UGC Sanctioned Major Research Project Entitled · Often, such wastes also contain significant...

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

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];

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.

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.


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).


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


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.


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).


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


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.


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


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


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


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


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


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


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


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


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).


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


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


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)


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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International Journal of Toxicology and Applied Pharmacology Universal Research Publications. All rights reserved

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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

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