The present study investigates the potential environmental impacts of ZnO-NPs, considering their escalating usage in diverse applications. The detrimental impact of varying concentrations of Zn nanoparticles and Zn ions on E. eugeniae, highlights their toxic effects. Under certain conditions, a wide spectrum of zinc concentrations can manifest in the environment. Metals have varying degrees of influence on soil organisms, according to Edwards and Arancon et al. (2022). The study's dosages were established according to the LC50 value (662 mg kg− 1 soil) of Zn nanoparticles for Eisenia fetida over a 28-day exposure period following Panda et al (1999). It aligns with the findings of previous studies conducted by Lebedev et al. (2015), Yausheva et al. (2016), and Zhang et al. (2022). The study administered multiple doses of zinc, specifically 0, 100, 250, 500, and 750 mg kg− 1, to replicate the typical levels of zinc concentration found in natural soil with precision. Lebedev et al. (2015) employed a soil sample with an LC50 range of 828 to 995 mg kg− 1 in their study. Thus, in alignment with the aforementioned prior studies, we have selected diverse dosage concentrations, specifically the minimum (100 mg kg− 1), intermediate (250–500 mg kg− 1), and maximum (750 mg kg− 1). The investigation assessed the impact of extended exposure to ZnO-NP/ZnCl2 on earthworms in relation to their viability, reproductive abilities, behaviour, bioaccumulation, and neurotoxicity. The application of the highest concentrations of Zn in nanoparticles, specifically 750 mg kg− 1, resulted in a survival rate of 63.3%. In contrast, the survivability rate was found 53.3% after a 28-day exposure period when the same concentrations of Zn were administered in ionic form. The lethality rate was observed to be 30% upon exposure to ZnO-NPs, whereas treatment with ZnCl2 at a dosage of 750 mg kg− 1 resulted in a lethality rate of 43.3% on the 21st day, specifically during the third week. The findings of the research are consistent with previous studies that have posited the higher toxicity of ions in comparison to nanoparticles (Filipiak et al., 2021 and and Hu et al., 2020). In a study by Bicho et al. (2017), Enchytraeus crypticus was subjected to CuCl2 and CuO nanoparticles at concentrations of 400 and 800 mg Cu kg− 1, respectively, in an experimental setting. The entire population did not survive in LUFA 2.2 soil, whereas no fatalities were detected in the presence of nanomaterials at concentrations of 1600 and 3200 mg Cu kg− 1 soil. Earthworms were exposed to various concentrations of necessary elements, such as Co and Cu, in a study carried out by Irizar et al. (2015). The study's conclusions showed that in under-regulated settings, earthworms' body weight, growth, production of cocoons, and reproductive development all decreased.
The study found that the EC50 value of ZnCl2 was 268.6 mg kg− 1 for reproduction, indicating a negative impact on the production of juvenile worms. In contrast, the EC50 value for the NP form was 533.4 mg kg− 1 in the experimental setting. The determination of metal bioaccumulation is contingent upon various factors, such as size and concentration (Alves et al., 2019). The highest concentration of Zn accumulation was occurred in the tissues of worms exposed to 500 and 750 mg kg− 1 of ZnO-NPs at the end of the experiment. It was notably elevated throughout the gut of the exposed worms, as depicted in Fig. 9. This trend was particularly prominent at the highest concentration of ZnO-NPs (750 mg kg− 1). Additionally, the presence of nanoparticles within the cytoplasm of the worm was observed in both dispersed and clustered arrangements (Fig. 9). In general, the findings indicate that the accumulation of Zn follows a concentration-dependent pattern.
The possibility of a connection between the bioaccumulation characteristic and the accumulation of 20 nm Au-NPs at high concentrations was previously investigated by Bocca et al. (2020). The study found that rather than the particles themselves, these particles were more prominently deposited in the soil's pure water. Furthermore, the hydrodynamic diameter of the 55 nm NPs was also larger. He et al. (2020) found that plants are capable of absorbing Au ion (Au1± & Au3±) from soil undergo a sequential reduction of one metal ion into another within their tissues (Gardea-Torresdey et al., 2005). The transformation of ZnO into Zn metal by earthworms is a possibility that can be considered in the present study. Alhussan et al. (2021) reported that endocytosis was the mechanism through which 50 nm NP was internalised by HeLa cells. The consequences of exposure to Au-NPs through drinking water were examined in a recent mice study. In the range of 10–58 nm, it was also found that uptake tended to decline as particle size increased. According to Sani et al. (2021), improved approaches must be developed further in order to study the aggregation state at extremely low concentrations and determine the mechanisms influencing nanoparticle uptake in soil and tissues.
In the context of enzymatic studies, it has been observed that living organisms possess specific enzymes that are capable of binding zinc within body tissues through the aid of metallothionein proteins, thereby facilitating the rapid elimination of Zn (Slobodian et al., 2021). As demonstrated in a prior investigation conducted by Singh et al. (2022), the application of ZnO-NPs and ZnCl2 has the potential to significantly increase the activity of SOD, CAT, and LPO, as well as the total GSH content, following a 28-day exposure period (p ≤ 0.05). The AChE enzyme, a neuro-biomarker, is a delicate enzyme that is present in the neural systems of both vertebrates and invertebrates and functions as an efficient neurological enzyme (Wong-Guerra et al., 2021 and Jankowska et al., 2023). The study revealed that the highest dose of ZnO-NPs and ZnCl2 resulted in elevated levels of the AChE enzyme. Specifically, a dosage of 750 mg kg− 1 was found to be associated with this increase. The enzyme exhibited higher activation levels subsequent to exposure to ZnCl2, whereby the degree of activation was contingent upon the concentration of the aforementioned substance. It has been observed that on the 28th day, the activation of AChE in earthworms gets boosted by metals, thereby elevating its catalytic efficacy. Excessive activation of the enzyme may pose a potential hazard. Romani et al. (2003) reported an increase in AChE activity (Vm/Km) in Sparus auratus following exposure to sublethal doses of copper. The assessment of the impact of metallic substances on AChE as an environmental biomarker is of utmost importance, despite its intricate nature (Vieira et al., 2021). This holds particularly true in settings that are polluted with diverse chemical substances. Metal ions have been found to have an impact on the enzyme acetylcholinesterase (AChE), however, the conclusions of these studies have been inconsistent (Romani et al., 2003; Wong-Guerra et al., 2021; Zatta et al., 2003). Researches have demonstrated that being exposed to organophosphate and carbamate pesticides can result in the suppression of AChE activity in both controlled experiments and factual conditions (Zheng et al., 2013; Wu et al., 2020). The study indicates that avoidance responses persisted during a 48-hour exposure to ZnO-NPs and ZnCl2, even when regulated. The exposure to ZnCl2 resulted in higher levels of avoidance behaviour in comparison to ZnO-NPs at all concentrations. The avoidance behaviour can be ascribed to the immediate effect of unbound Zinc ions on sensory perception, rather than being indicative of endogenous Zinc toxicity. Chaudhuri et al. (2021) reported that chemoreceptive organs located in the outer area of the prostomium and proximal segments of earthworms demonstrated a responsive behaviour towards metal ions. The statement mentioned pertains to the Free Ion Activity Model (FIAM), which proposes that the reaction of unbound metallic ions with the substrate's membrane leads to metal reactions in biological systems (Wu et al., 2019).
In general, the manifestation of sublethal reproduction impairment and avoidance behaviour indicated that Zinc exposure had a greater impact on these endpoints compared to Zn accumulation in the affected tissues. The rise in zinc levels in worms after being exposed to ZnO-NPs could suggest the manifestation of avoidance behaviour. Additionally, the accumulation of Zn does not necessarily rely on a specific concentration of zinc within the organism's body. According to the postulated finding, the act of avoiding a certain behaviour is a rapid reaction to a sensory effect that is actively attributed to free Zn, rather than the magnitude of internal zinc toxicity. The excessive presence of avoidance behavior has the potential to result in increased AChE enzyme activity subsequent to ZnCl2 exposure, which could develop as neurotoxicity. The exposure to ZnO-NPs did not exhibit any protective effect in relation to the high bioaccumulation of Zn contents. The worms exposed to ZnO-NPs exhibited the highest accumulation of Zn, while those exposed to ZnCl2 demonstrated a significant reduction in survivability and the subsequent impact on reproductive efficiency. The results of the study imply that zinc has the ability to produce significant internal and external thresholds for avoidance behaviour. In addition, the possible impact of zinc on reproductive mechanisms may offer a powerful approach for assessing the environmental risks associated with soils contaminated by this specific element.