Indian Journal of Experimental Biology Vol. 38, October 2000, pp. 1058-1061

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Pesticide induced alterations of non-protein nitrogenous constituents in the serum of a fresh water cat fish, Clarias batrachus(Linn.)

B.Jyothi* & G.Narayan

Department of Zoology, Osmania University, Hyderabad 500 007, India

Received 8 September 1999; revised 29 May 2000

A freshwater cat fish was exposed to sublethal concentrations of two pesticides - carbaryl, a carbamate and phorate, an organophosphorus pesticide for 24, 72, 120 and 168 hr. The alterations in the serum profile of non protein nitrogen compounds demonstrated an increase in urea, uric acid and creatinine throughout the experimental period.

Due to the biodegradability and short residence time in the environment, organophosphate pesticides are being increasingly used in recent years. Carbamates are comparatively less toxic with lower degree of persistence in soil and water have found wide application as insecticides and nematicides. Because of their low persistence, repeated application of these two groups of pesticides is being practiced for control of pests in agriculture and thereby large quantities of these find their way into the waterbodies. The pesticides selected in the present study are phorate, a soil . and systemic organophosphate insecticide, commercial formulation thimet (o,o-diethyl S-ethyl­thiomethyl phosphorodithiate), and carbaryl a broad spectrum insecticide, commercial formulation being Sevin(l-napthyl methyl carbamate). Of 91,913 tons of pesticides used in India during 1995-96, phorate accounted for 3900 tons and carbaryl, 550.

The toxic effects of these pesticides are due to the inhibition of acetyl cholinesterase (AChE) by the formation of an enzyme inhibitor complex I . Such inhibition of AChE in fish on exposure to pesticides is widely reported2

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. In addition to AChE inhibition, haematobiochemical parameters like blood urea, uric acid and creatinine serve as important indices of general condition and metabolic defects since blood reflects the condition of the life processes . Non­protein nitrogen (NPN) was found increased in fish exposed to various toxicants. Bansal et ai.s observed non significant elevation in Labeo rohita under chlordane toxicity. Verma et ai.6 observed increase in NPN in Saccobranchus fossilis with chlordane.

* Address for correspondence:- G I, Navodaya Apts., Snehapuri, Nacharam. Hyderabad 500 076.India. Fax: 040-7 I 73807

Gupta 7 recorded elevated NPN in Ciarias batrachus and Cirrhinus mrigala with aldrin and Swascofix CD-38. Dalela et al. 8 reported similar observations in Mystus vittatus under the toxic stress of thiotox, dichlorvas, carbofuran and their combination.

Popular freshwater fish Ciarias batrachus(Linn) was selected because it is known for its agility and rich protein content. Adult healthy fish weighing 60-80g and 18-20cm long were selectively collected from the local market. The fish were acclimatized in the laboratory for about 10 days, and were fed ad libitum with boiled eggs, ' fish-meal' and beef liver. Probit analysis of Finne/ was followed for 96 hr to determine LC50s for phorate (0.8 ppm) and carbaryl (46ppm). Commercial formulation of phorate (thimet; 10% w/w granules) were obtained from Cyanamid India Ltd. Bombay and that of carbaryl (sevin; 50% WDP powder) was obtained from Bhopal pesticides Pvt.Ltd. Bhopal. To avoid metabolic differences due to differential feeding , fish were starved one day prior to experimentation. Sublethal concentrations of ph orate and carbaryl was calculated as one-third of LC50. Fish were exposed to sub-lethal concentration of phorate (0.27 ppm) and carbaryl (15 .3 ppm) over a period of 168 hr. Initially fish were sacrificed after 24 hr and thereafter at every 48 hr interval. After 24, 72, 120 and 168 hr blood was collected from the caudal vein and allowed to clot at room temperature. Clot free blood was centrifuged at 3000 rpm for 30 min. Serum was isolated from the blood and used for estimation of the parameters. The water was changed every day and requisite amount of pesticides added to keep their concentrations constant. A concurrent control was maintained under identical conditions.

Stangen kits were used for estimation of all the parameters. Serum urea was estimated by in vitro

NOTES 1059

quantitative determination following D.A.M. method lO•

The intensity of the color produced by urea was measured by Spectrophotometer at 520 nm. Serum uric acid was measured by phosphotungstic acid method. 11

,12 The tungsten blue colour whose absorbance is proportional to uric acid concentration was measured by Spectrophotometer at 710 nm. Creatinine was measured using alkaline picrate methodl3

,14 at 520 nm on Spectrophotometer. The serum concentrations of urea, uric acid and creatinine increased on exposure to both the pesticides at all sampling periods.( Table 1).

The nitrogen concentration of a protein free filtrate or blood or serum is related to renal function. In the present investigation the toxic effects of two pesticides on urea, uric acid and creatinine were studied as these biochemical factors are useful in understanding the metabolic disturbance leading to specific functional defects.

Urea- Urea is quantitatively the most important non protein nitrogenous constituent of blood. It is the chief end product of protein metabolism and excreted by kidneys. Hence, its blood concentration is directly

related to the protein content of the diet and the renal excretory capacity. The liver is the only important site of urea formation in most of the vertebrates. In any pathological condition that involves the liver, urea formation is upset evidenced by either increase or decrease. In teleost fish, the main end product of nitrogen metabolism is ammonia which accounts for 55 to 80 % of the total nitrogen excreted, while urea excretion is about 6 to 8 % 15. The amino acids which are precursors of purines also contribute to urea outputJ6

.

Common cause for increase in blood urea is inadequate excretion usually due to kidney damage and urinary obstruction l7

. Renal dysfunction with severe renal insufficiency or excessive bodily breakdown of proteins due to toxic stress may be responsible for the elevation of urea level. Thu~ urea estimation is mainly useful for the diagnosis of renal disease or damage. Jyothi and Narayan J8 observed reduction in the protein content with phorate which can be correlated to elevated urea levels in the present study . Jayantha Rao et al. J9 who recorded same observation, suggested protein degradation or

Table 1-Urea, creatinine and uric acid levels in the serum of Clarias batrachus exposed to 15.3 ppm carbaryl, and 270 ppb phorate

[Values are means ± SE of six individual observations]

EX20sed 2eriod (hr) Parameters Group 24 72 120 168

On exposure to 15.3 22m of carbarrl

Control 20.58±1.17 20.89±0.80 19.71+0.93 20.08+1.04 Urea Exposed 22.08± 1.19+ 25.38±0.97* 27.52±O.97·· 30. 17±1 .32" (mgJdl) Variation (%) +7.3 +21.49 +39.62 +50.25

Control 2.24±0.22 2.02±0.18 2.15±0.16 1.93±0.18 Creatinine Exposed 3.39±0.29* 3.82±0.29** 4.37±0.25*** 4.90±0.25*** (mgJdl) Variation(%) +51.34 +89 .11 +103 +154

Control 1.88+0.13 1.78+0.06 2.01+0.07 1.94+0.11 Uric Acid Exposed

- .. 2.47±0.09 2.75±O.07·" 3.35±O.09'" 3.84±O.07"·

(mgJdl) Variation(%) +31.38 +54.5 +76.62 +97.94

On exposure to 270 ppb of phorate

Control 20.58±1.17 20.89+0.80 19.71+0.93 20.08+1 .04 Urea Exposed 21.48± 1. 006+ 24.75:t0.98· 28.28±1.26" 30.69±O.91··· (mg/dl) Variation(% ) +4.37 +18.47 +43.48 +52.84

Control 2.24+0.22 2.02+0.18 2.15+0.16 1.93+0.18 Creatinine Exposed

- . 4.95±O.29·" 5.16±O.23"· 4.84±O.30·" 3.43±0.28

(mgJdl) Variation (%) +53.13 +145 +140 +151

Control 1.88+0.13 1.78+0.06 2.01+0.07 1.94+0.11 Uric Acid Exposed 2.99±O.07"· 3.13±O.07"· 4.23±O.08"· 4 .91±O.1l··· (mgJdl) Variation(%) +59.04 +75 .84 +110.4 +153.1

P Values: * < 0.05, ** < 0.01, *** < 0.001 , + - Not significant

1060 INDIAN J EXP BlOL, OCTOBER 2000

biochemical transformation of protein nitrogen into the other nitrogenous products. Elevated levels of urea implies 'uraemia' which is possibly due to the inability of the toxicated kidney to filter urea in adequate amounts. Goel et apo. observed increase in blood urea in fish Clarias batrachus due to alachrol toxicity further suggested that the elevation is due possibly to the anomalies in kidney functioning. Sharma21 studied effect of lithium in Clarias batrachus and suggested that the elevated blood urea levels imply uraemia which appears a sequel of malfunctioning of kidney. Sivaramakrishna and Radhakrishnayya22 studied the effect of mercury in fish Cyprinus carpio and observed in increase in blood and liver urea level. They ascribed it to part of excess ammonia converting into the less toxic urea in the liver during active operation of urea -ornithine cycle.

Creatinine - It is an anhydride of creatine which is present in muscles and is a spontaneous decompo­sition product of creatine and phosphocreatine. Free creatinine is not reutilized in the body's metabolism and thus functions solely as a waste product of creatine and excreted by kidneys. Since its daily production is directly proportional to the creatine content of the body, the concentration of blood creatinine is more or less constant than that of any of the common excretory substances. The constancy of creatinine formation and excretion makes creatinine a useful index of renal function, primarily of glomerular filtration23

. By virtue of its relative independence from such factors as diet(protein intake), degree of hydration and protein metabolism the serum creatinine is significantly more reliable screening test or index of renal function.

In the present investigation, creatinine showed more or less significant elevation trend with both the pesticides. An increase in creatinine may occur in any of the conditions in which blood urea is increased . Elevated level of creatinine also reflects the malfunction of kidney under stress, being functionally insignificant because of cellular damage after pesticide exposure24. In addition, elevated levels of serum creatinine and 'creatinuria' may be observed in extensive muscle destruction. Similar observations were made by Jayantha Rao etal l9 in fish Tilapia mossambica under phosphamidon stress and Gael and Kalpana Gupta25 in Heteropneustes fossilis due to zinc toxicity.

Uric acid - Uric acid is the major product of purine metabolism. In fish, uric acid serves as main

f 15 2627 h' h . . d d f source 0 urea . , w IC m tum IS pro uce rom purine nucleotides. Uric acid can also be formed as the direct end product of the proteins . In the stress and pathological conditions, generally the rate of catabolism of energy yielding molecules is higher. The elevation in the level of uric acid suggests the increased rate of purine and pyramidine catabolism. Rise in Urea, uric acid and ammonia levels indicates their formation in a rapid rate and thus in tum indicates the enhanced breakdown of proteins. Generally in pathological conditions the metabolic rate increases and increase m the level of excretory products takes place.

The increase in urea, uric acid and creatmme suggests a progressive protein degradation or biochemical transformation of the protein nitrogen into other nitrogenous products and renal malfunction as a manifestation of toxic response.

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NOTES 1061

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