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J. Environ. Sci. & Natural Resources, 5(2): 231- 237, 2012 ISSN 1999-7361
Metals and Histopathological Alterations in the Liver of Schizothorax
niger, Heckel From The Dal Lake of Kashmir Valley
1R. Yousuf, 1S. H. Mir, S. Tanveer1, M. M. Darzi2 and 2M. S. Mir
1Department of Zoology, University of Kashmir, Srinagar-190 006
2Department of Pathology, FVSC & AH, Shuhama Alustend (SKUAST-K), Srinagar- 190 006
Abstract: The study was conducted to evaluate the metal induced abnormalities in the liver of Schizothorax niger from Dal Lake
seasonally for a period of two years. The varied seasonal metal concentrations for copper (66.77 3.12 to 81.68 3.51 ppm), zinc
(73.81 2.52 to 97.84 4.62 ppm), iron (204.92 5.21 to 296.51 4.37 ppm) and manganese (01.13 0.02 to 08.30 1.00 ppm)
were observed during the entire period of study. The highest concentration of metals was observed in the summer seasons and the
lowest concentrations in the winter seasons during the study period. Further, histochemical analysis demonstrated enormous
amount of metals (Cu, Fe and Zn) in the liver of Schizothorax niger in summer seasons during the entire study period. The
subsequent effects of metals, demonstrated by wet digestion-based Atomic Absorption Method and histochemical methods
showed histological changes on the liver of Schizothorax niger. The liver showed disruption of the hepatic cords with congestion
and degenerative changes in hepatocytes that varied from mild in winter seasons to severe vascular degeneration in summer
season. From the present study it may be concluded that the metals in the environment are polluting the water bodies and their
subsequent deleterious effects harm the aquatic fauna particularly the sensitive native fish, Schizothorax niger which is one of the
reasons for its decline from the fresh water resources of the Kashmir Valley.
Key Words: Histology, Liver, Metals, Schizothorax niger
Introduction
The varieties of human activities acting upon the
natural environment result in the release of different
chemicals including metals. The sources of metals
include commercial fertilizers, sewage sludge’s,
urban wastes, liming material and agrochemicals and
other wastes used as soil amendments (Rao, 1998).
The exposure of bio-organisms to metals can cause
long-term and non-reversible effects (Cheng, 2003).
Fish species are widely used to biologically monitor
variation in environmental levels of anthropogenic
pollutants (Whyte et al., 2000; Schmitt, 2004).
The liver is an important organ involved in metabolic
processes and in detoxification of xenobiotics. In
some situations, materials may accumulate in the
liver to toxic levels and cause pathological alterations
(Meyers and Hendricks, 1985; Ferguson, 1989;
Braunbeck et al., 1990). The liver not only represents
a central organ concerning basic metabolism
(Gingerich, 1982), but is also a major site of the
accumulation, biotransformation and excretion of
xenobiotic compounds (Meyers and Hendricks,
1985). It is the first organ to be exposed by the portal
circulation to toxicants ingested by the body (Hibiya,
1982). Because of its unique position and proximity
to the venous drainage of the digestive tract, the liver
is susceptible to damage from absorbed toxic
materials (Leeson and Leeson, 1976). The high
degree of metabolic activity of hepatocytes renders
them vulnerable and toxins can easily affect them.
The harmful effects of ingested toxic substances are
primarily exerted within the liver cells (Lloyd, 1992).
Subsequently, hepatocytes respond to changes in the
external and internal environments by alterations in
both cellular structure and function (Wheater et al.,
1985). Since histopathological alterations are
recognized and commonly used diagnostic tools in
fish toxicological studies (Lloyd, 1992) the present
study was designed to study the metal-induced
toxicity to liver of Schizothorax niger in naturally
occurring water body of Dal Lake.
Materials and Methods
Collection of Fish Hosts
Fishes were collected from the Dal Lake with the help
of local fishermen and were brought alive in plastic
buckets to the laboratory for investigating the
different parameters.
Species and number of fish used
The study was conducted on Schizothorax niger ,
Heckel. Pooled specimens were collected from the
collection sites of the Dal Lake so as to make a
sample size of 25 fish (of either sex) with an average
length of 30-40 cms. The study was repeated for each
season for the year-I and again during Year-II.
Seasonal classification
The study was conducted in four seasons annually,
each with a duration of 3 months. The four seasons
included Spring (March-May), Summer (June-
August), Autumn (September-November) and Winter
(December-February).
232
Metal analysis of water
For detection of metals in water, the samples were
collected in conical flasks, filtered through
Whatman’s filter paper and processed in Atomic
Absorption Spectrophotometer (AAS) for estimation
of various metal concentrations employing Lindsay
and Norwell Method (1978).
Histochemical demonstration of metals
For detection of metals viz copper, iron and zinc in
fish gills different histochemical methods such as
Perl’s method for Iron; Dithiooxamide method for
copper; Dithizone method for zinc etc (Luna, 1968)
were used so as to ensure metal induced toxicity
specify the manganese detection method.
Histological procedure
Histological examination was done after fixing the
fishes in 10% formalin, processed and embedded in
paraffin wax. Tissue blocks were sectioned 5 µm
thick and stained with Harris haematoxylin and eosin
(H&E) (Luna 1968).
Results
Metal concentrations in water
In Dal Lake the concentration of copper was in the
range of 1.020 to 1.070 ppm, with maximum
concentration found in summer season (year-II) and
the minimum in winter season (year-I). The iron
concentration ranged between 0.110 to 0.191ppm.
The highest value was observed during summer
season (year-II) and the minimum in winter season
(year-I). The zinc concentration ranged between
0.150 to 0.542 ppm, with maximum value observed in
summer season (year-II) and the lowest values in
winter season (year-I). The manganese concentration
ranged between 0.021 to 0.083 ppm with maximum
value observed in summer season year-II and the
lowest values in winter season (year-I).
Metal concentrations in the liver
In Schizothorax niger, the mean concentration of
copper in the liver ranged from 66.77 3.12 to 81.68
3.51 ppm (Table I). The maximum value of 81.68
3.51 ppm was observed in summer (year-II) and the
lowest value of 66.77 3.12 was observed in winter
season (year-I).
The concentration of zinc in the liver was 73.81
2.52 to 97.84 4.62 ppm (Table II). The maximum
value of 97.84 4.62 ppm was observed in the
summer season of year-II and the minimum value of
73.81 2.52 ppm was observed in the winter seasons
of year-I.
The concentration of iron in liver varied between
204.92 5.21 to 296.51 4.37 ppm (Table III). The
maximum values of 296.51 4.37 ppm in liver was
BREI
N
CENTAUR
DALGATE
NISHAT
Fig. 1: Map of Dal Lake showing different collection sites such as Site I
(Dalgate), Site II (Centaur), Site III (Brein) and Site IV (Nishat)
J. Environ. Sci. & Natural Resources, 5(2) : 231 - 237, 2012
233
observed in the summer (year-II) and the minimum
values of 204. 92 5.21 ppm in liver was observed in
the winter season (year-I).
The concentration of manganese in liver ranged
between 01.13 0.02 to 08.30 1.00 ppm (Table VI).
The highest values of 08.30 1.00 ppm in liver was
observed in summer season of year-II. The lowest
value of 01.13 0.02 ppm in liver was observed in
the spring season of the year-I.
Histological changes
The liver showed disruption of the hepatic cords and
tubules with congestion and degenerative changes in
hepatocytes that varied from mild in winter seasons to
severe hapataytic?degeneration in summer season
(Fig. 2-3). Further, kupffer cell hyperplasia was
noticed in the liver of Schizothorax niger (Fig. 3).
Table 1: Showing Copper concentration in the liver of Schizothorax niger in different season of the study period in
Dal Lake
Values are expressed as mean SEM
Table 2: Showing zinc concentration in the gills of Schizothorax niger in different season of the study period in Dal
Lake
Values are expressed as mean SEM
Table 3: Showing iron concentration in the liver of Schizothorax niger in different Season of the study period in Dal
Lake
Values are expressed as mean SEM
Water
resources
Fish Host
Year
No.
Observed
Copper accumulation (ppm)
Spring
Summer
Autumn
Winter
Dal Lake
Schizothorax
niger
I
25
70.01 2.12
76.52 2.81
68.52
2.12
66.77
3.12
II
25
74.54 3.24
81.68 3.51
72.82
3.24
70.54
3.12
Water
resources
Fish Host
Year
No.
Observed
Zinc accumulation (ppm)
Spring
Summer
Autumn
Winter
Dal Lake
Schizothorax
niger
I
25
74.52 2.24
90.61 3.92
75.12
4.77
73.81
2.52
II
25
81.06 3.44
97.84 4.62
82.52
3.99
80.88
4.15
Water
resources
Fish Host
Year
No.
Observed
Iron accumulation (ppm)
Spring
Summer
Autumn
Winter
Dal Lake
Schizothorax
niger
I
25
227.91
6.52
284.31 4.29
228.36
6.55
204.92
5.21
II
25
241.20
6.96
296.51 4.37
242.54
4.85
234.56
5.95
J. Environ. Sci. & Natural Resources, 5(2) : 231 - 237, 2012
234
Table 4: Showing manganese concentration in the liver of Schizothorax niger in different season of the study period
in Dal Lake
Values are expressed as mean SEM
Fig 2: showing severe congestion and degenerative changes in hepatocytes (×100X).
Fig 3: showing kupffer cell hyperplasia in the liver (×100X).
Discussion
In Dal Lake the concentration of copper was in the
range of 1.020 to 1.070 ppm; iron 0.110 to 0.191ppm;
zinc 0.150 to 0.542 ppm and manganese 0.021 to
0.822 ppm. Metal accumulations can be attributed to
a variety of sources- such as from rocks, solids, dead
and decomposing vegetation, wet and dry fallout of
atmospheric particulate matter and from human
activities including the discharge of various treated
and untreated liquid wastes into the water bodies
(Lasheen, 1987). The concentration of metals in the
Dal Lake water can be attributed to its stagnant
waters. Seasonal differences were also observed with
higher concentrations during the summers, followed
by spring, autumn and winter. This may be due to
higher fallout of metals from the decomposing matter
and increase in temperature during the hot seasons,
which gradually reduce during the colder months.
Water
resources
Fish Host
Year
No.
Observed
Manganese accumulation (ppm)
Spring
Summer
Autumn
Winter
Dal Lake
Schizothorax
niger
I
25
01.13 0.02
07.13 0.99
03.16
0.98
02.81
0.05
II
25
02.74 0.03
08.30 1.00
04.74
0.11
03.55
0.07
J. Environ. Sci. & Natural Resources, 5(2) : 231 - 237, 2012
235
Further, it is generally accepted that heavy metal
uptake occurs mainly from water, food and sediment
(bottom feeders and burrowing animals) (Canli et al.,
1998). However, the metal uptake from water is much
higher than uptake from sediment (Mance, 1987;
Langston, 1990; Merian, 1991). It may be
emphasised, that the efficiency of metal uptake from
contaminated water and food may differ in relation to
ecological needs, metabolism and the contamination
gradients of water and food and sediment, as well as
other factors such as salinity, temperature and
interacting agents (Pagenkopf, 1983; Cusimano et al.,
1986; Heath, 1987; Canli and Furness, 1993; Goyer,
1991; Canli and Furness, 1995). Years-wise data
showed a higher heavy metal concentration in the
latter year than the preceding in both water bodies.
This clearly suggested an increase in pollution levels
in the water body.
Season-wise higher tissue concentrations of heavy
metals were observed in summer with decline in their
levels during spring, autumn and winter in a
decreasing order. Obviously, the progressive increase
in the metal levels in the tissues coincides with the
period of rising temperatures during summer. It is
generally accepted that metal accumulation in living
organisms is largely controlled by specific uptake,
detoxification and elimination mechanisms and
therefore depends significantly on the season (Cogun
et al., 2006). Seasonal differences in the heavy metal
accumulation in fish can be related to their metabolic
rate, which determines the physiological condition of
fish (Farkas et al., 2003). Laboratory experiments
have shown that changes in temperature can affect the
increase or decrease of heavy metal concentrations
because of changes in metabolic and excretion rates
(Hilmy et al., 1987; Yang and Chen, 1996). The
copper was found to be greater in amount in the fish
tissues during the present research study and can be
attributed to the fact that it has a tendency to
accumulate to a greater extent than other essential
elements, such as zinc and iron (Heath, 1987;
Roesijadi and Robinson, 1994). Fish are naturally
exposed to a variety of metals including both
essential and non-essential elements. Copper is one of
the essential metals that after absorption from gills
and intestines is transported by metallothionein into
the blood circulation and some of it accumulates in
different internal organs specially liver and kidneys
(Peyghan et al., 2003).
It is generally accepted that metal accumulation in
tissues of aquatic animals is dependent upon exposure
concentration and period as well as some other factor
such as salinity, temperature, interacting agents and
metabolic activity of tissue in concern. Similarly, it is
also known that metal accumulation in tissues of fish
is dependent upon the rate of uptake, storage and
elimination (Health, 1987; Langston, 1990; Roesijadi
and Robisnson, 1994).
In terms of zinc toxicity, the concentrations of the
metal within certain tissue may be associated with
mortalities (Zitko, 1979) and sub lethal effects such
as behavioral and physiological disruptions (Buikema
et al., 1982). The analysis of the zinc in different
tissues of fish hosts observed in the present study
during different seasons showed higher concentration
in summer. These observations are similar to findings
of Velcheva (2006), who reported higher zinc content
in summer and autumn than spring and winter in the
water and fish tissue of both Kardjali and Studen
Kladenets dam lakes in Bulgaria. Other studies have
shown that zinc possesses affinity to protein
sulfhydryl groups and its increased load in the
kidneys and liver lead to a release of a specific metal
protein, metallothioneine from these organs (Cosson,
1994; Vilella et al., 1999).
Fish acquire iron predominantly from the diet and its
uptake varies in different organs (Andersen, 1997;
Bury et al., 2001). The highest concentration of iron
in liver observed in the present study can be attributed
to the fact that liver is the main storage pool for iron
in fish (Van Dijk et al., 1975; Walker and Fromm,
1976). Further studies have shown that liver, which is
a major producer of metal-binding proteins, show
high concentration of metals (Roesijadi and
Robinson, 1994; Allen, 1994).
Manganese, which is required in trace amounts by the
fish hosts, was found to be predominant in summer
followed by autumn, spring and winter in both fishes.
Excess external concentration of manganese in the
medium could lead to high internal levels and thereby
interfering with enzymatic activity or other metabolic
functions (Gonzalez et al., 1990). However, its
concentration was found to be lower than other
observed metals viz. copper, iron and zinc. This can
be attributed to the fact that fish can regulate the
amount of manganese in their body (Kwasnik et al.,
1978).
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