Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chemicals using zebrafish behavior at 6 days of age. The behavioral paradigm simultaneously tests individual larval zebrafish under both light and dark conditions in a 96-well plate using a video tracking system. We have found that many variables affect the level or pattern of locomotor activity, including age of the larvae, size of the well, and the presence of malformations. Some other variables, however, do not appear to affect larval behavior including type of rearing solution (10% Hank's vs. 1:3 Danieau vs 60 mg/kg Instant Ocean vs 1× and 1:10× EPA Moderately Hard Water). Zebrafish larval behavior using a microtiter plate format may be an ideal endpoint for screening developmentally neurotoxic chemicals, but it is imperative that many test variables be carefully specified and controlled.

► We describe a paradigm for evaluating chemical neurotoxicity in larval zebrafish.
► Locomotion is studied in multi-well plates under alternating lighting conditions.
► We find that age of larva, well size and presence of malformations all affect activity.
► We also find that rearing solution does not influence activity.
► We conclude that many non-chemical variables may affect the behavioral assay.