The Use of an <i>In Chemico</i> Digestibility Assay to Reduce the <i>In Vivo</i> Fish Bioaccumulation Testing of Nanomaterials

Currently for engineered nanomaterials (ENMs), there are no alternatives to bioaccumulation testing in fish as prescribed by OECD TG 305. New approaches are being sought, in order to reduce the numbers of animals used and address the ethical concerns associated with the use of vertebrates in such testing. This study aimed to demonstrate the value of an alternative method - the <i>in chemico</i> digestibility assay - as a tool to assess the bioaccumulation potential of ENMs. Titanium dioxide ENMs, or their equivalent bulk form, were incorporated onto a commercial fish diet and the fish digestion process was simulated <i>in vitro</i> at pH 2 (simulated stomach phase) and pH 7.8 (simulated intestinal phase). Only the 1000 mg/kg dry weight feed treatment showed measurable Ti release in the stomach phase, with the results being similar for the TiO₂ ENM and the bulk form. More Ti from TiO₂ was released from the feed during the intestinal phase of digestion compared to the stomach phase, but there were no statistically significant differences between either the ENM or bulk form. There was higher incidental Ti release from untreated control feed in the intestinal phase compared to the stomach phase. On a percentage basis, the stomach generally showed a statistically significant digestibility of around 3% of the total Ti in the feed, compared to around 7% in the intestine. The <i>in chemico</i> digestibility assay shows potential for use in the determination of the bioaccessible fraction of ENMs, and could be a useful screening tool to identify ENMs of concern for bioaccumulation.