International Journal of Scientific Research in Environmental Sciences, 2(6), pp.

192-198, 2014
Available online at http://www.ijsrpub.com/ijsres
ISSN: 2322-4983; 2014 IJSRPUB
http://dx.doi.org/10.12983/ijsres-2014-p0192-0198


192
Full Length Research Paper

Effects of Dimethoate (EC 30%) on Gill Morphology, Oxygen Consumption and
Serum Electrolyte Levels of Common Carp, Cyprinus Carpio (Linn)

Ram Nayan Singh

Department of Zoology, Kamla Nehru Institute of Physical, And Social Sciences, Sultanpur up India
Email- rnsingh_zool@rediffmail.com

Received 25 February 2014; Accepted 24 April 2014

Abstract. Synthetic pesticides are an important group of aquatic pollutants affecting health of fishes. This study is an attempt
to assess the sub lethal effects of dimethoate, an organophosphate insecticide on gill morphology, ventilation rate, oxygen
consumption and electrolyte levels of common carp (C. carpio). Healthy juvenile fishes were exposed to a sub lethal
concentration of 0.96 mg/l (60% of 96 h LC
50
) of dimethoate; and at 24, 48 and 96 h exposure blood and gill tissues were
collected, oxygen consumption and ventilation rate was determined. The changes in the gills of exposed fish were
characterized by epithelial hyperplasia, lifting and degeneration of respiratory epithelium, lamellar bending and curling, fusion
and disintegration of secondary gill lamellae. The degree of damage showed direct correlation with the duration of exposure.
The electrolyte levels also fluctuated, levels of calcium decreased and phosphate levels increased during the exposure. Oxygen
consumption and ventilation rate showed significant decrease at all exposure durations. The study demonstrates high
sensitivity of fish gills to dimethoate, at short term and sub lethal exposure.

Keywords: gill; Cyprinus carpio; dimethoate; pesticide; LC
50


1. INTRODUCTION

Dimethoate, [IUPAC Name O, O dimethyl S - (N
methylcarbamoylmethyl) phosphoro-dithioate],
popular with common name - ROGOR, is a systemic
organophosphate insecticide used widely for
controlling insect pests of fruits, vegetables and crop
plants. It is highly selective as insecticide because the
relative rates of degradative enzymes viz, esterases
and amidases are slower in insects than in mammals
(Rose and Hodgson, 2004). Dimethoate is non-
photodegradable, undergoes very slow hydrolysis and
shows moderate persistence in water. In aquatic
environment persistence of dimethoate is pH
dependent being greater at acidic pH than alkaline. In
surface water half life of dimethoate is 156 day at pH
5, 68 day at pH 7, and 4.4 day at pH 9
(http://www.who.int/whopes/quality/en/Dimethoate_e
val_specs_WHO_June_2012.pdf). Like other
organophosphates dimethoate irreversibly inhibits
acetyl cholinesterase enzyme and works primarily as
nerve poison. Dimethoate is acutely toxic and
classified as possible human carcinogen by USEPA
based on tumor occurrence in mice. In the WHO acute
hazard ranking dimethoate is rated as moderately
hazardous. The United States Environment Protection
Agency in its interim re registration eligibility
decision for dimethoate has declared 0.43 / 0.84 mgL
-1

as the No Observed Adverse Effect Concentration
(NOAEC) and the Lowest Observed Adverse Effect
Concentration (LOAEC) for technical grade
dimethoate in fish.
Contamination of aquatic ecosystems by pesticides
causes acute and chronic poisoning of fish which
results in severe damage to vital organs like liver,
kidney and gills (Verma et al., 1982; Omitoyin et al.,
2006, Velmurugan et al., 2007). Gills in fishes are
comparatively more vulnerable to pollutant stress
because of their constant contact with contaminated
water and large surface area. They are not only the
major site of uptake of waterborne toxicant but also
the first and most important site of toxic impact
(Vigliano et al., 2006). Cellular and histological
changes in gills of fishes may reflect their decreased
fitness as well as adaptation to the polluted
environment. Condition of the gills has direct
relationship with the quality of water and any
deterioration in their structure and function has
adverse impact on the survival and reproduction of
fish. Therefore, condition of fish gill can be used as an
indicator of fish health and level of water pollution
Singh
Effects of Dimethoate (EC 30%) on Gill Morphology, Oxygen Consumption and Serum Electrolyte Levels of Common
Carp, Cyprinus Carpio (Linn)
193
(Evans, 1987; Poleksic and Mitrovic-Tuntundzic,
1994; Fernandes and Mazon, 2003).
The common carp, Cyprinus carpio is a popular
food fish preferred for culture due to its fast growth
rate, hardiness and prolific breeding in confined
water. This is an exotic carp, introduced in India in
1957, which has now well adapted to culture ponds
and even established in river systems of northern
India. At Sultanpur (Uttar Pradesh, India), this is the
most common major carp in fish markets, available
afresh round the year. Due to high economic
importance of common carp and scarcity of data on
branchial toxicity of dimethoate, this study was
undertaken to assess the effect of dimethoate on gills
of common carp.

2. MATERIALS AND METHODS

2.1. Fish Collection and Acclimatization

Fish were brought from ponds of Uttar Pradesh state
governments local hatchery (Bhojpur, Sultanpur) and
treated with 0.05% potassium permanganate for two
minutes. They were acclimatized to laboratory
conditions for two weeks into plastic pools of 500 liter
capacity. Fishes were fed ad lib rice bran mixed with
mustard oilcake in the ratio of 2:1, and three-fourth of
the pool water was renewed daily during
acclimatization.

2.2. Water quality measurements

The experiment was conducted under natural
photoperiod and temperature. Water quality was
measured as per APHA (2005). The temperature of
the experimental water was 23 1.5
0
C, pH was 7.2
0.4, Dissolved oxygen was 7.2 0.6 mg/l, free carbon
dioxide was 6.2 0.4 mg/l and total hardness as
calcium carbonate was 112 3.2 mg/l.




Table1: Effects of dimethoate exposure (0.96 mg/l) on O
2
consumption, ventilation rate, serum calcium and inorganic
phosphate levels of Cyprinus carpio.
Duration in hours 24 48 96
O
2
consumption
mg/g/h
Control
Exp.
0.350.42
0.310.64
s

0.360.56
0.290.32
s

0.360.32
0.240.24
s

Ventilation rate
/minute
Control
Exp.
1102.42
883.40
s

1082.66
822.96
s

1122.86
743.26
s

Serum Calcium
mg/l
Control
Exp.
10.071.52
10.460.68
10.101.58
8.92 0.64
10.091.02
8.21 0.42
s

Serum phosphate
mg/l
Control
Exp.
45.215.67
40.165.48
44.614.46
45.585.28
45.054.17
54.323.34
s

Note: Values are mean of 10 fish SD. The superscripts indicates significant difference from control at p<0.05.

2.3. Preparation of Stock solution and Exposure of
fish

The 96 hr LC
50
value of dimethoate for common carp
fingerlings was determined as 1.60 mg/l by Finneys
probit method (Singh et al., 2009). Based on 96 hr
LC
50
value, a sub lethal concentration of 0.96 mg/l
(60% of LC
50
) dimethoate was selected for the
experiment. Technical grade dimethoate (ROGOR
30% EC, Rallis India Ltd, Mumbai) was procured and
stock solution (1mg/ml) was prepared in absolute
alcohol. Glass troughs of 30 liter capacity were filled
with 25 liter of water in which 24 ml of stock solution
was mixed thoroughly before releasing the fishes. In
control trough 24 ml absolute alcohol was mixed in 25
liter of water. Sixty individuals of common carp (age -
four months, size, 17-22 cm, and weight, 50-65 gm)
were sorted and separated in six groups of ten fish
each irrespective of sex from the acclimatized stock.
Feeding was stopped 24 h before and was not resumed
during the course of the experiment. Ten fish were
released in each test and control trough. No mortality
occurred in test and control groups during the
experiment. At the 24, 48 and 96 h after starting the
experiment fish from test and control were sacrificed
and data was collected.

2.4. Measurement of oxygen consumption

DO probe (Century, CD501, India) was used for
measuring oxygen consumption of fish. For
measuring oxygen consumption, glass jars of two liter
capacity were filled with tap water and their DO was
determined. Then after keeping fish individually in
airtight jar for one hour, oxygen level of jar water was
measured (Fitch, 1975). The values obtained were
used to calculate oxygen consumption by each fish.
Same exercise was repeated for measuring oxygen at
24, 48 and 96 h duration with control and
experimental fish. Rate of opercular movement was
determined by counting after 24, 48 and 96 h.



International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 192-198, 2014
194
2.5. Blood analysis and Histopathology

Fish were first immobilized in ice and then dissected
out carefully so as to expose their heart for collection
of blood from conus arteriosus. Blood was collected
from heart in syringes which were kept in vertical
position in test tube stand for separation of serum.
Serum was then used for determination of calcium and
inorganic phosphate levels of blood by semiautomatic
analyzer of ERBA Diagnostics Mannheim Gmbh,
Germany. Significance of data was analyzed by
Students t test. Gills were removed and fixed in
bouins fluid for 24 h and processed for
histopathological study, according to Humasons
Animal Tissue Techniques (Humason, 1979). Paraffin
embedded tissues were molded into blocks and
sections were cut at 5 6 micron and stained in
haematoxylin and eosin. The slides were examined
under light microscope (Olympus CH 20i) and
photographed for histopathological effects (Roberts,
2012).


Fig. 1 (a): Photomicrograph of secondary gill lamellae (SGL) of control Cyprinus carpio with closely applied respiratory
epithelium (RE), pillar cell (PC) and blood cell (BC). (H. E. x 1000).


Fig. 1 (b): Photomicrograph of gill of Cyprinus carpio after 24 h exposure to 0.96 mg/l dimethoate showing hemorrhage
(HM), hyperplasia (HL), hypertrophy (HT) and disintegrating respiratory epithelium (DRE). (H. E. x 400)

3. RESULTS AND DISCUSSIONS

The regular and closely applied respiratory epithelium
of secondary lamellae of the fish gill is seen in control
(Figure 1a). After pesticide exposure earliest signs of
stress are seen as hemorrhage in primary filament and
localized damage of respiratory epithelium. The
respiratory epithelium of exposed fish shows damage
in the form of hyperplasia, lifting, degeneration and
fragmentation (Figure 1b); and secondary gill lamellae
exhibit bending, curling, fusion and fragmentation
with increased mucus secretion. At 48 h exposure
extensive lifting of respiratory epithelium and bending
of secondary gill lamellae and enhanced mucus
secretion is observed (Figure 1c). After 96 h exposure
degenerative changes in respiratory epithelium and
Singh
Effects of Dimethoate (EC 30%) on Gill Morphology, Oxygen Consumption and Serum Electrolyte Levels of Common
Carp, Cyprinus Carpio (Linn)
195
secondary gill lamellae are more pronounced. At 96 h
exposure respiratory epithelium is almost sloughed
away and secondary gill lamellae exhibit curling,
fusion and fragmentation (Figure 1d-e). Hemorrhage,
lifting and detachment of epithelium of primary
filament are also observed in some instances. Severity
of damage increases with increasing duration of
exposure.
The gills being delicate structures are easily
affected by contaminated water and rapidly exhibit
histopathological effects. This is well reflected in the
damage of respiratory epithelium and secondary gill
lamellae of exposed fish in the present study. Similar
damage of respiratory epithelium and secondary
lamellae in the form of epithelial hypertrophy,
hyperplasia, epithelial lifting and necrosis, and curling
of secondary lamellae and lamellar fusion have been
reported in pesticide exposed fish gills by several
workers (Machado and Fanta, 2003; Velmurugan et
al., 2007; Saravanan et al., 2010). Butchiram et al.
(2013) observed multiple changes including epithelial
hyperplasia, fusion of secondary lamellae,
degeneration of primary and secondary gill lamellae
and enhanced mucus production in Labeo rohita after
exposure to phenol. In addition, dilation of blood
sinuses of secondary gill lamellae and hemorrhage in
primary and secondary gill lamellae, as observed in
the present study, were reported by Rao et al. (2006)
in Gambusia affinis after exposure to profenofos.
Increased mucus secretion as observed in the exposed
carps has earlier been reported after pesticide
exposure in rainbow trout by Altinok and Capkin
(2007). Stress arising out of variation in environment
and pathologic agents are reported to induce
proliferation of mucus cells in gills and increase
mucus secretion as defense response (Richmonds and
Dutta, 1989).


Fig. 1 (c): Photomicrographs of gill lamellae of Cyprinus carpio exposed to 0.96 mg/l dimethoate for 48 h showing extensive
lifting of respiratory epithelium (ELRE), hyperplasia (HL) and bending of secondary gill lamellae (BSGL). (H.E. x 400)

Epithelial necrosis, hyperplasia, lifting and rupture
of respiratory epithelium and lamellar fusion could be
considered direct deleterious effects of the toxicant on
fish gill. Edema and epithelial lifting as defense
response can reduce entry of poisons by diffusion
(Barot and Bahadur, 2013). Hyperplasia has been
reported as the most remarkable cellular change in the
epithelium of secondary gill lamellae of Cyprinus
carpio exposed to simazine (Oropesa-Jimenez et al.,
2005). Hyperplasia shown by gills of exposed fishes
protects the body from diffusion of toxicants by
reducing the branchial surface and by increasing the
distance between blood and water in which pollutant
is dissolved (Oropesa-Jimenez et al., 2005).
Detachment of respiratory epithelium increases the
diffusion distance of respiratory gases thus lowering
oxygen uptake and metabolism. Overall, structural
changes in the gill of fishes appear to be adaptive
responses to pesticide stress (Laurent and Perry 1991).
Besides histopathological effects of dimethoate on
gill, adverse effects on oxygen uptake, ventilation rate
and electrolyte balance of the fish are also observed,
as given in table 1. Dimethoate exposure decreases
oxygen consumption and ventilation rate significantly
at all exposure durations. Similar observations have
been made by other workers in pesticide exposed
fishes (Velmurugan et al., 2007; Singh et al., 2009).
Respiratory distress is one of the early symptoms of
pesticide stress (Murthy, 1986). Depression in
ventilation rate and consequent fall in oxygen uptake
may be due to alteration in energy metabolism under
pesticide stress. Exposed fishes exhibit decline in
serum calcium but decline is statistically insignificant
up to 48 h which becomes significant (p<0.05) at 96 h
exposure. Phosphate level of serum shows decline at
24 h exposure but then trend is reversed and it
registers slight increase from control at 48 h which
becomes significant at 96 h exposure.

International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 192-198, 2014
196


Fig. 1 (d - e): after 96 h exposure to 0.96 mg/l dimethoate showing degenerating respiratory epithelium (DRE), sloughing off
respiratory epithelium (RES), disintegrating secondary lamellae (DSGL), fusion and curling of secondary gill lamellae (FSGL,
CGL) and separation of primary gill lamellae (SPGL). (H. E. x 400)

Hypocalcemia and hyperphosphatemia observed in
this study are in agreement with earlier reports in
Heteropneustes fossilis exposed to aldrin and
propoxur (Singh et al., 1997) and dimethoate (Das et
al., 2013) respectively. Thangavel et al. (2005) have
reported hypocalcemia and hypophosphatemia in
Oreochromis mossambicus exposed to dimecron and
ziram. Hypocalcemia is caused probably due to
diffusion loss of calcium through damaged gills, under
pollutant stress (Giles, 1984). Damage to fish gills
after exposure to pesticides have been reported by
several workers (Velmurugan et al., 2007; Peebua et
al., 2008). The damage in branchial epithelium may
also inhibit the uptake of Ca
+2
from the ambient water
causing hypocalcemia. Hypocalcemia may also be
attributed to defective intestinal calcium absorption
and impaired renal reabsorption resulting in hyper-
excitability and tremor in exposed carps. Increase in
inorganic phosphate in blood and skeletal muscle have
been observed in fish after endosulfan exposure by
Gill et al. (1991). They attributed hyperphosphatemia
to enhanced breakdown of high energy phosphates
related to an overall hyper metabolic state. Singh et al.
(1997) also reported consistent hyperphosphatemia
with hypocalcaemia and skeletal deformity in H.
fossilis exposed to insecticides.

4. CONCLUSION

This study shows that dimethoate is very toxic to
common carp and severely affects the structure and
function of respiratory system and ionic balance at sub
lethal concentration. Therefore, it is concluded that
dimethoate at sub lethal concentration, can cause
considerable deterioration to fish health. For this
reason, dimethoate use must be regulated otherwise
contaminated runoff from agricultural fields can
deteriorate fish health and significantly reduce fish
productivity of water bodies.

ACKNOWLEDGEMENT

Author is thankful to Head, Department of Zoology,
and Principal, KNIPSS, Sultanpur for providing
necessary facilities for carrying out this work.

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International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 192-198, 2014
198








Dr Ram Nayan Singh M.Sc., Ph.D.
Assistant Professor, Department Of Zoology, Knipss Sultanpur Up

Academic Accomplishments
M. Sc. In Zoology (1994), Csir Net/ Jrf (1998), Ph. D. (2010)
Teaching Experience Ug 13 Years, Pg 13 Years
Research Area Fish Toxicology
Research Publcations 8
Seminars/ Conferences Attended- 16