First or best? Contrasting approaches reveal different outcomes in tadpole (Hypsiboas lundii) escape performance under acute chlorpyrifos exposure

Escape swimming performance tests are widely used to assess how aquatic organisms respond to environmental stressors. Two analytical approaches are commonly applied: the first response (reaction to the initial simulated predation attempt) and the best response (the highest performance among successive attempts). However, whether these approaches lead to convergent or divergent conclusions remains unclear. In this study, Hypsiboas lundii tadpoles were exposed for 48 h to two environmentally relevant concentrations of the organophosphate insecticide chlorpyrifos (CPF; 0.6 and 1.0 μg L⁻¹). Escape performance (first and best responses) and enzymatic biomarkers (acetylcholinesterase, AChE; and carboxylesterase, CbE) were assessed in body and tail tissues. AChE activity remained unchanged, whereas CbE activity increased significantly in the body of tadpoles at the highest CPF concentration. Reduced escape performance was therefore attributed to non-cholinergic, bioenergetic mechanisms. Best response analysis revealed significant reductions in escape distance, burst speed, and escape time at the higher CPF concentration, whereas first response analysis detected no effects. Our findings demonstrate that the two analytical approaches can yield contrasting conclusions, underscoring the importance of methodological choices in behavioral ecotoxicology.