Development of a gene expression fish embryo test as an alternative for chronic fish tests: analysis of toxic effects using differential gene expression in the Danio rerio embryo test (Gene-DarT)

Acute and chronic fish tests are performed for the registration of chemicals, pesticides, biocides and pharmaceuticals. While the fish embryo test has been developed as an alternative to the acute fish test, so far no alternatives for chronic fish tests are available. Therefore, the fish embryo test with the zebrafish (Danio rerio) was extended by an additional endpoint, the analysis of differential expression of marker genes, genes that are sensitive to toxicants. The objective is to replace chronic fish tests, such as the fish early life stage test, or to reduce the number of fish used in these tests. In  subproject 3 (‘Establishment of the test system’), a test protocol for the gene expression Danio rerio embryo test (Gene-DarT) was established based on reverse transcriptase polymerase chain reaction (RT-PCR), gel electrophoresis and densitometric analysis of the gels. Using this protocol, the effects of 14 substances on expression of 7 marker genes were investigated. All tested substances significantly affected the expression of at least one marker gene with cytochrome P450 1A (cyp1a) and heme oxygenase 1 (hmox1) being most sensitive. For most tested substances, lowest observed effect concentrations (LOECs) derived with the Gene-DarT differ by a factor of less than 10 from LOEC-values of fish early life stage tests with zebrafish. However, for some substances, larger differences were observed. These results indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test, but that there is still a need to improve the Gene-DarT.