Biplab Sarkar • Arabinda Mahanty • Ashis Saha • Arttatrana Pal •
Partha Bandyapadhyay • Sampad Kumar Sarkar • Subhendu Adhikari •
S. Ayyappan
Received:
31 May 2013 / Revised: 14 September 2013 / Accepted: 23 October 2013 /
Published online: 26 November 2013
_ The National Academy of Sciences, India 2013
Abstract A short term histological study was
conducted
to
determine the impacts of technical grade synthetic
pyrethroid
insecticide, cypermethrin and carbamate pesticide,
carbofuran
on different phases of ovarian maturation
of
freshwater indigenous carp, Labeo rohita. Adult females
of
L. rohita were
exposed to sublethal doses of carbofuran
(0.06,
0.15 mg/L) and cypermethrin (0.16 and 0.40 lL/L)
for
4 weeks during the pre-spawning (March), spawning
(July)
and post-spawning (November) phase. In the
spawning
phase, the carp showed maximum ovarian damage
by
both the pesticides while the pre-spawning phase
was
the next impaired stage. Considering all the phases of
ovarian
maturation, cypermethrin exhibited greater level of
impact
than carbofuran in both of its doses. Gonadosomatic
indices
for all these phases were also measured. In
the
reproductive cycle of fish, reduction in gonado-somatic
index
occurred by both the pesticides in all of its doses and
the
order was spawning[pre-spawning[post-spawning.
It
is concluded that ovarian maturation in Indian carp is
affected
by both the pesticides.
Keywords : Carbofuran _ Cypermethrin _
Gonado-somatic
index _ Histology _ Labeo rohita _
Ovary
Introduction
In
recent years, insecticides are used extensively in agriculture
for
the pest control but their residues often reach
aquatic
ecosystems. As a result of the chemical contaminants,
many
freshwater ecosystems are faced with spatially
or
temporally alarming levels of these xenobiotic chemicals.
They
are transferred through phytoplankton to fish and
ultimately
to humans. Different synthetic pyrethroid pesticides
have
largely displaced organophosphorous and organochlorine
pesticides
in last two decades throughout the
world.
Fish sensitivity to pyrethroids may be explained by
their
relatively slow metabolism and elimination of these
compounds
[1]. Although these are not
persistent in the
environment,
their acute toxicity to fish is high [2]. Among
different
pyrethroids, cypermethrin is a synthetic pyrethroid
which
is used to control many pests and is discharged into the
aquatic
environment [3].
Several laboratory studies have
shown
that cypermethrin is extremely toxic to fish and
aquatic
invertebrates even at very low concentrations [4].
Fish
is highly sensitive to very low concentration of cypermethrin
(0.4–2.2
lg/L) and acute exposure of
cypermethrin
B.
Sarkar
National
Institute of Abiotic Stress Management, Baramati,
Pune,
India
A.
Mahanty
Central
Inland Fisheries Research Institute, Barrackpore,
Kolkata,
India
B.
Sarkar _ A. Mahanty _ A. Pal
School
of Biotechnology, KIIT University, Bhubaneswar, India
B.
Sarkar (&) _ A. Saha _ S. K. Sarkar _ S. Adhikari _
S.
Ayyappan
Central
Institute of Freshwater Aquaculture, Kausalyaganga,
Bhubaneswar,
India
e-mail:
biplabsarkar.niam@gmail.com;
biplab_puru@yahoo.co.in
P.
Bandyapadhyay
Aquaculture
Research Unit, Vidyasagar University, Midnapore,
West
Bengal, India
S.
Ayyappan
Director
General Office, Indian Council of Agriculture Research,
Krishi
Bhavan, New Delhi, India
Proc.
Natl. Acad. Sci., India, Sect. B Biol. Sci. (Oct–Dec 2014) 84(4):989–996
DOI
10.1007/s40011-013-0265-8
inhibits
enzymatic activities, total protein, soluble protein
and
structural protein contents in fish tissues [5]. Carbofuran
is
an insecticide widely used in crops including rice, strawberries,
alfalfa,
corn, grapes, soybeans, and wheat. Carbofuran
is
a cholinesterase inhibitor. Owing to its relatively
short
half-life in the environment, it is used as a substitute for
insecticides
known to persist for long period of time and
accumulate
in animal tissues [6].
Relatively high water solubility
of
carbofuran coupled with its relatively low
adsorption
on soils and sediments permits natural surface
waters
to become a repository for excessive amounts of the
insecticide,
escaping treated areas and being accidentally
introduced
into aquatic environments. Carbofuran’s persistence
is
directly related to the pH of the water. The toxicity,
metabolism,
and degradation of carbofuran have been
extensively
studied in aquaculture. Among freshwater
organisms,
fish is the most sensitive and LC50 values for
carbofuran
in different fish varieties range from 130 to
14,000
ppb in tests of 72–96 h [6].
Gonadal
maturation is an important phenomenon in fish
physiology
that ultimately triggers the reproductive success,
fertilization
and hatching process leading to maximization
of
fingerling number. This phase also determines
the
success of professional aquaculture practices. Carps
contribute
substantially to Indian aquaculture production.
Among
carps, rohu is the major cultivable and marketable
fish
in India. There are some reports on pesticide induced
toxicity
in different fish but this is restricted to gonad of
perches,
mullets, catfishes [7–11] and in model fish like
zebra,
gold fish etc. or in fingerlings of carp but very few
reports
are available on mature, Indian major carps like
rohu
due to their tough and hardy maintenance in the large
polyfibre
pool at wet lab system as the trials cannot be
conducted
in aquarium due to their large size. In other way,
pesticide
experiments cannot be conducted in open ponds.
The
present attempt to evaluate the pesticide toxicity in
mature
rohu will incorporate new database in environmental
toxicology
and fishery.
Material and Methods
Fish
Samples
Female
L. rohita (average
weight: 0.8–1 kg; average
length:
41 ± 2
cm and age over 1.5 years) were collected
from
stocking pond of Central Institute of freshwater
aquaculture
(CIFA), Bhubaneswar, India, at three different
time
periods round the year (March— pre-spawning phase;
July—spawning
phase; November— post spawning phase).
Fishes
were transferred to a circular polyfibre pool (volume
2,500
L) after treatment with 0.1 % KMnO4 solution to
avoid
any external infection and then acclimatized under
laboratory
conditions for 18 days using aeration facility
and
natural photoperiodic regime. Fish samples were fed
twice
a day at 3 % body weight by CIFACA (protein—
30.80
%; fat—5 %; carbohydrate—40.50 %; energy—
3,600
kcal kg/L), a special feed supplement for carps
developed
at the CIFA, Bhubaneswar, India.
Experimental
Design
Acclimatized
fishes were divided into five experimental
groups
having six fish in each group. The 96 h LC50 was
determined
for both the pesticides by static bioassay. Two
groups
were treated with two different sublethal concentrations
of
cypermethrin and another two groups were
treated
with two different sublethal concentration of carbofuran.
1/10th
and 1/4th of LC50 values
for both the pesticides
were
taken as sublethal doses (0.16 and 0.40 lL/L
for
cypermethrin; 0.06, 0.15 mg/L for carbofuran). The
fifth
group was kept as control. These doses were calculated
and
selected according to the earlier results of LC50
value
obtained from L. rohita by applying these two pesticides
and
by considering their predictive permissible limit
in
the aquatic environment as reported by EPA. Pesticides
were
applied only at the beginning of experiment. Experiment
was
continued for 28 days along with CIFACA feed
(3
% of body weight). Cypermethrin and carbofuran were
supplied
on request by respective manufacturing companies
(Rallis
India Limited and Hindustan Insecticides
Limited
respectively) with proper certificate of analysis.
The
concentrations of the pesticides in water were not
determined
further as known amount of both the pesticides
were
used in a definite quantity of water. Quality of the test
water
was monitored every week as per the protocols of
APHA
[12]. As experiments were conducted on
brood fish
which
required extensive experimental set up and maintenance,
replication
of experiments were conducted for two
consecutive
years. The results were taken as mean of the
two.
Histological
Processing
At
the end of the experimental period, weights of two fish
from
all individual groups were measured. The selection of
two
fish were done on the basis of comparatively higher
and
lower size fish within the homogenous group of six fish
in
each category. The fishes were then vivisected and
ovaries
were carefully removed. Gonado-somatic Index
(GSI) for each
fish was calculated as the weight of the
gonads
relative to the total body weight expressed as percentage
using
the formula:
GSI
= weight
of the ovary / weight
of the fish *100
To
calculate the significance level of this study, one way
ANOVA
(Duncan multiple range test) were performed
[13]. Test of significance were
examined at 5 % level.
Pieces
of ovary from the anterior, middle and posterior
regions
were pooled and fixed in aqueous Bouin’s fluid.
Paraffin
sections were cut at 5–6 lm
using a rotary
microtome
(ERMA, Japan) and stained with Harris haematoxylin
and
Eosin. Slides with best distinctive histological
features
were selected for comparison through
detailed
observations using a binocular compound microscope
(Zeiss
axiophot, West Germany) and photomicrographs
were
taken in an automatic photo micrographic
system.
Results and Discussion
Pre-Spawning
Phase
Ovarian
histology was observed after 28 days treatment
with
two doses of carbofuran and cypermethrin along with
control
animals. First dose (0.06 mg/L) of carbofuran
treatment
showed little degeneration in ooplasm with no
other
prominent change (Fig. 1a)
where as deformity in
follicular
structure such as degeneration of follicular wall,
ooplasm
and connective tissue was recorded in second dose
(0.15
mg/L) of treatment (Fig. 1b).
Similarly, first dose
(0.16
lL/L) of cypermethrin treatment
showed low atresia
and
no change in shape and structure of follicular wall
(Fig.
1c). Reduction in size and deformity
of oocytes,
necrosis
in ooplasm, disorganized nucleus and degeneration
of
follicular wall was observed in second dose
(0.40
lL/L) of treatment (Fig. 1d). In ovarian histology of
control
fishes, follicles were moderate. They contained
yolk
droplets, nucleus and a large number of spherical
follicles
(Fig. 1e).
Spawning
Phase
First
dose (0.06 mg/L) of carbofuran treatment showed
little
atresia with thick ovarian wall; no other prominent
changes
were observed (Fig. 2a).
In second dose (0.15 mg/L)
of
treatment, medium atresia, thick ovarian wall and
changes
in shape of follicles were noticed (Fig. 2b). Similarly,
first
dose (0.16 lL/L)
of cypermethrin treatment
showed
medium atresia of vitellogenic follicles and
reduction
in size and deformity in follicles (Fig. 2c).
Intense
atresia of vitellogenic degeneration of follicular
wall
and ooplasm, clumped cytoplasm and mature ovaries
in
a stage of regression were noticed in second dose
(0.40
lL/L) of treatment (Fig. 2d). As shown in Fig. 2e,
control
ovaries occupied the entire body cavity. Ovarian
walls
were very thin, almost transparent. Ovaries were
turgid
with a large number of translucent eggs. Follicles
contained
poorly defined nucleus with vacuolated cytoplasm
and
yolk globules (Fig. 2e).
Post-Spawning
Phase
First
dose (0.06 mg/L) of carbofuran treatment showed
very
little or no changes in ovarian wall except the
deformed
follicles (Fig. 3a).
In second dose (0.15 mg/L) of
treatment,
small degeneration in ooplasm and deformed
follicles
were noticed along with wide inter follicular space
(Fig.
3b). Similarly, first dose (0.16 lL/L) of cypermethrin
treatment
showed little degeneration in ooplasm with no
other
prominent changes (Fig. 3c)
where as medium atresia,
increase
in inter follicular space, degeneration of follicular
wall,
ooplasm, and shrunk up follicles were
observed
in second dose (0.40 lL/L)
of treatment
(Fig.
3d). Ovaries of control fishes were
flaccid, shrunken
and
sac like with reduced vascular supply. Some unspawned
large
follicles and many small follicles were noticed
with
dark stained nucleus (Fig. 3e).
Gonado-Somatic
Index
Dose
effect of cypermethrin and carbofuran on GSI of L.
rohita is presented in Table 1 and 2
respectively. It is
evident
from the results that the reduction of GSI was
maximum
at spawning stage at both the doses for both the
pesticides
in comparison to control. The reduction was also
more
at higher dosages as compared to lower.
Water
Quality
The
physico-chemical character of the water was analyzed
over
the study period i.e. water temperature 27–30 _C,
pH
7.8–8.3, dissolved oxygen 5–5.8 mL/L, hardness
80–110
mg/L as CaCO3,
alkalinity 135–148 mg/L as
CaCO3, calcium 25–30 mg/L, ammonia
nitrogen
0.02–0.05
mg/L.
A
fundamental contribution of ‘green revolution’ has
been
the development and application of insecticides for
the
control of a wide variety of insectivorous and herbaceous
pests
which would otherwise diminish the quantity
and quality of
food production. Most of insecticides have
been
known to be highly toxic to non-target organism like
fish
that inhabit natural environment close to agricultural
field.
The impact of insecticides or pesticides on fish
reproductive
system has been documented in various fish
species.
It
has been reported that histological features of the
teleost
fish ovary vis-a`-vis maturation of oocytes are
adversely
affected by different chemical biocides as
observed
presently in L. rohita [14–17]. Fishes exposed to
pesticides
lead to lowered steroidogenesis [17], inhibition
of
development of advanced oocytes and thus reducing the
number
of viable oocytes [18]
and fall in 32p uptake by
ovaries
[19]. Moreover, reduction in GSI seems
to be the
most
important and common effect in female fish due to the
exposure
to pesticides. Similar results have also been
observed
in the present study [20–23]. The increase in
follicular
atresia was next most obvious influence of pesticides
on
fish ovary. Both the pesticides inhibited growth
of
oocytes and raised incidences of follicular atresia as
evident
in the ovary of L. rohita exposed to carbofuran and
cypermethrin
and in the case of some other fishes also [8].
Effect
of pesticides on fish reproduction and their possible
mechanism
of action has been reported by Kumar et al. [24].
Low
dose of metacid-50 and carbaryl produced reproductive
damage
in northern pike as a result of homeostatic imbalance
in
gonadotropic hormone and gonadotropic releasing hormone
(GnRH) [25]. The lack of proper gonadotropic stimulation
also
caused atresia in this species. From these different
studies,
it has been accomplished that pesticides affect the
follicular
growth causing follicular atresia in fish ovary by
inhibiting
the secretion of gonadotropins from the pituitary
and
affecting the metabolic activities of the liver as the
growth
of vitellogenic follicles is closely related to synthesis
of
yolk in the liver [3].
Vitellogenesis
is a multistep phenomenon where organs
like
follicular epithelial layer (for mediating the exogenous
yolk
precursors to the oocyte), liver and muscle (for supply
of protein and
lipid-containing yolk precursor to the
oocyte)
and oocyte themselves (for endogenous deposition
of
yolk) are involved. It is regulated by an elaborate
endocrine
mechanism involving gonadotropic hormones
and
estrogen. Since the follicular epithelial cells either
remain
syncytial and thick or indistinct in the exposed fish,
the
possibility of their role in vitellogenesis what so ever
may
not be ruled out. So also in the case with thecal cells,
which
remain indistinct around most of the ovarian follicles
of
all the stages when exposed to the pesticides. The
histopathological
lesions in liver [3, 26], variations in its
biochemical
constituents especially protein [27, 28]
and
decreased
hepato-somatic index in fishes exposed to sublethal
concentrations
of different pesticides have been
reported.
Lipid is also required for the deposition of yolk in
the
oocytes. It declines significantly when the fish is
exposed
to pesticide. It has also been supported histochemically
by
Singh et al. [27]
and Medford and Mackay
[28]. Hence, these cumulative effects
cause different
alterations
of ovarian structure particularly ovarian atresia.
The
increasing atresia thus affects significantly the fecundity
of
the fish. Besides these, accumulation of pesticidal
metabolites
and impairment of enzymatic machinery in
ovarian
follicles appear to be significant for restraining
ovarian
dynamics.
The
impacts of pesticides on ovary are dose-dependent
[20], duration or time-dependent [29–31] and rely significantly
on
pesticidal quality or type [19] which has been
observed
in the present study. Cypermethrin affects more
than
Carobofuran because of its better pesticidal sensitivity.
It
may be mentioned that alteration in ovarian activity
due
to the pesticides may also be influenced by physiological,
metabolic
and cellular energy status of ovary of the
fish.
It is remarkable that ovarian damages are stage-specific
and
the effect is more where accumulation of yolk is
high.
Thus vitellogenic phase is the most affected stage in
ovarian
dynamics.
In
the present study, histomicrographic observations of
gonad and GSI
analysis of brood rohu exposed to different
concentration
of insecticides reported significant damage in
reproductive
system and thus this methodology may be a
useful
technique for monitoring the gonadal status of brood
fish in
the aquaculture farm and natural resources. However
further
experiments can be done to know the mRNA
expression
of different genes of reproductive hormone to
understand
the basic mechanism of adverse effect of
insecticides in fish
reproductive system.
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