Artificial sweeteners are broadly used as safe food additives and in pharmaceutical formulations. As these compounds are relatively stable and poorly removed by water treatment facilities, their environmental concentrations increase. Therefore, concerns about their potential risks to non-targeted aquatic biota have been raised. Although no lethal effects are usually observed in standard toxicity testing, recent effect studies suggest a possible neurotoxic mode of action.We tested the effects of commonly used sugar substitutes (sucralose and acesulfame, up to 1 mg/L) in a nontarget species Daphnia magna, by assessing biochemical (acetylcholinesterase activity, AChE), physiological (heart rate, HR) and behavioural (time spent on swimming and feeding) endpoints. We found a dose-dependent increase in AChE activity and inhibitory effects on HR and behavioural endpoints, with lower EC 50 values observed for acesulfame than sucralose, although all these values were within the reported AS levels in the wastewater. Moreover, a biphasic response was observed for acesulfame-exposed daphnids, with AChE inhibition at the lower concentrations (< 1 μg/L ) and stimulatory effects at the higher concentrations. Thus, the response propagated across the biological organization levels, with swimming inhibition associated with AChE stimulation. Whereas HR and swimming were positively related across the ASs and all concentrations tested, the relationship between AChE and HR was substance-specific, suggesting possible differences in the mode of action. The observed LOEC values for acesulfame were as low as 0.1 μg/L , these results suggest that artificial sweeteners may exert adverse effects on nontarget biota at environmentally relevant concentrations, which calls for a critical evaluation of current thresholds used in their risk assessment.