Table 2 Summary of the Influence and optimization of experimental parameters on behavioral test performance
Parameter type | Parameter | Influence on | Potential measures to exclude bias |
|---|---|---|---|
Biological or intrinsic | Developmental stage | Biotransformation, effect concentration, receptor availability stage specific behavior | Unify developmental stage for comparability; report on incubation temperature |
Time of day | Performance of control group, test sensitivity | Measure behavior at similar time of the day | |
Developmental malformations | Effect concentration, effect direction (hypo- or hyperactivity), swimming ability | Malformations should be analyzed in parallel with behavioral testing to indicate at which concentration they may contribute to the observed behavioral effects | |
Endpoint parameter | Effect concentration, effect direction (hypo- or hyper-activity) | Compare the same endpoints within a particular assay, i.e., distance moved for LMR and frequency for STC | |
Rearing conditions during exposure | Performance of control group, test sensitivity | Report all rearing conditions | |
Zebrafish strain | Control performance, test sensitivity | Consider strain effects during result comparison and interpretation | |
Technical or extrinsic | Exposure concentration | Effect concentration type (LOEC versus ECx) and accuracy, effect direction (hypo- or hyperactivity) | Avoid single concentration and include a full concentration–response analysis |
Exposure duration | Effect concentration, Internal bioavailable concentration, biotransformation, effect direction (hypo- or hyper-activity), observation of (neuro)developmental effects | Consider toxicokinetics/toxicodynamics and its influence on chemical concentration during test design | |
Duration of behavior analysis | Performance of unexposed group, test sensitivity | Differences in the duration of light–dark cycles may impact the sensitivity and outcome in LMR-L/D | |
Exposure well size | Effect direction (hypo- or hyperactivity), Performance of control group, test sensitivity | Select and use exposure wells of same dimension for reproducibility, i.e., 96 wells for neurotoxicity testing and 24 wells for diagnosing neuroactivity MoA | |
Material used for exposure vessel | Effect concentration, test sensitivity | Avoid plastic for lipophilic chemicals, measure/predict exposure concentration and install measures to ensure a stable exposure concentration if required. | |
Light conditions | Performance of unexposed group, test sensitivity | Light intensity, duration, and sequence of photo stimuli should be reported | |
Solvent concentration | Exposure concentration, side effects | Avoid DMSO and other solvents or use 0.01% concentration when necessary. Test solvent control |