ORIGINAL ARTICLE

Biomarkers of selected heavy metal toxicity and histologyof Chanos chanos from Kaattuppalli Island, Chennai, southeastcoast of India

Sivakumar Rajeshkumar • Samuvel Sukumar •

Natesan Munuswamy

Received: 29 December 2012 / Accepted: 10 November 2013

� 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 � Sediment � Heavy metal �C. chanos � 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

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

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 1382100N and long. 308200

3082800E) 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

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

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-1dry weight), lake water (lg L-1) and sediment

(lg g-1dry 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

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

Cu

µg

l-1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4

Pb

µg

l-1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4

Zn

µg

l-1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4

Cd

µg

l-1

1

1.05

1.1

1.15

1.2

1.25

1 2 3 4

Cu

µg

l-1

0

0.05

0.1

0.15

1 2 3 4

Pb

µg

l-1

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4

Zn

µg

l-1

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

1 2 3 4

Cd

µg

l-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

Cu

µg

l- 1

0

0.05

0.1

0.15

0.2

1 2 3 4

Cd

µg

l-1

0

0.05

0.1

0.15

0.2

1 2 3 4

Pb

µg

l-1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4

Zn

µg

l-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

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

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)

NHP

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

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

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-10.15–0.23 lg

L-1Padmini 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-1Nwaedozie (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

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 should pose no health 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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