Zebrafish Larval Light-Dark Preference Test

Embryo and larval exposures proceeded as per Section 2.6. Zebrafish larvae (at 7 dpf) were transferred into a 12-well plate with ERM. Twenty trials were conducted with n = 13–36 fish per treatment for the light/dark preference test (LDPT). A company-manufactured cover was placed over the plate to create light and dark zones in each well. Poorly tracked larvae were excluded from analysis and data from twenty distinct runs were blended into a single graph to reflect all runs. Buspirone hydrochloride at 60 µM (CAS number: 33386-08-2, Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control in this assay since it is an anxiolytic compound that has been validated in the LDPT with zebrafish larvae [27 (link)]. The assay methodology is described in detail in our previous publication [5 (link)].