Influence of a collapsed coastal landfill on metal levels in sediments and biota--a portent for the future?

In May 2008 a coastal landslide deposited landfill debris onto the shore near Lyme Regis, UK. Six months later, intertidal sediments and biota from the area were sampled to determine whether the landslip had affected distribution and bioavailability of metals in the area and if there were any biological effects. Highest sediment concentrations for the majority of metals occurred near the landslip zone and in several cases exceeded Threshold Effects or Probable Effects Levels (As, Cu, Hg, Ni, Pb, Zn). The 1 M HCl-extractable fraction of Cd, Pb and Zn in sediments also increased near the landslip. Metal bioaccumulation by intertidal biota showed variability between different species and metals, but there were several instances of increased accumulation near the landslip through increased availability from seawater, sediment and dietary sources. In most cases, metal concentrations in molluscs exceeded Oslo and Paris Commission (OSPAR) background concentrations (BCs) together with background assessment concentrations (BACs) at some sites. Kidney tissues in winkles (Littorina littorea) were measured for evidence of oxidative stress using the Total Oxyradical Scavenging Capacity (TOSC) assay. Responses to peroxynitrite, peroxyl and hydroxyl radicals suggested raised levels of TOSC in animals from the sites close to or east of the landfill waste. There have been very few studies of direct impact of landfills on the marine environment and this study could serve as a practical model for similar events driven by sea level rise.

Immunological function in marine invertebrates: responses to environmental perturbation.

The inception of ecological immunology has led to an increase in the number of studies investigating the impact of environmental stressors on host immune defence mechanisms. This in turn has led to an increased understanding of the importance of invertebrate groups for immunological research. This review discusses the advances made within marine invertebrate ecological immunology over the past decade. By demonstrating the environmental stressors tested, the immune parameters typically investigated, and the species that have received the greatest level of investigation, this review provides a critical assessment of the field of marine invertebrate ecological immunology. In highlighting the methodologies employed within this field, our current inability to understand the true ecological significance of any immune dysfunction caused by environmental stressors is outlined. Additionally, a number of examples are provided in which studies successfully demonstrate a measure of immunocompetence through alterations in disease resistance and organism survival to a realized pathogenic threat. Consequently, this review highlights the potential to advance our current understanding of the ecological and evolutionary significance of environmental stressor related immune dysfunction. Furthermore, the potential for the advancement of our understanding of the immune system of marine invertebrates, through the incorporation of newly emerging and novel molecular techniques, is emphasized.

Effects of a weak oestrogenic active chemical (4-tert-pentylphenol) on pair-breeding and F1 development in the fathead minnow (Pimephales promelas).

A fish full life-cycle (FFLC) is the most comprehensive test to determine reproductive toxicity of chemicals to fish and this is likely to apply equally to endocrine active chemicals (EACs). However, FFLC tests use large numbers of animals, are expensive and time consuming. Alternative chronic tests, to the FFLC, potentially include sensitive life-stage windows of effect, such as sexual differentiation, early gonadal development and reproduction. In this paper, a fish pair-breeding study was applied to assess the biological effects of a weak environmental oestrogen, 4-tert-pentylphenol (4TPP), on reproduction and subsequent development of the F1 generation. The results of this study were then compared with the results for a published FFLC study, with this chemical. Fathead minnows (Pimephales promelas) were held in pairs and their reproductive performance assessed over two concurrent 21-day periods, the first without exposure to the test chemical, followed by the second with exposure to the test chemical, in a flow-through system at 25+/-1 degrees C. Embryos from two pairs, per treatment, were subsequently grown up in clean water until 90 days post-hatch to assess developmental effects of the parental exposure on the F1 generation. Nominal (measured geometric mean, time weighted) test concentrations of 4TPP were 56 (48), 180 (173) and 560 (570) microg l(-1). A significant decrease in fecundity was observed in all 4TPP exposed fish (mean number of eggs spawned per pair and number of spawns per pair) when compared to the solvent control. Vitellogenin (VTG) was significantly elevated in F0 males exposed to 560 microg 4TPPl(-1). Somatic endpoints, secondary sexual characteristics (SSC) and gonadosomatic index (GSI) were not affected by the 4TPP exposure. In the F1 generation, there were no treatment-related effects on hatching success, survival, growth, SSC or GSI. Histological examination of the gonads of the F1 fish revealed no treatment-related effects on sex ratio, sexual differentiation or sexual development. However, plasma VTG concentrations were significantly elevated in F1 male fish, derived from parents that had previously been exposed to 4TPP at concentrations of > or = 180 microg l(-1). These data show that the reproductive performance test is suitable for detecting weak environmental oestrogenic chemicals and that exposure of adult fish to oestrogens can result in altered biomarker expression (VTG) of the F1 generation. Our findings indicate that the reproductive performance test was as sensitive for detecting effects on reproduction when compared with a published FFLC test for 4TPP.

Validation of the use of zebrafish larvae in visual safety assessment.

INTRODUCTION: The use of zebrafish (Danio rerio) larvae was investigated to predict adverse visual effects and to establish the potential application of this organism in early drug safety assessment. METHODS: Following a comparison of the effects of 4 compounds in TL and WIK strains of zebrafish larvae, a blinded validation set of 27 compounds was tested on WIK strain of larval zebrafish in the optomotor response (OMR) assay. Selected compounds were also tested in the optokinetic response (OKR) and locomotor assays. Larvae were exposed from 3-8 days post-fertilisation (d.p.f.) by immersion in embryo culture media (E3) containing the compound in 1% DMSO (v/v). At 8 d.p.f. toxicity was assessed and the OMR or OKR assays were undertaken at non-toxic treatment levels. Compounds were then rated as 'red', 'amber' or 'green' according to their effects on visual function prior to unblinding of the identities of the test compounds. RESULTS: Overall, the OMR assay revealed a good concordance between the effects of compounds in WIK strain zebrafish with the data available from other in vivo and in vitro models or the clinic: thirteen out of nineteen positive compounds produced the expected effect while six of the eight negative compounds were correctly predicted. This gave an overall predictivity of 70% with a sensitivity of 68% and a specificity of 75%. The two false positive compounds were further tested in locomotor and optokinetic response assays and it was shown that a motility defect, rather than an effect on vision, had given rise to the positive result in the OMR assays. Therefore, the OMR assay would best be employed with other techniques to identify false positives. Further studies on two of the false negatives at higher concentrations suggested that the initial concentrations tested were too low. Therefore, it should be ensured that the maximum tolerated concentration is tested in the OMR assay. A comparison of four standard compounds in the OMR assay in WIK and TL zebrafish wild type strains revealed no difference in sensitivity between the strains. DISCUSSION: Overall, these results suggest that the OMR assay in zebrafish could be useful in predicting the adverse effects of drugs on visual function in man and would support its potential as a screen for 'frontloading' safety pharmacology assessment of this endpoint in vivo.

Development of chronic tests for endocrine active chemicals. Part 1. An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas).

An extended early-life stage test (based on OECD test guideline 210) was developed to allow the evaluation of a weak environmental oestrogen, 4-tert-pentyphenol (4TPP), on sexual differentiation and gonadal development. Fathead minnow (Pimephales promelas) embryos were exposed to three concentrations of 4TPP (56, 180 and 560 microg l(-1)) in a flow-through system, at 25+/-1 degrees C, for <107 days post-hatch (dph). In addition, some embryos were exposed to 180 microg 4TPPl(-1) until 30 or 60 dph, after which they were exposed to dilution water only until 107 dph. At 30, 60 and 107 dph fish were evaluated for growth and gonadal development (via histology), and at 107 dph fish were also evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI) and plasma vitellogenin (VTG). There were no effects of 4TPP on hatching success or survival, however, there was a delay in the time taken for embryos to hatch (560 microg 4TPPl(-1)). No treatment-related effects were observed on fish growth, with the exception of at 107 dph when the condition factor in female fish was reduced in all 4TPP continuous exposure treatments. Plasma VTG was only elevated in female fish exposed to 180 microg 4TPPl(-1) and inhibition of gonadal growth (GSI) occurred only in females exposed to 560 microg 4TPPl(-1). Histological examination of the gonads revealed delays and disruption in male sexual differentiation and development (180 microg 4TPPl(-1)) and no testicular tissue was observed in any fish exposed to 560 microg 4TPPl(-1). Mixed gonads (predominately testes with a scattering of primary oocytes) were present in fish exposed to all doses of 180 microg 4TPPl(-1) at 107 dph. Feminisation of the reproductive ducts (formation of an ovarian like cavity) occurred in the testis of all males exposed to 180 microg l(-1), regardless of length of 4TPP exposure. Results indicate that the period of 30-60 dph appears to be the sensitive window for disruption of formation of the reproductive duct and this effect is not reversible when the fish are transferred to dilution water. The data also show that this integrative test is suitable for the detection of a weak environmental oestrogen and comparisons of these results with that of a fish full life-cycle, in medaka, indicate that this test could be a suitable surrogate for a fish full life-cycle.

Acute and chronic effects of carrier solvents in aquatic organisms: a critical review.

Recognising the scientific and regulatory need for testing relatively hydrophobic or 'difficult substances', the OECD currently recommends that selected organic solvents may be used in aquatic toxicity testing in order to help achieve more effective dispersion of the toxicant. The OECD recommends a maximum solvent concentration of 100 microl l(-1) (with specific gravity equivalents to 100 microl l(-1) in parentheses) for acetone (79 mg l(-1)), dimethylformamide (95 mg l(-1)), dimethylsulfoxide (1.10 mg l(-1)), ethanol (78.9 mg l(-1)), methanol (79.2 mg l(-1)) and triethylene glycol (1.12 mg l(-1)). While this recommendation is supported by historical data, we have recently observed evidence that some solvents may affect the reproduction of certain fish species, and also impact biomarkers of endocrine disruption. This review presents available data on the effects of solvents in aquatic organisms, supplemented by relevant information from mammalian studies (e.g. effects on liver enzyme induction potentially altering the metabolism of sex hormones). In conclusion, it is recommended that maximum effort should be given to avoiding the use of carrier solvents wherever possible, for example through the use of saturation columns or other physical methods (e.g. stirring or ultrasonification). Where solvent use is necessary, however, it is recommended that in reproduction studies with aquatic organisms, the maximum solvent concentration should not exceed 20 microl l(-1) of dilution water.

A strategy to reduce the use of fish in acute ecotoxicity testing of new chemical substances notified in the European Union.

This study explores the applicability of a fish acute threshold (step-down) test approach for the assessment of new chemical substances notified in the EU. The proposed approach basically implies replacing the fish LC50 toxicity test with a simple acute threshold test and thus reducing the number of fish used and also costs. The fish test would be performed only at one concentration, the lowest between the EC50 concentrations obtained with previous testing with algae and daphnia. When fish would be more sensitive than algae and daphnia, testing with fish would be continued at lower concentrations (step-down). From step-down test results the LC50 value can be obtained by applying the binominal method of interpolation. These data can be used together with algal and daphnid data to provide the same Predicted No Effect Concentration values. The acute aquatic toxicity data used in this evaluation were extracted from the New Chemicals Database of the European Chemicals Bureau. The results show that 53.6-71.2% reduction of the number of fish used would be possible when applying this new testing strategy and suggest its use for regulatory purposes.

Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development.

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow (Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25 degrees C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6) microg l(-1), for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6) microg l(-1) and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5) microg l(-1). After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6 microg l(-1), day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5 microg l(-1)), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6 microg l(-1)), but inhibited in female fish (6.0 microg l(-1)), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens.

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes).

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.

A copepod life-cycle test and growth model for interpreting the effects of lindane.

A full life-cycle test was performed to measure the effects of lindane (3.2-3,200 microg l(-1)) on the survival, development and reproduction of the freshwater copepod Bryocamptus zschokkei. This copepod survived at relatively high concentrations of lindane compared with other freshwater crustaceans with a 10 day LC50 of 241 microg l(-1) (95% CL of 141-440). 'Equiproportional development', which assumes that each moult stage represents a specific proportion of the total development time, and is not affected by processes that influence metabolism such as temperature and food quality, was used to determine the mode of action of lindane on development in B. zschokkei. Development to adult was significantly longer at 100 microg l(-1) lindane compared with the controls, however, development remained equiproportional regardless of lindane exposure. Increased development times, therefore, are not due to a direct effect of lindane on the moulting process but are due probably to reduced food intake or increased metabolism through the stress imposed by toxicant exposure. Although the survival data suggest that B. zschokkei is relatively tolerant of lindane exposure, reproduction was affected at low lindane concentrations. At 32 microg l(-1) lindane, significantly fewer eggs and viable offspring were produced per female compared with the solvent control. At very low lindane concentrations (3.2 and 10 microg l(-1)), there was a significant increase in the numbers of offspring produced per female compared with the controls and this is interpreted as a hormesis effect. In conclusion, a full life-cycle test demonstrated B. zschokkei is relatively sensitive to lindane compared with other freshwater crustaceans. Incorporating a copepod growth model (equiproportional development) into the life-cycle test design, provided information on the dominant mode of action of the toxicant.

Cloning and gene expression of P450 17alpha-hydroxylase,17,20-lyase cDNA in the gonads and brain of the fathead minnow Pimephales promelas.

P450 17alpha-hydroxylase,17,20-lyase (P450c17) is a key steroidogenic enzyme in the production of androgens and, therefore, is also indispensable for the production of oestrogens (that are produced from the aromatisation of androgens). In this study, P450c17 cDNA was cloned from the ovary of the fathead minnow (FHM) and its gene expression was examined in the gonads and brains of male and female FHM at different stages of gonadal development with a view to developing an understanding of its involvement in the reproductive physiology in this species. The FHM-P450c17 cDNA sequence cloned was 1812 bp in length, with an open reading frame of 1554 nucleotides encoding a protein of 518 amino acids. Amino acid identity of FHM-P450c17 with P450c17s in other animals was up to 81.8% in other teleosts (channel catfish), 62% in elasmobranches (spiny dogfish), 64% in birds (chicken), and up to 48.8% in mammals (human). FHM-P450c17 gene expression occurred in the ovary, testis, and also in the brain (both male and female) at all stages of sexual development studied. Expression in the brain was at least 30-fold lower than in the gonads, but consistent in all fish life stages studied. In the testis, FHM-P450c17 gene expression was negatively correlated with gonadal development, but there was no obvious association between P450c17 gene expression and sexual development in the ovary, or brain (in both males and females). The results from this study demonstrate the expression of P450c17 in the brain for the first time in fish. Enzymatic studies are now needed to investigate the possible role of P450c17 in neurosteroid production in teleosts.

Ecological risk assessment and testing for endocrine disruption in the aquatic environment.

Field observations of endocrine disruption (ED) in fish and other aquatic species have contributed to a global effort to establish test methods for detecting ED effects in wildlife species. For ecological risk assessment, validated tests are needed for Amphibia, fish and aquatic invertebrates, supported by a tiered approach incorporating mechanistic data and exposure characterisation. The potential for extrapolation of ED data from mammalian to aquatic species may be limited, however, due to significant physiological differences in function and regulation of hormone systems in (aquatic) lower vertebrates and invertebrates. Presently, the OECD is considering a tiered approach for ED risk assessment, incorporating a fish 14-day screening assay (Tier 1); fish development and reproduction tests (both Tier 2); and a fish full life-cycle test (Tier 3). For detection of (anti-)oestrogens, the yolk-precursor protein vitellogenin is an ideal biomarker of exposure and functionally equivalent biomarkers are being sought for (anti-)androgens in fish. At the two higher tiers, impacts are assessed in terms of apical endpoints (e.g. development, breeding behaviour and fecundity) and also gonadal histopathology. Validation of these higher tier tests should include comparison of sensitivity of biochemical and apical endpoints to optimise the value of biomarkers for predicting adverse health effects (e.g. impaired reproduction). The specificity of future OECD fish and amphibian test guidelines for endocrine disrupters needs further consideration through inclusion of mechanistic endpoints based on state-of-the-art molecular endocrinology.

Exposure to exogenous 17beta-oestradiol disrupts p450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas).

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (p450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17beta-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of p450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on p450aromB mRNA expression in the gonads; E2 up-regulated p450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, p450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on p450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.

Screening of environmental contaminants for ecdysteroid agonist and antagonist activity using the Drosophila melanogaster B(II) cell in vitro assay.

The B(II) bioassay was developed as a rapid and reliable tool for detecting potential insect growth regulators acting as ecdysteroid receptor (ant)agonists. Based on an ecdysteroid-responsive cell line from Drosophila melanogaster, this microplate assay is ideally suited to the evaluation of environmental contaminants as potential endocrine disrupters. Data are presented for about 80 potential environmental contaminants, including industrial chemicals, pesticides, pharmaceuticals, phytoestrogens, and vertebrate steroids, and are compared with data for known (ant)agonists. Apart from androst-4-ene-3,17-dione (a weak antagonist), vertebrate steroids were inactive at concentrations up to 10(-3) M. The vast majority of xenobiotics also showed no (ant)agonist activity. Among the industrial chemicals, antagonistic activity was observed for bisphenol A median effective concentration (EC50) of 1.0 x 10(-4) M and diethylphthalate (EC50 of 2.0 x 10(-3) M). Some organochlorine compounds also showed weak antagonistic activity, including o,p'-dichlorodiphenyldichloroethylene (DDE), p,p'-DDE, dieldrin, and lindane (EC50 of 3.0 x 10(-5) M). For lindane, bisphenol A, and diethylphthalate, activity is not associated with impurities in the samples and, for lindane and bisphenol A at least, the compounds are able to compete with ecdysteroids for the ligand binding site on the receptor complex, albeit at concentrations very much higher than those found in the environment. The only pharmaceutical showing any detectable antagonist activity was 17alpha-ethynylestradiol. In the context of recent publications on potential endocrine disruption in marine and freshwater arthropods, these findings suggest that, for some compounds (e.g., diethylstilbestrol), ecdysteroid receptor-mediated responses are unlikely to be involved in producing chronic effects. The B(II) assay has a potentially valuable role to play in distinguishing between endocrine-mediated, which normally occur at submicromolar concentrations, and pharmacological effects in insects and crustaceans.

Assessing the biological potency of binary mixtures of environmental estrogens using vitellogenin induction in juvenile rainbow trout (Oncorhynchus mykiss).

Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as the endpoint. The estrogenic potencies of estradiol-17beta (E2), 4-tertnonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L(-1) for E2, 6.1 and 6.4 microg L(-1) for NP, and 4.4 and 6.5 microg L(-1) for MXC. Concentration-response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor alpha agonist and an estrogen receptor beta antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range forwhich the expected response curve forthe binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.

Effects of the synthetic estrogen 17 alpha-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas).

A fish full life-cycle (FFLC) study was conducted for 17 alpha-ethinylestradiol (EE2) using the fathead minnow, Pimephales promelas. Newly fertilized embryos (< 24 h old) were exposed to five concentrations of EE2 (0.2, 1.0, 4.0, 16, and 64 ng/L nominal) in continuous flow-through conditions for 305 d at 25 +/- 1 degrees C. Exposure concentrations were verified by 14C-EE2 radiochemistry, supported by radioimmunoassay, and mean measured values were > or = 70% of nominal. For the F0 adult phase until 301 d posthatch, the no-observed-effect concentrations (NOECs) for growth, survival, and reproduction (as egg production) were all > or = 1.0 ng/L. The NOEC values for F1 embryo hatching success and larval survival (at 28 d posthatch) were both > or = 1.0 ng/L. While statistically detectable changes in F1 growth were evident at 0.2 ng/L, these were not considered to be biologically significant when compared with historical control data. Male fish exposed to EE2 at 4.0 ng/L failed to develop normal secondary sexual characteristics; on the other hand, assumed females exposed to this level of EE2 were able to breed when paired with males that had not been exposed to EE2. Histology of F0 control, 0.2-, and 1-ng/L exposed fish at 56 d posthatch indicated an approximate female-to-male (F:M) sex ratio of 50:50 (with no ovatestes observed in the control), while fish exposed to EE2 at 4.0 ng/L for 56 d posthatch had a F:M sex ratio of 84:5 (with ovatestes in 11% of fish). After 172 d posthatch, no testicular tissue was observed in any fish exposed to EE2 at 4.0 ng/L. At the same time point, plasma vitellogenin levels were significantly higher in fish exposed to EE2 at 16 ng/L. A lack of sexual differentiation occurred in males at concentrations > or = 4.0 ng/L. Taking into account these data, the overall no-observed-adverse-effect concentration was considered to be 1.0 ng/L.

Ecological risk assessment of endocrine disruptors.

The European Centre for Ecotoxicology and Toxicology of Chemicals proposes a tiered approach for the ecological risk assessment of endocrine disruptors, integrating exposure and hazard (effects) characterization. Exposure assessment for endocrine disruptors should direct specific tests for wildlife species, placing hazard data into a risk assessment context. Supplementing the suite of mammalian screens now under Organization for Economic Cooperation and Development (OECD) validation, high priority should be given to developing a fish screening assay for detecting endocrine activity in oviparous species. Taking into account both exposure characterization and alerts from endocrine screening, higher tier tests are also a priority for defining adverse effects. We propose that in vivo mammalian and fish assays provide a comprehensive screening battery for diverse hormonal functions (including androgen, estrogen, and thyroid hormone), whereas Amphibia should be considered at higher tiers if there are exposure concerns. Higher tier endocrine-disruptor testing should include fish development and fish reproduction tests, whereas a full life-cycle test could be subsequently used to refine aquatic risk assessments when necessary. For avian risk assessment, the new OECD Japanese quail reproduction test guideline provides a valuable basis for developing a test to detecting endocrine-mediated reproductive effects; this species could be used, where necessary, for an avian life-cycle test. For aquatic and terrestrial invertebrates, data from existing developmental and reproductive tests remain of high value for ecological risk assessment. High priority should be given to research into comparative endocrine physiology of invertebrates to support data extrapolation to this diverse fauna.

Assessment of developmental effects, cytotoxicity and genotoxicity in the marine polychaete (Platynereis dumerilii) exposed to disinfected municipal sewage effluent.

While sodium hypochlorite is widely used as a disinfectant for municipal sewage effluents and power station cooling waters discharged into coastal environments, there is limited information on the potential in vivo genotoxicity of such disinfection procedures to marine organisms. Using a recently developed test system based on the marine polychaete Platynereis dumerilii, we have evaluated impacts based on embryo-larval development, cytotoxicity and genotoxicity following exposure to disinfected settled (primary) effluent from a municipal sewage treatment works (STW). Sewage samples were collected from Newton Abbot STW, Devon, UK and then disinfected with sodium hypochlorite based on standard operational procedures. Exposure of polychaetes to dilutions of disinfected sewage in seawater (20 +/- 1 degree C) led to a marked reduction in normal embryo-larval development (7 h EC50 from 0.57-1.88% (v/v), n = 4), with a simultaneous increase in cytotoxicity. Following the calculation of the Maximum Tolerated Dose (MTD), based on developmental and cytotoxic effects, the organisms were also analysed for the induction of chromosomal aberrations. This investigation demonstrated the absence of genotoxicity in polychaetes exposed in vivo to sewage disinfected with sodium hypochlorite. These observations extend our previously published studies in which polychaetes exposed to non-disinfected sewage, while showing developmental toxicity and cytotoxicity, did not exhibit any evidence of cytogenetic damage.

Autosomal recessive inheritance of erythrokeratoderma variabilis.

Erythrokeratoderma variabilis is a rare genodermatosis conventionally regarded as autosomal dominant in inheritance. We describe the clinical features and light and electron microscopic findings in two affected siblings born to unaffected parents and suggest an autosomal recessive mode of inheritance in this family. We also briefly review the literature on this disorder.