###### Abstract  
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 _Danio rerio_ and 250-500-750-1000-1250 mg/kg for _Enchytraeus cryptic_. Besides the standard endpoints, additional endpoints were evaluated (_D. rerio_: behavior and biochemical responses; _E. crypticus_: extension of exposure duration (28 d (days) + 28 d) and organisms' sizes). For _D. rerio_, 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 \(S\)-transferase activity increased (2 mg/L), and, after 120 h, larvae distance swam increased (20 mg/L). For _E. crypticus_, 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. p 2021 11 2021  
recommended to apply on foliage against fungi that affect several crops, such as citrus, lettuce, and hops [11]. According to EFSA (2018) [10], FOS is a phosphonate compound; its structure and mode of action vary from most of the organophosphorus compounds applied as pesticides. EFSA also reported a data gap for the FOS risk to aquatic organisms and to non-target terrestrial plants. There is no information regarding the lethal and sub-lethal effects of FOS on the ecotoxicological model aquatic and terrestrial species, _Danio rerio_ and _Enchytraeus cryptic_, respectively. Considering the effects of other pesticides to _D. rerio_, a median lethal concentration (LC\({}_{50}\)) of 0.235 mg/L, a decreased hatching success, and several malformations were found on embryos after exposure to the insecticide sumithion [12]. Additionally, a strong inhibition of hatching by triazole fungicides [13] and diazimon insecticide [14] exposure was already reported in zebrafish embryos. For _E. crypticus_, organisms were not affected by the insecticide toxaphene [15] and fungicide prosaro [16]. However, the insecticide dimethoate increased the survival and limited the growth of enchytraeids, and fungicides mancozeb and amistar decreased the survival and reproduction [16; 17]. Zebrafish (_D. rerio_) is a widely used model organism for aquatic (eco)toxicology being employed for the assessment of effects at different levels of biological organization [18]. Particularly, the fish embryo toxicity (FET) test with zebrafish embryos has been shown to be a valuable alternative to the common fish toxicity test [19]. The FET test [20] may be seen as a refinement or even a potential replacement of animal experiments according to the 3 Rs principle (refinement, reduction, and replacement) [21], as the embryos are assumed to experience less or no pain compared to adult fish. In specific, zebrafish was already employed to assess the toxicity of several pesticides, such as imidalcoprid, atrazine, chlorpyrifos, butachlor, \(\lambda\)-cyhalothrin, sumithion, linuron, and propomacarb [12; 18; 22; 23]. Enchytraeids are ecologically important, soil-dwelling annelids, playing an essential role in organic matter decomposition and soil bioturbation [24]. Enchytraeid reproduction test (ERT) guidelines [25] have been established for the genus _Enchytraeus_. _E.