| Summary: | Worldwide, pesticides have contaminated the environment, affecting non-target species. The aim of this work was to evaluate the effects of fosetyl-Al (FOS) on model organisms. Based on the 3 Rs for animal research and described guidelines, the OECD 236 and 220 were applied with some modifications. The FOS test concentrations were 0.02–0.2–2–20–200 mg/L for <i>Danio rerio</i> and 250–500–750–1000–1250 mg/kg for <i>Enchytraeus crypticus</i>. Besides the standard endpoints, additional endpoints were evaluated (<i>D. rerio</i>: behavior and biochemical responses; <i>E. crypticus</i>: extension of exposure duration (28 d (days) + 28 d) and organisms’ sizes). For <i>D. rerio</i>, after 96 h (h), hatching was inhibited (200 mg/L), proteins’ content increased (2 and 20 mg/L), lipids’ content decreased (2 mg/L), glutathione <i>S</i>-transferase activity increased (2 mg/L), and, after 120 h, larvae distance swam increased (20 mg/L). For <i>E. crypticus</i>, after 28 d, almost all the tested concentrations enlarged the organisms’ sizes and, after 56 d, 1250 mg/kg decreased the reproduction. In general, alterations in the organisms’ biochemical responses, behavior, and growth occurred at lower concentrations than the effects observed at the standard endpoints. This ecotoxicological assessment showed that FOS may not be considered safe for the tested species, only at higher concentrations than the predicted environmental concentrations (PECs). This research highlighted the importance of a multi-endpoint approach to assess the (eco)toxic effects of the contaminants.
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