Assessment of titanium dioxide nanoparticle toxicity in the rainbow trout (Onchorynchus mykiss) liver and gill cell lines RTL-W1 and RTgill-W1 under particular consideration of nanoparticle stability and interference with fluorometric assays

Abstract

The increasing release of titanium dioxide (TiO2) nanoparticles (NPs) into the environment calls for a thorough assessment of their hazard to aquatic organisms. In vitro-assays based on continuous fish cell lines bear a high potential for determining the relative cytotoxic potency and mode of toxic action of environmental contaminants. Their application for testing of manufactured nanomaterials is however concomitant with technical challenges. The objective of this study was to determine the acute cytotoxicity of TiO2 NP in the rainbow trout (Onchorynchus mykiss) liver and gill cell lines RTL-W1 and RTgill-W1 under special consideration of so far widely ignored issues arising from poor NP stability and NP-dependent interference with fluorometric assays. We validated a protocol for the preparation of serum albumin-stabilized TiO2 NP dispersions in two growth media, which are frequently used in the culture of fish cells, Leibovitz-15 (L-15) and L-15/ex. The TiO2 NPs dispersions displayed high colloidal stability with their size distribution remaining unchanged over a concentration range that spans two orders of magnitude (1–100 μg/ml) and an incubation period of 72 h. The TiO2 NPs dispersions caused little cytotoxicity upon 24 h and 72 h exposure, but NP agglomerates/aggregates were found inside intracellular vesicles in both gill and liver cells. Furthermore we demonstrated that TiO2 NPs, which remained adsorbed to the plasma membrane and/or were internalized by the cell interfere with fluorometric cell viability assays by attenuating part of the incident and emitted light resulting in effect overestimation. We propose modifications in the original assay protocol that may allow quantifying and mathematically correcting for the level of interference at different exposure concentrations.