Ecotoxicological Effects of Glyphosate-based herbicide Sub lethal Concentration on DNA Damage, Antioxidant Response and Histo-physiological consequences in Common carp (Cyprinus carpio)

Ecotoxicological Effects of Glyphosate-based herbicide Sub lethal Concentration on DNA Damage, Antioxidant Response and Histo-physiological consequences in Common carp (Cyprinus carpio) | 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 Ecotoxicological Effects of Glyphosate-based herbicide Sub lethal Concentration on DNA Damage, Antioxidant Response and Histo-physiological consequences in Common carp (Cyprinus carpio) Abdul Muhsin, Dil Naz, Sawaira Nazir, Ahmed Ismail, Imran Khan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8022273/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 Glyphosate-based herbicides (GBHs), a class of organophosphate pesticides, are extensively applied worldwide and their residues pose significant ecological risks, particularly in aquatic ecosystems. This study evaluated the effects of sub-lethal GBH concentrations on DNA integrity, peripheral erythrocytes, and antioxidant enzyme responses such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in Cyprinus carpio (common carp). Forty fish were distributed into four groups: control (untreated) and three experimental groups (E1, E2, E3) exposed to 0.4, 0.8, and 1.5 ppm GBH, respectively, for 7, 14, 21, and 28 days. At the lowest concentration (0.4 ppm), GBH caused a mild reduction in antioxidant enzyme activities, maintaining reactive oxygen species (ROS) at a manageable level. However, higher concentrations (0.8 ppm and 1.5 ppm) led to excessive ROS generation, overwhelming the antioxidant defense system and depleting enzymatic activity. DNA damage, assessed through single-cell gel electrophoresis (comet assay), increased in a dose-dependent manner. Similarly, micronucleus frequency in peripheral erythrocytes rose with elevated GBH exposure. Histopathological examination of gills revealed lesions such as hyperemia, epithelial hypertrophy and atrophy, lamellar shortening, and pillar cell distortion. In conclusion, sub-lethal exposure to GBH (0.4ppm, 0.8 ppm &1.5 ppm) induces oxidative stress, genotoxicity, and histopathological alterations in C. carpio , highlighting its potential toxicity to aquatic organisms. Cyprinus carpio glyphosate-based herbicide DNA damage micronucleus antioxidant markers gills histopathology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Research Highlights Exposure to glyphosate, cause severe DNA damage and peripheral micronuclei Declining in an antioxidant markers leads to physiological disabilities Aquatic pollution cause disruption of the aquatic food chains and food webs 1. Introduction Environmental pollution has become a major global concern due to its detrimental effects on human health (Fereidoun et al., 2007). In recent decades, the growing levels of pollution have intensified worries about its public health implications (Msc & NJAGI, 2013). Compared to earlier periods in Earth’s history, modern humans experience significantly higher exposure to environmental contaminants (Schell et al., 2006). Elevated pollutant levels resulting from urban waste, fossil fuel combustion, and excessive pesticide use in agriculture have severe consequences for humans, terrestrial and aquatic organisms, plants, and even tropical ecosystems (Nriagu & Pacyna, 1988). Pesticides, though widely used to enhance crop yield by controlling pests, often affect non-target species as well. Their residues in aquatic ecosystems can lead to serious toxic effects in fish and other aquatic life. (Ruiz-Suárez et al., 2015). Pesticides' environmental impact and genotoxic effects on non-target organisms are a growing issue (Rasmussen et al., 2015). Organochlorine pesticides pose a higher risk to animals than pyrethroid, organophosphates, and carbamates (Kutz et al., 1991). According to Soso et al. (2007), glyphosate can enter water bodies through agricultural use or direct application to suppress macrophyte plants. Glyphosate's high solubility in water causes ongoing contamination of soils and aquatic systems (Soso et al., 2007). According to Tate et al. (1997), non-target organisms may experience developmental, morphological, physiological, and biochemical alterations (Tate et al., 1997). Fish are commonly used in biomonitoring studies globally to assess the genotoxicity induced by aquatic pollutants (Bhatnagar et al., 2016; Qu et al., 2017; Shah et al., 2021; Siraj et al., 2018). Glyphosate and glyphosate based herbicide (GBH) can accumulate in fish tissue (Campos et al., 2005; Sultana et al., 2014), and alter protein and lipid concentrations in the kidneys and liver (Murty & Devi, 1982). Glyphosate and GBH affect the renal and hepatic ATPase activity in the fish Channa punctatus (Singh et al., 2018). Glyphosate, a herbicide, suppresses the synthesis of aromatic amino acids in plants, including tryptophan, tyrosine, and phenylalanine (Santos et al., 2007). Glyphosate inhibits enzyme-5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants. Inhibiting EPSPS in plastids leads to an accumulation of shikimate-3 phosphate, which inhibits the formation of aromatic amino acids and therefore protein synthesis. Glyphosate targets only one enzyme, EPSPS, but also affects several physiochemical and physical processes (Baylis, 2000). The Comet assay can detect cross-linkage in DNA templet, broken helices both single and double, and an alkali reactive sites (Khisroon et al., 2015). Azqueta et al. (2019) found that the approach can assess DND damage in both in vitro and in vivo settings (Azqueta et al., 2019). According to Zegura and Filipič (2019), the comet assay is one of the most effective methods for studying toxicants on fish. DNA analysis is commonly used to assess genetic risks associated with xenobiotics (Siraj et al., 2018). Monitoring xenobiotics in local species helps assess environmental quality (Kousar & Javed, 2015). In view of the aforesaid challenges related with pesticides, current research work was conducted to examine the adverse effects of the glyphosate-based herbicide on DNA integrity, antioxidant response and gills histology of Common carp ( Cyprinus carpio ) using the comet assay and micronucleus assay. 2. Materials and Methods 2.1 Fish collection and acclimatization Forty healthy common carp ( Cyprinus carpio ) were obtained from Mardan Hatchery, KPK, Pakistan, with an average weight of 110.56 ± 4 g and length of 11.73 ± 2.12 cm. The fish were transported to the laboratory within four hours and transferred into glass aquaria. Prior to experimentation, they were screened to ensure the absence of microbial infections. Fish were acclimated for 10 days in aerated glass aquaria (15 L capacity) containing dechlorinated tap water maintained at 25–30°C, pH 7–8, and 100% dissolved oxygen. During acclimatization, they were fed daily at 2% of their body weight. 2.2 Experimental design and Dose concentration Four 50-L glass aquaria were arranged and connected to air pumps. Each aquarium was marked as C, E 1 , E 2 , and E 3 . Each aquarium contained 30L of water. Each aquarium was stocked with 10 fish (Fig. 1 ). Glyphosate-based herbicide was tested at three sub lethal concentrations (0.4ppm, 0.8ppm, and 1.5ppm) using a commercial formulation with 480g/L (41% w/w) as an active ingredient and diluted to the volume of water in the aquaria (30L) (Giaquinto et al., 2017; Kale et al., 2023; Ukaegbu et al., 2022). Aquarium E 1 received 0.4ppm of the test solution, whereas Aquarium E 2 received 0.8ppm and Aquarium E 3 received 1.5ppm. Aquarium C acted as a control group. During the experiment, 41% of the GBH containing water was removed and replaced with fresh water on a regular basis. Each aquarium received daily food at 2% of the fish's body weight, and hot water. The temperature of aquaria water was kept at 25–27°C, the pH was maintained at 7.5–7.9, and aerated oxygen was 100%. The experiment was lost for 28 days. 2.3 Collection of blood samples On days 7, 14, 21, and 28, three fish from each aquarium showing reduced activity or loss of balance were sampled. Blood was collected from the caudal vein using heparinized syringes to prevent clotting and transferred into Eppendorf tubes. On day 28, additional blood samples were drawn with 2.5 mL heparinized syringes, centrifuged at 6000 g for 10 minutes, and the serum was separated and stored at − 10°C for subsequent antioxidant analysis. Remaining fish tissues were disposed of in accordance with the ethical and biosafety guidelines of the University of Malakand. 2.4 Comet assay The comet assay was performed with minor modifications following the method of Khisroon et al. (2015). Slides were first coated with a layer of normal melting agarose (NMA) and allowed to solidify. For the second layer, 10 µL of blood diluted in 500 µL phosphate-buffered saline (PBS) was mixed with 75 µL of 0.5% low melting point agarose (LMPA) and spread over the pre-coated slide. After solidification on ice, an additional 85 µL of LMPA was added to fill gaps. The slides were lysed overnight at 4°C, then placed in a horizontal electrophoresis chamber containing cold buffer for 20 minutes before electrophoresis at 25 V and 300 mA for 25 minutes. Following neutralization and fixation in 70% ethanol, slides were stained with acridine orange (20 ug/mL). Comet images were captured at 400× magnification using a Nikon Eclipse 801 epi-fluorescence microscope (450–490 nm excitations) at the Institute of Zoological Sciences, University of Peshawar. DNA damage was evaluated as total comet score (TCS) for each sample following standard procedures (Collins, 2004). 2.5 Micronucleus assay For the micronucleus (MN) assay the blood samples collected at different days (7, 14, 21, & 28) were then transferred into EDTA tubes, and two drops of blood were immediately applied to clean, grease-free slides to create peripheral blood smear two for each fish specimen. Following a 24-hour air drying period, these slides were dipped in cold absolute methanol for 15 minutes and then allowed to air dry for an additional hour. After 30 minutes of Giemsa dye staining in phosphate buffer, the slides were cleaned and allowed to dry. A 100x oil immersion lens was used to view the prepared slides. Then total magnification becomes 10 × 100 = 1000 and frequency for MN was counted by using following formula: $$\:MN\%=\frac{Number\:of\:cells\:containing\:MN}{Total\:number\:of\:cells\:counted}⨯100\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(i\right)$$ 2.6 Estimation of Antioxidant markers Catalase activity was measured using the Takahara et al. (1960) method, as described earlier (Greenwald, 1985). This approach relied on the disappearance of H 2 O 2 at 240 nm. The reaction mixture contained 1ml of 0.05M H 2 O 2 in 0.1M sodium phosphate buffer (pH 7.4) at 25˚C, 1.9ml of distilled water, and 0.1 ml of gills homogenate. The change in absorbance was measured using a spectrophotometer at 240nm (Nanomoles). However, Catalase activity was measured in micromole H 2 O 2 used per minute per milligram of protein (µmol mm − 1 mg protein − 1 ) (Takahara et al., 1960). Misra and Fridovich (1972), approach was used to measure superoxide dismutase (SOD) activity in blood. The assay measured SOD's ability to prevent epinephrine-adrenochrome from oxidizing and transitioning to an alkaline pH. Dilute 0.5ml of tissue homogenate with 0.5ml of distilled water. Add 0.25ml of ice-cold ethanol and 0.15ml of ice-cold chloroform. The material was thoroughly blended using a cyclo mixer for 5 minutes before centrifugation at 2500 rpm. To the 0.5ml supernatant, add 1.5ml of carbonate buffer (0.05M, pH 10.2) and 0.5ml of EDTA solution (0.49M). To activate the reaction, add 0.4ml of epinephrine (3mM) and measure the change in optical density/minute at 480nm against the blank reagent using spectrophotometer. SOD activity was measured as the number of enzyme units per mg protein. The enzyme unit was defined as the change in optical density/minute at 50% inhibition of the epinephrine-to-adrenochrome transition (Misra & Fridovich, 1972). The activity of Glutathione Peroxidase (GPx) was measured using the method published by Paglia and Valentine (1967), with revisions by Lawrence and Burke (1978). The reaction mixture included 50mM potassium phosphate buffer (pH 8.3), 1mM EDTA, 1mM sodium azide, and 0.2mM nicotinamide adenine reductase. The reaction began with the addition of 1.5mM cumene hydroperoxide. The enzyme activity was determined using the rate of NADPH oxidation. The reagents were combined, and absorbance was measured at 340nm. Enzyme activity was given as mmol/minute/milligram protein (mmol min − 1 mg protein − 1 ) (Lawrence & Burk, 1976; Paglia & Valentine, 1967). 2.7 Histopathological assessment For histological analysis, at day 28th the dissection of each fish was performed to collect gills tissues from all experimental groups. All collected gills were cleansed with distilled water and immersed immediately in 10% formalin, a widely used fixative for tissue preservation. The organs were left in the fixative solution for a period of 15 hours. Subsequently, the specimens underwent dehydration by immersion in gradient ethanol solutions (60%, 70%, 80%, 90%, and 100%) respectively. To remove excess fats from the specimens, xylene was employed as a clearing agent with 2 times dip. For the preparation of microtome sections, tissue blocks were embedded in paraffin wax. Thin sections (4µm thickness) of tissue were then obtained using semi-automatic microtomes for subsequent light microscopic analysis (Comanescu et al., 2012). 2.8 Statistical analysis All data from the research were presented as means and standard deviations (SD). For the analysis of group comparisons, one-way ANOVA followed by Tukey test was performed using IBM SPSS software, version 27. The level of significance was set at P < 0.05. 3. Results 3.1 Comet assay results at various days Table 1 presents the DNA damage observed on day 7, showing significant differences between treated groups and the control (aP ≤ 0.020 for 0.4 ppm, bP ≤ 0.035 for 0.8 ppm, and cP ≤ 0.021 for 1.5 ppm). Table 2 illustrates day 14 results, where DNA damage was highly significant (aP ≤ 0.001, bP ≤ 0.000, cP ≤ 0.003) compared to the control. Similarly, Table 3 indicates that on day 21, DNA damage increased markedly in all treatment groups (aP ≤ 0.001, bP ≤ 0.000, cP ≤ 0.000). On day 28 (Table 4 ), the DNA damage remained significantly elevated in exposed groups (aP ≤ 0.002, bP ≤ 0.000, cP ≤ 0.000) relative to the control group. Table 1 Mean ± S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 7 days at concentrations of 0.4, 0.8, and 1.5 ppm/day Experimental group Class-0 Class-1 Class-2 Class-3 Class-4 TCS Control 97.3 ± 1.5 0.7 ± 1.5 0.7 ± 0.6 1.6 ± 0.6 0.0 ± 0.0 6.3 ± 1.1 E 1 -0.4ppm 68.4 ± 11.5 14.6 ± 2.5 5.0 ± 1.2 9.0 ± 2.0 3.0 ± 1.3 64.0 ± 15.5 a E 2 -0.8ppm 36.0 ± 15.7 28.7 ± 9.4 20.0 ± 12.2 10.7 ± 11.1 5.3 ± 6.5 114.7 ± 34.9 b E 3 -1.5ppm 32.7 ± 17.5 24.0 ± 14.6 15.3 ± 10.7 18.3 ± 3.5 9.7 ± 5.6 127.0 ± 21.6 c S.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP ≤ 0.001, bP ≤ 0.01, and cP ≤ 0.05. Table 2 Mean ± S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 14 days at concentrations of 0.4, 0.8, and 1.5 ppm/day Experimental group Class-0 Class-1 Class-2 Class-3 Class-4 TCS Control 93.4 ± 2.5 5.2 ± 1.6 1.6 ± 2.7 0.0 ± 0.0 0.0 ± 0.0 5.3 ± 2.2 E 1 -0.4ppm 41.0 ± 6.0 31.0 ± 12.3 15.0 ± 1.0 9.3 ± 9.2 3.7 ± 2.5 89.7 ± 10.5 a E 2 -0.8ppm 25.7 ± 6.9 39.3 ± 2.8 20.3 ± 7.8 10.6 ± 3.0 4.1 ± 0.0 135.3 ± 8.9 b E 3 -1.5ppm 15.7 ± 3.5 24.3 ± 8.8 24.8 ± 4.6 22.7 ± 9.0 12.5 ± 10.6 171.0 ± 34.1 c S.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP ≤ 0.001, bP ≤ 0.01, and cP ≤ 0.05. Table 3 Mean ± S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 21 days at concentrations of 0.4, 0.8, and 1.5 ppm/day Experimental group Class-0 Class-1 Class-2 Class-3 Class-4 TCS Control 96.0 ± 2.0 1.7 ± 4.1 1.3 ± 0.5 1.0 ± 0.6 0.0 ± 0.0 8.3 ± 1.3 E 1 -0.4ppm 22.3 ± 7.4 36.7 ± 11.5 15.7 ± 2.5 14.3 ± 9.5 12.0 ± 6.6 159.7 ± 25.8 a E 2 -0.8ppm 10.0 ± 12.6 24.7 ± 1.8 21.3 ± 7.5 27.7 ± 4.5 16.3 ± 1.1 212.7 ± 21.7 b E 3 -1.5ppm 7.7 ± 12.5 12.3 ± 9.0 25.0 ± 2.8 30.0 ± 7.5 25.0 ± 3.8 245.3 ± 12.6 c S.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP ≤ 0.001, bP ≤ 0.01, and cP ≤ 0.05. Table 4 Mean ± S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 28 days at concentrations of 0.4, 0.8, and 1.5 ppm/day Experimental group Class-0 Class-1 Class-2 Class-3 Class-4 TCS Control 95.0 ± 4.1 3.0 ± 1.0 2.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 6.8 ± 3.1 E 1 -0.4ppm 13.6 ± 5.5 16.4 ± 9.3 20.0 ± 3.1 13.0 ± 6.9 37.0 ± 20.8 229.3 ± 34.4 a E 2 -0.8ppm 10.0 ± 3.7 15.6 ± 8.9 17.4 ± 6.1 18.0 ± 4.9 39.0 ± 2.3 251.0 ± 17.4 b E 3 -1.5ppm 5.7 ± 2.1 7.3 ± 5.1 16.3 ± 12.7 22.7 ± 7.0 48.0 ± 15.6 297.7 ± 26.8 c S.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP ≤ 0.001, bP ≤ 0.01, and cP ≤ 0.05. 3.2 Micronucleus assay results at different days Figure 2 illustrates micronuclei formation on day 7, showing significant increases in treated groups compared to the control ( a P ≤ 0.001 for 0.4 ppm, b P ≤ 0.01 for 0.8 ppm, and c P ≤ 0.05 for 1.5 ppm). Figure 3 presents’ day 14 data, indicating a highly significant rise in micronuclei frequency ( a P ≤ 0.0002, b P ≤ 0.005, c P ≤ 0.009). On day 21 (Fig. 4 ), micronuclei formation remained significantly elevated in all treated groups ( a P ≤ 0.001, b P ≤ 0.000, c P ≤ 0.000). Similarly, Fig. 5 shows that by day 28, micronuclei frequency were still markedly higher in exposed fish ( a P ≤ 0.002, b P ≤ 0.000, c P ≤ 0.000) compared with the control group. The overall micronucleus assay of erythrocytes at different days was plotted as shown in (Fig. 6 ). These findings indicate that the micronuclei numbers in each experimental group exposed to GBH with increase in GBH sub lethal concentrations and duration of exposure. 3.3 Assessment of antioxidant markers GBH significantly ( P ≤ 0.05, P ≤ 0.01, & P ≤ 0.001) decline GPx level as shown as compared to the control group (Fig. 7 ). Higher SOD concentration was seen higher in control group, but significantly reduced in GBH treated group as dose concentration increases. The CAT concentration also sequentially reduced more promptly in higher doses groups as compared to the control group. (Significant difference relative to the control group at a P ≤ 0.001, b P ≤ 0.01, and c P ≤ 0.05) 3.4 Gills histopathological assessment The photomicrograph presented in Fig. 8 shows noteworthy alterations in gills when exposed to different concentrations of glyphosate based herbicide for 28 days. Control group have gills architecture of primary and secondary gills lamella, normal oval chloride and pillar cells. The alteration was more prominently observed in E 3 group exposed to the highest concentration of GBH as compared to E 1 group. These changes are hyperemia, lamellar fusion, shortening and congestion of secondary lamella, epithelial lifting, and distortion of chloride cells, congestion of pillar cells, atrophy and hypertrophy of lamellar cells. The aforementioned alterations shows severity in higher doses groups ( E 2 & E 3 ). The photomicrograph in Fig. 9 represents additional peripheral micronuclei in RBCs. In control group, red blood cells showed normal disc shape without any additional micronucleus (blue arrow), E 1 -group RBCs have single peripheral micronucleus (red arrow), E 2 -group RBCs have double peripheral micronuclei (black arrow), and E 3 -group RBCs have more peripheral micronuclei (green arrow). The photomicrograph in Fig. 10 represents the DNA adducts in different GBH treated group’s blood cells. Control group has no DNA adduct tail, but in dose-dependent groups the DNA adduct formation was observed more clearly in high GBH treated group. 4. Discussion The genotoxic potential of various pesticides has been widely examined across different animal models, including fish, amphibians, birds, and mammals (Shah et al., 2021; Siraj et al., 2018; Suliman et al., 2020), with varying levels of DNA damage reported. Fish are frequently used as bio indicators due to their ecological significance, economic value, and sensitivity to low concentrations of toxicants (Ali et al., 2008; Osman et al., 2007). Present study indicates glyphosate-based herbicide exposure induced time and dose-dependent DNA damage in Cyprinus carpio . These findings align with earlier reports showing that glyphosate exposure significantly increases DNA damage (Shao et al., 2012; Sharma et al., 2007). The observed DNA damage in this study may result from the formation of DNA adducts, single- and double-strand breaks, or DNA–protein/DNA–DNA cross-links, potentially induced by glyphosate or its metabolites (Lu et al., 2000). Glyphosate-based herbicides (GBHs) are also known to generate reactive oxygen species (ROS) in a time- and concentration-dependent manner, contributing to oxidative DNA damage (Shao et al., 2012; Sharma et al., 2007). Sebastian and Raghavan (2016) demonstrated that glyphosate induces ROS-mediated genotoxicity in human and mouse cells by enhancing the activity of proteins and enzymes involved in the non-homologous end joining (NHEJ) DNA repair pathway, which is primarily responsible for repairing double-strand breaks and long junction deletions. Based on these findings, it can be inferred that glyphosate or its metabolites may damage DNA either directly—through adduct formation and alkali-labile sites—or indirectly by inducing ROS (Sebastian & Raghavan, 2016). Thus, both ROS generation and adduct formation likely contribute to the genotoxic effects of GBH observed in this study. The MN assay is chiefly used to identify the biological effects of aquatic pollutants on aquatic species; the genotoxic effect of pollutants is responsible for the induction of micronuclei in peripheral erythrocytes (Tripathy, 2020). According to the MN assay, the current investigation found that the GBH was genotoxic to Cyprinus carpio ( C. carpio ) peripheral erythrocytes. The development of MN in erythrocytes indicates that genetic damage was greater in treated fish groups than in control groups. Our findings are consistent with those of D'Costa et al. (2018) in Danio rerio subjected to monocrotophos (an organophosphate pesticide) and Naqvi et al. (2016) when they treated O. mossambicus with several pesticides (D’Costa et al., 2018; Naqvi et al., 2016). Grass carp erythrocytes exposed to copper, lead, and chromium also showed comparable genotoxic effects (Shah et al., 2021). Similarly, giving Labeo rohita ( L. rohita ) thiamethoxam causes a marked rise in the frequency of micronuclei (Ghaffar et al. 2020). According to Ucar et al. (2020), Oncorhynchus mykiss ( O. mykiss ) treated with fipronil similarly showed a comparable increase in micronucleus frequency (Uçar et al., 2021). One of the most essential biochemical indicators of antioxidant effects is the tissues' SOD level, which is directly linked to GPx and CAT activity (Ai et al., 2011). In the present study GBH markedly decline the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in dose-dependent treated groups (Fig-7). This is might be due to the lipid preoxidation and production of malondialdehyde (MDA) which cause the oxidant/antioxidant imbalances. To protect against such stress, these three are the most vital inducible antioxidant enzymes. A modest change in oxidative stress triggered the expression of these enzymes. A severe oxidative stressor, however, can inhibit these enzymes' functions, leading to the loss of compensatory mechanisms. In general, water-soluble defenses like glutathione and enzymes like CAT, GRd, GPx, GST, and SOD make up antioxidant defenses (Di Giulio et al., 1989; Kappus, 1985). These enzymes' ability to initiate under oxidative stress is one of their key characteristics, which may be crucial for adaptability to stress brought on by pollutants. SOD may be the first enzyme to fight off produced oxy-radicals since it catalyzes the conversion of superoxide radicals to H 2 O 2 and O 2 (Kappus, 1985). Increased SOD and CAT activity levels show that the adaption equilibrium has formed. Thus, these two may be the most promising biomarkers of heavy metal toxicity and are in charge of the first adaptive response to oxidative stress. While GPx catalyzes the reduction of both hydrogen peroxide and lipid peroxides to H 2 O and O 2 , it is thought to be an effective protective enzyme against lipid peroxidation. The peroxisomal enzyme CAT transforms excess H 2 O 2 to H 2 O and O 2 (Winston & Di Giulio, 1991). Glyphosate enters fish within few minutes of exposure, with gills being the first organ to respond due to their roles in respiration, osmoregulation, nitrogenous waste elimination, and acid-base balance (Benli et al., 2008; Cengiz & Unlu, 2006). Histopathological analysis showed noteworthy gill damage, including chloride cell hyperplasia, hyperemia, epithelial lifting, curling of secondary lamella, lamellar fusion, and shortening of secondary lamella, with increasing with glyphosate concentrations. These findings are consistent with previous studies on Nile tilapia and immature blunt snout bream (Benli et al., 2008; Rivenson et al., 2019). 5. Conclusion It was concluded that increase in aquatic agrochemical pesticides have a negative effects on fish physiology and genetics. In the present research due to chronic exposure to the dose-dependent glyphosate-based herbicide sub lethal concentrations, the key adverse effects of GBH on common carp fish were the significant reduction in body antioxidant markers and elevation of oxidative stress, DNA adducts formation, peripheral micronuclei formation in erythrocytes, and severe damage of the gills tissues. Exposure to GBH, the degree of DNA injury, and micronuclei formation in erythrocytes was triggered depends on both GBH concentration and on duration of exposure. Declarations Acknowledgments Department of Zoology, University of Malakand was highly acknowledged for providing space and ethical approval. Funding This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. KFU254472]. Author Contributions AM: Designed study, writing and editing-original draft, statistical analysis, DN: Review the article, SN: conduct experiment, IK & SN: sample collection and data analysis, AI, WE, MA & ATM: data analysis, AA & HA: Funding acquisition. Ethics declaration Ethics Approval All the experimental work was conducted in accordance with the ethical guideline approved by the ethical committee of the Department of Zoology, University of Malakand, KPK Pakistan with (Ref. no. E-SA-11-2009). Consent to participate This is not applicable. Consent to publish This is not applicable. Conflicts of Interest The authors have no conflict of interest. Data Availability Data presented in this study are available on fair request to the corresponding author. 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1","display":"","copyAsset":false,"role":"figure","size":103497,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for exposure of grass carp to glyphosate\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/1b095e3ab309181ab6d08112.png"},{"id":98649021,"identity":"c4c51e36-b314-44d8-a0ef-0f72df744212","added_by":"auto","created_at":"2025-12-19 22:10:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":628199,"visible":true,"origin":"","legend":"\u003cp\u003eMicronucleus Assay of grass carp erythrocytes at day 7\u003csup\u003eth\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/26321477d0585dfa2c5bff7d.png"},{"id":98775347,"identity":"c49fb477-ab67-468d-8af0-d48639d25811","added_by":"auto","created_at":"2025-12-22 12:19:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":440411,"visible":true,"origin":"","legend":"\u003cp\u003eMicronucleus Assay of grass carp erythrocytes at day 14\u003csup\u003eth\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/a8f50d5e36575b140a4b4bb6.png"},{"id":98775355,"identity":"dbb5171e-ff3c-47b8-b841-7f2d95896f71","added_by":"auto","created_at":"2025-12-22 12:19:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":294913,"visible":true,"origin":"","legend":"\u003cp\u003eMicronucleus Assay of grass carp erythrocytes at day 21\u003csup\u003est\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/e372e635e79d005b88e8f1b8.png"},{"id":98649030,"identity":"a5bf7af6-7639-49f6-97b6-2779d8e12a1a","added_by":"auto","created_at":"2025-12-19 22:10:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":288756,"visible":true,"origin":"","legend":"\u003cp\u003eMicronucleus Assay of grass carp erythrocytes at day 28\u003csup\u003eth\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/25947dcf10b79b658d482d95.png"},{"id":98649028,"identity":"522cd5da-1967-4cff-8563-6e76667afeea","added_by":"auto","created_at":"2025-12-19 22:10:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105102,"visible":true,"origin":"","legend":"\u003cp\u003eMN/100 cells count of different treated groups at day 7\u003csup\u003eth\u003c/sup\u003e, 14\u003csup\u003eth\u003c/sup\u003e, 21\u003csup\u003est\u003c/sup\u003e, and 28\u003csup\u003eth\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/b4fc872b4a8afdf31b514d87.png"},{"id":98775668,"identity":"155f5f6a-753a-4537-ba2c-22b83cbf3b7d","added_by":"auto","created_at":"2025-12-22 12:20:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":178143,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant markers of grass carp exposed to dose dependent concentrations of GLY\u003c/p\u003e\n\u003cp\u003e(Significant difference relative to the control group at \u003csup\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eP ≤\u003c/strong\u003e\u003c/em\u003e 0.001, \u003csup\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eP ≤\u003c/strong\u003e\u003c/em\u003e 0.01, and \u003csup\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eP ≤\u003c/strong\u003e\u003c/em\u003e 0.05)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/a7fdfdf971d91aa79e283e1f.png"},{"id":98649039,"identity":"34181e8d-f42d-4f69-bbc5-9dbe38a7c047","added_by":"auto","created_at":"2025-12-19 22:10:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4975956,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological photomicrographs (400 x) of gill of Common carp under control condition, glyphosate-treated laboratory condition, and glyphosate-treated field condition. (A) Control; normal structure of primary gill lamellae ‘‘PGL’’ and secondary lamella ‘‘SGL’’ under light microscopy. (B) E\u003csub\u003e1-\u003c/sub\u003eGroup; congestion of blood vessel ‘‘CBV’’, distortion of chloride ‘‘DC’’ and pillar cells ‘‘PC’’, distorted ‘‘PGL’’ \u0026amp; ‘‘SGL’’, lamellar atrophy ‘‘Atp’’, and hypertrophy ‘‘Htp’’. (C) E\u003csub\u003e2\u003c/sub\u003e-Group; atrophy ‘‘Atp’’ and hypertrophy ‘‘Htp’’ and inter lamellar space between ‘‘SGL’’. (D) E\u003csub\u003e3\u003c/sub\u003e-Group; severe congestion of secondary gills lamella ‘‘CSGL’’, more inter lamellar spaces between ‘‘SGL’’, curling of ‘‘SGL’’, severe ‘‘Atp’’ \u0026amp; ‘‘Htp’’.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/663e07cd6ab2353b578d8eac.png"},{"id":98775369,"identity":"eaf6bdac-524d-413e-8290-94a17dd10fb2","added_by":"auto","created_at":"2025-12-22 12:19:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2007403,"visible":true,"origin":"","legend":"\u003cp\u003eBlood smear of photomicrograph of Common carp. (A) Control; normal blood cell and nuclei (blue arrow), (B) E\u003csub\u003e1\u003c/sub\u003e-Group; blood cells with additional single micronuclei (MN) (red arrow), (C) E\u003csub\u003e2\u003c/sub\u003e-Group blood cells with two additional micronuclei (MN) (black arrow), \u0026amp; (D) E\u003csub\u003e3\u003c/sub\u003e-Group; blood cells with more micronuclei (MN) (green arrow).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/bc033ff048ad2d8703005faf.png"},{"id":98775240,"identity":"c8b85234-6b72-45aa-bb37-f53fc3d08da3","added_by":"auto","created_at":"2025-12-22 12:18:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2551389,"visible":true,"origin":"","legend":"\u003cp\u003eBlood smear (stained with acridine orange fluorescent dye) photomicrograph of Common carp. (A) Control; normal blood cell and no DNA adduct tail (blue arrow), (B) E\u003csub\u003e1\u003c/sub\u003e-Group; blood cells with DNA adduct tail (red arrow), (C) E\u003csub\u003e2\u003c/sub\u003e-Group blood cells with more DNA adduct tail (black arrow) and (D) E\u003csub\u003e3\u003c/sub\u003e-Group; blood cells with maximum number of DNA adduct tail in each blood cell (purple arrow).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/507595279189399b7ba88e47.png"},{"id":101943348,"identity":"9ca1d50f-6ec2-414b-9a15-0959f6cbe540","added_by":"auto","created_at":"2026-02-05 09:41:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18145278,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/286001e7-d679-4702-ad23-80a67d5ba251.pdf"},{"id":98649023,"identity":"631f69ab-0d21-4960-9918-624667086c6f","added_by":"auto","created_at":"2025-12-19 22:10:18","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022273/v1/7ef08f493ebd0959a0c58a0a.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ecotoxicological Effects of Glyphosate-based herbicide Sub lethal Concentration on DNA Damage, Antioxidant Response and Histo-physiological consequences in Common carp (Cyprinus carpio)","fulltext":[{"header":"Research Highlights ","content":"\u003cul\u003e\n \u003cli\u003eExposure to glyphosate, cause severe DNA damage and peripheral micronuclei\u003c/li\u003e\n \u003cli\u003eDeclining in an antioxidant markers leads to physiological disabilities\u003c/li\u003e\n \u003cli\u003eAquatic pollution cause disruption of the aquatic food chains and food webs\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eEnvironmental pollution has become a major global concern due to its detrimental effects on human health (Fereidoun et al., 2007). In recent decades, the growing levels of pollution have intensified worries about its public health implications (Msc \u0026amp; NJAGI, 2013). Compared to earlier periods in Earth\u0026rsquo;s history, modern humans experience significantly higher exposure to environmental contaminants (Schell et al., 2006). Elevated pollutant levels resulting from urban waste, fossil fuel combustion, and excessive pesticide use in agriculture have severe consequences for humans, terrestrial and aquatic organisms, plants, and even tropical ecosystems (Nriagu \u0026amp; Pacyna, 1988).\u003c/p\u003e \u003cp\u003ePesticides, though widely used to enhance crop yield by controlling pests, often affect non-target species as well. Their residues in aquatic ecosystems can lead to serious toxic effects in fish and other aquatic life. (Ruiz-Su\u0026aacute;rez et al., 2015). Pesticides' environmental impact and genotoxic effects on non-target organisms are a growing issue (Rasmussen et al., 2015). Organochlorine pesticides pose a higher risk to animals than pyrethroid, organophosphates, and carbamates (Kutz et al., 1991). According to Soso et al. (2007), glyphosate can enter water bodies through agricultural use or direct application to suppress macrophyte plants. Glyphosate's high solubility in water causes ongoing contamination of soils and aquatic systems (Soso et al., 2007). According to Tate et al. (1997), non-target organisms may experience developmental, morphological, physiological, and biochemical alterations (Tate et al., 1997).\u003c/p\u003e \u003cp\u003eFish are commonly used in biomonitoring studies globally to assess the genotoxicity induced by aquatic pollutants (Bhatnagar et al., 2016; Qu et al., 2017; Shah et al., 2021; Siraj et al., 2018). Glyphosate and glyphosate based herbicide (GBH) can accumulate in fish tissue (Campos et al., 2005; Sultana et al., 2014), and alter protein and lipid concentrations in the kidneys and liver (Murty \u0026amp; Devi, 1982). Glyphosate and GBH affect the renal and hepatic ATPase activity in the fish \u003cem\u003eChanna punctatus\u003c/em\u003e (Singh et al., 2018). Glyphosate, a herbicide, suppresses the synthesis of aromatic amino acids in plants, including tryptophan, tyrosine, and phenylalanine (Santos et al., 2007). Glyphosate inhibits enzyme-5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants. Inhibiting EPSPS in plastids leads to an accumulation of shikimate-3 phosphate, which inhibits the formation of aromatic amino acids and therefore protein synthesis. Glyphosate targets only one enzyme, EPSPS, but also affects several physiochemical and physical processes (Baylis, 2000).\u003c/p\u003e \u003cp\u003eThe Comet assay can detect cross-linkage in DNA templet, broken helices both single and double, and an alkali reactive sites (Khisroon et al., 2015). Azqueta et al. (2019) found that the approach can assess DND damage in both in vitro and in vivo settings (Azqueta et al., 2019). According to Zegura and Filipič (2019), the comet assay is one of the most effective methods for studying toxicants on fish. DNA analysis is commonly used to assess genetic risks associated with xenobiotics (Siraj et al., 2018). Monitoring xenobiotics in local species helps assess environmental quality (Kousar \u0026amp; Javed, 2015).\u003c/p\u003e \u003cp\u003eIn view of the aforesaid challenges related with pesticides, current research work was conducted to examine the adverse effects of the glyphosate-based herbicide on DNA integrity, antioxidant response and gills histology of Common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e) using the comet assay and micronucleus assay.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Fish collection and acclimatization\u003c/h2\u003e \u003cp\u003eForty healthy common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e) were obtained from Mardan Hatchery, KPK, Pakistan, with an average weight of 110.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4 g and length of 11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12 cm. The fish were transported to the laboratory within four hours and transferred into glass aquaria. Prior to experimentation, they were screened to ensure the absence of microbial infections. Fish were acclimated for 10 days in aerated glass aquaria (15 L capacity) containing dechlorinated tap water maintained at 25\u0026ndash;30\u0026deg;C, pH 7\u0026ndash;8, and 100% dissolved oxygen. During acclimatization, they were fed daily at 2% of their body weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design and Dose concentration\u003c/h2\u003e \u003cp\u003eFour 50-L glass aquaria were arranged and connected to air pumps. Each aquarium was marked as \u003cb\u003eC, E\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e, \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e, and \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e. Each aquarium contained 30L of water. Each aquarium was stocked with 10 fish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Glyphosate-based herbicide was tested at three sub lethal concentrations (0.4ppm, 0.8ppm, and 1.5ppm) using a commercial formulation with 480g/L (41% w/w) as an active ingredient and diluted to the volume of water in the aquaria (30L) (Giaquinto et al., 2017; Kale et al., 2023; Ukaegbu et al., 2022). Aquarium \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e received 0.4ppm of the test solution, whereas Aquarium \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e received 0.8ppm and Aquarium \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e received 1.5ppm. Aquarium \u003cb\u003eC\u003c/b\u003e acted as a control group. During the experiment, 41% of the GBH containing water was removed and replaced with fresh water on a regular basis. Each aquarium received daily food at 2% of the fish's body weight, and hot water. The temperature of aquaria water was kept at 25\u0026ndash;27\u0026deg;C, the pH was maintained at 7.5\u0026ndash;7.9, and aerated oxygen was 100%. The experiment was lost for 28 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Collection of blood samples\u003c/h2\u003e \u003cp\u003eOn days 7, 14, 21, and 28, three fish from each aquarium showing reduced activity or loss of balance were sampled. Blood was collected from the caudal vein using heparinized syringes to prevent clotting and transferred into Eppendorf tubes. On day 28, additional blood samples were drawn with 2.5 mL heparinized syringes, centrifuged at 6000 g for 10 minutes, and the serum was separated and stored at \u0026minus;\u0026thinsp;10\u0026deg;C for subsequent antioxidant analysis. Remaining fish tissues were disposed of in accordance with the ethical and biosafety guidelines of the University of Malakand.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Comet assay\u003c/h2\u003e \u003cp\u003eThe comet assay was performed with minor modifications following the method of Khisroon et al. (2015). Slides were first coated with a layer of normal melting agarose (NMA) and allowed to solidify. For the second layer, 10 \u0026micro;L of blood diluted in 500 \u0026micro;L phosphate-buffered saline (PBS) was mixed with 75 \u0026micro;L of 0.5% low melting point agarose (LMPA) and spread over the pre-coated slide. After solidification on ice, an additional 85 \u0026micro;L of LMPA was added to fill gaps. The slides were lysed overnight at 4\u0026deg;C, then placed in a horizontal electrophoresis chamber containing cold buffer for 20 minutes before electrophoresis at 25 V and 300 mA for 25 minutes. Following neutralization and fixation in 70% ethanol, slides were stained with acridine orange (20 ug/mL). Comet images were captured at 400\u0026times; magnification using a Nikon Eclipse 801 epi-fluorescence microscope (450\u0026ndash;490 nm excitations) at the Institute of Zoological Sciences, University of Peshawar. DNA damage was evaluated as total comet score (TCS) for each sample following standard procedures (Collins, 2004).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Micronucleus assay\u003c/h2\u003e \u003cp\u003eFor the micronucleus (MN) assay the blood samples collected at different days (7, 14, 21, \u0026amp; 28) were then transferred into EDTA tubes, and two drops of blood were immediately applied to clean, grease-free slides to create peripheral blood smear two for each fish specimen. Following a 24-hour air drying period, these slides were dipped in cold absolute methanol for 15 minutes and then allowed to air dry for an additional hour. After 30 minutes of Giemsa dye staining in phosphate buffer, the slides were cleaned and allowed to dry. A 100x oil immersion lens was used to view the prepared slides. Then total magnification becomes 10 \u0026times; 100\u0026thinsp;=\u0026thinsp;1000 and frequency for MN was counted by using following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:MN\\%=\\frac{Number\\:of\\:cells\\:containing\\:MN}{Total\\:number\\:of\\:cells\\:counted}⨯100\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(i\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Estimation of Antioxidant markers\u003c/h2\u003e \u003cp\u003eCatalase activity was measured using the Takahara et al. (1960) method, as described earlier (Greenwald, 1985). This approach relied on the disappearance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 240 nm. The reaction mixture contained 1ml of 0.05M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 0.1M sodium phosphate buffer (pH 7.4) at 25˚C, 1.9ml of distilled water, and 0.1 ml of gills homogenate. The change in absorbance was measured using a spectrophotometer at 240nm (Nanomoles). However, Catalase activity was measured in micromole H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e used per minute per milligram of protein (\u0026micro;mol mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Takahara et al., 1960).\u003c/p\u003e \u003cp\u003eMisra and Fridovich (1972), approach was used to measure superoxide dismutase (SOD) activity in blood. The assay measured SOD's ability to prevent epinephrine-adrenochrome from oxidizing and transitioning to an alkaline pH. Dilute 0.5ml of tissue homogenate with 0.5ml of distilled water. Add 0.25ml of ice-cold ethanol and 0.15ml of ice-cold chloroform. The material was thoroughly blended using a cyclo mixer for 5 minutes before centrifugation at 2500 rpm. To the 0.5ml supernatant, add 1.5ml of carbonate buffer (0.05M, pH 10.2) and 0.5ml of EDTA solution (0.49M). To activate the reaction, add 0.4ml of epinephrine (3mM) and measure the change in optical density/minute at 480nm against the blank reagent using spectrophotometer. SOD activity was measured as the number of enzyme units per mg protein. The enzyme unit was defined as the change in optical density/minute at 50% inhibition of the epinephrine-to-adrenochrome transition (Misra \u0026amp; Fridovich, 1972).\u003c/p\u003e \u003cp\u003eThe activity of Glutathione Peroxidase (GPx) was measured using the method published by Paglia and Valentine (1967), with revisions by Lawrence and Burke (1978). The reaction mixture included 50mM potassium phosphate buffer (pH 8.3), 1mM EDTA, 1mM sodium azide, and 0.2mM nicotinamide adenine reductase. The reaction began with the addition of 1.5mM cumene hydroperoxide. The enzyme activity was determined using the rate of NADPH oxidation. The reagents were combined, and absorbance was measured at 340nm. Enzyme activity was given as mmol/minute/milligram protein (mmol min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg protein\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Lawrence \u0026amp; Burk, 1976; Paglia \u0026amp; Valentine, 1967).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histopathological assessment\u003c/h2\u003e \u003cp\u003eFor histological analysis, at day 28th the dissection of each fish was performed to collect gills tissues from all experimental groups. All collected gills were cleansed with distilled water and immersed immediately in 10% formalin, a widely used fixative for tissue preservation. The organs were left in the fixative solution for a period of 15 hours. Subsequently, the specimens underwent dehydration by immersion in gradient ethanol solutions (60%, 70%, 80%, 90%, and 100%) respectively. To remove excess fats from the specimens, xylene was employed as a clearing agent with 2 times dip. For the preparation of microtome sections, tissue blocks were embedded in paraffin wax. Thin sections (4\u0026micro;m thickness) of tissue were then obtained using semi-automatic microtomes for subsequent light microscopic analysis (Comanescu et al., 2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data from the research were presented as means and standard deviations (SD). For the analysis of group comparisons, one-way ANOVA followed by Tukey test was performed using IBM SPSS software, version 27. The level of significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Comet assay results at various days\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the DNA damage observed on day 7, showing significant differences between treated groups and the control (aP\u0026thinsp;\u0026le;\u0026thinsp;0.020 for 0.4 ppm, bP\u0026thinsp;\u0026le;\u0026thinsp;0.035 for 0.8 ppm, and cP\u0026thinsp;\u0026le;\u0026thinsp;0.021 for 1.5 ppm). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates day 14 results, where DNA damage was highly significant (aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.000, cP\u0026thinsp;\u0026le;\u0026thinsp;0.003) compared to the control. Similarly, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e indicates that on day 21, DNA damage increased markedly in all treatment groups (aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.000, cP\u0026thinsp;\u0026le;\u0026thinsp;0.000). On day 28 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the DNA damage remained significantly elevated in exposed groups (aP\u0026thinsp;\u0026le;\u0026thinsp;0.002, bP\u0026thinsp;\u0026le;\u0026thinsp;0.000, cP\u0026thinsp;\u0026le;\u0026thinsp;0.000) relative to the control group.\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\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 7 days at concentrations of 0.4, 0.8, and 1.5 ppm/day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClass-0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClass-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClass-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClass-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClass-4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTCS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e97.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e1\u003c/sub\u003e-0.4ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e68.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e-0.8ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e114.7\u0026thinsp;\u0026plusmn;\u0026thinsp;34.9\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e3\u003c/sub\u003e-1.5ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;17.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e127.0\u0026thinsp;\u0026plusmn;\u0026thinsp;21.6\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.01, and cP\u0026thinsp;\u0026le;\u0026thinsp;0.05.\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\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 14 days at concentrations of 0.4, 0.8, and 1.5 ppm/day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClass-0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClass-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClass-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClass-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClass-4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTCS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e1\u003c/sub\u003e-0.4ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5\u003cb\u003ea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e-0.8ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e135.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e3\u003c/sub\u003e-1.5ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e171.0\u0026thinsp;\u0026plusmn;\u0026thinsp;34.1\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.01, and cP\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 21 days at concentrations of 0.4, 0.8, and 1.5 ppm/day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClass-0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClass-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClass-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClass-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClass-4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTCS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e96.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e1\u003c/sub\u003e-0.4ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e159.7\u0026thinsp;\u0026plusmn;\u0026thinsp;25.8\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e-0.8ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e212.7\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e3\u003c/sub\u003e-1.5ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e245.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.6\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.01, and cP\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D values of comet classes and total comet score (TCS) in 100 blood cells of control and GBH-exposed fish after 28 days at concentrations of 0.4, 0.8, and 1.5 ppm/day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClass-0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClass-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClass-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClass-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClass-4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTCS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e1\u003c/sub\u003e-0.4ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;20.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e229.3\u0026thinsp;\u0026plusmn;\u0026thinsp;34.4\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e-0.8ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e39.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e251.0\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e3\u003c/sub\u003e-1.5ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e48.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e297.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.8\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS.D: standard deviation; TCS: total comet score; ppm: parts per million. Significant differences from the control group are indicated as aP\u0026thinsp;\u0026le;\u0026thinsp;0.001, bP\u0026thinsp;\u0026le;\u0026thinsp;0.01, and cP\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Micronucleus assay results at different days\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates micronuclei formation on day 7, showing significant increases in treated groups compared to the control (\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.001 for 0.4 ppm, \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.01 for 0.8 ppm, and \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.05 for 1.5 ppm). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents\u0026rsquo; day 14 data, indicating a highly significant rise in micronuclei frequency (\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.0002, \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.005, \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.009). On day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), micronuclei formation remained significantly elevated in all treated groups (\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.001, \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.000, \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.000). Similarly, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that by day 28, micronuclei frequency were still markedly higher in exposed fish (\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.002, \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.000, \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026le; 0.000) compared with the control group. The overall micronucleus assay of erythrocytes at different days was plotted as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings indicate that the micronuclei numbers in each experimental group exposed to GBH with increase in GBH sub lethal concentrations and duration of exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Assessment of antioxidant markers\u003c/h2\u003e \u003cp\u003eGBH significantly (\u003cem\u003eP\u0026thinsp;\u0026le;\u003c/em\u003e\u0026thinsp;0.05, \u003cem\u003eP\u0026thinsp;\u0026le;\u003c/em\u003e\u0026thinsp;0.01, \u0026amp; \u003cem\u003eP\u0026thinsp;\u0026le;\u003c/em\u003e\u0026thinsp;0.001) decline GPx level as shown as compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Higher SOD concentration was seen higher in control group, but significantly reduced in GBH treated group as dose concentration increases. The CAT concentration also sequentially reduced more promptly in higher doses groups as compared to the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(Significant difference relative to the control group at \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP \u0026le;\u003c/b\u003e 0.001, \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP \u0026le;\u003c/b\u003e 0.01, and \u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP \u0026le;\u003c/b\u003e 0.05)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Gills histopathological assessment\u003c/h2\u003e \u003cp\u003eThe photomicrograph presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows noteworthy alterations in gills when exposed to different concentrations of glyphosate based herbicide for 28 days. Control group have gills architecture of primary and secondary gills lamella, normal oval chloride and pillar cells. The alteration was more prominently observed in \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e group exposed to the highest concentration of GBH as compared to \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e group. These changes are hyperemia, lamellar fusion, shortening and congestion of secondary lamella, epithelial lifting, and distortion of chloride cells, congestion of pillar cells, atrophy and hypertrophy of lamellar cells. The aforementioned alterations shows severity in higher doses groups (\u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u0026amp; \u003cb\u003eE\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e). The photomicrograph in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e represents additional peripheral micronuclei in RBCs. In control group, red blood cells showed normal disc shape without any additional micronucleus (blue arrow), E\u003csub\u003e1\u003c/sub\u003e-group RBCs have single peripheral micronucleus (red arrow), E\u003csub\u003e2\u003c/sub\u003e-group RBCs have double peripheral micronuclei (black arrow), and E\u003csub\u003e3\u003c/sub\u003e-group RBCs have more peripheral micronuclei (green arrow). The photomicrograph in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e represents the DNA adducts in different GBH treated group\u0026rsquo;s blood cells. Control group has no DNA adduct tail, but in dose-dependent groups the DNA adduct formation was observed more clearly in high GBH treated group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe genotoxic potential of various pesticides has been widely examined across different animal models, including fish, amphibians, birds, and mammals (Shah et al., 2021; Siraj et al., 2018; Suliman et al., 2020), with varying levels of DNA damage reported. Fish are frequently used as bio indicators due to their ecological significance, economic value, and sensitivity to low concentrations of toxicants (Ali et al., 2008; Osman et al., 2007). Present study indicates glyphosate-based herbicide exposure induced time and dose-dependent DNA damage in \u003cem\u003eCyprinus carpio\u003c/em\u003e. These findings align with earlier reports showing that glyphosate exposure significantly increases DNA damage (Shao et al., 2012; Sharma et al., 2007).\u003c/p\u003e \u003cp\u003eThe observed DNA damage in this study may result from the formation of DNA adducts, single- and double-strand breaks, or DNA\u0026ndash;protein/DNA\u0026ndash;DNA cross-links, potentially induced by glyphosate or its metabolites (Lu et al., 2000). Glyphosate-based herbicides (GBHs) are also known to generate reactive oxygen species (ROS) in a time- and concentration-dependent manner, contributing to oxidative DNA damage (Shao et al., 2012; Sharma et al., 2007). Sebastian and Raghavan (2016) demonstrated that glyphosate induces ROS-mediated genotoxicity in human and mouse cells by enhancing the activity of proteins and enzymes involved in the non-homologous end joining (NHEJ) DNA repair pathway, which is primarily responsible for repairing double-strand breaks and long junction deletions. Based on these findings, it can be inferred that glyphosate or its metabolites may damage DNA either directly\u0026mdash;through adduct formation and alkali-labile sites\u0026mdash;or indirectly by inducing ROS (Sebastian \u0026amp; Raghavan, 2016). Thus, both ROS generation and adduct formation likely contribute to the genotoxic effects of GBH observed in this study.\u003c/p\u003e \u003cp\u003eThe MN assay is chiefly used to identify the biological effects of aquatic pollutants on aquatic species; the genotoxic effect of pollutants is responsible for the induction of micronuclei in peripheral erythrocytes (Tripathy, 2020). According to the MN assay, the current investigation found that the GBH was genotoxic to \u003cem\u003eCyprinus carpio\u003c/em\u003e (\u003cem\u003eC. carpio\u003c/em\u003e) peripheral erythrocytes. The development of MN in erythrocytes indicates that genetic damage was greater in treated fish groups than in control groups. Our findings are consistent with those of D'Costa et al. (2018) in Danio rerio subjected to monocrotophos (an organophosphate pesticide) and Naqvi et al. (2016) when they treated \u003cem\u003eO. mossambicus\u003c/em\u003e with several pesticides (D\u0026rsquo;Costa et al., 2018; Naqvi et al., 2016). Grass carp erythrocytes exposed to copper, lead, and chromium also showed comparable genotoxic effects (Shah et al., 2021). Similarly, giving \u003cem\u003eLabeo rohita\u003c/em\u003e (\u003cem\u003eL. rohita\u003c/em\u003e) thiamethoxam causes a marked rise in the frequency of micronuclei (Ghaffar et al. 2020). According to Ucar et al. (2020), \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (\u003cem\u003eO. mykiss\u003c/em\u003e) treated with fipronil similarly showed a comparable increase in micronucleus frequency (U\u0026ccedil;ar et al., 2021).\u003c/p\u003e \u003cp\u003eOne of the most essential biochemical indicators of antioxidant effects is the tissues' SOD level, which is directly linked to GPx and CAT activity (Ai et al., 2011). In the present study GBH markedly decline the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in dose-dependent treated groups (Fig-7). This is might be due to the lipid preoxidation and production of malondialdehyde (MDA) which cause the oxidant/antioxidant imbalances. To protect against such stress, these three are the most vital inducible antioxidant enzymes. A modest change in oxidative stress triggered the expression of these enzymes. A severe oxidative stressor, however, can inhibit these enzymes' functions, leading to the loss of compensatory mechanisms. In general, water-soluble defenses like glutathione and enzymes like CAT, GRd, GPx, GST, and SOD make up antioxidant defenses (Di Giulio et al., 1989; Kappus, 1985). These enzymes' ability to initiate under oxidative stress is one of their key characteristics, which may be crucial for adaptability to stress brought on by pollutants. SOD may be the first enzyme to fight off produced oxy-radicals since it catalyzes the conversion of superoxide radicals to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e (Kappus, 1985). Increased SOD and CAT activity levels show that the adaption equilibrium has formed. Thus, these two may be the most promising biomarkers of heavy metal toxicity and are in charge of the first adaptive response to oxidative stress. While GPx catalyzes the reduction of both hydrogen peroxide and lipid peroxides to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e, it is thought to be an effective protective enzyme against lipid peroxidation. The peroxisomal enzyme CAT transforms excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e (Winston \u0026amp; Di Giulio, 1991).\u003c/p\u003e \u003cp\u003eGlyphosate enters fish within few minutes of exposure, with gills being the first organ to respond due to their roles in respiration, osmoregulation, nitrogenous waste elimination, and acid-base balance (Benli et al., 2008; Cengiz \u0026amp; Unlu, 2006). Histopathological analysis showed noteworthy gill damage, including chloride cell hyperplasia, hyperemia, epithelial lifting, curling of secondary lamella, lamellar fusion, and shortening of secondary lamella, with increasing with glyphosate concentrations. These findings are consistent with previous studies on Nile tilapia and immature blunt snout bream (Benli et al., 2008; Rivenson et al., 2019).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIt was concluded that increase in aquatic agrochemical pesticides have a negative effects on fish physiology and genetics. In the present research due to chronic exposure to the dose-dependent glyphosate-based herbicide sub lethal concentrations, the key adverse effects of GBH on common carp fish were the significant reduction in body antioxidant markers and elevation of oxidative stress, DNA adducts formation, peripheral micronuclei formation in erythrocytes, and severe damage of the gills tissues. Exposure to GBH, the degree of DNA injury, and micronuclei formation in erythrocytes was triggered depends on both GBH concentration and on duration of exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Zoology, University of Malakand was highly acknowledged for providing space and ethical approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant\u0026nbsp;No. KFU254472].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAM: Designed study, writing and editing-original draft, statistical analysis, DN: Review the article, SN: conduct experiment, IK \u0026amp; SN: sample collection and data analysis, AI, WE, MA \u0026amp; ATM: data analysis, AA \u0026amp; HA: Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimental work was conducted in accordance with the ethical guideline approved by the ethical committee of the Department of Zoology, University of Malakand, KPK Pakistan with\u0026nbsp;(Ref. no.\u0026nbsp;E-SA-11-2009).\u003c/p\u003e\n\u003ch3\u003eConsent to participate\u003c/h3\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003ch3\u003eConsent to publish\u003c/h3\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData presented in this study are available on fair request to the corresponding author.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAi, Q., Xu, H., Mai, K., Xu, W., Wang, J., \u0026amp; Zhang, W. 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Sub-lethal effect of Round-up (a glyphosate-based herbicide) on juveniles of African cat fish (Clarias gariepinus). \u003cem\u003eSouth Asian Journal of Experimental Biology\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinston, G. W., \u0026amp; Di Giulio, R. T. (1991). Prooxidant and antioxidant mechanisms in aquatic organisms. \u003cem\u003eAquatic toxicology\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(2), 137\u0026ndash;161.\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":"Cyprinus carpio, glyphosate-based herbicide, DNA damage, micronucleus, antioxidant markers, gills histopathology","lastPublishedDoi":"10.21203/rs.3.rs-8022273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8022273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlyphosate-based herbicides (GBHs), a class of organophosphate pesticides, are extensively applied worldwide and their residues pose significant ecological risks, particularly in aquatic ecosystems. This study evaluated the effects of sub-lethal GBH concentrations on DNA integrity, peripheral erythrocytes, and antioxidant enzyme responses such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in \u003cem\u003eCyprinus carpio\u003c/em\u003e (common carp). Forty fish were distributed into four groups: control (untreated) and three experimental groups (E1, E2, E3) exposed to 0.4, 0.8, and 1.5 ppm GBH, respectively, for 7, 14, 21, and 28 days. At the lowest concentration (0.4 ppm), GBH caused a mild reduction in antioxidant enzyme activities, maintaining reactive oxygen species (ROS) at a manageable level. However, higher concentrations (0.8 ppm and 1.5 ppm) led to excessive ROS generation, overwhelming the antioxidant defense system and depleting enzymatic activity. DNA damage, assessed through single-cell gel electrophoresis (comet assay), increased in a dose-dependent manner. Similarly, micronucleus frequency in peripheral erythrocytes rose with elevated GBH exposure. Histopathological examination of gills revealed lesions such as hyperemia, epithelial hypertrophy and atrophy, lamellar shortening, and pillar cell distortion. In conclusion, sub-lethal exposure to GBH (0.4ppm, 0.8 ppm \u0026amp;1.5 ppm) induces oxidative stress, genotoxicity, and histopathological alterations in \u003cem\u003eC. carpio\u003c/em\u003e, highlighting its potential toxicity to aquatic organisms.\u003c/p\u003e","manuscriptTitle":"Ecotoxicological Effects of Glyphosate-based herbicide Sub lethal Concentration on DNA Damage, Antioxidant Response and Histo-physiological consequences in Common carp (Cyprinus carpio)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 22:10:09","doi":"10.21203/rs.3.rs-8022273/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":"3cf79aa4-0508-4135-8785-60547e648f4c","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-05T05:41:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 22:10:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8022273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8022273","identity":"rs-8022273","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}