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Abstract Spinosad and indoxacarb are two relatively new insecticides mainly used in agriculture to control insect pests. However, at their current application rates, non-target aquatic insect species may also be impacted. In this study, larvae of the non-biting midge Chironomus riparius were exposed in the laboratory to both insecticides and their effects evaluated at the organismal level, using standard ecotoxicological tests, and at the biochemical level, by monitoring specific oxidative stress, neuronal, and energy metabolism biomarkers. Chronic exposure to both insecticides compromised growth and emergence of C. riparius. Short-term exposures revealed alterations at biochemical level that might be related to the toxicological targets of both insecticides. Growth and development time were the most sensitive endpoints at individual level for both pesticides, while at the biochemical level, the electron transport system activity was the most sensitive biomarker for spinosad exposure, suggesting an increase in energy demands associated with the activation of defense mechanisms. Glutathione-S-transferase was the most sensitive biomarker for indoxacarb exposure, underlining the role of this enzyme in the detoxification of indoxacarb. Additionally, changes in lactate dehydrogenase and glutathione peroxidase activities were observed for both insecticides, and evidences of oxidative damage were found for spinosad. This study contributes to the growing knowledge on sublethal effects of novel insecticides on non-target aquatic invertebrates and strengthens the usefulness of biochemical biomarkers to support the interpretation of their potentially deleterious effects on aquatic insects near agricultural fields. Graphical abstract Unlabelled Image Highlights • Responses of C. riparius to spinosad and indoxacarb exposure were investigated. • Exposure to both insecticides compromised C. riparius life-history traits. • Both insecticides induced defense mechanisms and cellular oxygen consumption. • Non-target aquatic insect species may be impacted by spinosad and indoxacarb use. [ABSTRACT FROM AUTHOR]