Within human modified ecosystems the effects of individual stressors are difficult to establish amid co-occurring biological processes, environmental gradients and other stressors. Coupled examination of several endpoints across different levels of organisation may help elucidate the individual and combined effects of stressors and interactions. Malathion is a commonly used organophosphate pesticide that contaminates freshwaters and has strong negative effects on aquatic biota. However, both other stressors (e.g. increased sediment) and common ecosystem components (e.g. macrophytes and variable pH) can reduce the aqueous concentrations of malathion, reducing its toxic effects. We conducted a fully orthogonal bioassay to examine how pH (at 7 and 7.8) and sorptive processes (across two levels of kaoline clay 0 and 24 g L−1) affected aqueous malathion concentrations and toxicity in an aquatic invertebrate genus. Survival and acetylcholinesterase activity as a sub-organism response were examined in the mayfly Coloburiscoides spp. (Ephemeroptera; Coluburiscidae). Measured aqueous malathion concentrations decreased with increased pH and in the presence of kaolin clay. Survival declined with increasing malathion concentrations and exposure period. Results further identify that antagonism of malathion toxicity was associated with both pH (alkaline hydrolysis) and effects associated with sediment independent of pH (driven by sorptive processes). However, model predictions varied associated with target and measured concentrations and concentrations examined. Antagonistic effects were most apparent using subset target malathion concentrations because of the dominant effect of malathion at high concentrations. Acetylcholinesterase activity, identified repression occurred across all treatments and did not identify antagonistic interactions, but these results were similar to survival responses at the time points examined (i.e. 120 h). Examination of chemistry, acetylcholinesterase, and survival, affords greater understanding of stressor effects and their interactions. Measured malathion concentrations may underestimate effects on aquatic biota; not because of synergism among stressors, but because of strong effects despite antagonism.