Ingestion and the toxicological effects of virgin polyethylene (PE) and PVC microplastics in commercial freshwater fish, Tilapia (Oreochromis niloticus)

Ingestion and the toxicological effects of virgin polyethylene (PE) and PVC microplastics in commercial freshwater fish, Tilapia (Oreochromis niloticus) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ingestion and the toxicological effects of virgin polyethylene (PE) and PVC microplastics in commercial freshwater fish, Tilapia (Oreochromis niloticus) Kolandhasamy Prabhu, Saheli Singha, Sourav Bhattacharya, Suguna Anbukkarasu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6761113/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 8 You are reading this latest preprint version Abstract Microplastics (MPs), which are tiny particles measuring less than 5 mm, have emerged as a notable environmental issue due to their widespread presence in aquatic environments and their potential to harm aquatic organisms. In this study, the diet of tilapia ( Oreochromis niloticus) exposed them to two types of MP materials: PE and PVC fragments. The fish were exposed for three weeks (21 days), and various behavioural changes and mortality were noticed. Moreover, microplastics can impact the growth, reproduction, and survival of tilapia, as evidenced by reduced growth rates and observed behavioural changes in exposed fish. Such modifications might have important effects on the general condition and population dynamics of aquatic environments. In both the gill and gastrointestinal tract (GIT), the MPs fragments were accumulated. The GIT of tilapia fish revealed 12.6 items/individual from the collected PVC pieces; gills included 4.3 items/individual. Similarly, PE fragment accumulation in the GI tract of fish showed 1.18 items/individual, and the gills showed 2.0 items/individual. A dietary intake of microplastics led to increasing inflammatory alterations in the liver and intestines. This study assessed the levels of oxidative enzymes in exposed groups of fishes (control, PVC, and PE fragments). The MPs-exposed tilapia fish exhibited remarkable Changes in the enzyme level and the nutritional values which was compared to control groups. All things considered, microplastics seriously compromise the health and ecological processes of freshwater fish including tilapia. More study is required to completely understand these effects as well as develop feasible strategies for reducing the microplastics' hazard in freshwater habitats. PE and PVC fragments behavioural changes gill GIT oxidative enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION The ecological health of the oceans is under serious threats due to the massive influx of plastic debris each year. In 2016, approximately 335 million tons of plastic debris were produced globally, with an estimated 10% ending up in marine environments Alimba and Faggio (2019). It is estimated that by 2050, the oceans may be contained approximately 937 million tons of plastic, surpassing the estimated 895 million tons of fish biomass (MacArthur, 2017). Once in the ocean, plastics degrade through physical and chemical processes, forming microplastics (MPs) particles which is less than 5 mm and nanoplastics (NPs), which are smaller than 100 nanometres (Rai et al., 2021). Marine organisms frequently ingest MPs, which cannot be broken down by digestive enzymes. Microplastic (MP) ingestion can lead to a wide range of harmful effects at various biological levels such as subcellular and cellular disruption, altered gene expression, oxidative stress, compromised antioxidant defences, impaired cell division, disrupted fatty acid metabolism, reduced growth and reproduction, biodiversity loss, and population declines (Arienzo et al., 2021b). Ingestion is likely the most frequently observed effect linked to plastic debris, with over 270 different species reported (Laist, 1997). Moreover, the effects of MPs go beyond individual organisms, posing risks to entire ecosystems. MPs can also act as carriers for environmental pollutants (xenobiotics), facilitating the widespread dispersal of toxic substances through wind and ocean currents, thereby enhancing their environmental and ecological impact (Arienzo et al., 2021a). Freshwater ecosystem plays a pivotal role in the lifecycle of microplastics (MPs), acting simultaneously as sources, pathways, and final sinks for these particles (Wagner and Lambert, 2018). Despite this critical role, research on the toxicological impacts of MPs in freshwater organisms-especially fish-remains limited, with most existing studies concentrating on marine species (Li et al., 2018). Fish, however, are widely recognized as effective biomonitors for evaluating the ecological health of aquatic environments. MPs in water can be simply consumed by fish. Researchers have described the incidence of MPs in fish (Bilal et al., 2021; Bilal et al., 2023). MPs deposit in fish and have a wide range of negative impacts i.e., decreased feeding activity, impeded growth, energy interruption, oxidative stress, and even genotoxicity (Lu et al., 2016; Singh et al., 2022; Hassan et al., 2023) Microplastics (MPs) primarily threaten freshwater ecosystems, either directly or indirectly, due to their tiny and irregular form. Further, MPs have the potential to absorb heavy metals and organic pollutants from the surrounding medium, which can have a compounding effect on ecosystems. The toxicity patterns of microplastics have their own consequences and mechanisms and they are important factors to assessing environmental risks and human health consequences. MPs constitute a dual risk to freshwater ecosystems by causing direct damage as well as serving as transporters of additional contaminants (Ding et al., 2021). Plastic polymers employ thousands of various additives, which are probably found in all kinds of plastics. For instance, PVC contains 73% of these chemicals by volume, while PE and PP account for 10% (Murphy, 2001). In freshwater creatures, microplastics (MPs) cause physical injury, such as damage to cell walls and membranes, altered feeding and movement habits, nutrient absorption obstruction, decreased reproductive potential, and decreased growth or survival rates (Cai et al., 2019; Chen et al., 2019). The species tilapia Oreochromis niloticus was first brought to India in the latter part of 1987 (Singh and Lakra, 2011). The cultivation of O. niloticus is expanding quickly, particularly in West Bengal, Orissa, and Andhra Pradesh. The fish is found in many states, particularly those that are near the coast. The most commonly farmed tilapias are hybrids of O. niloticus and other native African species (Mohamed and Abood, 2021). Tilapia O. niloticus is among the simplest and most profitable fish to cultivate, largely due to their omnivorous nature (Eknath and Hulata, 2009). It can be sustained entirely on a plant-based diet. This species, along with other fish that consume vegetable matter, present a more ecologically balanced and environmentally sustainable method of supplying a plentiful amount of nutritious and tasty fish for human consumption (Singh et al., 2014). Devi et al. (2020) documented that the alien fish species Pirapitinga, Piaractus brachypomus , in the Vembanad Lake on India's southwest coast were consuming MPs. Pandey et al. (2023) indicated that MP pollution poses significant implications for environmental health and the accessibility of clean food for humans. Bhatt et al. (2024) studied MP consumption by five fish species with different feeding habits from River Alaknanda at Srinagar, namely, Schizothorax richardsonii Crossocheilus latius, Cyprinus carpio and Botia horii showed fibers (66%) were the predominant (Bhatt et al., (2023). Further, Anandhan et al. (2022) examined microplastics (315 MPs) in the gastrointestinal (GI) tracts of the five fish species from the Kollidam and Vellar rivers of Tamil Nadu, Southern India. The toxicity of polypropylene microplastic to O. mossambicus is assessed by (Jeyavani et al., 2023). The O. mossambicus consumed MPs, which caused homeostasis to fluctuate, reactive oxygen species (ROS) levels to rise, antioxidant parameters such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and glutathione peroxidase (GPx) to change, lipid molecules to oxidize more readily, and the neurotransmitter enzyme acetylcholinesterase (AChE) to become denaturated. The aim of the present research is to investigate the toxicological effects and ingestion of polyethylene (PE) and polyvinyl chloride (PVC) microplastics in the economically important freshwater fish, Oreochromis niloticus (Tilapia). The accumulation of microplastic particles in specific organs, such as the gastrointestinal tract and gills, was evaluated by exposing fish to them. In addition, the investigation assesses the physiological, biochemical, and behavioural modifications that may result from microplastic exposure, thereby offering an understanding of the potential health hazards that aquatic organisms might encounter. 2. MATERIALS AND METHODS 2.1. Fish exposure experimental setup The rectangular glass tanks of medium size (18 × 9 × 9 inches) with a capacity of around 24L were purchased from the local aquarium market in Tiruchirappalli, India. Then the glass tanks were carefully washed with 5% potassium permanganate and diluted 5% HCl before tilapia was added. Following washing, 20 L of filtered tap water were filled in every tank. A proper aeration system was fitted to ensure optimal oxygen levels. The temperature was maintained within a consistent range of 28ºC − 30ºC, while the pH level was kept between 7.5 and 9, both of which are optimal conditions for tilapia fish (Kaloyianni et al., 2021). 2.2. Domestication of freshwater fish Tilapia is a freshwater fish species, is highly economically value due to its strong environmental resilience and omnivorous feeding habit (Tesfahun and Temesgen, 2017 ). In the present study we selected Tilapia ( Oreochromis niloticus ), for microplastic exposure experiment. Tilapia fingerlings, were obtained from a local commercial fish seed supplier (Kamtchi Fish Farm, Thanjavur) approximately 21 days old, making them suitable for aquariums. Before commencing the experiment, all fish underwent an acclimatization period in a round plastic bucket, where water from both the bucket and the experimental tank was mixed using a small silicon tube. Following a day of acclimation, the fish were transferred to the experimental tank. Before introducing them into the tanks, we measured their initial length using a 30 cm scale and determined their initial weight using a digital weighing machine. After the 21-day experimental period, we measured their final weight and length to evaluate the effects of the microplastic diet. The water conditions in the tanks were maintained within a pH range of 7.5-9 and a temperature range of 28°C − 30°C. To prevent external contamination, the surface of each tank was covered with a mosquito net and secured with black paper clips. 2.3. Preparation of microplastics For the MPs exposure experiment, the fish feed was prepared by utilizing two varieties of microplastics: PVC and Polyethylene (PE) MPs fragments (Fig. S1 & S2). The small PVC fragments were created by cutting them with a hacksaw blade. The polymer composition of these fragments was determined through FTIR-ATR (Jasco, Model, 6600) analysis to be polyvinyl chloride and Polyethylene (PE). Following this, all fragments underwent a process of soaking in 75% v/v alcohol and subsequent ultrasonication with milli Q water to eliminate any potentially attached substances. The average length of the PVC fragments was determined to be 2–3 mm and polyethylene fragments were 0.5 to 1 mm size (Figure S1 ). 2.4. Microplastic food preparation Commercial fish food, specifically the Growfin pellet fish feed, was ensured to be completely devoid of microplastic (MPs) contamination. This fish food was initially soaked in filtered tap water to soften it. Subsequently, PVC and PE fragments were incorporated into the softened food mixture, which was then air-dried under hot air oven. The preparation of PVC-containing food was based on weight, with an adjustment of 10 mg of PVC fragments per 10 grams of fish feed, distributed across 10 pellets of commercial food pellets. Conversely, each pellet containing PE fragments consisted of 15 to 20 particles. Though slight variations may have occurred, on average, the fish were administered a concentration of 10 pellets per tank (equivalent to 1.6 pellets per gram of wet weight fish, including both food and MPs). This dosage translated to approximately 15–20 PVC particles and PE particles being fed to each fish. In the control group, fish were fed pellets devoid of any plastic fragments (Fig. S2). 2.5. Feeding strategy and observation Before the experiment commenced, the fish underwent a 48-hour fasting period. The experiment utilised in total of nine tanks: 3 serving as control groups and 3 each for the PVC and Polyethylene exposure groups. Each tank housed six individual fish, and three replicates were conducted simultaneously. Observations were made twice in a day for each group. In the control tanks, fish were provided with standard commercially available fish feed without MPs particles, whereas group 1 fish were fed with PVC-based pellets, and group 2 fish were fed with polyethylene-based pellets. The fish were fed twice daily, once in the morning at 10:30 am and once in the evening 5:00 pm. Through the initial observation, the feeding behaviour of fish was examined within the initial five minutes, with ingestion or rejection of food being recorded. 2.6. Processing of sample After 21 (3 weeks) days exposure, all fish were anesthetized with MS-22 (100 mg/L) and sacrificed for immediate analysis. Total length (cm) and wet weight (g) and condition factor (CF = w/L 3 ×100, where CF- condition factor, w – weight and L – standard length) of each fish was recorded (Table 1 ). Each fish was decapitated and the GIT, liver, brain and Gill were removed through a ventral incision. Heads of all fish and the gut and livers of the 12 fishes. The GITs of the fishes were frozen at -20ºC for further analysis. Table 1 Length, weight and condition factor of fish exposure experiments Exposure group No of Fish Exposure time Average Length (cm) Average weight (g) Condition factor Control 10 21 days 6.96 ± 0.29 4.96 ± 0.60 1.47 ± 0.32 PVC 10 21 days 7.2 ± 0.48 4.37 ± 0.32 1.17 ± 0.27 PE 10 21 days 6.34 ± 0.62 4.24 ± 0.31 1.66 ± 0.0.48 2.7. Observation of Behaviour The fish were monitored daily for a duration of 10 minutes, comprising 5 minutes during feeding. Simultaneously, one-minute videos were recorded from a frontal viewpoint using an Android device (Moto G72 model) (Supplementary file). Various behavioural alterations were observed, encompassing differences in feeding habits, swimming behaviours, aggression levels, and indications of stress. A comparative analysis of the behaviours of fish in the tank with polyethylene dust and fragment tanks was conducted against those of the control group. 2.8. Hydrogen peroxide treatment The entire digestive tracts, including the gills and gastrointestinal tracts (GITs), were processed to extract MPs that had been ingested. To target the accumulation of microplastics in specific organs, the gills and GITs were individually placed in clean glass bottles of 500 L capacity and digested separately. Microplastic extraction followed the method outlined by Jabeen et al. ( 2017 ). 2.9. Microscopic observation After the filtration, filter paper was observed under the microscope and images of the microplastics were taken with in camera. The microplastics were assessment by visually (Hidalgo-Ruz et al., 2012). 2.10. Biochemical analysis 2.10.1. Oxidative stress enzyme analysis After the 21 days exposure the fish were removed from the tanks. They were euthanized using progressive chilling to 2°- 4°C followed by decapitation. The fishes were dissected; gills, gut and tissue were removed for enzyme analysis. The weights of the gills and guts were determined using a digital weighing machine and then placed in labelled petri plates, which were already situated on an ice bag. To prevent any damage, the petri plates were immediately stored in a -4ºC deep freezer. 2.10.2. Preparation Homogenate phosphate Buffer: A phosphate buffer, was prepared to create a homogenized sample for the cell lysis and protein estimation. Combine 0.780 g of Sodium di basic, 0.709 g of Sodium mono basic, a small amount of DTT (Dithiothriotol), 0.6 g of EDTA, and 0.876 g of NaCl. Stir these reagents into distilled water until the total volume reaches 100 ml. Following this, incorporate 10 µl of PMSF and 5 µl of Triton into the solution. The weight of the gut and gills was determined using an electronic weighing machine. For every 100 mg of gut and gill tissue, 1 ml of buffer solution was added to 1.5 ml centrifugal test tubes using a 1000 µl pipette. The tubes were kept on ice bags to maintain a low temperature and prevent any damage. The samples were then homogenized using a disposable plastic pestle. After homogenization, the samples were labelled and stored in a single door Visi Cooler. The samples were then centrifuged at 5000 rpm for 15 minutes. The activity levels of three types of oxidative stress enzymes were evaluated in the tilapia fish: (i) SOD, (ii) GPX, (iii) MDA. 2.10.2.(i). Analysis of Superoxide Dismutase (SOD) The activity of SOD in fish tissues was measured previously described by Markland (Marklund et al., 1974 ). Ten 5 ml cryovial tubes were prepared and labelled with stickers indicating their contents of gut and gills samples of control, PVC and Polyethylene exposed fishes. A 0.1 ml (100 µl) sample was pipetted into each cryovial tube. To this, 250 µl of absolute alcohol, 0.15 ml of Chloroform, and 1 ml of H2O2 were added. The solution was then centrifuged at 2500 rpm for 15 minutes. The supernatant was collected and mixed with 2 ml of EDTA buffer and 0.5 ml of pyrogallol. The optical density (OD) value was subsequently measured at a wavelength of 420 nm using a UV-1900 UV-Vis Spectrophotometer, at various time intervals. 2.10.2.(ii). Estimation of Glutathione peroxidase (GPX) Glutathione peroxidase activity was assessed following the protocol by (Rotruck et al., 1973 ). Fish tissues were homogenized in phosphate buffer and then centrifuged at 2,500 rpm for 5 minutes. A 0.2 ml aliquot of the supernatant was transferred to a clean test tube, followed by the addition of an enzyme mixture consisting of 0.2 ml phosphate buffer, 0.2 ml of 0.4nM EDTA, and 0.1 ml sodium azide. The reaction mixture was thoroughly mixed and then incubated for two minutes at 37°C. Following this initial incubation, 0.2 ml of reduced glutathione and 0.1 ml of H2O2 were added to the mixture, which was then further incubated at 37°C for exactly 10 minutes. The reaction was terminated by adding 0.5 ml of 10% TCA, and the resulting color was measured at 412 nm. A standard graph was constructed using reduced glutathione. The results are reported as moles of GSH utilized per minute per milligram of protein. 2.10.2.(iii). Malondialdehyde (MDA). The degradation of lipid hydroperoxides leads to the production of bioactive aldehydes. MDA is one of the most critical bioactive aldehydes. Butyl hydroxytoluene (BHT) and EDTA are introduced to the reaction mixture and sample to mitigate the oxidation of the lipids that are artificially introduced during the TBA reaction. The samples are derived from fish tissue organs. In order to mitigate the dissociation of lipid hydroperoxides, the reaction mixture's temperature is decreased. The reaction pH is optimized to facilitate the hydrolysis of MDA, as it is frequently bound to protein as a Schiff-base compound. The MDA test, which we employed as an oxidative stress marker in fish that were exposed to nano-sized particles for these purposes, is described separately, as well as all the steps that led to the measurement. The freshly prepared homogenate of each tissue sample was used to estimate MDA in accordance with the method of (Buege and Aust, 1978 ). 2. 10.3. Estimation of Protein from gut and gill Protein was estimated by the Bradford ( 1976 ) adapted for use in a 96-well microplate with slight modifications. 2.10.4. Estimation of Protein, Lipid and Carbohydrate from fish tissues After removing the gut and gill fish muscles were cut by a sizer (cleaned with ethanol) and kept in three clean petri-plates. Control fish muscles, PVC tank fish muscles and polyethylene exposed fish muscles were kept in separate petri-plate. Labelled the petri-plates with the stickers and kept in hot air-oven for 35ºC for drying. After 5 days the dried fishes were grinded to make a fine powder with the help of a manual grinder pot and stored. Nutritional values ware estimated from this fish powder samples. The protein concentration of fish tissue was estimated by following (Bradford, 1976 ) and Carbohydrates by following colorimetric method of Yemm and Willis ( 1954 ). The total lipids were estimated using the method of Folch et al. ( 1957 ). All the samples were taken in triplicates and the concentrations were given as percentage of weight of the tissue. Estimation of carbohydrate \(\::\frac{\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{c}.\:\mathbf{o}\mathbf{f}\:\mathbf{s}\mathbf{t}\mathbf{a}\mathbf{n}\mathbf{d}\mathbf{a}\mathbf{r}\mathbf{d}\:\mathbf{x}\:\mathbf{O}\mathbf{D}\:\mathbf{o}\mathbf{f}\:\mathbf{t}\mathbf{h}\mathbf{e}\:\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\:}{\varvec{O}\varvec{D}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{s}\varvec{t}\varvec{a}\varvec{n}\varvec{d}\varvec{a}\varvec{r}\varvec{d}}\) Estimation of protein \(\:(\text{m}\text{g}/\text{g}\text{m})\::\frac{\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{c}.\:\mathbf{o}\mathbf{f}\:\mathbf{s}\mathbf{t}\mathbf{a}\mathbf{n}\mathbf{d}\mathbf{a}\mathbf{r}\mathbf{d}\:\mathbf{x}\:\mathbf{O}\mathbf{D}\:\mathbf{o}\mathbf{f}\:\mathbf{t}\mathbf{h}\mathbf{e}\:\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\:}{\varvec{O}\varvec{D}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{s}\varvec{t}\varvec{a}\varvec{n}\varvec{d}\varvec{a}\varvec{r}\varvec{d}}\) Estimation of lipid \(\::\frac{\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{c}.\:\mathbf{o}\mathbf{f}\:\mathbf{s}\mathbf{t}\mathbf{a}\mathbf{n}\mathbf{d}\mathbf{a}\mathbf{r}\mathbf{d}\:\mathbf{x}\:\mathbf{O}\mathbf{D}\:\mathbf{o}\mathbf{f}\:\mathbf{t}\mathbf{h}\mathbf{e}\:\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\:}{\varvec{O}\varvec{D}\:\varvec{o}\varvec{f}\:\varvec{t}\varvec{h}\varvec{e}\:\varvec{s}\varvec{t}\varvec{a}\varvec{n}\varvec{d}\varvec{a}\varvec{r}\varvec{d}}\) 2.10.5. Animal ethical approval We have obtained institutional ethical clearance to conduct a fish exposure experiment in the Department of Marine Science. The ethical clearance number is BDU/IAEC/P19/2024, dated March 23, 2024. 2.11. Data analysis Data analysis was conducted with Origin 16 and GraphPad 9.0. The length and weight differences were analyzed using one-way analysis of variance, followed by Tukey's HSD test. Additionally, we conducted ANOVA comparisons within each organ, accounting for varying exposure and control groups. The statistically significant difference was observed at p < 0.05 and p < 0.01. 3. RESULTS 3.1. Behaviour, ingestion and accumulation of MPs After 21-days of exposure, fish exhibited diverse alterations in their behaviour. Fish exposed to microplastics were contrasted with those in the control tank. Differences were observed in feeding patterns, aggression levels, pigmentation, mortality rates, and fin conditions. 3.1.1. Feeding behaviour The fish exhibited varying feeding behaviours, leading to their categorization into active and inactive feeders based on their response to food. In the control tank, fish promptly consumed their feed upon presentation. However, in the PVC-exposed group, fish showed decreased activity in feeding after the fifth or sixth day. While larger fish remained active, smaller ones took approximately 5.0 ± 2.0 seconds to start feeding. In the PE-exposed group, fish were moderately active initially, with all individuals actively consuming feed for the first ten days. However, starting from the eleventh day, smaller fish began to show slower feeding responses (after 10.0 ± 2.0 seconds) and decreased activity overall. 3.1.3. Aggressive nature The aggression levels of freshwater fishes during the exposure to microplastics such PVC fragments and polyethylene were examined. The group exposed to polyethylene exhibited increased aggression after feeding, whereas no signs of aggressiveness were observed in the control groups. 5.1.4. Swelling and fin rot Swelling of the body was observed in fish from the PVC-exposed group on the 5th day, and similarly, swelling occurred in the fish from the PE-exposed group on the 8th day. No swelling was observed in the control group fish. Additionally, fish from PE exposed group exhibited torn anal fins and tails due to aggressive behaviour (Fig.S3). 3.1.6. Growth rate Initially, we recorded the length and weight of the fish before introducing them to the experimental tank (Fig. S4). After 21 days of exposure, we again measured their final length and weight before dissecting them. The results revealed an increase in length for each fish in the control group. However, the increase in length was comparatively lower for the experimental fish in the PVC group (0.63 ± 0.160 cm) and the PE group (0.28 ± 0.044 cm) compared to the control tank fish (1.16 ± 0.141cm). Similarly, the weight of the experimental fish exposed to microplastics (PVC: 0.48 ± 0.15 g, PE: 0.22 ± 0.16 g) showed a slight change compared to the control tank fish (0.92 ± 0.84 g). These findings suggest that the ingestion of plastics has an impact on the growth of tilapia fish. 3.1.7. Accumulation of MPs Observations revealed the accumulation of PVC fragments and PE dust in the gut and gills of tilapia, as illustrated in Table 2 and Fig. S5. No microplastics were detected in the control groups. In the PVC exposure group, each individual ingested an average of 4.8 ± 2.7 MPs/ individuals in gastrointestinal (GIT) tract and 6.6 ± 2.07 MPs/individuals in gills. While in the PE group, the intake was 5.6 ± 2.6 MPs/individual in GIT tract 5.8 ± 0.84 MPs/individuals in gills. This data indicates that fish from the PVC group consumed a higher quantity of microplastics compared to those in the PE group. The fish exposed to PVC-MPs amended food groups showed significantly higher than the PE exposed groups (p = 0.00). Table 2 Ingestion and accumulation of microplastic particles in fish gills and gastrointestinal tract (GIT) Exposure Total no of fish MPs in GIT (No/ind.) MPs in gill (No/ind.) PVC fragment 10 4.8 ± 2.7 6.6 ± 2.07 PE fragments 10 5.6 ± 2.6 5.8 ± 0.84 3.2. Biochemical analysis of fish organs 3.2.1. Oxidative stress enzyme analysis- Superoxide Dismutase (SOD) Oxidative stress occurs due to an imbalance between the production of reactive oxygen species and the antioxidant defense system. The current findings indicate that the superoxide dismutase (SOD) levels in the gills were highest in the control group, followed by the PVC exposure group, and lowest in the PE exposure group, as depicted in the Fig. 1 . Similarly, the concentration of SOD values in the gastrointestinal tract (GIT) is illustrated in the Fig. 1 . The PVC exposure group exhibited the highest SOD values, followed by the PE exposure group, while the control group showed the lowest SOD values. 3.2.2. Glutathione peroxidase (GPX) in fish organs GPXs are vital in combating oxidative stress by acting as antioxidants. According to the findings, the levels of GPX in the gills and GIT are depicted in Fig. 2 . The PVC-exposed group exhibited the highest GPX values, and followed by the PE-exposed group. The highest GPX OD values of GIT tract showed 2.90 nmol of NADPH oxidized in PE exposed group and 2.55 nmol of NADPH oxidized in PVC exposed fish. Similarly, in the Gills, the highest GPX values were observed in the PVC-exposed group (1.28nmol of NADPH oxidized), followed by the PE-exposed group (1.23 nmol of NADPH oxidized). In both the gills and GIT, the control group displayed lower concentrations of GPX compared to the PVC and PE groups. 3.2.3. Malondialdehyde (MDA) The breakdown of lipid hydroperoxides leads to the generation of bioactive aldehydes, among which MDA holds significant importance. MDA has served as a reliable biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids for many years. Lipid peroxidation serves as an indicator of oxidative stress, which arises from imbalances in the oxidant and antioxidant equilibrium, often resulting in heightened levels of reactive oxygen species and subsequent oxidative damage. The current findings indicate that the PVC exposure groups exhibited higher levels of MDA in both the gastrointestinal tract (GIT) and gills compared to the control group (Fig. 3 ). 3.2.4. Protein estimation from gill, gut and tissue The protein estimations on samples taken from the gills, gut, and tissue of tilapia fish were showed in (Fig. 4 ). The results revealed higher protein concentrations in the control group fish compared to those in the PVC and PE exposure groups (648.34 µg/ml, 573.55 µg/ml, and 607.01 µg/ml respectively). This suggests that the ingestion of microplastics may lead to a decrease in protein concentration in the gut and gills. Additionally, protein estimation was performed on dried tissue samples, showing the highest protein concentration in fish tissue from the PE dust tank (317.013 µg/ml) and the lowest in fish from the PVC tank (208.21 µg/ml) (Fig. 4 ) 3.3. Calculation of protein, lipid and carbohydrates from fish tissue The proximate composition of fish tissue exposed to microplastics is illustrated in Fig. 5 . The highest concentration of protein was observed in the PE-exposed group (317.01 µg/g), followed by the control group (233.41 µg/g). Conversely, lipid content was highest in the control group compared to the exposure groups. Similarly, carbohydrate levels were higher in the PVC exposure group (1.56 µg/g) than in the PE exposure (1.21 µg/g) and control groups (0.69 µg/g). 4. DISCUSSION The findings of this experiment indicate that microplastics have deleterious effects on Oreochromis niloticus fish. These effects include significant impacts on growth, behavior, mortality rate, nutritional value, and concentrations of oxidative stress enzymes. The experiment revealed distinct behavioral differences among fish in the control group, those in tanks with PVC fragments, and those exposed to PE dust. Fish in the control group exhibited no signs of stress, while those consuming microplastics as part of their diet showed elevated stress levels, leading to altered behavior such as increased fighting tendencies, biting, and chasing, particularly among those exposed to PE dust. Mtega et al. ( 2023 ) documented similar behavioral changes in tilapia fish following microplastic exposure, including loss of body equilibrium, abnormal swimming patterns, and pigmentation after 21 days of experimentation. Additionally, Rios-Fuster et al. ( 2021 ) found that seabream fish in the control group displayed significantly more chasing and avoidance behaviors compared to those exposed to microplastics. Similarly, Kim et al. ( 2022 ) observed abnormal swimming patterns, such as wall-hugging and core-circling, in zebrafish from tanks with microplastic exposure. Tilapia fish are known for their robustness and adaptability to various environments, often enjoying a long lifespan. During the acclimatization process, not a single tilapia died, indicating their resilience to stressors. However, in the current study, during the initial four days of the 15-day experiment, all fish remained alive and appeared unstressed. On the fifth day, the first fish succumbed, marking the beginning of mortality among the experimental group. The 15-day exposure period revealed a higher mortality rate among fish that consumed microplastics. Interestingly, contrary to these findings, Naidoo and Glassom ( 2019 ) reported a decrease in mortality rates among small juvenile fish following exposure to microplastics. Jabeen et al. ( 2017 ) found no reported mortalities during microplastic exposure in goldfish. The exposure of microplastics not only effected on the behaviour of fish but al it affected the growth rate of the fishes. This experiment is showing that control fishes has grown after 15 days of exposure experiment and compared to control, fishes those intake microplastics (PVC, PE) showed not a noticeable growth rate. The length and weight were likely same as before the experiment. In some studies researcher described about the effect of microplastics on growth. According to Wang et al. ( 2022 ) the juvenile orange-spotted groupers decreased their growth significantly with increasing concentrations of PS-NP exposure (0.158%±0.032%, 0.095%±0.020%, and 0.074%±0.016% for 0, 300, and 3000µg/L PS-NP groups, respectively). All the studies, along with present experimental findings, highlight the significant impact of microplastic exposure on fish (Kumar et al., 2024 ; Ashokkumar et al., 2025 ). Each tank was supplied with 10 feed balls, each containing three microplastics (PVC and PE) with an average diameter of 2000µm. Following a 15-day exposure period, significant changes were observed in the fish from both the PVC and PE dust tanks due to the accumulation of plastic materials. Table-3 illustrates that on average, each fish consumed 16.9 ± 15.5 PVC microplastics from the PVC tank and 11.8 ± 15.5 PE microplastics individually. Table 2 depict the microplastics ingested by the fish, reflecting the consumption limited to the juvenile stage. Oxidative stress is assessed by examining the equilibrium between the activity of antioxidant enzymes and the level of lipid peroxidation. In this study, we conducted tests on superoxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) to understand the impact of microplastics on oxidative stress enzymes. SOD, short for superoxide dismutase, is a crucial enzyme involved in antioxidant defense mechanisms found in nearly all living cells. Its primary role is to facilitate the dismutation of the superoxide radical (O2-) into oxygen and hydrogen peroxide. This reaction is vital for safeguarding cells against the harmful effects of reactive oxygen species (ROS), byproducts of normal cellular metabolism that can inflict damage on DNA, proteins, and lipids if left unchecked. SOD plays a pivotal role in maintaining cellular health and preventing diseases associated with oxidative stress. Our experimental findings indicate that SOD levels are elevated in the gut of fish exposed to PVC, while they are lower in the gut of fish from the control group. However, the gills of fish from the control tank exhibit higher SOD levels. Dong et al. ( 2022 ) observed increased SOD activity in the liver of tilapia but reduced activity in the liver of zebrafish after seven days of microplastic exposure. Similarly, Huang et al. ( 2020 ) found higher SOD levels in the liver of zebrafish in the control group in their experiment. Glutathione peroxidase (GPX) is another essential enzyme found within fish organisms, playing a critical role in defending cells against oxidative harm. It belongs to a group of enzymes that aid in reducing organic hydroperoxides and hydrogen peroxide by utilizing glutathione. This mechanism prevents the formation of detrimental free radicals and lipid peroxides, thereby shielding cells and tissues from oxidative stress. In the GPX experiment, upon analysing the results, it becomes evident that GPX levels are higher in the gills of fish from PVC fragment 2 tank, as depicted in Graph b. Additionally, the present results illustrate that GPX is notably abundant in the gut of fish from the PE exposed group. Li et al. ( 2021 ) observed heightened GPX enzyme activity in the liver of juvenile yellow croaker exposed to PS-MPs. Conversely, Xu et al. ( 2021 ) noted a decrease in GPX activity in the liver of common carp larvae after exposure to PVC-MPs. Umamaheswari et al. ( 2021 ) reported a significant rise in GPX levels in zebrafish exposed to microplastic (1000 mg/kg). Malondialdehyde (MDA), is a reactive compound formed as a result of lipid peroxidation when lipids such as membrane phospholipids or cholesterol undergo oxidation by reactive oxygen species (ROS). This process involves a series of chemical reactions leading to the production of MDA. Serving as an indicator of oxidative stress, MDA is commonly measured in biological samples to assess the degree of lipid peroxidation. Changes in the activity of antioxidant enzymes and MDA levels signify lipid peroxidation in fish. In this study, the findings indicated increased levels of oxidative stress enzymes in the PVC fragment exposed group and minimal amounts in the control fish. Similarly, Xue et al. ( 2021 ) observed zebrafish exposed to microplastics for five days and found no significant alterations in MDA levels in the intestine, although there was a slight decrease in the gill of the fish. Ding et al. ( 2018 ) reported that the MDA content remained stable following microplastic exposure in O. niloticus. Tilapia, recognized for its nutritional richness and delectable taste, has become increasingly scarce due to widespread plastic pollution. The ingestion of microplastics by fish, whether knowingly or inadvertently, has raised concerns about its potential impact on their nutritional content. Hence, assessing the dietary value, including protein, lipid, and carbohydrate levels, in fish tissue, gut, and gill is imperative. Tilapia is renowned for its protein content and various nutrients, although its carbohydrate content is minimal, typically less than 1 gram. Experimental findings indicate that the liver of fish in the control tank exhibits higher protein concentrations (648.34µg/ml) compared to those in the PVC and PE tanks, suggesting a decline in protein concentration with exposure to microplastics. Conversely, protein concentrations in fish tissue from the PE dust tank are notably elevated. Carbohydrate concentrations are higher in the PVC fragment tank, while total lipid concentrations are greater in the control tissue. 5. CONCLUSION The widespread distribution of microplastics in freshwater environments and their subsequent consumption by aquatic creatures, notably Tilapia fish, has become a central focus of environmental investigations. The adverse impacts of microplastics on Tilapia, a commercially valuable freshwater species, are increasingly evident. Microplastics, being small and buoyant, are easily ingested by Tilapia, often mistaken for prey. Upon ingestion, they can cause physical harm, such as internal injuries and digestive obstructions. Furthermore, the contaminants commonly found alongside microplastics, like heavy metals and persistent organic pollutants, can accumulate in Tilapia, presenting potential health hazards to both predators and humans who consume these fish. The repercussions of microplastics extend beyond physical harm and toxin accumulation. Research indicates that exposure to microplastics can lead to altered growth patterns, shifts in feeding habits, and even reduced fertility in Tilapia. These alterations can have profound consequences, influencing population dynamics and the overall health of ecosystems. Nevertheless, the complete extent of microplastic effects on Tilapia and other freshwater species remains incompletely understood. Further comprehensive investigations are imperative to uncover the long-term repercussions of microplastic exposure on fish health, reproduction, and survival. Additionally, efforts should concentrate on devising effective methods to mitigate microplastic presence in freshwater ecosystems. In summary, the microplastic predicament represents a critical environmental issue demanding immediate attention. The well-being of freshwater environments, the sustainability of species like Tilapia, and ultimately, human health may hinge on our capacity to effectively tackle this global challenge. Declarations Acknowledgement The authors express their sincere thanks Department of Marine Science, Bharathidasan University, Tiruchirappalli for support, and research facility. Dr. P.K acknowledge to Saveetha Institute of Medical and Technical Sciences (SIMATS) for their support and providing research facilities. Funding declaration: We are thankful to the Dr. S. Kothari Post-Doctoral Cell, University grants Commission (UGC), govt. of India for granting Post-Doctoral Fellowship (No.F.4-2/2006 (BSR)/OT/20-21/0010 (89th) dt.14.09.2021). Ethics and Consent to Participate declarations: Not applicable Consent to Publish declaration: not applicable Competing Interest : No Competing Interest Ethics approval: We have obtained institutional ethical clearance to conduct a fish exposure experiment in the Department of Marine Science. The ethical clearance number is BDU/IAEC/P19/2024, dated March 23, 2024. Authors contribution statement Prabhu Kolandhasamy : writing – review & editing original draft. Data curation, Conceptualization. Saheli Singha : Data curation, writing first draft. Sourav Bhattacharya : supporting to conduct the experiment, data collection and validation. Suguna Anbukkarasu : review and editing the original daft of manuscript ; Sivaraj Sigamani: review and editing the original daft of manuscript; Rajendiran Rajaram : review & editing Methodology, Conceptualization. References Alimba, C.G., Faggio, C., 2019. Microplastics in the marine environment: Current trends in environmental pollution and mechanisms of toxicological profile. Environ Toxicol Pharmacol 68, 61-74. Anandhan, K., Tharini, K., Thangal, S.H., Yogeshwaran, A., Muralisankar, T., 2022. Occurrence of microplastics in the gastrointestinal tracts of edible fishes from South Indian Rivers. Bull Environ Contam Toxicol 109, 1023-1028. Arienzo, M., Ferrara, L., Trifuoggi, M., 2021a. The dual role of microplastics in marine environment: Sink and vectors of pollutants. J Mar Sci Eng 9, 642. Arienzo, M., Ferrara, L., Trifuoggi, M., 2021b. Research progress in transfer, accumulation and effects of microplastics in the oceans. J Mar Sci Eng 9, 433. Ashokkumar, V., Chandramughi, V., Mohanty, K., Gummadi, S.N., 2025. Microplastic pollution: Critical analysis of global hotspots and their impact on health and ecosystems. J Environ Manag 381, 124995. Bhatt, V., Badola, N., Chauhan, J.S., 2024. Microplastic in fishes: the first report from a Himalayan River—Alaknanda. Environ Sci Pollut 31, 1637-1643. Bilal, M., Qadir, A., Yaqub, A., Irfan, M., Aslam, M., Ullah, R., 2021. Microplastics Monitoring in Environmental Matrices of an Alpine River originating from Hindu Kush Mountain Range. Bilal, M., Yaqub, A., Hassan, H.U., Akhtar, S., Rafiq, N., Ali Shah, M.I., Hussain, I., Salman Khan, M., Nawaz, A., Manoharadas, S., 2023. Microplastic quantification in aquatic birds: biomonitoring the environmental health of the Panjkora river freshwater ecosystem in Pakistan. Toxics 11, 972. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254. Buege, J.A., Aust, S.D., 1978. [30] Microsomal lipid peroxidation. Methods in enzymology. Elsevier, pp. 302-310. Cai, L., Wu, D., Xia, J., Shi, H., Kim, H., 2019. Influence of physicochemical surface properties on the adhesion of bacteria onto four types of plastics. Sci Total Environ 671, 1101-1107. Chen, X., Xiong, X., Jiang, X., Shi, H., Wu, C., 2019. Sinking of floating plastic debris caused by biofilm development in a freshwater lake. Chemosph 222, 856-864. Devi, S.S., Sreedevi, A.V., Kumar, A.B., 2020. First report of microplastic ingestion by the alien fish Pirapitinga (Piaractus brachypomus) in the Ramsar site Vembanad Lake, south India. Mar Pollut Bull 160, 111637. Ding, J., Zhang, S., Razanajatovo, R.M., Zou, H., Zhu, W., 2018. Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environ Pollut 238, 1-9. Ding, R., Tong, L., Zhang, W., 2021. Microplastics in freshwater environments: sources, fates and toxicity. Water Air Soil Pollut 232, 1-19. Dong, R., Zhou, C., Wang, S., Yan, Y., Jiang, Q., 2022. Probiotics ameliorate polyethylene microplastics-induced liver injury by inhibition of oxidative stress in Nile tilapia (Oreochromis niloticus). Fish Shellfish Imm 130, 261-272. Eknath, A.E., Hulata, G., 2009. Use and exchange of genetic resources of Nile tilapia (Oreochromis niloticus). Rev Aqua 1, 197-213. Folch, J., Lees, M., Stanley, G.S., 1957. A simple method for the isolation and purification of total lipides from animal tissues. JBC 226, 497-509. Hassan, H., Mahboob, S., Masood, Z., Riaz, M., Rizwan, S., Al-Misne, F., Abdel-Aziz, M., Al-Ghanim, K., Gabol, K., Chatta, A., 2023. Biodiversity of commercially important finfish species caught by mid-water and bottom trawls from two different coasts of Arabian Sea: threats and conservation strategies. Braz J Biol 83, e249211. Huang, X., Li, Y., Wang, T., Liu, H., Shi, J., Zhang, X., 2020. Evaluation of the oxidative stress status in zebrafish (Danio rerio) liver induced by three typical organic UV filters (BP-4, PABA and PBSA). Int J Environ Res Public Health 17, 651. Jabeen, K., Su, L., Li, J., Yang, D., Tong, C., Mu, J., Shi, H., 2017. Microplastics and mesoplastics in fish from coastal and fresh waters of China. Environ Pollut 221, 141-149. Jeyavani, J., Sibiya, A., Stalin, T., Vigneshkumar, G., Al-Ghanim, K.A., Riaz, M.N., Govindarajan, M., Vaseeharan, B., 2023. Biochemical, genotoxic and histological implications of polypropylene microplastics on freshwater fish Oreochromis mossambicus: An aquatic eco-toxicological assessment. Toxics 11, 282. Kim, S.A., Kim, L., Kim, T.H., An, Y.-J., 2022. Assessing the size-dependent effects of microplastics on zebrafish larvae through fish lateral line system and gut damage. Mar Pollut Bullet 185, 114279. Kumar, V., Umesh, M., Chakraborty, P., Sharma, P., Sarojini, S., Basheer, T., Kaur, K., Pasrija, R., Barcelo, D., 2024. Origin, ecotoxicity, and analytical methods for microplastic detection in aquatic systems. TrAC 170, 117392. Laist, D.W., 1997. Impacts of marine debris: entanglement of marine life in marine debris including a comprehensive list of species with entanglement and ingestion records. Marine debris: sources, impacts, and solutions. Springer, pp. 99-139. Li, B., Liang, W., Liu, Q.X., Fu, S., Ma, C., Chen, Q., Su, L., Craig, N.J., Shi, H., 2021. Fish Ingest Microplastics Unintentionally. Environ Sci Technol 55, 10471-10479. Li, J., Liu, H., Paul Chen, J., 2018. Microplastics in freshwater systems: A review on occurrence, environmental effects, and methods for microplastics detection. Water Res 137, 362-374. Lu, Y., Zhang, Y., Deng, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H., 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. Environ Sci Tech 50, 4054-4060. MacArthur, E., 2017. The new plastics economy: Rethinking the future of plastics & catalysing action. Ellen MacArthur Foundation: Cowes, UK 68. Marklund, S., Ohlsson, P.-I., Opara, A., Paul, K.-G., 1974. The substrate profiles of the acidic and slightly basic horseradish peroxidases. Biochim Biophys Acta Enzymol 350, 304-313. Mohamed, A.-R.M., Abood, A.N., 2021. Trophic Interactions between Two Cichlid Species in Shatt Al-Arab River, Iraq. Biol App Environ Res 5, 74-85. Mtega, M.P., Mihale, M.J., Kilulya, K.F., 2023. Evaluation of Toxicological Risks and Effects of Microplastics on Nile Tilapia (Oreochromisniloticus) under in Vitro Laboratory Conditions. East Afr J Biophys Comput Sci 4, 54-68. Murphy, J., 2001. Additives for plastics handbook. Elsevier. Naidoo, T., Glassom, D., 2019. Decreased growth and survival in small juvenile fish, after chronic exposure to environmentally relevant concentrations of microplastic. Mar Pollut Bull 145, 254-259. Pandey, N., Verma, R., Patnaik, S., Anbumani, S., 2023. Abundance, characteristics, and risk assessment of microplastics in indigenous freshwater fishes of India. Environ Res 218, 115011. Rai, P.K., Lee, J., Brown, R.J., Kim, K.-H., 2021. Environmental fate, ecotoxicity biomarkers, and potential health effects of micro-and nano-scale plastic contamination. Journal of Hazardous Materials 403, 123910. Rios-Fuster, B., Arechavala-Lopez, P., García-Marcos, K., Alomar, C., Compa, M., Álvarez, E., Julià, M.M., Martí, A.S., Sureda, A., Deudero, S., 2021. Experimental evidence of physiological and behavioral effects of microplastic ingestion in Sparus aurata. Aqua Toxicol 231, 105737. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A., Hafeman, D.G., Hoekstra, W., 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science 179, 588-590. Singh, A., Verma, P., Srivastava, S.C., Tripathi, M., 2014. Invasion, biology and impact of feral population of Nile tilapia (Oreochromis niloticus Linnaeus, 1757) in the Ganga River (India). Asia Pacific J Res 1. Singh, A.K., Lakra, W.S., 2011. Risk and benefit assessment of alien fish species of the aquaculture and aquarium trade into India. Rev Aquac 3, 3-18. Singh, S., Kumar Naik, T.S.S., Anil, A.G., Dhiman, J., Kumar, V., Dhanjal, D.S., Aguilar-Marcelino, L., Singh, J., Ramamurthy, P.C., 2022. Micro (nano) plastics in wastewater: A critical review on toxicity risk assessment, behaviour, environmental impact and challenges. Chemosph 290, 133169. Tesfahun, A., Temesgen, M., 2017. Food and Feeding Habits of the African Big Barb Labeobarbus intermedius (Rüppell, 1836) in Ethiopian Water Bodies: A Review. J Nutr 6, 28-33. Umamaheswari, S., Priyadarshinee, S., Bhattacharjee, M., Kadirvelu, K., Ramesh, M., 2021. Exposure to polystyrene microplastics induced gene modulated biological responses in zebrafish (Danio rerio). Chemosphere 281, 128592. Wagner, M., Lambert, S., 2018. Freshwater microplastics: emerging environmental contaminants? Springer Nature. Wang, Q., Huang, F., Liang, K., Niu, W., Duan, X., Jia, X., Wu, X., Xu, P., Zhou, L., 2022. Polystyrene nanoplastics affect digestive function and growth in juvenile groupers. Sci Total Environ 808, 152098. Xu, X., Zhang, L., Xue, Y., Gao, Y., Wang, L., Peng, M., Jiang, S., Zhang, Q., 2021. Microplastic pollution characteristic in surface water and freshwater fish of Gehu Lake, China. Environ Sci Pollut Res Int 28, 67203-67213. Xue, Y.H., Feng, L.S., Xu, Z.Y., Zhao, F.Y., Wen, X.L., Jin, T., Sun, Z.X., 2021. The time-dependent variations of zebrafish intestine and gill after polyethylene microplastics exposure. Ecotoxicol 30, 1997-2010. Yemm, E., Willis, A., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57, 508. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Fish Physiology and Biochemistry → Version 1 posted Editorial decision: Revision requested 20 Aug, 2025 Reviews received at journal 19 Aug, 2025 Reviews received at journal 29 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers invited by journal 16 Jun, 2025 Submission checks completed at journal 13 Jun, 2025 First submitted to journal 09 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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17:08:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1479171,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6761113/v1/84593e0f-6272-437b-9a23-2f0370d7a37e.pdf"},{"id":85731889,"identity":"416cc52a-61c8-4636-9f77-19cd8234da75","added_by":"auto","created_at":"2025-07-01 07:27:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3355258,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6761113/v1/da0e6082f361d7a365a4f6e6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ingestion and the toxicological effects of virgin polyethylene (PE) and PVC microplastics in commercial freshwater fish, Tilapia (Oreochromis niloticus)","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe ecological health of the oceans is under serious threats due to the massive influx of plastic debris each year. In 2016, approximately 335 million tons of plastic debris were produced globally, with an estimated 10% ending up in marine environments Alimba and Faggio (2019). It is estimated that by 2050, the oceans may be contained approximately 937 million tons of plastic, surpassing the estimated 895 million tons of fish biomass (MacArthur, 2017). Once in the ocean, plastics degrade through physical and chemical processes, forming microplastics (MPs) particles which is less than 5 mm and nanoplastics (NPs), which are smaller than 100 nanometres (Rai et al., 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMarine organisms frequently ingest MPs, which cannot be broken down by digestive enzymes. Microplastic (MP) ingestion can lead to a wide range of harmful effects at various biological levels such as subcellular and cellular disruption, altered gene expression, oxidative stress, compromised antioxidant defences, impaired cell division, disrupted fatty acid metabolism, reduced growth and reproduction, biodiversity loss, and population declines (Arienzo et al., 2021b). Ingestion is likely the most frequently observed effect linked to plastic debris, with over 270 different species reported (Laist, 1997). Moreover, the effects of MPs go beyond individual organisms, posing risks to entire ecosystems. MPs can also act as carriers for environmental pollutants (xenobiotics), facilitating the widespread dispersal of toxic substances through wind and ocean currents, thereby enhancing their environmental and ecological impact (Arienzo et al., 2021a).\u003c/p\u003e\n\u003cp\u003eFreshwater ecosystem plays a pivotal role in the lifecycle of microplastics (MPs), acting simultaneously as sources, pathways, and final sinks for these particles (Wagner and Lambert, 2018). Despite this critical role, research on the toxicological impacts of MPs in freshwater organisms-especially fish-remains limited, with most existing studies concentrating on marine species (Li et al., 2018). Fish, however, are widely recognized as effective biomonitors for evaluating the ecological health of aquatic environments. MPs in water can be simply consumed by fish. Researchers have described the incidence of MPs in fish (Bilal et al., 2021; Bilal et al., 2023). MPs deposit in fish and have a wide range of negative impacts i.e., decreased feeding activity, impeded growth, energy interruption, oxidative stress, and even genotoxicity (Lu et al., 2016; Singh et al., 2022; Hassan et al., 2023)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroplastics (MPs) primarily threaten freshwater ecosystems, either directly or indirectly, due to their tiny and irregular form. Further, MPs have the potential to absorb heavy metals and organic pollutants from the surrounding medium, which can have a compounding effect on ecosystems. The toxicity patterns of microplastics have their own consequences and mechanisms and they are important factors to assessing environmental risks and human health consequences. MPs constitute a dual risk to freshwater ecosystems by causing direct damage as well as serving as transporters of additional contaminants (Ding et al., 2021). Plastic polymers employ thousands of various additives, which are probably found in all kinds of plastics. For instance, PVC contains 73% of these chemicals by volume, while PE and PP account for 10% (Murphy, 2001). In freshwater creatures, microplastics (MPs) cause physical injury, such as damage to cell walls and membranes, altered feeding and movement habits, nutrient absorption obstruction, decreased reproductive potential, and decreased growth or survival rates (Cai et al., 2019; Chen et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe species tilapia \u003cem\u003eOreochromis niloticus\u003c/em\u003e was first brought to India in the latter part of 1987 (Singh and Lakra, 2011). The cultivation of \u003cem\u003eO. niloticus\u003c/em\u003e is expanding quickly, particularly in West Bengal, Orissa, and Andhra Pradesh. The fish is found in many states, particularly those that are near the coast. The most commonly farmed tilapias are hybrids of \u003cem\u003eO. niloticus\u003c/em\u003e and other native African species (Mohamed and Abood, 2021). Tilapia \u003cem\u003eO. niloticus\u003c/em\u003e is among the simplest and most profitable fish to cultivate, largely due to their omnivorous nature (Eknath and Hulata, 2009). It can be sustained entirely on a plant-based diet. This species, along with other fish that consume vegetable matter, present a more ecologically balanced and environmentally sustainable method of supplying a plentiful amount of nutritious and tasty fish for human consumption (Singh et al., 2014).\u0026nbsp;Devi et al. (2020)\u0026nbsp;documented that the alien fish species Pirapitinga, \u003cem\u003ePiaractus brachypomus\u003c/em\u003e, in the Vembanad Lake on India\u0026apos;s southwest coast were consuming MPs.\u0026nbsp;Pandey et al. (2023)\u0026nbsp;indicated that MP pollution poses significant implications for environmental health and the accessibility of clean food for humans.\u0026nbsp;Bhatt et al. (2024)\u0026nbsp;studied MP consumption by five fish species with different feeding habits from River Alaknanda at Srinagar, namely, \u003cem\u003eSchizothorax richardsonii Crossocheilus latius, Cyprinus carpio\u003c/em\u003e and \u003cem\u003eBotia horii\u003c/em\u003e showed fibers (66%) were the predominant (Bhatt et al., (2023). Further,\u0026nbsp;Anandhan et al. (2022)\u0026nbsp;examined microplastics (315 MPs) in the gastrointestinal (GI) tracts of the five fish species from the Kollidam and Vellar rivers of Tamil Nadu, Southern India. The toxicity of polypropylene microplastic to \u003cem\u003eO. mossambicus\u003c/em\u003e is assessed by\u0026nbsp;(Jeyavani et al., 2023). The \u003cem\u003eO. mossambicus\u003c/em\u003e consumed MPs, which caused homeostasis to fluctuate, reactive oxygen species (ROS) levels to rise, antioxidant parameters such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and glutathione peroxidase (GPx) to change, lipid molecules to oxidize more readily, and the neurotransmitter enzyme acetylcholinesterase (AChE) to become denaturated. The aim of the present research is to investigate the toxicological effects and ingestion of polyethylene (PE) and polyvinyl chloride (PVC) microplastics in the economically important freshwater fish, Oreochromis niloticus (Tilapia). The accumulation of microplastic particles in specific organs, such as the gastrointestinal tract and gills, was evaluated by exposing fish to them. In addition, the investigation assesses the physiological, biochemical, and behavioural modifications that may result from microplastic exposure, thereby offering an understanding of the potential health hazards that aquatic organisms might encounter.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Fish exposure experimental setup\u003c/h2\u003e \u003cp\u003eThe rectangular glass tanks of medium size (18 \u0026times; 9 \u0026times; 9 inches) with a capacity of around 24L were purchased from the local aquarium market in Tiruchirappalli, India. Then the glass tanks were carefully washed with 5% potassium permanganate and diluted 5% HCl before tilapia was added. Following washing, 20 L of filtered tap water were filled in every tank. A proper aeration system was fitted to ensure optimal oxygen levels. The temperature was maintained within a consistent range of 28\u0026ordm;C \u0026minus;\u0026thinsp;30\u0026ordm;C, while the pH level was kept between 7.5 and 9, both of which are optimal conditions for tilapia fish (Kaloyianni et al., 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Domestication of freshwater fish\u003c/h2\u003e \u003cp\u003eTilapia is a freshwater fish species, is highly economically value due to its strong environmental resilience and omnivorous feeding habit (Tesfahun and Temesgen, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the present study we selected Tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e), for microplastic exposure experiment. Tilapia fingerlings, were obtained from a local commercial fish seed supplier (Kamtchi Fish Farm, Thanjavur) approximately 21 days old, making them suitable for aquariums. Before commencing the experiment, all fish underwent an acclimatization period in a round plastic bucket, where water from both the bucket and the experimental tank was mixed using a small silicon tube. Following a day of acclimation, the fish were transferred to the experimental tank. Before introducing them into the tanks, we measured their initial length using a 30 cm scale and determined their initial weight using a digital weighing machine. After the 21-day experimental period, we measured their final weight and length to evaluate the effects of the microplastic diet. The water conditions in the tanks were maintained within a pH range of 7.5-9 and a temperature range of 28\u0026deg;C \u0026minus;\u0026thinsp;30\u0026deg;C. To prevent external contamination, the surface of each tank was covered with a mosquito net and secured with black paper clips.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of microplastics\u003c/h2\u003e \u003cp\u003eFor the MPs exposure experiment, the fish feed was prepared by utilizing two varieties of microplastics: PVC and Polyethylene (PE) MPs fragments (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026amp; S2). The small PVC fragments were created by cutting them with a hacksaw blade. The polymer composition of these fragments was determined through FTIR-ATR (Jasco, Model, 6600) analysis to be polyvinyl chloride and Polyethylene (PE). Following this, all fragments underwent a process of soaking in 75% v/v alcohol and subsequent ultrasonication with milli Q water to eliminate any potentially attached substances. The average length of the PVC fragments was determined to be 2\u0026ndash;3 mm and polyethylene fragments were 0.5 to 1 mm size (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Microplastic food preparation\u003c/h2\u003e \u003cp\u003eCommercial fish food, specifically the Growfin pellet fish feed, was ensured to be completely devoid of microplastic (MPs) contamination. This fish food was initially soaked in filtered tap water to soften it. Subsequently, PVC and PE fragments were incorporated into the softened food mixture, which was then air-dried under hot air oven. The preparation of PVC-containing food was based on weight, with an adjustment of 10 mg of PVC fragments per 10 grams of fish feed, distributed across 10 pellets of commercial food pellets. Conversely, each pellet containing PE fragments consisted of 15 to 20 particles. Though slight variations may have occurred, on average, the fish were administered a concentration of 10 pellets per tank (equivalent to 1.6 pellets per gram of wet weight fish, including both food and MPs). This dosage translated to approximately 15\u0026ndash;20 PVC particles and PE particles being fed to each fish. In the control group, fish were fed pellets devoid of any plastic fragments (Fig. S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Feeding strategy and observation\u003c/h2\u003e \u003cp\u003eBefore the experiment commenced, the fish underwent a 48-hour fasting period. The experiment utilised in total of nine tanks: 3 serving as control groups and 3 each for the PVC and Polyethylene exposure groups. Each tank housed six individual fish, and three replicates were conducted simultaneously. Observations were made twice in a day for each group. In the control tanks, fish were provided with standard commercially available fish feed without MPs particles, whereas group 1 fish were fed with PVC-based pellets, and group 2 fish were fed with polyethylene-based pellets. The fish were fed twice daily, once in the morning at 10:30 am and once in the evening 5:00 pm. Through the initial observation, the feeding behaviour of fish was examined within the initial five minutes, with ingestion or rejection of food being recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Processing of sample\u003c/h2\u003e \u003cp\u003eAfter 21 (3 weeks) days exposure, all fish were anesthetized with MS-22 (100 mg/L) and sacrificed for immediate analysis. Total length (cm) and wet weight (g) and condition factor (CF\u0026thinsp;=\u0026thinsp;w/L\u003csup\u003e3\u003c/sup\u003e \u0026times;100, where CF- condition factor, w \u0026ndash; weight and L \u0026ndash; standard length) of each fish was recorded (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Each fish was decapitated and the GIT, liver, brain and Gill were removed through a ventral incision. Heads of all fish and the gut and livers of the 12 fishes. The GITs of the fishes were frozen at -20\u0026ordm;C for further analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLength, weight and condition factor of fish exposure experiments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExposure group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo of Fish\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExposure time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage Length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCondition factor\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Observation of Behaviour\u003c/h2\u003e \u003cp\u003eThe fish were monitored daily for a duration of 10 minutes, comprising 5 minutes during feeding. Simultaneously, one-minute videos were recorded from a frontal viewpoint using an Android device (Moto G72 model) (Supplementary file). Various behavioural alterations were observed, encompassing differences in feeding habits, swimming behaviours, aggression levels, and indications of stress. A comparative analysis of the behaviours of fish in the tank with polyethylene dust and fragment tanks was conducted against those of the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Hydrogen peroxide treatment\u003c/h2\u003e \u003cp\u003eThe entire digestive tracts, including the gills and gastrointestinal tracts (GITs), were processed to extract MPs that had been ingested. To target the accumulation of microplastics in specific organs, the gills and GITs were individually placed in clean glass bottles of 500 L capacity and digested separately. Microplastic extraction followed the method outlined by Jabeen et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Microscopic observation\u003c/h2\u003e \u003cp\u003eAfter the filtration, filter paper was observed under the microscope and images of the microplastics were taken with in camera. The microplastics were assessment by visually (Hidalgo-Ruz et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Biochemical analysis\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1. Oxidative stress enzyme analysis\u003c/h2\u003e \u003cp\u003eAfter the 21 days exposure the fish were removed from the tanks. They were euthanized using progressive chilling to 2\u0026deg;- 4\u0026deg;C followed by decapitation. The fishes were dissected; gills, gut and tissue were removed for enzyme analysis. The weights of the gills and guts were determined using a digital weighing machine and then placed in labelled petri plates, which were already situated on an ice bag. To prevent any damage, the petri plates were immediately stored in a -4\u0026ordm;C deep freezer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2. Preparation Homogenate phosphate Buffer:\u003c/h2\u003e \u003cp\u003eA phosphate buffer, was prepared to create a homogenized sample for the cell lysis and protein estimation. Combine 0.780 g of Sodium di basic, 0.709 g of Sodium mono basic, a small amount of DTT (Dithiothriotol), 0.6 g of EDTA, and 0.876 g of NaCl. Stir these reagents into distilled water until the total volume reaches 100 ml. Following this, incorporate 10 \u0026micro;l of PMSF and 5 \u0026micro;l of Triton into the solution. The weight of the gut and gills was determined using an electronic weighing machine. For every 100 mg of gut and gill tissue, 1 ml of buffer solution was added to 1.5 ml centrifugal test tubes using a 1000 \u0026micro;l pipette. The tubes were kept on ice bags to maintain a low temperature and prevent any damage. The samples were then homogenized using a disposable plastic pestle. After homogenization, the samples were labelled and stored in a single door Visi Cooler. The samples were then centrifuged at 5000 rpm for 15 minutes. The activity levels of three types of oxidative stress enzymes were evaluated in the tilapia fish: (i) SOD, (ii) GPX, (iii) MDA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2.(i). Analysis of Superoxide Dismutase (SOD)\u003c/h2\u003e \u003cp\u003eThe activity of SOD in fish tissues was measured previously described by Markland (Marklund et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). Ten 5 ml cryovial tubes were prepared and labelled with stickers indicating their contents of gut and gills samples of control, PVC and Polyethylene exposed fishes. A 0.1 ml (100 \u0026micro;l) sample was pipetted into each cryovial tube. To this, 250 \u0026micro;l of absolute alcohol, 0.15 ml of Chloroform, and 1 ml of H2O2 were added. The solution was then centrifuged at 2500 rpm for 15 minutes. The supernatant was collected and mixed with 2 ml of EDTA buffer and 0.5 ml of pyrogallol. The optical density (OD) value was subsequently measured at a wavelength of 420 nm using a UV-1900 UV-Vis Spectrophotometer, at various time intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2.(ii). Estimation of Glutathione peroxidase (GPX)\u003c/h2\u003e \u003cp\u003eGlutathione peroxidase activity was assessed following the protocol by (Rotruck et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Fish tissues were homogenized in phosphate buffer and then centrifuged at 2,500 rpm for 5 minutes. A 0.2 ml aliquot of the supernatant was transferred to a clean test tube, followed by the addition of an enzyme mixture consisting of 0.2 ml phosphate buffer, 0.2 ml of 0.4nM EDTA, and 0.1 ml sodium azide. The reaction mixture was thoroughly mixed and then incubated for two minutes at 37\u0026deg;C. Following this initial incubation, 0.2 ml of reduced glutathione and 0.1 ml of H2O2 were added to the mixture, which was then further incubated at 37\u0026deg;C for exactly 10 minutes. The reaction was terminated by adding 0.5 ml of 10% TCA, and the resulting color was measured at 412 nm. A standard graph was constructed using reduced glutathione. The results are reported as moles of GSH utilized per minute per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2.(iii). Malondialdehyde (MDA).\u003c/h2\u003e \u003cp\u003eThe degradation of lipid hydroperoxides leads to the production of bioactive aldehydes. MDA is one of the most critical bioactive aldehydes. Butyl hydroxytoluene (BHT) and EDTA are introduced to the reaction mixture and sample to mitigate the oxidation of the lipids that are artificially introduced during the TBA reaction. The samples are derived from fish tissue organs. In order to mitigate the dissociation of lipid hydroperoxides, the reaction mixture's temperature is decreased. The reaction pH is optimized to facilitate the hydrolysis of MDA, as it is frequently bound to protein as a Schiff-base compound. The MDA test, which we employed as an oxidative stress marker in fish that were exposed to nano-sized particles for these purposes, is described separately, as well as all the steps that led to the measurement. The freshly prepared homogenate of each tissue sample was used to estimate MDA in accordance with the method of (Buege and Aust, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1978\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e2. 10.3. Estimation of Protein from gut and gill\u003c/h3\u003e\n\u003cp\u003eProtein was estimated by the Bradford (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1976\u003c/span\u003e) adapted for use in a 96-well microplate with slight modifications.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.10.4. Estimation of Protein, Lipid and Carbohydrate from fish tissues\u003c/h2\u003e \u003cp\u003eAfter removing the gut and gill fish muscles were cut by a sizer (cleaned with ethanol) and kept in three clean petri-plates. Control fish muscles, PVC tank fish muscles and polyethylene exposed fish muscles were kept in separate petri-plate. Labelled the petri-plates with the stickers and kept in hot air-oven for 35\u0026ordm;C for drying. After 5 days the dried fishes were grinded to make a fine powder with the help of a manual grinder pot and stored. Nutritional values ware estimated from this fish powder samples. The protein concentration of fish tissue was estimated by following (Bradford, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1976\u003c/span\u003e) and Carbohydrates by following colorimetric method of Yemm and Willis (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). The total lipids were estimated using the method of Folch et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). All the samples were taken in triplicates and the concentrations were given as percentage of weight of the tissue.\u003c/p\u003e \u003cp\u003eEstimation of carbohydrate \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\::\\frac{\\mathbf{C}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{s}\\mathbf{t}\\mathbf{a}\\mathbf{n}\\mathbf{d}\\mathbf{a}\\mathbf{r}\\mathbf{d}\\:\\mathbf{x}\\:\\mathbf{O}\\mathbf{D}\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{t}\\mathbf{h}\\mathbf{e}\\:\\mathbf{s}\\mathbf{a}\\mathbf{m}\\mathbf{p}\\mathbf{l}\\mathbf{e}\\:}{\\varvec{O}\\varvec{D}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{s}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{d}\\varvec{a}\\varvec{r}\\varvec{d}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eEstimation of protein \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:(\\text{m}\\text{g}/\\text{g}\\text{m})\\::\\frac{\\mathbf{C}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{s}\\mathbf{t}\\mathbf{a}\\mathbf{n}\\mathbf{d}\\mathbf{a}\\mathbf{r}\\mathbf{d}\\:\\mathbf{x}\\:\\mathbf{O}\\mathbf{D}\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{t}\\mathbf{h}\\mathbf{e}\\:\\mathbf{s}\\mathbf{a}\\mathbf{m}\\mathbf{p}\\mathbf{l}\\mathbf{e}\\:}{\\varvec{O}\\varvec{D}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{s}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{d}\\varvec{a}\\varvec{r}\\varvec{d}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eEstimation of lipid \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\::\\frac{\\mathbf{C}\\mathbf{o}\\mathbf{n}\\mathbf{c}.\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{s}\\mathbf{t}\\mathbf{a}\\mathbf{n}\\mathbf{d}\\mathbf{a}\\mathbf{r}\\mathbf{d}\\:\\mathbf{x}\\:\\mathbf{O}\\mathbf{D}\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{t}\\mathbf{h}\\mathbf{e}\\:\\mathbf{s}\\mathbf{a}\\mathbf{m}\\mathbf{p}\\mathbf{l}\\mathbf{e}\\:}{\\varvec{O}\\varvec{D}\\:\\varvec{o}\\varvec{f}\\:\\varvec{t}\\varvec{h}\\varvec{e}\\:\\varvec{s}\\varvec{t}\\varvec{a}\\varvec{n}\\varvec{d}\\varvec{a}\\varvec{r}\\varvec{d}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.10.5. Animal ethical approval\u003c/h2\u003e \u003cp\u003eWe have obtained institutional ethical clearance to conduct a fish exposure experiment in the Department of Marine Science. The ethical clearance number is BDU/IAEC/P19/2024, dated March 23, 2024.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Data analysis\u003c/h2\u003e \u003cp\u003eData analysis was conducted with Origin 16 and GraphPad 9.0. The length and weight differences were analyzed using one-way analysis of variance, followed by Tukey's HSD test. Additionally, we conducted ANOVA comparisons within each organ, accounting for varying exposure and control groups. The statistically significant difference was observed at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Behaviour, ingestion and accumulation of MPs\u003c/h2\u003e\n \u003cp\u003eAfter 21-days of exposure, fish exhibited diverse alterations in their behaviour. Fish exposed to microplastics were contrasted with those in the control tank. Differences were observed in feeding patterns, aggression levels, pigmentation, mortality rates, and fin conditions.\u003c/p\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1. Feeding behaviour\u003c/h2\u003e\n \u003cp\u003eThe fish exhibited varying feeding behaviours, leading to their categorization into active and inactive feeders based on their response to food. In the control tank, fish promptly consumed their feed upon presentation. However, in the PVC-exposed group, fish showed decreased activity in feeding after the fifth or sixth day. While larger fish remained active, smaller ones took approximately 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 seconds to start feeding. In the PE-exposed group, fish were moderately active initially, with all individuals actively consuming feed for the first ten days. However, starting from the eleventh day, smaller fish began to show slower feeding responses (after 10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 seconds) and decreased activity overall.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.3. Aggressive nature\u003c/h2\u003e\n \u003cp\u003eThe aggression levels of freshwater fishes during the exposure to microplastics such PVC fragments and polyethylene were examined. The group exposed to polyethylene exhibited increased aggression after feeding, whereas no signs of aggressiveness were observed in the control groups.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e5.1.4. Swelling and fin rot\u003c/h2\u003e\n \u003cp\u003eSwelling of the body was observed in fish from the PVC-exposed group on the 5th day, and similarly, swelling occurred in the fish from the PE-exposed group on the 8th day. No swelling was observed in the control group fish. Additionally, fish from PE exposed group exhibited torn anal fins and tails due to aggressive behaviour (Fig.S3).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.6. Growth rate\u003c/h2\u003e\n \u003cp\u003eInitially, we recorded the length and weight of the fish before introducing them to the experimental tank (Fig. S4). After 21 days of exposure, we again measured their final length and weight before dissecting them. The results revealed an increase in length for each fish in the control group. However, the increase in length was comparatively lower for the experimental fish in the PVC group (0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.160 cm) and the PE group (0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044 cm) compared to the control tank fish (1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.141cm). Similarly, the weight of the experimental fish exposed to microplastics (PVC: 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 g, PE: 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 g) showed a slight change compared to the control tank fish (0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 g). These findings suggest that the ingestion of plastics has an impact on the growth of tilapia fish.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.7. Accumulation of MPs\u003c/h2\u003e\n \u003cp\u003eObservations revealed the accumulation of PVC fragments and PE dust in the gut and gills of tilapia, as illustrated in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. S5. No microplastics were detected in the control groups. In the PVC exposure group, each individual ingested an average of 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 MPs/ individuals in gastrointestinal (GIT) tract and 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07 MPs/individuals in gills. While in the PE group, the intake was 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 MPs/individual in GIT tract 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 MPs/individuals in gills. This data indicates that fish from the PVC group consumed a higher quantity of microplastics compared to those in the PE group. The fish exposed to PVC-MPs amended food groups showed significantly higher than the PE exposed groups (p\u0026thinsp;=\u0026thinsp;0.00).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIngestion and accumulation of microplastic particles in fish gills and gastrointestinal tract (GIT)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExposure\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal no of fish\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMPs in GIT\u003c/p\u003e\n \u003cp\u003e(No/ind.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMPs in gill\u003c/p\u003e\n \u003cp\u003e(No/ind.)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePVC fragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE fragments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Biochemical analysis of fish organs\u003c/h2\u003e\n \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1. \u003cem\u003eOxidative stress enzyme analysis- Superoxide Dismutase (SOD)\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eOxidative stress occurs due to an imbalance between the production of reactive oxygen species and the antioxidant defense system. The current findings indicate that the superoxide dismutase (SOD) levels in the gills were highest in the control group, followed by the PVC exposure group, and lowest in the PE exposure group, as depicted in the Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Similarly, the concentration of SOD values in the gastrointestinal tract (GIT) is illustrated in the Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The PVC exposure group exhibited the highest SOD values, followed by the PE exposure group, while the control group showed the lowest SOD values.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. Glutathione peroxidase (GPX) in fish organs\u003c/h2\u003e\n \u003cp\u003eGPXs are vital in combating oxidative stress by acting as antioxidants. According to the findings, the levels of GPX in the gills and GIT are depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The PVC-exposed group exhibited the highest GPX values, and followed by the PE-exposed group. The highest GPX OD values of GIT tract showed 2.90 nmol of NADPH oxidized in PE exposed group and 2.55 nmol of NADPH oxidized in PVC exposed fish. Similarly, in the Gills, the highest GPX values were observed in the PVC-exposed group (1.28nmol of NADPH oxidized), followed by the PE-exposed group (1.23 nmol of NADPH oxidized). In both the gills and GIT, the control group displayed lower concentrations of GPX compared to the PVC and PE groups.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3. Malondialdehyde (MDA)\u003c/h2\u003e\n \u003cp\u003eThe breakdown of lipid hydroperoxides leads to the generation of bioactive aldehydes, among which MDA holds significant importance. MDA has served as a reliable biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids for many years. Lipid peroxidation serves as an indicator of oxidative stress, which arises from imbalances in the oxidant and antioxidant equilibrium, often resulting in heightened levels of reactive oxygen species and subsequent oxidative damage. The current findings indicate that the PVC exposure groups exhibited higher levels of MDA in both the gastrointestinal tract (GIT) and gills compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.4. Protein estimation from gill, gut and tissue\u003c/h2\u003e\n \u003cp\u003eThe protein estimations on samples taken from the gills, gut, and tissue of tilapia fish were showed in (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The results revealed higher protein concentrations in the control group fish compared to those in the PVC and PE exposure groups (648.34 \u0026micro;g/ml, 573.55 \u0026micro;g/ml, and 607.01 \u0026micro;g/ml respectively). This suggests that the ingestion of microplastics may lead to a decrease in protein concentration in the gut and gills. Additionally, protein estimation was performed on dried tissue samples, showing the highest protein concentration in fish tissue from the PE dust tank (317.013 \u0026micro;g/ml) and the lowest in fish from the PVC tank (208.21 \u0026micro;g/ml) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Calculation of protein, lipid and carbohydrates from fish tissue\u003c/h2\u003e\n \u003cp\u003eThe proximate composition of fish tissue exposed to microplastics is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The highest concentration of protein was observed in the PE-exposed group (317.01 \u0026micro;g/g), followed by the control group (233.41 \u0026micro;g/g). Conversely, lipid content was highest in the control group compared to the exposure groups. Similarly, carbohydrate levels were higher in the PVC exposure group (1.56 \u0026micro;g/g) than in the PE exposure (1.21 \u0026micro;g/g) and control groups (0.69 \u0026micro;g/g).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe findings of this experiment indicate that microplastics have deleterious effects on Oreochromis niloticus fish. These effects include significant impacts on growth, behavior, mortality rate, nutritional value, and concentrations of oxidative stress enzymes. The experiment revealed distinct behavioral differences among fish in the control group, those in tanks with PVC fragments, and those exposed to PE dust. Fish in the control group exhibited no signs of stress, while those consuming microplastics as part of their diet showed elevated stress levels, leading to altered behavior such as increased fighting tendencies, biting, and chasing, particularly among those exposed to PE dust. Mtega et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) documented similar behavioral changes in tilapia fish following microplastic exposure, including loss of body equilibrium, abnormal swimming patterns, and pigmentation after 21 days of experimentation. Additionally, Rios-Fuster et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that seabream fish in the control group displayed significantly more chasing and avoidance behaviors compared to those exposed to microplastics. Similarly, Kim et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed abnormal swimming patterns, such as wall-hugging and core-circling, in zebrafish from tanks with microplastic exposure.\u003c/p\u003e \u003cp\u003eTilapia fish are known for their robustness and adaptability to various environments, often enjoying a long lifespan. During the acclimatization process, not a single tilapia died, indicating their resilience to stressors. However, in the current study, during the initial four days of the 15-day experiment, all fish remained alive and appeared unstressed. On the fifth day, the first fish succumbed, marking the beginning of mortality among the experimental group. The 15-day exposure period revealed a higher mortality rate among fish that consumed microplastics. Interestingly, contrary to these findings, Naidoo and Glassom (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported a decrease in mortality rates among small juvenile fish following exposure to microplastics. Jabeen et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found no reported mortalities during microplastic exposure in goldfish.\u003c/p\u003e \u003cp\u003eThe exposure of microplastics not only effected on the behaviour of fish but al it affected the growth rate of the fishes. This experiment is showing that control fishes has grown after 15 days of exposure experiment and compared to control, fishes those intake microplastics (PVC, PE) showed not a noticeable growth rate. The length and weight were likely same as before the experiment. In some studies researcher described about the effect of microplastics on growth. According to Wang et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) the juvenile orange-spotted groupers decreased their growth significantly with increasing concentrations of PS-NP exposure (0.158%\u0026plusmn;0.032%, 0.095%\u0026plusmn;0.020%, and 0.074%\u0026plusmn;0.016% for 0, 300, and 3000\u0026micro;g/L PS-NP groups, respectively). All the studies, along with present experimental findings, highlight the significant impact of microplastic exposure on fish (Kumar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ashokkumar et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Each tank was supplied with 10 feed balls, each containing three microplastics (PVC and PE) with an average diameter of 2000\u0026micro;m. Following a 15-day exposure period, significant changes were observed in the fish from both the PVC and PE dust tanks due to the accumulation of plastic materials. Table-3 illustrates that on average, each fish consumed 16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5 PVC microplastics from the PVC tank and 11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5 PE microplastics individually. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depict the microplastics ingested by the fish, reflecting the consumption limited to the juvenile stage.\u003c/p\u003e \u003cp\u003eOxidative stress is assessed by examining the equilibrium between the activity of antioxidant enzymes and the level of lipid peroxidation. In this study, we conducted tests on superoxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) to understand the impact of microplastics on oxidative stress enzymes. SOD, short for superoxide dismutase, is a crucial enzyme involved in antioxidant defense mechanisms found in nearly all living cells. Its primary role is to facilitate the dismutation of the superoxide radical (O2-) into oxygen and hydrogen peroxide. This reaction is vital for safeguarding cells against the harmful effects of reactive oxygen species (ROS), byproducts of normal cellular metabolism that can inflict damage on DNA, proteins, and lipids if left unchecked. SOD plays a pivotal role in maintaining cellular health and preventing diseases associated with oxidative stress.\u003c/p\u003e \u003cp\u003eOur experimental findings indicate that SOD levels are elevated in the gut of fish exposed to PVC, while they are lower in the gut of fish from the control group. However, the gills of fish from the control tank exhibit higher SOD levels. Dong et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed increased SOD activity in the liver of tilapia but reduced activity in the liver of zebrafish after seven days of microplastic exposure. Similarly, Huang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found higher SOD levels in the liver of zebrafish in the control group in their experiment.\u003c/p\u003e \u003cp\u003eGlutathione peroxidase (GPX) is another essential enzyme found within fish organisms, playing a critical role in defending cells against oxidative harm. It belongs to a group of enzymes that aid in reducing organic hydroperoxides and hydrogen peroxide by utilizing glutathione. This mechanism prevents the formation of detrimental free radicals and lipid peroxides, thereby shielding cells and tissues from oxidative stress. In the GPX experiment, upon analysing the results, it becomes evident that GPX levels are higher in the gills of fish from PVC fragment 2 tank, as depicted in Graph b. Additionally, the present results illustrate that GPX is notably abundant in the gut of fish from the PE exposed group. Li et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed heightened GPX enzyme activity in the liver of juvenile yellow croaker exposed to PS-MPs. Conversely, Xu et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) noted a decrease in GPX activity in the liver of common carp larvae after exposure to PVC-MPs. Umamaheswari et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported a significant rise in GPX levels in zebrafish exposed to microplastic (1000 mg/kg).\u003c/p\u003e \u003cp\u003eMalondialdehyde (MDA), is a reactive compound formed as a result of lipid peroxidation when lipids such as membrane phospholipids or cholesterol undergo oxidation by reactive oxygen species (ROS). This process involves a series of chemical reactions leading to the production of MDA. Serving as an indicator of oxidative stress, MDA is commonly measured in biological samples to assess the degree of lipid peroxidation. Changes in the activity of antioxidant enzymes and MDA levels signify lipid peroxidation in fish. In this study, the findings indicated increased levels of oxidative stress enzymes in the PVC fragment exposed group and minimal amounts in the control fish. Similarly, Xue et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed zebrafish exposed to microplastics for five days and found no significant alterations in MDA levels in the intestine, although there was a slight decrease in the gill of the fish. Ding et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that the MDA content remained stable following microplastic exposure in \u003cem\u003eO. niloticus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTilapia, recognized for its nutritional richness and delectable taste, has become increasingly scarce due to widespread plastic pollution. The ingestion of microplastics by fish, whether knowingly or inadvertently, has raised concerns about its potential impact on their nutritional content. Hence, assessing the dietary value, including protein, lipid, and carbohydrate levels, in fish tissue, gut, and gill is imperative. Tilapia is renowned for its protein content and various nutrients, although its carbohydrate content is minimal, typically less than 1 gram. Experimental findings indicate that the liver of fish in the control tank exhibits higher protein concentrations (648.34\u0026micro;g/ml) compared to those in the PVC and PE tanks, suggesting a decline in protein concentration with exposure to microplastics. Conversely, protein concentrations in fish tissue from the PE dust tank are notably elevated. Carbohydrate concentrations are higher in the PVC fragment tank, while total lipid concentrations are greater in the control tissue.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe widespread distribution of microplastics in freshwater environments and their subsequent consumption by aquatic creatures, notably Tilapia fish, has become a central focus of environmental investigations. The adverse impacts of microplastics on Tilapia, a commercially valuable freshwater species, are increasingly evident. Microplastics, being small and buoyant, are easily ingested by Tilapia, often mistaken for prey. Upon ingestion, they can cause physical harm, such as internal injuries and digestive obstructions. Furthermore, the contaminants commonly found alongside microplastics, like heavy metals and persistent organic pollutants, can accumulate in Tilapia, presenting potential health hazards to both predators and humans who consume these fish. The repercussions of microplastics extend beyond physical harm and toxin accumulation. Research indicates that exposure to microplastics can lead to altered growth patterns, shifts in feeding habits, and even reduced fertility in Tilapia. These alterations can have profound consequences, influencing population dynamics and the overall health of ecosystems. Nevertheless, the complete extent of microplastic effects on Tilapia and other freshwater species remains incompletely understood. Further comprehensive investigations are imperative to uncover the long-term repercussions of microplastic exposure on fish health, reproduction, and survival. Additionally, efforts should concentrate on devising effective methods to mitigate microplastic presence in freshwater ecosystems. In summary, the microplastic predicament represents a critical environmental issue demanding immediate attention. The well-being of freshwater environments, the sustainability of species like Tilapia, and ultimately, human health may hinge on our capacity to effectively tackle this global challenge.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere thanks Department of Marine Science, Bharathidasan University, Tiruchirappalli for support, and research facility. Dr. P.K acknowledge to Saveetha Institute of Medical and Technical Sciences (SIMATS) for their support and providing research facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to the Dr. S. Kothari Post-Doctoral Cell, University grants Commission (UGC), govt. of India for granting Post-Doctoral Fellowship (No.F.4-2/2006 (BSR)/OT/20-21/0010 (89th) dt.14.09.2021). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e: No Competing Interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e We have obtained institutional ethical clearance to conduct a fish exposure experiment in the Department of Marine Science. The ethical clearance number is BDU/IAEC/P19/2024, dated March 23, 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrabhu Kolandhasamy\u003c/strong\u003e: writing \u0026ndash; review \u0026amp; editing original draft. Data curation, Conceptualization.\u0026nbsp;\u003cstrong\u003eSaheli Singha\u003c/strong\u003e: Data curation, writing first draft. \u003cstrong\u003eSourav Bhattacharya\u003c/strong\u003e: supporting to conduct the experiment, data collection and validation.\u0026nbsp;\u003cstrong\u003eSuguna Anbukkarasu\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003ereview and editing the original daft of manuscript\u003cstrong\u003e; Sivaraj Sigamani:\u003c/strong\u003e review and editing the original daft of manuscript;\u003cstrong\u003e\u0026nbsp;Rajendiran Rajaram\u003c/strong\u003e: \u0026nbsp;review \u0026amp; editing Methodology, Conceptualization.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlimba, C.G., Faggio, C., 2019. Microplastics in the marine environment: Current trends in environmental pollution and mechanisms of toxicological profile. Environ Toxicol Pharmacol 68, 61-74.\u003c/li\u003e\n \u003cli\u003eAnandhan, K., Tharini, K., Thangal, S.H., Yogeshwaran, A., Muralisankar, T., 2022. Occurrence of microplastics in the gastrointestinal tracts of edible fishes from South Indian Rivers. Bull Environ Contam Toxicol 109, 1023-1028.\u003c/li\u003e\n \u003cli\u003eArienzo, M., Ferrara, L., Trifuoggi, M., 2021a. The dual role of microplastics in marine environment: Sink and vectors of pollutants. J Mar Sci Eng 9, 642.\u003c/li\u003e\n \u003cli\u003eArienzo, M., Ferrara, L., Trifuoggi, M., 2021b. Research progress in transfer, accumulation and effects of microplastics in the oceans. J Mar Sci Eng 9, 433.\u003c/li\u003e\n \u003cli\u003eAshokkumar, V., Chandramughi, V., Mohanty, K., Gummadi, S.N., 2025. Microplastic pollution: Critical analysis of global hotspots and their impact on health and ecosystems. J Environ Manag 381, 124995.\u003c/li\u003e\n \u003cli\u003eBhatt, V., Badola, N., Chauhan, J.S., 2024. Microplastic in fishes: the first report from a Himalayan River\u0026mdash;Alaknanda. Environ Sci Pollut 31, 1637-1643.\u003c/li\u003e\n \u003cli\u003eBilal, M., Qadir, A., Yaqub, A., Irfan, M., Aslam, M., Ullah, R., 2021. Microplastics Monitoring in Environmental Matrices of an Alpine River originating from Hindu Kush Mountain Range.\u003c/li\u003e\n \u003cli\u003eBilal, M., Yaqub, A., Hassan, H.U., Akhtar, S., Rafiq, N., Ali Shah, M.I., Hussain, I., Salman Khan, M., Nawaz, A., Manoharadas, S., 2023. Microplastic quantification in aquatic birds: biomonitoring the environmental health of the Panjkora river freshwater ecosystem in Pakistan. Toxics 11, 972.\u003c/li\u003e\n \u003cli\u003eBradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.\u003c/li\u003e\n \u003cli\u003eBuege, J.A., Aust, S.D., 1978. [30] Microsomal lipid peroxidation. Methods in enzymology. Elsevier, pp. 302-310.\u003c/li\u003e\n \u003cli\u003eCai, L., Wu, D., Xia, J., Shi, H., Kim, H., 2019. Influence of physicochemical surface properties on the adhesion of bacteria onto four types of plastics. Sci Total Environ 671, 1101-1107.\u003c/li\u003e\n \u003cli\u003eChen, X., Xiong, X., Jiang, X., Shi, H., Wu, C., 2019. Sinking of floating plastic debris caused by biofilm development in a freshwater lake. Chemosph 222, 856-864.\u003c/li\u003e\n \u003cli\u003eDevi, S.S., Sreedevi, A.V., Kumar, A.B., 2020. First report of microplastic ingestion by the alien fish Pirapitinga (Piaractus brachypomus) in the Ramsar site Vembanad Lake, south India. Mar Pollut Bull 160, 111637.\u003c/li\u003e\n \u003cli\u003eDing, J., Zhang, S., Razanajatovo, R.M., Zou, H., Zhu, W., 2018. Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environ Pollut 238, 1-9.\u003c/li\u003e\n \u003cli\u003eDing, R., Tong, L., Zhang, W., 2021. Microplastics in freshwater environments: sources, fates and toxicity. Water Air Soil Pollut 232, 1-19.\u003c/li\u003e\n \u003cli\u003eDong, R., Zhou, C., Wang, S., Yan, Y., Jiang, Q., 2022. Probiotics ameliorate polyethylene microplastics-induced liver injury by inhibition of oxidative stress in Nile tilapia (Oreochromis niloticus). Fish Shellfish Imm 130, 261-272.\u003c/li\u003e\n \u003cli\u003eEknath, A.E., Hulata, G., 2009. Use and exchange of genetic resources of Nile tilapia (Oreochromis niloticus). Rev Aqua 1, 197-213.\u003c/li\u003e\n \u003cli\u003eFolch, J., Lees, M., Stanley, G.S., 1957. A simple method for the isolation and purification of total lipides from animal tissues. JBC 226, 497-509.\u003c/li\u003e\n \u003cli\u003eHassan, H., Mahboob, S., Masood, Z., Riaz, M., Rizwan, S., Al-Misne, F., Abdel-Aziz, M., Al-Ghanim, K., Gabol, K., Chatta, A., 2023. Biodiversity of commercially important finfish species caught by mid-water and bottom trawls from two different coasts of Arabian Sea: threats and conservation strategies. Braz J Biol 83, e249211.\u003c/li\u003e\n \u003cli\u003eHuang, X., Li, Y., Wang, T., Liu, H., Shi, J., Zhang, X., 2020. Evaluation of the oxidative stress status in zebrafish (Danio rerio) liver induced by three typical organic UV filters (BP-4, PABA and PBSA). Int J Environ Res Public Health 17, 651.\u003c/li\u003e\n \u003cli\u003eJabeen, K., Su, L., Li, J., Yang, D., Tong, C., Mu, J., Shi, H., 2017. Microplastics and mesoplastics in fish from coastal and fresh waters of China. Environ Pollut 221, 141-149.\u003c/li\u003e\n \u003cli\u003eJeyavani, J., Sibiya, A., Stalin, T., Vigneshkumar, G., Al-Ghanim, K.A., Riaz, M.N., Govindarajan, M., Vaseeharan, B., 2023. Biochemical, genotoxic and histological implications of polypropylene microplastics on freshwater fish Oreochromis mossambicus: An aquatic eco-toxicological assessment. Toxics 11, 282.\u003c/li\u003e\n \u003cli\u003eKim, S.A., Kim, L., Kim, T.H., An, Y.-J., 2022. Assessing the size-dependent effects of microplastics on zebrafish larvae through fish lateral line system and gut damage. Mar Pollut Bullet 185, 114279.\u003c/li\u003e\n \u003cli\u003eKumar, V., Umesh, M., Chakraborty, P., Sharma, P., Sarojini, S., Basheer, T., Kaur, K., Pasrija, R., Barcelo, D., 2024. Origin, ecotoxicity, and analytical methods for microplastic detection in aquatic systems. TrAC 170, 117392.\u003c/li\u003e\n \u003cli\u003eLaist, D.W., 1997. Impacts of marine debris: entanglement of marine life in marine debris including a comprehensive list of species with entanglement and ingestion records. Marine debris: sources, impacts, and solutions. Springer, pp. 99-139.\u003c/li\u003e\n \u003cli\u003eLi, B., Liang, W., Liu, Q.X., Fu, S., Ma, C., Chen, Q., Su, L., Craig, N.J., Shi, H., 2021. Fish Ingest Microplastics Unintentionally. Environ Sci Technol 55, 10471-10479.\u003c/li\u003e\n \u003cli\u003eLi, J., Liu, H., Paul Chen, J., 2018. Microplastics in freshwater systems: A review on occurrence, environmental effects, and methods for microplastics detection. Water Res 137, 362-374.\u003c/li\u003e\n \u003cli\u003eLu, Y., Zhang, Y., Deng, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H., 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. Environ Sci Tech 50, 4054-4060.\u003c/li\u003e\n \u003cli\u003eMacArthur, E., 2017. The new plastics economy: Rethinking the future of plastics \u0026amp; catalysing action. Ellen MacArthur Foundation: Cowes, UK 68.\u003c/li\u003e\n \u003cli\u003eMarklund, S., Ohlsson, P.-I., Opara, A., Paul, K.-G., 1974. The substrate profiles of the acidic and slightly basic horseradish peroxidases. Biochim Biophys Acta Enzymol 350, 304-313.\u003c/li\u003e\n \u003cli\u003eMohamed, A.-R.M., Abood, A.N., 2021. Trophic Interactions between Two Cichlid Species in Shatt Al-Arab River, Iraq. Biol App Environ Res 5, 74-85.\u003c/li\u003e\n \u003cli\u003eMtega, M.P., Mihale, M.J., Kilulya, K.F., 2023. Evaluation of Toxicological Risks and Effects of Microplastics on Nile Tilapia (Oreochromisniloticus) under in Vitro Laboratory Conditions. East Afr J Biophys Comput Sci 4, 54-68.\u003c/li\u003e\n \u003cli\u003eMurphy, J., 2001. Additives for plastics handbook. Elsevier.\u003c/li\u003e\n \u003cli\u003eNaidoo, T., Glassom, D., 2019. Decreased growth and survival in small juvenile fish, after chronic exposure to environmentally relevant concentrations of microplastic. Mar Pollut Bull 145, 254-259.\u003c/li\u003e\n \u003cli\u003ePandey, N., Verma, R., Patnaik, S., Anbumani, S., 2023. Abundance, characteristics, and risk assessment of microplastics in indigenous freshwater fishes of India. Environ Res 218, 115011.\u003c/li\u003e\n \u003cli\u003eRai, P.K., Lee, J., Brown, R.J., Kim, K.-H., 2021. Environmental fate, ecotoxicity biomarkers, and potential health effects of micro-and nano-scale plastic contamination. Journal of Hazardous Materials 403, 123910.\u003c/li\u003e\n \u003cli\u003eRios-Fuster, B., Arechavala-Lopez, P., Garc\u0026iacute;a-Marcos, K., Alomar, C., Compa, M., \u0026Aacute;lvarez, E., Juli\u0026agrave;, M.M., Mart\u0026iacute;, A.S., Sureda, A., Deudero, S., 2021. Experimental evidence of physiological and behavioral effects of microplastic ingestion in Sparus aurata. Aqua Toxicol 231, 105737.\u003c/li\u003e\n \u003cli\u003eRotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A., Hafeman, D.G., Hoekstra, W., 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science 179, 588-590.\u003c/li\u003e\n \u003cli\u003eSingh, A., Verma, P., Srivastava, S.C., Tripathi, M., 2014. Invasion, biology and impact of feral population of Nile tilapia (Oreochromis niloticus Linnaeus, 1757) in the Ganga River (India). Asia Pacific J Res 1.\u003c/li\u003e\n \u003cli\u003eSingh, A.K., Lakra, W.S., 2011. Risk and benefit assessment of alien fish species of the aquaculture and aquarium trade into India. Rev Aquac 3, 3-18.\u003c/li\u003e\n \u003cli\u003eSingh, S., Kumar Naik, T.S.S., Anil, A.G., Dhiman, J., Kumar, V., Dhanjal, D.S., Aguilar-Marcelino, L., Singh, J., Ramamurthy, P.C., 2022. Micro (nano) plastics in wastewater: A critical review on toxicity risk assessment, behaviour, environmental impact and challenges. Chemosph 290, 133169.\u003c/li\u003e\n \u003cli\u003eTesfahun, A., Temesgen, M., 2017. Food and Feeding Habits of the African Big Barb Labeobarbus intermedius (R\u0026uuml;ppell, 1836) in Ethiopian Water Bodies: A Review. J Nutr 6, 28-33.\u003c/li\u003e\n \u003cli\u003eUmamaheswari, S., Priyadarshinee, S., Bhattacharjee, M., Kadirvelu, K., Ramesh, M., 2021. Exposure to polystyrene microplastics induced gene modulated biological responses in zebrafish (Danio rerio). Chemosphere 281, 128592.\u003c/li\u003e\n \u003cli\u003eWagner, M., Lambert, S., 2018. Freshwater microplastics: emerging environmental contaminants? Springer Nature.\u003c/li\u003e\n \u003cli\u003eWang, Q., Huang, F., Liang, K., Niu, W., Duan, X., Jia, X., Wu, X., Xu, P., Zhou, L., 2022. Polystyrene nanoplastics affect digestive function and growth in juvenile groupers. Sci Total Environ 808, 152098.\u003c/li\u003e\n \u003cli\u003eXu, X., Zhang, L., Xue, Y., Gao, Y., Wang, L., Peng, M., Jiang, S., Zhang, Q., 2021. Microplastic pollution characteristic in surface water and freshwater fish of Gehu Lake, China. Environ Sci Pollut Res Int 28, 67203-67213.\u003c/li\u003e\n \u003cli\u003eXue, Y.H., Feng, L.S., Xu, Z.Y., Zhao, F.Y., Wen, X.L., Jin, T., Sun, Z.X., 2021. The time-dependent variations of zebrafish intestine and gill after polyethylene microplastics exposure. Ecotoxicol 30, 1997-2010.\u003c/li\u003e\n \u003cli\u003eYemm, E., Willis, A., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57, 508.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PE and PVC fragments, behavioural changes, gill, GIT, oxidative enzymes","lastPublishedDoi":"10.21203/rs.3.rs-6761113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6761113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroplastics (MPs), which are tiny particles measuring less than 5 mm, have emerged as a notable environmental issue due to their widespread presence in aquatic environments and their potential to harm aquatic organisms. In this study, the diet of tilapia (\u003cem\u003eOreochromis niloticus)\u003c/em\u003e exposed them to two types of MP materials: PE and PVC fragments. The fish were exposed for three weeks (21 days), and various behavioural changes and mortality were noticed. Moreover, microplastics can impact the growth, reproduction, and survival of tilapia, as evidenced by reduced growth rates and observed behavioural changes in exposed fish. Such modifications might have important effects on the general condition and population dynamics of aquatic environments. In both the gill and gastrointestinal tract (GIT), the MPs fragments were accumulated. The GIT of tilapia fish revealed 12.6 items/individual from the collected PVC pieces; gills included 4.3 items/individual. Similarly, PE fragment accumulation in the GI tract of fish showed 1.18 items/individual, and the gills showed 2.0 items/individual. A dietary intake of microplastics led to increasing inflammatory alterations in the liver and intestines. This study assessed the levels of oxidative enzymes in exposed groups of fishes (control, PVC, and PE fragments). The MPs-exposed tilapia fish exhibited remarkable Changes in the enzyme level and the nutritional values which was compared to control groups. All things considered, microplastics seriously compromise the health and ecological processes of freshwater fish including tilapia. More study is required to completely understand these effects as well as develop feasible strategies for reducing the microplastics' hazard in freshwater habitats.\u003c/p\u003e","manuscriptTitle":"Ingestion and the toxicological effects of virgin polyethylene (PE) and PVC microplastics in commercial freshwater fish, Tilapia (Oreochromis niloticus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 07:27:43","doi":"10.21203/rs.3.rs-6761113/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-20T04:32:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T01:22:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-29T07:32:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78414636546120399749607419493622822751","date":"2025-07-09T17:41:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51251935819735048115431118073287008166","date":"2025-06-16T06:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T06:42:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-13T18:33:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2025-06-09T17:14:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5f8e877b-8a55-4a13-9c04-191aa8e51c89","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:23:31+00:00","versionOfRecord":{"articleIdentity":"rs-6761113","link":"https://doi.org/10.1007/s10695-025-01596-7","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2025-10-22 16:16:19","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-07-01 07:27:43","video":"","vorDoi":"10.1007/s10695-025-01596-7","vorDoiUrl":"https://doi.org/10.1007/s10695-025-01596-7","workflowStages":[]},"version":"v1","identity":"rs-6761113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6761113","identity":"rs-6761113","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}