Subscribe Now Subscribe Today
Research Article
 

Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata



Shefali Srivastava, Anupam Pati Tripathi, V.K. Singh and D.K. Singh
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Snails of family Lymnaeidae act as an intermediate host of Fasciola species, which caused fasciolosis nearly in all the continents of world. Eugenol and quercetin are active molluscicides against snail Lymnaea acuminata. Laboratory evaluation is done to determine the effect of abiotic factors on the toxicity of these molluscicides in the year 2010-2011. Highest toxicity of eugenol (24 h LC50 10.03 mg L–1) and quercetin (24 h LC50 129.13 mg L–1) was observed in the month of May. Significant correlation was observed between LC50 values of eugenol and quercetin with temperature/pH/dissolved oxygen/carbon dioxide. In vivo acetylcholinesterase (AChE), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were measured in the nervous tissue of treated snails in each month of year 2010-2011.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Shefali Srivastava, Anupam Pati Tripathi, V.K. Singh and D.K. Singh, 2015. Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata. Research Journal of Environmental Toxicology, 9: 332-341.

DOI: 10.3923/rjet.2015.332.341

URL: https://scialert.net/abstract/?doi=rjet.2015.332.341
 
Received: July 25, 2015; Accepted: September 04, 2015; Published: October 10, 2015



INTRODUCTION

Fasciolosis is one of the world’s most prevalent zoonotic disease caused by trematode parasite of the genus Fasciola that infects over 17 million people causing significant morbidity and mortality (Mas-Coma et al., 2005; WHO., 2006). Fasciolosis becomes hyperendemic and problematic in regions, where animal reservoir and snail vectors are easily available (Hassan et al., 2011). Fasciolosis caused serious economic losses to animal husbandry in the Northern part of India. (Upadhyay and Singh, 2011a) Treatment of Fasciola requires high or multiple doses of drugs (Abdul-Samie et al., 2010). Snail is considered to be one of the weakest links in Fasciola. So management of snail population below a threshold level is advocated for effective control of fasciolosis (Mello-Silva et al., 2006). Heavy use of synthetic molluscicides for control of snail population has created serious problem for the aquatic animals. Alternatively, in recent years much attention has been focused on the use of molluscicides of plant origin. Plant molluscicidal appears to be a simple, inexpensive and ecologically safe (Al-Daihan, 2010; Upadhyay and Singh, 2011a,b). Eugenol and quercetin are potent molluscicides found in Syzygium aromaticum and Allium cepa (Singh et al., 1997; Kumar and Singh, 2006).

Earlier studies have shown that various abiotic factors such as temperature, pH, dissolved oxygen, carbon dioxide and conductivity of water affects the toxicity of the synthetic molluscicides (Singh and Singh, 2009). Acetylcholinesterase (AChE), acid phosphatase (ACP) and alkaline phosphatase (ALP) are the sensitive enzymes in the nervous tissue of L. acuminata. These enzymes are mainly are inhibited by several potent plant molluscicides (Jaiswal et al., 2008; Kumar et al., 2009; Upadhyay and Singh, 2011b). The prospective of the present study is to reveal whether seasonal changes in abiotic factors, viz. temperature, pH, dissolved oxygen and carbon dioxide and conductivity of the test water can influence the activity of AChE, ACP and ALP assayed in each month of the year 2010-2011 as well as toxicity of eugenol and quercetin.

MATERIALS AND METHODS

Test materials: Eugenol is a phenylpropene, an ally chain substitute of guiacol, quercetin is a plant derived flavonoid (3, 3, 4, 5, 7-penta hydroxyl flavones), purchased from Sigma Chemicals Co. U.S.A. Temperature, pH and electrical conductivity were measured by thermometer, digital pH meter and conductivity meter, respectively. Dissolved oxygen and carbon dioxide were estimated according to the method of APHA (2005).

Bioassays for LC50: Adult snail Lymnaea acuminata (length 2.30±0.25 cm) were collected from the different ponds, pools and lake near the Gorakhpur university campus. These experimental snails were acclimatized in dechlorinated tap water for 72 h. Ten experimental snails were kept in a glass aquarium containing 3 L of dechlorinated tap water. Snails were exposed to different concentration of eugenol and quercetin. A set of control animals were kept in an equal volume of dechlorinated tap water under similar conditions. Lethality of the concentrations was observed by number of mortal snails after 24, 48, 72 and 96 h. No behavioral response towards the needle probe is taken as the evidence of the death of snails. Simultaneously with the toxicity test at every 24-96 h temperature, pH, dissolved oxygen, carbon dioxide and conductivity were also measured. Bioassay for the determination of LC50 was performed in each month of the year. Lethal concentration (LC50) values, lower and upper confidence limits (LCL and UCL) and slope values were calculated by the method of POLO computer programme of Robertson et al. (2007). The product moment coefficient was determined between LC50 and temperature/pH/conductivity/ dissolved oxygen/carbon dioxide of water in each month to observe any significant correlation.

Enzyme assays: In a glass aquarium containing 3 L of dechlorinated water, twenty experimental snails were exposed to sublethal concentration (60% of 24 h LC50) of eugenol and quercetin in each month of the year. After the 24 h exposure the snails were washed with water and the nervous tissue was dissected out for the measurement of AChE, ACP and ALP activity.

Acetylcholinesterase: Acetylcholinesterase (AChE) activity was measured according to the method of Ellman et al. (1961) as modified by Singh and Agarwal (1983). Fifty milligram of nervous tissue was homogenized in 1.0 mL of 0.1 M phosphate buffer pH 8.0 for 5 min in an ice bath and centrifuged at 1000 g for 30 min at 4°C. Supernatant was used as enzyme source. The change in optical density at 412 nm was recorded for 3 min after every 30 sec interval. Enzyme activity was expressed as micro moles "SH" hydrolysed/min/mg protein.

Phosphatases: Acid (ACP) and alkaline (ALP) phosphatases activities was measured by the method of Bergmeyer (1967) as modified by Singh and Agarwal (1989). Tissue homogenate (2%w/v) was prepared in ice cold 0.9% NaCl and centrifuged at 5000 g for 20 min at 4°C. The 4-nitro phenyl phosphate disodium was used as substrate. The acid (ACP) and alkaline phosphate (ALP) have been expressed as micro moles substrate hydrolysed/30 min/mg protein.

Protein estimation: Protein was estimated by the method of Lowry et al. (1951).

Statistical analysis: Results have been expressed as Mean ±SE of six replicates. Rank correlation was applied in between LC50 and corresponding changes in enzyme activity in different months of the year 2010-2011 (Sokal and Rohlf, 1973). One way analysis of variance were applied between enzymatic activity in different month of the year.

RESULTS AND DISCUSSION

There was significant (p<0.05) variation in the toxicity of eugenol and quercetin against the Lymnaea acuminata in each month of the year 2010-2011. Highest toxicity of eugenol (24 h LC50 10.03 mg L–1) (Table 1) and quercetin (24 h LC50 129.13 mg L–1) (Table 2) was observed in the month of May and lowest in the month of January (24 h LC50 40.69 mg L–1) (Table 1) and quercetin (24 h LC50 430.67 mg L–1) (Table 2). A significant positive correlation between LC50 and water pH/carbon dioxide, contrary a negative correlation between LC50 and dissolved oxygen/temperature was observed in different months of year 2010-2011 (Table 1). There was no marked correlation in between the LC50 and conductivity of water. The slope values were steep and separate estimation of LC50 based on each of six replicates was found to be within the 95% confidence limits of LC50. The t-ratio is greater than 1.96 and the heterogeneity factor is less than 1.0. The g-value was less than 0.5 at all probability levels. There was a positive rank correlation between LC50 of eugenol in different months and corresponding AChE/ACP/ALP activity in the nervous tissue of treated snail L. acuminata. No significant rank correlation was observed between LC50 of quercetin and corresponding ACP/ALP activity in the nervous tissue of treated snail.

Maximum inhibition of AChE (52.38% of control) was observed when snails were exposed to 60% 24 h LC50 of quercetin followed by eugenol (66.66% of control). Maximum inhibition of ALP (60.15% of control) was observed when snails were exposed to 60% 24 h LC50 of eugenol followed by quercetin (90.73% of control) (Table 3). Maximum inhibition of ACP (82.08% of control) was observed when snails were exposed to 60% 24 h LC50 of eugenol (Table 3) followed by quercetin (90.72% of control) (Table 4).

Abiotic factors interact with each other to influence a physiological activity of animals (Gertseva and Gertseva, 2006). Pesticides become more lethal when there is an indulgence of abiotic conditions along with variation in pH and temperature (Relyea, 2006). The toxicity of molluscicides may get influenced by the abiotic factors such as temperature, pH, dissolved oxygen, carbon dioxide and conductivity (Singh and Singh, 2009). As we know when temperature is high the toxicity is also high as compare to higher LC50 values in winter seasons. Temperature influences the activity of the molluscicides (Oliveira-Filho et al., 1999). Higher temperature diminishes the solubility of dissolved oxygen and thus decreases the availability of this essential gas. In normal condition metabolic demand for oxygen increases substantially with temperature (Portner, 2002). Lymnaea acuminata is very sensitive to dissolve content of water (Janse, 1981). The temperature and the dissolved oxygen shows inverse correlationship when temperature increases the oxygen dissolving capacity of water system are reduced. At low temperature the water holds more Oxygen (O2) (Ingram et al., 1997). Dissolved oxygen is utilized by aquatic organism to accommodate metabolism and excreted as carbon dioxide (CO2). At high temperature the rate of metabolism in snail body increases consequently, it causes release of more carbon dioxide in water. Higher concentration of carbon dioxide effect the pH of water (Toews et al., 1995; Berge et al., 2006). The high concentration of carbon dioxide in water may synergize the lethality of molluscicides.

Eugenol (Kumar and Singh, 2006; El-Din, 2006) and Quercetin (Singh et al., 1997; Lahlou, 2004) is potent molluscicides.

Table 1:
Alterations in toxicity (LC50 mg L–1) of eugenol against Lymnaea acuminata and different environmental factors in pond water in different months of the year 2010-2011
Image for - Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata
Each experiment was replicated six times and values are the mean of six replications, Temperature, pH, DO: Dissolved oxygen, DCO2: free carbon dioxide and conductivity were measured at intervals of 24 h up to 96 h. Product moment correlation coefficient in between LC50 and different abiotic parameters indicate significant (p<0.05) positive (+) and negative(*) correlation. Testing significance of the regression coefficient May 3.68+, June 8.86++, July 9.16+, Aug 11.89+, Sep12.74++, Oct 14.24++, Nov 8.73+, Dec 7.36++, Jan 14.44+, Feb 10.36+, Mar 6.10++, Apr 4.95+. +: Linear regression between X and Y. ++: Non linear regression between log X and Y. Months show highest and lowest toxicity of eugenol against Lymnaea acuminata

Table 2:
Alterations in toxicity (LC50 mg L–1) of quercetin against Lymnaea acuminata and different environmental factors in pond water in different month of the year 2010-2011
Image for - Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata
Each experiment was replicated six times and values are the mean of six replications, Temperature, pH, DO: Dissolved oxygen, DCO2: free carbon dioxide and conductivity were measured at intervals of 24 h up to 96 h. Product moment correlation coefficient in between LC50 and different abiotic parameters indicate significant (p<0.05) positive (+) and negative (*) correlation. Testing significance of the regression coefficient May 90.79++, June 9.87+, July 13.84++, Aug 13.12+, Sep 8.87+,Oct 17.79+, Nov 16.37++, Dec 17.42+, Jan 7.42++, Feb 50.67+, Mar 13.76+, Apr 6.28+. +: Linear regression between X and Y. ++: Non linear regression between log X and Y. Months show highest and lowest toxicity of quercetin against Lymnaea acuminata

Table 3:
Effect of 24 h exposure of 60% of 24 h LC50 of eugenol in different months of the year 2010-2011 on acetylcholinesterase, alkaline and acid phosphatase activity in the nervous tissue of L. acuminata
Image for - Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata
Values are Mean±SE of six replicates. Value in parenthesis indicates % enzyme activity with untreated control taken as 100%. Rank correlation coefficient in between LC50 and AChE/ALP/ACP activity in treated group indicate significant (p<0.05) positive (+) correlation a, Significant (p<0.05) when one way ANOVA was applied in between the enzymatic activity in different month of the year in control as well as treated group

Table 4:
Effect of 24 h exposure of 60% of 24 h LC50 of Quercetin in different months of the year 2010-2011 on acetylcholinesterase, alkaline and acid phosphatase activity in the nervous tissue of L. acuminata
Image for - Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata
Values are Mean±SE of six replicates. Value in parenthesis indicates (%) enzyme activity with untreated control taken as 100%.Rank correlation coefficient in between LC50 and AChE/ALP/ACP activity in treated group indicate significant (p<0.05) positive (+) correlation a, Significant ( p<0.05) when one way ANOVA was applied in between the enzymatic activity in different month of the year in control as well as treated group.

The time dependant effect of these plant products may be due to the uptake of active moiety which progressively increases the amount of active components in snail body with increase in exposure period or it may be possible that the active component(s) could change into more toxic forms in the aquarium water or in the snail body.

The enzyme acetylcholinesterase occurs in the outer basal lamina of nerve synapses (Taylor, 1980) neuromuscular junction and in certain other tissues (Hall, 1973). Acetylcholinesterase (AChE) inhibition results in the accumulation of acetylcholine at nerve synapses, so that the post snaptic membrane is in a state of permanent stimulation producing paralysis, ataxia and general lack of co-ordination in neuromuscular system and eventual death. Earlier it has been reported that sublethal treatment 60% of 24 h LC50 of eugenol caused significant inhibition of AChE activity (70.47% of control) in nervous tissue of Lymnaea acuminata (Kumar et al., 2009). Earlier it has been shown proved that quercetin is toxic to L. acuminata (Singh et al., 1997).

Eugenol has a radio protective potential (Tiku et al., 2004). It also acts as mixed type inhibitors (Chaieb et al., 2005). It acts as the sex attractant in fruit flies (Mahmood et al., 2002). It was observed that treatment of methanol (200 ppm) and ethanol (600 ppm) extracts of S. aromaticum were toxic against larvae of Culex pipiens. They reported that these extracts caused complete inhibition of adult emergence. Kim et al. (1998) observed that aqueous extract of S. aromaticum induced systematic and phylaxis in rats (IC50-31.25 mg kg–1). Quercetin is as effective as N-acetylecysteine in protecting rat peripheral lymphocytes against DNA damage (Muthukumaran et al., 2008). It improved immune function against external stress and also acts as powerful antioxidant and free radical scavenger (Shin et al., 2010).

Acid phosphatase (ACP) a lysosomal enzyme (Aruna et al., 1979), which plays an important role in catabolism, pathological necrosis, autolysis and phgocytosis (Abou-Donia, 1978) and alkaline which plays a critical role in protein synthesis (Pilo et al., 1972) and shell formation. Positive rank correlation coefficient between LC50 value and corresponding AChE/ACP/ALP inhibition indicate that toxicity of eugenol is due to simultaneous inhibition of these enzymes. It indicates that changes in the activity of AChE/ACP/ALP in the nervous tissue of snail can also be the cause of mortality of these snails.

CONCLUSION

The present study conclusively shows that variant abiotic factors can significantly alter the toxicity of eugenol. It also results for the most suitable period to control the snail in India. It is more potent and cost effective during these months rather than spending the rest months of the year. Out of both the molluscicides namely eugenol and quercetin, eugenol is prove to be more potent because of low values of LC50 in comparison to quercetin. Hence eugenol can be used as potent molluscicides in the month of May rather than spending throughout the year against vector snail Lymnaea acuminata.

REFERENCES

1:  Abdul-Samie, R.E., E.O. Soliman, H. El-Nemr and A. Masou, 2010. Study of Il1b, Il4, Il5 and IgE before and after Mirazid Therapy in children with intestinal schistosomiasis and fascsioliasis. N. Y. Sci. J., 3: 116-122.

2:  Abou-Donia, M.B., 1978. Increased acid phosphatase activity in hens following an oral dose of leptophos. Toxicol. Lett., 2: 199-203.
CrossRef  |  Direct Link  |  

3:  Al-Daihan, S., 2010. Effect of plant molluscicides on selected enzymes related to energy metabolism in Biomphalaria arabica snails molluscan hosts to Schistosoma mansoni in Saudi Arabia. J. Egypt. Soc. Parasitol., 40: 187-195.
PubMed  |  

4:  APHA., 2005. Standard Methods for the Examination of Water and Wasterwater. 21st Edn., APHA, AWWA, WEF, ISBN-13: 9780875530475, Washington, DC., USA., Pages: 55

5:  Aruna, P., C.S. Chetty, R.C. Naidu and K.S. Swami, 1979. Acid phosphatase activity in Indian apple snail, Pila globosa (Swainson), during aestivation and starvation stress. Proc. Indian Acad. Sci., 88B: 363-365.
Direct Link  |  

6:  Berge, J.A., B. Bjerkeng, O. Pettersen, M.T. Schaanning and S. Oxnevad, 2006. Effects of increased sea water concentrations of CO2 on growth of the bivalve Mytilus edulis L. Chemosphere, 62: 681-687.
CrossRef  |  Direct Link  |  

7:  Bergmeyer, U.H., 1967. Methods of Enzymatic Analysis. Academic Press, New York, Pages: 1129

8:  Chaieb, E., A. Bouyanzer, B. Hammouti and M. Benkaddour, 2005. Inhibition of the corrosion of steel in 1M HCl by eugenol derivatives. Applied Surf. Sci., 246: 199-206.
CrossRef  |  Direct Link  |  

9:  El-Din, A.T., 2006. Molluscicidal effect of three monoterpenes oils on schistosomiasis and fascioliasis vector snails in Egypt. J. Egypt Soc. Parasitol., 36: 599-612.
PubMed  |  

10:  Ellman, G.L., K.D. Courtney, V. Andres Jr. and R.M. Featherstone, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7: 88-95.
CrossRef  |  Direct Link  |  

11:  Gertseva, V.V. and V.I. Gertsev, 2006. A conceptual model of fish functional relationships in marine ecosystems and its application for fisheries stock assessment. Fisher. Res., 81: 9-14.
CrossRef  |  Direct Link  |  

12:  Hall, Z.W., 1973. Multiple forms of acetylcholinesterase and their contribution in end plate regions of rat diaphragm muscle. J. Neurobiol., 4: 343-361.

13:  Hassan, A.A., A.E. Mahmoud, R.A. Hassan and E.M. Huseein, 2011. Evaluation of Euphorbia aphylla, Ziziphus spina-christi and Enterolobium contortisiliquum as molluscicidal agents. J. Am. Sci., 7: 511-520.
Direct Link  |  

14:  Ingram, B.A., J.H. Hawking and R.J. Shield, 1997. Aquatic Life in Fresh Water Ponds: A Guide to Identification and Ecology in Life in Aquaculture Ponds and Farm Dams in South-Eastern Australia. Co-operative Research Centre for Freshwater Ecology, Albury, NSW., Australia

15:  Jaiswal, P., V.K. Singh and D.K. Singh, 2008. Enzyme inhibition by molluscicidal component of Areca catechu and Carica papaya in the nervous tissue of vector snail Lymnaea acuminata. Pestic. Biochem. Physiol., 92: 164-168.
CrossRef  |  

16:  Janse, C., 1981. The effect of oxygen on gravity orientation in the pulmonate snail Lymnaea stagnalis. J. Comp. Physiol., 142: 51-59.
CrossRef  |  Direct Link  |  

17:  Kim, H.M., E.H. Lee, S.H. Hong, H.J. Song, M.K. Shin, S.H. Kim and T.Y. Shin, 1998. Effect of Syzygium aromaticum extract on immediate hypersensitivity in rats. J. Ethnopharmacol., 60: 125-131.
CrossRef  |  Direct Link  |  

18:  Kumar, P. and D.K. Singh, 2006. Molluscicidal activity of Ferula asafoetida, Syzygium aromaticum and Carum carvi and their active components against the snail Lymnaea acuminata. Chemosphere, 63: 1568-1574.
CrossRef  |  PubMed  |  Direct Link  |  

19:  Kumar, P., V.K. Singh and D.K. Singh, 2009. Kinetics of enzyme inhibition by active molluscicidal agents ferulic acid, umbelliferone, eugenol and limonene in the nervous tissue of snail Lymnaea acuminata. Phytother. Res., 23: 172-177.
CrossRef  |  PubMed  |  Direct Link  |  

20:  Lowry, O.H., N.J. Rosenbrough, A.N. Farrand and R.J. Randall, 1951. Protein estimation with folin phenol reagent. J. Biol. Chem., 193: 265-275.
Direct Link  |  

21:  Mahmood, T., S.I. Hussain, K.M. Khokhar, M. Ahmad and Hidayatullah, 2002. Studies on menthly eugenol as a sex attractant for fruit fly Dacus zonatus (Saund) in relation to abiotic factors in peach orchard. Asian J. Plant Sci., 1: 401-402.
CrossRef  |  Direct Link  |  

22:  Mas-Coma, S., M.D. Bargues and M.A. Valero, 2005. Fascioliasis and other plant-borne trematode zoonoses. Int. J. Parasitol., 35: 1255-1278.
CrossRef  |  Direct Link  |  

23:  Mello-Silva, C.C., M.C. de Vasconcellos, J. Pinheiro and M.L.A. Rodrigues, 2006. Physiological changes in Biomphalaria glabrata say, 1818 (Pulmonata: Planorbidae) caused by sub-lethal concentrations of the latex of Euphorbia splendens var. hislopii N.E.B (Euphorbiaceae). MemOrias Instituto Oswaldo Cruz, 101: 3-8.
CrossRef  |  Direct Link  |  

24:  Lahlou, M., 2004. Study of the molluscicidal activity of some phenolic compounds: Structure-activity relationship. Pharmaceut. Biol., 42: 258-261.
CrossRef  |  Direct Link  |  

25:  Muthukumaran, S., A.R. Sudheer, N. Nalini and V.P. Menon, 2008. Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes. Redox Rep., 13: 217-224.
CrossRef  |  Direct Link  |  

26:  Oliveira-Filho, E.C., R.R. De-Carvalho and E.J. Paumgartten, 1999. The influence of environmental factors on the molluscicidal activity of Euphorbia milii latex. J. Environ. Sci. Health Part B: Pestic. Food Contam. Agric. Wastes, 34: 289-303.
CrossRef  |  Direct Link  |  

27:  Pilo, B., M.V. Asnani and R.V. Shah, 1972. Studies on wound healing and repair in pigeon liver. III. Histochemical studies on acid and alkaline phosphatase activity during the process. J. Anim. Morphol. Physiol., 19: 205-212.

28:  Portner, H.O., 2002. Climate variations and the physiological basis of temperature dependent biogeography: Systemic to molecular hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol., 132: 739-761.
CrossRef  |  Direct Link  |  

29:  Relyea, R.A., 2006. The effects of pesticides, pH and predatory stress on amphibians under mesocosm conditions. Ecotoxicology, 15: 503-511.
CrossRef  |  Direct Link  |  

30:  Robertson, J.L., R.M. Russell, H.K. Preisler and N.E. Savin, 2007. Bioassay with Arthropods. 2nd Edn., CRC Press, New York, USA., ISBN-13: 9781420004045, Pages: 224

31:  Shin, H.S., J.H. Yoo, T.H. Min, K.H. Lee and C.Y. Choi, 2010. The effects of quercetin on physiological characteristics and oxidative stress resistance in olive flounder, Paralichthys olivaceus. Asian-Australasian J. Anim. Sci., 23: 588-597.
Direct Link  |  

32:  Singh, V. and D.K. Singh, 2009. The effect of abiotic factors on the toxicity of cypermethrin against the snail Lymnaea acuminata in the control of fascioliasis. J. Helminthol., 83: 39-45.
CrossRef  |  Direct Link  |  

33:  Singh, S., V.K. Singh and D.K. Singh, 1997. Molluscicidal activity of some common spice plants. Biol. Agric. Hortic., 14: 237-249.
CrossRef  |  Direct Link  |  

34:  Singh, D.K. and R.A. Agarwal, 1983. Inhibition kinetics of certain organophosphorus and carbamate pesticides on acetylcholinesterase from the snail Lymnaea acuminata. Toxicol. Lett., 19: 313-319.
CrossRef  |  Direct Link  |  

35:  Singh, D.K. and R.A. Agarwal, 1989. Toxicity of piperonyl butoxide-carbaryl synergism on the snail Lymnaea acuminata. Internationale Revue Gesamten Hydrobiologie Hydrographie, 74: 689-699.
CrossRef  |  Direct Link  |  

36:  Sokal, R.R. and F.J. Rohlf, 1973. Introduction to Biostatistics. Freeman, San Francisco, USA., pp: 171

37:  Taylor, P., 1980. Anticholinesterase Agents. In: The Pharmacological Basis of Therapeutics, Goodman, A.S., A.G. Gilman, T.W. Roll and F. Murad (Eds.). Macmillan Publishing Co., New York, pp: 110

38:  Tiku, A.B., S.K. Abraham and R.K. Kale, 2004. Eugenol as an in vivo radioprotective agent. J. Radiat. Res., 45: 435-440.
CrossRef  |  Direct Link  |  

39:  Toews, K.L., R.M. Shroll, C.M. Wai and N.G. Smart, 1995. pH-defining equilibrium between water and supercritical CO2. Influence on SFE of organics and metal chelates. Anal. Chem., 67: 4040-4043.
CrossRef  |  Direct Link  |  

40:  Upadhyay, A. and D.K. Singh, 2011. Molluscicidal activity of Sapindus mukorossi and Terminalia chebula against the freshwater snail Lymnaea acuminata. Chemosphere, 83: 468-474.
CrossRef  |  Direct Link  |  

41:  Upadhyay, A. and D.K. Singh, 2011. Inhibition kinetics of certain enzymes in the nervous tissue of vector snail Lymnaea acuminata by active molluscicidal components of Sapindus mukorossi and Terminalia chebula. Chemosphere, 85: 1095-1100.
CrossRef  |  Direct Link  |  

42:  WHO., 2006. Report of the WHO informal meeting on use of triclabendazole in fascioliasis control. World Health Organization, Geneva, Switzerland.

©  2021 Science Alert. All Rights Reserved