The environmental quality of freshwater ecosystem has deteriorated markedly
over the last two decades. Pollution of water bodies is assuming alarming proportions
with increased population, industrialization, urbanization and intensive agriculture
(Venkataraman et al., 2007). It includes not
only the influences of other plants and animals present but also those of the
physico-chemical and materiological factors (Velmurugan
et al., 2007). Water pollution affects and kill all forms of wild
life. Humans are the biggest threat to fish. Previously, reported due to this
most organisms present in fresh waters face not only nutritional hazards but
also marked diurnal and seasonal oscillations as well as man-made changes in
the environment extremes may toxic even the physiological activities to the
limit (Ehiagbonare et al., 2009). The effects
of increasing pollution stress upon fresh water environments have become a cause
of concern to man due to feed back to our own health, economic welfare and degradation
in recreational facilities (Oluah, 2008). It is therefore
necessary to evolve environmental management strategies and action plans for
integration of environmental consideration in development activities of the
It increase turbidity and may cause discoloration of water with possible adverse
effects on fisheries and recreational activities. Some type of particulate matter
may damage gill surfaces of fishes and vertebrate (Mohamed,
2006). Also, the materials settling at the bottom may alter the composition
of sediment affecting benthic organisms. If the solids are organic, aerobic
conditions may develop in the sediment overlying water layer, affecting in the
organisms. Furthermore, Oluah (2008) assessed the pollution
effects involves not only the chemical and physical character of the aquatic
ecosystems but the biological parameters. Haematology is a reliable indicator
of the physiological condition of the fish. This has been the incentive to the
scientific interest and development of fish heamatology as chemical tool in
monitoring fish health programmers (Gill et al.,
1991). These also review background effects of water pollution and toxicity
studies. A wide array of factors, both within and without the organisms affects
the peripheral haematological make up of fishes (Mahajan
and Dheer, 1983). Previously, Koca et al. (2008)
reported sex, season and maturity stages are known to affect haematological
values of fishes. The effects of biocides on target organism particularly on
fishes may be exhibits to many ways. Majority of biocides produce detrimental
and sometimes fatal side effects on fish fauna. Many researchers have been studied
the bioassay of water pollutants of A. testudiensis. Even though, the
water pollution properties extensive use in agricultural fields especially fields
results in pesticide pollution (Kamal et al., 2007).
Hence, the bioassay of water pollution and its histopathological effect on liver
was investigated on A. testudiensis (Bloch.) which were a fresh water
table fish with very high environment resistance and common in paddy fields.
MATERIALS AND METHODS
Five live fishes of equal size belonging to the species A. testudeneus where collected from the Parvathiputhanar canal located in Trivandrum. The fishes of same species where also collected from the fresh water Karamana river located in Trivandrum. They were brought into the laboratory and kept in glass tank at room temperature. Karamana River is maintained in another tank named as control. The fishes kept tank P. P were maintained as experimental fishes.
Haematological studies: The fishes were sacrificed and the body was cut open. Blood samples were collected from both control and experimental fishes. The samples were oxalated for preventing clotting. The following parameters of blood were studied.
Haematocrit or packed cell volume (PCV): Determined by centrifugalize samples of blood taken in the Wintrob tube. Haemoglobin content was determined using haemometer. Total count of RBC, WBC and Thrombocytes was made using the haemocytometer. Blood smear preparations were made and stained with Leishmanns stain to study the details of RBC and Thrombocytes. Differential count of Leucocytes was also made.
Studies on changes in the level of glycogen: The tissues were isolated
from the body of fishes Parvathiputhanar and Karamana river and kept in separate
Petri dish. From the sample and control fishes the following organs such as
liver kidney and intestine were selected to identify the preliminary study of
glycogen content present these three organs. Tissue were cut into small pieces
and fixed separately in absolute alcohol. Paraffin sections were prepared and
histochemical localization of glycogen was done by PAS technique. Photomicrographs
were also prepared for detailed observation. The work had been carried out in
January 2008 to June 2009 during the fourth semester M.Sc course in the department
of Zoology Mar Ivanios College TVM.
Haematological studies: The following parameters of blood of A. testudineus
such as PCV, haemoglobin, total count of RBC, WBC and Thrombocytes differential
count of Lymphocytes, Granulocytes and macrophages of both control and experimental
fishes were compared. The observation and result were summarized in Table
The Haematocrit (PCV) was found to be 22% haemoglobin content was 7.5 g mL-1 of blood Erythrocytes were nucleate and oval in shape. Length of RBC varied from 12-14 m and their width varied from 8.5-9.5 m erythrocytes numbered 2.3x106 mm-3 of blood.
Leucocytes: Leucocytes were numerous than erythrocytes. Leucocytes count was 0.14x106 mm-3 blood. Three kinds of leucocytes were identified. Lymphocytes x106 lymphocytes were most abundant leucocytes there were two kind of lymphocytes-small and large. Nucleus was round shaped. Lymphocytes constitute 65% of the total WBC. Diameter of these cells ranged from 4.2 to 8.2 m. The nucleus was seen deep violet eighth Leishmann Stain. Then the granulocyte possessed prominent granules in the cytoplasm. They formed 29% of the total WBC. Among the granulocytes Neutrophills were identified by virtue of their multilobed nucleus. The diameter of granulocyte varies from 9-12 m.
Macrophages: Large in size arranging in diameter from 30-35 m.
Thrombocytes: Spindle shaped cells having a length of 12-15 m Tejendra the cytoplasm was granulat and deeply basophilic in center and pale homogeneous periphery. Cytoplasm purplish in colour. Thrombocytes numbered 0.071x106 mm-3 blood.
Haematological observation between fresh water and polluted water living A. testudeneus: Haematocrit (PCV) was found decreased to 16%. A similar reduction was also observes in the haemoglobin content (4.8 g 100/mL).
Erythrocytes: Erythrocytes had undergone several changes in shape and
size. Shape varied from oval to irregular (Fig. 1) Plasma
membrane ruptures and was disintegrating in many RBCs.
|| Study of blood cells characterization of the pollutant and
fresh water fish of A. testudiensis
|| Blood cells and its physiological on A. testudineus
from Karamana river (fresh water)
Nuclei was without change, nuclear size renamed more or less uniform length
and width of the RBCs decreased to 1.7106 mm-3 blood.
Leucocytes WBC count decreased to 0.11x38106 mm-3 blood. When compared the Lymphocytes count there were a significant reduction in number observed than the control river fish A. testudiensis of lymphocytes could be Diameter remained unchanged. Large lymphocytes were more abundant. Differential count of lymphocytes was 55% showing a decrease of 10% over control fishes.
Granulocytes: There was a significant decrease in the number of granulocytes (8% WBC) and the difference between the control fish 21%. Though, size and shape of the cells remain unchanged.
Macrophages: There was a slight increase in the diameter in the diameter of macrophages (32-38 m) macrophages formed 37% of WBC showing an increase of 31% over control fishes.
Thrombocytes: A reduction in the number of thrombocytes was observed (0.055x106 mm-3 blood) (Fig. 1, 2).
Changes of glycogen content: The control slides were compares with experimental
sections after localizing glycogen in liver, intestine and kidney. In the control
slides, all regions of liver gave intense PAS positive reaction for glycogen.
There was preponderance of glycogen in the outer region. The middle and inner
region also colored deep pond. In the experimental sections, the reactions were
slight to moderate. Many degenerating patches of parenchyma cells ranging from
small to moderate size were also observed. The intestine showed intense PAS
positive reactions including the serosa, muscularis, mucosa and villi on the
control slides. Though, slight to abundant reactions were obtained in the experimental
sections (Table 2). The serosa gives a slight PAS positive
||Blood cells and its physiological on A. testudineus
from Parvathyputhanar river (polluted water)
|| Preliminary analysis of glycogen content from the 13 different
regions of A. testudiensis (PAS)
The glycogen concentration was moderate in the muscularis and abundant in the
mucosa and villi. All regions of the kidney gave intense action for PAS in the
control slides. In the experimental slides outer region showed slight PAS positive
reaction. The middle region was moderate while the inner region showed abundant
PAS positive reaction.
Histopathological studies: The pathological change in the blood cells
of the fish from the polluted and non polluted river living fish has been observed.
Haemorrhage, blood congestion and necrotic cells were generally observed in
the liver tissue. Mononuclear cell focal infiltration was observed (Fig.
3b). The lesions in the kidneys of the fish exposed to acidic water in the
unused lignite mine included degeneration of the epithelial cells of the renal
tubules, degeneration of the glomeruli, hypertrophy of the epithelial cells
of the renal tubules narrowing of the tubular lumen and glomerular contraction
in the Bowmans capsule (Fig. 3).
||Histological view of liver, kidney and intestine from the
Parvathiputhanar (polluted) river A. testudiensis
||Histological view of liver, kidney and intestine from the
Karamana (fresh water) river A. testudiensis
Such an observation doesnt present in the kidneys of the fresh water
fish (Fig. 4).
One of the commonest pollutants of the fresh water ecosystem in all developing
or undeveloped countries from sewage, raw or treated. In reasonable quantities,
sewage can be harmless or even beneficial. The nitrate and phosphates in sewage
fertile water, leading to increased growth of microscopic plant life, phytoplanktons
in the water. This serve as the food for minute animals which is their turn
end up as food for fish and other animals (Sarkar et
al., 2006). Particulate matter in the sewage water which depress according
to its settling velocity can also influence this aquatic environment in many
ways (Omitoyin, 2006). Assessment or interpretation of
predicted inputs represent a vital activity in an environment impact study (Saloom
and Duncan, 2005). Significant impacts on physical, chemical, biological
environmental and socio-economic components of the environment are being caused
human activities (Stone and Thormforde, 2003). The ecosystem
under consideration in the Parvathiputhanar canal an artificial canal dug out
for the purpose of inland water transport is now increasingly being used for
the disposal of various waste materials. The present study is attempts undertaken
to evaluate the variations in the biological characteristics include morphology,
physiology, haematology and glycogen content of fishes of Parvathiputhanar a
highly polluted river and fresh water river Karamana river.
The important of the hydrographic studies in aquatic environments is well realized
in view of their value in assessing the biological potentialities of an ecosystem.
The haematological Variations in the Parvathiputhanar fish is due to this canal
slightly remained on the alkaline (7.1-8.5) because it one end having the influx
of water from Veli lake and Poonthura water lake and also the garbage and sewages
from the town and villages. The morphology and difference physiology of the
fishes both in controlled and experimentalised shows variance due to environmental
stresses. The experimental fish from Parvathiputhanar was highly adapted to
the polluted condition. It can be clearly identified when the fishes are acclimated
in the laboratory (Ademoroti, 1996). But the fishes were
rejected it and very interested to feed earthworm. The regular feeding was stopped
for two months. Meanwhile, the water has been changed every week. There was
no infection identified and it survived. But the fishes from Karamana river
were died after one day acclimated in the laboratory.
A poisoned fish become a more susceptible to bacterial and fungal infection.
Anemia develops during infection (Biggs et al., 2005).
The reduction in the total RBC count (1.78107 mm-3 blood)
in the poisoned fish also reflects the anemia accompanies in bacterial in bacterial
and fungal infection. The RBCs had undergone several morphological changes after
exposure of the fish to the pesticide. The poison getting in to the blood by
absorption to integument may be causing changes in the permeability of RBCs.
Still the physiological upset was bound to affect the manifestation of blood
itself as perceivable in the circulatory blood. All species from polluted waters
showed significantly lower erythrocyte numbers, haematocrit, haemoglobin and
thrombocytes percentage and significantly higher Mean Cell Volume (MCV), leucocyte
numbers and lymphocyte percentage, compared with the controls. The glycogen
depletion observer in the liver, kidney and intestine hence the present study
shows that glycogenolysis has occurred in these organs (Jamuna
and Noorjahan, 2009). Freshwater fish A. scandens was exposed to
sublethal concentration of lead nitrate (10 ppm) for a period of 15 days as
a results revealed that the glycogen level reduced significantly in the liver
and muscle during exposure this kind of similar observation has been made by
Jamuna and Noorjahan (2009). For instance, the depletion
of liver, kidney and intestine glycogen observed in the present study corroborates
with the findings of the earlier investigators.
Previously, Tulasi et al. (1992) reported exposure
of the freshwater fish A. testudineus to a sublethal (5 ppm) concentration
of lead nitrate for a period of 30 days during the preparatory phase of its
annual reproductive cycle reduced the total lipids, phospholipids and cholesterol
levels in the liver and ovary tissues while the free fatty acid levels were
increased and lipase activity was elevated. All the parameters in the blood
were found to be increased.
Furthermore, Oluah (2008) studied the Haemoglobin,
haematocrit and erythrocyte counts in fish exposed to wastewater were significantly
lower than in a control group. There was a significant increase in the total
leucocytes count in treated fish, which also suffered microcytic anaemia. The
observed changes in the haematological parameters of the fish exposed to brewery
wastewater may represent part of the physiological processes by which the wastewater
exerts its deleterious impacts on the fish. The similar kind of results also
explained by Roy and Bhattacharya (2006) reported irregularities
in the renal tubule including apoptotic and necrotic cells were also common.
Corresponding with the histopathological lesions, dose-dependent disturbances
in liver and renal functions. Liver diffuse necroses, cordial disarrangement,
individualization of hepatocytes, etc there significant changes induced by cypermethrin
were hyperplasia, disintegration of hepatic mass, focal coagulative necrosis,
From the two different water bodies collected fish A. testudiensis shows
necrosis of tubular epithelium, cloudy swelling of epithelial cells of renal
tubules, narrowing of the tubular lumen and contraction of the glomerulus and
expansion of space inside the Bowmans capsule were observed in the kidney tissues
of fish after exposure. Hepatic lesions in fish living with polluted water (Parvathyputhanar)
be characterized by hypertrophy of hepatocytes, cloudy degeneration, congestion,
karyolysis and karyohexis dilatation of sinusoids and focal necrosis been observed
from polluted water living fish In order to that the intestinal lesions included
infiltration of eosinophils into the lamina propria and atrophy of epithelial
cells Fig. 3. The present study proves its toxic potential
in terms of the damages in organ level observed from polluted canal. In natural
condition pollutants will be less than the present study, but continuous usage
of the pesticide might lead to the concentration that was used in the experimental
condition (Venkataraman et al., 2007).
Figure 2 indicates that the blood smear and its changes of
pollutant free water living fish of Anabas testudensis (Karamana river).
Qualitative study of Haematological parameters of the A. testudineus
from various two water bodies of Parvathiputhanar (fresh water) and polluted
water from Karamana river. Study of the intestine, liver and intestine in the
control fish showed a typical structural organization of the internal cell organelles.
Fish exposed to acid water had several histological alterations namely desquamation
of lamellar epithelium, fusion of the lamellae and lamellar aneurisms (Ogbeibu
and Edutie, 2006). The gill abnormalities observed in this present study
were similar to previous studies of low environmental pH on fish gill morphology,
which showed separation of the epithelial layers of secondary gill lamellae,
deformation of secondary lamellae and degeneration of chloride cells accompanied
by hyperplasia of undifferentiated cells in the primary lamellae (Gill
et al., 1991). Heavy metal compounds associated with acidification
have also been associated with a reduction of both carbonic anhydrase and Na+,
K+ ATPase activities in salmonids, even at a relatively high pH (pH
5.0). The lamellar fusions are defense mechanisms that reduce the branchial
superficial area in contact with the external surroundings, furthermore this
mechanism also increase the diffusion barrier to the pollutant (Mishra,
1991). As in higher vertebrates, the kidneys of fish perform an important
function relate to electrolyte and water balance and the maintenance of a stable
internal environment (Das et al., 1990).
An attempt to demonstrated the changes of the morphological, haematological parameters and the changes in the level of glycogen in the liver, kidney and intestine of A. testudineus from a highly polluted Parvathiputhanar river and fresh water river karamana where evaluated. The haematocrit and haemoglobin content drops considerable in experimental fishes. There is a significant reduction in the total count of RBC in experimental fishes believed to a resulting from anemia accompanying bacterial infection. There is a significant reduction in the total count of WBC except the macrophages. Granulocytes and lymphocytes reduce in number. Macrophages increase in number also thrombocytes reduced in number. The accumulation of pesticides in the Parvathiputhanar river flows to Thiruvallam and cause mass death of fishes. Since, the morphological variation and histological changes in liver, kidney and intestine of fishes (A. testudineus) from Parvathiputhanar and Karamana river shows remarkable variations depends upon the polluted materials in the water content. There is a depletion of glycogen in the liver, kidney and intestine of A. testudineus from the Parvathiputhanar river due to influx of pesticides.
The authors gratefully acknowledged to our Malankara Catholic College Correspondent Fr. Prem Kumar (M.S.W) and Superintentend Rev. Sr. Archana Das given encouragement and support for preparation of this research manuscript. The corresponding author wish to extend my thanks to Dr. J. Ezhil and Dr. Manju Arthi for the supports of histological results of this research work.