Comparative assessment of behavioural alterations induced by common laboratory solvents in zebrafish (Danio rerio) larvae.

Organic solvents are routinely used to dissolve poorly water-soluble chemicals in zebrafish-based assays. However, their intrinsic biological activity may confound behavioural endpoints and compromise data interpretation. This study systematically evaluated the effects of seven commonly used laboratory solvents, dimethyl sulfoxide (DMSO), ethanol (EtOH), methanol (MeOH), acetone (Ace), acetonitrile (ACN), isopropanol (IPA), and ethyl acetate (EtOAc), at four concentrations (1, 0.5, 0.1, 0.05%; v/v), on locomotor behaviour of zebrafish Danio rerio larvae at 120 h post fertilization. Larval activity was quantified using automated video tracking under alternating light-dark conditions, assessing distance moved, velocity, acceleration, mobility states, and turning behaviour. Behavioural responses were strongly solvent- and concentration-dependent. EtOH, MeOH, Ace, and IPA induced hyperactivity at ≥ 0.5%, while DMSO showed biphasic effects, stimulating activity at 0.5% but suppressing it at 1%. ACN demonstrated a significant inhibitory response even at 0.5%, while EtOAc was tolerated only at ≤ 0.1%. Principal component analysis (PCA) and hierarchical clustering on principal components (HCPC) further revealed coherent behavioural signatures shaped not only by solvent identity and concentration but also strongly by light-dark transitions, underscoring the critical importance of consistent illumination conditions in zebrafish behavioural assays. Overall, ≤ 0.1% of Ace and EtOAc, and ≤ 0.05% of DMSO, EtOH, MeOH, ACN, and IPA did not significantly alter locomotion, suggesting their suitability for zebrafish behavioural assays. These findings establish reference points for solvent selection and concentration thresholds in zebrafish behavioural assays under the specific experimental conditions tested, thereby supporting reproducibility and reliability in scientific research.