Integrated Biomarker Responses Reveal Cypermethrin-Induced Stress and Organ Damage in Nile Tilapia: Insights into Hepatic, Neural, and Hematological Toxicity

Integrated Biomarker Responses Reveal Cypermethrin-Induced Stress and Organ Damage in Nile Tilapia: Insights into Hepatic, Neural, and Hematological Toxicity | 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 Integrated Biomarker Responses Reveal Cypermethrin-Induced Stress and Organ Damage in Nile Tilapia: Insights into Hepatic, Neural, and Hematological Toxicity Maria Binte Moin, Shaon Kumar Mondol, Sadia Ibnat, Zakir Hossain This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7200428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pesticides sprayed near waterbodies, without proper precautions, can cause detrimental effects on the fish population. Pesticides' polarity and water solubility determine the bioaccumulation of its in fish. A ten-day-long study was conducted to assess the toxico-physiological response in nile tilapia fish in four triplicated treatments, such as control (0 ppm) and three treatments following 25%, 50%, and 75% of \(\:{LC}_{50}\) of cypermethrin. The recommended dose for cypermethrin was 0.886 ppm, whereas the determined \(\:{LC}_{50}\) value is much lower, which is 0.668 ppm. The fish were sampled at the end of the experiments, and a substantial drop in RBC count was noted, corroborated by a reduction in hemoglobin levels after 7 days. On the other hand, elevated WBC count occurred as a reaction of the defense system. Similarly, ameliorated antioxidant levels were found to safeguard cells from oxidative stress. However, Total Antioxidant capacity (T-AOC) demonstrates a notable decline, which measures the overall ability of cells or tissues that neutralize free radicals and ROS. It represents the overall failure of the antioxidant defense system to counteract sustained oxidative pressure. Serum calcium levels exhibited a dose-dependent decline, indicating how calcium ions mediate cellular reactions under stress. Enzymes are reliable markers of the general health of fish. Cypermethrin exposure significantly elevated the activity of both plasma glutamic oxaloacetic transaminase and plasma glutamic pyruvic transaminase enzymes in Oreochromis niloticus after 7 days, presumably due to damage to muscle and hepatic tissues, as evidenced by histopathological observations of the liver cells. In addition, a notable rise in Aspartate aminotransferase, Alanine transaminase, and Alkaline phosphatase was identified, indicating metabolic disruptions. Histological studies of the intestine and liver aligned with the biochemical disruptions. The findings suggest that even sublethal doses can induce physiological alterations, underscoring the need for cautious pesticide application. Oxidative stress immuno-toxicity hepatic enzymes histopathology biochemical analysis pyrethroids sublethal exposure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction A diverse range of pesticides is employed worldwide to enhance production and harvesting efficiency, exerting environmental pressure on ecosystems both on land and in water (Stanley et al . 2016). In addition, as pests gain adaptability and resistance to pesticides, increasing quantities and newer chemical compounds are being used for crop protection each year. Around 4.1 million tonnes of chemicals were employed globally in 2018, which is predicted to increase due to the surging demands of an overgrowing population (FAO 2021). Bangladesh is no exception in this regard. In 2020, 65,142 tonnes of pesticides were applied to vegetables and crops in Bangladesh (FAO 2021). Inadequate supervision and inefficiency in farmer training have resulted in the contamination of rivers by several pesticides. Most herbicides, insecticides, and fungicides lack selectivity in their targets. So, they not only interact with the targeted animals but also interfere with the molecular and cellular function of non-target species (Felix et al. 2019). The indiscriminate use of chemicals leads to environmental degradation and poses serious threats to aquatic species (Velisek et al. 2012). Massive concerns exist over the gradual deterioration of open-water fish stocks in Bangladesh, adversely affected by several toxicant exposures. An adverse relationship exists between the solubility of pesticide compounds and the bioaccumulation of these chemicals in fish. The exposure of aquatic organisms to synthetic pyrethroid pesticides, such as permethrin, deltamethrin, tetramethrin, and cypermethrin, may lead to substantial toxicological consequences (Coats et al. 1989). Pyrethroids are preferred over organophosphates, carbamates, and organochlorines due to their greater efficiency, lower toxicity, and easy biodegradability (Sharaf et al. 2010). Although Synthetic pyrethroids are claimed to be safe and environmentally benign due to their selective pesticidal toxicity, low persistence, and minimal toxicity to mammals and birds, they exhibit significant damage to incidental animals, including fish, lobsters, prawns, mayfly nymphs, and numerous species of zooplankton (Oudou et al. 2004). Pyrethroids at first target the peripheral and central nervous system axons by interacting with sodium channels, so they are usually categorized as neurotoxins. It mediates the interaction between calcium ions and the intracellular membrane. Consequently, obstructing the calcium removal process from nerve terminals leads to less spontaneous neurotransmitter release (Wang 2008). Pyrethroids can enter the outer layer of the skin and bind directly with a carrier protein in the bloodstream or lymphatic system. As a result, the spread-out pyrethroids and the cells on the outer layer of the skin immediately upregulate the central nervous system by interacting with the sensory organs of the peripheral nervous system (Neal et al. 2010). Additionally, pyrethroids may enter the body by inhalation in vapor form, even if in minimal amounts. Furthermore, they might infiltrate the blood or hemolymph via the digestive system during the digestive process (Neal et al. 2010). The detrimental effects of pyrethroids on fish may be related to their biotransformation properties. Pyrethroid compounds are prone to being affected by the non-aqueous components of cells due to their lipophilic characteristics (Prusty et al . 2015). Despite the low concentration of these chemicals in water, they may be rapidly absorbed by the gills (Moore and Waring 2001). Cypermethrin is a fourth generation halogenated synthetic pyrethroid with strong reactivity, which makes it more resistant to degradation (Jaensson et al. 2007; Sharma et al. 2021; Ullah et al. 2018). Cypermethrin prohibits sodium ions from being transferred through the cell membrane, as they remain stable in neutral and acidic solutions (Tiwari et al. 2012). Nile tilapia ( Oreochromis niloticus ) is a freshwater fish species that is extensively cultivated worldwide owing to its rapid development, adaptability to many settings, and significant tolerance to low water quality. It has been widely considered a model organism in toxicity, physiology, and genetics research as a bioindicator for the health of freshwater ecosystems. This study aims to assess the impact of acute cypermethrin pesticide intoxication on several hematological and biochemical markers in Nile tilapia. Comprehending pesticide toxicity in nile tilapia aids in developing environmental rules, directing sustainable pesticide use, and safeguarding aquatic biodiversity. 2. Materials and methods 2.1. Experimental animal and rearing condition Healthy O. niloticus fishes of both sexes, with an initial average length and weight of 4.78 ± 1.14 cm and 6.89 ± 1.09 g, were collected from the Bangladesh Fisheries Research Institute, Mymensingh. Only the active and healthy O. niloticus were selected for acclimatization and were kept in a cistern for 7 days. The fish were fed with commercial food pellets, with water quality being monitored periodically. The fish were examined carefully for any pathological symptoms before starting the experiment. Water containing 0.1 mg/L of potassium permanganate solution was used for disinfecting purposes. 2.2. Exposure study In the initial experiment, ten fish were stocked in each aquarium of 120 L water capacity, which were filled up to 20 L. Fish were subjected to five distinct concentrations of pesticide to determine the lethal dosage of cypermethrin for O. niloticus . Once the lethal dose was established, the fish was subjected to three sublethal doses, i.e., 25%, 50%, and 75% of \(\:{LC}_{50}\) for further study. Each aquarium was provided with these concentrations of cypermethrin and was labeled as T0, T1, T2, and T3. T0 was kept as a control group. 2.3. \(\:{\varvec{L}\varvec{C}}_{50}\) dose determination The acute toxicity \(\:\:{LC}_{50}\) of cypermethrin at 96h for O. niloticus , was determined in our laboratory using the semi-static method (OECD method 2001). The nile tilapia (10 in 20 L of test medium) was exposed to five concentrations of cypermethrin to determine \(\:{LC}_{50}\) dose. In the short-term conclusive testing, concentrations of the test chemical varied from 0.2 ppm to 1.0 ppm, as the recommended agricultural dosage for cypermethrin 10EC was determined to be 0.86 ppm, taking into account a water volume of 20 L in the rice field. These concentrations of cypermethrin were measured with a micropipette, then put into water in a glass jar (each having three replications), and then homogeneously mixed with water using a gentle mixing motion. The test medium was replaced every 24 hours with the corresponding quantities of the toxicant, without aeration. Mortality was documented every 12 hours, with deceased fish being removed upon observation, while consistently recording the number for 96 hours to assess acute toxicity. 2.4. Estimation of hematological indices 2.4.1. Morphological study of erythrocytes Fresh blood samples were collected from T0 (0 ppm), T1 (0.168 ppm), T2 (0.334 ppm), and T3 (0.51 ppm) groups of cypermethrin-exposed fish for evaluating erythrocytic cellular and nuclear abnormalities after 7 days of exposure. Methanol-fixed blood smears of O. niloticus were stained with Wright’s Giemsa stain and analyzed using immersion oil microscopy (OLYMPUS-CX41, 400×). 2.4.2. Evaluation of hematological indices To evaluate the physiological effects of cypermethrin, hematological parameters were examined only for the control group and the highest treatment group, T3 (0.51 ppm). This selective comparison was predicated on the notion that the greatest pesticide dosage would manifest the most significant physiological changes, hence yielding greater insights into stress response. Blood was drawn from the caudal vein and heart using disposable needles and stored in Eppendorf tubes on ice. After going through centrifugation at 8000 rpm for 15 minutes, the resulting serum was stored at 4℃ for subsequent analysis. Red blood cell (RBC) and white blood cell (WBC) counts were conducted utilizing a hemocytometer (OPTIA B-350, Italy) in accordance with established dilution protocols (five µl of blood was diluted in 995 µl of RBC solution for the RBC count, and for the WBC count, same amount of blood was diluted in 195 µl of WBC solution). Glucose levels were assessed via a glucometer (Health Assure®, Taiwan), while hemoglobin (Hb) was quantified using Easy Mate® GHb strips. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were computed utilizing conventional hematological equations. MCHC = (Hb ÷ PCV) × 100; MCV = (PCV ÷ RBC) × 10; MCH (pg) = (Hb × 10) ÷ RBC 2.5. Estimation of antioxidant activity Antioxidant responses were assessed solely in the T3 (0.51 ppm) and control groups (0 ppm) to ascertain potential oxidative damage resulting from maximal pesticide exposure. Blood serum samples from O. niloticus was utilized to measure antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), peroxidase (POD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) after 7days. Serum was acquired by homogenizing blood in a cold phosphate buffer (pH 6.5, 0.2 M, 1:4 w/v), and afterwards subjected to centrifugation at 10,000 rpm for 15 minutes at 4°C. The measurement of SOD activity was conducted using the Giannopolitis and Ries approach, which relies on the photoreduction of NBT (Giannopolitic and Ries 1977). The CAT activity was assessed according to Shangari and O’Brien by observing H₂O₂ degradation at 240 nm (Shangari and Brien 2006). The GST activity was assessed via Mannervik’s approach by observing the production of the GSH-CDNB conjugate at 340 nm (Mannervik et al. 1985). POD activity was evaluated from serum and acetone powder using a phosphate buffer extraction, and subsequently analyzed via spectrophotometry. T-AOC was quantified by the reduction of Fe³⁺, with absorbance measured at around 520 nm. MDA concentrations were quantified using a TBA test at 532 nm following serum treatment with TBA reagent and subsequent centrifugation, with values derived from a standard curve. 2.6. Histological examination of liver and intestine O. niloticus were subjected to three doses (T1, T2, and T3) of cypermethrin for 10 days in glass aquaria, alongside a control group in water devoid of pesticide (T0). Liver and intestinal samples were obtained from all groups and stored in 10% neutral buffered formalin. Tissues underwent dehydration, clearing, and infiltration by an automated tissue processor (SHANDON, CITADEL 1000) adhering to a typical 21-hour protocol utilizing alcohol, xylene, and paraffin. Tissues were processed, embedded in paraffin, sectioned at 5 µm with a Leica JUNG RM 2035 microtome, and affixed to glass slides. Sections were stained with hematoxylin and eosin (H&E) using usual techniques and viewed under a light microscope for cellular abnormalities. 2.7. Serum calcium ( \(\:{\varvec{C}\varvec{a}}^{2+}\) ) analysis Serum calcium level was quantified in three treatment groups relative to the control group using method developed by (Reza et al. 2013) refined from (WHO 2006). Blood was extracted from the caudal veins of fish after 7 days of exposure, and serum was isolated using centrifugation (10,000×g, 15 minutes, 4°C). A 2000 µl working reagent comprising 8-hydroxyquinoline, O-cresol phthalein complexone, and distilled water was combined with 100 µl of serum. Absorbance was quantified at 540 nm utilizing a Spectronic Genesys TM5 spectrophotometer, whilst calcium concentrations were ascertained by a reference curve. 2.8. Stress enzyme activity measurement 2.8.1. Acetylcholinesterase enzyme (AChE) activity measurement of cypermethrin-treated fish O. niloticus were subjected to three sub-lethal concentrations of cypermethrin for 7 days. During sample day, three fish were sacrificed, and brain tissues were harvested, weighed, and homogenized in sodium phosphate buffer (pH 8.0) to attain a concentration of 20 mg/ml. The homogenate underwent centrifugation at 2000 rpm for 10 minutes at 4°C, and the pellet was subsequently rinsed with 0.15 M KCl. The tissue was re-homogenized in a 10% w/v Tris-HCl buffer (0.1 M, pH 7.4) and subjected to centrifugation at 5000 rpm for 10 minutes. The supernatant was utilized for acetylcholinesterase estimation. A reaction mixture comprising 2.7 ml of phosphate buffer, 100 µl of supernatant, and 100 µl of Ellman’s reagent (0.16 mM DNTB) was incubated at 37°C for 10 minutes. The reaction commenced with the addition of 100 µl of acetylcholine iodide. Absorbance was measured at 412 nm. The total protein was quantified using 10% trichloroacetic acid, employing bovine serum albumin as the reference. Results were quantified as nmol/min/mg protein, utilizing the formula: R = 5.74 (10 − 4 ) ΔA/C 0 Where, R = rate in moles substrate hydrolyzed per min per g of tissue; ΔA = change in absorbance per min; C 0 = original concentration of tissue. 2.8.2. Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) analysis For PGOT and PGPT assays, O. niloticus were subjected similarly to three sub-lethal dosages of cypermethrin for 7 days. Blood was obtained from the caudal vein, and plasma was isolated using centrifugation at 11,000 rpm for 15 minutes. Enzyme activity was assessed according to the methodology of Reitman and Frankel (Reitman and Frankel 1957). A phosphate buffer (0.1 M, pH 7.4) was formulated, to which 2 mM α-ketoglutaric acid and 200 mM dl-aspartate were added for PGOT. For PGPT, 2 mM α-ketoglutaric acid and 200 mM dl-alanine were used. One milliliter of each substrate solution was pre-incubated at 40°C for 10 minutes, followed by the addition of 0.2 milliliters of plasma. The reaction was conducted for 60 minutes (PGOT) or 30 minutes (PGPT), and thereafter terminated with 1 ml of 2,4-dinitrophenylhydrazine reagent (1 mM in 1N HCl). After 20 minutes, 10 ml of 0.4 N NaOH was introduced, and the absorbance was measured at 505 nm after 30 minutes. A standard curve was established utilizing pyruvate and α-ketoglutarate. The enzyme activity (U/ml) was determined using: A = C×V / m Here, A = activity of the material in units per mass of material C = desired concentration of the final solution. V = final or total volume of the solution m = mass of the solute dissolved in the solution 2.9. Liver enzyme analysis Liver enzymes in the highest treatment group (T3) and control group (T0) were analyzed following the instructions of the Nanjing Jiancheng Bioengineering Institute (China) kit protocol after 7 days of exposure. To measure aspartate aminotransferase (AST) activity, the blood sample was added to the reaction mixture containing aspartate and α-ketoglutarate. The mixture was incubated at 37°C for a set time. After that, a color-developing reagent, 2,4-dinitrophenylhydrazine, to detect oxaloacetate was added. The absorbance at the specified wavelength (typically 505 nm) was measured. It is usually expressed in U/L. Alanine aminotransferase (ALT) Activity measurement was done in the same process however, absorbance was measured at 510nm. To determine alkaline phosphatase (AKP) activity, the sample was mixed with the substrate solution containing p-nitrophenyl phosphate and incubated at 37°C. The reaction was stopped by typical sodium hydroxide. Absorbance was measured at 405 nm. 2.10. Water quality analysis Water quality parameters were measured using specific instruments: Temperature was recorded with a Celsius thermometer, a multi-parameter DO meter (Multi 340 Iset, DO-5509; China) used for dissolved oxygen, a digital pH meter (HANNA-HI98107 pHep®, Romania) for pH, and a portable TDS meter (HANNA-HI98302 DiST®2, Romania) for TDS. In addition, ammonia and total alkalinity levels were evaluated every three days utilizing the API® ammonia and alkalinity test kit (HANNA-HI3811, Romania) throughout the experiment. 2.11. Statistical analysis The probit analysis was performed considering p < 0.01 statistical significance, as well as the one-way variance analysis (ANOVA) was performed by using SPSS ver. 26.0 computer software program. 3. Result The water quality parameters in the cypermethrin-treated aquariums were maintained within acceptable ranges throughout the experiment. The recorded values for temperature, pH, DO, total alkalinity, and ammonia were 29.17 ± 1.67℃, 8.09 ± 0.49, 4.26 ± 0.19 mg/L, 108.30 ± 0.51 mg/L, and 0.07 ± 0.04 mg/L, respectively. These parameters remained stable, ensuring no external environmental stress influenced the observed physiological responses. 3.1. \(\:{\varvec{L}\varvec{C}}_{50}\) of cypermethrin 10EC for Oreochromis niloticus In order to study the acute 96h toxicity of cypermethrin, ten O. niloticus fish were exposed to five different pesticide concentrations (0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.0 ppm). A control group without pesticide was maintained. No mortality was observed at 0.2 ppm, whereas 100% mortality occurred at 1.0 ppm (Table 1 ). Cypermethrin's calculated 96h acute \(\:{LC}_{50}\:\) value (95% confidence limits) using a static bioassay system to nile tilapia ( O. niloticus ) was found to be 0.668 ppm (Table 1 ). Table 1 Acute 96h toxicity of cypermethrin in Oreochromis niloticus Concentration Total mortality 24h 48h 72h 96h 0.2 ppm 0 0 0 0 0.4 ppm 0 0 1 0 0.6 ppm 0 1 2 0 0.8 ppm 1 3 3 0 1.0 ppm 0 6 3 1 3.2. The effects of cypermethrin pesticide on the hematological changes of O. niloticus 3.2.1 Morphological alterations of erythrocytes of fish blood upon cypermethrin exposure Several erythrocyte abnormalities in nile tilapia fish were observed. As the doses of cypermethrin increased, so did these abnormalities' frequency and severity. The control group showed normal, regular-shaped erythrocytes. However, even at the lowest dose of cypermethrin exposure, abnormalities like large lymphocytes, small lymphocytes, swollen cells, and sickle cells were observed. Fish exposed to 0.334 ppm (T2) showed nuclear abnormalities, including binucleated erythrocytes and micronuclei. At the highest dose (T3), cypermethrin exposure revealed monocytes, teardrop-like cells, and nuclear-fragmented erythrocytes were revealed from blood samples of fish. The frequencies of erythrocyte nuclear abnormalities followed an increasing trend with increasing cypermethrin concentrations (Fig. 1 ). 3.2.2 Hematological parameters The highest treatment group, T3 (0.51 ppm), was compared with the control group to identify any kind of changes in the blood parameters. A significant decrease ( p < 0.05) in RBC count was found in the treated group 3. The hemoglobin level of the highest pesticide-treated fish was significantly lower ( p < 0.05) than the control group, and was 9.97 ± 0.06 g/dl and 12.24 ± 0.06 g/dl, respectively, in the treatment and control groups, supporting the result of RBC count. On the contrary, the WBC count of T3 was recorded as significantly greater than the control value. Additionally, the T3 group exhibited significantly higher ( p < 0.01) total serum protein and blood glucose levels than the control group. The mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) showed a significant decrease ( p < 0.01) in T3-treated fish compared to the control group. Conversely, hematocrit and mean corpuscular hemoglobin concentration (MCHC) were significantly elevated ( p < 0.01) in the T3 group. The results of the hematological indices are given in Table 2 . Table 2 Hematological parameters of Oreochromis niloticus Parameters Control (M ± SD) Treatment (M ± SD) RBC (×10 6 /µL) 3.56 ± 0.17 2.51 ± 0.13* WBC (×10 3 /µL) 210.97 ± 6.6 320.62 ± 9.22** Hemoglobin (g/dL) 12.24 ± 0.06 9.97 ± 0.06 * Hematocrit (%) 30.71 ± 0.81 41.40 ± 0.98** MCH (Pg/cell) 54.77 ± 2.45 51.82 ± 3.89** MCV (fl/cell) 140.47 ± 4.56 138.77 ± 5.36** MCHC (g/dL) 34.91 ± 3.11 35.40 ± 4.21** Glucose (mg/dL) 7.91 ± 1.11 15.40 ± 2.21** Serum protein (mg/dL) 34.91 ± 3.11 35.40 ± 4.21** * (Asterisk) indicates significant difference (* p < 0.05 vs control and ** p < 0.01 vs control) 3.3. Antioxidant activity Fish evolved enzymatic and non-enzymatic antioxidant defense mechanisms to neutralize reactive oxygen species (ROS) produced during cellular metabolism or due to exposure to environmental contaminants. SOD, CAT, POD, GST, and MDA activities of O. niloticus serum increased significantly ( p < 0.05) in highest concentration (T3) group of fish compared to the control group (Fig. 4.3) However, the GST level was the highest, 286.77 ± 7.36 µmol in the treatment 3 group among the all-tested antioxidant enzymes. The total antioxidant activity (T-AOC) level was found to be 6.51 ± 0.25 U/ml in treatment group 3, which was significantly ( p < 0.05) lower than the control value of 21.81 ± 2.3 U/ml (Fig. 2 ). 3.4. Histopathological changes in pesticide-treated fish 3.4.1 Histopathological changes in the intestine The histology of the intestine of fish treated with three doses of cypermethrin was compared to the control group. Intestinal changes such as lamina propria alterations, hemorrhage, and absorptive vacuoles were found in fish treated with 0.168 ppm cypermethrin (T1). As the concentration of cypermethrin escalated, the incidence of changes correspondingly rose. The presence of a cracked clay appearance, fusion of villi, and distorted goblet cells was observed in the 0.334 ppm treated group (T2). At the highest exposure dose (T3), severe necrosis in the enterocytes, along with goblet cell loss, sloughing of the epithelial layer, and damage to the brush border were seen. In contrast, the control group exhibited well-structured epithelial cells, long and intact villi, and a clear epithelial lining was observed (Fig. 3 ). 3.4.2 Histopathological changes in liver The liver is the primary detoxification organ in fish, so it was heavily affected by toxicant exposure. Liver showed the following histopathological changes in the treated groups. These changes compromised the liver’s ability to detoxify and regulate metabolism (Fig. 4 ). 3.5. Effect of cypermethrin in serum \(\:{\varvec{C}\varvec{a}}^{2+}\) For the assessment of serum \(\:{Ca}^{2+}\) , Fish were exposed to three sublethal doses of pesticide. Increased doses of pesticide resulted in a decline in \(\:{Ca}^{2+}\) level. In the control group, the calcium level was 10.149 ± 0.042 mg/µl, whereas in T1, T2, and T3 the value was, respectively, 9.663 ± 0.1985 mg/µl, 8.549 ± 0.1557 mg/µl, and 6.213 ± 0.372 mg/µl, which are significantly different ( p < 0.05) from the control group (Fig. 5 ) 3.6. Effects of cypermethrin on neurotransmitters and stress-indicating enzymes 3.6.1 Acetylcholinesterase (AChE) activity Acetylcholinesterase (AChE) is an important bioindicator of neurotoxicity. To assess AChE activity in the brain, O. niloticus was exposed to three different dosages of pesticide. This result indicated that exposure to cypermethrin at such sublethal doses was a source of stress to O. niloticus (Fig. 6 ). 3.6.2 Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) level The PGOT and PGPT of blood serum are used as biomarkers for liver function and stress in organisms, particularly in fish when exposed to toxicants. At greater doses of cypermethrin, their level rises in response to cellular damage in the liver and muscles. As a consequence, the values of PGOT in the treatment groups were significantly different ( p < 0.05) of the control group (Fig. 7 ) On the other hand, the level of PGPT was as follows 477.847 ± 15.319 U/ml, 518.33 ± 13.018 U/ml, and 539 ± 12.124U/ml in T0, T1, T2 and T3, respectively which was also significantly ( p < 0.05) different from the PGPT level as 208.667 ± 33.65 U/ml in the control group (Fig. 7 ). 3.7. Liver enzyme analysis Liver enzymes like ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and AKP (Alkaline Phosphatase) were measured in the highest treatment group and the control group The ALT, AST, and AKP levels in treatment group 3 were significantly ( p < 0.05) higher compared to the control group. AST level was 60.11 \(\:\pm\:\) 4.99 U/L and 85.38 \(\:\pm\:\) 6.11 U/L in control and treatment 3, respectively. AKP value was the lowest among these three liver enzymes. In the control group, the value of AKP was 40.01 \(\:\pm\:\) 3.65 U/gprotein, which increased upon cypermethrin exposure and reached 56.45 \(\:\pm\:\) 4.00 U/gprotein in the T3 group. Among the three liver enzymes, the ALT value was the highest in the treatment group. ALT value was 112.39 \(\:\pm\:\) 5.75 U/L in the control group, which increased and reached 149.21 \(\:\pm\:\) 7.45 U/L in treatment 3 (Fig. 7 ). 4. Discussion 4.1. Acute toxicity test of cypermethrin for O. niloticus With a 1600 cubic inch area in mind, the appropriate dosage of cypermethrin 10EC was 0.86 ppm. Nevertheless, fish treated with lower doses than advised also experienced physiologic effects, leading to hyperactivity and even fish death. In the current study, findings showed that 0.668 ppm of cypermethrin is the lethal level for O. niloticus after 96 hours of exposure (Table 1 ). Kenthao et al . 2020 revealed that the \(\:{LC}_{50}\:\) for cypermethrin-exposed nile tilapia, is 3.24 µg/l at 96 h and 4.23 µg/l at which 70% mortality occurs. The \(\:{LC}_{50\:\:}\) of cypermethrin for tilapia was 2.2 µg/l (Bradbury and Coats 1989). These results are higher than the present study's findings. Chemical composition, exposure conditions, water quality, size, and age affect the toxicity of cypermethrin. The majority of cypermethrin products consist of a combination of its isomers. The mixture's composition, which is the ratio of cis and trans isomers, determines the effectiveness of cypermethrin. Yuniari et al. (2016) identified that at a concentration of 0.042 ppm, 0.065 ppm, and 0.087 ppm, death of the fish starts happening, with up to 70% mortality at a concentration of 0.087 ppm, when the highest mortality was observed. This discrepancy highlights the complex relationship between toxicants' properties and the reaction of an organism's biology. The decrease in fish survival at the highest dose is ascribed to their incapacity to acclimate to environmental contaminants. As a result, the fish are unable to mitigate the harmful impacts of the pollutants in the testing media. 4.2. Effects of cypermethrin pesticide on the hematological changes in O. niloticus Hematological changes are often the first identifiable and measurable reactions to environmental modifications (Wendelaar 1997). Hematological profiles can shed light on the organism's internal environmental condition. 4.2.1 Morphological alterations of erythrocytes in cypermethrin-treated fish The current investigation detected many morphological changes in blood erythrocytes. These modifications demonstrate the reduced erythrocyte levels resulting from the harmful effects of pesticides on hematopoietic tissues and circulating erythrocytes. Several morphological alterations, including teardrop-shaped cells, sickle cells, swollen cells, monocytes, and large and small lymphocytes, were seen (Fig. 1 ). The formation of micronuclei and lobed nucleus erythrocytes in the current study is in line with the findings of Hussain et al . (2014). These Alterations may come from increased synthesis of caspase-activated DNAase. It leads to the formation of cleavage of cytoskeletal proteins (vimentin, gelsolin, and fodrin) and nuclear proteins owing to oxidative stress affecting the mitochondria (Hussain et al. 2014; Hussain et al. 2012; Campos et al. 2012). The cytopathogenic changes may occur from elevated lipid peroxidation, leading to enhanced production of intracellular reactive oxygen and nitrogen species [ 26 , 27 , 28 ]. Previous research has suggested that nuclear changes in erythrocytes may result from chromosomal aberrations caused by several hazardous chemicals (Hussain et al. 2012). The lobed and bilobed nuclei of erythrocytes in the present research may result from the disruption of tubulin polymerization, oxidation of mRNA, and nitration of proteins, inhibiting intracellular metabolism (Hussain et al. 2014; Campos et al. 2012) 4.2.2 Hematological indices Cypermethrin exists as methyl ethyl carboxylate. It interacts with Fe in hemoglobin in the alkylation process, potentially substituting oxygen in blood cells (Ajani and Awogbade 2012), which leads to decreased erythrocytes. While White blood cells are the primary constituents that protect the organism during injury, hemorrhage, and the transit of foreign antigens into the body (Vermurugan et al. 2016). As a result, an increase in the WBC count is observed as a response to hypersensitivity of immune cells. Hematological parameters and recovery pattern were studied in Labeo rohita upon exposure to cypermethrin and carbofuran by(Adhikari et al. 2004). It resulted in significantly ( p < 0.05) lower values for erythrocyte count (RBC), hemoglobin content (Hb), and hematocrit when compared with the control group. In contrast, there was a significant ( p < 0.05) increase in leukocyte count (TLC) in the pesticide-treated group. MCV and MCH decreased in response to both pesticides during their study. Significant changes in the blood components of L. rohita upon treatment with cypermethrin were noted in the findings ofKhan et al. (2018) which correspond to the present study's findings. These changes included a reduction in the Hb level, HCT, MCV, MCH, and MCHC. It can be concluded that cypermethrin intoxication resulted in elevated levels of certain blood components that fight against toxicants, such as the number of white blood cells (×10 3 /mm 3 ) and red blood cells (×10 6 /mm 3 ). Conversely, the decrease in hemoglobin and several other blood constituents can result from the suppression of hemoglobin formation, osmoregulatory impairment, and the death of erythrocytes in hematopoietic organs, as previously found in Catla catla (Vani et al. 2011). These reductions might be attributed to erythroblastosis, causing anemia (Saleh and Marie 2016). 4.3 Antioxidant enzyme activity Antioxidant enzymes assist fish in managing environmental stressors by modulating their activity, with the degree of this modulation determined by the stressor's strength, the species, and the exposure method. Increased lipid peroxidation can affect the activities of several protective enzymatic and nonenzymatic antioxidants, which are well known as the bio-indicators of increased oxidation. Superoxide dismutase (SOD) and catalase (CAT) function as a principal defense mechanism, safeguarding biological macromolecules from oxidative harm. SOD, a category of metalloenzymes, serve as the primary barrier against the detrimental effects of superoxide radicals in aerobic organisms (Kohlen and Nyska 2002). When reactive oxygen species start generating, SOD tries to neutralize them. In such a condition, an enhancement in its activity is observed as an induced adaptive response. CAT is an enzyme located in peroxisomes and facilitates the removal of hydrogen peroxide \(\:{H}_{2}{O}_{2}\) , which is metabolized to molecular oxygen and water (Van et al . 2003; Yilmaz et al. 2006). The SOD intervenes in the first transformation by disputing the superoxide free radicals \(\:{O}_{2}\:\) into \(\:{H}_{2}{O}_{2}\) (Fig. 2 ), whereas CAT converts it into \(\:{H}_{2}O\:\) and \(\:\:{O}_{2}\) (Dorval et al. 2003). The current work suggests that the activation of SOD activity in hepatic tissue may result from increased \(\:{O}_{2}\:\) production which is regarded as the primary defense mechanism against oxidative stress. Similar findings have been reported in various fish models exposed to organophosphate pesticides. In addition, Üner et al . (2001) demonstrated that malondialdehyde (MDA) increased in fish liver and kidney following cypermethrin exposure. On the other hand, in a study carried out in Channa punctatus ,Sayeed et al. (2003) reported that deltamethrin exposure increased MDA levels in the fish liver, kidney, and gills. In our investigation, the increased MDA levels may be ascribed to the production of free radicals after cypermethrin introduction, indicating a possible association between cypermethrin toxicity and lipid peroxidation (Fig. 2 ). The upwards peroxidase (POD) activity detected in the liver may correlate with its function in the detoxification of harmful substances. The generation of ROS during the biotransformation of toxicants can cause cellular damage via oxidative mechanisms (Van et al. 2003). The POD activity scavenges the ROS by converting hydrogen peroxide (Hinton et al. 2008). The increased GST activity may be ascribed to the augmented metabolism of lipoperoxides produced during the Fenton reaction or the biotransformation of toxicants, indicating an adaptive response in fish. 4.4 Histopathological changes 4.4.1 Histopathological changes observed in the intestine The intestinal tract is a crucial component of the fish digestive system, significantly contributing to the digestion and absorption of nutrients. It is extremely responsive to any absorbed hazardous substances and serves as a significant biomarker organ for evaluating ecotoxicology. In the study, Khan et al. (2018) noticed changes in intestinal tissues of L. rohita , such as predominantly necrosis, hemorrhages, overproduction of goblet cells in villi, fusion, detachment, and shortening of villi, which are similar to the report reported earlier by Hasan et al . (2015) for acute endosulfan toxicity. The deterioration of villi, mucosal folds disintegration, vacuolations, hypertrophy, and necrosis in C. carpi o and Cirrhinus mrigala treated with atrazine and fenvalerate was observed (Velmurugan et al. 2016). Severe mucosal secretion occurs due to distress, enabling fish to cope with ecological stress (Samanta et al. 2016). Abnormal histopathological alterations, for example, shortening of villi with inflammation, rupture of cells, degeneration changes in tips of villi, curved villi, hemorrhage, necrosis, numerous vacuoles, dilation in the blood vessels, completely damaged villi, and loss of architecture in a number of fish species were also identified (Velmurugan et al 2016; Cengiz and Unlu 2006) The findings of the present study, including degenerative enterocytes, hemorrhage, absorptive vacuole, cracked clay appearance, fusion of villi, and hemorrhage in the lamina propria area, are consistent with the study of the above findings (Fig. 3 ). 4.4.2 Histopathological changes observed in the liver The liver is an essential organ that facilitates the metabolism of carbohydrates, proteins, and lipids, in addition to detoxifying toxic compounds. The buildup of pesticides and their metabolites in hepatocytes frequently results in considerable histological changes and structural modifications in the organ (Sharma et al. 2012). In the current investigation, the melano-macrophage center, focal area of necrosis, hemorrhage, and pyknosis were found (Fig. 4 ). Edema around the hepatocyte and focal areas in between them were observed. Biliary duct epithelial detachment and vacuolar degeneration were also seen. Similar observations were noted, such as dissolution of the cell membrane, blood mobbing and congestion, pyknosis, necrosis, hyperplasia, and vacuolations of hepatocytes in L. rohita after exposure to cypermethrin (Saleh and Marie 2016; Murussi et al . 2016). The modifications of liver revealed in the present trials are in accordance with the results in Nile tilapia, i.e., O. niloticus (Coimbra et al. 2007), and rainbow trout, i.e., Oncorhynchus mykiss treated with varying concentrations of endosulfan (Authman et al. 2015), Common carp treated with chlorpyrifos, C. carpio treated with buprofezin fipronil (Pal et al. 2012), C. catla exposed to α-cypermethrin (Muthuviveganandavel et al. 2013) 4.5 Serum \(\:\:{\varvec{C}\varvec{a}}^{2+}\) activity This study observed reduced serum calcium levels in fish exposed to cypermethrin. This is supported by earlier investigators' reports of reduced blood/plasma calcium levels in fish exposed to toxicants (Fig. 5 ). The decline indicates exhaustion of the Ca 2+ depots and their reduced uptake from gills and kidney tissues (Velmurugan et al. 2016; Cengiz and Unlu 2006). Cypermethrin in the ambient medium comes in direct contact with the gills and ruptures the chloride cell membrane. Injury to the gill epithelium hinders Ca 2+ absorption from the ambient water, resulting in hypocalcaemia and respiratory discomfort, accompanied by hyperexcitability and body tremors in Heteropneustes fossilis (Pandey et al. 2009) 4.6 Effects of cypermethrin on neurotransmitter and stress-indicating enzyme 4.6.1 Acetylcholinesterase (AChE) activity Assessing AChE activity in the environment may offer considerable benefits compared to exclusive dependence on analytical chemistry. Pesticides inhibit the function of cholinesterase enzymes, which play a major role in the hydrolysis of the neurotransmitter acetylcholine (ACh), enabling its elimination from the synaptic cleft (Mineau 1991). Acetylcholine functions as both a preganglionic and postganglionic neurotransmitter in the parasympathetic nervous system and as a preganglionic neurotransmitter in the sympathetic nervous system. Inhibition of cholinesterase by toxicants leads to the accumulation of acetylcholine at the nerve synapse, hence compromising normal nervous system function (Fig. 6 ). This produces rapid twitching of voluntary muscles, followed by paralysis (Mineau 1991). Once bound, pesticides are considered irreversible inhibitors, as recovery usually depends on new enzyme synthesis, AChE (Fulton and Key 2001). The inhibition of AChE is the principal mechanism by which organophosphorus pesticides manifest their toxicity, so linking this biomarker directly to the compound's harmful mode of action (Fulton and Key 2001). In the present study, a significant decline in the AChE enzyme was noted in comparison to the control group. 4.6.2 Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) level Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) are essential for mobilizing L-amino acids for gluconeogenesis and a pivotal connection between carbohydrate and protein metabolism during altered physiological, pathological, and environmental stress conditions (Ramaswamy et al. 1999). In the present study, activities of GOT and GPT were found to be significantly elevated in O. niloticus irrespective of concentrations of cypermethrin when compared with the control. The observed increase in GOT activity indicates that molecular rearrangements involving amino acids, linked to the citric acid cycle at two junctures—oxaloacetate and α-ketoglutaric acid—were modified. Similarly, the increase in GPT indicates that the exposed fish required intensive glycogenesis to cope with the energy crisis (Bhavan et al. 2015). It has also been suggested that stress leads to elevation of the transamination pathways (Li et al. 2011). These results have also been evident from the histopathological observations of the liver cells. Furthermore, modifications in gill and blood cells may exacerbate the condition by hindering the respiratory processes of the test organism. In reaction to toxic stress, the organism compensates by raising its metabolic rate. Nonetheless, given that the liver functions as the primary organ for the metabolism of vital macromolecules in vertebrates, any impairment to the hepatic system might result in considerable physiological and biochemical disruptions, including changes in GOT and GPT activity (Egnatchik et al. 2019; Malarvizhi et al. 2012; Sancho et al. 2009)Extended exposure to, or elevated levels of, organophosphate compounds may further inhibit GOT and GPT activities by interfering with cellular processes, finally resulting in fish mortality (Saravanan et al. 2013). 4.7. Liver enzyme activity During injury or liver damage, the cells are destroyed, which results in the release of ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and AKP (Alkaline Phosphatase) into plasma, and their high concentration in plasma is considered an indicator of abnormal physiology. An increase in their concentrations shows stress-based tissue impairment (Palanivelu et al. 2005). The findings of ALT, AST, and AKP often suggest degenerative alterations and diminished liver function, since the toxicant adversely affects hepatocytes, leading to tissue destruction and the release of cellular enzymes into the blood serum. Therefore, increases in these enzyme activities in the serum of O. niloticus are mainly due to the leakage of these enzymes from the liver cytosol into the bloodstream as a result of damage caused by cypermethrin, which indicates the hepatotoxic effect of toxicants. The present results are in agreement with the findings of Jee et al. (2005), who found an increase in activities of serum ALT, AST, and LDH in Korean rockfish ( Sebastes schlegeli ) exposed to cypermethrin. 5. Conclusion The present study reveals that cypermethrin exposure caused substantial changes in all evaluated biochemical and physiological parameters, highlighting the toxicological effects of this pesticide. The pesticide-treated fish demonstrated a significant reduction in red blood cells (RBC) and hemoglobin levels, perhaps signifying compromised oxygen delivery and anemia. The reduction in antioxidant enzyme activity, including SOD, CAT, MDA, GST, and elevated T-AOC implies increased oxidative stress, while the suppression of AChE signifies possible neurotoxicity. The increased levels of PGOT and PGPT indicate liver injury; in addition, the disruption of calcium homeostasis highlights the extensive impact of cypermethrin on physiological activities. The histopathological alterations indicate the systemic toxic effects of cypermethrin on essential organs, underscoring its capacity to induce physiological malfunction and enduring harm to aquatic organisms. These findings underscore the hazards of cypermethrin exposure to aquatic creatures, stressing the necessity for regulated application and the creation of safer alternatives to reduce environmental and ecological harm. Declarations Ethics approval This research work was approved by the Animal Welfare and Experimental Ethics Committee, Bangladesh Agricultural University, Mymensingh 2202 (AWEEC/BAU/2023, 18). The study complied with the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines. Funding This work was funded by the Bangladesh Agricultural University Research System (BAURES) through a research project (Project No. 2017/283/BAU) Competing interests The authors declare that they have no financial or intellectual competing interests with any person or organization. Author contributions Zakir Hossain : supervision, conceptualization, manuscript writing, reviewing, editing, and funding acquisition; Maria Binte Moin : conducting the experiment , methodology, data collection, analysis, and manuscript writing, editing; Sadia Ibnat : manuscript writing, reviewing; Shaon Kumar Mondol : manuscript writing, reviewing. 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Sancho E, Fernández-Vega C, Andreu E, Ferrando MD, Effects of propanil on the European eel Anguilla anguilla and post-exposure recovery using selected biomarkers as effect criteria, Ecotoxicology and Environmental Safety 72(3) (2009) 704–713, https://doi.org/10.1016/j.ecoenv.2008.09.008 . Saravanan M, Ramesh M, Petkam R, Alteration in certain enzymological parameters of an Indian major carp, Cirrhinus mrigala exposed to shortand long-term exposure of clofibric acid and diclofenac, Fish Physiology and Biochemistry 39(6) (2013) 1431–1440, https://doi.org/10.1007/s10695-013-9797-3 . Palanivelu V, Vijayavel K, Balasubramanian SE, Balasubramanian MP, Influence of insecticidal derivative ( cartap hydrochloride ) from the marine polycheate on certain enzyme systems of the fresh water fish Oreochromis mossambicus , Journal of Environmental Biology 26(2) (2005) 191–195. Jee JH, Masroor F, Kang JC, Responses of cypermethrin-induced stress in haematological parameters of Korean rockfish, Sebastes schlegeli (Hilgendorf), Aquaculture Research 36(9) (2005) 898–905, https://doi.org/10.1111/j.1365-2109.2005.01299.x . Additional Declarations No competing interests reported. Supplementary Files Articlehighlight.docx Proofofethicsapproval.docx Graphicalabstract.tiff Cite Share Download PDF Status: Posted Version 1 posted 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7200428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505302104,"identity":"aed992cc-3ba6-4892-a204-d8c03ce8e34e","order_by":0,"name":"Maria Binte Moin","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Binte","lastName":"Moin","suffix":""},{"id":505302105,"identity":"d9cb89b7-ba68-4bc9-afb8-1e0b64da8c31","order_by":1,"name":"Shaon Kumar Mondol","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shaon","middleName":"Kumar","lastName":"Mondol","suffix":""},{"id":505302106,"identity":"99b6ecac-237c-45a0-8b5e-8bb81a69a251","order_by":2,"name":"Sadia Ibnat","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sadia","middleName":"","lastName":"Ibnat","suffix":""},{"id":505302107,"identity":"ea3e7677-b265-431d-b2ba-a172c8c93544","order_by":3,"name":"Zakir Hossain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYFACNoYDDAxyCQzsPWAuYwMDD4MEEVqMExh4zpCghQGsRSKHSC3y7ccSD1cwGOTJR749+JmHwUZ2wwHegzfwaWHsSTtw8AyDQbHh7bxkaR6GNOMNB/iSLfBpYWZIbzjYwPAncePsHAOglsOJGw7wmOF1GBv/c5AWg8SNM88Y/+Zh+E9YC48E0GEgLfMleMyAthwgrEVC4lnCwQYDg2IDnrw0yzkGycYzDxPwi3x/mvHHhgpgiLWfPXzjTYWdbN/xXvwhBgEGQHQAygCGCJFAvoFYlaNgFIyCUTDiAADYOEme4YrPewAAAABJRU5ErkJggg==","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Zakir","middleName":"","lastName":"Hossain","suffix":""}],"badges":[],"createdAt":"2025-07-24 01:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7200428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7200428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90200610,"identity":"1b1c1553-0fc3-4bc4-9279-ae8d6acbc6b0","added_by":"auto","created_at":"2025-08-29 18:30:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":225160,"visible":true,"origin":"","legend":"\u003cp\u003ePhotographs of analysis of the changes of morphology of erythrocytes in blood smear at 7d after exposure to cypermethrin (by 100×oil immersion observation). A. Control B. Treatment 1(0.168 ppm exposure), C. Treatment 2 (exposed to 0.334 ppm), and D. Treatment 3 (exposed to 0.51 ppm). Morphological alterations such as LL (large lymphocyte), SS (small lymphocyte), SC (swelled cell), Sk (sickle cell), BE (binucleated erythrocyte), MN (micronucleated erythrocyte), M (monocyte), Tr (teardrop like cell) and NrFC (nuclear fragmented erythrocyte) were observed.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/164c7f665fef42a610f9ccf9.jpg"},{"id":90200609,"identity":"1ff19ff9-11dd-4235-93ac-e49c7c189005","added_by":"auto","created_at":"2025-08-29 18:30:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1539777,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant defence mechanism and oxidative stress biomarkers in \u003cem\u003eOreochromis niloticus\u003c/em\u003e following 7 days' exposure to cypermethrin. a. SOD-superoxide dismutase; b. CAT-catalase; c. GST-glutathione S-transferase; d. POD-peroxidase; e. MDA-malondialdehyde; f. T-AOC- total antioxidant capacity. Data has been presented as mean ± SE (one-way ANOVA test). Asterisks indicate a significant difference between treatment and control (*\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/e5772442e7371b02260c57ae.png"},{"id":90200615,"identity":"7c57ea15-39e7-4773-aafe-5bc4a7f7d899","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98109,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological examination of nile tilapia fish intestine. A. 0 ppm group revealing normal villi. B. Cypermethrin (0.168 ppm) group revealing hemorrhage (H) and absorptive vacuole (AV). C. Cypermethrin (0.334 ppm) group showing fusion of villi (FV), and distorted goblet cells (DGC). D. Cypermethrin (0.51 ppm) group exposed severe necrosis in the enterocytes with goblet cells (GC), brush border (BB), and sloughing of epithelium (SoE) was also observed\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/77b436b84cb9693ead0d47f0.jpg"},{"id":90200612,"identity":"cb0ca0b6-5c7a-46ad-b73e-dc1330edfbca","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120878,"visible":true,"origin":"","legend":"\u003cp\u003eHistological structure of the liver of nile tilapia showing histopathological alterations due to 10-day cypermethrin exposure at different concentrations. A: Liver section from a control group of fish without pesticide, showing normal liver structure; B: A liver section from the 0.168 ppm group shows melano-macrophage center (MC), hemorrhage (H); C: Lipid droplet (LD) and pyknotic nucleus (PN) was observed in this representative section from fish exposed to 0.334 ppm D: Sections from fish exposed to 0.51 ppm of cypermethrin are characterized by sinusoidal congestion (SC), vacuoler degeneration (VD) and hemorrhage (H)\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/f8493133f30a54748383ca2e.jpg"},{"id":90200636,"identity":"6b774811-fa08-4c1b-8328-1672adb479fd","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":344390,"visible":true,"origin":"","legend":"\u003cp\u003eAlteration in serum Ca2+ concentration expressed in mg/µl is represented. This alteration has also been considered a secondary toxic mechanism of pyrethroids related to osmoregulation disorders. Data has been presented as mean ± SE (one-way ANOVA test).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/eacf62dcaeb38a146887bdbd.png"},{"id":90200626,"identity":"c3f5d004-771b-455f-8e69-18e46f556b8a","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":510316,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of cypermethrin exposure on acetylcholinesterase (AChE) enzyme activity in \u003cem\u003eOreochromis niloticus \u003c/em\u003eafter 7 days. The bar graph shows AChE activity (nmol/min/mg protein) in the brain tissue of fish exposed to varying concentrations of cypermethrin (0.16, 0.34, and 0.51 ppm) compared to the control group. A significant dose-dependent decrease in AChE activity was observed in all treatment groups (*\u003cem\u003ep\u0026lt;0.05), \u003c/em\u003eindicating neurotoxicity. The top right diagram illustrates the AChE inhibition mechanism. Cypermethrin binds to the enzyme, blocking the breakdown of acetylcholine, which leads to continuous nerve stimulation and ultimately results in the organism's death. Data have been presented as mean ± S.E.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/7a7afae8df4ae3ab4e33460e.png"},{"id":90200631,"identity":"1a0c3b0d-9303-4486-9b20-9cfec57c2d5a","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":471021,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in liver enzyme biomarkers in response to cypermethrin exposure. The activities of plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) were analyzed in control and three treatment groups, whereas other hepatic biomarkers, ALT, AST, and AKP, were compared only between control and the highest treatment group (T3). All enzymes showed a dose-dependent increase in response to cypermethrin exposure when compared to the control group. Data has been presented as mean ± SE (one-way ANOVA test). Asterisks indicate a significant difference between treatment and control (*\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/4794d71fe4367bd890acb84d.png"},{"id":91472808,"identity":"1c170bdc-b2ed-443b-aa5f-421068548dce","added_by":"auto","created_at":"2025-09-16 22:31:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5109642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/39cc21a7-5e61-48b8-844c-cc9de9b1ff45.pdf"},{"id":90200618,"identity":"3f4856b9-fece-47d8-970a-1bd0082636c0","added_by":"auto","created_at":"2025-08-29 18:30:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15756,"visible":true,"origin":"","legend":"","description":"","filename":"Articlehighlight.docx","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/3254b03bf4500284da4eddea.docx"},{"id":90200955,"identity":"96a3da61-794e-420c-b413-3123a5bc2708","added_by":"auto","created_at":"2025-08-29 18:38:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14581,"visible":true,"origin":"","legend":"","description":"","filename":"Proofofethicsapproval.docx","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/0842cdaa1471ff58b43a5074.docx"},{"id":90201455,"identity":"75eef8ce-5fb9-43ba-9dbe-fbf2c3d5d43f","added_by":"auto","created_at":"2025-08-29 18:46:12","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":281995,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7200428/v1/2c5f8784e063d5bd6d3239aa.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Biomarker Responses Reveal Cypermethrin-Induced Stress and Organ Damage in Nile Tilapia: Insights into Hepatic, Neural, and Hematological Toxicity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA diverse range of pesticides is employed worldwide to enhance production and harvesting efficiency, exerting environmental pressure on ecosystems both on land and in water (Stanley \u003cem\u003eet al\u003c/em\u003e. 2016). In addition, as pests gain adaptability and resistance to pesticides, increasing quantities and newer chemical compounds are being used for crop protection each year. Around 4.1\u0026nbsp;million tonnes of chemicals were employed globally in 2018, which is predicted to increase due to the surging demands of an overgrowing population (FAO 2021). Bangladesh is no exception in this regard. In 2020, 65,142 tonnes of pesticides were applied to vegetables and crops in Bangladesh (FAO 2021). Inadequate supervision and inefficiency in farmer training have resulted in the contamination of rivers by several pesticides.\u003c/p\u003e\u003cp\u003eMost herbicides, insecticides, and fungicides lack selectivity in their targets. So, they not only interact with the targeted animals but also interfere with the molecular and cellular function of non-target species (Felix \u003cem\u003eet al.\u003c/em\u003e 2019). The indiscriminate use of chemicals leads to environmental degradation and poses serious threats to aquatic species (Velisek \u003cem\u003eet al.\u003c/em\u003e 2012). Massive concerns exist over the gradual deterioration of open-water fish stocks in Bangladesh, adversely affected by several toxicant exposures. An adverse relationship exists between the solubility of pesticide compounds and the bioaccumulation of these chemicals in fish. The exposure of aquatic organisms to synthetic pyrethroid pesticides, such as permethrin, deltamethrin, tetramethrin, and cypermethrin, may lead to substantial toxicological consequences (Coats \u003cem\u003eet al.\u003c/em\u003e 1989).\u003c/p\u003e\u003cp\u003ePyrethroids are preferred over organophosphates, carbamates, and organochlorines due to their greater efficiency, lower toxicity, and easy biodegradability (Sharaf \u003cem\u003eet al.\u003c/em\u003e 2010). Although Synthetic pyrethroids are claimed to be safe and environmentally benign due to their selective pesticidal toxicity, low persistence, and minimal toxicity to mammals and birds, they exhibit significant damage to incidental animals, including fish, lobsters, prawns, mayfly nymphs, and numerous species of zooplankton (Oudou \u003cem\u003eet al.\u003c/em\u003e 2004).\u003c/p\u003e\u003cp\u003ePyrethroids at first target the peripheral and central nervous system axons by interacting with sodium channels, so they are usually categorized as neurotoxins. It mediates the interaction between calcium ions and the intracellular membrane. Consequently, obstructing the calcium removal process from nerve terminals leads to less spontaneous neurotransmitter release (Wang 2008). Pyrethroids can enter the outer layer of the skin and bind directly with a carrier protein in the bloodstream or lymphatic system. As a result, the spread-out pyrethroids and the cells on the outer layer of the skin immediately upregulate the central nervous system by interacting with the sensory organs of the peripheral nervous system (Neal \u003cem\u003eet al.\u003c/em\u003e 2010). Additionally, pyrethroids may enter the body by inhalation in vapor form, even if in minimal amounts. Furthermore, they might infiltrate the blood or hemolymph via the digestive system during the digestive process (Neal \u003cem\u003eet al.\u003c/em\u003e 2010).\u003c/p\u003e\u003cp\u003eThe detrimental effects of pyrethroids on fish may be related to their biotransformation properties. Pyrethroid compounds are prone to being affected by the non-aqueous components of cells due to their lipophilic characteristics (Prusty \u003cem\u003eet al\u003c/em\u003e. 2015). Despite the low concentration of these chemicals in water, they may be rapidly absorbed by the gills (Moore and Waring 2001). Cypermethrin is a fourth generation halogenated synthetic pyrethroid with strong reactivity, which makes it more resistant to degradation (Jaensson \u003cem\u003eet al.\u003c/em\u003e 2007; Sharma \u003cem\u003eet al.\u003c/em\u003e 2021; Ullah \u003cem\u003eet al.\u003c/em\u003e 2018). Cypermethrin prohibits sodium ions from being transferred through the cell membrane, as they remain stable in neutral and acidic solutions (Tiwari \u003cem\u003eet al.\u003c/em\u003e 2012).\u003c/p\u003e\u003cp\u003eNile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) is a freshwater fish species that is extensively cultivated worldwide owing to its rapid development, adaptability to many settings, and significant tolerance to low water quality. It has been widely considered a model organism in toxicity, physiology, and genetics research as a bioindicator for the health of freshwater ecosystems. This study aims to assess the impact of acute cypermethrin pesticide intoxication on several hematological and biochemical markers in Nile tilapia. Comprehending pesticide toxicity in nile tilapia aids in developing environmental rules, directing sustainable pesticide use, and safeguarding aquatic biodiversity.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental animal and rearing condition\u003c/h2\u003e\u003cp\u003eHealthy \u003cem\u003eO. niloticus\u003c/em\u003e fishes of both sexes, with an initial average length and weight of 4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 cm and 6.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 g, were collected from the Bangladesh Fisheries Research Institute, Mymensingh. Only the active and healthy \u003cem\u003eO. niloticus\u003c/em\u003e were selected for acclimatization and were kept in a cistern for 7 days. The fish were fed with commercial food pellets, with water quality being monitored periodically. The fish were examined carefully for any pathological symptoms before starting the experiment. Water containing 0.1 mg/L of potassium permanganate solution was used for disinfecting purposes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Exposure study\u003c/h2\u003e\u003cp\u003eIn the initial experiment, ten fish were stocked in each aquarium of 120 L water capacity, which were filled up to 20 L. Fish were subjected to five distinct concentrations of pesticide to determine the lethal dosage of cypermethrin for \u003cem\u003eO. niloticus\u003c/em\u003e. Once the lethal dose was established, the fish was subjected to three sublethal doses, i.e., 25%, 50%, and 75% of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\)\u003c/span\u003e\u003c/span\u003e for further study. Each aquarium was provided with these concentrations of cypermethrin and was labeled as T0, T1, T2, and T3. T0 was kept as a control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{L}\\varvec{C}}_{50}\\)\u003c/span\u003e\u003c/span\u003e dose determination\u003c/h2\u003e\u003cp\u003eThe acute toxicity\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{LC}_{50}\\)\u003c/span\u003e\u003c/span\u003e of cypermethrin at 96h for \u003cem\u003eO. niloticus\u003c/em\u003e, was determined in our laboratory using the semi-static method (OECD method 2001). The nile tilapia (10 in 20 L of test medium) was exposed to five concentrations of cypermethrin to determine \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\)\u003c/span\u003e\u003c/span\u003e dose. In the short-term conclusive testing, concentrations of the test chemical varied from 0.2 ppm to 1.0 ppm, as the recommended agricultural dosage for cypermethrin 10EC was determined to be 0.86 ppm, taking into account a water volume of 20 L in the rice field. These concentrations of cypermethrin were measured with a micropipette, then put into water in a glass jar (each having three replications), and then homogeneously mixed with water using a gentle mixing motion. The test medium was replaced every 24 hours with the corresponding quantities of the toxicant, without aeration. Mortality was documented every 12 hours, with deceased fish being removed upon observation, while consistently recording the number for 96 hours to assess acute toxicity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Estimation of hematological indices\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Morphological study of erythrocytes\u003c/h2\u003e\u003cp\u003eFresh blood samples were collected from T0 (0 ppm), T1 (0.168 ppm), T2 (0.334 ppm), and T3 (0.51 ppm) groups of cypermethrin-exposed fish for evaluating erythrocytic cellular and nuclear abnormalities after 7 days of exposure. Methanol-fixed blood smears of \u003cem\u003eO. niloticus\u003c/em\u003e were stained with Wright\u0026rsquo;s Giemsa stain and analyzed using immersion oil microscopy (OLYMPUS-CX41, 400\u0026times;).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Evaluation of hematological indices\u003c/h2\u003e\u003cp\u003eTo evaluate the physiological effects of cypermethrin, hematological parameters were examined only for the control group and the highest treatment group, T3 (0.51 ppm). This selective comparison was predicated on the notion that the greatest pesticide dosage would manifest the most significant physiological changes, hence yielding greater insights into stress response. Blood was drawn from the caudal vein and heart using disposable needles and stored in Eppendorf tubes on ice. After going through centrifugation at 8000 rpm for 15 minutes, the resulting serum was stored at 4℃ for subsequent analysis. Red blood cell (RBC) and white blood cell (WBC) counts were conducted utilizing a hemocytometer (OPTIA B-350, Italy) in accordance with established dilution protocols (five \u0026micro;l of blood was diluted in 995 \u0026micro;l of RBC solution for the RBC count, and for the WBC count, same amount of blood was diluted in 195 \u0026micro;l of WBC solution). Glucose levels were assessed via a glucometer (Health Assure\u0026reg;, Taiwan), while hemoglobin (Hb) was quantified using Easy Mate\u0026reg; GHb strips.\u003c/p\u003e\u003cp\u003eMean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were computed utilizing conventional hematological equations.\u003c/p\u003e\u003cp\u003eMCHC = (Hb\u0026thinsp;\u0026divide;\u0026thinsp;PCV) \u0026times; 100;\u003c/p\u003e\u003cp\u003eMCV = (PCV\u0026thinsp;\u0026divide;\u0026thinsp;RBC) \u0026times; 10;\u003c/p\u003e\u003cp\u003eMCH (pg) = (Hb \u0026times; 10)\u0026thinsp;\u0026divide;\u0026thinsp;RBC\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Estimation of antioxidant activity\u003c/h2\u003e\u003cp\u003eAntioxidant responses were assessed solely in the T3 (0.51 ppm) and control groups (0 ppm) to ascertain potential oxidative damage resulting from maximal pesticide exposure. Blood serum samples from \u003cem\u003eO. niloticus\u003c/em\u003e was utilized to measure antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), peroxidase (POD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) after 7days. Serum was acquired by homogenizing blood in a cold phosphate buffer (pH 6.5, 0.2 M, 1:4 w/v), and afterwards subjected to centrifugation at 10,000 rpm for 15 minutes at 4\u0026deg;C. The measurement of SOD activity was conducted using the Giannopolitis and Ries approach, which relies on the photoreduction of NBT (Giannopolitic and Ries 1977). The CAT activity was assessed according to Shangari and O\u0026rsquo;Brien by observing H₂O₂ degradation at 240 nm (Shangari and Brien 2006). The GST activity was assessed via Mannervik\u0026rsquo;s approach by observing the production of the GSH-CDNB conjugate at 340 nm (Mannervik \u003cem\u003eet al.\u003c/em\u003e 1985). POD activity was evaluated from serum and acetone powder using a phosphate buffer extraction, and subsequently analyzed via spectrophotometry. T-AOC was quantified by the reduction of Fe\u0026sup3;⁺, with absorbance measured at around 520 nm. MDA concentrations were quantified using a TBA test at 532 nm following serum treatment with TBA reagent and subsequent centrifugation, with values derived from a standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Histological examination of liver and intestine\u003c/h2\u003e\u003cp\u003e\u003cem\u003eO. niloticus\u003c/em\u003e were subjected to three doses (T1, T2, and T3) of cypermethrin for 10 days in glass aquaria, alongside a control group in water devoid of pesticide (T0). Liver and intestinal samples were obtained from all groups and stored in 10% neutral buffered formalin. Tissues underwent dehydration, clearing, and infiltration by an automated tissue processor (SHANDON, CITADEL 1000) adhering to a typical 21-hour protocol utilizing alcohol, xylene, and paraffin. Tissues were processed, embedded in paraffin, sectioned at 5 \u0026micro;m with a Leica JUNG RM 2035 microtome, and affixed to glass slides. Sections were stained with hematoxylin and eosin (H\u0026amp;E) using usual techniques and viewed under a light microscope for cellular abnormalities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Serum calcium (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{C}\\varvec{a}}^{2+}\\)\u003c/span\u003e\u003c/span\u003e) analysis\u003c/h2\u003e\u003cp\u003eSerum calcium level was quantified in three treatment groups relative to the control group using method developed by (Reza \u003cem\u003eet al.\u003c/em\u003e 2013) refined from (WHO 2006). Blood was extracted from the caudal veins of fish after 7 days of exposure, and serum was isolated using centrifugation (10,000\u0026times;g, 15 minutes, 4\u0026deg;C). A 2000 \u0026micro;l working reagent comprising 8-hydroxyquinoline, O-cresol phthalein complexone, and distilled water was combined with 100 \u0026micro;l of serum. Absorbance was quantified at 540 nm utilizing a Spectronic Genesys TM5 spectrophotometer, whilst calcium concentrations were ascertained by a reference curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.8. Stress enzyme activity measurement\u003c/b\u003e\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. Acetylcholinesterase enzyme (AChE) activity measurement of cypermethrin-treated fish\u003c/h2\u003e\u003cp\u003e\u003cem\u003eO. niloticus\u003c/em\u003e were subjected to three sub-lethal concentrations of cypermethrin for 7 days. During sample day, three fish were sacrificed, and brain tissues were harvested, weighed, and homogenized in sodium phosphate buffer (pH 8.0) to attain a concentration of 20 mg/ml. The homogenate underwent centrifugation at 2000 rpm for 10 minutes at 4\u0026deg;C, and the pellet was subsequently rinsed with 0.15 M KCl. The tissue was re-homogenized in a 10% w/v Tris-HCl buffer (0.1 M, pH 7.4) and subjected to centrifugation at 5000 rpm for 10 minutes.\u003c/p\u003e\u003cp\u003eThe supernatant was utilized for acetylcholinesterase estimation. A reaction mixture comprising 2.7 ml of phosphate buffer, 100 \u0026micro;l of supernatant, and 100 \u0026micro;l of Ellman\u0026rsquo;s reagent (0.16 mM DNTB) was incubated at 37\u0026deg;C for 10 minutes. The reaction commenced with the addition of 100 \u0026micro;l of acetylcholine iodide. Absorbance was measured at 412 nm. The total protein was quantified using 10% trichloroacetic acid, employing bovine serum albumin as the reference. Results were quantified as nmol/min/mg protein, utilizing the formula:\u003c/p\u003e\u003cp\u003eR\u0026thinsp;=\u0026thinsp;5.74 (10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) ΔA/C\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eWhere, R\u0026thinsp;=\u0026thinsp;rate in moles substrate hydrolyzed per min per g of tissue;\u003c/p\u003e\u003cp\u003eΔA\u0026thinsp;=\u0026thinsp;change in absorbance per min;\u003c/p\u003e\u003cp\u003eC\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;original concentration of tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) analysis\u003c/h2\u003e\u003cp\u003eFor PGOT and PGPT assays, \u003cem\u003eO. niloticus\u003c/em\u003e were subjected similarly to three sub-lethal dosages of cypermethrin for 7 days. Blood was obtained from the caudal vein, and plasma was isolated using centrifugation at 11,000 rpm for 15 minutes. Enzyme activity was assessed according to the methodology of Reitman and Frankel (Reitman and Frankel 1957). A phosphate buffer (0.1 M, pH 7.4) was formulated, to which 2 mM α-ketoglutaric acid and 200 mM dl-aspartate were added for PGOT. For PGPT, 2 mM α-ketoglutaric acid and 200 mM dl-alanine were used. One milliliter of each substrate solution was pre-incubated at 40\u0026deg;C for 10 minutes, followed by the addition of 0.2 milliliters of plasma. The reaction was conducted for 60 minutes (PGOT) or 30 minutes (PGPT), and thereafter terminated with 1 ml of 2,4-dinitrophenylhydrazine reagent (1 mM in 1N HCl). After 20 minutes, 10 ml of 0.4 N NaOH was introduced, and the absorbance was measured at 505 nm after 30 minutes. A standard curve was established utilizing pyruvate and α-ketoglutarate. The enzyme activity (U/ml) was determined using:\u003c/p\u003e\u003cp\u003eA\u0026thinsp;=\u0026thinsp;C\u0026times;V / m\u003c/p\u003e\u003cp\u003eHere,\u003c/p\u003e\u003cp\u003eA\u0026thinsp;=\u0026thinsp;activity of the material in units per mass of material\u003c/p\u003e\u003cp\u003eC\u0026thinsp;=\u0026thinsp;desired concentration of the final solution.\u003c/p\u003e\u003cp\u003eV\u0026thinsp;=\u0026thinsp;final or total volume of the solution\u003c/p\u003e\u003cp\u003em\u0026thinsp;=\u0026thinsp;mass of the solute dissolved in the solution\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Liver enzyme analysis\u003c/h2\u003e\u003cp\u003eLiver enzymes in the highest treatment group (T3) and control group (T0) were analyzed following the instructions of the Nanjing Jiancheng Bioengineering Institute (China) kit protocol after 7 days of exposure. To measure aspartate aminotransferase (AST) activity, the blood sample was added to the reaction mixture containing aspartate and α-ketoglutarate. The mixture was incubated at 37\u0026deg;C for a set time. After that, a color-developing reagent, 2,4-dinitrophenylhydrazine, to detect oxaloacetate was added. The absorbance at the specified wavelength (typically 505 nm) was measured. It is usually expressed in U/L. Alanine aminotransferase (ALT) Activity measurement was done in the same process however, absorbance was measured at 510nm. To determine alkaline phosphatase (AKP) activity, the sample was mixed with the substrate solution containing p-nitrophenyl phosphate and incubated at 37\u0026deg;C. The reaction was stopped by typical sodium hydroxide. Absorbance was measured at 405 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Water quality analysis\u003c/h2\u003e\u003cp\u003eWater quality parameters were measured using specific instruments: Temperature was recorded with a Celsius thermometer, a multi-parameter DO meter (Multi 340 Iset, DO-5509; China) used for dissolved oxygen, a digital pH meter (HANNA-HI98107 pHep\u0026reg;, Romania) for pH, and a portable TDS meter (HANNA-HI98302 DiST\u0026reg;2, Romania) for TDS. In addition, ammonia and total alkalinity levels were evaluated every three days utilizing the API\u0026reg; ammonia and alkalinity test kit (HANNA-HI3811, Romania) throughout the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe probit analysis was performed considering \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 statistical significance, as well as the one-way variance analysis (ANOVA) was performed by using SPSS ver. 26.0 computer software program.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result","content":"\u003cp\u003eThe water quality parameters in the cypermethrin-treated aquariums were maintained within acceptable ranges throughout the experiment. The recorded values for temperature, pH, DO, total alkalinity, and ammonia were 29.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67℃, 8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49, 4.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 mg/L, 108.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 mg/L, and 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mg/L, respectively. These parameters remained stable, ensuring no external environmental stress influenced the observed physiological responses.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{L}\\varvec{C}}_{50}\\)\u003c/span\u003e\u003c/span\u003e of cypermethrin 10EC for \u003cem\u003eOreochromis niloticus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eIn order to study the acute 96h toxicity of cypermethrin, ten \u003cem\u003eO. niloticus\u003c/em\u003e fish were exposed to five different pesticide concentrations (0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm, and 1.0 ppm). A control group without pesticide was maintained. No mortality was observed at 0.2 ppm, whereas 100% mortality occurred at 1.0 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cypermethrin's calculated 96h acute \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\:\\)\u003c/span\u003e\u003c/span\u003evalue (95% confidence limits) using a static bioassay system to nile tilapia (\u003cem\u003eO. niloticus\u003c/em\u003e) was found to be 0.668 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eAcute 96h toxicity of cypermethrin in \u003cem\u003eOreochromis niloticus\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eTotal mortality\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e96h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.2 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.4 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.6 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.8 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.0 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\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=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2. The effects of cypermethrin pesticide on the hematological changes of \u003cem\u003eO. niloticus\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Morphological alterations of erythrocytes of fish blood upon cypermethrin exposure\u003c/h2\u003e\u003cp\u003eSeveral erythrocyte abnormalities in nile tilapia fish were observed. As the doses of cypermethrin increased, so did these abnormalities' frequency and severity. The control group showed normal, regular-shaped erythrocytes. However, even at the lowest dose of cypermethrin exposure, abnormalities like large lymphocytes, small lymphocytes, swollen cells, and sickle cells were observed. Fish exposed to 0.334 ppm (T2) showed nuclear abnormalities, including binucleated erythrocytes and micronuclei. At the highest dose (T3), cypermethrin exposure revealed monocytes, teardrop-like cells, and nuclear-fragmented erythrocytes were revealed from blood samples of fish. The frequencies of erythrocyte nuclear abnormalities followed an increasing trend with increasing cypermethrin concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Hematological parameters\u003c/h2\u003e\u003cp\u003eThe highest treatment group, T3 (0.51 ppm), was compared with the control group to identify any kind of changes in the blood parameters. A significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) in RBC count was found in the treated group 3. The hemoglobin level of the highest pesticide-treated fish was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the control group, and was 9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g/dl and 12.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g/dl, respectively, in the treatment and control groups, supporting the result of RBC count. On the contrary, the WBC count of T3 was recorded as significantly greater than the control value. Additionally, the T3 group exhibited significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) total serum protein and blood glucose levels than the control group. The mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) showed a significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/em\u003e in T3-treated fish compared to the control group. Conversely, hematocrit and mean corpuscular hemoglobin concentration (MCHC) were significantly elevated (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/em\u003e in the T3 group. The results of the hematological indices are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHematological parameters of \u003cem\u003eOreochromis niloticus\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl (M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTreatment (M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRBC (\u0026times;10\u003csup\u003e6\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWBC (\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e210.97\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e320.62\u0026thinsp;\u0026plusmn;\u0026thinsp;9.22**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 *\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHematocrit (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e30.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCH (Pg/cell)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e54.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e51.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCV (fl/cell)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e140.47\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e138.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCHC (g/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e34.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucose (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerum protein (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e34.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e* (Asterisk) indicates significant difference (* \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control and ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs control)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Antioxidant activity\u003c/h2\u003e\u003cp\u003eFish evolved enzymatic and non-enzymatic antioxidant defense mechanisms to neutralize reactive oxygen species (ROS) produced during cellular metabolism or due to exposure to environmental contaminants. SOD, CAT, POD, GST, and MDA activities of \u003cem\u003eO. niloticus\u003c/em\u003e serum increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) in highest concentration (T3) group of fish compared to the control group (Fig.\u0026nbsp;4.3) However, the GST level was the highest, 286.77\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36 \u0026micro;mol in the treatment 3 group among the all-tested antioxidant enzymes. The total antioxidant activity (T-AOC) level was found to be 6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 U/ml in treatment group 3, which was significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) lower than the control value of 21.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 U/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Histopathological changes in pesticide-treated fish\u003c/h2\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Histopathological changes in the intestine\u003c/h2\u003e\u003cp\u003eThe histology of the intestine of fish treated with three doses of cypermethrin was compared to the control group. Intestinal changes such as lamina propria alterations, hemorrhage, and absorptive vacuoles were found in fish treated with 0.168 ppm cypermethrin (T1). As the concentration of cypermethrin escalated, the incidence of changes correspondingly rose. The presence of a cracked clay appearance, fusion of villi, and distorted goblet cells was observed in the 0.334 ppm treated group (T2). At the highest exposure dose (T3), severe necrosis in the enterocytes, along with goblet cell loss, sloughing of the epithelial layer, and damage to the brush border were seen. In contrast, the control group exhibited well-structured epithelial cells, long and intact villi, and a clear epithelial lining was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Histopathological changes in liver\u003c/h2\u003e\u003cp\u003eThe liver is the primary detoxification organ in fish, so it was heavily affected by toxicant exposure. Liver showed the following histopathological changes in the treated groups. These changes compromised the liver\u0026rsquo;s ability to detoxify and regulate metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effect of cypermethrin in serum \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{C}\\varvec{a}}^{2+}\\)\u003c/span\u003e\u003c/span\u003e\u003c/h2\u003e\u003cp\u003eFor the assessment of serum \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e, Fish were exposed to three sublethal doses of pesticide. Increased doses of pesticide resulted in a decline in \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e level. In the control group, the calcium level was 10.149\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042 mg/\u0026micro;l, whereas in T1, T2, and T3 the value was, respectively, 9.663\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1985 mg/\u0026micro;l, 8.549\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1557 mg/\u0026micro;l, and 6.213\u0026thinsp;\u0026plusmn;\u0026thinsp;0.372 mg/\u0026micro;l, which are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Effects of cypermethrin on neurotransmitters and stress-indicating enzymes\u003c/h2\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.6.1 Acetylcholinesterase (AChE) activity\u003c/h2\u003e\u003cp\u003eAcetylcholinesterase (AChE) is an important bioindicator of neurotoxicity. To assess AChE activity in the brain, \u003cem\u003eO. niloticus\u003c/em\u003e was exposed to three different dosages of pesticide. This result indicated that exposure to cypermethrin at such sublethal doses was a source of stress to \u003cem\u003eO. niloticus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.6.2 Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) level\u003c/h2\u003e\u003cp\u003eThe PGOT and PGPT of blood serum are used as biomarkers for liver function and stress in organisms, particularly in fish when exposed to toxicants. At greater doses of cypermethrin, their level rises in response to cellular damage in the liver and muscles. As a consequence, the values of PGOT in the treatment groups were significantly different (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eOn the other hand, the level of PGPT was as follows 477.847\u0026thinsp;\u0026plusmn;\u0026thinsp;15.319 U/ml, 518.33\u0026thinsp;\u0026plusmn;\u0026thinsp;13.018 U/ml, and 539\u0026thinsp;\u0026plusmn;\u0026thinsp;12.124U/ml in T0, T1, T2 and T3, respectively which was also significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) different from the PGPT level as 208.667\u0026thinsp;\u0026plusmn;\u0026thinsp;33.65 U/ml in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Liver enzyme analysis\u003c/h2\u003e\u003cp\u003eLiver enzymes like ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and AKP (Alkaline Phosphatase) were measured in the highest treatment group and the control group The ALT, AST, and AKP levels in treatment group 3 were significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) higher compared to the control group. AST level was 60.11 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 4.99 U/L and 85.38 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 6.11 U/L in control and treatment 3, respectively. AKP value was the lowest among these three liver enzymes. In the control group, the value of AKP was 40.01 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 3.65 U/gprotein, which increased upon cypermethrin exposure and reached 56.45 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 4.00 U/gprotein in the T3 group. Among the three liver enzymes, the ALT value was the highest in the treatment group. ALT value was 112.39 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 5.75 U/L in the control group, which increased and reached 149.21\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 7.45 U/L in treatment 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Acute toxicity test of cypermethrin for \u003cem\u003eO. niloticus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eWith a 1600 cubic inch area in mind, the appropriate dosage of cypermethrin 10EC was 0.86 ppm. Nevertheless, fish treated with lower doses than advised also experienced physiologic effects, leading to hyperactivity and even fish death. In the current study, findings showed that 0.668 ppm of cypermethrin is the lethal level for \u003cem\u003eO. niloticus\u003c/em\u003e after 96 hours of exposure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Kenthao \u003cem\u003eet al\u003c/em\u003e. 2020 revealed that the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\:\\)\u003c/span\u003e\u003c/span\u003e for cypermethrin-exposed nile tilapia, is 3.24 \u0026micro;g/l at 96 h and 4.23 \u0026micro;g/l at which 70% mortality occurs. The \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50\\:\\:}\\)\u003c/span\u003e\u003c/span\u003eof cypermethrin for tilapia was 2.2 \u0026micro;g/l (Bradbury and Coats 1989). These results are higher than the present study's findings. Chemical composition, exposure conditions, water quality, size, and age affect the toxicity of cypermethrin. The majority of cypermethrin products consist of a combination of its isomers. The mixture's composition, which is the ratio of \u003cem\u003ecis\u003c/em\u003e and \u003cem\u003etrans\u003c/em\u003e isomers, determines the effectiveness of cypermethrin. Yuniari \u003cem\u003eet al.\u003c/em\u003e (2016) identified that at a concentration of 0.042 ppm, 0.065 ppm, and 0.087 ppm, death of the fish starts happening, with up to 70% mortality at a concentration of 0.087 ppm, when the highest mortality was observed. This discrepancy highlights the complex relationship between toxicants' properties and the reaction of an organism's biology. The decrease in fish survival at the highest dose is ascribed to their incapacity to acclimate to environmental contaminants. As a result, the fish are unable to mitigate the harmful impacts of the pollutants in the testing media.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Effects of cypermethrin pesticide on the hematological changes in \u003cem\u003eO. niloticus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eHematological changes are often the first identifiable and measurable reactions to environmental modifications (Wendelaar 1997). Hematological profiles can shed light on the organism's internal environmental condition.\u003c/p\u003e\u003cdiv id=\"Sec35\" class=\"Section3\"\u003e\u003ch2\u003e4.2.1 Morphological alterations of erythrocytes in cypermethrin-treated fish\u003c/h2\u003e\u003cp\u003eThe current investigation detected many morphological changes in blood erythrocytes. These modifications demonstrate the reduced erythrocyte levels resulting from the harmful effects of pesticides on hematopoietic tissues and circulating erythrocytes. Several morphological alterations, including teardrop-shaped cells, sickle cells, swollen cells, monocytes, and large and small lymphocytes, were seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The formation of micronuclei and lobed nucleus erythrocytes in the current study is in line with the findings of Hussain \u003cem\u003eet al\u003c/em\u003e. (2014). These Alterations may come from increased synthesis of caspase-activated DNAase. It leads to the formation of cleavage of cytoskeletal proteins (vimentin, gelsolin, and fodrin) and nuclear proteins owing to oxidative stress affecting the mitochondria (Hussain \u003cem\u003eet al.\u003c/em\u003e 2014; Hussain \u003cem\u003eet al.\u003c/em\u003e 2012; Campos \u003cem\u003eet al.\u003c/em\u003e 2012). The cytopathogenic changes may occur from elevated lipid peroxidation, leading to enhanced production of intracellular reactive oxygen and nitrogen species [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous research has suggested that nuclear changes in erythrocytes may result from chromosomal aberrations caused by several hazardous chemicals (Hussain \u003cem\u003eet al.\u003c/em\u003e 2012). The lobed and bilobed nuclei of erythrocytes in the present research may result from the disruption of tubulin polymerization, oxidation of mRNA, and nitration of proteins, inhibiting intracellular metabolism (Hussain \u003cem\u003eet al.\u003c/em\u003e 2014; Campos \u003cem\u003eet al.\u003c/em\u003e 2012)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section3\"\u003e\u003ch2\u003e4.2.2 Hematological indices\u003c/h2\u003e\u003cp\u003eCypermethrin exists as methyl ethyl carboxylate. It interacts with Fe in hemoglobin in the alkylation process, potentially substituting oxygen in blood cells (Ajani and Awogbade 2012), which leads to decreased erythrocytes. While White blood cells are the primary constituents that protect the organism during injury, hemorrhage, and the transit of foreign antigens into the body (Vermurugan \u003cem\u003eet al.\u003c/em\u003e 2016). As a result, an increase in the WBC count is observed as a response to hypersensitivity of immune cells.\u003c/p\u003e\u003cp\u003eHematological parameters and recovery pattern were studied in \u003cem\u003eLabeo rohita\u003c/em\u003e upon exposure to cypermethrin and carbofuran by(Adhikari \u003cem\u003eet al.\u003c/em\u003e 2004). It resulted in significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower values for erythrocyte count (RBC), hemoglobin content (Hb), and hematocrit when compared with the control group. In contrast, there was a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in leukocyte count (TLC) in the pesticide-treated group. MCV and MCH decreased in response to both pesticides during their study. Significant changes in the blood components of \u003cem\u003eL. rohita\u003c/em\u003e upon treatment with cypermethrin were noted in the findings ofKhan \u003cem\u003eet al.\u003c/em\u003e (2018) which correspond to the present study's findings. These changes included a reduction in the Hb level, HCT, MCV, MCH, and MCHC. It can be concluded that cypermethrin intoxication resulted in elevated levels of certain blood components that fight against toxicants, such as the number of white blood cells (\u0026times;10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e) and red blood cells (\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e). Conversely, the decrease in hemoglobin and several other blood constituents can result from the suppression of hemoglobin formation, osmoregulatory impairment, and the death of erythrocytes in hematopoietic organs, as previously found in \u003cem\u003eCatla catla\u003c/em\u003e (Vani \u003cem\u003eet al.\u003c/em\u003e 2011). These reductions might be attributed to erythroblastosis, causing anemia (Saleh and Marie 2016).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Antioxidant enzyme activity\u003c/h2\u003e\u003cp\u003eAntioxidant enzymes assist fish in managing environmental stressors by modulating their activity, with the degree of this modulation determined by the stressor's strength, the species, and the exposure method. Increased lipid peroxidation can affect the activities of several protective enzymatic and nonenzymatic antioxidants, which are well known as the bio-indicators of increased oxidation. Superoxide dismutase (SOD) and catalase (CAT) function as a principal defense mechanism, safeguarding biological macromolecules from oxidative harm. SOD, a category of metalloenzymes, serve as the primary barrier against the detrimental effects of superoxide radicals in aerobic organisms (Kohlen and Nyska 2002). When reactive oxygen species start generating, SOD tries to neutralize them. In such a condition, an enhancement in its activity is observed as an induced adaptive response. CAT is an enzyme located in peroxisomes and facilitates the removal of hydrogen peroxide \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{H}_{2}{O}_{2}\\)\u003c/span\u003e\u003c/span\u003e, which is metabolized to molecular oxygen and water (Van \u003cem\u003eet al\u003c/em\u003e. 2003; Yilmaz \u003cem\u003eet al.\u003c/em\u003e 2006). The SOD intervenes in the first transformation by disputing the superoxide free radicals \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{O}_{2}\\:\\)\u003c/span\u003e\u003c/span\u003einto \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{H}_{2}{O}_{2}\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas CAT converts it into \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{H}_{2}O\\:\\)\u003c/span\u003e\u003c/span\u003eand\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{O}_{2}\\)\u003c/span\u003e\u003c/span\u003e (Dorval \u003cem\u003eet al.\u003c/em\u003e 2003). The current work suggests that the activation of SOD activity in hepatic tissue may result from increased \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{O}_{2}\\:\\)\u003c/span\u003e\u003c/span\u003eproduction which is regarded as the primary defense mechanism against oxidative stress. Similar findings have been reported in various fish models exposed to organophosphate pesticides. In addition, \u0026Uuml;ner \u003cem\u003eet al\u003c/em\u003e. (2001) demonstrated that malondialdehyde (MDA) increased in fish liver and kidney following cypermethrin exposure. On the other hand, in a study carried out in \u003cem\u003eChanna punctatus\u003c/em\u003e,Sayeed \u003cem\u003eet al.\u003c/em\u003e (2003) reported that deltamethrin exposure increased MDA levels in the fish liver, kidney, and gills. In our investigation, the increased MDA levels may be ascribed to the production of free radicals after cypermethrin introduction, indicating a possible association between cypermethrin toxicity and lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The upwards peroxidase (POD) activity detected in the liver may correlate with its function in the detoxification of harmful substances. The generation of ROS during the biotransformation of toxicants can cause cellular damage via oxidative mechanisms (Van \u003cem\u003eet al.\u003c/em\u003e 2003). The POD activity scavenges the ROS by converting hydrogen peroxide (Hinton \u003cem\u003eet al.\u003c/em\u003e 2008). The increased GST activity may be ascribed to the augmented metabolism of lipoperoxides produced during the Fenton reaction or the biotransformation of toxicants, indicating an adaptive response in fish.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Histopathological changes\u003c/h2\u003e\u003cdiv id=\"Sec39\" class=\"Section3\"\u003e\u003ch2\u003e4.4.1 Histopathological changes observed in the intestine\u003c/h2\u003e\u003cp\u003eThe intestinal tract is a crucial component of the fish digestive system, significantly contributing to the digestion and absorption of nutrients. It is extremely responsive to any absorbed hazardous substances and serves as a significant biomarker organ for evaluating ecotoxicology. In the study, Khan \u003cem\u003eet al.\u003c/em\u003e (2018) noticed changes in intestinal tissues of \u003cem\u003eL. rohita\u003c/em\u003e, such as predominantly necrosis, hemorrhages, overproduction of goblet cells in villi, fusion, detachment, and shortening of villi, which are similar to the report reported earlier by Hasan \u003cem\u003eet al\u003c/em\u003e. (2015) for acute endosulfan toxicity. The deterioration of villi, mucosal folds disintegration, vacuolations, hypertrophy, and necrosis in \u003cem\u003eC. carpi\u003c/em\u003eo and \u003cem\u003eCirrhinus mrigala\u003c/em\u003e treated with atrazine and fenvalerate was observed (Velmurugan \u003cem\u003eet al.\u003c/em\u003e 2016). Severe mucosal secretion occurs due to distress, enabling fish to cope with ecological stress (Samanta \u003cem\u003eet al.\u003c/em\u003e 2016). Abnormal histopathological alterations, for example, shortening of villi with inflammation, rupture of cells, degeneration changes in tips of villi, curved villi, hemorrhage, necrosis, numerous vacuoles, dilation in the blood vessels, completely damaged villi, and loss of architecture in a number of fish species were also identified (Velmurugan \u003cem\u003eet al\u003c/em\u003e 2016; Cengiz and Unlu 2006) The findings of the present study, including degenerative enterocytes, hemorrhage, absorptive vacuole, cracked clay appearance, fusion of villi, and hemorrhage in the lamina propria area, are consistent with the study of the above findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003e4.4.2 Histopathological changes observed in the liver\u003c/h2\u003e\u003cp\u003eThe liver is an essential organ that facilitates the metabolism of carbohydrates, proteins, and lipids, in addition to detoxifying toxic compounds. The buildup of pesticides and their metabolites in hepatocytes frequently results in considerable histological changes and structural modifications in the organ (Sharma \u003cem\u003eet al.\u003c/em\u003e 2012). In the current investigation, the melano-macrophage center, focal area of necrosis, hemorrhage, and pyknosis were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Edema around the hepatocyte and focal areas in between them were observed. Biliary duct epithelial detachment and vacuolar degeneration were also seen. Similar observations were noted, such as dissolution of the cell membrane, blood mobbing and congestion, pyknosis, necrosis, hyperplasia, and vacuolations of hepatocytes in \u003cem\u003eL. rohita\u003c/em\u003e after exposure to cypermethrin (Saleh and Marie 2016; Murussi \u003cem\u003eet al\u003c/em\u003e. 2016). The modifications of liver revealed in the present trials are in accordance with the results in Nile tilapia, i.e., \u003cem\u003eO. niloticus\u003c/em\u003e (Coimbra \u003cem\u003eet al.\u003c/em\u003e 2007), and rainbow trout, i.e., \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e treated with varying concentrations of endosulfan (Authman \u003cem\u003eet al.\u003c/em\u003e 2015), Common carp treated with chlorpyrifos, \u003cem\u003eC. carpio\u003c/em\u003e treated with buprofezin fipronil (Pal \u003cem\u003eet al.\u003c/em\u003e 2012), \u003cem\u003eC. catla\u003c/em\u003e exposed to α-cypermethrin (Muthuviveganandavel \u003cem\u003eet al.\u003c/em\u003e 2013)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Serum \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{\\varvec{C}\\varvec{a}}^{2+}\\)\u003c/span\u003e\u003c/span\u003eactivity\u003c/h2\u003e\u003cp\u003eThis study observed reduced serum calcium levels in fish exposed to cypermethrin. This is supported by earlier investigators' reports of reduced blood/plasma calcium levels in fish exposed to toxicants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The decline indicates exhaustion of the Ca\u003csup\u003e2+\u003c/sup\u003e depots and their reduced uptake from gills and kidney tissues (Velmurugan \u003cem\u003eet al.\u003c/em\u003e 2016; Cengiz and Unlu 2006). Cypermethrin in the ambient medium comes in direct contact with the gills and ruptures the chloride cell membrane. Injury to the gill epithelium hinders Ca\u003csup\u003e2+\u003c/sup\u003e absorption from the ambient water, resulting in hypocalcaemia and respiratory discomfort, accompanied by hyperexcitability and body tremors in \u003cem\u003eHeteropneustes fossilis\u003c/em\u003e (Pandey \u003cem\u003eet al.\u003c/em\u003e 2009)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section2\"\u003e\u003ch2\u003e4.6 Effects of cypermethrin on neurotransmitter and stress-indicating enzyme\u003c/h2\u003e\u003cdiv id=\"Sec43\" class=\"Section3\"\u003e\u003ch2\u003e4.6.1 Acetylcholinesterase (AChE) activity\u003c/h2\u003e\u003cp\u003eAssessing AChE activity in the environment may offer considerable benefits compared to exclusive dependence on analytical chemistry. Pesticides inhibit the function of cholinesterase enzymes, which play a major role in the hydrolysis of the neurotransmitter acetylcholine (ACh), enabling its elimination from the synaptic cleft (Mineau 1991). Acetylcholine functions as both a preganglionic and postganglionic neurotransmitter in the parasympathetic nervous system and as a preganglionic neurotransmitter in the sympathetic nervous system. Inhibition of cholinesterase by toxicants leads to the accumulation of acetylcholine at the nerve synapse, hence compromising normal nervous system function (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This produces rapid twitching of voluntary muscles, followed by paralysis (Mineau 1991). Once bound, pesticides are considered irreversible inhibitors, as recovery usually depends on new enzyme synthesis, AChE (Fulton and Key 2001). The inhibition of AChE is the principal mechanism by which organophosphorus pesticides manifest their toxicity, so linking this biomarker directly to the compound's harmful mode of action (Fulton and Key 2001). In the present study, a significant decline in the AChE enzyme was noted in comparison to the control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section3\"\u003e\u003ch2\u003e4.6.2 Plasma glutamic oxaloacetic transaminase (PGOT) and plasma glutamic pyruvic transaminase (PGPT) level\u003c/h2\u003e\u003cp\u003eGlutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) are essential for mobilizing L-amino acids for gluconeogenesis and a pivotal connection between carbohydrate and protein metabolism during altered physiological, pathological, and environmental stress conditions (Ramaswamy \u003cem\u003eet al.\u003c/em\u003e 1999). In the present study, activities of GOT and GPT were found to be significantly elevated in \u003cem\u003eO. niloticus\u003c/em\u003e irrespective of concentrations of cypermethrin when compared with the control. The observed increase in GOT activity indicates that molecular rearrangements involving amino acids, linked to the citric acid cycle at two junctures\u0026mdash;oxaloacetate and α-ketoglutaric acid\u0026mdash;were modified. Similarly, the increase in GPT indicates that the exposed fish required intensive glycogenesis to cope with the energy crisis (Bhavan \u003cem\u003eet al.\u003c/em\u003e 2015). It has also been suggested that stress leads to elevation of the transamination pathways (Li \u003cem\u003eet al.\u003c/em\u003e 2011). These results have also been evident from the histopathological observations of the liver cells. Furthermore, modifications in gill and blood cells may exacerbate the condition by hindering the respiratory processes of the test organism. In reaction to toxic stress, the organism compensates by raising its metabolic rate. Nonetheless, given that the liver functions as the primary organ for the metabolism of vital macromolecules in vertebrates, any impairment to the hepatic system might result in considerable physiological and biochemical disruptions, including changes in GOT and GPT activity (Egnatchik \u003cem\u003eet al.\u003c/em\u003e 2019; Malarvizhi \u003cem\u003eet al.\u003c/em\u003e 2012; Sancho \u003cem\u003eet al.\u003c/em\u003e 2009)Extended exposure to, or elevated levels of, organophosphate compounds may further inhibit GOT and GPT activities by interfering with cellular processes, finally resulting in fish mortality (Saravanan \u003cem\u003eet al.\u003c/em\u003e 2013).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section2\"\u003e\u003ch2\u003e4.7. Liver enzyme activity\u003c/h2\u003e\u003cp\u003eDuring injury or liver damage, the cells are destroyed, which results in the release of ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and AKP (Alkaline Phosphatase) into plasma, and their high concentration in plasma is considered an indicator of abnormal physiology. An increase in their concentrations shows stress-based tissue impairment (Palanivelu \u003cem\u003eet al.\u003c/em\u003e 2005). The findings of ALT, AST, and AKP often suggest degenerative alterations and diminished liver function, since the toxicant adversely affects hepatocytes, leading to tissue destruction and the release of cellular enzymes into the blood serum. Therefore, increases in these enzyme activities in the serum of \u003cem\u003eO. niloticus\u003c/em\u003e are mainly due to the leakage of these enzymes from the liver cytosol into the bloodstream as a result of damage caused by cypermethrin, which indicates the hepatotoxic effect of toxicants. The present results are in agreement with the findings of Jee \u003cem\u003eet al.\u003c/em\u003e (2005), who found an increase in activities of serum ALT, AST, and LDH in Korean rockfish (\u003cem\u003eSebastes schlegeli\u003c/em\u003e) exposed to cypermethrin.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study reveals that cypermethrin exposure caused substantial changes in all evaluated biochemical and physiological parameters, highlighting the toxicological effects of this pesticide. The pesticide-treated fish demonstrated a significant reduction in red blood cells (RBC) and hemoglobin levels, perhaps signifying compromised oxygen delivery and anemia. The reduction in antioxidant enzyme activity, including SOD, CAT, MDA, GST, and elevated T-AOC implies increased oxidative stress, while the suppression of AChE signifies possible neurotoxicity. The increased levels of PGOT and PGPT indicate liver injury; in addition, the disruption of calcium homeostasis highlights the extensive impact of cypermethrin on physiological activities. The histopathological alterations indicate the systemic toxic effects of cypermethrin on essential organs, underscoring its capacity to induce physiological malfunction and enduring harm to aquatic organisms. These findings underscore the hazards of cypermethrin exposure to aquatic creatures, stressing the necessity for regulated application and the creation of safer alternatives to reduce environmental and ecological harm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was approved by the\u0026nbsp;Animal Welfare and Experimental Ethics Committee, Bangladesh Agricultural University, Mymensingh 2202 (AWEEC/BAU/2023, 18).\u0026nbsp;The study complied with the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Bangladesh Agricultural University Research System (BAURES) through a research project (Project No. 2017/283/BAU)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or intellectual competing interests with any person or organization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZakir Hossain\u003c/strong\u003e: supervision, conceptualization, manuscript writing, reviewing, editing, and funding acquisition;\u0026nbsp;\u003cstrong\u003eMaria Binte Moin\u003c/strong\u003e: conducting the experiment\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003emethodology, data collection, analysis, and manuscript writing, editing; \u003cstrong\u003eSadia Ibnat\u003c/strong\u003e: manuscript writing, reviewing; \u003cstrong\u003eShaon Kumar Mondol\u003c/strong\u003e: manuscript writing, reviewing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript and agreed to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStanley J, Preetha G, Pesticide Toxicity to Fishes: Exposure, Toxicity and Risk Assessment Methodologies, \u003cem\u003ePesticide Toxicity to Non-Target Organisms\u003c/em\u003e (2016) 411\u0026ndash;497, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-94-017-7752-0_7\u003c/span\u003e\u003cspan address=\"10.1007/978-94-017-7752-0_7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u003cem\u003eFAO\u003c/em\u003e (2021\u003cem\u003e) FAOSTAT\u003c/em\u003e: Pesticides Use and Trade. 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Saravanan M, Ramesh M, Carbamazepine (CBZ) induced enzymatic stress in gill, liver and muscle of a common carp, \u003cem\u003eCyprinus carpio\u003c/em\u003e, \u003cem\u003eJournal of King Saud University-Science\u003c/em\u003e 24(2) (2012) 179\u0026ndash;186, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jksus.2011.01.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jksus.2011.01.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSancho E, Fern\u0026aacute;ndez-Vega C, Andreu E, Ferrando MD, Effects of propanil on the European eel \u003cem\u003eAnguilla anguilla\u003c/em\u003e and post-exposure recovery using selected biomarkers as effect criteria, \u003cem\u003eEcotoxicology and Environmental Safety\u003c/em\u003e 72(3) (2009) 704\u0026ndash;713, \u003cspan class=\"ExternalRef\"\u003e\u003cspan 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of insecticidal derivative (\u003cem\u003ecartap hydrochloride\u003c/em\u003e) from the marine polycheate on certain enzyme systems of the fresh water fish \u003cem\u003eOreochromis mossambicus\u003c/em\u003e, \u003cem\u003eJournal of Environmental Biology\u003c/em\u003e 26(2) (2005) 191\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJee JH, Masroor F, Kang JC, Responses of cypermethrin-induced stress in haematological parameters of Korean rockfish, \u003cem\u003eSebastes schlegeli\u003c/em\u003e (Hilgendorf), \u003cem\u003eAquaculture Research\u003c/em\u003e 36(9) (2005) 898\u0026ndash;905, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2109.2005.01299.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2109.2005.01299.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oxidative stress, immuno-toxicity, hepatic enzymes, histopathology, biochemical analysis, pyrethroids, sublethal exposure","lastPublishedDoi":"10.21203/rs.3.rs-7200428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7200428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePesticides sprayed near waterbodies, without proper precautions, can cause detrimental effects on the fish population. Pesticides' polarity and water solubility determine the bioaccumulation of its in fish. A ten-day-long study was conducted to assess the toxico-physiological response in nile tilapia fish in four triplicated treatments, such as control (0 ppm) and three treatments following 25%, 50%, and 75% of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\)\u003c/span\u003e\u003c/span\u003e of cypermethrin. The recommended dose for cypermethrin was 0.886 ppm, whereas the determined \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{LC}_{50}\\)\u003c/span\u003e\u003c/span\u003evalue is much lower, which is 0.668 ppm. The fish were sampled at the end of the experiments, and a substantial drop in RBC count was noted, corroborated by a reduction in hemoglobin levels after 7 days. On the other hand, elevated WBC count occurred as a reaction of the defense system. Similarly, ameliorated antioxidant levels were found to safeguard cells from oxidative stress. However, Total Antioxidant capacity (T-AOC) demonstrates a notable decline, which measures the overall ability of cells or tissues that neutralize free radicals and ROS. It represents the overall failure of the antioxidant defense system to counteract sustained oxidative pressure. Serum calcium levels exhibited a dose-dependent decline, indicating how calcium ions mediate cellular reactions under stress. Enzymes are reliable markers of the general health of fish. Cypermethrin exposure significantly elevated the activity of both plasma glutamic oxaloacetic transaminase and plasma glutamic pyruvic transaminase enzymes in \u003cem\u003eOreochromis niloticus\u003c/em\u003e after 7 days, presumably due to damage to muscle and hepatic tissues, as evidenced by histopathological observations of the liver cells. In addition, a notable rise in Aspartate aminotransferase, Alanine transaminase, and Alkaline phosphatase was identified, indicating metabolic disruptions. Histological studies of the intestine and liver aligned with the biochemical disruptions. The findings suggest that even sublethal doses can induce physiological alterations, underscoring the need for cautious pesticide application.\u003c/p\u003e","manuscriptTitle":"Integrated Biomarker Responses Reveal Cypermethrin-Induced Stress and Organ Damage in Nile Tilapia: Insights into Hepatic, Neural, and Hematological Toxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 18:30:07","doi":"10.21203/rs.3.rs-7200428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dcd91872-786a-4344-933b-25010a34e019","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-16T22:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 18:30:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7200428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7200428","identity":"rs-7200428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}