Lighting-driven behavioral responses in zebrafish for water toxicity monitoring and ecological risk assessment

Zebrafish exhibit innate phototaxis, making light stimulation a promising tool for water toxicity monitoring. This study systematically investigated the effects of light color (red, yellow, green, blue), intensity (0-2400 lx), and stimulation duration (1-3 min) on zebrafish phototactic and locomotor behaviors, and established an optimized photobehavioral assay. The results showed that zebrafish exhibited strong positive phototaxis toward red light (612 nm, peak 78.74%) and clear negative phototaxis toward blue light (450 nm, peak -36.23%), with peak behavioral responses at 700 lx and 300 lx, respectively. An optimized intermittent stimulation protocol (red light: 2 min stimulation / 15 min interval; blue light: 1 min stimulation / 15 min interval) effectively maintained behavioral sensitivity while minimizing habituation. A "startle-phototaxis-adaptation" behavioral sequence was identified, consisting of an initial luminance-driven startle (first minute) followed by wavelength-specific phototaxis. Validation with Cd²⁺ exposure revealed concentration-dependent suppression of phototaxis: at 2 TU, the red-light phototaxis rate decreased from 78.9% to -55.9%. With high repeatability, this photobehavioral assay acts as a sensitive, ecologically meaningful proof-of-concept tool, using Cd²⁺ as a model toxicant to support subsequent aquatic pollutant risk evaluation.