Because the bioavailability of a trace metal, and consequently its toxicity, is dependent on the physical and chemical form of the metal, we have presented a detailed assessment of how speciation of copper would be expected to affect its toxicity. Principles of chemical speciation are applied to demonstrate that inorganic forms will be in constant proportion to each other and to free copper ion during the course of the titration of a sample of natural water with copper or in the various treatments in a toxicity test conducted at constant pH and alkalinity. Binding of copper to dissolved organic matter or to suspended particulate matter may render the copper nonbioavailable. We have considered a simple complexation model to describe the complexation of copper to soluble ligands. Naturally occurring dissolved organic matter is present at concentrations only slightly greater than that of copper. Consequently, titration of water with copper results in a nonlinear relationship between the concentration of copper present as free copper ion plus inorganic copper species. The effects of stability constant of the complex, concentration of ligand, and the total copper concentration are evaluated. We have related bioavailable copper to the concentration of free copper ion plus inorganic copper complexes, which is valid if the pH and alkalinity of the waters used to develop a criteria are not different. On the basis of limited field data for the complexation of copper in Narragansett Bay water, we do not expect that significant differences in water quality criteria (WQC) would result if the criteria were to be based on free copper ion plus inorganic copper complexes rather than total copper concentrations. We examined the effect of speciation of copper in different waters as related to empirical or theoretically calculated water effect ratios (WER). We show that, on the basis of sound chemical principles, it would be expected that the most sensitive organisms would have the greatest WER. This prediction is confirmed by the empirical observations available. For insensitive organisms, knowledge of the concentration of ligand is sufficient to reasonably predict the WER. However, for the more sensitive organisms that give higher WERs, it is necessary to measure or calculate the speciation of copper to predict the WER. Use of predicted WERs may replace use of empirically derived WERs as is now part of regulatory guidance for derivation of site‐specific WQC. if correspondence has been demonstrated.