Carbaryl and fenitrothion are two insecticides sharing a common mode of action, the inhibition of the acetylcholinesterase (AChE) activity. Their use is now regulated or banned in different countries, and the environmental levels of both compounds in aquatic ecosystems have decreased to the range of pg/L to ng/L. As these concentrations are below the non-observed-adverse-effect-concentrations (NOAEC) for AChE inhibition reported for both compounds in aquatic organisms, there is a general agreement that the current levels of these two chemicals are safe for aquatic organisms. In this study we have exposed zebrafish, Japanese medaka and Daphnia magna to concentrations of carbaryl and fenitrothion under their NOAECs for 24-h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR) and its habituation have been evaluated. Both pesticides increased the HR in the three selected model organisms, although the intensity of this effect was chemical-, concentration- and organism-dependent. The exposure to both pesticides also led to a decrease in BLA and an increase in VMR in all three species, although this effect was only significant in zebrafish larvae. For SR and its habituation, the response profile was more species- and concentration-specific. The results presented in this manuscript demonstrate that concentrations of carbaryl and fenitrothion well below their respective NOAECs induce tachycardia and the impairment of ecologically relevant behaviors in phylogenetically distinct aquatic model organisms, both vertebrates and invertebrates, emphasizing the need to include this range of concentrations in the environmental risk assessment.Read less <