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Year : 2015  |  Volume : 47  |  Issue : 2  |  Page : 206--211

Hematobiochemical and pathological alterations due to chronic chlorpyrifos intoxication in indigenous chicken

Shameem Ara Begum1, Tirtha Nath Upadhyaya1, Taibur Rahman1, Debesh Chandra Pathak1, Kavita Sarma2, Chandana Choudhury Barua3, RS Bora4,  
1 Department of Veterinary Pathology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, India
2 Department of Anatomy and Histology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, India
3 Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, India
4 Department of Animal Production and Management, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, India

Correspondence Address:
Shameem Ara Begum
Department of Veterinary Pathology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati


Objective: The present study investigates the effect of oral administration of chlorpyrifos (CPF) in indigenous chicken. Materials and Methods: The birds were divided into two groups I and II. Group I served as control and group II was treated with CPF (0.36 mg/kg) orally daily up to 12 weeks. Blood samples were assayed for hemoglobin (Hb), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count, and biochemical constituents like alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholinesterase (CHE), total protein and uric acid. Representative pieces of tissues from liver and kidney were collected weekly for histopathological examination. Results: A significant (P < 0.01) increase of Hb, TEC, TLC, and heterophil percent and decrease of lymphocyte percent was observed. Serum ALP, AST, ALT, and uric acid increased significantly and CHE values decreased significantly in CPF treated birds. The protein level remained similar. Uric acid level was found to be increased significantly in the treated group. The results indicate that chronic CPF intoxication produces hematological, biochemical, and pathological changes in treated birds.

How to cite this article:
Begum SA, Upadhyaya TN, Rahman T, Pathak DC, Sarma K, Barua CC, Bora R S. Hematobiochemical and pathological alterations due to chronic chlorpyrifos intoxication in indigenous chicken.Indian J Pharmacol 2015;47:206-211

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Begum SA, Upadhyaya TN, Rahman T, Pathak DC, Sarma K, Barua CC, Bora R S. Hematobiochemical and pathological alterations due to chronic chlorpyrifos intoxication in indigenous chicken. Indian J Pharmacol [serial online] 2015 [cited 2021 Mar 6 ];47:206-211
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Chlorpyrifos (CPF) (O, O-diethyl-O-[3, 5, 6-trichloro-2-pyridyl] phosphorothioate), an organophosphorus compound, particularly affects the cholinesterase (CHE) system. CPF affects the nervous system of the pest by inhibiting the breakdown of acetylcholine (ACh), a neurotransmitter. Resulting accumulation of ACh in the synaptic cleft causes overstimulation of the neuronal cells, which leads to neurotoxicity and eventually death. [1] Toxicological studies of CPF in chickens focused on the sub-acute effects on plasma or serum enzymes and other biochemical parameters, [2] examination of delayed neurotoxicity, [3] foodborne toxicity, [4] developmental effects, [5] and pathology of long-term exposure. [6]

In the present context, the hematological, biochemical, and pathological effect of chronic exposure to CPF was evaluated in indigenous chickens.

 Materials and Methods


Three-month-old unsexed 24 indigenous chickens procured from All India Coordinated Research Project on Poultry, College of Veterinary Science (CVSc), A. A. U., Khanapara Guwahati-781022 were wing banded, weighed, and reared in the Department of Pathology, CVSc with ad libitum supply of feed and water. The experimental trials were approved by the Institutional Animal Ethics Committee (No. 770/ac/CPCSEA/FVSc/AAU/IAEC/11-12/128).

Chemical (Insecticide)

Commercial products of CPF (20%) used in this study were procured from Excel Crop Care Private Limited, Mumbai, India.

Experimental Protocol

Thirty-two chickens were randomly segregated into two groups of 16 each and fasted for 6 h prior to dosing. Group I served as control and received distilled water p.o. for 90 days. Group II served as CPF group. CPF was diluted in a tenfold serial dilution with distilled water to obtain a concentration of 0.2 mg/ml (10−4 ). Fresh preparations were orally administered daily using oral gavage. Group II was administered 0.36 mg/kg b.w. CPF (1.8 ml of 10−4 dilution) daily up to 90 days. Doses were calculated on weekly body weight basis and administered accordingly. The birds were closely watched for the presence of clinical signs, if any, and sacrificed at weekly interval till the end of the experiment.


Hemoglobin (Hb), total erythrocyte count (TEC), and total leukocyte count were estimated with the help of automated hematology cell counter (Model-ms4e). Differential leukocyte count was analyzed as per standard method. [7]

Biochemical Estimations

Blood samples were collected via wing vein or jugular vein puncture at 0 day and weekly intervals up to 12 th week. In control group, blood was collected at the same time and same day of collection as in case of treated group; the serum was rapidly separated and processed for determination of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), CHE, total protein and uric acid using commercial kits (Siemens Diagnostics India Ltd) on ultraviolet-visible spectrophotometer.

Histopathological Examination

Representative samples irrespective of lesions from the liver and kidney were collected weekly in 10% neutral formalin. After washing in running water and dehydration in alcohol, tissues were embedded in paraffin, and 5 μm paraffin sections cut and stained with hematoxylin and eosin as per standard method. [8]

Statistical Analysis

All data were expressed as mean ± standard error. The statistical significance of the mean differences between control and treated groups was analyzed by one-way ANOVA. Statistical calculations were performed with the SPSS 11.5 computer program (SPSS Inc., Chicago, Illinois, USA). The value of (P < 0.05) was taken as the cut-off value to consider differences statistically significant.


Immediately after oral dosing the chickens developed increased thirst which disappeared gradually, except for reduced feed intake and gradual reduction in body weight gain which showed an increase of 39% and 23.1%, respectively, for the control and treated birds over a period of 12 weeks. The difference in weight gain was found to be statistically significant (P < 0.05). Birds appeared to be active and alert. After 2 months of treatment, some of the birds exhibited slightly staggering gait, leg weakness, tremor, and diarrhea. Some developed curled toes with pale mucous membrane and prominent keel bone. These symptoms disappeared subsequently towards the end of the experiment.

Effects of CPF on hematological parameters are tabulated in [Table 1]. There was significant (P < 0.05) increase in Hb and TEC; heterophil percent was increased, and lymphocyte percent was decreased in the treatment group compared to control. There was no variation in the monocyte, eosinophil, and basophil percent in both the groups.{Table 1}

Alkaline phosphatase, AST, ALT, CHE, total protein, and uric acid in chickens fed CPF are presented in the [Table 2]. When compared to the control and CPF groups ALP, AST, and ALT values differed significantly (P < 0.05) in the CPF treated group. There was significant inhibition of CHE (P < 0.05) in CPF intoxicated chickens compared to the control chickens. Significant increase in the uric acid level was observed in the CPF treated group. The levels of total protein remained unaltered in both groups.{Table 2}

Histopathologically, during the 1 st month of treatment, the liver of CPF treated birds showed mild congestion with scattered hemorrhages and mild mononuclear cell infiltration. By 2 nd month, changes were more prominent, and consisted of degeneration of hepatocytes with focal areas of hepatocellular necrosis [Figure 1] in the CPF-treated birds. Mild to moderate proliferation of biliary epithelial cells around the portal veins with formation of new bile ducts was observed [Figure 2]. Gradually by 3 rd month, changes were more severe in the CPF treated group and were characterized by dilatation of sinusoids, marked congestion, disruption of hepatic cords, focal to diffuse hemorrhage, and mononuclear cell infiltration in the hepatic parenchyma, particularly around the blood vessels of the portal region [Figure 3].{Figure 1}{Figure 2}{Figure 3}

Renal changes were found to be mild up to 3 rd week of the experiment. From 4 th week onwards changes became prominent and were characterized by congestion, focal to diffuse hemorrhage, tubular degeneration, necrosis, and cellular swelling [Figure 4]. Glomeruli showed congestion and necrosis [Figure 5] with dilatation of Bowman's space and vacuolar degeneration.{Figure 4}{Figure 5}


The increased Hb concentrations and TEC observed in the present study might be due to severe diarrhea causing dehydration resulting in hemoconcentration. [9],[10] The gradual lymphopenia might be due to decrease production of lymphocyte in the lymphoid tissues resulting from the stress of intoxication. [11]

The significant heterophilia might be due to ingestion of CPF, which caused pathological stress in chickens due to noninflammatory disorders resulting in the rise of glucocorticoid level in circulatory blood. [9] Similar observations were recorded earlier in chickens due to CPF toxicity. [12]

The increased ALT, AST, and ALP values might be attributed to the liver damage in the toxicant fed birds. The results suggest that administration of CPF caused necrotic changes in the liver, as seen in histopathological study, thus causing leakage of the enzyme into the blood. Significant increase in AST and ALT was reported in goats fed with CPF. [13] AST is found in liver, skeletal muscle, heart, kidney, and brain in the variable amount between species. It is the last enzyme to rise after muscle or liver damage. [14] Increased levels of these enzymes have also been reported in the serum of birds in CPF toxicity. [15]

Reduced CHE activity is a reliable indicator of organophosphorus (OP) poisoning and a biomarker of absorption of OP insecticides. [16] Significant inhibition of CHE activity was reported in CPF intoxicated chicken. [15] CPF act through their active oxon metabolites and inhibits the target CHE. Plasma and other tissue CHE are important for assessing the extent of poisoning induced by organophosphates. Plasma CHE inhibition by 20-30% usually indicates exposure to organophosphate, whereas 50% inhibition or more is associated with serious poisoning and adverse effects. [17]

The fall in total protein could be due to the stress enhancing effect of CPF, or general toxic action that leads to decrease in weight in the treated birds.

The rapid increase in uric acid concentration might be due to acute renal disorders, as observed in histopathological study. The transient renal failure was due to both a direct action of the OP, causing tubular cell necrosis, and to a secondary mechanism that follows the cholinergic crisis, causing hypovolemic shock and rhabdomyolysis. [18] Mononuclear cell infiltrations in liver suggested onset of immunological response by the host. Lesions observed in liver in the present study are consistent with several workers. [19] Hepatotoxic effect of CPF reported in layer chickens [15] corroborate with the findings of the present study. Histopathological lesions of kidneys were indicative of nephrotoxicity of CPF and its metabolites as kidneys are the major route for elimination of CPF. [15] Since the renal tubules are particularly sensitive to toxic influences, in part because they have high oxygen consumption and vulnerable enzyme systems, and in part, because they have complicated transport mechanisms that may be used for transport of toxicants and may be damaged by such toxicants. The presence of necrosis may be related to depletion of ATP, which finally leads to death of the cells. [20]

Thus, it is concluded that exposure to CPF produces hematological, biochemical and pathological alterations in indigenous chicken. There is an obvious correlation between hematological, plasma biochemical, and histopathological lesions observed in the liver and kidneys of the CPF treated chickens. However, the exact mechanism that caused cell damage leading to hematological, biochemical, and pathological alterations needs to be elucidated.


Authors are grateful to Dean, College of Veterinary Science, Assam Agricultural University, Khanapara for financial help and providing necessary facilities to carry out the research work.


1Malik G, Dhahiya JP, Gera S. Biochemical studies on chlorpyriphos toxicity in broiler chicken. Indian J Anim Sci 2004;74:473-6.
2Richardson RJ, Moore TB, Kayyali US, Randall JC. Chlorpyrifos: Assessment of potential for delayed neurotoxicity by repeated dosing in adult hens with monitoring of brain acetylcholinesterase, brain and lymphocyte neurotoxic esterase, and plasma butyrylcholinesterase activities. Fundam Appl Toxicol 1993;21:89-96.
3Karanth S, Pope C. Carboxylesterase and A-esterase activities during maturation and aging: Relationship to the toxicity of chlorpyrifos and parathion in rats. Toxicol Sci 2000;58:282-9.
4Malik G, Agarwal VK, Gera S, Dahiya JP. Studies on growth pattern and feed efficiency in broiler chickens following chlorpyriphos intoxication. Haryana Vet 2001;40:38-40.
5Geller AM, Abdel-Rahman AA, Peiffer RL, Abou-Donia MB, Boyes WK. The organophosphate pesticide chlorpyrifos affects form deprivation myopia. Invest Ophthalmol Vis Sci 1998;39:1290-4.
6Krishnamoorthy P, Vairamuthu S, Balachandran C, Muralimanohar B. Pathology of chlorpyriphos and T-2 toxin on broiler chicken. Vet Arh 2007;77:47-57.
7Schalm OW, Jain NC, Carrol EJ. Veterinary Haematology. 3 rd ed. Philadelphia: Lea and Febiger; 1995. p. 664-7.
8Luna LG. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3 rd ed. London: McGrew-Hill; 1968.
9Benjamin MM. Outline of Veterinary Clinical Pathology. 3 rd ed. Iowa USA: The Iowa State University Press; 1976.
10Sastry GA. Veterinary Clinical Pathology. New Delhi: CBS Publishers and Distrbutors Private Limited; 1983.
11Coles EH. Veterinary Clinical Pathology. 3 rd ed.  Philadelphia: W. H. Sounders and Co.; 1980. p. 217-21.
12Malik G, Dhahiya JP, Sandeep G, Mshra SK. Clinicopathological Studies on Chlorpyriphos Intoxication in Broiler Chicken. Abstr. In XIX Annual Conference of Indian Association of Veterinary Pathologists, College of Veterinary and Animal Science, CSKHPKV, Palampur, Himachal Pradesh, India; 2002.
13Kaur H, Srivastava AK, Garg SK, Prakash D. Subacue oral toxicity of chlorpyriphos in goats with particular reference to blood biochemical and pathomorphological alteration. Indian J Toxicol 2000;2:83-90.
14Forbes NA. Avian information sheet no 3-Avian clinical pathology. Lansdown Veterinary Surgeons; 2001. Available from: http//www. [Last accessed on 2005 Sep 18].
15Kammon AM, Brar RS, Banga HS, Sodhi S. Patho-biochemical studies on hepatotoxicity and nephrotoxicity on exposure to chlorpyrifos and imidacloprid in layer chickens. Vet Arh 2010;80:663-72.
16Mohammad FK, Al-Badrany YM, Al-Jobory MM. Acute toxicity and cholinesterase inhibition in chicks dosed orally with organophosphate insecticides. Arh Hig Rada Toksikol 2008;59:145-51.
17Wilson BW. Cholinesterase inhibition. In: Wexler P, editor. Encyclopedia of Toxicology. San Diego USA: Academic Press; 1998. p. 326-40.
18Betrosian A, Balla M, Kafiri G, Kofinas G, Makri R, Kakouri A. Multiple systems organ failure from organophosphate poisoning. J Toxicol Clin Toxicol 1995;33:257-60.
19Choudhary N, Sharma M, Verma P, Joshi SC. Hepato and nephrotoxicity in rat exposed to endosulfan. J Environ Biol 2003;24:305-8.
20Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, et al. Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: Possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 1996;12:2045-50.