**1. Introduction**

Engineered nanoparticles (ENPs), which are extensively used in industrial and domestic products, are emitted into the environment [1]. This has raised concerns due to their potential toxicity to biota [1,2]. Zinc oxide nanoparticles (ZnO-NPs), with a global production of approximately 550–33,400 tons, are one of the most widely used ENPs in the electrical and manufacturing industries and personal care products [3,4]. In addition, due to their biocompatibility and cost-effectiveness, ZnO-NPs have been increasingly used in the biomedical field, such as in antibacterial, antifungal, antiviral, antidiabetic, and wound healing applications [5]. These advantages have also resulted in the growing usage of ZnO-NPs in dentistry [6]. Moreover, ZnO-NPs exhibit luminescent properties, making ZnO-NPs an excellent candidate for bioimaging [7]. ZnO-NPs are also commonly used as medicine in livestock, aquaculture, and pet animals despite the occasionally occurring side effects [8]. ZnO-NPs are also used in the synthesis of hybrid nanomaterials, which show excellent sorbent properties for the removal of pollutants [9–11]. As a result, the production, manufacturing, and consumption of ZnO-NPs result in their emission via industrial effluents [12]. The amount of ZnO-NPs entering sewage treatment plants by the European Union is estimated to be 1.05 million kg per year (1.7–45 μg/L in the effluent of sewage treatment plants) [12,13]. Considering that environmental ZnO-NPs levels are likely to rise due to the growing demand [13], this contaminant is a cause for concern because of its potential adverse effects on biota [14].

**Citation:** How, C.M.; Huang, C.-W. Dietary Transfer of Zinc Oxide Nanoparticles Induces Locomotive Defects Associated with GABAergic Motor Neuron Damage in *Caenorhabditis elegans*. *Nanomaterials* **2023**, *13*, 289. https://doi.org/ 10.3390/nano13020289

Academic Editor: Alexey Pestryakov

Received: 27 December 2022 Revised: 6 January 2023 Accepted: 8 January 2023 Published: 10 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Dietary transfer is an important pathway for pollutant transfer and accumulation, which might cause harmful effects in organisms at higher trophic levels [15]. The inclusion of dietary transfer as a factor in ecotoxicological risk assessment for aquatic species is recommended considering the significant toxicity caused by dietary exposure [16]. Due to the long-lasting and non-biodegradable properties of metals, the effects of the accumulation of metal-based ENPs due to dietary transfer require urgen<sup>t</sup> investigation [2]. ENPs, such as ZnO, silver (Ag), and titanium dioxide (TiO2), are transferred through food chains and accumulate in the higher trophic levels [17–19]. The enhanced accumulation of metal-based ENPs, including Ag, silicon dioxide (SiO2), tin oxide (SnO2), cerium oxide (CeO2), and magnetite (Fe3O4), has several sublethal adverse effects on reproduction, development, and locomotion [20–22]. Although ZnO-NPs are one of the most widely used ENPs, studies related to their toxic effects induced by dietary intake are limited. In addition, whether ZnO-NPs from dietary transfer affect other target tissues, such as the neurons, remains unclear.

Metal-based ENPs accumulate in various organs or tissues after ingestion and induce toxicity predominantly via reactive oxygen species (ROS) [23]. Recent research has suggested that neuromuscular defects are induced by ENPs in the nematode *Caenorhabditis elegans* [24,25]. Furthermore, locomotion in zebrafish and *C. elegans* is known to be adversely affected by ZnO-NPs [26,27]. Previous studies showed that D-type GABAergic motor neurons, which control locomotive behaviors, are potential targets of toxicants, including quantum dots and heavy metals [28,29]. The altered behaviors and neurotoxicity may reduce the fitness of organisms and result in ecotoxicity [30]. However, the involvement of the dietary transfer of ZnO-NPs in neurotoxicity and its underlying mechanisms remain unknown.

To investigate the toxic effects and mechanisms of the dietary transfer of ZnO-NPs, we used *C. elegans* as the model organism and *Escherichia coli* OP50 as its food source to establish a dietary transfer assay model. *C. elegans* is regarded as a useful model in environmental toxicology and neurotoxicology due to its short life cycle, ease of maintenance, and availability of mutants and transgenic strains [31]. A 72 h exposure period, which covers all developmental stages until puberty, may be considered long-term exposure in *C. elegans* [32]. Our study aims to investigate the toxic effects of the long-term dietary transfer of ZnO-NPs in the *E. coli*–*C. elegans* food chain model by examining their accumulation in these organisms and assessing the impairment of locomotive behaviors and D-type GABAergic motor neurons.
