Protective Effect of Supplementation with Powdered Mulberry Leaves on Glyphosate-Induced Toxicity in Catfish ( Clarias gariepinus )

| The prolonged-release of agricultural waste has led to an increase in the level of pesticides especially glyphosate which was reached in aquatic environments. Research has been carried out on catfish to detect the impacts of glyphosate and the protective effect of adding mulberry leaves powder against glyphosate on cat fish. Eighty immature catfish ( Clarias gariepinus ) were allocated into 4 groups; (G1) was maintained in the glyphosate-free water (G2) was exposed to 0.053 mg glyphosate (Glyph) /l water (1/10 96hr LC 50 ), (G3) was exposed to M. albus leaves powdered (5% ration supplementation) and (G4) was exposed to Glyph (0.053 mg/L) + M. albus (5% from ration supplementation). At day 21 of the experiment, fish were taken for biochemical and histopathological examination and to determine its residues in muscle. Biochemical results indicated an elevation in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), Urea, creatinine, and malondialdehyde (MDA) in G2 group and reduction in G3 and G4 treated groups. While, Total protein (TP), albumin, Ca, Na, Mg ions, and reduced glutathione (GSH) significantly reduced in G2 group and elevate in G3 and G4 treated group. Several histopathological lesions were observed in the brain, gills, kidney, labyrinth organ, liver, and spleen in G2 and were improved after using M. albus in treated groups. The outcome of this study indicated that M.albus has to ameliorate the negative impacts of glyphosate on catfish through its antioxidant activity and the biologically active component which lead to improving in cat fish intoxicated with gyphosate.


fiSh aNd glaSS aquaria
A round of (80) seemingly healthy immature catfish (Clarias gariepinus) including both sexes with bodyweight (150±20 g) has been collected from private farm fish in Tanta province. Before the beginning of the experiment, catfish were acclimatized for two weeks in eight glass fish tanks (100×50×50 cm/each) filled with tap water free chlorines under experimental conditions (natural photoperiod 11.58/12.38h and temperature 25.8±1.8 o C). The aeration process was constant and fixed in all fish tanks using the air pump. Commercially, the ration was used for catfish usually at 3% of body weight daily contain 25% protein. Dissolved oxygen, temperature, ammonia, and pH of experimental water was used according to WHO (2001).

ExpEriMENtal procEdurES
Twenty-one day for subacute toxicity was done by using (1/10 96hr LC 50 ) of glyphosate was added to tested catfish (20 fish for each). The study was done in aquarium each contains 100 liters of water and 10 fish. Two aquariums were used for each group and the requested concentration was kept and replenished daily by siphoning and substituted by the same amount of water. A control group (G1) (20 catfish) was put in glyphosate free fish tanks along the time of the experimental, glyphosate (Glyph), G2 was exposed to (1/10 96 hr LC 50 ) glyphosate in concentration 0.053 mg/L (Ayanda et al., 2015). G3 (M. albus) was exposed to M. albus leaves powdered (5% ration supplementation) (Hou et al., 2020) and finally G4 was exposed to Glyph (0.053 mg/L) + M. albus (5% from ration supplementation). At day 21 of the experiment, the fish were taken for biochemical, histopathological examination and residue determination in muscle. The tested fish were regularly followed-up daily along the experimental period. The experimental protocols and procedures were approved by Animal Health Research Institute (AHRI), Tanta Branch, Agriculture Research Centre (ARC), Giza, Egypt with approved number: (AHRI; 31122019).

SaMpliNg prEparatioN
Throughout the study end, blood samples were collected from the caudal vein of catfish in a test tube and set for 30 min for coagulation followed by centrifugation (3000 rpm for 15 min) and preserved at −20 °C until using for measuring the liver and kidney enzymes and electrolytes level. Immediately, the liver was removed and cleaned with NE US Academic Publishers

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October 2021 | Volume 9 | Issue 10 | Page 1720 sterile saline was used for detection of oxidant/ antioxidant parameters, samples from gills, liver, kidneys, labyrinth organ, spleen, and brain were used for pathological examination.
The fish muscles were analyzed for determination of glyphosate residue as described by Sun et al. (2017).

oxidaNt/aNtioxidaNt dEtEctioN iN hEpatic hoMogENatES
Malondialdehyde (MDA) is the indicator of oxidative stress and the reduced glutathione (GSH) which is considered a biomarker of antioxidant parameters were determined spectrophotometrically as recorded by Ohkawa et al. (1979) and Beutler (1963

prEparatioN of thE StaNdard SolutioNS
The stock solution of Glyphosate standard was prepared in deionized water at a concentration of 1000 ppm, and this standard solution was stored in dark bottles at 4℃. Sodium tetraborate in deionized water (50mM/L) and FMOC dissolved in acetonitrile (1g/L) were used for the derivatization process. The standards for plotting of the calibration curve were prepared in the blank fish muscle at range (0.1-2 µg/kg) then extracted as described below in sample preparation.

SaMplE prEparatioN
Glyphosate were extracted and purified from fish muscle as described by Sun et al. (2017).

chroMatographic coNditioNS
The parameters of the HPLC method were illustrated in a Table 1. Fish of all experimental groups were subjected to postmortem examination and then fresh tissue specimens from gills, liver, spleen. kidneys, labyrinth organ and brain were quickly put in neutral formalin solution 10% for at least one day for pathological examination according to Suvarna et al. (2013).

StatiStical aNalySiS
Data of this study were analyzed for detection differences among the groups through using one way a nova computer program (ANOVA) of the Statistical Analysis System was found by Tukey's HSD post hoc test using statistical (Sas, 2001). The study results are represented as the mean ± standard error with differences P ≤ 0.05 is statistically significant.

livEr aNd kidNEy fuNctioN
The adverse effect of a glyphosate-based herbicide on catfish and the potential protective role of M. albus leave powder on serum liver and kidney function biomarkers are recorded. In comparison with control, serum ALT and AST enzyme activities were significantly elevated in the glyphosate intoxicated group and significantly reduced in M. alba and in the treated group (M. alba/Glyh). While, there was a statically decrease in total protein and albumin in the intoxicated glyphosate group, and there is no statistically significant detection were recorded in M. alba and treated group in relation to control. But there is

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October 2021 | Volume 9 | Issue 10 | Page 1721 a significant elevation in the glyphosate intoxicated group after using M.alba in the treated group. Creatinine and urea were significantly elevated in the intoxicated group with glyphosate and a significant reduced in the treated group with M. alba and in the combination (Glyh+M. albus) treated group were observed in Table 2.

SEruM ElEctrolytES dEtErMiNatioN
Calcium, sodium, and magnesium electrolyte concentrations were statistically reduced in glyphosate intoxicated group concerning with control and a significant increase in M. alba and in the combination-treated group (GLyh+M. albus) in all mineral except Ca concentration were reduced in relation to control and increased in relation to the intoxicated group as in Table 2.

Mda aNd gSh dEtEctioN
In comparison with control, there is a significant elevation in MDA in the intoxicated group with glyphosate, and a significant reduction in M. alba and the combinationtreated group (M. alba/ Glyph) was observed. In contrast, GSH the indicator of the antioxidant system was observed significantly reduced in the intoxicated group with glyphosate. While there is a significant increase was observed in M. alba/ Glyph treated group in relation to the intoxicated group as in Table 3.

glyphoSatE rESiduE iN fiSh MuSclE by hplc
According to USP (2019) the method was validated and the validation results were summarized in Table 4. The standard curve of glyphosate concentration versus peak area was detected in Supplementary Figure S1. The obtained results showing that the used method was accurate, precise and robust as detected in Figures 1 and 2).
The levels of glyphosate in fish muscle were mentioned showing no significance in glyphosate intoxicated groups and glyphosate with mulberry in the treated group as shown in Figure 3.

thE MacroScopical ExaMiNatioN
Macroscopical examination of the control and Mulberry groups showed normal macroscopical picture of the internal organs. While the Glyphosphate group exhibited hyperemic gills with mucous secretions. The kidneys and livers were pale in color with congested vasculatures. The labyrinth organ exhibited hard in texture and the spleens were pale in color. On the other hand, the Mulberry treated group showed no marked gross lesions.

thE hiStopathological ExaMiNatioN
The histopathological examination of catfish brain in comparison with the control group (C) which was appeared with normal basophilic neurons, normal brain parenchyma, and normal white matter nerve fibers in the brain as in Supplementary Figure S2C. The intoxicated group with glyphosate Supplementary Figure S2 G(a) the brain revealed many pathological lesions as severe acidophilic necrosis of neurons with cavitation (motheaten appearance) around the neurons with thrombosis of blood capillaries. The brain in Supplementary Figure

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October 2021 | Volume 9 | Issue 10 | Page 1722     The protected group showed improvement in gill filaments structure with the still presence of mild gill necrosis and some chondrocytes in the central part of filaments Supplementary Figure S3 (T). in addition to the chart of the gills of fish intoxicated with glyphosate recorded hyperplasia and adhesion, mucus cell, chondrocytes in central filaments and degeneration and necrosis, while that lesion was disappeared from CTR and another treated group and improved in treated group with (Glyh+M. albus) Figure 5.
The labyrinth organ of control which revealed a normal central elastic cartilage and peripheral maize like structure epithelial cells with normal blood capillaries and the suparbranchial organ tree-like structure from the second and fourth-gill arches allows the catfish to take oxygen directly from the air for short period Supplementary Figure S4 (C). the labyrinth organ has a pathological lesion in the intoxicated group with glyphosate as the following conversion of elastic cartilage into fibrocartilage with necrosis and degeneration of epithelial lining the outer structure Supplementary Figure   In addition, the kidney of fish intoxicated with glyphosate has degeneration, necrosis sloughing of epithelial renal, glomerular atrophy, blood dilation and folding, blood capillary thickness, thrombus in blood capillary, complete necrosis of tissue and interstitial fibrosis, while this lesion were disappear from CTR and other treated group and improved in treated group with (Glyh+M. albus).as presented in Figure 7.
The hepatic tissue of the control showed a normal

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October 2021 | Volume 9 | Issue 10 | Page 1724 thickness Supplementary Figure   that the spleen intoxicated with glyphosate have enhanced depletion of lymphocytes in white pulb, but the increase lymphocytes in white pulp with dilated blood vessels and increase in periarterial lymphatic sheath were recorded in mullberry group, and treated group (Glyh+M. albus) Figure 9.  The eco-environment is widely affected by different types of pesticides from different sources. The disturbance in the aquatic environment may be due to several factors one of them is the elevation of herbicides concentrations due to the huge using in different agricultural practices with a large amount, and the fish is considered an avital monitor in ecotoxicology research (Glusczak et al., 2011). The presence of high-level glyphosate in the aquatic environment has received much interest in the research study due to its detrimental consequences on the living organism. Numerous studies have reported oxidative stress effect which was occurred during glyphosate intoxication is mainly an effect on biological systems (Sánchez et al.,

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October 2021 | Volume 9 | Issue 10 | Page 1725 2017). Accordingly, glyphosate is likely to provoke the deregulation of many biological metabolic pathways that may lead to the destruction of DNA, lipids, and proteins from ROS generation that elevate the level of oxidative damage and lowering cellular antioxidant levels in aquatic organisms (Boligon et al., 2015;Chaufan et al., 2014). Current biochemical studies have already demonstrated that glyph has increased the level of oxidative stress biomarker MDA and reduced the antioxidant level of reduced glutathione mostly in glyphosate-based herbicides intoxicated group due to exhaustion of GSH in a conjugation reaction or lowering through its biosynthesis pathway (Turkmen et al., 2019). Free radical scavengers with antioxidant activity in the cells act on ameliorating the damaging effects provoked by reactive free radicals. Apart from this oxidative stress indicator, hepatic, renal, and cardiac function are useful bioindicators of exposure to the pollutants as glyphosate-based insecticides in the eco-environment (Kalender et al., 2010). In comparison with the control catfish group in this study, serum AST and ALT levels significantly elevated in glyphosate intoxicated group due to hepatocyte damage resulted from liver injury ( Jasper et al., 2012;Youness et al., 2016). Reducing albumin and total protein levels in serum in this study due to reducing the oxidative phosphorylation process, that can lead to reducing protein absorption and protein synthesis followed by enhancing the catabolic process (Ghanbari et al., 2016). Serum creatinine and urea are helpful biomarkers in assessment in vivo and in vitro renal functions studies which were elevated in the group intoxicated with glyphosate as recorded by Çavuşoğlu et al. (2011). This may be due to impairment in the renal tubular destruction and impaired glomerular filtration in catfish that exposed to glyphosate insecticides in water (Çavuşoğlu et al., 2011). The electrolytes concentration in fish is responsible for the osmoregulation process during the stress condition (Ogamba et al., 2011). So that, the electrolytes are essential for all types of tissue function, the presence of alkali metal ions as (Na + , K + , Ca 2+) are vitally important for the stimulation of the enzyme activity. On the other hand, the critical losses of body electrolyte may reduce the osmotic concentration and leads to circulatory failure. The lower concentration of Na + and Ca + in the glyphosate exposure group due to the proximal tubules damage resulted from renal toxicity. And also, the higher serum (K+) concentration in the glyphosate exposed group due to the induction of oxidative damage (Ambali et al., 2010).
Mulberry leaves have a long history of using in traditional medicine in different countries. A wide range of nutritive content like amino acids, minerals, fatty acids, vitamins as well as quercetin, anthocyanins, chlorogenic, polysaccharides acid, and rutin that have been detected in mulberry depending on the cultivars and maturity stages (Yuan and Zhao, 2017). Mulberry leaves act as beneficial sources for antioxidant molecules like phenols, terpenoids, flavonoids, and flavonols (Iqbal et al., 2012). Taking responsibility for a significant decrease in MDA level and the elevated GSH level in the treated groups with M.albus could be attributable to its antioxidant properties in lowering peroxidative activity in the cells. It also includes various polyhydroxy alkaloids such as stilbenoids which are supposed to act as resveratrol and oxyresveratrol, flavonoids going to act as quercetin and kaempferol, rutin, and medicinal imino-sugars (Song et al., 2009). The dietary adding M. albus has led to an improvement in GOT and GPT enzyme activities in hepatic tissue and reduced the oxidative destruction through reducing ROS generation and cellular oxidation component and increasing antioxidant molecules activity , and the hepatoprotective activity resulted from antioxidant activity in alkaloid as flavonoids anti-carcinogenic activities (Majinda et al., 2011). The improvement in the levels of Ca and Na in the Glyph/M. albus group may be due to the high nutritive value of the dried leaves of M. albus that are mainly contain fibers, protein, carbohydrates, ash and are rich in β-carotene, ascorbic acid, calcium, iron, sodium, zinc and vital polyunsaturated fatty acids linoleic acid and essential amino acids which were required for normal metabolism and growth (Sarkar et al., 2018).
Bioaccumulation of glyphosate in animal tissues is not expected to be high due to its high water solubility, accompanied by a very low log Pow (-3.2), and its ionic character (European Commission, 2002). However, the bioaccumulation study showed that glyphosate accumulated in catfish muscle in both groups (Glyphosate and Glyphosate+Mulberry groups). These results agree with Contardo-Jara et al. (2009). Mulberry extract not significantly decrease the concentration of glyphosate in fish muscle.
Glyphosate-based herbicide in this study has led to a serious histopathological lesion in the gills, brain, labyrinth organ, hepatic, renal, and spleen. Neuronal changes in the brain were reported that detect a degenerative focus in neuronal bodies in the telencephalon of fish and necrotic changes in an intoxicated group with glyphosate (Ramírez-Duarte et al., 2008). Additionally, it can disrupt the barrier function through inducing opening in the blood-brain barrier that lead to changes in glucose metabolism in neuronal cell and glucose up taken in microvascular endothelial of the brain (Martinez and Al-Ahmad, 2019). The thrombus formation in brain blood vessels and an elevation of the white blood cells and platelets were recorded due to oxidative stress damage in the current result and this supported by Naz et al. (2019). The neuro-protective effect of mulberry leaves on brain histopathological lesion in a treated group

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October 2021 | Volume 9 | Issue 10 | Page 1726 due to its increasing antioxidant activity which reduces ROS formation and improving the blood-brain barrier permeability, with elevation in neuron density in the subregion of the hippocampus and reverses action Aβ25-35which induced neuronal injury and apoptosis inhibition through down regulation mRNA levels of an apoptotic protease-activating factor (Apaf1) (Rebai et al., 2017).
Gills inflammation, degeneration, and necrosis were detected in the group intoxicated with glyphosate (Ramírez-Duarte et al., 2008) which was improved after consuming the mulberry leaves which lead to improve the respiratory function in the brachia by elevating GOT activities and hemoglobin level in RBCs of crucian carp . While the histopathological changes in the labyrinth elastic cartilage in glyphosate induced toxicity group which is converted to fibrocartilage due to a severe depletion of serum Mn. Chondroitin sulfate synthesis depends on Mn (manganese), and its deficiency leads to osteoporosis and osteomalacia (Samsel and Seneff, 2015). Glyphosate also induces stiffening in the cell membrane and the appearance of cytoskeleton structures at a subcellular level of the skin which improved due to antioxidant activity and manganese content of mulberry leaves (Heu et al., 2012).
The histopathological changes in kidney tissue as degeneration, necrosis and atrophy in the renal tubular epithelium and interstitial fibrosis kidneys, thrombosis in the lumen of renal blood induced by glyphosate as supported by Naz et al. (2019). The treated group with mulberry revealed high normal intact renal glomeruli, healthy tubular epithelium, and normal renal blood vessels due to the improving the renal damage which lowering mALB/Cre (microalbumin/creatinine) ratio in urine and regenerate the tubular epithelial cells and the epithelial thickening of renal cystic that finding supported by . The liver of catfish which is intoxicated with glyphosate showed thickening of the portal vein and fibroblastic proliferation and hyalinization into the wall. The injury of both walls of the blood vessel and blood sinusoid due to glyphosate metabolites (aminom-ethyl phosphoric acid (AMPA)) and formaldehyde which are found in environments in soil and rivers which supported by Saleh et al. (2018). The mulberry extract helps to improve liver injury induced by alcohol through decreasing liver injury biomarkers levels (AST, ALT and ALP), triglyceride (TG), and cholesterol (TC). It also reduces lipid accumulation and lipogenesis, improves the transportation of fatty acid and oxidation process, decreased oxidative stress, and facilitated anti-inflammation as reported by Tang et al. (2013). The spleen in the intoxicated fish with glyphosate revealed depletion of lymphocytes in white pulp impacted with lymphocytes the protected group exhibited increased lymphocytes in white pulp the mulberry group showed an increased population of lymphocytes in white pulp and that in agreement with  who found thymus and spleen in mulberry treated groups were greater in comparison with control.

CONCLUSION AND RECOMMENDATIONS
The current modeling analysis derived from a research study on glyphosate-based herbicides induced toxicity in catfish via induction of ROS and the possible protective rules of M. albus leaves on glyphosate toxicity were discussed in this study. Hepato-nephron toxicity was induced by glyphosate is due to oxidative damage which is attributed to serum biochemical parameters and histopathological abnormalities and also, it was accumulated in the muscle tissue. The improvements in liver and kidney parameters of catfish may be due to mulberry leaves powder. It was concluded that the mulberry leaves herbal extract is being ecofriendly antioxidant compound with a highly natural protective effect against glyphosate induced toxicity. Finally, we recommended the following study to use different levels from M.albus leaves powder and increase the duration of study to be more than three months to record better result for using M. albus in different species.

ACKNOWLEDGMENTS
Many thanks for Professor Dr. Mahmoud Arafa (Arafa M.) Biochem. Department of Animal Health Research Institute in Dokki to assist in the preparation and estimation of blood biochemistry.

NOVELTy STATEMENT
The prolonged -relased of agriculture wastes has led to elevate the level of pesticides especially glyphosate which was reached to aquatic environment. research has been carried out on catfish to detect the toxic impacts of glyphosate and the protective effect of adding Mullberry leaves against glyphosate intoxication in cat fish.

AUTHOR'S CONTRIBUTION
El-Sheshtawy Sm and Samak DH Contributed experemiental desigh and biochemical analysis ,Abd-Elhafeez Ms determined glyphosate residue in catfish muscle by HPLC and statistical analysis of the method validation parameters and Nada MM contributed histopathological Examination. All authors participated in writing the manuscript.