Determination of the median effective concentration (EC50) of Cu on Mytilus galloprovincialis larvae by diffusive gradient in thin films (DGT) has been shown to effectively reduce the need to consider dissolved organic carbon (DOC) concentration and quality. A standard toxicity test protocol was used to validate previously modeled protective effects, afforded to highly sensitive marine larvae by ligand competition, in 5 diverse site waters. The results demonstrate significant narrowing of M. galloprovincialis toxicological endpoints, where EC50s ranged from 3.74 to 6.67 μg/L as CDGT Cu versus 8.76 to 26.8 μg/L as dissolved Cu (CuDISS ) over a DOC range of 0.74 to 3.11 mg/L; Strongylocentrotus purpuratus EC50s were 10.5 to 19.3 μg/L as CDGT Cu versus 22.7 to 67.1 μg/L as CuDISS over the same DOC range. The quality of DOC was characterized by fluorescence excitation and emission matrices. The results indicate that the heterogeneity of competing Cu binding ligands, in common marine waters, minimizes the need for class determinations toward explaining the degree of protection. Using conservative assumptions, an M. galloprovincialis CDGT Cu EC50 of 3.7 µg/L and corresponding criterion maximum concentration CDGT Cu of 1.8 µg/L, for universal application by regulatory compliance-monitoring programs, are proposed as a superior approach toward both integration of dynamic water quality over effective exposure periods and quantification of biologically relevant trace Cu speciation. Environ Toxicol Chem 2020;39:873-881. © 2020 SETAC.