The aim of this study was to assess the toxicity of runoff waters in an agricultural multipollution context through an in-depth assessment of copper bioavailability and toxicity. Runoff waters were screened for major ions, metals and diuron. The potential environmental impact of these runoff waters was evaluated using the conventional 72-h growth inhibition test with the green alga Pseudokirchneriella subcapitata. The results suggested that the toxicity detected in the calcareous vineyard field was due to the presence of diuron, whereas the non-calcareous runoff waters were non-toxic. Chemical speciation modelling by MINEQL revealed that most of the copper present in the non-toxic natural runoff waters was complexed by organic matter. These samples were spiked with copper, and then the toxicity and the electrochemically bioavailable copper fraction were measured. Differential pulse anodic stripping voltammetry (DPASV) was used to detect labile complexes and free copper. This combined approach highlighted the presence of some labile copper complexes in samples reaching the EC10—these could have contributed to the copper toxicity