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 Ali2
’*
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:mhdyousif@yahoo.com
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‫اﻟﻔﺎﴍ‬ ‫ﺟﺎﻣﻌﺔ‬–‫ﻣﺠﻠﺔ‬‫اﻟﻌﻠﻮم‬‫اﻟﺘﻄﺒﻴﻘﻴﺔ‬–‫اﻷول‬ ‫اﻟﻌﺪد‬–‫ﻳﻮﻧﻴﻮ‬2013‫م‬
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 Extracts
2-6-1 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 % (78-80 b.r), Chloroform 95 % (60-62 b.r),
Petroleum ether 90 % (40-60 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 Tests
2-7-1 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 Extracts
lethal 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,cd)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, Lethal
concentration that killed 90% of snails (LC90 ) = 132.40ppm, Fiducial limits with 95% confidence limits = ± 2.014, Regression
coefficient (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% confidence
limits = ± 1.184, Regression coefficient (r2
) =0 .966.
Table 1, d- Petroleum ether extract
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, Lethal
concentration that killed 90% of snails (LC90 ) = 33.45ppm, Fiducial limits with 95% confidence limits = ± 1.363, Regression
coefficient (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 concentraƟon = 30͕ Regression equaƟon = y = 7.932X Ͳ ϭ͘ϵϯϮ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ y ʹ ŽĞĨĨŝĐŝĞŶƚ
сϬ͘ϲϳϲ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ z ʹ ŽĞĸĐŝĞŶƚ сϬ ͘ϱϱϲ͕ ĞƚŚĂů concentraƟon that killed 50% of snails (LCϱϬ Ϳ с ϳ͘ϰϴ ƉƉŵ͕ ĞƚŚĂů
concentraƟon that killed 90% of snails (LCϵϬ Ϳ с ϭϭ͘ϯϱ ƉƉŵ͕ ŝĚƵĐŝĂů ůŝŵŝƚƐ ǁŝƚŚ ϵϱй ĐŽŶĮĚĞŶĐĞ ůŝŵŝƚƐ с цϬ ͘ϳϴϲ͕ ZĞŐƌĞƐƐŝŽŶ
ĐŽĞĨĨŝĐŝĞŶƚ ;ƌ
Ϯ
Ϳ сϬ ͘ϵϳϮ͘
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% - -
ŽŶĐĞŶƚƌĂƚŝŽŶ DŽƌƚĂůŝƚǇ WƌŽďŝƚ
;ƉƉŵͿ ŽŐ dŽƚĂů ;йͿ dĂďƵůĂƚĞĚ ĂůĐƵůĂƚĞĚ
ϭϮ ϭ͘ϬϳϵϮ ϯϬ ϭϬϬй Ͳ Ͳ
ϭϬ ϭ͘ϬϬϬϬ Ϯϱ ϴϯй ϱ͘ϵϱ ϲ͘ϭϯ
ϴ Ϭ͘ϵϬϯϭ ϭϳ ϱϳй ϱ͘ϭϴ ϱ͘ϱϭ
ϲ Ϭ͘ϳϳϴϮ ϴ Ϯϳй ϰ͘ϯϵ ϰ͘ϳϮ
ϰ Ϭ͘ϲϬϮϭ Ϯ ϳй ϯ͘ϱϮ ϯ͘ϲϭ
Ϯ Ϭ͘ϯϬϭϬ Ϭ Ϭй Ͳ Ͳ
;ŽŶƚƌŽůͿ Ͳ Ϭ Ϭй Ͳ Ͳ

85
Table 2, b- Ethanolic extract
EƵŵďĞƌ ŽĨ ƐŶĂŝůƐ ƚĞƐƚĞĚ ĨŽƌ ĞĂĐŚ concentraƟon = 30͕ Regression equaƟon = y = 8.877X Ͳ ϱ͘ϴϮϰ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ y ʹ ŽĞĨĨŝĐŝĞŶƚ
с Ϭ͘ϴϰϴ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ z ʹ ŽĞĸĐŝĞŶƚ с Ϭ͘ϵϲϴ͕ Lethal concentraƟon that killed 50% of snails (LCϱϬ Ϳ с ϭϴ͘ϰϱ ƉƉŵ͕ ĞƚŚĂů
concentraƟon that killed 90% of snaiůƐ ;ϵϬ Ϳ с Ϯϰ͘ϬϬ ƉƉŵ͕ ŝĚƵĐŝĂů ůŝŵŝƚƐ ǁŝƚŚ ϵϱй ĐŽŶĮĚĞŶĐĞ ůŝŵŝƚƐ с ц ϭ͘ϭϮϮ͕
ZĞŐƌĞƐƐŝŽŶ ĐŽĞĨĨŝĐŝĞŶƚ ;ƌ
Ϯ
Ϳ сϬ ͘ϵϳϯ͘
Table 2, c- Chloroform extract
Number of snails tested for each concentraƟon = 30, Regression equaƟon = y = 12.797X Ͳ ͘ϱϳϰ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ y ʹ ŽĞĨĨŝĐŝĞŶƚ
с ϭ͘ϴϲϴ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ z ʹ Coeﬃcient =0 .762, Lethal concentraƟon that killed 50% of snails (LCϱϬ Ϳ с Ϯ͘ϴϴ ƉƉŵ͕ ĞƚŚĂů
ĐŽŶĐĞŶtraƟon that killed 90% of snails (LCϵϬ Ϳ с ϯ͘ϲϵ ƉƉŵ͕ ŝĚƵĐŝĂů ůŝŵŝƚƐ ǁŝƚŚ ϵϱй ĐŽŶĮĚĞŶĐĞ ůŝŵŝƚƐ с цϬ ͘ϯϲϯ͕ ZĞŐƌĞƐƐŝŽŶ
ĐŽĞĨĨŝĐŝĞŶƚ ;ƌϮ
Ϳ сϬ͘ϵϬϰ͘
Number of snails tested for each concentraƟon = 30͕ Regression equaƟon = y = 16.575X Ͳ ϯ͘Ϯϳϰ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ y ʹ
ŽĞĸĐŝĞŶƚ с Ϯ͘Ϯϱϰ͕ ^ƚĚ͘ ƌƌŽƌ ŽĨ z ʹ ŽĞĸĐŝĞŶƚ с ϭ͘Ϯϭϰ͕ Lethal concentraƟon that killed 50% of snails (LCϱϬ Ϳ с ϯ͘ϵϴ ƉƉŵ͕
Lethal concentraƟon that killed 90% of snails (LCϵϬ Ϳ с ϰ͘ϱϳ ƉƉŵ͕ ŝĚƵĐŝĂů ůŝŵŝƚƐ ǁŝƚŚ ϵϱй ĐŽŶĮĚĞŶĐĞ ůŝŵŝƚƐ с ц Ϭ͘ϰϱϰ͕
ZĞŐƌĞƐƐŝŽŶ ĐŽĞĨĨŝĐŝĞŶƚ ;ƌ
Ϯ
Ϳ сϬ ͘ϴϵϱ͘
Ϯϱ ϭ͘ϯϵϳϵ ϯϬ ϭϬϬ͘Ϭй Ͳ Ͳ
ϮϬ ϭ͘ϯϬϭϬ ϮϮ ϳϯй ϱ͘ϲϭ ϲ͘ϭϬ
ϭϱ ϭ͘ϭϳϲϭ ϭϭ ϯϳй ϰ͘ϲϳ ϰ͘ϴϳ
ϭϬ ϭ͘ϬϬϬϬ ϯ ϭϬй ϯ͘ϳϮ ϯ͘ϭϱ
ϱ Ϭ͘ϲϵϵϬ Ϭ Ϭй Ͳ Ͳ
ϰ Ϭ͘ϲϬϮϭ ϯϬ ϭϬϬй Ͳ Ͳ
ϯ͘ϱ Ϭ͘ϱϰϰϭ Ϯϵ ϵϳй ϲ͘ϴϴ ϲ͘ϳϱ
ϯ Ϭ͘ϰϳϳϭ Ϯϯ ϳϳй ϱ͘ϳϰ ϱ͘ϵϮ
Ϯ͘ϱ Ϭ͘ϯϵϳϵ ϭϰ ϰϳй ϰ͘ϵϮ ϰ͘ϵϱ
Ϯ Ϭ͘ϯϬϭϬ ϱ ϭϳй ϰ͘Ϭϱ ϯ͘ϳϲ
ϭ͘ϱ Ϭ͘ϭϳϲϭ Ϭ Ϭй Ͳ Ͳ
ϭ Ϭ͘ϬϬϬϬ Ϭ Ϭй Ͳ Ͳ
;ŽŶƚƌŽůͿ Ͳ Ϭ Ϭ͘ϬϬй Ͳ Ͳ
ϰ͘ϱ Ϭ͘ϲϱϯϮ ϯϬ ϭϬϬй Ͳ Ͳ
ϰ Ϭ͘ϲϬϮϭ Ϯϴ ϵϯй ϲ͘ϰϴ ϲ͘ϱϵ
ϯ͘ϱ Ϭ͘ϱϰϰϭ ϮϮ ϳϯй ϱ͘ϲϭ ϱ͘ϵϲ
ϯ Ϭ͘ϰϳϳϭ ϭϯ ϰϯй ϱ͘Ϭϴ ϱ͘Ϯϯ
Ϯ͘ϱ Ϭ͘ϯϵϳϵ ϱ ϭϳй ϰ͘ϴϮ ϰ͘ϯϳ
Ϯ Ϭ͘ϯϬϭϬ Ϭ Ϭй Ͳ Ͳ
ϭ͘ϱ Ϭ͘ϭϳϲϭ Ϭ Ϭй Ͳ Ͳ

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)
Coefficient = 1.139, Std. Error of Y – Coefficient = 1.301, Lethal concentration that killed 50% of snails (LC50 ) = 17.05
ppm, 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 concentration
that 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, Lethal
concentration 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
ppm, 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,
) =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 –
ppm, 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
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,
) =0 .951.
Coefficient = 0.439, Std. Error of Y – Coefficient =0 .384, Lethal concentration that killed 50% of snails (LC50 ) = 9.97
ppm, 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% - -
Coefficient =0.814, Std. Error of Y – Coefficient =0 .668, Lethal concentration that killed 50% of snails (LC50 ) = 7.00
) =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, Lethal
concentration that killed 90% of snails (LC90 ) = 5.08 ppm, Fiducial limits with 95% confidence limits = ±0 .485,
) =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, Regression
coefficient (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
) =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):-
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,
) =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
Coefficient =0.732, Std. Error of Y – Coefficient =0.641, Lethal concentration that killed 50% of snails (LC50 ) = 7.50
) = 0.956.
) = 0.928.
) = 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
24
26 27.69
7.01
33.45
4.57
7.27
109.23
87.11
5.8
23.63
3.98
5.3
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).
ǆƚƌĂĐƚŝŽŶ
ĐŽŶƐĞŶƚƌĂƚŝŽŶƐ ;ƉƉŵͿ

93
127.28
10.45
24
29.06
5.08
11.51 11.13
4.46
13.46
3.75
5.48
22.5
105
4.21
9.97
33.671.95
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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