Molluscicidal Activity of Cannabis sativa, Acacia nilotica and Tinospora cordifolia Extracts Against Vector Snail Lymnaea acuminata

Received 08 Feb, 2023

Accepted 07 Apr, 2023

Published 27 May, 2023

Background and Objective: Fresh water snails of family lymnaeidae serve as vectors for parasitic trematode Fasciola hepatica which causes a serious zoonotic disease fascioliasis. The study aimed to investigate the possibility of indigenous plants Cannabis sativa, Acacia nilotica and Tinospora cordifolia to serve as natural molluscicides to control the population of vector snails. Materials and Methods: Aqueous extracts were prepared from freshly collected aerial parts of these plants. Healthy and acclimatized snails were exposed against different concentrations of the plant extracts continuously up to 96 hrs to analyse the toxicity of these extracts. Results: Mortality of snails after exposure of different combinations of the extracts indicates that the extracts from indigenous plants C. sativa, A. nilotica and T. cordifolia shows very promising molluscicidal activity against Lymnaea acuminata. Leaves of Cannabis sativa showed highest toxicity. Conclusion: This study obviously indicates that these indigenous plant extracts can be used as a potent natural molluscicides.

INTRODUCTION

Fascioliasis is an important zoonotic disease caused by two trematodes, Fasciola hepatica and Fasciola gigantica^1-3. These parasitic trematodes are of considerable veterinarian and medical importance. Fascioliasis has recently proved to be an important public health problem, with human cases reported in 91 countries and the global burden of fascioliasis estimated to be 2.7 million^4,5. Population in the developing countries are likely to be most affected by facioliasis as the COVID-19 pandemic impacts their health symptoms and efforts to control Fasciola decline^6-8. Yattoo et al.⁹ reported two cases of human fascioliasis from Kashmir Valley in India.

Species of freshwater snails from the family Lymnaeidae are well known for their role as the intermediate host for Fasciola species. The most important host for F. hepatica is Lymnaea acuminata (Fig. 1 and 2) and Lymnaea rufescence in Indian subcontinent^8-12 and Lymnaea trunculata in Europe, Asia, Africa and North America^8,13,14. The control of fascioliasis depends on the understanding of the ecology, biology and distribution of the intermediate host snails. Controlling the population of the host snails below the threshold level can be a rapid and effective means of reducing fascioliasis^3,14.

Fig. 1: Lymnaea acuminata

Fig. 2: Lymnaea acuminata (Dead)

Various synthetic molluscicides have been widely used for effective control of harmful snails and such molluscicides includes copper sulphate, trifenmorph, Bayluscide, sodium pentachlorophenate, copper pentachlorophenate and niclosamide^15,16. Significant success has been achieved by the use of these synthetic chemicals for the elimination of harmful snails. Nonetheless, the use of these synthetic molluscicides for controlling snails causes serious environmental pollution¹⁷.

There are large numbers of pharmacological effects of C. sativa, A. nilotica and T. cordifolia have been reported that C. sativa shows anti-oxidant activity^18,19, larvicidal activity against mosquito larvae²⁰ and anti-microbial activity^21,22. Revathi et al.²³ reported anti-cancer, anti-microbial and anti-oxidant properties of A. nilotica. Anti-cancer, anti-viral infection, inflammation, immunomodulatory role and neurological and anti-diabetes properties of T. cordifolia have already been reported²⁴.

The search indigenous of plants with molluscicidal activity is considered more sustainable than the use of synthetic molluscicides. The present study aimed to evaluate the molluscicidal activity of Cannabis sativa, Acacia nilotica and Tinospora cordifolia to explore the full potential of these indigenous plants as potent molluscicides.

MATERIALS AND METHODS

The experiment was carried out in the Malacology Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur in August to November, 2022.

Collection of plant materials: Aerial parts of Cannabis sativa (leaf, stem), Acacia nilotica (leaf, bark) and Tinospora cordifolia (leaf, stem) were collected freshly from local areas and identified by the Department of Botany, DDU Gorakhpur University, Gorakhpur, India.

Animal collection: Adult Lymnaea acuminata (1.35±0.2 cm in length) were collected from freshwater ponds of Gorakhpur District, Uttar Pradesh, India. The snails were acclimatized for 72 hrs in laboratory conditions. Ten snails were then allocated to each of the groups and immersed in 3 L untreated dechlorinated tap water at 23±1°C. The pH, free carbon dioxide, dissolved oxygen and bicarbonate alkalinity were 7.1-7.3, 5.2-6.3, 6.6-7.3 and 102-104 mg L^–1, respectively. The dead snails were removed from the aquaria to avoid any contamination.

Plant extract preparation: The extraction of plant parts was performed by the method of Singh and Agarwal²⁵ with some modifications. Freshly collected leaves and stems of C. sativa were washed thoroughly, then finely cut into small pieces and then separately mixed with water (50 g leaves/stem+50 mL water) then filtered with Whatman No. 1 filter paper. The filtered extract thus obtained was evaporated under a vacuum. The residues thus obtained were used for the determination of toxicity. The same procedure was performed with A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) extraction.

Molluscicidal activity test: Molluscicidal evaluation of plant extracts was performed according to WHO guidelines as modified by Singh and Agarwal²⁵. Ten uninfected snails were kept in a glass aquarium containing 3 L deionized water. In each setup, snails were prevented from crawling out of the aquaria employing a fine mesh placed above the water surface. Snails were challenged for 96 hrs with various concentrations of different plant part extracts singly and in various binary combinations (Table1). Six replicates were set up for each concentration. Control experiments were performed with deionized water without any treatment (negative control). These snails were neither fed nor disturbed during the exposure period. Mortality of snails was recorded at an interval of 24 up to 96 hrs. Death of the snails was determined and confirmed by lack of reaction to irritation of foot with needle prove to elicit typical withdrawal movements and contraction of their body in the shell (Fig. 2).

Statistical analysis (p<0.05): The LC₅₀ values, Lower Confidence Limits (LCL), Upper Confidence Limits (UCL), t-ratio heterogeneity factor, slope values and g-values were calculated by polo software program (PoLo Plus LeOra software version 2.0).

Ethical consideration: All applicable protocols of institutional, national or international guidelines for the care and use of animals were followed during this experiment.

RESULTS

The toxicity of extracts of C. sativa (leaf, stem), A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) against Lymnaea acuminata was time and concentration-dependent. The LC₅₀ determined after 24 hrs treatment was highest in C. sativa leaf extract (304.31 mg L^–1) (Table 2). Cannabis sativa stem extract was more toxic (24 hrs LC₅₀ = 404.39 mg L^–1) than aerial part extracts of A. nilotica and T. cordifolia (Table 2). There was a significant (p<0.05) negative correlation between the LC₅₀ and the exposure period. The order of 24 hrs toxicity of different plant parts extracts singly against L. acuminata were C. sativa leaf>C. sativa stem>A. nilotica leaf>T. cordifolia leaf>A. nilotica bark>T. cordifolia stem (Table 2).

The binary combination (1:1) of C. sativa leaf+A. nilotica leaf was more toxic (24 hrs LC₅₀ = 174.76 mg L^–1) than other combinations in the 1:1 ratio (Table 3) and their single treatments. The order of toxicity of different binary combinations (1:1) against L. acuminata was C. sativa leaf+A. nilotica leaf>C. sativa stem+A. nilotica bark>T. cordifolia leaf+C. sativa leaf>T. cordifolia leaf+A. nilotica leaf>T. cordifolia stem+C. sativa stem>T. cordifolia stem+A. nilotica bark (Table 3).

Table 1:

Concentration of different plant products (crude extracts) used for toxicity determination against Lymnaea acuminata

Binary combinations	Molluscicide	Concentration (mg L–1)
	Cannabis sativa leaf	150, 200, 250, 300
	Cannabis sativa stem	250, 300, 350, 400
	Acacia nilotica leaf	200, 300, 400, 500
	Acacia nilotica bark	350, 450, 550, 650
	Tinospora cordifolia leaf	300, 400, 500, 600
	Tinospora cordifolia stem	450, 550, 650, 750
1:01	C.S. Leaf+A.N. leaf	110, 130, 150, 170
	C.S. Stem+A.N. bark	125, 150, 175, 200
	T.C. Leaf+C.S. leaf	100, 150, 200, 250
	T.C. Stem+C.S. stem	125, 175, 225, 275
	T.C. Leaf+A.N. leaf	125, 175, 225, 275
	T.C. Stem+A.N. bark	100, 175, 250, 325
1:05	C.S. Leaf+A.N. leaf	30, 50, 70, 90
	C.S. Stem+A.N. bark	70, 90, 110, 130
	T.C. Leaf+C.S. leaf	30, 50, 70, 90
	T.C. Stem+C.S. stem	50, 70, 90, 110
	T.C. Leaf+A.N. leaf	30, 50, 70, 90
	T.C. Stem+A.N. bark	50, 70, 90, 110
5:01	C.S. Leaf+A.N. leaf	10, 30, 50, 70
	C.S. Stem+A.N. bark	30, 50, 70, 90
	T.C. Leaf+C.S. leaf	50, 70, 90, 110
	T.C. Stem+C.S. stem	90, 110, 130, 150
	T.C. Leaf+A.N. leaf	50, 70, 90, 110
	T.C. Stem+A.N. bark	90, 110, 130, 150
C.S. leaf: Cannabis sativa leaf, C.S. stem: Cannabis sativa stem, A.N. Leaf: Acacia nilotica leaf, A.N. bark: Acacia nilotica bark, T.C. leaf: Tinospora cordifolia leaf and T.C. stem: Tinospora cordifolia stem

Table 2:

Concentration of different plant products (crude extracts) used for toxicity determination against Lymnaea acuminata

	Limits
Treatment	Exposure period	Effective dose (mg L–1)	LCL	UCL	Slope value	‘t’ ratio	Heterogeneity factor
C. sativa leaf	24 hrs	LC50 = 304.31	271.84	375.92	4.33±0.86	5.03	0.24
	48 hrs	LC50 = 248.92	226.48	283.91	4.05±0.78	5.18	0.22
	72 hrs	LC50 = 197.54	172.66	218.64	3.73±0.76	4.91	0.25
	96 hrs	LC50 = 160.96	135	178.48	4.51±0.82	5.48	0.43
C. sativa stem	24 hrs	LC50 = 404.39	373.82	469.31	6.31±1.26	5.01	0.23
	48 hrs	LC50 = 350.89	328.72	384.67	5.86±1.14	5.1	0.23
	72 hrs	LC50 = 304.80	278.24	327.73	5.26±1.11	4.71	0.17
	96 hrs	LC50 = 264.00	273.29	281.8	7.02±1.23	5.69	0.37
A. nilotica leaf	24 hrs	LC50 = 535.98	463.48	704.34	3.63±0.70	5.16	0.24
	48 hrs	LC50 = 429.67	378.26	524.62	3.14±0.60	5.17	0.27
	72 hrs	LC50 = 323.46	285.36	364.42	3.33±0.58	5.57	0.53
	96 hrs	LC50 = 247.89	208.51	278.4	3.62±61	5.93	0.67
A. nilotica bark	24 hrs	LC50 = 675.75	607.06	833.45	4.81±0.98	4.91	0.02
	48 hrs	LC50 = 547.54	504.19	613.08	4.64±0.87	5.29	0.24
	72 hrs	LC50 = 453.52	407.75	494.14	4.45±0.85	5.18	0.38
	96 hrs	LC50 = 375.73	315.32	414.6	4.57±0.90	5.08	0.55
T. cordifolia leaf	24 hrs	LC50 =593.41	583.69	700.31	4.97±0.90	5.5	0.22
	48 hrs	LC50 =487.88	444.18	552.55	4.06±0.77	5.52	0.27
	72 hrs	LC50 =385.22	335.75	425.22	3.85±0.76	5.04	0.26
	96 hrs	LC50 =321.59	277.69	325.62	5.20±0.86	6.05	0.41
T. cordifolia stem	24 hrs	LC50 = 772.62	706.98	919	5.81±1.18	4.92	0.19
	48 hrs	LC50 = 651.25	606.67	719.66	5.39±1.05	5.1	0.19
	72 hrs	LC50 =552.65	504.35	593.78	5.30±1.03	5.1	0.19
	96 hrs	LC50 = 484.06	436.51	517.17	6.81±1.14	5.95	0.55
Mortality was determined every 24 up to 96 hrs, each set of experiment was replicate six times, C. sativa: Cannabis sativa, A. nilotica: Acacia nilotica, T. cordifolia: Tinospora cordifolia, LCL: Lower confidence limit, UCL: Upper confidence limit, Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments

Table 3:

Toxicity of binary combinations (1:1) of crude extract (aqueous) of plant parts of C. sativa, A. nilotica and T. cordifolia against snail Lymnaea acuminata

	Limits
Treatment	Exposure period	Effective dose (mg L–1)	LCL	UCL	Slope value	‘t’ ratio	Heterogeneity factor
C. sativa leaf+	24 hrs	LC50 = 174.76	159.5	215.3	5.40±1.12	4.18	0.18
A. nilotica leaf	48 hrs	LC50 = 148.01	137.86	163.79	5.30±1.2	4.39	0.16
	72 hrs	LC50 = 121.81	103.64	132.28	4.66±1.1	3.89	0.31
	96 hrs	LC50 = 112.26	100.69	119.66	7.79±1.37	5.67	0.34
C. sativa stem+	24 hrs	LC50 = 193.53	178.6	225.74	5.40±1.18	4.57	0.29
A. nilotica bark	48 hrs	LC50 = 165.74	154.22	180.17	5.30±1.2	4.8	0.17
	72 hrs	LC50 = 141.06	127.2	150.82	4.66±1.1	5.18	0.15
	96 hrs	LC50 = 129.38	116.92	137.76	7.77±1.37	6	0.4
T. cordifolia leaf+	24 hrs	LC50 = 281.41	233.35	427.35	2.83±0.64	4.37	0.18
C. sativa leaf	48 hrs	LC50 = 226.43	190.61	322.13	2.32±0.58	3.98	0.14
	72 hrs	LC50 = 153.80	126.07	180.72	2.40±0.56	4.25	0.14
	96 hrs	LC50 = 114.90	93.2	130.7	3.50±0.61	5.69	0.49
T. cordifolia stem+	24 hrs	LC50 = 324.50	270.5	502.53	3.21±0.76	4.2	0.24
C. sativa stem	48 hrs	LC50 = 234.66	206.3	289.24	3.02±0.67	4.46	0.16
	72 hrs	LC50 = 153.64	116.9	177.67	2.65±0.65	4.02	0.3
	96 hrs	LC50 = 127.72	103.01	144.51	4.18±0.74	5.62	0.42
T. cordifolia leaf+	24 hrs	LC50 = 308.03	258.1	471.88	3.00±0.73	4.09	0.21
A. nilotica leaf	48 hrs	LC50 = 237.76	209.54	294.33	3.07±0.68	4.51	0.2
	72 hrs	LC50 = 169.35	142.8	191.28	3.10±0.66	4.67	0.25
	96 hrs	LC50 = 134.98	113.26	150.44	4.45±0.74	5.96	0.37
T. cordifolia stem+	24 hrs	LC50 = 390.27	304.1	695.39	2.14±0.56	4.24	0.19
A. nilotica bark	48 hrs	LC50 = 229.25	191.29	291.68	2.09±0.45	4.64	0.16
	72 hrs	LC50 =147.22	113.8	174.61	2.27±0.44	5.08	0.31
	96 hrs	LC50 = 117.68	88	140.08	2.65±0.47	5.6	0.42
Mortality was determined every 24 up to 96 hrs, each set of experiment was replicate six times, C. sativa: Cannabis sativa, A. nilotica: Acacia nilotica, T. cordifolia: Tinospora cordifolia, LCL: Lower confidence limit, UCL: Upper confidence limit, Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments

The binary combination (1:5) of C. sativa leaf+A. nilotica leaf was found to be more toxic (24 hrs LC₅₀ = 82.32 mg L^–1) than other combinations (Table 4). The order of toxicity of different binary combinations (1:5) against L. acuminata was C. sativa leaf+A. nilotica leaf>T. cordifolia leaf+A. nilotica leaf>T. cordifolia leaf+C. sativa leaf>T. cordifolia stem+A. nilotica bark>T. cordifolia stem+C. sativa stem>C. sativa stem+A. nilotica bark (Table 4).

The binary combination (5:1) of C. sativa leaf+A. nilotica leaf was more toxic (24 hrs LC₅₀ = 82.32 mg L^–1) than other combinations (Table 5). The order of toxicity of different binary combinations (5:1) against L. acuminata was C. sativa leaf+A. nilotica leaf>C. sativa stem+A. nilotica bark>T. cordifolia leaf+A. nilotica leaf>T. cordifolia leaf+C. sativa leaf>T. cordifolia stem+A. nilotica bark>T. cordifolia stem+C. sativa stem (Table 5).

It is evident from the results that extracts of C. sativa (leaf, stem), A. nilotica (leaf, bark) and T. cordifolia (leaf, stem) are toxic against the snail Lymnaea acuminata. The toxicity of C. sativa leaf is highest in comparison to the stem and other parts. Binary combinations of extracts of plant pats show increased toxicity than they are used separately.

The slope values given in Tables 2-5 were steep. Based on each of the six replicates the separate estimates of LC values were found to be within 95% confidence limit of LC₅₀. The t-ratio was higher than 1.96 and the heterogeneity factor was less than 1.0. The g-value was less than 0.5 at all the probability levels (90, 95 and 99). There was a significant negative regression (p<0.05) between exposure time and LC₅₀ of the exposures (Table 2-5).

Table 4:

Toxicity of binary combinations (1:5) of crude extract (aqueous) of plant parts of C. sativa, A. nilotica and T. cordifolia against snail Lymnaea acuminata

	Limits
Treatment	Exposure period	Effective dose (mg L–1)	LCL	UCL	Slope value	‘t’ ratio	Heterogeneity factor
C. sativa leaf+	24 hrs	LC50 = 82.32	69.53	110.88	4.49±0.51	4.86	0.38
A. nilotica leaf	48 hrs	LC50 = 56.97	46.87	69.89	2.09±0.47	4.42	0.21
	72 hrs	LC50 = 36.67	25.85	44.29	2.24±0.48	4.64	0.2
	96 hrs	LC50 = 31.70	24.29	37.21	3.23±0.54	5.96	0.42
C. sativa Stem+	24 hrs	LC50 = 128.45	115.92	155.92	4.51±0.93	4.85	0.15
A. nilotica bark	48 hrs	LC50 = 104.87	96.96	115.29	4.89±0.87	5.57	0.13
	72 hrs	LC50 = 86.29	75.26	94.7	4.08±0.85	4.79	0.21
	96 hrs	LC50 = 71.75	61.33	78.65	5.51±0.96	5.7	0.43
T. cordifolia leaf+	24 hrs	LC50 = 93.24	77.17	135.3	2.51±0.53	4.71	0.14
C. sativa leaf	48 hrs	LC50 = 63.34	55.07	74.96	2.74±0.49	5.35	0.14
	72 hrs	LC50 = 43.37	33.74	51.14	3.33±0.47	4.87	0.17
	96 hrs	LC50 = 33.65	26.31	39.2	3.19±0.52	6.02	0.46
T. cordifolia stem+	24 hrs	LC50 = 116.19	100.6	155.9	3.51±0.75	4.67	0.33
C. sativa stem	48 hrs	LC50 = 89.69	79.17	108.28	3.02±0.67	4.5	0.16
	72 hrs	LC50 = 64.40	52.47	73.24	2.95±0.66	4.45	0.33
	96 hrs	LC50 = 54.80	46.27	60.92	4.46±0.74	5.98	0.75
T. cordifolia leaf+	24 hrs	LC50 = 88.53	73.46	127.06	2.39±0.51	4.62	0.17
A. nilotica leaf	48 hrs	LC50 = 58.92	49.71	71.05	2.32±0.48	4.83	0.17
	72 hrs	LC50 = 42.65	32.01	50.93	2.16±0.47	4.56	0.23
	96 hrs	LC50 = 32.84	24.87	38.74	2.98±0.52	5.74	0.41
T. cordifolia stem+	24 hrs	LC50 = 114.54	98.12	160.29	3.11±72	4.33	0.13
A. nilotica bark	48 hrs	LC50 = 85.52	73.95	105.43	2.60±0.65	3.95	0.25
	72 hrs	LC50 = 62.68	50.34	71.43	2.95±0.66	4.44	0.23
	96 hrs	LC50 = 54.94	46.24	61.17	4.36±0.73	5.93	0.29
Mortality was determined every 24 up to 96 hrs, each set of experiment was replicate six times, C. sativa: Cannabis sativa, A. nilotica: Acacia nilotica, T. cordifolia: Tinospora cordifolia, LCL: Lower confidence limit, UCL: Upper confidence limit, Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments

Table 5:

Toxicity of binary combinations (5:1) of crude extract (aqueous) of plant parts of C. sativa, A. nilotica and T. cordifolia against snail Lymnaea acuminata

	Limits
Treatment	Exposure period	Effective dose (mg L–1)	LCL	UCL	Slope value	‘t’ ratio	Heterogeneity factor
C. sativa leaf+	24 hrs	LC50 = 84.43	63.03	146.81	1.76±0.34	5.05	0.35
A. nilotica leaf	48 hrs	LC50 = 50.59	38.77	74.84	1.45±0.28	5.08	0.17
	72 hrs	LC50 = 21.88	14.58	29.01	1.35±0.26	5.08	0.24
	96 hrs	LC50 =13.65	8.96	17.87	1.77±0.28	6.23	0.43
C. sativa stem+	24 hrs	LC50 = 89.57	74.15	129.72	3.29±0.52	4.6	0.28
A. nilotica bark	48 hrs	LC50 = 64.03	53.99	80.03	2.22±0.48	4.61	0.17
	72 hrs	LC50 = 44.95	35.63	52.82	2.36±0.47	4.93	0.27
	96 hrs	LC50 = 37.05	30.1	42.51	3.22±0.51	6.22	0.39
T. cordifolia leaf+	24 hrs	LC50 = 119.25	104.08	155.39	3.99±0.80	4.95	0.2
C. sativa leaf	48 hrs	LC50 = 90.66	80.95	107	3.38±0.68	4.93	0.18
	72 hrs	LC50 = 66.03	56.67	73.64	3.51±0.67	5.21	0.18
	96 hrs	LC50 = 54.42	45.84	60.57	4.45±0.74	6	0.31
T. cordifolia stem+	24 hrs	LC50 = 162.24	144.24	215.24	4.63±1.12	4.1	0.11
C. sativa stem	48 hrs	LC50 = 131.28	121.99	146.16	5.21±1.05	4.95	0.14
	72 hrs	LC50 = 100.37	85.39	109.14	4.61±1.03	4.46	0.17
	96 hrs	LC50 = 93.57	83.51	100.32	6.97±1.18	5.91	0.39
T. cordifolia leaf+	24 hrs	LC50 = 113.90	99.97	146.24	3.84±0.77	4.98	0.12
A. nilotica leaf	48 hrs	LC50 = 92.26	82.24	109.89	3.35±0.68	4.87	0.11
	72 hrs	LC50 = 64.60	54.48	72.44	3.35±0.67	5	0.19
	96 hrs	LC50 = 52.98	44	9.27	4.39±0.75	5.85	0.4
T. cordifolia stem+	24 hrs	LC50 = 150.60	135.84	189.7	4.45±0.80	4.12	0.2
A. nilotica bark	48 hrs	LC50 = 120.21	108.69	134.32	4.05±1.01	4	0.16
	72 hrs	LC50 = 101.69	89.89	109.75	5.24±1.05	4.99	0.33
	96 hrs	LC50 = 91.73	80.4	99.04	6.56±0.16	5.63	0.45
Mortality was determined every 24 up to 96 hrs, each set of experiment was replicate six times, C. sativa: Cannabis sativa, A. nilotica: Acacia nilotica, T. cordifolia: Tinospora cordifolia, LCL: Lower confidence limit, UCL: Upper confidence limit, Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments

DISCUSSION

The results shown in Table 2 indicate that the extracts from indigenous plants C. sativa, A. nilotica and T. cordifolia shows very promising molluscicidal activity against Lymnaea acuminata that act as intermediate host of human and livestock disease fascioliasis. The highest toxicity was shown by extracts from leaves of Cannabis sativa. It is very obvious from this study that when used in different binary combinations, these plant extracts show enhanced toxicity against L. acuminata.

It has been reported that Cannabis sativa contains about 104 cannabinoids and 140 members of terpenes²⁶. Mantzoukas et al.²⁷ reported the larvicidal activity of cannabidiol oil from C. sativa. It seems that cannabinoids present in Cannabis sativa caused the mortality of L. acuminata. The bark extract of Acacia nilotica contains terpenoids, tannins, alkaloids, saponins and glycosides²⁸ and saponins have been reported as potent molluscicides²⁹. The chemical components of Tinospora cordifolia include terpenoids (tinosporide, tinosporaside and ecdysterone), alkaloids (tinosporine and magnoflorine), lignans, steroids, giloin and tinosponon³⁰ and terpenoids are found to be potent larvicidal agents³¹. It seems that terpenoids present in T. cordifolia extracts caused the mortality of snail L. acuminata.

The toxicity of the binary combination (1:5) of C. sativa leaf+A. nilotica leaf extract is 3.69 and 6.51 times higher than C. sativa leaf and A. nilotica leaf, respectively. The toxicity of binary combination (1:5) of C. sativa stem+A. nilotica bark is 3.14 and 5.26 times higher than C. sativa stem and A. nilotica bark, respectively used alone. The toxicity of T. cordifolia leaf+C. sativa leaf (1:5) is 6.36 and 4.33 folds higher than T. cordifolia leaf and C. sativa leaf, respectively. A binary combination (1:5) of T. cordifolia stem+C. sativa stem is 6.64 and 3.48 times more toxic than T. cordifolia stem and C. sativa stem, respectively. Similarly, the binary combination (1:5) of T. cordifolia leaf+A. nilotica leaf is 6.70 and 6.05 times more toxic than the T. cordifolia leaf and A. nilotica leaf, respectively. Likewise, the toxicity of binary combination (1:5) of T. cordifolia stem+A. nilotica bark is 6.74 and 5.89 times higher than T. cordifolia stem and A. nilotica bark, respectively.

The steep slope values indicate that an increase in concentrations causes increased mortality in the snails. The t-ratio value greater than 1.96 indicates that the regression is significant, the value of heterogeneity factor less than 1.0 indicates that replicate lines would fall within 95% confidence limit and thus the model fitsthe data adequately.

Synthetic molluscicides are costly and cause adverse effects on the environment. The use of plant molluscicides not only may eliminate the burden of expensive synthetic molluscicides, but also could prevent the environment. The present study shows that C. sativa, A. nilotica and T. cordifolia may be used as potent molluscicides. Further, investigations are required to elucidate the mode of action in the snail body, if used as molluscicides.

CONCLUSION

The indigenous plant extracts were found to be effective and efficient in killing the vector snails after time and concentration dependent treatment. The experimental results revealed that plant based molluscicides are a better substitute for the harmful synthetic molluscicides. Further investigations are required to reveal the phytoconstituent and its mode of action responsible for the toxicity.

SIGNIFICANCE STATEMENT

Use of molluscicides to control population of the vector snails is an approach to control fascioliasis. Synthetic molluscicides are costly and cause adverse effects on the environment. This study discovered the potentiality of indigenous plants as cost-effective and eco-friendly molluscicides.

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