The efficacies of three concentrations of 2-phenoxyethanol from Aqua-sedTM were examined to determine the suitability for euthanasia of butterfly splitfin (Ameca splendens). Thirty fish were randomly assigned to be immersed in one of three Aqua-SedTM concentrations: 0.5, 2, and 6 ml/L. Opercular rates and caudal fin stroke rates were quantified, and time to the cessation of physiological measures including heartbeat, caudal fin strokes, reaction to external stimuli, righting reflex, swimming, and operculation were recorded. To test for the effects of concentration of Aqua-sedTM on all times to cessation of physiological measures, a linear mixed model was performed followed by pairwise comparisons with a Bonferroni correction. A proportions test compared the number of fish that lost their heartbeat across Aqua-sedTM concentrations. Varying anesthetic induction times were observed for all three Aqua-sedTM concentrations; time to cessation of physiological measures was fastest with 6 ml/L, followed by 2 then 0.5 ml/L, but only concentrations of 2 and 6 ml/L induced anesthesia in less than the recommended 180 s. Aqua-sedTM concentration had a significant effect on time to cessation of physiological measures (P < 0.01), but not on time to cessation of a heartbeat (P > 0.05). Pairwise comparisons indicate time to cessation of caudal fin strokes, reaction to external stimuli, righting reflex, and swimming significantly decreased as concentration increased (P < 0.01). After 60 min of immersion in Aqua-sedTM the number of fish without a heartbeat differed significantly across concentrations (P < 0.01); 20% of fish immersed in 0.5 ml/L, 90% of fish immersed in 2 ml/L, and 90% of fish immersed in 6 ml/L had no heartbeat. A minimum dose of 2 ml/L of Aqua-sedTM, and an immersion time of 60 min for euthanasia of butterfly splitfin is recommended, with a secondary euthanasia measure to confirm death when the heartbeat remains.
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