1. Introduction

Fasciolosis a zoonotic disease of domestic herbivorous caused by the digenetic trematode of Fasciola hepatica and Fasciola gigantica (Singh and Agarwal, 198; Mas-Coma et al., 2005; Jigyasu and Singh, 2010). Human fasciolosis is endemic different parts of the world. In human endemic areas, fasciolosis mainly affects children and female, with fluke infecting even at very early age (Mas-Coma et al., 1999; 2009). Human infection has been reported in 51 different countries from 5 continents (Estaben et al., 1998). The incidence of fasciolosis in domestic herbivorous of Eastern Uttar Pradesh, India is very high. Singh and Agarwal (1981) reported that 94% of buffaloes slaughtered in local slaughter houses of Gorakhpur district are infected with F. gigantica. Fresh water snails Lymnaea acuminata act as the intermediate host of the F. hepatica and F. gigantica. One of the possible approaches to control this problem is to interrupt the life cycle of the liver fluke by eliminating the snail at threshold level. Synthetic molluscicides have been widely used for the effective control of vector snail/pest control has create the problem of acute and chronic toxicity to non target animal (Agarwal and Singh, 1988, Shafer et al., 2005).

Some time they are toxic to near target animal may have long term detrimental effect on the environment. Bio-pesticides are use toxic eco-friendly and also reduce the pollution problems caused by conventional pesticides (Singh et al., 1996a). Cow urine has been used for a long time in India to control pest, vector and pathogen. Cow urine is known to causes weight loss, and reverses certain cardiac and kidney problems, indigestion, stomach ache, edema, etc (Krishnamurthi et al., 2004). In Ayurveda, clinical effects of cow urine were described to counter kaph and pitta dosha (Krishnamurthi et al., 2004). It has been reported that use of different preparation of cow urine and dung cause viral, bacterial & fungal disease of plants (Sridhar 2002, Jarald et al., 2008). Kalkude et al., (2010) observed cow urine concentrate a potent antimicrobial and antihelmintic activity. Tripathi et al., (2010) has been reported that the Haryana breed cow urine have potent molluscicides and the sub lethal treatment of the cow urine powder with combination of different plant derived molluscicides caused significant reduction in fecundity hatchability and survival of snail Lymnaea acuminata. Recently, we have observed that molluscicidal activity of freeze-dried cow urine of different Indian breeds against Lymnaea acuminata (Kumar et al., 2011). In the present study describes the molluscicidal activity of different Indian breeds freeze-dried cow urine (FCU) such as, Sahiwal, Geer and Tharparkar with combination of different plant molluscicides against the harmful snail Lymnaea acuminata.

2. Materials and Methods

2.1. Cow Urine Preparation

Cow urine was collected locally Gorakhpur in sterilized bottles from green grass grazing 3- year old different Indian breeds such as Sahiwal, Geer and Tharparkar. No other food materials were given to the experimental animals.

1- Sahiwal, Geer and Tharparkar cow urine kept for 15 in sun light 8h/days.

Table 1. Toxicity of freeze-dried cow urine (FCU) kept for 15 days in sunlight (8h/day) of different breeds of cow Sahiwal, Geer, Tharparkar and its binary combination (1:1) with A. squamosa, C. sinensis, F. asafoetida and A. indica against L. acuminata at different exposure period. Exposure period Treatment LC50 mg/l (w/v) Limits LCL- UCL Slope value t- ratio g- value Heterogeneity 24h Sh CU+AS 97.27 81.80-134.47 2.98±0.58 5.15 0.14 0.21 Gr CU+AS 148.04 130.70-186.16 3.83±0.75 5.07 0.14 0.30 Th CU+AS 158.06 136.98-210.64 3.64±0.75 4.81 0.16 0.18 Sh CU+CS 161.62 139.91-216.26 3.78±0.77 4.85 0.16 0.24 Gr CU+CS 159.09 139.27-205.18 4.06±0.79 5.11 0.14 0.23 Th CU+CS 150.18 133.03-186.94 4.07±0.07 5.26 0.13 0.43 Sh CU+FA 159.22 138.59-209.26 3.85±0.78 4.94 0.15 0.22 Gr CU+FA 183.31 164.36-227.26 4.91±0.99 4.93 0.15 0.32 Th CU+FA 132.87 121.52-151.28 4.81±0.77 6.24 0.09 0.22 Sh CU+AI 118.81 103.93-151.24 3.69±0.70 5.26 0.13 0.27 Gr CU+AI 119.30 104.10-152.83 3.63±0.69 5.23 0.14 0.17 Th CU+AI 120.02 103.45-159.79 3.29±0.67 4.88 0.16 0.11 48h Sh CU+AS 73.24 62.75-92.73 2.53±0.49 5.07 0.14 0.25 Gr CU+AS 122.41 108.80-146.62 3.24±0.68 4.70 0.17 0.18 Th CU+AS 102.26 75.25-130.36 2.47±0.52 4.68 0.17 0.12 Sh CU+CS 124.35 110.39-150.10 3.27±0.68 4.71 0.17 0.24 Gr CU+CS 135.34 119.56-168.60 3.36±0.70 4.77 0.16 0.14 Th CU+CS 128.82 114.99-154.62 3.51±0.70 5.00 0.15 0.38 Sh CU+FA 128.79 113.96-158.29 3.22±0.69 4.65 0.17 0.11 Gr CU+FA 160.07 143.84-194.59 3.80±0.84 4.50 0.19 0.28 Th CU+FA 111.23 99.91-126.43 3.57±0.68 5.20 0.14 0.12 Sh CU+AI 93.08 82.33-110.99 3.11±0.62 5.00 0.15 0.17 Gr CU+AI 87.73 61.58-79.15 3.39±0.62 5.45 0.12 0.22 Th CU+AI 102.41 87.66-137.84 2.52±0.61 4.11 0.22 0.10 72h Sh CU+AS 52.31 44.35-60.67 2.63±0.48 5.43 0.13 0.33 Gr CU+AS 95.29 80.40-108.74 2.89±0.67 4.30 0.20 0.28 Th CU+AS 90.67 89.03-117.07 3.04±0.67 4.50 0.18 0.24 Sh CU+CS 99.96 87.94-112.48 3.35±0.68 4.93 0.15 0.21 Gr CU+CS 99.95 87.83-112.57 3.33±0.68 4.89 0.16 0.23 Th CU+CS 94.46 83.07-104.90 3.64±0.68 5.29 0.13 0.45 Sh CU+FA 101.49 88.80-115.29 3.17±0.67 4.68 0.17 0.13 Gr CU+FA 130.63 119.34-143.30 4.45±0.82 5.40 0.13 0.35 Th CU+FA 92.61 80.02-103.52 3.40±0.68 4.98 0.15 0.17 Sh CU+AI 71.20 61.98-79.81 3.35±0.62 5.39 0.13 0.27 Gr CU+AI 70.71 61.58-79.15 3.39±0.62 5.45 0.12 0.22 Th CU+AI 75.98 62.79-90.17 2.38±0.59 3.97 0.24 0.14 96h Sh CU+AS 36.19 28.91-41.82 3.12±0.51 6.01 0.10 0.72 Gr CU+AS 78.99 65.94-88.21 3.83±0.72 5.30 0.13 0.39 Th CU+AS 81.52 70.36-89.93 4.21±0.73 5.73 0.11 0.63 Sh CU+CS 80.23 68.66-88.77 4.15±0.73 5.66 0.12 0.37 Gr CU+CS 81.87 71.60-89.80 4.50±0.74 6.02 0.10 0.61 Th CU+CS 77.56 67.01-85.36 4.64±0.76 6.06 0.10 0.61 Sh CU+FA 81.09 68.82-90.07 3.92±0.72 5.42 0.13 0.55 Gr CU+FA 106.91 93.86-116.58 4.70±0.83 5.60 0.12 0.76 Th CU+FA 77.93 68.56-85.05 5.12±0.79 6.42 0.09 0.44 Sh CU+AI 58.92 50.74-65.23 4.23±0.68 6.17 0.10 0.55 Gr CU+AI 58.74 49.60-65.60 3.85±0.66 5.80 0.11 0.37 Th CU+AI 59.82 50.60-66.80 3.77±0.66 5.71 0.11 0.52 Six batches of 10 snails were exposed different concentration of the above molluscicides with different breeds of cow urine. Significant negative regression (P<0.05) was observed between exposure time and LC50 of treatments. Ts-testing significant of the regression coefficient: ShCU+AS-16.24+; GrCU+AS-13.67+; ThCU+AS-8.22++; ShCU+CS-9.47+; GrCU+CS-11.94+; ThCU+CS-11.12+; ShCU+FA-9.55++; GrCU+FA-30.56+; ThCU+FA-16.64+; ShCU+AI-9.30+; GrCU+AI-16.34++; ThCU+AI-15.12+. +: linear regression between x and y; ++: non–linear regression between log x and log y. Abbreviation; Sh Sahiwal, Gr Geer, Th Tharparkar, AS Annona squamosa, CS Camellia sinensis FA Ferula asafoetida, AI Azadirachta indica.

2-Sahiwal, Geer and Tharparkar cow urine kept for 15 in laboratory condition.

3- Binary combinations (1:1) of plant molluscicides such as Annona squamosa (Annonaceae) seed powder, Azadirachta indica (Meliaceae) oil, Ferula asafoetida (Apiaceae) root latex and Camellia sinensis (Theaceae) leaf with freeze dried cow urine (FCU) of different breeds cow urine kept for 15 days in laboratory and sun light (8 h/days).

2.2. Plants Used

Plants used in this works were collected locally, and identified at the herbarium of the Botany Department, DDU Gorakhpur University, Gorakhpur, India where voucher herbarium specimens (# No 3228 for Annona squamosa and 3815 for Ferula asafoetida) are on deposit. A. Indica oil was obtained from Indian Herbs Saharanpur, UP, India. Annona Squamosa seed powders were prepared by using the methods of Singh and Singh (2001).

Table 2. Toxicity of freeze-dried cow urine (FCU) kept for 15 days in laboratory condition of different breeds of cow Sahiwal, Geer, Tharparkar and its binary combination (1:1) with A. squamosa, C. sinensis, F. asafoetida and A. indica against L. acuminata at different exposure period. Exposure period Treatment LC50 mg/l (w/v) Limits LCL-UCL Slope value t- ratio g- value Heterogeneity 24h Sh CU+AS 81.40 70.87-101.26 3.11±0.55 5.62 0.12 0.16 Gr CU+AS 132.95 111.89-192.10 3.25±0.70 4.62 0.17 0.29 Th CU+AS 151.50 133.20-192.81 3.84±0.67 5.06 0.15 0.23 Sh CU+CS 118.90 102.98-155.77 3.38±0.67 5.00 0.15 0.24 Gr CU+CS 148.19 130.34-188.32 3.70±0.74 4.98 0.15 0.29 Th CU+CS 153.48 133.96-199.83 3.68±0.75 4.90 0.16 0.20 Sh CU+FA 136.09 115.07-192.37 3.54±0.73 4.84 0.16 0.22 Gr CU+FA 141.15 127.39-166.56 4.48±0.77 5.78 0.11 0.23 Th CU+FA 125.47 108.25-166.64 3.58±0.71 5.05 0.15 0.29 Sh CU+AI 111.99 97.91-142.28 3.34±0.66 5.07 0.14 0.11 Gr CU+AI 107.13 94.31-132.89 3.35±0.65 5.16 0.14 0.20 Th CU+AI 90.67 75.25-130.36 2.47±0.52 4.68 0.17 0.12 48h Sh CU+AS 62.77 53.56-76.29 2.41±0.48 4.97 0.15 0.15 Gr CU+AS 90.65 81.85-103.31 3.80±0.63 5.94 0.10 0.33 Th CU+AS 127.39 111.18-162.93 2.82±0.68 4.14 0.22 0.15 Sh CU+CS 90.23 80.31-105.30 3.27±0.62 5.24 0.14 0.26 Gr CU+CS 121.16 107.90-143.95 3.28±0.68 5.78 0.16 0.31 Th CU+CS 136.53 118.46-181.33 2.89±0.69 4.19 0.21 0.18 Sh CU+FA 99.56 87.28-123.43 2.99±0.62 4.81 0.16 0.16 Gr CU+FA 118.73 108.18-134.04 4.16±0.71 5.84 0.11 0.20 Th CU+FA 100.29 87.58-125.96 2.90±0.62 4.66 0.17 0.19 Sh CU+AI 89.66 78.85-106.85 2.94±0.61 4.79 0.16 0.13 Gr CU+AI 87.52 77.56-102.00 3.15±0.61 5.10 0.14 0.17 Th CU+AI 68.34 57.30-88.96 2.14±0.48 4.43 0.19 0.24 72h Sh CU+AS 44.44 35.69-51.82 2.49±0.48 5.19 0.14 0.29 Gr CU+AS 82.30 72.99-94.06 3.25±0.61 5.26 0.13 0.42 Th CU+AS 91.93 76.40-104.69 2.89±0.67 4.29 0.20 0.21 Sh CU+CS 69.04 58.83-77.96 3.12±0.61 5.08 0.14 0.22 Gr CU+CS 121.16 107.90-143.95 3.28±0.68 5.78 0.16 0.31 Th CU+CS 91.15 80.48-100.42 3.96±0.70 5.66 0.12 0.38 Sh CU+FA 75.41 64.50-87.42 2.94±0.60 4.45 0.19 0.19 Gr CU+FA 97.59 86.92-108.05 3.80±0.69 5.50 0.12 0.14 Th CU+FA 72.55 62.46-82.24 3.06±0.61 4.99 0.15 0.20 Sh CU+AI 66.79 56.29-75.43 3.13±0.61 5.06 0.15 0.24 Gr CU+AI 68.49 58.18-77.34 3.12±0.61 5.07 0.14 0.15 Th CU+AI 46.85 38.21-54.60 2.48±0.48 5.17 0.14 0.30 96h Sh CU+AS 34.30 26.77-39.98 3.09±0.52 5.91 0.11 0.52 Gr CU+AS 67.79 59.02-75.44 3.60±0.63 5.69 0.11 0.44 Th CU+AS 75.76 63.35-84.42 4.16±0.75 5.53 0.12 0.59 Sh CU+CS 57.48 49.96-63.29 4.65±0.65 6.47 0.09 0.48 Gr CU+CS 80.89 68.84-89.76 3.99±0.72 5.54 0.12 0.29 Th CU+CS 80.00 69.28-88.04 4.44±0.75 5.89 0.11 0.88 Sh CU+FA 58.18 48.49-65.30 3.70±0.66 5.58 0.12 0.48 Gr CU+FA 77.52 66.87-85.36 4.62±0.76 6.03 0.10 0.33 Th CU+FA 56.16 47.09-62.80 4.03±0.68 5.86 0.11 0.47 Sh CU+AI 56.73 48.46-62.95 4.34±0.70 6.19 0.10 0.39 Gr CU+AI 55.37 46.55-61.83 4.18±0.70 5.98 0.10 0.42 Th CU+AI 35.95 28.44-41.69 3.05±0.51 5.90 0.11 0.74 Six batches of 10 snails were exposed different concentration of the above molluscicides with different breeds of cow urine. Significant negative regression (P<0.05) was observed between exposure time and LC50 of treatments. Ts-testing significant of the regression coefficient: ShCU+AS-10.98+; GrCU+AS-10.44++; ThCU+AS-10.49+; ShCU+CS-10.78++; GrCU+CS-3.88+; ThCU+CS-5.87+; ShCU+FA-8.93++; GrCU+FA-57.01+; ThCU+FA-13.84+; ShCU+AI-8.74+; GrCU+AI-15.94+; ThCU+AI-9.59+. +: linear regression between x and y; ++: non–linear regression between log x and log y. Abbreviation; Sh Sahiwal, Gr Geer, Th Thaparkar, AS Annona squamosa, CS Camellia sinensis FA Ferula asafoetida, AI Azadirachta indica.

2.3. Test Animal

The adult snails Lymnaea acuminata (2.60 ± 0.30 cm length) were collected from the local ponds and pools in Gorakhpur. The collected snails were storage 72h in laboratory condition for acclimatized before the treatment.

2.4. Experiment

Toxicity experiment was performed by the methods of Singh and Agarwal (1984). Ten experimental animals were kept in a glass aquaria containing 3 liter of dechlorinated tap water at 22°C to 24°C. The pH of the water was 7.1-7.3 and dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 6.5-7.2 mg/l, 5.2-6.3 mg/l and 102.0-105.0 mg/l, respectively. Dead animals were removed immediately from the aquaria to avoid any contamination. Snails were exposed to binary combinations (1:1) of plant derived molluscicides such as A. squamosa seed powder, A. indica oil, F. asafoetida root latex and C. sinensis with freeze-dried powder (FCU) of different breeds cow urine kept for 15 days in laboratory condition or sun light (8 h/days). Six aquaria were set up for each concentration. Mortality was recorded at 24h interval up to 96h exposure period. Snail mortality was established by contraction of the body within the shell and absence of response to a needle probe was taken as evidence of death.

2.5. Statistical Analysis

Lethal concentration value (LC₅₀) upper and lower confidence limits (UCL and LCL) and slope value, t-ratio, g-value, and heterogeneity were calculated according to the POLO computer programme of Robertson et al., (2007). The regression coefficient was determined between exposure time and different values of LC₅₀ (Sokal and Rohlf 1973).

2.6. Result

The fresh cow urine of different breeds of cow was pale yellow in colour, it kept for 15 days in sun light (8h/day) the colour was converted into dark brown, while it kept for 15 days in laboratory condition it light brown in colour. Toxicity of binary combination (1:1) of plant molluscicides and freeze-dried cow urine (FCU) of different breeds of cow such as Sahiwal, Geer, and Tharparkar kept for 15 days in sunlight (8h/day) and laboratory condition against snail L. acuminata was time and concentration dependent.

The toxicity of binary combination (1:1) of Sahiwal freeze-dried cow urine+A. squamosa kept for 15 days in sunlight (8h/day) (24h LC₅₀ -97.27mg/l) was more effective than the Geer (24h LC₅₀-148.04mg/l) and Tharparkar freeze-dried cow urine (24h LC₅₀-158.06mg/l), respectively (Table-1). Binary combination of Sahiwal freeze-dried cow urine+A. squamosa (24h LC₅₀ -81.40mg/l) kept for 15 days in laboratory condition was more toxic than Geer (24h LC₅₀ -132.95mg/l) and Tharparkar (24h LC₅₀ -151.50mg/l), respectively (Table-1). Toxicity of 96h LC₅₀ of Sahiwal freeze-dried cow urine+A. squamosa (96h LC₅₀ -34.30mg/l) kept for 15 days in laboratory condition is more toxic than the other breed of cow (FCU) (Table-2). The 24h LC₅₀ of Tharparkar cow urine kept for 15 days in sunlight and laboratory condition with F. asafoetida (132.87mg/l and 125.47 mg/l) was lower than the Sahiwal (159.22mg/l and 136.09mg/l) and Geer (183.31mg/l and 141.15mg/l) cow urine kept for 15 days in sunlight and laboratory condition, respectively (Table-1, 2).

The other binary combination of Sahiwal cow urine kept for 15 days in sunlight with A. indica oil (24h LC₅₀ -118.81mg/l and 96h LC₅₀ -58.92mg/l) Tharparkar cow urine+A. indica oil (24h LC₅₀-120.02mg/l and 96h LC₅₀-59.82mg/l) and Geer cow urine+A. indica oil (24h LC₅₀-119.30mg/l and 96h LC₅₀-58.74mg/l), respectively almost similar have same toxicity against L. acuminata. Tharparkar cow urine kept for 15 days in sun light with C. sinensis (24h LC₅₀-150.18mg/l) was more effective than the Sahiwal (24h LC₅₀-161.62mg/l) and Geer cow urine (24h LC₅₀-159.09mg/l), respectively (Table- 2).

The slope values were steep and separate estimation of LC based on each of the six replicates was found to be within 95% confidence limits of LC₅₀. The t- ratio was 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 (90, 95 and 99).

3. Discussion

The result clearly demonstrate that the freeze-dried cow urine (FCU) of different Indian breeds kept for 15 days in sunlight (8h/day) and laboratory condition and its binary combination (1:1) with plant molluscicides such as A. squamosa seed powder, A. indica oil, F. asafoetida root latex and C. sinensis leaf are potent molluscicide. It has been reported by various scientist that cow urine fermented and distillate cow urine cause insecticidal, fungicidal, antimicrobial, antihelmintic and molluscicidal activity (Adane and Gautam, 2003; Tripathi et al., 2006; Kalkude, et al., 2010; Kumar et al., 2011). Urea is major component present in urine and is the end product of protein metabolism (Kumar et al. 2011). It is a strong antibacterial agent. Similarly, uric acid is also present in urine is a strong antibacterial. It may be possible that in the present study cow urine also acts as bio-enhancer with the plant products so that the active molluscicidal components of these plants reaches higher titer in snail body. It was natural disinfectant and anti-septic qualities. In traditional medicines cow urine was consumed as an effective and simple medicine. It contains 24 types of salt as well as iron, calcium, phosphorus, carbonic acid, potash and lactose. Its main constituent show disinfectant activity is carbolic acid, which is mixture of phenol and cresol (Kelly, 1997, Khanuja et al., 2002).

Cow urine kept for 15 days in sunlight (8h/day) and laboratory condition of Sahiwal+A. squamosa (96h LC₅₀ -36.19mg/l and 34.30mg/l) was more toxic than the other combination. It has been reported that the toxicity of annonaceous acetogenins found cytotoxic and antitumor activity (Fang et al., 1993). It behaves as bioenhancer and enhances the antimicrobial effect of antibiotics many fold (Khanuja et al., 2002). Adane and Gautam (2003) have been reported insecticidal and fungicidal activity of fermented cow urine. Cow urine distillate act as antimicrobial against clinical pathogens (Sathasiyam et al., 2010). Tripathi et al., (2010) have been reported that the sublethal doses of cow urine and their formulation with plant derived molluscicides caused a significant reduction in fecundity, hatchability and survival of snail L. acuminata. Mikolajczak et al., (1989) annonaceous acetogenins using for the control of pest. In the present observation the toxicity of Sahiwal cow urine kept for 15 days in sunlight and laboratory condition with squamosa is more pronounced at 96h exposure period. It indicate that the cow urine prolong and enhance the toxicity of A. squamosa up to 96h, while other binary combination Geer cow urine kept for 15 days in sunlight and laboratory condition with A. indica oil (96h LC₅₀ -58.74mg/l, 55.37mg/l), respectively were more toxic than other combination. The active component of A. indica oil is azadirachtin, toxic to the snail L. acuminata was effective only 24h (Singh et al., 1996b). Azadirachtin, the active component of Azadirachta indica, is not stable in water after 24h (Szeto and Wan, 1996). Increase in the toxicity of binary combination of urine of different breeds of cow urine with plant molluscicides may be due to the inhibition of enzyme which detoxify the active molluscicidal agents in plants or it may be possible that in binary combination of two molluscicidal components act at different sites in the snail body which ultimately result come in higher toxicity against the snail L. acuminata.

It is evident that the steep slope value that a small increase in the binary combination of plant derived molluscicides such as A. squamosa seed powder, A. indica oil, F. asafoetida root latex and C. sinensis with freeze-dried powder of different breeds cow urine kept for 15 days laboratory and sun light 8 h/days was causes a higher mortality in snail. A t- ratio value greater than 1.96 indicates that the regression is significant. Heterogeneity factor value is less than 1.0 denoted that in the replicate test of random sample, the concentration response line would fall within 95% confidence limits and thus the model fits our data adequately. The index of significant of protein estimation g-value indicates that the value of mean is within the limits at all probability level (90, 95 and 99%).

Present studies demonstrate that the different breeds of freeze-dried cow urine (FCU) and plant molluscicides are potent molluscicides. They kill the intermediate host of Fasciola hepatica and Fasciola gigantica. This concept is an advanced safe technology for the control of harmful snail without spending more active molluscicides directly in the aquatic environment.

ACKNOWLEDGMENTS

One of the authors (Shiv Kumar) is thankful to RajivGandhi National Fellowship (RJNF), University Grants Commission (UGC), and New Delhi letter No. F. 14-2(SC) /2010 (SA-III) for financial assistance.