Oxidative Stress in Tilapia guineensis Exposed to Glyphosate in the Laboratory

Okenwa U, Eze BU, Nwosu P and Ogolo C

Published on: 2024-07-26

Abstract

Roundup is among the most widely used glyphosate herbicides in Nigeria for a range of weed control in agricultural operations. A biomarker test was conducted on Sarotherodon melanotheron plasma to evaluate the oxidative impact of the herbicide using a portion of the antioxidants. Specific antioxidants, including glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), lipid peroxidase (LPO), and Glutathione (GSH), were measured in the plasma of S.melanotheron exposed to Roundup in order to evaluate oxidative stress in fish exposed to different concentrations of the chemical: 0.05, 0.10, 0.15, 0.20, and 0.25 mg/l. Both juvenile and adult Sarotherodon melanotheron had their blood samples drawn, and Randox test kits were utilized for the study. The results of the antioxidant analysis showed that SOD and GSH values dropped significantly (P<0.05), although CAT and LPO were significantly increased in both sizes when compared to the control. Compared to the adult fish, the juvenile fish displayed more pronounced alterations, and these changes were concentration-dependent. The results obtained align with the integrated application of oxidative stress parameters in the assessment of pollution risk to aquatic ecosystems.

Keywords

Glyphosate; Toxicants; Toxicology; Aquatic Environment; Oxidative Stress

Introduction

Aquatic organisms are commonly used for organizing and protecting aquaculture habitats and for conducting toxicity studies [1]. Aquatic pollution has been rising recently due to the widespread and careless usage of primary fertilizers like nitrogen and phosphorus. Numerous human activities are to blame for the higher concentrations of pollutants, such as pesticides, heavy metals, and industrial chemicals, in the aquatic environment. The elevated concentration of organic pollutants in the aquatic ecosystem results in a decrease in oxygen levels, hence increasing the mortality rate and hindering the regular functioning of exposed aquatic organisms. Research and accurate data collection are essential when it comes to detrimental effects on the survival rate, physicochemical conditions of aquatic organisms, and unpredictable changes in their health [2]. Aquatic species, such as fish, plants, invertebrates, and vertebrates, are important natural resources that lessen the severe stress brought on by human activity [3]. Research indicates that the primary causes of pollution in the aquatic environment are residential activities, agricultural runoff, and industrial effluents [4,5]. This pollution has a detrimental impact on the health of the ecosystem.

Aquatic organisms have suffered varying degrees of injury as a result of the increased release of industrial, agricultural, and home pollutants into the aquatic environment [6]. The use of pesticides in agriculture worries environmental and health specialists greatly because some of these chemicals damage surrounding aquatic systems even when sprayed in designated locations due to rain and flooding. Particularly impacted are fish [7,8]. Furthermore, a significant buildup and increased potential for poisoning aquatic life can be caused by pesticides' high solubility, frequent application, unintentional spills, and discharge from untreated effluents, and spray drift [9]. These pollutants' molecules in water can attach themselves to suspended particles, build up in sediment, or be consumed by aquatic life. In addition to having an impact on aquatic organisms' physiology and survival, these substances have the ability to interact with their genetic makeup to cause mutations and/or cancer [10].

It is well recognized that consuming tainted fish, zooplankton, phytoplankton, aquatic weeds, sediments, and agricultural goods can expose humans to harmful substances such heavy metals, pesticide residues, herbicides, fungicides, and other industrial wastes [11]. Of all the invertebrates, fish and other aquatic species are the most sensitive. They exhibit almost all of the physical and biochemical alterations caused by exposure to dangerous compounds, and they are frequently used as alert systems to monitor the normal state of the aquatic ecosystem. Among the several organisms in the aquatic food chain, fish are the most important and well-known species [12] due to their ability to absorb, digest, and concentrate harmful compounds found in the water. Aquatic organisms are especially susceptible to increased metal exposure because oxidative stress can lead to the development of highly reactive oxygen species [13]. The production of oxygen reactive radicals, which interact with proteins, lipids, and nuclei to cause genetic, cellular, and metabolic abnormalities that ultimately cause the living thing to die, is a representation of the mechanism of metal toxicity [14]. Genetic, cellular, and metabolic reactions are brought on by oxidative stresses.

Oxidative stress is defined as a disruption of the prooxidant-antioxidant equilibrium in favor of the former, which may cause damage [15]. The cause is either an increase in reactive oxygen species (ROS), a breakdown in antioxidant defense mechanisms, or an inability to repair oxidative damage [16]. As they interact with the aquatic environment, fish face a number of pressures. Reactive oxygen species (ROS), which can cause oxidative stress, are regularly produced by the organism in response to environmental stressors [17]. Elevated reactive oxygen species (ROS) have the potential to interact with biological macromolecules, leading to various consequences such as lipid peroxidation, DNA damage, and alterations in the functions of various antioxidant enzymes, including reduced glutathione, glutathione reductase, catalase, superoxide dismutase, and glutathione peroxidase [18]. According to Somdare et al. [19], oxidative stress has been linked to a number of diseases, including cancer, respiratory issues, and neurological issues.

Antioxidants can halt the oxidative stress caused by the dangerous chemicals [20]. Antioxidants found in their enzyme systems and low molecular weight proteins are essential to fishes' antioxidant defense mechanisms. Fish exposed to heavy metals may experience dose- and time-dependent oxidative stresses due to their high production of reactive oxygen species, high peroxidase activity, and low glutathione levels [21]. Prior studies have demonstrated that a number of harmful effects, such as reduced growth, altered immune responses, oxidative stress induction, genotoxic effects, and metabolic alterations, can be caused by specific pesticides and toxicants [22,23,24]. The aim of this study was to determine the level of antioxidant enzyme activity in the plasma of Tilapia guineensis after exposure to different dosages of lab-based Round Up.

Materials And Methods

Experimental Site

The study was conducted at the African Regional Aquaculture Center, an outstation of the Nigerian Institute for Oceanography and Marine Research, in Buguma, Rivers State, Nigeria. Seven days were spent acclimating 180 T. guineensis, of which 90 were juvenile and adult fish that were collected during low tide from ponds. The fish were transported in six open 50-liter plastic containers to the laboratory.

 Preparation of Test Solutions and Exposure of Fish

The chemical glyphosate, which is classified as a weak acid, was employed in the current investigation. Hydrogen ions can be given to other molecules by weak acids. I bought the herbicide Roundup, which contains glyphosate as its primary ingredient, from a store in Port Harcourt, Nigeria. T. guineensis were treated to the substance in triplicate at concentrations of 0.00 mg/L as a control, 0.05, 0.10, 0.15, 0.20, and 0.25 mg/L. Each test tank had five fish, chosen at random. The trial went on for fifteen days. Every day, fresh water was added to the tanks. The fish were fed commercial feed twice a day at a rate of 3% body weight.

Analytical Procedure

Using a small needle to make a caudal puncture, 2 milliliters of fresh blood were drawn at the conclusion of each experimental session and placed into heparinized sample vials. Samples of blood were immediately centrifuged at 5000 rpm for 15 minutes. Before being analyzed, plasma specimens were pipetted into eppendorf tubes, separated, and kept in a refrigerated at -20°C [25]. A Jenwayvisible spectrophotometer (Model 6405) with a universal microplate reader was used to read the findings. The approach of Beechey et al. [26] was utilized to assess the antioxidant activity in centrifuged plasma using spectrophotometric analysis. The APHA methods were also used to determine the parameters of water quality [27].

Statistical Analysis

The mean and standard deviation of the mean were used to express all the data. The data analysis was conducted using SPSS Version 22, a statistical software. Two-way ANOVA was used to separate the means, and at 5% (P<0.05), the two means were deemed significant.

Results

With the exception of DO, where lower values were obtained at greater herbicide concentrations, the water quality metrics (Table 1) were all within the same range. Table 2 shows how glyphosate affects the antioxidants in the plasma of juvenile T. guineensis. It was shown that as the herbicide concentration increased, the values of SOD and GSH dropped. On the other hand, CAT and LPO considerably raised in comparison to the control values. The antioxidant levels of adult fish exposed to the herbicide showed a similar pattern (Table 3), with SOD and GSH values declining as pesticide concentrations increased. On the other hand, CAT and LPO considerably rose in comparison to the control values.

Table1: Physico-Chemical Parameters of Water in Experimental Tanks of T.guineensis Exposed To Glyphosate Formulations.

Concentrations (mg/l) DO (mg/l) Temperature (oC) pH NH3 (mg/l)
0 6.43±0.89 b 29.88±2.51 a 6.68±1.99 a 0.02±0.01 a
0.05 5.54±0.19 b 29.85±7.11 a 6.67±1.54 a 0.02±0.01 a
0.1 5.22±0.01 b 29.83±1.09 a 6.67±1.71 a 0.02±0.01 a
0.15 5.01±0.31 b 29.89±3.65 a 6.65±0.86 a 0.02±0.01 a
0.2 4.77±0.55 a 29.87±5.52 a 6.67±0.41 a 0.03±0.01 a
0.25 4.02±0.45 a 29.81±7.99 a 6.65±0.43 a 0.03±0.01 a

Means within the same column with different super scripts are significantly different (P<0.05)

Table 2: Antioxidants Levels in Juvenile Sizes of T.guineensis Exposed to Glyphosate Formulations.

Concentration (mg/L) CAT (mmol/protein) GSH (mmol/protein) SOD (mmol/protein) LPO (mmol/protein)
0 60.55±9.44 a 7.87±1.03 c 12.88±0.57 b 7.02±0.77a
0.05 63.02±4.77 a 5.66±1.23 c 9.04±0.88 a 9.77±0.55 a
0.1 71.33±7.89 b 4.88±1.01 b 7.73±0.88 a 12.81±3.19b
0.15 76.77±9.99 b 3.99±1.02 b 6.71±1.07 a 14.03±2.09 b
0.2 81.31±6.66 c 2.65±0.88 a 4.66±0.88 a 17.02±7.07 b
0.25 86.01±7.91 c 1.64±0.48a 3.54±0.71 a 18.99±6.88 b

Means within the same column with different super scripts are significantly different (P<0.05)

Key: CAT- Catalase; SOD- Superoxide dismutase; LPO- Lipid peroxidase; GSH-Glutathione

Table 3: Antioxidants Levels in Adult Sizes of T.guineensis Exposed to Glyphosate Formulations.

Concentration (mg/l) CAT (mmol/protein) GSH (mmol/protein) SOD (mmol/protein) LPO (mmol/protein)
0 81.41±7.77 a 7.44±0.72 b 19.88±4.01 b 10.22±1.22 a
0.05 84.88±9.99 a 6.54±1.77 b 16.44±2.44 b 13.88±0.76 a
0.1 92.77±8.68 a 5.61±1.22 a 14.02±2.17 b 15.05±1.77 a
0.15 97.88±9.71 a 5.45±1.04 a 12.37±2.02 a 17.04±1.81 a
0.2 112.90±9.01 b 5.13±0.41 a 11.88±2.66 a 19.69±0.72 b
0.25 129.65±8.37 b 5.07±0.79 a 10.92±1.01 a 22.04±1.88c

Means within the same column with different super scripts are significantly different (P<0.05)

Key: CAT- Catalase; SOD- Superoxide dismutase; LPO- Lipid peroxidase; GSH-Glutathione

Discussion

When physiological conditions are demanding, mild oxidative stress produces reactive oxygen species (ROS) as a compensatory reaction. Removing these ROS can protect organisms from oxidative damage [Dar et al., 28]. Antioxidant activity may rise or fall in response to chemical stress, contingent upon the kind, length, and vulnerability of the exposed species. The decline in GSH values seen in this investigation could be the consequence of either increased peroxidase activity or direct scavenging of radicals, given that fish plasma fulfills a range of functions related to the metabolism of toxicants [29,30]. The two T.guineensis sizes used in this experiment demonstrated dose- and time-dependent increases in LPO. This may have to do with the capacity of glyohosate formulations to generate reactive oxygen species (ROS), which may then interact with the macromolecules in the fish to damage cells and alter antioxidants. The current study's findings about the increase in LPO brought on by gluphodate and the oxidative stress that ensued are consistent with those of Salah et al. [31], who reported that grass carp treated to zinc and mercury showed an increase in LPO. Following fenthion therapy, a rise in LPO leading to oxidative stress has also been seen in the species Rana ridibunda [32]. High amounts of LPO were seen in the plasma of C. gariepinus exposed to sub-lethal doses of deltamethrin [33]. Carassius carassius treated to endosulfan exhibited a notable LPO amplification in its blood, as reported by Dar et al. [34]. The lipid membrane is one of the targets of ROS that occurs through lipid peroxidation (LPO) [35]. As a result, LPO estimate has proven to be a useful tool for identifying oxidative stress brought on by contaminants in aquatic animals [36]. As bioindicators of oxidative stress, fish exposed to various toxins may show increased LPO and activation of antioxidant enzymes [37].

Oxidative stress is brought on by reactive oxygen metabolites (ROS), which are produced by glyphosate's toxicity [38]. Production of ROS can interact with lipid membranes in cells, causing lipid peroxidation, DNA damage, and disruption of physiological processes [39, 40]. According to reports, glyphosate can cause oxidative stress and cytoplasmic membrane toxicity, which can both negatively impact cellular function [41,42]. Malondialdehyde (MDA) concentrations are a sign of lipid peroxidation [43], glutathione (GSH) is an antioxidant molecule [44], and an imbalance in the generation and elimination of ROS causes GSH to become activated in order to counterbalance MDA concentrations [45]. The fish exposed to this study had higher plasma levels of LPO and CAT activity. 

Superoxide dismutases (SODs) are the first line of defense against free radicals because they may catalyze the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen [46]. In both sizes of T.guineensis, it was suppressed by glyphosate e exposure when compared to the control fish in the current experiment. The study's findings indicate that the treated fish's plasma had lower SOD levels, which may indicate a reduction in the tissues' ability to resist free radicals that produce oxygen. Similar findings on decreased SOD have been seen in the tissues of Oreochromis niloticus exposed to heavy metal ingestion [47].

This investigation's findings demonstrated that the plasma of T. guineensis treated with glyphosate had significantly (P<0.05) higher CAT activity after 15 days of exposure. The increase in CAT seen in this study may be a physiological reaction to the decrease in ROS production. Similar results have been observed in tilapia (O. niloticus) exposed to pesticides in the laboratory [48]. Antioxidant defense enzymes such as SOD and CAT protect aquatic organisms against free radicals that can lead to oxidative stress, which is why these enzymes are so beneficial. The present data showed that SOD activity generally decreased as CAT activity increased. It was also shown that CAT was the most sensitive antioxidant enzyme when compared to the other antioxidant enzymes. The spike in CAT activity may be related to coping with the increased oxidative stress caused by chemical exposures, while the reduction in CAT activity may be related to the possible direct binding of metal ions to the -SH groups on the enzyme molecule. Many fish species similarly showed increased CAT activity after being exposed to pesticides [49]. The sensitivity of SOD and CAT activity to metal exposures was also corroborated by our previous research [50]. Diminished SOD activity could indicate damage to the antioxidant systems caused by pesticides. As the pesticide concentration increased, there was a discernible drop in the GSH values. It's possible that the fish's cells are lowering their GSH levels to protect themselves from the glyphosate-induced oxidative stress. Similar drops in GSH levels have also been seen in fish exposed to other toxicants [51]. Regardless of fish size, the decrease in GSH in the exposed fish may indicate the presence of superoxide radicals and the antioxidant's limited capacity to combat oxidative stress. One idea states that because of their organ-specific reactivity, fish tissues exposed to pesticides exhibited reduced GSH levels [52]. Fish's defense mechanism that protects them from oxidative stress is most likely the cause of their low GSH level. Reduced GSH levels brought on by pesticides might result from GSH's capacity to bind to toxins and counteract their negative effects. At first, GSH offered prompt protection against oxidative stress by either directly purifying the ROS that oxidative stress created or by using the GSH redox cycle [53].

Conclusion and Recommendations

According to the results of the present investigation, glyphosate caused oxidative stress, as evidenced by decreases in SOD and GSH levels and increases in CAT and LPO levels. The regulatory authorities may find it helpful to combine the use of oxidative stress biomarkers with a fish model for assessing the risk of contaminants in aquatic ecosystems. To better understand the mechanisms of action leading to the development of oxidative stress, more research on the toxicokinetics and dynamics of glyphosate is required. To assess the toxicity of pesticides in aquatic habitats, these traits can be used as biomarkers. More investigation is needed to evaluate the pesticide's residual effects in different fish body tissues in order to better understand the physiological significance of the methyl parathion status in natural populations.

References

  1. Abdelmagid AD, El Asely AM, Said AM. Evaluation of Foeniculum vulgare impact on glyphosate hepato-toxicity in Nile tilapia: biochemical, molecular and histopathological study. Aquac Res. 2021; 52: 5397-5406.
  2. Abdelmagid AD, Said AM, Gawad EAA, Shalaby SA, Dawood MAO. Propolis nanoparticles relieved the impacts of glyphosateinduced oxidative stress and immunosuppression in Nile tilapia. Environ Sci Pollut Res. 2022; 29: 19778-19789.
  3. Abdel-Warith A-WA, Younis EM, Al-Asgah NA, Gewaily MS, ElTonoby SM, Dawood MAO. Role of Fucoidan on the growth behavior and blood metabolites and toxic effects of atrazine in Nile Tilapia Oreochromis niloticus. Linnaeus, 1758. Animals. 2021; 11: 1448.
  4. Acar U, Inanan BE, Navruz FZ, Yilmaz S. Alterations in blood parameters, DNA damage, oxidative stress and antioxidant enzymes and immune-related genes expression in Nile Tilapia Oreochromis niloticus exposed to glyphosate-based herbicide. Comp Biochem Physiol c Toxicol Pharmacol. 2021; 249: 109147.
  5. Al-Ghanim KA, Mahboob S, Vijayaraghavan P, Al-Misned FA, Kim YO, Kim HJ. Sub-lethal efect of synthetic pyrethroid pesticide on metabolic enzymes and protein profle of non-target Zebrafsh, Danio rerio. Saudi Journal of Biological Sciences. 2020; 27: 441-447.
  6. Blahova J, Cocilovo C, Plhalova L, Svobodova Z, Faggio C. Embryotoxicity of atrazine and its degradation products to early life stages of zebrafsh Danio rerio. Environ Toxicol Pharmacol. 2020; 77: 103370.
  7. Paray BA, El-Basuini MF, Alagawany M, Albeshr MF, Farah MA, Dawood MAO. Yucca schidigera usage for healthy aquatic animals: potential roles for sustainability. Animals. 2021; 11: 93.
  8. Peillex C, Pelletier M. The impact and toxicity of glyphosate and glyphosate-based herbicides on health and immunity. J Immunotoxicol. 2020; 17: 163-174.
  9. Munro C, Lasley B. Non-radiometric methods for immunoassay of steroid hormones. Prog Clin Biol Res. 1988; 285: 289-329.
  10. Polakof S, Panserat S, Soengas JL, Moon TW. Glucose metabolism in FSH: a review. J Comp Physiol B. 2012; 182: 1015-1045.
  11. Muhammad UA, Yasid NA, Daud HM, Shukor MY. Glyphosate herbicide induces changes in the growth pattern and somatic indices of crossbred red Tilapia O. niloticus × O. mossambicus. Animals. 2021; 11: 1209.
  12. Naiel MAE, Shehata AM, Negm SS, Abd El-Hack ME, Amer MS, Khafaga AF, et al. The new aspects of using some safe feed additives on alleviated imidacloprid toxicity in farmed fsh: a review. Rev Aquac. 2020; 12: 2250-2267.
  13. Ramaiah SK. A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food Chem Toxicol. 2007; 45: 1551-1557.
  14. Rossi AS, Fantón N, Michlig MP, Repetti MR, Cazenave J. Fish inhabiting rice felds: bioaccumulation, oxidative stress and neurotoxic effects after pesticides application. Ecol Ind. 2020; 113: 106186.
  15. Batool Y, Abdullah, Naz H, Abbas K. SubLethal Effect of Waterborne Cadmium Exposure on Glutathione S-Transferase and Total Protein Contents in Liver of Carnivorous Fish, Wallago attu. B Life Environ. Sci. 2018; 55: 21-25.
  16. Talas ZS, Orun I, Ozdemir L, Erdogan K, Alkan A ,Yilmaz I. Antioxidative role of selenium against the toxic effect of heavy metals (Cd+2, Cr+3)on liver of rainbow Trout Oncorhnchus mykiss. Fish Physiol.Biochem. 2008; 34: 217-222.
  17. Livingstoone DR. Contaminant stimulated reactive oxygen species production and oxidative damagein aquatic organisms. Mar. Pollution Bull. 2001; 42: 656-666.
  18. Zang J, Shen H, XuTL, Wang, XR, Li WM, Gu YF. Effect of long term exposure of low level diesel oil on the antioxidant defense system of fish, Environ.Contam. Toxicol. 2003; 71: 234-239.
  19. Korni FMM, Khalil F. Effect of ginger and its nanoparticles on growth performance, cognition capability, immunity and prevention of motile Aeromonas septicaemia in Cyprinus carpio fngerlings. Aquac Nutr. 2017; 23: 1492-1499.
  20. Jesudoss VAS, Santiago SVA, Venkatachalam K, Subramanian P. Chapter 21 - Zingerone Ginger Extract: Antioxidant Potential for Efficacy in Gastrointestinal and Liver Disease. Gastrointestinal Tissue. Academic Press. 2017; 289-297.
  21. Langiano VdC, Martinez CBR. Toxicity and efects of a glyphosate-based herbicide on the Neotropical FSH Prochilodus lineatus. Comp Biochem Physiol C Toxicol Pharmacol. 2008; 147: 222-231.
  22. Korni FMM, Abo El-Ela FI, Moawad UK, Mahmoud RK, Gadelhak YM. Prevention of Edwardsiellosis in Clarias gariepinus using ginger and its nanoparticles with a reference to histopathological alterations. Aquaculture. 2021; 539: 736603.
  23. Lackner R. “Oxidative stress” in fish by environmental pollutants. 1998; 203-224.
  24. Kavitha P, Venkateswara RJ. Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquitofsh, Gambusia afnis after lethal effect of an organophosphate pesticide, monocrotophos. Pestic Biochem Physiol. 2007; 87: 182-188
  25. Jiraungkoorskul W, Upatham ES, Kruatrachue M, Sahaphong S, Vichasri-Grams S, Pokethitiyook P. Biochemical and histopathological efects of glyphosate herbicide on Nile tilapia Oreochromis niloticus. Environ Toxicol. 2003; 18: 260-267.
  26. Ellman GL. Tissue Sulfhydryl Groups. Arch Bio¬chem Biophys. 1959; 82: 70-77.
  27. Beechey RB, Hubbard SA, Linnett PE, Mitchell AD, Muvn EA. A simple and rapid method for the preparationof adenosinetriphosphatasefrom submitochondrial particles. Biochem. J. 1975; 48: 533-537.
  28. APHA (American Public Health Association) Standard Methods for the Examination of water and wastewater 19thEdn. Washington DC. 1998.
  29. Dar SA, Yousuf AR, Balkhi MH, et al. Investigation on the genotoxicity of endosulfan to freshwater cyprinid fish Crucian carp Carassius carassius L. using the micronucleus and chromosomal aberration as biomarkers. Nucleus. 2014; 57: 87-98.
  30. Simonata J, Guedes C, Martinez C. Biochemical, physiological and histological changes in Neotropical fish Prochildus lineatus exposed to diesel oil. Ecotoxicol. Environ. Saf. 2008; 69: 112-120.
  31. Jiminez BD, Stegeman JJ. Detoxification enzymes as indicators of environmental stress on fish. Am. Fish. Soc. Symp. 1990; 8: 67-79.
  32. Saleh PM, Martinez GA, Chaves AR and Anon MC. Peroxidase from strawberry fruit Fragaria ananassa Duch: partial purification and determination of some properties. J. Agric. Food Chem. 1995; 43: 2596-2601.
  33. Kanter A, Celik I. Acute effects of fenthion on certain oxidative stress biomarkers in various tissues of frogs (Rana ridibunda). Toxicol Ind Health. 2012; 28: 369-376.
  34. Amin KA, Hashem KS. Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol. BMC Vet Res. 2012; 8: 45-52.
  35. Dar SA, Yousuf AR, Balkhi M, et al. Assessment of endosulfan induced genotoxicity and mutagenicity manifested by oxidative stress pathways in freshwater cyprinid fish crucian carp Carassius carassius L. Chemosphere. 2015; 120: 273-283.
  36. Kumar P, Kumar R, Nagpure NS, et al. Genotoxicity and antioxidant enzyme activity induced by hexavalent chromium in Cyprinus carpio after in vivo exposure. Drug Chem Toxicol. 2013; 36: 451-460.
  37. Dabas A, Nagpure NS, Ravindra K. Assessment of tissuespecific effects of cadmium on antioxidant defense system and lipid peroxidation in freshwater murrel, Channa punctatus. Fish Physiol Biochem. 2013; 38: 468-482.
  38. Lakra WS, Nagpure NS. Genotoxicological studies in fishes: a review. Indian J Anim Sci. 2009; 79: 93-98. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium deficient rat liver. Biochem Biophys Res Commun. 1976; 71: 952-958.
  39. Li ZH, Velisek J, Zlabek V. Chronic toxicity of verapamil on juvenile rainbow trout Oncorhynchus mykiss: effects on morphological indices, hematological parameters and antioxidant responses. J Hazard Mat. 2011; 185: 870-880.
  40. Firat O, Kargin F. Biochemical alteration induced by Zn and Cd individually or in combination in the serum of Oreochromis niloticus.Fish.Physiol. Biochem. 2010; 36: 647-653.
  41. Durmaz H, Sevgiler Y, Uner V. Tissues specific antioxidative and neurotoxic responses to diazinon in Oreochromis niloticus. Pestisid. Biochem.and Physio. 2006; 84: 215-226.
  42. Sunaina A, Ansari BA. Oxidative stress biomarkers in assessing arsenic tri oxide toxicity in the Zebrafish, Danio rerio. Int. J. Fish. Aquat. Syst. 2016; 4: 8-13.
  43. Taweel A, Othman MS and Ahmad A. Heavy metals concentration in different organs of tilapia fish (Oreochromis niloticus) from selected areas of Bangi, Selangor, Malaysia. Afr. J. Biotech. 2011; 10: 11562-11566.
  44. Tripathi G, Shasmal J. Concentration related responses of chlorpyriphos in antioxidant, anaerobic and protein synthesizing machinery of the freshwater fish, Heteropneustes fossilis. Pesticide Biochem. Physiol. 2011; 99: 215-220.
  45. Sun X, Li J, Zhao H, Wang Y, Liu J, Shao Y, et al. Synergistic effect of copper and arsenic upon oxidative stress, inflammation and autophagy alterations in brain tissues of Gallus gallus. J. Inorg. Biochem. 2018; 178: 54-62.
  46. Faheem M, Sulehria AQK, Tariq MI, Khadija A, Fiaz Saeed M. Effect of sublethal dose of cadmium chloride on biochemical profile and catalase activity in freshwater fish Oreoochromi niloticus. Biologia. 2012; 58: 73-78.
  47. Banaee M, Akhlaghi M, Soltanian S, Sureda A, Gholamhosseini A, Rakhshaninejad M. Combined efects of exposure to sub-lethal concentration of the insecticide chlorpyrifos and the herbicide glyphosate on the biochemical changes in the freshwater rayfish Pontastacus Leptodactylus. Ecotoxicology. 2020; 29: 1500-1515.
  48. Cardoso AJdS, dos Santos WV, Gomes JR, Martins MTS, Coura RR, Oliveira MGdA, et al. Ginger oil, Zingiber officinale, improve palatability, growth and nutrient utilisation efciency in Nile tilapia fed with excess of starch. Anim Feed Sci Technol. 2021; 272: 114756.
  49. Brum A, Cardoso L, Chagas EC, Chaves FCM, Mourino JLP, Martins ML. Histological changes in Nile tilapia fed essential oils of clove basil and ginger after challenge with Streptococcus agalactiae. Aquaculture. 2018; 490: 98-107.
  50. Chung S, Ribeiro K, Melo JFB, Teixeira DV, Vidal LVO, Copatti CE. Essential oil from ginger infuences the growth, haematological and biochemical variables and histomorphometry of intestine and liver of Nile tilapia juveniles. Aquaculture. 2021b; 534: 736325.
  51. Dawood MAO, AbdEl-kader MF, Moustafa EM, Gewaily MS, Abdo SE. Growth performance and hemato-immunological responses of Nile tilapia Oreochromis niloticus exposed to deltamethrin and fed immunobiotics. Environ Sci Pollut Res. 2020b; 27: 11608-11617.
  52. Fazelan Z, Vatnikov YA, Kulikov EV, Plushikov VG, Yousef M. Efects of dietary ginger Zingiber ofcinale)administration on growth performance and stress, immunological, and antioxidant responses of common carp Cyprinus carpio reared under high stocking density. Aquaculture. 2020; 518: 734833.
  53. Gewaily MS, Shukry M, Abdel-Kader MF, Alkafafy M, Farrag FA, Moustafa EM, et al. Dietary Lactobacillus plantarum relieves Nile tilapia Oreochromis niloticus juvenile from oxidative stress, immunosuppression and inflammation induced by deltamethrin and Aeromonas hydrophila. Front Mar Sci. 2021; 8: 203.
  54. Hassan MA, Hozien ST, Abdel Wahab MM, Hassan AM. Ameliorative effect of selenium yeast supplementation on the physiopathological impacts of chronic exposure to glyphosate and or Malathion in Oreochromis niloticus. BMC Vet Res. 2022; 18: 159.