effect of sublethal concentrations of cypermethrin on ...acid in 10 mg in sorenson phosphate buffer...

Sreenivasan R S, et al., 2017/ Effect of Sublethal Concentrations of Cypermethrin on Acetyl

RESEARCH ARTICLE

International Research Journal of Pharmaceutical and Biosciences

Pri -ISSN: 2394 - 5826

http://www.irjpbs.com e-ISSN: 2394 - 5834

Effect of Sublethal Concentrations of Cypermethrin on Acetyl Cholinesterase Activity in the Brain of the Fresh Water Female Field Crab, Spiralothelphusa hydrodroma(Herbst) Sreenivasan.R.S.1*, Krishna Moorthy.P.2 and Deecaraman.M.3 1*Department of Biochemistry, Kaveripakkam College of Arts and Science, Kaveripakkam-632508, 2Department of Bioinformatics, Bharath University, Chennai-600073, 3Department of Industrial Biotechnology, Dr.MGR University, Chennai-600095, INDIA.

Article info Abstract

Article history: Received 24 DEC 2016 Accepted 27 FEB 2017

*Corresponding author: rssreenivasan2012@ gmail.com

Copyright©2017 irjpbs

The fresh water field crab, Spiralothelphusa hydrodroma is an important food source in parts of South India. The impact of cypermethrin on the variations in the acetyl cholinesterase enzyme activity of brain of the experimental female crab was carried out. The toxicity of the cypermethrin on the crab was estimated using LC₅₀. A quantitative enzymatic study of acetyl cholinesterase in the brain was undertaken. Keywords: Brain, cypermethrin, LC₅₀, Spiralothelphusa hydrodroma.

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International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 4 (1) 56


INTRODUCTION

Pesticides are toxic substances, non-biodegradable and accumulate in the food chain. Mostly they affect the nervous system causing tumors in living organisms. They are not only neurotoxic but also affect other systems and have shown a high degree of impact on metabolism by inhibiting enzymes like acetyl cholinesterase(1,2). In biological systems, the biocatalysts play a vital role in the metabolic pathways. Animal exposed to stress conditions alter their physiological status with the help of enzymes. Toxicants like pesticides are known to secrete hyper or hypo level of enzymes. The trace metal concentration in Queensland Estuarine crabs, Australoplax tridentate and Scylla serrata have been observed(3). Various chemicals entering the aquatic ecosystem through human activities, either accidentally or by design may cause adverse effects on the aquatic biota, including deleterious changes which disrupt metabolic activity at the biochemical levels(4). The pesticide derivatives are known to alter the physico-chemical characteristics of water; these in turn interfere and interact with various physiological activities of organisms. Changes in metabolic rate among organisms exposed to pollution stress have been used as indicator of stress condition. When any aquatic animal is exposed to polluted medium, a sudden stress is developed for which the animals should meet more energy demand to overcome the toxic stress. (5)reported on the toxic effects of sublethal concentration of copper sulphate, on certain biologically important enzymes in Saccobranchus fossilis. The present work was that the effect of cypermethrin on the brain of Spiralothelphusa hydrodroma.

MATERIALS AND METHODS

Spiralothelphusa hydrodroma were collected from fresh water ponds and paddy fields on the outskirts of Walajapet, Vellore District, Tamil Nadu, India. The crabs were brought to the laboratory in large plastic trough and maintained in normal daylight illumination in the laboratory thereby providing normal acclimatization. The crabs were fed with uncooked oats. For all experiments, the crabs were used with carapace length ranging from 3 to 4.5 cm and breadth ranging from 5 to 6.5 cm. The water level was maintained carefully so that the crabs were partially immersed. Acute toxicity study was carried out to determine the potency of cypermethrin for static but renewal type of bioassay was adopted in the present investigation to estimate the LC₅₀ values. The cypermethrin was used as commercial preparation. The experiment was carried out to find the range of concentrations for confirmatory evaluation. The mortality was recorded for the crab at 24, 48, 72 and 96 hr exposure to cypermethrin; the LC₅₀ values were estimated as 2.027, 1.698, 1.452 and 1.315 ppm respectively were corrected for natural response by Abbott’s formula(6).

The acetyl cholinesterase activity in the brain was estimated by the modified acetylthiocholine / DTNB procedure suggested by(7). The colorimetric determination of thiocholine with 5-5-dithiobis-2- nitrobenzoic acid (DTNB), after enzymatic hydrolysis of choline esters, acetyl thiocholine (enzyme), thiocholine acetate, thiocholine and dithionitro benzoate resulted in yellow colour. 500 mg of brain tissue was weighed accurately and transferred to a tissue homogenizer, to which 10 volumes of 0.9% sodium chloride solution was added and homogenized in an ice bath. The homogenate was centrifuged at 3000 rpm for 10 minutes. The supernatant of 0.5 ml was pipetted into a 25 ml volumetric flask and the volume was made up with freshly prepared reagent (dithiobis nitrobenzoic acid in 10 mg in Sorenson phosphate buffer of 100 ml). Aliquot of 4 ml was pipetted into two test tubes. Into the first tube, 2 drops of the inhibitor solution was added, then 1 ml of substrate solution



was added in both the tubes. Then they were incubated for 10 minutes at 30oC. The solution in the first tube containing inhibitor was used as a blank, to set zero in the photocolorimeter using 420 nm. The absorbance of the second tube indicated the direct measurement of acetyl cholinesterase activity. The values thus obtained were expressed as moles substrate hydrolysed / min / mg tissue. Table 1: The LC50 values and regression equations for Spiralothelphusa hydrodroma treated with cypermethrin

Exposure periods (hours)

LC₅₀ (ppm)

Upper confidence limits (ppm)

Lower confidence limits (ppm)

Regression results Slope

function r2

24 2.027 2.561 1.739 Y = - 0.932 X + 0.468 2.973 0.99

48 1.698 1.938 1.345 Y = - 0.658 X + 0.281 3.265 0.98

72 1.452 1.883 1.136 Y = - 0.724 X + 0.391 4.121 0.99

96 1.315 1.763 1.118 Y = - 0.611 X + 0.324 4.973 0.99

Chronic study on the effect of cypermethrin on the crab was conducted by exposing to two sublethal, safe concentrations for 20 and 40 days. According to(8), 1/10th and 1/3rd of the 96 hr LC₅₀ value represent lower and higher sublethal concentrations respectively. Hence lower (0.1315 ppm) and higher (0.4383 ppm) sublethal concentrations of the insecticide were arbitrarily used. At the end of the treatment period, the control and treated crabs were dissected and the brain was collected for enzymatic assay. Acetyl cholinesterase was estimated following the techniques adopted(7). One way analysis of variance (ANOVA) was performed based on the methods of(9). RESULTS In the present study, quantitative changes in acetyl cholinesterase were observed in brain. Each experiment was repeated for three times with different individuals and the mean value was taken. The acetyl cholinesterase (AChE) activity was evaluated in the brain of S.hydrodroma, treated with lower and higher sublethal concentrations of cypermethrin. In the control crabs, the AChE activity in the brain was found to be 111.23 and 111.50 moles substrate hydrolysed / min / mg tissue respectively for 20 and 40 days. In the lower sublethal concentration of cypermethrin the AChE activity was 78.38 and 66.72 moles substrate hydrolysed / min / mg tissue, whereas in higher sublethal concentration the AChE activity was 71.26 and 54.33 moles substrate hydrolysed / min / mg tissue for 20 and 40 days of exposure period respectively. The decrease in the AChE activity was statistically significant P<0.01 in both the exposure period for treated crabs.



Table 2: Effect of lower and higher sublethal concentrations of cypermethrin on acetyl cholinesterase activity

Brain Duration of Exposure

F-Value P-Value 20 days 40 days

Control 111.23 111.50 0.0113 NS

Lower Sublethal Concentration 78.38 66.72 48.26 ** <0.01

% Change Over Control 28.53 40.16

Higher Sublethal Concentration 71.26 54.33 18.36 ** <0.01

% Change Over Control 35.93 51.27

Mean ± SD of six individual observations Values are expressed in moles substrate hydrolysed / min / mg tissue NS – not significant * * indicates significance at 0.01 level


0

2 0

4 0

6 0

8 0

10 0

12 0

mol e s

subst r a

t e

hy dr ol y

se d /

mi n /

mg of

t i ssue

Brain

Cont rol 111.23 111.5

Low 78.38 66.72

High 71.26 54.33

20 Days 40 Days


DISCUSSION Water is one of the basic requirements of all aquatic as well as terrestrial lives for growth and survival. Aquatic systems are contaminated by disposal of various abiotic factors. Pesticides by virtue of their design and application induce a broad spectrum biocidal effect influencing most of the organisms(10). These pollutants also destroy the quality of aquatic ecosystems and render it unfit for various aquatic organisms, particularly fresh water crabs. In the present study, the strategy of energy production adopted by Spiralothelphusa hydrodroma were assayed by tracking changes in the acetyl cholinesterase activity in its brain under stress at lower and higher sublethal concentrations of cypermethrin revealed highly fascinating informations. (11)reported that the maximum inhibition of acetyl cholinesterase activity in T.mossambica in response to the cypermethrin toxicity. In the present study, the activity of acetyl cholinesterase was found to decrease in the experimental crabs when compared with that of the control crabs. Acetyl cholinesterase activity also significantly decreased in the brain tissue of treated crabs when compared to the control crabs. The results of the present study are well in accordance with that of previous investigations in the increased activity of acetyl cholinesterase in cypermethrin treated crabs. REFERENCES

1. Matsumura, F., 1975. Toxicology of insecticides. Plenom Press, New York.

2. O’ Brien, R. D., 1967. Insecticides: Action and metabolism. Academic Press, New York.

3. Mortimer, M.R., 2000. Pesticide and trace metal concentrations in Queensland Estuarine crabs. Marine Pollut. Bull., 41(7-12): 359-366.

4. Hirth, D.F., 1964. Enzyme damage due to heavy metal intoxication. Munch, med. Wschr, 106: 985-988.

5. Verma, S.R., I.P.Tonk, A.K.Gupta and R.C.Dakia, 1981. Invivo enzymatic alterations in certain tissues of Saccobranchus fossilis following an exposure to four toxic substances. Environ. Pollut., 26: 121-127.

6. Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomo., 18: 265-267.

7. Voss, G. and K. Sachsse, 1970. Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetyl thiocholine / DTNB procedure. Toxicol. Appl. Pharmacol., 16: 764-772.

8. Sprague, J.B., 1971. Measurement of pollutant toxicology of fish III: Sublethal effects and safe concentrations. Wat. Res., 5: 245-266.

9. Winer, B.J., 1971. Statistical principles in experimental design, 2nd edition. McGraw-Hill, New York.

10. Ware, G.W., 1980. Effect of pesticides on non-target organisms. Residue Rev., 173-201.

11. Reddy, A.T., K. Ayyanna and K. Yellamma, 1991. Sensitivity of brain cholinesterase to cypermethrin toxicity in fresh water teleost, Tilapia mossambica. Biochem. Int., 23(5): 959-962.


effect of sublethal concentrations of cypermethrin on ...acid in 10 mg in sorenson phosphate buffer...

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