Design, sensitivity analysis, and testing of a Caenorhabditis elegans nanoecotoxicity assay
Shannon Hanna, Gregory A. Cooksey, Bryant C. Nelson, John T. Elliott, Elijah J. Petersen
Increasing production and usage of engineered nanomaterials (ENMs) has generated widespread interest in measuring their environmental and human health effects. However, the lack of standardized methods for these measurements has often led to contradictory results. Our goal in this study was to assess the feasibility of using a standardized C. elegans growth inhibition- based toxicity test for dissolved chemicals for assessing nanomaterial toxicity. First, we improved the precision of the assay through development of a robust, semi-automated microscopy imaging and image analysis approach. Then, cause-and-effect analysis was used to catalog potential sources of variability in the assay protocol. Sensitivity analysis of five key experimental factors identified by the cause-and-effect analysis revealed that bacterial feed concentration and plate shaking had significant effects on growth inhibition by a reference toxicant, benzylcetyldimethylammonium chloride (BAC-C16). Other factors such as organism maintenance, ionic strength, and bacterial viability showed minimal effect on the test method. Bacterial concentration was inversely proportional to experimental EC50 values, while shaking the plates during the assay decreased nematode growth in control groups by 22% and decreased nematode sensitivity to BAC-C16. Testing this assay with polystyrene nanoparticles (PSNPs) revealed that the variability in the PSNP EC50 values were larger compared to those of BAC-C16. Our study details the modifications that can be made to adapt the standard C. elegans toxicity assay for use with ENMs with minimal change in assay results.