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

Abstract

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.

Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study

“…The NP dispersion step of the protocol is not precisely defined and the timing between the dispersion step and the dosing step is not specified, which can influence the extent of agglomeration during the exposure or the structure/composition of the protein corona. If variable precipitation of the NP agglomerates occurred during the separate rounds, the cells may have been effectively exposed to different dosing conditions (Petersen et al, 2014). Improved dispersion procedures or the use of more automation in preparing the dosing plate could reduce the variability observed within the laboratory when using NH 2 -PS NP and serum.…”

Interactions between engineered nanomaterials and agricultural crops: implications for food safety

“…However, the most advanced instrumentation platforms used for these types of studies are costly and of limited accessibility (Szakal et al, 2014). As such, a multidisciplinary and orthogonal approach is needed, including the development of new or hybridized measurement techniques, in order to promote the understanding of ENMs-crop interactions (GardeaTorresdey et al, 2014;Petersen et al, 2014).…”

Control experiments to avoid artifacts and misinterpretations in nanoecotoxicology testing

“…However, results from toxicological studies, such as measurements of their cell toxicity, often differ substantially among laboratories and have questionable reliability (1)(2)(3). Moreover, numerous studies have identified artifacts (i.e., incorrect test results) during nanoecotoxicology studies as a result of the unique behaviors of ENMs (3). One important aspect of experimental design is the inclusion of control experiments to identify potential artifacts and increase reliability of the test results.…”

“…This information is modified from a recent publication on the avoidance of artifacts and misinterpretations in nanoecotoxicology testing, and the reader is referred to that manuscript for a more complete discussion of this topic (3). While many of these same control experiments are relevant for measurements on the potential human health impacts of ENMs, the focus in this protocol is on measurements of potential ecological effects.…”