International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Toxicity of Neem Leaf Extracts (Azadirachta indica) on DNA
and RNA Content in Different Organs of Grass carp
Ctenopharyngodon idella
Nutan D. Rathod
#Assistant Professor, Department of Zoology, Moolji Jaitha College, Jalgaon, India.
Abstract–The objective of this study was to determine
the toxicity of the neem leaf extracts from the neem
tree, Azadirachta indica; on common edible fish grass
carp (Ctenopharyngodon idella) as well as
investigation of its sublethal concentration for 24, 48,
72 and 96 hrs were observed on DNA and RNA
contents in selected tissues like gills, liver, kidney and
muscle of sample fish. Nucleic acids like DNA and
RNA react differently with the various concentration
and period of exposure. An overall decrease in nucleic
acid was noted in these contents which is statistically
significant.
Keywords— Azadirachta indica, toxicity, DNA, RNA,
Ctenopharyngodon idella.
I. INTRODUCTION
Azadirachta indica is a common medicinal plant in
India having high a wide spectrum of biological
activity and well known for its insecticidal properties
and one of the most promising natural compounds
(Winkaler et al., 2007; ICAR, 1993). Neem is called
„Arista‟ in Sankrit - a word that means „perfect,
complete and imperishable‟. This eco-friendly native
tree of India is perhaps most researched tree in the
world. The neem tree (Azadirachta indica) has been
the subject of at least 153 patents worldwide. Neem
has been used worldwide in traditional medicine for
various therapeutic purposes: anti-bacterial, antifungal, anti-viral and anti-fertility properties. Neem
has been used in India and other southeast countries
for thousands of years in agriculture, food storage and
incurring many diseases (Biswas et al., 2002; Das et
al., 2002). Water-soluble extract of Azadirachta indica
leaves was found to possess significant hypoglycemic,
hypolipidemic, hepatoprotective, antifertility and
hypotensive activities. Many researcher studies have
proven that neem has been used for insecticides,
fungicides and miticides. Neem plant and their
product have played a significant role. Neem has
proved to be one of the most effective for its various
products as biopesticides and reduce the chemical load
in the environment (Tiwari and Singh, 2003). The
aqueous extract of neem leaves and other neem-based
products have been extensively used in fish farms as
alternative for the control of fish parasites and fish fry
predators (Martinez, 2002). Although neem extract is
considered of low toxicity towards non-target aquatic
life, while long exposure to low concentrations of the
crude extract of A. indica delayed the growth of fish
ISSN: 2231-5381
(Hassanein and Hanan, 2008). However, the impact
of neem extracts with reference to grass carp
Ctenopharyngodon
idella
is
very
limited.
Ctenopharyngodon idella is an important Indian grass
carp and widely cultivated in different parts of India
for edible and economic importance. Therefore, in this
study we aimed to evaluate the toxic effects of
aqueous leaf extract of A. indica on certain
biochemical parameters of Ctenopharyngodon idella.
The present study is also aimed at establishing the safe
limits of aqueous extracts of neem on water quality. In
order to achieve the desired results administrative and
technical measures based on well planned research
investigations are urgently necessary with increasing
public concern.
II. MATERIALS AND METHODS
A. Study Area
The Jalgaon district is situated in latitude 210 00‟
18.39” N and longitude 750 32‟ 47.99” E and is a part
of Maharashtra, India shown in Figure 1. (JalaSRI
Watershed surveillance Research Institute, Jalgaon,
ArcGIS 9.3.1). Mehrun Lake is located in latitude
20058‟29.80” N to 20059‟04.85”N and Longitude
75033‟39.90” E to 75034‟16.85” E. The lake is nearly
0.710355 Km2 and Perimeter is 3.93595 Km. It is an
important irrigation and drinking water source, and
bird nesting and visiting area.
Figure1: Map showing the Jalgaon, Maharashtra,
India. (Study area: latitude 210 00‟ 18.39” N and
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
longitude 750 32‟ 47.99” E)-JalaSRI Watershed
surveillance Research Institute, Jalgaon, ArcGIS 9.3.1.
experimental fish. The water of appropriate
concentration of the neem leaf extract from the
experimental aquaria as well as of control was change
once in every twenty-four hours. Each treatment had
B. Sampling and Sample Preparation
Sampling was collected from the Mehrun Lake three replicates till the percentage mortality in each of
were selected for the present study. Selected common the tests was constant. Acute toxicity tests were
edible fish grass carp (Ctenopharyngodon idella) were conducted for 24, 48, 72 and 96 hours duration for
capture from Mehrun Lake, were selected and water neem leaf extract. After completion of the exposure
sample meant for physico-chemical analysis (Table-1). period, dead fishes were counted. From the number of
The freshwater fish Ctenopharyngodon idella with a dead fishes percentage mortality was calculated and it
length ranging from 5 to 8 cm and weight ranging was then converted in to probit kill values by means of
from 10  5 gms were collected and acclimatized for probit table and plotted against the logarithmic values
a week in Laboratory condition. The physico- of the respective concentration. While plotting
chemical parameters like pH, EC, air temperature, actually on the graph paper it was observed that very
water temperature, dissolved oxygen (Winkler‟s few of the values diverged from the straight line
technique), free CO2, carbonate, bicarbonate, total passing through most of them. To take an account of
alkalinity and acidity were studied frequently such diverting values, regression equations were work
following the standard procedures described in APHA, out by the statistical method described by Bailey,
(1998).
(1965). The regression equation between log
concentration (in ppm) (x) and probit mortality (y)
C. Preparation of aqueous Neem leaf extract
The Neem plant Azadirachta indica - fresh green was statistically calculated using the formula
leaves were collected during April and May from area (according to Finney, 1971) (Y = a ± b x) The Neem
of the botanical garden of M. J. College College, leaf extract is the least toxic of all the neem leaf
Jalgaon. Azadirachta indica (A. Indica) leaves were extracts its LC50 values for 24, 48, 72 and 96 hours are
dried and finely chopped then dissolved in tap water, 0.827, 0.671, 0.606 and 0.415 ppm respectively.
500 gms / L, for 24 hours at room temperature (Cruz Concentrations of metal level which significantly
et al., 2004). The mixture was filtered and the extract effects of neem leaf extract on sample fish were
was used immediately in the experiments in different observed (Table 1, 2 and 3) and Graph 1 and 2.
dilutions.
D. Biochemical parameters
Estimation of DNA and RNA was carried out as
III. TABLE I
described by the method of Schneider, (1957) using
diphenylamine and orcinol regents respectively.
Parameters
Site-I
Site-II
SiteHomogenates (10 mg . ml, w/v) were prepared in 5%
TCA at 900C, centrifuged at 5000 rpm for 20 min and
III
supernant was used for estimation. Calf thymus DNA
7.3
7.2
pH
7.2
and yeast RNA were used as standard for DNA and
6.2
5.8
RNA respectively. Both DNA and RNA have been
EC
5. 5
expressed as mg . mg tissue.
0
0
27 C
AIR TEMP
27 0C 27 C
Table -2: The DNA content (mg/mg) in selected
WATER TEMP
25 0C
25 0C
25 0C
OXYGEN CONTENT
3.32
3.36
3.35
FREE CARBON DIOXIDE
9.86
9.75
9.52
TOTAL ALKALINITY
72
75
76
(BROMOCRESOLINDICATOR)
ACIDITY
3.78
3.78
3.78
(PHENOL PHTHALEIN
METHODE)
All Parameter are expressed in mg/L except pH and
temperature.
D. Determination of LC50 value of neem leaf
extracts
For the experiment, the classical method used for
the evaluation of the toxicity of aqueous neem leaf
extract is to exposed groups of ten fishes in each
successive batch to different concentrations. The first
group was controls were maintained along with
ISSN: 2231-5381
tissues of freshwater fish.
Tissues
Control
Neem Leaf extracts
24 hrs
48 hrs
72 hrs
96 hrs
Liver
3.69
2.48
1.92
1.58
1.22
S.D
0.773
0.624
1.284
1.624
1.822
P' value
0.01
0.01
0.01
0.01
%
-32.79
-47.96
-57.18
-66.93
Muscle
2.83
2.13
1.74
1.35
1.03
S.D
0.89
0.26
0.27
0.22
0.24
P' value
0.01
0.01
0.001
0.001
%
-24.73
-38.51
-52.29
-63.6
Gills
1.86
1.34
1.22
0.95
0.81
S.D
0.065
0.033
0.063
0.13
0.015
P' value
0.001
0.001
0.001
0.001
%
-27.95
-34.41
-48.92
-56.45
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Kidney
2.73
2.34
2.08
1.78
1.68
S.D
0.062
0.024
0.068
0.12
0.15
P' value
0.001
0.001
0.001
0.001
%
-14.28
-23.81
-34.79
-38.46
Graph-2
DNA expressed as mg . mg tissue
Graph-1
4
3
2
Liver
1
Muscle
0
7
6
5
4
3
2
1
0
Liver
Muscle
Gills
Kidney
24 hrs 48 hrs 72 hrs 96 hrs
Gills
24 hrs48 hrs
Kidney
72 hrs 96 hrs
Control
Neem Leaf extracts
Control
Neem Leaf extracts
Table -2: The RNA content (mg/mg) in selected
tissues of freshwater fish.
Tissues
Control
Neem Leaf extracts
24 hrs
48 hrs
72 hrs
96 hrs
Liver
6.89
5.36
4.72
4.02
3.44
S.D
0.32
0.05
0.11
0.26
0.38
0.001
0.001
0.001
0.001
-22.21
-31.49
-41.65
-50.07
P' value
%
Muscle
5.23
3.32
2.41
2.22
1.13
S.D
0.31
0.11
0.25
0.28
0.48
0.001
0.001
0.001
0.001
-36.52
-53.91
-57.55
-78.39
P' value
%
Gills
3.86
3.34
2.84
2.64
2.43
S.D
0.19
0.37
0.012
0.016
0.12
0.001
0.001
0.001
0.001
-13.47
-26.42
-31.61
-37.05
P' value
%
Kidney
4.52
4.11
3.86
3.42
2.76
S.D
0.28
0.27
0.49
0.012
0.17
0.01
0.01
0.001
0.001
-9.07
-14.6
-24.33
-38.93
P' value
%
RNA expressed as mg . mg tissue
ISSN: 2231-5381
RESULTS AND DISCUSSION
Biochemical studies were done to understand the
changes in the glycogen, protein, lipid and nucleic
acid content in the gills, liver, kidney and muscle
tissues of grass carp (Ctenopharyngodon idella). Prior
to death, darkening of the exposed fish, erratic
swimming, and respiratory distress were observed.
The alterations were not uniform in all the tissues. In
case of control fish, biomolecules like DNA and RNA
of the 4 tissues are in the order as DNA: L > M > K >
G; and RNA: L > M > K > G.
Biochemical variations in relation to pesticide
toxicity indicate alteration in nucleic acid synthesis.
The level of DNA and RNA was found to be
decreased in the gills, liver, kidney and muscle of
Ctenopharyngodon idella. Despite the importance,
information on impact of neem extracts on DNA and
RNA are meager. Impairment of nucleic acid
metabolism results in deranged protein metabolism.
The decrease in nucleic acid suggests the decrease in
protein synthesis and further damage to the liver,
which is the major metabolic organ of drug
detoxification (Ramalingam et al., 2000). This is
because the animal requires more energy to overcome
the stress upon exposure to the neem extracts. From
the present investigation, it can be concluded that the
marked decrease in the DNA content. In case of DNA,
most decrease was found in the liver and least in the
kidney. These results are in accordance with the
results recorded by Hassanein (2004). Furthermore,
these results may also be due to damage occurred in
the DNA. This suggestion is supported by the marked
decrease DNA observed during the present study, as
well as the results of Akudugu et al. (2001) who
reported that DNA is a critical target for neem
intoxication. RNA plays significant role in protein
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
synthesis. Hence, any alteration in RNA content
reflects on protein levels of the animals. Thus, this
RNA content can be show a relationship with the rate
of protein synthesis and thereby the protein level of
the animal. The former observations on protein
showed a greater decrease in the level after the
treatment with neem leaf extracts, which correlate
with the present results of RNA. This reduction in
RNA level thus reduces the protein level. In
conclusion, data collected in the present investigation
may suggest that these plant extracts are potential
inhibitor of DNA synthesis. Thus, it is possible that
these pesticides will have inhibited the enzyme
necessary for DNA synthesis. On compilation of the
results, it appears that the disruption of DNA synthesis
might have affected RNA synthesis and consequently
protein synthesis.
The above information is significant and essential
because the environmental factors and ecosystem in to
which the pesticides are introduced will determine
whether the material is toxic. It is therefore, necessary
to study the effect of synthetic pesticides, herbicide
and natural plant products (Neem leaf extract) on
commercially
important
freshwater
fish.
Establishment of suitable guidelines for the protection
of aquatic organisms against pollution is governed by
many factors. Some of these factors are the amounts
and types of chemical wastes present, the
interpretation of chemical residue analyses and the
degree of fluctuation in physical and chemical
environmental conditions. The resources of fishes that
can sustain regular and very productive fisheries are of
scientific interest. The significance of fishes in future
would be greater as a potential source for human
consumption and also in scientific research.
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