1. The study analyzed levels of heavy metals like copper, lead, zinc, and cadmium in water, sediment, and tissues of milkfish (Chanos chanos) collected from Kaattuppalli Island and a reference site near Kovalam coast in India. 2. Heavy metal concentrations were highest in sediment, water, and fish tissues from Kaattuppalli Island, which receives untreated industrial waste. 3. Histological examination found lipid accumulation in the liver and increased mucous cells in the gills of fish from Kaattuppalli Island, indicating effects of heavy metal contamination.

1. ORIGINAL ARTICLE
Biomarkers of selected heavy metal toxicity and histology
of Chanos chanos from Kaattuppalli Island, Chennai, southeast
coast of India
Sivakumar Rajeshkumar • Samuvel Sukumar •
Natesan Munuswamy
Received: 29 December 2012 / Accepted: 10 November 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Bioaccumulation of heavy metals and its
associated histological perturbations were studied in vari-
ous tissues of Chanos chanos collected from Kaattuppalli
Island, and they were compared with those of fish collected
from the Kovalam coast. The concentration of four heavy
metals: copper, lead, zinc and cadmium were determined in
water, sediment and different tissues of fish (gills, liver and
muscle). The result showed a marked difference between
the two sites as well as significant variations within the
water, sediment and biota samples. The decreasing trend of
metals from both Kaattuppalli Island and Kovalam coast
was in the order of Cd [ Cu [ Pb [ Zn. Overall, the
highest metal concentration was found in the sediment,
water and biota collected from Kaattuppalli Island. The
accumulation in the gills and liver of C. chanos was found
to be quite high in comparison with that of muscle. These
tissues were further investigated by light microscopy and
the results were compared with the tissues from the refer-
ence site (Kovalam coast). The presence of large lipid
droplets in the liver and increase of mucous cells in the
gills were some of the most noticeable alterations observed
that were related to heavy metal contaminants. It is con-
cluded that histopathological biomarkers provide reliable
and discriminatory data to augment heavy metal pollution
in Kaattuppalli Island. Therefore, long-term monitoring is
necessary to assess the eco-health of the Kaattuppalli
Island environment by choosing a bio-indicator species like
C. chanos, which provide accurate, reliable measurements
of environmental quality.
Keywords Water Á Sediment Á Heavy metal Á
C. chanos Á Histology
Introduction
In the marine environment, toxic metals are accumulating
in water, sediments and marine organisms; and they are
subsequently transferred to man through the food web.
Thus, it has become increasingly important to determine
and assess levels of heavy metals in marine organisms due
to nutritional and safety considerations. This monitoring is
important especially for edible marine organisms due to
their being a potential dietary source of protein (Blasco
et al. 1999). According to Zyadah and Chouikhi (1999),
knowledge of the distribution of metals in isolated tissues
of marine organisms is useful in order to identify specific
organs that may be particularly selective and sensitive to
the accumulation of heavy metals. Fish have been proposed
as sentinel species for the biomonitoring of land-based
pollution because they may accumulate hydrophobic
organic compounds in their tissues, directly from water,
sediments and/or through their diets. Heavy metals accu-
mulate in tissues and may pose a health risk to those who
frequently consume fish. In the organism, xenobiotic
compounds undergo a series of biotransformation reactions
S. Rajeshkumar (&)
Unit of Toxicology, Faculty of Agriculture and Forestry,
University of Guyana Berbice Campus, Johns, Corentyne,
Berbice, Guyana, South America
e-mail: biorajsiva@gmail.com; rajesh.kumar@uog.edu.gy
S. Sukumar
Department of Zoology, Madras Christian College, Tambaram,
Chennai 600059, Tamil Nadu, India
N. Munuswamy
Unit of Aquaculture and Cryobiology, Department of Zoology,
University of Madras, Maraimalai Campus,
Chennai 600025, Tamil Nadu, India
e-mail: munuswamynm@yahoo.com
123
Environ Earth Sci
DOI 10.1007/s12665-013-2975-x

2. catalysed by different enzymatic systems, and their acti-
vation may provide additional evidence for pollution
exposure (Mormede and Davies 2001).
The presence of metals in aquatic ecosystems is due to
the natural interactions between water, sediments and
atmosphere (Sankar et al. 2006). Heavy metals may enter
an aquatic ecosystem from different natural and anthro-
pogenic sources, including industrial or domestic sewage,
storm runoff, leaching from landfills, shipping and harbour
activities and atmospheric deposits (Nair et al. 2006). The
study of organisms as pollutant monitors has several
advantages over the chemical analysis of abiotic compo-
nents (Fernandes et al. 2007). Organisms can only accu-
mulate the biologically available forms of pollutants that
are always present in the environment; thus, they enable
the continuous monitoring of pollutants.
Sediments, not only act as a carrier of contaminants, but
they also act as potential secondary sources of contami-
nants in aquatic systems (Calmano et al. 1990). Marine
organisms, in general, accumulate contaminants from the
environment and, therefore, have been extensively used in
marine pollution monitoring programmes (Uthe et al. 1991;
UNEP 1993). In many countries, significant alterations in
industrial development lead to increased discharge of
chemical effluents into the ecosystem, causing damage to
marine habitats. Due to their toxicity and accumulative
behaviour, heavy metals which are discharged into the
marine environment, damage both marine species diversity
and ecosystems (Agah et al. 2009). Anthropogenic activi-
ties continually increase the quantity of heavy metals in the
environment, especially in aquatic ecosystems where
anthropogenic activities are increasing at an alarming rate,
and have become a serious world-wide problem (Malik
et al. 2010). Therefore, heavy metals can be bioaccumu-
lated and biomagnified via the food chain, and they can
finally be assimilated by human consumers resulting in
health risks (Agah et al. 2009).
Intensive industrial and agricultural activities have
inevitably increased the levels of heavy metals in natural
waters (Jordao et al. 2002). For these reasons, it is
important to determine the concentrations of heavy metals
in commercial fish in order to evaluate the possible risk of
fish consumption to human health (Cid et al. 2001).
Accumulation patterns of contaminants in fish and other
aquatic organisms depend both on uptake and elimination
rates (Guven et al. 1999). The wide diversity of human
activities introduces pollutants into the environment and
their magnitude makes the assessment of environmental
impact a subject of utmost importance (Marcovecchio
2004). Heavy metals are taken up through different organs
of the fish, and many metals are concentrated at different
levels in different organs of the body (Rao and Padmaja
2000). Fish forms an important part of human food and it
is, therefore, not surprising that numerous studies have
been carried out on metal pollution in different species of
edible fish (Lewis et al. 2002). Industrial and mining
wastes can create a potential source of heavy metal pol-
lution in the aquatic environment (Lee and Stuebing 1990).
Under certain environmental conditions, heavy metals
might accumulate up to toxic concentrations and cause
ecological damage (Guven et al. 1999).
An increasing number of researchers now incorporate
histopathological biomarkers in practical ecological risk
assessment methodology (Wester et al. 2002). Histopa-
thological analysis has already been tested and proposed as
an efficient and sensitive tool in the monitoring of fish
health and environmental pollution in natural water bodies
(Teh et al. 1997). Studies of histopathological biomarkers
are linked to the notion that they reflect fish health more
realistically than biochemical biomarkers and can thus be
better extrapolated to community and ecosystem-level
effects of toxicity (Au et al. 1999). Cells have evolved
different networks of cellular stress responses to adapt
during environmental changes and survive by combating a
wide variety of stress (Padmini and Usha Rani 2010).
Earlier studies have reported that the exposure of fish to
pollutants (agricultural, industrial and sewage) resulted in
several pathological alterations in different tissues of fish
(Abbas and Ali 2007). The liver, as the major organ of
metabolism, comes into close contact with xenobiotics
absorbed from the environment, and liver lesions are often
associated with aquatic pollution. Histopathological chan-
ges were observed in the gills of many fish as a result of
exposure to different toxicants (Camargo and Martinez
2006). On the northern side the Island is also connected to
the brackish Pulicat Water Lake, which once nurtured rich
fauna and flora, including mangroves (Rajeshkumar and
Munuswamy 2013). Overexploitation, mismanagement and
improperly treated industrial effluents from more than 25
industries were continuously discharged into the North
Chennai Coastal region, creating a great challenge to the
ecosystem balance (Kamala-Kannan et al. 2008). Earlier
studies in North Chennai coastal waters recorded an ele-
vated level of Cu, Cd and Pb concentrations in water,
sediment and plant samples. The Cd concentration in water
samples was 0.01 mg L in both the seasons. The average
concentration of Cd in sediments during the premonsoon
was 6.25 l g g-1
; however, in the postmonsoon, it was
7.38 l g g-1
(Periakali and Padma 1998).
In this study, the concentration of Cu, Pb, Zn and Cd in
water, sediments, and C. chanos (gills, liver and muscle)
from Kaattuppalli Island was determined. Earlier studies
had also confirmed the histological alteration and differ-
ential expression of HSP70 in different tissues of fish
collected from two different sites of Kaattuppalli Island
(Rajeshkumar and Munuswamy 2011). However, in this
Environ Earth Sci
123

3. context, the present study was carried out to detect early
biological effects which could be both indicative of pos-
sible deterioration in the ecological status of the estuary
and useful in monitoring environmental quality trends. The
specific objectives were to determine the distribution of
heavy metals in water, sediment and various fish tissues of
Kaattuppalli Island and Kovalam coast, Chennai, India.
Materials and methods
Study area
Kaattuppalli Island (lat. 138140
1382100
N and long. 308200
3082800
E) is a narrow longitudinal Island, situated in the
eastern coastal plain, north of Chennai, separated from the
mainland by the backwaters on the eastern aspect,
extending from the brackish water Lake Pulicat in the
north, to the Buckingham Canal in the west, the Ennore
Creek in the south and the Bay of Bengal in the east.
Covering a total of about 40 km2
, the island is about
12.5 km long, with an average width of 3 km; a bridge
over the Buckingham Canal connects the Island with the
main land. This island was chosen as the test (contami-
nated) site as it receives untreated sewage from Royapuram
sewage outfall, untreated/treated industrial effluents from
the North Chennai Thermal Power Plant, Ennore port
activities, the Manali Industrial Belt, which houses many
chemical industries like fertilizer, oil refineries, sugar,
chemicals, etc., in addition to fishing and navigational
activities that take place in the area. The navigational
activities take place in the other nearby industries and
untreated edging activities in this area such as dust pollu-
tion to the coast by quarrying process (Padmini and Usha
Rani 2010). The rapid development of Chennai city in the
last two decades has put additional stress on the local
aquatic environment. The main source of metal input to
Kaattuppalli Island is via the discharge of waste water
effluents, leachates, chemicals, paints, fertilizers and
petroleum refining industry waste from the northern part of
the city. A major portion of the effluent input is also from a
coal-powered thermal power plant situated very close to
the creek which drains the effluents directly into it (Padma
and Periakali 1998). However, in recent years, the dis-
charge from major industries, including fertilizers, rubber
factories, steel rolling, motor vehicles, oil refineries and
operations of the second major harbour for coal import,
which includes a thermal power plant situated nearby, has
imparted severe stress on the estuarine ecosystem. Due to a
mounting population and the development of major
industries during the past three decades, the ecosystem
surrounding Ennore creek has been severely disturbed by
heavy metal pollution (Jayaprakash et al. 2005). The
reference site taken for this study is the Kovalam coast,
which is located 40 km south of Chennai (128490
N, 80850
E). The temperature and salinity of this estuary ranges
between 25 and 28 °C and 24 and 26 ppm, respectively. It
was chosen as the unpolluted site for the present investi-
gation as it is surrounded by high vegetation and it is free
from industrial or urban pollution (Padmini and Usha Rani
2009). Hence, this site has been selected as a reference site
to compare the results with results obtained from the pol-
luted Kaattuppalli Island (Fig. 1).
Heavy metal analysis
Surface water
Heavy metals (Cu, Pb, Zn and Cd) were determined in
unfiltered samples based on the liquid–liquid extraction
method as described by Mentasti et al. (1989) and Jayap-
rakash et al. (2005). In this method, 100 mL of unfiltered
water sample was placed in an acid cleaned separating
funnel. Its pH was adjusted with concentrated nitric acid.
After the addition of 2 mL of ammonium pyr-
olidinedithiocarbamate (APDC), the chelates were extrac-
ted into 10 mL of methyl iso-butyl ketone (MIBK) under
agitation. The aqueous phase was removed and the metals
present in the IBMK were back titrated with concentrated
nitric acid and distilled water. The acidic extractants were
evaporated on a low-temperature hot plate to remove traces
of the organic solvent. The final metal concentrations were
determined by atomic absorption spectrophotometry (Per-
kin-Elmer AA700).
Sediment
For heavy metal (Cu, Pb, Zn and Cd) analysis the sedi-
ments were dried at 60 °C in an oven and disaggregated in
an agate mortar, before chemical treatment. For each
sample, 1 g of sediment was digested with a solution of
concentrated HClO4 (2 mL) and HF (10 mL) to near dry-
ness. Subsequently, a second addition of HClO4 (1 mL)
and HF (10 mL) was effected and the mixture evaporated
to near dryness. Finally, HClO4 alone was added and the
sample was evaporated until white fumes appeared. The
residue was dissolved in concentrated HCl and diluted to
25 mL (Tessier et al. 1979). The final metal concentrations
in sediments were determined by atomic absorption spec-
trophotometer (Perkin-Elmer, AA700).
Sampling and analytical procedure
C. chanos (Milk fish), a natural inhabitant of the island,
was chosen as the experimental animal for the study with
reference to the Food and Agriculture Organization (FAO)
Environ Earth Sci
123

4. species identification sheets (Fischer and Bianchi 1984).
Milk fish, with an average length of 30–32 cm, were col-
lected from Kaattuppalli Island and the Kovalam coast
using baited minnow traps and brought to the laboratory on
the same day. Samples of gills, liver and muscle from each
specimen were dissected, washed with distilled water,
weighed, packed in polyethylene bags and stored at
-20 °C prior to analysis. Frozen samples were thawed at
room temperature and known quantities of the samples
were oven-dried at 90 °C for 24 h. At complete dryness the
tissues were homogenized separately with pestle and
mortar. The dried powder tissue samples were then
weighed accurately to approximately 2 g. The samples
were transferred to a 25-mL conical flask, to which 10 mL
of a 4:1 (v/v) nitric acid and perchloric acid mixture were
added. Each conical flask was then covered with a watch
glass and allowed to react overnight at room temperature.
Then the samples were digested to near dryness by evap-
orating liquid at 90 °C on a hot plate. The samples were
then cooled to room temperature. The digested samples
were then filtered through Whatman No. 1 filter paper and
collected into 50-mL beakers. The filters were rinsed
thoroughly with deionized water. Contents of the beakers
were quantitatively transferred to the 10 mL volumetric
flasks, and brought to volume with ultrapure water. Ele-
ment concentrations of the samples were determined by
atomic absorption spectrometry (Perkin-Elmer, AA 700)
and are expressed as lg g-1
dry weight of tissue (Kingston
and Jassie 1988). The accuracy of the analytical procedures
was verified by analysis of appropriate CRMs using the
Fig. 1 Location map of the
study area—Kaattuppalli Island
and Kovalam coast
Environ Earth Sci
123

5. same digestion and analytical methods. Quantitative results
were obtained for each metal in each CRM (Table 1).
Histology
Samples of gills, liver and muscle were quickly removed
from the fish and fixed in a 5 % neutral buffered formal-
dehyde solution (pH 7.0). After fixation, the tissues were
dehydrated through a graded alcohol series and embedded
in paraffin wax. Tissue sections of 6–8 lm thickness were
taken and stained with hematoxylin and eosin. Photomi-
crographs were taken at varying magnifications using a
Leica 2,500 microscope from Germany.
Statistical analyses
Two-way analysis of variance was performed using a SPSS
7.2 version statistical package to determine significant
differences in heavy metal concentrations in the water,
sediment and tissues and between sites. A probability level
below p 0.05 was considered as statistically significant.
Results
In the present study, the heavy metal concentrations in
water, sediment and C. chanos collected from Kaattuppalli
Island was studied and compared to those of samples col-
lected from the Kovalam coast, which was taken as a ref-
erence site.
Concentrations of metals in water
The abundance of metals in the Island water decreased in
the following order: Cu [ Pb [ Cd [ Zn (Table 2;
Fig. 2a). The minimum level of copper was recorded at the
Kovalam coast during the monsoon and the maximum level
was recorded at Kaattuppalli Island during the premon-
soon. In general, the highest mean value in water was
observed during the premonsoon and the lowest during the
postmonsoon. The Pb concentration in Kaattuppalli Island
water was highest during the premonsoon. The estimated
maximum concentration of cadmium in the water was
observed during the premonsoon in Kaattuppalli Island and
minimum concentration during the postmonsoon at the
Kovalam coast. The zinc concentration in the water was
found to be at maximum during the premonsoon seasons at
Kaattuppalli Island and the minimum during the post-
monsoon. In general, when comparing the findings at the
stations during the postmonsoon, relatively high concen-
trations of heavy metals were recorded during the monsoon
at Kaattuppalli Island and lower concentration at the
Kovalam coast.
Concentrations of metals in sediment
The concentration of metals in the sediments decreased in the
order: Cu [ Zn [ Cd [ Pb (Table 2; Fig. 2b). The maxi-
mum Cu concentrations were recorded at Kaattuppalli Island
during the monsoon and the minimum concentrations during
Table 1 Measured and certified values of heavy metal concentration,
as mg kg-1
dry weight, in standard reference material BCSS and
DORM (dog fish muscle)
Reference material Certified value Measured value Recovery %
BCSS-1
Copper 19 18.1 95.4
Lead 22.7 21.6 96.2
Zinc 119 115.6 96.2
Cadmium 0.25 0.24 96.0
DORM-2
Copper 2.34 ± 0.16 2.32 99.1
Lead 0.065 ± 0.007 0.065 100
Zinc 25.5 ± 2.3 25.2 98.4
Cadmium 0.043 ± 0.008 0.042 99.5
Table 2 Metal concentrations from all sampling stations and seasons
in C. chanos (lg g-1
dry weight), lake water (lg L-1
) and sediment
(lg g-1
dry weight)
Metal No. of
sample
Range Mean Standard
deviation
Muscle Cu 12 0.020–0.447 1.206 ±0.168
Zn 12 0.100–0.132 0.324 ±0.052
Cd 12 0.011–0.034 0.153 ±0.011
Pb 12 0.017–0.038 0.058 ±0.016
Gills Cu 12 0.023–0.027 0.124 ±0.011
Zn 12 0.087–0.323 0.490 ±0.099
Cd 12 0.031–0.037 0.127 ±0.014
Pb 12 0.018–0.028 0.139 ±0.118
Liver Cu 12 0.484–0.620 1.904 ±0.232
Zn 12 1.209–2.537 3.880 ±0.813
Cd 12 0.020–0.026 0.137 ±0.010
Pb 12 0.007–0.028 0.127 ±0.010
Water Cu 12 0.564–2.618 2.075 ±1.108
Zn 12 0.246–0.370 1.136 ±0.160
Cd 12 0.129–0.406 1.185 ±0.214
Pb 12 0.274–0.736 1.412 ±0.318
Sediment Cu 12 0.072–5.337 3.765 ±2.265
Zn 12 0.730–2.644 1.900 ±1.175
Cd 12 0.039–1.106 0.367 ±0.489
Pb 12 0.065–0.124 0.178 ±0.048
Results are mean value of three replications
Environ Earth Sci
123

6. the premonsoon at stations on the Kovalam coast respec-
tively. The Pb concentration in sediments was found to be a
maximum during the premonsoon at Kaattuppalli Island and
a minimum during the postmonsoon. In general, the highest
mean value was observed during the premonsoon. The
maximum concentrations were observed during the mon-
soon and the minimum concentrations during the premon-
soon in sediments of Kaattuppalli Island, and the lowest
mean value was observed during the monsoon at the Kova-
lam coast. The zinc concentration in the sediment was a
maximum during the premonsoon seasons at Kaattuppalli
Island and at minimum during the postmonsoon at the
Kovalam coast.
Concentrations of metals in C. chanos
The heavy metal accumulations in the tissues of C. chanos,
sampled from Kaattuppalli Island and Kovalam coast are
presented in Table 2 and Fig. 2c. The relative abundance
of metals in the gills, liver and muscle of C. chanos were in
the order Zn [ Pb [ Cd [ Cu; Zn [ Cu [ Cd [ Pb and
Cu [ Zn [ Cd [ Pb respectively. Among the four metals
studied, Pb concentrations were low, whereas Zn, Cd and
Cu concentrations were high in different tissues of the fish
C. chanos. A high degree of organ specificity was pro-
nounced in these organisms, where gill and liver exhibited
greater accumulation compared to the muscle. Thus, it
0
0.2
0.4
0.6
0.8
1
1.2
1 2 3 4
Cuµgl-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4
Pbµgl-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4
Znµgl-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4
Cdµgl-1
1
1.05
1.1
1.15
1.2
1.25
1 2 3 4
Cuµgl-1
0
0.05
0.1
0.15
1 2 3 4
Pbµgl-1
0
0.2
0.4
0.6
0.8
1
1.2
1 2 3 4
Znµgl-1
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
1 2 3 4
Cdµgl-1
Sampling station Sampling station
(a) Water (b) Sediment
Sampling station
Sampling station
Sampling station
Sampling station
Sampling station
Sampling station
Sampling station
(c) Chanos chanos
0
0.5
1
1.5
2
2.5
1 2 3 4
Cuµgl-1
0
0.05
0.1
0.15
0.2
1 2 3 4
Cdµgl-1
0
0.05
0.1
0.15
0.2
1 2 3 4
Pbµgl-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4
Znµgl-1
Muscle Gills Liver
Sampling station
Sampling station
Sampling station
Muscle Gills Liver
Muscle Gills Liver
Muscle Gills Liver
Fig. 2 Local distribution of heavy metal concentrations: a water (lg L-1
), b sediment (lg g-1
dry weight) and c C. chanos (lg g-1
dry weight).
Each column represents the mean of the values recorded at a station during all seasonal samplings; bars represent the standard error
Environ Earth Sci
123

7. seems increasingly apparent that industrialization and other
multifaceted activities of humans have caused the deteri-
oration of such aquatic island ecosystems.
Histological observations
Muscle
The section of muscle of fish from the reference site
exhibited normal arrangements of muscle fibres and muscle
bundles with well-organized connective tissues (Fig. 3a).
In contrast, the fish collected from Kaattuppalli Island
exhibited degenerative and necrotic changes in the muscle
bundles. The deformities observed in the muscle tissue
include connective tissue damage, splitting of muscle fibres
and formation of edema between muscle bundles (Fig. 3b).
Gills
The histoarchitecture of the gills of fish collected from
Kovalam coast showed the primary lamellae arranged in
double rows, projecting towards the lateral side with a
series of alternately arranged secondary lamellae
(Fig. 4a). This is common for unaffected teleost gills. The
gills of fish collected from Kaattuppalli Island showed
aneurysm or nodule formation in the secondary lamellae
and hypertrophy is observed with the enlargement of the
tissues. The lamellae fused together and necrosed with
mucoid depositions along the surface. Damage was pro-
nounced with swelling of lamellae and epithelial lifting in
the interfilamentar regions. The cartilaginous rod at the
core of primary lamellae was seen to be disrupted
(Fig. 4b).
MF
MB
(a)
MB
(b)
MF
Fig. 3 a Section through the muscle of fish collected from Kovalam
coast showing a normal arrangement of muscle fibre (MF) and muscle
bundles (MB) with uniform connective tissues (CT). b Muscles of fish
collected from Kaattuppalli Island showing loss of necrosis (N) in the
muscle bundles, connective tissue damage (CT) and splitting of
muscle fibres (SMF) and breakdown of muscle bundles (MB) (scale
bar 50 lM)
SL
PL
(a)
IL
EF
FL
EL
PL
SL
N
(b)
IL
Fig. 4 a Gills of fish Kovalam coast showing normal arrangement of
primary lamellae (PL) and secondary lamellae (SL). b Section
through gill of fish collected from Kaattuppalli Island showing
filamentary epithelium (EF) proliferation, lamellar fusion (FL),
ruptured epithelial layer (EL), Lifting of lamellae (PL), lamellar
swelling (S) and necrosis in the interfilamental region (N) (scale bar
50 lM)
Environ Earth Sci
123

8. Liver
Sections through the fish liver from the reference site
exhibited normal parenchymal architecture of hepatocytes,
which contained homogenous cytoplasm with a centrally
placed nucleus. The liver is composed of masses of hepa-
tocytes organized in distinct lobules interrupted by sinu-
soids and endothelial cells lining the sinusoidal lumen
(Fig. 5a). Fish liver collected from Kaattuppalli Island
showed vacuolization in the hepatocytes and proliferation
of fibroblast. There was an increase in fat vacuolation and
granular degeneration. Hepatocellular necrosis was obvi-
ous in the hepatocytes. The hepatocytes were shrunk with
engorged sinusoidal blood spaces and granular degenera-
tions became evident in most of the hepatocytes (Fig. 5b).
Discussion
In the present study, the concentrations of heavy metals
and their impact on histological changes in C. chanos
inhabiting the Kaattuppalli Island and Kovalam coast were
assessed. Knowledge of heavy metal kinetics in fish is
important for natural resource management and the use of
fish for human consumption (Karadede et al. 2004). Some
authors have previously demonstrated the pollution stress
status of Kaattuppalli Island and accumulation of heavy
metals in fish (Rajeshkumar and Munuswamy 2011). The
present study documents seasonal variations and degree of
heavy metal contamination of water, sediments and biota
from both less polluted and polluted sites of Kaattuppalli
Island. Trace metals, such as zinc, chromium, manganese,
cadmium, cobalt, etc., play a biochemical role in aquatic
life, with their excess being both toxic and nonbiodegrad-
able (Nurnberg 1982). Heavy metal contamination of the
environment is recognized as a serious pollution problem
(Singh and Chandel 2006). Variability in metal concen-
trations in marine organisms depends on many factors, both
environmental and purely biological (Phillips and Rainbow
1993).
The accumulation of heavy metals in tissues of aquatic
organisms may cause various physiological defects and
mortality (Karakoc 1999). In surface water, heavy metals
are typically present only at very low concentrations often
in combination with other inorganic contaminants (Eckw-
ert and Kohler 1997). The concentration of metals observed
in this study were comparable to concentrations reported in
different estuaries. The increased distribution of heavy
metals in the Kaattuppalli Island sediment and water may
be due to the discharge of heavy metal containing effluents
even though effluents from the industries surrounding the
study area are treated. Seasonal variation in metal distri-
bution is influenced by strong hydrodynamic and physico-
chemical conditions prevailing in the estuary (Padmini and
Kavitha 2005). In Kaattuppalli Island, the concentration of
metals was observed to be significantly higher during
summer than during the monsoon. These low seasonal
values may be attributed to freshwater input following rain
as well as the release of surplus water from the Poondi
reservoir into the sea via Ennore creek. Higher values in
summer were due to evaporation raising the metal con-
centrations (Murthy and Rao 1987). In an earlier study, low
metal concentrations were observed during winter and
higher concentrations during summer (Caccia et al. 2003).
The high metal concentration in the tissues of fish
inhabiting Kaattupppalli Island is probably related to a high
influx of metals as a result of pollution from the sur-
rounding industries; thereby increasing bioavailability to
the fish. Nammalwar (1992) reported that the concentra-
tions of Hg, Cd, Cu, Zn, Ni, Pb and Fe in various tissues of
Liza macrolepis inhabiting the Ennore Estuary were above
permissible safe levels. Padmini and Kavitha (2005)
N
HP
V
CV
(b)
HP
CV
(a)
N
Fig. 5 a Section through liver tissue of fish collected from Kovalam
coast showing normal hepatocytes with central vein (CV), hepatic
plate (HP) and nuclei (N). b Liver of fish collected from Kaattuppalli
Island showing damage and structural changes with rupture of central
vein (CV) and irregular hepatic plate (HP) with more number of
vacuoles (V) (scale bar 50 lM)
Environ Earth Sci
123

9. reported that the tissue of C. chanos is subject to severe
stress as it is manages to survive in highly contaminated
habitats. This contamination may cause oxidative stress in
this fish, which in turn can lead to decreased reproduction,
susceptibility to infection and sudden death of fish in large
numbers (Padmini et al. 2004).
The average concentration of iron in sediment samples
during summer was 211.42 lg L-1
and in the post mon-
soon it was 76.193 lg L-1
. The cadmium concentration in
water samples was 1.253 mg L-1
in both seasons. The
average concentration of cadmium in sediments during the
premonsoon was 0.120 lg g-1
and in the post monsoon
0.233 lg g-1
. The levels of heavy metal in fish also varied
with respect to species and different aquatic environments
(Kalay and Canli 1999). The concentration of metals
increased more markedly at the polluted sites than its
counterpart during summer rather than the monsoon. This
increase during summer (Apr–Sep) in polluted sites may be
due to maximum evaporation of water leading to increased
concentration of metals. However, during winter (October–
March) lower values may be due to increased fresh water
input following rain (Murthy and Rao 1987).
Consistent with these findings, evidence was provided
that heavy metal contaminants differentially modulate the
structure of vital organs of C. chanos inhabiting Kaattup-
palli Island. During direct contact with contaminants, most
of the chemicals were taken up into the organism by dif-
fusion or actively through semi-permeable membranes of
the gills and gut epithelia (Fanta et al. 2003). Once metals
passed through the penetration barriers, they were trans-
ferred to the blood stream. From the results, it became
obvious that the bioaccumulation was pronounced in the
gills and the liver compared to the muscle. This was also
confirmed experimentally in L. macrolepis (Chen and Chen
1999). Relatively high concentrations of heavy metals in
the liver and the gills were also found in different species
of fish in River Tigris and Lake Ataturk Dam (Karadede
and Unlu 2000). The concentrations of metals in the gills
reflect the concentrations of metals in habitat waters,
whereas the concentrations in the liver indicate longer
lasting storage of metals (Rao and Padmaja 2000). The
adsorption of metals on the gill surfaces, as the first target
for pollutants in water, could also have an important
influence on total metal levels in the gills (Heath 1987).
The trace metal concentrations varied in the surface waters,
sediments and biota of both the Kovalam coast and Ka-
attuppalli Island. Trace metal concentrations increased in
water and sediment samples of the polluted sites of Ka-
attuppalli Island. The concentrations of Cu, Cd, Pb and Zn
were found to be higher during summer than the monsoon
seasons. However, the concentration of metals such as Cu,
Pb and Zn in water were found to be higher in summer at
Kaattuppalli Island than at Kovalam coast. Similarly, Cu
and Cd were found to be higher in sediment samples in
summer at Kaattuppalli Island than at the Kovalam coast.
Overall, heavy metal accumulations were found to be
higher during summer in the polluted sites of Kaattuppalli
Island.
This present study also provides information on the
accumulation of heavy metals in the candidate fish C.
chanos from different sampling sites in Kaattuppalli Island.
The relative abundance of metals in the gills, liver and
muscle of fish were observed in the order Zn [ Cu [
Pb [ Cd; Zn [ Pb [ Cu [ Cd; Zn [ Cu [ Cd [ Pb,
respectively. Of the metals studied, Pb, and Cd concen-
trations were low, whereas those of Zn, Cd and Cu were
high in different tissues. Overall, the heavy metal accu-
mulation was high during summer at Kaattuppalli Island
and low during the monsoon season. Studies carried out
with different fish species have shown that heavy metals
accumulate mainly in metabolic organs such as the liver
that store metals for detoxification by producing metallo-
thioneins (Hogstrand and Haux 1991). Thus, the liver and
the gills are more often recommended as environmental
indicator organs of water pollution than other fish organs.
This is possibly attributable to the tendency of liver and
gills to accumulate pollutants at different levels from their
environment (Al-Yousuf et al. 2000; Canli and Atli 2003).
The accumulation of lead, zinc and copper is great in the
gills due to body’s defense mechanism, and this organ
forms the principal route for entry of pollutants from water.
The metal concentrations of muscle tissues are important
for the edible parts of the fish. The mean concentrations of
heavy metals in the fish collected from the Kovalam coast
were lower than the maximum permissible limits proposed
by FAO (1983). However, the metal concentrations in the
fish obtained from Kaattuppalli Island were higher than the
permissible limits. The concentrations of cadmium in the
fish from the Ennore Estuary exceeded the upper limit of
1.0 g for fish used for human consumption set by the EU
(2001). Of the metals, the highest mean value was for Cu in
the liver and the lowest for Cd in muscle tissues. There are
several possible reasons for the lower accumulation of
metals in muscles. Firstly, the muscle does not come into
direct contact with the toxicant medium because it is totally
covered by the skin which helps the organism to avoid the
penetration of the toxicant. Similar results have been
reported for a number of fish species and show that the
muscle is not active in accumulating heavy metals (Ka-
radede and Unlu 2000). Similarly, the maximum levels of
Cd and Cu were recorded in the liver of L. macrolepis
collected from the coastal waters off Ann-Ping (Chen and
Chen 2001). The results showed greater accumulation than
reported for the mullet, M. cephalus, in the Gulf of Antalya
(Yazkan et al. 2002). Epithelial cell lifting, epithelial
hypertrophy and hyperplasia, slight deformations of the
Environ Earth Sci
123

10. lamellae, and fusion of adjacent lamellae were more pre-
valent and more pronounced in the fish collected from
Kaattuppalli Island. Several histological lesions observed
in the present study were similar to those observed in trout
(Bernet et al. 2004). Lamellar fusion was found in con-
taminated sole specimens; this change could be a protective
effect for diminishing the amount of vulnerable gill surface
area (Mallatt 1985).
The results of the present study also illustrated the
excessive production of mucous secretion from the surface
of the secondary lamellae. They are normally found in the
filaments; however, the mucus can be found on the respi-
ratory epithelium of fish exposed to stress conditions,
which may mean that the mucous layer protects lamellar
surfaces against infectious agents, toxic agents and parti-
cles in suspension. The liver can be studied in environ-
mental monitoring due to its high sensitivity to
contaminants. Heavy metals at sublethal levels are known
to affect the structure and functioning of cellular compo-
nents, leading to the impairment of vital functions of many
Table 3 Comparison of metal concentrations in water, sediment and fish species observed by different authors at southeast coast, Chennai, India
Sample Description Metals Author
Cu Cd Pb Zn
Perna viridis
Muscle Ennore Estuary 3.289 lg L-1
0.416 lg L-1
0.761 lg L-1
4.658 lg L-1
Arockia Vasanthi et al.
(2013)Gills Ennore Estuary 3.098 lg L-1
0.315 lg L-1
0.892 lg L-1
3.378 lg L-1
Digestive gland Ennore Estuary 3.098 lg L-1
0.315 lg L-1
0.892 lg L-1
3.378 lg L-1
Sediment Pulicat Lake 6.81 lg L-1
Padma and Periakali (1998)
Lake water Pulicat Lake 0.01 mg L-1
Water Ennore Estuary 0.01–0.03 mg
L-1
0.15–0.23 lg
L-1
Padmini and Geetha (2007)
Mugil cephalus
(muscle)
Ennore Estuary 1.258 lg L-1
1.67 lg L-1
Lake water Pulicat Lake 9.24 mg L-1
0.36 mg L-1
5.76 mg L-1
32.46 mg
L-1
Nwaedozie (1998)
Mugil cephalus Pulicat Lake
Gills 8.46 mg g-1
0.63 mg g-1
13.00 mg g-1
13.90 mg g-1
Liver 11.20 mg g-1
1.10 mg g-1
15.45 mg g-1
16.49 mg g-1
Crassostrea
madrasensis
Pulicat Lake
Gills 19.04 mg g-1
0.68 mg g-1
12.45 mg g-1
15.02 mg g-1
Carangoidal
malabaricus
Pulicat Lake Prabhu Dass Batvari et al.
(2007)
Gills 0.348 mg g-1
0.962 mg g-1
0.159 mg g-1
Liver 0.408 mg g-1
1.608 mg g-1
0.365 mg g-1
Muscle 0.040 mg g-1
0.673 mg g-1
0.098 mg g-1
Belone stronglurus
Gills 0.333 mg g-1
0.971 mg g-1
0.247 mg g-1
Liver 0.379 mg g-1
1.943 mg g-1
0.443 mg g-1
Muscle 0.046 mg g-1
0.479 mg g-1
0.113 mg g-1
Lake water Pulicat Lake 0.567 lg L-1
2.88 lg L-1
Kamala-Kannan et al. (2008)
Sediment 64.21 lg L-1
8.32 lg L-1
Ulva lactuca 38.07 lg L-1
11.56 lg L-1
Water Kaattuppalli
Island
3.664 1.253 1.866 1.462 Present study
Sediment 6.642 1.181 0.255 3.800 Present study
Chanos chanos Present study
Gills 1.518 1.148 0.126 0.661
Liver 2.102 1.173 0.151 1.230
Muscle 1.138 0.125 0.085 0.391 Present study
Present study
Environ Earth Sci
123

11. marine organisms (Maharajan et al. 2011). The histopa-
thological alterations observed in the liver were sinusoid
dilation with blood congestion, hydropic swelling of
hepatocytes, and fibrocyst proliferation. These pathological
changes are consistent with those reported by Kendall
(1977) and Sastry and Gupta (1978) using fish exposed to
methyl mercury and mercuric chloride, respectively. In the
liver of C. chanos, there was an increase in the number of
lipid droplets, which were larger in size compared to those
in control specimens. These lipids could possibly indicate
an alteration of lipid metabolism or a partial change in their
morphology. The disruption in the endothelial lining of the
sinusoids and membranous inclusions near the sinusoids
due to stress were observed in the fish from Kaattuppalli
Island. The hepatocytes of fish inhabiting the polluted
Ennore Estuary showed oxidative stress to the organism,
the condition being mediated by redox cycling of the heavy
metals, the important contaminants of the estuary (Padmini
and Usha Rani 2009). The morphological perturbations of
gills, liver and muscle are results of a defensive mechanism
or adaptive changes to heavy metal contamination in the
study area (Au 2004). Our findings leave us to suppose that
the structural modifications in the tissues at the contami-
nated sites might be associated to change at the membrane
level that implied in-tissue perturbations. Relatively high
concentrations of heavy metals were found in the liver and
the gills of the species examined, caught from Kaattuppalli
Island, and suggest the possibility of using these two
organs as bio-indicators for metals present in the sur-
rounding environment. However, it is believed that moni-
toring of these species should be repeated on similar-sized
populations on more occasions and over a longer period to
test whether the results and associated correlations were
sufficiently consistent and robust for monitoring purposes.
The heavy metal concentrations of C. chanos might
have been due to the fact that these metals are weakly
bound to the suspended particulate fraction. A low chloride
concentration and decreased pH might also have enhanced
the solubility and mobility of metals and thus increased
their availability (Kamala-Kannan and Krishnamoorthy
2006). In addition, the variations were also affected by the
total concentrations in water and sediments; some metals
were found to be scavenged from surface sediments by
algal tissues and in some cases from the suspended parti-
cles. The seasonal variation of Cd and Pb in sediments
followed the same pattern. A comparison of metal con-
centration in the tissues of C. chanos with those in the same
or other species of the genus from other areas (Kamala-
Kannan et al. 2008) reveals that the concentration of Pb, Cd
and Cu were higher than those from other areas; also, the
Cu in C. chanos in the study area exceeded the normal
level in marine macrophytes, and the maximum
concentration was detected in fish species from a Cu pol-
luted area (Prabhu Dass Batvari et al. 2007) (Table 3).
Conclusion
This study provides primary information on the distribution
of metal concentrations in the water, sediment and different
tissues of C. chanos in the polluted Kaattuppalli Island and
unpolluted sites of the Kovalam coast. Based on the results it
was clear that the concentrations of some of the metals
exceeded the prescribed standard limit and burdened the fish
tissues especially the gills, liver and the muscle of C. chanos.
Therefore, it can be concluded that these metals in the edible
parts of the species examined shouldpose nohealth problems
for consumers. However, in the future, bioaccumulation of
analysed metals in this study can be a possible risk for the
consumption of these species because of industrial practices
in the vicinity, the establishment of a number of industries
along the river, as well as developmental activities along the
Coast of Kaattuppalli Island. Further studies are necessary in
order to evaluate the ecological significance of this con-
tamination as well as monitoring programmes for assessment
and management purpose.
Acknowledgements Financial assistance to Dr. S. Rajeshkumar
(SRF), from the Ministry of Environment and Forests (MOEF),
Government of India, New Delhi (Ref No: F.N.J.22012/18/
2007W.dt12thNov2007.) is gratefully acknowledged.
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