**2. Materials and Methods**

### *2.1. Natural Water Preparation and Sampling*

We collected natural water samples from different water bodies, including the Erren River, Zengwun Reservoir, and Cheng Kung Lake located at the campus of National Cheng Kung University (NCKU), in spring (May) and autumn (September) in Taiwan. Among them, the Erren River was considered a polluted water body, whereas Zengwun reservoir water was the source of drinking water for the public. Natural water was collected in sterilized sampling bags. Before storage, all the original water samples were filtered with a 53 μm filter to remove large impurities and microorganisms, and then the water samples were stored at 4 ◦C.

### *2.2. Preparation and Characterization of AgNPs and ZnONPs*

The principle of AgNP synthesis was based on the NaBH4 reduction of the Ag ion. First, the silver nitrate solution (50 mL, 20 mM), sodium citrate solution (40 mL, 80 mM). and 890 mL deionized water were fully mixed. Then, a NaBH4 solution (20 mL, 100 mM) was added slowly to promote the Ag ion reduction. The solution was stirred vigorously for 2 h, allowing a full reductive reaction. After synthesizing a sufficient amount of AgNP solution, it was centrifuged at 7500× *g* for 30 min, and the supernatant was collected. Furthermore, the solution was centrifuged again at 12,500× *g* for 2 h, and the precipitate was removed. The synthesized AgNP solution was stored at 4 ◦C in the dark.

We dissolved zinc acetate dehydrate (Zn(CH3COO)2(H2O)) in 3.35 mM ethanol and stirred the solution vigorously at 60 ◦C to synthesize ZnONPs. Furthermore, the solution was slowly titrated with a 6.59 mM potassium hydroxide (KOH) solution into a zinc acetate dehydrate solution along the edge of the beaker and reacted for 1.5 h. Next, the solution was incubated at room temperature for 2 h. The synthetic ZnONP solution was centrifuged at 10,000 rpm for 10 min, and then the supernatant was removed. The precipitate was collected and subsequently washed twice with 50 mL of ethanol. Then, 0.25 mL of (3-aminopropyl)triethoxysilane (APTES), 0.5 mL of deionized water, and 0.05 mL of ammonia (25 wt%) were mixed immediately with the ZnONP solution and reacted at room temperature for 20 min. After the reaction, the solution was centrifuged at 10,000 rpm for 15 min, and then we collected the precipitates. The precipitates were washed 2 times with ethanol and resuspended in water to obtain amine-coated ZnONPs. The aim of the surface modification was to improve the dispersion of nanoparticles in various solutions. Precisely, the formal name of ZnONPs we applied in this study was aminopropyl silicacoated ZnONPs, which is abbreviated as ZnONPs. The primary size and morphology of NPs were observed by transmission electron microscopy (TEM, JEOL Co., Akishima, Tokyo, Japan). The elemental analysis of NPs and NPs spiked in natural water were detected through electron dispersive X-ray (EDX) (JEOL Co., Akishima, Tokyo, Japan). The hydrodynamic diameter and polydispersity index were analyzed via dynamic light scattering (DLS, Delsa™ Nano C, Beckman Coulter, Inc., Brea, CA, USA). The zeta potential of the NPs was measured by phase analysis light scattering (PALS, Delsa™ Nano C, Beckman Coulter, Inc., Brea, CA, USA). The NP stability was investigated by ultraviolet visible spectroscopy (UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA).

### *2.3. Fish Husbandry and Egg Spawning*

Zebrafish embryos (*Danio rerio*) were obtained from the Taiwan Zebrafish Core Facility. Zebrafish were raised and maintained in a thermostatic culture system at 28–30 ◦C with a photoperiod of 14 h of light/10 h of darkness. Fish were fed twice a day with brine shrimp. Male and female fish were placed in the mating box. Spawning was triggered when the light was turned on in the morning. At 4 hpf, zebrafish embryos were collected and rinsed several times to remove residues on the surface. The dead, unfertilized, and abnormal embryos were removed. Healthy embryos were randomly selected for experiments.

### *2.4. The Acute Toxicity Test on Zebrafish Embryos*

The fish embryo acute toxicity test conducted in this study was based on OECD Test Guideline TG No. 236, released in 2013. All experiments were performed using a semi-static system. First, the fertilized embryos were gently collected with a dropper at 4 hpf, and healthy embryos were randomly divided and placed in 12-well plates (10 embryos/well). Embryos (30 embryos/group) were treated with AgNPs and ZnONPs. The positive controls (4 mg/L 3,4-dichloroaniline) were observed until 96 hpf, and mortality and deformities were recorded daily. The AgNPs and ZnONPs solutions were refreshed every 24 h until the end of the experiment to avoid ENP aggregation during exposure. All acute toxicity assays were performed in triplicate.
