Chlorophyllin Treatment Against the Snail Lymnaea acuminata: A new tool in Fasciolosis Control

Objective: To observe the toxicity of chlorophyllin against Lymnaea acuminata to control fasciolosis caused by liver fluke fasciola gigantica, very prominent in eastern region of Uttar Pradesh. Materials and Methods: Ten snails Lymnaea acuminata were placed in a glass aquarium containing 3 L of dechlorinated tap water. These snails were treated with different concentrations of chlorophyllin. Chlorophyll was extracted from spinach with the help of macerated leaves and kept for 2 h in 100% ethanol at 55oC. Results: The results of the experiment showed that the photodynamically active chlorophyllin, at low concentration was able to kill the snails under exposure of solar radiation in summer season instead of winter season. In winter, extracted chlorophyllin toxicity against L. acuminata in sunlight (96 h LC50 91.82 mg L-1) /laboratory condition (96 h LC50 921.93 mg L -1) was less than pure chlorophyllin in sunlight (96 h LC50 12.05 mg L -1) /laboratory condition (96 h LC5019.22 mg L -1), respectively. In summer, pure chlorophyllin was more toxic in sunlight (96 h LC50 3.90 mg L -1) than laboratory condition (96 h LC50 7.18 mg L -1). Pure chlorophyllin is more than five times toxic than synthetic molluscicides. Treatment of chlorophyllin caused no toxic effect against the fish (Colisa fasciatus). The result presented in this paper is found very beneficial and ecologically safe, as a photodynamic substance chlorophyllin, which found in every green plant. Conclusion: Phytotherapy of snails by photodynamic water soluble chlorophyllin to control fasciolosis can be used as potent molluscicides with low cost and easily biodegradable.


INTRODUCTION
2][3] Human fasciolosis and it outbreaks in the last two decades have changed the status of fasciolosis from a zoonosis to an emerging health problem in tropics. 4,5Fasciola gigantica is the causative agent of fasciolosis in eastern Uttar Pradesh, India. 6,7Snails are the important links in transmission of fasciolosis.The snail Lymnaea acuminata is the intermediate host of F. gigantica. 8,9An obvious preventive method to reduce the incidence of fasciolosis is to control the population of carrier snail.Indiscriminate use of synthetic molluscicides such as Carbamates and organophosphates has created several environmental hazards. 10So that, plant molluscicides are advocated as they are easily biodegradable, cheap and easier to handle by native users. 11,12lorophyll is found in all green plants.Chlorophyll product chlorophyllin is extremely toxic against mosquito larvae in sunlight. 13,14Recently, Singh and Singh 7 noted the cercaricidal activity of chlorophyllin against Fasciola gigantica larvae.The present study reports the molluscicidal activity of chlorophyllin against host snail L. acuminata.

Experimental Animal
Adult L. acuminata of average size (2.25 ± 0.30 in length) were collected locally from ponds, lakes and low-lying submerged fields of the district Gorakhpur, UP, India.Gorakhpur lies between latitude 26° 46´ N and longitude 83° 22´ E at an altitude of 95 meter above the sea level.The collected snails were kept in glass aquarium containing de-chlorinated tap water for 72 h for acclimatization.The animals were kept in de-chlorinated tap water at room temperature (22-25ºC).The pH, dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 7.1-7.3;6.5-7.3 mg L -1 ; 5.2-6.3mg L -1 and 102-105 mg L -1 , respectively.Water was changed once every 24 h and dead animals were removed to prevent the water from being contaminated by decaying tissue.

Preparation of Chlorophyllin
Chlorophyllin was prepared by the method of Wohllebe et al. 14 Chlorophyll was extracted from spinach with the help of macerated leaves for 2 h in 100% was 2.67 to 4.84 times higher in summer than winter.The toxicity of pure chlorophyllin in laboratory condition (96 h LC 50 7.18 mg L -1 ) was less effective than sunlight (96 h LC 50 3.90 mg L -1 ) (Table 1 and 2).No mortality in snails was noted in group III control 1 and contr.ol 2 as well as fish treated with 24 h LC 90 against L. acuminata.No mortality in fish population may be either due to rapid detoxification of chlorophyllin in fish body or the concentration range used for the snails is safe against fish.The slope values were steep and separate estimations of LC 50, based on each of the six replicates, were found within the 95% confidence limits of LC 50 .The t-ratio values were greater than 1.96 indicating a significant regression of each dose response line.The heterogeneity factor was less than 1.0, demonstrating the log-dose-probit lines are within the 95% confidence limits and thus the model fitted our data.Value of g less than 0.5 indicated that mean was within the limit at all probability levels of 90, 95 and 95%.

DISCUSSION
The results of the present study indicate that the chlorophyllin extracted from spinach is a potent source of plant molluscicides.Toxicity of chlorophyllin is time and concentration dependent, as evident from negative ethanol at 55ºC.To avoid transformation of chlorophyll into pheophytin by the acidic content of the cell vacuoles 1.0 mg CaCO 3 / g leaves were added as a buffer.The extract was subsequently filtered and equal volume of petroleum benzene was added.After shaking the mixture the chlorophyll moved into the lipophillic benzene phase.The two phases were separated in separatory funnel and about 1 ml methanolic KOH was added to 50 ml of the benzene phase.The chlorophyll came into contact with the methanolic KOH and was transformed into water-soluble chlorophyllin (this process occurs due to the breakage of the ester bond between the chlorophyllin and the phytol tail by saponification).

Toxicity-Determination
Toxicity experiments were done according to the method of Singh and Agarwal. 15Ten experimental snails were placed in a glass aquarium containing 3 L of de-chlorinated tap water.The experiment was setup with two groups and in the I group the control 1 snails were kept in laboratory condition (winter/summer) after 4 h of dark incubation for 96 h with no chlorophyllin, and control 2 put into the sunlight condition (winter/summer) and all the condition was same as in control 1.Further in the II group the treatment 1 were kept in laboratory condition (mercury light intensity 150W/m 2 , winter/summer) and treatment 2 in sunlight condition (light intensity 900W/m 2 in winter and 1200W/m 2 in summer).Light intensity was measure with the help of digital lux meter (Mextech LX-1010B) in flux.Thereafter, it has been converted in irradiance W/m 2 .Snail mortality was recorded at every 24 h upto 96 h.Each treatment was replicated six times.Dead animals were removed immediately from the aquarium to avoid any contamination of the water.Snail mortality was confirmed by the contraction of the body within the shell and absence of any response to a needle probe.Toxicity determinations were also studied against non-target animal fish Colisa fasciatus.A group III of ten fishes were taken in a 6 L dechlorinated tap water and all the conditions were same as in group I and group II.The fishes were treated with 24 h Lethal Concentration 90 (LC 90 against L. acuminata) for 96 h in the same experimental condition as in snail treatment.The slope value of the probit line was also estimated.This program ran chi-square tests for goodness of fit of the data to the probit model.If the model fits, the calculated value of chi-square is less than the chi-square table value for appropriate degree of freedom.If the model does not fit, the LC 50 value for the particular population may not be reliably estimated and is adjusted with the heterogeneity factor as correction factor when the value of Pearson's chi-square statistics is significant at P= 0.05.The index of significance for potency estimation (g-value) was used to calculate 95% confidence intervals for potency (relative potency is equivalent to tolerance ratio).Parallelism of the probit regression lines implies a constant relative potency at all levels of response.POLO-PC (LeOra software) of Robertson et al 16 was used to test equality and parallelism of the slope of the probit lines was calculated by using the probit analysis programme.The regression co-efficient was determined between exposure time and different values of LC 50 by the method of Sokal and Rohlf 17.

RESULTS
The toxicity of chlorophyllin against L. acuminata was concentration and time-dependent.In winter, toxicity of extracted and pure chlorophyllin in laboratory condition (96 h LC 50 Extracted-921.93mg L -1 , Pure-19.22 mg L -1 ) was lower than the sunlight (96 h LC 50 extracted-91.82mg L -1 , Pure-12.05 mg L -1 ).Whereas, in summer toxicity of extracted and pure chlorophyllin in laboratory condition (96 h LC 50 Extracted-257.11mg L -1 , Pure-7.18 mg L -1 ) was lower than the sunlight (96 h LC 50 extracted-24.95mg L -1 , Pure-3.90 mg L -1 ) (Table 1 and 2).Toxicity of extracted chlorophyllin against L. acuminata was 1.75 to 3.68 times higher in summer than winter.Whereas, pure chlorophyllin toxicity +: linear regression between x and y; ++ non linear regression between log x and log y.
Abbreviations: Chl, chlorophyllin; Pure Chl, pure chlorophyllin.LCL, lower confidence limit; UCL, upper confidence limit; regression between exposure period and LC 50 of chlorophyllin.Earlier, Wohllebe et al 18 have noted that chlorophyllin can kill the different stages of the protozoan parasite Ichthyophthirius mulftifiliis in fresh water fish species.It is reported that chlorophyllin was able to kill four different species, a small crustacean (Daphnia similis), a unicellular alga (Euglena gracilis) and two species of fish (Astyanax bimaculatus and Cyprynus carpio) the vector of parasitic diseases. 19Although it has been reported 19 that chlorophyllin is toxic against Astyanax bimaculatus and Cyprynus carpio, yet in our observation it is not toxic against C. fasciatus.Both extracted and pure chlorophyllin are more effective in summer than winter.Water in winter season holds more oxygen 20 Singh et al. 21reported that in 2010 summer water temperature was in between 20°C to 35°C in year 2010.The reported dissolved oxygen concentration in this water was 2.0 to 4.0 mg L -1 , which caused higher mortality of snails.Earlier it was reported in my own laboratory 22 that in warm water dissolved oxygen concentration in summer season is low (2.0 -4.0 mg L -1 ) and in winter season it is high (5.0 -7.0 mg L -1 ).In the present experiment when we have treated the same conditions while toxicity the chlorophyllin, it may be possible that their effect along with toxic singlet oxygen production in the body of the snail after the treatment of chlorophyllin enhances the snail mortality.
Whereas in winter, when water temperature and corresponding dissolved oxygen concentration was in between 18°C to 25°C and was 5.0 to 7.3 mg L -1 , respectively 21 .Higher toxicity of chlorophyllin in sunlight in comparison to laboratory condition is due to the more production of toxic singlet oxygen.Photodynamic reactions of chlorophyllin lead to the formation of the highly reactive singlet oxygen, which can react with various biomolecules. 23The toxicity of pure chlorophyllin is much more than extracted chlorophyllin because it is isolated from extraction of spinach.
And it has high concentration of chlorophyll in comparison to extracted chlorophyllin which consist other component such as carotene, xanthophylls, and many other components too. 24The HPLC (High performance liquid chromatography) was done in my own laboratory by Chaturvedi and Singh 25 identification of active components in the extracted and pure chlorophyllin.
Chlorophyllin is a derivative of chlorophyll, 26 in which magnesium atom at the centre of the ring is replaced with copper and phytol tail is lost. 27ue to loss of phytol ring chlorophyllin is more soluble and stable in water than chlorophyll. 28Chlorophyllin is accumulated in the intestine of exposed mosquito larvae and cercaria larva due to its higher solubility. 29,7ecently, Chaturvedi and Singh, 30 also observed the toxicity of chlorophyllin against Lymnaea acuminata at different wavelengths of visible light.Photosensitization of this chlorophyllin produces reactive oxygen substances causing cell death. 31Very earlier, Singh et al. 32 was studied the larvicidal activity of photodynamic product pheophorbide a in different wavelength of light as well as in sunlight against cercaria larvae.The 96 h LC 50 of toxicity of pure chlorophyllin (3.90 mg L -1 ) in sunlight is lower than active molluscicidal component Papain (9.74 mg L -1 ), Quercetin (65.91 mg L -1 ), thymol (10.71 mg L -1 ), and Citral (16.68 mg L -1 ) [33][34][35] and synthetic molluscicides phorate (15 mg L -1 ), carbaryl (14.4 mg L -1 ) and formothion (8.5 mg L -1 ). 36Extracted and pure chlorophyllin can be used as safe molluscicides as there was no mortality in fish even at 24 h LC 90 (against L. acuminata).
Evidence from the steep slope shows values indicate that a small increase in the concentration of the different treatments causes a marked mortality in snails.A t-ratio value greater than 1.96 indicates that the regression is significant.Values of heterogeneity factor less than 1.0 denote that in the replicate test 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-value indicates that the value of the mean is within the limits at all probability levels (90, 95 and 99) less than 0.5.

CONCLUSION
It can be stated that water soluble chlorophyllin extracted from green plant leaves, can be used as potent molluscicides.Due to the photodynamic nature of chlorophyllin, it has the potential to control fasciolosis by killing the snails.Use of photodynamic chlorophyllin against snails, is one of the effective biotechnological tool for effective control of fasciolosis.The use of chlorophyllin is safe, as it is not toxic to fish sharing the same habitat.