**1. Introduction**

Currently, engineered nanomaterials and nanoparticles (ENPs) are used in a wide variety of industrial and commercial applications, including catalysts, environmental remediation, personal care products, and cosmetics. In addition, ENPs also show grea<sup>t</sup> promise in medicine, such as imaging and drug delivery [1]. Examples of two widely utilized ENPs are silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) [2]. AgNPs are used in cosmetics, textiles, antibacterial agents, the food industry, paints, and medical

**Citation:** Lee, Y.-L.; Shih, Y.-S.; Chen, Z.-Y.; Cheng, F.-Y.; Lu, J.-Y.; Wu, Y.-H.; Wang, Y.-J. Toxic Effects and Mechanisms of Silver and Zinc Oxide Nanoparticles on Zebrafish Embryos in Aquatic Ecosystems. *Nanomaterials* **2022**, *12*, 717. https://doi.org/ 10.3390/nano12040717

Academic Editor: Vivian Hsiu-Chuan Liao

Received: 28 January 2022 Accepted: 19 February 2022 Published: 21 February 2022

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devices, whereas ZnONPs are widely used in sunscreen, cosmetics, paints, and antibacterial ointments [3,4]. The global usage of both AgNPs and ZnONPs has increased exponentially, thereby increasing the amount of AgNPs and ZnONPs that enter the aquatic environment through various routes [3]. The continuous release of ENPs into the environment from consumer products, industrial waste, and sewage sludge raises concerns about their distribution, behavior, and characteristics in natural water and adverse effects on ecosystems [5]. Nevertheless, the comprehensive toxicological effects of AgNPs and ZnONPs present in the environment have still not been studied in detail. Thus, an analysis of the effects of AgNPs and ZnONPs on ecological systems and human health is important.

In general, a detailed understanding of the behavior of ENPs in aquatic environments is crucial to determine their final distribution and the associated risks [5–7]. Due to their small size, some ENPs, such as AgNPs and ZnONPs, release ions in aquatic systems that are responsible for the major causes of toxicity. However, they also have a confined solid phase similar to other poorly soluble compounds in the aquatic system [7]. In a real water system, natural organic matter (NOM), certain biological macromolecules, and the environmental conditions, including pH and ionic strength, will modify the ENP behavior [5]. Predicting the fate of ENPs in natural surface water systems continues to be a challenge because ENPs can heteroaggregate with natural organic and mineral suspended matter. For example, NOM has been proposed to dominate ENP surface chemistry, and inorganic compounds such as sulfate also play important roles in the modifications of ENP structures and in reducing their toxicity [5]. Transformations of ENPs, such as agglomeration, aggregation, dissolution, and surface change, by interacting with natural water components might substantially alter their environmental release and toxicity [2]. Thus, defining the behavior of ENPs in aquatic systems and their toxic effects on living organisms can facilitate the establishment of scientific foundations for the risk assessment of ENPs in aquatic ecosystems.

The zebrafish (*Danio rerio*) embryo (ZFE) is an ideal model to assess the hazards of both conventional chemicals and ENPs in (eco)toxicology [8]. This model possesses advantages, including rapid development and optical transparency, allowing easy observations of phenotypic responses at lethal, acute, and sublethal toxicological endpoints. In addition, large amounts of embryos can be generated rapidly at low cost, permitting them to serve as a high-throughput assay for the study of developmental processes upon exposure to ENPs [8]. We have recently reported the possible targets and mechanisms of the toxic effects of AgNPs on a ZFE model. Our major finding is that exposure to AgNPs alters lysosomal activity (an indicator of autophagy) and leads to a greater number of apoptotic cells distributed among the developmental organs of the embryo [8,9]. From the (eco)toxicological perspective, the toxic effects induced by AgNPs and ZnONPs, including cytotoxicity, hematotoxicity, immunotoxicity, hepatotoxicity, and embryotoxicity, either in vitro or in vivo, are quite similar [10]. The well-known toxicity induced by ZnONPs is predominantly mediated by the formation of ROS. Excessive ROS generation may damage mitochondria, which subsequently leads to inflammasome activation and cell death through apoptosis and autophagy, which might be a novel mechanism modulating ZnONP-induced inflammatory and cytotoxic effects [11]. ENPs-induced oxidative stress is considered the initiator of the disruption of mitochondrial membrane potential and apoptosis and/or autophagy. Although extensive research has been conducted on ENP applications and toxic mechanisms, research on the environmental transport behavior and ecotoxicity of emerging materials such as AgNPs or ZnONPs is still limited and is needed for sustainable environmental implementation.

We conducted experiments in both complex natural original waters (lake, reservoir, and river) and filtrates from natural waters produced with different pore sizes of filters (1 μm, 0.45 μm, 0.22 μm, and 0.1 μm) to understand the behavior, characteristics, and embryotoxicity of AgNPs and ZnONPs in aquatic systems. Although results obtained from natural waters may more realistically approximate actual ecosystems, they are often unable to provide sufficient information due to the complexity of water chemistry. The

results obtained from filtrates of natural waters and MQ water (control group), which have a reduced complexity, can improve our understanding of the processes involved but are not necessarily representative of natural systems. The objective of this study was to characterize aggregates of AgNPs and ZnONPs in natural waters and determine whether they are toxic to developing zebrafish embryos. Different toxic effects and mechanisms were investigated here to evaluate ENP toxicity by measuring the survival rate, hatching rate, body length, oxidative stress, apoptosis, and autophagy. The results from those experiments are essential for a better understanding of AgNPs and ZnONPs in aquatic ecosystems and provide important underlying mechanisms for ecological risk assessments of them and other nanoparticles.
