Menthol as a sustainable alternative anaesthetic for adult zebrafish (Danio rerio)

The use of anaesthetics has become pivotal in promoting ethical, sustainable, and responsible welfare practices in aquaculture and research procedures. However, species-specific reactions and side-effects have arisen from the use of the most common anaesthetics (MS-222 and eugenol). This study investigated the potential of menthol, a naturally derived and environmentally friendly compound, as an alternative immersion anaesthetic for adult zebrafish (Danio rerio) through concentration-response assays and the evaluation of cardiorespiratory, locomotion and aversive-related effects. Concentrations below 25 mg/L did not induce anaesthetic states while concentrations of 50 and 75 mg/mL were considered the most effective, as they successfully induced anaesthesia with induction and recovery times below 400 s. These values were comparable to those observed with MS-222. In addition, no mortality was observed at these concentrations despite the transient depression of the cardio-respiratory system. The video tracking analyses showed no significant behavioural changes for the lowest concentrations while relatively narrow behavioural safety margins were noticed for the highest concentrations (> 150 mg/L). In addition, concentrations above 75 mg/L induced aversive behaviours which do not support their use for fish anaesthesia. Based on these findings, a short-exposure (10-min) was conducted using menthol concentration of 50 mg/L, resulting in cortisol values equal to those of the control group. Overall, this study shows that a menthol concentration of 50 mg/L can be used as an alternative and more sustainable anaesthetic for zebrafish while higher concentrations may compromise fish welfare.