell line (MVLN cells). The leachate was processed with two different membrane-employing processes operating in parallel. One process consists of aerobic biological degradation, ultrafiltration, and subsequent adsorption to activated carbon. The second process was a reverse osmosis treatment of the raw leachate. Both processes are common in the treatment of landfill leachate. Here, the efficacy of the two processes to remove "estrogenicity" was compared. Both treatment processes removed more than 97% of the estrogenic activity, calculated as estradiol equivalents (EEQs), but they were not equally effective. After adsorption to activated carbon, no estrogenicity was detected, whereas concentrated effluent of the reverse osmosis treatment still elicited an estrogenic response in the bioassay. On the basis of chemical analysis, it is proposed that bisphenol A was responsible for the majority of estrogenic activity in the raw and treated leachate. Although the contribution of treated leachate to the estrogenic load on the aquatic environment seems to be low compared to that of sewage treatment works, the high estrogenic activity in raw landfill leachate stresses the necessity for the appropriate treatment of these leachates.     

Gene expression profiling for understanding chemical causation of biological effects for complex mixtures: a case study on estrogens

Gene expression profiling offers considerable potential for identifying chemical causation of effects induced in exposures to complex mixtures, and for understanding the mechanistic basis for their phenotypic effects. We characterized gene expression responses in livers and gonads of fathead minnow (Pimephales promelas) exposed (for 14-21 days) to estrogenic wastewater treatment works final effluents with varying potencies and assessed the extent to which these expression profiles mapped with those induced by individual steroid estrogens present in the effluents (17beta-estradiol and 17alpha-ethinylestradiol) and, thus, were diagnostic of estrogen exposure. For these studies, we adopted a targeted approach (via real-time PCR) with a suite of 12 genes in liver and 21 genes in gonad known to play key roles in reproduction, growth and development (processes controlled by estrogens) and responses were compared with effects on phenotypic end points indicative of feminization. Gene responses to effluent were induced predominantly in a linear (monotonic) concentration-dependent manner but were complex with many genes responding differently between tissue types and sexes. The gene expression profiles for the estrogenic effluents and the individual steroid estrogens had many common features. There were marked differences in the profiles between the two effluents, however, that were not explained by differences in their estrogenic potencies, suggesting that these may have arisen as a consequence of differences in the contents of other chemicals, which may act directly or indirectly with the estrogen-response pathway to alter estrogen-induced gene expression. These data demonstrate that the patterns of gene expression induced by estrogenic effluents, although complex, can be diagnostic for some of the estrogens they contain and provide insights into the mechanistic basis for the phenotypic effects seen.     

Predicting concentrations of organic chemicals in fish by using toxicokinetic models

Quantification of chemical toxicity continues to be generally based on measured external concentrations. Yet, internal chemical concentrations have been suggested to be a more suitable parameter. To better understand the relationship between the external and internal concentrations of chemicals in fish, and to quantify internal concentrations, we compared three toxicokinetic (TK) models with each other and with literature data of measured concentrations of 39 chemicals. Two one-compartment models, together with the physiologically based toxicokinetic (PBTK) model, in which we improved the treatment of lipids, were used to predict concentrations of organic chemicals in two fish species: rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas). All models predicted the measured internal concentrations in fish within 1 order of magnitude for at least 68% of the chemicals. Furthermore, the PBTK model outperformed the one-compartment models with respect to simulating chemical concentrations in the whole body (at least 88% of internal concentrations were predicted within 1 order of magnitude using the PBTK model). All the models can be used to predict concentrations in different fish species without additional experiments. However, further development of TK models is required for polar, ionizable, and easily biotransformed compounds.     

Mixture toxicity of reactive chemicals by using two bacterial growth assays as indicators of protein and DNA damage

The mixture toxicity of reactive chemicals was investigated with a set of bioanalytical tests that quantify not only the toxic effects but also allow the identification of the preferred target of reactive chemicals in bacterial cells. Softer electrophiles such as acrylates react preferentially with thiol groups in proteins and peptides, and harder electrophiles such as epoxides preferentially attack DNA. In addition, some compounds, e.g., benzyl chloride, have no preference for a biological target and damage both DNA and proteins. A thiophenol was used as a model compound representing nucleophiles. We explored if the paradigms of mixture toxicity also hold true for reactive chemicals. Compounds with the same targets and the same modes of action should act concentration additive in mixtures, and compounds with different modes of action should act according to the concept of independent action. In addition, we investigated the potential for interaction of compounds of mixtures of electrophiles or electrophiles plus nucleophiles, which might lead to synergistic or antagonistic effects. The toxicity of mixtures of electrophiles with a single preferred target was consistent with the prediction for concentration addition. Unfortunately, the predictions for independent action did not differ much from those for concentration addition; therefore it was not possible to differentiate between these two models. Mixtures of two groups with different preferred target sites clearly showed concentration addition. In contrast, mixtures of compounds with multiple targets, i.e., compounds that show nonspecific reactivity toward any biological nucleophile, exhibited effects that lay distinctly between the predictions for concentration addition and independent action. We observed neither synergism (higher toxicity than predicted by concentration addition) nor antagonism (lower toxicity than predicted by independent action) for mixtures of electrophiles. Binary combinations of different electrophiles with the nucleophile 4-chlorothiophenol yielded smaller effects than those expected from the prediction for independent action. The degree of antagonism was correlated with the reaction rate constant of the electrophile with the thiol group of glutathione, which indicates that the interaction between the mixture components occur in the toxicokinetic phase and is purely a result of chemical reactivity between the mixture components. Overall, we conclude that the concepts of mixture toxicity apply not only for baseline toxicity and receptor-mediated mechanisms, as has been shown in a large number of studies, but also for reactive mechanisms of toxicity, provided that one has checked beforehand that no chemical reactions occur between the mixture components.     

Lessons from endocrine disruption and their application to other issues concerning trace organics in the aquatic environment

In the past 10 years, many thousands of research papers covering the many different aspects of endocrine disruption in the environment have been published. What has been learned from all this research? We have tried to reduce this very large volume of research into a relatively small number of "lessons". Hence, this paper is not a typical review, but instead it summarizes our personal opinions on what we consider are the major messages to have come from all this research. We realize that what has been a lesson to us may have been obvious from the outset to someone more knowledgeable on that particular aspect of the burgeoning field of endocrine disruption. In addition, it is inevitable that others will consider that we have "missed" some lessons that they would have expected to find included in our list. If so, we encourage them to submit them as responses to our paper. Our own lessons range widely, from the design and interpretation of data from fieldwork studies, through some key messages to come out of the very many laboratory studies that have been conducted, to issues around the sources and fates in the environment of endocrine-disrupting chemicals, and finally to the key role of sewage treatment in controlling the concentrations of these chemicals in the aquatic environment. Having (hopefully) learned our lessons, we have then applied them to the difficult issue of how best to approach future concerns about the potential impacts of other new and emerging contaminants (e.g., pharmaceuticals) on wildlife.