The environmental pollution plays a key role in the occurrence of many diseases affecting plants, animals and humans. Irrational use of pesticides results in environmental pollution. In agriculture and veterinary practices, pesticides are used in the form of herbicides, insecticides and fungicides. One such group of pesticides is fungicides, which acts on the fungal pathogens. Among the fungicides, the most commonly used ones are Carbendazim belonging to benzimidazole family. Carbendazim which is widely used broad spectrum fungicide, to control fungi on various food crops, vegetables, fruits, cereals, ornamental plants and medicinal herbs in pre- and post- harvest treatment^1,2. The European Commission has categorized Carbendazim into their priority list as one among the endocrine disrupting chemicals³ and ⁴categorized Carbendazim as hazardous chemical. In India, it is one among the top five pesticides with an annual consumption of 1992 metric tonnes⁵ and it stands in second position after mancozeb in terms of most consumed carbamates. Inhalation, dietary intake and dermal contact were acting as the common routes of exposure of Carbendazim in birds⁶. Vitamin E acts as potential scavenger of reactive oxygen species (ROS) and plays a protective role against the pesticide toxicity and showing its antioxidant property. ⁷reported that Carbendazim induces oxidative stress leading to the generation of free radicals and alteration in antioxidant or oxygen free radical scavenging enzymes in rats and also reported the amelioration effect of vitamin E against Carbendazim⁸.

Day old female chicks were procured and maintained in the deep litter system. The chicks were vaccinated as per the standard schedule and maintained in ambient conditions with adlibitum normal feed and water for acclimatisation. The experiment was carried out from 6^th week to 12 weeks of age for 42 days with prior approval of Institutional Animal Ethics Committee [(IAEC), reference number: 281/go/ReBi/S/2000/CPCSEA/CVSc/TPTY/14/pathology/2021]. At the beginning of 6^th week birds were randomly divided into 6 groups.

The experimental birds were observed daily for exhibition of clinical signs if any during the entire period of experiment. They were sacrificed, at fortnight intervals. The blood samples were collected in EDTA and clot activating vials for haematological and serum biochemical parameters respectively. Haemoglobin (Hb) and packed cell volume (PCV) were estimated by Sahli’s acid haematin method and micro-haematocrit method respectively whereas, total erythrocytes count (TEC) and total leukocytes counts (TLC) were measured by Nambiar’s method^9,10. For serum biochemical analysis of total protein, albumin, triglycerides, total cholesterol, SGPT and SGOT Excel biochemical kits were used.

Approximately 5 g of liver and kidney pieces were collected at each slaughter of birds separately in chilled phosphate buffer (pH 7.2) and stored at -20°C for antioxidant assay. The collected tissues were blotted to dry, thawed and homogenized at 4°C in 3 volumes of 0. 25 M sucrose containing 0.07M phosphate buffer (pH 7.2), 10mM EDTA and 0.1% TritonX-100. The, post mitochondrial supernatant was prepared by centrifugation at 12 000 x G, for 15 min at 4°C and used immediately for measuring the enzyme activities. Total protein estimation was carried out according to the modified method described by using Folinphenol reagent¹¹ and was used in the assay of Catalase, Superoxide dismutase (SOD) and Glutathione-S-transferase (GST) antioxidant assay in liver and kidney in the present study. The activity of catalase was carried out by following the protocol mentioned¹². All the data generated for various parameters were subjected to statistical analysis^13,14 by applying one-way ANOVA by using SPSS (22^nd Version) differences between means tested and significance level was set at P<0.05.

The representative tissue samples of liver, kidney were collected and given cuts at different places for proper fixation by changing with fresh l0% neutral buffered formalin (NBF). Further, the tissues were trimmed into small size sections and kept overnight under running tap water for washing. Then the tissues were subjected to dehydration in ascending grades of alcohol and cleared in xylol and further transferred to paraffin for embedding. Paraffin blocks were prepared and 5µ size sections were cut by rotatory microtome. The sections were lifted on clear, grease free slides and kept overnight for drying. Sections were stained by using routine Hematoxylin and Eosin staining method¹⁵.

The birds in the experimental groups exhibited dullness, decreased feed intake and water intake within few hours after exposure to Carbendazim in 8,10 and 12 weeks of age. These alterations in the behaviour of experimental group birds might be due to toxic effect of Carbendazim. The decrease in the concentration of the haemoglobin might be due to the decrease in the total erythrocytes count (TEC) because of the effect of Carbendazim which inhibit haematopoiesis by interfering with mitotic division. The results are in accordance with^16,17 in Japanese quails and in laboratory mammals^18-20. The decrease in the TEC count might be due to increase in the destruction of RBC or decrease in the erythropoiesis activity by interfering with the mitotic division during hemopoiesis. The reports in Japanese quails^16,17 and in laboratory mammals^18-22 were in accordance with the above results. The alteration in PCV though observed but not significantly might be due to water loss or dehydration depending on the extent. The decrease in TLC count in the CBZ exposed groups indicates the immunosuppressive effect of Carbendazim. The results are in accordance with reports of^23,24 in Japanese quails and in laboratory mammals^21,25,26.

The significant decrease in the concentration of the serum total protein and albumin in CBZ exposed groups was due to indirect effect of Carbendazim on plasma protein i.e., CBZ affects the DNA synthesis by interfering with metabolic activities involving purine leading to inhibition of DNA synthesis resulting in disruption of mRNA transcription and in turn inhibit protein translation. As reported by^6,16 in Japanese quails and by^22,27 in laboratory mammals were in accordance with the results of present study.

The increase in the concentration of triglycerides in the serum of CBZ treated groups might be due to vital interactions between the micronutrients, nutritional factors with Carbendazim or hepatic damage evidenced histologically, serum enzymes and antioxidant parameters of the present study. The reports are in accordance to results of²⁸ in Japanese quails and in laboratory mammals^19,27,29.

The increase in the levels of serum total cholesterol in CBZ treated groups is due to stimulation of catecholamine in turn leads to lipolysis and increase in fatty acids production which was evidenced in the gross and histological alterations in liver³⁰, serum biochemical and antioxidant parameters of the present study. Results were in accordance with the reports in Japanese quails²⁴ and in laboratory mammals^22,25,27,29.

The elevated SGOT and SGPT are indicators of hepatocellular damage and dysfunction which in turn increases the cell membrane permeability to transaminases. The reports were in accordance with the results in Japanese quails^23,24,33 and in laboratory mammals^{18-20,26-27,29,31-32}.

The decrease in the activity of the catalase indicates the reduced peroxisomes and affects the catabolism of long chain fatty acids and reactive oxygen species especially hydrogen peroxide. These results were supported by serum biochemical parameters and are in agreement with the results stated in Japanese quails^23,24,33 and in laboratory mammals by^7,20,34. The slight improvement was noticed in the amelioration groups in comparison with CBZ treated groups at the end of 12^th week in both liver and kidney indicated the amelioration effect of vitamin E against CBZ toxicity^7,23. The SOD enzyme ensures the protection from toxic action of reactive oxygen species (nascent oxygen, hydrogen peroxide) and decrease in the SOD activity suggests an increased superoxide radical production supported by serum biochemical parameters. The results are in accordance with reports of^23,24,33 in Japanese quails and in laboratory mammals by^7,20,34. In amelioration groups, slight increase in the SOD activity on comparison with CBZ treated groups by the end of 8^th, 10^th and 12^th week were in agreement with studies of ^7,23.

GST protects cellular macromolecules against noxious chemicals and oxidative damage by conjugating toxic electrophilic molecules. Thus, the decrease in the GST activity of liver and kidney indicates inhibition of GST-mediated detoxification process in the CBZ treated groups supported by serum biochemical parameters^7,23,24. The slight improvement in the amelioration groups was observed in comparison to the CBZ groups were also reported by^7,23.

The findings of the present study indicated that CBZ at higher dose showed significant changes in haematological and serum biochemical parameters and antioxidant profile. The co-administration of vitamin E showed partial amelioration against CBZ toxicity.

indicate significant difference among groups I, II, III, IV, V and VI (P < 0.05)

indicate significant difference among groups I, II, III, IV, V and VI (P < 0.05)

indicate significant difference among groups I, II, III, IV, V and VI (P < 0.05)

References