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Chapter IV Discussion

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Organism’s behaviour provides a link between the physiology and ecology and its

environment [Little & Brewer, 2001]. It is an attempt to adjust to external and internal stimuli to

meet the challenges of surviving in an altered surrounding. Behaviour represents an integrated

response of fish species to toxicant induced stress [Kane et al., 2005]. The development of

behavioral methods in fish as an important tool in aquatic toxicology has been standardized. The

fast and abrupt movement of fish in toxic media is to escape from such changes. The most

observed visible abnormal behaviour in the present study were quick incessant jumping showing

surface to bottom movement and gulping of air, restlessness, loss of equilibrium, increased

opercular activities and resting at the bottom. Such stressful and erratic behaviour of fish during

the experimental period indicates respiratory impairment due to the presence of heavy metal salts

in water on the gills. These observations were similar to those by Omoniyi et al., [2002];

Rahman et al., [2002] and Aguigwo, [2002]. Variation in spontaneous activity and respiratory

responses are sensitive indicators of sublethal exposure. Scherer [1992] and Macleod & Passah,

[1973] reported that loss of appetite, weight, equilibrium, erratic swimming, nervousness and

gradual onset of inactivity as a result of inorganic mercury intoxication. During the acute toxicity

tests of the pesticide malathion, Labeo rohita were seen to exhibit several behavioural responses,

such as fast jerking, frequent jumping, erratic swimming, spiraling, convulsions and tendency to

escape from the aquaria [Thenmozhi et al., 2010] Following this state of hyper excitability, the

fish became inactive and lost orientation. There was loss of equilibrium and paralysis which

ultimately resolved in death of fish. Similar observations were noticed in behavior of Gambusia

affinis in response to the sub-lethal exposure to chlorpyrifos [Rao et al., 2005].

Gulping of air may help to ease respiratory stress and avoid contact of the toxicated

medium. Surfacing phenomenon may be due to elevated demand for oxygen during the exposure

periods. Fish exposed to mercury sank to the bottom with reduced opercular movements, failing

to fight stress in both the sublethal exposures due to toxicity on gills .Similar effects were seen

by Omitoyin et al., [2006] and Aguigwo, [2002] on fish exposed to pesticide. Alteration in

normal behavioral pattern by exposure to toxicants poses serious risks to fish populations.

Behavioral disturbances such as off feed and restlessness were also observed. Oliveira Ribeiro et

al., [1995] reported that olfactory organs were affected by mercury intoxication, that changed the

normal behaviour of the fish .


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A heavy mucous as seen on the surface in all exposed fishes in the present study was

also reported by acute toxicity study in Catla catla exposed to fenvalerate [Susan et al, 2010], in

guppy exposed to delta methrin [Viran et al., 2003], in Heteropneustes fossilis and Cyprinus

carpio exposed to synthetic pyrethroid cypermethrin [Saha & Kaviraj, 2003 and Calta & Ural,

2004] in fingerling of European catfish exposed to organophosphorus pesticide diazinon

[Köprücü et al., 2006] and also in rohu exposed to sodium cyanide [Dube & Hosetti, 2010]. The

formation of a layer of excessive mucus observed in this study could have increased the

respiratory problem [Tiwari & Singh, 2005 and Jothivel & Paul, 2008]. The thin mucus layer

covered the delicate and sensitive gills thereby hindering active gaseous exchange and could

therefore, be responsible for the exhibited respiratory distress and death [Omoniyi et al., 2002]

Histopathology has been successfully employed as a diagnostic tool in medical and

veterinary sciences since the first cellular investigations were carried out in the mid- nineteenth

century. Considerable developments have taken place in all aspects of cellular biology with the

result that many sophisticated techniques only recently revised for mammalian histologists, are

now also available for the fish histopathology. Inspite of this, more information regarding their

use and implication in aquatic health is needed, especially with regard to establishing

histopathology as a reliable biomarker of exposure.

Fish gills are the first target of waterborne heavy metals because they come in immediate

contact with it. Fingerlings exposed to copper, nickel and mercury causing lamellar epithelial

lifting, their proliferation, lamellar axis vasodilation, telangiectasis of secondary gill lamellae

confirm the occurrence of edema independent of heavy metal ions levels, as in other fish species.

Such histological alterations have earlier been observed in tiger shrimp and common carp [Chen

& Lin, 2001 and De Boeck et al., 2001] , in rainbow trout after drugs exposure [Schwaiger et al.,

2004] and in trouts exposed to nickel [Pane et al., 2004]. Complete loss of secondary gill

lamellae as seen in fish exposed to higher concentration of copper, nickel and mercury in the

present study has also been observed earlier in sea bass fry [Krishnani et al., 2003]. Edema with

lifting of lamellar epithelium is a defense mechanism as separated lamellar epithelium increases

the distance across which waterborne heavy metals which diffuse to reach the bloodstream.

Earlier studies revealed that epithelial edema is a frequent lesion observed in gill of fish rainbow

trout exposed to copper [Van et al., 2004]. The production of excessive mucus and the gill


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lesions suggest that heavy metal irritates the gills and increases respiratory diffusion distress as

has been observed earlier [Nowak, 1992]. Mercury ions appear to be ion regulatory toxicants and

have much greater potency to stimulate proliferation of mucous cells of gills and the secretion of

mucus into water [Olsan et al., 1973]. Teleangiectasis seen in fish exposed to higher

concentration of copper and nickel causes acute respiratory problems. Haemorrhage in fish

exposed to all above mentioned metal ions interrupts the circulation of the deoxygenated blood

into the secondary lamellae. As a result, oxygen uptake is hindered and causes hypoxia. The

epithelial lifting and lamellar fusion are defense mechanisms that reduce the branchial superficial

area in contact with the outer surroundings. These mechanisms also increase the diffusion barrier

to the pollutants [Van et al., 2004]. Hyperplasia is thus an adaptation to protect underlying

tissues from any toxicants.

The renal lesions are good indicators of environmental pollution as the kidney of fish

performs electrolyte and water balance [Ortiz et al., 2003]. The kidney is a target of toxicants,

which interrupt its functions and cause temporary or permanent damage to homeostasis [Miller,

2002]. In trunk kidney, most alterations were seen in the tubular cells rather than in glomeruli.

Dilation in the lumen of tubules and infiltration of mononuclear cells in interstitium, marked

cellular infiltrations of mononuclear cells in the interstitial region, are explained as a defense

mechanism in the fish to counter toxic metabolites by Das & Mukherjee, [2000]. Similar

pathological changes observed in trunk kidney of catla in the present study were also observed in

Channa punctatus [Mishra & Mohanty, 2009], on Prochilodus lineatus [Camargo & Martinez,

2007], in Lates calcarifer [Thophon et al., 2003], in Catla catla [Patel & Bahadur, 2010] and on

Carassius auratus gibelio [Staicu et al., 2008]. These findings were similar to those described by

Bhatnagar et al., [2007] and Ayoola & Ajani, [2008]. Damaged and shrunken glomeruli were

seen in fingerlings exposed to copper, nickel and mercury salt and these decrease the total

filtering surface. Swelling of tubules and destruction of lining cells inhibit re-absorption. The

histopathological effects of mercury in fish kidney are similar to those in mammals. Total

destruction of cells and their cytoplasm are also indicative of hindered tubular reabsorption and

end-stage renal failure. As a result, the processes glomerular filtration and urine formation are

affected and severe tubulonecrosis and glomerular disintegration occur. The histopathological

alterations seen in fish due to exposure to nickel resulted in respiratory, osmoregulatory and

circulatory impairment. The changes in the size of cells and narrow lumen could be a


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consequence of changed kidney function. Pathological changes, observed in the present study are

severe enough to cause impairment in kidney functioning. The degenerative changes such as

altered metabolic activity and damaged nephrons may result in impaired osmotic and ionic

regulation since the renal tubular epithelium has a major function in excretion of divalent ions

[Gupta & Srivastava, 2006]. Glomerular perturbations and necrosis of proximal tubular

epithelium may be the hallmarks of piscine renal toxicity. Nephrotoxic lesions, including

degenerative changes (e.g. vacuolization) and desquamation of the tubular epithelium, dilation of

the tubular lumina and necrosis of tubular epithelium have earlier been noted in fish exposed to

PCBs, organochlorine and organophosphate insecticides, herbicides, petroleum hydrocarbons

and phenols [Meyers & Hendricks, 1985].

The histopathological alterations seen in liver in present study have also been reported in

Oreochromis niloticus [Kaoud & El-Dahshan., 2010], in Labeo rohita [Loganathan et al., 2006],

in Gambusia affinis [Cengiz & Unlu, 2006], in Poecilia sphenops [Tekkan et al., 2009] and

Cirrhinus mrigala [Velmurugan et al., 2009]. Similar changes were observed in the liver of

Catla catla exposed to chlorpyrifos [Weisman & Miller, 2006] and in Molly Fish (Poecilia

sphenops) exposed to sodium perchlorate [Tekkan et al., 2009]. On the other hand, liver

alterations such as necrosis have been found at different levels of severity depending on the

contaminant, exposure time and dose [Oliveira Ribeiro, 2002].

Alterations in liver are useful markers as it is the prime target organ of various xenobiotics

and accumulates them. It is a major storage spot of lipids and the site of biotransformation. Liver

alterations observed in present work were alarming. Liver is in the path of blood vessels that

transport substances from the digestive system and so liver has the first chance to metabolize

these substances. It is also the first organ exposed to ingested toxicants. If the detoxification

pathways become overloaded with harmful substances, a buildup of toxicants may occur in the

liver cells [Cabots, 2000]. A constant exposure to toxicants may cause damage to liver tissue

[Nero et al., 2005].

Degeneration of hepatocytes in periportal zones implies the influence of toxic compounds

in the digestive tract. The biochemical changes in liver profile relate to hepatocytes damage with

significant changes as hyperplasia, disintegration of hepatic mass, focal coagulative necrosis in

fish exposed to cypermethrin [Sarkar et al., 2005]. Present studies show that alterations in


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number, size and shape of hepatocyte nucleus occur due to contaminants. Alteration in the size

of nucleus has also been previously reported by Paris-Palacios et al., [2000] in Brachydanio rerio

exposed to sublethal concentrations of copper sulphate. Braunbeck et al., [1990] referred that

alterations in size and shape of nucleus are signs of increased metabolism and may be of

pathological origin. Necrosis of some portions of liver tissue observed almost in all above three

metal ions exposure probably resulted from the excessive work required by the fish to get rid of

the toxicant from its body during the process of detoxification very similar to the observation by

Rahman et al.,[2002]. Marked steatosis in fish exposed to mercury is the result of inability of fish

to mobilize stored fat which as a result continued to increase. These anomalies are more severe

and have been associated with the exposure of Channa punctatus to mercurial fungicide [Ram &

Sathyanesan, 1987], Brachyodanio rerio to copper [Paris-Palacios et al., 2000], Salvelinus

alpines to mercury [Oliveira Ribeiro et al., 2002], Lates calcarifer to mercury [Krishnani et al.,

2003], Corydoras paleatus contaminated by organophosphate pesticides [Fanta et al., 2003] and

in Ctenopharyngodon Idella to mercury [Khan et al., 2004]. The high proportion of fibrotic

tissue within the lobules and peribilliary connective tissue points toward hepatic cirrhosis. The

histological alterations in liver suggest that the exposed fish faced metabolic crisis causing by

serious tissue damage. Vacuoles in the cytoplasm of the hepatocytes contain fat and glycogen

deposits related to the normal metabolic function of the liver. Depletion of the glycogen in the

hepatocytes is observed in stressed animals [Hinton & Laurén, 1990 and Wilhelm Filho et al.,

2002], as glycogen acts as a reserve of glucose to supply higher energetic demand occurring in

such situations [Panepucci et al., 2001]. Pacheco and Santos, [2002] described increased

vacuolization of hepatocytes as a signal of degenerative process that suggests metabolic damage,

possibly related to the exposure to contaminated water. These alterations are more severe and

have been associated with the exposure of the rohu by azo dye [Barot & Bahadur, 2011]. The

present study shows that the histopathological changes in liver affects the metabolism directly

and diminishes its life fitness. Changes in liver tissue are linked with histological abnormalities

of kidney and gill. Once absorbed, toxicant is transported by blood circulation to liver for

transformation and/or storage, and if transformed in the liver it may be excreted through the bile

or pass back into blood for possible excretion by kidney or gill. Hepatocytes in all exposed fish

were vacuolated. This is due to inability of fish to mobilize stored fat which thus continued to

increase even after exposure. Fatty change in liver cells is a common response associated with


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exposure to a variety of different chemicals to fish [Meyers & Henderick, 1985]. Increased

hepatic copper levels resulting in vacuolated hepatocytes suggest that fish can redistribute

accumulated copper for excretion through liver as has been observed by Shaw & Handy, [2006]

and Clearwater et al., [2002]. A constant exposure to toxicants may damage to liver tissue [Nero

et al., 2005]. The fibrosis, steatosis, hyperemia and necrosis were the changes similar to those

reported for fish caught in contaminated water or exposed to various chemicals in laboratory

conditions [Brand et al., 2001; Koehler, 2004; Olojo et al., 2005; Camargo & Martinez, 2007;

Wahbi & El-Greisy, 2007 and Aniladevi et al., 2008]. Moderate cytoplasmic degeneration in

hepatocytes, formation of vacuoles, rupture in blood vessels and pyknotic nuclei seen in catla

match were similar to liver alterations studies of Tilapia mossambica exposed to fenvalerate

[Tilak et al., 2005]. The loss of stored lipid substances in hepatocytes in fish exposed to acute

water-borne and trophic doses of inorganic mercury suggests an increase of metabolism as a

quick and primary response of the cells. The evidence of multiple necrotic sites in liver exposed

to a low single dose of methyl mercury explains again to high toxic capacity.

Histopathological manifestations associated with the brain to sublethal exposure of

copper, nickel and mercury ions in the present study match with the study on Lates calcarifer fry

exposed to various concentrations of mercury [Krishnani et al., 2003], in rohu exposed to

hexachlorocyclohexane [Das & Mukherjee, 2000], in Clarias gariepinus [Omitoyin et al., 2006],

in Clarias gariepinus exposed to cypermethrin [Ayoola & Ajani, 2008], in Labeo rohita exposed

to surfactants [Patel et al., 2009], and in Oreochromis niloticus juvenile [Ayoola, 2008], in

Cyprinus carpio [Sepici-Dinc-el et al., 2009] and in Labeo rohita exposed to Zinc [Loganathan

et al., 2006]. The brain indicated severe congestion and generalised spongiosis showing severe

damage. This agrees with the findings of Omitoyin et al., [2001] and Ayoola & Ajani [2008].

Change in the histological structure of brain affects the overall health and behaviour of fish. The

toxicants bioaccumulate in this fatty tissue thereby disrupt normal physiology of the

experimental animal. The present study indicates that mercury is most toxic of all the metal

studied. The present experimental trials revealed that heavy metals may also be neurotoxic as

evidenced by the histopathological changes characterized by vacuolation of brain parenchyma

and moderate swelling of pyramidal cells of the cerebrum. The skull of all the fingerlings

became translucent making brain visible probably due to the decalcification [Patel & Bahadur,

2011]


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Any damage to the lining of intestine can be a good indicator to the toxicity of the

xenobiotic to that particular biological system. Some of the intestinal alterations seen in the

present study in copper and nickel exposure of fish were also reported in Salvelinus alpines

[Oliveira Ribeiro et al., 2002], in L. rohita juveniles [Kumar et al., 2005], in Labeo rohita

[Reyad & Salah, 2008 and Bhatnagar et al., 2007] and in the intestine of Mystus tengara (Ham.)

due to CdCl2 toxicity [Kothari et al., 1990]. According to Bhatnagar et al., [2007], the observed

irritation and destruction of the mucosa of the intestine hampered absorption. Degeneration and

necrosis of villous epithelium in the intestine of mercury exposed fish observed in present study

was same as seen in Tilapia zillii and Solea vulgaris exposed to contaminated drainage water

[Fatma, 2009], on Capoeta capoeta capoeta exposed to toxic effects of cobalt

parahydroxybenzoate [Yılmaz et al., 2008] and in some marine fishes [Marzouk et al., 2009].

The pathological manifestations in the intestine of catla are in agreement with those observed in

Tilapia [Soufy et al., 2007], in Oreochromis niloticus [Hanna et al., 2005] and in Gambusia

affinis [Cengiz & Unlu, 2006]. Degeneration of the intestinal villi in catla decreased its

absorptive surface area, which ultimately resulted in less efficient food utilization [Patel &

Bahadur, 2011].

The study of blood parameters supports prognoses of morbid conditions in fish

populations [Tavares-Dias & Moraes, 2004] and therefore, contributes to a better understanding

of comparative physiology, feeding conditions and other related parameters. Formation of

micronuclei in cells occurs due to structural and/or numerical chromosomal aberrations arising

during mitosis [Heddle et al., 1991]. Except for their small size, they resemble the major nucleus.

Micronuclei and nuclear abnormality tests in fish are generally performed in enucleated blood

erythrocytes mainly due to technical feasibility. It is well known that heavy metals interfere the

regular chromosome segregation during cell division mainly by inhibition of polymerization of

actin tubules, an essential structure of the mitotic spindle. Probable underlying mechanisms are

interactions with motor protein functions, leading to aneugenicity and generation of reactive

oxygen, leading to clastogenicity. The micronucleus formation is a subcellular process resulting

from induced chromosomal breaks or cell spindle malfunction. The presence of micronuclei seen

in this study has also been reported in Prussian carp treated with selenium, mercury, methyl

mercury and their mixtures [Al-Sabti, 1994], in cultured gilthead seabream due to seasonality

[Strunjak-Perovic et al., 2009] and in winter flounder [Hughes & Hebert., 1991]. The


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micronucleus seen in present study were similar to those with Puntius altus exposed to cadmium

and ascorbic acid [Jiraungkoorskul et al., 2007] and marine fish turbot and Atlantic cod treated

with crude oil [Barsiene et al., 2006] and in rohu exposed to azo dyes [Barot & Bahadur, 2011].

The anisocytosis, cell membrane deformation, vacuolization in the nucleus and cytoplasm

of erythrocytes as well as changes in the nucleus observed in the present work were also seen in

Barbus conchonius exposed to mercuric chloride [Tejendra & Jaglish, 1985], Clarias batrachus

treated by a carbamate pesticide [Patnaik & Patra, 2006]. Poikilocytosis seen in present work is

also seen in a freshwater fish Gambusia affinis exposed to textile wastewaters (untreated and

treated) [Sharma et al, 2007], in gilthead sea bream [Strunjak-Perovic et al.,2009], in Gobius

niger due to pollution [Tejendra & Jaglish, 1985]. Swelling of RBC observed in copper exposed

fingerlings might be due to the increase in regulatory volume mechanism of the cell as has been

suggested earlier by Weaver et al., [1999]. These haematological manifestations as good

indicators of toxicity of heavy metals were supported in the previous work on Catla catla

[Chavda, et al 2010] exposed to pathogens. Increase of erythrocyte size is generally considered a

response against stress and could be a consequence of numerous factors.

The count of red blood cells is quite a stable index and the fish body tries to maintain this

count within the limits of certain physiological standards using various physiological

mechanisms of compensation. As compared to control group, all the blood samples of exposed

fingerlings showed decreased RBC count, Hb and Hct. Reduced erythrocyte count and

haemoglobin content in all exposed fingerlings causes anaemia. The decreased level of RBC,

hemoglobin and hematocrit marked in present study revealed the hematotoxic effects of heavy

metals. This has also been shown by others using heavy metals such as cadmium, chromium,

nickel and lead on Cyprinus Carpio [Rajamanickam & Muthuswamy, 2009], in Oreochromis

hybrid exposed to aluminium [Bhagwant & Bhikajee, 2000], in Labeo rohita treated with

chromium [Vutukuru, 2005]. Hb and Hct decreased in Clarias lazera exposed to vanadium [Zaki

et al., 2007]. RBC count and Hb also decreased in Clarias batracus exposed to mercuric chloride

while WBC count increased [Maheswaran et al., 2008]. Declined RBC and Hb and raised WBC

count were also reported in Cyprinus carpio exposed to chlorpyrifos [Ramesh & Saravanan,

2008].However, very slight fluctuations (increase/decrease) were recorded in the WBC count


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when compared with the control. Decreased hematocrit and haemoglobin values together with

decreased and distorted erythrocytes are obvious signals of anaemia [Ololade & Oginni, 2010].

Increased WBC seen in present study was also found in African catfish exposed to

chlorpyrifos [Okechukwu et al., 2007]. But a reduction in WBC count was found in African

catfish, Clarias gariepinus, fingerlings exposed to nickel [Ololade & Ogini, 2010]. Long-term

exposure (3 months) to 0.1 and 0.2 mg/l concentrations of copper decreased the leucocyte count

in blood [Vosyliene, 1996] which matches with present result seen in copper exposed catla. This

may be due to the release of epinephrine during stress which is capable of causing the

contraction of spleen and a decrease of leucocytes count, thus weakening the immune system

[Svoboda, 2001 and Witesta, 2003]. An increase in the leucocyte count is mostly observed

during the first days of stress reaction when fish tries to restore disturbed homeostasis, however

later a decrease of leucocytes count can be observed, which shows the weakening of the immune

system.

Increase in WBC as observed in fish fed with compounded feed is attributed to increase

in production of leucocytes in haematopoietic tissue of kidney and perhaps spleen. Lymphocytes

are the most numerous cells which function in the production of antibodies and chemical

substances serving as defense against infection. The primary consequence of observed changes

in leucocyte count in stressed fish is suppression of the immune system and increased

susceptibility to disease [Ayoola, 2011].

Change in leucocytes synthesis manifests in the form of leucocytosis with heterophilia

and lymphopenia which are characteristics of leucocytic response in animals exhibiting stress.

The increase in WBC count can be correlated with an increase in antibody production which

helps in survival and recovery of fish exposed to the pesticides Lindane and malathion [Joshi et

al., 2002]. In the present study, increase in WBC count indicates hypersensitivity of leucocytes to

chlorpyrifos and these changes could be immunological reactions to produce antibodies to cope

up with stress induced by chlorpyrifos.

Declined haemoglobin impairs oxygen supply to various tissues resulting in slow

metabolic rate and hypoxia that promotes erythropoiesis. Nussey et al., [1995] noted that the

erythrocytosis could be triggered by shortage of oxygen during metal ion exposure. This would


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impose oxygen debit in fish, in that way promoting anaerobic respiration as a result of high

carbon dioxide level in the blood. Under the existing situation, the fish would start to produce

immature erythrocytes as a compensatory and adaptive reaction to deal with the challenge in an

attempt to transport more oxygen to the tissues [Oluah & Ulasi, 2010]. Significantly lower values

of RBC, Hb and Hct were reported as a result of possible disruption of haematopoiesis. Reduced

Hb may reflect metabolic adjustment according to reduced need for oxygen by change in blood

pH. Haemoglobin concentrations reflect the supply of an organism with oxygen and the

organism itself tries to maintain them as much stable as possible. Haematological indices

(erythrocyte count, concentration of haemoglobin and percent of haematocrit) are secondary

responses of an organism to heavy metals.

Reduction in hemoglobin values, indicated anemia in the heavy metal exposed fingerlings

could be due to erythropoiesis, hemosynthesis and osmoregulatory dysfunction or due to increase

in the rate of erythrocyte destruction in hematopoietic organs [Jenkins et al., 2003 and Sheth &

Saxena, 2003]. In the present study, decrease in RBC count might have resulted from severe

anemic state or hemolysing power of toxicant particularly on the red cell membrane. The

decrease in hemoglobin content in present study results from rapid oxidation of hemoglobin to

methaemoglobin or releases oxygen radical brought about by the toxic stress of the

pesticidechlorpyrifos. It is increasingly recognized that xenobiotics capable of undergoing redox

cycling can exert toxic effects via the generation of oxygen free radicals. Matkovics et al., [1981]

observed in cyprinus carpio a quick decrease in hemoglobin content in response to Paraquate

toxicity and suggested that it might presumably through methaemoglobin formation and a direct

response of oxygen radicals.

An increase in Hct can result from recruitment of erythrocytes from the spleen of fish

[Yamamoto, 1987; Yamamoto & Itazawa, 1989 and Wells & Weber, 1990]. Erythrocytes are

stored in the spleen and are expelled into the systemic circulation by contraction of the spleen

[Nilsson & Grove, 1974]. An increase in Hct can also result from erythrocyte swelling

[Nikinmaa, 1983 and Wells & Weber, 1990] and from the movement of water out of the plasma,

which results in haemoconcentration.

In this study, the concentration of haemoglobin in the red blood cells were much lower in

the exposed fishes than in the control fish, thereby depicting an anaemic condition. Increase in


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MCH and MCHC was attributed to direct or feedback responses of structural damage to red

blood cells membranes resulting in haemolysis and impairment in haemoglobin synthesis and

stress-related release of red blood cells from the spleen and hypoxia, induced by exposure to

toxicant [Shah, 2006].

These parameters could be effectively used as potential biomarkers of heavy metal

toxicity to the freshwater fish in the field of environmental biomonitoring. Heavy metals may

alter properties of hemoglobin by decreasing its affinity towards oxygen binding capacity

rendering the erythrocytes more fragile and permeable, which probably results in swelling

deformation and damage [Witeska & Kosciuk, 2003]. The results are in good agreement with

earlier work that reported a significant decrease in RBCs, hemoglobin and hematocrit of fresh

water fish exposed to heavy metals [Vutukuru, 2005 and Shalaby, 2007]. The manifestations in

these blood indices may be attributed to a defense reaction against toxicity through the

stimulation of erythropoiesis. The related decrease in hematological indices proved the toxic

effect of heavy metals that affects both metabolic and hemopoietic activities of Catla catla.

The distinct decrease in the level of haemoglobin and increase in the mean corpuscular

volume (MCV) which matches with Oreochromis hybrid exposed to aluminium [Bhagwant &

Bhikajee., 2000] clearly suggests that a haemodilution mechanism being operational. The

decrease in MCV with a low haemoglobin content indicates that red blood cells shrink, either

due to hypoxia or a microcytic anaemia. The macrocytosis is probably an adaptive response

through the influx of immature erythrocytes from the haematopoietic tissues to the peripheral

blood to make up the reduced RBC number and decreased haemoglobin concentration. These

findings further support the hypothesis that haemodilution is a probable cause for decrease in Hb

content in metal ion -dosed fishes. The MCHC is a good indicator of red blood cell swelling

[Wepener et al., 1992]. The decrease in the MCHC observed in the present study has also been

revealed on Oreochromis hybrid exposed to aluminium [Bhagwant & Bhikajee , 2000], in the

Clarias gariepinus due to effect of tobacco leaf dust [Kori-Siakpere & Oboh, 2011], and

Cyprinus carpio exposed to trichlorfon [Al-Ghanim et al., 2008] is probably an indication of red

blood cell swelling and/or to a decrease in haemoglobin synthesis. Other blood parameters such

as MCV and MCH increased considerably in all exposed fingerlings compared to the control

match with Clarias gariepinus (Burchell, 1822) exposed to lead [Adeyemo, 2007]


144

The significant decrease in MCHC after the 7days exposure period is probably an

indication of swelling of red blood cells / a decreased red blood cells swelling / a decrease in

haemoglobin synthesis. Bhagwant & Bhikajee, [2000] reported that prolonged reduction in

haemoglobin content is deleterious to oxygen transport and any blood dyscrasia and degeneration

of the erythrocytes could be ascribed as pathological conditions in fish exposed to tobacco leaf

dust. Also, fluctuations in the mean corpuscular haemoglobin (MCH) and mean corpuscular

volume (MCV) in the study clearly indicate that the concentration of haemoglobin in RBC was

much lower in the exposed fish that in the control thereby depicting an anaemic condition.

MCHC decrease indicates swelling of RBC.

These alterations have been attributed to direct or feedback responses of structural

damage to RBC membranes resulting in haemolysis and impairment in haemoglobin synthesis,

stress related release of RBCs from the spleen and hypoxia, which was induced by exposure to

lead. This study therefore gives an insight into toxic effect of lead on fish.The observed depiction

in the hemoglobin and hematocrit values in the fish could be attributed to the lysis of

erythrocytes.

Haemolysis is associated with the destruction of RBCs, and the formation of

methaemoglobin indicates a change to the ferric state. Both haemolysis and methaemoglobin

formation diminish the oxygen-carrying capacity of blood [Witeska & Kosciuk, 2003]. This

study suggests that waterborne heavy metals, initially bound to the gills and subsequently

deposited in other tissues, might affect the fish, even if toxic agents were removed from the

water. An increase in hematocrit levels could be explained as a typical stress response in metal-

exposed fish. A significant drop in leukocyte count, especially in fish exposed to different

metals, indicated the high sensitivity of the immune system to metal impact. It has been known

that copper and zinc induce a decrease in white blood cell count in fish [Witeska & Kosciuk,

2003; Dick & Dixon,1985; Vosyliene, 1996; Mishra & Srivastava, 1980 and Svobodova et al,

1993]. The hematological changes during the chronic toxicosis of edifenphos showed a

significant decrease mostly during the entire period in Hb, RBCs count and Hct. This reveals the

prominent anemic effect of edifenphos confirmed further by the blood indices. The chocolate

discoloration of parynchymatus organs is seen, as hemoglobin may be converted into

methemoglobin with resultant hemolysis and reduced blood oxygen carrying capacity causing


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respiratory distress to the fish. The severity of anemia also is magnified by the hypoproteinemic

effect showed by edifenphos. The haemolytic and destructive effects of pesticides on blood cells

were supported by El- Boushy, [1994] and Robert, [2001]. The RBCs, Hb and Hct reduced in

Oreochromis niloticus exposed to cadmium were less than that of the control. The RBCs count

decreased significantly in fish exposed to cadmium at 15 and 45 days. These parameters

returned to the normal values and increased significantly in fish exposed to cadmium with

EDTA. MCV increased significantly in fish exposed to cadmium alone, while the MCH and

MCHC decreased significantly in fish exposed to cadmium only when compared with the control

[Shalaby, 2007]. The calculated blood indices play a role in anemia diagnosis in most animals

[Coles, 1986]. The changes in these blood indices (increase MCV, decrease of MCH and

MCHC) may be because of attributed to a defense against cadmium toxicity through the

stimulation of erythropiosis [Moussa, 1999]. These results indicate that EDTA is effective in

removing cadmium from water , and reducing cadmium bioaccumulation in fish. Particulate

organic matter which can scavenge metal from water and help to reduce metal from fish. These

results are in agreement with Santschi, [1988] who studied that any agent that can remove

cadmium from water helps to reduce the bioaccumulation of this metal in fish.

Phosphatases play a major role in moulting physiology of many fishes. The functional

activity of these enzymes was found to increase during the exposure with heavy metals as an

adaptive response in mitigating the metal toxicity. The alkaline phosphatase is composed of

several isoenzymes present in practically all tissues of the body, especially in cell membranes.

Theset catalyse the hydrolysis of monophosphate esters and have wide substrate specificity.

Increased stimulation of alkaline phosphatase has previously been found in such pathological

processes as liver impairment, kidney dysfunction and bone disease [Kopp & Hetesa, 2000 and

Yang & Chen, 2003]. Alkaline phosphatase splits various phosphorous esters and its activity is

dependent on cellular damage. In the present investigation rise in the activities of phosphatases

in gills and kidney observed was seen similar to observed in Channa punctatus [Agrahari &

Krishna, 2009]. Increased level of alkaline phosphatase in Ni exposed Catla catla has previously

been found in Cyprinus carpio exposed heavy metal salt solution [Rajamanickam and

Muthuswamy, 2008] indicating its adaptive response to its leakage into the blood stream due to

the metal toxicity. This results are in accordance with the results on fresh water fish by Zikic

[1997]. Alterations in alkaline phosphatase and acid phosphatase activities in tissues and serum

http://scialert.net/asci/author.php?author=Adel%20M.E.&last=Shalaby


146

have been reported in pesticide treated fish [Palanivelu et al., 2005]. Increase in the levels of

ALP may be indicative of renal and liver damage [Bhattacharya et al., 2005 and Gill et al.,

1990]. ALP is basically a membrane bound enzyme. Increase in ALP activity in the organs of

nonylphenol (NP)-treated fish showed that nonylphenol could interact directly with the plasma

membrane and brought about alteration in its functions [Bhattacharya et al., 2008] same as in the

present results. The functional activity of this enzyme was increased during the exposure with

heavy metals as an adaptive response in mitigating the metal toxicity. Rise in ACP activity in

brain as observed in the present study has also been reported in stress and mercuric chloride

exposed Channa punctatus [Sastry & Sharma, 1980,1981]. In our study, ACP level in liver was

reduced in the exposed fingerlings probably due to the suppressed lysosomal activity in the target

organs. Decreased ACP level in liver was also found in Channa punctatus exposed to

monocrotophos [Agrahari & Krishna, 2009] and in Oreochromis mossambicus exposed to novel

organophosphorus insecticide [Rao, 2006a].

The impairment in the activities of acid and alkaline phosphatases could be part of an

overall biochemical manifestation of toxicity. It has been reported that even a minute quantity of

xenobiotics will affect the enzyme activity.

Transaminases play an important role in carbohydrate and amino acid metabolism in the

tissues of fish and other organisms [Atroshi, 2000]. ASAT and ALAT are the most important

enzymes acting as transaminases involved in amino acid metabolism and are known to be

sensitive to metal exposures [Almeida et al., 2001; Levesque et al., 2002 and Gravato et al.,

2006]. Also, the alanine aminotransferase has a part in transforming protein to glycogen, which

is the major reserve fuel of the body during the stress-induced toxicity in the liver ASAT and

ALAT are two key enzymes which are clinically important metabolic transaminases. These liver

specific enzymes are sensitive markers of hepatotoxicity/ histopathologic changes and can be

assessed within a shorter time [Balint, 1997]. In the present study, an increase in ALAT and

ASAT activities was observed only in kidney and decreased in liver and gills of Catla catla.

Similar results have also been reported in Channa punctatus exposed to MCP [Agrahari &

Krishna, 2009] and in different fish species such as Clarias albopunctatus and Carassius

auratus gibelio [Oluah,1998,1999 and Zikic et al., 2001]. This is in accordance with the findings

of Rao, [2006a] who reported similar enzymatic changes in Oreochromis mossambicus due to


147

monocrotophos stress. Elevation in the transaminases indicates the utilization of amino acids for

the oxidation or for glucogenesis [Philip, 1995] and is used to determine liver damage. ASAT

and ALAT activities diminished significantly below the control levels in the present work

probably due to cytolysis and enzyme leakage into the blood. Because of less availability of

blood enough plasma could not be obtained for enzyme assays, and therefore,, no data support

this assumption. All the three metals increased aspartate transaminase activity, but decreased

alanine transaminase activity in gill, liver, brain and intestine. Liver is the major site of metal

storage and excretion in fish. Due to its major role in metabolism and sensitivity to metals in the

environment, particular attention has been given to liver in toxicological investigations [Parvez

et al., 2006]. Significant increase in ASAT activity and decreases in ALAT activity seen in

present study match in Oreochromis niloticus with Oner et al., [2009] and may depend upon the

liver damage following metal stress and the effects are observed maximally at initial exposure

.Although ASAT level enhances by day 20 and day 30, the metal effects tend to decline. Various

responses of ASAT and ALAT activity have earlier been recorded for different metal species,

their concentrations and exposure durations [Zikic et al., 2001 and Vutukuru et al., 2007]. The

enhancement of the aminotransferase activity may occur in order to counter the energy demand

during metal stress, however decrease in its activity may be observed as a result of high metal

accumulations in the tissues. Thus, aminotransferases can be measured to assess the levels of

contamination in the environment and toxicity of metals before the appearance of detrimental

effects. In the present study, the increase in ASAT activity was observed in the liver of Catla

catla exposed to copper and mercury as has also been observed in Clarias lazera exposed to

vanadium [Zaki et al., 2007]. Alanine aminotransferase is a key metabolic enzyme released on

the damage of hepatocytes. The enzyme showed a decreasing level on the first day and from then

onwards its level increased steadily in the injured liver, indicating its adaptive response to the

leakage into the blood stream due to the metal toxicity. Significant increase in ASAT activity in

fish exposed to mercury could be due to possible leakage of enzymes across damaged plasma

membranes and/or the increased synthesis of enzymes by the liver. Increasesed ASAT activity in

liver seen in present study was also observed in Labeo rohita treated with arsenic and chromium

by Vutukuru et al., [2007]. ASAT level is a good index for the health status of liver

parenchymatous tissue necrosis as the main source of ASAT. Increased activity ALAT and

ASAT in kidney in the present work is similar with that of Channa punctatus (Bloch) exposed


148

to a chloroacetanilide herbicide [Tilak et al., 2009]. The increased trend in both ASAT and

ALAT activity indicates enhanced conversion of amino acids into keto acids than normally

utilized for energy synthesis [Tilak et al., 2003, 2009].

These results indicate that under the influence of different heavy metals ions or in a state

of stress, the damage of tissues and organs may occur with concomitant elevation and liberation

of transaminases into the circulation.

Stimulation of lactate dehydrogenase, an enzyme associated with the anaerobic pathway

of carbohydrate metabolism and has a fundamental role in anaerobic pathway of energy

production in the cell, catalysing the reversible conversion of pyruvate to lactate [Vassault,

1983]. In environmental studies it has been used, for example, to diagnose hypoxia [Wu and

Lam, 1997] and alterations in the processes of energy production [De Coen & Janssen, 2003].

LDH is an important glycolytic enzyme in biological systems and is induced by oxygen stress.

The level of LDH was found to increase in the gills and brain and decrease in the liver, kidney in

this study is in accordance with the findings on Channa punctatus [Agrahari & Krishna, 2009]

and Oreochromis mossambicus [Rao, 2006a] exposed to an organophosphorus insecticides. The

higher LDH activity was observed in brain and gill but the activity however decreased in liver.

LDH is also a marker of tissue damage and its elevated activity has been reported in liver

necrosis in fish [Ramesh et al.,1993]. This enzyme was released from the liver after its cellular

damage and failure due to organophosphorus insecticides intoxication [Ceron, et al 1997]. LDH,

ASAT, ALAT and ALP are released in acute and chronic liver disorders. These enzymes are thus

biomarkers of acute hepatic damage and their bioassay can serve as a diagnostic tool for

assessing necrosis of liver cells [Coppo et al., 2001-2002]. Decrease in enzyme activity may also

be related to hormonal level in the fish body. There might be decrease in the hormonal level

which would suppress enzyme activity in fish [Tomake, 1998].

Fish during depuration study demonstrated recovery in swimming behaviour. It can be

suggested that the respiratory stress caused by exposure to heavy metal salts results in the shut

down of routine swimming behaviour. It can also be presumed that the response was evoked

because locomotor behavior was reduced in response to low oxygen available. This is supported

by the evidence presented by Wilson et al., [1994b] who found damaged gill in juvenile rainbow

trout exposed to aluminium. Neville, [1985] observed similar moderation of swimming activity


149

in rainbow trout which was highly correlated with survival. These examples demonstrate the

potential advantages of pronounced changes in swimming behaviour in response to limitations in

oxygen uptake. This would have a considerable impact on energy metabolism and reduce the

possibility of exceeding anaerobic thresholds and therefore accumulating oxygen debt. Such a

strategy would have obvious benefits to fish with compromised respiratory capacity. The

advantages of such behavioural changes may not be solely linked to metabolism or energetic.

The adoption of less active swimming behaviour as in the present experiment could also form

part of a behavioural strategy to cope with heavy metal exposure.

Fish gills are the first target of waterborne pollutants such as heavy metal ions because

they come into direct and long-lasting contact with the water. In the depuration period, partial

recovery of gill structure was seen. Similar to these findings, some other fish also represent the

recovery pattern if reacclimatized in pollutant free water. The restored alterations observed in the

surface of gills of Catla catla in the present study match with the results on C. carpio, A. facetum

and A. fasciatus. exposed to cadmium during depuration [Ferrari et al., 2009]. The toxic effects

of lead on Catla catla were reduced by the treatment of DMSA which signified detoxification of

metal and helped to recover gill rays [Palaniappan et al., 2008]. The tropical fish Prochilodus

scrofa and juvenile fathead minnows exhibited gill recovery after copper ion exposure when

transferred into fresh water [Cerqueira & Fernandes, 2002 and Tate-Boldt & Kolok, 2008].

Transient structural changes in gills were also reported in Pimephales promelas, Prochilodus

scrofa and Oreochromis mossambicus exposed to copper [Pratap & Wendelaar Bonga, 1993;

Cerqueira & Fernandes, 2002 and Tate-Boldt & Kolok, 2008] and in Catla catla exposed to lead

[Palaniappan et al., 2008]. Kidney also showed recovery on reacclimatization. Ultra structure of

kidney and liver of silver carp also signify a reversible pattern after the depuration period of

microcystin bloom from water [Li et al., 2007]. Cypermethrin-induced histopathological

alterations in liver of Labeo rohita were reversible on withdrawal from cypermethrin [Sarkar et

al., 2005]. As mentioned earlier, liver has immense capacity to recover itself. Liver of fingerlings

reacclimatized into dechlorinated metal free water also showed good recovery as it is the major

site for the detoxification of all xenobiotics. Cyprinus carpio exposed to HgCl2 showed

histopathological recovery in liver after withdrawal of the HgCl2 treatment [Masud et al., 2009].

The liver cells of control fish exhibited normal histological appearance at the time of termination

of experiment. The hepatocytes of recovery group exhibited cytoplasmic vacuolization and


150

granulation with prominent nuclei suggesting resumption of the normal biosynthetic activities

almost to the level of control group. The hepatocytes showed the sign of regeneration. Common

carp, Cyprinus carpio and Nile tilapia showed good recovery in the histology of gills, liver and

intestine when reacclimatized in copper free water [Ajani & Akpoilih, 2010 and Shaw & Handy,

2006]. Recovery of brain in catla was also seen in rohu exposed to azo dyes [Barot, 2011].

Recovery patterns were also noticed in rainbow trout fingerlings [Fisk et al., 2000]. A little

recovery was observed in overall structure of intestine [Patel & Bahadur, 2011].

Above mentioned recovery in the target organs’ histology might be an indication of the

adapted immune system of exposed fingerlings. Changes observed in this study were reversible

and of moderate intensity. However, these may affect fish health, make them more sensitive to

environmental changes and the parameters evaluated can be used to monitor heavy metal toxicity

in Catla catla.

Heavy metal exposure is known to induce changes in blood parameters in fish [Heath,

1995]. Hematological studies have assumed greater significance because these parameters were

to be used as an effective and sensitive index to monitor physiological and pathological changes

induced by natural or anthropometric factors. The changes in red blood cells suggest a

compensatory response to respiratory surface reduction of gills in order to maintain oxygen

transference from water to tissues, allowing the fish to survive during stress. The direct effects of

copper on blood parameters are usually associated with increased erythrocytes crumbling or in

the case of more sensitive species, damage of the hemopoietic system [Svobodova et al., 1994].

There are numerous reports on the short-term effects of Cu exposure on fish hematology

[Mazon, 2002] but there are only a few papers that explore chronic copper effects and

physiology mechanisms of fish depuration using hematological parameters as sensitive index to

monitor the depuration capacity.

Nussey [2010] observed that erythrocytosis could be triggered by shortage of oxygen

during the exposure of lead. This would impose oxygen debt in fish, thereby promoting

anaerobic respiration resulting in high carbon dioxide level in blood. Under this prevailing

circumstance, the fish would begin to produce immature erythrocytes as a compensatory and

adaptative response to cope with the challenge in an attempt to deliver more oxygen to the

tissues.


151

The elevation of leukocyte count observed in mercury washed fingerlings reveals the

effect of the mixture of copper and zinc on Oncorhynchus mykiss [Bagdonas & Vosyliene,

2006]. Similar to this finding, a minute recovery in the haemoglobin value has also been reported

in common carp on the transfer into nitrite free water [Ajani & Akpoilih, 2010 and Shaw &

Handy., 2006]. Similar post exposed haematological parameters were reported by Singh and

Reddy, [1990] in Indian catfish, Heteropneustes fossilis (Bloch) reacclimatized in copper free

water. The cypermethrin exposed rohu also showed such a recovery pattern after post exposure

into freshwater for the period of 80 days [Adhikari et al., 2004].

Hypoxia as a result of reduction in respiratory actions leads in physiological changes in

blood factors to combat with lowering in oxygen in circulation for breathing and survival.

Elevating Hct and hemoglobin was similar to the reports for other anesthetized fish [Park et al.,

2009, Pirhonen & Schreck, 2003, Sandodden et al., 2001, Seol et al., 2007 and Gomes et al.,

2001]. WBCs were measured to evaluate clove essence effect on fish immune system. It showed

a decline trend associated with arresting in anesthetic in stage III in Acipenser persicus [Imanpur

et al., 2010].

Increase in haemoglobin and MCHC in the acclimatized fish might provide better

buffering of acidosis during short term increase in energy requirement as well as maintaining

oxygen carrying capacity. As haemoglobin is the principal buffering protein in blood, increase in

its content and MCHC could be part of a physiological strategy to cope with increased incidence

of acidosis. Acclimatization appears to allow a rapid response takes place is not yet understood

[Allin & Wilson, 2000].

In contrast, the aluminium-acclimatized fish demonstrated acclimatization in all of these

parameters by day 14 and no elevation of haematocrit or RBC numbers at the end of the pulse

exposure was observed. They did, however, demonstrate increases in the haemoglobin content of

the blood and MCHC, the trend opposite to the aluminium washed fish. Impairement of gas

exchange in fish exposed to acid and aluminium is known to result in hypoxemia, mixed

respiratory and metabolic acidosis, and ultimately complete respiratory failure [Walker et al.,

1988 and Witters et al., 1990]. Neville, [1985] also noted that respiratory acidosis in fish exposed

to acid and aluminium was counteracted by elevated levels of haemoglobin.


152

During the recovery period, the changes in red blood cells suggest a compensatory

response of this species to heighten the blood’s O2 carrying capacity. In conclusion, the changes

in the blood cell count reflect the responses to the effects of stress caused by copper and, after

transference to clean water, most of the changes are evidence of compensatory responses that

enable fish to recover from copper-related damage.

The recovered histopathological manifestations observed during depuration period were

clearly supported by the enzymatic activities in the respective target organs. The use of

enzymatic indices has been advocated to provide an early warning of potentially damaging

changes appeared relatively before the clinical symptoms produced by toxicants in stressed fish

[ Hedayati et al., 2010].

The recovery in the ACP, ALP, ALAT and ASAT enzymes activities seen in this present

study has also been reported in gill, liver and kidney, while LDH level recovered in liver, brain

and gills of insecticide RPR-V exposed Oreochromis mossambicus after 7 days recovery period

[Rao, 2006a]. ALAT and ASAT were restored in gills and liver of fish Clarias batrachus if

depuration was done after cypermethrin exposure [Begum, 2005]. Freshwater fish, Clarias

batrachus showed ALAT and ASAT recovery in liver and brain on reacclimatization in

carbofuran free water [Begum, 2004]. Hepatic ALAT and LDH levels were re-established in

Anguilla anguilla during depuration of propanil for 96 h [Sancho et al., 2009]. LDH activity was

also restored in Clarias batrachus when transfered in endosulfan washed dechlorinated

freshwater [Tripathi & Verma, 2004]. Indian catfish, Heteropneustes fossilis showed changes in

blood chemistry and were restored on the post exposure to copper sulphate [Singh & Reddy,

1990]. The carp showed functional enzyme activity recovered after the removal of copper

sulphate from the exposed medium [Karan et al., 1998]. In malathion depurated fish, the levels

of acid phosphatase and alkaline phosphatase activities progressively increased indicating a

probable recovery from the disruption of internal organ. It was more or less similar with present

observations [Thenmozhi, 2010].

El-Dermerdash, [2001] stated that HgCl2 intoxication significantly decreases the ACP and

ALP activities in rats. In liver, it is closely connected with lipid membrane in the canalicular

zone, so that any interference with the bile flow, whether extra-hepatic or intra-hepatic leads to


153

decrease in ACP and ALP activities. Inhibition of ACP and ALP activities is due to increased

necrosis in the tissues like hepatocytes [ Thenmozhi, 2010].

Finally, from this study, it was concluded that the toxic effects of heavy metals were time

and concentration dependent. But exposure during depuration study, the changes more or less

recovered to reestablish normal physiology of body. Here, all performed studies (behavioral,

histopathological, hematological and enzymatic) well correlated with one another so as to

understand post exposure recovery pattern in copper, nickel and mercury exposed fingerlings.

Biosorption is the ability of biological materials to accumulate heavy metals from

wastewater through metabolically mediated or physico-chemical pathways of uptake [Naddafi et

al., 2007; Fourest et al.,1992,1994]. The equilibrium of sorption is an important physico-chemical

parameter for the evaluation of the sorption process The equilibrium sorption studies determine

the capacity of the sorbent. The results indicate that the time taken to reach equilibrium is longer

as the concentration of the Cu (II) and Ni(II) increases. It also shows that at higher

concentrations the amount of Cu (II) and Ni(II) adsorbed is much higher than at lower

concentrations. The equilibrium is established when the concentration of adsorbate in the bulk

solution is in dynamic balance with that of the interface [Amarasinghe et al., 2007]. The number

of adsorption sites or surface area increases with the weight of adsorbent and hence results in a

higher percent of metal removal at a high dose.

Biosorption of metal ions using calcium alginate beads was rapid during the first 20 min

and 30 min for copper and nickel, respectively. Afterwards, the adsorption of metal ions almost

reached a plateau. These results show that the rate of adsorption of both the metal ions rapidly

increased and finally attained equilibrium state. Biosorption of mercury was not carried out due

to it being colourless. Retention of metal ions was proportional to contact time when

concentration of metal ions and adsorbent mass were kept fixed. For both the metal ions, the

adsorption rate decreased after the equilibrium time. The removal rate of adsorption is rapid

initially but it gradually decreases with time until it reaches equilibrium [Pandey et al., 2009]. In

other words, desorption of metal ions occurs after equilibrium time and it might be due to the

saturation at the surface of the adsorbents with metal ions. This result matches with copper and

nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide

(EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the


154

immobilized C. luteola TEM05 from aqueous solutions [Ozdemir et al., 2005]. At low ion

concentrations the ratio of surface active sites for the metal ions in solution is high and hence

metal ion may interact with the adsorbent and be removed from the solution. Similar results were

seen during removal of Copper (II) from aqueous solutions using teak (Tectona grandis L.f)

leaves [Rathnakumar et al., 2009]. The removal of Cu (II) increases with time and attains

saturation after a given contact time.

One of the parameters that strongly affect the biosorption capacity is the adsorbent

amount. In present study, the rate of adsorption was dependent on quantity of calcium alginate

beads. The metal ions removal rate increased with the increased adsorbent dose from 0.15g to

0.6g. then it became stable. Similar trend has also been reported during adsorption of copper and

nickel ions on chitosan coated PVC beads by Popuri et al., [2009] , Cu(II) sorbed by STL sorbent

by Bajpai & Jain, [2010], during removal of Cu by tea waste as a low cost adsorbent

[Amarasinghe & Williams, 2007], and also during removal of azo dye on pure chitosan and

chitosan coated calcium alginate beads [Barot, 2011]. However, the uptake capacity of metal ion

per unit mass of adsorbent decreases with increase in its dose and may be due to lower capacity

at higher dosage. The drop in adsorption capacity is basically due to sites remaining unsaturated

during the adsorption reaction.

pH is one of the most important environmental factor influencing not only site

dissociation, but also the solution chemistry of the heavy metals; hydrolysis, complexation by

organic and/or inorganic ligands. Redox reactions, precipitation are strongly influenced by Ph. It

strongly influences the speciation and the biosorption availability of the heavy metals [Chen et

al., 2008]. The pH of solution is an important controlling parameter in the adsorption process.

The adsorption of copper ions and nickel ions was maximum at pH 7 and pH 5, respectively.

Similar results were also seen during biosorption of copper and nickel on the immobilized

biomass [Al-Saraj et al., 1999; Blanco et al., 1999 and Yan & Viraraghavan, 2001] and during

biosorption of copper by composite membranes [Genç et al., 2003]. Maximum uptake of Ni(II)

ions occurs at pH 5 which matches with removal of nickel ions from water using chitosan coated

PVC beads [Popuri et al., 2009]. The adsorption capacity increased with increase in pH of the

solution. The low level of metal ion uptake at lower pH could be attributed to the increased

concentration of hydrogen ions which compete along with Cu(II) and Ni(II) ions for binding


155

sites. As the pH is lowered, the overall surface charge on the beads becomes positive, which will

inhibits the approach of positively charged metal cations. At pH above the isoelectric point, there

is a net negative charge on the surface and the ionic point of ligands such as carboxyl, hydroxyl

and amino groups are free so as to promote interaction with the metal cations [Quek et al., 1998].

This would lead to electrostatic attractions between positively charged cations such as Cu(II) and

Ni(II) and negatively charged binding sites [Popuri et al., 2009]. The metal ion removal is fast

and highly effective during the initial phase. Subsequently it decreases, as a consequence of the

progressive saturation of the binding sites.

The adsorbate ions would undergo lateral repulsion with time at these pH values,

accounting for a low degree of sorption. As the pH of the system increases, the number of

negatively charged sites increases and the number of positively charged surface sites decreases.

Negatively charged sites on the adsorbent favor adsorption of metal ions. In all cases, the

maximum heavy metals adsorption occurred when the pH was between 5.0 and 7.0. It has been

confirmed in previous studies [Bailey et al., 1999 and Schiewer & Volesky, 1997] that ionic

strength plays an important role in metal ion uptake by biosorbents. As alginate is a highly

charged polymer, negative charges due to the ionized functional group of the alginate (–COO)

will interact with anions in the proximity of the polymer making their concentration in the

vicinity of the surface lower than that of the bulk solution. The basic mechanism involved in the

heavy metal ions sorption form dilute solutions using biosorbents such as alginate is considered

to be ion exchange due to the electrostatic interactions between the metal cations and the anionic

carboxy groups existing in the polymeric matrix of the polysaccharide. Since binding of a metal

species onto a well defined number of available binding sites of the material results in decreased

availability of binding sites for other metal cation existing in the solution, the mechanism

involved is strongly competitive. Therefore, metal species with greater affinity for the carboxylic

groups will be preferentially sorbed over species with lower affinity for the sorbent. The increase

in biosorption levels with an increase in pH can be explained by the surface charge of the

adsorbent and the H+ ions present in the solution. At low pH values, the surface of adsorbent

would also be surrounded by hydronium ions, which decrease the Cu(II) interaction with binding

sites of the by greater repulsive forces and therefore lower adsorption. In contrast, when the pH

was increased, the competing effect of hydrogen ions decreased. With an increase in pH,

percentage sorption and metal uptake (q) also increased. At lower pH values (pH ≤ 5.0),


156

biosorption was not favourable and also the H+ ions competes strongly with metal ions for active

sites [Matheickal et al., 1999].

The adsorption rate of both the metal ions decreased with the rise in temperature. Similar

results were also reported during removal of copper (II) from aqueous solutions using teak

(tectona grandis l.f) leaves [Rathnakumar et al., 2009], during the adsorption of azo dyes [Barot,

2011] and the dye brilliant green [Nandi et al.,2009]. In constrast to our results, the adsorption of

copper ions increasd with increasing in temperature [Pandey et al., 2009], adsorption of Ni(II),

Cu(II) and Fe(III) from aqueous solutions using activated carbon[Edwin Vasu, 2008] and

adsorption of a dye using sludge adsorbent and activated carbon fibers [Chiang et al., 2009]. The

increased adsorption at higher temperatures could be due to one or more of the following

reasons. Acceleration of some originally slow step(s)[Khalid et al.,1998]; creation of some new

activation sites on the adsorbent surface [Khalid et al.,1999]; and decrease in the size of the

adsorbing species[Johnson, 1990]. This could well occur due to progressive desolvation of the

adsorbing ion as the solution temperature increases. This indicates that the adsorption is

accompanied by a chemical reaction. It is restricted to just one layer of molecules on the surface,

but may be followed by additional layers of physically adsorbed molecules [Latif & Fanous,

2004].

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