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J. Env. Bio-Sci., 2019: Vol. 33 (2):235-242 ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

SUSCEPTIBILITY OF DIFFERENTIAL LEUCOCYTE COUNTS OF THE FRESH WATER

CATFISH , CLARIAS BATRACHUS TO LEAD
V. K. Verma1, N Gupta2* and D. K Gupta3
1.
Department of Environment and Science, Future Institute of Engineering and Technology, Bareilly
2.
C.S.J.M. University Kanpur, U.P. (India) *
3.
Department of Biotechnology, Bareilly College, Bareilly
[Corresponding Author Email*: guptagrawal@rediffmail.com]

Received: 11-04-2019 Accepted: 02-12-2019

Expansion of industrial and domestic activities has resulted in increasing heavy metals deposition in natural water. The presence
and loading of heavy metals deposition in water is a serious issue with respect to environmental pollution. The present
communication provides a brief account of the lethal effects of lead (Pb) on Differential Leucocyte Count (DLC) of the fresh water
catfish, Clarias batrachus. Acclimatized fish were treated with 0.5 ppm (Group B), 1.00 ppm (Group C), and 3.00ppm (Group D), of
Pb during 7, 14, 21 and 28 days time interval and their values compared with the control (Group A) group. Significant changes in DLC
were observed in all concentrations of lead. The small lymphocytes were found to decrease in lead treated groups as compared
to the control ones. The lowest decrease in the numbers of small lymphocytes was 36% during 28 days exposure in Group B and
C. Large lymphocytes reduced significantly to 22% during 28 days exposure in Group D. The neutrophils significantly increased in
all the exposure groups reaching to a maximum of 28% during 28 days exposure of two groups (Group C and Group D). The value
of monocytes also increased in all treated groups during final exposure time (28 days) showing the same value (6%) but the
maximum number of monocytes (8%) were observed in Group C during 21 days time interval. Maximum number of eosinophils (6%)
were recorded in Group B (21 and 28 days) while the numbers of basophils observed also showed minor fluctuations. The results
of the present study indicate that Pb is highly toxic and causes undesirable effects on the haematological profile of living fish.
Maximum 166.6 percentage increase was found in monocytes exposed in Group C treated with 1.00 ppm of Pb for 21 days
exposure period.
Key words: Clarias batrachus, leucocytes, lead, pollution
Study of several researches have shown that heavy metal 2001; Godwin et al., 2003; Osman et al., 2007).
toxicity caused several haematological and biochemical
Blood parameters have been commonly used to observe and
changes in animals in vivo as well as in vitro conditions. The
follow fish health, since variations in blood tissue of fish are
toxicity of heavy metals have been shown by several workers
caused by environmental stress (Shah and Altindag,
during the last decade (Adeyemo et al., 2010; Abedi et al.,
2005).Various workers have shown that the use of
2013; Hedayati and Ghaffari, 2013; Thangam et al., 2014;
haematological parameters as indicators of metal toxicity can
Moosavi and Shamushaki, 2015; Mehta, 2017; Kaur et al.,
provide information on the physiological response of fish due
2018). Among all heavy metals, lead is the most important
to the close association of the circulatory system with the
toxic heavy element in the environment. Due to its important
external environment (Gupta et al., 2002; Adyemo et al., 2010).
physio-chemical properties, its use can be retraced to historical
Fish live in very intimate contact with their environment, and
times. Globally, it is an abundantly distributed, important yet
are therefore very susceptible to physical and chemical changes
dangerous environmental chemical (Mahaffey, 1990; Gabriel
which may be reflected in their blood components (Wilson
et al., 2007). Several reports have indicated that Pb can cause
and Taylor, 1993). Furthermore, haematological indices are of
neurological, hematological, gastrointestinal, reproductive,
different sensitivity to various environmental factors and
circulatory, immunological, histopathological and
chemicals (Lebedeva et al., 1998; Vosyliene, 1999a,b). Several
histochemical changes, all of them being related to the dose
researches have focused on adverse effects on fishes (Adakole,
and time of exposure to Pb (Mirhashemi et al., 2010). Fish
2012; Ikeogu et al., 2013; Tenekhy, 2015; Mohiseni et al.,
accumulate toxic chemicals such as lead nitrate directly from
2016; Raja and Puvaneshwari, 2017; Samual et al., 2018).
water and diet, and contaminant residues may ultimately reach
Inspite of recent interest in the immune systemof fish species,
concentrations hundreds or thousands of times above those
very little attention has been paid to the structural features of
measured in the water, sediments and food (Labonne et al.,
NAAS Rating (2019)-4.43
 VERMA, GUPTA AND GUPTA
fish blood cells (Esteban et al., 2000).The use of immune
system parameters in order to assess alterations in fish
experiencing heavy metal exposure and the interest generated
in defense mechanisms stem from the need to develop health
management tools to support a rapidly growing aquaculture
industry (Adyemo et al., 2010, Dutta et al., 2015). The aim of
present study was to evaluate the sublethal effects of lead on
the alteration in nuclear morphology of leucocytes of the fresh
water catfish Clarias batrachus.
MATERIALS AND METHODS
Clarias batrachus (n= 64) were collected from different fish
markets of Bareilly district and acclimatized for 7 days to
laboratory conditions. 16 healthy and large sized fishes
(weighing 30-80 gms) were selected for each experimental
group. One control and 3 chronic levels of lead-exposed groups
were designed for the experiments as follows:
Group 'A'-Control group.
Group 'B'-Exposed to low concentration (0.5ppm) of Pb
Group 'C'-Exposed to intermediate concentration (1.00ppm)
of Pb
Group 'D'-Exposed to high concentration (3.00ppm) of Pb
Thin blood smears were prepared on clean slides, immediately
air-dried and fixed in methanol for 2-5 minutes. The films were
then stained with Giemsa (Qualigens) in phosphate buffer
solution (pH 6.8), the solutions were mixed uniformly in the
ratio of 1:7 by means of a soft brush, stained for 40 mins and
washed in running tap water for 2-4 mins to drain the excess
stain. They were allowed to stand on end to dry and mounted
in DPX.The cells were counted using high power microscope
in a strip running along the whole length of the film by means
of manual / electronic blood cell counter. At least 100 cells
were counted and the leucocytes differentiated by the
morphological characteristics of the nucleus and characterized
as small lymphocytes, large lymphocytes,
monocytes,neutrophils, eosinophils and basophils according
to Wintrobe (1981).
RESULTS AND DISCUSSION
Significant changes in different leucocyte counts (Figs 1-6)
were observed in all concentrations of lead. The small
lymphocytes decreased in lead treated groups as compared
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to the control ones. The lowest decrease in the numbers of
small lymphocytes was 36% during 28 days exposure in Group
B and C. Large lymphocytes reduced significantly to 22%
during 28 days exposure in Group D. The maximum percentage
fell to 13.6% during 28 days exposure period in Group B and
C in small lymphocyte count (Fig. 7) and 33.3% during 28
days exposure period in Group D in large lymphocytes counts
(Figs. 2,8).
The neutrophils significantly increased in all the exposure
groups reaching to a maximum of 28% during 28 days exposure
of two groups (Group C and Group D) and the maximum (75%)
changes occurred in Group C during 28 days exposure period
(Fig. 4, 9). The value of monocytes increased in all treated
groups during final exposure time (28 days) showing the same
value (6%) but the maximum number of monocytes (8%) was
observed in Group C during 21 days time interval, the
percentage change rose to 133.33% during 21 days exposure
period in Group D (Fig. 3, 10). Maximum numbers of
eosinophils (6%) were recorded in Group B (21 and 28 days)
(Fig. 5, 11). The number of basophils also showed minor
fluctuations. The percentage changes were 100% in all the
groups (B, C and D) after 28 days exposure to Pb (Fig. 12.)
(Table 1).
Water pollution has become a global problem, the heavy metals
have long been recognized as serious pollutants of the aquatic
environment, they can enter water bodies through
anthropogenic activities like mining, industrial, agricultural and
domestic discharge in streams, ponds, lakes, river and ground
water (Obasohan et al., 2008). Pb is not necessary for
biological functions of living organisms even at low
concentration. In the 18th and 19th century, lead was illegally
added to wine both as a sweetener to make it appear fresh.
(Mai, 2006). Its poisoning is believed to be primarily responsible
for the collapse of the Roman Empire,in which lead acetate
was used in wine and its prolonged use was considered to
have caused as dementia, it also causes anaemia (Cohen et
al. 1981). Now it is being discharged in aquatic systems mainly
from petroleum, dye, paint, mining and other human activities,
which have toxic effects and can bioaccumulate as well as
biomagnify (Sorenson, 1991; Heath, 1995). Hematological
changes have been considered to be good indicators of fish
health and are strongly related to the way fish respond to
environmental variations, thus they constitute an ideal tool for
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Table 1: Differential leucocyte counts of C. batrachus exposed to various concentration of Pb (Lead)

Sl. No GroupA GroupB GroupC GroupD

DLC (Control) (0.5ppmPb) (1.00ppmPb) (3.00ppmPb)
%
7 14 21 28 7 Days 14 21 28 7 14 21 28
Days Days Days Days Days Days Days Days Days Days Days
1 Small 44 42 40 37 36 40 39 37 36 41 39 38 38
Lymphocytes
2 Large 33 30 29 27 26 31 27 25 26 30 27 24 22
Lymphocytes
3 Neutrophils 16 19 20 23 25 20 22 25 28 20 22 25 28
4 Monocytes 3 4 5 5 6 4 6 8 6 5 6 7 6
5 Eosinophils 3 5 5 6 6 5 5 3 3 3 4 5 5
6 Basophils 1 0 1 2 1 0 1 2 1 1 2 1 1
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(239) SUSCEPTIBILITY OF DIFFERENTIAL LEUCOCYTE COUNTS OF THE FRESH WATER CATFISH
 VERMA, GUPTA AND GUPTA
in vitro or in situ toxicological studies (Al-Akel and Shamsi,
2000; Hamidipoor et al., 2015; Seriani et al., 2015). The
differential counting of blood cells is considered to be a suitable
tool for assessing the health status of fish, and this approach
has been employed in fish farms after drug administration and
parasite infestations, as well as for other purposes (Dias et
al., 2011; Alabbasi et al., 2017). It is believed that neutrophils
and monocytes have phagocytic stir which might elucidate
their increased proportion during infectious condition.Darvish
et al. (2001) observed that neutrophils and monocytes
increased in channel catfish exposed to KMnO4 solution and
our findings fall on similar lines.
The differential counts (Rey and Guerrero, 2007; Tripathi, 2014)
were examined at different lead concentrations and time
intervals. All differential leucocytes were affected due to toxicity
of exposed heavy metal, both the lymphocytes decreased at
maximum concentration during exposure period. Shah (2006)
on Tinca tinca and Adyemo (2007) on Clarias batrachus
observed that the immune system weakened due to decrease
in leucocytes but differential leucocytes like lymphocytes
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percentage increased and neutrophil values decreased
corresponding to the increase in the concentration of lead.
Mastan et al. (2008) and Adeyemo et al. (2010) also held
similar views on lead-exposed fish, the differential leucocyte
count deviating significantly from the normal values,
lymphocytes and eosinophils increased while neutrophils and
monocytes of Clarias batrachus and C. gariepinus decreased.
When Rutilus rutilus were exposed to lead and cadmium for a
period of 4 days, Pb reduced lymphocyte percentage and
neutrophils and monocytes increased while the cadmium
elevated the lymphocyte values (Akbari, 2010). Furthermore,
Witeska et al. (2010) also observed the that lead suppressed
the immune system of fish. The results of Hedayati and Gaffari
(2013) proved that the neutrophils and monocytes raised while
lymphocytes decreased. Khalesi et al. (2017) studied the lethal
effects of Pb and Cd on common carp, remaining unchanged
in lymphocytes values but neutrophil percentage showed a
more significant rise in response to both the heavy metals.
CONCLUSION
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Fig. 1: Blood film of Clarias batrachus showing small lymphocytes, Fig. 2: Blood film of Clarias batrachus showing
large lymphocytes, Fig. 3: Blood film of Clarias batrachus showing monocyte,

Fig. 4: Blood film of Clarias batrachus showing neutrophils, Fig. 5: Blood film of Clarias batrachusshowing eosinophils,
Fig. 6: Blood film of Clarias batrachus showing basophils.