## 1 Introduction  
In recent years, international communities such as Organization for Economic Co-operation and Development (OECD) and the European Union (EU) have been working on test guidelines regarding the physico-chemical properties and hazards of manufactured nanomaterials (MNs), while internationally expanding the scope of MNs to advanced materials [1; 2; 3]. As nanomaterials are being increasingly used, there has been a growing concern regarding environmental safety [4]. Of all the MNs, metallic nanomaterials, in particular, show aggregation resulting from increased ionic strength of the test medium and owing to the presence of various ionic substances, consequently hindering accurate toxicity tests [5; 6]. From an environmental perspective, the amount of nanomaterials entering water systems decreases with nanomaterial aggregation and precipitation, which consequently partially increases the exposure to the surrounding area where aggregation and precipitation occur. By contrast, improved particle mobility will facilitate dispersion in water systems over a long range, and therefore, the possibility of long-distance pollution should also be considered [7]. While inorganic nanomaterials are well-aggregated in the presence of ions, ZnO nanomaterials exposed to various ions such as sodium chloride and calcium chloride in water systems increase aggregation with the increase in the strength of ions [8; 9]. In addition, as the repulsive force varies depending on the type of ions, the different types of ions exposed will show different aggregation tendencies for the particles [10]. Therefore, for accurate toxicity studies, it is necessary to conduct a test using additional dispersants that strengthen stability with increased repulsive force. OECD has suggested the development of acute toxicity guidelines to conduct toxicity evaluation of the soluble chemicals. However, for the insoluble nanomaterials with unique physicochemical and toxic properties, the applicability of the test guidelines (TGs) should be further considered. OECD TG 318 suggested a testing method for improving dispersion by adding natural organic matters (NOMs) as a dispersant, and recommended that the concentrations of the stock solution be determined and the concentrations of the test media be maintained through an hourly dispersion measurement of the nanomaterials [11]. In a previous study, the method of dispersing nanomaterials using ultrasonic waves and dispersants was used to address the problem of aggregation. However, excessive exposure to ultrasonic waves can lead to the oxidized nanomaterials generating increased amounts of ions, thereby increasing the oxidative stress of the exposed species, as well as the toxic effects of the dispersants that are used for surface modification of the nanomaterials [12]. To reduce these adverse effects, several studies have been undertaken using NOM, which are ubiquitous complex mixtures of organic matters that exist in nature and can improve the dispersion of nanoparticles (NPs). However, compared to Ag NPs, other metallic nanomaterials such as ZnO NPs have not been widely investigated to confirm their toxic effects in the presence or absence of NOM. According to existing research, when NOMs are used as dispersants, the toxicities of Ag NPs are likely to be reduced. [PERSON] et al. [13] also showed that the presence of NOM resulted in a 2.5-6.7 fold decrease in toxicities for 20 nm Ag NPs and a 2.7-3.1 fold decrease in toxicities for 100 nm Ag NPs. Furthermore, [PERSON] et al. [14] and [PERSON] et al. [15] proved that NOM reduced the toxicities of Ag NPs, stating that the possible causes of the reduced toxicities were the different ionization mechanisms of Ag NPs with and without NOM. As shown in Table 1, for ZnO NPs in the absence of NOM, half effective concentration (EC\({}_{50}\)) (72 h) for algae ranged from 37 to 68 ug/L [16; 17], whereas EC/LC\({}_{50}\) (48 h) for water fleas ranged from 0.62 to 7.5 mg/L [18; 19; 20; 21; 22; 23; 24; 25; 26].