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Applications in environmental risk assessment of biochemical analysis on the Indian fresh water fish, Labeo rohita exposed to monocrotophos pesticide
Accepted Manuscript Title: Applications in environmental risk assessment of biochemical analysis on the Indian fresh water fish, Labeo rohita exposed to monocrotophos pesticide Author: S. Binukumari K.Anusiya Devi J. Vasanthi PII: DOI: Reference:
S1382-6689(16)30217-4 http://dx.doi.org/doi:10.1016/j.etap.2016.08.014 ENVTOX 2602
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
19-6-2015 13-8-2016 21-8-2016
Please cite this article as: Binukumari, S., Devi, K.Anusiya, Vasanthi, J., Applications in environmental risk assessment of biochemical analysis on the Indian fresh water fish, Labeo rohita exposed to monocrotophos pesticide.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Applications in environmental risk assessment of Biochemical analysis on the Indian Fresh Water fish, Labeo rohita exposed to Monocrotophos pesticide S. Binukumari*, K. Anusiya Devi, J. Vasanthi PG and Research Department of Zoology, Kongunadu Arts and Science college, Coimbatore-641029, Tamilnadu, India _________________________________________________________________________________________________________________
Highlights
Repeated exposure to acute and chronic toxicity of monocrotophos pesticide is observed. Biochemical analysis has been analysed in the freshwater fish, Labeo rohita Decrease in protein, carbohydrate and lipid were noted when compared to control. The ecological risk of these pesticides should be strictly controlled.
ABSTRACT __________________________________________________________________________________ Pesticides are widely used in modern agriculture to aid in the production of high quality food. However, some pesticides have the potential to cause serious health and environmental damage. Repeated exposure to sub-lethal doses of pesticides can cause physiological and behavioral changes in fish that reduce populations such as abandonment of nests and broods, decreased immunity to disease and increased failure to avoid predators. Monocrotophos is one of the organophosphorus pesticide used in this study. The median lethal concentration (LC50) of Monocrotophos to fish L. rohita for 96 hour was found to be 45.1 ppm. In sublethal concentration (1/10th of LC50 96 hour value, 4.51ppm) fishes were exposed for 24, 48, 72, 96 hours and 10, 20 and 30 days. Organs of fishes were sacrificed and tested for biochemical analysis. A significant decrease in protein, carbohydrate and lipids were observed throughout the study period when compared to the control. It is essential for assessing the ecological risk of these pesticides. Keywords: Monocrotophos, biochemical, acute toxicity, chronic toxicity
__________________________________ 1.
Introduction
Different industries like distilleries, cotton mills, tanneries, paper mills, jute mills, fertilizers, plants and chemical plants pass out their effluents in adjoining rivers, ponds and other water resources. Apart from these, the run-off water from agricultural fields carries a lot of pesticides, herbicides, fungicides and weedicides (Alam, 2002). Pesticides are one of the major classes of toxic substances used in India for management of pest in agricultural lands and control of insect vectors of human disease. The runoff from treated areas enters the river and aquaculture ponds that are supplied by rivers. Such rivers and the
adjacent aquaculture ponds are likely to be contaminated by pesticide (Begum, 2004). Pesticides and herbicides at high concentration are known to reduce the survival, growth and reproduction of fish and produce many visible effects on fish (Rahman et al., 2002). Proteins, carbohydrates and lipids are involved in major physiological events, therefore the assessment of the content can be considered as a diagnostic tool to determine the physiological phase of organism (Martin and Arivoli, 2008). Proteins are important organic substance required by organisms in tissue building and play an important role in energy metabolism (Yeragi et al., 2003). Biochemical changes of tissues can be used as an indicators of physiological stress and health of fish (Ali Muhammad Yousafzai, 2004).
* Corresponding author.Mob.: 09894721285. E-mail address: [email protected] (S. Binukumari) India. The procured bulk samples of Labeo _____________________________ rohita were transported to the laboratory in well aerated polythene bag and acclimatized to 2. Materials and methods the laboratory conditions under natural photo period for one week in large plastic containers 2.1. Collection and maintanence of fish at (26 ± 5oC). The tank was previously washed with potassium permanganate to prevent any Fingerlings of the fresh water fish, Labeo fungal infection. The fishes were maintained rohita ranging in weight from 4g to 8g and in dechlorinated tap water of the quality used measuring (4 cm to 6 cm in length) were in the test and water was renewed every day to procured from Aliyar Fishfarm, Tamilnadu, provide freshwater rich in oxygen. During the
periods of acclimatization they were fed everyday with oil cake mixed with rice flour. Unhealthy fish and those with infections were removed. Feeding was stopped one day prior to the experiment to maintain same state of metabolic requirements. Fish belonging to both sexes were selected for the present investigation. All the precautions, laid down on recommendations of the toxicity tests to aquatic organisms are followed Anon, (1975). The tap water free from contaminants was used as dilution water for the present study. The physico-chemical analysis of water used in the experiments was carried out using the method of APHA, (2005); temperature 27.2 ± 0.9 (oC), pH 7.1 ± 0.1, dissolved oxygen 5.4 ± 0.4 (mg /l), total hardness 180 ±1.9 (mg /l), salinity 0.3 ± 0.1 (ppt). Continuous artificial aeration was maintained throughout the acclimatization and exposure periods. 2.2.
Toxicant
Monocrotophos one of the organophosphorus insecticides extensively used in agriculture and animal husbandry (Rao, 2004). Monocrotophos is a brownish yellow liquid with a sharp smell that irritates the eyes and skin. The IUPAC name is dimethyl (E)-1methyl-2-(methyl-carbamoyl) vinyl phosphate. Molecular formula is C7H14NO5P and molecular weight is 223.2.
median lethal concentration using a minimum of 6 fishes for each concentration and the mortality was recorded for every 24 hrs up to 96 hrs. It was found as for 48 hrs using probit analysis method (Finney, 1971). From the stock solution various sublethal concentrations were prepared for bioassay studies. 2.4.Sublethal toxicity Seven groups of fishes were exposed to (1/10th of the pesticide ‘Monocrotophos’ for 24, 48, 72 and 96 hrs, 10, 20 and 30 days. Another group was maintained as control. All the groups received the same type of food and other conditions were maintained similarly. At the end of each exposure period, fishes were sacrificed for further analysis. 2.5. Preparation of tissue samples After each exposure periods tissues such as liver, gill, muscle, brain and kidney were dissected and removed. The tissues (10 mg) were homogenized in 80% methanol, centrifuged at 3500 rpm for 15 minutes and the clear supernatant was used for the analysis of biochemical parameters (protein, lipid and carbohydrates). 2.6. Estimation of total protein Total protein concentration was estimated by the method of Lowry et al. (1951) based on the following principle.
2.3. Determination of 96 h LC50 value of MC
2.6.1. Principle
The concentrations of the pollutant at which 50 percent of the test animals die during a specific test period of time is referred to as median lethal concentration (LC50) (or) median tolerant limit. In aquatic toxicology the traditional LC50 test is often used to measure the potential risk of a chemical (Jack de Bruijin et al., 1991). Batches of 10 healthy fishes were exposed to different concentrations of pesticide, Monocrotophos to calculate the LC50 value. One more set of fishes are maintained as control in tap water. To find the wide range of concentration 100-600 ml were chosen and the number of dead or affected fishes was counted at regular intervals up to 48 hrs. The level of the dissolved oxygen, pH, alkalinity and hardness were monitored and maintained constant. Appropriate narrow range of concentration was used to find the
In alkaline medium protein in the sample form a complex with copper ions. The amino acids containing aromatic groups, tyrosine and tryptophan present in copper protein complex react with Folin Cliocalteu phenol reagent to give blue color due to the reduction of phosphomolybdate. The intensity of the color developed is proportional to the concentration of protein present in the sample. The value is expressed as mg/g of tissues. 2.7. Estimation of carbohydrate Quantitative estimation of carbohydrate in the tissues was done following the method by Hedg’s and Hofreiter, (1962). 2.7.1. Principle Carbohydrates are first hydrolyzed into simple sugars using dilute hydrochloric acid. In hot
acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms with anthrone, a green colored product with an absorption maximum at 630 nm
The amount of protein estimated in different tissues of the fish, L. rohita subjected to short term and long term exposures are presented in Figs.1 and 2.
2.8. Estimation of lipid
The amount of protein in gill tissue was 1.88, 1.61, 1.57 and 1.35 mg/g in the fishes exposed to short term duration of Monocrotophos pesticide after 24, 48, 72 and 96 hours respectively. The fishes which were exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days were found to contain 1.01, 0.99 and 0.68 mg/g while the control fish contained 2.00 mg/g of protein.1.97, 1.66, 1.34 and 1.21 of protein were present in the liver tissue respectively after 24, 48, 72 and 96 hrs of short term exposure of the fishes. The protein content in the fishes that were subjected to long term exposure was 1.12, 0.94 and 0.75 mg/g respectively. The mean control value was 2.16 mg/g. The muscle protein levels in the fishes that were subjected to short term and long term exposure in 4.51 per cent of Monocrotophos pesticide were 1.74, 1.16, 1.13 , 0.97, 0.91, 0.88 and 0.74 mg/g respectively. The mean control value was 1.76 mg/g. Kidney recorded 1.91, 1.46, 1.34 and 1.01 mg/g of protein in fishes exposed to short term duration of Monocrotophos pesticide for 24, 48, 72 and 96 hours respectively. 0.97, 0.74 and 0.69 mg/g were recorded in the kidney of fishes exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days. The mean control value was 2.01 mg/g. 3.21, 3.05, 2.97 and 2.71 of protein were present in the brain tissue respectively after 24, 48, 72 and 96 hours of short term exposure of the fishes. The protein content in the fishes that were subjected to long term exposure was 2.42, 2.12 and 1.98 mg/g respectively. The mean control value was 3.61 mg/g. Blood recorded 2.15, 1.94, 1.37 and 1.24 mg/g of protein in fishes exposed to short term duration of Monocrotophos pesticide for 24, 48, 72 and 96 hours respectively.1.12, 0.99 and 0.86 mg/g were recorded in the kidney of fishes exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days. The mean control value was 2.83 mg/g. 3.2.2. Total Carbohydrate content The amount of carbohydrate in the tissues estimated after subjecting the fishes to short term and long term exposure periods of
Estimation of Lipid was estimated by the method of Richmond, (1973) based on the following principle. 2.8.1. Principle Cholesterol esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3- one and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide oxidatively copies with 4-aminoantipyrine and phenol to produce red color dye which has absorbance maximum at 510nm (500-530). The intensity of red color is proportional to the amount of total cholesterol in the specimen. Cholesterol esterase Cholesterol esters
-----------------------
Cholesterol + fattyacids
(1)
Cholesterol oxidase Cholesterol + O2
------------------------
H2O2 + Cholest + 4-en-3one
(2)
2H2O2 + 4-aminoantipyrine + Phenol Peroxidase --------- Redquinoeimine dye + H2O (3) 2.9. Statistical analysis The data were analyzed statistically at t< 0.01. To test their significance the t values were calculated by Student’s t-test. _____________________________________
3. Results 3.1. LC 50 value-96 hour In the present study the 96 hour LC50 value of MC to L. rohita was determined to be 45.1ppm. 3.2.1.Total Protein content
the Monocrotophos pesticide are presented in Figs. 3 and 4. The gill of the fishes exposed to 4.51 per cent Monocrotophos pesticide for 24 hrs, 48 hrs, 72 hrs and 96 hrs was found to contain 15.70, 12.10, 10.60 and 8.80 mg/g of carbohydrate respectively. In the case of long term exposed fishes the values were 6.70, 5.80 and 4.10 mg/g after 10, 20 and 30 days respectively. The fishes maintained as control were found to contain a mean of 17.60 mg/g in their gill tissue. Liver tissue was found to contain 9.60, 7.80, 6.74 and 4.94 mg/g of carbohydrate respectively in 24, 48, 72 and 96 hours exposures in 4.51 per cent concentration of Monocrotophos pesticide. Under treatment of Monocrotophos pesticide for 10, 20 and 30 days exposures, the values were 4.12, 3.41 and 3.18 mg/g respectively. The mean carbohydrate content in the liver of the control was 12.40 mg/g. The mean carbohydrate content in the muscle of the control fish was 18.60 mg/g. The amount of carbohydrate in the fishes exposed to 24, 48, 72 and 96 hours in 4.51 per cent Monocrotophos pesticide were 15.10, 11.40, 11.21 and 10.44 mg/g respectively. The amount of carbohydrate in long term treatment were 6.74, 3.81 and 2.41 mg/g under 10, 20 and 30 days exposures in 4.51 per cent pesticide concentration.26.40, 20.10, 12.60 and 10.81 mg/g of carbohydrate were found in the kidney tissue of 24, 48, 72 and 96 hours treated fishes respectively. Values of carbohydrate estimated in the fishes exposed to long term periods (10, 20 and 30 days) in 4.51 per cent Monocrotophos pesticide were 7.94, 5.60 and 2.31 mg/g in their kidney tissue. The mean control value was 31.00 mg/g. The mean carbohydrate content in the brain of the control fish was 30.40 mg/g. The amount of carbohydrate in the fishes exposed to 24, 48, 72 and 96 hours in 4.51per cent Monocrotophos pesticide were 24.60, 19.70, 15.60 and 13.40 mg/g respectively. The amount of carbohydrate in long term treatment were 9.50, 6.82 and 5.71 mg/g under 10, 20 and 30 days exposures in 4.51per cent pesticide concentration. Blood tissue was found to contain 29.00, 25.60, 21.40 and 19.60 mg/g of carbohydrate respectively in 24, 48, 72 and 96 hours exposures in 4.51 per cent concentration of Monocrotophos pesticide. Under treatment of Monocrotophos pesticide for 10, 20 and 30 days exposures, the
values were 17.10, 14.70 and 9.40 mg/g respectively. The mean carbohydrate content in the liver of the control was 34.70 mg/g. 3.2.3. Total Lipid content The amount of lipid in the tissues estimated after exposing the fishes to short term and long term periods of the Monocrotophos pesticide are presented in Figs. 5 and 6. The Lipid content in the gill tissue of fishes exposed to short term periods in term of 24, 48, 72 and 96 hours were 11.50, 10.40, 9.80 and 8.00 mg/g respectively. The fishes exposed to long term periods of 10, 20 and 30 days in 4.51 per cent Monocrotophos pesticide contained 7.80, 6.70 and 5.60 mg/g of lipid in their gill respectively against an average of 15.70 mg/g in the control. Liver tissue was found to contain 15.40, 10.50, 9.41 and 7.60 mg/g of lipid in short term exposure periods of 24, 48, 72 and 96 hours. The fishes subjected to long term periods were found to contain 6.41, 5.44 and 4.10 mg/g of lipid. The mean control value was 21.6 mg/g. The amount of lipid in the muscle tissue were 16.30, 16.10, 14.21 and 13.20 mg/g in the fishes exposed to 4.51 per cent Monocrotophos pesticide after 24, 48, 72 and 96 hours exposure periods. However the fishes exposed to longer durations were found to contain 10.61, 9.50 and 7.60 mg/g of lipid. The control fishes were found to contain 20.10 mg/g of lipid in their muscles. Kidney recorded 37.40 mg/g in the control fishes. The fishes exposed for short term periods were found to contain 31.20, 26.40, 19.50 and 11.20 mg/g of lipid. However those exposed to longer durations contained 10.12, 9.60 and 5.51 mg/g. The Lipid content in the brain tissue of fishes exposed to short term periods in term of 24, 48, 72 and 96 hours were 56.80, 41.40, 34.30 and 29.50 mg/g respectively. The fishes exposed to long term periods of 10, 20 and 30 days in 4.51 per cent Monocrotophos pesticide contained 24.10, 20.70 and 17.50 mg/g of lipid in their gill respectively against an average of 64.70 mg/g in the control. Blood recorded 42.80 mg/g in the control fishes. The fishes exposed for short term periods were found to contain 37.40, 33.50, 29.30 and 22.50 mg/g of lipid. However those exposed to longer durations contained 19.40, 12.60 and 11.80 mg/g. _____________________________________
4. Discussion 4.1.Biochemical response of Labeo rohita In the present study, it was found that the biochemical constituents of the fish were greatly reduced in the fish, L. rohita exposed to short and long term exposure of Monocrotophos pesticide for 24, 48, 72, 96 hours 10, 20 and 30 days. The decrease in biochemical constituents may be due to the pollution stress to which the fishes were subjected to the energy needed to face the polluted environment is met from all the three major biochemical compounds such as protein, carbohydrate and lipid. 4.1.1. Protein content of tissues Pang-Hung et al. (2008) stated that proteins are important organic substances required by organisms in tissue building and play an important role in energy metabolism. The result of the present study showed significant decrease in protein content in the tissues studied (Figs.1 and 2). Per cent decrease in 24, 48, 72 and 96 hrs was found to be more in blood -56.18 during 96 hours and less in muscle -1.14 during 24 hours. Per cent decrease in 10 days, 20 days and 30 days was found to be more in blood -69.61 during 30 days and less in brain -32.96 during 10 days. Ghousia and Vijayaraghavan, (1995) reported that decrease in protein content of dimethoate intoxicated fish, Clarias batrachus indicated physiological adaptability of the fish to compensate for pesticide stress. Rajyashree, (1996) also observed decline in protein level in liver, muscles, gills and brain during carbamide exposure of the fish, L. rohita. Das et al. (1999) observed that decrease in the protein content of various tissues like kidney and muscles and slight increase in the protein content of brain and gills in cypermethrin treated fish, Channa punctatus. Susan et al. (1999) have also reported a significant decrease in protein content under sublethal concentrations of pyrethroid fenvalerate in the gills of the fish, Catla catla. Protein depletion in the muscle might be due to muscular atrophy (Bhatnagar, 1999). Decreased protein level may be attributed to stress mediated immobilization of these compounds to fulfill an increased element for energy by the fish to cope with environmental
condition exposed by the toxicant (Jankins and Smith, 2003). A reduction in protein content was also observed in Cyprinus carpio and Labeo rohita exposed to cypermethrin as reported by David et al. (2004). The depletion of protein fraction in gill, liver, kidney and muscle might have been due to their degradation and possible utilization for metabolic purpose (Venkataramana et al. 2006). Kannan et al. (2010) reported the decreased protein content on gill, brain and muscle of Mystus vittatus when exposed to mercuric chloride. Present findings are in good agreement with the above findings by different workers. The present study clearly supports the earlier workers that tissue proteins are used to meet the increased energy demand posed by stress. 4.1.2. Carbohydrate content of tissues Carbohydrate is an essential component of living cells and sources of energy for animals. The results of the present findings showed a significant decrease in carbohydrate content in all the tissues studied (Figs. 3 and 4). Per cent decrease in 24, 48, 72 and 96 hrs recorded were ranged from -10.79 to -50.00 in gills, 22.58 to -60.16 in liver, -18.81 to -78.16 in muscle, -14.83 to -65.12 in kidney, -19.07 to -55.92 in brain and -16.42 to -43.51 in blood. The muscle showed the highest per cent decrease -78.16 in carbohydrate content during 96 hours. Per cent decrease in 10, 20 and 30 days recorded were ranged from -61.93 to 76.70 in gills, -66.77 to -74.35 in liver, -63.76 to -87.04 in muscle,-74.38 to -92.54 in kidney, -68.75 to -81.21 in brain and -50.72 to -72.91 in blood. The kidney showed the highest per cent decrease -92.54 in carbohydrate content during 30 days. Nasreen et al. (1994) observed that the depletion in the carbohydrate content in all the tissues to phenol exposure in the fish, Channa punctatus. A similar decrease trend in carbohydrate was reported by Arasta et al. (1996) in the fish, Mystus vittatus on exposed to pesticide. This could have happened by rapid glycogenolysis and inhibition of glycogenesis phosphorylase and depression of glycogen transferase (Jha and Jha, 1995). A significant decrease in carbohydrate content of the muscle tissue in the present study is similar to the earlier observations in the tissues of muscle, liver and gills in L. rohita treated with monovalent mercury (Rajasubramaniam et al., 2006).
Remia et al. (2008) reported that the elevation of carbohydrates might be due to the stress induced by the Monocrotophos as physiology of organisms with the help of corticosteroids in the fish, Tilapia mossambicus. Logaswamy and Remia, (2009) reported that sublethal concentration of certain organophosphate pesticides caused glycogenolysis,which produced hyperglycemia in the African food fish, Tilapia mossambica. The fish showed stress condition during exposure period as fast swimming, fast opercular movements, dashing with the walls of aquarium and reduced feeding in the present study. Thus, during such type of stress conditions, the glycogen reserves are depleted to meet energy demand as studied by Tiwari and Singh, (2009). Saradhamani and Selvarani, (2009) reported a similar trend of decrease in carbohydrate of the fresh water fish, Tilapia mossambica on exposed to metribuzin. Sreenivasa and Indirani, (2010) reported that the decreased level of carbohydrate content of cardiac muscle, testes and liver in fish, Oreochromis mossambicus on exposed to dimethoate. The above findings are in conformity with the present work. According to Chezhian et al. (2010) observed that the decreased level of glycogen may be due to the induced activation of adrenal pituitary glucocorticoid hormones, which stimulate the hepatic glucose production thereby elevating blood glucose level to meet the critical need of energy under effluent stress in the Estuarine fish, Lates calcarifer. Tissue specific depletion of carbohydrates as observed in the present study may be due to its rapid utilization to meet the energy demands under the impact of pesticide. 4.1.3. Lipid content of tissues Lipid is an important normal body constituent used in the structure of cell membranes, synthesis of bile acid and synthesis of steroid hormones. The results presented in (Figs. 5 and 6) shows a significant decrease in lipid content in the studied tissues of fish, L. rohita. Generally, the decreases in lipid contents in all tissues were found to be increased with the hours of exposure. The per cent decrease was found to be greater in all tissues during 96 hrs exposure -49.04 in gill, -64.81 in liver, -34.32 in muscle, -70.05 in kidneys, -54.40 in brain and -47.42 in blood. The kidney showed the
highest per cent decrease -70.05 in lipid content. The per cent decrease was found to be greater in all tissues during 30 days exposure 64.33 in gills, -81.01 in liver, -62.18 in muscle, -43.72 in kidneys, -72.95 in brain and -72.42 in blood. The liver showed the highest per cent decrease -81.01 in lipid content. Srinivas et al., (1991) has showed decreased lipid content in Tilapia mossambicus on exposed to Atrazine. (Shakoori et al., 1996) reported that the lipid decrease may be due to utilization of fatty deposits instead of glucose for energy purpose of the fish, Etenopharyngodon idella on exposed to fenvalerate. Decrease in lipid content may be due to decline in the lipid synthesizing capacity and due to an increase in the hydrolysis of hepatic lipid to combat stress condition (Virk and Sharma, 1999). (Leela et al. (2000) observed that total lipid content of liver, muscle and gill of Tilapia mossambica was decreased under the stress of phosalone. The alterations of lipid content may be due to its utilization in cortical steroidogenesis and also impairment in the synthesis of lipid. Similar observation was made in Glassogobius giuris (Sreenivasa, 2002). Arockia and Milton, (2006) have showed declining trend of lipid content in the tissues like brain, gill, kidney, liver and muscles upon exposure to carbamate in the fish, Oreochromis mossambicus. However, Remia et al. (2008) reported that the decline of lipid may be due to utilization of fatty deposits instead of glucose for energy purpose of the fish, Tilapia mossambica on exposed to Monocrotophos. Saradhamani and Selvarani, (2009) showed significant decrease in lipid content in the tissues of the fresh water fish, Tilapia mossambica on exposed to metribuzin. Decrease in the lipid content was noticed in the fish, Mystus vittatus on exposed to mercuric chloride by Kannan et al. (2010). The present study reveals that the changes in protein, carbohydrate and lipid in pesticides treated fishes will naturally affect the nutritive values of these animals and all the metabolites studied are found to be sensitive biochemical indicators, which reflect changes in the normal activities of various functional systems. _____________________________________ 5. Conclusion
In the present study, it is concluded that MC has a profound influence on the biochemical analysis of an Indian major carp, L. rohita and MC is toxic to aquatic organisms. The results imply a better understanding on the toxicological endpoint of this specific pesticide and provide significant information on safe levels in the aquatic environment. _____________________________________ Conflict of Interest None declared. _____________________________________ Acknowledgement Authors are gratefully thankful to staff members for their valuable suggestions and Head of the Department of Zoology for providing facilities to carryout this research work successfully.
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Rahman, M. Z., Hossain, Z. M., Ellah, M. F. R.,Ahmed, G. U., 2002. Effect of Diazinon 60EC on Anabustestudineus, Channa punctatus and barbadesgomonous. Naga. The ICLARM quarterly 25, 8-11. Rajasubramaniam, V., Sasikumar, K. and Subhasini, S., 2006. Effect of Monovalent mercury on liver, muscle glycogen and blood glucose levels of freshwater teleost , L. rohita. J. Eco. Toxical. Environ. Monit 16, 215-220. Rajyashree, M., 1996. Carbamide induced alterations in some metabolic aspects of fish, Labeo rohita. J. Ecotoxicol. Environ. Monit 6 (1), 41-44. Rao, J V., 2004. Effects of monocrotophos and its analogs in acetylcholinesterase activity’s inhibition and its pattern of recovery on euryhaline fish, Oreochromis mossambicus. Ecotox. Environ Safe 59, 217–222. Remia, K. M., Logaswamy, S., Logankumar K., Rajmohan, D., 2008. Effect of an insecticides (Monocrotophos) on some biochemical constituents of the fish Tilipia Mossambica. Poll.Res 27, 523526. Richmond, W., 1973. Preparation and properties of a cholesterol oxidase from Nocardia Sp. And its application to the enzymatic assay of total Lipid in serum.Clin.chem. 19, 1350-1356. Saradhamani, N., Selvarani, B. J., 2009. A study on the effect of herbicide Metribuzin on the biochemical constituents of the freshwater fish,Tilapia mossambica Peters (Pisces: Curr. Biotica, Cichlidae), Curr.Biotica 3, 220-231. Shakoori, A. R., Mughal, A. L., Iqbal, M., 1996. Effect of sublethal doses of Fenvalarate (A synthetic pyrethroid) administered continuously for 4 weeks on the blood, liver, muscles of freshwater fish. Etenopharyngodon idella, Bull. Environ.Contam. Toxicol 57, 487-499. Sreenivasa, V., 2002. Studies on the effects of pesticide on Cardiac and body muscles of fresh water gobiid fish, Gloossogobius giuris (Ham). Ph.D, thesis, Bangalore University. India. Sreenivasa. V., Indirani, R., 2010. Impact of Dimethoate on biochemical
constituents in the fish, Oreochromis mossambicus. J. Ecotoxicol. Environ. Monit 20(2),151-156. Srinivas, T., Prasad, T. A. V., Rafi, G. M. D., Reddy, D. C., 1991. Effect of Atrazine on some aspects of lipid metabolism in freshwater fish. Biol.Inter 23, 603609. Susan, A. T., Veeraiah, K., Tilak, K. S., 1999. Biochemical and enzymatic changes in the tissues of Catla catla exposed to the pyrethroid fenvalerate. J. Ecobiol 11(2), 109-116. Tiwari S., Singh, A., 2009. Changes in some biochemical parameters in the liver and muscle of Colisa fasciatus due to toxicity of ethanolic extract of Narium indicum Mill (Lal Kaner) latex. Natural Product Radiance., 8(1), 4854. Venkataramana, G. V., Sandhya Rani, P. N., Murthy, P. S., 2006. Impact of Malathion on the biochemical parameters of gobiid fish, Glossogobius giuris (Ham). Journal of Environmental Biology 27(1), 119122. Virk K, S., Sharma, R. C., 1999. Biochemical changes induced by Nickel and Chromium in the liver of Cyprinus carpio. Poll. Res 18, 217-222. Yeragi, S. G., Ranaand, A. M., Koli, V. A., 2003. Effect of Pesticide on protein metabolism of mudskipper, Boleophtalmus dussumieri. J. Ecotoxicol. Environ. Monit 13, 211214.
30
Values in mg/g
Values in mg/g
40 4 3 2 1 0
20 10 0
ORGANS ORGANS CONTROL
24hrs
48hrs
24hrs
72hrs
96hrs
48hrs
Fig.3 – Changes in the carbohydrate content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
4 3
Values in mg/g
Values in mg/g
Fig.1 – Changes in the protein content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
CONTROL
2 1 0
40 30 20 10 0
ORGANS CONTROL 20 DAYS
10 DAYS 30 DAYS
Fig.2 – Changes in the protein content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure
ORGANS CONTROL
10 DAYS
20 DAYS
30 DAYS
Fig.4 – Changes in the carbohydrate content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure.
Values in mg/g
70 60 50 40 30 20 10 0
ORGANS CONTROL
24hrs
72hrs
96hrs
48hrs
Fig.5 – Changes in the lipid content in a fresh water fish, L. rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
Values in mg/g
70 60 50 40 30 20 10 0
ORGANS CONTROL 20 DAYS
10 DAYS 30 DAYS
Fig.6 – Changes in the lipid content in a fresh water fish, L. rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure.
S1382-6689(16)30217-4 http://dx.doi.org/doi:10.1016/j.etap.2016.08.014 ENVTOX 2602
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
19-6-2015 13-8-2016 21-8-2016
Please cite this article as: Binukumari, S., Devi, K.Anusiya, Vasanthi, J., Applications in environmental risk assessment of biochemical analysis on the Indian fresh water fish, Labeo rohita exposed to monocrotophos pesticide.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Applications in environmental risk assessment of Biochemical analysis on the Indian Fresh Water fish, Labeo rohita exposed to Monocrotophos pesticide S. Binukumari*, K. Anusiya Devi, J. Vasanthi PG and Research Department of Zoology, Kongunadu Arts and Science college, Coimbatore-641029, Tamilnadu, India _________________________________________________________________________________________________________________
Highlights
Repeated exposure to acute and chronic toxicity of monocrotophos pesticide is observed. Biochemical analysis has been analysed in the freshwater fish, Labeo rohita Decrease in protein, carbohydrate and lipid were noted when compared to control. The ecological risk of these pesticides should be strictly controlled.
ABSTRACT __________________________________________________________________________________ Pesticides are widely used in modern agriculture to aid in the production of high quality food. However, some pesticides have the potential to cause serious health and environmental damage. Repeated exposure to sub-lethal doses of pesticides can cause physiological and behavioral changes in fish that reduce populations such as abandonment of nests and broods, decreased immunity to disease and increased failure to avoid predators. Monocrotophos is one of the organophosphorus pesticide used in this study. The median lethal concentration (LC50) of Monocrotophos to fish L. rohita for 96 hour was found to be 45.1 ppm. In sublethal concentration (1/10th of LC50 96 hour value, 4.51ppm) fishes were exposed for 24, 48, 72, 96 hours and 10, 20 and 30 days. Organs of fishes were sacrificed and tested for biochemical analysis. A significant decrease in protein, carbohydrate and lipids were observed throughout the study period when compared to the control. It is essential for assessing the ecological risk of these pesticides. Keywords: Monocrotophos, biochemical, acute toxicity, chronic toxicity
__________________________________ 1.
Introduction
Different industries like distilleries, cotton mills, tanneries, paper mills, jute mills, fertilizers, plants and chemical plants pass out their effluents in adjoining rivers, ponds and other water resources. Apart from these, the run-off water from agricultural fields carries a lot of pesticides, herbicides, fungicides and weedicides (Alam, 2002). Pesticides are one of the major classes of toxic substances used in India for management of pest in agricultural lands and control of insect vectors of human disease. The runoff from treated areas enters the river and aquaculture ponds that are supplied by rivers. Such rivers and the
adjacent aquaculture ponds are likely to be contaminated by pesticide (Begum, 2004). Pesticides and herbicides at high concentration are known to reduce the survival, growth and reproduction of fish and produce many visible effects on fish (Rahman et al., 2002). Proteins, carbohydrates and lipids are involved in major physiological events, therefore the assessment of the content can be considered as a diagnostic tool to determine the physiological phase of organism (Martin and Arivoli, 2008). Proteins are important organic substance required by organisms in tissue building and play an important role in energy metabolism (Yeragi et al., 2003). Biochemical changes of tissues can be used as an indicators of physiological stress and health of fish (Ali Muhammad Yousafzai, 2004).
* Corresponding author.Mob.: 09894721285. E-mail address: [email protected] (S. Binukumari) India. The procured bulk samples of Labeo _____________________________ rohita were transported to the laboratory in well aerated polythene bag and acclimatized to 2. Materials and methods the laboratory conditions under natural photo period for one week in large plastic containers 2.1. Collection and maintanence of fish at (26 ± 5oC). The tank was previously washed with potassium permanganate to prevent any Fingerlings of the fresh water fish, Labeo fungal infection. The fishes were maintained rohita ranging in weight from 4g to 8g and in dechlorinated tap water of the quality used measuring (4 cm to 6 cm in length) were in the test and water was renewed every day to procured from Aliyar Fishfarm, Tamilnadu, provide freshwater rich in oxygen. During the
periods of acclimatization they were fed everyday with oil cake mixed with rice flour. Unhealthy fish and those with infections were removed. Feeding was stopped one day prior to the experiment to maintain same state of metabolic requirements. Fish belonging to both sexes were selected for the present investigation. All the precautions, laid down on recommendations of the toxicity tests to aquatic organisms are followed Anon, (1975). The tap water free from contaminants was used as dilution water for the present study. The physico-chemical analysis of water used in the experiments was carried out using the method of APHA, (2005); temperature 27.2 ± 0.9 (oC), pH 7.1 ± 0.1, dissolved oxygen 5.4 ± 0.4 (mg /l), total hardness 180 ±1.9 (mg /l), salinity 0.3 ± 0.1 (ppt). Continuous artificial aeration was maintained throughout the acclimatization and exposure periods. 2.2.
Toxicant
Monocrotophos one of the organophosphorus insecticides extensively used in agriculture and animal husbandry (Rao, 2004). Monocrotophos is a brownish yellow liquid with a sharp smell that irritates the eyes and skin. The IUPAC name is dimethyl (E)-1methyl-2-(methyl-carbamoyl) vinyl phosphate. Molecular formula is C7H14NO5P and molecular weight is 223.2.
median lethal concentration using a minimum of 6 fishes for each concentration and the mortality was recorded for every 24 hrs up to 96 hrs. It was found as for 48 hrs using probit analysis method (Finney, 1971). From the stock solution various sublethal concentrations were prepared for bioassay studies. 2.4.Sublethal toxicity Seven groups of fishes were exposed to (1/10th of the pesticide ‘Monocrotophos’ for 24, 48, 72 and 96 hrs, 10, 20 and 30 days. Another group was maintained as control. All the groups received the same type of food and other conditions were maintained similarly. At the end of each exposure period, fishes were sacrificed for further analysis. 2.5. Preparation of tissue samples After each exposure periods tissues such as liver, gill, muscle, brain and kidney were dissected and removed. The tissues (10 mg) were homogenized in 80% methanol, centrifuged at 3500 rpm for 15 minutes and the clear supernatant was used for the analysis of biochemical parameters (protein, lipid and carbohydrates). 2.6. Estimation of total protein Total protein concentration was estimated by the method of Lowry et al. (1951) based on the following principle.
2.3. Determination of 96 h LC50 value of MC
2.6.1. Principle
The concentrations of the pollutant at which 50 percent of the test animals die during a specific test period of time is referred to as median lethal concentration (LC50) (or) median tolerant limit. In aquatic toxicology the traditional LC50 test is often used to measure the potential risk of a chemical (Jack de Bruijin et al., 1991). Batches of 10 healthy fishes were exposed to different concentrations of pesticide, Monocrotophos to calculate the LC50 value. One more set of fishes are maintained as control in tap water. To find the wide range of concentration 100-600 ml were chosen and the number of dead or affected fishes was counted at regular intervals up to 48 hrs. The level of the dissolved oxygen, pH, alkalinity and hardness were monitored and maintained constant. Appropriate narrow range of concentration was used to find the
In alkaline medium protein in the sample form a complex with copper ions. The amino acids containing aromatic groups, tyrosine and tryptophan present in copper protein complex react with Folin Cliocalteu phenol reagent to give blue color due to the reduction of phosphomolybdate. The intensity of the color developed is proportional to the concentration of protein present in the sample. The value is expressed as mg/g of tissues. 2.7. Estimation of carbohydrate Quantitative estimation of carbohydrate in the tissues was done following the method by Hedg’s and Hofreiter, (1962). 2.7.1. Principle Carbohydrates are first hydrolyzed into simple sugars using dilute hydrochloric acid. In hot
acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms with anthrone, a green colored product with an absorption maximum at 630 nm
The amount of protein estimated in different tissues of the fish, L. rohita subjected to short term and long term exposures are presented in Figs.1 and 2.
2.8. Estimation of lipid
The amount of protein in gill tissue was 1.88, 1.61, 1.57 and 1.35 mg/g in the fishes exposed to short term duration of Monocrotophos pesticide after 24, 48, 72 and 96 hours respectively. The fishes which were exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days were found to contain 1.01, 0.99 and 0.68 mg/g while the control fish contained 2.00 mg/g of protein.1.97, 1.66, 1.34 and 1.21 of protein were present in the liver tissue respectively after 24, 48, 72 and 96 hrs of short term exposure of the fishes. The protein content in the fishes that were subjected to long term exposure was 1.12, 0.94 and 0.75 mg/g respectively. The mean control value was 2.16 mg/g. The muscle protein levels in the fishes that were subjected to short term and long term exposure in 4.51 per cent of Monocrotophos pesticide were 1.74, 1.16, 1.13 , 0.97, 0.91, 0.88 and 0.74 mg/g respectively. The mean control value was 1.76 mg/g. Kidney recorded 1.91, 1.46, 1.34 and 1.01 mg/g of protein in fishes exposed to short term duration of Monocrotophos pesticide for 24, 48, 72 and 96 hours respectively. 0.97, 0.74 and 0.69 mg/g were recorded in the kidney of fishes exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days. The mean control value was 2.01 mg/g. 3.21, 3.05, 2.97 and 2.71 of protein were present in the brain tissue respectively after 24, 48, 72 and 96 hours of short term exposure of the fishes. The protein content in the fishes that were subjected to long term exposure was 2.42, 2.12 and 1.98 mg/g respectively. The mean control value was 3.61 mg/g. Blood recorded 2.15, 1.94, 1.37 and 1.24 mg/g of protein in fishes exposed to short term duration of Monocrotophos pesticide for 24, 48, 72 and 96 hours respectively.1.12, 0.99 and 0.86 mg/g were recorded in the kidney of fishes exposed to long term duration of Monocrotophos pesticide for 10, 20 and 30 days. The mean control value was 2.83 mg/g. 3.2.2. Total Carbohydrate content The amount of carbohydrate in the tissues estimated after subjecting the fishes to short term and long term exposure periods of
Estimation of Lipid was estimated by the method of Richmond, (1973) based on the following principle. 2.8.1. Principle Cholesterol esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3- one and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide oxidatively copies with 4-aminoantipyrine and phenol to produce red color dye which has absorbance maximum at 510nm (500-530). The intensity of red color is proportional to the amount of total cholesterol in the specimen. Cholesterol esterase Cholesterol esters
-----------------------
Cholesterol + fattyacids
(1)
Cholesterol oxidase Cholesterol + O2
------------------------
H2O2 + Cholest + 4-en-3one
(2)
2H2O2 + 4-aminoantipyrine + Phenol Peroxidase --------- Redquinoeimine dye + H2O (3) 2.9. Statistical analysis The data were analyzed statistically at t< 0.01. To test their significance the t values were calculated by Student’s t-test. _____________________________________
3. Results 3.1. LC 50 value-96 hour In the present study the 96 hour LC50 value of MC to L. rohita was determined to be 45.1ppm. 3.2.1.Total Protein content
the Monocrotophos pesticide are presented in Figs. 3 and 4. The gill of the fishes exposed to 4.51 per cent Monocrotophos pesticide for 24 hrs, 48 hrs, 72 hrs and 96 hrs was found to contain 15.70, 12.10, 10.60 and 8.80 mg/g of carbohydrate respectively. In the case of long term exposed fishes the values were 6.70, 5.80 and 4.10 mg/g after 10, 20 and 30 days respectively. The fishes maintained as control were found to contain a mean of 17.60 mg/g in their gill tissue. Liver tissue was found to contain 9.60, 7.80, 6.74 and 4.94 mg/g of carbohydrate respectively in 24, 48, 72 and 96 hours exposures in 4.51 per cent concentration of Monocrotophos pesticide. Under treatment of Monocrotophos pesticide for 10, 20 and 30 days exposures, the values were 4.12, 3.41 and 3.18 mg/g respectively. The mean carbohydrate content in the liver of the control was 12.40 mg/g. The mean carbohydrate content in the muscle of the control fish was 18.60 mg/g. The amount of carbohydrate in the fishes exposed to 24, 48, 72 and 96 hours in 4.51 per cent Monocrotophos pesticide were 15.10, 11.40, 11.21 and 10.44 mg/g respectively. The amount of carbohydrate in long term treatment were 6.74, 3.81 and 2.41 mg/g under 10, 20 and 30 days exposures in 4.51 per cent pesticide concentration.26.40, 20.10, 12.60 and 10.81 mg/g of carbohydrate were found in the kidney tissue of 24, 48, 72 and 96 hours treated fishes respectively. Values of carbohydrate estimated in the fishes exposed to long term periods (10, 20 and 30 days) in 4.51 per cent Monocrotophos pesticide were 7.94, 5.60 and 2.31 mg/g in their kidney tissue. The mean control value was 31.00 mg/g. The mean carbohydrate content in the brain of the control fish was 30.40 mg/g. The amount of carbohydrate in the fishes exposed to 24, 48, 72 and 96 hours in 4.51per cent Monocrotophos pesticide were 24.60, 19.70, 15.60 and 13.40 mg/g respectively. The amount of carbohydrate in long term treatment were 9.50, 6.82 and 5.71 mg/g under 10, 20 and 30 days exposures in 4.51per cent pesticide concentration. Blood tissue was found to contain 29.00, 25.60, 21.40 and 19.60 mg/g of carbohydrate respectively in 24, 48, 72 and 96 hours exposures in 4.51 per cent concentration of Monocrotophos pesticide. Under treatment of Monocrotophos pesticide for 10, 20 and 30 days exposures, the
values were 17.10, 14.70 and 9.40 mg/g respectively. The mean carbohydrate content in the liver of the control was 34.70 mg/g. 3.2.3. Total Lipid content The amount of lipid in the tissues estimated after exposing the fishes to short term and long term periods of the Monocrotophos pesticide are presented in Figs. 5 and 6. The Lipid content in the gill tissue of fishes exposed to short term periods in term of 24, 48, 72 and 96 hours were 11.50, 10.40, 9.80 and 8.00 mg/g respectively. The fishes exposed to long term periods of 10, 20 and 30 days in 4.51 per cent Monocrotophos pesticide contained 7.80, 6.70 and 5.60 mg/g of lipid in their gill respectively against an average of 15.70 mg/g in the control. Liver tissue was found to contain 15.40, 10.50, 9.41 and 7.60 mg/g of lipid in short term exposure periods of 24, 48, 72 and 96 hours. The fishes subjected to long term periods were found to contain 6.41, 5.44 and 4.10 mg/g of lipid. The mean control value was 21.6 mg/g. The amount of lipid in the muscle tissue were 16.30, 16.10, 14.21 and 13.20 mg/g in the fishes exposed to 4.51 per cent Monocrotophos pesticide after 24, 48, 72 and 96 hours exposure periods. However the fishes exposed to longer durations were found to contain 10.61, 9.50 and 7.60 mg/g of lipid. The control fishes were found to contain 20.10 mg/g of lipid in their muscles. Kidney recorded 37.40 mg/g in the control fishes. The fishes exposed for short term periods were found to contain 31.20, 26.40, 19.50 and 11.20 mg/g of lipid. However those exposed to longer durations contained 10.12, 9.60 and 5.51 mg/g. The Lipid content in the brain tissue of fishes exposed to short term periods in term of 24, 48, 72 and 96 hours were 56.80, 41.40, 34.30 and 29.50 mg/g respectively. The fishes exposed to long term periods of 10, 20 and 30 days in 4.51 per cent Monocrotophos pesticide contained 24.10, 20.70 and 17.50 mg/g of lipid in their gill respectively against an average of 64.70 mg/g in the control. Blood recorded 42.80 mg/g in the control fishes. The fishes exposed for short term periods were found to contain 37.40, 33.50, 29.30 and 22.50 mg/g of lipid. However those exposed to longer durations contained 19.40, 12.60 and 11.80 mg/g. _____________________________________
4. Discussion 4.1.Biochemical response of Labeo rohita In the present study, it was found that the biochemical constituents of the fish were greatly reduced in the fish, L. rohita exposed to short and long term exposure of Monocrotophos pesticide for 24, 48, 72, 96 hours 10, 20 and 30 days. The decrease in biochemical constituents may be due to the pollution stress to which the fishes were subjected to the energy needed to face the polluted environment is met from all the three major biochemical compounds such as protein, carbohydrate and lipid. 4.1.1. Protein content of tissues Pang-Hung et al. (2008) stated that proteins are important organic substances required by organisms in tissue building and play an important role in energy metabolism. The result of the present study showed significant decrease in protein content in the tissues studied (Figs.1 and 2). Per cent decrease in 24, 48, 72 and 96 hrs was found to be more in blood -56.18 during 96 hours and less in muscle -1.14 during 24 hours. Per cent decrease in 10 days, 20 days and 30 days was found to be more in blood -69.61 during 30 days and less in brain -32.96 during 10 days. Ghousia and Vijayaraghavan, (1995) reported that decrease in protein content of dimethoate intoxicated fish, Clarias batrachus indicated physiological adaptability of the fish to compensate for pesticide stress. Rajyashree, (1996) also observed decline in protein level in liver, muscles, gills and brain during carbamide exposure of the fish, L. rohita. Das et al. (1999) observed that decrease in the protein content of various tissues like kidney and muscles and slight increase in the protein content of brain and gills in cypermethrin treated fish, Channa punctatus. Susan et al. (1999) have also reported a significant decrease in protein content under sublethal concentrations of pyrethroid fenvalerate in the gills of the fish, Catla catla. Protein depletion in the muscle might be due to muscular atrophy (Bhatnagar, 1999). Decreased protein level may be attributed to stress mediated immobilization of these compounds to fulfill an increased element for energy by the fish to cope with environmental
condition exposed by the toxicant (Jankins and Smith, 2003). A reduction in protein content was also observed in Cyprinus carpio and Labeo rohita exposed to cypermethrin as reported by David et al. (2004). The depletion of protein fraction in gill, liver, kidney and muscle might have been due to their degradation and possible utilization for metabolic purpose (Venkataramana et al. 2006). Kannan et al. (2010) reported the decreased protein content on gill, brain and muscle of Mystus vittatus when exposed to mercuric chloride. Present findings are in good agreement with the above findings by different workers. The present study clearly supports the earlier workers that tissue proteins are used to meet the increased energy demand posed by stress. 4.1.2. Carbohydrate content of tissues Carbohydrate is an essential component of living cells and sources of energy for animals. The results of the present findings showed a significant decrease in carbohydrate content in all the tissues studied (Figs. 3 and 4). Per cent decrease in 24, 48, 72 and 96 hrs recorded were ranged from -10.79 to -50.00 in gills, 22.58 to -60.16 in liver, -18.81 to -78.16 in muscle, -14.83 to -65.12 in kidney, -19.07 to -55.92 in brain and -16.42 to -43.51 in blood. The muscle showed the highest per cent decrease -78.16 in carbohydrate content during 96 hours. Per cent decrease in 10, 20 and 30 days recorded were ranged from -61.93 to 76.70 in gills, -66.77 to -74.35 in liver, -63.76 to -87.04 in muscle,-74.38 to -92.54 in kidney, -68.75 to -81.21 in brain and -50.72 to -72.91 in blood. The kidney showed the highest per cent decrease -92.54 in carbohydrate content during 30 days. Nasreen et al. (1994) observed that the depletion in the carbohydrate content in all the tissues to phenol exposure in the fish, Channa punctatus. A similar decrease trend in carbohydrate was reported by Arasta et al. (1996) in the fish, Mystus vittatus on exposed to pesticide. This could have happened by rapid glycogenolysis and inhibition of glycogenesis phosphorylase and depression of glycogen transferase (Jha and Jha, 1995). A significant decrease in carbohydrate content of the muscle tissue in the present study is similar to the earlier observations in the tissues of muscle, liver and gills in L. rohita treated with monovalent mercury (Rajasubramaniam et al., 2006).
Remia et al. (2008) reported that the elevation of carbohydrates might be due to the stress induced by the Monocrotophos as physiology of organisms with the help of corticosteroids in the fish, Tilapia mossambicus. Logaswamy and Remia, (2009) reported that sublethal concentration of certain organophosphate pesticides caused glycogenolysis,which produced hyperglycemia in the African food fish, Tilapia mossambica. The fish showed stress condition during exposure period as fast swimming, fast opercular movements, dashing with the walls of aquarium and reduced feeding in the present study. Thus, during such type of stress conditions, the glycogen reserves are depleted to meet energy demand as studied by Tiwari and Singh, (2009). Saradhamani and Selvarani, (2009) reported a similar trend of decrease in carbohydrate of the fresh water fish, Tilapia mossambica on exposed to metribuzin. Sreenivasa and Indirani, (2010) reported that the decreased level of carbohydrate content of cardiac muscle, testes and liver in fish, Oreochromis mossambicus on exposed to dimethoate. The above findings are in conformity with the present work. According to Chezhian et al. (2010) observed that the decreased level of glycogen may be due to the induced activation of adrenal pituitary glucocorticoid hormones, which stimulate the hepatic glucose production thereby elevating blood glucose level to meet the critical need of energy under effluent stress in the Estuarine fish, Lates calcarifer. Tissue specific depletion of carbohydrates as observed in the present study may be due to its rapid utilization to meet the energy demands under the impact of pesticide. 4.1.3. Lipid content of tissues Lipid is an important normal body constituent used in the structure of cell membranes, synthesis of bile acid and synthesis of steroid hormones. The results presented in (Figs. 5 and 6) shows a significant decrease in lipid content in the studied tissues of fish, L. rohita. Generally, the decreases in lipid contents in all tissues were found to be increased with the hours of exposure. The per cent decrease was found to be greater in all tissues during 96 hrs exposure -49.04 in gill, -64.81 in liver, -34.32 in muscle, -70.05 in kidneys, -54.40 in brain and -47.42 in blood. The kidney showed the
highest per cent decrease -70.05 in lipid content. The per cent decrease was found to be greater in all tissues during 30 days exposure 64.33 in gills, -81.01 in liver, -62.18 in muscle, -43.72 in kidneys, -72.95 in brain and -72.42 in blood. The liver showed the highest per cent decrease -81.01 in lipid content. Srinivas et al., (1991) has showed decreased lipid content in Tilapia mossambicus on exposed to Atrazine. (Shakoori et al., 1996) reported that the lipid decrease may be due to utilization of fatty deposits instead of glucose for energy purpose of the fish, Etenopharyngodon idella on exposed to fenvalerate. Decrease in lipid content may be due to decline in the lipid synthesizing capacity and due to an increase in the hydrolysis of hepatic lipid to combat stress condition (Virk and Sharma, 1999). (Leela et al. (2000) observed that total lipid content of liver, muscle and gill of Tilapia mossambica was decreased under the stress of phosalone. The alterations of lipid content may be due to its utilization in cortical steroidogenesis and also impairment in the synthesis of lipid. Similar observation was made in Glassogobius giuris (Sreenivasa, 2002). Arockia and Milton, (2006) have showed declining trend of lipid content in the tissues like brain, gill, kidney, liver and muscles upon exposure to carbamate in the fish, Oreochromis mossambicus. However, Remia et al. (2008) reported that the decline of lipid may be due to utilization of fatty deposits instead of glucose for energy purpose of the fish, Tilapia mossambica on exposed to Monocrotophos. Saradhamani and Selvarani, (2009) showed significant decrease in lipid content in the tissues of the fresh water fish, Tilapia mossambica on exposed to metribuzin. Decrease in the lipid content was noticed in the fish, Mystus vittatus on exposed to mercuric chloride by Kannan et al. (2010). The present study reveals that the changes in protein, carbohydrate and lipid in pesticides treated fishes will naturally affect the nutritive values of these animals and all the metabolites studied are found to be sensitive biochemical indicators, which reflect changes in the normal activities of various functional systems. _____________________________________ 5. Conclusion
In the present study, it is concluded that MC has a profound influence on the biochemical analysis of an Indian major carp, L. rohita and MC is toxic to aquatic organisms. The results imply a better understanding on the toxicological endpoint of this specific pesticide and provide significant information on safe levels in the aquatic environment. _____________________________________ Conflict of Interest None declared. _____________________________________ Acknowledgement Authors are gratefully thankful to staff members for their valuable suggestions and Head of the Department of Zoology for providing facilities to carryout this research work successfully.
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30
Values in mg/g
Values in mg/g
40 4 3 2 1 0
20 10 0
ORGANS ORGANS CONTROL
24hrs
48hrs
24hrs
72hrs
96hrs
48hrs
Fig.3 – Changes in the carbohydrate content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
4 3
Values in mg/g
Values in mg/g
Fig.1 – Changes in the protein content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
CONTROL
2 1 0
40 30 20 10 0
ORGANS CONTROL 20 DAYS
10 DAYS 30 DAYS
Fig.2 – Changes in the protein content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm;30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure
ORGANS CONTROL
10 DAYS
20 DAYS
30 DAYS
Fig.4 – Changes in the carbohydrate content in a fresh water fish, L.rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure.
Values in mg/g
70 60 50 40 30 20 10 0
ORGANS CONTROL
24hrs
72hrs
96hrs
48hrs
Fig.5 – Changes in the lipid content in a fresh water fish, L. rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of short term exposure.
Values in mg/g
70 60 50 40 30 20 10 0
ORGANS CONTROL 20 DAYS
10 DAYS 30 DAYS
Fig.6 – Changes in the lipid content in a fresh water fish, L. rohita treated with sublethal concentration of MC (4.51 ppm; 30 days). Bars represent means and standard deviation of six individual observations. Significant at p<0.01 (based on t-test) of long term exposure.