Rajkumar - International Journal of Advanced Life Sciences

International Journal of Advanced Life Sciences (IJALS)
ISSN
2277 – 758X
Rajkumar ` and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
Acute effects of chromium on bioaccumulation and biochemical
profile of Mugil cephalus (Linnaeus, 1758)
J.S.I Rajkumar* and Samuel Tennyson **
*Department of Advanced Zoology and Biotechnology, Loyola College, Chennai 600034,
Tamil Nadu, India. ** Department of Zoology, Madras Christian College,
Chennai-600059, Tamil Nadu, India
Email : jsirajkumar@gmail.com
Corresponding Author
Dr. J.S.I Rajkumar
Department of Advanced Zoology
and Biotechnology, Loyola College,
Chennai 600034,
Tamil Nadu, India.
Email : jsirajkumar@gmail.com
Article History
Received on 14 December, 2012;
Revised in revised form 25 January,
2012; Accepted 5 February, 2013
Abstract
Acute toxicity of chromium and its effects on survival, accumulation
and biochemical status of the flathead mullet, Mugil cephalus was determined
in the present study. Acute toxicity tests were performed for a period of 96-
hour, using 10, 20, 40, 80 and 160 mg/l concentrations of potassium
dichromate and the 96 h LC 50 value was found to be 65.01 mg/l. Appreciable
changes in the biochemical profiles such as total protein, catalase, lipid
peroxidation, acetylcholinesterase and glutathione-S-transferase contents
of the fish were also observed. The activity of catalase and acetylcholinesterase
significantly decreased (P

International Journal of Advanced Life Sciences (IJALS)
ISSN
2277 – 758X
Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
components of these systems involve reduced glutathione
(GSH) and certain antioxidant enzymes including free
radical scavenging enzymes, such as catalase (CAT)
and glutathione S-transferase (GST). Under oxidative
stress conditions, ROS can be reduced by GSH, with
the concomitant formation of the oxidized disulphide and
oxidized glutathione (GSSG). The aquatic environment
which acts as a sink for many active contaminants is
responsible for inducing oxidative stress and is also of
ecological significance (Oliveira et al., 2010). Changes
in the activity of enzymes and other biomarkers are the
possible tools in aquatic toxicological research. Fish are
specific indicators of different environmental compartments
in relation to their habitat and food web position
and they exhibit different rates of bioaccumulation with
respect to xenobiotics (Kord et al., 2010). Therefore,
the present study was undertaken to investigate the
acute toxic effects of heavy metal on the biochemical
and bioaccumulation profile of juvenile specimens of
Mugil cephalus through static renewal bioassay using
chromium as the contaminant.
Materials and Methods
Juvenile specimens of Mugil cephalus collected
from the Kovalam creek (12°14'16.99" N, 80°14' 52.94" E,
Tamilnadu, India) were immediately transported to the
laboratory in air-filled plastic bags. Test organisms were
acclimatized in 1000 L FRP tanks with aerated natural
filtered seawater for a period of eight days with 28 ppt
salinity, temperature of 29 ±2 °C, dissolved oxygen of
5.5 mg/l and pH of 7.9. M. cephalus was fed with
pellets of rice bran and oil cake. Stock solutions of
chromium were freshly prepared by dissolving the
potassium dichromate (K 2 Cr 2 O 7 ) in deionized (double
distilled) water and fresh stock solutions were prepared
daily. These solutions were serially diluted to arrive at
different experimental concentration.
The experimental method includes static renewal
(24-hr renewal) test by following the method of USEPA
(2002a). Five concentrations (10, 20, 40, 80 and 160 mg/l)
in a geometric series including control were prepared for
the test. Toxicant and seawater were replaced on daily
basis. Each series of test chambers consisted of triplicates
with ten animals in a 10 L glass trough. Test chambers
were loosely covered to prevent loss of test animals. All
the experiments were conducted at salinity of 29 PSU,
temperature of 29 ±2 °C, dissolved oxygen of 5.6 mg/l
and pH of 7.8. Daily observations were recorded for
survival and mortality. The criterion for determining death
was the absence of movement when the animals were
gently stimulated. Dead animals were removed at each
observation and survivors were counted. Maximumallowable
control mortality was ten per cent for a 96-
hour period of testing (USEPA, 2002b). Computerized
probit analysis program was carried out for the calculation
of 96-hour LC 50 values and the upper and lower 95 per
cent confidence levels were also calculated.
Biochemical estimation
Sample preparation : Tissue samples of test animals were
pooled and made in duplicates. For the analysis, 1g tissue
was homogenized in chilled pestle and mortar with 5ml
homogenization buffer (0.25M sucrose, 10 mMTris, 1
mM EDTA and pH 7.4) and centrifuged at 5,000 rpm
for 15 minutes at 4°C. The resulting supernatant was the
homogenate which was used for the estimation of various
biochemical assays using Hitachi (UV-700) spectrometer.
Lipid peroxidation (LPO) : Lipid peroxidation level
was assayed by measuring Malondialdehyde (MDA), a
decomposed product of polyunsaturated fatty acids.
Hydroperoxides were determined by the thiobarbituric
acid reaction as described by Ohkawa et al. (1979). The
absorbance was read at 532 nm after removal of any
flocculated material by centrifugation. The amount of
thiobarbituric acid reactive substance (TBARS) was
calculated by using an extinction coefficient of 1.56 x
105/M/cm and was expressed as nmol TBARS formed
/mg protein.
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International Journal of Advanced Life Sciences (IJALS)
ISSN
2277 – 758X
Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
Catalase (CAT) : Catalase (CAT) activity was measured
at 240 nm by determining the decay of hydrogen peroxide
(H 2 O 2 ) levels followed by Beers and Sizer (1952) and
was expressed as µmol of H 2 O 2 consumed / min /mg /
protein.
Acetylcholinesterase (AChE) : Acetylcholinesterase
(AChE) activity was determined using Ellman’s reagent,
DTNB (5, 5’-dithio-bis (2- nitrobenzoic acid); 0.5mM)
and acetylthiocholine iodide (ACTI) as substrate (Alves
et al., 2002). The rate of change of absorbance at 412 nm
was recorded over two minutes at 25°C. Blank samples
were read to make sure that there was no non-specific
esterase or other background activity and was expressed
as nmol ACTI /min/mg/protein.
Glutathione S-transferase (GST) : Activity of GST
was assayed at 340 nm by measuring the increase in
absorbance using 1-chloro-2, 4-dinitrobenzene (CDNB)
as the substrate (Habig et al., 1974). The results were
expressed as nM of GSH and CDNB conjugate formed
/min/mg protein. Values expressed as nanomoles of
reduced glutathione and CDNB conjugate formed /min
/mg protein. The protein concentration of each of the
sample extract was determined according to Lowry
et al. (1951) using bovine serum albumin as the standard
and values expressed as mg protein/g tissue.
Bioaccumulation
The soft tissues of the test organisms were
removed using a teflon scalpel, rinsed with distilled
water and was frozen at - 80°C untill analysis. During
the course of analysis, the tissue was washed with
distilled water and dried at 95°C in hot air oven and
grinded to a fine powder with pestle and mortar. Metal
analysis was carried out according to the methodology
adopted by UNEP (1984). To ensure the accuracy and
precision in the sample analysis, certified reference
material (DOLT-3, Dogfish liver certified reference
material for trace metal, from national research council
Canada) was used (DOLT-3, 1999). Nearest gram of
the dried tissue powder was transferred to a Teflon
crucible. To the tissue was added 8-10 ml of concentrated
acid (60 per cent nitric acid (HNO 3 ): 70 per cent perchloric
acid (HClO 4 ), such that the tissue was totally wet with
slight excess of acid and was kept at room temperature
(29 ±2°C) for 12 hours. The digested samples were
heated slowly to 180°C on a hot plate, till the sample
volume was reduced to 2-3 ml. The resulting colorless
solution was made up to 25ml in a standard glass flask
and stored in 50 ml Polyethylene-tereftalate (PET)
bottles and was analyzed for metals in Varian Spectra
AA 220FS Atomic absorption spectrophotometer
(AAS). Suitable internal chemical standards (Merck
Chemicals, Germany) were used to calibrate the
instrument. All the reagents used were analytical grade
of high purity. The results were expressed as µg/g dry
weight.
Results and Discussion
During the toxicity test, temperature was
maintained at 28 °C ±0.3, salinity at 28 ±1.2 PSU, pH
was 7.78 and dissolved oxygen was maintained with 4.9
mg/l. The LC 50 value was 65.01 mg/l (42.44–120.88). The
rate of survival was found to be 25 per cent in 160 mg/l
and rate of mortality was 75 per cent. The mortality
ranged from 15 to 75 per cent and increased with a
corresponding increase in the toxicant concentration
and also duration of the exposure demonstrating both
time and concentration dependent responses. The rate
of survival percentages are presented in Figure 1 and 2.
Vutukuru (2005) reported an LC 50 value of 111.45 mg/l
to freshwater fish, Labeo rohita in acute toxicity test
with potassium dichromate. The behavior and condition
of the fishes in both the control and test solution was
observed. The test organism showed a marked change in
their behavior when exposed to different concentrations
of the test solution viz., 80 and 160 mg/l and the test
fish showed rapid swimming. Behavioral symptoms of
acute toxicity like copious secretion of mucus, loss
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International Journal of Advanced Life Sciences (IJALS)
ISSN
2277 – 758X
Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
of scales, discolouration, surfacing and darting movements
were observed in M. cephalus exposed to higher concentration
of potassium dichromate 160 mg/l.
Concentrations of chromium used in the present
study are regarded to be high for M. cephalus. Such
concentrations do not occur permanently in surface
waters. However, due to accidental industrial discharges of
heavy metals into the aquatic environment, fish may
have shorter or longer contact with such concentrations
of heavy metals (Rajkumar and Samuel, 2012). This
may be dangerous for fish, especially for juveniles that
are considered to be more vulnerable to intoxication
caused by heavy metals than embryos or older individuals
(Rajkumar and Milton, 2011). The biochemical profile,
catalase, acetylcholinesterase, glutathione S-transferase
and lipid peroxidation are represented in the form of
correlation matrix in Table - 1. The use of biochemical
indicators in environmental pollution studies, particularly
in this field is of high toxicological relevance. Presence of
low concentration of scavenging enzymes in the
juveniles makes them susceptible to oxidative damage
when attacked by ROS (Runnalls et al., 2007).
M. cephalus exposed to exposure concentrations
experienced severe Oxidative stress (OS) characterized
by significant changes in the levels of OS biomarkers,
which had also been observed in brain samples of the
mullet (Padmini and Kavitha, 2005). Konradt and
Braunbeck (2001) have reported that the general
esterases are the good biomarkers to differentiate the levels
of contaminants. Maintenance of enzyme activity may
serve as a quality criterion of the cells and enzymatic
changes may be regarded as important markers for the
presence of hazardous substances (Oost et al., 2003).
Livingstone et al. (1992), found induction of catalase in
dab (Limanda limanda) exposed to contaminated
sediments. Mullet (Mugil sp.) from contaminated Spanish
areas revealed increased activities of antioxidant (catalase)
and detoxifying GST enzymes (Rodriguez-Ariza et al.,
1995). The metabolism of toxic compounds frequently
results in the formation of ROS, which significantly
contribute to their toxicity (Monferran et al., 2008).
Changes in GST activity reflect detoxification
process in fish exposed to toxic compounds (Ballesteros
et al., 2009). Increase of GST was an observed activity
in fish exposed to chromium in the present study. This
induction in GST activity could indicate a defensive
response against oxidative stress damage produced by
adverse conditions in fish. Increased levels of LPO
have been observed in fish under experimental conditions,
upon exposure to different xenobiotics (Bhattacharya
et al., 2007). There are evidences that heavy metals
produce increased LPO levels in M.cephalus (Talas et
al., 2008). The concurrent use of several biomarkers is
important to minimize the misinterpretation in cases of
complex situations of pollution (Linde-Arias et al.,
2008). The reduction in protein content might be also
due to the proteolysis process for energy production
and utilization owing to the decreased food intake of
crabs under stress (Elumalai and Balasubramanian, 1999).
The results obtained in present study show the existence
changes in GSH content and its data may indicate a
faster rate of GSH utilization or degradation, which
could be responsible for the observed lower GSH content.
Moreover, increase of GSH content may be related to
prevention of oxidative challenge (Dandapat et al., 2000).
Aquatic organisms maintain high content of
GSH in tissues and increased content has the function
of protection (Thomas and Juedes, 1992). High content
of GSH could be a consequence of its increased synthesis
due to high cysteine accessibility, which is necessary
for GSH synthesis. GSH content increased after
treatment with cadmium (Son et al., 2001); this could
provide the first line of defense against the influence of
toxic heavy metals. MacFarlane et al. (2006a) found
that heavy metals induce increase of GSH content in
the crab Parasesarma erythodactyla, while decrease of
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International Journal of Advanced Life Sciences (IJALS)
ISSN
2277 – 758X
Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
Fig.-1. Fraction of survivors with time profile of
M. cephalus to chromium
Fig. -2. Fraction of survivors with concentration
profile of M. cephalus to chromium
Table - 1. Correlation matrix of Biochemical profile of M. cephalus exposed to chromium in acute toxicity test
Conc. P LPX GST ACHE CAT B W.C.
Conc. 1
P -0.79 1
LPX 0.90 -0.93 1
GST 0.96 -0.65 0.78 1
ACHE -0.80 0.75 -0.91 -0.68 1
CAT 0.99 -0.83 0.93 0.92 -0.81 1
B 0.97 -0.79 0.94 0.93 -0.90 0.96 1
W.C. 1.00 -0.78 0.90 0.97 -0.79 0.99 0.97 1
Conc.-Concentration; LPX-lipid peroxidation; GST-Gluthathione S-Transferase; ACHE-Acetylcholinesterase;
CAT- Catalase; P-Protein; B- Bioaccumulation; W.C.- water concentration P

International Journal of Advanced Life Sciences (IJALS)
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Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
GSH content in crab Scylla serrata was reported by
Vijayavel and Balasubramanian (2006). It has been
established that GSH content are related to the survival
rate of mussels (Pena-Llopis et al., 2003). Although
pollution influence on GSH content is regulated by a
feedback mechanism, the GSH levels can be used as a
potential biomarker in fish (Oost et al., 1996).
Bioaccumulation of chromium in the test organism
increased linearly with increase in concentration (Fig.-3).
Heavy metals may affect organisms by accumulating in
their bodies or by transferring to the next trophic level
of the food chain. The toxicity of heavy metals accumulated
in the living organisms may become highly toxic when
the accumulated rate increases the safer level (Yildirim
et al., 2009). Aquatic organisms exposed to a higher
concentration of trace metals in water may take up
substantial quantities of these metals (Kord et al., 2010).
In the aquatic environment, the metals are accumulated
either directly from the surrounding water or by ingestion
of food (Kumar and Mathur, 1991). However, when
metal contaminated aquatic organisms are transferred to
clean water, metal depuration occurs (Ekpo et al., 2008).
Uptake and elimination are two of the most important
factors in metal metabolism. Metal accumulation in the
tissues of fish varies according to the rates of uptake,
storage and elimination (Evers et al., 2007). Toxicity
and bioaccumulation potential of a xenobiotic are greatly
affected by the rate of elimination from the organism.
An unaltered chemical like copper element can be
eliminated rapidly, residues will not accumulate and
tissue less likely damaged (Subathra and Karuppasamy,
2008). Environmental factors such as salinity, pH,
hardness and temperature also play a significant role in
metal accumulation. Ecological needs, size and age of
individuals, life cycle and life history, feeding habits
and the season of capture were also found to affect
experimental results from the tissues (Aksoy et al.,
2009). Contamination of aquatic ecosystems with heavy
metals has been receiving increased worldwide concern
(Tsangaris et al., 2007). Ideally, these assessment tools
promote the sustainability of ecosystems and pinpoint
early symptoms of exposure in order to prevent the
progression of environmental degradation whilst conditions
are still reversible. Protection can be accomplished if
causes associated with effects are both quantifiable and
can be used to generate preventive guidelines. The
guidelines should be protective of as many species as
possible and should be flexible to ensure the generation
of new data that would provide maximum protection.
The challenge that still remains for ecologists and
ecotoxicologists is the definition of what effects on the
ecosystem are acceptable or unacceptable in relation to
the most sensitive endpoints on the species level. Thus
developments in risk assessment models should focus
on the translation from laboratory species to field
communities.
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International Journal of Advanced Life Sciences (IJALS)
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Rajkumar and Samuel Tennyson , IJALS, Volume (6) Issue (2) Feb - 2013. RESEARCH ARTICLE
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Corresponding Author : Dr.J.S.I Rajkumar, Department of Advanced Zoology and Biotechnology, Loyola College,
Chennai 600034, Tamil Nadu, India. Email : jsirajkumar@gmail.com. ©2013, IJALS. All Rights Reserved.
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