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The effects of pH (5.3–8.7), water hardness (CaCO₃ at 25–500 mg/L), dissolved organic carbon (DOC) concentration (1.6–18.4 mg/L), and DOC source on the chronic toxicity of copper to Daphnia magna were investigated by using a multifactorial, central composite test design. Natural dissolved organic matter (DOM) was collected at three sites in Belgium and The Netherlands by using reverse osmosis. For a total number of 35 toxicity tests performed, 21‐d no‐observed‐effect concentrations (NOECs) of copper based on reproduction ranged from 29.4 to 228 μg/L and 21‐d concentrations of copper causing 50% reduction of reproduction (EC50s) ranged from 41.5 to 316 μg/L. Statistical analysis revealed that DOC concentration and pH had a significant effect on copper toxicity but hardness (at the levels tested) did not. In general, an increase in pH or DOC resulted in a linear increase of 21‐d NOEC and EC50 …