Comparative live-imaging of in vivo EROD (ethoxyresorufin-O-deethylase) induction in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) embryos after exposure to PAHs and river sediment extracts

Abstract

The measurement of EROD (ethoxyresorufin-O-deethylase) activity to determine the induction of CYP1A after exposure to dioxin-like substances is a well-established biomarker in fish. For reasons of animal welfare and implementations of new chemicals regulations (REACh), in vivo methods using zebrafish (Danio rerio) and medaka (Oryzias latipes) embryos have recently been developed to quantify CYP1A induction, which is visualized as mean intensity of the autofluorescent resorufin formed in living anaesthetized embryos.In the present study, concentration ranges of three PAHs (benzo[a]pyrene, β-naphthoflavone, benzo[k]fluoranthene) as examples of known CYP1A inducers as well as extracts of two well-characterized sediment samples of the lower Neckar river (Southern Germany) were used to determine the suitability of the fathead minnow (Pimephales promelas) embryo for the in vivo EROD assay. Data for zebrafish embryos were generated for comparison. Fathead minnow embryos were principally suitable to show in vivo EROD induction via live-imaging. Since in fathead minnow embryos both signal area and fluorescence intensities are lower than in zebrafish embryos, the induction potentials of the three model PAHs and the environmental samples proved to be species-dependent.Among the three PAHs tested, benzo[k]fluoranthene lead to the strongest EROD signal followed by β-naphthoflavone and benzo[a]pyrene in comparison to the positive control. Whereas benzo[k]fluoranthene and β-naphthoflavone showed a dose-response relationship for the EROD induction, benzo[a]pyrene failed to induce a significant signal in fathead minnow embryos. If compared to the model PAHs, the extracts of both sediments from the lower Neckar River induced stronger EROD signals in both fathead minnow and zebrafish embryos. Observations thus documented fathead minnow embryos to be as suitable for biomonitoring purposes as are zebrafish embryos.