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Frontiers in Aquaculture ·

Volume 5 - 2026 | https://doi.org/10.3389/faquc.2026.1771660

Ecotoxicological effects of low-density polyethylene microplastic on Heteropneustes fossilis: behavioral, hematological, biochemical, and histopathological impacts

  • Rohit Kumar Gautam1,2
  • Mahender Singh2
  • Bulbul Ali1
  • Neelam Sharma1
  • Ashish Sahu2
  • Abha Mishra1*corresponding author
  1. 1Department of Zoology, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India
  2. 2ICAR-National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, India
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Abstract

Microplastics (MPs) ingestion has been demonstrated in several living organisms, indicating the widespread dispersion of these tiny particles throughout aquatic ecosystems. In the present study, we elucidate the concentration-dependent ecotoxicological effects of low-density polyethylene microplastics (LDPE MPs) on the commercially important freshwater fish Heteropneustes fossilis. Experimental set up [one control and different concentration (100, 500, 1000 μg/L) of LDPE MPs exposed groups] had six fish per group for seven days duration. Exposed fish specimens were evaluated for behavioral, hematological (direct, indirect parameters), biochemical (glycogen, lipid, and protein), antioxidant stress enzymes (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD; and lipid peroxidation, LPO), and histopathological changes in major vital organs compared to the control experimental group. The hematological indices (RBC counts, Hb, Hct, and MCHC) showed a significant decline after exposure to MPs. The biochemical changes were tissue-specific, following the order: Muscle > Gill > Intestine > Brain > Kidney > Liver. Histopathological damage was recorded as maximum in the gills, followed by the intestine, stomach, liver, and kidney. The least damage was in the brain. The antioxidant enzymes (SOD, CAT, and POD) showed a decline, whereas LPO levels increased at higher MPs concentrations, indicating oxidative stress compared to the control group. The study demonstrated that LDPE MPs exert adverse effects on target organs and significantly alter the immune response in MPs exposed experimental fish, H. fossilis.

Keywords

1. Introduction

Plastics have been found to persist in different environments for many years due to their non-biodegradable properties, thus becoming an unmanageable threat to the ecosystem (Multisanti et al., 2025). Polyethylene (PE) is a commonly used synthetic plastic polymer composed of carbon and hydrogen atoms. Based on molecular branching and density, it can be categorized into two types: high-density polyethylene (HDPE) and low-density polyethylene (LDPE) (Shivasharana and Kesti, 2019). HDPE contains a low branching structure, characterized by a linear molecular arrangement, which enables it to align and pack closely due to its high density, crystallinity, and mechanical strength (Brown et al., 2007). Although showing similar impacts as HDPE, LDPE affect particle fragmentation, surface area, and interaction with biological membranes (Sahu et al., 2024) due to its highly branched molecular structure, tight chain packaging, lower density, crystallinity, and increased flexibility (Shivasharana and Kesti, 2019; Murat et al., 2020). These physicochemical differences are relevant to the observed biological endpoints, as they can modulate bioavailability, cellular uptake, oxidative stress responses, and enzyme activity. LDPE is a widely used and environmentally prevalent polymer in aquatic ecosystems due to its extensive use as single-use plastics and high fragmentation potential (Song et al., 2024). Consequently, LDPE MPs are frequently reported in freshwater environments, where their bioavailability poses risks and causes a direct threat to aquatic organisms and an indirect threat to humans (Chen et al., 2021).

In general, MPs are synthetic plastic particles with a size of less than 5 mm (Wu et al., 2017). MPs are considered primary when they are intentionally manufactured as products or ingredients (Lee et al., 2024; Song et al., 2024). They are termed secondary when they result from the breakdown of primary MPs through UV radiation and mechanical abrasion (Sun et al., 2022; Jang et al., 2024; Huber et al., 2022). As a persistent environmental pollutant, MPs adversely affect aquatic ecosystem from freshwater to seawater (Yang et al., 2021). MPs can adsorb and transport toxic contaminants, further emphasizing environmental and health concerns (Adamu et al., 2024; Burgos-Aceves et al., 2024; Burgos-Aceves et al., 2025). Such pollutants harm aquatic biodiversity and human health due to bioaccumulation and trophic transfer (Aliko et al., 2022; Simionov et al., 2023; Banaee et al., 2025a).

Bioaccumulation of MPs has been reported in different organs of aquatic animals, such as the brain, gills, liver, kidney, gut, and muscles (Su et al., 2019; Barboza et al., 2020; Hamed et al., 2020; Hurt et al., 2020). It has been found associated with hazardous effects primarily related to concentration-dependent physiological stress (Jin et al., 2018; Yu et al., 2018), such as behavioral alterations (Limonta et al., 2019), hematological disruptions (Hamed et al., 2019), neurotoxicity (Bakhasha et al., 2025), reproductive impairment (Li et al., 2020a), oxidative stress (Banaee et al., 2025b), and biochemical metabolism disruption (Banaee et al., 2023; Gholamhosseini et al., 2023). Several studies have reported intestinal deterioration, metabolic alterations, microbiota imbalance, histopathological changes, altered gene expression, and disturbances in energy distribution (Xie et al., 2021; Lu et al., 2022; Mbugani et al., 2022).

MPs act at two levels within biological systems: (1) direct or physical effects that influence organismal health, and (2) indirect or chemical effects resulting from the leaching of plastic additives or adsorbed environmental pollutants (Hasan et al., 2024; Mahmood et al., 2024; Emecheta, 2024; Iftikhar et al., 2024). Several studies have been reported the effects of MPs on various aquatic resources, including fish such as Nile tilapia (Oreochromis niloticus) (Hamed et al., 2019), zebrafish (Danio rerio) (Bhagat et al., 2020), silver carp (Hypophthalmichthys molitrix) (Zu et al., 2023), and marine medaka (Oryzias melastigma) (Ye et al., 2021), as well as shellfish such as bivalves and molluscs (Martyniuk et al., 2023; Impellitteri et al., 2023; Wang et al., 2023). Hence, analyzing the routes of accumulation and exposure is crucial for elucidating the toxic effects of environmental pollutants on fish fauna (Van Der Oost et al., 2003).

Fishes are among the most important aquatic animals due to their high economic value and sensitivity to pollutants, and are widely used in ecotoxicological studies (Abarghouei et al., 2021). The Singhi (H. fossilis) belongs to the Heteropneustidae family, which is one of the most cultivable candidate fish species in the aquaculture sector. Additionally, H. fossilis was used as a model in this experimental ecotoxicological study to evaluate organ-specific effects of LDPE MPs. This study provides the first assessment of LDPE MPs impacts in H. fossilis, establishing it as a novel and environmentally relevant model for MPs toxicity research. The species is of high economic and nutritional value, being rich in omega-3 fatty acids, and its suitable size and hardy nature make it ideal for laboratory-based toxicological studies (Ali et al., 2025).

In this study, exposure to increasing concentrations of LDPE MPs will induce concentration-dependent behavioral changes, hematological parameters (RBC, WBC, Hct, Hb, MCV, MCH, and MCHC), oxidative stress biomarkers (CAT, SOD, and POD), lipid peroxidation (LPO), biochemical markers (glycogen, lipid, and protein levels), and histopathological alterations in major vital organs. We selected the above parameters as integrative measures of physiological, metabolic, and tissue-level effects of LDPE MPs induced toxicity in this fish. The findings will further underscore the understanding of MPs-related ecological risk assessments for conservation strategies in freshwater ecosystems and fauna.

2. Materials and methods

Chemical

For the experiment, LDPE MPs (1–5 µm) were purchased from Nanochemazone, Chemazone Inc., Canada (CAS No. 9002-88-4; Batch No. NCZ-AE-120/0223A), and other chemicals and reagents used for biochemical estimations were of analytical grade (Labline and Gyan Scientific Traders) and purchased from scientific suppliers.

Fish collection and acclimatization

Adult healthy fish, H. fossilis (21-26 ± 2.5 g in wet weight, 19.5 - 22 ± 1.5 cm in body length) were collected from the local fishermen. Fish were brought to the laboratory and treated with potassium permanganate (KMnO 4) to eliminate dermal or parasitic infections (Hagar et al., 2019). Fish were acclimatized for seven days under natural photoperiod and temperature. Groundwater was used throughout the acclimatization and experimental period (photoperiod 14:10 h light: dark; temperature 20–25 °C; dissolved oxygen 6 mg/L; pH 6.9–7.1; ammonia 0.05 ± 0.01 mg/L; nitrite 0.06 mg/L; conductivity 260.8 µS/cm), measured using a multi parameter device (HI98194, HANNA). The fish were fed twice daily at 3% of their body weight per day (Muin and Taufek, 2024) with commercial pellet feed (Nutrila, Growel Feeds Pvt. Ltd., India). Water was replaced with fresh, fecal matter and uneaten food were siphoned out daily.

Experimental setup

Fish were acclimated, and all experimental procedures were conducted in strict accordance with the guidelines of the Institutional Animal Ethics Committee (IAEC), under approval number 909/GO/Re/S/05/CCSEA, dated 22/12/2023, as sanctioned by the Committee for Control and Supervision of Experiments on Animals (CCSEA).

The acclimatized fish were divided into four groups: group I as control (free of MPs) and groups II, III, and IV as experimental groups with different concentrations of LDPE MPs 100, 500, and 1000 µg/L, respectively (Ding et al., 2018) with surfactant Tween 80 (0.1% of the total volume) was used (Balakrishnan et al., 2019). Each group comprised 12 fish (total 48 fish). For parameters, histology, and stress enzyme analysis, six fish were used. Remaining six fish were used for hematology, and biochemical analysis. Analysis was performed in duplicates from one animal (Table 1). The experimental setup was maintained for 7 days to assess the effects of MPs exposure. Water in the experiment setup was replaced daily with the same dose of MPs to maintain concentration.

Behavioral observation and tissue sampling

Fish were closely monitored throughout the exposure period (seven days) for any signs of infection or abnormal behavior compared to control. Fish behavior observations were recorded three times daily (morning, afternoon, and evening) (Critchell and Hoogenboom, 2018; Li et al., 2024) and expressed in ordinal way (Supplementary Table 1).

After seven days of the experiment, blood samples were collected from the caudal vein of six fish per group using heparinized syringes (1 mL) for hematology parameter studies in duplicate from one animal. Fish were anaesthetized using 0.02% clove oil (Matin et al., 2009) and sacrificed by decapitation. Vital tissues (brain, gill, liver, kidney, intestine, and muscle) were dissected out and stored at −20 °C for energy biomolecule analysis in duplicate from one animal.

Remaining six fish from the experimental groups were sacrificed as above. Small pieces of dissected out fish tissue samples (gill, liver, kidney, intestine, stomach, brain) were preserved in Bouin’s fixative for histological studies. Furthermore, remaining tissues (brain, gill, liver, kidney, intestine, and muscle) were stored at −20 °C for stress enzyme analysis in duplicate from one animal.

Hematological parameters

Hematological indices, including red blood cells (RBCs), white blood cells (WBCs) with (manual Neubauer chamber counting method), hemoglobin (Hb) (Sahli hematometer), and hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) (by indirect formula method), were analyzed (Pandey et al., 2022).

Antioxidant enzyme assay

The tissue was dissected and stored at -20°C; subsequently, 100mg of tissue was homogenized in 1 mL of 0.01 M phosphate buffer (pH 7.0), and then centrifuged at 2,500 rpm for 10 minutes at 4 °C. Then, approximately 200 µL of supernatant was carefully collected and used for biochemical analyses. Different antioxidant stress enzymes were estimated using the following methods: LPO (Zeb and Ullah, 2016), CAT (Li and Schellhorn, 2007), SOD (Stankovic et al., 2013), and POD (Anoopkumar-Dukie et al., 2001). Enzyme activity was expressed with respect to constant tissue and compared with the control for the LDPE MPs effect analysis.

Biomolecule analysis

For further analysis, protein, glycogen, and total lipid contents were estimated following the methods of Waterborg (2009); Kannadhasan (2011); Axelsson and Gentili (2014), respectively. Absorbance was measured using a UV–VIS (Ultraviolet-Visible spectroscopy) spectrophotometer (Eppendorf, Hamburg, Germany).

Histopathology study

After 24 hours of tissue fixation, the samples were processed for paraffin block preparation and sectioned into 5 µm-thick slices using a rotary microtome (Weswox Optik ARMT-1090A). The microsections were double-stained with hematoxylin and eosin for microscopic examination and image capture at 20 X magnification using an Olympus CX41 microscope (Q-imaging, 3.3 RTV camera).

Statistical analysis

Behavioral data expressed in ordinal form and histopathology data expressed qualitatively in microphotographs. Data of biochemical, hematology, and stress enzyme were analyzed using SPSS (Statistics version 30.0.0, IBM Corp., USA). Values are presented as mean ± standard error of the mean (SEM). Before performing one-way analysis of variance (ANOVA), data normality was assessed using the Shapiro–Wilk test and homogeneity of variances was evaluated using Levene’s test. Significant differences among groups were analyzed using one-way ANOVA with (P < 0.001). The F-value in ANOVA represents the variance in between groups to within groups, a higher F-value represents significant mean differences among the groups. Intergroup comparisons were performed using Tukey’s post hoc test (P < 0.05). Groups sharing the same letter or numbers indicate no significant differences, while different letters or numbers registered significant differences.

3. Results

Behavioral changes in exposed fish

No mortality occurred during the entire experiment. Only behavioral patterns of MPs exposed fish had been observed to show several changes in comparison to untreated fish. The LDPE MPs exposed fish registered decrease in schooling, but increase in opercular beats (beats per minute, bpm), body coloration (dark), mucus secretion, air gulping, erratic swimming (swimming with jerk) and resting behavior with increase in concentration and duration of MPs exposure to fish, when compared to control group’s fish behavior (Supplementary Table 1).

Hematological parameters

The hematological parameters show significant alterations upon exposure to different concentrations of LDPE MPs compared to the control group (RBCs, F = 80.261; Hb, F = 82.758; Hct, F = 80.252) Significant concentration dependent decline in RBCs, Hb, and Hct levels was observed with increasing concentrations (100 - 1000 µg/L; P < 0.05) (Table 2). While MCHC and MCH levels (MCHC, F = 8.377; MCV, F = 1.264; F = 7.682; WBCs, F = 191.278; P < 0.001) also significantly decreased across all exposure levels compared to the control (P < 0.05). However, MCV remained unchanged across all concentration groups (P > 0.05). WBCs counts were increased at higher concentrations.

Antioxidant stress enzyme assay

CAT activity in target tissues shows significant variations compared to the control group (brain, F = 6.639; gill, F = 12.209; liver, F = 7.271; kidney, F = 9.213; intestine, F = 9.814; muscle, F = 7.505; P < 0.001; Figure 1A). In the brain and gills, CAT activity increased significantly at 500 µg/L, followed by decline at 1000 µg/L. However, the CAT activity in the liver, kidney, and intestine shows no significance at 100 and 500 µg/L. However, at 1000 µg/L, a significant decrease in activity was observed compared to the lower concentrations, although this reduction shows no statistical significance when compared to the control (P > 0.05). In contrast, the muscle exhibited a significant increase at 1000 µg/L concentration, while there was no significant difference compared to 500 µg/L. Also, there were no significant differences between the control and 100 or 500 µg/L groups either (P > 0.05; Figure 1A).

SOD activity in tissues shows significant variations compared to the control group (brain, F = 9.662; gill, F = 5.338; liver, F = 5.883; kidney, F = 7.575; intestine, F = 7.815; muscle, F = 7.982; P < 0.001; Figure 1B). In the brain, liver and kidney, SOD activity show no significant increase at 100 µg/L, followed by a significant increase at 500 µg/L compared to the control. However, activity declined at 1000 µg/L, compared to the control. The gills show no significant increase in SOD activity, which decreased further at the highest MPs concentration of exposed fish. Conversely, the intestine shows a substantial increase in SOD activity at all exposure levels, with no significant changes between them compared to the control. Muscle shows significant increase at 100 µg/L. However, decreased at 500 and 1000 µg/L concentration, compared to control (P < 0.05; Figure 1B).

POD activity in target organs shows significant changes upon exposure to MPs compared to the control group (brain, F = 16.922; gill, F = 18.639; liver, F = 45.720; kidney, F = 16.037; intestine, F = 12.046; muscle, F = 21.262; P < 0.001; Figure 1C). In the brain, kidney, intestine, and muscle, POD activity increased significantly at 100 µg/L, but show no significant increase in 500 µg/L. However, activity declined at 1000 µg/L (P < 0.05), with no significant difference from control group. In contrast, the gills show no significant difference at 100 µg/L compared to the control. POD activity increases at higher concentrations compared to the control, although the difference is not statistically significant from that at 100 µg/L. In the liver, POD activity shows no significant change at 100 µg/L, but increased at 500 µg/L, followed by significant decrease at 1000 µg/L compared to the control group (P < 0.05; Figure 1C).

LPO activity was significantly increased in MPs exposed target organs compared to the control (brain, F = 54.4275; gill, F = 9.908; liver, F = 78.251; kidney, F = 22.435; intestine, F = 42.771; Muscle, F = 49.800; P < 0.001; Figure 1D). LPO activity in the brain and intestine followed a similar trend, remaining stable at 100 µg/L (compared to the control), but showing significant increase at 500 µg/L, which further elevated with increasing MPs concentrations up to 1000 µg/L. However, LPO shows significant increase at 500 and 1000 µg/L concentrations of MPs. In gills, the LPO activity increased at 100 µg/L, and was not significant in the 500 and 1000 µg/L concentration groups. In the liver and kidneys, LPO enzyme activity increased significantly at 100 µg/L and remained stable at 500 µg/L. Furthermore, significantly increased at 1000 µg/L (P < 0.05). However, the muscle shows significant increase in the activities with the increase in the concentrations.

Biochemical analysis

The glycogen content was significantly different in all groups of each tissue (brain, F = 47.44; gill, F = 29.98; liver, F = 65.60; kidney, F = 52.19; intestine, F = 37.476; muscle, F = 14.601; P < 0.001; Figure 2A). Glycogen content of all tissues was more in the control group, while exposed MPs tissues shows lower content. The control group show higher glycogen content across all tissue samples, while the exposed MPs tissues show low content. The brain and kidney show significant decrease in glycogen levels in each concentration compared to the control group. However, no changes were shown between 500 and 1000 µg/L concentrations (P < 0.05). Similarly, gills show significant decrease in glycogen levels compared to the control, with no significant difference among the exposed groups. The liver showed significant decline in glycogen content with increased concentration (P < 0.05). Intestine show no significant decrease at 100 µg/L, compared to the control. While it shows significant decline at 500 and 1000 µg/L, compared to the control. However, there were no significant difference between the 500 and 1000 µg/L concentrations. Glycogen levels in muscle declined significantly with increasing MPs concentrations, with no significant difference between the 100 and 500 µg/L groups (P > 0.05).

The total lipid content in the tissue samples significantly depleted when compared to the control group (brain, F = 396.580; gill, F = 104.659; liver, F = 70.680; kidney, F = 46.492; intestine, F = 10.190; muscle, F = 18.034; P < 0.001; Figure 2B). In the brain, gill, liver, and kidney shows decline in lipid content, with significant differences among all exposure concentrations (P < 0.05). However, the intestine and muscle also shows significant reduction in lipid content, with no significant difference between the 500 and 1000 µg/L groups (P > 0.05). Similarly, in muscle tissue, lipid levels decreased slightly, but shows no significant difference between the 500 and 1000 µg/L groups (P > 0.05).

The protein content in tissues was significantly reduced compared to the control group (brain, F = 28.194; gill, F = 24.624; liver, F = 49.828; kidney, F = 13.348; intestine, F = 27.983; muscle, F = 23.178; P < 0.001; Figure 2C). In the brain, protein levels show concentration-dependent decline, with significant differences among all exposure groups except between 100 and 500 µg/L (P < 0.05). In gills, protein content gradually declined, with no significant difference observed between the control and 100 µg/L groups. However, a major reduction was found at 500 and 1000 µg/L, although the difference between these concentrations was not statistically significant (P > 0.05). The liver shows a sharp and significant decrease in protein content at 100 µg/L compared to the control, which continued to decline with increasing MPs concentrations. However, no significant difference was found between 500 and 1000 µg/L. In the kidney, protein levels decreased gradually at lower concentrations and declined sharply at the highest exposure level (1000 µg/L).

In both intestine and muscle, no significant changes were found at 100 µg/L compared to the control. Similarly, the 100 µg/L experimental group also shows no significant changes at 500 µg/L. However, both tissues show significant reduction at 1000 µg/L, indicating further depletion of protein levels.

Histology

In the control group, brain tissue shows normal histoarchitecture with neurons, distinct glial cells, neuropil, and blood capillaries (Figure 3a). In the 100 µg/L concentration group, neurons were damaged, including pyknosis of neurons, edema, and hypertrophied neurons. Necrosis and detachment were observed in large neurons (Figure 3b). At the 500 µg/L concentration group, spongiosis and degeneration of blood vessels were increased, as well as necrosis in large neurons (Figure 3c). In 1000 µg/L MPs-exposed group, the brain shows severe spongiosis, hemorrhage, degeneration of blood vessels, and pronounced necrosis and detachment of large neurons (Figure 3d).

In the control group, gills show normal structure with primary and secondary lamellae with distinct epithelial cells and capillaries (Figure 3e). In the 100 µg/L group, epithelial cells lifting and fusion of secondary lamellae were observed, along with necrosis and degeneration of lamellar structures in gills (Figure 3f). In the 500 µg/L group, necrosis of secondary lamellae, edema, and dilated congested blood vessels were seen along with the lamellar fusion (Figure 3g). At 1000 µg/L, hyperplasia of epithelial cells, vacuolization, and further dilation of congested blood vessels were observed. The necrosis of secondary lamellae was more damaged in this group (Figure 3h).

In the control group, the liver shows normal hepatocytes, normal sinusoids, and distinct kupffer cells (Figure 3i). In the 100 µg/L MPs exposure group, few histological changes, such as sinusoid dilation, pyknosis, and increased kupffer cells, were observed (Figure 3j). In the 500 µg/L concentration group, hepatocyte hypertrophy was evident along with pyknosis and sinusoid dilation, as well as increase in kupffer cell density (Figure 3k). In 1000 µg/L, severe cytoplasmic vacuolization and hepatocyte necrosis were observed, with continued proliferation of kupffer cells and hepatocyte hypertrophy (Figure 3l).

In control, kidney tissue shows normal histoarchitecture with distinct proximal and distal tubules, clear brush borders, and intact bowman’s capsules (Figure 4a). At 100 µg/L concentration, tissues of the kidney show lobulated glomerular tufts, vacuolization of glomeruli, necrosis and lymphocytic infiltration (Figure 4b). At 500 µg/L, damage intensified with increased vacuolization, expanded necrotic areas, and dilated renal tubules (Figure 4c). In 1000 µg/L group, kidney show severe atrophy of glomeruli, hemorrhage, elliptical glomerular structures, degeneration of renal tubules, and obliteration of bowman’s space (Figure 4d).

In control group, stomach shows normal architecture with organized mucosa, submucosa, and gastric glands (Figure 4e). At 100 µg/L concentration, degeneration of mucosa, necrotic patches, vacuolization, and hemorrhage were found in stomach (Figure 4f). The 500 µg/L group show moderate degeneration, including necrotic patches and breakdown of microvilli (Figure 4g). In the 1000 µg/L group, more changes such as extensive necrotic patches, aggregation of gastric glands, hemorrhage, widespread vacuolization, and damaged microvilli were reported (Figure 4h).

In the control group, the intestine shows intact histoarchitecture, including muscularis externa, muscularis interna, mucosa, and submucosa layers (Figure 4i). At 100 µg/L experimental group, vacuolization in the muscularis externa, necrosis, collapsed brush border, and atrophy of goblet cells were observed (Figure 4j). In the 500 µg/L group, epithelial columnar degeneration, mucosal atrophy, necrosis, and vacuolated submucosa were evident, along with a collapsed brush border (Figure 4k). At 1000 µg/L, more intestinal damage including hyperplasia of the submucosa, crypt degeneration, collapsed brush border, and extensive vacuolization of the submucosa were observed (Figure 4l).

4. Discussion

Although there has been extensive research on the effects of MPs in fish, but the available studies are fragmented and organ-specific. In this study, the potential effects of LDPE MPs (1–5 µm) exposure on adult H. fossilis were investigated over a 7- days acute period under controlled conditions via waterborne exposure. The results revealed significant changes in behavioral, hematological parameters, oxidative stress biomarkers, lipid peroxidation, biochemical markers, and histopathological alterations in different vital organs of fish in a concentration dependent manner of MPs.

Behavior

The concentration-dependent behavioral changes (such as opercular beats, mucus secretion, air gulping, and erratic swimming) were observed in the LDPE MPs-exposed fish individuals. Several researchers have also reported behavioral changes in teleost fish species exposed to different types of MPs polymers (Su et al., 2019; Wang et al., 2019; Barboza et al., 2020). When food was provided in isolation, fish exposed to MPs dispersed throughout the tank and exhibited reduced feeding performance, whereas those in the control group actively congregated around the feeding area. These results suggest that the exposed fish were not consistently attracted to food or attempted to catch food as often as the control group (Hamed et al., 2020; Bhagat et al., 2020).

Chronic stress can compromise predator avoidance in natural environments, thus reducing fitness and destabilizing population dynamics in freshwater habitats (Limonta et al., 2019; Wang et al., 2019; Yu et al., 2018; Hamed et al., 2020). Notably, these behavior responses may be due to the physical and chemical toxicities of MPs. Physical blockage of digestive or respiratory tracts by MPs can impair nutrient absorption and gas exchange, triggering energy reallocation and metabolic strain (Bhat et al., 2024; Hasan et al., 2024). Concurrently, leached additives or adsorbed pollutants from LDPE MPs (e.g., plasticizers, PAHs) may disrupt neuroendocrine pathways, explain erratic swimming and changes coloration (Kim et al., 2021; Lin et al., 2023). Ye et al. (2021) found metabolic disturbances in the liver of marine medaka (Oryzias melastigmas) at higher MPs doses; no mortality was observed in this study, suggesting possible species-specific tolerance or different effects of MPs.

Hematology

In this study, LDPE MPs disrupt different hematological parameters, major indicators of cytotoxicity, environmental stress, and fish health (Hamed et al., 2019). The RBCs count, Hb, and Hct levels showed a significant, concentration-dependent decline with increasing concentrations of LDPE-MPs exposure. Choi et al. (2021) reported that the sharp surfaces of MPs can physically damage RBC membranes, compromise structural integrity and lead to cell lysis. Moreover, higher concentrations of MPs in the blood were associated with increased levels of hemolysis. Moreover, Hamed et al. (2019); Ismail et al. (2021) reported a significant decrease in the RBCs count and Hb and Hct levels of O. niloticus when exposed to MPs and suggested that the changes in hematological parameters were due to hematopoietic tissue damage and RBCs hemolysis, both caused by MPs toxicity. Similar findings were reported by Vijayaraghavan et al. (2022) in green chromide. Hence, the observed changes in RBCs count and Hb and Hct levels might result in blood hemolysis and blood cell destruction due to MPs toxicity. Red cell indices (such as MCV, MCH, and MCHC) are important biomarkers that provide useful information on the Hb content and size of RBCs (Ogueji et al., 2018). Deviations in red cell indices from the normal physiological range generally indicate microcytic or macrocytic anemia (Barbara et al., 2011).

In the present study, reduced MCH and MCHC levels suggest hypochromic anaemia, potentially stemming from impaired iron metabolism or Hb synthesis in hematopoietic organs, such as the kidney or spleen (Jo et al., 2025). Stable MCV values rule out erythrocyte swelling or shrinkage and emphasize functional Hb deficits. Conversely, increased WBC count indicates leukocytosis, reflecting immune system activation due to inflammation and cytokine-driven stimulation of leucopoiesis (Do Nascimento et al., 2023; Hamed et al., 2019; Das et al., 2023). However, a decline in WBCs at higher doses in C. gariepinus suggested a species-specific threshold for immune exhaustion versus activation (Soliman et al., 2023). Based on the findings of the present study, LDPE MPs adverse effect on hematological parameters in exposed groups (Supplementary Table 1) but had no significant effect on MCV.

Antioxidant

In the current study, exposure to LDPE MPs induces a concentration-dependent and tissue-integrated modulation of antioxidant enzyme activities (including CAT, SOD, POD, and LPO) in H. fossilis, reflecting a coordinated yet ultimately vulnerable oxidative defence system. MPs toxicity impairs ROS metabolism in fish and induces oxidative stress due to imbalance in ROS production/elimination (Bhagat et al., 2020). CAT, SOD, and POD are common antioxidant enzymes in the body, capable of scavenging free radicals and protecting cells from oxidative damage (Ding et al., 2023).

CAT is an antioxidant enzyme that protects cells from oxidative stress by decomposing H 2 O 2 into H 2 O and O 2 (Hamed et al., 2019). In our experiments CAT activity decline at higher concetration of MPs in target organs (such as the brain, gills, liver, kidney, and intestine.) MPs induced ROS initially activates antioxidant defenses but suppresses enzymes under excessive oxidative stress, while muscle shows greater resilience due to lower metabolic activity. Barboza et al. (2018) reported a significant increase in CAT activities in exposed European seabass (Dicentrarchus labrax). Similarly, Solomando et al. (2021) suggested that an increase in the activity of these enzymes serves to detoxify the toxicity of MPs in the gilthead seabream (Sparus aurata).

SOD is the first detoxifying antioxidant enzyme that decomposes ROS into H 2 O and H 2 O 2 (Xia et al., 2020). In this study, biphasic SOD response reflects initial ROS-mediated antioxidant activation followed by enzyme inhibition under excessive oxidative stress, with tissue-specific differences driven by metabolic activity and exposure intensity. Ding et al. (2018) reported an increase SOD in O. niloticus exposed to polystyrene, which indicates the activation of the antioxidant system against ROS formation and oxidative stress. Liu et al. (2024) reported an increase SOD activity in grass carp (Ctenopharyngodon idella), exposed to polystyrene, which was attributed to the activation of antioxidant responses to reduce oxidative loss due to MPs-induced stress.

POD helps detoxify reactive oxygen in fish species by reducing H 2 O 2 using various electron donors (Ugya et al., 2020). In this study, POD response indicates adaptive upregulation at lower MP exposure to detoxify ROS, followed by enzymatic suppression under excessive oxidative stress at higher concentrations. Tissue-specific differences reflect variations in metabolic activity, antioxidant reserves, and direct MPs exposure. Jayaseelan et al. (2014) reported a decline in POD activity in Mozambique tilapia (O. mossambicus). Some studies have also shown that changes in antioxidant enzyme activity are correlated with the concentration of pollutants (Subaramaniyam et al., 2023). Moreover, POD in the liver and kidney of C. idella may be related to their different physiological functions (Li et al., 2020b).

LPO is a useful biomarker of oxidative damage and cellular function in aquatic organisms, particularly in response to oxidative stress (Ajima et al., 2017). When fish are exposed to MPs, it raises ROS levels. Furthermore, it causes inflammation and cell death. The formation of oxygen-free radicals triggered the LPO activity. In the present study, elevated LPO reflects MP-induced ROS overproduction leading to oxidative damage of membrane lipids. Tissue-specific and concentration-dependent patterns indicate differences in oxidative susceptibility, detoxification capacity, and MPs exposure intensity. Similar to our findings, MPs exposure dramatically increases LPO activity in Dicentrarchus labrax (Barboza et al., 2018) and O. niloticus (Hamed et al., 2021). It increases the end product of LPO (MDA) levels, resulting in tissue damage (cell membrane damage) due to oxidative stress.

Biomolecule

The concentration-dependent depletion of macromolecules, including glycogen, lipids, and proteins, across tissues upon exposure to LDPE MPs induced metabolic disruption, likely driven by oxidative stress and reallocation of energy for detoxification. The progressive depletion suggests sustained activation of gluconeogenesis or glycogenolysis, as well as β-oxidation of lipids to meet energy demands under MPs-induced stress (Sokolova et al., 2012). Similarly, sharp declines at higher concentrations indicate compromised mitochondrial function in the gills (Lu et al., 2018) and activation of the stress response in busy organs (Wright et al., 2013). However, the less affected muscle shows declines linked to prolonged energy expenditure for cellular repair (Jeong et al., 2017). Overall, MPs-induced oxidative stress (Wright et al., 2013) likely triggers ATP-dependent antioxidant defence, depleting energy reserves, mainly glycogen. Prioritizing energy for stress mitigation over storage underscores physiological trade-offs, compromising long-term energy homeostasis. Sokolova et al. (2012) suggested a threshold beyond which energy reserves stabilize, due to adaptive suppression of catabolism or exhaustion of mobilizable reserves.

Histology

In our experiment, histological alterations were observed in the exposed fish individuals. The brain lesions are attributable to MPs-induced neurotoxicity mediated by oxidative stress, inflammation, and disruption of neuronal membranes and vascular integrity (Abdelnaeim Hussein and Cao, 2018). These alterations are similar to other experimental studies, such as in tilapia (Hamed et al., 2021), goldfish (Karami et al., 2017), and zebrafish (Romano et al., 2020).

In the study, gills alterations result from direct MPs contact, inducing oxidative stress, inflammation, and impaired gas-exchange surface integrity (Wang et al., 2019; Hu et al., 2020; Yang et al., 2020). These changes caused by mechanical damage or responses to leached MPs (Hu et al., 2020). The separation of epithelium from the basal membrane is a symptom of disorders related to osmoregulation and can act as a protective mechanism to increase the distance from toxicants (Movahedinia et al., 2012), the fusion of lamellae causes an overall reduction in surface area (Farrell et al., 2010), higher concentration of MPs leads to lamellar aneurysms causes congestion with blood (Hamed et al., 2021).

In the liver, exposure to LDPE MPs caused alter the histoarchitecture of hepatic tissue, such as sinusoid dilation, pyknosis, and increased Kupffer cells, hepatocyte hypertrophy, cytoplasmic vacuolization and hepatocyte necrosis (Da Costa Araújo et al., 2020; Xia et al., 2020; Yang et al., 2020). Moreover, the presence of sinusoidal dilatation and inflammatory cell infiltration may be due to physical blockage of hepatic capillaries by LDPE MPs translocated to the liver, primarily the smaller caliber ones (Brancatelli et al., 2018). The inflammatory response may have been induced by the presence of circulating and accumulated MPs in the liver tissue, which would be followed by increased production of reactive oxygen species in adherent immune cells and by the response to combat oxidation caused by hepatocytes (Von Moos et al., 2012).

The renal lesions are driven by MPs-induced oxidative stress and inflammation, leading to impaired filtration, tubular degeneration, and structural collapse of glomerular architecture. Zhu et al. (2020) also reported that dietary exposure to polystyrene MPs in maturing O. latipes resulted in glomerulopathy (expansion and congestion of glomerular capillaries), increased glomerular size (i.e., glomerulomegaly), and expansion of Bowman’s space. It is reasonable that elevated blood pressure puts mechanical stress on the glomerular walls, which may lead to injury (Ichimura et al., 2013; Espinosa et al., 2017).

The stomach showed different alterations that were similar to other MPs experiments, which demonstrated histopathological changes in the stomach. The observed gastric damage is mediated by MPs-induced oxidative stress and inflammation following direct mucosal contact and epithelial barrier disruption (Ghosh and Chakrabarti, 2015; Samanta et al., 2016; Moawad et al., 2017).

Intestinal damage likely results from MPs induced oxidative stress and inflammation, which disrupts epithelial integrity, induces cell death, and impairs mucosal and submucosal structures (Ahrendt et al., 2020; Iheanacho and Odo, 2020; Zhu et al., 2020). The increased number and size of goblet cells observed in this study indicate an adaptive response to enhance mucus secretion, which serves as the first line of defense against foreign particles (Peda et al., 2016). Moreover, the presence of numerous leukocyte infiltrations is a typical response to stress conditions (Peda et al., 2016). Karami et al. (2016) suggested that the sharper texture of those MPs was probably the cause of such damage. Moreover, breakdown of the epithelial layer could allow cytotoxic substances from the gut lumen to enter the blood, potentially resulting in functional alterations in different internal organs (Mobin et al., 2000). Although all of the histopathological lesions observed in this study were concentration-dependent.

5. Conclusions

The LDPE MPs induced concentration-dependent behavioral alterations, hematological disruptions, oxidative stress, and biomolecule depletion in most vital tissues of H. fossilis. The histological microphotographs supported that MPs caused neuronal and cellular damage. During the study period, there was nil mortality due to MPs; only significant reductions in health parameters supported compromised fish quality due to MPs exposure. Therefore, urgent mitigation strategies should be adopted to prevent bio-stock damage.

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Data availability

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics approval

The animal study was approved by Institutional Animal Ethical Committee (IAEC). Approval number -909/GO/Re/S/05/CCSEA, Committee for Control and Supervision of Experiments on Animals (CCSEA). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

RG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. MS: Conceptualization, Data curation, Formal analysis, Investigation, Resources, Supervision, Validation, Visualization, Writing – review & editing. BA: Conceptualization, Data curation, Formal analysis, Resources, Software, Writing – review & editing. NS: Data curation, Formal Analysis, Software, Writing – review & editing. AS: Data curation, Formal analysis, Visualization, Writing – review & editing. AM: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.

Acknowledgements

Institutional financial support in the form of a fellowship is thankfully acknowledged.

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Citation

Gautam, R. K., et al. (2026). Ecotoxicological effects of low-density polyethylene microplastic on Heteropneustes fossilis: behavioral, hematological, biochemical, and histopathological impacts. Frontiers in Aquaculture, Volume 5. https://doi.org/
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5 - 2026

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Copyright © 2026 Gautam, Singh, Ali, Sharma, Sahu and Mishra.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Abha Mishra

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