Neonicotinoids are systemic, broad-spectrum insecticides that are largely applied on crops for human consumption. Recently added to the EU watch list as a drug to be monitored (2018/840/ EC), acetamiprid (ACE) is a chlorinated nicotine pesticide primarily used to prevent Lepidoptera and Hemiptera pests. Environmental transformation of acetamiprid produces metabolites that can exhibit very high persistency in soil and high mobility in groundwaters. This combined with high water solubility, facilitates the transport and contamination of nearby freshwater compartments. Additionally to the active ingredients (a.i.), insecticides used in agricultural production contain several additives, solvents and excipients that are listed as inert but can exhibit relevant toxicological properties. The main objective of this work was to assess the toxicity of acetamiprid as an active ingredient (96% purity) and its commercial formulation EPIK SG® (20% p/p a.i.) to freshwater non-target organisms of different trophic levels. Worst-case scenarios of toxic exposure were evaluated, by carrying short-term toxicity assays with primary producers (Raphidocelis subcapitata, 72h-growth inhibition assay), primary consumers (Daphnia magna, 48h immobilization assay) and secondary consumers (Hydra viridissima, 96h mortality assay; Xenopus laevis and Pelophylax perezi, 96h FETAX assay, 96h tadpole acute toxicity assay). Behaviour assays were also performed with Xenopus laevis tadpoles. Mortality and sublethal endpoints were evaluated and concentrations causing 20 and 50% of lethal (LCx) and sublethal effects (ECx) were estimated. Primary producers were the less sensitive species to EPIK SG^® and acetamiprid (EC_{50, EPIK SG®} = 517.8 mg/L of a.i., EC_{50, ACE} = 747.9 mg/L for R. subcapitata growth rate). H. viridissima revealed the highest lethality to EPIK SG® (LC₅₀ = 22.04 mg/L of a.i.) and P. perezi to acetamiprid (LC₅₀ = 134.4 mg/L). Behaviour testing proved to be much more sensitive than traditional toxicity tests. EPIK SG® was the most toxic compound to all freshwater species tested, showing that formulation additives can exhibit a higher toxicity by themselves and/or enhance the toxicity of active ingredients. These findings highlight the necessity to invest in alternative models of conventional formulas, minimizing the toxicity and persistence in the environment, contributing towards more sustainable practices.