Zebrafish startle habituation: How readout choice influences the interpretation of chemical effects in neurotoxicity screening

Zebrafish behavioral profiling is increasingly used in environmentally oriented New Approach Methodologies (NAMs) for chemical hazard assessment, including neuroactive chemicals. However, interpreting acoustic/vibrational startle habituation requires understanding how different quantification strategies influence toxicological readouts. In this study, we performed a controlled cross-platform comparison of two common habituation metrics in 5-dpf zebrafish eleutheroembryos following acute chemical exposure. Using embryos from the same spawning and shared stock solutions, we evaluated: (i) a centroid-based workflow (30 fps), quantifying post-stimulus displacement as area under the curve (AUC), and (ii) a high-speed kinematic workflow (1000 fps), classifying individual startle events to calculat%habituation. Eight neuroactive reference chemicals were tested to benchmark both approaches and to improve interpretation of an environmentally relevant neurobehavioral screening endpoint. The high-speed kinematic workflow showed high directional concordance with expected modulation patterns for 6/8 compounds and provided detailed resolution of C-start mechanics. Meanwhile, the centroid-based workflow, a robust and widely accessible standard in the field, reproduced expected directions for 3/8 compounds, capturing robust modulations for specific neuroactive profiles while showing lower sensitivity to other chemicals, such as ketamine, despite their marked effect on habituation kinetics. Notably, results for compounds such as nimodipine showed that both assays are essential for identifying complete loss of the motor response, thereby avoiding misinterpretation of performance deficits as altered plasticity. Overall, our findings show that while both platforms capture behavioral shifts, readout choice substantially influences the sensitivity and interpretation of chemical effects. This study supports fit-for-purpose endpoint selection, strengthening environmental hazard assessment and prioritization.