Nanoparticles (NPs) of copper oxide (CuO), zinc oxide (ZnO) and especially nanosilver are intentionally used to fight the undesirable growth of bacteria, fungi and algae. Release of these NPs from consumer and household products into waste streams and further into the environment may, however, pose threat to the ‘non-target’ organisms, such as natural microbes and aquatic organisms. This review summarizes the recent research on (eco)toxicity of silver (Ag), CuO and ZnO NPs. Organism-wise it focuses on key test species used for the analysis of ecotoxicological hazard. For comparison, the toxic effects of studied NPs toward mammalian cells in vitro were addressed. Altogether 317 L(E)C50 or minimal inhibitory concentrations (MIC) values were obtained for algae, crustaceans, fish, bacteria, yeast, nematodes, protozoa and mammalian cell lines. As a rule, crustaceans, algae and fish proved most sensitive to the studied NPs. The median L(E)C50 values of Ag NPs, CuO NPs and ZnO NPs (mg/L) were 0.01, 2.1 and 2.3 for crustaceans; 0.36, 2.8 and 0.08 for algae; and 1.36, 100 and 3.0 for fish, respectively. Surprisingly, the NPs were less toxic to bacteria than to aquatic organisms: the median MIC values for bacteria were 7.1, 200 and 500 mg/L for Ag, CuO and ZnO NPs, respectively. In comparison, the respective median L(E)C50 values for mammalian cells were 11.3, 25 and 43 mg/L. Thus, the toxic range of all the three metal-containing NPs to target- and non-target organisms overlaps, indicating that the leaching of biocidal NPs from consumer products should be addressed.

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.