Effects of Cypermethrin on the Hematological Parameters, Biochemical Components of Blood and Histopathological Changes in Different Organs of Chirruh Snow Trout (Schizothorax esocinus)

Naveed Akhtar1*, Muhammad Fiaz Khan1*, Sadia Tabassum1, Munawar Saleem Ahmad2 and Khan Dil Badshah3 1Department of Zoology, Hazara University, Mansehra, Pakistan 2Department of Zoology, University of Swabi, Pakistan 3Department of Chemistry, Government Postgraduate College, Haripur, Pakistan Article Information Received 10 April 2019 Revised 04 June 2019 Accepted 20 June 2019 Available online 03 August 2020


INTRODUCTION
I nsecticides are chemicals practiced for averting and monitoring pests, containing trajectories of humanoid or animal infections. They have been practiced for regulating undesirable floras or faunas triggering injury to the manufacture, handling, storing, advertising of foodstuff, agronomic possessions, timber goods, animal materials, which might be given to animals for controlling pests or additional insects attached to their bodies (WHO, 2002).
Pyrethroid and organophosphates are among the widely used pesticides. Pyrethroids are imitated type of pyrethrins (Soderlund et al., 2002). Pyrethroid have two varieties which differ in biochemical organization; in variety I allethrin, tetramethrin, resmethrin, bioremethrin, and permethrin are present, whereas variety II have cypermethrin, cyfluthrin, cyphenothrin and deltamethrin O n l i n e

F i r s t A r t i c l e
health status (Dorucu and Girgin, 2001;Ranzani-Paiva et al., 2005;Pimpao et al., 2007). Fish is highly at health risk to pyrethroid exposure (Moraes et al., 2013) and fish toxicity increases with increase in value of toxin per liter (Guner, 2009). Pesticides are accumulated in tissues like liver and muscle that result in dysfunction of organ and ultimately result is loss of fish (Srivastava and Kaushik, 2001). Many enzymes in fish are important for normal biological role and disruption in in the activity of enzymes act as primary indicator of contaminant toxicity (Jensen et al., 1991). The aim of this study was to assess the toxic effects of sublethal concentrations of cypermethrin on hematology, biochemistry, histopathology and DNA damage in freshwater fish Schizothorax esocinus.

MATERIALS AND METHODS
This research was conducted in Department of Zoology, Post Graduate College Haripur. All the guidelines were followed provided by the ethical committee of Post Graduate College Haripur.

Test species and laboratory care
Fish specimens of chirruh snow trout (Schizothorax esocinus n=25) were collected from river Swat and were transported to laboratory in a small glass aquarium having aerated oxygen. Fish were acclimatized for one week before starting the experiment. Five specimens were kept in each aquarium. Persistent oxygen was bubbled via air pumps in aquaria.

Experimental design
Cypermethrin ([S, R]-N-α-cyno-3-phenoxybenzyl-(IR, IS, cis, trans)-2, 2-dimethyl-3, (2, 2-dichlorovinyl, cyclopropane carboxylate), used in this study was purchased from a local market. Four different concentrations i.e., 1.00 ppb, 2.00 ppb, 3.00 ppb and 4.00 ppb of CYP were formulated from a commercial devising carrying 32% active ingredients. Five fishes were stocked in each aquarium of 100 L water capacity which were filled up to 60 L. Each aquarium was provided with different concentration of CYP and were labeled as, C0, C1, C2, C3 and C4. C0 was kept as control group, C1 had 1.00 ppb, C2 had 2.00 ppb, C3 had 3.00 ppb and C4 had 4.00 ppb CYP. Fishes were exposed to CYP for 96 h to these concentrations. After 96 h, fishes were anesthetized in clove oil. Blood was taken from caudal vein using 1 ml disposable syringe for hematology, electrolytes, metabolites, enzymes and DNA damage study. Fish was sacrificed to take tissues of brain, gills, kidney, liver, intestine and muscles.

Hematological parameters
WBCs and RBCs were measured manually by following the protocol provided by Hrubec et al. (1996). Hemoglobin was assessed spectrophotometrically by cyanomethaemoglobin technique described by (Drabkin, 1964). MCV, MCH, MCHC, platelets, monocytes, lymphocytes and neutrophils (10 9 /L) were estimated according to Jain (1986). Blood smears, were stained with Wright's Giemsa stain and used for the differential cell count as described by Hrubec et al. (1996).

Biochemical parameters
For biochemical analysis, the clotted blood samples were centrifuged to separate serum which was used for estimation of biochemical parameters using Chem Reader SBA-733 Plus (Semi-auto Chemistry analyzer, Advanced Japanese Technology). Biochemical parameters included in this study were; Electrolytes (Calcium, Potassium, Phosphorus and Sodium), Metabolites (Total Protein, Glucose, Creatinine, Urea, Cholesterol and Triglycerides) and Enzymes (Lactate Dehydrogenase (LDH) Alanine, Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST) and Aminotransferase (ALT)).

Histopathology
Gills, brain, liver, intestine, kidney and muscles tissues were dissected out of fish, fixed in 10% formalin followed by dehydration by passing through different grades of alcohol. The tissues were embedded in 4-5 µm thick sections were cut and stained hematoxylin and eosin.

Comet assay
The genotoxicity was executed by following the technique used by Singh et al. (1988). Blood samples were mixed in saline solution of 1000 μl. Using 10 uL of saline solution slides were prepared and (0.5%) of low melting agarose of 120 μl at 37 °C. In lysis solution consisting (1 mL of Triton X-100, 10 mL of DMSO and 89 mL of lysing solution stock, pH 10.0 -stock solution: 2.5 M of NaCl, 100 mM of EDTA100, 10 mM to 1 L of Tris) slides were kept for 60 minutes in the refrigerator. Slides after 60 minutes, slides were kept in a horizontal electrophoresis system at 25 V, 300 mA for 20 minutes. For about 15 minutes slides were neutralized with 0.4M of Tris having pH 7.5, and stable in ethanol for 10 minutes. Those cells in which no DNA damage occurred move consistently, whereas those cells having DNA damage, they showed fragments of dissimilar masses, and minor cells move quicker in electrophoresis, so make the tail of a comet.

Statistical analysis
Data was analyzed using SPSS software (Version 24.0). Comparison was made among all of the four experimental groups using one-way ANOVA followed by LSD. Variables were stated as means and standard deviations. p<0.05 was statistically significant. Table I show the effect of CYP on blood parameters.

Hematological parameters
Decrease is observed was WBCs, RBCs, Hb, HCT, MCV, MCH, MCHC and platelets levels of all treated groups . Monocytes showed increase in all treated groups. Lymphocytes showed decrease in number in all treated groups. Neutrophils counts showed increase in all groups. Figure 1 shows effect of different concentration on level of electrolytes in the fish serum. Increase was observed in sodium level and potassium level in all treated groups, whereas calcium and phosphorous level showed

O n l i n e F i r s t A r t i c l e
decreased level in all treated groups. Figure 2 shows effect of different concentration cypermethrin on triglycerides, cholesterol and urea which decreased in all treated groups. Creatinine showed no changes. Glucose level showed increase in all treated groups. Total protein level decreased in all treated groups. Figure 3 shows increase in ALP, ALT, AST and LDH activities in all treated groups after cypermethrin administration.

Comet assay
After the exposure of S. esocinus to CYP damage occurd in peripheral blood erythrocytes. Genotoxicity increased with increase in the concentration of CYP. CI has DNA damage of 0.001% which showed low DNA damage. C2 has DNA damage of 23.87% showing higher DNA damage. C3 has of DNA damage of 23.67% showing higher DNA damage. C4 has DNA damage of 63.62% showing higher DNA damage (Fig. 10).

DISCUSSION
This study was assessed to estimate the toxic potential of cypermethrin, on chirruh snow trout S. esocinus. Blood characteristics of fish express major deviation, and various ecological and lethal toxins can persuade broad variations (Zahran et al., 2018). Reduction in blood parameters of exposed fishes to insecticides, designate low RBC count results because we of blood formation, ion-change irregularity and proliferation in WBCs damage in blood producing tissues (Jenkins et al., 2003;Seth et al., 2003). In present study reduction occurred in WBC value. Other studies show increase in WBC counts in fishes exposed to different concentrations of cypermethrin (Akinrotimi et al., 2012;Masud et al., 2013). In our study decrease is observed in RBCs, Hb, HCT, MCV, MCH and MCHC exposed to different concentrations of CYP. Our findings are similar to previous studies conducted by (Masud et al., 2013;Velmurugan et al., 2016) in which they reported reduction of RBC, hemoglobin, hematocrit, MCV, MCH and MCHC level after exposure to different concentrations of CYP. In our study lymphocytes decreased whereas monocytes and neutrophils increased in all treated groups. Similar findings are reported by (Akinrotimi et al., 2012) in which they reported increase in neutrophils level. Montanha et al. (2014) reported no such changes in neutrophil, lymphocytes, monocyte.
Changes in biological parameters in due to ecological trauma is supported by different researchers in many species Channa punctatus (Topal et al., 2014), Labeo rohita (Kishor et al., 2014), Cirrhinus mrigala (Sreekala and Zutshi, 2012). In our study increase is observed in sodium level and potassium level, whereas decrease is observed in calcium and phosphorous level in all treated groups. Similar findings are reported by (Atamanalp et al., 2002) in which he reported decrease in calcium and phosphorous after exposure of fish to cypermethrin. In our study decrease is observed in triglycerides, cholesterol and urea in all treated groups. Similar findings are reported by (Orun et al., 2014) in which they reported decrease (p<0.05) in the levels of triglycerides, whereas other researchers reported higher values of cholesterol, urea and triglycerides in fish exposed to different concentration of CYP (Bhanu and Deepak, 2015;Ojutiku et al., 2013). This variation may be due to change in species. In our study increase is observed in glucose level in all treated groups. Similar findings are reported by (Bhanu and Deepak, 2015) in which he reported increase in glucose level after exposure of C. carpio to CYP. In our study decrease is observed in total protein level in all treated groups. Similar results are reported by (Bhanu and Deepak, 2015) in which they reported decrease in proteins level of fish after exposure to CYP. In our study increase is observed in ALP, ALT, AST and LDH activity in all treated groups. Similar findings are reported by (Ojutiku et al., 2013) by reporting increase in Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) in fish after exposure to CYP (Ojutiku et al., 2013). Similarly, increase is reported in Aspartate aminotransferase (AST) in fish treated with concentrations of 1.5 and 2.5 of cypermethrin (Montanha et al., 2014). Similarly, increase in aspartate aminotransferase (AST), lactate dehydrogenase (LDH) is reported in fish after exposure to CYP (Velisek et al., 2006).
In fish, histology variation serves as powerful and precise tags in valuation of fish status after exposure to contaminants (Van der Oost et al., 2003). Gills are the first target of fishes on exposure to insecticides and are major index for water excellence (Qureshi et al., 2016). Gills besides absorbing oxygen is busy in other vital tasks like exchange of ions, acid base equilibrium and removal of hazardous substances from body, therefore environmental pollutants destroy such important tissues badly and enforce unwanted noteworthy problems on body of fish (Bantu et al., 2017). In our study gills exposed to different concentrations of cypermethrin show shortening of lamellae, erosion of gills arch, blood congestion, necrosis, lamellar curling, hypertrophy of epithelial cells, degenerative changes, shortening of secondary gills lamellae, degeneration of epithelial cells and lamellar destruction. Other researchers reported similar findings as (Khan et al., 2018) reported lamellar disorganization, disruption of cartilage, epithelial lifting, loss, fusion, curling and shortening of secondary gills lamellae, atrophy and blood congestion. Common carp exposed to different concentrations of atrazine show alteration in gills including severe lamellar teleangiectasis, dilated lamellar capillaries filled with erythrocytes, rupture of dilated lamellar capillary and pooling of the blood with formation of thrombi (Blahova et al., 2014). Gills of Nile thilapia treated with chlorpyrifos indicate, abnormality and merging of secondary lamellae, mild hyperplasia of epithelial cells, accumulation of mucus, severe epithelial hyperplasia, lamellar necrosis, degradation in respiratory epithelial cells and connective tissue cells (Zahran et al., 2018).
Intestine is a major organ of fish, having chief part in ingestion and adjustment of food. It might be used as tag organ in ecotoxicology due to its delicate nature to all type of lethal substances (Khan et al., 2018). In our study intestine exposed to different concentrations of cypermethrin show blood congestion, necrosis, destructive changes, ulceration of mucosa, loss of structural integrity, pyknosis, shrinkage of mucosa and necrosis of tip. Rohu (L. Rohita) exposed to different concentrations of cypermethrin show alterations in intestines including, cup cells construction in villi, shortening of villi, fusion of villi, necrosis, hemorrhages and pyknosis (Khan et al., 2018).
Kidney of fish have key work in homeostasis, erythropoieses and removal of injurious substances as it is main tissues which is damaged by toxins in water atmosphere (Thophon et al., 2003;Neelima et al., 2015). In our study kidney exposed to different concentrations of cypermethrin show vacuolation, interstitial hemorrhage, hyperactivated MM, interstitial hemorrhage and multifocal granulomas. Nile thalipia exposed to different concentrations of chlorpyrifos show lesions in kidney including, interstitial amassing of eosinophilic watery liquid, hyperactivated MM, interstitial fluid, adequate vacuolization of epithelial lining proximal and distal tubules, interstitial hemorrhage, extensive aggregation of MNCs, austere vacuolization of epithelial lining proximal and distal tubules as well as enlarged interstitial space (Zahran et al., 2018).
Liver is among the main organs in body due to its key function in metabolism of protein, carbohydrates and fats. Accumulation of insecticides and their byproducts in liver results in important histology modification and variation in liver (Sharma et al., 2012) In our study liver exposed to different concentrations of cypermethrin show disappearance of hepatic cell wall, densely packed hepatocytes, bile pigment, necrosis, leukocyte infiltration and pyknosis. Others researchers reported similar changes in liver of L. rohita treated with cypermethrin, like termination of cell membrane, blood congestion, congestion, pyknosis, necrosis, hyperplasia and vacuolations of hepatocytes (Khan et al., 2018). Common carp exposed to atrazine show lesions in live including vacuolar degeneration of hepatocytes (Blahova et al., 2014). Liver of Nile thilapia exposed to chlorpyrifos show sporadic region with peculiar cores, mobbed blood vessels with perivascular MNCs accumulation, vacuolization of pancreatic cells, reduction and necrosis of pancreatic cells and MNCs aggregation nearby hepatopancreas (Zahran et al., 2018).
In our study brain exposed to different concentrations of cypermethrin show pyknosis, degenerative changes, necrosis and blood congestion. Brain of cyprinus carpio treatd with 2, 4-D NA salt, glyphosate and paraquat present, swelling of pyramidal cells with binucleated nucli, austere necrosis of neuron of cerebrum showing damage of nissl substances, mild vacular variations with hollow spaces, tegmentum was completely disappeared and number of neuroglial cells decreased (Deivasigamani, 2015). Similarly, C. carpio exposed to phorate show, mild degenerative changes, mild structural damage, degenerative changes, structural degeneration and congestion of neural cells in brain (Lakshmaiah, 2017). Cyprinus carpio exposed to quinalphos show, mild degenerative changes in neural cells, structural damage, necrotic changes in neural cells and intracellular edema, increased necrotic condition of neural cells and cytoplasmic vacuolization observed, slight degenerative changes and vacuolization (Chamarthi et al., 2014).
In our study muscles exposed to different concentrations of cypermethrin show muscles necrosis and fragmentation of sarcoplasm. Muscles of Tilapia nilotica exposed to pendimethlain show, muscle necrosis with calcified muscles, muscle necrosis with fragmentation of sarcoplasm, muscle necrosis with calcified muscles and mononuclear cell infiltration (Abd-Algadir et al., 2011).
Mutilation in DNA is due to significance oxidative stress, xenobiotic metabolites or xenobiotic (Santos and Martinez, 2012). Neotropical fish Prochilodus lineatus treated with different concentrations of atrazine result in increased DNA destruction (Santos and Martinez, 2012). Cyprinus carpio treated with fipronil and buprofezin in combinations result in increased genotoxicity in whole blood DNA content (Bantu et al., 2017). Genotoxicity of C. carpio exposed to oxadiazon was conducted through comet assay, which result in significantly higher DNA damage (21.3%, 22.9%, and 28.4%, respectively) (Zanjani et al., 2017). In our study S. esocinus exposed to different concentration of CYP cause damage in DNA of peripheral blood erythrocytes. Low DNA damage (0.001%) was observed at 1 ppb exposure whereas higher DNA damage (63.62%) was observed at 4 ppb concentration. It is evident that genotoxicity increased with increase in the concentration of CYP.

O n l i n e F i r s t A r t i c l e
Effects of Cypermethrin 9

CONCLUSION
The present study reveals that cypermethrin is toxic to Schizothorax esocinus even at low concentration. Cypermethrin promote hematological, biochemical, histopathological alterations and genotoxicity in S. esocinus. It is harmful to aquatic life; therefore, its use should be minimized and alternative techniques should be adopted.

ACKNOWLEDGMENT
This study was supported by higher education commission of Pakistan (10108).

Statement of conflict of interest
The authors declare no conflict of interest.