Defining Moribund States in Zebrafish to Refine Fish Acute Toxicity Tests

In acute fish toxicity tests, mortality has traditionally served as the primary endpoint. However, in accordance with the “3Rs” principle—replacement, reduction, and refinement—there is a growing need to minimize the suffering and pain experienced by test fish. In this study we aimed to establish a behavioral framework for identifying the moribund state in zebrafish (Danio rerio), providing a humane and ethically refined alternative endpoint. Continual observation of zebrafish exposed to ten representative chemicals allowed the documentation of twelve clinical signs, with severities of the signs evaluated using the death/clinical-sign ratio, which represents the proportion of fish exhibiting a given sign that subsequently died. The signs “immobility,” “immobility at surface,” and “lethargy” emerged as strong predictors of imminent death, each exhibiting a death/clinical-sign ratio of 1.0 across all tested chemicals and concentrations, indicating that all fish exhibiting these signs died within the 96-hr test period. Furthermore, the survival times from the onset of these signs to death were sufficiently short to justify their definition as moribund states. Accordingly, we defined these signs as moribund endpoints and propose that fish exhibiting any of them should be euthanized during the test period. Notably, these findings align with previously reported results in Japanese medaka, indicating the potential cross-species applicability of these moribund endpoints.