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81
National Journal of Life Sciences, Vol. 7(1) 2010 : 81-84
*Author for correspondence (email : csjawale@hotmail.com)
HAEMATOLOGICAL CHANGES IN THE FRESH WATER FISH,
EXPOSED TO SUB-LETHAL CONCENTRATION OF PISCICIDAL COMPOUNDS
FROM (FAM : SOLANACEAE)
C. S. JAWALE1* and L. B. DAMA2
ABSTRACT : The sub-lethal concentration of the active principal of two piscicidal plants viz. Cestrum nocturnum
(2.8 mg-1) and Cestrum diurnum (3.2 mg-1) induced the haematological changes in a freshwater fish Cyprinus carpio.
Behavioural activities such as erratic swimming, loss of reflex, hyperventilation increased surfacing and jerk move-
ments were observed. The total number of erythrocytes, leukocytes, and haemoglobin concentration and packed cell
volume declined while erythrocytes sedimentation rate and related parameters like MCH, MCHC, MCV altered with
exposure. Since the plant toxins influence cell integrity and hemopoiesis, the oxygen carrying capacity of blood was
decreased, consequently thermodynamic and metabolic activity in fish was disturbed, therefore Cestrum species seems
to be Karyocytophilic and Cytokaryophilic toxins to the fish.
Key words : Cestrum nocturnum, Cestrum diurnum, Cyprinus carpio, Piscicidal plants.
INTRODUCTION
The applications of haematological studies to the in-
vestigation of animal and human disease progress is well ac-
cepted and considered to be routine procedure in diagnostic
methods. There have been attempts to apply haematological
parameter to the study of abnormal physiological processes
in fish (Celik,2004 and Gabrielet al.,2009); However the hae-
matological parameters in relation with toxicant stress to In-
dian fishes were studied in recent past by various ichthyolo-
gists (Ghazaly,1991 and Raizada & Singh,1980), while the
studies on the effect of piscicidal compounds of plants on fish
are scanty (Bhatt & Singh,1985; Bhatt et al.,1987; Bhatt &
Farswan,1992; Olufayo,2009 and Gabriel et al.,2009). Pisci-
cidal (Fish poison) compounds of plant origin were studied
by various workers for their use in aquaculture to remove pred-
atory and weed fishes (Konar,1973; Chowdhary,1968 and
Singh et al.,1996). But very less efforts have been made to
evaluate the mechanism of poisoning in fish through plant pi-
scicides. Cestrum species are recently reported to be a bio-
cidal plant (Patil and Jawale,2002). Despite its poisonous na-
ture, it also showed remarkable pharmacological activities
(Roy & Chatterjee,1962 and Chatterjee & Ray,1964). Ces-
trum species are well evaluated for its phytochemicals like
saponins, alkaloid, hydrocarbons, tannins, sterols, fatty acids
and essential oil by various workers (Riaz & Chaudhari,1993;
Haraguchi et al.,1999 and Ahmad et al.,1993). Therefore to
evaluate the mechanism of poisoning, the present study was
carried out on haematological changes in Cyprinus carpio
induced by plant piscicides of Cestrum nocturnum and Ces-
trum diurnum (Family : Solanaceae) at different sub-lethal
doses which are useful in detection and diagnosis of fish death.
MATERIAL AND METHODS
Piscicidal plant Cestrum nocturnum and Cestrum di-
urnum were cultivated in garden, their essences were collect-
ed, shed dried, and powdered mechanically. The saponins of
C.nocturnum and C.diurnum were isolated by the method of
Chakravarti et al. (1963,1962). The leaves were extracted with
ethanol. the extract were hydrolysed with NaCl and HCl mixed
with aqueous butanol and repeatedly crystallised with aque-
ous alcohol. The concentrated residue was evaporated to dry-
ness to yield crystalline saponins of m.p. 242°C and 269°C
from C.diurnum and C.nocturnum respectively. The isolated
compounds were dried and 1% aqueous solution of each com-
pound was prepared to be use. The healthy freshwater fish
C.carpio were collected from the nearby rearing ponds and
kept in tank having a continues supply of dechlorinated water
and standard food. Water temperature was maintained at 20±
3°C. Fish mortality rate was less than 1% and fish were main-
tained in the tank for the period of upto six weeks. Ten fishes
were exposed to the pre-determined sub-lethal concentration
of each toxicant such as 2.8 mg-l (C.nocturnum), 3.2 mg-1
(C.diurnum) for 48 hrs (Jawale,2002). A control set was also
maintained. After 48 hrs alive fishes from each set were bleed
by cardiac puncture. Bleeding was accomplished in less than
one minute after each fish was removed from the holding tank.
Blood was collected in heparinized syringe, using a 27 gauge
hypodermic needle. Physical resistant without anesthetic was
used in blood collection. Various haematological parameters
such as haemoglobin content (Hb), total erythrocyte count
(TEC), packed cell volume (PCV), total leukocyte count (TLC)
were estimated using methods described by Dacie and Lewis
(1963). Absolute value were determined using the formulae
Received 01.04.2010 Accepted 05.06.2010
ISSN : 0972-995X
82
jerk, escaping the toxicant, uncontrolled, imbalanced swim-
ming, convulsion, surfacing, engulf of air and suffocation be-
haviour. The mean values for the haematological parameters
of C.carpio studied are shown in Table.1. There was signifi-
cant alteration in the haematological values after 48 h. expose
to plant toxicant. The result indicates that the haemoglobin
percentage, packed cell volume, total number of erythrocytes
and leukocytes were decline, while erythrocytes sedimenta-
tion rate increased with C.nocturnum andC.diurnum as com-
pared to control fish. Other absolute values (MCV, MCHC
and MCH) also altered in response to the changes in above
parameters.
as, mean cell haemoglobin concentration (MCHC) was cal-
culated by dividing the haemoglobin content in gm/1000 ml
by the PCV/1000 ml of blood. (MCH) was determined from
the haemoglobin value (Hb) and from the erythrocyte count.
Results were express as mean ± standard error of mean (SEM)
and differences between means were considered to be signif-
icant when P<0.05.
RESULTS AND DISCUSSION
Fish shows remarkable alteration in the behaviours on
exposure to toxicant such as, erratic swimming movements,
Fish being a very successful group of vertebrate, has
adapted a wide range of environmental conditions, however
at the same time, they are highly sensitive to the environmen-
tal stress. The results of present study indicated that the entry
of plant compounds into the blood stream of fish, promotes
their ill effects on various blood parameters. The reduction in
red cell count and haemoglobin percentage indicates the oc-
currence of acute anaemia. Such anaemia in fishes is known
to induce by various toxicants (Agrawal et al.,1982,1983).
The anaemia might be due to deficiency of iron, leading to
decrease in haemoglobin synthesis (Sharma & Gupta,1982;
Olufayo,2009 and Nanda & Behera,1996). It can also be in-
ferred that erythropenia might be due to the disturbance in
metabolism of the haemopoietic organs (Shammi and
Quayyum,1982) or due to increased in the rate of erythro-
cytes destruction (Agrawal and Srivastava,1980).
Plant toxicants of saponin group are reported to be
haemolytic in nature (Georgeet al.,2002). Bhatt and Faraswan
(1992) had observed the ruptured cell membrane, nuclear
membrane, reduction in size of nuclei and vacuolated cyto-
plasm in the RBCs of fish exposed toAesculus indica (Colebr),
Engelhardtia colebrookiana (Linn), Lyonia ovalifolia(Wall)
and Zanthoxylem alatum (Roxb) containing plant chemicals.
Olufayo (2009), elucidate that haemolysis and injury
in the blood cell made the fish inefficient to take up the oxy-
gen required for its survival. As red cell number and haemo-
globin concentration reflects the oxygen carrying efficiency
of the blood and need of oxygen, in carrying out normal be-
haviours of the fish. Agrawal et al. (1982), described lym-
phocytopanea, haemorrhages and oedema, decreases the total
number of erythrocytes, leukocytes and haemoglobin percent-
age in intoxicated fishes.
The change in MCV, MCHC and MCH in plant toxi-
cant treated fishes suggested the destruction of RBCs, which
result in reduced oxygen carrying capacity and ultimately death
of fish. The reduced WBCs and RBCs concentration reflect
the plant toxicant induced cell lysis. It was apparent at this
level, the gills and body of the toxicated fish become pale
whitish or colour less in contrast to the normal individuals in
which the gills were dark reddish. Thus saponins of
C.nocturnum and C.diurnum could be considered as Karyo-
cytophilic and Cytokaryophilic toxins to the fish. The increased
erythrocyte sedimentation rate (ESR) and decreased packed
cell volume (PCV) accounted for the degradation of blood
proteins in the fish as observed by Bhatt (1985) on injecting
certain plant alkaloid and tannins.
Parameter Control Fish treated with
Body length (cm) 13.23 ± 0.20 13.40 ± 0.42 12.14 ± 0.01
Body wt. (gm) 26.39 ± 0.00 28.61 ± 0.00 24.40 ± 0.07
Hb (gm/100 ml) 12.8 ± 0.02 4.40 ± 0.04 6.20 ± 0.02
PCV (%) 36.07 ± 0.03 18.02 ± 0.05 22.58 ± 0.12
MCH (pg) 45.28 ± 0.76 119.69 ± 0.34 102.39 ± 0.02
MCHC (gm/lit) 33.28 ± 0.12 26.28 ± 0.09 23.28 ± 0.02
MCV (um3) 132.27 ± 0.54 412.40 ± 0.59 422.10 ± 0.72
Total no. of RBC (1000/mm3) 4.72 ± 0.36 2.32 ± 0.27 3.03 ± 0.18
Total no. of WBC (1000/mm3) 4.30 ± 0.02 2.89 ± 0.28 3.32 ± 0.42
ESR (mm/h) 1.8962 ± 0.063 3.79 ± 0.072 3.01 ± 0.081
Table 1 : Mean values of body length, body weight and various haematological parameters of control
and treated individuals of exposed to sublethal concentration of
(2.8 mg-1) and (3.2 mg-1) for 48 hours.
*Mean values with standard deviation (+SD).
Legend : PCV - Packed cell volume, Hb - Haemoglobin, MCHC - Mean corpuscular haemoglobin concretion
ESR - Erythrocyte sedimentation rate.
82 JAWALE and DAMA
83
Thus the active ingredient of both piscicidal plants
definitely influenced the dynamicity of haemopoesis in fish,
and therefore the oxygen carrying capacity of the blood was
gradually decreases. At this level because of high CO2 and
low O2 tension, blood was quit unable to compensate the def-
icit oxygen budget of the cell and organs of fish; which intern
affected the thermodynamics and metabolic activities of the
fish (Faraswan,1989). Consequently such condition might have
proved a contributory factor to anaemia and ultimately death
in the fishes toxified with C.nocturnum and C.diurnum.
Further work is in progress to evaluate C.nocturnum
as fish toxicant to be used in aquaculture to remove predatory
and weed fishes during pond reclamation.
ACKNOWLEDGEMENTS
Authors are thankful to the Head, Department of Zool-
ogy, Dr. B. A. Marathwada University, Aurangabad and Prin-
cipal of H.P.T. Arts and R.Y.K. Science College, Nashik for
providing necessary facilities.
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