Effect of stem-bark extract of some common plants on non-target fresh water fish Channa marulius (Ham.)

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1 Indian J. Fish., 50(4) : , Oct.-Dec., Note Effect of stem-bark extract of some common plants on non-target fresh water fish Channa marulius (Ham.) D. SINGH AND A. SINGH* National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow (U.P.), India * Department of Zoology, DDU Gorakhpur University, Gorakhpur (U.P.), India ABSTRACT Stem-bark extracts of four different plants (Euphorbia royleana, Jatropha gossypifolia, Nerium indicum and Thevetia peruviana) caused mortality and biochemical changes in freshwater non-target fish Channa marulius. It was found that dilute stem-bark powder was effective in killing the fishes. The ranking of toxicity of four plants was N. indicum > E. royleana > J. gossypifolia and T. perucviana. Biochemical changes were observed in gonad tissue, after exposure to 40% and 80% of LC50 (24h) of stem-bark extract of all plants. Withdrawal experiments, showed recovery in total protein, total free amino acid, while partial slow recovery observed in glycogen level after 144 h on termination of experiment. It has been suggested that the alterations at both level (metabolic and enzyme) is due to activation of protein kinase C. These extracts show mortality at higher concentration and recovery after 7 th day of withdrawal. So, the lower doses of purified form of stem-bark can be used as bio-pesticides for the control of aquatic weeds without environmental hazards. The extensive use of synthetic pesticides and synthetic pyrethroids affect the aquatic environment due to some properties such as high toxicity to target and no-target organism and long-term persistence in the environment (Sastry and Shukla, 1993; Uma Devi, 1997). Their hazardous nature has prompted the scientists, to find out new options for the control of weed and aquatic pest. In recent time, the use of plant/ natural products has gained more popularity all over the world. These plant products are focus of attention as a suitable alternative of synthetic pesticides due to some ideal properties such as low cost, easy availability and biodegradability in the nature (Marston and Hostettman, 1985). A large number of pesticides of plant origin are in use in aquatic ecosystem for the control of aquatic weeds, pest and harmful snails (Mohapatra and Nayak, 1998). Several group of compounds (saponins, tannins, alkaloids, alkenyl phenols and phorbal esters) have been found to be toxic to fresh water target and non-target animals (Singh et al., 1996).

2 D. Singh and A. Singh In present communication, an attempt has been made to assess the effect of stem-bark of four plants on non-target freshwater fish Channa marulius. C. marulius (17.5±1.20 cm) were collected from their natural habitat, kept in glass aquaria containing 50L dechlorinated tap water. Prior to experiment, fishes were allowed to acclimatize to laboratory conditions for seven days. Diseased, injured and dead fish (if any) were removed as soon as possible. Water was changed every 24h. Stem-bark extract of E. royleana, J. gossypifolia, N. indicum and T. peruviana were collected from Botanical garden of D.D.U. Gorakhpur University, Gorakhpur. The stem-bark extracts of all plants were dried at 37 C and pulverised in mortar/ pestle. Dried powder was used for the experiment. Toxicity experiments were performed by the method of Singh and Agarwal (1988). Ten fishes were kept in glass aquaria, containing 6L-dechlorinated tap water. Six aquaria were set up for each dose. Control animals were kept in similar condition without any treatment. Mortality was recorded after every 24h up to 96h by prodding with glass rod. values, upper and lower confidence limits and slope value were calculated (Russel et al., 1977). Student t test was applied to find out significant (P<0.05) differences between treated and control animals (Sokal and Rohalf, 1973). Physico-chemical parameters were determined by the method of APHA/AWWA/WPCF, Biochemical changes were measured in fish after exposure to 40 and 80% of (24h) of stem-bark of all plants. After termination of exposure period, gonad tissue dissected and homogenates were prepared for the measurement of total protein, total free amino acid, glycogen and protease. Protein content of gonad tissue was determined by the 526 method of Lowry et al. (1951). Standard curves were prepared with different concentrations of bovine serum albumin. Estimation of total free amino acids in gonad tissue was done by the method of Spies (1957). Standard curves using the same procedure were drawn with known amounts of Glycine. Estimation of glycogen level in gonad tissues was done by the method of Van der Vies (1954) as modified by Mahendru and Agarwal (1982). In withdrawal experiments, the fishes were exposed to 40 and 80% of the (24h) for 96h. One half of the animals were sacrificed for measurement of all biochemical parameters (total protein, total free amino acid, glycogen and protease). The other half set of animals were transferred to freshwater, which was changed every 24h up to 6 days. Following this, all parameters were measured in same tissue. Control animals were kept in stem-bark free dechlorinated tap water without any treatment. Temperature, ph and carbon dioxide ranged between C, and 2-8 respectively while dissolved oxygen, alkalinity and hardness of water were 6.5 ml/l, 9.6 ppm and 140 ppm respectively. C. marulius becomes hyper active soon after exposure to stem-bark extract of all plants. Fish showed some irregular and jerky movements. Fish started experiencing respiratory distress and were observed for gulping air at surface, followed by loss of equilibrium and death. Similar behavioural responses were also observed in organophosphate and carbamate pesticide exposed fishes (Gill et al., 1991). These compounds are cholinergic inhibitors and known nerve poisons (Stansely, 1993). Gill et al. (1991) described that these behavioural anomalies are due to inhibition of cholinergic impulse by the hydrolysis of neurotransmitter acetylcho-

3 Effect of stem-bark extract on fresh water fish 527 line released during synaptic transmission. This possibility cannot be ruled out in case of plant extracts caused behavioural changes. Extremely dilute stem-bark solutions were effective in killing C. marulius and N. indicum and had strongest toxic activity (Table 1). There is a negative correlation between values and exposure periods. Twentyfour hour values of stembark extract of E. royleana, J. gossypifolia, N. indicum and T. peruviana were 0.096, 3.98, 1.048, and 4.56 g/ll whereas 96 h values were 0.042, 3.16, and 4.32 g/l respectively (Table 1). Toxicity experiments show that stem-bark extract of the four plants are lethal to C. marulius. The most obvious sign of distress in the treated fish were restlessness, laboured breathing, loss of equilibrium and lethargy. Change in behaviour of exposed fish was the rapid opercular movement, frequent surfacing, muscular tetany, expansion of pectoral fin. In progressive toxic effects, fish sink to bottom of the aquaria and might lead to death. Singh and Agarwal (1984, 1990) have reported that E. royleana and J. gossypifolia not only cause inhibition of acetylcholinesterase but also reduce the endogenous level of other known neurotransmitter i.e. 5-hydroxytrytamine, epinephrine and dopamine in nervous tissue of freshwater snail Lymnaea acuminata in the time and dose dependent manner. Table 2 indicates a significant (P<0.05) dose dependent decrease in protein level in gonad tissue of C. marulius exposed to stem-bark of E. royleana. Like E. royleana, the stem-bark extracts of J.gossypifolia, N. indicum and T. peruviana showed same trend of result in gonad tissue of C. marulius. Table 2, shows TABLE 1. Toxicity caused by stem bark extract of four plants against non-target freshwater fish C. marulius at different exposure periods. Exposure periods Name of Plant Parameter 24h 48h 72h 96h (g/l) E. royleana LCL UCL Slope value 3.41± ± ± ±0.30 (g/l) J. gossypifolia LCL UCL Slope value 1.60± ± ± ±2.40 (g/l) N. indicum LCL UCL Slope value 2.42± ± ± ±0.51 (g/l) LCL T. peruviana UCL Slope value 3.59± ± ± ±7.62 Mortality was determined every 24h. Batches of 10 fishes were exposed to four different concentration of above treatment Concentrations given are the final concentration (w/v) in the aquarium water. LCL = lower confidence limit, UCL = Upper confidence limit.

4 D. Singh and A. Singh the result of recovery in C. marulius after 144h of withdrawal. There was a complete recovery in protein and amino acid level, while slow recovery observed in glycogen. E. royleana for 24h or 96h caused significant (P<0.05) increase of protease level in gonad tissue (Table 3). Treatment with 40% of 24h, of stembark increased the level to 115% of controls, while 80% of 24h stem-bark increased to 120% of control values. After 96h, 40% and 80% of (24h) of stem-bark extract increased the protease 528 while after 96h, the enhancement observed was 132 to 152% respectively (Table 3). N. indicum and T. peruviana showed same trend like both euphorbiales. (Table 3). Increase in mortality with increase in exposure period could be affected by several factors, which may be acting separately or conjointly. For example, uptake of pesticides is time dependent, which leads to a progressive increase of pesticides in body. Stability (lifespan of pesti- TABLE 2. Changes in total protein, total free amino acid and glycogen level in gonad tissue of C. marulius after exposure to 40% and 80% of (24h) of stem-bark extract of different plants and recovery after 7 th day of withdrawal. 40% of 24h 80% of 24h 7th day after Name of plant Parameter Control withdrawal P (µg/mg) 127.6±1.00(100) 93.8±1.09(73) 73.2±1.31*(57) 125.3±1.46ª(97) E. royleana AA (µg/mg) 17.6±0.78(100) 27.3±0.73(155) 30.5±1.01*(173) 20.0±0.63ª(110) G (mg/g) 1.73±0.01(100) 1.12±0.03(65) 0.88±0.02*(51) 1.23±0.02ª(71) P (µg/mg) 128.3±1.08(100) 98.3±1.54(76) 82.5±1.62*(64) 124.5±1.35ª(97) J. gossypifolia AA (µg/mg) 18.6±0.70(100) 25.5±1.35(137) 28.1±0.77*(151) 21.2±0.95ª(104) G (mg/g) 1.75±0.01(100) 1.26±0.03(72) 1.08±0.01*(62) 1.32±0.01ª(75) P (µg/mg) 130.8±1.53(100) 93.0±1.06(71) 78.6±0.96*(60) 113.6±1.20ª(88) N. indicum AA (µg/mg) 19.0±0.69(100) 29.5±0.68(155) 35.8±1.31*(188) 25.3±0.83(128) G (mg/g) 1.75±0.015(100) 1.11±0.03(63) 0.87±0.01*(50) 1.29±0.07ª(72) p (µg/mg) 128.3±1.28(100) 96.1±1.24(74) 79.5±1.19*(61) 125.6±1.00ª(97) T. peruviana A (µg/mg) 18.5±0.73(100) 26.0±0.40(140) 29.7±1.28*(160) 22.5±1.40ª(125) G (mg/g) 1.68±0.01(100) 1.17±0.01(69) 0.98±0.07*(58) 1.24±0.01ª(75) Value is mean ± SE (Standard error) of six replicates. Value in parenthesis is % change with control taken as 100%. Data were analysed through Student t test. *, Significant (P<0.05), when treated groups were compared with controls. ª, Significant (P<0.05), when student t test was applied between control and withdrawal exp. P = Protein; AA= Amino acid; G= Glycogen level up to 140% and 162% of control, respectively. 40% and 80% of (24h) of stem-bark extract of J. gossypifolia for 24h or 96h also caused significant enhancement in protease level in gonad tissue. Thus 24h exposure to 40% of (24h) altered the protease level to 110% of the control. Similarly, 80% of (24h) of stem-bark extract significantly increased protease level to 117% in 24h, cides) in environment and the rate of their detoxification in animal body also affect the mortality and exposure period relationship (Matsumura, 1985). Kinghorn and Evans (1975) demonstrated that the toxicological action of the euphorbiales may be due to the presence of a group of diterphenephorbal esters, while alkaloids, sterols and triterpenoids present in plants of family Apocynaceae

5 Effect of stem-bark extract on fresh water fish 529 are responsible for toxicity (Wealth of India. Vol. 1995). Stem-bark of all plants altered the metabolic as well enzyme level in gonad tissue of C. marulius. Protein is the major constituent of fishes. The reduction in protein level indicates that total protein may undergo proteolysis, which result in production of free amino acid and used in TCA cycle for energy production (Elista Rani and Balasubramanian, 1999). The decrease in the total protein in gonad tissue, suggest the existence of high protein hydrolytic activitiy due to evaluation of protease as observed in the present study (Table 3). It was earlier reported by Singh and Singh (2001), that lattices of these plants decreased RNA level in gonad tissue during stress condition. Inhibition of RNA at transcription level, affect the protein and free amino acid level (Begum and Vijayraghawan, 1996). Evaluation in free amino acid content in gonad tissue was thus a degradation of protein and impaired incorporamay be due to decreased glycogen synthesis or due to increased glycogen utilization through the glycolytic pathway (Omar et al., 1978). Under bark exposure, an enhanced protease activity was observed in gonad tissue of C. marulius. Sahib (1975), also reported increase in protease activity due to stressor. Increase in protease activity corroborates with enhancement in the free amino acid level The increased protease activity in liver tissue is further supported by a remarkable decrease in total protein and an increase in free amino acid level in goandal tissue (Table 2). There is an enhanced protease activity and consequent elevated free amino acid level in fish tissue exposed to sub lethal concentrations of Cypermethrin (Malla Reddy and Basamohideen, 1995). It is evident that after 144h of transfer into fresh water, the effects were reversible and the level of proteins and amino acids recovered completely, while glycogen level increased but still it was insignificantly different TABLE 3. Level of neutral protease in the gonad tissue of Channa marulius after exposure to 40% and 80% of (24h) of stem-bark extract of all plants for 24h and 96h. Neutral protease activity Name of Expo (µ moles of tyrosine equivalents/ mg protein/h) Plant periods Control 40% of of 80% of of (24h) (24h) 24h ±0.0168(100) ±0.0202(115) ±0.0107ª(120) E. royleana 96h ±0.2017(100) ±0.0165(140) ±0.0136ª(162) 24h ±0.0184(100) ±0.0166(110) ±0.0166ª(117) J. gossypifolia 96h ±0.0136(100) ±0.0049(132) ±0.0068ª(152) 24h ±0.0068(100) ±0.0184(114) ±0.0186ª(118) N. indicum 96h ±0.0166(100) ±0.0137(138) ±0.0168ª(158) 24h ±0.1659(100) ±0.0142(112) ±0.0092ª(118) T. peruviana 96h ±0.0049(100) ±0.0068(135) ±0.0138ª(153) As given in table 2 tion of amino acid to polypeptide chain. During stress condition amino acids are reported to enter Trans animation pathway (Singh et al., 1996) to be utilized for energy. Reduction in glycogen content from control as glycogen is a storage material. Metabolism of this biochemical pool will be lower and therefore, the slower recovery can be explained. Similar effects were also observed at meta-

6 D. Singh and A. Singh bolic level in Lymnaea acuminata and Indoplanorbis exustus (Yadav, 2001). In conclusion, stem-bark extract caused mortality and biochemical changes at higher doses. However, they are safe at low concentrations, hence stem-bark extract of four plants at lower doses can be employed as bio-pesticides or bio-toxins for the control of aquatic pest and snails. These plant extracts are not only cheap but also environmentally safe particularly to non-target animals. References APHA Standard Methods for the Analysis of Water and Wastewater. Begum, V. and S. Vijayraghawan Alteration in protein metabolism of muscle tissue in the fish Clarius batrachus (Linn) by commercial grade dimethoate. Bull. Environ. Cont. Toxicol., 57: Elista Rani, E. and M.P. Balasubramanian Effect of mixture of pesticides and fertilizer. Gill, T.S., J. Pandey and H. Tewari Individual and combined toxicity of common pesticides to teleost Puntius conchonius (Ham). Indian. J. Exp. Biol., 29(2): Kinghorn, A.D. and F.J. Evans A biological screen of selected species of the genus euphorbia for skin irritant effects. Pl. Med., 28: Lowery, O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randell Protein measurement with foline phenol reagent. J. Biol Chem., 193: Mahendru, V.K. and R.A. Agarwal Changes in metabolism in various organs of the snail Lymnaea acuminata, following exposure to trichlorfon. Acta Pharm. et Toxicol., 48: Malla Reddy, P. and Md. Bashamohideen Alteration in protein metabolism in selected tissue of fish Cyprinus carpio during sub-lethal concentration of 530 Cypermethrin. Enviorn. Monit. Assess., 36: Marston A. and K. Hostettman Plant molluscicides. Phytochem., 24: Mastumura, F Toxicology of insecticides. 2 nd Plenum Press. New Yark. p Mohapatra, B.C. and G.B. Nayak Assessment of toxicity of ripe fruit pulp of Hingan, B. royburghii on different fishes. J. Aquacult. Trop., 6: Moore, S. and W.H. Stein Methods in Enzymology, Vol. II, Colowick and Kaplan (Eds.)), Academic press, New York. Omkar, D.K. Singh and R.A. Agarwal Carbamate and organo phosphorous pesticides against snails. Pesticides (August) Russel, R.M., J.L. Robertson and N.E. Savin POLO: a new computer programme for probit analysis. Bull. Entamological Soc. Amer., 23: Sahib, A Neurohumoral transmission and autonomic nervous system. In: Pharmacological Basis of Therapeutics, A.G. Gilman T.W. Rall A.S. Nies and P. Taylor, (Eds.), P Pergamon Press, New York. Sastry, K.V. and V. Shukla Up take and distribution of cadmium in tissue of Channa marulius. J. Enviorn. Biol., 14(2): Singh, A. and R.A. Agarwal Possibility of using latex of euphorbiales for snail control. Sci. of Tot. Enviorn., 77: Singh, A. and R.A. Agarwal Molluscicidal and Anicholinesterase activity of euphorbiales. Biol. Agric. Hortic., 7: Singh, A., D.K. Singh, T.N. Mishra and R.A. Agarwal Molluscicides of plant origin. Biol. Agric & Hortic., 13: Singh, D. and A. Singh Toxicity of latex of Thevetia peruviana and Nerium

7 Effect of stem-bark extract on fresh water fish 531 indicum: effect on metabolism of fish Channa punctatus. J. Med. Arom. Pl. Sci., 22/4A & 23/1A: Singh, D.K. and R.A. Agarwal Alteration of biogenic amine level in snail L. acuminata by the latex of E. royleana. Taxi. Lett., 21: Sokal, R.R. and R.J. Rohalf 1973, Introduction to bio-stastic. (Freeman(Ed.), 368 pp. Spies, J.R Colorimetric procedures for amino acids. In: Methods of Enzymology, S.P. Calowick and N.O. Kaplon. (Eds.). Academic press, New York. 468 pp. Stansely, W Field result using cholinesterase reactivation techniques to degnase acute anicholinesterase poisoning in birds and fish. Arch. Enviorn Cont Toxi; 25: Uma Devi Heavy metal toxicity to gastropod, Morula granulata: tolerance to copper, mercury, zinc and cadmium. J. Enviorn Biol., 18 (3): Van der Vies J Two methods for glycogen determination of in liver. Bioch. J., 57: Wealth of India A dictionary of Indian raw material and industrial products. Publications and Information Directorate, CSIR New Delhi Vol. X: sp-w. Yadav, R.P Molluscicidal effects of some common Euphorbiales on target snails and non-target freshwater fish Channa punctatus. (Ph.D. thesis), D.D.U. Gorakhpur University.

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World Journal of Zoology 10 (1): 47-53, 2015 ISSN 1817-3098 IDOSI Publications, 2015 DOI: 10.5829/idosi.wjz.2015.10.1.91102 Molluscicidal Effect of Medicinal Plant Euphorbia tirucalli on the Harmful Snails