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There are currently a variety of applications for multiwalled carbon nanotubes (MWCNTs), but considerable concerns exist regarding their release into the environment. Their potential accumulation by aquatic organisms could lead to transfer throughout food chains. Considering the divergences in experimental data published on the ecotoxicity of carbon nanotubes, further research is required. The dispersion of MWCNTs in aqueous culturing media of organisms as well as the determination of concentrations are relevant aspects to obtain accurate ecotoxicity results. Ultraviolet-visible spectroscopy is one of the most reported techniques to analyze concentration quickly and economically, but the methodologies to prepare dispersions and selecting the wavelengths for ultraviolet-visible measurements have not yet been clearly defined. The present study demonstrates that dispersion procedures influence absorbance, and an approach to determine the most appropriate measurement wavelength is proposed. Ecotoxicity tests with MWCNTs were performed on Vibrio fischeri bacteria, and divergences in the results were observed with respect to those previously reported. The present study contributes to the attempt to overcome the lack of standardization in the environmental assessment of MWCNTs.