## 3 Results and Discussion
### Ecotoxicological Effect of NOM on ZnO  
The sensitivity test was conducted with potassium dichromate (K\({}_{2}\)Cr\({}_{2}\)O\({}_{7}\)), a reference material, to verify the reliability of the growth inhibition test with _Pseudokirchneriella subcapitata_. The results showed that the growth rate (\(\upmu\)) of algae decreased as the concentration of the reference material increased, indicating that the EC\({}_{50}\) value, the average coefficient of variation (C.V.) of the specific growth rate, and the C.V. of the average specific growth rate fell to the acceptable limits of 137.7 \(\pm\) 2.0 \(\upmu\)g/L, 30.9% (within 35% of critical limit), and 5.6% (within 7%), respectively. In the algae growth inhibition test of ZnO NPs, based on the EC\({}_{50}\) values with and without NOM, it is shown that both the control and positive control groups with NOM have negligible difference in the growth rates compared to the initial inoculation volume after 72 h of exposure; this suggests that NOM did not have any direct impact on the algae growth (Figure S1). In addition, the average C.V. of the specific growth rate due to the control group's interval indicated an appropriate range (Figure S1). The EC\({}_{50}\) (72 h) of ZnO NPs was 137.7 \(\pm\) 2.0 \(\upmu\)g/L, and the EC\({}_{50}\) (72 h) with NOM was 397.6 \(\pm\) 12.3 \(\upmu\)g/L, which was a threefold decrease in algae growth inhibition, resulting in decreased toxicity effects (Figure 7). Figure 7: Algae growth inhibition by ZnO NPs with/without NOM. In the sensitivity test of water fleas, the 24 h-immobilization rate using potassium dichromate (K\({}_{2}\)Cr\({}_{2}\)O\({}_{7}\)) as a reference material increased to 1.41 mg/L (EC\({}_{50}\)) with the concentrations falling into an acceptable range of 0.6 to 2.1 mg/L, suggesting that it was suitable to conduct the acute toxicity test. In the water flea acute toxicity test of ZnO NPs, immobilized individuals were observed in all concentrations greater than 1.25 mg/L, particularly at immobilization rates of 40%, 80%, and 100% in concentrations of 1.25, 2.5, and 5 mg/L, respectively, with 48 h-EC\({}_{50}\) at 1.55 mg/L (Figure 8). The EC\({}_{50}\) of ZnO NPs dispersed with NOM was 12.92 mg/L, which is an 8.3 fold increase, indicating a decrease in toxicity owing to the presence of NOM (Figure 8). In the study conducted by [PERSON] et al., when _Daphnia magna_ were exposed to ZnO NPs for 48 h, the opposite trend was observed as their toxicity increased [20] to 1.5 mg/L (EC\({}_{50}\)) with addition of NOM from a value of 4.7 mg/L (EC\({}_{50}\)) without NOM. We attributed this trend to the different surface property of ZnO NPs that caused increased dissolution in the presence of NOM, resulting in increased toxicities as well. In the case of Ag NPs, similar tendencies were observed as toxicity decreased to a value greater than 100 mg/L in the presence of NOM from 41.3 mg/L (EC\({}_{50}\)). Based on the findings, it was confirmed that NOM did not directly affect toxicity in water fleas, because no immobilization was observed in the control and positive control groups. In the acute toxicity test of ZnO NPs with _Oryza latipes_, no deaths were detected at the lowest concentration (26.9 mg/L) in the control group without NOM. However, mortality rates were observed as 10%, 85%, and 95% at 38.4, 58.2, and 61.8 mg/L, respectively, indicating that 96 h-LC\({}_{50}\) was 47.31 mg/L (Figure 9). However, in the presence of NOM, no mortalities were seen at any concentration level with LC\({}_{50}>100\) mg/L, suggesting that the toxicity was significantly alleviated in the presence of NOM. Figure 8: _Daphnia magna_ immobility by ZnO NPs with/without NOM. Based on the above results, it was confirmed that the toxicity values (EC\({}_{50}\), LC\({}_{50}\)) for algae, water fleas, and fish was lowered in the presence of NOM. Note that the control tests showed that no direct toxic effects were observed from NOM for all cases.