Despite the consensus about the importance of chemical speciation in controlling the bioavailability and ecotoxicity of trace elements, detailed speciation studies during laboratory ecotoxicity testing remain scarce, contributing to uncertainty when extrapolating laboratory findings to real field situations in risk assessment. We characterized the speciation and ecotoxicological effects of CrIII and CrVI in the OECD medium for algal ecotoxicity testing. Total and dissolved (< 0.22 µm) Cr concentrations showed little variability in media spiked with CrVI, while dissolved Cr concentration decreased by as much as 80% over 72 hours in medium amended with CrIII. Analyses by ion chromatography ICP-MS highlighted the absence of redox interconversion between CrIII or CrVI both in the presence and absence of algal cells (Raphidocelis subcapitata). Furthermore, the concentration of ionic CrIII dropped below detection limits in less than 2 hours with the corresponding formation of carbonate complexes and Cr hydroxides. Precipitation of CrIII in the form of colloidal particles of variable diameters was confirmed by nanoparticle tracking analysis, spICP-MS and single particle counting. In terms of time weighted dissolved (< 0.22 µm) Cr concentration, CrIII was 4 to 10 times more toxic than CrVI. However, CrIII ecotoxicity could arise from interactions between free ionic CrIII and algae at the beginning of the test, from the presence of Cr-bearing nanoparticles or a combination of the two. Future ecotoxicological studies must pay more attention to Cr speciation to reliably compare the ecotoxicity of CrIII and CrVI.