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

India and other developing countries (Ansari and
The long-term ecological hazards associated with the Razdan, 2001; Jana-Kara et al., 1995; Yadav et al.,
use of organochlorine, organophosphate, and carba- 2001). Deltamethrin is known to be toxic to fish and
mate pesticides propelled the introduction of a new various other aquatic organisms (Bradbury and Coats,
generation of pesticides with a lesser degree of persis- 1989; Haya, 1989; Mittal et al., 1994). Reports reveal
tence. In this direction, synthetic pyrethroids have that deltamethrin has led to an ecocatastrophe in Lake
emerged as viable substitutes. The synthetic pyrethroids Balaton, Hungary, where 30 ton of eels (Anguilla
are less persistent and less toxic to mammals and birds. anguilla) died (Nemcsok et al., 1999).
One of the pyrethroids that has found wide acceptability Effects of deltamethrin on nervous, respiratory, and
is deltamethrin ((S)a-cyano-3-phenoxybenzyl-(1R)-cis-3- hematological systems in fishes are reported and its
(2.2-dibromovinyl)-2,2-dimethylcyclopropane carboxy- tumorigenicity in rodents is reported (Csillik et al., 2000;
late). It is extensively used in agriculture and forestry Kumar et al., 1999; Shukla et al., 2001; Srivastav et al.,
because of its high activity against a broad spectrum of 1997). Unlike most animals, in which pyrethroids
insect pests (Villarini et al., 1998). It is also used as an including deltamethrin have a short life and are readily
metabolized, fish are reported to be deficient in enzymes


Corresponding author. Fax: +91-11-26059663. that hydrolyze pyrethroids (Haya, 1989). A high rate of
E-mail address: [email protected] (S. Raisuddin). 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). ing an equal amount of solvent (acetone) in which
These factors seem to contribute to the various toxic deltamethrin concentration was prepared.
effects of deltamethrin in fish, including oxidative stress.
Study of deltamethrin-induced oxidative stress and its 2.3. Preparation of postmitochondrial supernatant
influence on various antioxidants of fish and other (PMS)
aquatic organisms could provide useful information on
the ecotoxicological consequences of deltamethrin use. After the exposure was complete, liver, kidneys, and
Our studies have shown that the antioxidants of fish gills of the sacrificed fish were quickly removed, cleaned
could be used as biomarkers of exposure to aquatic of extraneous tissue, and immediately perfused with ice-
pollutants (Ahmad et al., 2000). While investigating cold saline. The tissues were homogenized in chilled
ecotoxicity of endosulfan we have observed that phosphate buffer (0.1 M, pH 7.4) containing KCl
oxidative stress and induction of antioxidants may (1.17%), using a Potter–Elvehjem homogenizer. The
prove to be a biomarker of exposure to endosulfan in supernatant was centrifuged at 10,500g for 30 min at
aquatic animals (Pandey et al., 2001). A review of the 41C to obtain the PMS, which was used for various
literature reveals that there is a paucity of information biochemical analyses.
on deltamethrin-induced oxidative stress and its effect
on antioxidants in fish. The present study was, therefore, 2.4. Lipid peroxidation (LPO)
undertaken to investigate deltamethrin-induced oxida-
tive stress and its effects on various enzymatic and Lipid peroxidation was determined by the procedure
nonenzymatic antioxidants in fish. An attempt has also of Utley et al. (1967) with some modifications as
been made to assess usefulness of these parameters as adopted by Fatima et al. (2000). Briefly, the tissues
biomarkers of exposure to deltamethrin. 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
2. Materials and methods
3 mL. The rate of LPO was expressed as nanomoles
of thiobarbituric acid reactive substance (TBARS)
2.1. Experimental setup and acclimation
formed/h/mg of protein using a molar extinction
coefficient of 1.56  105 M1 cm1.
Studies were conducted in freshwater fish, Channa
punctatus (Bloch), obtained from local water bodies.
2.5. Antioxidant enzymes
They weighed 23–50 g and their length was in the range
13–17 cm. Fish were maintained in glass aquaria each
Catalase (CAT) activity was assayed by the method of
containing 60 L water following the standard fish
Claiborne (1985) with some modifications as described
maintenance procedure during acclimation and expo-
by Ahmad et al. (2000). The assay mixture consisted of
sure (Clesceri et al., 1998). Fish (15 fish in one
0.1 M phosphate buffer (pH 7.4), 0.019 M hydrogen
aquarium) were acclimatized for 15 days before use.
peroxide, and 10% PMS in a final volume of 3 mL.
Water was kept oxygen-saturated by aeration and its
Catalase activity was calculated in terms of nmol H2O2
temperature was maintained at ambient laboratory
consumed/min/mg protein. Glutathione peroxidase
temperature (25721C). Water changes were made every
(GPx) activity was assayed according to the method
24 h to minimize contamination from metabolic wastes.
described by Mohandas et al. (1984) with some
Fish were fed autoclaved goat liver five times a week.
modifications (Ahmad et al., 2000). The assay mixture
consisted of 0.1 M phosphate buffer, 1 mM ethylenedia-
2.2. Exposure mine tetraacetic acid (EDTA) disodium salt, 1mM
sodium azide, glutathione reductase (1 IU/mL), 1 mM
In a preliminary experiment the 96 h LC50 value of reduced glutathione (GSH), 0.2 mM nicotinamide
deltamethrin (Hoechst Schering Agro Evid Limited adenine dinucleotide phosphate reduced (NADPH),
Ankleshwar, India) was evaluated according to Clesceri 0.25 mM hydrogen peroxide, and PMS (10%) in a total
et al. (1998) in Channa punctatus. The LC50 was found to volume of 2 mL. The enzyme activity was calculated as
be 1.5 mg/L. Subsequently, one group of fish (n ¼ 6) was nmol NADPH oxidized/min/mg of protein, using a
exposed to 0.75 mg/L of deltamethrin (1/2 of 96-h LC50 molar extinction coefficient of 6.22  103 M1 cm1.
value) for 48 h. The duration of exposure was selected Glutathione S-transferase (GST) activity was deter-
based on the fact that discernible toxic effects are mined by the method of Habig et al. (1974) with some
reported to appear after 48 h exposure to deltamethrin modifications as proposed by Ahmad et al. (2000). The
in fishes (Csillik et al., 2000). Concurrently, a control reaction mixture consisted of 0.1 M phosphate buffer,
group of fish (n ¼ 6) was exposed to tapwater contain- 1 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitro-
 ARTICLE IN PRESS
I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301 297

benzene (CDNB), and PMS (10%) in a total volume Control Deltamethrin

of 2 mL. The enzyme activity was calculated as nmol 100
CDNB conjugates/min/mg protein using a molar *
extinction coefficient of 9.6  103 M1 cm1.

(nmol TBARS formed/h/mg protein)

80
2.6. Non-enzymatic antioxidants
60 **
Reduced glutathione, thiols, and ascorbic acid (AsA) *
were studied as nonenzymatic antioxidants. GSH was
determined in PMS by the method of Jollow et al. 40
(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 20
15 min at 41C. The assay mixture contained supernatant,
0.1 M phosphate buffer, and 5,50 -dithio-bis (2-nitroben-
0
zoic acid) (DTNB) (stock=100 mM in 0.1 M phosphate Liver Kidney Gill
buffer) in a total volume of 3 mL. The optical density of
Organ
reaction product was read at 412 nm on a spectro-
photometer and results were expressed as nmol GSH/g Fig. 1. LPO values in liver, kidneys, and gills of control and
deltamethrin-exposed fish. The values are expressed as means7SE
tissue. Total (T-SH), protein-bound (P-SH), and non-
(n ¼ 6). LPO values are expressed as nanomoles of TBARS formed/h/
protein-bound thiol (NP-SH) groups in the PMS were mg of protein. The significance levels observed are
Po0:01 and


determined using the method of Sedlak and Lindsay Po0:001 when compared with control group values.
(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 3.2. Effect of deltamethrin on antioxidant enzymes
estimated by the method of Roe (1954) with some
modification. The reaction mixture was incubated at The exposure to deltamethrin caused a significant
601C for 1 h after 2,4-dinitrophenylhydrazine reagent (Po0:01) decrease in the activity of catalase in all the
(Sigma) was added. The absorbance was taken at organs (Tables 1–3). The decrease was in the order of
540 nm and the results were expressed as mg of AsA/g 45%, 43%, and 33% in liver, kidney, and gill,
tissue. respectively. A significant (Po0:0120:001) increase
was recorded in the actvities of GST and GPx (Tables
2.7. Protein estimation 1–3) in liver and kidney, while there was a significant
decrease (Po0:001) in the activities of GST and GPx
Protein contents in various samples were estimated by (Tables 1–3) in gills.
the method of Lowry et al. (1951) using Folin’s reagent
and bovine serum albumin (BSA) as standard. 3.3. Effect of deltamethrin on nonenzymatic antioxidants

2.8. Statistical analysis With respect to nonenzymatic antioxidants signifi-

cantly greater (Po0:001) values of GSH were observed
The statistical analysis of data was done using in the liver, kidneys and gills of deltamethrin-exposed
Student’s t-test. The significance of the results was fish when compared with values for control fish (Tables
ascertained at Po0:05: 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,
3. Results respectively.
The levels of total thiol, nonprotein thiol, and
3.1. Effect of deltamethrin on lipid peroxidation protein thiol (Tables 1–3) increased significantly
(Po0:0520:001) in liver, while a significant decrease
Fig. 1 shows levels of LPO in various tissues of fish of (Po0:0520:001) was observed in gill. There was a
control and deltamethrin-exposed groups. The LPO 19%, 7%, and 20% increase in the levels of T-SH,
levels were significantly (Po0:0120:001) increased in all NP-SH, and P-SH in the case of kidney, but the increase
the tissues, namely liver, kidney, and gill. The increase in was not significant.
the mean values of TBARS in liver, kidney, and gill were The ascorbic acid level increased significantly
39%, 21%, and 182%, respectively, over control values. (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 1994). The present findings implicate a role of oxidative
of liver there was an increase of 16%, which was not stress and free radical formation in these effects. Studies
significant when compared with the values for control in rodents have demonstrated that absorbed deltame-
fish. 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
4. Discussion slightly higher residues occurred. The major metabolic
reactions ascribed to deltamethrin metabolism are
A single 48 h exposure to deltamethrin-induced oxidations mediated by the microsomal monooxygenase
oxidative stress in all the tissues, as depicted by elevated system. The degradation pathways in cows, poultry, and
levels of LPO in tissues. Deltamethrin is known to alter fish are almost similar to those in rodents (Casida et al.,
cell metabolism in various ways, with a potential 1983; IPCS, 1989, Kulkarni and Hodgson, 1984; Shono
genotoxic risk, including DNA damage and micronu- et al., 1979). However, comparative in vivo and in vitro
cleus induction in human lymphocytes (Scassellati et al., metabolic studies have shown that fish have a lower
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I. Sayeed et al. / Ecotoxicology and Environmental Safety 56 (2003) 295–301 299

capacity to metabolize and eliminate pyrethroid insecti- xenobiotic (deltamethrin). However, the induction
cides (Glickman and Lech, 1981; Glickman et al., 1982). pattern of GSH under long-term exposure conditions
This is reflected in the present investigation, where needs to be investigated.
deltamethrin induced peroxidative damage in all the It has been observed that when normal enzymatic
tissues and the gills in particular during a short-term defenses are stressed, secondary defenses such as vitamin
subacute exposure regimen. C prevent the autooxidation chain reaction. Ascorbic
The extent of LPO is determined by the balance acid has long been linked as an essential factor to
between the production of oxidants and the removal and ameliorate some of the toxic effects of oxygen radicals
scavenging of those oxidants by antioxidants (Filho, (Bendich et al., 1986; Burton and Ingold, 1989; Meister
1996; Halliwell, 1987; Lopez-Torres et al., 1993). Our and Anderson, 1983). In this investigation, AsA levels
investigations on endosulfan and paper mill effluent in were increased in the kidney and liver, while the
fish have shown that gills are the organs most sensitive opposite was the case in the gills. Elevated AsA levels
to the LPO induced by xenobiotics and their antioxidant have been reported in the livers of fish exposed to waste
potential is also weak compared to that of other organs water from paper and pulp mills (Andersson et al., 1988;
(Fatima et al., 2000; Pandey et al., 2001). Gills are the Larsson et al., 1988). Increased utilization of AsA by
primary sites for the absorption of deltamethrin. It is, fish exposed to a variety of stresses has been reported
therefore, obvious that we noted a high level of LPO (Tucker and Halver, 1986). The available literature,
coupled with depletion of antioxidant enzymes in the however, indicates that varied responses are observed as
gills. Lipid peroxidation has been extensively used as a far as AsA content in fish exposed to different pollutants
marker of oxidative stress (Huggett et al., 1992). In the is concerned (Mayer et al., 1978; Thomas, 1987; Thomas
present investigation, we also showed that LPO estima- et al., 1982). In our opinion, this parameter has a low
tion could provide useful information about the reliability level from a biomonitoring viewpoint.
exposure to aquatic pollutants. However, the response As is common with many pyrethroids, deltamethrin
may be nonspecific in terms of type of pollutants. has a high toxicity to fish under laboratory conditions.
Use of GST and other detoxification enzymes of However, in the field, under normal conditions of use,
phase II biotransformation as biomarkers of exposure deltamethrin does not exhibit the same level of toxicity
to organic xenobiotics has gained credence in aquatic in fish. This may be due partly to rapid adsorption of
pollution biomonitoring (Livingstone, 1998; Payne et al., deltamethrin in sediment, uptake by plants, and
1987; Rodriguez-Ariza et al., 1991). Deltamethrin evaporation in the air (Haug and Hoffman, 1990). To
exposure induced activity of antioxidant enzymes GPx date no study is reported showing movement of
and GST in liver and kidney. The activities of GPx and sediment-adsorbed deltamethrin in the food chain and
GST, unlike other organs, were depleted in gill. Catalase its consequences. However, studies conducted on aqua-
activity decreased in all organs. Radi et al. (1985) have tic invertebrates showed an adverse effect on the
reported induction of GPx activity in fish resulting from functioning of the bivalve community in the aquatic
exposure to environmental pollutants. In deltamethrin- environment (Kontreczky et al., 1997). Similarly, a
exposed fish, the levels of GPx and GST in liver and series of studies conducted in Hungary on eels and other
kidney were found elevated, apparently to provide fish species show that deltamethrin may adversely affect
protection against ROS damage. Increase in GST the fish community (Csillik et al., 2000; Lang et al.,
activity was concomitant to increase in glutathione 1997; Nemcsok et al., 1999).
content in liver and kidney. The activity of CAT was The present investigation unequivocally establishes
found to be significantly decreased in all the organs. oxidative-stress-inducing effects of deltamethrin in fish.
This decrease in catalase activity could be due to the flux Use of deltamethrin in agricultural pest control and
of superoxide radicals, which have been reported to malaria control programs has tremendously increased in
inhibit CAT activity (Kono and Fridovich, 1982). In our several countries including India. In India, deltame-
previous study involving endosulfan a similar observa- thrin-impregnated bednets have been recommended in
tion was made (Pandey et al., 2001) where activities of malaria-infested areas. The present study warrants a
all other antioxidants were elevated, barring CAT, thorough ecotoxicological investigation of the use of
which recorded a decrease. deltamethrin.
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 5. Conclusions
against oxidative-stress-induced toxicity in aquatic
animals is well established (Hasspielar et al., 1994). Synthetic pyrethroids, including deltamethrin, be-
Our results show an increase in glutathione levels in cause of their beneficial qualities, have attracted farmers
various tissues. This demonstrates a protective response and health departments to use these compounds in pest
in fish toward exposure to an oxidative-stress-inducing 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 Csillik, B., Fazakas, J., Nemcsok, J., Knyihar-Csillik, E., 2000. Effect
reported to be the most toxic, as it is neither fully of pesticide deltamethrin on the mauthner cells of the Lake Balaton
metabolized nor quickly detoxified, and therefore fish. Neurotoxicology 21, 343–352.
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