INTRODUCTION

INTRODUCTION

Trace metals, essent ial or non-essent ia l are

distributed and redistributed naturally in the environment by both

geologic and biologic cycles. Weathering and disintegration of rocks

bring them into the soil, streams and rivers. Ultimately they are

carried to the oceans where they are deposited as sediments.

Geologic disturbances in earth's crust finally bring these deposits

up as sedimentary rocks from the soil or water. These elements

are taken up by plants in the living system and passed on to higher

trophic levels. When living beings die, they decay and their

decomposi t ion release the heavy metal elements in the

environment. Some of these elements may become air-borne in

the process from where they are returned to the soil or water

underneath as dry fall-outs or along with rains.

Trace metals play a very important role in living

organisms. These metals may serve as biostimulaters, catalysts,

co-factors etc. and form indispensible structural component of a

number of macro molecules. This is demonstrated by the fact that

a number of trace metals occur regularly in a living system in a

definite amount and proportions.

If they are deficient or absent altogether, the

organism fails to grow normally. However, even those trace metals

which are essential for growth of an organism may become

harmful if their concentration is raised a little.

The toxic action of most of the trace metals is due to

stems from the fact that they are capable of interacting and

forming strong bonds with metabolically active groups within a

living system.

Trace metals in general, cause only local pollution

problems. Environmental significance of the enhanced levels of

these metals is judged in terms of the degree of toxicity, the

extent of exploitation of the element, their application and

consequent mobilization into the air, water and soil.

Problems caused by trace metals are multifold;

1. Higher levels of trace elements are injurious to plants, animals

and microbial components of the biosphere.

2. Their persistence in the environment and subsequent transfor­

mation into more toxic state are distinct possibilities.

3. Many trace elements and heavy metals are rendered lipophilic

in nature as a consequence of formation of conjugates with

organic molecules. This provides them a free access into a

biological system and makes them more dangerous as they can

be readily taken up by living organisms.

4. Bio-concentration and magnification in the biosphere may

confront us with highly toxic levels of trace metals. This may

substantially damage our food supplies, water resources and

agricultural land.

5. Synergistic effects, when two or more than two, such elements

are involved may greatly enhance the toxicity of trace elements.

6. Carcinogenic, teratogenic and mutagenic effects may occur even

at low concentration which often requires detection.

Some heavy metals, notably Cu, Zn, Co, Cr are

essential to life when present in traces, therefore they are

essential trace metals. However, some heavy metals have no

metabolic role in l iving cells but are found into the living

organisms because of their availability in the environment. The

ecologically more significant heavy metals Hg, Pb, Zn, Cu, Ni and

As play similar distruptive role when enter in the body system in

higher than the required amounts.

Metals are used extensively in the workplace and

exposure of the employee can result from numerous industrial

operations, including welding, grinding, soldering, painting,

smelting and storage battery manufacturing.

Bioaccumulation of heavy metals in aquatic ecosystem :

In aquatic animals the external surfaces are

generally speaking, much more structurally and physiologically

delicate than comparable liquid - exposed surfaces in terristrial

animals. Thus a particular metal could be toxic to an aquatic

animal because of its surface acting as well as whatever internal

effect it might have. Metals which are most likely to be internally

toxic only are those that are readily absorbed and have little, if any

surface activity.

In natural waters, these are typically found as lipid -

soluble, organo-metallic complexes that are readily permeable to

biological membranes. Most other metal contaminants of aquatic

environments tend to occur as water soluble cations and so are

at least potentially active at the surface of fish. Indeed, the

surface activity of some metals and probably of H* as well

(McDonald, 1989). may be all that is needed to explain their

respective toxicities.

Aquatic environments are final collectors of all kinds

of pol lutants. Elements in water ref lect air borne metal

contamination and pathway. Life in water bodies as opposed to

terrestrial conditions is characterized by stronger relation between

aquatic organisms and factors of the environment due to the high

mobility of polluting substances in water. Fish, in comparison with

invertebrates, are more sensitive to many toxicants and are the

convenient test-objects for water quality assessment.Pathological

changes in fish allow us to define toxicity of the environment and

understand the potential danger of pollutant inputs.

Combined effects of metal mixtures and their

toxicological properties are dependent on such factors as pH, Ca**

and organic ligand concentration.

Poisoning by metals can be due to both toxic effects

and from larger - term consequences related to bioaccumulation,

including mutagenic, embryotoxic, gonadotoxic and carcinogenic

effects; and toxicological properties depend on metal speciation,

combination of elements.

Toxicity of metals :

Fisheries plays an important role in India's economy

as they form about 2 % of the Gross Domestic product. The

minimum protein requirement is 12.5 million tones of fish, which is

met by inland fish production. Nearly 40% of the world's annual

fresh water fish harvest contain farmed fish. India also produces a

good amount of fish and fish by-products which are very useful in

the international food market.

Increasing disposable heavy metals by the industries

into the water bodies causes a great threat to the fishes. Many

studies have been done in relation and sub-lethal levels, behav­

iour, histological, pathological, biochemical and physiological

changes to the toxicity of various heavy metals like.Cd, Zn, Cu, Ni

in the fishes.

Fish are located at the end of the aquatic food chain

and may accumulate metals and pass them to human beings

through food, causing chronic and acute diseases.

Heavy metals are very toxic because, as ions or in

compound forms, they are soluble in water and may be readily

absorbed into living organisms. After absorption, these metals can

bind to vital cellular components such as structural proteins,

enzymes and nucleic acids and interfere with their functioning.

Heavy metals stored in specific tissues or organs

within a biological system. These tissues or organs can be thought

of as a storage depots for the heavy metals concerned and the

phenomenon is known as bio-accumulation which may at times

involve active absorption of heavy metals from food or from the

surrounding environment.

Absorption

Free form

Bound form

Excretion

1 Storage depots

Bio-transformation

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Fig:1.1, The fate of toxic agents in a biological system.

Bioaccumulation of heavy metals in Fish :

Fish appears to possess most of the metabolic

capabilities of terrestrial animals in dealing with heavy metals. The

rates of the metabolic processes are generally much slower in the

former than in the later, and the temperature optimum is as a rule

lower in fishes. The excretion of parent compounds is usually the

main excretory mechanism for many heavy metals in fishes, but a

number of metabolites are also formed and excreted in many

instances.

The accumulation of metals in fish can reflect an

integrated dose of metals from the water over a long period of time,

upto the entire life cycle of the fish (Moissenko et al., 2001)

1. Copper:

Copper is widely distr ibuted in nature in the

elemental state,as Sulfides, Arsenities, Chlorides, and Carbonates.

The total flux of copper to the atomosphere is

approximately 75,000 metric tons per year of which 5000 - 13,000

tons are deposited into ocean. Atmospheric emissions are the main

route of entry to the environment. Approximately 75 % of

atmospheric emissions are from anthropogenic sources. Through

processes of wet and dry deposition, atmospheric copper enters

the hydrological cycle either by deposition into soil and subsequent

erosion into water bodies or direct deposition into rivers, lakes and

oceans. Production of nonferrous metals is the largest single

emission source, followed by wood combustion and iron/steel

production.

C (U

rv ^

Fluvial Sediment

ATMOSPHERE

Estuarine Water

Oceanic Sediment

^ iii CD —1

fn CD LU

Esturarine Sediment

Diagram :- The movement of trace metals through the hydrocycle

Water is the primary means of transport of metals

through the environment. The movement of the trace metal through

the hydrocycle, the outer ring represents the particulate

transport, the inner ring the movement of soluble trace metals and

the center indicates the influence of the atmosphere on the entire

cycle.

1.1 Chemistry :

• I . The copper (I) ion has the electronic structure 3d^° so that its

compounds are diamagnetic and colourless except where color

results from the anion or charge transfer bands. Tetrahedral

coordination dominates with two and three coordinators much less

common.

Copper (II)

The copper (II) is the most important form of copper.

Most Copper (II) compounds are fairly readily oxidized to cupric

compounds. There is a large number of salts of various anions,

many of which are water soluble, in addition to a variety of

complexes. With the d^ electronic configuration, cupric compounds

are normally paramagnetic with a single unpaired electron.

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Copper (III)

Copper (III) can occur in crystalline compounds and

in complexes. Copper (III) with a d^ shell is isoelectronic with nickel

(II).

1.2 Uses of copper :

1)lndustrial and Agricultural uses .

2)Nutrient supplementation.

3)The use of copper to control the growth of organisms.

4)The use of copper in biologically important chemicals.

5)Copper in dental amalgams and mouth rinses.

6)Copper in wood preservatives.

1.3 Sources :

Copper is an essential trace element which is

ubiquitous in earth's crust .The main sources of copper and it's

sulphide and oxide are from which the metal is extracted by

roasting , smelting and electrolyte refining. There are substantial

deposits of copper in Rajasthan in India, which have been

estimated to contain about 200 million ton's of copper.

II

Copper is used widely in dyes, paints, pigments,

ceramics, in many pesticides and some therapeutical preparations

as well Copper contamination of the environment is largely due to

it's release by industrial units producing non- feeous metals,

fertilizers, disposal of failings or the solid wastes from mines and

from fly ash produced by combustion of coal and organic matter.

1.4 Copper in aquatic system :

In aquatic environments, copper can exist in three

broad categories ; particulate, colloidal, and soluble. Speciation

of copper in natural waters is determined by the physico-

chemical, hydrodynamic characteristics and the biological state of

the water. The speciation of copper is directly linked to the

bioavailability and toxicity of Copper, copper is most toxic in the

free ion form prevalant at pH < 7. Various processes in the aquatic

environment act to bind copper to inorganic and organic ligands

which precipitate out of solution. Copper may also bind to

particulates which settle out of the water column. These processes

act to sequester copper in the sediment in a bound form making it

less available and therefore less toxic to marine and fresh water

organisms. As well, the presence of various complexing agents in

the water column such as bicarbonate, carbonate, and hydroxide

ions have a marked effect on the toxicity of copper.

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1.5 Toxicity :

The wide spread use of copper sulphate to control

aquatic vegetation may result in significant copper levels in water.

Fishes are apparently susceptible to copper toxicosis,

and trace concentration 1/10 to 1/20 of accepted standard for

drinking water, can be lethal to fish in regions where surface

water is very soft ( Sprague, 1968).

The 48 hr. LCg^ (lethal concentration 50 ) in rainbow

trout ( Salmo gaivedneri) was found to be 750 f.ig/1 (Brown ef a/.

1970). The acute (10 days) lethal concentration of copper for brook

trout ( Salvelines fontinalis) was about 0.05 \XQI\ recorded by

Sprague (1968). The absolute copper concentration that is toxic

depends upon physical and chemical characteristics of the water,

especially pH and calcium concentration. The toxicity of copper to

fish has been reviewed by Doudoroff and Katz (1953) and Makee

and Wolf (1963). Copper is one of the most toxic heavy metals to

fish (Mance, 1987). The 96h Lc^^ values of copper ranged from

0.06 - 0.34 mg L'' for Juveniles of the Japanese eel, Anguilla

japonica . (Yang and Chen, 1996) to 1.97 mg/ l ' for juveniles of the

pompano Trac/i/noftys caro//nue (Birdsong and Avaul, 1971).

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1.6 Bioaccumulation :

1.6.1: Fish

High concentration of copper have been recorded in

seabed sediments (EPD , 1996 ) Fish (Chan, 1995 ), and

biomoniters such as Oysters and mussels ( Chan, 1988; Phillips

and Rainbow, 1988; Phillips, 1989; Chu et al., 1990; Cheung and

Wong, 1992) collected in Hong Kong.

The gills are the first target organ of several heavy

metals like Cu and Zn because of their very large interface area

between external and internal fish environments. Performing vital

function such as gas exchange, ion osmoregulation and N2

excret ion, the gi l ls are part icular ly sensi t ive to adverse

environmental conditions, and the changes on gill epithelia have

been considered good indicators of the effects of xenobiotics on

fish.

Mohammed ( 1994) noted that the values of copper

in muscle tissue ranged from, 0.13 to 0.57 mg/kg in different

species with maximum value in parrot fish.

The maximum concentration was found in liver and

kidney followed by gut and gills. The minimum values were recorded

in muscle tissue. When compared with the muscle tissue, the

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concentration of copper in the liver was 23 times in Beda fish; 4.6

times in parrot fish; and 13.72 times in Safi fish.

1.6.2 Plants :

In plants low levels of copper are essential for

normal activity of a number of enzymes and for chlorophyll

synthesis. However, at a slightly higher concentration copper is

the most toxic elements after mercury. Inhibition of growth occurs

at concentrations less than 0.1 ppm in majority of plant species.

Uptake of copper by plants is accelerated in the presence of

calcium and magnesium ions but diminishes with fall in pH.

The Cu residues in aquatic plants collected from

polluted surface waters generally range from 10 to 100 mg/kg dry

weight.

The accumulation of heavy metals in soil, water,

aquatic weed and fish samples of sewage fed ponds was studied

by Pandey et al. ( 1995 ).

Soil is not only a medium for plants to grow or a pool

to dispose of undesirable materials, but also a transmeter of many

pollutants to surface water, ground water, atmosphere and food.

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Therefore accumulated pollutants in surface soil can be transported

to different environmental compartments, eg. deep soil, water,

plants, and dust particles. Soil pollution may threaten human health

not only through its effects on the hygiene quality of food and

drinking water but also through its effects on air quality ( Chen et

al., 1997 ).

1.6.3. Invertebrates :

Hare and Tessier, (1998) tested larvae of three

species of the wide spread lacustrine insect chaoborns as

potential biomonitor of the trace metals Cd, Cu, Pb and Zn.

Maximum concentration of Cu in the soft tissues of invertebrate

like molluscs.

2. Zinc :

Zinc is distr ibuted throughout fresh water and

marine aquatic environment and occurs in all organisms.The

disposal of industrial and muncipal wastes in the aquatic

environment has made zinc toxicosis a potential problem for aquatic

organisms.

2.1 Chemistry:

Zinc is classified as a borderline metal, meaning that

it forms bonds with oxygen as well as nitrogen and sulphur donar

atoms. Under aerobic conditions, Zn ' ' is the predominant species

at acidic pH, but is replaced by Zn (0H)2at pH 8 - 1 1 , and Zn (0H)3

/Zn (OH), 2-at pH > 11.

An aerobic conditions leads to the formation of ZnS

regardless of pH within the range 1 - 14.

Zinc binds readily with many organic l igands,

particularly in the presence of nitrogen or sulfur donor atoms.

Stability constants ( KO) for zinc/humic acid complexes are vari­

able, typically ranging from 2.3 to 5.9. This means that the fate of

zinc will vary among waterways, depending on the type of humic

material present in the system. Zinc also shows variable

behaviour in binding to suspended paerticulates, depending on pH.

2.2 Uses :

1. Zinc is used extensively in machine building, and automotive

industries as a component.

2. Zinc sulphate is used in the production of plastics.

3. Zinc chromate is a wood preservatives.

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4. Zinc carbonate is used as a dietary supplement for farm

animals

5. Several organozinc compounds are useful as fungicides,

tropical antibiotics and lubricants.

2.3 Sources :

The total amount of zinc discharged to fresh waters

from anthropogenic sources comes to 77-373 x 10 ^ metric tons/yr.

There are several major sources including the discharge of

domestic waste water, coal burning, power plants, manufacturing

process involving metals, and atmospheric fallout.

Approximately 34 % of all emission of zinc to the

atmosphere come from natural sources, the reminder originating

from metal production, burning of coal and oil, and fertilizer and

cement production.

2.4 Zinc in aquatic system :

Zinc is an abundant element and contr ibutes

approximately 0.04 g/kg of the earth's crust. Its occurance in

sewage is expected because of its extensive use in making

household appliances and by leaching from galvanised pipes.

Generally the concentration of zinc was found to be quite low in

animal tissues, water and soil. The average values of zinc were

0.064 mg/g in soil, 0.041 ppm in water, 0.022 mg/g in fish, 0.067

ppm in raw sewage, 0.025 ppm in treated sewage and 0.03 mg/

g in aquatic weed. Rao et al. (1993) reported accumulated of zinc

in storm water and lake which raged between 0.02 and 0.036 ppm.

Duttamunshi and Singh (1989) estimated the concentration of zinc

in water, soil and fish samples of river Subarnakha at Ghatsila.

They reported average value of zinc for soil, water and fish

samples as 0.182 mg/g for soil, 0.246 ppm for water and 0.117

ppm for fish.

Toxicity of zinc to plants is highly variable, with

EC^Q. Effective concentration ranging from < 0.01 to >100mg/l. This

extreme variabi l i ty is due to - (1). The effect of different

physicochemical conditions on uptake, and (2). The ability of many

species to adapt to high zinc levels. As early as 1980, Allen et al.

noted that algal {Microcystis aeruginosa) growth decreased by more

than an order of magnitude as the quantity of chelater in solution

declined. In fact toxicity was not related to total Zn but predicted

to free metal concentration.

2.5 Toxicity :

Sediments are a primary sink for zinc, residues

in excess of 1000 mg/kg dry weight have been found in the vicinity

of mines and smelters. Relatively high concentrations can also be

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found near major municipalities and coal burning power stations.

Zinc is very toxic in the ionic form [ Zn OH * ],

however increase in water hardness causes decreased zinc

toxicity. There is an avoidance response ( water hardness 112 mg/

I (CaC03), agonistic behaviour by dormant individuals and egg

production is reduced. Zinc interferes with the respiratory surface

and causes historical gill damage.

Human destruct ive inf luence on the aquatic

environment is in the form of sublethal pollution, which results in

chronic stress conditions that have a negative effect on aquatic

life. Under normal circumstances, metals which are mainly

benef ic ia l , and essent ia l , such as copper and zinc, may

become pollutants when present in excess by exhibiting toxic

effects on organism ( Devenport, 1985 ; Tort ef a/.; 1987,

Mason, 1991). A stress factor or stressor is an environmental

change that is severe enough to require a "physiological" response

on the part of a fish in an ecosystem ( Wedemeyer and Mcleay,

1981). Adaptation to the stressor will occur, if the stress response

can re-establish a satisfactory relationship between the changed

environment and the fish.

The pollution load from the pulp and paper industries

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leading to zinc contamination, on various rivers has estimated by

Subramanyam and Rao (1973). Most of our rivers are being

polluted by effluents released from these industries. Agrawal (1996)

has indicated the location specific problems of river pollution,

such as complete deoxygenation of water of lb river upto at least

three km downstream at Brijranagar during summer due to the

discharge of liquid wastes by orient paper mill, Orissa. The

instances of large scale fish mortality in river Gomati due to

distillery effluents containing zinc have been reported ( Joshi 1990).

Among the physiological aspects, energy metabolism

has key role as fish have to expend more energy to overcome the

toxic stress. However, long term effects of sublethal concentration

of heavy metals like Zinc, copper on different aspects of

physiology of fishes are not well understood. Hence, it was thought

to investigate the impact of long term exposure of the fish, Channa

punctatus to various heavy metal compounds like Zinc sulphate

and copper sulphate.

2.6 Bioaccumulation :

2.6.1 Fish

Total Zinc in the muscle tissue of fish is generally

much lower than residues in plants or invertebrates. Most reports

place total Zn at <50 mg/Kg weight, with a range of < 1-100 mg/kg.

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much higher levels ( 100 - 300 mg/ kg ) can be found in the liver

and kidney of many fish species.

The concentration of zinc in different fishes differed

with respect to different organs. Liver and kidney accumulated more

zinc and the least accumulation was found in the muscle tissue in

fishes.(Mohammed M.AI-Mohanna: 1994).

Caged rainbow trout were exposed to various heavy

metal salts for a month. Gills, spleen, kidney, muscle and

vertebral bones were examined for metal content after 7, 15 and

30 days of exposure. Cd accumulated mostly in spleen and mus­

cle; Cu in kidneyand the highest Zn levels were measured in gills.

( Camusso et al. 1995 ).

The zinc accumulation in the organs of fish (Cirrhinus

mrigala) revealed highest value ( 1.904 mg/g ) in the vertebral

bones, followed by kidney ( 1.723 mg/g ) and skin ( 1.602 mg/g ).

The lowest accumulation ( 0.606 mg/g ) was in the brain ( Mwachiro

etal. 1997 ).

2.6.2. Plants :

Aquatic plants are well known accumulators of

metals and are therefore used for biomonitoring or biogeochemical

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prospecting the extent of metal accumulation depends somewhat

upon the plant species. The importance of aquatic plants in this

aspects has drawn much attention, since the analysis of the plants

can give an indication of the water environment to which it has

been exposed. Water hyacinth ( Eichhornia crassipes ) is used in

pollution treatment systems and is reported to remove heavy

metals like Ni, As, Cd, Pb, Hg, Cu, Mn, Zn from aquatic system.

(Panda, 1996).

Total Zn residues in aquatic plants collected from

polluted surface waters generally range from 100 to 500 mg/kg dry

weight, but considerable higher levels have been found in the

vicinity of base metal mince.

Aquatic plants and organisms accumulate metal

internally. The concentrations observed in some aquatic plants have

such high values that complete internal location of the metals is

unlikely. During extracellular exchange, trace metals bind to ligand

on the insoluble polymeric material within the plant cell wall.

The maximum concentrations of Zn upto 10 mg/kg in

the aquatic moss Fontinalis squomosa is on record (Panda, 1996).

Heavy metal pollution in river and its impact on

aquatic ecosystem is a dynamic process. Assessing the impact on

whole ecosystem and constructing an ecological model are multi -

disciplinary scientific problems and pose a major challange to the

understanding of heavy metal pollution (Meng Chang et al., 2001).

2.6.3 Invertebrate :

Although total Zn in the soft tissues of invertebrates

generally ranges from 50 to 500 mg/kg dry weight, residues of over

3g/kg may be found in exceptional cases. Maximum concentrations

in molluscs are usually as sociated with the digestive glands,

kidney, gill and gonad rather than the foot or muscle tissue.

The number of invertebrates were affected by

consumption of food contaminated with high levels of heavy

metals like mercury, copper, zinc, incorporated through magnified

acumulation of the metal in the food chain.

As crustacean gills are important in respiration and

osmoregulation, hence, cellular damage of gill tissue may lead to

serious consequences. Evidently, the immature stage of the

marine crab, 0- regulosus are externaly sensitive to heavy metal

stress ( Sambasiva, 1988 ).

The present work was carried out to understand the

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accumulation of Cu and Zn in the aquatic ecosystem and their

impact on the biochemical parameters of the mudskipper, Channa

punctatus (Bloch).

The problem was investigated by considering the fol­

lowing aspects.

1. Bio-accumulation of the trace metals ( Copper and Zinc ) in

various components of the ecosystem i.e. water, aquatic plant,

soil and various tissues of fish, Channa punctatus.

2. Effects of sublethal and lethal concentrations of the two heavy

metal compounds ( copper sulphate and zinc sulphate ) on

some biochemical parameters of the fish,C/?anna punctatus

( Bloch).

3. Recovery studies with respect to bio-accumulation of heavy

metals.

4. Recovery studies with respect to bio-chemical status of the

fish.

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