Determining the appropriateness of timely euthanasia in acute fish toxicity testing using observed clinical signs to predict time of death

Applying euthanasia before death in acute fish toxicity tests contributes significantly to animal welfare by serving as a "refinement" of the test method. A previous study defined three severe signs as moribund states in Japanese medaka (Oryzias latipes) based on the severity classification of clinical signs for the purpose of euthanizing fish during acute fish toxicity testing before death. However, this approach limits the range of signs that can be used to justify euthanasia. It has also been suggested that predicting the time of death can help determine the appropriate timing for euthanasia in such tests, if the survival time for each clinical sign is known (i.e., the time from onset of a clinical sign to death). In this study, we used video recording equipment to assess the survival times for 12 clinical signs observed in response to eight chemical substances, with the aim of ensuring timely euthanasia for fish during acute toxicity tests using Japanese medaka. We found that euthanasia could be applied to a broader range of signs and was appropriate for many fish exhibiting clinical signs by predicting the latest time of death from the maximum survival time for each clinical sign. Case studies with five chemicals showed that this method allowed euthanasia to be applied in a timely manner to many individuals exhibiting clinical signs, without any deviation from the lethal concentration 50 values obtained using the original test method.