Neutral red retention time assay in determination of toxicity of nanoparticles

• Neutral red retention time assay used haemolymph of five pooled mussels.
• Assay was miniaturised for reading in a plate reader, facilitating many samples and replicates.
• Copper, chromium and cobalt nanoparticles were toxic while gold and titanium were not.

The neutral red retention time (NRRT) assay is useful for detecting decreased lysosomal membrane stability in haemocytes sampled from bivalves, a phenomenon often associated with exposure to environmental pollutants including nanomaterials. Bivalves are popular sentinel species in ecotoxicology and use of NRRT in study of species in the genus Mytilus is widespread in environmental monitoring. The NRRT assay has been used as an in vivo test for toxicity of carbon nanoparticles (Moore MN, Readman JAJ, Readman JW, Lowe DM, Frickers PE, Beesley A. 2009. Lysosomal cytotoxicity of carbon nanoparticles in cells of the molluscan immune system: An in vivo study. Nanotoxicology. 3 (1), 40–45). We here report application of this assay adapted to a microtitre plate format to a panel of metal and metal oxide nanoparticles (2 ppm). This showed that copper, chromium and cobalt nanoparticles are toxic by this criterion while gold and titanium nanoparticles are not. As the former three nanoparticles are often reported to be cytotoxic while the latter two are thought to be non-cytotoxic, these data support use of NRRT as a general in vitro assay in nanotoxicology.