Global Advanced Research rch Journal of Environmental Science and Toxicology (ISSN: 2315

2315-5140)
5140) Vol. 2(1) pp. xxx-xxx,
xxx January, 2013
Available online http://garj.org/garjest/index.htm
Copyright © 2013 Global Advancedanced Research Journals

Full Length Research Paper

Haematological approach in mice to assess the impact

of pollution
P. B. Reddy1 and Deepali Amb2
1
PG Department of Zoology, Govt.PG Arts and Science College, Ratlam
2
PG Department of Zoology
Zoology, Govt.PG College, Nimach, India
Accepted January 22, 2013

The use of biomarkers has become attractive and useful for monitoring environmental quality and
health of organism inhabiting polluted ecosystems. Among them, blood biochemical parameters could
be a promissory indicator of health condition. The present investigation
estigation is aimed to study the
haematological changes of Musculus albinus as biomarkers in combination with chemical analysis of
the water from different sites along the whole course of the River Chambal at Nagda. Water analysis
showed that many attributestes of the water quality, in particular in the downstream were far beyond
admissible levels. For the present experiment 30 healthy six months old Swiss albino male mice
Musculus albinos weighing (30+2 gm) were used. The animals were allowed to drink the river
ri water from
different stations during a 96 hours period. It is clear from the results that the water quality induces
changes in the values of many hematological parameters. The main hematological alteration results
includes significant decrease in haematocrit and hemoglobin concentration, and no significant decrease
in red blood cell counts
ts and erythrocyte sedimentation rates in the mice of Group II. Plasma glucose
levels decreased in the fish of both Station 2 and 3. The hematological indices of MCHC, MCH and MCV
were also followed similar trend indicating that the Chambal River is contacontaminated
minated which induced
several haematological alternations.

Keywords: pollution, biomarkers, stress, Chambal River

INTRODUCTION

Water pollution is known to be detrimental to human
health and aquatic ecosystems, since surface waters are
used both as a source of drinking water and for
agricultural, recreational and religious activities around
the world (Ohe et al., 2004). Water pollution is most
commonly associated with the discharge of effluents from
sewers or sewage treatment plants, drains and factories
*Corresponding Author's E-mail: reddysir@yahoo.co.in
to the water body of rivers, seas and marines. In the
attempt to define and measure the presence and effects
of pollutants in rivers and oceans, the biological markers
have attracted a great deal of interest (Reddy and
Renusingh, 2011, Reddy and Baghel, 2012).
2012)
Biomarkers in different animals have been used within
environmental
vironmental monitoring programs to estimate the deg-
deg
radation of aquatic ecosystems (Rand, 1995; Seriani et
al., 2009). Hematological changes in animals including
human may be used for assessing the t effects of
contaminants, because blood parameters respond to low
doses of pollutants (França et al., 2007, Seriani et al.,
2009, Seriani et al., 2010). The use of haematological
and biochemical biomarkers in aquatic organisms is very
important in order to address the broad spectrum of
industrial, agricultural, commercial and domestic
chemicals entering the environment and especially the
aquatic environment and being taken up into the tissues
of aquatic organisms. Like in man, changes in the blood
parameters of mice, which occur because of injuries or
infections of some tissues or organs, can be used to
determine and confirm the dysfunction or injuries of the
organs or tissues.
In this context, the present study evaluated the quality
of the water of Chambal River at Nagda (M.P) through
the analysis of physical-chemical parameters of the
water, and the analysis of blood parameters in of
Musculus albinus.
MATERIAL AND METHODS
Study area
Nagda is very close to tropic of cancer at 23’27N and
75’25 and 517 meters above MSL.Nagda is a city and
municipality in Ujjain district in the Indian state of Madhya
Pradesh. River Chambal receives water from different
Industries and sewage from Nagda town. Waste after
coming from the factory complex runs in a channel for
about 3km and joins River Chambal near Juna Nagda.
Description of Study stations
Station 1
This station is located at upstream at Methwasa village.
Human activities are reduced here to bathing and fishing.
This station was taken as the reference station (control)
owing to the absence of discharge coming into the River
from industries.
Station 2
It is located near Chamunda mata temple. It is close to
Nagda town and 4 km away from station 1.Motor vehicles
are constantly washed. Also devotees of Goddess
immerse their pantheon (pooja material) directly into the
River.
Station 3
This station is located near Mukteswar temple near Juna
Nagda. The discharges of industrial complex and
domestic waste are drained into this station. It is poorly
vegetated. It is about 2 km away from station 2.
Experimental Design
For the present experiment 30 healthy six months old
Swiss albino male mice Musculus albinos weighing (30+2
gm) were procured from the veterinary college, Mhow.
These mice were acclimated for two weeks before
experimentation in well-ventilated animal cages (270 X
220 X 140mcm.) and under standard laboratory
conditions (photoperiod and temperature). The acute
toxicity of industrial effluent to mice was determined using
a standard 24-h static renewal technique. (USEPA, 1987)
and it was found to be 35%. The animals were randomly
divided into three groups that receiving water from three
different sampling sites of the Chambal River (five
animals per group) and one control group (n = 4). River
water samples were administered orally (as drinking
water; Horst et al., 2010) for a 96 hours period to animals
in the treatment groups. The control group received
dechlorinated tap water. All animal groups were fed a
standard laboratory diet, kept under the same
temperature conditions (25 ± 1 ºC). After 96 hours of
treatment, the animals were sacrificed and blood was
collected for haematological study.
Water analysis
In the three selected sites, water samples were collected
in winter (December 2010). The water samples were
collected in plastic or glass bottles at about 10 cm below
the surface. All collected water samples were refrigerated
at 4 ºC, transported to the laboratory and analyzed within
24 hours, as is required for the analysis of non-preserved
samples according to APHA (1998). The following
parameters were assessed, based on the Standard
Methods for the Examination of Water and Wastewater
(APHA, 1998): pH, chemical oxygen demand (COD),
conductivity, chlorides, hardness, total dissolved solids,
nitrite, turbidity, dissolved oxygen (DO), and BOD.
Haematological studies
After termination of the experiment the mice were
sacrificed and blood was collected into a sterilized tubes.
At least three replicated were taken in clean sterilized test
tubes. Blood samples transferred to tubes containing
ethylenediamine tetra acetic acid -potassium (EDTA-K2)
as an anticoagulant.
1. Erythrocytes (RBC) were counted immediately after
blood collection in hemocytometer (Improved Neubauer.
Weber scientific Ltd.) according to Wintrobe (1934).To
measure hematocrit (Hct), ammonium heparinized
haematocrit capillary tubes were filled with blood and
centrifuged for 5-min at 5000 x g in a micro capillary
centrifuge. The percentage of haematocrit was
determined by the use of a micro capillary reader.
 Table 1. Summary of physico chemical parameters of Chambal River from different stations

S.No Parameter Station I Station II Station III

o
1 Temperature, C 22.1±.8 22.8±.0.6 22.4±0.2NS
2 pH 7.1±.0.2 8.8±.0.4 9.4±.0.6**
3 COD mg/l 12.1±.0.4 39.1±.1.3** 38.1±.1.1
4 BOD mg/l 1.0±.0.02 26.6±.1.7** 54.1±.3.4**
5 Total suspended solids, mg/l 16.1±.1.1 147.23±.14.2** 136.3±.8.9
6 Total Dissolved solids (TDS mg/l) 110.10±.8.6 402.31±.17.5** 389.15±.13.4
7 Electrical conductivity, (u mho/cm) 85±.7.3 190±.12.8** 290±12.6.
8 Hardness, mg/l 200±.12.4 800±.21.1** 982±23.4.
9 Dissolved oxygen, ml/l 7.2±.0.5 6.1±.0.11* 4.8±.0.31**
NS= Not significant, * P> 0.5, ** P> 0.01 significant

Table 2. Summary of different haematological indices of Musculus albinus

PARAMETER GI G II GII
Haematocrit% 39.1±.082 24.1±.011** 25.21±.072**
Hb.gm/100ml 14.5±0.6 10.1±0.33** 9.3±0.28***
RBC(million/ul) 3.62±0.22 1.24±0.08* 1.28±0.09**
WBC/10/L 4.4±0.11 5.12±0.31* 4.9±0.21NS
MCHC (%) 34.2±2.34 30.91±3.3 32.11±3.2NS
MCV (ug) 248.1±16.1 230 ±12.8 215.1±1.4**
MCH(g) 94.1±10.2 79.4±8.11NS 64.1±10.2**
Plasma glucose mg/L 282.1±6.54 200.11±8.1* 180.21±7.7**
NS= Not significant, * P> 0.5, ** P> 0.01 significant

Hemoglobin concentration was measured by the Hematological studies

cyanmethaemoglobin method (Larsen and Snieszko,
1961) using a commercially available kit (Span, India).
Mean cell hemoglobin concentration (MCHC), mean cell
hemoglobin (MCH), and Mean cell volume (MCV) were
calculated using the formulae mentioned by Dacie and
Lewis (2001).
RESULTS
Physico chemical parameters
A summary of physico chemical parameters obtained in
Chambal River for all the different stations are shown in
Table1. Also indicated are mean ± standard error values
and the maximum and minimum values for each
parameter ’T ‘test is used to detect a significant
difference among each station.
Water temperature from different study areas did not
show much variation. But it was observed that pH
fluctuated between 7.1 to 9.4 in all the stations. Only
Control samples ( Station1) have pH with permissible
limit, while other samples have higher pH values . The
EC and turbidity values were found high in station 2 and
3.But DO level much decreased in station 2 and 3. BOD
and COD values in experimental stations 2 and 3 were
high.
The Summary of various haematological indices of
Musculus albinus were shown in Table 2.The main
hematological alteration results includes significant
decrease in haematocrit and hemoglobin concentration,
and no significant decrease in red blood cell counts and
erythrocyte sedimentation rates in the mice of Group II .
(Table 2). Plasma glucose levels decreased in the mice
of both Group II and III. The hematological indices of
MCHC, MCH and MCV were also followed similar trend.
Results of the hemoglobin and haematocrit values in the
mice of Group II and III decreased significantly (P <0.01)
compared to those of the control. Though there was a
decrease in red blood cell and erythrocyte sedimentation
rate values of mice of Group II and III , but the decrease
was not much significant (P >0.05) compared to the
control fish. Similar trend was followed by WBC.
DISCUSSION
The physico chemical parameters exhibited considerable
variations from sample to sample. Water temperature is a
critical parameter for aquatic life and has an impact on
other water quality parameters such as dissolved oxygen
concentrations, and bacteria activity in water. It may be
due to fact that water has great specific heat capacity.
Hence radiation received by water body hardly brings
about serious fluctuations.
pH affects many chemical and biological processes in
the water and this is important to the survival and
reproduction of fish and other aquatic life. These results
are in accordance with the results of (Nosheen et al,
2002), who reported that textile wastes are highly
alkaline. pH effluents affects physico chemical properties
of water which in turn adversely affects aquatic life, plants
and humans, This also changes soil permeability which
results in polluting underground resource of water(Rump
and Krist, 1992).
The EC values were found high in station 2 and 3 may
be due to dissolved salts present in the effluent.
Conduction is function of water, hence a standard
temperature usually25’C is specified in reporting
conductivity. Higher turbidity increases water
temperatures because suspended particles absorb more
heat. This, in turn, reduces dissolved oxygen (DO)
concentrations because warm water holds less DO than
cold water. Higher turbidity also reduces the amount of
light that can penetrate the water, which reduces
photosynthesis and DO production. The influx of
industrial effluents significantly leads to increase in total
dissolved solids in station 2 and 3. TDS value of majority
of the samples is much higher than the permissible limits
which predicts the presence of excess materials and
dissolved matter in textile effluent. These values are very
close to the findings of Francis et al (2008).High TDS are
one of the major source of sediments which reduce the
light penetration into water and ultimately decrease the
photosynthesis. Consequently it reduces the DO level
(Tyagi and Mehra, 1990). Earlier studies from Reddy and
Baghel (2012) showed high loads of domestic and
industrial sewage in the lower reaches of the Chambal
River.
Both, BOD and COD values in experimental stations 2
and 3 were high. In addition, the high levels of TDS and
suspended solids in the water systems increased the
BOD and COD, which depleted DO in the water
ecosystem. The levels of TDS in broad sense therefore
reflect the pollutant burden on the aquatic system. It
shows that effluents have highly oxygen demanding
waste (Kumar, 1989) which causes the depletion of DO
which is a fundamental requirement for aquatic life.
Results clearly indicate that water samples from all
experimental stations including upstream were showed
high concentrations of total hardness. In the present
study water was very hard and crossed the permissible
limits. It is well known that hardness is not caused by a
single substance but by variety of dissolved ions,
predominantly calcium and magnesium. The high
concentration of total hardness in water samples may be
due to dissolution of ions from sediment rocks, seepage
and run off from soil. In the present study, total hardness
was positively correlated with chloride, calcium
and magnesium (Reddy and Baghel, 2012). The strong
correlation ship between these parameters could be due
to climate factors or industrialization.
The hematological indices of mean corpuscular
volume and mean corpuscular hemoglobin content (MCV
and MCHC) were also decreased but were not
statistically significant (P >0.05). The mean corpuscular
hemoglobin (MCH) decreased in the mice of both
experimental groups but the decrease was only
significant (P <0.05) in Group II.
Studies have shown that when the water quality was
contaminated by toxicants, any physiological changes will
be reflected in the values of one or more of the
hematological parameters (Nussey et al., 1995). In the
light of the present study, the significant decrease in the
HCT observed in collected mice could be attributed to the
destruction of the erythroblast, thereby, limiting their
synthesis. Similar trends in erythrocytes in fishes
exposed to various toxicants and pathogens have been
observed by other researcher (Mc Leafy, 1975; Smit et al.
1979; Koyama and Ozaki, 1984; Srivastava and Narain,
1985; Van der Merwe, 1992).
The MCV gives an indication of the status or size of
the erythrocytes and reflects an abnormal or normal cell
division during erythropoiesis. On the other hand, the
decrease in MCV that observed in this study after
infection coupled with low hemoglobin content indicated
that the erythrocytes have shrunk, either due to hypoxia
or a microcytic anemia. Thus, microcytosis might be due
to the decrease in the haematocrit during exposure.
Similar pattern has been detected in fish (Labeo
umbratus) after exposure to various pollutants (Nussey et
al., 2000).
In this study the fluctuation in the MCH was observed
but MCHC had increment .It was indicated that the
concentration of hemoglobin in the red blood cells were
much lower in the infected mice than in the control group,
thereby, depicting an anemic condition. The macrocytosis
is probably an adaptive response through the influx of
immature erythrocytes from the hematopoietic tissues to
the peripheral blood to make up the reduced erythrocytes
number and decreased hemoglobin concentration
(Rehulka et al., 2005).
These findings further support the hypothesis that
hemodilution is a probable cause for decrease in
hemoglobin content in experimental mice. The MCHC is
a superior indicator of erythrocytes swelling (Wepener et
al., 1992). The MCHC, which is the ratio of blood
hemoglobin concentration as opposed to the haematocrit,
was not influenced by the blood volume nor by the
number of cells in the blood but could be interpreted
incorrectly only when new cells, with a different
hemoglobin concentration, were released into blood
circulation (Sovlo et al., 1981). The significant decrease
in the MCHC in this study was an indication of
erythrocytes swelling and/or due to a decrease in
hemoglobin synthesis. Buckley et al. (1976) repor-
ted that prolonged reduction in hemoglobin content was
deleterious to oxygen transport and any blood dyspraxia
and degeneration of the erythrocytes could be attributed
as pathological conditions in animals exposed to
toxicants or infectious diseases.
The incorporation of ecologically relevant biomarkers
into routine environmental management programs has
been advocated as a practical means of linking
environmental degradation with its causes (Galloway et
al., 2004). Humans, aquatic life and other living
organisms are constantly exposed to contaminants from
factories, refineries, sewage treatment plants, fertilizers
and pesticides washed into rivers, or urban water
supplies. Therefore, the assessment of the effects of
contaminated water on blood indices of mice is of great
importance for a possible prediction of such effects on
humans. The use of a group of biomarkers covering
different levels of biological organization provides
knowledge about at what level pollutants interact with the
body and at what level the body is more susceptible to
the action of pollutants. The results of studies such as the
present one are essential to the design of effective
strategies targeting the rehabilitation of biodiversity in
aquatic ecosystems.
Acknowledgements - This study was supported in part
by a research grant from UGC, CRO, and Bhopal to
Dr.P.B.Reddy (UGC, code-107011, F.No. MS-
38/107011/CRO).
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