A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos.

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.