Aquatic Exposure to Nanoparticles: Fish as Bio indicators of Potential Human Health Hazards

The creation and use of tailored nanoparticles in a variety of scientific and industrial domains have greatly expanded due to the speedy development of nanotechnology. This is because of their distinctive physicochemical characteristics, such as high surface reactivity, large surface area, and varied functional potential, metal and metal oxide nanoparticles in particular have attracted a lot of attention (Asghar et al., 2015). These qualities make them ideal for use in biomedical sciences, electronics, catalysis, and environmental clean-up. Zinc oxide (ZnO) nanoparticles have emerged as especially important among the other nanomaterials due to their properties that can be regulated by manipulating the size and shape of the particles. ZnO nanoparticles have been extensively explored for diverse technological applications, including antimicrobial treatments, magnetic systems, electronic devices, catalysis, drug delivery, water purification, air filtration, and photocatalytic degradation of pollutants and hazardous dyes (S. Chakrabarti and B. Dutta, 2004). In addition to these industrial uses, their multifunctional properties have enabled their application in biological and medical fields such as biosensing, bioimaging, gene delivery, targeted drug delivery, and nanomedicine (Rasmussen et al., 2010). Furthermore, ZnO nanoparticles are widely used as antimicrobial agents in products such as ointments, lotions, mouthwashes, and surface coatings to inhibit microbial growth (Jones et al., 2008). They are also utilized as dietary supplements in humans and livestock, as zinc plays a crucial role in enhancing immune function and exerting anti-inflammatory effects (Prasad, 2008; Rincker et al., 2005) However, the rapid expansion of nanotechnology-based industries has led to the continuous release of engineered nanoparticles into the environment, particularly into aquatic ecosystems. According to the Environmental Protection Agency (EPA), ZnO nanoparticles are considered priority nanomaterials of environmental concern due to their high production volume and widespread applications (Lai et al., 2023). Numerous studies have investigated the toxicological effects of ZnO nanoparticles, particularly in mammalian cell lines (Yang et al., 2009; Xia et al., 2008). These studies indicate that ZnO nanoparticles can induce oxidative stress through the generation of reactive oxygen species (ROS), leading to cellular damage, disruption of enzymatic activities, and structural alterations in tissues and macromolecules (Boudou, A. and Ribeyre, F.,1997). More ever, exposure to ZnO nanoparticles has been related with histopathological changes in vital organs such as the gills, liver, and kidneys. Fish are widely recognized as effective bioindicators for assessing environmental contamination due to their sensitivity to pollutants and their ecological relevance. Among fish organs, the gills represent a critical interface between the organism and its surrounding environment, as they are directly exposed to waterborne contaminants. Structurally and functionally, fish gills share similarities with the lung epithelium (Handy et al., 2008), including a large surface area, thin epithelial lining, and direct interaction with the external environment. This resemblance provides a valuable model for understanding respiratory toxicity and predicting potential human health risks. ZnO nanoparticles are known to generate reactive oxygen species, which can cause epithelial damage, inflammation, and impairment of normal physiological functions in both aquatic organisms and mammalian systems (Oberdörster et al., 2005). Therefore, histopathological alterations observed in fish gills can serve as an important indicator for assessing nanoparticle-induced toxicity and may provide insights into potential respiratory and systemic effects in humans.  In this context, the present study aims to evaluate the acute toxicity (LC??) of ZnO nanoparticles and to investigate their dose-dependent histopathological effects on the gills of the freshwater fish Trichopodus trichopterus. The results obtained from the current study will be instrumental in increasing the knowledge about the toxicity of nanoparticles in the aquatic environment and help to understand their possible implications on environmental and human safety.

2. Materials and Methods

2.1. Chemicals and Nanoparticle Preparation

Zinc oxide nanoparticles (ZnO-NPs) (< 20 nm) were synthesized following the method reported in our earlier study (Chandak et al., 2026). In our previous work, physical and chemical characterization of ZnO-NPs was performed. Stock suspensions of ZnO-NPs were prepared in distilled water and sonicated (40 kHz, 100 W) for 30 minutes before treatment to ensure uniform dispersion of the nanoparticles.

2.2. Experimental Animals and Maintenance

Healthy adult specimens of Trichopodus trichopterus (average length: 7.60 ± 0.09 cm; weight: 7.90 ± 0.15 g) were collected from a local source. Fish were acclimatized under laboratory conditions for two weeks prior to experimentation in dechlorinated tap water. During acclimation, fish were maintained in aerated glass aquaria under controlled conditions (temperature: 24.18 ± 0.14°C; pH: 7.4 ± 0.06; dissolved oxygen: 4.8 ± 0.19 mg/L) with a 12:12 h light-dark cycle. Fish were fed a standard commercial diet and dried worms ad libitum. All experimental procedures were conducted in accordance with CPCSEA guidelines, New Delhi.

2.3. Acute toxicity Assessment (96 h LC50)

Acute toxicity of ZnO-NPs was assessed by exposing fish to a range of concentrations (0, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, and 25 mg/L) under static conditions for 96 hours. Ten fish were employed at each concentration. Sonication was conducted for 30 minutes prior to treatment to keep the suspension homogeneous.  Fish were not fed during the exposure period in order to prevent nanoparticle adsorption to feed and feces. Mortality was recorded at 12-hour intervals, and dead fish were removed to avoid contamination.

2.4. Sublethal Exposure Study

During the experiment for sub chronic toxicity testing, fishes were kept for 14 days under semi-static condition in the presence of sub lethal concentration of ZnO-NPs at doses of 1, 5, and 10 mg/L. Control fishes, without any nanoparticle treatment, were also maintained. Six fishes per group were used (n=18) and the experiment was conducted in three replicates.

2.5.  Histopathological Examination

Fish were randomly chosen at the end of the exposure period and sacrificed for histological analysis. Gill samples were dissected from the fish and were placed in Bouin’s fixative for 24 hours. Afterwards, the specimens underwent a dehydration process via graded series of ethanol, cleared in xylene, and embedded in paraffin wax. Sections of approximately 8 µm thickness were cut with rotary microtome and stained using hematoxylin and eosin (H&E). Histological observations were carried out using a Zeiss Primo Star microscope equipped with an Axiocam 105 digital camera.

2.6. Statistical Analysis

LC?? value was determined after 96 hours via Probit analysis and was reported at 95% confidence limits. Data were reported as mean ± SEM. Differences between treated and control group were statistically analyzed with ANOVA coupled with Dunnnet test for a value of p < 0.05. Statistical analysis was done with SPSS version 20 and Graphpad Prism software.

3. Results and Discussion

The present study demonstrates that exposure to zinc oxide nanoparticles (ZnO-NPs) induces significant toxic effects in Trichopodus trichopterus under controlled laboratory conditions. Water quality parameters remained stable throughout the experimental period, ensuring that the observed effects were primarily due to nanoparticle exposure. A clear concentration-dependent increase in mortality was recorded, with a 96 h LC?? value (Table1 and figure 1) of 20.44 mg/L, indicating moderate toxicity of ZnO nanoparticles to the test species.

The LC?? value obtained in this study is consistent with previously reported findings in different fish species, although variations have been observed depending on species sensitivity, nanoparticle size, and exposure conditions (Saha et al., 2022; Aziz et al., 2020; Asghar et al., 2015). Such variability highlights the importance of species-specific evaluation in nanotoxicological studies.

Table1.LC 50 Fish acute toxicity of ZnO NPs (mg/L)

Figure 1.  96 h percent mortality and probit mortality of T. trichopterus exposed to ZnO NPs

Figure. 3.Effect of ZnO NPs on SGL length and width of T. trichopterus exposed to ZnO NPs for 14 days

Importantly, the toxic effects observed in fish models have broader implications beyond aquatic systems. The ability of nanoparticles to induce oxidative stress, epithelial damage, and tissue dysfunction suggests potential risks to higher organisms, including humans, upon exposure. Therefore, fish-based toxicity studies provide valuable insights into nanoparticle-induced biological effects and contribute to the evaluation of potential risks to human health

CONCLUSION:

This study revealed that zinc oxide nanoparticles have strong toxic effect on T. trichopterus.

This reflected higher mortality rates and histopathological damage in the gills as the concentration of nanoparticles increases. The observed changes, such as epithelial hyperplasia, lamellar fusion, necrosis, aneurism, and overall structural disruption suggest serious impairment of respiratory and osmoregulatory functions.  These results highlight the ecological dangers posed by nanoparticles pollution in aquatic ecosystem which can indirectly reflected in food chain. Nanoparticles pollution in aquatic ecosystems confirm that fish gills are sensitive and reliable indicators of environmental stress. Moreover, the gill damage observed after nanoparticle exposure indicates that these fish models could help predict potential respiratory and systemic toxicity of nanoparticles in humans. Overall, the findings emphasize the need for careful monitoring and regulation of nanoparticles release into aquatic environments and add to the growing understanding of their effects on both ecosystems and human.