Towards unbiased interpretations of interactive effects in ecotoxicological studies

Ecotoxicological research has increasingly focused on the interactive effects of chemical mixtures on biological models, emphasising additive, synergistic, or antagonistic interactions. However, these combination studies often test chemicals at unique concentrations (e.g. x:y), limiting our understanding of the effects across the full spectrum of possible combinations. Evidence from human toxicology suggests that interactive effects among chemicals can vary significantly with total concentration (e.g. x:y vs. 2x:2y), their ratio (e.g. x:2y vs. 2x:y), and the magnitude of the tested effect (e.g. LC10vs. LC50). Our non-exhaustive review of studies on binary mixtures in bee ecotoxicology reveals that such parameters are frequently neglected. Of the 60 studies we examined, only two utilised multiple total concentrations and ratios, thus exploring a broad range of possible combinations. In contrast, 26 studies tested only a single concentration of each chemical, resulting in incomplete interpretations of the potential interactive effects. Other studies utilised various concentrations and/or ratios but failed to capture a broad spectrum of possible combinations. We also discuss potential discrepancies in interactive effects based on different metrics and exposure designs. We advocate for future ecotoxicological studies to investigate a wider spectrum of chemical combinations, including various concentrations and ratios, and to address different levels of effects.