Evaluation of thymol application for anaesthesia of adult zebrafish

The use of anaesthetics has become extremely important in promoting responsible welfare practices in aquaculture and research. However, adverse reactions and side effects have been described for commonly used anaesthetics such as MS-222 and eugenol. This study examined thymol as a new anaesthetic for adult zebrafish (Danio rerio), a species for which no previous thymol anaesthesia studies have been reported. Concentration-response tests (25-200 mg/L) were conducted using 10 individual animals per concentration, along with assessments of cardiorespiratory, locomotion, and aversive-related effects. Concentrations below 25 mg/L did not induce anaesthetic effects while 50 and 75 mg/L were most effective, inducing deep anaesthesia with average induction times of around 300 s and recovery times of between 400 and 500 s. Induction times at these concentrations were comparable to those observed with MS-222 and eugenol; however, recovery periods were longer. No mortality was observed at these concentrations, although transient cardiorespiratory depression was noted. Video tracking analyses revealed no significant behavioural changes at the lowest concentrations. In contrast, concentrations above 150 mg/L showed narrow behavioural safety margins and induced aversive responses, rendering them unsuitable for fish aesthesia. Based on these findings, a short 10-min exposure to 50 mg/L thymol resulted in cortisol levels comparable to eugenol and lower than in the control group. These findings support the use of 50 mg/L thymol as an effective and welfare-conscious anaesthetic for adult zebrafish. However, further research is recommended to elucidate thymol's physiological effects and its mechanism of action in this species.