Friday, June 21, 2019

Impact of Cypermethrin and Carbofuran on the Ovarian Cycle of the Indian Major Carp, Labeo rohita (Hamilton)

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 _

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
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 [711] 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

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

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
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 [1417]. 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 [2023]. 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 [2931] 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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