Vol.2, No.10, 1148-1154 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.210142
Effect of abiotic factors on the molluscicidal activity
of oleoresin of Zingiber officinale against the snail
Lymnaea acuminata
Vijya Singh, Pradeep Kumar, Vinay Kumar Singh, Dinesh Kumar Singh*
Malacology Laboratory, Department of Zoology, D. D. U., Gorakhpur University, Gorakhpur, India; *Corresponding Author:
Received 28 April 2010; revised 30 May 2010; accepted 3 June 2010.
ABSTRACT
Earlier it has been observed that oleoresin of
Zingiber officinale is a potent molluscicide ag-
ainst Lymnaea acuminata. This snail is the
vector of Fasciola species, which cause ende-
mic fascioliasis in eastern Uttar Pradesh. As
this snail breeds and maintain their population
constant through out the year, so that the pre-
sent study has been designed to find out the
effect of variations in some environmental fac-
tors in different seasons, on the molluscicidal
activity of oleoresin of Zingiber officinale and its
relative effect on certain enzymes viz., acetyl-
cholinesterase, acid and alkaline phosphatases
in the nervous tissue of the snail Lymnaea acu-
minata. In this study temperature, pH, dissolve
oxygen, free carbon dioxide, conductivity of the
water in control, as well as molluscicide treated
water, was measured simultaneously. LC50 value
of oleoresin was determined in each month of
the year. Toxicity of oleoresin in June-July (24 h
LC50 16.54-14.28 mgL-1) is highest. Acetylcholi-
nesterase, acid and alkaline phosphatases ac-
tivity in the nervous tissue of the snails treated
with sub-lethal concentration of oleoresin was
simultaneously measured. Sig- nificant positive
rank correlation, in between the acetylcholi-
nesterase or acid phosphatase activity and LC50
of oleoresin was observed. The pre- sent study
conclusively shows that variant abi- otic factors
can significantly alter the toxicity of oleoresin of
Z. officinale in L. acuminata. The most suitable
period for control of L. acuminata is June-July.
Keywords: Environmental factors;
Acetylcholinesterase; Oleoresin; Temperature; pH
1. INTRODUCTION
It has been reported that, oleoresin of Zingiber offici-
nale is a potent molluscicide [1,2]. Fresh water snail
Lymnaea acuminata is the intermediate host of liver
fluke Fasciola gigantica, causing an endemic fascio-
liasis in the cattle population of eastern region of the
state of Uttar Pradesh in India [3,4]. An effective method
to reduce the incidence of fascioliasis is to control the
population of vector snails and, thereby, break the life
cycle of these flukes [5-8]. Earlier studies have shown
that oleoresin of Zingiber officinale has a powerful mol-
luscicidal action on the snail L. acuminate [1,2]. It has
also been conclusively shown that acetylcholinesterase
(AChE), acid and alkaline phosphatase (ACP and ALP)
in the nervous tissue of L. acuminata are very sensitive
parameters influenced by molluscicides [7-10]. The aim
of the present study was to explore the possibility
whether seasonal changes in abiotic factors, viz tem-
perature, pH, dissolved oxygen and carbon dioxide, and
conductivity of test water can influence the level of
AChE, ACP and ALP assayed in each month of the year
2006-2007 following exposure to sublethal concentra-
tions (40% and 80%) of 24 h LC50 of oleoresin of Z.
officinale.
2. MATERIALS AND METHODS
2.1. Test Materials
Oleoresin was obtained by extraction of prepared
dried rhizomes of Z. officinale with alcohol. The re-
moval of the solvent under vacuum yields oleoresin of Z.
officinale [1,11]. Temperature, pH and conductivity of
water were measured by thermometer and digital pH and
conductivity meters, respectively. Dissolved O2 and CO2
were estimated according to the methods prescribe by
APHA [12].

V. Singh et al. / Natural Science 2 (2010) 1148-1154
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2.2. Bioassays for LC50
Adult L. acuminata (length 2.25 ± 0.2 cm) were col-
lected from Ramgarh Lake, located in almost adjacent to
this university campus. Snails were acclimatized in
de-chlorinated tap water for 72 h. The snails were ex-
posed to different concentrations of oleoresin in glass
aquaria containing 3 litres of de-chlorinated water. Ten
experimental animals were kept in each aquarium. Con-
trol animals were kept in equal volumes of de-chlorin-
ated tap water under similar conditions. Mortality of
snails was observed after 24, 48, 72, 96 h. No response
to a needle probe was taken as evidence of death. Dis-
solved O2, CO2 and conductivity, temperature and pH of
treated and control group of water was measured simul-
taneously with toxicity test at every 24 h of period to 96.
Bioassays for the determination of LC50 value was per-
formed in each month of the year. Lethal concentration
(LC50) values, lower and upper confidence limits (LCL
and UCL) and slopes value were calculated by the
method of POLO computer program of Robertson et al.
[13]. The Product moment correlation coefficient was
determined between LC50 and temperature / pH / con-
ductivity / dissolved O2 / CO2, of water in each of the
twelve months in order to observe any significant corre-
lation according to the method of Sokal and Rohlf [14].
2.3. Enzyme Assays
Twenty snails, kept in glass aquaria containing 5 litres
of dechlorinated water, were exposed to 40% and 80%
of 24 h LC50 of oleoresin in each month. Six such
aquaria were set up for each concentration. After 24 h
treatment, the snails were washed with water and the
nervous tissue was dissected out from the buccal mass
for the measurement of enzyme AChE, ACP and ALP
activities.
2.3.1. Acetylcholinesterase
Acetylcholinesterase (AChE) activity was measured
according to the method of Ellman et al. [15] as modi-
fied by Singh et al. [16]. Fifty mg of nervous tissue was
homogenized in 1.0 ml of 0.1 M phosphate buffer pH
8.0 for 5 minute in an ice bath and centrifuged at 1000 g
for 30 minute at 4℃. Supernatant was used as enzyme
source. The change in optical density at 412 nm was
recorded for 3 minute after every 30 second interval.
Enzyme activity was expressed as µ mol “SH” hydro-
lyzed / min / mg protein.
2.3.2. Phosphatases
Acid (ACP) and alkaline (ALP) phosphatases activi-
ties were measured by the method of Bergmeyer [17] as
modified by Singh and Agarwal [18]. Tissue homogenate
(2% w/v) was prepared in ice cold 0.9 % NaCl and cen-
trifuged at 5000 g for 20 minute at 4℃. The 4-nitro-
phenyl phosphate disodium was used as substrate. The
acid (ACP) and alkaline phosphatases (ALP) activity has
been expressed as µmole substrate hydrolyzed /30 min/
mg protein.
2.3.3. Protein Estimation
Protein was estimated by the method of Lowry et al.
[19].
2.4. Statistical Analysis
Results have been expressed as mean ± SE of six rep-
licates. Rank correlation was applied in between control
and corresponding changes in the enzyme activity in
different months of the year [14].
3. RESULTS
There was a significant (P < 0.05) time dependent
variation in the toxicity of oleoresin Z. officinale in dif-
ferent months of the year against L. acuminata (Table 1);
highest toxicity was observed in months of June and July
(24 h LC50 14.28-16.54 mgL-1) and lowest (24 h LC50
124.09-126.27 mgL-1) during January and February. A
significant positive correlation (r = 0.89; P = 0.001) be-
tween LC50 and water pH was noted for each month and
at each interval of 24 h exposure (Table 1). A similar
finding between LC50 and dissolved O2 (r = 0.82; P =
0.001) was found. Contrastively, significant negative
correlation between LC50 and dissolved CO2 (r = 0.86; P
= 0.001) and with water temperature (r = 0.91; P = 0.001)
was noted. No marked correlation was observed between
the LC50 and conductivity of water. High temperature
(36℃), and free CO2 (30.0 ppm), low pH (7.11) and
dissolved oxygen (1.0 ppm) increases the toxicity of
oleoresin against L. acuminata. The slope values were
steep and separate estimations of LC50 based on each of
the six replicates were found to be with in the 95% con-
fidence limits of LC50. The t-ratio is greater than 1.96
and the heterogeneity factor is less than 1.0. The g value
was less than 0.5 at all probability levels.
There was significant positive rank correlation (τ =
0.666; P = 0.02 – 40% of 24 h LC50, τ = 0.636, P= 0.02 –
80% of 24 h LC50) between LC50 of different months and
corresponding anti AChE activity in the sub-lethal
treatment (40% and 80% of 24 h LC50) of nervous tissue
of snail L. acuminata. Maximum inhibition in AChE
activity (56.09% of control) was observed in snails ex-
posed to 80% of 24 h LC50 of oleoresin in month of July
(Table 2). There was no significant positive rank corre-
lation between LC50 of different months and alkaline
phosphatase activity in the sub-lethal treatment (40%
and 80% of 24 h LC50) of nervous tissue of snail L. acu-
minata (Table 3). Like AChE, there was significant posi-
tive rank correlation between LC50 and acid phosphatas

V. Singh et al. / Natural Science 2 (2010) 1148-1154
1150
Table 1. Alterations in toxicity (LC50 mL-1) of oleoresin of Z. officinale against L. acuminata and different environmental factors in different months of the yea
r
2006-07.
Each experiment was replicated six times and values are the mean of six replications. Temperature, pH, dissolved oxygen, free carbon dioxide and conductivity were measured intervals of 24 h to 96 h. Product moment correlation coefficient in between the LC50 and different parameters indicate significant (P < 0.05) (+) positive / (*) negative correlation.

V. Singh et al. / Natural Science 2 (2010) 1148-1154
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Table 2. Effect of 24 h exposure of 40% and 80% of 24 h LC50 of oleoresin of Z. officinale in different months of the year 2006-07
on acetylcholinesterase activity in the nervous tissue of L. acuminata.
Months 24 h LC50 mgL-1 AChE-µ mole “SH” hydrolyzed / min / mg protein
Control
a 40% of 24h LC
50 80% of 24h LC50
August 39.67
0.093 ± 0.01
(100)
0.091 ± 0.01
(97.84)
0.088 ± 0.01
(94.62)
September 54.54
0.139 ± 0.01
(100)
0.136 ± 0.01
(97.84)
0.132 ± 0.01
(94.96)
October 55.92
0.101 ± 0.02
(100)
0.099 ± 0.01
(98.02)
0.097 ± 0.02
(96.04)
November 58.79
0.106 ± 0.01
(100)
0.103 ± 0.02
(97.17)
0.101 ± 0.01
(95.28)
December 62.18
0.109 ± 0.00
(100)
0.107 ± 0.01
(98.17)
0.105 ± 0.01
(96.33)
January 126.27
0.190 ± 0.00
(100)
0.189 ± 0.01
(99.47)
0.186 ± 0.01
(97.89)
February 124.09
0.176 ± 0.01
(100)
0.175 ± 0.00
(99.43)
0.171 ± 0.00
(97.16)
March 74.93
0.147 ± 0.02
(100)
0.142 ± 0.01
(96.60)
0.134 ± 0.01
(91.16)
April 24.88
0.141 ± 0.02
(100)
0.137 ± 0.01
(93.66)
0.133 ± 0.01
(84.51)
May 21.67
0.104 ± 0.01
(100)
0.101 ± 0.02
(75.92)
0.099 ± 0.03
(70.37)
June 16.54
0.087 ± 0.03
(100)
0.083 ± 0.02
(62.19)
0.074 ± 0.02
(59.75)
July 14.28
0.082 ± 0.02
(100)
0.073 ± 0.01
(63.41)
0.071 ± 0.01
(56.09)
Values are mean ± SE of six replicates. Value in parenthesis indicates % enzyme activity with untreated control taken as 100%. Rank correlation coefficient in
between LC50 and AChE activity in treated group indicate significant (P < 0.05) positive (+) correlation. a, Significant (P < 0.05) when one way of ANOVA was
applied in between the enzyme activity in different months of the year in control group without treatment.
Table 3. Effect of 24 h exposure of 40% and 80% of 24 h LC50 of oleoresin of Z. officinale in different months of the year 2006-07
on alkaline phosphatase activity in the nervous tissue of L. acuminata.
Months 24 h LC50 mgL-1 ALP-µ moles / 30 min / mg protein
Control 40% of 24 h LC50 80% of 24 h LC50
August 39.67
2.27 ± 0.01
(100)
2.17 ± 0.01
(95.59)
2.14 ± 0.01
(94.27)
September 54.54
3.34 ± 0.01
(100)
3.31 ± 0.01
(99.10)
3.27 ± 0.01
(97.90)
October 55.92
3.27 ± 0.01
(100)
3.23 ± 0.02
(98.77)
3.19 ± 0.02
(97.55)
November 58.79
3.36 ± 0.02
(100)
3.32 ± 0.02
(98.80)
3.27 ± 0.00
(97.32)
December 62.18
3.11 ± 0.00
(100)
3.06 ± 0.02
(98.39)
3.02 ± 0.02
(97.10)
January 126.27
2.89 ± 0.01
(100)
2.85 ± 0.00
(98.62)
2.81 ± 0.01
(97.23)
February 124.09
3.07 ± 0.01
(100)
3.02 ± 0.01
(98.37)
2.98 ± 0.01
(97.06)
March 74.93
3.10 ± 0.00
(100)
3.07 ± 0.00
(99.03)
3.02 ± 0.01
(97.41)
April 24.88
3.45 ± 0.01
(100)
3.42 ± 0.02
(99.13)
3.37 ± 0.02
(97.68)
May 21.67
2.94 ± 0.02
(100)
2.89 ± 0.00
(98.29)
2.86 ± 0.03
(97.28)
June 16.54
1.86 ± 0.03
(100)
1.74 ± 0.02
(93.55)
1.64 ± 0.02
(88.17)
July 14.28
2.36 ± 0.01
(100)
2.23 ± 0.01
(94.49)
2.16 ± 0.02
(91.52)
Values are mean ± SE of six replicates. Value in parenthesis indicates % enzyme activity with untreated control taken as 100%. Rank correlation coefficient in
between LC50 and ALP activity in treated group indicate non significant (P < 0.05) positive correlation. a, Significant (P < 0.05) when one way of ANOVA was
applied in between the enzyme activity in different months of the year in control group without treatment.

V. Singh et al. / Natural Science 2 (2010) 1148-1154
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(ACP) (τ = 0.606; P = 0.05 – 40% of 24 h LC50, τ = 0.606;
P = 0.05 – 80% of 24 h LC50) activity in the nervous
tissue of L. acuminata exposed to sub-lethal treatments
of oleoresin in different months. Maximum inhibition in
ACP activity (91.00% of control) was observed in snails
exposed to 80% of 24 h LC50 in July (Table 4).
4. DISCUSSION
It is clear from result section that toxicity of oleoresin
varies with changes in abiotic environmental factors in
the water. Effect of abiotic variants in aquatic environ-
ment i.e. pH [20], Temperature [21] on the toxicity of
different pesticides have been reported. The temperature
of water is a significant factor, which alters the toxicity
of oleoresin in each month of the year. When the water
temperature is higher in summer season June-July, the
toxicity of oleoresin is maximum. Contrarily, in winter
season, the temperature of water is low and toxicity of
oleoresin is less as evident by higher LC50 value. Tem-
perature of environment in which the animal resides is a
crucial factor, when toxicity of pesticides is determined
[22-25]. Osterauer and Kohler [26] reported that the tox-
icity of diazinon against zebra fish strongly increased at
elevated temperature. Dissolved oxygen is also one of
the factors, which alter the toxicity of oleoresin. Water
in winter season holds more oxygen [27] and as a result,
less mortality of snails occurs during this period. At higher
water temperature dissolved oxygen concentration de-
creases which is reflected by higher mortality of the
snails. Dissolved oxygen is one of the major components,
which is required by snails during metabolic activity
[28,29]. Consequently, at higher temperature, increasing
rate of metabolism in snail body may release more CO2,
which affects the pH of water [30,31] As the time dura-
tion increases concentration of CO2 increases in the wa-
ter (released by snails) and it also affects the pH of water.
Murphy [32] reported that pesticides belonging to or-
ganophosphate and carbamate groups are very sensitive
to change in pH. Earlier Vasconcellos [33] observed the
influence of pH variation on the molluscicidal activity of
Euphorbia splendens latex. According to them mollus-
cicidal activity was maximum at pH 5.0 - 6.0 and mini-
mum at pH 7.0 - 8.0. Toxicity of oleoresin is highest at
high temperature, CO2 of water as well as low pH, dis-
solved O2 of water. The low concentration of dissolved
O2 act as physical stressor on aquatic animals [34] and in
the absence of sufficient dissolved O2; the snails appear
to become more sensitive against the molluscicide. The
pungent moieties of oleoresin are gingerol, zingirone and
shogaol [35]. It is conceivable that, the active mollus-
cicidal component, present in the oleoresin might get
converted into a more toxic form in the aquarium water
Table 4. Effect of 24 h exposure of 40% and 80% of 24 h LC50 of oleoresin of Z. officinale in different months of the year 2006-07
on acid phosphatase activity in the nervous tissue of L. acuminata.
Months 24 h LC50 mgL-1 ACP-µ moles/ 30min / mg protein
Control 40% of 24 h LC50 80% of 24 h LC50-
August 39.67
3.15 ± 0.00
(100)
3.04 ± 0.01
(95.51)
2.91 ± 0.01
(92.38)
September 54.54
3.64 ± 0.01
(100)
3.58 ± 0.01
(98.35)
3.52 ± 0.01
(96.70)
October 55.92
3.31 ± 0.01
(100)
3.29 ± 0.01
(99.40)
3.24 ± 0.00
(97.89)
November 58.79
3.34 ± 0.02
(100)
3.32 ± 0.02
(99.40)
3.28 ± 0.01
(98.20)
December 62.18
3.28 ± 0.01
(100)
3.23 ± 0.01
(98.48)
3.19 ± 0.01
(97.26)
January 126.27
3.29 ± 0.01
(100)
3.27 ± 0.00
(99.70)
3.21 ± 0.02
(97.87)
February 124.09
3.40 ± 0.00
(100)
3.39 ± 0.01
(99.70)
3.33 ± 0.01
(97.94)
March 74.93
3.46 ± 0.01
(100)
3.40 ± 0.00
(98.27)
3.34 ± 0.01
(96.53)
April 24.88
3.26 ± 0.01
(100)
3.19 ± 0.01
(97.85)
3.09 ± 0.02
(94.79)
May 21.67
3.45 ± 0.02
(100)
3.38 ± 0.02
(97.97)
3.31 ± 0.02
(95.94)
June 16.54
2.95 ± 0.03
(100)
2.86 ± 0.01
(96.95)
2.72 ± 0.03
(92.20)
July 14.28
3.00 ± 0.01
(100)
2.82 ± 0.01
(94.00)
2.73 ± 0.01
(91.00)
Values are mean ± SE of six replicates. Value in parenthesis indicates % enzyme activity with untreated control taken as 100%. Rank correlation coefficient in
between LC50 and ACP activity in treated group indicate significant (P < 0.05) positive (+) correlation. a, Significant (P < 0.05) when one way of ANOVA was
applied in between the enzyme activity in different months of the year in control group without treatment.

V. Singh et al. / Natural Science 2 (2010) 1148-1154
115
1153
or in the snail body due to variant environmental factors
in the month of June and July. Earlier, it has been shown
that the treatment of oleoresin of Z. officinale caused
significant inhibition of AChE, ALP and ACP activity in
the nervous tissue of L. acuminata [9]. The high anti
AChE and ACP activity of oleoresin of Z. officinale was
observed in months of June-July. The enzyme ALP plays
a critical role in protein synthesis [36] and secretary ac-
tivity [37] is comparatively less inhibited than AChE.
Acid phosphatase (ACP), a lysosomal enzyme [38], plays
an important role in autolysis and phagocytosis, patho-
logical necrosis, and overall catabolism [8,10,39] was
reduced significantly. Earlier, it has been observed that
increased activity of ACP causes breakdown of existing
protein in L. acuminata [18], but inhibition of ACP ac-
tivity in this study indicates that it is not used in break-
down of cellular protein. The rank correlation coefficient
applied between the LC50 values of different months and
the corresponding inhibition in enzyme activity, point
out a positive correlation between the LC50 and the inhi-
bition of AChE and ACP. Whereas there was no correla-
tion in between LC50 and ALP activity indicate that ALP
is not altered by action of oleoresin in different months.
Accurate prediction of molluscicide fate and toxicity
in aqueous environment against snails are hindered due
to lack of information that how abiotic factors of aque-
ous environment affect the biological activity and related
toxicity of molluscicides. Abiotic factors are not only
correlated with the lethality of molluscicide, but with
each other also. The present study conclusively shows
that variant abiotic factors can significantly alter the
toxicity of oleoresin of Z. officinale in L. acuminata. It is
also obvious that the most suitable period for the control
of this snail in India is the month of June, July. It is sug-
gested that the treatment of a water body with oleoresin
of Z. officinale for the control of L. acuminata and ulti-
mately fascioliasis, is not only more potent and cost ef-
fective during these months than spending more money
by using higher concentrations of this molluscicide dur-
ing the rest ten months of the year.
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