**3. Results**

### *3.1. The Physical and Chemical Properties of AgNPs and ZnONPs*

Two types of nanoparticles were synthesized in this study to evaluate the environmental toxicity of ENP-spiked natural water. We synthesized citrate-coated AgNPs and amine-modified ZnONPs and fully characterized their physicochemical properties. As shown in Figure 1, The TEM images present the mean primary sizes of AgNPs and ZnONPs, which were 13 and 27 nm, respectively, and the morphology was spherical (Figure 1a,c). Element analysis indicated that AgNPs contained silver and ZnONPs were composed of zinc and oxygen (Figure 1b). The Si element came from a synthesized process which used APTES, and the Cu came from carbon support films on copper grids, which were used in TEM analysis. Dynamic light scattering (DLS) results revealed that the hydrodynamic diameters of the AgNPs and ZnONPs were 16 nm and 43 nm, respectively, and the NPs exhibited a homogeneous dispersion (Figure 1c). In addition, AgNPs and ZnONPs represented negative ( −18.6) and positive surface charges (+25.4), respectively (Figure 1c).


**Figure 1.** Physical and chemical properties of AgNPs and ZnONPs. (**a**) TEM images of AgNPs and ZnONPs. (**b**) EDX analysis of AgNPs and ZnONPs. (**c**) Analysis of the physicochemical properties of AgNPs and ZnONPs. AgNPs, silver nanoparticles; ZnONPs, zinc oxide nanoparticles; TEM, transmission electron microscopy; EDX, electron dispersive X-ray; DLS, dynamic light scattering; PALS, phase analysis light scattering; UV–Vis, ultraviolet visible spectroscopy.

### *3.2. The Deposition of AgNPs and ZnONPs Spiked in Natural Water*

Several studies have shown that the physicochemical characteristics of ENPs change when they are spiked in natural water bodies [12,13]. We obtained water from NCKU Lake, Zengwun Reservoir, and Erren River to determine the physicochemical properties of Ag/ZnONPs in natural water. NCKU Lake is located at National Cheng Kung University and possesses a high species richness. Zengwun Reservoir is a relatively clean water source that provides drinking water. The Erren River is one of the contaminated rivers in Taiwan. To date, the Erren River is still considered moderately polluted.

AgNPs and ZnONPs were spiked into the water samples from NCKU Lake, Zengwun Reservoir, and Erren River, and deposition was observed after 30 min. For the NCKU Lake water sample, the AgNPs were still suspended in water, but the color quickly changed. ZnONPs spiked in NCKU Lake water were deposited. In addition, water from the Zengwun Reservoir and Erren River caused a substantial aggregation of AgNPs and ZnONPs upon spiking into the natural water (Figure 1a,b). Furthermore, the physicochemical characteristics of these nanoparticles spiked in natural water were analyzed by determining their particle size, hydrodynamic diameter, and dispersity, as summarized in Figure 2c–e. Both AgNPs and ZnONPs spiked in NCKU Lake water exhibited a larger particle size and hydrodynamic diameter and decreased dispersity (Figure 2c,e). After spiking in the Zengwun Reservoir and Erren River water, AgNPs and ZnONPs aggregated and were not suspended in solution, and thus the data were undetectable (Figure 2c,e). These results were attributed to the chemical reaction of these nanoparticles with complex water components after spiking in natural water, which led to aggregation and precipitation.

**Figure 2.** Deposition of AgNPs and ZnONPs spiked in natural water. The deposition of 5, 10, 15, 20, 25, 50, 75, and 100 μg/mL (**a**) AgNPs and (**b**) ZnONPs spiked in natural water from NCKU Lake, Zengwun Reservoir, and Erren River. Ag/ZnONPs aggregated and deposited at the bottom of the vessel. The arrow indicates Ag/ZnONPs deposition. (**c**) The physical and chemical properties of AgNPs and ZnONPs spiked in natural water. (**d**,**<sup>e</sup>**) TEM images of AgNPs and ZnONPs spiked in natural water. AgNPs and ZnONPs aggregated after spiking in natural water samples. AgNPs, silver nanoparticles; ZnONPs, zinc oxide nanoparticles; TEM, transmission electron microscopy.

### *3.3. Elemental Mapping of Natural Water*

Furthermore, we performed elemental mapping to determine the elements present in natural water that reacted with AgNPs and ZnONPs. NCKU Lake, Zengwun Reservoir, and Erren River water samples were spiked with AgNPs and ZnONPs. We found that NCKU Lake, Zengwun Reservoir, and Erren River water precipitants contained abundant sulfur (S) and chlorine (Cl) and that Ag perfectly colocalized with them (Figure 3a–c). For ZnONP-spiked water samples, zinc was accompanied by magnesium (Mg) and potassium (K) in NCKU Lake water (Figure 3d). Furthermore, Zengwun Reservoir water samples contained calcium (Ca) and chlorine that colocalized with zinc (Figure 3e). Last, the Erren River water contained sulfur and calcium that overlapped with zinc (Figure 3f). Based on these results, we determined that both AgNPs and ZnONPs undergo complex chemical reactions with the components in natural water.

### *3.4. The Mortality of AgNPs and ZnONPs Spiked in Nature Water*

First, we observed the survival of embryos after exposure to the three water samples to determine whether natural water exerted adverse effects on embryos. Compared with the positive control group (3,4-DCA), the original water samples that were not spiked with AgNPs and ZnONPs did not cause obvious mortality at 24, 48, and 96 hpf. Even in the sample from the polluted Erren River, the survival rate was still high (Figure S1a–d). When the original water samples were filtered through membranes with different pore sizes of 1 μm, 0.45 μm, 0.22 μm, and 0.1 μm, the filtrates also did not produce significant adverse effects. The survival rates of all groups were still greater than 80% (Figure S1a–d).

According to previous studies, natural substances, such as humic acid, in natural water mitigated the toxic effects of NPs [13]. We designed additional experiments to confirm whether the substances in natural water resulted in a reduction in toxicity. First, the larger substances in natural water were removed with a 0.45 μm filter in advance and then the samples were spiked with AgNPs and ZnONPs (Figure 4a,b). Second, the original natural water was spiked with AgNPs and ZnONPs first and then passed through a 0.45 μm filter (to remove deposited ENPs) (Figure 4a,b). Third, we spiked 1 and 10 μg/mL AgNPs and ZnONPs in MQ water, respectively, for comparison and observed a substantial increase in mortality (Figure 4c,d). Accordingly, ZnONPs spiked in the natural water samples from NCKU Lake, Zengwun Reservoir, and Erren River exerted minor toxic effects, even in the 10 μg/mL groups (Figure 4e,f). In terms of the deposited NP removal groups, the elimination of AgNPs and ZnONPs deposits in natural water reversed the mortality, especially in the 10 μg treatment group (Figure 4g–j). In summary, although AgNPs and ZnONPs spiked into natural water exhibited a dramatically reduced toxicity, they still exerted adverse effects on the ecological environment at higher concentrations.

**Figure 4.** Lethality of AgNPs and ZnONPs spiked in natural water. The study design included two processes: (**a**) NP spiking and (**b**) deposited NP removal. The embryos were treated with (**c**) 1 μg/mL AgNPs, (**d**) 10 μg/mL AgNPs, (**e**) 1 μg/mL ZnONPs, and (**f**) 10 μg/mL ZnONP-spiked natural water. The mortality of embryos exposed to (**g**,**h**) 1 μg/mL and 10 μg/mL AgNP-spiked water, as well aI (**i**,**j**) 1 μg/mL and 10 μg/mL ZnONP-spiked natural water in which the aggregates were removed by a 0.45 μm filter. AgNPs, silver nanoparticles; ZnONPs, zinc oxide nanoparticles; 3,4-DCA, 3,4-dichloroaniline.

### *3.5. ZnONPs Caused Developmental Toxicity*

We measured body length to explore the relationship between zebrafish embryo development and substances in the natural water environment. Interestingly, the filtrates of water samples from NCKU Lake, Zengwun Reservoir, and Erren River that were passed through 1 μm, 0.45 μm, 0.22 μm, and 0.1 μm filters did not alter the body length of the zebrafish compared with the control group (MQ water) (Figure S2). The body length of the

zebrafish even increased after exposure to filtrates of natural water compared to the control group, but significant differences were not observed between the filtrate groups. In addition to the survival rate and body length, the hatching rate is also an indicator of developmental toxicity. When ZnONPs were spiked into the three different natural water samples and then passed through a 0.45 μm filter, we observed a significantly later hatching time at 96 hpf in embryos exposed to a higher concentration of 10 μg/mL (Figure 5b). Nonetheless, the embryos exposed to ZnONPs still hatched successfully without deformities. The AgNPs did not induce a delayed hatching time in this study (data not shown).

**Figure 5.** Developmental toxicity of ZnONPs. Hatching rate of zebrafish embryos exposed to ZnONPs. ZnONPs spiked in NCKU Lake, Zengwun Reservoir, and Erren River water. Hatching rate at (**a**) 72 hpf and (**b**) 96 hpf after exposure to different natural water samples. ZnONPs spiked in different water samples resulted in later hatching times in all groups. The significance between each different concentration groups were represented by \* sign. (\* *p* < 0.05). ZnONPs, zinc oxide nanoparticles; hpf, hours post-fertilization.

### *3.6. AgNPs and ZnONPs Induced Programmed Cell Death*

Several studies have revealed that AgNPs and ZnONPs lead to cytotoxicity by inducing programmed cell death [8,14,15]. We applied the TUNEL assay to investigate the cytotoxic effects of AgNPs and ZnONPs spiked in deionized (MQ) or natural water at 72 hpf. DNase I served as a positive control group and resulted in a significant increase in the number of apoptotic cells (Figure 5a,b). Groups treated with AgNPs and ZnONPs spiked in the MQ water exhibited an increased number of apoptotic cells, and the signals were mainly located in the yolk sac (Figure 5a,b). Surprisingly, groups treated with both AgNPs and ZnONPs spiked in natural water also exhibited substantial cytotoxicity. These results provide evidence that ENPs in natural water exert cytotoxic effects by activating apoptosis.

### *3.7. AgNPs and ZnONPs Induced ROS Production*

Excessive oxidative stress is an important cellular mechanism of ENPs and is recognized as an initial form of cell damage [8,14,16,17]. Next, we investigated whether AgNPs and ZnONPs spiked in different water samples would increase the ROS level in zebrafish embryos. We used dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye to detect ROS generation in embryos exposed to these nanoparticles at 72 hpf. As expected, the H2O2 groups (positive control) displayed increased ROS generation (Figure 6a,b). Similarly, 10 μg/mL AgNPs and ZnONPs spiked in the MQ water increased ROS generation, and the signals were primarily observed in the intestine (Figure 6a,b). As mentioned above, we concluded that ENPs spiked into the natural water increase ROS level in zebrafish embryos.

**Figure 6.** Cytotoxicity of AgNPs and ZnONPs. Zebrafish embryos were exposed to natural water after passing through a 0.45 μm filter and then spiked with NPs. After 72 hpf, zebrafish embryos were subjected to a TUNEL assay. Fluorescence images and intensity of (**a**) AgNPs and (**b**) ZnONPs. Apoptotic cells are represented by red fluorescence signals. The fluorescence intensity was assigned as + (weak), ++ (middle), and +++ (strong). Both AgNPs and ZnONPs spiked in natural water produced increased apoptosis signals in the 10 μg/mL NP groups. AgNPs, silver nanoparticles; ZnONPs, zinc oxide nanoparticles; hpf, hours post-fertilization.

### *3.8. AgNPs and ZnONPs Induced Autophagy*

According to recent studies, AgNPs and ZnONPs cause dysfunctional autophagy in vitro [14,18]. Hence, we used the LysoTracker RED probe to evaluate lysosomal activity in zebrafish embryos incubated with AgNPs and ZnONPs spiked in different water samples. Treatment with 10 μg/mL AgNPs and ZnONPs in the MQ water activated autophagy, and the signals were detected in the yolk sac (Figure 7a,b). For natural water, AgNPs spiked

in Zengwun Reservoir natural water apparently increased the activation of autophagy (Figure 7a). Some lysosomal activity was also observed in the other groups. Therefore, AgNPs and ZnONPs spiked in natural water induce autophagy.

**Figure 7.** ROS generation induced by AgNPs and ZnONPs. Zebrafish embryos were exposed to natural water after passing through a 0.45 μm filter and then spiked with (**a**) AgNPs and (**b**) ZnONPs. After 72 hpf, zebrafish embryos were stained with DCFH-DA (green). The fluorescence intensity was assigned as + (weak), ++ (middle), and +++ (strong). The H2O2-positive control group showed ROS generation. Both AgNPs and ZnONPs spiked in natural water resulted in increased ROS signals. AgNPs, silver nanoparticles; ZnONPs, zinc oxide nanoparticles; DCFH-DA, dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species.
