Assay development and validation. Image of zebrafish in wells of a 96-well plate at (A) 0 h and (B) 24 h of incubation in Alamar Blue®. (C) A confocal microscopy image of Alamar Blue within a zebrafish after 1 h of exposure to an assay solution. (D) Alamar Blue assay results varying the number of fish/well and the temperature of incubation (n=7–8, *p<0.05, relative to 22°C). (E) BD Oxygen Biosensor assay results varying the number of fish/well (n=5). (F) Correlation of relative change in fluorescence measured by Alamar Blue assays and BD Oxygen Biosensor assays conducted with 1, 3, and 5 fish. (G) Alamar Blue assay results with incubation time (0–24 h) and the addition of 0.1% dimethyl sulfoxide (DMSO) to an assay medium (n=8, *p<0.05, relative to no DMSO). (H) BD Oxygen Biosensor assay results as a function of incubation time (0.5–4 h,a,b indicate data points that differ significantly [p<0.05]).