Toxicity of Chlorophyllin against Lymnaea acuminata at Different Wavelengths of Visible Light

Fasciolosis is a water and food-borne disease caused by the liver fluke Fasciola hepatica and Fasciola gigantica. This disease is widespread in different parts of the world. Lymnaeidae and Planorbidae snails are the intermediate hosts of these flukes. Snail population management is a good tool to control fasciolosis because gastropods represent the weakest link in the life-cycle of trematodes. Chlorophyll can be extracted from any green plant. Chlorophyllin was prepared from spinach in 100% ethanol by using different types of chemicals. The chlorophyll obtained from spinach was transformed into waterCorresponding author: vinaygkpuniv@gmail.com Divya Chaturvedi and Vinay Kumar Singh 26 soluble chlorophyllin. In the present paper, toxicity of chlorophyllin against the snail Lymnaea acuminata was time and concentration dependent. The toxicity of extracted and pure chlorophyllin at continuous 4 h exposure of sunlight was highest with lethal concentration (LC50) of 331.01 mg/L and 2.60 mg/L, respectively, than discontinuous exposure of sunlight up to 8 h with LC50 of 357.04 mg/L and 4.94 mg/L, respectively. Toxicity of extracted chlorophyllin was noted in the presence of different monochromatic visible lights. The highest toxicity was noted in yellow light (96 h, LC50 392.77 mg/L) and the lowest in green light (96 h, LC50 833.02 mg/L). Chlorophyllin in combination with solar radiation or different wavelength of monochromatic visible lights may become a latent remedy against the snail L. acuminata. It was demonstrated that chlorophyllin was more toxic in sunlight. Chlorophyllin is ecologically safe and more economical than synthetic molluscicides which have the potential to control the incidence of fasciolosis in developing countries.


INTRODUCTION
Fasciolosis is one of the most debilitating zoonotic diseases.Fresh water snail Lymnaea acuminata is the vector of liver flukes Fasciola gigantica, which causes endemic fasciolosis in cattle populations in Northern parts of India (Singh & Agarwal 1981;Singh & Singh 2016).About 94% buffaloes slaughtered in Gorakhpur, UP, India, are infected with F. gigantica (Singh & Agarwal 1981;Sunita et al. 2016).No continent is free from fasciolosis (Soliman 2008).The epidemiology of human fasciolosis has changed in recent years and significantly increased in the last two decades (Mas-Coma et al. 2005, 2009).Snail control is one of the best methods to eliminate fasciolosis.Mollusciciding is still in the centre of efforts to control fasciolosis.Synthetic molluscicides have been widely used for the effective control of carrier snails (Singh et al. 2010;Singh et al. 2012).Now it has been realised that synthetic molluscicides are toxic to nontarget animals and have a long term detrimental effect on the aquatic environment (Shafer et al. 2005;Upadhyay & Singh 2011).The molluscicides of plant origin are gaining importance than their synthetic counterparts (Singh et al. 1996).The derivates of chlorophyll is a very promising substance for snail control.Chlorophyllin can be easily extracted from any green plant.It is very economical and biodegradable.Molluscicidal activity of chlorophyllin has been reported against Lymnaea stagnalis, Biomphalaria spp.and Physa marmorata (Mahmoud et al. 2013).Previously, it has been reported that chlorophyllin is a potent larvicide (Wohllebe et al. 2009).Recently, it has been reported by Singh and Singh (2015) that the combination of monochromatic visible light with chlorophyllin has effective larvicidal activity against F. gigantica.Visible light spectrum initiates the orientation and locomotion of exposed snails towards a light source (Sakakibara et al. 2005).It was also noted that the L. acuminata snail monitors the intensity variation of visible light (Tripathi et al. 2013(Tripathi et al. , 2014)).Kumar and Singh (2015) reported that the toxic effect of chlorophyllin against Lymnaea acuminata in the presence of red light and sunlight.The aim of this present study is to evaluate the photo-toxicodynamic activity of extracted/pure chlorophyllin against fresh water snail L. acuminata in sunlight and in different wavelengths of monochromatic visible light.

Pure Compound
Chlorophyllin was purchased from Sigma Chemical Co.(St.Louis, Missouri, USA).

Experimental Animal
The fresh water snail, L. acuminata (2.35±0.30cm in length) was collected locally from ponds, lakes, and low lying submerged fields in Gorakhpur and was used as the test animals.The collected snails were acclimatised for 72 h in laboratory conditions.The experimental animals were kept in a glass aquarium containing dechlorinated tap water at room temperature (22°C-25°C).The pH, dissolved oxygen, free carbon dioxide, and bicarbonate alkalinity were 7.1-7.3,6.5-7.3 mg/L, 5.2-6.3mg/L, and 102-105 mg/L, respectively.Dead animals were removed immediately to prevent the water from being contaminated by decaying tissue.

Preparation of Extracted Chlorophyllin
Preparation of chlorophyllin was done according to the method of Wohllebe et al. (2011).Chlorophyll was isolated from spinach using 100% ethanol (for about 2 h at 55°C).Then, CaCO3 (about 1 mg/gm plant material) was added as a buffer, to prevent the transformation of chlorophyll into pheophytin.Before adding benzene the extract was irradiated with solar radiation for 1-2 h.The extract was filtered and 50 mL benzene was added.After addition of benzene, the mixture was shaken well and as a result the chlorophyll moved into the lipophilic benzene phase.The two phases were separated in separatory funnel and about 1.0 mL methanolic KOH was added to 50 mL of the benzene phase.Upon agitation 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.After separation of the methanolic KOH phase and the benzene phase, most of the chlorophyllin was found in the KOH phase.The extract was stored in a dark flask at room temperature.Only fresh chemicals were used in the course of these experiments.

Design of Photo Toxicity Experiments
Light experiment was set up according to the method of Tripathi et al. (2013).Xenon arc lamp (500 W) was used as the visible light source.Spectral response from 400 nm to 650 nm were produced with the help of the interference colour filters.Exposure of monochromatic light at different wavelengths and fixed intensity (500 Wm -2 ) was used to study their effects on snail's mortality.
A glass aquarium (70 cm diameter and 15 cm height) was filled with 3 L of dechlorinated tap water.Ten L. acuminata snails were placed in each aquarium.Then, all the 10 snails were exposed to different concentrations of extracted chlorophyllin and monochromatic visible light.Each treatment of extracted chlorophyllin and monochromatic visible light was replicated six times.Experiment was done at a room temperature of 22°C-25°C.

Toxicity Experiment
Toxicity experiments were done according to the method of Singh and Agarwal (1984).A total of 10 snails were placed in a glass aquarium containing 3 L of dechlorinated tap water.In all 3 sets (treated/control) of experiment, 10 snails were used at different concentrations of extracted/pure chlorophyllin (Table 1).For the first set of experiments, snails were treated with extracted chlorophyllin (600 mg/L, 700 mg/L, 800 mg/L, and 900 mg/L) in laboratory conditions (no exposure to sunlight or monochromatic visible light).Mortality of snails in laboratory condition was recorded at 24 h up to 96 h.In the second set of experiments, the extracted and pure chlorophyllin (200 mg/L, 300 mg/L, 500 mg/L, and 700 mg/L, and 2 mg/L, 4 mg/L, 6 mg/L, and 8 mg/L, respectively) was continuously exposed for 4 h in sunlight (10:00 a.m. to 2:00 p.m.).Mortality was recorded at every 1 h interval up to 4 h, and in another experiment the extracted and pure chlorophyllin (200 mg/L, 300 mg/L, 400 mg/L, and 500 mg/L and 3 mg/L, 5 mg/L, 7 mg/L, and 9 mg/L, respectively) was discontinuously exposed for 8 h in sunlight and a break of 1 h was given after each 2 h exposure to sunlight up to 8 h.Mortality was recorded at every 1 h interval up to 8 h.During the break period, the animals were placed under laboratory condition for 1 h.The third set of experiments was set up to observe the mortality of extracted chlorophyllin (300 mg/L, 500 mg/L, 700 mg/L, and 900 mg/L) treated snails in the presence of different wavelength of monochromatic visible lights in place of sunlight, while other controlled variables were held constant.Mortality of snails in laboratory condition was recorded at 24 h up to 96 h.For the first, second, and third sets of experiments, three types of control groups, I, II, and III, were also set up.In control group I no treatment of chlorophyllin was given in laboratory condition.In control group II, snails were only exposed to sunlight without any treatment of chlorophyllin.In control group III, snails were only exposed to monochromatic visible lights in laboratory condition without chlorophyllin treatment.Each treatment (treated/control) was replicated 6 times.Dead animals were removed from the aquarium immediately to avoid any contamination of water.Snail mortality was established by the contraction of the body within the shell and absence of response to a needle probe was taken as evidence of death.
The experiment was designed to observe the effect of extracted chlorophyllin in laboratory conditions (Table 2).Thereafter, the second set of experiments was designed to observe any effect of continuous exposure of sunlight or interrupted exposure of sunlight against chlorophyllin treated snails (Tables 3 and 4).In another experiment, the effect of different monochromatic visible lights on extracted chlorophyllin toxicity against L. acuminata was recorded (Table 5).All the above experiments indicate that continuous exposure of chlorophyllin in sunlight causes more toxicity against snails than laboratory condition.Interruption of light exposure also affects toxicity of chlorophyllin whereby the use of chlorophyllin in a cloudy environment will certainly affect the chlorophyllin's activity against snails.Toxicity observed in different spectral bands also indicate that variation in wavelength of visible light has a significant effect on the toxicity of chlorophyllin.Longer wavelength range red and yellow was more effective against chlorophyllin treated snails.
The lethal concentration values, lower and upper confidence limits (LCL, UCL), slope values, t-ratio, g-values, and heterogeneity factors were calculated using POLO computer software (Petaluma, CA, USA) (Robertson et al. 2007).The regression co-efficient between exposure time and different values of lethal concentration (LC50) was determined (Sokal & Rohlf 1996).Pure Chl -pure chlorophyllin; LCL -lower confidence limit, UCL -upper confidence limit.Significant negative regression (p<0.05) was observed between exposure time and LC50 treatments.Ts -testing significance of the regression coefficient -Ext Chl -3.72 + to -2.23 + and 265.8 ++ to 1268 ++ was observed.+ Linear regression between X and Y. ++ Non-linear regression between X and Y.  Notes: Six batches of 10 snails were exposed to different concentrations.Mortality was determined at 2 h to 8 h each with 1 h intervals.Concentrations given are the final concentration (w/v) in the glass aquarium water.Ext Chlextracted chlorophyllin, Pure Chl -pure chlorophyllin, LCL -lower confidence limit, UCL -upper confidence limit.Significant negative regression (p<0.05) was observed between exposure time and LC50 treatments.Ts -testing significant of the regression coefficient -Ext Chl -66.85 + to -42.51 + and 0.0 ++ to 1268 ++ and Pure Chl -1.81 + to -0.59 + and 0.0 ++ to 25.10 ++ was observed.+ Linear regression between X and Y. ++ Non-linear regression between X and Y.
Table 5: Toxicity of chlorophyllin in the presence of different spectra of light against L. acuminata.

RESULTS
The molluscicidal activity of extracted/pure chlorophyllin was tested at different times of exposure to various light spectra and chlorophyllin concentrations against the host snail L. acuminata (Table 1).A significant (p<0.05)negative regression was observed between the exposure time and the LC50 of the treatments (Tables 2-5).The toxicity of extracted chlorophyllin at 96 h, LC50 was 666.56 mg/L in laboratory condition (Table 2).Continuous 4 h exposure of sunlight in experimental aquarium containing chlorophyllin caused significant mortality in snails.Toxicity of extracted and pure chlorophyllin at continuous 4 h exposure of sunlight (4 h, LC50 331.01 mg/L and 2.60 mg/L, respectively) was more than break of 1 h after each 2 h exposure of sunlight up to 8 h (8 h, LC50 357.04 mg/L and 4.94 mg/L, respectively) (Tables 3 and 4).Toxicity experiment was conducted in laboratory condition with extracted chlorophyllin in the presence of monochromatic visible light.The highest toxicity in yellow light (96 h, LC50 392.77 mg/L) and lowest in green light (96 h, LC50 833.02 mg/L) was observed (Table 5).There was no mortality in control groups I, II, and III.It was observed that both extracted/pure chlorophyllin was more toxic in sunlight than in laboratory conditions.The slope values were steep and the separate estimation of the LC50 based on each of the six replicates was within the 95% confidence limits of LC50.The t-ratio was greater than 1.96, the heterogeneity factor was less than 1.0, and the g-value was less than 0.5 at all probabilities (90, 95, and 99) levels (Tables 2-5).

DISCUSSION
Chlorophyllin, a product of chlorophyll is a very effective photodynamic substance to control host L. acuminata.Recently, the toxicity of chlorophyllin in different wavelengths of visible light against redia and cercaria larvae of F. gigantica has been reported by Singh and Singh (2015).The larvicidal efficiency of chlorophyllin was demonstrated by Wohllebe et al. (2009) from spinach against mosquito larvae (Chaoborus crystallinus).Even at low concentrations, chlorophyllin was able to kill mosquito larvae and other small animals within a few hours in sunlight (Wohllebe et al. 2009).Chlorophyllin was also able to kill the protozoan parasite Ichthyophthirius multifiliis (Fouquet) of fresh water fish species (Wohllebe et al. 2012;Hader et al. 2016).Erzinger et al. (2011) also noted that mosquito larvae were killed by chlorophyllin.
Results of the present study indicate that the chlorophyllin extracted from the spinach is a potent molluscicide.Chlorophyllin toxicity against L. acuminata was time and concentration dependent, by the negative regression between exposure period and LC50 values of the different treatments.The timedependent toxic effect of tested plant products may be due to the uptake of active compounds by the snails, which progressively accumulated in the body with an increase time in the exposure period.It is also possible that the active compound could change into more toxic forms in the aquarium water or in the snail ' s body due to the sunlight.Four hours of continuous exposure of sunlight treatment was more effective as compared to the intermittent sunlight treatment of 2 h exposure followed by 1 h break.In sunlight, solubilised chlorophyllin transferred its excitation energy to oxygen, which produced singlet oxygen and other reactive oxygen species (ROS), which have the potential to kill the vector organism (He & Hader 2002;Tominaga et al. 2004).
In another set of toxicity test, extracted chlorophyllin treatments were given in combination of various wavelengths of monochromatic visible light at fixed intensity (500 Wm -2 ).Table 5 shows that the yellow light spectrum recorded the highest toxicity of extracted chlorophyllin against L. acuminata (96 h, LC50 392.77 mg/L) than other wavelengths of light.Higher toxicity of extracted chlorophyllin was also noted in other monochromatic visible lights than extracted chlorophyllin treatment in laboratory conditions (96 h, LC50 666.56 mg/L) without any exposure to monochromatic visible lights.Chlorophyllin is a photodynamic substance (Wohllebe et al. 2009).Consequently, toxicity of the chlorophyllin was higher in sunlight or in monochromatic visible light than in laboratory conditions.All the monochromatic visible lights have sufficient energy, which can elicit the response of the photodynamic product of chlorophyllin.Absorbed photon produces ROS.Even though the toxicity of chlorophyllin in laboratory conditions is also noted against L. acuminata, exposure to sunlight and monochromatic light of any wavelengths caused comparatively higher mortality of snails within a few hours.It was also reported that the retina of snails contain only one type of photo pigment, rhodopsin; they can differentiate only gradation of light intensity but not the colour of light (Chernorizov & Sokolov 2010).Earlier, it has been reported that Lymnaea stagnalis's eye has two types of ocular photoreceptors and three types of statocyst hair cells (Sakakibara et al. 2005).Type-A photoreceptor had more spectral sensitivity between 480 nm and 500 nm.Type-T photoreceptor had a much broader spectral sensitivity between 450 nm and 600 nm.At low intensity, Type-A photoreceptor and at higher intensity, Type-T photoreceptor are more sensitive.Probably, it seems that in the treatment of extracted chlorophyllin in exposure to the same intensity of visible monochromatic light, there is a significant variation in their toxicity as evident from different LC50 of chlorophyllin.Obviously, it also indicates that variation in wavelength of light has significant effect on mortality, as evident from higher toxicity of extracted chlorophyllin in yellow light.
The steep slope value indicates that a small increase in the concentration of molluscicide caused higher mortality.A t-ratio value greater than 1.96 indicates that the regression is significant (p<0.05).A heterogeneity factor value less than 1.0 denotes that in the replicate test of random samples, the concentration response is limited and thus, the model fits the data adequately.The index of significance of the potency estimation g indicates that the value of the mean is within the limit at all probability levels (90, 95, and 99) since it is less than 0.5.

Table 1 :
Concentration of extracted and pure chlorophyllin used in toxicity experiment against L. acuminata.

Table 2 :
Toxicity of extracted chlorophyllin in laboratory condition against L. acuminata.
Notes: Six batches of 10 snails were exposed to different concentrations.Mortality was determined at 24 h to 96 h.Concentrations given are the final concentration (w/v) in the glass aquarium water.Ext Chl -extracted chlorophyllin;
Notes: + Linear regression between X and Y. ++ Non-linear regression between X and Y.

Table 4 :
Toxicity of chlorophyllin against L. acuminata with discontinuous (1 h interval) exposure in sunlight.