Mutation Research 514 (2002) 223–231

Genotoxic effects of malathion: an organophosphorus

insecticide, using three mammalian bioassays in vivo
S. Giri a,∗ , S.B. Prasad b , A. Giri a , G.D. Sharma c,1
a Genetic Toxicology Laboratory, Department of Life Science, Assam University, P.O. Box 11, Silchar 788011, India
b Cell and Tumor Biology Laboratory, Department of Zoology, North-Eastern Hill University, Slillong 793022, India
c Microbial Ecology Laboratory, Department of Life Science, Assam University, P.O. Box 11, Silchar 788011, India

Received 22 August 2001; received in revised form 14 November 2001; accepted 14 November 2001

Abstract
The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and
sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10 mg/kg) of malathion tested in the present study,
induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not
affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations,
two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology
following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase
was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in
average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and
may be regarded as a potential germ cell mutagen also. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Malathion; Organophosphorus insecticide; Chromosome aberration; Sister chromatid exchange; Sperm abnormality; Replication
index

1. Introduction Malathion [S-1,2-bis(ethoxycarbonyl)ethyl O,O-

According to a 1997 market estimate, approxi-
mately 5684 million pounds of pesticide active ingre-
dients are applied annually throughout the world [1].
The World Health Organization (WHO) [2] reported
that roughly three million pesticide poisonings occur
annually and result in 220,000 deaths worldwide.
Many of these chemicals are mutagenic [3,4], linked
to the development of cancers [5], or may lead to
developmental deficits [6].
∗ Corresponding author.
E-mail address: girisarbani@yahoo.com (S. Giri).
1 Present address: Nagaland University, Lumami, Kohima
797001, India.
dimethyl phosphorodithioate; CAS Registry No.
121-75-5] is a commonly used organophosphorus
insecticide. It has been employed in major eradi-
cation programs [7]. Malathion was implicated in
the epidemic malathion poisoning of 2800 Pakistani
spray men during a malaria control program [8]. The
large-scale use of malathion in various eradication
programs has raised concern over its potential to
cause genetic damage [9].
The genotoxic data on malathion is so far equivocal.
In experimental animal studies using very high doses
(ranging between 50 and 2000 mg/kg), malathion is
reported to produce chromosomal aberrations and mi-
cronuclei [9–11]. On occupational settings, pesticide
applicators exposed to technical grade malathion and

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 3 4 1 - 2
224 S. Giri et al. / Mutation Research 514 (2002) 223–231
other insecticides are reported to exhibit higher levels
of chromosome aberrations and SCEs [12]. Malathion
has been reported to produce chromosome aberrations
in plants also [13]. However, negative results in assays
for dominant lethals in mice [14]; chromosome loss
and non-disjunction in male germ cells [15], somatic
mutations in white–zeste eye spot test [16], and wing
spot test [17] in Drosophila melanogaster have also
been reported.
Most of the studies on the mutagenic effects of
malathion are supported from in vitro studies [3,18–
21]. However, in vivo test is especially relevant
for genotoxicity, because it allows consideration of
factors of in vivo metabolism, pharmacokinetics,
and DNA-repair process and is also useful in further
investigation of mutagenic effects detected by an in
vitro genotoxicity test [22]. In the present study, the
genotoxic potential of malathion, as revealed by bone
marrow chromosome aberration, SCEs and sperm
shape abnormality assays in mice is reported.
2. Materials and methods
2.1. Test chemicals
Malathion [S-1,2-bis(ethoxycarbonyl)ethyl O,O-
dimethyl phosphorodithioate; CAS Registry No.
121-75-5] (Fig. 1) was obtained from G.L. Indus-
tries (E) Ltd., Guwahati, India. Mitomycin C (MMC;
2 mg in 48 mg of NaCl) was obtained from Kyowa
Hakko Kogyo Co., Tokyo, Japan. Giemsa’s stain was
procured from Glaxo India Ltd., Mumbai. All other
chemicals used were of analytical grade. Malathion
and MMC were diluted with distilled water to the
required concentration before treatment.
2.2. Test animals
Swiss albino mice originally obtained from
Pasture Institute, Shillong, India, were maintained in
Fig. 1. Structure of malathion.
a closely inbreed colony. The animals were housed
in polypropylene cages (4–5 animals per cage with
sawdust as the bedding material) at 25 ± 5 ◦ C tem-
perature on a 12 h light/dark cycle. The animals were
supplied with dry food pellets commercially available
(Hindustan Liver Ltd., New Delhi), and water ad libi-
tum. Healthy animals of both sexes, in the age group
of 10–12 weeks were used for the experiments.
2.3. Dose
The highest sub-lethal acute dose (10 mg/kg) used
in the present study was standardized by trial. The
animals tolerated the highest dose with minimal toxic
symptoms. The toxic symptoms were mostly neuro-
logical. However, the animals recovered within 1 h of
the treatment. MMC (2 mg/kg) was used as a positive
control, and the untreated control mice were given
equal volumes of normal saline only.
2.4. Chromosome aberration assay
Three sub-lethal acute doses of malathion (2.5, 5
and 10 mg/kg) were administered through intraperi-
toneal (i.p.) route and the bone marrow cells were
fixed at 24 and 48 h after treatment. For sub-acute
exposure, the highest dose (10 mg/kg) was divided
into five equal parts and injected at 24 h successive
intervals. The animals were sacrificed after expiry
of 24 h of the last treatment. In addition, to have a
comparative idea on the route of exposure, the mid-
dle dose (5 mg/kg) and the sub-acute dose (2 mg for
5 days) were administered post-orally (p.o.) by gav-
age. Bone marrow cells were fixed at 24 h of the last
treatment.
Experimental animals were injected (i.p.) with
colchicine (4 mg/kg) 1.5 h prior to sacrifice. Bone
marrow cells were collected from both the femora
by flushing in KCl (0.56% at 37 ◦ C), incubated at
37 ◦ C for 18 min. The material was then centrifuged
at 1000 rpm for 5 min, fixed in acetomethanol (acetic
acid:methanol, 1:3). Centrifugation and fixation (in
cold) were repeated twice at an interval of 30 min.
The material, re-suspended in a small volume of the
fixative, was dropped on to chilled slides, flame-dried
and stained in the following day in 5% buffered
Giemsa (pH = 7.0). One hundred good metaphase
spreads were examined per animal and chromosome
 S. Giri et al. / Mutation Research 514 (2002) 223–231 225

Table 1
Frequency of chromosome aberrations in the bone marrow cells of mice induced by malathiona,b
Dose (mg/kg) Route Fixation Total Chromatid Isochromatid Exchanges SCU Total % Aberration
time (h) cells/n aberrationsc (mean ± S.D.)
Gaps Breaks Gaps Breaks

Control i.p. 24 300/3 1 – – – – – 0 00.00 ± 0.00

MMC (2.0) i.p. 24 300/3 26 78 2 11 4 1 94 31.30 ± 4.93
MMC (2.0) i.p. 48 300/3 9 49 2 5 2 1 57 19.00 ± 2.64
2.5 i.p. 24 300/3 4 5 1 – – – 5 01.66 ± 0.57∗ cde
2.5 i.p. 48 300/3 2 4 – 2 – – 6 02.00 ± 0.00∗
5 i.p. 24 300/3 4 10 2 2 – – 12 04.00 ± 1.00∗ df
5 i.p. 48 300/3 3 6 2 1 – 1 8 02.66 ± 0.57∗
10 i.p. 24 300/3 11 28 7 7 1 – 36 12.00 ± 1.73∗ abef
10 i.p. 48 300/3 5 12 3 4 1 – 17 05.66 ± 1.15∗ a
2.0 × 5 i.p. 24 300/3 8 14 5 2 1 – 17 05.66 ± 1.15∗ bc
MMC (2.0) p.o. 24 300/3 15 53 5 8 1 – 62 20.66 ± 4.50
5 p.o. 24 300/3 2 5 2 1 – – 6 02.00 ± 1.00∗
2.0 × 5 p.o. 24 300/3 8 12 3 2 – 1 15 05.00 ± 2.00∗
a Control: only normal saline was given. MMC: mitomycin C; SC: sister chromatid union; n: number of animals; i.p.: intraperitoneal;

p.o.: post-oral. Statistical analysis: Student’s t-test.

b Values having similar letters are significantly different from each other (P < 0.01 (a, b, f); P < 0.001 (c, e); P < 0.05 (d)).
c Values are excluding gaps.
∗ Significantly different from control (P < 0.01).

aberrations were classified as indicated in Table 1.
Gaps have not been considered for statistical analysis
due to their controversial genetic significance [23].
2.5. Sister chromatid exchange (SCE) assay
SCE assay was done as described by Goto et al. [24].
Ten minute before the various treatments, the mice
weighing between 20 and 22 g were sub-cutaneously
implanted with bromodeoxy uridine (BrdU) tablets
(50 mg) under light ether anesthesia. Before sacrifice,
the animals were treated with colchicine (4 mg/kg,
i.p.) for one and half hours. The animals were sac-
rificed by cervical dislocation after 24 h of the treat-
ment; both the femora were dissected out and cleaned
of any adhering muscles. Metaphase spreads were
prepared as described above and staining was done as
described by Goto et al. [24]. In brief, the slides were
treated for 10 min with Hoechst 33258 (50 ␮g/ml) in
dark at room temperature. The slides were then rinsed
in distilled water, mounted in 2× SSC (NaCl—sodium
citrate, pH = 6.8) and kept under direct sun light in
moist condition for 30–40 min depending upon the
intensity of sun light. Then the slides were rinsed and
stained in 3% buffered Giemsa (pH = 7.0) for 6 min.
Fifty well spread second division metaphases iden-
tifiable by their uniform differential staining pattern
containing 40 chromosomes were analyzed for SCE
per animal. Every switch of staining between the
sister chromatids was scored as one SCE.
In addition to SCEs, cells were analyzed for the
relative frequency of first division metaphases (M1,
identifiable by uniform staining of both the sister chro-
matids), second division metaphases (M2, identifiable
by differential staining of the sister chromatids), and
third and subsequent division metaphases (M3, iden-
tifiable by non-uniform pattern of staining). These
information were used to evaluate cell proliferation
kinetics as described by Tice and Hollaender [25].
Replication index (RI) or proliferation index is the
average number of replications completed by meta-
phase cells and is calculated as follows:
1 × (% first division metaphases) + 2
×(% second division metaphases) + 3
×(% subsequent division metaphases)
RI =
100
The average generation time (AGT) was determined
as the ratio of BrdU duration (h) and RI.
226 S. Giri et al. / Mutation Research 514 (2002) 223–231

2.6. Sperm abnormality assay frequencies of gaps were observed for all the doses
(2.5, 5 and 10 mg/kg) tested. A tentative assessment
Five equal sub-divisions of each dose were succes- of the distribution of breaks and gaps revealed that
sively injected (i.p.) at 24 h intervals to healthy and the distal regions of the long chromosomes were more
sexually matured male mice. The controls were given vulnerable to malathion.
an equal volume of normal saline. Animals treated Malathion induced a significantly higher frequency
with MMC (2 mg/kg) served as positive controls. of aberrations (P < 0.001) for all the three doses
The animals were sacrificed by cervical dislocation tested at both the time points (24 and 48 h) as com-
35 days after the first injection. Both the cauda epi- pared to the control (Table 1). The dose response
didymis were dissected out and placed in 1 ml of PBS curve at 24 h of i.p. treatment (Fig. 2A) shows a
(pH = 7.2). The sperms were released by mechanical linear increase in the frequency of aberrations with
disruption and washing of the epididymis. The suspen- increasing dose (r = 0.9734, P < 0.05). Although
sion was sieved through two layers of musolin cloth to the frequency of aberrations in the p.o. route was
remove the tissue debris. A drop of the suspension was
taken on a clean slide and a smear was made, air-dried
and fixed in absolute methanol for 10 min. The slides
were stained in the following day in 0.1% eosin Y
for 8–10 min. One thousand sperms per animal were
scored and the abnormalities were categorized as close
as to those described by Wyrobek and Bruce [26].
Sperm count was done as described by Rai and
Vijayalaxmi [27]. In brief, an aliquot (0.05 ml) from
the sperm suspension (1 ml) was diluted (1:40) with
PBS. A sample was taken in the Neubauer chamber
and the total sperm count in eight squires of 1 mm2
each was determined. The cell number was multiplied
by 5 × 104 and the sperm number was expressed as
cells/epididymis.

2.7. Statistical analysis

Student’s t-test was used for comparing the level

of significance in the results between the malathion
treated groups and the untreated control as well as
among the various treated groups. Regression analysis
was carried out to find out the dose–response relation-
ship.

3. Results

The metaphase analysis of the bone marrow cells

revealed various types of chromosomal aberrations
which consisted of chromatid and isochromatid types
of gaps and breaks, double minutes (included among
isochromatid breaks), exchanges, and sister chromatid
unions (SCU). Chromatid type breaks were noted to be
more frequent than others (Table 1). Relatively higher
Fig. 2. Incidence of chromosome aberrations in bone marrow cells
of mice following malathion exposure: (A) dose response curve
at 24 h of i.p. exposure showing dose-dependent increase in the
frequency of aberrations; (B) histogram showing time-dependent
change in the frequency of aberrations at 24 and 48 h of i.p.
exposure to malathion.
 S. Giri et al. / Mutation Research 514 (2002) 223–231 227

Table 2
Effect of malathion on cell cycle kinetics and the frequency of SCEs in the bone marrow cells of micea
Dose TM M1 M2 M3 % M1 % M1 RI AGT AGT Total SCE/M SCE/M
(mg/kg) (mean ± S.D.) (mean ± S.D.) cells (mean ± S.D.)
Control 181 18 154 9 9.94 10.46 ± 2.18 1.950 12.30 12.36 ± 0.12 50 2.80 3.06 ± 0.26
156 14 135 7 8.97 1.955 12.27 50 3.08
185 24 152 9 12.97 1.918 12.51 50 3.32
MMC (2.0) 240 91 146 3 37.91 42.09 ± 4.04 1.633 14.69 14.99 ± 0.28 50 9.16 9.30 ± 0.13
187 86 095 6 45.98 1.572 15.26 50 9.34
210 89 117 4 42.38 1.595 15.04 50 9.42
2.5 157 14 138 5 08.91 11.26 ± 2.63 1.942 12.35 12.59 ± 0.28 50 5.94 5.97 ± 0.21∗∗∗
195 21 169 5 10.76 1.917 12.51 50 5.78
163 26 134 3 14.11 1.858 12.91 50 6.20
5 166 24 139 3 14.45 15.41 ± 3.28 1.873 12.81 12.84 ± 0.20∗ 50 6.24 6.06 ± 0.17∗∗∗
173 22 147 4 12.71 1.895 12.66 50 6.04
152 29 119 4 19.07 1.835 13.07 50 5.90
10 185 35 142 3 18.91 1.772 13.07 50 6.24
170 29 138 3 17.05 1.847 13.54 50 5.90
205 39 162 4 19.02 18.32 ± 1.10∗∗ 1.829 12.99 13.21 ± 0.28∗∗ 50 6.36 6.16 ± 0.23∗∗∗
a
Control: only normal saline was given. TM: total metaphases; AGT: average generation time; M: metaphase; RI: replication index;
SCE: sister chromatid exchange. Statistical analysis: Student’s t-test (n = 3).
∗ Significantly different from control (P < 0.05).
∗∗ Significantly different from control (P < 0.01).
∗∗∗ Significantly different from control (P < 0.001).

relatively lower than the i.p. route, but these were
not significant (P > 0.05). Similarly, except for the
highest dose (10 mg/kg, P < 0.01), no significant
difference could be found in the time response study
(Fig. 2B). Further, the sub-acute (2 mg/kg for 5 days)
treatment produced lower effects (P < 0.01) than the
corresponding acute dose (10 mg/kg) (Table 1).
All the three acute doses (2.5, 5 and 10 mg/kg) of
malathion, induced significantly higher (P < 0.001)
frequency of SCEs as compared to the control value
(Table 2). However, no significant dose response could
be found for SCEs in the present study. The AGT was
significantly increased following 5 mg/kg (P < 0.05)
and 10 mg/kg (P < 0.01) of malathion exposure. The
frequency distribution of SCEs did not show any large
variation as most of the cells showed SCEs in the range
of 2–8 SCEs per cell (data not shown).
Following 2.5, 5 and 10 mg/kg of malathion treat-
ment, significant increases (P < 0.02, 0.001 and
0.001, respectively) in the frequency of sperm head

Table 3
Incidence of sperm shape abnormality induced by malathion and total sperm count in micea,b
Dose (mg/kg) No. of sperms No. of % Abnormal sperms Sperm count/epididymis
studies per animal abnormal sperms (mean ± S.D.) (mean ± S.D.) ×106
Control 3000/3 53 1.76 ± 0.35 6.42 ± 0.18
MMC (2.0) 3000/3 314 10.46 ± 0.65 5.16 ± 0.23
2.5 3000/3 89 2.96 ± 0.41∗ ac 6.44 ± 0.25
5 3000/3 158 5.20 ± 0.40∗∗ ab 6.21 ± 0.24
10 3000/3 227 7.56 ± 0.35∗∗ bc 6.22 ± 0.15
a Control: only normal saline was given. Statistical analysis: Student’s t-test.
b Values having similar letters are significantly different from each other (P < 0.02 (a, b); P < 0.001 (c)).
∗ Significantly different from the control (P < 0.05).
∗∗ Significantly different from the control (P < 0.01).
228 S. Giri et al. / Mutation Research 514 (2002) 223–231
abnormalities were noted as compared to the un-
treated control (Table 3). The amorphous type of head
morphology was more prevalent than other types.
However, no significant difference could be observed
in the total number of sperms per epididymis as
observed in the sperm count assay (Table 3).
4. Discussion
Malathion is a widely used pesticide with high
potential for human exposure. The genotoxic data
have been somewhat inconclusive with regard to
malathion exposure. It is found to be non-genotoxic in
a variety of test systems [14–17]. Positive results have
also been reported by many authors [3,9–12]. The
results of the present study indicate that malathion is
genotoxic in mice.
Malathion induced a variety of chromosomal
aberrations in the bone marrow cells (Table 1). The
dose-dependent increase in the frequency of chro-
mosome aberrations as observed presently (Fig. 2A),
indicate that the chemical might be available in greater
concentrations to the target cells at higher doses. The
significantly higher frequency of aberrations observed
at 48 h of the treatment could be due to residual ef-
fect of the chemical or its toxic metabolite(s). Similar
effects have been observed for carbofuran also [28].
In addition, for 2.5 and 5 mg/kg treated groups, no
significant difference in the frequency of chromo-
some aberrations at 24 and 48 h of the treatment was
found (Fig. 2B). This indicates that not only the par-
ent compound, but also its secondary metabolite(s)
may induce mutagenic effects. Malaxon, the oxida-
tion product of malathion is reported to be mutagenic
in human peripheral blood lymphocytes in vitro [21].
However, the significant difference (P < 0.01) in
the time response study in the groups treated with
10 mg/kg could be due to death and removal of highly
damaged cells from the population.
In the sub-acute (2 mg/kg for 5 days) treatment
group, significantly lower frequency of chromosome
aberrations was observed as compared to the equiv-
alent acute dose (10 mg/kg) (Table 1). However, the
higher frequency of chromosome aberrations in this
group than the acute dose of 2.5 (P < 0.01) and
5 mg/kg (P < 0.01) cannot be explained in the light
of residual effect and/or secondary genotoxic metabo-
lite(s) only. Therefore, the possibility of a cumulative
effect even for a shorter duration may not be ruled out.
SCEs arise from reciprocal exchanges of DNA at
apparently identical loci of the sister chromatids of
a duplicated chromosome in response to a damaged
DNA template [25,29]. It is suggested that there are at
least two forms of SCE induction. One is by damaging
DNA [30] and other is by inhibiting DNA synthesis
[31]. In eukaryotic cells, the frequency of SCEs is
reported to be increased following exposure to geno-
toxic agents that induce DNA damage capable of
interfering with DNA replication, but not strand
breakage only [29]. The significant increase (P <
0.001) in SCEs following malathion exposure in the
present study further supports the genotoxic poten-
tial of malathion. Malathion induces SCEs in other
test systems also [9]. Several studies have reported
that malathion is a very potent cell cycle inhibitor
[10–12,32]. In the present study, malathion induced a
small but significant delay in cell cycle in the 5 mg/kg
(P < 0.05) and 10 mg/kg (P < 0.01) treated group,
but not in the 2.5 mg/kg treated group. Further, we
failed to find any dose-dependent increase in the fre-
quency of SCEs. Our findings are in agreement with
earlier studies reporting lack of correlation between
the frequency of SCEs and dose [18,33]. We have ob-
served a clear dose-dependent increase in chromosome
aberrations in the present study (Fig. 2A) in contrast
to those reported earlier [11,34,35]. Therefore, the
cell cycle delay may only be partially responsible for
the lack of dose response observed here for SCEs, but
other factors may also be involved. The significance of
this finding is difficult to explain at present. However,
it appears that malathion may induce chromosome
aberrations and SCEs through different mechanisms.
These findings merit further investigations.
Sperm abnormality assay is a sensitive and reliable
endpoint and is widely used to identify germ cell
mutagens [25–27,36]. Induction of abnormal sperms
is presumed to be a result of naturally occurring errors
in the differentiation process, or the consequence of an
abnormal chromosome complement [37]. The charac-
teristics controlling sperm head shape are carried on
the autosomes and sperm abnormality test identifies
those agents, which cause small alterations in testic-
ular DNA [38,39]. The significant increase in sperm
head abnormality (Table 3) induced by malathion in-
dicates its probable potential as a germ cell mutagen.
 S. Giri et al. / Mutation Research 514 (2002) 223–231
It has been reported that exposure to pesticides results
in a decrease in sperm count [40,41]. However, in the
present study, malathion in the dose ranges tested did
not show any change in sperm count as compared to
the control.
The exact mechanism of the genotoxic effects of
malathion is not known. Organophosphorus insecti-
cides containing the P=S bond (called “thion”) are
converted to P=O (called “oxon”) by a system of
enzymes called microsomal mixed-function oxidases
(MFO) in which the enzyme cytochrome P-450 play
a major role [42]. The oxons are highly toxic com-
pounds, which account for the profound cytotoxic
effects of organophosphorus pesticides [43]. For
example, paraoxon and malaoxon are more potent
than parathion and malathion, respectively [42].
Organophosphorus compounds show alkylating
properties [44,45], and the methyl esters have a higher
alkylating potential than the ethyl esters [44]. Alkylat-
ing agents are known to cause DNA damage [46]. Most
genotoxic carcinogens are electrophillic by themselves
or activated to electrophillic intermediates that bind to
critical macromolecules [47]. The mutagenic activity
of malathion may be due to the existence of elec-
trophillic sites in the parent molecule or its metabolic
intermediates, which are capable of binding to nu-
cleophillic sites in DNA. In malathion, there are two
potential electrophillic sites—the alkyl group(s) and
the phosphoryl group [20]. Organophosphorus com-
pounds are reported to have the ability to bind to DNA
[45,48], and cause mutations [49,50]. Earlier stud-
ies have suggested only a weak interaction between
malathion and DNA. In these studies, malathion was
able to induce breakage in E. coli plasmid DNA [45],
alkylate nitrobenzylpyridine, a synthetic substrate
[51] and methylate DNA bases in vitro [52].
Malathion used in the present study is a techni-
cal grade product, and hence may contain impurities
formed during manufacturing and storage. Among the
impurities often found in malathion are malaoxon,
formed by oxidation of malathion, and isomalathion,
formed by isomerization of malathion [53]. Malaoxon
and isomalathion have been reported to be mutagenic
[9,20]. Therefore, possible contribution of impurities
to the observed genotoxic effect of malathion in the
present study cannot be ignored.
The present findings strongly indicate the potential
of technical grade malathion to induce genotoxicity
229
and may be regarded as a potential germ cell mutagen.
Therefore, application of malathion that exposes large
populations should be restricted.
Acknowledgements
We are thankful to the Head of the Department of
Life Science, Assam University, Silchar, for providing
laboratory facilities.
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