Negligible cytotoxicity induced by different titanium dioxide nanoparticles in fish cell lines

Titanium dioxide nanoparticles (TiO₂-NPs) have a wide number of applications in cosmetic, solar and paint industries due to their photocatalyst and ultraviolet blocking properties. The continuous increase in the production of TiO₂-NPs enhances the risk for this manufactured nanomaterial to enter water bodies through treated effluents or agricultural amendments. TiO₂-NPs have shown very low toxicity in a number of aquatic organisms. However, there are no conclusive data about their deleterious effects and on their possible mechanisms of toxic action. At this level, in vitro cell culture systems are a useful tool to gain insight about processes underlying the toxicity of a wide variety of substances, including nanomaterials. Differences in the physiology of different taxa make advisable the use of cells coming from the taxon of interest, but collecting data from a variety of cellular types allows a better understanding of the studied processes. Taking all this into account, the aim of the present study was to assess the toxicity of three types of TiO₂-NP, rutile hydrophobic (NM-103), rutile hydrophilic (NM-104) and rutile-anatase (NM-105), obtained from the EU Joint Research Centre (JRC) repository, using various fish cell lines (RTG-2, PLHC-1, RTH-149, RTL-W1) and rainbow trout primary hepatocytes. For comparative purposes, the effect of different dispersion protocols, end-point assays and extended exposure time was studied in a fish cell line (RTG-2) and in the rat hepatoma cell line (H4IIE). TiO₂-NPs dispersions showed a variable degree of aggregation in cell culture media. Disruption of mitochondrial metabolic activity, plasma membrane integrity and lysosome function was not detected in any cell line after exposure to TiO₂-NPs at any time and concentration ranges tested. These results are indicative of a low toxicity of the TiO₂-NPs tested and show the usefulness of fish cells maintained in vitro as high throughput screening methods that can facilitate further testing in the framework of integrated testing strategies.