UGC Sanctioned Major Research Project Entitled · Often, such wastes also contain significant...
Transcript of UGC Sanctioned Major Research Project Entitled · Often, such wastes also contain significant...
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A Report on
UGC Sanctioned Major Research Project Entitled
Submitted to,
By
Dr. M. David Ph.D.Principal Investigator
UGC Major Research Project, Professor
Department of Zoology Karnatak University, Dharwad- 580003
Karnataka, India 2019
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Dr. M. David Professor Department of Zoology Karnatak University (University with Potential for Excellence) Dharwad, Karnataka 580003, India Email: [email protected]; Ph: 0836- 2215230 Mobile: 09845709815
Declaration
I hereby declare that the project entitled “Toxicological endpoints of
sodium cyanide on freshwater fish Cyprinus carpio (Linnaeus) under
sublethal exposure” with F. No. 41-103/ 2012 (SR) comprises original work
carried out under my supervision at Department of Zoology, Karnatak
University, Dharwad, Karnataka, India. This report has not been submitted
for research organization or board.
Date:
Place: Dharwad
(PRICIPAL INVESTIGATOR)
mailto:[email protected];
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PREFACE
Environmental pollution has turned in to a global distress. The aquatic environment is
continuously being contaminated with toxic chemicals from industrial, agricultural and domestic
activities. Cyanides are one of the major classes of toxic substances used on a large scale in the
mining, electroplating, printed circuit board manufacturing, steel and chemical industries.
Consequently, these industries, discharge large quantities of cyanide-containing liquid waste.
Often, such wastes also contain significant amounts of heavy metals, viz., copper, nickel, zinc,
silver and iron. Cyanide readily combines with most major and trace metals, property that makes
it useful in extracting metals from ores. And because cyanide is carbon based compound, it reacts
readily with other carbon based matter, including living organisms. Owing to the highly reactive
nature of cyanide ions, metal complexes of variable stability and toxicity are readily formed. Since
the formation of metal cyanide complexes does not eliminate the toxicity of cyanide, these must be
removed from wastewaters prior to their discharge in the environment. Cyanide toxicity resulted
primarily from inhibition of cytochrome oxidase, the terminal enzyme of the mitochondrial electron
transport chain, resulting in histoxic hypoxia and impairment of energy production. In natural
waters, cyanide may affect fish populations by direct lethal or through sublethal toxic effects due
to the large pollution of industrial wastes.
The extent of cyanide toxicity in fish depends mainly on the rate of its detoxification in
vivo. Fish occupy a prominent position in the field of toxicology; in studies concerning both human
and ecological health. Sublethal concentrations that do not cause death over the short term but do
harm the individual, thus making it expend resources to survive in a state of altered equilibrium.
Much of the toxicological interest in cyanide has been focused on its rapid lethal action; however,
the most widespread problems arising from cyanide are from chronic/ sub chronic exposures. Thus
an attempt has been made in the present study to investigate the effect of sodium cyanide on
freshwater fish, Cyprinus carpio under sublethal exposures.
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
General introduction
Environmental pollution has become an issue of global concern (David and Kartheek,
2015). With growing industrialization, the additional effects on viable organisms has become
important than never before (David and Kartheek, 2014). Industrialization and large scale
production and introduction of toxic pollutants like pesticides into environment dates back to
mid-sixties of 20th century along with inputs for propagating green revolution package in
Indian agriculture (Bhardwaj and Sharma, 2013). However, the process of metal extraction and
purification dates back to thousands of years, during which, large amount of heavy metals, and
cyanide residues were known to be released in to environment. Nevertheless, the large scale
industrialization and the rapid destruction of environment could be observed only in recent
times. The primary reason of environmental chemical pollution could be due to the dependency
on chemical compounds which has gradually resulted in contaminating environment.
Sodium cyanide, which is the simple salt of sodium and cyanide components is
an extremely toxic, respiratory toxicant which might hamper the fish health through the
impairment in the metabolism, sometimes leading to death. This is because, most of the
cyanide absorbed is detoxified by enzymatic combination with sulfur, and thus the
detoxification process imposes a nutritional cost (Prashanth and Neelagund, 2007).
Concentrations of cyanides and lethal and sublethal toxicities of cyanides to the fishes
are well documented (Dube and Hosetti 2010). Further it is also essential to evaluate
the sublethal effects of cyanides on the fishes, since they form very important members
of the food chain. Consequently it was considered as a matter of warranted interest to
elucidate biochemical effects in the fish. Evaluation of biochemical activities in an
organism helps to identify disturbances in its metabolism.
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Cyprinus carpio (C. carpio) is known for its importance as a staple food and due
to the fact that the toxic effects of sodium cyanide on the fish species, particularly, C.
carpio has been scarcely studied so far. Even though few studies have been reported on
NaCN impact on C. carpio (David and Kartheek 2014) the toxicity studies in this fish
through different approaches like ROS, embryo toxicity, histopathology etc., is almost
ignored. Fishes in particular C.carpio is of priority in toxicity studies due to its high
nutritive value by which it is of primary importance in southern India, especially in and
around the mining areas of Hutti gold mines, where millions of gallons of water
containing high concentrations of sodium cyanide are recycled and let in to streams
after the utilization for ore extraction. This species is known to be resistant and
adaptable to different temperatures (Castagnolli and Cyrino 1986), however, the toxic
gestures of liver in fish C. carpio towards sodium cyanide are found to be very limited.
Hence in the present investigation, an effort is made to elucidate the toxic effects
of sodium cyanide on freshwater fish C.carpio following its exposure to sublethal
concentrations.
Materials and Methods
Healthy Cyprinus carpio were procured from the State Fisheries Department,
Dharwad, India and were acclimatized to laboratory conditions for 15 days at 23-27 °C.
Further they were held in dechlorinated tap water in large cement tanks which was
previously washed with potassium permanganate to free the walls from any microbial
growth. Fish were fed regularly and 12–16 h of photoperiod was maintained during
acclimation. Water was renewed daily, whose physico-chemical characteristics were
analyzed following the methods mentioned in APHA (1989).
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Characteristics of the water were determined by the methods mentioned in
APHA (1989). Water was renewed every day with 12-12 h dark and light cycle was
maintained and fish were fed (ad libitum) daily with commercial dry feed pellets (Nova,
Aquatic P. Feed) during acclimatization and test periods. But for acute toxicity test,
feeding was stopped two days prior exposure to the test medium. During acclimation
batches with less than 5% of mortalities were only considered for further
experimentations.
Experimental toxicant and experimental design
Sodium cyanide of 95% purity was procured from Loba Chemie Pvt. Ltd.,
Mumbai, India. Stock solution was prepared by dissolving sodium cyanide in double
distilled water in a standard volumetric flask. Water was renewed every day over test
periods. Henceforth, the replacement of the water medium was followed by the addition
of the desired dose of the test compound. The fish were exposed in batches of 10 to a
fixed concentration of sodium cyanide with 20 L of water in three replicates for each
concentration. One fifth (0.2 mg/L), one tenth (0.1mg/L) and one fifteenth (0.066mg/L)
and one twentieth (0.05mg/L) of the LC50 (1mg/L) was selected as sub lethal
concentrations for studies and the duration of exposure were 10 and 20 days. Further,
the fish were allowed to undergo a recovery period of 14 days. This study was
conducted under OECD Guideline for static-renewal test conditions (1992). At the end
of 10 and 20 days and that of post recovery of 14 days, fish were sacrificed and were
sampled for further studies.
Mortality was recorded every 24 h and the dead fish were removed when observed,
every time noting the number of fish death at each concentration up to 96 h for
estimation of acute toxicity (LC50). Statistical analysis using ANOVA was employed
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
for comparing mean mortality values. Time of exposure was the repeated measure factor
while treatment (concentration and control) was the second factor. The LC50 was
calculated using probit analysis (Finney, 1953), which has been recommended by
OECD guideline as an appropriate statistical method for toxicity data analysis (Lilius,
et. al, 1994). After linearization of the concentration response curve by logarithmic
transformation of concentrations (log+2), 96 h LC50 with 95% confidence limits and
slope function were calculated to provide a consistent presentation of the toxicity data.
Tissues selected
Biochemical parameters: Gill, Muscle and Liver.
Histopathology: Liver, kidney and spleen
Ultra-structural pathology: Liver, kidney and gill
Fishes were exposed to their respective concentrations of sodium cyanide and
were maintained in these concentrations up to the stipulated period of exposure. At the
end of exposure the fishes were stunned to death and the target organs were dissected
out from each animal using sterilized instrument. The organs were transferred into ice-
jacketed micro beakers containing fish ringer solution. The fish ringer was prepared as
per the composition given by Ekenberg, (1958). An equilibration time of 15 minutes
was allotted to the organs to regain normalcy from a state of shock, if any, due to the
handling and dissecting procedures. The experiment was repeated for six times and
results were analyzed.
Protein metabolism
The levels of soluble, structural and total proteins, free amino acids, ammonia,
urea and glutamine were estimated in the gill, muscle, liver of fish under this study.
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Estimation of soluble, structural and total proteins: The soluble, structural and the total
proteins in the organs were estimated using the Folin-Phenol reagent method as
described by Lowry et al., (1951).
Estimation of free amino acids:
Free amino acid levels in the tissues were estimated by the ninhydrin method as
described by Moore and Stein (1954).
Estimation of protease activity:
Protease activities in the tissues were estimated by the Ninhydrin method as
described by Davis and Smith (1955).
Ionic composition and associated ATPases
The levels of sodium, potassium and calcium ions and the activities of Na+- K+
ATPase, Mg2+ ATPase and Ca2+ ATPases were estimated in gill, liver and muscle of
fishes.
Estimation of sodium, potassium and calcium ions: The weighed organs were wet ashed
in 50:50 (V/V) concentrated perchloric acid and nitric acid (Dall, 1967).
Na+- K+, Mg2+ and Ca2+ ATPase activities (ATPase phosphorylase EC. 3.6.1.3.):
Na+- K+, Mg2+ and Ca2+ ATPase activities were estimated separately in the
organs by the method described by Watson and Beamish, (1981) with slight
modification. The inorganic phosphates liberated were estimated by the method of Fiske
and Rao, (1925).
Superoxide dismutase activity
Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured by
methodology as described by Kakkar et al. (1984).
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Catalase activity
Catalase (CAT; EC 1.11.1.6) activity was determined by measuring the decrease
of hydrogen peroxide concentration at 240 nm according to Luck (1963).
Glutathione peroxidase activity
Glutathione peroxidase (GPx; EC 1.11.1.9) activity was measured by the method
of Paglia and Valentine (1967).
Glutathione S-transferase activity
The glutathione S-transferase activity was assayed by the method of Habig et al.
(1974).
Malondialdehyde
Malondialdehyde (MDA) the secondary product of lipid peroxidation was
estimated in the tissue homogenates utilizing the colorimetric reaction of thiobarbituric
acid (TBA) (Buege and Aust, 1978).
Statistical analysis
The antioxidant activities are reported as the mean ± standard error of the mean
(SEM) obtained from triplicates. The data were subjected to one-way analysis of
variance and further subjected to Tukey’s test for post hoc analysis by defining the
significance level at p< 0.05.
Ultrastructure of liver and kidney
Treated and control liver of fish were isolated by perfusion and fixed in buffered
glutaraldehyde (2.5%; pH 7.2). The organs were stored in the sodium cacodylate buffer
at 4 oC (pH 7.4, 0.1M), washed in buffer and post fixed in the 1% osmium tetraoxide
for one and half to two hours. Then again washed with buffer, dehydrated in alcoholic
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
series for 1 h, stained enblock in 2% uranyl acetate, in 90% methanol for 1 h and cleared
in propylene oxide for 10-15 min and in-filtered with araldite: propylene (1:1) mixture
for overnight. Then in-filtered again with fresh araldite (3 changes with a gap of 3-4 h)
and embedded in the same media in a beam capsule. The blocks were cut in Leica LKB
Broma ultramicrotome. Ultrathin sections were cut at 100-300 Å, mounted on copper
grids and stained with 1% aqueous uranyl acetate and lead citrate (Reynolds, 1963). The
stained sections were scanned in Jeol – TEM 100 C X II electron microscope for
ultrastructural observations.
Embryo toxicity
Embryo-larval toxicity testing was performed according to OECD guidelines
210 (Fish, Early-life Stage Toxicity Test). Fertilized eggs of common carp were
obtained from State fisheries department, Neersagar, Dharwad. Eggs were produced
according to standard methods of artificial reproduction (Kocour et al. 2005). A semi-
static trial with solution replacement twice daily was used. Replacement was conducted
with care to avoid interfering with development of embryos and larvae.
Scoring Eggs for Embryo-toxicity
At the end of each experiment, eggs were inspected under a compound
microscope. Double blind scoring was performed to reduce bias by covering the
treatment vials with tin foil and randomly assigning a numbers to each vial. The vials
were shuffled and selected randomly for scoring. The eggs were scored for signs and
severity of embryo-toxicity. Signs of pathology included: pericardial and yolk sac
edema, abnormal eye development, fin rot, spinal deformities, body and yolk
haemorrhaging, and craniofacial deformities.
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Calculating Embryo-toxicity Score
Individual fish scores for signs of pathology were averaged to give embryo-
toxicity scores for live fish in each treatment. Mortalities after day 0 were assumed to
be associated with embryo-toxicity due to NaCN exposure. Dead fish were assigned the
maximum score for embryo-toxicity (11) plus 0.5; therefore the highest possible
embryo-toxicity score when mortality was considered was 11.5.
Objective 1
Collection and maintenance of fish
Healthy Cyprinus carpio were procured from the State Fisheries Department,
Dharwad, India and were acclimatized to laboratory conditions for 15 days at 24± 2 °C.
Each fish was investigated for presence of any parasite and made sure that no such
contaminants were accompanied. The fish were transferred to cement tank with volume
capacity of 3000 litres. Further they were held in dechlorinated tap water in large cement
tanks which was previously washed with potassium permanganate to free the walls from
any microbial growth. Fish were fed regularly and 12–12 h of photoperiod daily during
acclimation.
Objective 2
The water used for the experimentation throughout the tenure was of freshwater
source. The dechlorination process of stored water was carried out by constant aeration.
The fish were later transferred to the large tanks containing this dechlorinated water.
Fish were later separated as per the requirements of number of concentrations required
and additionally a control group was maintained. Care was taken so as to avoid
conditions of turbidity that might has otherwise resulted from frequent photosynthesis
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
process. The fish throughout the tenure was investigated for any kind of parasitic
infections and appropriate measures were taken to ensure that no such conditions
occurred.
Water was renewed daily, whose physico-chemical characteristics were analysed
following the methods mentioned in APHA (1989) and were found as mentioned below.
Values for quality assessment of water used for the present investigation.
Objection 3
Proteins are nitrogen-containing substances that are formed by amino acids
(Parma de et al., 2002). They serve as the major structural component of muscle and
other tissues in the body. In addition, they are used to produce hormones, enzymes and
hemoglobin. Proteins can also be used as energy; however, they are not the primary
choice as an energy source (Bashamohideen 1988). Amino acids are essential
intermediates in protein synthesis and its degradation products appear in the form of
different nitrogenous substances.
Parameter Values obtained
Temperature 24 ± 1º C
pH 7.1 ± 0.3
Dissolved oxygen 6.1 ± 0.4 mg/L
Total hardness 37.3 ± 3.1 mg/L
Salinity Nil
Specific gravity 1.003
Calcium 21.31± 0.27 mg/L
Phosphate 0.9 ± 0.04 mg/L
Magnesium 0.85 ±0.3 mg/L
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
The results obtained for changes in protein levels demonstrated the decline in terms of
their concentration for both liver and gill organs. The overall decline which was seen under the
presence of NaCN indicated the severe toxicity to fish metabolism. It is to be noted that the
decline observed for total, soluble and structural protein was found to be duration and
concentration dependent. The highest decline was obtained for gill tissue at 20 days of exposure
while the lowest variation was noted for 1/20th concentration. The fish under 1/10th sublethal
concentration also suffered from decline in terms of their protein concentration. However, the
intensity of the change was found to be much less as compared to the 1/5th concentration. The
results present suggest the decline was the result of NaCN intoxication to the fish C. carpio
which was on purpose exposed to four sublethal concentrations respectively.
The effects of chronic cyanide toxicity on the growth, metabolism, reproduction,
and histopathology of freshwater fish has been investigated (Dixon and Leduc 1981)
and the results of the present work indicate that sublethal cyanide exposure led to
alterations in some blood biochemical parameters that may help to determine mode of
action of toxicant and organ dysfunction following cyanide exposure.
Based on the present results, cyanide exposure at the dose of 0.2 mg/L caused
significant increase in glucose concentration in common carp that is reminiscent to the
results reported previously in swine and rats (Tulsawani 2005). On the other hand, no
alterations in plasma glucose were observed following chronic cyanide exposure in
goats, rats and rabbits (Soto-Blanco and Gorniak 2003). Based on the present study
results, increased creatinine concentration was observed following cyanide exposure
that might be associated with kidney damage due to cyanide exposure. In line with this
finding significant increases in serum creatinine concentrations have been reported
following KCN administration in rats (Elsaid and Elkomy 2006) and pigs (Manzano et
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
al. 2007). It has been also reported that degenerative changes in the kidney sections of
the cyanide-fed rabbits may be responsible for the significant increases in serum urea
and creatinine observed in these animals (Okolie and Osagie 1999).
Objective 4
Biochemical markers of contamination are important indices used in fish toxicity
tests and for field monitoring of aquatic pollution (David and Kartheek, 2015). The
concern of aquatic pollution arises as the, potential harmful chemicals or substances
such as heavy metals, pesticides and hydrocarbons are dumped either or released into
the water bodies (Ullah et al., 2014). When these pollutants flow into water bodies in
higher concentration than permissible limits then these result in the form of heavy
mortalities of all life form residing in those aquatic systems such as fish and shell fish
etc. while in lower concentration these lead to bio accumulation of these pollutants and
ultimately go through the food web to human beings (Abedi et al., 2013). This issue is
attention seeking and should be treated and focused properly and attentively in order to
ensure safer fish consumption on priority basis (Ullah et al., 2014).
The results for ionic content in tissues of fish under control and exposure regime
indicated significant difference. The cause of the present condition was attributed to the
intervention of NaCN content in the physiological and metabolic participation. The
results obtained for changes in ionic content and their respective ATPases indicated the
catastrophic action of NaCN on fish metabolism in terms of their concentration in the
respective organs. The decline in ionic content as highest was noted at E1 under 1/5th
sublethal exposure, followed with E2. This was followed with the group of fishes under
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
1/10th sublethal group which also showed certain degree of variation towards the ionic
balance. The next two sublethal exposures did not show much variation which could be
speculated due to the lesser concentration of NaCN content. The ATPases level also
varied in similar fashion with respect to the concentration of toxicant and time of
exposure. The last group under which the fish were exposed to 1/20th sublethal
concentration did not indicate the significant variation in any of the exposure durations
which further indicates the safer levels of concentration that the fish could possibly
endure and survive without losing the equilibrium in terms of ionic content and its
respective ATPases.
Experiments have shown higher effect of pesticides in protein contents, in
different tissue such as gills, liver, blood, intestine and muscle of various fish species,
such as nickel caused decrease in protein level of Heteropneustes fossilis (Nanda et al.,
2000), nickel chloride caused appreciable decrease in gonads, liver and muscles of
Anabus testudineus ( Jha and Jha, 1995), phenyl mercuric acetate caused reduction of
protein level in muscles and liver of Channa punctatus (Karuppasamy, 2000) while the
same species showed low protein level when exposed to oleandrin and arsenic.
Objective 5
Cyanides are components of electroplating solutions, fertilizers, fumigant
mixtures, metal polishes and rodenticides. As oxidative damage is mediated by free
radicals, it was necessary to investigate the status of endogenous antioxidant enzymes
under different treatment conditions. Aerobic organisms possess antioxidant defence
systems that deal with ROS (Mates et al., 1999). SOD catalyzes the conversion of the
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
highly reactive superoxide anion to O2 and to H2O2 (Mates et al., 1999). Taken together
the mechanism of injuries resulting from inhibition of mitochondrial respiration
includes a cytotoxic pathway that arises partly from an energy deficit but also from
reductive stress which releases non protein bound iron from intracellular pools and
induces cytotoxic ROS formation (Niknahad et al., 1995). Thus it is necessary to
investigate the disastrous effects of cyanide on the antioxidant status.
The antioxidant enzyme status in the fish exposed to different sublethal
concentrations of NaCN for 10 days (E1) and 20 days (E2) and fish that underwent a
recovery period of 14 days (R) was found to significantly vary (p
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
of blunting the low levels of oxidant stress while the role of CA is more significant in
protection against severe oxidative stress (Mates, 2000). Therefore, decrease in the
activity of CA in KCN treated cells may be due to elevated levels of superoxide anion
radical as a result of a reduction in SOD activity (David and Kartheek, 2016). Oxidative
species such as hydrogen peroxide, superoxide, or free radical intermediates play a
crucial role upstream of apoptosis activation. Oxidative stress is a key factor in cyanide-
mediated apoptosis, partly by activation of redox-sensitive transcription factor NF-kB
(Shou et al., 2000).
Cyanide exposure is associated with calcium-dependent production of hydroperoxides
and lipid peroxides, and its treatment has been shown to cause relatively prolonged
elevation of conjugated dienes in kidney (Ardelt et al., 1994).
Cyanide is known to induce oxidative stress by depleting the cellular thiol (Gunasekar
et al., 1996).
Objective 6
Transmission electron microscopy (TEM) has become a powerful tool for
characterizing the structure of materials, both inorganic and organic. In the quest to
attain atomic resolution and to link this information to the materials macroscopic
properties, we have witnessed major improvements in technology and methodology
over the past decades. With inorganic solids, the characterization of interfaces or of
defects at the atomic level has become almost routine. Histological studies on fish have
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
revealed that various toxicants have produced pathological changes in the tissues such
as macrobiotic changes in the liver, tubular damage of kidneys, gill and lamellar
abnormalities (Ramalingam, 2000). Review of Hodgson and Meyer, (2010) reported
wide array of structural features identified as alerts for hepatotoxicity are present among
the diverse chemical structures comprising the pesticides. Liver toxicity exist among
members of the various use classes including acaricides, algicides, fungicides,
herbicides, insecticides, molluscicides, nematocides and rodenticides.
The results from the present investigation indicate the toxic effect of NaCN on
different tissues of the exposed fish. The damage which was observed in the exposed
fish was found to be concentration and duration dependent. The changes that were
observed in the sections suggested the possible destruction of mitochondrial complexes
in liver of fish. The cytoplasmic destruction is indicated by the occurrence of more and
more vacuoles which are found to be in exposed fish, unlike control. The same have
been shown in the Plate 1 - 4.
The ultrastructural observation on C. carpio treated with NaCN showed
abundant vesicle structures, interruption and loss of basement membrane, swelling of
nuclear envelope with different electron dense nuclei, mitochondria with mixed electron
density matrix,myelin-like structures, lysosomes, autophagic vacuoles and high
electron density of microvilli grounds suggesting a high metabolic activity. Different
alterations were observed at the ultrastructural level for C. carpio, including
cytoplasmatic vacuolation of epithelial cells from convoluted proximal tubules and
increased autophagic vacuoles. Other cells showed less abundant mitochondria
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
interdigitations on the area nuclei near the basal border. Myelin-like figures were
observed in the capillary endothelial cell cytoplasm.
Ultrastructural observations revealed disorganization hepatocytes and the
organelles within the cytoplasm at lethal and sublethal concentration of cypermethrin
exhibiting acute and subacute effects on the liver, leading to the disorder and chaos in
cytoskeleton organization suggesting a strong toxic effect in hepatocytes. Observed
ultrastructural changes are molecular lesions as result of complex molecular interactions
with cypermethrin, consequently affecting cellular physiology. Since hepatocytes are
involved in various aspects of intermediate metabolism of proteins, carbohydrate and
lipids.
Loose arrangement of hepatocytes with increased sinusoidal space observed in
the lethal concentrations may affect the exchange between hepatocytes and sinusoids
(e.g., distribution and movement of nutrients, chemical signals or other physiological
components), important components of cell physiology and stability of the organ
function. Changes observed in the hepatocyte nuclei and condensation of
heterochromatin could be the result of the accumulation of cypermethrin in this organ
as observed in various contaminants. The morphological effects observed in nuclei are
evidenced that this organelle can accumulate pyrethroids more intensely than other
cellular compartments. This is a general type of cellular response to outer stress factors
and, hence, it is reasonable to expect that the observed collapse of the cellular
compartmentation in liver cells of fish exposed to xenobiotics (Gernhöfer et al., 2001).
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Objective 7
Rapid industrialization and economic development in Malaysia has resulted in
increased water pollution in the coastal areas. This issue has been the focus of numerous
studies. Pollutants deposited into water cause serious changes which in turn directly or
indirectly affect the ecological balance of the environment, creating extensive damage
and even mass mortality to the life and activities of aquatic organisms because of their
high toxicity and accumulative behavior (Matta et al., 1999). Heavy metal
contamination of the coastal environment continues to attract the attention of
environmental researchers because of its increasing input to coastal waters, especially
in developing countries. In fact, in recent decades, industrial and urban activities have
contributed to the increase of heavy metal contamination in the marine environment and
have directly influenced coastal ecosystems (Ong and Kamaruzzaman, 2009).
The present analysis for bioconcentration factor of NaCN in fish tissues revealed
that no traces of the toxicant was found to accumulate in the tissues of fish upon their
exposure to different sublethal concentrations at different tenures.
Many studies showed that freshwater fishes are the most cyanide-sensitive group
of aquatic organisms. Free cyanide at concentration of >5 g/l can cause negative impact
on the swimming and reproduction of fresh water fish. While at concentration of >20
g/l, the cyanide induces high fish mortality (Eisler and Wiemeyer, 2004; Hossein and
Reza, 2011). Cyanide exists in water in the form of free state (CN−and HCN), simple
cyanide (e.g., NaCN), complex cyanide and total cyanide. Almost all cyanide persists
in the undissociated form (HCN) in pH below 7. But at a pH value of11, all of the
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
cyanide appears as the free (CN) ion (Broderius et al., 1977). Toxicity of cyanide is
primarily determined by the concentration of non-dissociated HCN in the water column
(even in case of simple cyanides and metal cyanide complexes). Hydrocyanic acid
(HCN) is also more toxic than the free cyanide and the degree of its toxicity depends
on the solubility and the dissociation rate to form free cyanide (Sorokin et al., 2008).
Cyanide has low persistence in the aquatic environment and does not accumulate
in or store within the tissue of fish because of its rapid detoxification at sub-lethal doses
and death at lethal doses. The toxicity of cyanide is attributed to the presence of HCN
derived from dissociation of the complexes that penetrate cell walls (Pablo et al., 1996)
and cause fish mortality (Prashanth et al., 2011). When the fish is subjected to direct
contact with sodium cyanide, it shows some behavioral changes during its movement
(Shwetha and Hosetti, 2009). Such behaviors can be used as biological indicators which
provide a unique perspective linking the physiology and ecology of an organism and its
environment (Dube and Hosetti, 2010). Few studies have been done to evaluate the
effect of cyanide on behavior of fish. One of those studies, showed that sodium cyanide
has tremendous effect on the behavior of Clarias gariepinus resulting from depression
of the central nervous system which may be attributed to the combination of lactate
acidosis with cytotoxic hypoxia (Khalid and Shahid, 2012).
Objective 8
The quantity of pollutants entering the aquatic environment primarily through
effluent discharge from mining industries is highly dependent on the site activity and
demand. In addition, human usage, pharmacokinetic and physiochemical properties of
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
certain drugs, and sewage and wastewater treatment processes also add up to this
(Daughton and Ternes, 1999). Over the years, the widespread use of cyanide
compounds under the umbrella of different industrial sectors has led to the
contamination of surface water, ground water, and municipal wastewater (Kolpin et al.,
2002). Based on the results from the present study, it was evident that NaCN greatly
impacted on the growth rate, survival and hatching process of fish embryos. So far, it
has mainly been used in crop science, but the availability of dozens or hundreds of eggs
per female, and the fact that gametes can usually be gained by simple methods and used
for external and very controlled fertilization allows for such powerful experimental
designs in fish (Wedekind et al., 2001).
The recent developments in the literature significantly amend previous
suggestions about the use of fish embryos in toxicity tests (OECD, 1992) and have the
potential of turning embryo toxicity tests into a real and cost-effective alternative that
may partly replace the more problematic fish acute toxicity tests. Tests on fish embryos
can potentially solve many of the problems listed above. Many questions that are
currently studied with juvenile or adult fish, e.g. the relative toxicity of two chemicals,
or the specific toxicity of a chemical within a given ecosystem, could also be studied
on embryos with the appropriate test design (Aydin and Koprucu, 2005). Gamete donors
could be taken from natural populations of various species (Wedekind and Mu¨ller,
2005)
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Toxicological endpoints of sodium cyanide on freshwater fish Cyprinus carpio under sublethal exposures
Summary and Conclusion
Based on the results obtained from the present study, it was apparent that fish C.
carpio under NaCN stress undergoes partial abolition of functional regulation in
different organs through various mechanisms that are yet to be discovered. The
impairment in terms of histoarchitechture, and ultramicroscopy studies reveal the
toxicity of NaCN on fishes. Further, the recovery studies suggested the incomplete
recovery transition after duration of 14 days. The process clearly indicates the effort of
regulatory mechanism of fish to compensate the loss in harmonic manner and in turn
rescue and reimburse the imbalance caused by toxic insult. It is thus an intrinsic
oscillatory ability of fish which is viewed as a nutshell in the present investigation.
Further direct analysis under molecular evaluation methodologies may certainly throw
light on the principles behind the cascadic changes. The study may contribute in the
course of regulatory surveillance and monitoring of aquatic bodies with its applications
to aquaculture practices as well.
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IJPCBS2014,4(3),634-639Davidetal.ISSN:2249-9504
634
INTERNATIONALJOURNALOFPHARMACEUTICAL,CHEMICALANDBIOLOGICALSCIENCESAvailableonlineatwww.ijpcbs.com
SODIUMCYANIDEINDUCEDHISTOPATHOLOGICAL
CHANGESINKIDNEYOFFRESHWATERFISHCYPRINUS
CARPIOUNDERSUBLETHALEXPOSUREM.David*andRM.Kartheek
DivisionofEnvironmentalandmoleculartoxicology,DepartmentofP.G.StudiesinZoology,KarnatakaUniversity,Dharwad,Karnataka,India-580003.
1. INTRODUCTIONCyanideisextremelydestructivechemicalwhichcan kill both target and nontarget organismswhen expelled in the environment (Dube andHosetti, 2011). Industries dealing with metalplating and finishing, mining and extraction ofmetals such as gold and silver, production ofsynthetic fibres and the processing of coalgenerate large quantities of cyanide-containingwastes (Rocha-e-Silva et al., 2010).Consequently release of cyanide containingwastes into the environment has generatedconsiderable interest. Cyanide is found to behighly toxic to theaquaticorganisms,primarilydue to the formation of complexes with metalions that are present as enzyme cofactors. Thedischarge of toxic pollutants into waterwaysmay result in acute or chronic toxicity in fish(Ballantyne,1987;Leblanc,1997).Cyanidesareusedwidelyandextensively inthemanufactureof synthetic fabrics, plastics, in electroplatingbathsandmetalminingoperations,aspesticidalagentsandintermediatesinagricultural
chemical production, in predator controldevices, and constitute a hazard to aquaticecosystems in certain waste-receiving watersandto livestock(EPA1980;Towilletal.,1978).The toxicity of cyanide is a consequence of itshigh potency as a respiratory poison in allaerobic forms of life (Yen et al., 1995). Acutedoses of cyanide are usually fatal, due to themarked susceptibility of the nerve cells of therespiratory centre to hypoxia (Greer and Jo,1995).Kumar and Pant (1984) have stated thathistopathological studies are useful to evaluatethe pollution potential of various toxicants,which do not cause animal mortality over agiven period, but are capable of producingconsiderable original damage. Kidney serves asa major route of excretion of metabolites ofxenobiotics,andreceivesthelargestproportionofpostbranchialblood,andtherefore, itismorelikely to undergo histopathological alterationsundertoxicstress(Ortizetal.,2003).Althoughanumberofstudieshaveinvestigatedtheeffectof
ResearchArticle
ABSTRACT Sodium cyanide which has a wide range of applications in mining industries also has harmful effects on various organisms. The cyanide utilized by mining industries is let off into streams seriously compromises with the survival of the fish population. In the present study an attempt was made to analyze the toxicity of sodium cyanide to the kidney of freshwater fish Cyprinus carpio by histopathological aids. The results obtained suggested that sodium cyanide at a concentration of 0.2mg/L (1/5th of LC50) can impact the fish kidney in a catastrophic manner and cause the variation in histoarchitechture of kidney. Based on the outcome of the present study, it is therefore suggested that appropriate measures be taken for detoxification of sodium cyanide before it is discharged in to streams, as it can compromise the survival of aquatic habitat consequently resulting in the disturbances of aquatic ecosystem. Keywords: Freshwater fish, Nephrotoxicity, histopathology, sodium cyanide.
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Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: 2231-6876
BIOCHEMICAL CHANGES IN LIVER OF FRESHWATER FISH CYPRINUS CARPIO EXPOSED TO SUBLETHAL CONCENTRATION OF SODIUM CYANIDE M. David*, R.M. Kartheek Environmental and Molecular Toxicology Laboratory, Department of PG Studies in Zoology, Karnatak University, Dharwad, Karnataka, India- 580003.
Corresponding author Dr. M. David, Professor Environmental and Molecular Toxicology Laboratory Department of PG studies in Zoology Karnatak University, Dharwad, Karnataka India- 580003 [email protected]
Copy right © 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ARTICLE INFO ABSTRACT Article history Received 04/09/2014 Available online 20/09/2014
Keywords C. Carpio, Free Amino Acid, Protease Activity And Sodium Cyanide.
The present study deals with noxious effects of sodium cyanide on some biochemical aspects in the liver of freshwater fish Cyprinus carpio. Fish which were exposed to a sublethal concentration (0.l mg/L) of sodium cyanide, for a period of 10 and 20 days and further allowed to undergo a recovery of 14 days, resulted in variation of levels of protein, free amino acid and protease enzyme activity with respect to control. The changes were in a manner that suggested the possible existence an oscillatory phase in biochemical turnover towards a more synthetic phase leading to the establishment of convalescence and adaptation phenomena. Yet, sodium cyanide being toxic component needs attention towards its neutralization prior to its disposal. Hence, it is inferred that necessary care should be taken during its disposal, as it possesses a serious threat to fish.
Please cite this article in press as M David et al., Biochemical Changes in Liver of Freshwater Fish Cyprinus Carpio Exposed To Sublethal Concentration of Sodium Cyanide. Indo American Journal of Pharm Research.2014:4(09).
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64 International Journal of Toxicology and Applied Pharmacology 2014; 4(4): 64-69
ISSN 2249 9709 Original Article
SODIUM CYANIDE INDUCED BIOCHEMICAL AND HISTOPATHOLOGICAL CHANGES IN FRESH WATER FISH CYPRINUS CARPIO UNDER SUBLETHAL EXPOSURE
M. David*, Kartheek. R. M. Environmental and molecular toxicology laboratory, Department of P.G. Studies in Zoology,
Karnatak University, Dharwad, Karnataka, India- 580003 Email: [email protected]
Received 08 August 2014; accepted 23 September 2014 Abstract Sodium cyanide is an agent which has wide range of applications in mining industries. The utilized cyanide component is let off into streams which drastically affects the survival of the fish. In the present study an attempt was made to analyze the toxicity of sodium cyanide and correlate the relationship between levels of protein, free amino acid, protease activity and histopathology of fish liver. The results obtained suggested that sodium cyanide at a concentration of 0.2mg/L (1/5 th of LC50) can impact the fish liver catastrophically and cause the variation in biochemical and histopathological aspects as well. Based on the outcome of the present study, it is therefore suggested that appropriate measures be taken for detoxification of sodium cyanide before it is discharged in to streams, as it can compromise the survival of aquatic habitat consequently resulting in the disturbances of aquatic ecosystem.
© 2014 Universal Research Publications. All rights reserved. Keywords: Biochemical changes, fish, hepatotoxicity, histopathology, sodium cyanide.
INTRODUCTION Cyanide is an extremely toxic compound, which
has an ability to restrain the use of oxygen and finally resulting in death of the exposed organism [1]. It is extremely destructive chemical in nature and is the one which can kill organisms irrespective of whether being targeted or non-targeted when it is let off in environment [2]. Various sensitivities to cyanide have been reported in different fishes [3, 4]. Despite being a highly toxic substance, cyanide as a chemical has a number of applications in various industries like mining (silver and gold), electroplating, printed circuit boards and other chemical industries. The daily effluent discharge by cyanide users usually range from 200- 1000 liters for small scale industries and 1- 20 cubic meters or more for large scale industries. Toxicity of cyanide and its derivatives is well-known as a metabolic inhibitor [5]. The toxicity is derived mainly from its potency as a respiratory poison in aerobic organisms. The in-depth chemistry of cyanide and its chemical behaviour in streams and sediments is complex and its toxicity is influenced by several factors, including acidity or alkalinity [6]. Massive death of fish and other aquatic biota due to the accidental discharges of cyanide wastes have been reported earlier [7].
Cyanide is readily absorbed across gill membranes in fish and is a potent, rapid acting asphyxiant inducing tissue anoxia and cytotoxic hypoxia due to inhibition of
cytochrome c oxidase, an enzyme which has an important role to play in respiratory chain [7]. In addition to acute cyanide poisoning, chronic toxicity of cyanide has frequently been reported in recent years, and it is suggested that the most widespread problems arising from cyanide are from chronic dietary, industrial and environmental sources [8]. Reports by various authors around the world suggest fishes to be the most sensitive group of organisms towards wide range of toxicants including cyanides in the overall aquatic system [9, 10]. Furthermore, liver being an important organ of an individual is the first organ to face a toxic abuse through portal circulation and is usually subjected to severe damage upon toxicant exposure [11, 12]. Sodium cyanide (NaCN) is important component used in metallurgy and its highly toxic form is used increasingly by the international mining community to extract gold and other precious metals which require cycling of millions of liters of alkaline water containing high concentrations of potentially toxic sodium cyanide, free cyanide and metal. The release of cyanide from various industries has been estimated to be more than 14 million kg/year [13] which usually enter the aquatic system. Cyprinus carpio (common carp) is a widely used fish species in the evaluation of the toxic potential of agents in in-vitro studies as well as in environmental studies. Since fishes are considered to be important biomarkers of aquatic toxicity; the present study was carried to analyze the potential toxicity of sodium
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Open Veterinary Journal, (2015), Vol. 5(1): 1-5 ISSN: 2226-4485 (Print) ISSN: 2218-6050 (Online) Original Article
________________________________________________________________________________________________________ *Corresponding Author: Prof. Muniswamy David. Department of PG Studies and Research in Zoology, Karnatak University,
Dharwad, Karnataka, India- 580003. Email: [email protected] 1
_____________________________________________________________________________________ Submitted: 13/09/2014 Accepted: 01/01/2015 Published: 12/01/2015
Histopathological alterations in spleen of freshwater fish Cyprinus carpio
exposed to sublethal concentration of sodium cyanide
M. David* and R.M. Kartheek Environmental and Molecular Toxicology Laboratory, Department of PG Studies and Research in Zoology,
Karnatak University, Dharwad, Karnataka, India- 580003 _____________________________________________________________________________________________ Abstract Aquatic ecosystems in areas with intense mining activity are often subject to cyanide contamination; the present study was aimed to evaluate the harmful effects of sodium cyanide on histoarchitechtural aspect of spleen of freshwater fish Cyprinus carpio using an in vivo approach. The fishes were exposed to a sublethal concentration of 0.2 mg/L of sodium cyanide for duration of 10 and 20 days and were further allowed to undergo recovery for 14 days in a toxicant free medium. From the present investigation findings like occurrence of haemosiderin pigment, melanomacrophage centers, vacuolation and necrotic eosinophils were evident in all the fishes exposed to sodium cyanide. However, changes were more pronounced in fish subjected to 10 days of exposure, which was followed by 20 days of exposure and 14 days of recovery. The study revealed that there seemed to be the presence of homeostatic mechanism in fish that allows them to stabilize and overcome stress, which in present case is caused by sublethal concentration of sodium cyanide. Since the recovery phenomenon may be adaptive and even strategic, the present investigation also throws a light on adaptive behaviour of fish under stressful environments. Keywords: Histopathology, Melanomacrophage center, Recovery studies, Sodium cyanide, Spleen. _____________________________________________________________________________________________
Introduction One of the most important and poisonous substances known to man is cyanide. Cyanide is a noxious substance and possesses a property of killing both target and non-target organisms when discharged into the environment (Dube and Hosetti, 2011). The toxicity of cyanide is due to its influence as a respiratory poison in almost all forms of life (Yen et al., 1995). Acute doses of cyanide are usually lethal, due to marked susceptibility of the nerve cells of the respiratory centre leading to hypoxia (Greer and Jo, 1995). Chronic cyanide intoxication has been implicated in numerous anomalies such as ataxic neuropathy (Osuntokun, 1981), goitre (Cliff et al., 1986) and histopathology (Dixon and Leduc, 1981). Sodium cyanide being extremely toxic is also very functional in various fields and hence is used in large scale by the international mining community to purify gold and other precious metals through milling of high grade ores and heap leaching of low grade ores. This process adequately needs cycling of millions of gallons of alkaline solutions containing high concentrations of potentially toxic sodium cyanide, free cyanide and metal cyanide complexes, which th