An effective biological early warning system for the detection of water contamination should employ undemanding species that rapidly react to the presence of contaminants in their environment. The demonstrated reaction should be comprehensible and unambiguously evidential of the contamination event. This study utilized 96h post fertilization zebrafish larvae and tested their behavioral response to acute exposure to low concentrations of cadmium chloride (CdCl2) (5.0, 2.5, 1.25, 0.625mg/L) and permethrin (0.05, 0.029, 0.017, 0.01μg/L). We hypothesize that the number of larvae that show advanced trajectories in a group corresponds with water contamination, as the latter triggers avoidance behavior in the organisms. The proportion of advanced trajectories in the control and treated groups during the first minute of darkness was designated as a segregation parameter. It was parametrized and a threshold value was set using one CdCl2 trial and then applied to the remaining CdCl2 and permethrin replicates. For all cases, the method allowed distinguishing between the control and treated groups within two cycles of light: dark. The calculated parameter was statistically significantly different between the treated and control groups, except for the lowest CdCl2 concentration (0.625mg/L) in one replicate. This proof-of-concept study shows the potential of the proposed methodology for utilization as part of a multispecies biomonitoring system.