CHAPTER-5

HAEMATOLOGICAL STUDY
Introduction:

Haematology is the study of blood and is concerned primarily with the study

o f erythrocytes or red blood cells (RBC), leucocytes or white blood cells

(WBC) and thrombocytes and platelets. Haematological study reports the

enumeration of cells in circulation. The haematological parameters are altered

with the induction o f insecticides in the body. (Verma et al.,1979). Study of

the stained blood smear also helps in detection of morphological abnormalities

of various cells seen in the peripheral blood circulation. Blood is considered as

the important and sensitive indicator of any toxic effect and physiological

stress of various xenobiotic chemicals (Mishra and Srivastava;1983).

All the red blood cells are more or less of same size with smaller nucleus.

Mature erythrocyte is a round biconcave disc. Though the colour of the RBC

is yellow, due to the presence of haemoglobin, these are seen as red.

Haemoglobin is the red blood pigment exclusively found in erythrocytes, is

responsible for transportation o f oxygen from the lungs to all the cells of body

for tissue respiration. Heme is the most important porphyrin containing

compound, which is synthesized in the liver and erythrocyte producing cells of

bone marrow (erythroid cells). A decrease in haemoglobin concentration in

blood below normal values is a sign of anaemia.

Leucocytes are typical cells with amoeboid ability. These are divided into two

types, such as granulocytes or polymorphonuclear leucocytes and

agranulocytes or mononuclear lymphocytes. Further more the granulocytes are

63
divided into basophil.neutrophil and eosinophil and agranulocytes are divided

into lymphocytes and monocytes.

The fish is the first vertebrate, where a closed blood vascular system evolved

along with the different types of blood corpuscles (Joshi; 1973).Piscine

haematological studies are gaining momentum, all over the world almost

parallel to increase fresh water aquaculture at all commercial level

(Joshi;2005). Haematological parameters of blood are the major indicator of

stress condition of the fish, which are affected by the insecticides.

In most cases o f physical or chemical stresses to these fresh water fishes, the

basic haematologic value of TEC, TLC, Hb and PCV show a sudden rise in

the beginning, which usually tends to subside in the course of time, or that

these values may exhibit intermittent fluctuations. This state simply reflects

the internal struggle of various organs which they undergo to maintain the

homeostatic status in the internal milieu of the whole system and the unstable

hematological picture is just the reflection of the same. Haematological

parameters, apart from the serological parameters are now also being used in

the monitoring of the ambient water quality (Joshi; 1973, Joshi; 1992). Debral;

1983 and Sharma; 1984 revealed that the blood corpuscular size, stage of

maturity and their staining property are among the simplest parameters which

help under specific conditions of aquatic pollution. Thus fish blood shows

immediate impact to any sort of eco-biological stresses, whether it is occurred

under natural conditions or under experimental studies.

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Review of literature:

Few studies were conducted on biochemical and haematological changes of

several fish species after the treatment of a numbers of insecticides and heavy

metals by Shammi et a l, 1978, Verma et a l, 1979, Pandey et a l, 1979,

Srivastava and Sriwastwa; 1980, Dalela et al., 1981 and Qayyum and

Shammi; 1983, Chauhan et ah, 1983, Rai and Qayyum; 1984, Dick and

Dixon; 1985, Srivastava and Mishra; 1985, Lowe-Jinde and Niimi; 1986, Hase

et a l ,1987, Chakrabarty and Banerjee; 1988, Sharma.; 1991, Mohan; 2000,

Manoj et al., 1999, Sikic et a l,2001. They reported that insecticides and heavy

metals were very toxic to the fish species and induced changes in biochemical

and haematological parameters.

Pandey, et al., 1976 reported polycythemia due to urea stress in fish. Pandey et

a l, 1979 also reported that DDT, metacid and unizeb cause anemia whereas

treatment of endrin, urea and phenol brings about polycythemia. Srivastava

and Sriwastava;(1980) observed the cellular and nuclear hypertrophy, change

of shape, agglutination and bursting of erythrocytes under 500 pm urea stress

in C.mrigala fingerlings.

Panigrahi and Mishra; 1978 assessed the toxicological impact of Hg on fresh

water fish Anabas scandens and observed pathological, biochemical and

haematological alteration after the treatment with a sub-lethal dose. Srivastava

and Narain;1988 noted the haematological responses of H.fossilis to pollutant

fertilizer and pesticides.

65
In Clarias batrachus, diptarex intoxication brings about changes in

erythrocyte count and Hb concentration (Verma et al, 1979). Quyyum et ah,

1982 reported that RBC, WBC, PCY and Hb increased with increasing the

treatment period while ESR and clotting time were decreased in

Saceobranchus fossils. The effects of thiotox, dichlorvus and carbofuran on

certain organic and inorganic components in the blood of Mystus vittatus have

showed severe disturbance which ultimately alter the physiology of fishes

(Dalela et al., 1981).

Morphological changes in fish red cell under xenobiotic poisoning in fishes

exposed to chlorine, copper, lead and other pollutants were reported by

Buckley et al.,1976; Singh and Singh;1982; Hardig et n/,,1988; Sordyl;1990;

Vincent etal., 1996.

Goel et ah, 1981 studied the different haematological parameters and observed

change in RBC, WBC count, haemogloibin content, alkaline phosphatase and

amylase activity of the blood of H.fossilis exposed to malathion. Srivastava

and Mishra (1985) investigated the effect of malathion on blood parameters of

Hfossilis and reported erythropenia. Panigrahi et al., 1984 evaluated the toxic

effect of emisan-6 on the erythrocytes morphology in Anabus scandens.

Lowe-Jinde and Niimi (1986) observed decreasing trend of erythrocyte count

and increase in leucocyte count in Salmo gairdneri on exposure to chromium

and reported that these changes may be associated with immune response.

Similar observation was also sited by Dutta et al.,(1992) in malathion exposed

66
H.fossilis. Erythropenia was also observed by Dhembare and Pondhe (2000) in

Punctius sophore in malathion treatment.

Srikanta; 2007 noticed the changes of hematological parameters of fish. The

hemoglobin content, packed cell volume, mean corpuscular hemoglobin

concentration decreases significantly, while mean corpuscular volume and

lymphocyte count increases.

The above cited literature showed that the pesticides and different chemicals

had adverse effects on haematological parameters in different fish species.

Though various works on haematological parameters have been going in all

over the World but the effect o f endosulfan on Channa punctatus is still very

scanty. So these haematological parameters were included for the study after

96 hr of exposure to the endosulfan.

Methods:

Collection of blood samples:

After 96 hr of exposure to insecticides blood was collected from the heart and

caudal peduncle of fishes in 5 ml graduated syringe o f all the three groups,

causing minimum stress to all the fishes. Immediately blood was transferred in

to small vials containing EDTA as anticoagulant for determination of

Haemoglobin percentage, total RBC count, WBC count and differential count

of WBC. The study of WBC, RBC, Haemoglobin percentage were carried by

different standard methods and experiments were repeated thrice and data

67
were statistically analyzed along with student’s‘t’ test for the significance

(Bailey,1965).

Estimation of Haemoglobin percentage:

Haemoglobin was estimated by the method of acid haematin method, which

was also known by Sahil method.

Principle: The method o f estimation is based on the principle of making an

acid haematin solution of blood in the graduated tube and then comparing it

with the sealed comparison tube containing the standard acid haematin.

Procedure: The graduated tubes were first cleaned with distilled water and

thoroughly dried up before used. Then with the help of a dropper the N/10

HC1 solution was filled in the graduated tube up to 2 gm mark and with the

help of a micropipette fresh blood from the fishes were sucked up to the mark

of 20 cmm. The micropipette was wiped off very carefully with sterilized

cotton and the blood was added to the N/10 HC1 solution in the graduated

tube. The pipette was introduced in to the graduated tube very carefully so that

its lower mouth passed right up to the bottom in to HC1 solution. Then the acid

haematin solution was stirred thoroughly with the help of a glass rod and then

allowed to stand for 10 minutes. After that the acid haematin solution was

gradually diluted by adding distilled water in a drop wise manner so that its

colour was matched with that of the standard sealed tube. This was continued

till the colour of acid haematin solution just faded away as compared to the

standard comparison tube. Then the reading was taken.

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Calculation: Experiment was carried on three times for each sample and

recorded the reading. After that average was taken as a result. The reading was

taken as % of normal or in g/100 ml.

Determination of total RBC count: Total RBC count was calculated by

using haemocytometer. Hayem’s solution was used as RBC diluting fluid to

fix the RBC in the blood and to stop haemolysis.

Procedure: Collected blood from the fishes of the different experimental

groups were sucked in the RBC pipette up to 0.5 mark taking care so that air

bubbles could not entered in the pipette. The pipette was kept horizontally and

carefully wiped the outside o f the pipette with absorbent paper. Then the

pipette was transferred to the container of hayem’s solution which was

carefully sucked up to 101 marks. The dilution of blood was about 200 times.

Then the pipette was kept between the fore finger and thumb of the hand and

rotated several times for thorough mixing with the hayem’s solution. The red

bead in the pipette helped in mixing. After that for counting RBC, the glass

counting chamber which was cleaned with alcohol was covered with

coverslip. The coverslip was supported upon the side platform but remain

separated from the central platform by a distance o f 0.1 mm. 2-3 drops of

mixture was rejected. Then few drops of blood mixture were allowed to flow

in the narrow space between the coverslip and the counting chamber with the

help o f the tip o f the pipette between the coverslip and the platform. Blood

mixture remained filled up between the coverslip and the counting chamber

69
due to capillary action. When the counting chambers were properly filled with

blood mixture, it was kept aside for few minutes to settle down the RBC and

observed under the microscope and counting was done without disturbing the

settled RBC.

Calculation was done only in five smaller chambers, i.e. in the 1st, 5th, 13th,

21st and 25th. The RBCs lying on the lower and right sides of a square were

counted but upper and left sides were rejected for reduced the error of double

counting.

Calculation:

M u t e of cells counted X dilution X4000

M u te s of EBGs in 1 cubic mm of blood
Number of small square counted

Determination of total WBC count: Total WBC count was calculated by

using haemocytometer. WBC diluting fluid, which helped to haemolyse the

RBCs present in the blood and stained the nuclei o f the WBC for clear

recognition.

Procedure: The procedure of WBC counting was similar to RBC except in

case of calculation. The WBC counting chambers were different from RBCs

counting chambers and the WBC pipette was also varying from that RBCs

pipette.

Calculation: Total numbers of WBCs present in 1 cubic mm of

,. . Numbers of cells counted _ .

blood = --------------------------------------- x Dilution
Numbers of 1 sq. mm. counted

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Differential count of WBC: Generally staining method was adopted to

determine the relative proportion of WBC in the different experiment groups

of fishes.

Procedure: Blood was taken in the slide and prepared a thin smear of it. The

slide was then kept for few minutes for drying and stained the blood film with

Leishman’s stain. After 1 minute, few drops of distilled water were added to

the slide and were mixed the stain with distilled water. After that a greenish

metallic colour was appeared on the surface of the slide. The slide was kept

for 10 minutes and again washed with distilled water until the rose pink colour

appeared. Then the slide was examined under the microscope.

Calculation: To calculate the percentage of different types of WBC, the

following formula was adopted.

Numbers of different types

---------------------------- of WBC ,
±r-------------- xlOO
Total Numbers of WBC

Results and Discussion:

The results of haematological parameters were calculated after 96 hrs of

exposure to endosulfan, shown in the table no-5.1 and 5.2 and graph no-5.1.

71
 Tablc-5.1: Effect of sub-lethal concentration of endosulfan on some selected

haematological parameters of Channapanctatus at 96 hrs exposure.

Parameters Normal Control Treated

H b percentage (gm/lOOml) 8.88±0.34 8.67±0.41 5.40±0.46

Total RBC count (Million/ m m 3) 3.59±0.14 3.33±0.11 1.82±.07

Total WBC count (Million/ 2009.6±161.09 2079.2±100.55 3000±189.74
m m 3)
Table No-5.2 '.Differential count o f WBC (%)

Groups Normal Treated

Control

Parameters
Neutrophils 47.00±3.74 47.33±4.93 25.00±3.60
Basophils 2.67±0.47** 2.33±.57** 2.66±057
Eosinophils 4.67±1.25** 4.33±.08** 5.00±0.57
Lymphocytes 39.00±1.41 39.30±1.52 59.6ftfcl.52
Monocytes 4.33±0.47 4.33±0.47 1.33±0.47

N =3,± = SD.
Significant p<0.05; ** = not significant.

The haemoglobin percentages were found in the normal and control groups of

fishes respectively 8.88±0.34 gm/lOOml and 8.67±0.41 gm/lOOml, which was

reduced to 5.40±0.46 gm/lOOml in the treated group o f animal (Table No-5.1

and Graph No-5.1). The total numbers of RBC in normal control and treated

groups were found as 3.59±0.14 million/mm3, 3.33±0.11 million/ mm3 and

1.82±.07 million/ mm3. The total WBC counts were calculated as

2009.6±161.09 million/ mm3, 2079.2±100.55 million/ mm3 and 3000±189.74

million/ mm3 respectively in normal, control and treated groups of fishes.

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Hb % (gm 100ml)

NOR CON TRT

Graph No. 5.1 : Comparison for Haemoglobin percentage

in normal, control and treated group of fishes.
(NOR : Normal CON : Control TRT : Treated)

Graph No. 5.2 : Comparison for RBC Count in normal, control

and treated group of fishes.
(NOR : Normal CON : Control TRT : Treated)
 G r a p h N o . 5 . 3 : C o m p a r i s o n f o r W B C C o u n t i n n o r m a l ,

c o n t r o l a n d t r e a t e d g r o u p o f f i s h e s .

( N O R : N o r m a l C O N : C o n t r o l T R T : T r e a t e d )

6 0

5 0

4 0
□ NOR
P e r c e n t a g e f . l 3 0 ■ CON
□ TRT
2 0

10

0
NEU BAS EOS LYM MON

G r a p h N o . 5 . 4 : D i f f e r e n t i a l C o u n t o f W B C ( % )

( N E U : N e u t r o p h i l , B A S : B a s o p h i l , E O S : E o s i n o p h i l

L Y M : L y m p h o c y t e M O N : M o n o c y t e )

( N O R : N o r m a l C O N : C o n t r o l T R T : T r e a t e d )
The result showed 8.88±0.34 gm/lOOml of haemoglobin content in normal

group, which is similar to 8.43±.47 gm/lOOml of haemoglobin content in

control group, was reduced to 5,33±.5 gm/lOOml in treated group of fishes that

led to anaemic condition of the fish. This was possibly due to haemolysis and

disruption in iron metabolism that led to the defective haemoglobin synthesis

caused by the insecticide. Workers like Richardson et a l, (1974), Kumuda et

al, (1980), Rai and Qayyum; (1984), Singh et al, (1990) and Patil and Jabde;

(1998), also put forwarded similar opinion in different fishes exposed to

different chemicals and pesticides

Anaemic condition might be occurred due to the impact of endosulfan as

Talwar and Srivastava;2006 reported that many drugs such as oxidant drug,

which is associated with intracorpuscular defect by inhibiting G6PD (glucose

6 phosphate dehydrogenase). Again in case of lead poisoning, lead interferes

with heme synthesis as it inhibit ALA( a amino levulinic acid) synthetase,

ALA dehydrase and heme synthetase, causing hemolysis.

Richardson et a l, 1974 reported that anemic condition appeared due to defect

in iron metabolism caused by deficiency in intestinal absorption caused by

cadmium induced in Japanese quail. Kumuda et al., 1980 reported the

accumulation of cadmium in various organs of rainbow trout, particularity the

kidney & liver. Later on, the activity of these haemopoetic tissues may be

suppressed.

Baruffalde and Cucchi;1989 observed the changes in RBC on exposure to

DDT. The chlorinated hydrocarbon group produced anemia in animals by

73
hindering delivery of oxygen to tissue due to oxidation of haemoglobin to

methehaemoglobin, (Grothe and Eaton; 1975, Zeitoun;1977).

Total numbers of RBC showed decreasing trend from 3.59±0.14, 3.33±0.11

million/mm3 in normal and control group to 1.83±.07 million/ mm3 in the

treated group (Table No-5.1 and Graph No-5.2). The decreased trend of RBC

might be due to the haemolysis and shrinkage of the blood cells by the toxic

effect of insecticide. Mount and Putnicki; (1966) observed erythropenia and

leucopenia in fish o f Mississippi river induced by the organochlorine

insecticide endrin poisoning.

In differential count o f WBC, the neutrophils percentages were 47.00±3.74,

47.33±4.93 and 25.00±3.60 respectively in normal, control and treated groups

(Table No-5.1 and Graph No-5.4). The basophils percentages were counted as

2.67±0.47, 2.33±.57 and 2.66±057 respectively in the three groups.

Eosinophils percentages were counted as 4.67±1.25, 4.33±.08 and 5.00±0.57.

The lymphocytes percentage were calculated as 39.00±1.41, 39.30±1.5 and

59.60±1.52 and the monocytes percentage were 4.33±0.47 4.33±0.47

1.33±0.47 respectively in normal, control and treated groups o f fishes.

In the present experiment the structure of blood cells were also studied and

some adverse effects were observed on the exposure o f endosulfan. The

changes include rupture of cell membrane and nucleus deformity as shown

Plate no No-5.2, 5.4 and 5.5. These results were also supported by the finding

of many workers. Srivastava and Sriwastwa; (1980) also observed cellular and

nuclear hypertrophy, change in shape, agglutination and bursting of

74
 PLATE 5.1 : Blood cells offish (Control) (X100)
A - RBC
B- WBC

PLATE 5.2 : Blood cells offish (96 hr. treated with endosulfan)
A - Rupture of RBC membrane
B-WBC
 PLATE 5.3 : Blood cells offish (Control) (10 X 100)
A - Neutrophil, B - Lymphocyte, C - Monocyte, D - Basophil, E - RBC

>

0M
>%• tit#
.
B
*
%
m
ft

# *

PLATE 5.4 : Blood cells offish (96 hr. treated with endosulfan) (10 X 100)
A - Vacualation of RBC, B - Rupture of RBC,
C & E- Damaged WBC structure, D - Breakage of cell membrane
PLATE 5.5 : Blood cells offish (96 hr. treated with endosulfan) (10 X 100)
A - Breakage of RBC membrane
B - Vacualation of RBC
erythrocytes in C.mrigala fingerlings treated with urea. Chauhan et al; (1983),

Chakraborty and Banaijee; (1988)1 and Singh; (1990), also observed in a

similar findings in fish treated with pesticides and chemicals.

Thakur;1985 reported decrease in iHb percentage and RBC and increase in

total WBC in carbaryl treated fishes (Garra gotyla gotyla), which induced a

condition of anemia in the fish by lpss of haemoglobin from the blood and a
t

great increase in the number of leucocytes probably to combat against the

toxicant stress. Jauch;1981 observed significant decrease in RBC count in

Cichlid Heterotilapia multispinosa' and Tilapia mucostica treated with low

dose of lebaycid. Kanoh et a/.,(1982) reported decrease in total RBC counts in

!
i

rat exposed to sub-lethal malathion in the first 30 days but the count revived to

the normal level subsequently and remained at that level for the rest of the

year.

Rai and Qayyum (1984), noted gradual decrease in erythrocytes, Hb

concentration due to intoxication of lead poisoning in Catla. He stated that this

i

anaemic condition of fish can be attributed a haemolysis or erythropoietic

I
i

disorders which seem to be brought about by the lead poisoning. Similar

I

observation was reported by Dick and Dixon (1985) with copper exposure and
i
stated that this might be the result of stressor induced haemodilution. In order

to optimize gas exchange catecholamine released under stressful conditions

act to increase both blood flow to and permeability of fish tissue. These factors

also increase the exchange of water and ions resulting in water uptake in
i

freshwater (haemodilution) and water loss in salt water (haemoconcentration)

75
Randall etal, 1967, Rankin and Mante; 1971, Stevans; 1972, Patil & Jabde;
j
1998. !
t
i
i

The study showed changes in total numbers of WBC, which were increased in

numbers from 0.2009.6±161.09; 0.2079.2±100.55 to 0.3000±189.74

I
million/mm3 (Table No-5.1 and Graph No. 5.3). In the differential count of
i
WBC, lymphocytes and neutrophils revealed interesting changes leading to

neutropenia and leucopenia. Lymphocytes and monocytes were found to be

I

increased in numbers. On the other hand decreasing numbers of neutrophils

i
l

were observed. Basophils and eosinophils did not produce any significant
!

changes. Increased numbers of lymphocytes and monocytes might be

associated with immunological reaction to produce antibodies to cope with

stress induced by the insecticide. Decreased numbers of neutrophils indicated

i

infectious state of the fish body duejto immune response resulted by the toxic
I
effect of the insecticide. Similar results were recorded by Patil and Jabde,

(1998) observed the similar trend in Channa gachua exposed to mercury

l

chloride and they reported leucocytosis in the fish due to the result of a large

increase in lymphocytes. Blood performs a defense function in the body of an

organism and is the most important factor of immunity (Thakur et ah,1985).

The insusceptibility to infection was accomplished by the leucocytes in the

I

blood, which were capable o f phagocytosis. Leucocytes were also able to

i
render microorganism and their toxins in to harmless by producing immune
i
bodies and destroy foreign protein. From the analysis of blood parameters it is

76
indicated that Endosulfan is extremely toxic to most fishes and causes massive

mortality.

77