PUJZ_Wajid Ali et al (1).pdf

Punjab University Journal of Zoology 33(2): xxx-xxx (2018) http:dx.doi.org/10.17582/pujz/2018.33.2..... Research Article Study of Induce Histological and Biochemical Alterations in Hypophthalmicthyes molitrix after Acute Exposure to Deltamethrine Wajid Ali1,*, Asma Karim1, Muhammad Irfan2, Hafiz Abdullah Shakir3, Ghulam Mustafa4, Zeeshan Shafique1, Ijaz Anwar1 1 Department of Zoology Government College of Science, Wahdat Road, Lahore, Pakistan Department of Biotechnology, University of Sargodha, Sargodha, Pakistan 3 Department of Zoology, University of the Punjab, New Campus, Lahore, Pakistan 4 Department of Zoology, Govt. College University, Lahore, Pakistan 2 Article History Received: April 04, 2018 Revised: June 13, 2018 Accepted: June 20, 2018 Published: August 04, 2018 Authors’ Contributions ..............? Keywords Deltamethrine, Acute exposure, Silver carp, Serum, Biochemistry, Histology. Abstract | Pesticides have brought us great economic benefits, at the same time they are also root of serious ecological harms like non-target toxicity of organism. Synthetic pyrethroid (deltamethrine) is widely used due to its less toxicity and easy degradation in soil. In this study fresh water fish Hypophthalmicthyes molitrix were exposed to sub lethal concentrations of deltamethrine. Fish were divided into 4 groups and each group contains six fish. One of them is control and A, B and C were experimental groups. Experimental groups viz. A, B and C were exposed to 25, 50 and 75% of LC50, respectively for four days. After 96 h; sample of blood for biochemical analysis (blood glucose, cholesterol and total protein) and gills tissue for histology were taken. After acute exposure to pesticide, significant changes were observed in serum biochemistry and histology. Serum glucose and cholesterol were increased while total protein was decreased. Histopathological result reveled that gills tissue of experimental fish was damage severely resulting Necrosis, Damaged nuclei , rupturing of epithelial cells, Mucous cells and severe lamellar fusion .it was concluded that, deltamethrine is highly toxic for non-target aquatic organisms like silver carp (Hypophthalmicthyes molitrix). To cite this article: Ali, W., Karim, A., Irfan, M., Shakir, H.A., Mustafa, G., Shafique, Z. and Anwar, I., 2018. Study of induce histological and biochemical alterations in Hypophthalmicthyes molitrix after acute exposure to deltamethrine. Punjab Univ. J. Zool., 33(2): xxx-xxxx. http:dx.doi. org/10.17582/pujz/2018.3xxxxxxxxx ndustrial effluents, domestic sewage and pesticides are the important sources of aquatic pollution. Aquatic bodies that run through agricultural or industrial areas have high vulnerability of being polluted by pesticides through surface runoff and leaching. Aquatic ecosystems are greater part of natural environment. Aquatic ecosystems are continuously being polluted with a wide variety of environmental pollutants such as pesticides from industrial, agricultural and domestic activities (Okuku and Peter, 2011). Environmental pollution is a universal problem in a present society. Pesticides are used to get better crop production and other benefits. Extensive used pesticides has raised the questions about human health and non-target animals. Unfortunately, the application of these pesticides is highly toxic even at very small concentration to a number of non-target aquatic organisms such as fish (El-Sayed et al., 2007; Begum, 2005). Extensive use of insecticides has contaminates the ecosystems with toxicant. Pesticides are present in different concentrations in groundwater, crop samples and agricultural streams (Sharma et al., 2015; Mahboob et al., 2015; Yadav et al., 2015). *Corresponding author: Wajid Ali wajid_wajid1@yahoo.com Due to short biodegradation period and lack of the accumulative tendency in organisms, pyrethroid deltame- Introduction I December 2018 | Volume 33 | Issue 2 | Page 1 W. Ali et al. thrine, is extensively used (Laskowski, 2002). Their poisonous effect is in the parts per billion for non-target organisms (Bradbury and Coats, 1989b). Fish are considered to be indicators of environmental pollution because it is very sensitive to the presence of toxicant (Prusty et al., 2011). Fish during their life cycle may be exposed to a wide range of pesticides. When pesticides enter into the organs of fish they may considerably alter certain biochemical and physiological processes (Banaee et al., 2011). Pyrethroids are absorbed directly through gills into the circulation (Aydin et al., 2005). Pyrethroids cause 1000 times’ greater toxicity to fish as compare to other groups of birds and mammal (Bradbury and Coats, 1989a). In biological monitoring program It has been reported that biochemical changes are useful for observing water pollution, environmental quality and the health conditions of aquatic organisms (Kohler et al., 2007; Kori-Siakpere et al., 2008). Deltamethrine (Pyrethroid) is extensively used to control pest (Bradbury and Coats, 1989a). Sediment samples taken from the lake have confirmed the presence of deltamethrine (Balint et al., 1995). Histopathological investigations have long been recognized to be reliable biomarkers of stress in fish during lab experiment (van der Oost et al., 2003; Boran et al., 2012). Gills are the first target of water borne pollutants due to the constant contact with the external environment (Perry and Laurent, 1993). Since gills in fishes are considered the main passage for entrance of pollutants to the internal body organs (Takashima and Hibiya, 1995). Alterations found in this organ are normally easier to identify than functional ones (Fanta et al., 2003) and serve as warning signs of damage to animal health (Hinton and Laurén, 1990). This study was designed to check out acute toxic effect of pyrethroid deltamethrine on fish (Hypophthalmicthyes molitrix). In this experiment histopathology of gills and biochemical variable (glucose, cholesterol and total protein) was observed after acute exposure to deltamethrine. Materials and Methods Experimental animal Live silver carp (H. molitrix) were purchased from Himalayan Fish Hatchery Muredke, Sheikhupura. The average weight and length of fish specimen was 160.33±5.27 g and 9.96±0.138 cm, respectively. Fish were acclimatized with uninterrupted air supply with air pump in aquarium containing 40 liter tap water (without chlorination) for duration of two week at room temperature. After it fish were exposed to toxicant for 96 h. During this period dead fish were removed instantaneously. This study is performed at lab of department of zoology Government College of Science, Wahdat Road, Lahore, Pakistan. Pesticides Commercially synthesized deltamethrine (ParathyDecember 2018 | Volume 33 | Issue 2 | Page 2 roid) was used as toxicant and the concentration applied on experimental groups were described in our earlier reports (Karim et al., 2016a, b). Fishes were divided into four groups. One of them was a controlled group and A, B, C were experimental groups. Each group contain six fish (n=6). According to LC50 values three sub-lethal concentrations of deltamethrine were prepared. Three doses used for the experiment groups A, B, and C was as follows: Group A= 0.4 µg/ L (25% of LC50), Group B =0.8 µg/ L (50% of LC50), Group C=1.2 µg/L (75% of LC50). Biochemical and histological analysis After 96 h fish blood was drawn directly in sterilized syringes through cardiac puncture for biochemical studies of blood glucose, cholesterol and total protein. 2ml of blood was taken in serum vacutainer (devoid of any clotting factor) to separate the serum. At 4000rpm blood was centrifuged for twenty minutes. Then separated serum was stock up at -20°C until study. Small piece of gills tissue was fixed in 10% formalin after rinsing with saline solution for histological studies. The process of gills fixation was done by following standard protocol (Mumford, 2007). Statistical analysis The data ware statistically evaluated by One Way ANOVA test with SPSS 13 Statistical program and data were reported as Mean ± SEM with n=6. Results Glucose After 96 h exposure, the serum glucose level significantly increased. The concentration of glucose in control group was 54.2±0.80 mg/dl, when fish exposed to sub lethal concentrations of deltamethrine, glucose values increased significantly (P<0.000) in Group A (62.8±1.15 mg/dl), Group B (74.6±1.53 mg/dl) and Group C (63±1.30 mg/ dl) in acute exposure, respectively (Figure 1A). Cholesterol The concentration of cholesterol significantly increased in experimental groups as compared to control group. The concentration of cholesterol in control group was 177±5.08 mg/dl, when fish exposed to sub-lethal concentrations of deltamethrine, cholesterol values increased significantly (P>0.000) in Group A (212±4.06 mg/dl), Group B (227.6±3.26 mg/dl) and Group C (229±1.81 mg/dl) in acute exposure, respectively (Figure 1B). Total protein The concentration of total protein significantly decreased in experimental groups after 96 h exposure to deltamethrine. Concentration of total protein in control group was 6.64±0.23 mg/dl, when fish exposed to sub lethal concentrations of deltamethrine, total protein values decreased significantly (P<0.000) in Group A (5.25±0.27 mg/dl), Group B (4.4±0.20 mg/dl) and Group Histological and Biochemical Alterations in Hypophthalmicthyes molitrix C (4.22±0.23 mg/dl) in acute exposure, respectively (Figure 1C). Histopathology of gills Histopathological investigations are reliable biomarkers to check toxicity induce by pesticide. Normal aspect of control group gills showed primary lamella, secondary lamellae, mucous cell, chloride cell, pillar cell and epithelial cell. Histopathology of group A gills after acute exposure showed necrosis, damaged secondary lamella, damaged primary lamellae. Deltamethrine induce alteration in gills of group B after acute exposure resulting necrosis, degeneration, damaged nuclei, and rupturing of epithelial cells. Gills texture of Group C was altered severely causing necrosis, hypertrophy of mucous cells and lamellar severe fusion. These results reveled that deltamethrine badly effect the gills of experimental groups (A, B and C) as compare to control group (Figure 2). Figure 1: Glucose (A), cholesterol (B) and total protein (C) concentrations in control and experimental groups. Figure 2: Photomicrograph of Gills of Hypophthalmicthyes molitrix. section of control group showing primery lamella (PL), secondary lamellae (SL), mucous cells (MC), epithelial cells (EC) and pillar cells (PC). Group A showing necrosis (N), damaged secondary lamella (DSL), damaged primary lamellae (DPL). Group B showing necrosis (N), degeneration (D), damaged nuclei (DN) and rupturing of epithelial cells (REC). Group C showing necrosis (N), hypertrophy of mucous cells (HMC) and lamellar severe fusion (LSF). December 2018 | Volume 33 | Issue 2 | Page 3 W. Ali et al. Discussion The estimation of serum characteristics of blood plasma in fish has become a diagnostic tool to study the toxicological and pathological impacts of pollutant like pesticides (Suvetha et al., 2010). Glucose provides energy to organism but hyper-glycemia in blood due to pesticide exposure is a common marker of environmental stress in fish (Sepici-Dinçel et al., 2009). In agreement with our results (Ceron et al., 1997) observed significant hyperglycemia in common eel (Anguilla anguilla) after exposing it to sub-lethal concentrations of diazinon .Glycogen in hepatic tissue of fish was reduced and caused the Hyperglycemia against toxicant insult. After cypermethrin (pyrethroid) exposure to Indian major carp Labeo rohita, Increased blood glucose level was reported (Das et al., 2003) which are similar to our results. The low level of blood protein may be due to degradation or increased proteolytic activity or reduced protein production (Sulekha and Mercy, 2011; Shakoori et al., 1990). The important reason for low synthesis of total protein, immunoglobulin and albumin by the liver was exposure of diazinon which damaged the hepatocyte (Gokcimen et al., 2007). In agreement with our result a low level of serum total protein was reported in fish Oreochromis niloticus (One et al., 2008) and in Rhamdia quelen (Borges et al., 2007) in response to Cu and cypermethrin exposure, respectively. The increase in blood cholesterol level was due to less excretion of cholesterol in the bile duct and the large amount of cholesterol produced by liver as a result of stress. Similar increase in serum cholesterol was observed in fish after exposure to pollutants (Goel and Garg, 1980; Ghazaly, 1991). In Rhamdia quelen after cypermethrin exposure hypercholesteremia was observed (Borges et al., 2007). In agreement with our result, Yousef et al. (2003) reported that aggregation of pesticides in the liver increased the permeability of hepatic cells resulting into hypercholesteremia and disrupt lipid metabolism. Histopathology of gill is the appropriate bio indicator to pollution monitoring because gills come in immediate contact with the environment, tissue damages brought about by water borne pollutants can be easily observed (Dutta et al., 1993). Deltamethrine induce alteration like damaged nuclei, rupturing of epithelial cells, mucous cells and severe lamellar fusion. These types of lesions were seen (Cengiz and Unlu, 2006; Das and Mukherjee, 2000; Sahoo et al., 2017). Conclusion Result of this study concluded that deltamethrine induce serological and histological changes in silver carp so there is need to use biological control method in replacement of pesticide as alternative method to control pests. December 2018 | Volume 33 | Issue 2 | Page 4 Conflicts of interest The authors declare no conflicts of interest. References Aydin, R., Koprucu, K., Dörücü, M. and Köprücüss, P.M., 2005. Acute toxicity of synthetic pyrethroidcypermethrin on the common carp (Cyprinuscarpio L.) embryos and larvae. Aquacult. Int., 13: 451-458. https://doi.org/10.1007/s10499-005-0615-5 Balint, T., Szegletes, T., Szegletes, Z., Halasy, K. and Nemcsok, J., 1995. Biochemical and subcellular changes in carp exposed to the organophosphorus methidathion and the pyrethroid deltamethrin. Aquat. Toxicol., 33: 279-295. https://doi. org/10.1016/0166-445X(95)00029-4 Banaee, M., Mirvaghefei, A.R., Majaziamiri, B., Rafei, G.R. and Nematdost, B., 2011. Hematological and histopathological study of experimental diazinon poisoning in common carp fish (Cyprinus carpio). Iranian J. Nat. Res., 64: 1-14. Begum, G., 2005. In vivo biochemical changes in liver and gill of Clarias batrachus during cypermethrin exposure and following cessation of exposure. Pestic. Biochem. Physiol., 82: 185-196. https://doi. org/10.1016/j.pestbp.2005.02.006 Boran, H., Capkin, E., Altinok, I. and Terzi, E., 2012. Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout. Exp. Toxicol. Pathol., 64: 175-179. https://doi.org/10.1016/j. etp.2010.08.003 Borges, A., Scotti, L.V., Siqueira, D.R., Zanini, R., Do-Amaral, F., Jurinitz, D.F. and Wassermann, G.F., 2007. Changes in hematological and serum biochemical values in jundiá Rhamdia quelen due to sub-lethal toxicity of cypermethrin. Chemosphere, 69: 920-926. https://doi.org/10.1016/j.chemosphere.2007.05.068 Bradbury, S.P. and Coats, J.R., 1989. Toxicokinetics and toxicodynamics of pyrethroid insecticides in fish. Environ. Toxicol. Chem., 8: 373-380. https://doi. org/10.1002/etc.5620080503 Bradbury, S P. and Coats, J.R., 1989. Comparative toxicology of the pyrethroid insecticides. Rev. Environ. Contam. Toxicol., 108: 133-177. https://doi. org/10.1007/978-1-4613-8850-0_4 Cengiz, E.I. and Unlu, E., 2006. Sub-lethal effects of commercial deltamethrin on the structure of the gill, liver and gut tissues of mosquito fish, Gambusiaaffinis: A Microscopic study. Environ. Toxicol. Pharmaco., 2: 246-253. https://doi.org/10.1016/j. etap.2005.08.005 Ceron, J.J., Sancho, E., Ferrando, M.D., Gutierrez, C. and Andreu, E., 1997. Changes in carbohydrate metabolism in the eel Anguilla anguilla, during short-term Histological and Biochemical Alterations in Hypophthalmicthyes molitrix exposure to diazinon.Toxicol. environ. Chem., 60: 201210. https://doi.org/10.1080/02772249709358464 Das, B.K. and Mukherjee, S.C., 2003. Toxicity of cypermethrin in Labeo rohita fingerlings: biochemical, enzymatic and haematological consequences. Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol., 134: 109-121. https://doi.org/10.1016/S15320456(02)00219-3 Das, B.K. and Mukherjee, S.C., 2000. A histopathological study of carp (Labeo rohita) exposed to hexachlorocyclohexane. Vet. Arhiv., 70: 169-180. Dutta, H.M. and Meijer, H.J.M., 2003. Sublethal effects of diazinon on the structure of the testis of bluegill, Lepomis macrochirus: A microscopic analysis. Environ. Pollut., 125: 355-360. https://doi.org/10.1016/ S0269-7491(03)00123-4 El-Sayed, Y.S., Saad, T.T. and El-Bahr, S.M., 2007. Acute intoxication of deltamethrin in monosex Nile tilapia, Oreochromis niloticus with special reference to the clinical, biochemical and haematological effects. Environ. Toxicol. Pharmacol., 24: 212-217. https:// doi.org/10.1016/j.etap.2007.05.006 Fanta, E., Rios, F.S., Romao, S., Vianna, A.C.C. and Freiberger, S., 2003. Histopathology of the fish Corydoras paleatus contaminated with sublethal levels of organophosphorus in water and food. Ecotoxicol. environ. Safe., 54: 119-130. https://doi.org/10.1016/ S0147-6513(02)00044-1 Ghazaly, K.S., 1991. Physiological alterations in Clarias lazera induced by two different pollutants. Water Air Soil Pollut., 60:181-187. https://doi.org/10.1007/ BF00293973 Goel, K.A. and Garg, V., 1980. 2, 3’, 4-Triaminoazobenzene-induced hematobiochemical anomalies in fish (Channa punctatus). Bull. Environ. Contam. Toxicol., 25: 136-141. https://doi.org/10.1007/BF01985501 Gokcimen, A., Gulle, K., Demirin, H., Bayram, D., Kocak, A. and Altuntas, I., 2007. Effects of diazinon at different doses on rat liver and pancreas tissues. Pestic. Biochem. Physiol., 87: 103-108. https://doi. org/10.1016/j.pestbp.2006.06.011 Hinton, D.E. and Lauren, D.J., 1990. Liver structural alterations accompanying chronic toxicity in fishes: potential biomarkers of exposure. In: Biomarkers of environmental contamination (eds. J.F. McCarthy and L.R. Shugart). Lewis Publishers, pp. 17-52. Karim, A., Ahmad, N. and Ali, W., 2016a. Histopathological changes in spleen and kidney of silver carp (Hypophthalmicthys molitrix) after acute exposure to deltamethrin. Biologia, 62: 139-144. Karim, A., Ali, W., Ahmad, N., Irfan, M. and Shakir, H.A., 2016b. Histological and biochemical study of liver of silver carp (Hypophthalmichthys molitrix) after acute exposure to pyrethoride (deltamethrin). Punjab J. Zool., 31: 229-236. Köhler, H.R, Sandu, C., Scheil, V., Nagy-Petrică, E.M., December 2018 | Volume 33 | Issue 2 | Page 5 Segner, H., Telcean, I., Stan, G. and Triebskorn, R., 2000. Monitoring pollution in River Mureş, Romania, part III: Biochemical effect markers in fish and integrative reflection. Environ. Monitor. Assess., 127: 47-54. https://doi.org/10.1007/s10661-0069257-y Kori-Siakpere, O. and Ubogu, E.O., 2008. Sublethal haematological effects of zinc on the freshwater fish, Heteroclarias sp. (Osteichthyes: Clariidae). Afri. J. Biotechnol., 7: 2068-2073. https://doi.org/10.5897/ AJB07.706 Laskowski, D.A., 2002. Physical and chemical properties of pyrethroids. Rev. environ. Contam. Toxicol., 174: 49-170. https://doi.org/10.1007/978-1-47574260-2_3 Mahboob, S., Niazi, F., Alghanim, K., Sultana, S., AlMisned, F. and Ahmed, Z., 2015. Health risks associated with pesticide residues in water, sediments and the muscle tissues of Catla catla at Head Balloki on the River Ravi. Environ. Monitor. Assess., 187: 1-10. https://doi.org/10.1007/s10661-015-4285-0 Mumford, S., Heidel, J., Smith, C., MacConnell, B. and Blazer, V., 2007. Fish histology and histopathology. US Fish and Wildlife National Conservation Training Center, Amerika Serikat. Okuku, E.O. and Peter, H.K., 2011. Choose of heavy metals pollution biomonitors: A critic of the method that uses sediments total metals concentration as the benchmark. Int. J. environ. Res., 6: 313-22. Öne, M., Atli, G. and Canli, M., 2008. Changes in serum biochemical parameters of freshwater fish Oreochromis niloticus following prolonged metal (Ag, Cd, Cr, Cu, Zn) exposures. Environ. Toxicol. Chem., 27: 360-366. https://doi.org/10.1897/07-281R.1 Perry, SF. and Laurent, P., 1993. Environmental effects on fish gill structure and function. In: Fish ecophysiology (eds. J.C. Rankin and F.B. Jensen). Chapman and Hall, London, pp. 231-264. https://doi. org/10.1007/978-94-011-2304-4_9 Prusty, A.K., Kohli, M.P.S., Sahu, N.P., Pal, A.K., Saharan, N., Mohapatra, S. and Gupta, S.K., 2011. Effect of short term exposure of fenvalerate on biochemical and haematological responses in Labeo rohita (Hamilton) fingerlings. Pestic. Biochem. Physiol., 100: 124129. https://doi.org/10.1016/j.pestbp.2011.02.010 Sahoo, J., Nanda, S., Mahapatra, C.R., Panda, D. and Kund, G.C., 2017. Lethal toxicity of deltamethrin and histological changes in the vital organs of fingerlings of Labeo rohita. Int. J. Fish. aquat. Stud., 5: 506-513. Sepici-Dinçel, A., Benli, A.Ç., Selvi, M., Sarikaya, R., Şahin, D., Özkul, I.A. and Erkoç, F., 2009. Sublethal cyfluthrin toxicity to carp (Cyprinus carpio L.) fingerlings: biochemical, hematological, histopathological alterations. Ecotoxicol. environ. Safe., 72: 1433-1439. https://doi.org/10.1016/j.ecoenv.2009.01.008 W. Ali et al. Shakoori, A.R., Aziz, F., Alam, J. and Ali, S.S., 1990. Toxic effects of Talstar, a new synthetic pyrethroid, on blood and liver of rabbit. Pakistan J. Zool., 22: 289300. Sharma, R., Peshin, R., Shankar, U., Kaul, V. and Sharma, S., 2015. Impact evaluation indicators of an Integrated Pest Management program in vegetable crops in the subtropical region of Jammu and Kashmir, India. Crop Protect., 67: 191-199. https://doi. org/10.1016/j.cropro.2014.10.014 Sulekha, B.T. and Mercy, T.A., 2011. Pesticide induced changes in the proximate composition of a freshwater fish for estimating maximum allowable toxicant concentration of the pesticide under tropical conditions. Indian J. Fish., 58: 85-90. Suvetha, L., Ramesh, M. and Saravanan, M., 2010. Influence of cypermethrin toxicity on ionic regulation and gill Na+/K+-ATPase activity of a freshwater teleost fish Cyprinus carpio. Environ. Toxicol. Pharmacol., 29: 44-49. https://doi.org/10.1016/j. December 2018 | Volume 33 | Issue 2 | Page 6 etap.2009.09.005 Takashima, F. and Hibiya, T., 1995. An atlas of fish histology: Normal and pathological features. Tokyo, Japan. van der Oosts, R., Beyer, J. and Vermeulenn, P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: A review. Environ. Toxicol. Pharmacol., 13: 57-149. https://doi.org/10.1016/ S1382-6689(02)00126-6 Yadav, I.C., Devi, N.L., Syed, J.H., Cheng, Z., Li, J., Zhang, G. and Jones, K.C., 2015. Current status of persistent organic pesticides residues in air, water, and soil, and their possible effect on neighboring countries: A comprehensive review of India. Sci. Total Environ., 511: 123-137. https://doi.org/10.1016/j.scitotenv.2014.12.041 Yousef, M.I., El-Demerdash, F.M., Kamel, K.I. and Al-Salhen, K.S., 2003. Changes in some hematological and biochemical indices of rabbits induced by isoflavones and cypermethrin. Toxicology, 189: 223-234. https://doi.org/10.1016/S0300-483X(03)00145-8