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.

Do migratory or demographic disruptions rule the population impact of pollution in spatial networks?

Ecotoxicology supplies environmental quality criteria mainly based on the potential effects of contaminants on demographic rates of natural populations. Possible impacts through pollutant-induced disruptions of spatial behaviors are totally neglected. Should it be significant to take into account this "second way"? We developed the example of a hypothetical brown trout population living in a river network. We analyzed how behaviors of toxic avoidance or attraction during the spawning migration alter the impact of pollution. Attraction behaviors basically enhanced the bad effect of pollution. More interesting, avoidance behaviors can weakly lift the asymptotic population growth rate, while if there is density-dependent effects on recruitment, pollutant avoidance can actually lead to a substantial drop in equilibrium size. Our model allowed comparing the relative significance of migratory and demographic disruptions for explaining the population impacts of pollution; we thus stress on the need of increasing efforts to develop knowledge relative to toxicant-induced spatial behaviors and to integrate such effects in the definition of environmental quality criteria.

The measurement of cholinesterase activities as a biomarker in chub (Leuciscus cephalus): the fish length should not be ignored.

Biomarkers are early warning systems of the exposure of aquatic organisms to pollutants. Among them, the measurement of the cholinesterase (ChE) activities in fish muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides. As such it has been used in numerous field studies both in marine and continental waters. Cyprinids (chub, Leuciscus cephalus) were sampled in river sites (France) in relatively clean and polluted areas. We performed the statistical analysis of the ChE activities and we generally observed a statistical relationship between ChE activities and fish length, the larger fish having the lower ChE activities. We concluded that the great majority of the significant differences in ChE activities between sites could be due in fact to differences in fish length between field samples. We stress the importance of taking into account the fish length whenever differences in ChE levels between field sites must be interpreted.

Multibiomarker responses in fish from the Moselle River (France).

The response of wild fish to pollutants was studied using two biomarkers in chub (Leuciscus cephalus) at five stations in the Moselle River (France) in 1998 and in 1999. The induction of cytochrome P450 1A was quantified by the ethoxyresorufin O-deethylase (EROD) activity in the liver and the level of DNA single-strand breaks was determined in erythrocytes using the comet assay. EROD activity was observed to be up to 10-fold induced in both males and females from the downstream stations in comparison to the fish from the upstream station. Levels of DNA damage did not parallel EROD induction. Chemical analyses did not clearly explain the responses of the studied biomarkers, confirming the great difficulty in relating chemical and biological information in the field. This study confirms the difficulty in assessing the biological effects of mixtures of pollutants and points out the usefulness of a large array of biomarkers.

Statistical analysis of regulatory ecotoxicity tests.

ANOVA-type data analysis, i.e.. determination of lowest-observed-effect concentrations (LOECs), and no-observed-effect concentrations (NOECs), has been widely used for statistical analysis of chronic ecotoxicity data. However, it is more and more criticised for several reasons, among which the most important is probably the fact that the NOEC depends on the choice of test concentrations and number of replications and rewards poor experiments, i.e., high variability, with high NOEC values. Thus, a recent OECD workshop concluded that the use of the NOEC should be phased out and that a regression-based estimation procedure should be used. Following this workshop, a working group was established at the French level between government, academia and industry representatives. Twenty-seven sets of chronic data (algae, daphnia, fish) were collected and analysed by ANOVA and regression procedures. Several regression models were compared and relations between NOECs and ECx, for different values of x, were established in order to find an alternative summary parameter to the NOEC. Biological arguments are scarce to help in defining a negligible level of effect x for the ECx. With regard to their use in the risk assessment procedures, a convenient methodology would be to choose x so that ECx are on average similar to the present NOEC. This would lead to no major change in the risk assessment procedure. However, experimental data show that the ECx depend on the regression models and that their accuracy decreases in the low effect zone. This disadvantage could probably be reduced by adapting existing experimental protocols but it could mean more experimental effort and higher cost. ECx (derived with existing test guidelines, e.g., regarding the number of replicates) whose lowest bounds of the confidence interval are on average similar to present NOEC would improve this approach by a priori encouraging more precise experiments. However, narrow confidence intervals are not only linked to good experimental practices, but also depend on the distance between the best model fit and experimental data. At least, these approaches still use the NOEC as a reference although this reference is statistically not correct. On the contrary, EC50 are the most precise values to estimate on a concentration response curve, but they are clearly different from the NOEC and their use would require a modification of existing assessment factors.

Responses of chub (Leuciscus cephalus) populations to chemical stress, assessed by genetic markers, DNA damage and cytochrome P4501A induction.

Indicators of effects at the population level (genetic variation using allozymes) and early indicators of pollution (EROD activity and DNA strand break formation) were analysed in chub (Leuciscus cephalus) living in weakly and heavily contaminated stations of the Rhône River watershed. The genetic erosion was mainly detected in a fish population living in a contaminated small river system, through modifications in allelic and genotypic frequencies for PGM-2 locus and could be linked to a genetic bottleneck and to the reduced gene flow from upstream unable to maintain or restore the genetic diversity. In a contaminated large river system, the genetic diversity for PGM-2 and other loci was maintained and was probably the consequence of a high gene flow from upstream, linked to a sustained drift of larvae and juveniles in the system. A convergent increase of the frequency of the 90 allele at PGM-2 was observed in two contaminated stations compared with the reference station, this trend being confirmed on a more extensive geographic scale over the Rhône River basin. A high level of EROD activity was detected in both contaminated sites but only the fish in the large river system showed a significant DNA damage level compared to the reference population. The low DNA damage level and high hepato-somatic ratio characterized the impacted population of the small river system and could be associated to a chronic high-level exposure of fish to pollutants which selected individuals exhibiting a high level of DNA damage repair. In the two contaminated systems, some genotypes at the PGM-2 and EST-2 loci showed a low level of DNA damage and/or a high EROD activity and may be considered as being tolerant to pollutants. A higher tolerance of the most heterozygous fish was also detected in the contaminated large system and confirmed that a high level of heterozygosity may be necessary for survival in such a system.

Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus).

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Cytochrome P450 1A induction in gudgeon Gobio gobio : Laboratory and Field Studies.

The induction of cytochrome P450 1A was studied in gudgeon (Gobio gobio), a common European cyprinid, using both farm-raised and field-caught fish. The effects of sex, reproductive status and past exposure to xenobiotics were assessed. When exposed to beta-naphthoflavone (bNF), reared gudgeon showed a dose-dependent increase of EROD activity with a plateau observed at doses from 20 mg kg-1 (females) and 5 mg kg-1 (males). The sexual difference in EROD activity was related to the gonadosomatic index (GSI) of the female whatever the level of induction. Dose and sex effects were confirmed by the immunodetection of CYP1A protein. More than 1 month was necessary for EROD activity to decrease to baseline levels. A second bNF injection after 32 days gave similar levels of induction, suggesting that EROD induction by bNF was not impaired by a pretreatment. Wild fish were brought from two sites in the Rhone river basin: a low contaminated site (Ain) and a highly contaminated site (Rhone). Wild gudgeon were highly induced by bNF in laboratory conditions, except males from the Rhone site which exhibited EROD levels as high as the EROD plateau found in laboratory conditions. A 2- month depuration period in clean water was necessary for EROD activity in wild gudgeon to decrease to baseline levels. These results provide better knowledge of the main factors of modulation of the induction in gudgeon as well as on the influence of the history of exposure to inducers.

Impact assessment of a wastewater treatment plant effluent using the fish biomarker ethoxyresorufin-O-deethylase: field and on-site experiments.

The impact of a wastewater treatment plant (WWTP) effluent was assessed with the fish biomarker ethoxyresorufin-O-deethylase (EROD) using field and on-site laboratory experiments. EROD activity was measured in chub (Leuciscus cephalus) and stone loach (Noemacheilus barbatulus) caught at three sites of the Chalaronne River (southeast France). Liver somatic index (LSI) and organochloride bioaccumulation in muscle were estimated for chub only. In September, EROD activity and LSI of chub increased significantly between the sites above and below the WWTP effluent discharge. EROD induction detected in chub was confirmed by on-site tank experiments. EROD levels were determined in juvenile rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) exposed to different concentrations of the WWTP effluent and river water for 16 days. After a 4-day exposure, EROD activities of the carp exposed to the effluent increased significantly compared with the control. The response was linked to the effluent concentration and was stable with exposure time. WWTP effluent induced EROD activity, whereas organic and metal analyses, performed on fish muscle and sediment, did not indicate any difference between upstream and downstream of the discharge.

Statistical analysis of cyprinid ethoxyresorufin-O-deethylase data in a large French watershed.

A comparison of ethoxyresorufin-O-deethylase (EROD) data collected in 1995 in various sites in the Rhône watershed (France) was carried out to quantify the influence of factors such as contamination and biological parameters on EROD levels and within-group variabilities. Three species of cyprinids were collected and fish chemical contamination was measured. A log transformation of EROD data provided both normalization and homogeneity of variances. The influence of female sexual maturation on the variability and EROD dimorphism was quantified. A relationship with contaminant bioaccumulation was observed. A comparison with EROD data collected during previous studies by the same laboratory was made to validate the results.