DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls

[Display omitted] •Solvents are frequently used during zebrafish toxicity testing but their effects are unknown.•DMSO affected behavior at a concentration of ≥0.55%•Different zebrafish strains showed different basal activity, but the same behavioral response to DMSO.•DMSO had an additive and interaction effects on behavior when co-exposed with positive controls. Embryonic and larval zebrafish (Danio rerio) behavior is commonly used to identify neurotoxic compounds. Here, we investigated whether sub-lethal exposures to the common solvents dimethyl sulfoxide (DMSO, 0.01–1%) and methanol (MeOH, 0.01–1%), or the anti-fungal agent methylene blue (MB, 0.0001–0.0005%), can influence larval behavior in a simple light/dark paradigm conducted in 96-well plates. In addition, we tested whether the media volume within the behavioral arena or the zebrafish strain, AB wild type, AB Tübingen (AB/TU), or Tüpfel long-fin (TL), could also influence larval behavior. Following the single exposures, we co-exposed larvae to DMSO and either MB or two other compounds with known behavioral effects in larval zebrafish, flutamide and perfluorooctanesulfonic acid (PFOS). We found ≥0.55% DMSO and 0.0005% MB significantly affected larval behavior, but there was no effect of MeOH. Similarly, TL showed less movement compared to AB and AB/TU strains, whereas lower media volumes also significantly reduced larval movement. However, all strains responded similarly to DMSO and MB. In the co-exposure studies, we found either additive or interaction effects between DMSO and either MB, flutamide, or PFOS, depending on the behavioral endpoint measured. In addition, media volume had no effect on the DMSO concentration response curve, but again we observed additive effects on behavior. In conclusion, methodology can lead to alterations in baseline locomotor activity and compounds can have additive or interaction effects on behavioral endpoints. However, we found no evidence that strain effects should be a concern when deciding on solvents for a simple light/dark behavioral test in larval zebrafish.