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The most up-to-date regulatory guidelines for establishing acute and chronic numeric limits for copper in freshwaters are based on a biotic ligand model for various species, but the model for Cu lacks data on dietary uptake. In addition, some common macroinvertebrate toxicity assay parameters are less representative of the ecosystem. We investigated the effects of diet and its type in the experimental setup and as an exposure pathway to an established amphipod (crustacean) Hyalella azteca (H. azteca) for Cu toxicity assays. We also investigated another overlooked aspect, the organic matter (OM) source. Our experiments compared the toxicity of pre-equilibrated and unequilibrated natural diets and a laboratory-favored diet in effluent and stormwater sources of organic matter adjusted to standard water characteristics. The experiments indicated a more toxic effect of the pre-equilibrated diet and natural dietary sources, and less toxic effects in the presence of effluent OM compared with stormwater OM, shifting LC50 or EC20 values by as much as 67% compared with the controls. The use of a pre-equilibrated natural diet in toxicity assays provides the advantage of producing toxicity data more representative of field conditions. Considering organic matter type, especially in dietary exposures, will better predict toxicity, accounting for copper complexation with OM from different sources and partitioning to the food supply. Adapting these ecologically relevant parameters in whole effluent toxicity testing or other assays will also provide safer regulatory oversite of discharges to surface waters.