Biochemical response of anthracene and benzo [a] pyrene in

milksh Chanos chanos

L. Palanikumar
n
, A.K. Kumaraguru, C.M. Ramakritinan, M. Anand
Department of Marine and Coastal Studies, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai 625021, India
a r t i c l e i n f o
Article history:
Received 2 June 2011
Received in revised form
29 August 2011
Accepted 30 August 2011
Keywords:
Anthracene
Benzo [a] pyrene
C. chanos
Acute toxicity
Bioaccumulation
Biomarkers
a b s t r a c t
Polycyclic aromatic hydrocarbons (PAHs) are common toxic pollutants found in the aquatic environ-
ment, and the assessment of their impact on biota is of considerable concern. The aim of the present
research was to study the acute toxicity, bioaccumulation and biochemical response of milksh Chanos
chanos (Forsskal) to two selected PAHs: anthracene and benzo [a] pyrene. Acute toxicity test results
were evaluated by the Probit analysis method and 96 h LC
50
values for C. chanos exposed to anthracene
was 0.030 mg l
1
and 0.014 mg l
1
for benzo [a] pyrene. Bioaccumulation concentration of anthracene
was high when compared to benzo [a] pyrene. Biomarkers indicative of neurotoxicity (acetylcholines-
terase, AchE), oxidative stress (lipid peroxidation, LPO and catalase, CAT) and phase II biotransforma-
tion of xenobiotics (glutathione S transferase, GST and reduced glutathione, GSH) were measured to
assess effects of selected PAHs. Anthracene and benzo [a] pyrene increase LPO and CAT level of C. chanos
suggesting that these PAHs may induce oxidative stress. Both the PAHs inhibited AchE indicating that
they have atleast one mechanism of neurotoxicity in common: the disruption of cholinergic transmis-
sion by inhibition of AChE. An induction of C. chanos glutathione S-transferase (GST) activity was found
in sh exposed to benzo [a] pyrene, while an inhibition was observed after exposure to anthracene.
These results suggest that GST is involved in the detoxication of benzo [a] pyrene, but not of
anthracene.
& 2011 Elsevier Inc. All rights reserved.
1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are the most wide-
spread organic pollutants. In addition to their presence in fossil
fuels, they are also formed by incomplete combustion of fuels
such as wood, coal, diesel, fat, tobacco, or incense. PAHs are found
wherever there is oil pollution and combustion wastes. Sediments
of many marine and freshwater harbors and even remote ocean
locations are contaminated with PAHs (Oliva et al., 2010). PAHs
and their halogenated forms are chemically stable, and due to
their lipophilic nature they can easily penetrate biological mem-
branes and accumulate in organisms. PAHs are important envir-
onmental pollutants because of their ubiquitous presence and
carcinogenicity (Tuvikene, 1995).
Anthracene is a commercially important PAH produced in
large quantities and extensively used as a reagent in organic
synthesis (Archer et al., 1979). Anthracene has also been used
frequently as a model PAH for studies of environmental fate and
transport in aquatic systems (Ausmus et al., 1980) or physiologi-
cal disposition in aquatic biota (Roubal et al., 1977). Benzo [a]
pyrene (B [a] P) is classied as potent carcinogen and/or mutagen
(Shaw and Connell, 1994). Sublethal amounts of B [a] P are
commonly found in marine environments especially after oil spill
accidents (Banni et al., 2010). Marine sh readily take up
lipophilic organic contaminants such as B [a] P from the marine
environment, with a variety of physiological effects (Walker and
Livingstone, 1992), which is a drawback in the consumption of
toxic marine sh as food.
Aquatic animals have often been used in bioassays to monitor
water quality of efuents and surface waters (Brungs et al., 1978).
Marine sh readily take up lipophilic organic contaminants such
as anthracene and B [a] P from the marine environment, with a
variety of physiological effects (Walker and Livingstone, 1992),
which is a drawback in the consumption of toxic marine sh as
food. PAHs have been found to induce adverse effects on sh
growth (Hannah et al., 1982; Ostrander et al., 1990; Jifa et al.,
2006; Kim et al., 2008), reproduction (Thomas, 1990; White et al.,
1999; Monteverdi and DiGiulio, 2000) and survival (Collier
and Varanasi, 1991; Hawkins et al., 1991). Furthermore, after
biotransformation, these compounds may originate reactive pro-
ducts that bind to DNA and may cause mutations or other
alterations on the genetic material (Hall and Glover, 1990;
Marvin et al., 1995; Woodhead et al., 1999; Wahidulla and
Rajamanickam, 2009).
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/ecoenv
Ecotoxicology and Environmental Safety
0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.08.028
n
Corresponding author.
E-mail address: palanikumarl@gmail.com (L. Palanikumar).
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]
Acute toxicity is the major subject of research for evaluating
the impact of toxic effect of chemicals on shes (Johnson and
Finley, 1980). The use of acute toxicity for testing the potential
hazards of chemical contaminants to aquatic animals is well
documented (Henderson et al., 1960; Sanders and Cope, 1966;
Hutchinson et al., 2006). Flow through toxicity tests were
believed to provide a better estimate of toxicity than static or
renewal toxicity tests because they provide a greater control of
toxicant concentrations, minimize changes in water quality,
reduce accumulation of waste products in test exposure waters
(Rand et al., 1995; ASTM, 1997; Welsh et al., 2008).
Many PAH accumulation studies have been carried out to
determine the adverse effects of PAHs (Boleas et al., 1998; Wetzel
and Van Vleet, 2004). Bioaccumulation results are integrated with
biochemical and toxicological data, providing more information
on the possible classes of contaminants, which cause adverse
biological effects (Gaudy et al., 1991).
In recent years, a relatively new concept in aquatic environ-
mental study is the analysis of changes in various physiological
and biochemical parameters in resident biota. The use of so-called
biomarker has been adopted from epidemiology or molecular
toxicology (Verlecar et al., 2006). In sh, PAHs in general, are
subject to biotransformation in a rst step by enzymes of the
phase I enzymes. The rst step in the xenobiotics metabolism is
usually catalyzed by cytochrome P450-dependent monooxy-
genases (phase I) and their products are subsequently coupled
to endogenous metabolites (phase II) (Landis and Yu, 1995; Oliva
et al., 2010). Therefore the role of PAHs detoxication deserves
further research.
The enzymatic activities of glutathione S-transferases (GST), a
family of multi-functional enzymes involved in phase II of
biotransformation are related to cellular antioxidant defences
due to the conjugation of electrophilic xenobiotics and oxidized
components with glutathione (GSH) (Fitzpatrick et al., 1995). It is
known that oxidative damage is an important mechanism of
toxicity induced by PAHs (Altenburger et al., 2003). The catalase
activity (CAT) was selected as an oxidative stress biomarker, since
it is an important enzymatic antioxidant defences (Livingstone,
2001). Acetylcholinesterase activity is usually used in biomoni-
toring programs as biomarker of exposure to organophosphorus
pesticides and metals (Banni et al., 2005).
Important aspect regarding the toxicity of petrochemical is the
potential that some of these compounds and mixtures seem to
have to inhibit the activity of acetylcholinesterase (AChE) and,
thus, to disrupt cholinergic neurotransmission (Vieiria et al.,
2008). In fact, several recent studies performed with invertebrates
and sh reported inhibition of this enzyme after exposure to fuel
oil and/or to PAHs (Moreira et al., 2004; Zapata-Pe rez et al., 2004;
Barsiene et al., 2006). However, no effects on AChE in sh exposed
to PAHs have been reported (Jifa et al., 2006). Therefore, this is
also a subject that needs further research, since this enzyme has
been used in biomonitoring studies (Lehtonen and Schiedek,
2006; Monteiro et al., 2007).
Fish play a major role for the ow of energy in aquatic
ecosystems. They are exposed continuously to contaminants in
the natural habitat and constitute an important part of human
diet, especially in the coastal region (Jha, 2004). Fishes have been
the most popular test organisms because they are presumed to be
the best understood organisms in the aquatic environment. Fishes
exposed to PAHs in water column throughout their life cycle can
serve as natural indicators of PAH contamination in surface
waters (Logan, 2007).
Milksh Chanos chanos (Forsskal, 1775) is an important tropical
marine sh and is cultured in the Philippines, Indonesia, India and
Taiwan producing about 330,000 tonnes every year (Rabanal, 1988).
The milksh has been the subject of numerous studies of varied
extent and depth by investigators in relation to biology, aquaculture,
hatchery context and toxicity (Bagarinao, 1994; Magesh and
Kumaraguru, 2006). In view of the above, the present investigations
were carried out to assess acute toxicity, bioaccumulation and
biomarker enzyme effects of anthracene and B [a] P on ngerlings
of the milksh C. chanos (Forsskal) of the Gulf of Mannar, Southeast
Coast of India.
2. Materials and methods
2.1. Experimental animals
Health and live specimens of a milksh ngerlings C. chanos (Forsskal)
(average size and weight 2.8270.07 cm; 1.5570.10 g) collected from Kundukal
and Chinnapalam regions (Latitude 09116.26
0
N and Longitude 079112.88
0
E) of
Pamban coast, Gulf of Mannar, Southeast coast of India. Initial disinfection
treatment was carried out using benzyl konium chloride (1 mg l
1
) for 1 h and
KMnO
4
solution (1 mg l
1
) for 1 h and then healthy individuals were separated,
acclimatized for ten days in a large glass aquaria containing aged, ltered seawater
(calcium hardness 385.4 mg l
1
, magnesium hardness 1422.0 mg l
1
, dissolved
oxygen 6.44 mg l
1
, silicates 6.44 mg l
1
, in-organic phosphate 3.32 mg l
1
, nitrite
nitrogen 1.72 mmol l
1
, nitratenitrogen 4.40 mmol l
1
and ammonia 0.12 mg l
1
).
During this period, the shes were fed on live brine shrimp (Artemia sp) nauplii
(Cruz and Tamse, 1989) and starved 24 h prior to and during the experiment. Every
effort as suggested by Bennett and Dooley (1982) was made to maintain optimal
conditions during acclimatization. The study has been approved and partially
funded by Ministry of Earth SciencesICMAM, Chennai.
2.2. Determination of acute concentrations of anthracene and benzo [a] pyrene
The acute toxicity bioassay procedure based on standard methods (Sprague,
1973; OECD, 1993; APHA/AWWA/WEF, 1998) was conducted to determine the LC
50
values of anthracene (CAS no 120-12-1) and benzo [a] pyrene (CAS no 50-32-8)
(Sigma-Aldrich Co, USA). Preliminary range nding tests was performed and
denitive range concentrations were chosen i.e., 0.011, 0.023, 0.047, 0.094 and
0.188 mg l
1
for anthracene and 0.002, 0.004, 0.007, 0.015 and 0.031 mg l
1
for
benzo [a] pyrene. The concentrations dissolved in test medium were estimated
(UNESCO, 1984). The unltered experimental medium samples (1 L) were spiked
with the following internal standards: anthracene (1 mg) and benzo [a] pyrene
(1 mg). The samples were consecutively extracted with 50 mL of hexane and 25 mL
of dichloromethane. The organic extracts were combined, dried over anhydrous.
Na
2
SO
4
, rotary-evaporated and fractionated in a glass column lled with neutral
alumina (1 g) and silica gel (1 g), both ve percent water deactivated. Two fractions
were eluted, the rst one with 2 mL of hexane, which contained the aliphatic
compounds and the second with 10 mL of dichloromethane:hexane (30:70 v/v),
which contained the PAHs.
The second fraction was analyzed by Capillary Gas ChromatographyHigh
Resolution Mass Spectrometry (GC/MS) system of Shimadzu GC/MS 2010 gas
chromatograph. Separation took place in a DB-XLB column (60 m0.25 mm
0.25 mm) (Agilent, Wilmington), temperature programmed from 50 1C (4 min) to
200 1C at 6 1C min
1
and nally to 325 1C at 4 1C min
1
, holding this temperature
for 10 min (Gonzalez et al., 2006). Shimadzu data system was used for PAH
analysis. The recoveries of spiked PAHs standards ranged from 65to 102 percent.
The range of concentrations present in test medium was 0.011, 0.022, 0.046, 0.094
and 0.176 mg l
1
for anthracene and 0.001, 0.004, 0.007, 0.014 and 0.031 mg l
1
for benzo [a] pyrene. 0.05 percent acetone and seawater was maintained as
solvent control and negative control. In control medium, concentrations of
anthracene and benzo [a] pyrene was nil. The experiments were carried out in
replicate for a period of 96 h under ow-through test system and mortality of
organisms were noted at an interval of 24 h.
2.3. Determination of PAHs bioaccumulation in sh tissues
Dry and powdered whole body tissues of anthracene and benzo [a] pyrene
exposed and control sh were used for estimation. All solvents used were HPLC
grade (Merck India Ltd., Mumbai). The extraction procedure was based on the
standard methods of Al-Omair and Helaleh (2004). The Capillary Gas
ChromatographyHigh Resolution Mass Spectrometry (GC/MS) system of Shi-
madzu GC/MS 2010 gas chromatograph, equipped with auto-sampler, 30 m 0.25ID
RTXs-5sil fused silica capillary column (Agilent, Wilmington) and Shimadzu data
system was used for PAH analysis. Helium was used as the carrier gas and the
column head pressure was maintained at 10 psi to give an approximate ow rate
of 1 ml min
1
. The injector and the transfer lines were maintained at 290 1C and
250 1C, respectively. All injection volumes were 1 ml in splitless mode. The column
temperature was initially held at 70 1C for 4 min, ramped to 300 1C at a rate of
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 2
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
10 1C min
1
, then held at 300 1C for 10 min (Anyakora et al., 2005). The mass
spectrometer was used in electron ionization mode and all spectra were acquired
using a mass range of m/z 50400 and automatic gain control (AGC).
2.4. Estimation of biomarker enzymes
After the stipulated periods of treatment (96 h), the live sh were dissected
and tissues (head, gill and dorsal n muscles) were isolated in ice-cold condition
for further studies.
2.4.1. Protein
The protein content in different sh tissues was determined by the method
described by Lowry et al. (1951) using bovine serum albumin (BSA) as a standard.
2.4.2. Lipid peroxidation
Head, gill and dorsal n muscles were individually analyzed according to
Buege and Aust (1978). Lipid peroxidation (LPO) was measured by the generation
of thiobarbituric acid reactive species and quantied in terms of MDA equivalents.
Its absorbance was measured at 532 nm with Systronics make double beam UV
visible spectrophotometer Model 2201 series. Each sample was run by triplicate.
2.4.3. Catalase
Enzymatic activity was evaluated individually in head, gill and dorsal n
muscles of sh exposed to acute concentrations following the method described
by Bainy et al. (1996). Catalase activity (CAT) was measured by the rate of
hydrogen peroxide (H
2
O
2
) decomposition at 240 nm (Beutler, 1982) with Systro-
nics make double beam UV visible spectrophotometer Model 2201 series. Each
sample was run by triplicate.
2.4.4. Acetyl choline esterase
The activity of acetyl choline esterase (AChE) in head, gill and dorsal n
muscles of sh tissue was assayed according to the method of Ellman et al. (1961).
The reaction mixture (3 ml) contained sodium phosphate buffer (50 ml, pH 7.5),
5,5,dithiobis-(nitrobenzoic acid), (DTNB, 0.5 mM prepared in 10 mM phosphate
buffer, pH 7.5 and 15 mg sodium bicarbonate added per 10 ml of solution), the
substrate acetylthiocholine iodide (ATI, 0.5 mM) and enzyme protein (50
100 mg). For assays, the concentration of the substrate, DTNB and enzyme protein
in reaction mixture were chosen so as to give maximal reaction rate. The increase
in absorbance was recorded at 412 nm and 28 1C for 3 min in a Systronics make
double beam UV visible spectrophotometer Model 2201 series. Measurement was
made in triplicate for each tissue homogenate. Simultaneously two blanks were
also used. One containing phosphate buffer, DTNB and ATI but not enzyme protein
to determine hydrolysis of ATI and the second containing phosphate buffer, DTNB
and enzyme protein but not substrate (ATI) to correct for any non-AChE
dependent formation of thio nitro benzoic acid. The blank readings were
subtracted from the experimental absorbance increase per min. One unit of
enzyme activity has been dened as the amount of enzyme required to catalyze
the hydrolysis of one micro mole of the ATI into product per minute under
specied experimental conditions. The specic activity of enzyme is expressed as
units of enzyme activity per mg protein. The extinction coefcient of the yellow
anion (1.3610
4
M
1
cm
1
) was employed for calculating the enzyme activity
(Ellman et al., 1961).
2.4.5. Glutathione S transferase
Glutathione S transferase (GST) activity in head, gill and dorsal n muscles of
sh tissue was estimated according to the method of Habig et al. (1974). Reaction
mixtures contained 4.95 ml phosphate buffer (0.1 M, at pH 6.5):0.9 ml GSH
(10 mM):0.15 ml CDNB (60 mM). One ml of reaction mixture was added to
0.5 ml of the sample, with the nal concentration of 1mMGSH and 1mMCDNB
in the assay. The activity rate of GST was measured as the change in OD/min
at 340 nm (ext. coefft. 9600 M
1
cm
1
) in a Systronics make double beam
UV visible spectrophotometer Model 2201 series and expressed as nmol min
1
mg protein
1
.
2.4.6. Reduced glutathione
Reduced glutathione (GSH) content in head, gill and dorsal n muscles were
individually determined using a uorometric assay (Jasco make uorometer
Model 6000 series) according to the method of Hissin and Hilf (1976).
2.5. Statistical analysis
Median lethal concentration (LC
50
) values were calculated for 24, 48, 72 and
96 h time points for each test series using the Probit analysis software (Finney,
1971; USEPA, 1994). The differences in biomarkers in comparison to control for
each PAHs were assessed by one way analysis of variance (ANOVA), Dunnetts test
was employed to compare the signicant difference between control and different
exposure concentrations (Zar, 1996). Analysis of variance was carried out using
Graph Pad prism software version 5.0.
3. Results
3.1. Inuence of acetone
Acetone (0.05 percent) was used as a solvent in the present
study. Control groups received equal volume of acetone. The
results showed that the acute toxicity, bioaccumulation and
biomarker enzyme activity were not affected by acetone.
3.2. Acute toxicity
The calculated 24, 48, 72 and 96 h acute LC
50
values and their
respective 95 percent condence limits for anthracene and benzo
[a] pyrene exposed to ngerlings of C. chanos under ow-through
test system was shown in Table 1. Non-locomotor and locomotor
response in swimming behavior of sh was observed. More
immobilized response was observed in higher concentrations of
anthracene and benzo [a] pyrene when compared to lower
concentrations. Under acute effect, no activity was found in sh
after 80 h. At higher concentrations, sh showed erratic swim-
ming movement. The death of the sh was conrmed by cessation
of opercular movement. No mortality was observed in control and
solvent control group. Percentage mortality of C. chanos exposed
to different acute concentrations of anthracene and benzo [a]
pyrene is shown in Figs. 1 and 2. The mortality of sh increased
with increase in concentrations of anthracene and benzo [a]
pyrene as well as experimental duration.
3.3. Bioaccumulation
Accumulation of anthracene and benzo [a] pyrene in the
different concentrations are summarized in Table 2. In control
tissues, no accumulation was detected. Maximum increase in
accumulation was noticed with anthracene. This may be due to
exposure of sh to higher concentration of anthracene i.e.,
0.176 mg l
1
than that of benzo [a] pyrene i.e., 0.031 mg l
1
.
3.4. Biomarker
3.4.1. Lipid peroxidation
Signicant differences (Po0.05) were detected among treat-
ment and control sh. LPO level in C. chanos exposed to different
Table 1
Median lethal concentration (LC
50
) values of anthracene and benzo [a] pyrene to milksh C. chanos (n4) (Mean7SD).
Toxicants LC
50
(mg l
1
) R
2
value Slope line equation
24 h 48 h 72 h 96 h
Anthracene 0.43370.054 0.24170.029 0.09870.008 0.03070.004 0.959 Y0.5380.135x
Benzo [a] pyrene 0.08870.017 0.07170.018 0.04970.015 0.01470.001 0.971 Y0.1160.024x
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 3
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
concentrations of PAHs is shown in Fig. 3a and b. Greater increase
in LPO level was noticed in benzo [a] pyrene exposure. Of the
three tissues examined, maximum increase in LPO level was
noticed in dorsal n muscles of benzo [a] pyrene exposed animals
i.e., 73 percent in 0.031 mg l
1
.
3.4.2. Catalase
CAT activity in C. chanos increased with increase in concentra-
tions of anthracene and benzo [a] pyrene (Fig. 3c and d). Sig-
nicant differences (Po0.05) were observed between treatment
and control sh. Maximum increase in CAT level was noticed in
benzo [a] pyrene exposure. Of the three tissues tested, maximum
increase in CAT activity was noticed in dorsal n muscles of benzo
[a] pyrene exposed animals i.e., 65 percent in 0.031 mg l
1
.
3.4.3. Acetyl choline esterase
The AchE levels of sh exposed to different acute concentra-
tions of anthracene and benzo [a] pyrene decreased with increase
in concentrations (Fig. 3e and f). Signicant differences (Po0.05)
were detected among treatment and control sh. Maximum
decrease in AchE level was noticed in anthracene exposure. Of
the three tissues tested, maximum reduction in AchE activity was
noticed in gills of anthracene exposed animals i.e., 67 percent in
0.176 mg l
1
. However, reduction in AchE activity was also
noticed in the head and dorsal n muscles of anthracene exposed
animals.
3.4.4. Glutathione S transferase
Signicant differences (Po0.05) were detected among treat-
ment and control sh. Increase in GST activity was observed in
benzo [a] pyrene exposed animals, while decrease in GST activity
was noticed in anthracene exposed sh (Fig. 3g and h). Maximum
GST activity was noticed in the highest concentration of benzo [a]
pyrene used i.e, 4.73 mg l
1
. Of the three tissues tested, maximum
increase in GST activity was noticed in gills of benzo [a] pyrene
exposed animals i.e., 46 percent. However, increase in GST
activity was also noticed in head and gill tissues of benzo [a]
pyrene exposed animals. Maximum decrease in GST activity was
noticed in the highest concentration of anthracene i.e.,
11.17 mg l
1
. Of the three tissues tested, maximum decrease in
GST activity was noticed in gills of anthracene exposed sh i.e., 66
percent. This level was followed by dorsal n muscles and head
tissues.
R
2
= 0.570 (24h)
R
2
= 0.673 (48h)
R
2
= 0.791 (72h)
R
2
= 0.788 (96h)
0
10
20
30
40
50
60
70
Control
M
o
r
t
a
l
i
t
y


(
%
)
Concentration (mg.l
-1
)
24h 48h 72h 96h 24h 48h 72h 96h
Solvent control 0.001 0.004 0.007 0.014 0.031
Fig. 2. Percentage mortality of C. chanos exposed to different concentrations of benzo [a] pyrene.
Table 2
Bioaccumulation of anthracene and benzo [a] pyrene in whole body tissues of
milksh C. chanos (n3) (Mean7SD).
Anthracene Benzo [a] pyrene
Concentration
(mg l
1
)
Accumulation
(lg g
1
dry weight)
Concentration
(mg l
1
)
Accumulation
(lg g
1
dry weight)
Control Not detected Control Not detected
0.011 8.2070.07 0.001 2.070.08
0.022 15.270.01 0.004 3.870.08
0.046 29.770.01 0.007 5.970.03
0.094 54.670.01 0.014 9.070.01
0.176 94.970.01 0.031 15.670.01
R
2
= 0.593 (24h)
R
2
= 0.704 (48h)
R
2
= 0.777 (72h)
R
2
= 0.887 (96h)
0
20
40
60
80
100
Control
M
o
r
t
a
l
i
t
y

(
%
)
Concentration (mg.l
-1
)
24h 48h 72h 96h 24h 48h 72h 96h
Solvent control 0.011 0.022 0.046 0.094 0.176
Fig. 1. Percentage mortality of C. chanos exposed to different concentrations of anthracene.
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 4
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
3.4.5. Reduced glutathione
Increase in GSH level was observed in anthracene exposed
animals, while decrease in GSH level was noticed in benzo [a]
pyrene exposed sh (Fig. 3i and j). Signicant differences (Po0.05)
were detected among treatment and control sh. Maximum
increase in GSH level was noticed in the highest concentration of
anthracene i.e., 11.17 mg l
1
. Of the three tissues tested, maximum
increase in GSH level was noticed in the dorsal n muscles of
anthracene exposed sh i.e., 77 percent. While, decrease in GSH
activity was noticed in the head, gill and dorsal n muscles of
benzo [a] pyrene exposed sh. Maximum decrease in GSH activity
was noticed in the highest concentration of benzo [a] pyrene
exposure i.e., 4.73 mg l
1
. Of the three tissues tested, the maximum
decrease in GSH level was more or less the same in head and gill
tissues of benzo [a] pyrene exposed sh i.e., 30 and 29 percent,
respectively. This level was followed by that of dorsal n muscles.
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Control 0.011 0.022 0.046 0.094 0.176
L
P
O

l
e
v
e
l

(
m
M

M
D
A

f
o
r
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
Concentration (mg.l
-1
)
Concentration (mg.l
-1
)
head Gills Dorsal fin muscles
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Control 0.001 0.004 0.007 0.014 0.031
L
P
O

(
M
D
A

f
o
r
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
Head Gills Dorsal fin muscles
Fig. 3. (a) Effect of anthracene on LPO level in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n 3). *P o 0.05, statistically different from control.
(b) Effect of benzo [a] pyrene on LPO level in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o 0.05, statistically different from control. (c) Effect
of anthracene on CAT activity in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (d) Effect of benzo [a]
pyrene on CAT activity in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (e) Effect of anthracene on
AchE activity in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (f) Effect of benzo [a] pyrene on CAT
activity in head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (g) Effect of anthracene on GST activity in
head, gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (h) Effect of benzo [a] pyrene on GST activity in head,
gills and dorsal n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control. (i) Effect of anthracene on GSH level in head, gills and dorsal
n muscles of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different fromcontrol. (j) Effect of benzo [a] pyrene on GSHlevel in head, gills and dorsal n muscles
of C. chanos. Values are mean7SD (n3). *P o0.05, statistically different from control.
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 5
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
4. Discussion
In the present study, the acute toxicity of two well known PAHs
(anthracene and benzo [a] pyrene) was investigated using ow-
through bioassay test system. Of the two PAHs tested, benzo [a]
pyrene was found to be more toxic to C. chanos compared to
anthracene. The variations in LC
50
values in the present ndings
were observed when compared to the results reported by several
authors (Yu-qiong et al., 2007; Vieiria et al., 2008; Pilarczyk and
Correia, 2009) may be due to various environmental factors including
the physico-chemical parameters (Magesh and Kumaraguru, 2006)
and regional inuence.
There was no background PAHs levels in natural seawater that
was used for the preparation of toxicant medium and there were
no PAHs accumulation in control sh. C. chanos exposed to both
anthracene and benzo [a] pyrene concentrations found increase
in accumulation levels with increase in concentrations of PAHs.
This means that the accumulation of PAHs level was in bio
available concentration in the test medium i.e., higher the concen-
tration higher the accumulation (Anyakora et al., 2008; Banni
et al., 2010).
The LPO level of C. chanos exposed to anthracene and benzo [a]
pyrene was increased with increase in concentrations. The accu-
mulation of PAHs increases intermediate products in the sh
tissue (Pilarczyk and Correia, 2009) and the antioxidant enzymes
could not eliminate the intermediate product such as O
2

and
H
2
O
2
effectively, leading to higher LPO level (Cheung et al., 2004;
Pan et al., 2006).
Catalase (CAT) plays an important role in protection against the
generation of contaminant-mediated oxyradicals (Peters et al.,
1994). CAT catalyzes the transformation of reactive oxygen species
i.e., hydrogen peroxide, to water. The CAT activity of C. chanos
exposed to anthracene and benzo [a] pyrene increased with
increase in PAHs concentration in acute exposure. The percentage
increase in CAT activity was 53, 55 and 65 percent in head, gills
and dorsal n muscles of C. chanos exposed to the maximum
0.0
3.0
6.0
9.0
12.0
15.0
18.0
21.0
Control 0.011 0.022 0.046 0.094 0.176
C
A
T

a
c
t
i
v
i
t
y

(
H
2
O
2
c
o
n
s
u
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
C
A
T

a
c
t
i
v
i
t
y

(
H
2
O
2
c
o
n
s
u
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
Head Gills Dorsal fin muscles
0.0
3.0
6.0
9.0
12.0
15.0
18.0
21.0
24.0
Control
Concentration (mg.l
-1
)
Concentration (mg.l
-1
)
Head Gills Dorsal fin muscles
0.001 0.004 0.007 0.014 0.031
Fig. 3. (continued)
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 6
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
concentrations of anthracene and 60, 31 and 75 percent in head,
gills and dorsal n muscles in the maximum concentration of
benzo [a] pyrene. PAHs induce the production of O
2

, which is
converted to hydrogen peroxide (H
2
O
2
) by the action of SOD and
then that H
2
O
2
is converted into water by the action of CAT and/or
GPx (Vieiria et al., 2008). The activation of CAT may be due to
conversion of H
2
O
2
into water molecules by the detoxication
process of oxy-radicals against PAHs. This is in good agreement
with the ndings of Jifa et al. (2006), Sturve et al. (2006) and Vieira
et al. (2008).
Acetyl choline esterase (AchE) is responsible for hydrolyzing
the neurotransmitter, acetylcholine, into choline and acetic acid.
AchE has been used as one of the important indices in toxicity
studies because it catalyzes hydrolysis of acetylcholine at the
synaptic junctions thereby facilitating the nerve impulse trans-
mission from one cholinergic neurons to the next one (Gaitonde
et al., 2006; Yadav et al., 2009). In the present study, the AchE
activity of head, gills and dorsal n muscles in C. chanos exposed
to anthracene and benzo [a] pyrene was declined suggesting
similar disrupting cholinergic neurotransmission through inhibi-
tion of AchE (Canty et al., 2007; Douhri and Sayah, 2009).
Glutathione S-transferase (GST) is also one of the important
enzymes involved in the detoxication process, since they catalyze
the conjugation of both endogenous substances and xenobiotics
with glutathione (GSH) (Hasspieler et al., 1994). They can also bind,
store and/or transport a number of compounds that are conjugated
with GSH (Parkinson, 2001). In the present study, benzo [a] pyrene
activated the GST activity, while anthracene inhibited the same.
These results indicate that GST and/or mechanisms controlling its
production or GSH availability respond differently to distinct PAHs.
Benzo [a] pyrene is a well known AhR ligand (Billiard et al., 2006)
with high afnity to this receptor, while anthracene has a con-
siderable lower afnity to this receptor (Barron et al., 2004;
Incardona et al., 2006). Other possible explanations are: (1) that
anthracene directly binds to GST causing its inhibition and (2) since
anthracene considerably increases the activity of GSH, the levels of
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Control 0.011 0.022 0.046 0.094 0.176
A
c
h
E

a
c
t
i
v
i
t
y

(
m
m
o
l

A
C
T
I

f
o
r
m
e
d

m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
A
c
h
E

a
c
t
i
v
i
t
y

(
m
m
o
l

A
C
T
I

f
o
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m
e
d

m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
Concentration (mg.l
-1
)
Head Gills Dorsal fin muscles
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Control 0.001 0.004 0.007 0.014 0.031
Concentration (mg.l
-1
)
Head Gills Dorsal fin muscles
Fig. 3. (continued)
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 7
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
this molecule available may be not enough to assure function of
GST and, thus the enzyme is inhibited due to the lack of GSH that is
being used in the process dealing with oxidative stress (Incardona
et al., 2006). The induction of GST by benzo [a] pyrene suggests
that GSH conjugation is involved in benzo [a] pyrene removal
(Gowland et al., 2002; Vieiria et al., 2008).
Reduced glutathione (GSH), an antioxidant, preventing damage
to important cellular components caused by reactive oxygen species
such as free radicals and peroxides (Pompella et al., 2003). In the
present study, anthracene caused induction of GSH level, while
benzo [a] pyrene caused its inhibition. Anthracene considerably
induces the activation of GSH and the levels of the molecule
available may not be enough to assure function of GST and thus,
the enzyme is inhibited due to lack of GSH being used in the process
dealing with oxidate stress, while benzo [a] pyrene inhibits the GSH,
which indicates that GSH conjugation is formed by benzo [a] pyrene
removal, where induction of GST is noticed and similar mechanism
exists (Vieira et al., 2008). GSH levels in the present study have
shown elevated levels suggesting that antioxidant responses in the
C. chanos are the rst line of defense against xenobiotic exposure
(Cheung et al., 2001).
Anthracene and benzo [a] pyrene were found to signicantly
induce two anti-oxidant enzymes tested, namely LPO and CAT,
which are crucial in the detoxication of oxyradicals to non-
reactive molecules (Van der Oost et al., 2003; Vieira et al., 2008).
Different PAHs had opposite effects on GST activity. In real
scenarios, usually several PAHs are present in ecosystems con-
taminated by petrochemical products. Therefore, since different
PAHs may have opposite effects on GST activity of sh, care
should be taken when using this enzyme as a biomarker in sites
contaminated with several PAHs because the overall result may
lead to erroneous conclusions (Vieira et al., 2008).
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Control 0.011 0.022 0.046 0.094 0.176
G
S
T

a
c
t
i
v
i
t
y

(

m
o
l
e
s

o
f

G
S
H

c
o
n
j
u
g
a
t
e
d

f
o
r
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
G
S
T

a
c
t
i
v
i
t
y

(

m
o
l
e
s

o
f

G
S
H

c
o
n
j
u
g
a
t
e
d

f
o
r
m
e
d
.
m
i
n
-
1
.
m
g

p
r
o
t
e
i
n
-
1
)
Concentration (mg.l
-1
)
Head Gills Dorsal fin muscles
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Control 0.001 0.004 0.007 0.014 0.031
Concentration (mg.l
-1
)
Head Gills Dorsal fin muscles
g
h
Fig. 3. (continued)
L. Palanikumar et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]] 8
Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
5. Conclusion
From the study it can be concluded that C. chanos can be used
in the PAH pollution monitoring program. Head, gills and dorsal
n muscles play a vital role by exhibiting changes to varied
biomarker studies. They also offer several types of unique infor-
mation: (1) relationships between the individual responses of
exposed organisms to pollution and the potential harm to human
health based on the responses of wildlife to pollution and (2) the
development of effective measures for decontamination and
remediation of aquatic water bodies (Yadav and Trivedi, 2009).
Acknowledgment
One of us, Mr. L. Palanikumar, JRF is thankful to MoES-ICMAM,
Chennai for the award of fellowship. Authors thank Dr. R. Babu
Rajendran, Reader, Department of Environmental Biotechnology,
School of Environmental Sciences, Bharathidasan University,
Trichy for providing GCMS facilities.
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G
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Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028
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Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milksh Chanos chanos.
Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028