Molluscicidal activity of neem ( Azadirachta indica A.Juss

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journal of ETHNO PHARMACOLOGY

ELSEVI ER

Journal of Ethnopharmacology52 (1996) 35-40

Molluscicidal activity of neem (Azadirachta indica A.Juss) K e s h a v Singh, Ajay Singh, D . K . Singh* Department of Zoology, Universityof Gorakhpur, Gorakhpur-273009, U.P., India

Received 5 September 1995; revised 8 January 1996; accepted 8 January 1996

Abstract

Molluscicidal property of Azadirachta indica A. Juss (neem) against the snails Lymnaea acuminata and Indoplanorbis exustus was studied. It was observed that the molluscicidal activity of the leaf, bark, cake, neem oil and the neem-based pesticides, achook and nimbecidine, was both time- and dose-dependent. The toxic effect of pure azadirachtin against both the snails was greater than the synthetic molluscicides. Keywords: Azadirachta indica; Fascioliasis; Toxicity; Snails

1. Introduction

Fascioliasis is very common in eastern Uttar Pradesh. 94% of buffaloes slaughtered in the local slaughter houses are infected with Fasciola gigantica (Singh and Agarwal, 1981), which reduces the milk yielding years of cattle and causes the premature death of its victims (Agarwal and Singh, 1988). This fluke is transmitted by the snails Lymnaea acuminata and lndoplanorbis exustus, which are found in ponds and low-lying submerged fields. Grazing in water-logged areas and the use of water plants as fodder is the primary cause of fluke infection. Synthetic organic molluscicides, such as organophosphorus, carbamate and pyrethroids, are widely used for the control of harmful snails (Agarwal and Singh, 1988) inspite * Corresponding author.

of the fact that these molluscicides are now causing serious environmental hazards. Molluscicides of plant origin have gained greater importance since it is believed that natural products are ecologically sound and culturally more acceptable than synthetic ones. A large number of plant families which possess natural molluscicidal activity (Hostattmann and Lea, 1987; Alard et al., 1991; Singh and Singh, 1993, 1994) have been identified. Neem (Azadirachta indica A. Juss; Meliaceae) is a tree that has been long used in agriculture and medicines. Indian Ayurvedic literature is full of neem uses. Neem extract shows anti-inflammatory, anti-pyretic (Okpanyi and Ezeukwu, 1981; van der Nat et al., 1991), vaginal contraceptive (Sinha et al., 1984), insect growth inhibitor, and insecticidal actions (Banerjee and Rembold, 1992; Rembold and Annadurai, 1993). The aim of the present study was to conduct

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K. Singh et al. / Journal of Ethnopharmacology 52 (1996) 35-40

laboratory evaluation of molluscicidal activity of neem products and neem-based pesticides against the snails Lymnaea acuminata and Indoplanorbis exustus.

Table 1 Concentrations used for toxicity determination of different parts of neem and their pesticides against L. acuminata and L exustus

Neem leaf

2. Materials and methods Adult Lymnaea acuminata (2.25 + 0.2 cm in length) and Indoplanorbis exustus (0.85 ± 0.037 cm in length) were collected locally and used as test animals. Ten experimental animals were kept in glass aquaria containing 3 1 of dechlorinated tap water. Snails were exposed to different concentrations of neem leaf, cake, bark, oil and some neembased pesticides. Required amounts of neem leaf, bark and cake were crushed in water and water extract was used for treatment. The final concentration in aquarium water was taken as w/v concentration of leaf, bark and cake in extracted water. Neem oil (100 mg) was dissolved in 1.0 ml of the non-ionic emulsifier, Snehakshar, and this mixture (w/v) was used in treatment. Neem-based pesticides, nimbecidine, achook and pure azadirachtin, were introduced (w/v) into aquarium water directly. The concentrations (w/v) used for calculating lethal concentration (LC) values are shown in Table 1. Neem leaf, bark and cake were collected locally and identified by the herbarium of the Botany Department, University of Gorakhpur, Gorakhpur, and a specimen voucher number 2051 was deposited. Neem oil, achook (azadirachtin 300 ppm; azadiradione 500 ppm; nimbocinol and epinimbocinol 2000 ppm), nimbecidine (0.03% aza dirachtin, neem oil 90.57%; hydroxyei 5.0%; epichlorohydrate 0.50%; aromax 3.0%) and azadirachtin were provided by Indian Herbs Co. Pvt. Ltd., Shaharanpur, Godrej Soaps Ltd., Bombay and T. Stanes and Co. Ltd., Coimbatore, respectively. Six aquaria were set up for each test group. Control animals were held in similar conditions without treatment. Mortality was recorded every 24 h up to 96 h. Snail mortality was established by the contraction of the body within the shell; no response to a needle probe was taken as evidence of death. LC values, upper and lower confidence limits (UCL and LCL) and slope values were

L. acurninata w/v, g/l 0.75, 1.0, 1.5, 2.0 L exustus w/v, g/I 2.0, 3.0, 5.0, 7.0

Neem cake L.acuminata w/v, g/l 3.0 5.0, 7.0, 9.0 Lexustus w/v, g/l 3.0, 5.0, 7.0, 9.0

Neem bark L. acuminata w/v, g/l 1.5, 3.0, 3.5, 5.0 I. exustus w/v, g/l 0.9, 1.2, 1.5, 2.0

Neem oil L. acuminata w/v, mg/I 6.0, 8.0, 14.0, 17.0 L exustus w/v, mg/1 4.0, 6.0, 8.0, 14.0

Achook L. acuminata w/v, mg/I 70, 120, 150, 180 L exustus w/v, mg/l 50, 70, 90, 120

Nimbecidine L. acuminata w/v, mg/I 27, 90, 144, 270 L exustus w/v, mg/I 27, 45, 63, 99

Azadirachtin L. acuminata w/v, mg/l 0.075, 0.021, 0.35, 0.49 1. exustus w/v, mg/1 0.35, 0.49, 0.60, 0.70

calculated according to the method of the POLO computer program of Russell et al. (1977). The regression coefficient (r) was determined between exposure time and different values of LCs0 (Sokal and Rohlf, 1973). 3. Results Laboratory toxicity evaluation of leaf, bark, cake, and oil of neem and the neem-based pesticides, nimbecidine and achook, indicated that the molluscicidal activity against L. acuminata a n d / . exustus was both time- and dose-dependent. There was a significant regression coefficient between exposure time and LCs0 of neem products (Tables 2 and 3). The toxicity of neem oil against both the snails was higher with respect to water extracts of neem leaf, bark, cake, and neem-based pesticides (Tables 2 and 3). Toxicity of neem oil at all exposure periods was more pronounced against L exustus than L. acuminata. The toxicity of water extracts of leaf and bark was higher against L. acuminata

K. Singh et al. / Journal of Ethnopharmacology 52 (1996) 35-40

(Tables 2 and 3). However, the toxicity of neem cake is higher against I. exustus. Neem-based pesticides, achook and nimbecidine, were more toxic against I. exustus. The toxicity of azadirachtin against L. acuminata and I. exustus was higher with respect to all the neem products and neem-

37

based pesticides, but it did not show a timedependent effect (Tables 2 and 3). The order of 24 h toxicity against L. acuminata was azadirachtin > neem oil > achook > nimbecidine > bark > leaf > cake, whereas the order of 24 h toxicity against I. exustus was

Table 2 Toxicity (LCs0) of different parts of neem and pesticides of neem products against snail Lymnaea acuminata Parts of neem and pesticide

24 h Achook Nimbecidine Cake a LeaP' Bark a Oil Azadiraehtin 48 h Achook Nimbecidine Cake a LeaP" Bark a Oil Azadirachtin 72 h Achook Nimbecidine Cake a Leafa Bark a Oil Azadirachtin 96 h Achook Nimbecidine Cake a Leafa Barka Oil Azadirachtin

Effective dose (w/v) (rag/l)

Limits

Slope value

t ratio

g value

Heterogeneity

LCL (mgO)

UCL (mg/I)

158.56 166.58 8.52 2.56 1.86 17.35 0.35

141.27 131.88 7.31 2.09 1.65 14.58 0.28

187.84 223.37 4.02 3.56 2.28 23.58 0.47

3.48 1.47 2.92 2.40 3.85 2.75 1.82

4. 0.62 4- 0.23 4. 0.54 4- 0.40 4. 0.72 4- 0.51 4. 0.31

5.55 6.16 5.38 5.91 5.32 5.32 5.85

0.12 0.10 0.13 0.11 0.13 0.13 0.11

0.30 0.41 0.24 0.27 0.23 0.23 0.26

107.39 69.63 4.26 1.47 1.53 13.41 0.35

94.32 49.99 3.79 1.27 1.36 11.70 0.28

119.22 92.79 4.69 1.74 1.79 16.18 0.47

3.74 1.21 4.60 2.62 3.33 2.90 1.82

4. 0.57 4- 0.21 4- 0.66 4- 0.39 4- 0.67 4. 0.48 4. 0.31

6.46 5.59 8.19 6.59 4.92 5.92 5.85

0.09 0.12 0.05 0.08 0.15 0.10 0.11

0.33 0.61 0.36 0.28 0.13 0.19 0.26

78.66 36.68 1.82 0.91 1.18 8.99 0.35

64.48 20.08 1.43 0.66 1.02 7.00 0.28

89.39 52.13 2.20 1.11 1.31 10.84 0.47

3.92 1.31 3.38 2.01 3.59 2.02 1.82

4. 0.60 -4- 0.22 4. 0.30 4. 0.40 4- 0.68 4- 0.46 4. 0.31

6.45 5.86 7.88 5.01 5.25 4.39 5.85

0.09 0.11 0.06 0.15 0.13 0.19 0.11

0.41 0.25 0.75 0.30 0.18 0.20 0.26

65.37 20.97 t.47 0.59 0.99 5.76 0.35

48.58 9.48 1.28 0.29 0.86 4.26 0.28

77.20 32.38 1.67 0.80 1.08 6.85 0.47

3.70 1.44 3.53 1.73 5.06 3.01 1.82

-,,- 0.64 ± 0.24 + 0.37 ± 0.40 + 0.80 -,,- 0.53 -4- 0.31

5.73 5.82 9.46 4.31 6.30 5.93 5.85

0.11 0.11 0.04 0.20 0.09 0.12 0.11

0.57 0.52 0.75 0.63 0.33 0.43 0.26

ag~. Batches of 10 snails were exposed to four different concentrations of the above treatment. Mortality was determined every 24 h. Each set of experiment was replicated six times. Concentrations given are the final concentrations (mg/l or ag/l) in the aquarium water. Significant negative regression (P < 0.05) was observed between exposure time and LC50 of pesticides, except in the case of pure azadirachtin. t s, testing significance of the regression coefficient - - achook, 5.12 +; nimbecidine, 3.54÷÷; cake, 3.81 ÷÷; leaf, 5.24÷; neem oil, 21.34 +. +, linear regression coefficient. ++, nonlinear regression coefficient.

K. Singh et al. /Journal of Ethnopharmacology 52 (1996) 35-40

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azadirachtin > neem oil > nimbecidine > achook > cake > leaf > bark. The slope values given in the Tables 2 and 3 were steep and the separate estimations of LC based on each of the six replicates were found to be within the 95% confidence limits of LCs0. The 't' ratio is greater than 1.96 and the heterogeneity

factor less than 1. The g-value is less than 0.5 at all probability levels. 4. Discussion and conclusion It is evident from the above results that the studied parts of neem have time- and dose-

Table 3 Toxicity (LCs0) of different parts of neem and pesticides of neem products against snail lndoplanorbis exustus Parts of neem and pesticide

24 h Achook Nimbecidine Cake a Leaf a Bark a Oil Azadirachtin 48 h Achook Nimbecidine Cake a Leafa Bark a Oil Azadirachtin 72 h Achook Nimbecidine Cake a Leafs Bark a Oil Azadirachtin 96 h Achook Nimbecidine Cake a Leafs Bark a Oil Azadirachtin

Effective dose (w/v) (mg/l)

Limits

Slope value

t ratio

g-value

Heterogeneity

LCL (mg/l)

UCL (mg/l)

97.40 88.24 4.04 4.16 4.83 8.80 0.53

87.91 70.61 3.60 3.71 3.73 7.49 0.49

112.43 136.03 4.56 4.69 7.58 10.91 0.57

3.92 1.95 3.55 3.57 1.90 2.37 0.30

± 0.65 ± 0.43 ± 0.45 ± 0.45 q- 0.34 ± 0.44 ± 0.81

5.96 4.54 7.78 7.83 5.49 5.40 6.55

0.10 0.18 0.06 0.06 0.12 0.13 0.08

0.22 0.22 0.37 0.19 0.26 0.17 0.15

78.35 49.70 . 3.08 2.69 4.48 0.53

69.70 42.30 . 2.75 2.20 3.77 0.49

88.09 57.82 . 3.40 3.46 5.04 0.57

3.47 2.61 . 4.24 1.89 4.06 0.30

± 0.62 ± 0.43

5.57 6.06

0.12 0.10

0.32 0.35

0.49 0.32 0.63 0.81

8.50 5.84 6.14 6.55

0.05 0.11 0.09 0.08

0.35 0.16 0.29 0.15

43.76 24.59 . 1.83 1.44 1.67 0.49

63.62 38.08 . 2.33 2.19 2.69 0.57

3.26 2.66 . 3.76 1.99 2.09 0.30

± 0.64 ± 0.46

5.06 5.77

0.14 0. I 1

0.34 0.49

0.42 0.32 0.26 0.81

8.86 6.06 7.77 6.55

0.04 0.10 0.06 0.08

0.49 0.18 0.82 0.15

40.56 12.46 . 1.53 1.06 0.99 0.49

53.75 29.16 . 1.98 1.57 1.56 0.57

0.59 2.25 . 3.57 2.54 2.00 0.30

± 0.92 ± 0.48

6.07 4.64

0.10 0.17

0.26 0.50

0.05 0.08 0.06 0.08

0.51 0.41 0.64 0.15

55.57 32.17 . 2.07 1.81 2.19 0.53 48.34 22.29 . 1.76 1.33 1.26 0.53

.

.

.

. ± ± ± ±

.

. ± ± ± ±

.

. ± ± ± ±

0.42 0.37 0.25 0.81

. 8.49 6.80 7.78 6.55

ag~. Batches of 10 snails were exposed to four different concentrations of the above treatment. Mortality was determined every 24 h. Each set of experiment was replicated six times. Concentrations given are the final concentrations (mg/l or a, g/l) in the aquarium water. Significant negative regression (P < 0.05) was observed between exposure time and LC50 of pesticides, except in the case of pure azadirachtin. ts, testing significance of the regression coefficient - - Achook, 7.34+; nimbecidine, 4.65+; leaf, 6.33++; ncem oil, 4.63 +. +, Linear regression coefficient. ++, Nonlinear regression coefficient.

K. Singh et al. /Journal of Ethnopharmacology 52 (1996) 35-40

dependent molluscicidal property. Toxicity of the water extract of neem cake against I. exustus is higher than against L. acuminata, whereas toxicity of water extract of leaf and bark is higher against L. acuminata. Data on molluscicidal properties of neem is lacking. In a preliminary study, Muley (1978) reported that 0.5% water extract of dried fruit of A. indica is toxic against snail Melania scarbra. Ayoub and Yankov (1985, 1986) observed that water extract (100 ppm) of neem bark is active against Biomphalaria pefifferi and Bulinus truncatus. Molluscicidal activity of the standard molluscicide, niclosamide (24 h LCs0, 11.8 mg/l) is only 1.4 times higher than neem oil (24 h LCs0, 17.35 mg/1) (Singh and Agarwal, 1984). Among the neem-based pesticides, nimbecidine is more toxic against both the snails except at 24 h exposure against L. acuminata. The high toxicity of nimbecidine with respect to achook may be due to the presence of more limonoids in neem oil, as 90.57% of neem oil was used in the preparation of nimbecidine, whereas in achook only four limonoids, i.e., azadirachtin, azadiradione, epinimbocinol and nimbocinol, were used. It has been reported that neem oil contains meliantriol, azadirone, azadiradione, epiazadiradione, nimbinin, vepinin, nimbidinin, salannolide, azadirachtin, nimbin and other limonoids (van der Nat et al., 1991).Its 96 h toxicity against L. acuminata is 1.39, 2.44 and 4.76 times lower than the synthetic pesticides, phorate, formothion and carbaryl, respectively (Singh and Agarwal, 1983). A toxicity study of azadirachtin, an active component of neem oil and other neem products, indicates that the active component is more toxic to these snails, as the 24 h LCs0 (0.35 mg/l) of azadirachtin against L. acuminata is about 33 times higher than standard molluscicide niclosamide (24 h LC50, 11.8 mg/l) (Singh and Agarwal, 1984). However, the toxic effect of azadirachtin is short-lived. Its maximum effect is observed only within 24 h, due either to the fact that pure azadirachtin is not stable in water after 24 h or it is metabolised by the snail within 24 h. The time-dependent effect of neem products and neem-based pesticides which contains azadirachtin indicates that azadirachtin used along with other chemical components of the neem is stable even up to 96 h.

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It is clear from the steep sl6pe values that a small increase in the dose of the different parts of the neem plant and neem-based pesticides causes large mortality in snails. A 't' ratio value greater than 1.96 indicates that the regression is significant. Heterogeneity factor values less than 1.0 denote that in the replicate tests of random samples, the concentration response lines would fall within 95% confidence limits and thus the model fits the data adequately. The index of significance of potency estimation, g, indicates that the value of the mean is within the limits at all probability levels (90%, 95% and 99%). In conclusion, it is our belief that the use of neem products and neem-based pesticides against harmful snails would be less expensive and less hazardous to the environment than synthetic molluscicides.

Acknowledgements Authors are thankful to Indian Herbs, Research and Supply Co. Pvt. Ltd., India, Godrej Soaps Ltd., India, T. Staines and Co. Ltd., India for supplying samples of neem oil, achook and nimbecidine-pure azadirachtin, respectively.

References Agarwal, R.A. and Singh, D.K. (1988) Harmful gastropods and their control. Acta Hydrochimistry Hydrobiology 16, 113-138. Alard, F., Geerts, S. and Triest, E.T.L. (1991) Toxicite D' Ambrosia Maritima L. Plante Molluscicide, sur les organismes Aquatiques Non-cibles. Toxicon 29, 745-750. Ayoub, S.M.H. and Yankov, L.K. (1985) Potential molluscicidesfrom some tannin containing plants growing in the Sudan. Fitoterapia 56, 371-375. Ayoub, S.M.H. and Yankov, L.K. (1986) The molluscicidal factor of tannin-bearing plants. International Journal of Crude Drug Research 24, 16-18. Banerjee,S. and Rembold,H. (1992)Azadirachtin A interferes with control of serotonin pools in the neuroendocrinesystem of locusts. Naturwissenschaften79, 81-84. Hostettmann, K. and Lea, P.J. (1987)Biologicallyactive natural products. Oxford SciencePublication, Oxford. Muley, E.V. (1978)Biologicaland chemicalcontrol of the vector snail Melania scabra (Gastropoda: Prosobranchiata). Bulletin of Zoological Survey of lndia 1, 1-5. Okapanyi, S.N. and Ezeukwu,G.C. (1981)Anti-inflammatory and anti-pyretic activities of Azadirachta indica. Planta Medica 41, 34-39. Rembold, H. and Annadurai, R.S. (1993)Azadirachtin inhibits

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proliferation of Sf 9 cells in monolayer culture. Zeitschrift Fur Naturforschung 48C, 495-499. Russell, R.M., Robertson, J.L. and Savin, N.E. (1977) POLO: A new computer program for probit analysis. Bulletin of Entomological Society of America 23, 209-213. Sinha, K.C., Riar, S.S., Tiwary, R.S., Dhawan, A.K., Bardhan, J., Thomas, P., Kain, A.K. and Jain, R.K. (1984) Neem oil as a vaginal contraceptive. Indian Journal of Medical Research 79, 131-136. Singh, A. and Singh, D.K. (1994) Pestoban, a potent herbal molluscicide. Biological Agriculture and Horticulture 10, 175-178. Singh, D.K. and Agarwal, R.A. (1983) In vivo and in vitro studies on synergism with anticholinesterase pesticide in the snail Lymnaea acuminata. Archive of Environmental Contamination and Toxicology 12, 483-487.

Singh, D.K. and Agarwal R.A. (1984) Correlation of the anticholinesterase and molluscicidal activity of the latex of Euphorbia royleana Bioss. on Lymnaea acuminata. Journal of Natural products 47, 702-705. Singh, D.K. and Singh, A. (1993) Garlic Allium sativum, a potent new molluscicide. Biological Agriculture and Horticulture 9, 121-124. Singh, O. and Agarwal, R.A. (1981) Toxicity of certain pesticides to two economic species of snails in northern India. Journal of Economic Entomology 74, 568-571. Sokal, R. and Rohlf, J. (1973) Introduction to Biostatistics. W.H. Freeman and Co., San Francisco. van der Nat, J.M., van der Sluis, W.G., de Silva, K.T.D. and Labadie, R.P. (1991) Ethnopharmacognostical survey of Azadirachta indica A. Juss (Meliaceae). Journal of Ethnopharmacology 35, 1-24.