Increases in the concentrations of major ions (Na+, K+, Ca2+, Mg2+, Cl−) in freshwater are a growing concern for ecosystem health. These increases may originate from anthropogenic activities such as road deicing, fracking spills, mining, and fertilizer application and have detrimental effects on freshwater organisms through disturbances in ionoregulation and acid–base balance. The cladoceran Daphnia magna is adapted for active ion uptake and reduction of ion loss to maintain osmotic balance, but alterations in ionic composition of the environmental water are associated with toxicity. In the present study, hemolymph ion concentrations were measured using ion‐selective microelectrode techniques. Increases in the hemolymph concentrations of Na+ and K+ correspond to elevations in the concentrations of these ions in ambient water. Water concentrations associated with sustained increases in hemolymph ion concentrations correlate well with median lethal concentration values from previous toxicology studies, indicating that Na+ and K+ concentrations in hemolymph may predict toxicity. When water K+ concentration is increased, a simultaneous increase in water Na+ concentration mitigates the increase in hemolymph K+ concentration, a finding which is consistent with the reported mitigation of K+ toxicity by Na+. When ambient concentrations of K+, Na+, and Cl− are increased, not only is there a rise in hemolymph ion concentration but hemolymph pH is altered and pH regulation appears to be prioritized over regulation of hemolymph Na+, K+, and Cl− in D. magna. Environ Toxicol Chem 2021;40:366–379. © 2020 SETAC