Comparative nanotoxicity of polyethylene, silver, and copper oxide nanoparticles in African catfish (Clarias gariepinus): impacts on antioxidant defense, immunity, and tissue integrity

The rapid expansion of nanotechnology has increased nanoparticle release into aquatic systems, raising ecological safety concerns. This study compares the toxicological impacts of polyethylene (PE-NPs), silver (Ag-NPs), and copper oxide nanoparticles (CuO-NPs) on Clarias gariepinus, focusing on nano-specific biological effects. Transmission electron microscopy characterized PE-NPs as irregularly shaped with a 40.4 nm average diameter, Ag-NPs as spherical (95 nm), and CuO-NPs as semi-spherical (63.4 nm). Catfish were exposed to 100 mg/L of each NP type over 15 days, revealing significant reductions (p < 0.0001) in antioxidant enzymes glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (TAC). Immunological parameters, including lysozyme (LYZ) activity, immunoglobulin M (IgM), and phagocytic activity (PA), were also significantly reduced (p < 0.0001), indicating immune suppression. Histopathological analysis revealed pronounced tissue-specific damage: PE-NPs caused dilated blood sinusoids in liver cells; Ag-NPs led to necrosis and hydropic degeneration; CuO-NPs resulted in melanomacrophage centers and increased mitotic activity. In kidney tissues, PE-NPs caused vacuolation, Ag-NPs induced degeneration and hemorrhages, and CuO-NPs displaced epithelial linings. Collectively, these findings demonstrate that exposure to these nanoparticles induces significant biochemical and histological disturbances in Clarias gariepinus, emphasizing the need for nano-specific risk assessments in aquatic environments.

The rapid growth of nanotechnology over recent decades has made nanoparticles (NPs) essential materials across a wide range of industries, from medicine and electronics to environmental management (Dutta and Das, 2021; Khan et al., 2022). Characterized by dimensions falling within the 1–100 nm range, these engineered materials exhibit distinctive physicochemical attributes that confer substantial functional advantages over their bulk counterparts (Fubini et al., 2010; Khan et al., 2019). Despite these compelling benefits, mounting evidence suggests that the escalating incorporation of NPs into industrial processes poses considerable threats to ecological integrity, particularly upon their introduction into freshwater and marine environments via industrial effluents (Batley et al., 2013). Specifically, polyethylene nanoparticles (PE-NPs), silver nanoparticles (Ag-NPs), and copper oxide nanoparticles (CuO-NPs) have the potential to cause adverse effects such as the production of reactive oxygen species, DNA damage, suppression of immune markers, and genetic damage (Naz et al., 2020; Noga et al., 2023; Shi et al., 2023). Furthermore, Ag and Cu ions can be released from Ag-NPs and CuO-NPs and have been demonstrated to possess hazardous properties, including the suppression of respiratory enzymes, cell membrane damage, and the disruption of DNA replication (Ferdous and Nemmar 2020; Ma et al., 2022).

The toxicity mechanisms of these nanoparticles differ substantially. Ag-NPs and CuO-NPs primarily induce oxidative stress through reactive oxygen species (ROS) generation and can release toxic Ag + and Cu 2+ ions, which disrupt respiratory enzymes and cell membranes (Naz et al., 2020). In contrast, PE-NPs, being polymeric and less chemically reactive, likely exert toxicity through physical interactions (e.g., membrane disruption, endosomal damage) and mechanical stress rather than ion-mediated pathways (Awashra and Młynarz, 2023; Chen et al., 2026). Immunologically, all three NPs can suppress lysozyme activity and phagocytosis, but metal-based NPs cause more severe effects due to additional pro-inflammatory signaling (Liu et al., 2022).

Although the present study focuses on a freshwater species (Clarias gariepinus) under freshwater conditions, the toxicological mechanisms elucidated including oxidative stress, immunosuppression, and histopathological damage are conserved across aquatic taxa and environments. Furthermore, nanoparticles released into freshwater systems ultimately reach estuarine and marine environments via river discharge (Jolaosho et al., 2025). Understanding freshwater nanotoxicity therefore provides a critical baseline for predicting marine ecosystem risks, particularly because salinity influences NP aggregation, dissolution, and bioavailability. Our findings thus have implications for both freshwater and marine pollution assessment.

While individual toxicological profiles of PE-NPs, Ag-NPs, and CuO-NPs have been reported separately, no previous study has systematically compared their effects side-by-side using a multi-biomarker approach in the same fish species under identical experimental conditions. The present study therefore provides a novel comparative assessment of these three physicochemically distinct nanoparticle types: polymeric, noble metal, and metal oxide in Clarias gariepinus, offering critical insights into how material composition dictates nanotoxicity outcomes.

Polyethylene (PE-NPs), silver (Ag-NPs), and copper oxide (CuO-NPs), are among the most studied due to their extensive industrial use (Haider and Kang, 2015; Dey et al., 2022; Lai et al., 2022). PE-NPs are widely used in packaging, Ag-NPs in medical and water treatment products, and CuO-NPs in catalysts and electronics (Haider and Kang, 2015; Dey et al., 2022; Gawande et al., 2016), leading to their release into aquatic environments. The unique properties of NPs, high surface area to volume ratio, reactivity, and membrane permeability confer industrial advantages but also raise ecological and toxicological concerns (Ahmad et al., 2022; Altammar 2023). Their small size and reactivity enable them to interact with biological systems in ways that larger particles cannot, potentially leading to unforeseen toxicological effects (Shvedova et al., 2010; Maynard et al., 2011). Recent investigations have demonstrated that PE-NPs, Ag-NPs, and CuO-NPs can interfere with the antioxidant defense mechanisms, alter immunological markers, and cause histological alterations in fish (Brandts et al., 2022; Bakr et al., 2023; Subaramaniyam et al., 2023; Xuan et al., 2023). An essential function of the antioxidant defense system in fish is to reduce oxidative stress, which is frequently worsened by NP exposure (Subaramaniyam et al., 2023). Degradation of cellular components caused by oxidative stress can result in compromised physiological activities and heightened vulnerability to illnesses.

Immunological measures, including the count of white blood cells, immunoglobulins M (IgM) levels, and generation of cytokines, are crucial indications of fish health (Kataoka and Kashiwada, 2021; Hamed et al., 2025). Lysozyme (LYZ) activity, immunoglobulin M (IgM) levels, and phagocytic activity (PA) were selected as key immunological endpoints because they represent complementary arms of the fish immune system: LYZ reflects innate humoral immunity, PA indicates cellular immune function, and IgM levels quantify adaptive humoral responses. Together, these biomarkers provide a comprehensive assessment of immunocompetence following NP exposure. Immunosuppression or hyperactivation from NP exposure can impair the fish’s capacity to combat infections and sustain general wellbeing. Furthermore, histopathological alterations in essential organs such as the liver, gills, and kidneys are a definitive sign of the harmful effects of different NPs (Ostaszewska et al., 2016; Kakakhel et al., 2021; Hamed et al., 2024). This emphasizes the need to assess these substances in aquaculture environments thoroughly.

The African catfish is extensively used in aquaculture because of its remarkable capacity to withstand challenges, grow quickly, and thrive in diverse environmental settings (Sayed et al., 2022; Manyise et al., 2024). Comprehending the impact of NP exposure on this species is crucial for guaranteeing the continued use of sustainable aquaculture methods. Waterborne micro-plastic and NPs can infiltrate aquatic ecosystems from many origins, such as industrial effluent, agricultural runoff, and nanotechnology-derived goods, resulting in possible bioaccumulation and detrimental impacts on aquatic organisms (Issac and Kandasubramanian, 2021; Wang et al., 2024).

This study compared the toxicological effects of PE-NPs, Ag-NPs, and CuO-NPs (100 mg/L, 15 days) on African catfish across three endpoints: (1) antioxidant enzyme activities (GST, SOD, CAT, TAC), (2) immunological parameters (LYZ, IgM, PA), and (3) liver and kidney histopathology, to enable comparative hazard ranking among these nanoparticle types. By understanding these effects, we can better assess the ecological risks associated with NP pollution and inform regulatory measures to protect aquatic life and ecosystem health. The findings from this research will contribute to the growing body of knowledge on the environmental impacts of NPs, particularly their effects on aquatic organisms. By elucidating the mechanisms of toxicity and identifying the specific adverse effects of different types of NPs, this study aims to provide valuable insights for developing strategies to mitigate the environmental risks of NP pollution.

Chemicals

The CuO-NPs were obtained in powder form with a purity of 99%, a molecular weight of 79.55, and an average particle size of < 50 nm. They were sourced from Sigma-Aldrich (United Kingdom). Ag-NPs were acquired as powdered spherical particles with a purity of 99.9%, a molecular weight of 107.87, and an average particle size of < 100 nm from Sigma-Aldrich (St. Louis, MO, United States). PE-NPs with sizes < 100 nm and irregularly shaped particles were sourced as raw powders from Toxemerge Pty Ltd., Australia. The NPs were characterized chemically using transmission electron microscopy (TEM). Comprehensive physicochemical characterization of these nanoparticles was previously reported by our group (Bakr et al., 2023). Additional characterization data are also available from the manufacturers’ product specifications and pamphlets (Sigma-Aldrich; Toxemerge Pty Ltd.).

Sample collection

Approximately 200 African catfish (Clarias gariepinus), weighing around 200 ± 25 g and measuring 25 ± 5 cm in length, were sourced from a local fish farm. They were confirmed to be free of any infections or signs of illness. Experimental protocols for fish handling, acclimation, and euthanasia followed standard ichthyological guidelines (OECD, 2019). The exposure design (15-day semi-static renewal with daily 40% water change) followed established protocols for sublethal nanotoxicological assessment in fish (Hamed et al., 2021; Bakr et al., 2023). Before the experiments began, the fish underwent a 14-day acclimatization period in 120-L tanks. During this period, they were kept in dechlorinated tap water at a temperature of 26 °C ± 2 °C and a pH range of 7.2–7.6. Oxygen levels were kept above 80%, and the natural photoperiod of 12 h of darkness followed by 12 h of light was maintained. The fish were fed commercial food twice daily, in accordance with the feeding regimen outlined in Test Guideline No. 203 for Fish, Acute Toxicity Testing (OECD, 2019). This feed contained 30% protein, and to maintain water quality, 40% water changes were conducted daily to minimize the accumulation of fish waste and ensure consistent dosing of any test substances.

Exposures

After the acclimatization phase, the fish were randomly assigned to one of four groups, each containing 30 fish, and further divided into triplicate tanks. Groups 1, 2, 3, and 4 served as the control, PE-NPs (100 mg/L), Ag-NPs (100 mg/L), and CuO-NPs (100 mg/L) 15-day exposure, respectively (Bakr et al., 2023; Hasan et al., 2024). The selected concentration of 100 mg/L for each nanoparticle type was chosen as a hazard-identification dose to reliably induce measurable sublethal biochemical, immunological, and histological responses within the 15-day exposure period. This concentration exceeds typical environmental levels (reported in the ng/L–µg/L range) and is therefore intended for comparative hazard ranking among NP types under standardized laboratory conditions, rather than for direct extrapolation to field conditions. During the experiment, the fish were fed commercial food twice a day, and 40% of the water was replaced daily to uphold water quality and ensure precise dosing, following standard protocols for sublethal nanoparticle exposure studies (OECD, 2019; Hamed et al., 2021).

Following a 15-day exposure period, six fish from each group were randomly chosen for analysis. To minimize stress, the fish were humanely euthanized using ice, as it induces rapid loss of consciousness prior to death (Wilson et al., 2009). Blood samples were collected into non-heparinized tubes by cutting the tail and drawing blood from the caudal vein (n = 6). These samples were then used to evaluate immunological responses, oxidative damage, and antioxidant defense enzyme levels.

Micronucleus test and erythrocyte alterations

After exposure, blood smears were prepared by making a caudal incision and transferring the blood onto clean microscopic slides. The smears were allowed to air-dry at room temperature and then fixed in absolute methanol for 10 min. Following fixation, the slides were stained with hematoxylin and eosin. Micronucleated and morphologically altered erythrocytes were examined using a 40X objective and a 10X eyepiece.

Immunological parameters

Serum was obtained from blood samples by centrifuging at 5,000 rpm for 10 min. Then the serum samples (n = 6 per treatment, with 2 fish from each replicate tank) were stored at −80 °C until immunological biomarker analysis. Lysozyme (LYZ) activity (mg/mL) was measured using the turbidity assay method described by (Parry et al., 1965) and following the procedure established by (Overkamp et al., 1988). Serum levels of IgM (µg/mL) were determined using an ELISA kit (CSB-E12045Fh). Phagocytic activity (PA) (%) of leukocytes was assessed using a modified method from (Siwicki et al., 1994). The nitro blue tetrazolium test (NBT) assay was performed based on the method described by (Freeman and King, 1972).

Assessment of antioxidant defence enzyme activity

Superoxide dismutase (SOD) activity was measured in serum in triplicate using a microplate reader (Biochrom Ltd.) at 450 nm, following the method of (Nishikimi et al., 1972). Catalase (CAT) activity was also quantified in triplicate serum using a UV–VIS spectrophotometer at 240 nm as described by (Aebi, 1984). Total antioxidant capacity (TAC) was assessed in serum in triplicate using the colorimetric spectrophotometry method outlined by (Koracevic et al., 2001), with measurements taken using a Spectronic 21 (Moton Roy Co., Madison, WI, United States). Glutathione S-transferase (GST) activity was measured in serum in triplicate according to the procedure of (Saliu and Bawa-Allah, 2012), with readings taken at 340 nm using a microplate reader (Biochrom Ltd.).

Histopathological studies

Freshly sacrificed fish underwent dissection to extract their liver and kidney tissues, which were then fixed in 10% neutral buffered formalin. A standard paraffin embedding technique was employed, and the samples were sectioned at a thickness of 5 μm. Harris’s hematoxylin and eosin (H&E) stain, following the protocol outlined by Bancroft and Gamble (2008), was utilized to visualize tissue structure. Histopathological changes were assessed semi-quantitatively using a scoring system adapted from previous studies (Nibamureke and Wagennar, 2021).

Statistical analysis

Statistical analysis for this study involved using appropriate tests to assess the significance of differences observed in various parameters between the control and treatment groups exposed to different treatments. Descriptive statistics were used to summarize the data, including mean values and standard errors (SE) where applicable. To determine statistical significance, a one-way analysis of variance (ANOVA) followed by Fisher’s LSD test as a post hoc analysis was employed for comparing multiple groups. The level of significance was set at P < 0.05. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc.), and all results were interpreted considering their biological relevance and significance.

A sample size of six fish per treatment group (n = 6) was selected based on previous nanotoxicological studies (Hamed et al., 2021; Bakr et al., 2023), which demonstrated that this sample size provides sufficient statistical power (≥ 80%) to detect biologically meaningful differences in biochemical and histological endpoints at α = 0.05. The use of triplicate tanks with two fish sampled per tank further accounted for within-treatment variability. Fish were distributed among triplicate tanks per treatment group (30 fish per group total; 10 fish per tank). For biochemical and immunological analyses, six fish per treatment were randomly selected (two fish from each of the three replicate tanks). Prior to pooling data across tanks, a one-way ANOVA was performed with tank as a fixed factor; no significant tank effect was detected (p > 0.05 for all endpoints), justifying the use of individual fish as independent replicates. This approach is consistent with previous nanotoxicological studies (Hamed et al., 2021; Bakr et al., 2023). Prior to one-way ANOVA, assumptions of normality were assessed using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test. All data met these assumptions (p > 0.05). When significant differences were detected (p < 0.05), Fisher’s LSD post hoc test was applied for pairwise comparisons.

NPs characterization

TEM micrographs of PE, Ag, and CuO NPs are illustrated in Figures 1a–c. Figure 1a shows that the PE-NPs have irregular particles with an average diameter of 40.4 nm. The TEM image of Ag-NPs in Figure 1b reveals spherical particles with a diameter of 95 nm. Meanwhile, Figure 1c displays CuO-NPs, which have semi-spherical particles with an average diameter of 63.4 nm.

The percentages of morphological alterations in RBCs

In the control group, red blood cells (RBCs) exhibited a typical shape with a centrally located nucleus. However, in all exposed groups, smears showed poikilocytosis in RBCs after the designated exposure periods. Various morphological alterations were observed, including tear-drop cells, spinocytes, crenated cells, acanthocytes, eccentric nuclei, kidney-shaped cells, and schistocytes. The percentages of cell alterations and nuclear abnormalities in RBCs significantly increased in all exposed groups compared to the control, with the highest frequencies observed in the CuO-NP group (Figure 2). After 15 days of exposure to PE-NPs, the percentages of cell alterations and nuclear abnormalities were 98.41 ± 1.9 (p < 0.0001) and 9.9 ± 0.56 (p < 0.0001), respectively, compared to the control group (control cell alterations: 4.2 ± 0.3; nuclear abnormalities: 0.8 ± 0.1). Similarly, Ag-NPs induced percentages of 100.4 ± 1.6 and 9.6 ± 0.51, respectively, while CuO-NPs caused the highest frequencies at 104.8 ± 1.1 and 10.1 ± 0.81, respectively.

Immunological parameters

The immunological parameters of catfish, including LYZ, IgM, and PA, showed a significant decrease after exposure to PE-NPs, Ag-NPs, and CuO-NPs, as illustrated in Figure 3. These decreases were statistically significant (p < 0.0001), indicating a substantial negative impact on the immune function of the catfish compared to the control group. Among the three nanoparticle types, CuO-NPs caused the most severe immunosuppression across all measured parameters, followed by Ag-NPs, while PE-NPs had the smallest effect.

Assessment of antioxidant defence enzyme activity

Antioxidant enzyme activities (GST, SOD, CAT, TAC) were significantly reduced after exposure to all three NP types (p < 0.0001) as depicted in Figure 4, with the greatest decreases observed in the CuO-NP group.

Histology of liver

In the control group, polygonal hepatocytes with clear boundaries and central vesicular nuclei were observed, along with very narrow blood sinusoids (Figure 5A). In the PE-NPs-exposed groups, hepatocytes displayed homogenous, deeply stained acidophilic cytoplasm with vesicular nuclei, and dilated blood sinusoids were noted (Figure 5B). In the groups treated with Ag-NPs, polygonal hepatocytes with faint acidophilic cytoplasm, increased mitotic figures, and some areas of necrosis and hydropic degeneration was observed (Figure 5C). In the CuO-NPs treated groups, hepatocytes showed severe alterations, characterized by homogenous acidophilic cytoplasm, increased melanomacrophage centres, mitotic figures, and shrunken blood sinusoids (Figure 5D).

Histology of kidney

In the control groups, the convoluted tubules exhibited a normal morphological appearance with intact cuboidal lining epithelium, acidophilic homogenous cytoplasm, vesicular nuclei, and haemopoietic tissues (Figure 6A). In all treated groups, severe disruption of renal structures was observed. The renal structures showed the presence of melanomacrophage centers, deformation of renal glomeruli, a shift of the epithelial lining of tubules from their basement membrane, and aggregates of tubular lining cells with pyknotic nuclei in their core due to edema. In the PE-NPs -exposed groups, vacuolation of cells at their base was observed (Figure 6B). In the Ag-NPs group, severe degeneration made it difficult to differentiate between degenerated tubules and haemopoietic tissues, and hemorrhages were noted (Figure 6C). In the Cuo-NPs treated group, entire tubules showed displacement of their lining from the basal membrane to their core (Figure 6D).

This study applied a multi-biomarker approach to assess the effects of polyethylene (PE-NPs), silver (Ag-NPs), and copper oxide nanoparticles (CuO-NPs) on African catfish (Clarias gariepinus). Impacts on hematological, biochemical, immunological, and histopathological endpoints were evaluated following a 15-day of exposure to 100 mg/L of each nanoparticle type. TEM revealed distinct morphologies: irregular PE-NPs (40.4 nm), spherical Ag-NPs (95 nm), and semi-spherical CuO-NPs (63.4 nm), consistent with previous reports (Lee and Jun 2019; Mariano et al., 2021; Cuarán-Rosero et al., 2024). These microstructural characteristics are essential for understanding the physicochemical properties and potential applications of each nanoparticle type in fields such as catalysis, environmental remediation, and biological sciences (Malatesta, 2021).

Furthermore, evaluation of cellular and nuclear abnormalities in red blood cells is a reliable indicator for assessing genotoxicity and cytotoxicity in aquatic organisms (Witeska et al., 2023). In this study, exposure to all three nanoparticle types significantly increased the percentage of altered RBCs and nuclear abnormalities compared to the control group. Notably, the order of toxicity was CuO-NPs > Ag-NPs > PE-NPs. This pattern aligns with the chemical reactivity of these materials, whereby metallic nanoparticles demonstrate a greater capacity to induce oxidative stress and cellular damage (Manke et al., 2013). While Ag-NPs and CuO-NPs can interfere with cellular activity by generating reactive oxygen species (ROS) and directly interacting with nucleic acids, PE-NPs likely exert predominantly mechanical effects due to their less reactive polymeric nature (Zhang et al., 2014; Onodera et al., 2015; Feng et al., 2018; Ma et al., 2022; Encinas-Gimenez et al., 2024). Similar findings have been reported in other fish species, where microplastic and metal nanoparticle exposures induced eryptosis and poikilocytosis (Hamed et al., 2021; Soliman et al., 2023). Consequently, alterations in RBC morphology suggest reduced efficiency in oxygen transport and potential impacts on immunological function and overall growth (Wells et al., 2005).

In addition, all three nanoparticle types significantly affected immunological parameters in C. gariepinus, including LYZ activity, IgM levels, and PA. Among the three, PE-NPs had the least impact, resulting in only modest decreases in these parameters. This observation suggests that the relatively mild immunosuppression caused by PE-NPs was primarily due to physical stress rather than chemical toxicity (Covello et al., 2024; Harikrishnan et al., 2024). In contrast, both Ag-NPs and CuO-NPs caused pronounced declines in LYZ activity, IgM levels, and PA, indicating potent immunosuppressive effects likely mediated by oxidative stress and direct cellular injury (El-Kassas et al., 2018; Zheng et al., 2023). Interestingly, CuO-NPs produced the most severe effects, significantly reducing all assessed immunological parameters. This finding aligns with previous reports that metallic nanoparticles, particularly CuO-NPs, present a substantial risk to the immune system, thereby impairing the fish’s capacity to fight infections and maintain overall health (Malhotra et al., 2020; Tulinska et al., 2022).

Regarding oxidative stress, antioxidant enzymes play a critical role in protecting cells against oxidative damage. SOD facilitates the dismutation of superoxide anions to hydrogen peroxide (H 2 O 2) and oxygen (Wang et al., 2018), while CAT subsequently breaks down H 2 O 2 to water and oxygen, preserving cellular redox balance (Baker et al., 2023; Anwar et al., 2024). Additionally, GST is involved in detoxification and the maintenance of reduced glutathione levels (Gallego-Ríos et al., 2021). In the present study, exposure to PE-NPs, Ag-NPs, and CuO-NPs for 15 days resulted in significant reductions in GST, SOD, CAT, and TAC compared to the control group (p < 0.0001). These decreases indicate that the antioxidant defense system was overwhelmed, potentially leading to oxidative stress. Importantly, we did not measure direct oxidative damage markers such as lipid peroxidation (MDA), protein carbonyls, or 8-hydroxy-2′-deoxyguanosine (8-OHdG). Therefore, while our findings are consistent with oxidative stress, they should be interpreted as indicative rather than confirmatory. Future studies should include direct oxidative damage endpoints to establish causality.

The most pronounced reductions were observed in the CuO-NP and Ag-NP groups, which is consistent with the higher chemical reactivity of metal-based nanoparticles (Naguib et al., 2020; Metryka et al., 2022; Singh et al., 2022). Similar findings have been reported in other aquatic organisms exposed to metal nanoparticles (Gomes et al., 2015; Hu and Palić, 2020; Subaramaniyam et al., 2023). It has been hypothesized that the greater toxicity of CuO-NPs and Ag-NPs compared to PE-NPs may be partially attributed to the release of copper and silver ions, which could enhance the bioavailability of these metals in aquatic organisms (Wang et al., 2013; Radwan et al., 2019; Jiang et al., 2017; Saleeb et al., 2020; Wu et al., 2020; Mat Lazim et al., 2023). However, because the present study did not directly measure ion release in water or fish tissues, this mechanism remains speculative and requires further investigation. A central question in metal-based nanotoxicology is whether observed effects are attributable to the nanoparticles themselves or to dissolved metal ions. For Ag-NPs and CuO-NPs, both particle-specific and ion-mediated mechanisms likely contribute. Dissolved Ag + and Cu 2+ ions can inhibit respiratory enzymes, disrupt cell membranes, and generate reactive oxygen species (ROS) (Vyas et al., 2026). However, nanoparticle-specific effects including direct particle-cell membrane interactions, endosomal damage, and the ‘proton sponge’ effect, may also occur. Several studies have demonstrated that Ag-NPs and CuO-NPs exhibit greater toxicity than their ionic counterparts at equivalent mass concentrations, suggesting nano-specific effects (Lee and Jun 2019). Nevertheless, because we did not measure free ion concentrations in exposure water or fish tissues, the relative contribution of ion release versus particle-specific effects remains to be determined in our study.

Regarding the histopathological changes, it is important to note that there was a significant impact on the hepatic structure of the C. gariepinus fish when subjected to nanoparticles. The histological changes observed in the PE-NP group were less severe, involving dilated blood sinusoids and slight vacuolization of the liver cells (Umamaheswari et al., 2021; Yin et al., 2022; Haldar et al., 2023). In the Ag-NP group, more pronounced changes were observed, including hepatocyte necrosis, hydropic degeneration, and increased mitotic figures, all of which are indicative of oxidative stress and direct cellular injury (Yousef et al., 2022; Noga et al., 2023). The CuO-NP group showed the most severe alterations, characterized by melanomacrophage centers, increased mitotic activity, and shrunken blood sinusoids, thereby demonstrating strong cytotoxic and hepatotoxic properties (Das et al., 2024). Taken together, these findings indicate a clear gradient of liver injury severity, with metallic CuO-NPs and Ag-NPs presenting higher risks than PE-NPs (Zhang et al., 2022; Sun et al., 2024).

Similarly, renal histology was notably affected by nanoparticle exposure. PE-NPs induced modest alterations, including vacuolation of renal tubular cells (Ahmed et al., 2022). In contrast, Ag-NP exposure led to severe degeneration of renal tubules, hemorrhage, and difficulty distinguishing between degenerated tubules and hematopoietic tissues, indicating serious kidney damage consistent with oxidative stress (Terentyuk et al., 2009; Nosrati et al., 2021). The most severe effects were again observed with CuO-NPs, including displacement of tubular epithelial linings from the basement membrane, edema, and the presence of melanomacrophage centers. The greater severity observed in proximal compared to distal tubules may be attributed to the higher reabsorptive and active transport activity of proximal tubules, which likely leads to greater accumulation of nanoparticles in these segments (Schrand et al., 2010). Therefore, metallic nanoparticles, particularly CuO-NPs, present a higher risk to renal function than PE-NPs.

Given these findings, it is important to consider the broader context of aquatic contamination. Aquatic contamination by toxic pollutants released through anthropogenic activities is widely recognized as a major environmental threat. Aquatic organisms can accumulate heavy metals such as copper and silver, as well as polyethylene nanoparticles and other contaminants, through water uptake and ion exchange across biological membranes. Differences in the accumulation of copper oxide, silver, and polyethylene nanoparticles in catfish may reflect variations in species characteristics and exposure conditions. (Jamil Emon et al., 2023; Gündoğdu et al., 2024). Nevertheless, several limitations of this study should be acknowledged. A key limitation is that we did not directly measure nanoparticle bioaccumulation in fish tissues or the release of dissolved ions from Ag-NPs and CuO-NPs into the exposure medium. Such measurements would help clarify whether the observed toxicity is primarily due to the nanoparticles themselves or to released ions. Consequently, future studies should include speciation analyses to assess the potential transformation of metal-based nanoparticles into ionic forms in water and within fish tissues.

A further limitation concerns nanoparticle characterization. Our analysis was confined to TEM-derived primary size and morphology, without measuring hydrodynamic diameter, polydispersity index, zeta potential, or aggregation behavior in the actual exposure medium (dechlorinated tap water). Additionally, we did not quantify the dissolved Ag + and Cu 2+ fractions released over the 15-day exposure period. These parameters are critical as they govern NP stability, bioavailability, and toxic potential. That said, comprehensive physicochemical data including DLS and zeta potential for the same nanoparticles have been previously published by our group (Bakr et al., 2023). Thus, the key shortcoming is the lack of parallel characterization under our specific experimental conditions. Going forward, studies should incorporate full physicochemical profiling in the relevant exposure matrix alongside direct measurement of ion release. We also suggest the use of Fourier-transform infrared spectroscopy (FTIR) to identify surface functional groups on PE-NPs, as these may modulate surface reactivity and toxicity. Moreover, extended-duration investigations are necessary to assess chronic effects, bioaccumulation, and trophic transfer of these nanoparticles through aquatic food webs, given the implications for human exposure via fish consumption. Finally, although the 100 mg/L concentration employed here exceeds typical environmental levels (ng/L–µg/L), it remains suitable for comparative hazard identification under controlled laboratory settings. Subsequent research should adopt longer exposure windows and environmentally realistic concentrations (µg/L to low mg/L) to better evaluate chronic risks.

This study provides a comparative hazard assessment of polyethylene (PE-NPs), silver (Ag-NPs), and copper oxide nanoparticles (CuO-NPs) in African catfish, demonstrating a clear toxicity ranking of CuO-NPs > Ag-NPs > PE-NPs across multiple biomarkers. From an ecological perspective, even short-term exposure to these nanoparticles induces significant oxidative stress, immunosuppression, and histopathological damage, raising concerns about the health of freshwater fish populations in contaminated environments. The pronounced suppression of lysozyme activity, IgM levels, and phagocytic capacity suggests that NP exposure could increase disease susceptibility in cultured catfish, with potential economic consequences for aquaculture operations. Regulatorily, our findings indicate that metal-based nanoparticles warrant closer monitoring and stricter discharge limits compared to polymeric NPs, particularly in waters supporting fish farming. Additionally, the observed histopathological changes in liver and kidney tissues may serve as sensitive biomarkers for routine ecotoxicological surveillance. We recommend that aquaculture facilities near industrial or urban effluents implement regular NP contamination screening and consider water pretreatment strategies. Future research should prioritize: (i) dose-response studies using environmentally relevant concentrations (µg/L to low mg/L), (ii) direct measurement of dissolved ion release and bioaccumulation, (iii) chronic exposure assessments to evaluate trophic transfer and human health risks, and (iv) inclusion of additional physicochemical characterization (e.g., FTIR for PE-NPs) to better understand toxicity mechanisms.