An in Vivo Testing System for Endocrine Disruptors in Fish Early Life Stages Using Induction of Vitellogenin

An enzyme-linked immunosorbent assay (ELISA) based on carp (Cyprinus carpio) vitellogenin (c-VTG, an estrogendependent precursor of egg yolk) was developed to quantify VTG in the fathead minnow (Pimephales promelas) and was employed to establish an in vivo testing system for estrogens in fish early life stages. The working range of the c-VTG ELISA was between 1 and 50 ng/ml (20%–80% binding). The interand intraassay variations were 9.7 6 3% and 8.9 6 3.4% (n 5 15), respectively. Plasma and whole body homogenates from females and from estrogenized male fathead minnow diluted parallel with the c-VTG standard. Measurements of plasma VTG fractionated on sodium dodecyl sulphate gels and quantified using densitometry were compared with measurements with the same plasma samples using the c-VTG ELISA and they were similar for the carp and fathead minnow. The plasma concentration of VTG in the male fathead minnow was 25 6 6 ng/ml, whereas in females, plasma concentrations of VTG ranged from 5.3 6 1 mg/ml to 182 6 7.6 mg/ml, depending on the state of maturity. Exposure of fathead minnows to estradiol-17b in the water (nominal concentrations of 25 ng/L, 50 ng/L, and 100 ng/L) from 24 h postfertilization to 30 d posthatch induced VTG synthesis in a dose-dependent manner. Shorter periods of exposure to 100 ng/L estradiol-17b (24 h postfertilization to 10 or 20 d posthatch) similarly induced a vitellogenic response. This work demonstrates that fathead minnow are sensitive to estrogen and are able to synthesize VTG very early in development; this provides the basis for the adaptation of existing fish earlylife-stage tests for the in vivo testing of estrogenic chemicals. Keywords—Estrogenic Vitellogenin Biomarker Fathead minnow Aquatic