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 (AFT) 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 the AFT testing before death. However, this approach limits the range of sign 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 survival time (time from the onset of a clinical sign to death) for each clinical sign is known. 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 fish 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 to many individuals exhibiting clinical signs in a timely manner, without any deviation from the LC50 values obtained using the original test method.