Nanomaterials are known to aggregate in the presence of ions. Similarly, the aggregation of zinc oxide nanoparticles (ZnO NPs) exposed to various ions such as sodium chloride and calcium chloride in water systems increases with the ionic strength. Therefore, for accurate toxicity studies, it is necessary to conduct a test using natural organic matters (NOMs) as additional dispersants that strengthen stability with increased repulsive forces. The three types of ecotoxicity tests based on the dispersion stability test using NOM showed that the toxicities of the three test samples decreased in the presence of NOM. To determine how NOM improved dispersion and reduced toxicities, we analyzed the ionization degree of ZnO NPs with and without NOM and found that the solubility was below 2 mg/L with a negligible change over time, implying that the ionization effect was low. The absolute value of the surface charge of particles increased in the presence of NOM, resulting in increased repulsive electrostatic forces and steric hindrance, causing less aggregation and more dispersion. Additionally, although the NOM used in the test is considered an effective dispersant that does not have a toxicological effect on aquatic organisms, the presence of NOM resulted in reduced toxicities and should be further investigated to establish it as a standard test method.

In order to investigate the effects of metallic nanoparticles (NPs) on the performance of in vitro bioassay, zinc oxide NP (ZnO NP), aluminum oxide NP (Al₂O₃ NP), bare silver NP (Ag NP), and Ag NP capped with citrate (Ag^cit NP) were evaluated with yeast (Saccharomyces cerevisiae Y190) two-hybrid system (YES assay), carrying Japanese medaka estrogen receptors (mERs) in the presence of 17β-estradiol (E2, 10⁻⁶ M), a reference chemical for estrogenic activity. The distribution of NPs in the yeast was also examined by field-emission transmission electron microscopy (FE-TEM). The results show that TEM analysis revealed that NPs were present inside the yeast and accumulated deep inside the cell organelles, suggesting that cell death was caused by NPs. However, despite no significant change of mortality, the E2 estrogenic activities in yeast exposed to ZnO NP and Al₂O₃ NP were dose-dependently reduced. For Ag NP and Ag^cit NP, such phenomenon observed in the exposure of ZnO NP and Al₂O₃ NP did not occur. From the observations, we found that ZnO NP and Al₂O₃ NP in the environmental media could result in underestimated estrogenicity of endocrine-disrupting compounds when evaluated by YES assay.