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Silver nanoparticles (AgNPs) are highly toxic to aquatic organisms, however, there is no consensus whether the toxicity is caused solely by released Ag-ions or also by reactive oxygen species (ROS). Here, the effects of protein-coated AgNPs (14.6 nm, Collargol) were studied on viability, oxidative stress and gene expression levels in wild type strains (CU427 and CU428) of ciliate Tetrahymena thermophila. Viability-based 24 h EC50 values of AgNPs were relatively high and significantly different for the two strains: ∼100 mg/L and ∼75 mg/L for CU427 and CU428, respectively. Similarly, the expression profiles of oxidative stress (OS) related genes in the two strains were different. However, even though some OS related genes were overexpressed in AgNP-exposed ciliates, intracellular ROS level was not elevated, possibly due to efficient cellular antioxidant defence mechanisms. Compared to OS related genes, metallothionein genes were upregulated at a considerably higher level (36 versus 5000-fold) suggesting that Ag-ion mediated toxicity mechanism prevailed over OS related pathway. Also, comparison between Ag-ions released from AgNPs at EC50 concentration and the respective EC50 values of AgNO3 indicated that Ag-ions played a major role in the toxicity of AgNPs in T. thermophila. The study highlights the importance of combining physiological assays with gene expression analysis in elucidating the mechanisms of action of NPs to reveal subtle cellular responses that may not be detectable in bioassays. In addition, our data filled the gaps on the toxicity of AgNPs for environmentally relevant and abundant organisms. The parallel study of two wild type strains allowed us to draw conclusions on strain to strain variability in susceptibility to AgNPs.

Currently, silver nanoparticles (AgNPs) are being increasingly used as biocides in various consumer products and if released in the environment they can affect non-target organisms. Therefore, understanding the toxicity mechanism is crucial for both the design of more efficient nano-antimicrobials and for the design of nanomaterials that are biologically and environmentally benign throughout their life-cycle. Here, the ciliate Tetrahymena thermophila was used to elucidate the mechanisms of action of AgNPs by analysing the gene expression profile by RNA-seq and the transcriptomic effects of AgNPs were compared to those induced by soluble silver salt, AgNO3. Exposure to AgNPs at sublethal concentrations for 24 h induced phagocytosis, transport pathways, response to oxidative stress, glutathione peroxidase activity, response to stimulus, oxidation-reduction, proteolysis, and nitrogen metabolism process. Based on gene set enrichment analysis (GSEA), some biological processes appeared targets of both toxicants. In addition to many similarities in affected genes, some effects were triggered only by NPs, like phagocytosis, glutathione peroxidase activity, response to stimulus, protein phosphorylation and nitrogen metabolism process. This research provides evidence that AgNPs compared to AgNO3 at the same concentration of dissolved silver ions dysregulate a higher number of cellular pathways. These findings confirm that AgNPs can induce toxicity not only due to soluble silver ions released from the particles but also to particle intrinsic features.