Nanoparticles and colored substances can inhibit algal growth by light shading and chemical toxicity. This study presents two complementary approaches to account for shading in algal growth inhibition tests of engineered nanomaterials (ENMs) and colored substances. The first approach distinguishes between shading effects and toxicity by varying the light path in parallel algal growth inhibition tests. This Multiple Path-Length (MPL) test was applied to TiO2 ENMs and the colored substances sodium picramate and Rhodamine B. A left shifting of concentration-response curves, with increasing light path lengths, indicated shading for Rhodamine B, sodium picramate and TiO2 ENMs. EC50-values obtained at the shortest light path length were generally found best suited to quantify the toxicity of ENMs and colored substances. The second approach addresses shading at the cellular level, where particles can attach to the cell surface and affect photo-pigment content and composition. Pigments associated with photosystem I and II were determined at varying light intensities and concentrations of TiO2 ENMs. The photo-pigments that increased in response to physical shading, decreased after TiO2 ENMs exposure. This indicates that toxicity rather than cellular shading dominated the response of algae exposed to TiO2 ENMs. Additional tests were conducted with the nanomaterials CeO2 and goethite to evaluate the applicability of this approach to other ENMs. On this basis, we recommend MPL testing for determining EC50-values that are not confounded by shading in the test solution, and the pigment-based approach for investigating shading on the cellular level.
Keywords: Green algae; ecotoxicology; exposure; test artifacts; titanium dioxide.