Eng6

Jfuas No.1 June 2013

73

Molluscicidal and Ovicidal Activities of Alternanthera

nodiflora Leaves and Flowers Aqueous and Organic Solvent

Extracts against Bulinus truncatus Snails

Mohammed yousif Adam1’*, Faysal Sawi Ali

2’*

1. Department of Science, Faculty of Education for Basic Teachers, University of

Alfashir, Sudan.

2. Department of Biology, Faculty of Education, University of Khartoum, Sudan.

*Corresponding author:[email protected]

والعضويةال المائية للمستخلصات القاتلة أتأثيرات وراقمنترنكاتسأو بولينس وبيض قواقع على نوديفلورا الترنانثرا زهار

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الفارش األول–التطبيقيةالعلوممجلة–جامعة م2013يونيو–العدد

74

Abstract

The plant Altrenanthera nodiflora (Locally called Abutagia)was

tested for the molluscicidal and ovicidal potency of leaves and flowers

using distilled water successive extract and increasing polarity

solvents viz ethanol, chloroform and petroleum ether for 12hours

Soxhlet extracts, on adult, one day old juveniles and one day old egg-

masses of B.truncatus snails. Results obtained showed that

chloroform extracts of A. nodiflora leaves and flowers was the most

potent, followed by petroleum ether, distilled water and(finaly)

ethanol extracts . B. truncatus one day old juvenile snails were most

susceptible, followed by one day old egg-masses and finally the adult

snails to the effects of the flower leave extracts of A. nodiflora . A.

nodiflora flowers chloroform and petroleum ether extracts were more

potent than the leaves on B. truncatus different stages. Statistical

analysis showed high significant differences at 0.05 levels between

LC50% and LC90% of A. nodiflora leaves and flowers solvents

extracts on all stages of B. truncatus snails.

Key Worrds: Altrenanthera nodiflora, Organic Solvents, Extracts,

Bulinus truncatus

Introduction

Snails act as intermediate hosts of various trematode parasites

causing schistosomiasis and fascioliasis in human and livestock

(Brown,1980). Bulinus truncatus is the intermediate host of blood

fluke schistosoma haematobium. The World Health Organization


75

estimated that approximately 250 million people are suffering from

the infection and that more than 600 million people reside in areas

where schistosomiasis is transmitted (Cheesbrugh, 1987). In Sudan

schistosomiasis was considered as one of the most important public

health problems after malaria (Bella etal., 1980).

The control of molluscan metazoans using the synthetic

molluscicides during the last decades have resulted in environmental

hazards risk to the aquatic animals, built up of resistance in major

vector species and the increased cost of molluscicides (Mott, 1987) .

Therefore, environmentally safer, low cost and indigenous methods of

control are needed.

Plant molluscicides can prove to be an ideal source of low cost,

safe and effective molluscicides (Morston and hostettmann, 1985).

Molgad et al, (1999) has expressed hope that plants showing

molluscicidal properties could be used on self- help basis to control

schistosomiasis in rural areas. Many plants were tested for their

molluscicidal activities all over the world (Kloos and McCllough,

1987). In Sudan, various attempts have been made to investigate the

molluscicidal effects of many plants ( El –Kheir and El-Tohami, 1979;

Abdel Aziz, 1982; Ali, 1997; Salha, 1999; Atta Elmannan, 2001;

Somia, 1999 2003; Elamin et al, 2005; Howiada, 2005 and Ali,

2007).

The objectives of the current study is to investigate the

molluscicdal and ovicidal activities of Alternanthera nodiflota leaves


76

and flowers extracts using different solvents against B. truncatus egg

masses, juvenile and adult snails

Materials and Methods

2-1 Study Area

The study was carried in Alfashir from August 2007 to September

2008. Al- Fashir is located in North Darfur State, Sudan, at latitude

˚13 37 N and longitude ˚25 22 E. The climate is semi-arid with an

annual rainfall, between 100-500 mm.

2-2 The candidate plant

Alternanthera nodiflota (R. Br., Prodr.Fl. Nov. Holl. I: 417

(1810)) is an annual herb known locally as Abutagia, the common

name is Joyweed. Leave of the plant is opposite, linear-lanceolate.

The Flower head is white and found in axillary clusters. Fruits are

circumscissiles. It is commonly grown on wet places, weed of

cultivation on silt soils leaves and flowers aqueous extracts proved to

be most potent against Bulinus truncatus snails Used locally as a

treatment of dysuria and schistosomiasis (Plate1)


77

Plate(1) Plant Species: Alternanthera nodiflora

2-3 Collection and Preparation of Plant Samples

A. nodiflora was collected from the Valleys and Goz lands around

Alfashir city between August-October 2007 during the rainy season,

when the plants are flowering and fruiting. Collected plant were

transported to the laboratory in Alfashir University. In the laboratory,

collected plants were assorted by part (leaves and flowers and dried

under shade. Thereafter, the plant part was ground in a wooden mortar

with a pestle. The powdered plant material was then put in labeled air

tight plastic bags and kept in desiccators for further use. Plant

specimens were identified by Dr. Abd-El-Gabar Nasir Gumma, a

taxonomist from Biology Department, Faculty of Education,

University of Khartoum.

2-4 Collection and Maintenance of Snail Samples

Snails were collected from Golo reservoir. Snail’s collections

were performed early morning at 8.00-10.00 a.m., using a flat dip-net

scoop (Demian and Kamel, 1972). The collected snails were put in a


78

plastic bucket containing some dam water with few leaves. The snails

were then transported to the laboratory for identification and

maintenance. The snails used in this study were idenified as Bulinus

truncatus according to key stablished by Danish Bilharzias

Laboratory(DBL, 1983). The healthy snails were maintained in

aquaria of plastic troughs (12cm depth×30cm diameter with a capacity

of about 6 liters) according to the method recommended by

DBL(1989). Dry Eruka sativa (Girgeer) leaves were dried and

powdered after the removal of the mid-rib.The powdered dry Girgeer

was used for feeding the snails three times a week. Snails fed on dry

ribs of the leaves did not survive. Water was changed twice a month

or when necessary. The dead snails were removed as soon as possible

from the troughs to prevent water fouling.

2-5 Collection and Preparation of Egg-masses and Juvenile Snails

About 10 adult B. truncatus snails taken from the stock aquaria

were transferred to plastic troughs (12cm depth×30cm diameter)

containing 2 litres of water. Then, 2-3 pieces of polythene sheets

(about 5cm× 15cm) were put in each plastic trough. The snails were

fed with Girgeer leaves and allowed to lay eggs. The polythene sheets

were checked for egg-masses after 24 hours. After this period the

snails were transferred to another new plastic trough and the same

steps repeated.

Polythene sheet containing the egg-masses were easily

located and isolated by cutting the plastic around each egg.-masses

with a scalpel (about 0.5-1.0 cm from the egg-mass). The egg-masses,


79

each attached to a piece of polythene, were immersed three times in

different Petri-dishes containing clean well water to remove any

debris and transferred to containers containing 200 ml of

dechlorinated tap water, the dishes were covered.

The egg-masses were divided to four groups, the first group

was the one day old egg-masses (group A), and they were treated

immediately after collection. The 2nd groups of the one day old egg-

masses were transferred to containers with 200ml of dechlorinated tap

water and left untreated as a control groups for one day old egg-

masses (group C).

The third group was left to hatch, then treated immediately

after hatching as one day old juveniles (group B); while some of one

day old juveniles were left as an untreated control group (group D).All

the groups of egg-masses were exposed to artificial light for about 8

hours daily.

2-6 preparation of the Extracts2-6-1 Successive Organic Solvents Extracts (SOSE)

Five grams of powdered dry leaves and flowers of Alternanthera

nodiflora were saperatly extracted for 12 hours in a Soxhlet apparatus

using: Ethanol 80 % (78-80Ċ b.r), Chloroform 95 % (60-62Ċ b.r),

Petroleum ether 90 % (40-60Ċ b.r).After extraction, the solvents were

removed by rotary evaporation and the volume adjusted to 20ml

(250000ppm).


80

2-6-2 Aqueous Successive Extracts

The distilled water solvent was extracted using successive

extraction method reported by Skoog and West (1982). Five grams of

A. nodiflora leaves and flowers were separately soaked in plastic

containers containing 50ml of distilled water, with vigorous shaking.

Each solution was allowed to stand for 3 hours then immediately

sieved by passing through three layers of 1mm² pores plastic meshes.

The mark was again soaked in new 50ml distilled water and allowed

to stand for 3 hours and sieved. The process of soaking and sieving

was done four times during the 12 hours extracting period. The

suspensions of each were mixed together and filtered with filter paper.

The mark was washed several times with distilled water and filtered.

All filtrates of each plant part sample were mixed together and final

volume was adjusted to200ml by adding distilled water to get stock

solution of 25000 ppm.

2-7 Molluscicidal and Ovicidal Potency Tests2-7-1 Adult Snails tests

The molluscicidal potency tests were investigated according to the

standard method prescribed by WHO (1971).To prepare the working

solutions, stock solutions of the leaves and flowers extracts prepared

with different solvents extractions were diluted using simple dilution

procedure. Thirty healthy adult Bulinus truncatus snails of uniform

size were immersed. The exposure period was 24 hours. The recovery

period was 24 hours. (LD100) were determined. All the snails which


81

at the end of the recovery period remained without response against

mechanical prodding, were considered dead. Three replicates for each

test were used with control groups using dechlorinated tape water.

Dead snails were counted and recorded.

2-7-2 Ovicidal Potency tests (Group A’- One Day Old Egg-masses)

The ovicidal potency tests were carried out according to the method

recommended by WHO (1965). To prepare 1000ppm stock solution, 0.8ml

of each solvent extract (5gm/20ml) was separately completed to 200ml by

adding dechlorinated tap water. While, 8ml stock solution from the distilled

water extract (5gm/200ml) was completed to 200 ml by adding

dechlorinated tap water to prepare 1000 ppm stock solution. From the later

stock solution, further dilutions were made to prepare the working solutions.

Three replicates of three egg-masses were put in transparent plastic

containers, 8cm depth 5.5cm diameter, containing 200ml of working

solution at room temperature (25-30˚C). Control groups were prepared by

putting three replicates of three egg masses in plastic containers containing

200ml of dechlorinated tap water (group C).

The dead egg masses were counted and recorded. When all the

egg masses killed, further dilutions were made to determine the least

concentration that killed 100% of the egg masses (LC100). Cell division

and the behavior of the embryos or the movements of juveniles of group (C)

were examined under the microscope (10×4 power lens) and the

observations were recorded daily until all control groups and group B

hatched.


82

2-7-3 One Day Old Juvenile Snails Potency Tests

One day old juvenile snails immediately hatched designed as

group ‘B’, were collected separately, using a magnifying lens, with a

fine drawing brush and observed under a dissecting microscope (10х4

power) for mortality. To prepare 1250ppm stock solution of the

organic extracts, one ml of each extract (5gm/20ml) was separately

completed to 200ml by adding dechlorinated tap water. Also, to

prepare 1000 ppm stock solution of A. nodiflora leaves and flowers

distilled water extract 5 gm/ 200 ml, 8 ml of the distilled water extract

stock solution was completed to 200 ml by adding dechlorinated tap

water. From the later stock solution, further dilutions were made to

prepare the working solutions. Thirty juvenile snails were tested using

the method descriped by(WHO, 1965). The control group ‘D’ was

subjected to 200ml dechlorinated tap water. There were three

replicates for each. The exposure period and the recovery was 24

hours, thereafter, mortality counts were recorded.

2-9 Statistical Analysis

The data of the present study were analyzed by probit analysis

(Finney, 1936). Dosage-mortality curves are used. The results of such

analysis are plotted on probit paper. A regression line is fitted to

dosage-mortality data graphed on probit paper (Sokal and Rohlf,

1973).


83

4- Results

4-1 Molluscicidal and Ovicidal Potency Tests of Alternanthera

nodiflora Leaves Extractslethal concentrations(LC50) of A. nodiflora leaf extrat for B. truncatus

adult snails, when 5gm of leaves powder of were extracted, successively for

12 hours with distilled water and using Soxhlet with, ethanol, chloroform

and petroleum ether solvents were: 87.11, 109.23, 17.50 and 23.63 ppm,

respectively, while LC90 values were: 107.64, 132.40, 27.50 and 33.45

ppm, respectively(Tables 1: a, b, c & d).

Tables 1(a,b,c&d)Molluscicidal Activity of Alternanthera nodi flora Leaves Extracts

on Bulinus truncatus Adult Snails (successive extraction for12hrs:-

Table 1, a- Distilled Water extract

Concentration Mortality Probit

(ppm) Log Total (%) Tabulated Calculated

110 2.0414 30 100.0% - -

100 2.0000 26 87% 6.13 6.57

90 1.9542 20 67% 5.44 5.74

80 1.9031 14 47% 4.92 4.80

70 1.8451 8 27% 4.39 3.74

60 1.7782 2 7% 3.52 2.51

50 1.6990 0 0% - -

(Control) - 0 0.00% - -

Number of snails tested for each concentration = 30, Regression equation = y = 10.30X - 14.64, Std. Error of X –

Coefficient =0.415, Std. Error of Y – Coefficient =0 .784, Lethal concentration that killed 50% of snails (LC50 ) =

87.11ppm, Lethal concentration that killed 90% of snails (LC90 ) = 107.64ppm, Fiducial limits with 95%confidence limits = ± 1.929, Regression coefficient (r2) =0.992.

Table 1, b- Ethanolic

Number of snails tested for each concentration = 30, Regression equation = y = 14.04X - 23.46, Std. Error of X – Coefficient =0.733, Std. Error of Y – Coefficient = 1.477, Lethal concentration that killed 50% of snails (LC50 ) = 109.23ppm, Lethalconcentration that killed 90% of snails (LC90 ) = 132.40ppm, Fiducial limits with 95% confidence limits = ± 2.014, Regressioncoefficient (r2) =0 .987.



135 2.1303 30 100.0% - -

125 2.0969 26 87% 6.13 6.52

115 2.0607 20 67% 5.44 5.63

105 2.0212 12 40% 4.75 4.66

95 1.9777 5 17% 4.05 3.60

85 1.9294 2 7% 3.52 2.41

75 1.8751 0 0% - -

0(Control) - 0 100.0% - -


84

Table1,c- Chloroform Extract

-

Number of snails tested for each concentration = 30, Regression equation = y = .618 X - 3.815 , Std. Error of X –

Coefficient = .357,Std. Error of Y – Coefficient =0 .430, Lethal concentration that killed 50% of snails (LC50 ) =

17.50ppm, Lethal concentration that killed 90% of snails (LC90 ) = 27.50ppm, Fiducial limits with 95% confidencelimits = ± 1.184, Regression coefficient (r2) =0 .966.

Table 1, d- Petroleum ether extractConcentration Mortality Probit


40 1.6021 30 100% - -

35 1.5441 28 93% 6.48 5.96

30 1.4771 21 70% 5.52 5.68

25 1.3979 14 47% 4.92 5.36

20 1.3010 8 27% 4.39 4.96

15 1.1761 2 7% 3.52 4.44

10 1.0000 0 0% - -

(Control) - 0 0.00% - -Number of snails tested for each concentration = 30, Regression equation = y = 6.140X - 3.447, Std. Error of X – Coefficient =0.566, Std. Error of Y – Coefficient =0 .775, Lethal concentration that killed 50% of snails (LC50 ) =23.63ppm, Lethalconcentration that killed 90% of snails (LC90 ) = 33.45ppm, Fiducial limits with 95% confidence limits = ± 1.363, Regressioncoefficient (r2) =0 .959.

The LC50 of one day old juvenile B. truncatus snails were: 7.48, 18.45,

2.88 and 3.99 ppm, respectively, and the LC90 were11.35, 24.00, 3.69 and

4.57 ppm, respectively as shown in table2: a, b, c & d.

Tables 2(a, b, c, d): Molluscicidal Activity of Alternanthera nodiflora leaves Extracts

on Bulinus truncatus One day old Juvenile Snails (Successive extraction for 12hrs):-

Table 2, a- Distilled water extract

Number of snails tested for each concentra�on = 30� Regression equa�on = y = 7.932X � ��

�� ! concentra�on that killed 50% of snails (LC�� " � ��#$ %%&� �� !

concentra�on that killed 90% of snails (LC�� " � �� %%&� '��(�� ! !�&�) *�� + ��,�� !�&�) � -� ��$�� .�/ �))��

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30 1.4771 30 100.0% - -

25 1.3979 26 87% 6.13 6.60

20 1.3010 21 70.0% 5.52 5.76

15 1.1761 14 47% 4.92 4.69

10 1.0000 6 20.0% 4.16 3.18

5 0.6990 0 0.0% - -

(Control) - 0 0.00% - -

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85

Table 2, b- Ethanolic extract

1(&2� �� )� �!) �)�� concentra�on = 30� Regression equa�on = y = 8.877X � ��$�#� ��

� ��$#$� �� $� Lethal concentra�on that killed 50% of snails (LC�� " � �$�#� %%&� �� !

concentra�on that killed 90% of snai!) 0�� " � �#�� %%&� '��(�� ! !�&�) *�� + ��,�� !�&�) � - ��

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Table 2, c- Chloroform extract

Number of snails tested for each concentra�on = 30, Regression equa�on = y = 12.797X � ��#� ��

� ��$�$� �� Coefficient =0 .762, Lethal concentra�on that killed 50% of snails (LC�� " � ��$$ %%&� �� !

��tra�on that killed 90% of snails (LC�� " � �� %%&� '��(�� ! !�&�) *�� + ��,�� !�&�) � -� �� .�/ �))��

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Table 2, d- Petroleum ether extract

Number of snails tested for each concentra�on = 30� Regression equa�on = y = 16.575X � ��#� ��

�� #� �� #� Lethal concentra�on that killed 50% of snails (LC�� " � ��$ %%&�

Lethal concentra�on that killed 90% of snails (LC�� " � #�� %%&� '��(�� ! !�&�) *�� + ��,�� !�&�) � - ��#�#�

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86

On the other hand, LC50 of one day old egg masses B. truncatus

snails on the same solvents were : 17.05, 18.54, 5.30 and5.80 ppm,

respectively, while the LC90 of embryos in egg masses were: 23.44,

26, 7.00 and 7.27 ppm, respectively(Table1).

Tables 3 (a, b, c & d): Molluscicidal Activity of Alternanthera nodiflora leaves Extracts

on One day old Egg Masses Bulinus truncatus (Successive extraction for12hrs)


Number of snails tested for each concentration = 30, Regression equation = y = 9.121X - 5.876, Std. Error of X –Coefficient = 1.139, Std. Error of Y – Coefficient = 1.301, Lethal concentration that killed 50% of snails (LC50 ) = 17.05ppm, Lethal concentration that killed 90% of snails (LC90 ) = 23.44 ppm, Fiducial limits with 95% confidence limits = ±1.122, Regression coefficient (r2) =0.955.


Number of snails tested for each concentration = 30, Regression equation = y = 22.287X - 38.478, Std. Error of X – Coefficient =4.087, Std. Error of Y – Coefficient = 7.857, Lethal concentration that killed 50% of snails (LC50 ) = 18.54 ppm, Lethal concentrationthat killed 90% of snails (LC90 ) = 26 ppm, Fiducial limits with 95% confidence limits = ± 1.921, Regression coefficient (r2) =0.881.

Table3, c- Chloroform extract



7 0.8451 30 100% - -

6 0.7782 28 93% 6.48 6.38

5 0.6990 19 63% 5.33 5.72

4 0.6021 11 37% 4.67 4.92

3 0.4771 4 13% 3.87 3.89

2 0.3010 0 0% - -

1 0.0000 0 0% - -

(Control) - 0 0.00% - -Number of snails tested for each concentration = 30, Regression equation = y = 8.805X - .842, Std. Error of X – Coefficient

= 1.311, Std. Error of Y – Coefficient =0.782, Lethal concentration that killed 50% of snails (LC50 ) = 5.30 ppm, Lethalconcentration that killed 90% of snails (LC90 ) = 7.01 ppm, Fiducial limits with 95% confidence limits = ±0.540,Regression coefficient (r2) = 0.900.



25 1.3979 30 100% - -

20 1.3010 26 87% 6.13 6.36

15 1.1761 14 47% 4.92 5.11

10 1.0000 5 17% 4.05 3.34

5 0.6990 0 0% - -

(Control) - 0 0% - -



30 2.0414 30 100.0% - -

25 2.0000 26 87% 6.13 6.01

20 1.9542 17 57% 5.18 5.57

15 1.9031 9 30% 4.48 5.01

10 1.8451 3 10% 3.72 4.21

5 1.7782 0 0% - -

(Control) - 0 0% - -


87


Number of snails tested for each concentration = 30, Regression equation = y = 12.361X - 4.797, Std. Error of X –Coefficient = 1.732, Std. Error of Y – Coefficient = 1.345, Lethal concentration that killed 50% of snails (LC50 ) = 5.80ppm, Lethal concentration that killed 90% of snails (LC90 ) = 7.27 ppm, Fiducial limits with 95% confidence limits =±0.763, Regression coefficient (r2) = 0.911.

4-2 - Molluscicidal and Ovicidal Potency Tests of Alternanthera

nodiflora Flowers Extracts

The results showed that, the LC50 of B. truncatus adult snails,

when 5gm of the flowers of A. nodiflora were extracted continuously

for 12 hours using distilled water successive extract and ethanol,

chloroform and petroleum ether solvents using Soxhlet were: 105,

22.50, 7 and 9.97 ppm, respectively, while the lethal dose that killed

90% (LC90) were 127.28, 29.06, 11.13 and 13.46 ppm,

respectively(Tables4: a, b, c & d).Tables 2(,a, b, c, d ): Molluscicidal Activity of Alternanthera nodiflora Flowers

Extracts on Bulinus truncatus Adult Snails(Successive extraction for12hrs) :-


Number of snails tested for each concentration = 30, Regression equation = y = 13.232X – 21.742, Std. Error of X –Coefficient = 0.940, Std. Error of Y – Coefficient = 1.873, Lethal concentration that killed 50% of snails (LC50 ) = 105ppm,Lethal concentration that killed 90% of snails (LC90 ) = 127.28ppm, Fiducial limits with 95% confidence limits = ± 1.992,Regression coefficient (r2) =0 .975.



9 0.9542 30 100% - -

8 0.9542 29 97% 6.88 7.22

7 0.8451 23 77% 5.74 6.44

6 0.7782 16 53% 5.08 5.96

5 0.6990 9 30% 4.48 5.40

4 0.6021 2 7% 3.52 4.71

3 0.4771 0 0% - -

(Control) - 0 0% - -



130 2.1139 30 100.0% - -

120 2.0792 24 80% 5.84 5.25

110 2.0414 17 57% 5.18 5.15

100 2.0000 11 37% 4.67 5.04

90 1.9542 5 17% 4.05 4.92

80 1.9031 1 3% 3.12 4.78

70 1.8451 0 0.0% - -

(Control) - 0 0.00% - -


88

Table4, b- Ethanolic extract

Number of snails tested for each concentration = 30, Regression equation = y = 86.86 X - 0.679, Std. Error of X –Coefficient = 1.315, Std. Error of Y – Coefficient = 1.655, Lethal concentration that killed 50% of snails (LC50 ) = 22.50ppm, Lethal concentration that killed 90% of snails (LC90 ) = 29.06 ppm, Fiducial limits with 95% confidence limits = ±1.239, Regression coefficient (r2) = 0.897.

Table 4, c- Chloroform extracts

Number of snails tested for each concentration = 30, Regression equation = y = 8.041X - 1.814, Std. Error of X –Coefficient =0 .915, Std. Error of Y – Coefficient = 750, Lethal concentration that killed 50% of snails (LC50 ) = 7 ppm,Lethal concentration that killed 90% of snails (LC90 ) = 11.13 ppm, Fiducial limits with 95% confidence limits = ±0.786,Regression coefficient (r2) =0 .951.


Number of snails tested for each concentration = 30, Regression equation = y = 7.330X - 1.820, Std. Error of X –Coefficient = 0.439, Std. Error of Y – Coefficient =0 .384, Lethal concentration that killed 50% of snails (LC50 ) = 9.97ppm, Lethal concentration that killed 90% of snails (LC90 ) = 13.46 ppm, Fiducial limits with 95% confidence limits = ±0.841, Regression coefficient (r2) = 0.982.

The LC50 of one day old juvenile B. truncatus snails, when 5gm

of the A. nodiflora flowers were extracted continuously for 12 hours

using distilled water, ethanol, chloroform and petroleum ether solvents



35 1.5441 30 100.0% - -

30 1.4771 27 90% 6.28 5.53

25 1.3979 19 63% 5.33 5.17

20 1.3010 10 33% 4.56 4.73

15 1.1761 5 17% 4.05 4.16

10 1.0000 0 0% - -

5 0.6990 0 0% - -

(Control) - 0 0.00% - -



12 1.0792 30 100% - -

10 1.0000 27 90% 6.28 6.56

8 0.9031 20 67% 5.44 5.71

6 0.7782 11 37% 4.67 4.61

4 0.6021 4 13% 3.87 3.07

2 0.3010 0 0% - -

(Control) - 0 0.00% - -



14 1.1461 30 100% - -

12 1.0792 26 87% 6.13 6.15

10 1.0000 20 67% 5.44 5.68

8 0.9031 12 40% 4.75 5.10

6 0.7782 5 17% 4.05 4.35

4 0.6021 1 3% 3.12 3.30

2 0.3010 0 0% - -

(Control) - 0 0.00% - -


89

were: 7.00, 3.88, 1.95 and 3.00 ppm, respectively, and the LC90 were

10.45, 5.08, 2.28 and 3.75 ppm, respectively(Tables5: a, b, c d).

Tables 5 (a, b, c & d): Molluscicidal Activity of Alternanthera nodiflora Flowers

Extracts on Bulinus truncatus One day old Juvenile Snails (Successive extraction for

12hrs).




12 1.0792 30 100% - -

10 1.0000 25 83% 5.95 6.22

8 0.9031 18 60% 5.25 5.61

6 0.7782 11 37% 4.67 4.81

4 0.6021 3 10% 3.72 3.70

2 0.3010 0 0% - -

(Control) - 0 0% - -Number of snails tested for each concentration = 30, Regression equation = y = 8.577X - 2.180, Std. Error of X –Coefficient =0.814, Std. Error of Y – Coefficient =0 .668, Lethal concentration that killed 50% of snails (LC50 ) = 7.00ppm, Lethal concentration that killed 90% of snails (LC90 ) = 10.45 ppm, Fiducial limits with 95% confidence limits = ±0.786, Regression coefficient (r2) =0 .965.


Number of snails tested for each concentration = 30, Regression equation = y = .286 X - 9.004, Std. Error of X – Coefficient= 0.519, Std. Error of Y – Coefficient = 0.282, Lethal concentration that killed 50% of snails (LC50 ) = 3.88 ppm, Lethalconcentration that killed 90% of snails (LC90 ) = 5.08 ppm, Fiducial limits with 95% confidence limits = ±0 .485,Regression coefficient (r2) =0 .987.


Number of snails tested for each concentration = 30, Regression equation = y = 3.381 X – 10.236, Std. Error of X – Coefficient=0.825, Std. Error of Y – Coefficient =0.225, Lethal concentration that killed 50% of snails (LC50 ) = 1.95 ppm, Lethal

concentration that killed 90% of snails (LC90 ) = 2.28 ppm, Fiducial limits with 95% confidence limits = ±0.122, Regressioncoefficient (r2) =0.981.



6 0.7782 30 100% - -

5 0.6990 28 93% 6.48 6.59

4 0.6021 20 67% 5.44 5.71

3 0.4771 11 37% 4.67 4.59

2 0.3010 2 7% 3.52 3.00

1 0.0000 0 0% - -

(Control) - 0 0% - -



2.5 0.3979 30 100% - -

2 0.3010 28 93% 6.48 6.47

1.5 0.1761 14 47% 4.92 5.19

1 0.0000 5 17% 4.05 3.38

.5 -0.3010 0 0% - -

(Control) - 0 0.00% - -


90


Number of snails tested for each concentration = 30, Regression equation = y = 26.346X - 12.944, Std. Error of X –Coefficient = 3.026, Std. Error of Y – Coefficient = 1.966, Lethal concentration that killed 50% of snails (LC50 ) = 3.00ppm, Lethal concentration that killed 90% of snails (LC90 ) = 3.75 ppm, Fiducial limits with 95% confidence limits = ±0.643, Regression coefficient (r2) =0.938.

On the other hand the LC50 of one day old egg masses B. truncatus

snails, when 5gm of the A. nodiflora flowers were extracted continuously

for 12 hours using distilled water, ethanol, chloroform and petroleum ether

solvents were: 18.02, 7.50, 3.67 and 4.21 ppm, respectively, while the lethal

dose that killed 90% of one day old egg-masses of B.truncatus snails

(LC90) were 24.00, 11.51, 4.22 and 5.48 ppm, respectively as indicated in

tables (Table6: a, b, c & d)

Tables 6 (a, b, c & d): Molluscicidal Activity of Alternanthera nodiflora Flowers

Extracts on One day old Egg

Masses Bulinus truncatus (Successive extraction for12hrs):-


Number of snails tested for each concentration = 30, Regression equation = y = 8.313X - 5.161, Std. Error of X –Coefficient = 1.110, Std. Error of Y – Coefficient = 1.336, Lethal concentration that killed 50% of snails (LC50 ) = 18.02 ppm,Lethal concentration that killed 90% of snails (LC90 ) = 24.00 ppm, Fiducial limits with 95% confidence limits = ± 1.184,Regression coefficient (r2) =0.933.



4 0.7782 30 100% - -

3.5 0.7404 28 93% 6.48 6.50

3 0.6990 20 67% 5.44 5.71

2.5 0.6532 11 37% 4.67 4.76

2 0.6021 4 13% 3.87 3.61

1.5 0.5441 0 0% - -

1 0.4771 0 0% - -

(Control) - 0 0% - -



30 1.4771 30 100% - -

25 1.3979 29 97% 6.88 6.39

20 1.3010 21 70% 5.52 5.93

15 1.1761 13 43% 4.82 3.33

10 1.0000 5 17% 4.05 4.49

5 0.6990 0 0% - -

(Control) - 0 0% - -


91


Number of snails tested for each concentration = 30, Regression equation = y = 7.611X - 1.763, Std. Error of X –Coefficient =0.732, Std. Error of Y – Coefficient =0.641, Lethal concentration that killed 50% of snails (LC50 ) = 7.50ppm, Lethal concentration that killed 90% of snails (LC90 ) = 11.51 ppm, Fiducial limits with 95% confidence limits =±0.841, Regression coefficient (r2) = 0.956.


Number of snails tested for each concentration = 30, Regression equation = y = 12.530X - 1.281, Std. Error of X –Coefficient = 1.564, Std. Error of Y – Coefficient = 0.746, Lethal concentration that killed 50% of snails (LC50 ) = 3.67ppm, Lethal concentration that killed 90% of snails (LC90 ) = 4.22 ppm, Fiducial limits with 95% confidence limits =

±0.454, Regression coefficient (r2) = 0.928.


Number of snails tested for each concentration = 30, Regression equation = y = 18.678X - 7.436, Std. Error of X –

Coefficient = 3.673, Std. Error of Y – Coefficient = 2.386, Lethal concentration that killed 50% of snails (LC50 ) = 4.21ppm, Lethal concentration that killed 90% of snails (LC90 ) = 5.48 ppm, Fiducial limits with 95% confidence limits =±0.643, Regression coefficient (r2) = 0.838.

Comparing the LC50 and LC90 values of the different solvents

used in these tests, the chloroform extract showed the higher activity,

followed by petroleum ether; ethanol and lastly the distilled water

extract. Statistical analysis showed high significant differences at 0.05



14 1.1461 30 100.0% - -

12 1.0792 29 97% 6.88 6.96

10 1.0000 23 77% 5.74 6.49

8 0.9031 16 53% 5.08 5.92

6 0.7782 9 30% 4.48 5.18

4 0.6021 2 7% 3.52 4.14

2 0.3010 0 0% - -

(Control) - 0 0% - -



4.5 0.6532 30 100% - -

4 0.6021 26 87% 6.13 6.72

3.5 0.5441 21 70% 5.52 6.14

3 0.4771 15 50% 5.00 5.46

2.5 0.3979 8 27% 4.39 4.66

2 0.3010 1 3% 3.12 3.69

1.5 0.1761 0 0% - -

(Control) - 0 0.00% - -



6 0.7782 30 100% - -

5.5 0.7404 27 90% 6.28 6.85

5 0.6990 23 77% 5.74 6.49

4.5 0.6532 16 53% 5.08 6.08

4 0.6021 10 33% 4.56 5.63

3.5 0.5441 4 13% 3.87 5.12

3 0.4771 0 0% - -

(Control) - 0 0% - -


92

107.64

11.35

23.44

132.4

2426 27.69

7.01

33.45

4.577.27

109.23

87.11

5.8

23.63

3.985.3

2.88

17.5

7.48

18.5418.5417.05

3.69

0

20

40

60

80

100

120

140

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Distilled Water Ethanol Chloroform Petroleum

Adult LC50 , LC90

Juveniles LC50 , LC90

Egg Mass LC50 , LC90

levels between LC50 and LC90 values of A. nodiflora leaves and

flowers material extracts of different solvents used in these tests

against one day old egg-masses of B. truncatus snails.

Finally, on comparing the age dependent susceptibility of B.

truncatus adult snails; one day old juvenile and one day old egg-masses

to A. nodiflora leaves and flowers extracts with the different solvents

showed that the juvenile snails were the most susceptible, followed by

one day old egg-masses and then the adult snails for both leaves and

flowers with different solvents as indicated in (figure 1 and 2).

Figure 1: Age dependent susceptibly of B. truncatus to A. nodiflora leaves extracts

with different solvents (Comparison of LC50 and LC90 values).

��

��


93

127.28

10.45

24

29.06

5.08

11.51 11.13

4.46

13.46

3.755.48

22.5

105

4.21

9.97

33.67

1.95

77 7.5

3.88

18.02

2.28

0

20

40

60

80

100

120

140

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Adult Juveniles Egg

Masses

Distilled Water Ethanol Chloroform Petroleum

Adult LC50 , LC90

Juveniles LC50 , LC90

Egg Mass LC50 , LC90

Figure 2: Age dependent susceptibly of B. truncatus to A. nodiflora flowers extracts

with different solvents (Comparison of LC50 and LC90 values).

Discussion

Schistosomiasis is still one of the most important public health

problems after malaria. Today, mollusciciding remains an important

aggressive strategy in the control of the snail hosts of schistosomiasis.

A. nodiflora plant has showed low values of LC100 for the both

leaves and flowers extract. It was therefore selected for further study

to test the potency of solvent extracts on adult, one day old juvenile

and one day old egg masses of B. truncatus snails

Data obtained from this preliminary study on A. nodiflora leaves

and flowers solvents extracts against different age stages of B.

truncatus snails has showed a good potent molluscicidal and ovicidal

activity. The mortality caused by A. nodiflora leaves and flowers


94

solvents extracts using the successive and Soxhlet methods showed a

clear significant correlation between dose and mortality, because an

increase in concentration of the working solution resulted in more

intake or entry of molluscicidal extract into the body of the snail

different stages. This trend is also independent upon several factors

such as, rate of penetration, nature of slope, variability and maximal

effects of active moieties (Goodman etal., 1985).

One of the problems envisaged in the use of plant extracts, in the

control of snails is the choice of the solvent for extracting the plant

material. Thus, the molluscicidal activity of A. nodiflora leaves and

flowers solvents extract, on the age stages of B. truncatus snail

revealed that the chloroform extract has the highest molluscicidal

activity on all stages at low concentration. LC90 0f A. nodiflora leaves

extracts were: 87.11, 109.23, 17.50 and 23.63 ppm on the adult snails;

7.48, 18.45, 2.88 and 3.98 ppm on one day old juveniles; 17.05, 18.54,

5.30 and 5.80 ppm on one day old egg-masses, respectively, while

LC90 of A. nodiflora flowers solvents extracts were: 1o5, 22.50, 7 and

9.97 ppm on the adult snails; 7.00, 3.88, 1.95 and 3.00 ppm on one

day old juveniles; 18.02, 7.50, 3.67 and 4.21 ppm, on one day old egg

masses respectively. The different potencies of the different parts may

be due to differences in the concentration and/or the type of the active

ingredient(s) present in each part, whereas the use of chloroform

provided the majority of the high active extracts in the bioassay with

B. truncatus. These observations can be rationalized in terms of the


95

polarity of the compounds being extracted by each solvent and in

addition to their intrinsic bioactivity.

According to World Health Organization, crude organic extracts

should present LC90 below 20 ppm to be considered as good

molluscicide candidate for direct application in infested water (WHO,

1993). However, it is possible that extracts active between 20 and 100

ppm could contain small amounts of very active components, which

could be isolated and/or concentrated using simple procedures, or

even obtained from other plants known to produce it in larger

amounts. Therefore, the results obtained using A. nodiflora seem to be

a promising plant molluscicide candidate and deserves further studies

in order to identify and characterize its molluscicidal components.

When we compare the results of molluscicidal potency of A.

nodiflora leaves and flowers in different solvents extraction (12 hours

Soxhlet and successive extracts) on B. truncatus stages, the one day

old juveniles were the most susceptible, followed by one day old egg-

masses and then the adult snails (Figures 1,2). The highest

susceptibility of one day old juvenile B. truncatus snails compared to

one day old egg-masses and adult snails might be attributed to the

high sensitivity of the juveniles to any external stimuli. On the other

hand, the less susceptibility of the adult snails may be due to a sort of

resistance and or adaptability to harmful environmental factors by

these snails, while the less susceptibility of the egg-masses to

molluscicides compared to one day old juvenile snails is attributed to

the gelatinous material around them which prevents or delays direct


96

exposure of embryos to molluscicides unless they possess high

capability of penetration. Somia (2003) found that the juveniles of

Biomphalaria pfeiferi were more susceptible to aqueous P. crispa

leaves extract than adult snails. Ali, (2007) found that the juveniles of

B. truncatus were more susceptible to P. crispa aqueous successive

extract than the adults.

The results obtained with A. nodiflora leaves and flowers in

distilled water successive extracts on B. truncatus adult snails, for 24

hours exposure were: LC90 87.11 and 105 ppm, respectively, this

shows A. nodiflora to be a powerful molluscicide compared with

aqueous extracts of many plants studied before. Somia, (2003)

reported LC90 685.7 ppm of aqueous extract of P. crispa leaves

against Biomphalaria pfeiferi adult snails, LC90 for one day old

juveniles was 77.86 ppm and LC90 for one day old egg-masses was at

200 ppm . Ali (2007) reported that P. crispa un-sieved residue of

leaves aqueous successive extract on B. truncatus adult snails LC90

240 ppm, while LC90 51.99 ppm for one day old juvenile snails.

Aqueous extract from Jatropha curcas L. (Euphorbiaceae) performed

poorly against snails transmitting Schistosoma mansoni and

Schistosoma haematobium, 500 ppm caused 50% mortality. 1000 ppm

of leaves and flowers water extracts of Ambrosia maritima caused

100% mortality of Bulinus truncatus. A. maritima is not toxic to cattle,

sheep, fish and man. Balanites aegyptiaca fruits extract on B.

truncatus snails caused 100% mortality at 9 ppm (Elamin et al., 2005).

Phtolocca dodecandra ‘endod’ revealed molluscicidal potency against


97

Planorbid snails within 24 hours (15-30ppm), but toxic to small fishes,

tadpoles, leeches at concentrations that required for snail control

(Kloos and McCullough, 1982). Aqueous extract of Corton

marostachys seeds (habit-el-mollok) was nearly as effective as major

synthetic molluscicides against B.truncatus(LC90 = 1 ppm), the

extract has ovicidal potency against the latest ages of B. pfeiferi eggs,

but the seeds were reported to have a high toxicity to humans

(Daffalla and Amin, 1976).

The results of the present investigations on A. nodiflora leaves and

flowers chloroform extracts for 12hours Soxhlet extract on B.

truncatus adult snails, for 24 hours exposure were: LC 50 (27.50

ppm), LC90 (17.50 ppm) and LC50 (11.13 ppm), LC90 (7 ppm),

respectively. These results are promising in comparison with other

solvents extracts of some previously studied plants such as: Cussonia

spicata stem bark with n-butanol extract on Biomphalaria glabrata

adult snails was 12.5 ppm, aqueous extract of Jatropha aceroids seeds

on B. truncatus adult snails have LC100 at 100 ppm, while the

chloroform successive extract of seeds revealed LC100 of 16.5 ppm

(Howida, 2005). Gnidia kraussiana roots and stem petroleum ether

successive extracts on B. truncaues adult snails revealed LC50 (0.02

ppm), LC100 (0.07 ppm) and LC50 (0.05 ppm), LC100 (0.3 ppm),

respectively, while for the leaves, the ethanol extract was the most

potent, revealing LC50 (3 ppm), LC100 (5 ppm) (El-Kheir and El-

Tohami, 1979). P. crispa leaves ethanol extract was used against B

truncates adult snails (Salha, 1999), Ali (1997), also repoted LC95


98

(1844.6ppm) for the fresh P. crispa leaves acetone extract on B.

pfeiferi adult snails, while for two months stored acetone extract

LC95 was 1979.2 ppm. Sukumaran et al. (2002), tested Sapindus

trifoliatus extracts on Lymnaea luteola egg-masses and adult snails,

the results showed that N-butanol extract was more toxic than the

methanol extract and the eggs were more susceptible than the adults.

Generally, the herb A. nodiflora appears to be one of the most

promising plants for the control of schistosomiasis. The plant is a

perennial herb that grows fast by seeding and it is not edible to

animals. It grows around water ponds or inside shallow water bodies

and highly saturated lands (locally called Aradi Al-Neil) in the form

of communities. The herb meets most of the important criteria for an

ideal molluscicide. It is an indigenous widely distributed plant in the

Sudan especially in areas where schistosomiasis is endemic. It is easy

to collect and prepare for use. Its active ingredients can be easily

extracted with most solvents including water.

On conclusion, the present investigation suggests further studies

on non- targeted animals and the field utilization or community self-

help snail control of A. nodiflora as an alternative plant molluscicide

that has to be assessed through a variety of approaches to confirm the

possibilities of its implementation in the field in the control of

schistosomiasis.


99

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