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Novel physicochemistries of engineered nanomaterials (ENMs) offer considerable commercial potential for new products and processes, but also the possibility of unforeseen and negative consequences upon ENM release into the environment. Investigations of ENM ecotoxicity have revealed that the unique properties of ENMs and a lack of appropriate test methods can lead to results that are inaccurate or not reproducible. The occurrence of spurious results or misinterpretations of results from ENM toxicity tests that are unique to investigations of ENMs (as opposed to traditional toxicants) have been reported, but have not yet been systemically reviewed. Our objective in this manuscript is to highlight artifacts and misinterpretations that can occur at each step of ecotoxicity testing: procurement or synthesis of the ENMs and assessment of potential toxic impurities such as metals or endotoxins, ENM storage, dispersion of the ENMs in the test medium, direct interference with assay reagents and unacknowledged indirect effects such as nutrient depletion during the assay, and assessment of the ENM biodistribution in organisms. We recommend thorough characterization of initial ENMs including measurement of impurities, implementation of steps to minimize changes to the ENMs during storage, inclusion of a set of experimental controls (e.g., to assess impacts of nutrient depletion, ENM specific effects, impurities in ENM formulation, desorbed surface coatings, the dispersion process, and direct interference of ENM with toxicity assays), and use of orthogonal measurement methods when available to assess ENMs fate and distribution in organisms.

Identification and Avoidance of Potential Artifacts and Misinterpretations in Nanomaterial Ecotoxicity Measurements