Vitellogenin content in European eel (Anguilla anguilla) in Flanders, Belgium.

As part of a large-scale monitoring program of bioaccumulating contaminants in the European eel (Anguilla anguilla) in Flanders (Belgium), we investigated potential effects of xenoestrogens in these fish. The present paper describes the results of the plasma vitellogenin (VTG) content, measured in 142 eels sampled at 20 different locations, in relation to the internal pollution levels. To validate the blood VTG assays, a small number of eels (n=8) was exposed to 10 microg ethinylestradiol/l (EE2) for 9 days. In this experiment, VTG was detected as a protein with a molecular weight of 214 kDa and confirmed by Western blotting. Compared with the solvent controls, significantly higher concentrations of VTG were measured in EE2 exposed eel. However, the VTG content was relatively low compared with other fish species exposed to high concentrations of estrogens. The plasma VTG content of eels from the field study was very low, despite a very high internal load of endocrine disrupters. These results, together with previously published studies, suggest that immature yellow European eel might not be the best sentinel species to study the effects of estrogenic compounds on VTG levels of wild fish populations.

Estrogenic and toxic effects of methoxychlor on zebrafish (Danio rerio).

Although zebrafish (Danio rerio) have been suggested as a good candidate for screening potential endocrine disruptors, little information is available on the effects of weak estrogens on this species. We investigated the sensitivity of different life stages of zebrafish toward toxic and estrogenic properties of methoxychlor (MXC). Short-term tests with adults resulted in a sex-specific 96-h lethal concentration for 50% (LC50) of the test animals of 36 microg/L for males and 129 microg/L for females. To determine the estrogenic capacity of MXC, adult zebrafish were exposed to 0, 0.5, 5, and 50 microg MXC/L for 14 d. Induction of vitellogenin ([VTG] measured with protein electrophoresis and Western blot) in males was detected at 5 and 50 microg MXC/L. Females, however, did not exhibit higher blood VTG concentrations at the tested MXC concentrations. In a second series of experiments, juvenile zebrafish were exposed to 0, 0.05, 0.5, and 5 microg MXC/L for 33 d. Survival, length, weight, and condition of larvae were examined as indicators of toxic stress and the VTG content in whole body homogenates of juveniles was measured to determine xenoestrogenic effects. No effects of the tested concentrations of MXC were observed. Finally, the effect of MXC on zebrafish eggs, exposed to 0, 1, 10, and 32 microg MXC/L, was examined. Hatching and survival of hatched zebrafish were affected at 10 and 32 microg MXC/L. This study demonstrated that adult male zebrafish are sensitive toward the estrogenic effects of MXC. However, the use of VTG induction to detect effects of (xeno)estrogens in early life stages has to be further investigated, as low concentrations of VTG were detectable in exposed as well as unexposed juvenile fish.

In vitro and in vivo estrogenicity and toxicity of o-, m-, and p-dichlorobenzene.

The estrogenicity of o-, m-, and p-dichlorobenzene (DCB) was evaluated with a yeast estrogen screen (YES) and zebrafish (Danio rerio) vitellogenin (VTG) assays. With the YES, p-DCB and m-DCB were found to be estrogenic in a concentration-responsive manner. The relative potency measured with the YES (relative to 17beta-estradiol) was 2.2 x 10(-7) for p-DCB and 1.04 x 10(-8) for m-DCB. Following acute toxicity tests with the zebrafish, plasma VTG production was measured to examine the in vivo estrogenic activity of the three compounds after a 14-d exposure. Adult zebrafish were exposed to different concentrations of o-, m- and p-DCB, ranging from 0.1 to 32 mg/L; ethynylestradiol ([EE2]; 5 ng/L, 10 ng/L, 50 ng/L, and 100 ng/L) was used as a positive control. After exposure, blood samples were taken and protein electrophoresis was performed to determine the relative VTG content. Gonadosomatic indices (GSI) and condition factors (CF) were also calculated. Elevated VTG levels and decreased female GSIs were found in fish exposed to > or = 5 ng EE2/L and in fish exposed to > or = 10 mg p-DCB/L. Low GSIs coincided with high levels of VTG in the blood of female zebrafish. This relation was not only found in fish exposed to EE2 but also in controls and fish exposed to DCB. Therefore, a direct or indirect effect of VTG on the GSI is suggested rather than a direct toxic effect of the tested compounds on the gonads.

Xenoestrogenic effects of ethinylestradiol in zebrafish (Danio rerio).

To assess the estrogenic effects of ethinylestradiol on zebrafish, zebrafish at different developmental stages (embryos, juveniles, and adults) were exposed to the synthetic hormone ethinylestradiol (EE2) in concentrations of 1, 10, and 100 ng/L for up to 33 days. Survival, hatching, length, weight, growth, condition, hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) production were examined. Exposure of zebrafish juveniles and embryos to 100 ng EE2/L for up to 33 days had significant effects on survival, growth, and hatching. Two VTG fragments with molecular weights of approximately 140 and 170 kDa were detected with protein electrophoresis and Western blotting in the blood of exposed males and exposed and unexposed females, as well as in whole-body homogenates of exposed and unexposed juveniles. Significantly higher VTG concentrations (compared to controls) were measured in adults exposed to 10 and 100 ng EE2/L for 14 days, but not in fish exposed to 1 ng EE2/L. This study demonstrated that (1) zebrafish juveniles, larvae, and embryos are sensitive to the toxic effects of the endocrine disrupter EE2; (2) the effects on VTG production in adults are detected after exposure to environmentally relevant concentrations of EE2; (3) unexposed juvenile zebrafish produce measurable concentrations of VTG.