Acute toxicity of chlorpyrifos (CP) and its principal metabolite 3,5,6-trichloro-2-pyridinol (TCP) alone and in combination have been evaluated using a test battery comprising aquatic organisms from different trophic levels: luminescent marine bacteria Aliivibrio fischeri, freshwater unicellular alga Pseudokirchneriella subcapitata, and cladoceran Daphnia magna. As expected, D. magna was the more sensitive organism to the compounds tested, being CP more toxic than its metabolite. On the contrary, TCP was found to be more toxic than its parental compound to A. fischeri and P. subcapitata. In all cases, the mixture of CP and its metabolite was more toxic than the compounds tested separately, multiplying between 5 and 200 times CP toxicity level and up to 15 times TCP toxicity level. These results indicate that the co-existence of parent chemical and its degradation product in the environment can result in a synergic interaction involving high risk to the aquatic ecosystems. Graphical abstract.