Environmental exposure to single and combined ZnO and TiO₂ nanoparticles: Implications for rainbow trout gill immune functions and microbiota

ZnO and TiO₂ nanoparticles (NPs) are widely employed for their antibacterial properties, but their potential environmental impact is raising concerns. This study aimed to assess their single and combined effects at environmentally relevant concentrations (210 μg L^-1) on rainbow trout (Oncorhynchus mykiss) gills microbiota and immune functions. 16S rRNA gene sequencing performed after 5 and 28 days of exposure suggests that TiO₂ NPs had a more immediate impact on bacterial diversity, while prolonged exposure to the mixture altered community composition. Changes in the relative abundance of potential pathogenic genera such as Candidatus Piscichlamydia and Flavobacterium were observed. Additionally, while the expression of the pro-inflammatory cytokine il1β, and antibacterial compounds (c3) was downregulated by TiO₂ NPs and the mixture, ZnO NPs affected immune (mpo) and tight junction proteins (zo1). These results highlight the differences in the toxicity mechanisms existing between the single NPs and their combination, which showed higher toxicity to the gill bacterial community, but not to immune mechanisms. Furthermore, they suggest that exposure to environmental concentrations of NPs could potentially affect fish mucosal immunity and associated microbiota, highlighting the need for further research on the toxicity of NP mixtures.