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In this sudy, the combined effects of pH, water hardness, and dissolved organic carbon(DO) concentration and type on the chronic (72‐h) effect of copper on growth inhibition of the green alga Pseudokirchneriella subcapitata were investigated. Natural dissolved organic matter (DOM) was collected at three sites in Belgium and The Netherlands using reverse osmosis. A full central composite test design was used for one DOM and a subset of the full design for the two other DOMs. For a total number of 35 toxicity tests performed, 72‐h effect concentration resulting in 10% growth inhibition (E_bC10s) ranged from 14.2 to 175.9 μg Cu/L (factor 12) and 72‐h E_bC50s from 26.9 to 506.8 μg Cu/L (factor 20). Statistical analysis demonstrated that DOC concentration, DOM type, and pH had a significant effect on copper toxicity; hardness did not affect toxicity at the levels tested. In general, an increase in pH resulted in …