## 3 Results and Discussion
### Measurement of Properties
#### 3.2.5 Analysis of Ionic Effect  
With aqueous complexation reactions with metal ions, NOM could not permeate through the 3 kDa filter, although the 30 kDa filter was permeable, thus facilitating NOM separation. All samples were pretreated as described in Section 2.3 and diluted 10 times. To express the measured concentration correctly, the diluted concentration was multiplied 10 times. Therefore, using two types of ultra-4-centrifugal filters, 3 and 30 kDa, we could determine whether aqueous complexes were formed [47; 48].  
As seen in Figure 6, the presence of NOMs resulted in different ionic concentrations of the solutions that passed through the 3 and 30 kDa filters at 96 h for DI water, 72 h for OECD medium, 48 h for ISO medium, and 96 h for dechlorinated tap water. The ionic concentrations of the test mediums were 2.10, 0.28, 0.12, and 0.61 mg/L in the presence of NOM, whereas they were 0.51, 0.11, 0.06, and 0.30 mg/L in the absence of NOM, respectively. In other words, all the test mediums showed higher ion concentrations when NOM was present, suggesting that NOM formed complexes with Zn\({}^{2+}\). As complex formation with NOM depends on the types of ions (monovalent and divalent), with more significant effects from divalent cations, the formation of different complexes prepared in different media depends on the ion species.  
Figure 6: Dissolution of ZnO NPs with/without NOM in DI water (**a**), OECD medium (**b**), ISO medium (**c**), and dechlorinated tap water (**d**).  
The ZnO NPs dispersed in DI water and the test medium was detected with a low ion concentration of less than 3 mg/L in all samples [43; 49]. The degree of ionization is known to increase with the effects of pH, ion concentration, and temperature, and, in particular, the pH effect is significant [37]. As the pH range of the ecotoxicity test medium with ZnO NPs did not significantly change, ranging from 7.53 to 7.99 (Table S1), we concluded that the effect of pH on the degree of ionization was not significant. The concentration of ZnO ions in DI water was higher than the concentrations in other mediums due to a low pH of 5.78 in DI water, resulting in increased solubility [14]. There is little change in the ionization over time, and the change in ionic amount during the exposure period under the OECD test method indicated a low value regardless of the presence of NOM (Figure 6). The concentration differences between the solutions with and without NOM that passed through the 30 kDa filter were not significant. When the solutions from the last samples of the OECD and ISO mediums passed through the 30 kDa filter, their ion concentrations with NOM were 0.51 mg/L for the OECD medium and 0.40 mg/L for the ISO medium, whereas for those without NOM, they were 0.45 mg/L for the OECD medium and 0.55 mg/L for the ISO medium. As ZnO demonstrated few ionic changes with NOM, we inferred that the ionic effect on toxicity was also not significant.  
As the NOM forms aqueous complexes, a solution obtained from filtering using the 30 kDa filter paper was expected to exhibit a higher concentration than the 3 kDa filter paper; however, the ionized concentration was low and could not be verified. With slight ionic changes, ZnO NPs is not expected to exhibit a large ionic effect during the toxicological evaluation.