Chapter I

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Introduction

All living organisms have their specific environment with which they

continuously interact and remain adapted. The environment embraces all abiotic

and biotic conditions influencing the organisms. Abiotic environment of all

organisms on the earth includes atmosphere, lithosphere, hydrosphere and physical

factors like light, temperature, pH, etc. Communities of living organisms interact

among themselves. The physical components of the environment experience

increasing impacts from human activities. Every organism alters environment in

some ways but men are unique in inducing both the extent and rate of changes. The

ecosystem can be terrestrial and aquatic. Terrestrial biomes are plants and animals

combined with relatively few common features of climate, soil, vegetation type

and structure. The climate and soil interact to determine the types of species that

can survive. It is worth mentioning that soil mostly acts as nutrient reservoirs,

allowing them to be recycled within the system.

Water of an aquatic ecosystem is vital in environment and covers about

70% of the earth’s surface. It occurs in all spheres of the environment—in the

oceans as salt water, on land as surface water in lakes and rivers, underground as

ground water, in the atmosphere as water vapour, solid ice and as in municipal

water distribution systems as described in chart. Water is an essential part of all

living systems and is the medium from which life evolved and exists. Aquatic

ecosystem e.g., pond, lake, river etc. interacts with organisms and their

environment for nutrients and shelter. This also includes areas such as flood plains,

wetlands with water for all or part of the year, thermal springs, ice fields and some

highly polluted water that can support large populations of bacteria.

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Salt water

River and pond water

Estuarine water

Ground water

Water vapour

ice

Water has several properties to support life and yet they are

disadvantageous to organisms in facilitating absorption and accumulation of

pollutants. Nonconservative components such as dissolved gases (e.g., O2, CO2,

H2S, N2), nutrients (phosphates and nitrates), silica, trace metals, colloids and

particulate matters are generally the limiting factors in growth of organisms.

However, aquatic ecosystems are home to a rich collection of flora and fauna. The

dominance of human being is mainly because of their ability to modify

environment around them. This has led to development on one side and destruction

of natural habitat on the other [1]. Growth of industries is a vital link factor for

improving the quality of human life. However, there has been a growing concern in

last few years over the environmental degradation due to ecologically unplanned

industrialization and urbanization. Wastes with toxic chemicals, released

constantly into water bodies from industries are polluting surface and ground water

sources [2]. Pollution is thus the price paid towards development through scientific

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achievements to improve human life style. In simple words pollution is an

anthropogenic contribution to nature through evolved technologies for the human

welfare. Natural ingredients are replaced or damaged by dangerous unnatural ones.

These may be gases, solids or liquids which cause imbalance to the system and to

create numerous health hazards to animals and man by damaging the ecosystem,

e.g., industries polluting air have set imbalance in composition of air and made it

unworthy to breathe - causing problems to human and animal life while extensive

use of pesticides on crops has set imbalance in ecosystems of lands besides causing

water pollution [3].

A vast array of industries can cause pollution. The high concentration of

chemicals is acutely toxic to aquatic organisms and causes damage to aquatic

environment and fish kill. Due to current promising environmental alarm resulting

in pollution control and remedial measures, surface waters are relatively clean

though the water pollution continues to be an ongoing concern. A classified list of

industries causing different types of pollution is given below:

• Textile industries [4-25] • Petroleum based industries [26,27] • Pesticide / fertilizer industries [28-56] • Detergent industries [57-80]

All other industries cause moderately less pollution and are less harmful.

Most of these industries are established for the progress of human society. Hence,

it is undisputable to have them for human existence. The only requirement is to

make them pollution free. The cause of pollution is not the industry itself, but also

the technology adopted.

Pollution causing agents (pollutants) may be gases, liquids, solids having

different chemical composition and are the byproducts of manufacturing processes.

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These vary with the nature of materials used as well as technology adopted.

According to the physical state these are grouped as under:

A. Gaseous Pollutants: During the process of manufacture, most industries

produce and release some familiar gaseous pollutants like:

1. Carbon monoxide, a dangerous gas with a high potential to cause cancer and respiratory ailments. Generally, it is evolved, when a fuel is burnt under limited supply of oxygen.

2. Carbon dioxide, is also a pollutant, when in large quantity as it can reduce the oxygen concentration in air. It is produced when a fuel is burnt in adequate oxygen. Normally, the atmospheric air has 0.03% carbon dioxide.

3. Sulphur dioxide, is another pollutant to form sulphuric acid, when mixed with water vapour. Sulphuric acid is the main cause of acid rain.

4. Hydrogen sulphide, is a hazardous pollutant gas with many deleterious effects on respiratory tract. It is released from many industries as a part of sulphonation process [166].

5. Cyanide chemicals, when these are used in manufacturing process, hydrogen cyanide is formed as a byproduct and is a highly corrosive and poisonous gas leading to eye irritations, respiratory problems as well as other complications.

6. Ammonia gas in high dose can cause death. In many industries, ammonia gas is used as reducing agent. It is a corrosive gas with a high potential to cause throat irritations, burning of pharynx, oesophagus, etc. It can also cause digestive problems - when excess gas is inhaled.

B. Liquid and Solid Pollutants: These are evolved during the manufacture of

pesticides, medicines, dyes, plastic, paper and other chemicals, refinement and

processing. Some of them are dangerous and cause serious implications on

drinking water quality.

The kinds of liquid pollutants can be:

(i) Acids – [hydrochloric, acetic, fumaric, sulphuric, nitric, benzoic] (ii) Bases – [hydroxides of sodium, calcium, magnesium] (iii) Carboxylic compounds (iv) Nitrogenous wastes

Some well known pollutants studied so far for their toxic effects on diverse group

of animals are listed in table 1.1.

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Table: 1.1 The literature survey on the effects of some toxicants on different animals

No. Toxicants Animals References 1. Dyes Microbes 11

Arthropoda Fish

9,10 8,12-25,106

Mammals 5-7 2. Oil spill Protozoa

Fish 26 26,27

3.

Pesticides

Protozoa Arthropoda Fish Amphibians

29 28,30 28,-56,145,146 35

4.

Surfactants Protozoa 60,77,80,82 Arthropoda Fish Mammals

61,69,71,72, 59,63-72,74-82 77

5.

Heavy metals Protozoa 86 Arthropoda Fish

84,86,87, 83-105,107-144,

6.

Organic/inorganic Compounds

Fish 81,82,147-160

7. Drugs/ Mutagens/ Carcinogens

Fish 161-165

Agriculture is primary activity serving as the basis of many others e.g., food

based industries, textile industries, agro-processing industries, oil industries, sugar

industries, etc. It is a source of large pollutants released into our environment in the

form of synthetic chemicals such as fertilizers, pesticides, growth promoters and

growth retarders. Fertilizers are synthetic chemicals that increase growth and yield.

Fertilizers commonly used in agriculture include urea, diammonium phosphate,

potassium chloride, potassium sulphate, gypsum, ammonium phosphate etc. and

these leave large amount of residues in the soil that is retained and create

imbalance in soil ecosystem or flown with water to cause water pollution.

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Sometimes, excess application of fertilizer results into gaseous release of pollutants

like ammonia by volatilization.

Pesticides are synthetic chemicals and about 5% of applied pesticide

reaches the pests while rest enters the ecosystem and remains in plant bodies and

may accumulate in edible part of crops. These can also enter to the ground water

and may cause water pollution. Some important pesticides used in agriculture are:

Organo-chlorine chemicals are highly dangerous pesticides that persist in plant and soil for long period to cause innumerable ecological and health hazards later, e.g. DDT, BHC, endosulfan, heptachlor and chlordane [34]. Organo-phosphorus chemicals are also dangerous pesticides with a high potentiality to kill many useful bacteria besides killing the pests. They act as a source of soil and ground water pollution, e.g. phorate, methyl parathion [31,32]. Carbamates are used as insecticides, acaricides or nematicides to kill insects, mites and nematodes respectively. They can kill many aerobic and anaerobic bacteria in soil. They can easily escape in underground water if excess dosage is used, e.g. carbofuran, aldicarb and carbaryl [35]. Ethylene di bromide (EDB) is a volatile liquid used to produce gaseous poisons to kill stored grain pests. It leaves traces of harmful chemicals and causes food pollution. Phenoxy alkanoates are the weedicides causing serious damages to the ecosystem, as they kill variety of plants apart from weeds. They can also remain in soil and cause water pollution when they fail to degrade in soil medium, e.g. 2, 4-D, 2, 4, 5-T [38].

Many growth regulators like gibberellic acid, naphthalene acetic acid,

maleic hydrazide etc. are used in agriculture to suppress or enhance the growth of

crops or to initiate flowering or to increase the fruit size. They are also prone to

affect edible part of the crop as they can remain in fruits, grains when extensively

used.

The environmental pollution is becoming a global problem of which water

pollution is most dangerous. The major sources of pollution are industrial

wastewaters and effluents discharged to common drainage. These effluents pollute

not only water bodies nearby but also contaminate soil. Ground water also causes

pollution of drinking water and hence the health of human beings is badly affected.

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General information about water pollution is presented in flow chart with an

emphasis on drinking water pollution.

1 Drinking water 1 City waste 1 Dangerous 2 Ground water 2 Agricultural waste 2 Mild /rectifiable 3 River water 3 Industrial waste 3 Negligible 4 Sea water

Classification of water pollution depending upon the utility and location of

water is an important consideration for environmental problem. Drinking water

pollution is caused by source and its route of flow. Thus, the ground and river

water pollution may lead to drinking water pollution. It is observed that sea water

is significantly polluted, although no pollutant is directly added to sea (except

accidental ship leakage, oil spill or sinking of ship etc). This is because all polluted

rivers carry the pollutants to sea and due to continuous accumulation, the

concentration in sea water increases with time. Water pollution can be point source

or non - point source, i.e. it can be caused near the source of pollution or pollutants

are far away from the water bodies. Besides human activities, Nature also

contributes to water pollution. The concentration of pollutants generally decreases

as water moves to longer distances/depths and as the time passes. Not all pollutants

are immediately harmful, but they may cause ill effects with time. Some pollutants

like DDT and sanitary wastes in water can be extremely dangerous even in minute

quantity. However, pollutants like cadmium, copper or ammonia compounds can

be harmful in high concentration. Some other pollutants (nuclear wastes) have long

term effects though no short term effects are noticed. However, the sensitivity to

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pollutants in drinking water is more connected to physiology of human/animal

body than concentration of pollutant within safe range.

Water pollution in special perspective to understand the causes can be

classified as Main source of pollution - where pure water is mixed with pollutant

for the first time and Secondary source of pollution - where polluted water is

further polluted when water is traversing on the surface or below the surface of

soil. When the same pollutant is added to water two or more times from main and

secondary source, the concentration of pollutant increases, but with distance

traversed the pollutant concentration decreases.

People have been using natural colorants for painting and dyeing of their

skins, clothes and surroundings since long. Organic natural colorants have also a

timeless history of application, especially as textile dyes. These dyes, all aromatic

compounds, originate from plants (e.g. the red dye alizarin from madder and indigo

from wood), insects (e.g. the scarlet dye kermes from the shield-louse), fungi and

lichens.

Dyes contain chromophores, and auxochromes, electron donating

substituents that intensify the colour of chromophore. Based on chemical structure

of chromophore, 20-30 different groups of dyes can be classified. Azo,

anthraquinone, phthalocyanine and triarylmethane dyes are quantitatively the most

important groups. Others are diarylmethane, indigoid, azine, oxazine, thiazine,

xanthene, nitro, nitroso, methine, thiazole, indamine, indophenol, lactone,

aminoketone and hydroxyketone dyes and dyes of unknown structure (stilbene and

sulphur dyes). The vast list of commercial colorants is classified in terms of colour,

structure and application method in the Colour Index (C.I.) by the society of dyers

and colourists and the American Association of Textile Chemists and Colorists.

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The C.I. lists represent 10,500 different dyes; some of them are currently produced.

Each dye is given a C.I., the generic name determined by its application

characteristics and colour.

Azo dyes are aromatic compounds linked together by azo (-N=N-)

chromophores, and represent the largest class of dyes used in textile and other

industries. Most of the dyes applied in textile industries are azo compounds. The

release of these compounds into the environment is undesirable, not only because

of their intense colour at low concentration, but also because their products are

toxic to life. To remove azo dyes from wastewater is an equally important problem.

As azo dyes represent the largest class of organic colorants listed in the C.I

as shown in the following chart.

Their relative share among reactive, acid and direct dyes is even higher. It

is presumed that they make the majority of dyes discharged by textile industries.

Azo dyes Pigment

dye

Mordant dye

Basic dye

Direct dye

Reactive dye

Acid dyes Solvent dyes

Azoic & naphthol dyes

Disperse dye

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Many dyes are visible in water at concentrations as low as 1 mg /L. Textile

wastewaters, typically with dye content in the range from 10-200 mg/L [167] are

therefore usually coloured and present an aesthetic problem. As dyes are designed

to be chemically and photolytically stable, they are highly persistent in natural

environments. The release of dyes may therefore present an eco-toxic hazard and a

danger of bioaccumulation that eventually affects man by transport through the

food chain.

Acid dyes, the second largest class of dyes are anionic compounds chiefly used for dyeing fabrics such as wool, polyamide, silk and modified acryl. They bind to the cationic NH4+ ions of those fibers. Most acid dyes are azo, anthraquinone or triarylmethane compounds.

Reactive dyes, a class has groups that form covalent bonds with OH-, NH-

or SH- in fibers of cotton, wool, silk, nylon etc. The use of reactive dyes has increased ever since their introduction. Most reactive dyes are of azo or metal complex azo compounds. While dyeing with reactive dyes, hydrolysis of the reactive groups is an undesired side reaction that lowers the degree of fixation. In spite of the addition of high quantities of salt to raise fixation, it is estimated that 10 to 50% does not react with the fabrics and remain hydrolyzed in the water phase. The problem of coloured effluents is therefore mainly identified with the use of reactive dyes.

Direct dyes have high affinity for cellulose fibers. Direct dyes are generally with more than one azo bond or phthalocyanine, stilbene or oxazine compounds. Basic dyes are cationic compounds used to dye acid-group containing synthetic fibers. They bind to the acid groups of fibers. Basic dyes are diarylmethane, triarylmethane, anthraquinone or azo compounds and represent 5% of all dyes listed in the Colour Index. Mordant dyes are fixed to fabric by the addition of a mordant that combines with dye and fiber. As mordant dyeing is probably the oldest way, their use is gradually decreasing. They are used with wool, leather, silk, paper and modified cellulose fibers. Most mordant dyes are azo, oxazine or triarylmethane compounds. Disperse dyes are scarcely soluble that penetrate synthetic fibers such as cellulose acetate, polyester, polyamide, acryl, etc. This diffusion requires swelling of the fiber, either due to high temperatures (>120 °C) or with the help of chemical softeners. Dyeing takes place in dye baths with fine disperse solutions of these dyes. They are usually small azo or nitro compounds (yellow to red), anthraquinones (blue and green) or metal complex azo compounds (all colours).

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Pigment dyes has a small but increasing fraction of pigments and is the most widely applied group of colorants. About 25% of all commercial dyes listed in the C.I. are in the form of pigment dyes. Pigment dyeing is achieved from a dispersed aqueous solution and therefore requires the use of dispersing agents. Pigments are usually used together with thickeners in print pastes for printing diverse fabrics. Most of them are azo compounds (yellow, orange and red) or metal complex phthalocyanines (blue and green). Vat dyes are water-insoluble, widely used for dyeing cellulose fibers. The dyeing method is based on the solubility of vat dyes in their reduced (leuco) form. Reduced with sodium dithionite, the soluble leuco vat dyes impregnate the fabric. Oxidation is done later to bring back the dye in its insoluble form. Almost all vat dyes are anthraquinones or indigoids. Indigo itself is a very old example of a vat dye, with about 5000 years of history. Azoic dyes and Ingrain dyes (naphthol dyes) are the insoluble products of a reaction between a coupling component (usually naphthols, phenols or acetoacetylamides as azoic coupling components) and a diazotized aromatic amine (as azoic diazo components). This reaction is carried out on the fiber. All naphthol dyes are azo compounds. Sulphur dyes are complex polymeric aromatics with heterocyclic S-containing rings. Though representing about 15% of the large-scale dye production, sulphur dyes are not so much used all over the world. Dyeing with sulphur dyes involves reduction and oxidation, comparable to vat dyeing. They are mainly used to dye cellulose fibers. Solvent dyes are non-ionic dyes that are used for dyeing substrates in which they can dissolve, e.g. plastics, varnishes, inks, waxes and fats. Their utilization is habitually increased for textile-processing. Most of these are diazo compounds with some molecular rearrangement. Fluorescent brighteners mask the yellowish tint of natural fibers by absorbing ultraviolet light and weakly emitting visible blue. They are not dyes in the usual sense as they lack intense colour. Based on chemical structure, several different classes of fluorescent brighteners are discerned: stilbene derivatives, coumarin derivatives, pyridoxines, 1,2-ethene derivatives, naphthalimides and aromatic or heterocyclic ring structures. Many of them contain triazinyl units and water-solubilising groups [168]. Apart from the above mentioned dye classes, the C. I. also lists food dyes

and natural dyes. Food dyes are not used as textile dyes while the use of natural

dyes (mainly anthraquinone, indigoid, flavenol, flavone or chroman compounds) is

limited as mordant, vat, direct, acid or solvent dyes in textile industries.

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From the studies it is evident that azo dyes are also important pollutants and

to study their toxicity will be the point of concern for researchers. Though dyes

make our lives colourful, they are the cause to make our lives more miserable. As

most of them are synthetic and nonbiodegradable, they accumulate into water

bodies, aquatic flora and fauna respectively and cause detrimental effects as

pollutants. Toxicants to which subjects are exposed may be in several different

physical forms, such as vapors or dusts that are inhaled, liquids that can be

absorbed through the skin, or solids ingested orally. Matrix, a medium with which

the toxicant may be associated have a strong effect upon the toxicity. There are

numerous variables related to the ways in which organisms are exposed to toxic

substances. The most crucial factors related to toxicology, dose, the duration of

exposure per incident and the frequency of exposure are also important to study.

The rate of exposure and total time period over which the organism is exposed are

both important situational variables. In this study azo dyes are toxicants while

water is the matrix. Dyestuff toxicity has been investigated in numerous

researches. These toxicity studies i.e. mortality, genotoxicity, mutagenicity and

carcinogenicity diverge from tests with aquatic organisms viz. fish, algae, bacteria,

etc. to compare with mammals.

Furthermore, research has been carried out to see the effects of dyestuffs

and dye containing effluents on the activity of both aerobic and anaerobic bacteria

in wastewater treatment systems. The acute toxicity of dyestuffs is generally low.

Algal growth tested with commercial dyestuffs was generally not inhibited at dye

concentrations below 1 mg/l. The most acute toxic dyes for algae are anionic basic

dyes [169]. Fish mortality tests showed that 2% out of numerous commercial

dyestuffs tested had LC50 values below 1 mg/l. The most acutely toxic dyes for fish

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are azo dyes, especially those with a triphenylmethane structure. Fish also seems to

be relatively sensitive to many acid dyes. Mortality tests with rats showed that only

1% out of 65 % commercial dyestuffs tested had LD50 values below 250 mg/kg of

body weight [170]. The chance of human mortality due to acute dyestuff toxicity is

probably very low. However, acute sensitization reactions by human to dyestuffs

often occur. Some disperse dyestuffs have been found to cause allergic reactions,

i.e. eczema or contact dermatitis [171]. Chronic effects of food colorants of azo

dyes have been studied for several decades. The effects of occupational exposure

of dyestuffs to workers in dye manufacturing and utilising industries have received

attention. Azo dyes as such are seldom mutagenic or carcinogenic, except for some

with free amino groups [172]. However, reduction of azo dyes leads to formation

of aromatic amines (mutagens and carcinogens). In mammals, metabolic reduction

of azo dyes is mainly due to bacterial activity in the anaerobic parts of the lower

gastrointestinal tract. Various other organs, especially the liver and the kidneys can

also reduce the concentration of azo dyes. Lower aquatic invertebrates and

vertebrates are more susceptible to these non biodegradable azo dyes, as they

slowly accumulate in water bodies and finally reach into aquatic organisms and

affect the various organs.

The bioaccumulation tendency of dyestuffs in fish has been

comprehensively investigated by the Ecological and Toxicological Association of

Dyes (ETAD). The bioconcentration factors (BCF) of 75 dyes from different

application classes were determined and related to the partition coefficient (n-

octanol/water) of each different compound. Water soluble dyes with low partition

coefficient, i.e. ionic dyes like some of acid, reactive and basic dyes are less

bioaccumulated [173,174].

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Aquatic animals are often particularly affected due to elevated exposures

arising from their living immersed in the surface water. They show very serious

changes like having highly permeable skin and gills and other inherent

sensitivities. Fish are the sole vertebrates with anamniotic eggs that undergo

development in surface waters and therefore the embryonic stages of these animals

are also highly sensitive to chemical pollutants [175]. These issues contribute to

the lower water quality guidelines to protect aquatic life and human health.

Fish occupies a prominent position in the study of toxicology mainly

concerning both human and ecological health. There are several reasons to select

fish as a model animal. Fish belongs to the most diverse class of vertebrates;

whose species identified till date is greater than the combined number of other

classes [176]. It also reflects the great diversity of aquatic systems that inhabit

from fresh water to hyper saline waters, with temperatures ranging from below

freezing to > 45°C, with pressures ranging from 1 to 1000 atm, other variabilities

in solar radiation, oxygen concentrations, ionic-organic matter compositions,

bottom environments etc. This habitat diversity has long driven the use of fishes

for scientific inquiries into influences of environmental variables on the evolution,

genetics, and adaptations of organisms. Aquatic systems, due to their tendency to

accumulate relatively high concentrations of chemicals discharging from

surrounding terrestrial systems, are highly vulnerable. Thus, they are often time

repositories for a large array of stressor chemicals. In discussing exposure sites for

toxicants, it is useful to consider the major routes and sites of exposure,

distribution and introduction of toxicants in the body as shown in table 1.2.

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Table: 1.2 Major routes & sites of exposure to pollutants.

Through ingestion Through inhaled air Through dermal exposure

It will affect all the related organs of the gastrointestinal track and change the cell metabolism as well as affect the kidney and bladder.

It will enter through the nose and affect the whole pulmonary system. In case of fish, it will enter through the gills.

It will enter through the skin and affect all the integ- umentory derivatives as well as blubber layer of the skin.

The major routes of accidental or intentional exposure to toxicants in fish

and other animals are the skin (percutaneous route), the lungs (inhalation,

respiration, pulmonary route) and the mouth (oral route). The pulmonary system of

other animals is the most likely to inhale toxic gases or very fine, respirable solid

or liquid particles inside the body while in fish the whole branchial system is most

likely to receive dissolved, non-biodegradable toxicants like dyes, pesticides,

fertilizers, heavy metals, surfactants etc. Fish plays important role in setting these

water quality guidelines for freshwater and marine systems. U. S. Environmental

Protection Agency (EPA) provides specific recommendations for freshwater and

marine species to be used for acute and chronic toxicity tests that are used in

establishing the chemical safety assessments [177]. The toxicity tests provide

information concerning relative acute toxicities of various chemicals, relative

sensitivities of different species to selected chemicals and the sublethal effects of

chemicals and their mixtures.

Toxicity tests also play important role in ecological assessments. In

addition to toxicity testing for ecological effects, fish is the most employed

organisms for biomonitoring. This is likely due to ability of aquatic systems to

receive and accumulate environmental contaminants. Fish-targeted biomonitoring

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includes approaches for detecting aquatic pollution and hence are known as

biomarkers.

Aquatic toxicology also deals with an approach to consider sublethal

concentration of toxicants that not cause death but harm the individual to altered

state. Biomarkers of exposure response and genetic susceptibility have been

studied and these helped to come across the questions of bioavailability of an

organism. Biomarkers illustrate multiple organs, tissues and cellular sites of action

and the spectrum of responses that were possible due to exposure of toxicants.

Surat (Gujarat) is famous worldwide for its dyeing and printing industries

that sometimes discharge effluents into nearby ponds and drains, without further

treatment. The problem is aggravated because water is directly used for irrigation

purpose and is discharged into the river. These effluents contain highly toxic dyes,

bleaching agents, salts, acids, alkalis, surfactants and some toxic heavy metals like

cadmium, copper, zinc, chromium, iron etc. Though dye is one of the prime

requirement, it creates inevitable changes in our lives. They accumulate into the

water bodies, aquatic flora and fauna as most of them are synthetic and

nonbiodegradable and hence cause detrimental effects on them; azo dyes are one of

these. Recent toxicological research with fish has pursued the study of mechanism

of action to respond to toxicant and has provided tools for eco-toxicological

investigations [178-180]. Due to intensifying strain that mankind put on natural

resources and the environment, there is an urgent need to develop approaches that

recognize and solve these problems. New and advanced dye removal technique is

required to decrease the effects of toxic azo dyes on aquatic ecosystem as well as

on human health [181-185].

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Several factors that determine the technical and economic feasibility of dye

removal are:

1. dye type 2. wastewater composition 3. dose and costs of required chemicals 4. operation costs (energy and material) 5. environmental fate and handling costs of generated waste products Various physical, chemical and biological pretreatment, main treatment and

post treatment techniques can be employed to remove colour from dye containing

wastewaters. The physicochemical techniques include membrane filtration,

coagulation/flocculation, precipitation, flotation, adsorption, ion exchange,

ultrasonic mineralisation, electrolysis, advanced oxidation (chlorination, bleaching,

ozonation, fenton oxidation and photocatalytic oxidation) and chemical reduction.

Biological techniques include bacterial, fungal biosorption and biodegradation in

aerobic, anaerobic, anoxic or combined anaerobic/aerobic treatment processes.

In general, each technique has its limitations. The use of one specialized

process may not be sufficient to achieve complete dye removal. The strategies

therefore consist of a combination of different techniques. Membrane filtration,

nano-filtration and reverse osmosis using membranes can be applied as main or

post treatment processes for separation of salts and larger molecules including dyes

from dye effluents.

Filtration is generally not suitable as the membrane pore size is too large to

prevent dye molecules passing through [178] but it can be successful as

pretreatment for nano-filtration or reverse osmosis [186]. Membrane filtration is a

quick method with low requirement. Some of the concentrated compounds,

including non-reactive dyes can be reused [187,188]. This reuse, then, applies

generally for smaller waste flows [189]. Disadvantage of membrane filtration

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technique is that the generated concentrate must be processed further [190]. The

capital costs of membrane filtration are therefore usually high.

Coagulation is often applied in the treatment of textile wastewater, either

to partly remove Chemical Oxygen Demand (COD) and colour from the raw

wastewater before further treatment [192-194], to polish the final effluents of

biologically or otherwise treated wastewater [178, 194, 195]. The principle of

process is the addition of a coagulant for rapid coagulation. So formed flocs

subsequently settle down or float and are removed from the water. Various

inorganic coagulants are used, mostly lime, magnesium, iron and aluminium salts.

Inorganic compounds are, however, generally not very suitable to remove highly

soluble dyes from solution [183,196] unless large quantities are dosed [180].

Hence, they produce considerable volume of toxic sludge that must be incinerated

or handled otherwise. This presents a serious drawback of the process [183].

Recently developed organic polymers have been proven highly effective as dye

coagulants (even for coagulation of reactive dyes) as sludge production associated

with polymer dosing is relatively low [197,198]. The polymers used for colour

removal are, however, cationic and may be toxic to aquatic life even at very low

concentrations (less than 1 mg/l). In biological wastewater treatment plants, some

cationic polymers have been found to inhibit the nitrification process [199].

In sorption and ion exchange, activated carbon can be used to remove

dyes from wastewater, either by adsorption of anionic dyes or by adsorption

combined with ion exchange for cationic dyes. Sorption techniques produce waste

sludge, i.e. dye-saturated material that should be disposed off. Activated carbon is

capable of adsorbing many different dyes with high adsorption capacity [200-203].

It is expensive as well as its regeneration is also costly because desorption of dye is

Chapter I

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not easily achieved [178, 204]. Various other (mostly low-cost) adsorbents

investigated as an alternative to activated carbon are listed in the table 1.3.

Table: 1.3 Literature survey on different bioadsorbents.

No. Types [Biomass] Materials References 1 Non-

modified cellulose (plant) biomass

Corn/maize cob [205 -208] Maize stalk [209] Wheat straw [207] Linseed straw [209] Rice husk [204,209] Wood chip [207,210,211] Sawdust [208,209,212,213] Bark [204,214,215] Coirpith [216] Banana pith [217] Bagasse pith [206,218-221] Palm fruit bunch particle [222-224] Peat moss [208,225] Peat [203,209,210] Sugar beet pulp & sugar industry mud [209]

Cotton waste [204] Cellulose [226]

2 Modified cellulose biomass

Carbonised coirpith [216] Carbonised coconut-tree sawdust [227] Chemically modified sunflower stalk [228] Polyamide-epichlorohydrin-cellulose [229- 231]

Carbamoyl and quaternised-cellulose [232-236] Sugarcane bagasse [ resin] [237]

3 Bacterial biomass Aeromonas [238] Actinomycetes [239] Activated sludge [240, 241] Dried and powdered biogas waste slurry [242- 243]

4 Fungal biomass [ 244 - 249] 5 Yeast biomass [250] 6 Chitin A material that can be found in shell [ 251,252] 7 Chitosan Deacylated chitin [253, 254]

Cross-linked chitosan fiber [255 – 268] 8 Soil material Sand [208]

Silica [269] 9. Charcoal Barbecue, bone and wood [208, 209,270] 10 Activated

biomass Bauxite and alumina [201, 213]

These materials show high dye removal capacity [203] even higher than

activated carbon. Materials such as rice husks, bark, cotton waste and hair have a

high capacity for binding basic (cationic) dyes but hardly remove dyes from other

Chapter I

20

classes. Acid and reactive dyes are generally most difficult to remove. Chitin and

chitosan have extremely high acid and reactive dye binding capacity [251-254]. To

evaluate the feasibility of a potential dye adsorbent, not only cost and dye-binding

capacity should be considered, but also its adsorption kinetics, regeneration

properties, requirements and limitations with respect to environmental conditions

(like pH, temperature and salt concentration) should be taken care off. Cross-linked

chitosan and sodium alginate are considered suitable materials with respect to the

adsorption capacity. Most non-modified biological materials possess low

adsorption capacity while adsorbents with a high adsorption capacity like chitin,

chitosan and polyamide-epichlorohydrin-cellulose have the drawback of very slow

kinetics [271]. Despite a large number of publications on dye adsorption, full-scale

application is limited to combinations, e.g. combined adsorption and

biodegradation in activated carbon amended activated sludge systems [272-275] or

anaerobic bioreactors [276], or combined sorption and coagulation by a synthetic

clay slurry [201].

Biological techniques are also used for the adsorption of dye. These are

based on microbial biotransformation of dyes. As dyes are designed to be stable

and long-lasting colorants, they are usually not easily biodegraded. Partial or

complete biodegradation of dyes by pure and mixed cultures of bacteria, fungi and

algae have been demonstrated. In case of bacterial biodegradation, the chemical

structures of dyes should be considered. Investigations to bacterial dye

biotransformation have so far mainly been focused to the most abundant chemical

class, the azo dyes. The electron- withdrawing nature of the azo linkages obstructs

the susceptibility of azo dye molecules to oxidative reactions. Therefore, azo dyes

generally resist aerobic bacterial biodegradation [240, 242, 277-279]. Only bacteria

Chapter I

21

with specialized azo dye reducing enzymes were found to degrade azo dyes under

fully aerobic conditions. In contrast, breakdown of azo linkages by reduction under

anaerobic conditions is much less specific. Anaerobic treatment must be

considered merely as the first stage of complete azo dye degradation. The second

stage involves conversion of the produced aromatic amines. This can be achieved

by biodegradation under aerobic conditions. Chitosan is a preferable adsorbent in

most textile industries. Chitosan is a linear polysaccharide composed of randomly

distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-

glucosamine (acetylated unit). Its chemical structure [264] is given below:

It has a number of commercial and possible biomedical uses. Commercial

chitosan is derived from the shells of shrimp and other sea crustaceans [280].

Chitosan and its derivatives have been used in non-viral gene delivery. Trimethyl

chitosan and quaternised chitosan have been used to transfect breast cancer cells.

At approximately 50% trimethylation, the derivative is most efficient at gene

delivery but is toxic. Oligomeric derivatives are relatively non-toxic and have good

gene delivery properties [281].

Chitosan is used primarily as a plant growth enhancer and as a substance

that boosts the ability of plants to fight against fungal infections. It is approved for

use outdoors and indoors on many plants grown commercially and by consumers.

Because of its low toxicity and abundance in the natural environment, chitosan is

not expected to harm people, pets, wildlife or the environment when used

according to labelled directions [282]. It can also be used in water processing

Chapter I

22

engineering as a part of a filtration process. It causes the fine sediment particles to

bind together and is subsequently removed with the sediment during sand

filtration. It also removes phosphorus, heavy minerals and oils from the water. It is

an important additive in the filtration process. Sand filtration apparently can

remove up to 50% of the turbidity alone while the sand filtration along with

chitosan removes 99% turbidity from the water [283].

Chitosan in combination with bentonite, gelatin, silica gel or other fining

agents is used to clarify wine, beer etc. It improves flocculation and removes yeast

cells, fruit particles and other substances that cause hazy wine. Chitosan combined

with colloidal silica is a popular fining agent for white wines as it does not require

acidic tannins (found primarily in red wines) to flocculate with. It is a partially

acetylated glucosamine biopolymer found in the cell wall of some fungi such as the

Mucorales strains. However, it mainly results from deacetylation of chitin [284-

286]. The degree of deacetylation (% DA) determined by NMR spectroscopy in

commercial chitosans is in the range 60-100 %. Chitosan is positively charged and

soluble in acidic to neutral solution with a charge density dependent on pH and

the % DA-value. In other words, it is bioadhesive and readily binds to negatively

charged surfaces such as mucosal membranes. It enhances the transport of polar

drugs across epithelial surfaces, and is biocompatible and biodegradable. Purified

qualities of chitosans are available for biomedical applications. This biopolymer is

also a known sorbent, effective in the uptake of transition metals since the amino

groups on chitosan chains serve as coordination sites [286]. In contrast to chitin,

the adsorption ability of dyes and metals with chitosan is superior due to its higher

content of amino functional groups [284,287].

Chapter I

23

Conventional procedures to remove dye from wastewater such as activated

sludge, bacterial and chemical oxidation, ozonisation or physical methods like

membrane filtration, ion exchange or electrochemical techniques are either

expensive or inadequate [278,288-293]. Solid phase extraction, commonly known

as sorption, is the recent technology to remove dye from wastewater. Activated

carbon is widely used as adsorbent [206] for this purpose but high procuring and

processing cost restricted its use in many countries. This promoted search for

alternative cost effective adsorbent. Recently different low cost adsorbents

including some industrial and agricultural wastes [294-298] such as fly ash, fuller’s

earth, waste red mud, bentonite clay, metal hydroxide sludge, peat, pith, cotton

waste, rice husk, teakwood bark etc. have been used but their effectiveness is

limited and inferior to that of activated carbon. Adsorption capacity of the

biological materials like coir pith [299], banana and orange peel particularly

towards anionic dye is very poor. Biological materials generally contain high

amount of cellulose and adsorb anionic dye in a reversible process as against basic

dyes where adsorption occurs there coulombic attraction and ion exchange process.

The biopolymer chitosan has gained importance in environmental biotechnology

due to its very good adsorption capacity towards dyes and metal ions [301,302].

Moreover, chitosan can be obtained at industrial scale by chemical deacetylation of

crustacean chitin, byproduct of seafood industry. Further, chitosan in the form of

swollen beads has been found to exhibit better adsorption capacity than flakes

[303]. Objective of the present investigation is to study the efficacy of chitosan and

chitosan coated calcium alginate in the form of hydrobeads to remove Direct Green

6 (DG 6) and Acid Orange 7 (AO 7), members of anionic azo dyes, from their

aqueous solutions. The brief outline of the work has been given later.

Chapter I

24

The overall aim of the present investigation is to determine the toxicity of

some azo dyes on fish. For this, two model azo dyes viz., DG 6 and AO 7 were

chosen and their effects on (i) behavioural, (ii) histopathological, (iii) biochemical

and (iv) haematological parameters in fish are investigated. The widely consumed

Indian major carp, Labeo rohita fingerlings were taken for these studies. Labeo

rohita is considered as one of the most commercially important freshwater fish in

India as a food source for human.

Knowledge of acute toxicity of xenobiotics is very helpful in predicting and

preventing acute damage to aquatic life in receiving waters as well as in regulating

toxic waste discharges. In view of this, short-term acute toxicity tests were

performed on Labeo rohita fingerlings over a period of 96 h to determine the lethal

concentration (LC50) value so as to elucidate the acute effects of above mentioned

azo dyes (DG 6 and AO 7).

Labeo rohita fingerlings were obtained from the Government Fish Farm,

Valod, Gujarat and healthy fingerlings were acclimatized in the laboratory

conditions. For that, physicochemical parameters of water such as dissolved

oxygen, alkalinity, hardness, pH and temperature were measured by well

established methods (APHA) [304] and maintained throughout the period of

acclimatization and experiments. Fingerlings were fed once daily and kept with

continuous aeration. Water along with the excreta was changed from the aquaria

every 48 h.

LC50 values of both the dyes for Labeo rohita were determined by standard

method [305]. On the basis of LC50 values found, three different sublethal

concentrations of the dyes were selected. Fingerlings were divided into groups of

ten and were tested for each concentration and toxicological effects were observed

Chapter I

25

after a fixed interval from the day of exposure. Controls without toxicant were also

run simultaneously. The behaviour of fingerlings was noted every 24 h upto end of

the study. After each exposure, fingerlings were sacrificed. The blood and organ

samples were collected for haematological, histopathological and biochemical

analysis. A comparison was always done with the control. Data obtained from the

experiments were analyzed statistically and the results expressed as mean ± S.E.

The significance of results was evaluated using student’s t’ test at p<0.05.

The chapter wise classification of the work presented in the thesis is briefly

summarized as follows:

Chapter 1

This chapter gives a general overview of the environment, water resources,

water pollution, dyes and their toxicity. An updated literature search on the effect

of water pollutants particularly dyes is provided. Previous studies on dyes affecting

different organs of fish are cited. It thoroughly emphasizes on some questions viz.,

why fish was chosen for the research work, and why Labeo rohita only? As azo

dyes are most important class of the dyes and are nonbiodegradable, these were

selected as toxicants. The chapter also discusses the remedial part by using

bioadsorbents to treat the textile wastewater. A brief description of work reported

in different chapters is also provided.

Chapter 2

Histopathology is an important tool for determining the action of any

toxicant at organ and tissue/cell level providing data concerning organ/tissue

damage. This chapter illustrates the behavioral and histopathological aspects of the

study. Fingerlings were exposed for a definite time period to all the concentrations

of both the dyes and then were sacrificed.

Chapter I

26

The organs selected for the study were on the basis of some important

criteria given below:

i. Gills serve a variety of physiological functions, including respiratory gas exchange, osmoregulation, nitrogen excretion and control of acid–base balance. Because of these functions, fish gills have the following features important for exchange of toxic chemicals between a fish and its environment. In case of fish, gills are the most effective sites for absorption of chemicals present in water as they remain in direct contact of water and vascularization of gills is very high compared to other tissues/organs. ii. Liver accumulates xenobiotics; the hepatocytes biotransform these compounds and transport them to bile for elimination. iii. Kidney plays a major role for excretion of toxic materials taken inside the body. iv. Brain is a fatty organ responsible for overall control and coordination in the body. v. Intestine is an important site of absorption of toxicants in fish. Above mentioned target organs viz., gills, liver, kidney, brain and intestine

were immediately taken out and fixed in the formal saline, embedded in the

paraffin wax. These organs were specially selected as they are examined in

histological assessments as part of fish health related studies. Thin sections of

organs (5-6µm) were cut. Haematoxylin and Eosin staining of sections was done

to assess the damage done to various organ systems and tissues at cellular level.

The lesions observed during the sub chronic trials were concentration and time

dependent. In case of gills, the observed changes were erosion of secondary gill

lamellae and central axis of primary gill lamella, lifting of epithelium and the

curved tip of the secondary gill lamellae. Major changes were seen mostly in the

liver like appearance of disposition of hepatic cords, perinuclear space, ruptured

blood sinusoids and atrophy of the cells. Kidney was also one of the target organs

and greatly affected and the changes were destruction of glomerulus, increase of

perinuclaer space, shrinkage of glomerular tubules etc. Some minor changes were

also seen in brain of exposed fingerlings like disjointment of all layers of optic

tectum, space around mononuclear cells, migration and clumping of these cells

Chapter I

27

towards margins. Intestinal villi were also affected because of accumulation of

dyes in intestine.

Chapter 3

Based on the changes found in histopathological observations, biochemical

estimation was also done. As necrosis was observed during histopathological

studies, activities of enzymes acid phosphatase (ACP) and alkaline phosphatase

(ALP) were also determined. Studies on the alteration in nitrogen metabolism

include the estimation of alanine aminotransferase (ALAT) and aspartate

aminotransferase (AST). The enzyme lactate dehydrogenase (LDH) is associated

with the cellular metabolism. It catalyses the reversible oxidation of lactic acid and

pyruvic acid at the terminal step of glycolysis. LDH acts as pivotal enzyme

between the glycolytic pathway and tricarboxylic acid cycle. The activity of

enzymes ACP, ALP, ALAT, AST and LDH were measured colorimetrically using

known procedures. The activity of enzymes in all organs after the exposure to

dyes, and post exposure levels of activity were also determined and compared to

that of the normal fish. In the liver and kidney, activities were decreased for all the

enzymes while in case of gills and intestine ACP and ALP till the end of the

exposure. LDH showed very minor change in its activity in all the organs except

brain. Remarkable increase in the LDH activity in the brain was observed. The

results from the biochemical estimation of target enzymes were observed and

comparatively analysed in all the organs after histopathological examination.

Chapter 4

Blood is a pathophysiological reflector of whole organism and hence

haematological parameters have been used as diagnostic parameters for

investigation of physiological changes. It has been reported that haematological

Chapter I

28

changes occur due to toxicity of pollutants. To confirm the effects of azo dyes with

these earlier observations, the haematology study was carried out. This chapter

describes the haematological changes on exposure to azo dyes. Blood samples

from the exposed fingerlings were collected in the EDTA bulb by puncturing the

heart as well as from the caudal vein and then were processed for the complete

blood cell (CBC) count and blood smear. The changes in the parameters like White

Blood Cell (WBC) count, Red Blood Cell (RBC) count, Haemoglobin Content

(Hb), Packed Cell Volume (PCV), Mean Corpuscular Volume (MCV), Mean

Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin

Concentration (MCHC) were compared with the blood taken from control

fingerling. Morphological changes as well as occurrence of micronuclei in the

RBC were also investigated by using standard methods. In all the haematological

studies, Hb and RBCs were greatly decreased while WBCs were increased.

Moreover, typical variations of micronuclei were observed in some RBCs.

Chapter 5

It is also important to check the reversibility of their toxicity with time

termed as recovery. This chapter describes the recovery phenomenon. Aquatic

organisms detoxify the organic xenobiotics by metabolism and are capable of

maintaining cell homeostasis. At the end of all exposures of each dye

concentrations, rest of the live fingerlings were again kept in the dye free

freshwater aquaria for 10 days to see recovery in all aspects like behaviour,

histopathological, biochemical, haematological changes etc. All the reversible

changes were compared with the normal and exposed fingerlings respectively.

When allowed a change in freshwater, all fingerlings showed good recovery in

terms of behaviour, histopathology and enzyme activity. But in same fingerlings

Chapter I

29

some haematological parameters remained unaltered i.e. they did not recover with

time.

Chapter 6

This chapter enlightens on an important part of this study i.e. adsorption.

The nonbiodegradable azo dyes were removed from the water by using appropriate

bioadsorbents like chitosan and sodium alginate. Chitosan beads and chitosan

coated calcium alginate beads were prepared, dried and stored in the incubator.

These beads were suspended in 50 ml of each dye solution (10ppm). The

adsorption of both the dyes on beads was observed at different pH, temperature

and adsorbent dose by using spectrophotometer. Adsorption with the help of

chitosan showed good results in case of DG 6 dye compared to AO 7 dye.

Chapter I

30

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