This study was conducted to investigate the sub-acute toxic effects of Chlorpyrifos, using male rabbits as an animal model for mammals. Lara-909 (containing 50% chlorpyrifos) was given @71.5 mg chlorpyrifos /kg body weight per day orally for 14 days. Blood samples were collected at day 0, 7 and day 15 for analysis. Intoxicated rabbits showed dullness, anorexia, dehydration, excessive salivation, muscle contractions, twitching with staggering gait and progressive loss of weight. Mortality over 14 days was 83%. In comparison to control group, mean values of Hb, MCH, MCHC and creatinine were significantly increased while lymphocyte count and albumin were significantly decreased on day seven of the experiment. The mean values of total protein, globulin, glucose, ALT, AST, and BUN did not differ significantly from control values.
Organophosphorus insecticides have been widely recognized as a health hazard due to their widespread use and release into the environment (Al-Haj et al., 2005). Besides fatalities, caused by high dose, exposure of animals to low dose organophosphorus insecticides has been found to have widespread effect on body including organ specific lesions in central nervous system, liver and kidneys; and generalized effects like immunosuppression, teratogenesis, carcinogenesis and metabolic disorders (Lengyl et al., 2005). Chlorpyrifos (CPF) is widely used in agriculture and domestic pest control (Gralewicz et al., 2002). Experimental studies have revealed that chlorpyrifos has dose related effects on liver, testes, spermatozoa, and nervous system besides causing wide ranges of clinico-pathological alterations (Akhtar et al., 2009; Solati et al., 2012). Since chlorpyrifos is widely used as insecticide by agriculturists in Kashmir valley, it remains a potential health hazard for animals and humans. Hence a study was conducted to study pathological effects of sub-acute chlorpyrifos toxicity in an animal model using rabbits.
Materials and Methods
A total of 12 gray giant male rabbits (1 to 1.5 Kg weight) were maintained in cage system under standard laboratory conditions. These were acclimatized to rearing conditions for one week before start of the experiment. The experimental protocol was approved by the Institutional Animal Ethics Committee. The rabbits were randomly allocated to two groups of six rabbit’s each- Group-I (control) and Group-II (Chlorpyrifos intoxicated). For inducing toxicity, Lara-909 (containing 50% chlorpyrifos) was administered orally @71.5 mg chlorpyrifos /kg body weight per day for 14 days (Fikes, 1992). Blood samples were collected at days 0, 7 and day 15 for hematological and biochemical analysis. Hematology was done using MS4 multi-species haematological analyser (MELET SCHLOESING Laboratoires-9 Chaussee Jules Cesar-Evolic 402-95520 OSNY France). Glucose, total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine were analyzed using diagnostic kits (Aspen Laboratories Pvt. Ltd, Rapid Diagnostic Group of Companies, Karnal Road Industrial Area, Delhi, India) and semi-automatic blood chemistry analyser (model ERBA CHEM-PRO). Data were processed by SPSS statistical software. The significant differences between control and Chlorpyrifos intoxicated groups was determined using t- test. ANOVA followed by DMRT was used for multiple comparisons. The differences were regarded significant at p <0.05.
Results and Discussion
The clinical signs observed in chlorpyrifos intoxicated rabbits (group II) were dullness, depression, partial to complete anorexia, lacrimation, bradypnea, mydriasis, muscle contractions, staggering and prostration. Rabbits fell suddenly revealing uneasiness and paddling of legs followed by unconsciousness with urination and defecation before death. Out of six intoxicated rabbits only one survived after 14 days experimental period. The clinical signs observed in present study were in concordance with those reported by Mehta et al. (2006) in calves. However, Mansour and Mossa (2010) did not observe any appreciable change in rats. The nervous signs observed prior to death might be attributed to acetyl cholinesterase (ACh) inhibition leading to accumulation of ACh in synaptic junctions (Karanth et al., 2006). Recumbency, wry-neck, and intermittent rolling on back, as seen in the present study, has been observed in terminal stages in rabbits following intoxication with a number of toxicants and had been attributed to neurotoxicity (Klassen, 2008). There was progressive loss of body weight in group II and mean body weight was significantly lower (P≤0.05) than that of group I at day 7. The mean body temperature decreased non-significantly over the period of toxicity and did not differ significantly from control values (Table1).
Table1: Effect of Chlorpyrifos (@71.5mg/Kg b.w.) on body weight and temperature in rabbits (Mean ± SE)
|Parameter||Time period||Group I (Control)||Group II (Chlorpyrifos intoxicated)|
|Body Weight (Kg)||0 day||1.59± 0.03a1||1.53 ± 0.05a1|
|7 day||1.73± 0.04a1||1.36± 0.16a2|
|14 day||1.86± 0.04 b||1.2 ± 0.0*|
|Temperature (°F)||0 day||101.26 ± 0.26a1||101.55 ± 0.24a1|
|7 day||101.13 ± 0.32a1||100.20 ± 0.51a1|
|14 day||101.2 ± 0.3a||90.00 ± 0.0*|
For each parameter the values within a row with different numerals as superscript differ significantly (p<0.05). The values in a column with different alphabets as superscript differ significantly (p<0.05)
Reduced body weight could be attributed to the overall increased degradation of lipids and proteins (Breslin et al., 1996; Farag et al., 2003).The mean values of haemoglobin (Hb) mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in group II rabbits at day 7 were significantly (P≤0.05) increased as compared to day 1 and respective control values. The mean packed cell volume (PCV), total erythrocyte counts (TEC), and mean corpuscular volume (MCV) values did not differ significantly from respective control values or over the time of exposure (Table 2). No significant changes were observed in total or differential leukocyte counts of group II as compared to group I except significantly decreased lymphocyte counts at day 7 (P ≤ 0.05) (Table 3). Contrary results were reported by Akhtar et al.(2009) and Savithri et al. (2010) reported decrease in haemoglobin concentration and PCV following chlorpyrifos intoxication in species. The difference might be attributed to shorter experimental period in the present study compared to their studies. Although chlorpyrifos intoxication has been incriminated for impaired haematopoiesis (Rahman et al., 1990) and increased rate of erythrocytes destruction (Savithri et al., 2010), no significant alterations in TEC were observed over 14 day period during the present study. However this needs to be weighed against haemoconcentration (Ambali et al., 2010). Contrary to present findings, leukocytosis with lymphocytosis, monocytosis, eosinophilia, basophilia and neutropenia has been observed in chlorpyrifos intoxicated rats which has been attributed to stimulation of lymphopoiesis or enhanced release of lymphocytes from lymph myeloid tissue (Savithri et al., 2010; Das and Mukherjee 2003). In general the variations with the earlier reports might be attributed to differences in the study period besides some other variants.
Table 2: Effect of sub acute chlorpyrifos toxicity haemoglobin and erythrocyte indices in rabbits (Mean ± SE)
|Parameter||Time Period||Group I||Group II|
|Hb (g/dl)||0 day||13.46± 0.38a1||13.10± 0.45a1|
|7 day||12.42± 0.44a1||14.80± 0.86b2|
|14 day||12.76± 0.20a||14.7±0.0*|
|PCV (%)||0 day||40.33± 0.74a1||40.21± 2.36a1|
|7 day||41.08± 0.86a1||39.28± 3.26a1|
|14 day||41.23± 0.56a||31.4±0*|
|TEC(106/cmm)||0day||6.10± 0.18a1||6.21± 0.44a1|
|7 day||6.17± 0.21a1||6.11± 0.53a1|
|MCV (fl)||0day||66.28± 1.64a1||64.23± 2.21a1|
|7 day||66.91± 2.35a1||64.46± 0.63a1|
|MCH (pg)||0day||19.62± 0.52a1||21.53± 1.53a1|
|7day||20.20± 0.91a1||24.76± 2.05a2|
|MCHC (%)||0day||29.73± 1.23a1||33.54± 2.03a1|
|7 day||30.32± 1.46a1||38.39± 2.92a2|
|14 day||28.55± 0 .35a||41.4±0.0*|
For each parameter the values within a row with different numerals as superscript differ significantly (p<0.05). The values in a column with different alphabets as superscript differ significantly (p<0.05). *Reading from single surviving rabbit
Table 3: Effect of Sub acute chlorpyrifos toxicity on Total and differential leukocyte counts (Mean ± SE)
|Parameter||Time Period||Group I (Control)||Group II (Chlorpyrifos intoxicated)|
|TLC(103/cmm)||0 day||9.29± 0.92a1||10.38± 1.44a1|
|7 day||9.40± 0.24a1||10.61± 1.43a1|
|14 day||9.99± 0.11a||14.78±0.0*|
|Heterophill (%)||0 day||32.36± 1.79a1||34.20± 3.26a1|
|7 day||31.93±2.88a1||38.32± 1.53a1|
|14 day||31.33± 2.14a||41.6±0.0*|
|Lymphocytes (%)||0 day||58.25±3.98a1||49.40± 6.16a1|
|7 day||59.40±3.47a1||43.84± 4.07a2|
|Monocytes (%)||0 day||7.06±2.88a1||11.70±2.27a1|
|Eosinophils (%)||0 day||1.75±0.25a1||3.98±0.94a1|
|Basophils (%)||0 day||0.56±0.20a1||0.71±0.15a1|
For each parameter the values within a row with different numerals as superscript differ significantly (p<0.05). The values in a column with different alphabets as superscript differ significantly (p<0.05).*Reading from single surviving rabbit*Reading from single surviving rabbit
The biochemical parameters did not alter significantly except increased creatinine and decreased albumin at day 7 in group II compared to group I (Table 4).
Table 4: Effect of Sub acute Chlorpyrifos toxicity on Biochemical parameters in rabbits (Mean ± SE)
|Parameter||Time Period||Group I (Control)||Group II (Chlorpyrifos intoxicated)|
|TotalProtein (g/dl)||0 day||5.81±0.40a1||6.52±0.25a1|
|Albumin (g/dl)||0 day||3.79±0.38a1||3.37±0.17a1|
|Globulin (g/dl)||0 day||2.01±0.14a1||3.15±0.39a1|
|Glucose (mg/dl)||0 day||122.28±7.39a1||106.98±10.17a1|
|AST (IU/l)||0 day||15.49±3.96a1||20.38±1.42a1|
|ALT (IU/l)||0 day||16.54±4.24a1||18.93±4.13a1|
|Creatinine (mg/dl)||0 day||0.87 ± 0.06a1||0.89 ± 0.05a1|
|7 day||0.89 ± 0.05a1||1.08 ± 0.05b2|
|14 day||0.82 ± 0.03a||0.61±0.0*|
|BUN (mg/dl)||0 day||18.78±3.41a1||15.02 ±1.39a1|
For each parameter the values within a row with different numerals as superscript differ significantly (p<0.05). The values in a column with different alphabets as superscript differ significantly (p<0.05). *Reading from single surviving rabbit*Reading from single surviving rabbit
Increase in total protein and globulin values without any alteration in albumin levels has been reported in rats intoxicated and has been attributed to liver and kidneys dysfunctions (Ambali et al., 2010). Contrary to our observations, Akhtar et al. (2009) reported significant decrease in glucose values. Some authors have reported increase in AST and ALT (Solati et al., 2012; Mansour and Mossa, 2010) while others have reported decrease in these values (Ambali et al., 2010). The discrepancy may be attributed to species differences besides other technical variants. Increased creatinine values are indicative of nephropathy and have been attributed to impairment of the renal glomerular function and tubular damage (Ambali et al., 2010; Mansour and Mossa, 2010).
Subacute chlorpyrifos intoxication in rabbits caused progressive clinical effects and progressive loss of body weight with 83% mortality over a period of 14 days. The hematological alterations and biochemical parameters were non-diagnostic except decreased lymphocyte count and albumin, and increased Hb, MCH, MCHC and creatinine on day 7 as compared to control group. It is suggested that such changes may have long lasting post-effects in survivors, if any.