Environmental risk assessments (ERAs) of genetically engineered (GE) plants rely on robust, high-quality, and relevant ecotoxicological studies on non-target invertebrates. Although ERAs also consider evidence from published literature, the quality of these studies and their suitability for ERA are often unclear. This review focuses on aquatic invertebrates and comprises 51 articles identified through systematic literature searches. The review provides a detailed critical appraisal of studies and synthesizes recommendations for future research.Studies using purified Bt proteins reported effects on aquatic species only at very high concentrations, which possibly reflect non-specific toxicity rather than Bt protein-specific effects. Adverse effects reported for Bt plant material were frequently inconsistent. Methodological shortcomings indicate plant-background effects or experimental artifacts rather than Bt protein effects. Studies reporting no effects are difficult to interpret when exposure levels are unknown or test durations are short. Artifacts may arise when test substances are insufficiently characterized, when negative control treatments differ from test treatments in aspects other than the test compound, or when results are inconsistent across treatments and temporal repetitions. For laboratory studies involving GE plants other than Bt, as well as field studies, the available evidence does not indicate adverse effects, although methodological limitations were also identified for those kind of studies. Future research using plant material should be designed to disentangle effects of the gene product from plant background effects. The critical appraisal scheme developed in this review provides guidance for robust test protocols using purified proteins and GE plants and is also applicable to studies on terrestrial non-target species.