Assessment of the changes in some diagnostics enzymes in fish Gambusia affinis treated with pesticide chlorpyrifos

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ISSN Print: 2278 – 2648 IJRPP |Volume 3 | Issue 4 | Oct-Dec-2014
ISSN Online: 2278-2656 Journal Home page: www.ijrpp.com
Research article Open Access
Assessment of the changes in some diagnostics enzymes in fish
Gambusia affinis treated with pesticide chlorpyrifos
Neelam Sharma,
Department of Zoology, Government College, Ajmer (Rajasthan) India.
*Corresponding author: Neelam Sharma,
E-mail id: drsharmaneelam1@gmail.com
ABSTRACT
The acute toxicity of an organophosphorus pesticide chlorpyrifos on enzyme activities of fish Gambusia affinis was
evaluated under statistic conditions. The fish is constantly exposed to pesticides, which are used extensively to
control agricultural pests. Evaluation of toxic effect of chlorpyrifos on fish was carried out. Effect of sub lethal
(LC50), 1/3rd
and 1/10th
LC50 concentrations of chlorpyrifos at 15, 30 and 45 days of exposure was carried out in the
sensitive organs such as liver. An alteration in the Acid phosphatase (ACP), Alkaline phosphatase (ALP), Lactate
dehydrogenase (LDH) and Adenosine triphosphatase enzyme (ATPase) was carried out. The enzyme ACP, ALP and
LDH was elevated throughout the experimented period and the ATPase activity was inhibited along the
experimental period at three different concentrations of chlorpyrifos.
Keywords: ACP, ALP, LDH, ATPase, Chlorpyrifos, Gambusia affinis and Liver.
INTRODUCTION
The application of various pollutants like pesticides
in the aquatic environment and their deposition in the
biotic system is known to cause several structural and
functional changes in the biota. A major part of the
world’s food is being supplied from fish source, so it
is essential to secure the health of fishes [1]. In India
as much as 70% of the chemical formulations
employed in agricultural practices are believed to
affect non-target organisms and to find their way to
fresh water fish, ultimately polluting them [2].
Pesticides are extensively used to protect agricultural
crops against the damages caused by pests. However,
this may reach other ecological compartments as
lakes, ponds and rivers through rain and wind,
affecting many other organisms away from the
primary target. Chlorpyrifos (CPF), O, O-diethyl-O-
(3, 5, 6-trichloro-2-pyridyl) phosphorothioate is a
broad spectrum organophosphate (OP) pesticide used
extensively for the management of domestic and
agricultural pests. CPF is commercially used for
more than a decade to control foliar insects
(Arthropoda) that affect agricultural crops, and
subterranean termites [3]. Fish species are sensitive
to enzymatic as well as hormone disruption which is
caused due to stress and pesticide effect. The use of
biochemical measurements in organisms as indicators
of pollution, give information about the adaptive or
deleterious responses in organisms exposed to a
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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certain amount of chemicals. Such analysis provides
early warning signals before other toxicological
points, including death is evident [4] Chlorpyrifos is
the second largest selling OP agro chemical in India.
Dursban is the most toxic organophosphorus
compound for fish and is more toxic than
organochlorine compounds.[5] Effects of insecticides
on the survival chance, biochemical parameters,
tissues and organs, development and growth, nervous
system, behavior, genetic and immune system of fish.
The information given the study facilitates the
evaluation of potential toxic hazard resulting from
exposure to different levels of these compounds. [6]
The teleost fish Gambusia affinis (Common name-
Mosquito fish) was selected for the present study due
to its wide availability and suitability as model for
toxicity testing and also due to sustainability in
laboratory conditions. The fish shows a well adaptive
nature with the changing environment. Hence, the
present study is the impact of the chlorpyrifos
pesticide on the non-target organisms and also
various enzymatic changes in liver of fish Gambusia
affinis.
MATERIAL & METHODS
Test chemicals
All the chemicals used in the present study of
analytical grade and were used without further
purification. The commercial grade 36% E.C
chlorpyrifos, a light yellow liquid was selected for
the present study.
Animal maintenance and sub-lethal study
The fish, Gambusia affinis was collected from Local
pond of Jhalra, near Ajmer District. Fishes were
transported to laboratory in large aerated fiber glass
and maintained in aquarium tanks containing well
aerated dechlorinated tap water (with physic-
chemical characteristics: (temperature 24±2°C, pH
7.1±0.2 at 24°C, dissolved oxygen 9.6±0.8 mg/L,
carbon dioxide 6.3±0.4 mg/L, total hardness 23.4±3.4
mg as CaCO3/L, phosphate 0.39±0.002 μg/L,
salinity) in different 50 L plastic tanks, for 20 days.
Water was renewed every day and a 12-12 hrs.
Photoperiod was maintained during acclimatization
and test periods. The fishes were fed on commercial
pelleted diet once a day, throughout the tenure of the
chronic experiment. The fish weight (0.5-1.0 gm),
length (3.0-4.5 cm) from both sexes were selected
randomly for the experimental studies. Mortality of
fishes was recorded in each group for 96 hr. The
regression equations were established by using probit
- mortality and log of concentration of pesticide and
LC50 value was determined. The present work
investigates the enzymes activities (ALP, ACP, LDH
& ATPase) in liver and kidney of fish Gambusia
affinis treated with pesticides; Chlorpyrifos. Fish
were divided into four groups containing 10
individuals each, Group I: Control, Group II:
Exposed to 1/10th of LC50 value, Group III: Exposed
to 1/3rd of LC50 value, Group IV: Exposed to Sub
lethal concentrations of 96 hrs-LC50 value of
chlorpyrifos. Each group was exposed to 0.284 (Sub
lethal), (1/10th of sub lethal) 0.028, and (1/3rd of sub
lethal) 0.094 ppm of Chlorpyrifos.
The fish were exposed to this concentrations for 15,
30 and 45th days and a control group was maintained
at an identical environment. The fish was dissected
out from all treated groups on 15, 30 and 45th days.
The liver taken out for enzymatic studies, weighted
tissue was washed in ice-cold isotonic saline. Various
biochemical parameters of liver were selected as
indicators of toxicity. Saline extract was prepared by
homogenizing a weighed piece of liver in measured
quantity of 0.89% (ice cold) saline glass
homogenizer. The homogenate was centrifuged at
4000 rpm for 15 minutes, to obtain clear supernatant.
The supernatant was kept at -20°C. It was used for
enzyme estimation of Alkaline Phosphatase (ALP),
Acid Phosphatase (ACP), Lactate dehydrogenase
(LDH) and ATPase estimation.
Enzyme assays
The ALP and ACP enzyme activity was estimated by
U.V. Kinetic Method (2008) and expressed as IU/L
[7] [8]. Lactate dehydrogenase activity estimated by
Bruns and Bergmeyer’s method, 1965, (IU/L) [9].
ATPase estimated by Koch’s method, 1970 [10].
ATPase colorimetric assay kit employs a 96-well
plate format with all reagents necessary for
measuring ATPase activity and expressed as nmol
Pi/min/mg protein.
Statistical analysis

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The experiments were repeated thrice and data was
analyzed by the Student ‘‘t’’ test. Significant
differences from exposure values p>5% was accepted
as levels of significance. Values are expressed mean
± SE of observations. Values are significant at x
P<0.05, y P<0.01, z P<0.001.
OBSERVATIONS
Mortality studies showed that the sub lethal level,
LC50 of Gambusia affinis for 96 hr. exposure was
0.284 ppm for Chlorpyrifos. The minimum effective
doses was exposed to 0.284 (Sub lethal), (1/10th of
sub lethal) 0.028, and (1/3rd of sub lethal) 0.094 ppm
of chlorpyrifos were calculated for experimental
purposes. Enzyme estimations of ACP, ALP, LDH
and ATPase activity in liver of control as well as in
test Gambusia affinis were done. Observations are
shown in below Tables:
Table 1: Changes in Acid phosphatase level in liver of Gambusia affinis during control and post-treatment
with three different concentrations of Chlorpyrifos at different periods (15, 30, 45th days) of exposure
Days of Exposure
Control Exposure Concentration
1/10th
of LD50 1/3rd
of LD50 LD50
15 Days 4.52±0.018 x
7.41±0.018 x
7.55±0.005y
7.87±0.005y
30 Days 5.43±0.027 x
7.52±0.012 x
7.89±0.005y
8.04±0.008y
45 Days 6.18±0.012 y
7.88±0.018 x
8.06±0.005y
8.13±0.005y
Value expressed in (IU/L) (Mean ± SD)
Table 2: Changes in Alkaline phosphatase level in liver of Gambusia affinis during control and post-treatment
with three different concentrations of Chlorpyrifos at different periods (15, 30, 45th days) of exposure
Days of Exposure
Control Exposure Concentration
1/10th
of LD50 1/3rd
of LD50 LD50
15 Days 8.02±0.018 x
9.31±0.015 x
9.66±0.005y
9.78±0.005y
30 Days 8.35±0.03 x
9.40±0.013 x
9.77±0.005y
9.88±0.005y
45 Days 8.69±0.018 x
9.54±0.011 y
9.89±0.005y
9.97±0.005y
Value expressed in (IU/L) (Mean ± SD).
Table 3: Changes in Lactate Dehydrogenase activity in liver of Gambusia affinis during control and post-
treatment with three different concentrations of Chlorpyrifos at different periods (15, 30, 45th days) of
exposure
Days of Exposure
Control Exposure Concentration
1/10th
of LD50 1/3rd
of LD50 LD50
15 Days 1.55±0.01x
2.00±0.01x
2.17±0.01x
2.25±0.01x
30 Days 1.78±0.01x
2.11±0.01x
2.34±0.01x
2.52±0.01x
45 Days 1.81±0.01x
2.54±0.01x
2.62±0.01x
2.79±0.01x
Value expressed in (IU/L) (Mean ± SD).

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Table 4: Changes in ATPase activity in liver of Gambusia affinis during control and post-treatment with three
different concentrations of Chlorpyrifos at different periods (15, 30, 45th days) of exposure
Days of Exposure
Control Exposure Concentration
1/10th
of LD50 1/3rd
of LD50 LD50
15 Days 1.85±0.01x
1.23±0.01x
1.13±0.01x
1.02±0.01x
30 Days 1.42±0.01x
1.11±0.01x
1.01±0.01x
0.79±0.01x
45 Days 1.33±0.005Y
0.88±0.005Y
0.83±0.005Y
0.72±0.005Y
Value expressed in (nmol Pi/min/mg protein) (Mean ± SD).
RESULT & DISSCUSION
The liver has a key role in xenobiotic metabolism and
excretion, digestion and storage, and the production
of yolk protein. Thus, alterations in structure are
expected under certain toxic conditions. The present
investigation indicates that toxicity of chlorpyrifos to
fish Gambusia affinis and significant alterations in
enzyme estimation in liver tissue. Table (1,2,3)
showed the level of ACP, ALP and LDH activity
were statistically increased, while in table 4 ATPase
activity were significantly decreased in treated fish
Gambusia affinis after exposed to different
concentration at 15, 30 and 45 days of exposure. All
these results are agreement with different previous
researchers which indicated that the exposure to
chlorpyrifos and other pesticides led to induce severe
biochemical and physiological disturbance in
experimental fish. The results showed a significant
decrease in ATPase activity while there is
significantly increased showed ACP, ALP and LDH
enzymes activity. Significantly, such biochemical
changes in fish are aimed at maintaining equilibrium
in the presence of chlorpyrifos pesticide which are
known to disrupt physiological and biochemical
process. In the present investigation, decrease was
more in sub lethal concentrations. It may be due to
detoxification enzymes hyperactivity and the
mechanism becomes apparently slow.
Acid phosphatases are hydrolytic lysosomal enzymes
and are released by the lysosomes for the hydrolysis
of foreign material; hence it has a role in certain
detoxification functions. Increased in ACP enzyme
activity in all the three conc. might be due to increase
in protease activity which causes damage to the
lysosomal membrane, thus permitting the leakage of
lysosomal enzyme into cytoplasm. Alteration in the
enzyme activity is due to adverse effect of
xenobiotics on the cell and its organelles. The present
study, the acid phosphatase activity of liver of
Gambusia affinis increased in all the three doses.
Such increase is also found as a function of exposure
period. The pronounced increase in protease activity
may be due to the damage caused to the lysosomal
membrane, thus permitting the leakage of lysosomal
enzyme into cytosol as suggested by Sherekar and
Kulkurni [11]. This increase in the hepatic alkaline
phosphatase activity was due to the cellular damages
caused by hepatotoxins or a response to overcome
toxicity of chlorpyrifos. Increase in ALP activity is
due to interruption in the secretion and flow of bile in
the liver. According to Mahendra and Agarwal
(1983) inhibition of alkaline phosphatase activity by
pesticide might be because, the enzyme serine
residue at their active sites is inhibited by
organophosphorus pesticide, quinalphos [12]. It has
been stated that organophosphorus compounds are
general inhibitors of serine containing enzyme [13].
Lactate dehydrogenase (LDH) levels showed a
significant increase in liver tissue of chlorpyrifos
treated fish groups after 15, 30 and 45 days of
exposure to chlorpyrifos. LDH activity increased
might be due to a shift from aerobic respiration to
anaerobic respiration promoting the glycolytic rate

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and the conversion of pyruvic acid to lactic acid,
under insecticide-induced stress. The tissues might
have tried to compensate the incapability of oxygen
consumption under stress condition. The increase in
LDH activity is due to the leakage of lysosomal cells
into the blood stream. An increased LDH activity has
been reported in different tissues of liver, muscle,
intestine, kidney, gill and brain of Channa punctatus
when exposed to low and high concentration of
phenyl mercuric acetate for short and long-term
exposure [14]. Khan and Sharma studied statistical
analysis in behavioral of the Gambusia affins
following sub-lethal exposure to chlorpyrifos; these
behavioral responses can be used as a tool in
biomonitoring programme to monitor ecotoxicity risk
of chlorpyrifos to the test species [15]. The ATPase
activity was depleted significantly at all three
concentrations over the different period of exposure
which was an indication of tissue hypoxic conditions.
In the present study, it has been found that increasing
dose exposure of chlorpyrifos caused decreased
activity of ATPases in liver. This could be due to
pesticide induced effect on cell membrane because of
their strong affinity for interaction with member
lipids. The major target molecules affected by
pesticides include ion dependent ATPases, which
lead to disturbances in ion homeostasis. The
inhibition of ATPases leads to decreased ATP
breakdown and reduced the availability of free
energy. Decrease of this enzyme may result from the
breakdown of the active transport mechanism and
also due to the disturbed Ca2+
homeostasis [16].
Altered Ca2+
levels due to the metal exposures
supported the decline of Ca2+
- ATPase activity in
this research [17]. Hence, results of the present
investigation conclude that organophosphate
chlorpyrifos has inhibitory and acceleratory effect on
the ACP, ALP, LDH and ATPase activities in liver of
an experimental fish Gambusia affinis.
CONCLUSION
The present study has showed the manner of response
and the induction of toxicological effects in fish
Gambusia affinis after exposure of (chlorpyrifos) at
different doses at the different period of exposure. It
is of importance to note here that the fish were
exposed to these doses for a period to disturb the
liver function. The increase in hepatic acid
phosphatase activity in intoxicated animals as
observed in the present investigation may be due to
the destruction of the lysosomal membrane which
resulted in the release of the enzyme. It is found that
chlorpyrifos (Dursban) disturb the chemical
constituents of the fish which leads to cell damages
and finally death of fishes.
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