Chapter IV Discussion
133
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 .
Chapter IV Discussion
134
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
Chapter IV Discussion
135
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
Chapter IV Discussion
136
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
Chapter IV Discussion
137
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
Chapter IV Discussion
138
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]
Chapter IV Discussion
139
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
Chapter IV Discussion
140
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
Chapter IV Discussion
141
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
Chapter IV Discussion
142
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
Chapter IV Discussion
143
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]
Chapter IV Discussion
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
Chapter IV Discussion
145
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
Chapter IV Discussion
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
Chapter IV Discussion
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
Chapter IV Discussion
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
Chapter IV Discussion
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
Chapter IV Discussion
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.
Chapter IV Discussion
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.
Chapter IV Discussion
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
Chapter IV Discussion
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
Chapter IV Discussion
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
Chapter IV Discussion
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),
Chapter IV Discussion
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].