Effects of dimethoate (30% EC) an organophosphate pesti- cide on

657

Journal of Environmental Biology, May 2013

Abstract

Organ histopathology and changes in biochemical parameters in fish are good biomarkers of aquatic

pollution. This study is an attempt to assess the effects of dimethoate, an organophosphate

insecticide on the liver of common carp (C. carpio). Healthy individual fish were exposed to 0.40 mg l1 (25%

of 96 hr LC50) concentration of dimethoate, for short term (96 hr). Liver of the exposed fish exhibited

alterations like disruption of regular arrangement of hepatocytes, congestion and rupture of vessels;

hemorrhage, cytoplasmic vacuolization, pyknotic nuclei and necrosis. Biochemical parameters

viz. total liver protein (p<0.001) and liver glycogen (p<0.001) registered a significant decrease and

blood glucose (p<0.001) exhibited significant increase throughout exposure.

Key words

Toxicity, Histopathology, Common carp, Organophosphate, Dimethoate

Publication Info

Paper received:

21 July 2011

Revised received:

12 January 2012

Re-revised received:

08 May 2012

Accepted:

26 July 2012

Introduction

Synthetic pesticides are mostly non selective but

highly effective chemicals (Breckenridge and Stevens,

2008). Their widespread use in agriculture and in health

and hygiene programs is one of the major causes of

aquatic pollution (Cope, 2004). Sometimes pesticides are

directly applied in water bodies but their residues mostly

reach into aquatic ecosystems through surface run off

(Jergentz et al., 2004) and affect the health of non target

organisms including fish. Ecological concerns arising out

of high persistence of organochlorines, have favored

extensive use of organophosphates for controlling

household, agricultural and public health pests due to

their less persistence and high efficacy (Oruc et al., 2006;

Jyothi and Narayan, 1999). This shift has resulted into

increased occurrence of organophosphates into water

bodies causing acute and chronic toxicity to fish fauna

(Aker et al., 2008; Rao et al., 2005; Velmurugan et al.,

2007; Pandey et al., 2009).

Different kind of biological responses can be used

as biomarkers to assess the toxic effects of pollutants.

Histopathology of fish liver is increasingly being utilized as

biomarkers of xenobiotic exposure (Fernandes et al., 2008)

along with biochemical parameters to study the stress

(Rawat et al., 2002). The present work therefore was an

effort to assess the impact of dimethoate on the

histopathology and biochemical parameters on liver of

common carp, Cyprinus carpio.

Materials and Methods

Fish caught from local government hatchery and

carefully packaged into aerated polythene bags were

brought to the laboratory. Fish were treated with 0.05%

KMnO4 for disinfecting and were further transferred into

plastic pools of 500 l capacity for two weeks for

acclimatization to laboratory conditions. The rice bran mixed

with mustard oil cake in the ratio of 2:1 was given to fish as

feed during acclimatization. Water of the pool was changed

daily and dead fish were removed immediately.

Effects of dimethoate (30% EC), an organophosphate pesti-cide on liver of common carp, Cyprinus carpio

Ram Nayan Singh

Department of Zoology, Kamla Nehru Institute of Physical and Social Sciences, Sultanpur- 228 118, India

*Corresponding Author email : [email protected]

© Triveni Enterprises, Lucknow (India ) Journal of Environmental Biology, Vol. 34, 657-661, May 2013

ISSN: 0254-8704CODEN: JEBIDPJournal of Environmental Biology

JEB

Journal Home page : www.jeb.co.in � E-mail : [email protected]

658


R.N. Singh

The experiment was conducted under natural

photoperiod and temperature (28-32ºC). Water quality was

measured as per APHA (2005). The temperature of the water

was 23 ± 1.5ºC, pH 7.2 ± 0.4, dissolved oxygen 7.2 ± 0.6 mg

l-1, free carbon dioxide 6.2 ± 0.4 mg l-1 and total hardness as

calcium carbonate 112 ± 3.2 mg l-1.

Based on 96 hr LC50 value 1.60 mg l-1 of dimethoate

(Rogor 30%EC, Rallis India Ltd, Mumbai) for C. carpio

(Singh et al., 2009), a dose of 0.40 mgl-1 (25% of 96 hr LC50)

was selected as sub lethal concentration. Fish (17-22 cm

length, and 50-65 gm weight) were starved for 24 hrs before

starting the experiment. Six fish each were exposed to 0.40

mgl-1 dose for 24, 48, 72 and 96 hr in each set and control

with six fish was run simultaneously for each interval.

Fish were sacrificed at intervals of 24, 48, 72 and 96

hr of exposure. Immediately the blood was collected from

sinus venosus and blood glucose was measured by Accu

Chek glucometer. The liver was removed and a portion fixed

for 24 hr in bouins fluid for histology and other portion

processed for estimating glycogen and total protein. For

estimating liver glycogen and total protein, 100 mg liver

tissue was transferred in to manual ice cooled homogenizers

containing 10% TCA. Glycogen was measured according

to the anthrone method of Van der Vies (1954). Total protein

was measured according to Lowry et al. (1951). For

measuring optical density digital spectrophotometer of MS

Electronics, India (Model, 305) was used. Significance of

the data was analyzed by student’s t – test.

The fixed liver tissues dehydrated in graded series

of alcohol were embedded in paraffin. A sections of 5-6

micron were cut and stained in haematoxylin and eosin. The

slides were examined under light microscope (Olympus CH

20i) and photographed for histopatholgical observations.

Results and Discussion

Liver in Cyprinus carpio is proportionally large and

spreads around alimentary canal in the abdominal cavity. In

the control liver, hepatocytes (H) appear polygonal, have

centrally placed spherical nucleus (N) with conspicuous

nucleolus (NU). They are arranged in regular cords along

sinuses forming parenchyma (Fig.1a). Histopathological

changes in the liver of exposed fish were conspicuous and

included disruption of regular arrangement of hepatocytes,

congestion and rupture of vein, extensive hemorrhage,

cytoplasmic vacuolization, indistinct cellular boundaries,

pyknotic nuclei, nuclear degeneration and necrosis.

Hemorrhage and cytoplasmic vacuolization appear as the

earliest effects in the liver of exposed fish. With increasing

duration of exposure symptoms like chromatin

condensation, nuclear degeneration and necrosis emerge.

After 24 hrs of exposure, cells appeared swollen, showed

increased cytoplasmic vacuolization and in some nucleus

was displaced towards periphery. Hemorrhage in

parenchyma was visible (Fig.1b). After 48 hrs of exposure,

cytoplasmic vacuolization of hepatocytes persisted and

hemorrhage became extensive. Chromatin in some

hepatocytes was condensed and cellular degeneration

started in some areas (Fig. 1c). After 72 hr, blood vessels

were obliterated and hepatocytes in the vicinity showed

nuclear degeneration and pyknotic nuclei. Hemorrhage and

cytoplasmic vacuolization persisted (Fig. 1d). After 96 hrs

of exposure, degenerative changes were pronounced,

shapes of hepatocytes were not clear as the cell boundaries

became indistinct. Nuclear degeneration and extensive

damage of hepatocytes leading to necrosis were discernible

(Fig. 1e).

Similar results have been reported in Channa

punctatus after exposure to alachlor (Butchiram et al., 2009).

Fig. 1 : Photomicrograph of liver of C. carpio (a) Control liver

exhibiting regular arrangement (hepatic cords - HC) of polyhedral

hepatocytes (H) with prominent spherical nucleus (N) in the centre

with a central nucleolus (NU) and also showing sinus (S) and blood

cells (BC) (b) Dimethoate (0.40 mgl-1) treated liver for 24 hr showing

corrugated glisson capsule (CGC), hepatocytes with increased

cytoplasmic vacuolization (V), some with eccentric nucleus (EN)

and conspicuous haemorrhage (HM).

a

b

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Dimethoate toxicity in Cyprinus carpio

Alterations like irregular shaped hepatocytes, cytoplasmic

vacuolization and laterally placed nuclei were also observed

in the siluriform Corydoras paleatus after exposure to

organophosphate pesticides for 96 hrs (Fanta et al., 2003).

Peebua et al. (2008) also reported lipid vacuoles, hepatocyte

swelling and pyknotic nuclei in Oreochromis niloticus

following alachlor exposure for 96 hrs. Ultrastructural

changes such as loss of structural integrity of rough

endoplasmic reticulum; swelling of mitochondria, cristae

regression; increased heterogeneity of lysosomal matrix and

increase in number of vacuoles have been reported in the

hepatocytes of Clarias garipienus from polluted water

(Abdel-Moneim and Abdel- Mohsen, 2010).

Histopathological changes in liver appear to arise from its

primary role in xenobiotic metabolism, which exposes it to

comparatively higher level of xenobiotics than in other

tissues and surroundings (Heath, 1995). Structural changes

such as cytoplasmic vacuolization, pyknotic nuclei,

cytoplasmic and nuclear degeneration and necrosis in liver

appeared as manifestation of stress on fish.

Total protein and glycogen content of liver registered

a significant decrease while blood glucose remained

consistently at significantly higher level than control

throughout exposure (Table 1). Liver protein; however,

showed slight but insignificant increase at 24 and 48 hr of

exposure. These results are in agreement with Singh and

Srivastava (1995) who reported reduction in liver glycogen,

liver protein and hyperglycemia in the teleost

Heteropneustes fossilis following exposure for four and eight

days to formothion and propoxur. Decrease in liver glycogen

and liver protein following toxicant exposure for 10 days

has also been reported in Channa punctatus (Tilak et al.,

2009) and in Colisa fasciatus after 96 hrs of exposure (Singh

et al., 2004). Depletion in liver glycogen and concurrent

increase in blood glucose may be the result of increased

glycogenolysis to meet the increased demand of energy

under pesticide stress. Increase in blood glucose level

demonstrates the metabolic response of fish to pesticide

stress (Velisek et al., 2006).

Decrease in glycogen and increased levels of lactic

acid in liver of Sarotherodon mossambicus occured

following individual and combined exposure of dimecron

and ziram (Thangavel et al., 2004). Depleted hepatic

glycogen and increased lactate levels indicate a shift toward

anaerobic metabolism caused by reduced oxygen

consumption under pollutant stress (Ramakritinan et al.,

2005). Singh et al. (2009) reported reduction in oxygen

consumption in Cyprinus carpio following exposure to

dimethoate.

Therefore decrease in hepatic protein, as observed

in the present study, may be due to its increased rate of

d

c

e

Fig. 1 : Photomicrograph of C. carpio liver treated with 0.40 mg

l-1 dimethoate showing (c) extensive hemorrhage (EHM) and

cytoplasmic vacuolization (V) of hepatocytes after 48 hrs (d)

obliteration of blood vessel (BVO) and hepatocytes with

vacuolization (V), chromatin condensation (CC), pyknotic nuclei

(PN) and degenerating nucleus (DN) after 72 hrs and (e) Extensive

degeneration of hepatocytes (EDH) and necrotic hepatocytes (NH)

with degenerating nucleus (DN) and indistinct cellular boundaries

(ICB) after 96 hrs (x 1000).

660


degradation and utilization for metabolic purposes. Tilak

et al. (2005) attributed decrease in protein to its mobilization

for gluconeogenesis to meet the energy demands.

Decrease in protein content of liver under pollutant stress

has also been reported in other fish such as Channa

punctatus (Naveed et al., 2010) and Labeo rohita

(Saravanan et al., 2010).

Present work showed that dimethoate is strongly

hepatotoxic and severely affects histology, carbohydrate

and protein metabolism of fish liver. Thus it is concluded

that liver histology and biochemical parameters can be used

as rapid and sensitive biomarkers of pesticide stress in fish.

Acknowledgments

The author wishes to express his gratitude to Dr.

Yashwant Singh Principal KNIPSS and to Dr. V.K. Das Ex-

Head of the Zoology Department for providing laboratory

facilities to carryout this work.

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Table 1 : Effect of 0.40 mgl-1 dimethoate on the levels of blood glucose, liver glycogen and liver protein in C. carpio

Time Blood glucose Liver glycogen Liver protein

(hrs) (mg dl-1) (mg g-1 f. wt.) (mg g-1 f. wt.)

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24 62.54±5.21 136.42**±2.60 68.67±4.84 42.22**±2.95 52.06±2.36 53.71 i±2.66

48 65.50±4.25 125.52**±3.45 69.64±3.04 36.06**±3.30 52.51±2,24 54.45 i±2.66

72 64.66±4.86 120.00**±2.84 69.40±1.93 28.84**±3.58 51.82±1.70 49.17*±1.75

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Dimethoate toxicity in Cyprinus carpio

Effects of dimethoate (30% EC) an organophosphate pesti- cide on

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