ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 56 (2003) 295–301

Oxidative stress biomarkers of exposure to deltamethrin in freshwater

fish, Channa punctatus Bloch
Iqbal Sayeed, Suhel Parvez, Suwarna Pandey, Bilal Bin-Hafeez, Rizwanul Haque,
and Sheikh Raisuddin

Ecotoxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
Received 23 April 2002; received in revised form 15 January 2003; accepted 20 January 2003

Abstract

The pyrethroid class of insecticides, including deltamethrin, are being used as substitutes for organochlorines and
organophosphates in pest-control programs because of their low environmental persistence and toxicity. Ecotoxicological
consequences of deltamethrin, particularly its effects on antioxidants in fish and other aquatic organisms, are not well understood.
We investigated the effect of deltamethrin (0.75 mg/L) on antioxidants in a freshwater fish, Channa punctatus Bloch, using standard
laboratory conditions. A single exposure for 48 h caused induction of various antioxidant enzymes and nonenzymatic antioxidants
in kidney and liver. The induction of these antioxidants was not very prominent in gills. In fact, certain antioxidants were found to
be depleted in gills. Catalase activity was decreased in all the tissues. Deltamethrin also induced lipid peroxidation in all the tissues,
gills showing the highest levels. Glutathione, which is an established nonenzymatic antioxidant in fish, was significantly (Po0:001)
increased in all the tissues. Ascorbic acid content increased in kidney and liver while it decreased in gills. The findings of the present
investigation show that deltamethrin has oxidative-stress-inducing potential in fish, and gills are the most sensitive organs. It is also
interesting to note that gills are the primary sites of deltamethrin absorption and their antioxidant potential is also very poor. The
various parameters studied in this investigation can also be used as biomarkers of exposure to deltamethrin. It is suggested that
appropriate ecotoxicological risk assessment should be made in the areas where deltamethrin is proposed to be used in pest control
activities.
r 2003 Elsevier Science (USA). All rights reserved.

Keywords: Deltamethrin; Fish; Oxidative stress; Antioxidants; Biomarkers; Biomonitoring; Pest control; Ecotoxicity

1. Introduction alternative pesticide in malaria control programs in

The long-term ecological hazards associated with the
use of organochlorine, organophosphate, and carba-
mate pesticides propelled the introduction of a new
generation of pesticides with a lesser degree of persis-
tence. In this direction, synthetic pyrethroids have
emerged as viable substitutes. The synthetic pyrethroids
are less persistent and less toxic to mammals and birds.
One of the pyrethroids that has found wide acceptability
is deltamethrin ((S)a-cyano-3-phenoxybenzyl-(1R)-cis-3-
(2.2-dibromovinyl)-2,2-dimethylcyclopropane carboxy-
late). It is extensively used in agriculture and forestry
because of its high activity against a broad spectrum of
insect pests (Villarini et al., 1998). It is also used as an


Corresponding author. Fax: +91-11-26059663.
E-mail address: sraisuddin@hotmail.com (S. Raisuddin).
India and other developing countries (Ansari and
Razdan, 2001; Jana-Kara et al., 1995; Yadav et al.,
2001). Deltamethrin is known to be toxic to fish and
various other aquatic organisms (Bradbury and Coats,
1989; Haya, 1989; Mittal et al., 1994). Reports reveal
that deltamethrin has led to an ecocatastrophe in Lake
Balaton, Hungary, where 30 ton of eels (Anguilla
anguilla) died (Nemcsok et al., 1999).
Effects of deltamethrin on nervous, respiratory, and
hematological systems in fishes are reported and its
tumorigenicity in rodents is reported (Csillik et al., 2000;
Kumar et al., 1999; Shukla et al., 2001; Srivastav et al.,
1997). Unlike most animals, in which pyrethroids
including deltamethrin have a short life and are readily
metabolized, fish are reported to be deficient in enzymes
that hydrolyze pyrethroids (Haya, 1989). A high rate of
absorption of deltamethrin through gills also makes fish

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0147-6513(03)00009-5
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296 I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301
a vulnerable target of its toxicity (Srivastav et al., 1997).
These factors seem to contribute to the various toxic
effects of deltamethrin in fish, including oxidative stress.
Study of deltamethrin-induced oxidative stress and its
influence on various antioxidants of fish and other
aquatic organisms could provide useful information on
the ecotoxicological consequences of deltamethrin use.
Our studies have shown that the antioxidants of fish
could be used as biomarkers of exposure to aquatic
pollutants (Ahmad et al., 2000). While investigating
ecotoxicity of endosulfan we have observed that
oxidative stress and induction of antioxidants may
prove to be a biomarker of exposure to endosulfan in
aquatic animals (Pandey et al., 2001). A review of the
literature reveals that there is a paucity of information
on deltamethrin-induced oxidative stress and its effect
on antioxidants in fish. The present study was, therefore,
undertaken to investigate deltamethrin-induced oxida-
tive stress and its effects on various enzymatic and
nonenzymatic antioxidants in fish. An attempt has also
been made to assess usefulness of these parameters as
biomarkers of exposure to deltamethrin.
2. Materials and methods
2.1. Experimental setup and acclimation
Studies were conducted in freshwater fish, Channa
punctatus (Bloch), obtained from local water bodies.
They weighed 23–50 g and their length was in the range
13–17 cm. Fish were maintained in glass aquaria each
containing 60 L water following the standard fish
maintenance procedure during acclimation and expo-
sure (Clesceri et al., 1998). Fish (15 fish in one
aquarium) were acclimatized for 15 days before use.
Water was kept oxygen-saturated by aeration and its
temperature was maintained at ambient laboratory
temperature (25721C). Water changes were made every
24 h to minimize contamination from metabolic wastes.
Fish were fed autoclaved goat liver five times a week.
2.2. Exposure
In a preliminary experiment the 96 h LC50 value of
deltamethrin (Hoechst Schering Agro Evid Limited
Ankleshwar, India) was evaluated according to Clesceri
et al. (1998) in Channa punctatus. The LC50 was found to
be 1.5 mg/L. Subsequently, one group of fish (n ¼ 6) was
exposed to 0.75 mg/L of deltamethrin (1/2 of 96-h LC50
value) for 48 h. The duration of exposure was selected
based on the fact that discernible toxic effects are
reported to appear after 48 h exposure to deltamethrin
in fishes (Csillik et al., 2000). Concurrently, a control
group of fish (n ¼ 6) was exposed to tapwater contain-
ing an equal amount of solvent (acetone) in which
deltamethrin concentration was prepared.
2.3. Preparation of postmitochondrial supernatant
(PMS)
After the exposure was complete, liver, kidneys, and
gills of the sacrificed fish were quickly removed, cleaned
of extraneous tissue, and immediately perfused with ice-
cold saline. The tissues were homogenized in chilled
phosphate buffer (0.1 M, pH 7.4) containing KCl
(1.17%), using a Potter–Elvehjem homogenizer. The
supernatant was centrifuged at 10,500g for 30 min at
41C to obtain the PMS, which was used for various
biochemical analyses.
2.4. Lipid peroxidation (LPO)
Lipid peroxidation was determined by the procedure
of Utley et al. (1967) with some modifications as
adopted by Fatima et al. (2000). Briefly, the tissues
were homogenized in chilled 0.1 M potassium chloride
solution. The assay mixture contained 0.67% thiobarbi-
turic acid (TBA), 10% chilled trichloroacetic acid
(TCA), and homogenate (10%) in a total volume of
3 mL. The rate of LPO was expressed as nanomoles
of thiobarbituric acid reactive substance (TBARS)
formed/h/mg of protein using a molar extinction
coefficient of 1.56  105 M1 cm1.
2.5. Antioxidant enzymes
Catalase (CAT) activity was assayed by the method of
Claiborne (1985) with some modifications as described
by Ahmad et al. (2000). The assay mixture consisted of
0.1 M phosphate buffer (pH 7.4), 0.019 M hydrogen
peroxide, and 10% PMS in a final volume of 3 mL.
Catalase activity was calculated in terms of nmol H2O2
consumed/min/mg protein. Glutathione peroxidase
(GPx) activity was assayed according to the method
described by Mohandas et al. (1984) with some
modifications (Ahmad et al., 2000). The assay mixture
consisted of 0.1 M phosphate buffer, 1 mM ethylenedia-
mine tetraacetic acid (EDTA) disodium salt, 1mM
sodium azide, glutathione reductase (1 IU/mL), 1 mM
reduced glutathione (GSH), 0.2 mM nicotinamide
adenine dinucleotide phosphate reduced (NADPH),
0.25 mM hydrogen peroxide, and PMS (10%) in a total
volume of 2 mL. The enzyme activity was calculated as
nmol NADPH oxidized/min/mg of protein, using a
molar extinction coefficient of 6.22  103 M1 cm1.
Glutathione S-transferase (GST) activity was deter-
mined by the method of Habig et al. (1974) with some
modifications as proposed by Ahmad et al. (2000). The
reaction mixture consisted of 0.1 M phosphate buffer,
1 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitro-
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I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301
benzene (CDNB), and PMS (10%) in a total volume
of 2 mL. The enzyme activity was calculated as nmol
CDNB conjugates/min/mg protein using a molar
extinction coefficient of 9.6  103 M1 cm1.
2.6. Non-enzymatic antioxidants
Reduced glutathione, thiols, and ascorbic acid (AsA)
were studied as nonenzymatic antioxidants. GSH was
determined in PMS by the method of Jollow et al.
(1974). PMS was precipitated with sulfosalicylic acid
(4.0%) of a ratio of 1:1. The samples were kept at 41C
for 1 h and then subjected to centrifugation at 1200g for
15 min at 41C. The assay mixture contained supernatant,
0.1 M phosphate buffer, and 5,50 -dithio-bis (2-nitroben-
zoic acid) (DTNB) (stock=100 mM in 0.1 M phosphate
buffer) in a total volume of 3 mL. The optical density of
reaction product was read at 412 nm on a spectro-
photometer and results were expressed as nmol GSH/g
tissue. Total (T-SH), protein-bound (P-SH), and non-
protein-bound thiol (NP-SH) groups in the PMS were
determined using the method of Sedlak and Lindsay
(1968) as adopted by Sayeed et al. (2000). The molar
extinction coefficient of 13,100 at 412 nm was used for
the determination of thiol content. The values were
expressed as mM/g of wet tissue. The AsA content was
estimated by the method of Roe (1954) with some
modification. The reaction mixture was incubated at
601C for 1 h after 2,4-dinitrophenylhydrazine reagent
(Sigma) was added. The absorbance was taken at
540 nm and the results were expressed as mg of AsA/g
tissue.
2.7. Protein estimation
Protein contents in various samples were estimated by
the method of Lowry et al. (1951) using Folin’s reagent
and bovine serum albumin (BSA) as standard.
2.8. Statistical analysis
The statistical analysis of data was done using
Student’s t-test. The significance of the results was
ascertained at Po0:05:
3. Results
3.1. Effect of deltamethrin on lipid peroxidation
Fig. 1 shows levels of LPO in various tissues of fish of
control and deltamethrin-exposed groups. The LPO
levels were significantly (Po0:0120:001) increased in all
the tissues, namely liver, kidney, and gill. The increase in
the mean values of TBARS in liver, kidney, and gill were
39%, 21%, and 182%, respectively, over control values.
297
Control Deltamethrin
100
*
(nmol TBARS formed/h/mg protein)
80
60 **
*
40
20
0
Liver Kidney Gill
Organ
Fig. 1. LPO values in liver, kidneys, and gills of control and
deltamethrin-exposed fish. The values are expressed as means7SE
(n ¼ 6). LPO values are expressed as nanomoles of TBARS formed/h/
mg of protein. The significance levels observed are
Po0:01 and


Po0:001 when compared with control group values.
3.2. Effect of deltamethrin on antioxidant enzymes
The exposure to deltamethrin caused a significant
(Po0:01) decrease in the activity of catalase in all the
organs (Tables 1–3). The decrease was in the order of
45%, 43%, and 33% in liver, kidney, and gill,
respectively. A significant (Po0:0120:001) increase
was recorded in the actvities of GST and GPx (Tables
1–3) in liver and kidney, while there was a significant
decrease (Po0:001) in the activities of GST and GPx
(Tables 1–3) in gills.
3.3. Effect of deltamethrin on nonenzymatic antioxidants
With respect to nonenzymatic antioxidants signifi-
cantly greater (Po0:001) values of GSH were observed
in the liver, kidneys and gills of deltamethrin-exposed
fish when compared with values for control fish (Tables
1–3). The percentage difference of the deltamethrin-
exposed group from the control group was observed
to be 52%, 338%, and 386% for liver, kidney, and gill,
respectively.
The levels of total thiol, nonprotein thiol, and
protein thiol (Tables 1–3) increased significantly
(Po0:0520:001) in liver, while a significant decrease
(Po0:0520:001) was observed in gill. There was a
19%, 7%, and 20% increase in the levels of T-SH,
NP-SH, and P-SH in the case of kidney, but the increase
was not significant.
The ascorbic acid level increased significantly
(Po0:001) in the case of kidney and decreased
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Table 1
Effect of deltamethrin on various biochemical parameters in the liver of Channa punctatus

Parameter (unit of expression) Control group Deltamethrin-exposed group

Catalase (nmol H2O2 consumed/min/mg protein) 174.19718.78 95.3278.19

Glutathione peroxidase (nmol NADPH oxidized/min/mg protein) 338.28723.76 654.04734.68



Glutathione S-transferase (nmol CDNB conjugates/min/mg protein) 376.54731.21 621.45749.77


Reduced glutathione (nmol GSH/g tissue) 2.1170.16 3.270.14



Total SH (mmol/gm tissue) 90.0375.87 177.4779.45



NP SH (mmol/gm tissue) 0.83770.065 1.05870.086

P SH (mmol/gm tissue) 98.3375.83 17679.33



Ascorbic acid (mg/g tissue) 455.6738.94 530.10742.15

Values are expressed as mean7SE (n ¼ 6);
Po0:05;

Po0:01;

Po0:001; when compared with control value; the values of control and
deltamethrin-exposed groups are based on 48 h exposure.

Table 2
Effect of deltamethrin on various biochemical parameters in the kidneys of Channa punctatus

Parameter (unit of expression) Control group Deltamethrin-exposed group

Catalase (nmol H2O2 consumed/min/mg protein) 98.4279.65 56.4673.75

Glutathione peroxidase (nmol NADPH oxidized/min/mg protein) 215.28711.70 328.73717.77



Glutathione S-transferase (nmol CDNB conjugates/min/mg protein) 311.48712.25 428.3677.63



Reduced glutathione (nmol GSH/g tissue) 0.30870.0048 1.3570.177



Total SH (mmol/gm tissue) 94.8474.65 113.3577.23
NP SH (mmol/gm tissue) 1.12470.14 1.20570.07
P SH (mmol/gm tissue) 93.7174.69 112.14577.25
Ascorbic acid (mg/g tissue) 267.54713.27 528.34735.73









Values are expressed as mean7SE (n ¼ 6); Po0:01; Po0:001; when compared with control value; the values of control and deltamethrin-
exposed groups are based on 48 h exposure.

Table 3
Effect of deltamethrin on various biochemical parameters in the gills of Channa punctatus

Parameter (unit of expression) Control group Deltamethrin-exposed group

Catalase (nmol H2O2 consumed/min/mg protein) 268.78721.37 179.15713.14

Glutathione peroxidase (nmol NADPH oxidized/min/mg protein) 356.87710.73 278.75710.32



Glutathione S-transferase (nmol CDNB conjugates/min/mg protein) 184.10711.23 97.077.82



Reduced glutathione (nmol GSH/g tissue) 0.14670.009 0.7170.054



Total SH (mmol/gm tissue) 82.6974.35 51.9072.67



NP SH (mmol/gm tissue) 0.53670.031 0.42570.027

P SH (mmol/gm tissue) 82.1574.37 51.4772.67



Ascorbic acid (mg/g tissue) 307711.00 23077.00

Values are expressed as mean7SE (n ¼ 6);
Po0:05;

Po0:01;

Po0:001; when compared with control values; the values of control and
deltamethrin-exposed groups are based on 48 h exposure.

significantly (Po0:001) in the case of gill. In the case
of liver there was an increase of 16%, which was not
significant when compared with the values for control
fish.
4. Discussion
A single 48 h exposure to deltamethrin-induced
oxidative stress in all the tissues, as depicted by elevated
levels of LPO in tissues. Deltamethrin is known to alter
cell metabolism in various ways, with a potential
genotoxic risk, including DNA damage and micronu-
cleus induction in human lymphocytes (Scassellati et al.,
1994). The present findings implicate a role of oxidative
stress and free radical formation in these effects. Studies
in rodents have demonstrated that absorbed deltame-
thrin is readily metabolized and excreted, elimination is
achieved within 2–4 days. The tissue residue levels are
reported to be generally very low, except in fat, where
slightly higher residues occurred. The major metabolic
reactions ascribed to deltamethrin metabolism are
oxidations mediated by the microsomal monooxygenase
system. The degradation pathways in cows, poultry, and
fish are almost similar to those in rodents (Casida et al.,
1983; IPCS, 1989, Kulkarni and Hodgson, 1984; Shono
et al., 1979). However, comparative in vivo and in vitro
metabolic studies have shown that fish have a lower
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I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301
capacity to metabolize and eliminate pyrethroid insecti-
cides (Glickman and Lech, 1981; Glickman et al., 1982).
This is reflected in the present investigation, where
deltamethrin induced peroxidative damage in all the
tissues and the gills in particular during a short-term
subacute exposure regimen.
The extent of LPO is determined by the balance
between the production of oxidants and the removal and
scavenging of those oxidants by antioxidants (Filho,
1996; Halliwell, 1987; Lopez-Torres et al., 1993). Our
investigations on endosulfan and paper mill effluent in
fish have shown that gills are the organs most sensitive
to the LPO induced by xenobiotics and their antioxidant
potential is also weak compared to that of other organs
(Fatima et al., 2000; Pandey et al., 2001). Gills are the
primary sites for the absorption of deltamethrin. It is,
therefore, obvious that we noted a high level of LPO
coupled with depletion of antioxidant enzymes in the
gills. Lipid peroxidation has been extensively used as a
marker of oxidative stress (Huggett et al., 1992). In the
present investigation, we also showed that LPO estima-
tion could provide useful information about the
exposure to aquatic pollutants. However, the response
may be nonspecific in terms of type of pollutants.
Use of GST and other detoxification enzymes of
phase II biotransformation as biomarkers of exposure
to organic xenobiotics has gained credence in aquatic
pollution biomonitoring (Livingstone, 1998; Payne et al.,
1987; Rodriguez-Ariza et al., 1991). Deltamethrin
exposure induced activity of antioxidant enzymes GPx
and GST in liver and kidney. The activities of GPx and
GST, unlike other organs, were depleted in gill. Catalase
activity decreased in all organs. Radi et al. (1985) have
reported induction of GPx activity in fish resulting from
exposure to environmental pollutants. In deltamethrin-
exposed fish, the levels of GPx and GST in liver and
kidney were found elevated, apparently to provide
protection against ROS damage. Increase in GST
activity was concomitant to increase in glutathione
content in liver and kidney. The activity of CAT was
found to be significantly decreased in all the organs.
This decrease in catalase activity could be due to the flux
of superoxide radicals, which have been reported to
inhibit CAT activity (Kono and Fridovich, 1982). In our
previous study involving endosulfan a similar observa-
tion was made (Pandey et al., 2001) where activities of
all other antioxidants were elevated, barring CAT,
which recorded a decrease.
Reduced glutathione is the main nonprotein thiol and
one of the main reductants found in cells (Siegers, 1989).
It possesses antioxidant properties and its protective role
against oxidative-stress-induced toxicity in aquatic
animals is well established (Hasspielar et al., 1994).
Our results show an increase in glutathione levels in
various tissues. This demonstrates a protective response
in fish toward exposure to an oxidative-stress-inducing
299
xenobiotic (deltamethrin). However, the induction
pattern of GSH under long-term exposure conditions
needs to be investigated.
It has been observed that when normal enzymatic
defenses are stressed, secondary defenses such as vitamin
C prevent the autooxidation chain reaction. Ascorbic
acid has long been linked as an essential factor to
ameliorate some of the toxic effects of oxygen radicals
(Bendich et al., 1986; Burton and Ingold, 1989; Meister
and Anderson, 1983). In this investigation, AsA levels
were increased in the kidney and liver, while the
opposite was the case in the gills. Elevated AsA levels
have been reported in the livers of fish exposed to waste
water from paper and pulp mills (Andersson et al., 1988;
Larsson et al., 1988). Increased utilization of AsA by
fish exposed to a variety of stresses has been reported
(Tucker and Halver, 1986). The available literature,
however, indicates that varied responses are observed as
far as AsA content in fish exposed to different pollutants
is concerned (Mayer et al., 1978; Thomas, 1987; Thomas
et al., 1982). In our opinion, this parameter has a low
reliability level from a biomonitoring viewpoint.
As is common with many pyrethroids, deltamethrin
has a high toxicity to fish under laboratory conditions.
However, in the field, under normal conditions of use,
deltamethrin does not exhibit the same level of toxicity
in fish. This may be due partly to rapid adsorption of
deltamethrin in sediment, uptake by plants, and
evaporation in the air (Haug and Hoffman, 1990). To
date no study is reported showing movement of
sediment-adsorbed deltamethrin in the food chain and
its consequences. However, studies conducted on aqua-
tic invertebrates showed an adverse effect on the
functioning of the bivalve community in the aquatic
environment (Kontreczky et al., 1997). Similarly, a
series of studies conducted in Hungary on eels and other
fish species show that deltamethrin may adversely affect
the fish community (Csillik et al., 2000; Lang et al.,
1997; Nemcsok et al., 1999).
The present investigation unequivocally establishes
oxidative-stress-inducing effects of deltamethrin in fish.
Use of deltamethrin in agricultural pest control and
malaria control programs has tremendously increased in
several countries including India. In India, deltame-
thrin-impregnated bednets have been recommended in
malaria-infested areas. The present study warrants a
thorough ecotoxicological investigation of the use of
deltamethrin.
5. Conclusions
Synthetic pyrethroids, including deltamethrin, be-
cause of their beneficial qualities, have attracted farmers
and health departments to use these compounds in pest
control. But these compounds are found to be highly
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300 I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301
toxic to fish. Among pyrethroids, deltamethrin is
reported to be the most toxic, as it is neither fully
metabolized nor quickly detoxified, and therefore
creates serious problem of residue accumulation, espe-
cially in fatty tissues. The evidence presented here,
together with other data from the literature, shows the
hazards of deltamethrin in fish. It is evident that from
the ecophysiological point of view, the effects of
deltamethrin on piscine models must be carefully
evaluated. Future studies should be carried out to
understand the underlying mechanisms involved in long-
term toxicity profile of deltamethrin. It is also envisaged
here that parameters of oxidative stress could be used in
aquatic pollution biomonitoring with varying degrees of
specificity.
Acknowledgments
Financial assistance from the Ministry of Environ-
ment and Forests (Government of India) is gratefully
acknowledged. The laboratory assistance of Mr. Razi
Ahmad and Mr. Mohammad Irshad is also acknowl-
edged.
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