This project aims to develop an alternative testing strategy to reduce the need for fish early life-stage toxicity tests (FELS) for the assessment of chronic toxicity of chemicals to fish, using the adverse outcome pathway (AOP) framework as a guidance for assay development. Currently, the FELS test (OECD TG 210) is one of the primary testing guidelines used to estimate the chronic toxicity of chemicals to fish. Results obtained using this TG are used to support risk assessment around the world. However, important limitations of this animal test are being recognized, including (1) the high numbers of fish used, (2) the relatively low-throughput, and (3) the lack of mechanistic information. In order to comply with the 3R principles, we are developing a non-animal testing strategy which includes both in vitro tests and in vivo whole organism alternative 120 hpf (hours post fertilization) ZFET (zebrafish embryo acute toxicity test) assays. One of the main research questions of this project is to what extent an assay development approach based on the AOP framework is capable of offering a mechanistic basis for selecting assays to develop an alternative testing strategy that allows the prediction of chronic FELS toxicity.