Previous studies have explored effects of imidacloprid and its metabolites on terrestrial species, such as bees, and indicated the importance of some active metabolites. However, the biotransformation of IMI and the toxicity of its metabolites to aquatic arthropods are largely unknown, especially the mechanisms driving species sensitivity differences and time-cumulative toxicity effects. To assess the potential effects of the metabolization of IMI and the toxicokinetics and toxicity of the metabolite(s) on aquatic arthropods, we first studied the acute toxicity of IMI and relevant metabolites to the mayfly species Cloen dipterum (sensitive to IMI) and the amphipod species Gammarus pulex (less sensitive to IMI). Secondly, toxicokinetic experiments were conducted using both the parent compound and imidacloprid-olefin (IMI-ole), a metabolite assessed as toxic in the acute tests and defined as bioactive. Of the four tested metabolites, only IMI-ole was readily biotransformed from the parent IMI and showed similar toxicity to C. dipterum as IMI. However, C. dipterum was hardly able to eliminate IMI-ole from its body. For G. pulex, IMI-ole was also the only detected metabolite causing toxicity, but the biotransformation of IMI to IMI-ole was slower and lower in G. pulex compared to C. dipterum, and G. pulex eliminated IMI-ole quicker than C. dipterum. Our results on internal kinetics of IMI and IMI-ole, and on biotransformation of IMI indicated that the metabolite IMI-ole was toxic and was rather persistent inside the body tissue of both invertebrate species, especially for C. dipterum. In conclusion, as IMI and IMI-ole have similar toxicity and IMI was replaced rapidly by IMI-ole which in turn was poorly eliminated by C. dipterum, the overall toxicity is a function of dose and time. As a result, no long-term threshold of effects of IMI may exist for C. dipterum as the poor elimination results in an ongoing increase of toxicity over time for mayflies as also found experimentally in previous published papers.