Bram J. Versonnen

Induction of vitellogenesis in 17α-ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison

Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE(2)) through injection (1, 10, 25 and 50 microg EE(2)/g fish/week) and via water exposure (1, 10 and 100 ng EE(2)/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 microg EE(2)/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE(2). Exposure of rainbow trout to EE(2) had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE(2)/l. Fish injected with 10, 25 and 50 microg EE(2)/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE(2) exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.

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.