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INTRODUCTION

Pesticides are stable compounds. Indiscriminate, liberal and

injudicious use of a pesticide by man to control the crop pests and

diseases for higher agricultural productivity has led to a slow but steady

deterioration of the aquatic ecosystem, since water is the ultimate sink.

These pollutants also destroy the quality of the aquatic media and render

it unfit for various aquatic organisms. Pesticides are useful tools in

agriculture and forestry, but their contribution to the gradual degradation

of the aquatic ecosystem cannot be ignored. Accumulated pesticides

induce generation of reactive oxygen species (ROS). The ROS attack

unsaturated fatty acids of the cell membrane that lead to the formation of

LPO (Viarengo, 1989). Increased formation of lipid peroxidation causes

alterations in the levels of GSH and activities of the antioxidant enzymes

such as SOD, CAT, GPx and GST which leads to oxidative stress. All the

bio-molecules of cell like nucleic acids, lipids, proteins and

polysaccharides are potential substrates of ROS (Manduzio et al., 2005).

Such an effect may be at cellular or even at molecular level but ultimately

it would lead to physiological, pathological and biochemical disorders

that may prove fatal to the organism (Patil et al., 1989; Jain, 2000). Many

investigators reported a variety of wreckage in various metabolic

processes in different species exposed to different kinds of pollutants

(Scott, 1967; Abel, 1974; Langstone, 1986; Lomte et al., 2000).

Qualitative and quantitative study of changes in major

biochemical components of organisms such as proteins, ascorbic acid,

DNA and RNA are useful to know different toxicants and defensive

mechanism of the body against toxic effects of pesticides. These

biochemical components are indices of pollution as they determine

nutritional status, health and vigor of an organism.

80

Halloway (1984) have state that ascorbic acid has potential role to

reduce the activity of free-radical induced reactions. Ascorbic acid is an

important dietary antioxidant and serves to protect against oxidative

damage to macromolecules such as lipids, protein, DNA and RNA which

are implicated in chronic diseases (Halliwell and Gutteridge, 1999).

The investigation regarding the physiological and biochemical

changes after pesticide exposure and its subsequent recovery in non target

aquatic species such as molluscs is insufficient. Hence in the present

study an attempt was made to investigate the effect of chronic treatment

of pesticides dicofol and dichlorovos and its subsequent recovery by

exogenous administration of L-ascorbic acid on the biochemical

composition of different tissues of fresh water bivalve, Parreysia

cylindrica. Some basic mechanisms of the mode of action of pesticides

can be studied on these model animals which can be applicable to other

higher forms. Vital organs viz. mantle, foot, gills, gonads, digestive

glands and whole soft body were used to as test organs.

Proteins:-

Proteins are encoded by genes, they are primary molecules that

make up cell structure and carry out cellular activities. Proteins are

designed to bind every conceivable molecule- from simple ion to large

complex molecule. They catalyze extraordinary range of chemical

reaction, provide structural rigidity to cell, control flow of material

through membrane, regulate concentration of metabolites and control

gene function. Amino acids are the monomeric building blocks of

proteins. Central α-carbon of amino acid adjacent to the carboxyl group is

bonded to four different chemical groups, an amino (-NH2) group, a

carboxyl (-COOH) group, a hydrogen atom (H) and one variable group

called a side chain or R-group. The structure of protein is described in

terms of four hierarchical level of organization as primary, secondary,

81

tertiary and quaternary structure. The function of nearly all protein

depends on their ability to bind other molecules or ligands with high

degree of specificity.

Any undesirable change in the environment affects the protein

level by changing the physiology of organism. Various toxicants like

heavy metals, pesticides etc are known to disturb the protein metabolism

in the body of organism. Young (1970) suggested that, dynamic

equilibrium mechanism in the internal environment of organism changes

the protein content of cell periodically by the degradation and synthesis.

Shakoori et al., (1976) studied the effect of malathion, dieldrin and endrin

on blood serum, proteins and free amino acids pool in Channa punctatus.

Ramanarao and Ramamurthy (1978) studied the protein content in the

tissue of Pila globosa after exposing to Sumithion. Yagana Bano et al.,

(1981) studied the change in protein content in the muscles of Clarias

batrachus due to DDT exposure. Nagy et al., (1981) studied that the

pesticides interfere with protein synthesis and degradation, resulting in

alteration of dynamic equilibrium. Sivaprasad et al., (1981) studied the

impact of methyl parathion on protein content in tissues of Pila globosa.

Ramalingam and Ramalingam (1982) studied the protein content in the

tissues of Sarotherodon mossambicus exposed to DDT, mercury and

malathion. Murty (1986) studied pesticide induced biochemical changes

like disturbance in metabolism, inhibition of important enzymes,

retardation of growth and decrease of fecundity and longevity in fish.

Mane and Muley (1989) studied alteration in protein content in

Lamellidens marginalis on exposure to endoslfan. Zambare (1991)

studied alteration in protein content of fresh water bivalve, Corbicula

striatella when exposed to heavy metals. Jadhav (1993) studied the

impact of pesticides on some physiological aspects of freshwater bivalve,

Corbicula striatella and decrease in protein content. Protein content in

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mantle, foot, gill, digestive gland and whole body tissue of freshwater

bivalve, Parreysia corrugata after acute and chronic exposure to copper

sulphate was studied by Deshmukh and Lomte (1995). The alteration in

protein content in Lamellidens marginalis on exposure to endosulfan was

studied by Mule and Mane (1995). Bais and Arasta (1995) studied the

effect of sublethal concentration of oldex on protein, lipid and glycogen

level in the catfish, Mystus vittatus. Singh and Agrawal, (1996) stated that

the toxicants may affect the hormonal balance which could directly or

indirectly affect the tissue protein levels. Gupta and Bhide (2001; 2004)

studied gradual decline in number of protein fractions as well as in the

intensities of some of the protein fractions in Lymnaea stagnalis when

exposed to Nuvan. Organophosphorous and carbamate pesticides caused

disruptive effects on carbohydrate and protein metabolism of the

freshwater snail, Lymnaea acuminate (Tripathi and Singh, 2002; 2003).

Jagatheeswari (2005) studied the biochemical changes induced by

pesticide, phosphalone in Cyprinus carpio at different concentrations.

Mohanty et al., (2005) analyzed and compared protein profiles in

different tissues namely, gills, foot and mantle of two fresh water

bivalves, Lamellidens corrianus and Lamellidens marginalis and found

protein markers which helps to study the molluscan taxonomy. Keshvan

et al., (2005) studied total protein content in freshwater crab,

Barytelphusa guerini on exposure to Hildan. Ghanbahadur et al., (2005)

studied the effect of Organophosphate (Nuvan) on protein contents of

gills and liver in, Rasbora daniconius. Borane and Zambare (2006)

studied the role of ascorbic acid on protein metabolism in different tissues

of an experimental model, the Channa orientalis after exposure to

cadmium chloride. Kharat et al., (2009) studied the impact of tributylin

chloride on total protein contents on freshwater prawn, Macrobrachium

kistnensis. Thenmozhi et al., (2009) studied that stress induced alterations

83

in crab in the carbohydrate, protein and lipid content decreased when

exposed to higher concentrations because of their utilization to meet the

energy requirement during the stress caused by cattle shed effluents.

Kamble et al., (2010) studied biochemical changes in the protein contents

in the tissues like gills, hepatopancreas, gonads, muscle, mantle and foot

of freshwater bivalve. Siddiqui et al., (2010) studied copper sulphate and

its effect on protein in some vital organs of freshwater crab, Barytelphusa

gureini. Shelke (2010) studied the effect of cadmium chloride on total

protein alterations in liver and gonads of freshwater fish,

Amblypharyngodon mola. Upadhye et al., (2010) studied the tissue

specific protein profiling of freshwater pearly mussel, Parreysia

corrugata. Inyang et al., (2010) studied the effects of sub lethal

concentrations of diazinon on total protein and transaminase activities in

Clarias gariepinus. Abdul et al., (2010) studied the impact of sublethal

concentration of Triazophos on regulation of protein metabolism in the

fish Channa punctatus (Bloch). Nandurkar and Zambare, (2010b) studied

the effect of Trimethorprim on protein content of the freshwater bivalves,

Lamellidens corrianus and Parreysia cylindrica. Patil (2011) studied

protein changes in different tissues of freshwater bivalve Parreysia

cylindrica after exposure to indoxacarb. Palanisamy et al., (2011) studied

the changes protein contents in the muscle of Mystus cavasius (Ham)

exposed to electroplating industrial effluent chromium.

Hughes (1974) reported that ascorbic acid is a diffusible biological

reductant when present in appropriate concentration and contributes to

the maintenance of the integrity of SH group of many proteins. L-

ascorbic acid is a strong antioxidant and may extent its protective effects

by chelating the toxicant or by precipitating free radicals and removing

them from the system (Tajmir and Riahi, 1991). Mahajan and Zambare,

(2001) reported the protection by ascorbic acid against the heavy metal

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induced alterations in protein levels in fresh water bivalve, Corbicula

striatella. Chandravathy and Reddy (1994) studied recovery trends in

protein content in fish Anabas scandens after transferring into pollutant

free water for 15 days and slowly limped back to normalcy. Many other

workers have studied the protective role of ascorbic acid against pesticide

induced changes in protein levels of aquatic animals (Kapila and

Ragothamam, 1999; Nagpure, 2004; Sinde, 2008). Ramanathan et al.,

(2003) studied protective role of ascorbic acid and a-tocopherol on

arsenic induced microsomal dysfunction. Gapat (2011) studied L-ascorbic

mediated protection against the pesticide induced biochemical changes in

fresh water bivalve, Lamellidens corrianus. Waykar and Pulate (2011)

studied the ameliorating effect of ascorbic acid against profenofos

induced changes in protein contents of the freshwater bivalve,

Lamellidens maginalis. Deshmukh (2012) studied effect of L-ascorbic

acid on copper induced alterations in protein contents of fresh water

bivalve model, Indonaia caeruleus.

Ascorbic acid:-

Ascorbic acid has two enantiomers, the L-ascorbic acid and the D-

ascorbic acid and their actions are antagonistic. Ascorbic acid plays a

very important role in tissue synthesis and growth processes and

obviously mediates rapid tissue repair in trouma and abnormal condition.

L-ascorbic acid by virtue of possessing reducing properties is known to

act radioprotective agent in several tissue including reproducing organ by

preventing radiation induced oxidation (Chinoy and Garg 1978). It plays

vital role as an antioxidant that serves protective function against

oxidative damage in tissues. Ascorbic acid serves important role in

distribution and elimination of trace minerals and toxic metals.

Antioxidant property of ascorbic acid helps to prevent free radical

85

formation from water soluble molecules, which may causes cellular

injuries and disease.

It plays an important role in the process of hydroxylation,

oxygenation and oxidation of corticosteroids (Chatterjee, 1967). Sinha et

al., (1978) proved rapid mobilization of fat by ascorbic acid and

formation of glucose from fat. It takes part in synthesis of collagen and

maturation of red blood corpuscles (Talwar, 1980). Ascorbic acid protects

the mammalian tissues against oxidative damage both at intracellular as

well as extracellular level (Chatterjee et al., 1995). Chinoy and

Seethalakshmi (1977) studied that ascorbic acid has significant role in

steriodogenesis in molluscs. Beside this significance, ascorbic acid takes

part in variety of other biochemical functions such as, biosynthesis of

amino acid carnitine and the catecholamines that regulate the nervous

system. It helps in degradation of histamine which is the inflammatory

component of many allergic reactions. Asthmatic patients treated with

vitamin ‘C’ had experienced much less difficulty in breathing (Miric and

Haxhiu, 1991). In chronic brucellosis vitamin ‘C’ was found to be

effective in setting the striking immune responses against the infection

(Boura, 1989). Dieler and Breitenbach (1971) suggested that, lymphoid

tissue regeneration and its differentiation takes place under the influence

of ascorbic acid. Interferons get enhanced in circulatory system after the

ingestion of ascorbic acid through diet (Siegel, 1974). According to

current evidence ascorbic acid may be mainly beneficial in reducing the

risk of developing cancer rather than in therapy (Gaby and Singh, 1991).

It is also found that, ascorbic acid has an important role in protecting the

lens. In combination with vitamin E, vitamin ‘C’ may provide protection

against cataracts (Robertson et al., 1991). Ascorbic acid acts as detoxifier

and may reduce the effect of toxicant through its anti – oxidant property.

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Its role in detoxification and immune system may protect the body from

various toxic pollutants in environment.

The study regarding the change in ascorbic acid content in

molluscs exposed to various toxins and stress situation is inadequate and

can be useful as an indicator for the study. Zambare (1991) studied the

effect of pollutants on ascorbic acid content in various tissues of fresh

water bivalve, Corbicula striatella. Waykar et al., (2001) studied effect of

cypermethrin on the ascorbic acid content in mantle, foot, gill, digestive

gland and whole body tissues of fresh water bivalve Parreysia cylindrica.

Desai and Sekhar (2002) reported gradual decrease in the levels of liver

protein and liver ascorbic acid due to proteolysis and liver glucose

breakdown respectively. Waykar and Lomte (2004) studied carbaryl

induced changes in the ascorbic acid content in different tissues of fresh

water bivalve Parreysia cylindrica. Pardeshi and Zambare (2005) studied

ascorbic acid content in various tissues viz. mantle, foot, gill, gonad and

digestive glands of the fresh water bivalve, Parreysia cylindica in

connection with reproduction. Vedpathak et al., (2007) studied ascorbic

acid content in freshwater bivalve, Indonaia caeruleus. Shinde (2008)

reported ascorbate effect on pesticide induced alterations in the ascorbic

acid content of Channa orientalis. Ahirao and Kulkarni (2011) studied

sublethal stress of pyrethroids on ascorbic acid contents in prostate glands

of fresh water snail, Bellamya bengalensis.

The recovery of ascorbic acid contents by ascorbic acid plays an

important role against dicofol and dichlorovos toxicity in fresh water

bivalve, Parreysia cylindrica. Chinoy et al., (1995) studied the impact of

fluoride on biochemical constituent of rat, as well as the therapeutic effect

of ascorbic acid in the amelioration of fluoride toxicity. Mahajan and

Zambare (2006) studied the effect of L- ascorbic acid supplementation on

arsenic induced alterations in the ascorbic acid levels of Lamellidens

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marginalis. Mahananada et al., (2010) studied the protective efficacy of

L.ascorbic acid against the toxicity of mercury in Labeo rohita.

Deshmukh (2012) studied effect of L-ascorbic acid on copper induced

alterations in ascorbic acid contents of fresh water bivalve model,

Indonaia caeruleus

DNA:-

Deoxyribonucleic acid (DNA) is the storehouse, or cellular library,

that contains all the information required to build the cell and tissues of

an organism. DNA the genetic material carries information to specify the

mono acid sequences in proteins. DNA is the chemical basis of heredity

and may be regarded as reserve bank of genetic information. DNA is

exclusively responsible for maintaining the identity of different species.

Further, every aspect of cellular function is under control of the DNA as

the genetic material carries information to specify mono acid sequences

in proteins (Satyanarayana et al., 1999). It is transcribed in to several type

of ribonucleic acid (RNA) including mRNA, tRNA and rRNA which

function in protein synthesis. Structurally DNA is linear polymer

composed of monomer called nucleotides, i.e. Four nitrogen bases, two

purines; adenine, guanine and two pyramidine cytosine, thymine. Double

helical structure of DNA consist of two polynucleotide strands that winds

together to form double helical structure.

Nucleic acid reflects the ability of an organism for synthesis of

important biomolecule like proteins. Different toxic levels and stressed

condition may alter or damage activity of nucleic acid. Genetic

information transformation and genome functioning is caused due to

nucleic acid composition of DNA and sequences of the nucleotides in the

DNA. Hence it becomes important to study the DNA and RNA under

stressed condition in various tissues (Khanduja, 1999). Quantitative

88

changes are reported in structure of DNA due to pesticide exposure and it

can be monitored using biochemical method (Bertini et al., 1998).

Structural changes in the DNA can be monitored using

biochemical methods and usually low quantitative changes are observed

on pollutant exposure. DNA strand scission can also be sensitively

monitored, and even more importantly, the specific nucleotide position

cleaved can be pin pointed by biochemical methods. This methodology

has been applied successfully in monitoring both the efficiency of DNA

strand scission by metal complexes and the specific sites cleaved, and

where the complexes are specifically bound on the helical strand, Bertini

et al., (1998).

Pesticide is known to cause DNA damage and related events, such

as DNA protein cross-links, micronuclei etc (Schaumloffel and Gebel,

1998), DNA strand breaks (Lynn et al., 1998; Liu and Jan, 2000), or

alterations in DNA repair enzymes (Hartwing, 1998). Supper oxide

scavengers such as Cu, Zn - SOD suppress arsenic induced DNA damage

(Hartwing, 1998; Lynn et al., 1998; Liu and Jan, 2000). Low-dose

exposures to pesticide are not likely to cause cancer in humans. Data on

effects related to mutation formation (Changes in DNA) indicates that

pesticide could increase frequencies of mutation in human eggs and

sperm. Black et al., (1996) found significant DNA strand breakage in

different tissues from Anodonta grandis exposed to toxicant. Low-dose

exposures to pesticide are not likely to cause cancer in humans. The

impact of toxic materials on the integrity and functioning of DNA has

been investigated in many organisms under field conditions (Sun et al.,

2010). Since the metabolic processes are under control of DNA.

Genotoxicants have the ability to alter DNA and their effects may be

particularly harmful as these agents can induce changes that may be

passed on to future generations and have an impact on populations long

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after the original exposure. Environmental contaminants have been

reported to induce DNA strand breaks in various mussel cells which can

damage their functions (Nicholson and Lam, 2005).

Pawar and Kulkarni (2000) studied the effect of cythion on DNA

levels of Paratelphusa jacquemonti. Tiwari and Singh (2003) studied the

effect of sublethal doses of methanol extract of E. Royleana latex on the

levels of total DNA in the liver and muscle tissues Channa punctatus.

Pandey et al., (2006) evaluated the genotoxic potential of endosulfan in

Channa punctatus. They exposed the fish to different doses of pesticides

and assessed the DNA damage in tissues like gill and kidney. Nwani et

al., (2010) studied mutagenic and genotoxic effects of carbosulfan in

freshwater fish, Channa punctatus. Bhosale et al., (2011) studied

biochemical alterations in DNA content of gill and gonad of Corbicula

striatella due to 5- fluorouracil toxicity. Pandey et al., (2011) studied

profenofos induced DNA damage in freshwater fish, Channa punctatus.

Thenmozi et al., (2011) studied subletal effects of malathion on DNA

content in different tissues of Labeo rohita. Deshmukh (2012) studied

effect of L-ascorbic acid on copper induced alterations in DNA contents

of fresh water bivalve model, Indonaia caeruleus.

The recovery of DNA contents by ascorbic acid was studied by

some workers. Fraga et al., (1991) studied the protective action of

ascorbic acid against endogenous oxidative damage in human sperm.

Suriyo and Anisur (2004) studied protective action of an anti-oxidant (L-

ascorbic acid) against genotoxicity and cytotoxicity in mice during p-

DAB induced hepatocarcinogensis. Greco et al., (2005) studied the effect

of antioxidants on sperm DNA damage. Singh and Rana (2007) studied

the inhibition of DNA damage by ascorbic acid in liver and kidney in rat

after arsenic toxicity. Nawale (2008) studied the protective effect of

caffiene and ascorbic acid on heavy metal induced depletion in DNA

90

content. Zongyuan et al., (2009) studied the protective effect of vitamin C

on renal DNA damage of mice exposed to arsenic.

RNA:-

Ribonucleic acid or RNA, is part of a group of molecules known as

the nucleic acids, which are one of the four major macromolecules (along

with lipids, carbohydrates and proteins) essential for all known forms of

life. Like DNA, RNA is made up of a long chain of components called

nucleotides. Each nucleotide consists of a nucleobase, a ribose sugar, and

a phosphate group. The sequence of nucleotides allows RNA to encode

genetic information. All cellular organisms use messenger RNA (mRNA)

to carry the genetic information that directs the synthesis of proteins.

Some RNA molecules play an active role in cells by catalyzing biological

reactions, controlling gene expression, or sensing and communicating

responses to cellular signals. One of these active processes is protein

synthesis, a universal function whereby mRNA molecules direct the

assembly of proteins on ribosomes. This process uses transfer RNA

(tRNA) molecules to deliver amino acids to the ribosome, where

ribosomal RNA (rRNA) links amino acids together to form proteins.

The chemical structure of RNA is very similar to that of DNA,

with two differences: (a) RNA contains the sugar ribose, while DNA

contains the slightly different sugar deoxyribose (a type of ribose that

lacks one oxygen atom), and (b) RNA has the nucleobase uracil, while

DNA contains thymine. Unlike DNA, most RNA molecules are single-

stranded and can adopt very complex three-dimensional structures.

Pesticides also interact with RNA polymerases. RNA polymerase

must bind site specifically to its DNA template, binds its nucleotide and

primer substrates, and form a new phosphodiester bond in elongating the

growing RNA. Zinc ion appears to be essential to the functioning to both

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RNA polymerases and DNA topoisomerases, (Giedroc and Coleman,

1989).

Eukaryatic RNA polymerases I, II and III are involved in the

synthesis of ribosomal, messenger and transfer RNAs, respectively. The

DNA dependent RNA polymerases I (Falchuk et al., 1977), II (Falchuk et

al., 1976) and III (Wandzilak and Benson, 1977) of the unicellular

eukaryote Euglena gracilis have all been showed to be zinc metallo

enzyme, each binding about 2 gram atoms of zinc.

Rao et al., (1998) studied the RNA levels in various tissues of

freshwater crab Barytelphusa cunicularis when exposed to Fluoride.

Ester Saball et al., (2000) observed the total tissue m-RNA of liver and

kidneys of control and HgCl2 treated rats. In any tissues, toxic influences

exert their effect first at the molecular and biochemical level (Robbins

and Angel, 1976), hence alteration in normal biochemical parameters

serve as the earliest indicators of toxic effect on tissues. These have been

referred to as reliable tools for evaluating the extent of hazard of any

chemicals much before any gross signs become apparent (Jha and

Pandey, 1989). Pawar and Kulkarni (2000) studied the effect of cythion

on RNA levels of Paratelphusa jacquemonti. Rathod and Kshirsagar

(2010) studied quantification of nucleic acid from fresh water fish

Punctius arenatus exposed to pesticides. Singh et al., (2010) studied

DNA and RNA alterations on cypermethrin exposure of fresh water

teleost fish Colisa fasciatus. Thenmozi et al., (2011) studied subletal

effects of malathion on RNA content in different tissues of Labeo rohita.

The recovery of RNA contents by antioxidant/ ascorbic acid was

studied by some workers. Tiwari and Singh (2003) studied the effect of

sublethal doses of methanol extract of E. Royleana latex on the levels of

total RNA in the liver and muscle tissues Channa punctatus. Gulbhile

(2006) studied the effect of ascorbic acid supplementation on mercury

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and arsenic induced RNA depletion in freshwater bivalve, Lamellidens

corrianus. Nawale (2008) studied the protective effect of caffeine and

ascorbic acid on heavy metal induced depletion in RNA content. Zongyan

et al., (2009) studied preventive effect of vitamin C on renal RNA

damage of mice exposed to arsenic

Investigation regarding the physiological and biochemical changes

and its subsequent recovery in non target aquatic species such as molluscs

is insufficient. Hence in the present study an attempt was made to

investigate the effect of chronic treatment of pesticides dicofol and

dichlorovos on biochemical contents of different tissues and its

subsequent recovery by exogenous administration of L-ascorbic acid in

fresh water bivalve, Parreysia cylindrica.

In present study, proteins, ascorbic acid, DNA and RNA levels in

the tissues after exposure to pesticides can be considered as the indices

for stress. Freshwater bivalve, Parreysia cylindrica was used as test

model to detect the role of ascorbic acid for the detoxification of

pesticides dicofol and dichlorovos. The biochemical contents such as

protein, ascorbic acid, DNA and RNA are studied as the indicators from

different tissues.

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MATERIALS AND METHODS

Medium sized, healthy, fresh water bivalve, Parreysia cylindrica

was collected from Girna Dam, 48 km away from Chalisgaon. Animals

were brought in laboratory and were acclimatized for a week to tap water.

The medium sized animals were selected for experiment. The animals

were exposed to chronic concentration of dicofol (0.04023 ppm), LC50/10

values of 96 hrs) and dichlorovos (0.09376 ppm) alone and along with 50

mg/l of L-ascorbic acid upto 21 days. Every day the solution was

changed.

Experimental design:

Set – I

For experimental studies the animals were divided into five groups –

A) Group ‘A’ was maintained as control.

B) Group ‘B’ animals were exposed to chronic dose of dicofol

(0.04023 ppm), LC50/10 values of 96 hrs.) upto 21 days.

C) Group ‘C’ animals were exposed to chronic dose of dicofol

(0.04023 ppm), along with 50 mg/l of L-ascorbic acid upto 21

days.

D) Group ‘D’ animals were exposed to chronic dose of dichlorovos

(0.09376 ppm), LC50/10 values of 96 hrs.) upto 21 days.

E) Group ‘E’ animals were exposed to chronic dose of dichlorovos

(0.09376 ppm), along with 50 mg/l of L-ascorbic acid upto 21

days.

Experimental design for recovery studies -

Set – II

1) Group ‘B’ animals from set – I were divided into two groups for

recovery studies.

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i) Animals pre-exposed to chronic dose of dicofol (0.04023 ppm)

were allowed to self cure normally in untreated fresh water up to

21 days.

ii) Animals pre-exposed to chronic dose of dicofol (0.04023 ppm)

were allowed to cure in 50 mg/l of L-ascorbic acid added fresh

water up to 21 days.

2) Group ‘D’ animals from set – I were divided into two groups for

recovery studies.

iii) Animals pre-exposed to chronic dose of dichlorovos (0.09376

ppm) were allowed to self cure normally in untreated fresh water

up to 21 days.

iv) Animals pre-exposed to chronic dose of dichlorovos (0.09376

ppm) were allowed to cure in 50 mg/l of L-ascorbic acid added

fresh water up to 21 days.

During experimentation animals were fed on fresh water algae.

After every 7th

,14th

and 21st days of interval animals from set-I and set-II

were taken out, dissected and tissues such as digestive glands, gonad,

gills, foot, mantle were separated and whole body mass of remaining

animals was taken. All tissues were dried at 70 – 800

C in an oven till

constant weights were obtained. The dried powders of different tissues of

control and experimental animals were used for the estimation of various

biochemical components (total protein, ascorbic acid, DNA and RNA).

The methods of estimation are as follows:

Protein estimation:-

Protein content of the tissues was estimated by Lowry’s method

(Lowry et al., 1951).10 mg of dry powder was homogenized small

amount of 10% TCA and the homogenate was diluted to 1o ml by 10%

TCA. Then it was centrifuged at 3000 rpm for 15 minutes. The

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supernatant was removed which was used for ascorbic acid estimation.

The protein precipitate at the bottom of centrifuged tubes was dissolved

in 10 ml 1.0 N NaOH solution. 0.1 ml of this solution of each powder was

taken in three test tubes containing 4.0 ml. freshly prepared Lowry’s ‘C’.

After adding 0.5 ml. Folin’s – phenol reagent, the test tubes were

incubated in dark at 370C for 30 minutes. The O. D. of blue colour

developed was read at 530 nm. The blank was prepared in same way

without dissolved protein precipitate.

The protein content in different tissues was calculated referring to

standard graph value and it was expressed in terms of mg protein/100 mg

of dry tissue. The Bovine serum albumen was used as a standard.

Ascorbic acid estimation:-

Ascorbic acid estimation was carried out by the method of Roe

(1967). 1.0 ml supernatant was taken in test tubes from the homogenate

which was already centrifuged for protein estimation. In these test tubes

0.25 ml. aliquot of hydrazine reagent was added. The reaction mixture

was kept in boiling water bath for 15 minutes. It was cooled and 3.0 ml

ice cold 85% H2SO4 was added drop wise with constant stirring. The

reaction mixture was kept at room temperature for 30 minutes. O. D. was

read at 530 nm.

A 1.0 ml. 10% TCA similarly treated was used as a blank, while

ascorbic acid was used as a standard. Amount of ascorbic acid in different

tissues was calculated from standard graph values. It was expressed as mg

of ascorbic acid per 100 mg of dry tissue.

DNA estimation:-

DNA content of the tissue was measured by using Diphenylamine

method of Burton (1956). 10mg of dry tissue powder was homogenized

by adding 10ml distilled water. Then it was centrifuged at 3000rpm for

10 minutes. The supernatant contains DNA was removed. After that 1ml

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supernatant was taken and 3ml diphenylamine reagent was added. Then

the solution in the test tube was boiling in water bath for 10 minutes.

After boiling the solution in the test tube was allowed to cool. Then the

optical density of the DNA was read at 595(620) nm filter.

RNA estimation:-

RNA content of the tissue was measured by following Orcinol

method of Volkin and Cohn (1954). 10mg of dry tissue powder was

homogenized by adding 10ml distilled water. Then it was centrifuged at

3000rpm for 10 minutes. The supernatant contains RNA was removed.

After that 1ml supernatant was taken and 3ml Orcinol reagent was added.

Then the solution in the test tube was in boiling water bath for 15

minutes. After boiling the solution in the test tube was allowed to cool.

Then the optical density of the RNA was read at 665(660) nm filter.

Each observation was confirmed by taking at least three replicates.

The difference in control and experimental animal group was tested for

significance by using student’s’ test (Bailey, 1965) and the percentage of

decrease or increase over control was calculated for each value.

97

OBSERVATIONS AND RESULTS

Changes in protein, ascorbic acid, DNA and RNA contents in

mantle, foot, gills, digestive glands, gonads and whole soft body tissues

of freshwater bivalve, Parreysia cylindrica after chronic treatment of

pesticide, dicofol and dichlorovos for 7, 14 and 21 days and its

subsequent recovery in normal water and in ascorbic acid medium for

period of 7, 14 and 21 days are studied and obtained results are

summarized in table No.3.1a to 3.8b and fig. no. 3.1.1 to 3.4.12.

Protein:

The variation in protein contents in different tissues i.e. mantle,

foot, gills, digestive glands, gonads and whole soft body tissues after

chronic exposure to dicofol and dichlorovos and its subsequent recovery

in normal water and in ascorbic acid medium for period of 7, 14 and 21

days are studied and obtained results are summarized in table no 3.1 a,b

and 3.2 a,b. and fig. no 3.1.1 to 3.1.12.

In the present study it was observed that the protein content of

mantle, foot, gills, digestive glands, gonads and whole soft body tissues

of experimental bivalves was decreased after chronic exposure to dicofol

and dichlorovos.

The percentage of decreased in protein content after chronic

treatment at 7, 14 and 21 days with dicofol was 33.23, 45.34 and 50.82 in

mantle, 24.66, 31.65 and 39.32 in foot, 44.92, 51.71 and 57.05 in gills,

49.01, 64.98 and 66.37 in digestive glands, 22.48, 32.46 and 47.67 in

gonad and 29.06, 42.77 and 48.82 in whole soft body.

The percentage of decreased in protein content after chronic

treatment of 7, 14 and 21 days with dichlorovos was 31.89, 38.90 and

43.22 in mantle, 17.25, 24.22 and 33.12 in foot, 37.53, 44.26 and 53.29 in

gills, 43.10, 57.17 and 62.44 in digestive glands, 29.15, 36.61 and 41.31

in gonad and 22.57, 37.87 and 44.04 in whole soft body.

98

In combined treatment of pesticide along with 50mg/l L-ascorbic

acid the total protein content in mantle, foot, gills, digestive glands,

gonads and whole soft body tissues was less decreased as compared to

bivalve treated with pesticide only.

The percentage of decreased in protein content after chronic

treatment of 7, 14 and 21 days with dicofol along with ascorbic acid was

24.40, 39.06 and 43.93 in mantle, 15.51, 24.12 and 30.11 in foot, 31.80,

40.31 and 45.83 in gills, 17.32, 27.94 and 47.11 in digestive glands,

24.12, 20.93 and 40.61 in gonad and 24.95, 35.90 and 42.26 in whole soft

body.

The percentage of decreased in protein content after chronic

treatment of 7, 14 and 21 days with dichlorovos with ascorbic acid was

19.81, 25.77 and 37.67 in mantle, 9.21, 18,42 and 23.91 in foot, 26.19,

34.73 and 39.06 in gills, 14.76, 28.53 and 41.21 in digestive glands,

21.85, 29.23 and 32.12 in gonad and 19.12, 28.04 and 34.85 in whole soft

body.

Animals pre-treated to chronic dose of pesticide dicofol for 21

days and are allowed to cure in normal water the increase in protein

content was noted. The percent increase in protein content after 7, 14 and

21 days was 15.33, 21.65 and 41.15 in mantle, 10.65, 18.09 and 25.31 in

foot, 13.87, 21.71 and 45.95 in gills, 16.02, 29.29 and 48.04 in digestive

glands, 12.14, 20.54 and 39.74 in gonad and 14.36, 23.40 and 46.53 in

whole soft body.

Animals pre-treated to chronic dose of pesticide dichlorovos for 21

days and are allowed to cure in ascorbic acid medium the increase in

protein content was noted. The percent decrease of protein content after

7, 14 and 21 days was 12.49, 18.21 and 32.65 in mantle, 6.32, 12.97 and

23.04 in foot, 11.21, 22.72 and 43.62 in gills, 15.50, 27.11 and 46.48 in

99

digestive glands, 11.69, 17.54 and 27.23 in gonad and 13.39, 21.70 and

44.04 in whole soft body.

Animal pre-treated to chronic treatment of dicofol and curing in

50mg/l L-ascorbic acid in water bivalve showed increase in protein

content in all soft body tissues of experimental bivalves. The percent

increase in protein content after 7, 14 and 21 days was 23.20, 48.33 and

73.36 in mantle, 20.32, 36.24 and 60.41 in foot, 23.47, 49.73 and 85.16 in

gills, 31.13, 53.22 and 91.04 in digestive glands, 22.27, 43.36 and 70.85

in gonad and 25.87, 48.70 and 79.87 in whole soft body.

Animal pre-treated to chronic treatment of dichlorovos and allowed

cure in 50mg/l L-ascorbic acid in water bivalve showed increase in

protein content in all soft body tissues of experimental bivalves. The

percent increase in protein content after 7, 14 and 21 days was 20.44,

45.22 and 71.30 in mantle, 14.17, 34.21 and 50.85 in foot, 25.51, 48.17

and 82.37 in gills, 30.34, 49.35 and 89.13 in digestive glands, 21.53,

40.71 and 68.18 in gonad and 23.73, 41.16 and 79.12 in whole soft body.

It was observed that after chronic exposure of dicofol and

dichlorovos there was significant decrease in the protein content in

mantle, foot, gills, digestive glands, gonads and whole soft body of

experimental bivalves as compared to those of control bivalves. Animal

pre-treated to chronic treatment of pesticides dicofol and dichlorovos and

were allowed to cure in 50mg/l L-ascorbic acid medium bivalve showed

increase in protein content in all soft body tissues of experimental

bivalves.

Ascorbic acid:

The variation in ascorbic acid contents in different tissues i.e.

mantle, foot, gills, digestive glands, gonads and whole soft body tissues

after chronic exposure to dicofol and dichlorovos and its subsequent

recovery in normal water and in ascorbic acid medium for period of 7, 14

100

and 21 days are studied and obtained results are summarized in table no.

3.3 a, b and 3.4 a,b. and fig. no. 3.2.1 to 3.2.12.

It was observed that the ascorbic acid content of mantle, foot, gills,

digestive glands, gonads and whole soft body in experimental bivalves

was decreased after chronic exposure to dicofol and dichlorovos.

The percentage of decreased in ascorbic acid content after chronic

treatment of 7, 14 and 21 days with dicofol was 29.86, 33.33 and 47.10 in

mantle, 26.27, 32.71 and 41.11 in foot, 29.20, 40.48 and 52.43 in gills,

32,82, 47.41 and 60.56 in digestive glands, 33.18, 42.43 and 45.78 in

gonad and 26.45, 37.80 and 52.44 in whole soft body.

The percentage of decreased in ascorbic acid content after chronic

treatment of 7, 14 and 21 days with dichlorovos was 25.84, 30.21 and

42.41 in mantle, 23.57, 27.56 and 36.74 in foot, 24.16, 35.59 and 47.83 in

gills, 30.02, 43.75 and 59.83 in digestive glands, 31.23, 40.78 and 45.61

in gonad and 22.76, 35.75 and 51.73 in whole soft body.

In combined treatment of pesticide along with 50 mg/l L-ascorbic

acid the total ascorbic acid content in mantle, foot, gills, digestive glands,

gonads and whole soft body was less decreased as compared to bivalve

treated with pesticide only.

The percentage of decrease in ascorbic acid content after chronic

treatment of 7, 14 and 21 days with dicofol along with ascorbic acid was

23.35, 29.41 and 37.17 in mantle, 18.43, 25.90 and 35.34 in foot, 25.44,

29.75 and 41.42 in gills, 28.40, 38.80 and 46.05 in digestive glands,

27.36, 34.22 and 36.90 in gonad and 21.78, 33.00 and 40.83 in whole soft

body.

The percentage of decrease in ascorbic acid content after chronic

treatment of 7, 14 and 21 days with dichlorovos along with ascorbic acid

was 19.47, 28.14 and 32.95 in mantle, 13.81, 22.03 and 30.37 in foot,

22.40, 27.80 and 36.29 in gills, 22.33, 35.08 and 42.49 in digestive

101

glands, 20.15, 28.34 and 31.19 in gonad and 17.35, 28.09 and 34.93 in

whole soft body.

During recovery in normal water after 21 days exposure to chronic

concentration of dicofol the percent increase in ascorbic acid content was

noted. The percent increase in ascorbic acid after 7, 14 and 21 days was

9.58, 12.64 and 23.48 in mantle, 11.12, 14.70 and 34.59 in foot, 16.61,

22.63 and 36.35 in gills, 11.39, 17.83 and 29.27 in digestive glands,

14.14, 19.55 and 39.48 in gonad and 12.43, 23.73 and 41.12 in whole soft

body.

During recovery in normal water after 21 days exposure to chronic

concentration of dichlorovos the percent increase in ascorbic acid content

was noted. The percent increase in ascorbic acid content after 7, 14 and

21 days was 10.16, 14.29 and 24.84 in mantle, 13.85, 17.23 and 35.33 in

foot, 16.38, 22.49 and 38.28 in gills, 14.90, 19.93 and 31.47 in digestive

glands, 11.05, 23.59 and 39.17 in gonad and 11.54, 24.61 and 43.24 in

whole soft body.

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dicofol the percent increase in

ascorbic acid content was noted. The percent increase in ascorbic acid

content after 7, 14 and 21 days was 22.10, 44.19 and 74.19 in mantle,

35.91, 53.19 and 75.29 in foot, 34.09, 50.01 and 90.42 in gills, 26.40,

44.40 and 83.30 in digestive glands, 40.83, 56.06 and 70.28 in gonad and

38.83, 47.58 and 77.46 in whole soft body.

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dichlorovos the percent increase in

ascorbic acid content was noted. The percent increase in ascorbic acid

content after 7, 14 and 21 days was 23.18, 46.73 and 75.85 in mantle,

33.71, 51.55 and 76.45 in foot, 37.37, 53.31 and 94.38 in gills, 24.65,

102

47.35 and 85.71 in digestive glands, 38.03, 55.77 and 84.61 in gonad and

37.16, 48.68 and 79.92 in whole soft body.

It was observed that after chronic exposure of dicofol and

dichlorovos there was significant decrease in the ascorbic acid content in

mantle, foot, gills, digestive glands, gonads and whole soft body of

experimental bivalves as compared to those of control bivalves. Animal

pre-treated to chronic treatment of pesticides dicofol and dichlorovos and

were allowed to cure in 50mg/l L-ascorbic acid medium bivalve showed

increase in ascorbic acid content in all soft body tissues of experimental

bivalves.

DNA:

The variation in DNA contents in different tissues i.e. mantle, foot,

gills, digestive glands, gonads and whole soft body tissues after chronic

exposure to dicofol and dichlorovos and its subsequent recovery in

normal water and in ascorbic acid medium for period of 7, 14 and 21

days are studied and obtained results are summarized in table no. 3.5 a,b

and 3.6 a,b and fig.no. 3.3.1 to 3.3.12.

It was observed that the DNA content of mantle, foot, gills,

digestive glands, gonads and whole soft body in experimental bivalves

decreased after chronic exposure to dicofol and dichlorovos.

The percentage of decrease in DNA content after chronic treatment

of 7, 14 and 21 days with dicofol was 26.07, 37.17 and 48.87 in mantle,

19.35, 27.51 and 40.18 in foot, 28.09, 43.37 and 52.41 in gills, 41.79,

50.76 and 65.71 in digestive glands, 37.02, 43.28 and 45.68 in gonad and

22.65, 31.92 and 46.85 in whole soft body.

The percentage of decrease in DNA content after chronic treatment

of 7, 14 and 21 days with dichlorovos was 20.30, 27.10 and 45.72 in

mantle, 12.65, 21.62 and 37.91 in foot, 25.94, 36.18 and 46.51 in gills,

103

32.09, 42.00 and 54.07 in digestive glands, 17.87, 28.15 and 39.48 in

gonad and 16.30, 27.29 and 40.27 in whole soft body.

In combined treatment of pesticide along with 50 mg/l L-ascorbic

acid the total DNA content in mantle, foot, gills, digestive glands, gonads

and whole soft body was less decreased as compared to bivalve treated

with pesticide only.

The percentage of decrease in DNA content after chronic treatment

of 7, 14 and 21 days with dicofol along with ascorbic acid was 14.21,

29.41 and 42.15 in mantle, 9.47, 18.26 and 33.24 in foot, 19.38, 31.44

and 48.40 in gills, 24.34, 34.12 and 49.73 in digestive glands, 25.85,

29.30 and 35.39 in gonad and 12.74, 30.51 and 40.97 in whole soft body.

The percentage of decrease in DNA content after chronic treatment

of 7, 14 and 21 days with dichlorovos along with ascorbic acid was 11.29,

25.03 and 40.30 in mantle, 6.89, 16.39 and 30.61 in foot, 15.86, 29.16

and 45.16 in gills, 20.19, 33.40 and 47.10 in digestive glands, 12.76,

24.42 and 32.17 in gonad and 10.87, 21.72 and 38.23 in whole soft body.

During recovery in normal water after 21 days exposure to chronic

concentration of dicofol the percent increase in DNA content was noted.

The percent increase in DNA content after 7, 14 and 21 days was 7.43,

14.70 and 28.08 in mantle, 5.25, 12.35 and 22.48 in foot, 12.85, 25.88

and 35.84 in gills, 10.30, 14 18 and 29.09 in digestive glands, 9.12, 17.16

and 31.89 in gonad and 8.51, 14.07 and 22.97 in whole soft body.

During recovery in normal water after 21 days exposure to chronic

concentration of dichlorovos the percent increase in DNA content was

noted. The percent increase in DNA content after 7, 14 and 21 days was

6.39, 12.02 and 25.09 in mantle, 4.33, 9.30 and 20.25in foot, 10.25, 21.48

and 30.57 in gills, 8.14, 12.17 and 31.75 in digestive glands, 8.26, 16.47

and 30.61 in gonad and 6.17, 12.60 and 20.35 in whole soft body.

104

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dicofol the percent increase in DNA

content was noted. The percent increase in DNA content after 7, 14 and

21 days was 25.12, 47.54 and 77.26 in mantle, 20.44, 39.71 and 65.59 in

foot, 33.48, 63.89 and 90.77 in gills, 28.17, 48.09 and 82.67 in digestive

glands, 33.49, 44.87 and 72.44 in gonad and 20.36, 43.38 and 74.75 in

whole soft body.

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dichlorovos the percent increase in

DNA content was noted. The percent increase in DNA content after 7, 14

and 21 days was 23.33, 46.52 and 74.84 in mantle, 18.42, 33.29 and

64.25 in foot, 27.54, 60.00 and 89.59 in gills, 27.72, 46.25 and 70.19 in

digestive glands, 29.65, 40.13 and 69.72 in gonad and 18.56, 38.60 and

71.24 in whole soft body.

It was observed that after chronic exposure of dicofol and

dichlorovos there was significant decrease in the DNA content in mantle,

foot, gills, digestive glands, gonads and whole soft body of experimental

bivalves as compared to those of control bivalves. Animal pre-treated to

chronic treatment of pesticides dicofol and dichlorovos and were allowed

to cure in 50mg/l L-ascorbic acid medium bivalve showed increase in

DNA content in all soft body tissues of experimental bivalves.

RNA:

The variation in RNA contents in different tissues i.e. mantle, foot,

gills, digestive glands, gonads and whole soft body tissues after chronic

exposure to dicofol and dichlorovos and its subsequent recovery in

normal water and in ascorbic acid medium for period of 7, 14 and 21 days

are studied and obtained results are summarized in table no. 3.7 a,b and

3.8 a,b and fig.no. 3.4.1 to 3.4.12.

105

It was observed that the RNA content of mantle, foot, gills,

digestive glands, gonads and whole soft body in experimental bivalves

decreased after chronic exposure to dicofol and dichlorovos.

The percentage of decrease in RNA content after chronic treatment

of 7, 14 and 21 days with dicofol was 18.76, 26.40 and 45.95 in mantle,

22.86, 30.84 and 37.28 in foot, 28.99, 36.85 and 52.00 in gills, 30.64,

44.29 and 58.47 in digestive glands, 20.36, 31.19 and 47.78 in gonad and

18.63, 28.37 and 43.00 in whole soft body.

The percentage of decrease in RNA content after chronic treatment

of 7, 14 and 21 days with dichlorovos was 10.60, 21.39 and 41.87 in

mantle, 14.51, 25.43 and 33.80 in foot, 17.98, 28.23 and 47.97 in gills,

27.14, 37.24 and 51.41 in digestive glands, 18.34, 27.35 and 35.08 in

gonad and 13.86, 21.14 and 38.56 in whole soft body.

In combined treatment of pesticide along with 50 mg/l L-ascorbic

acid the total RNA content in mantle, foot, gills, digestive glands, gonads

and whole soft body was less decreased as compared to bivalve treated

with pesticide only.

The percentage of decrease in RNA content after chronic treatment

of 7, 14 and 21 days with dicofol along with ascorbic acid was 14.14,

22.73 and 41.23 in mantle, 11.35, 25.49 and 31.65 in foot, 17.90, 32.17

and 44.23 in gills, 19.19, 34.93 and 46.63 in digestive glands, 18.16,

26.31 and 36.16 in gonad and 10.53, 22.92 and 38.24 in whole soft body.

The percentage of decrease in RNA content after chronic treatment

of 7, 14 and 21 days with dichlorovos along with ascorbic acid was 7.73,

18.41 and 31.77 in mantle, 9.62, 19.92 and 25.30 in foot, 11.05, 24.47

and 36.29 in gills, 17.98, 28.42 and 39.23 in digestive glands, 12.91,

21.29 and 29.11 in gonad and 8.83, 17.27 and 31.87 in whole soft body.

During recovery in normal water after 21 days exposure to chronic

concentration of dicofol the percent increase in RNA content was noted.

106

The percentage increase in RNA content after 7, 14 and 21 days was 8.46,

21.79 and 34.15 in mantle, 5.84, 12.30 and 25.86 in foot, 10.52, 17.05

and 39.30 in gills, 7.66, 14.26 and 35.48 in digestive glands, 6.16, 12.76

and 28.71 in gonad and 5.81, 11.72 and 31.05 in whole soft body.

During recovery in normal water after 21 days exposure to chronic

concentration of dichlorovos the percent increase in RNA content was

noted. The percentage increase in RNA content after 7, 14 and 21 days

was 7.74, 18.12 and 33.81 in mantle, 6.77, 13.70 and 23.57 in foot, 8.09,

15.34 and 38.66 in gills, 6.05, 14.20 and 36.29 in digestive glands, 9.17,

11.98 and 25.36 in gonad and 4.02, 9.87 and 29.65 in whole soft body.

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dicofol the percent increase in RNA

content was noted. The percentage increase in RNA content after 7, 14

and 21 days was 23.61, 42.48 and 78.54 in mantle, 17.25, 33.48 and

67.18 in foot, 21.87, 47.01 and 86.33 in gills, 24.00, 45.14 and 82.55 in

digestive glands, 20.73, 37.16 and 70.72 in gonad and 16.40, 40.64 and

74.20 in whole soft body.

During recovery in ascorbic acid (50mg/l) medium after 21 days

exposure to chronic concentration of dichlorovos the percent increase in

RNA content was noted. The percentage increase in RNA content after 7,

14 and 21 days was 21.95, 40.33 and 74.73 in mantle, 16.18, 31.37 and

61.88 in foot, 22.24, 44.07 and 82.87 in gills, 22.76, 41.74 and 79.80 in

digestive glands, 18.74, 34.81 and 65.16 in gonad and 14.95, 38.86 and

69.21 in whole soft body.

It was observed that after chronic exposure of dicofol and

dichlorovos there was significant decrease in the RNA content in mantle,

foot, gills, digestive glands, gonads and whole soft body of experimental

bivalves as compared to those of control bivalves. Animal pre-treated to

chronic treatment of pesticides dicofol and dichlorovos and were allowed

107

to cure in 50mg/l L-ascorbic acid medium bivalve showed increase in

RNA content in all soft body tissues of experimental bivalves.

108

Table No. 3.1.a. Total protein content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol without and

with ascorbic acid.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr. No.

Tissue

Control

(A)

Dicofol

(B)

Dicofol + A.A.(50 mg/lit)

(C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 44.8718

± 1.42

43.6821

± 1.16

43.1478 ± 1.52

29.9605* ± 1.56

(-33.23)

23.8772* ± 3.04

(-45.34)

21.2190** ± 4.17

(-50.82)

33.9216* ± 2.17

(-24.40)

26.6221** ± 2.49

(-39.06)

24.2241*** ± 1.34

(-43.93)

2 Foot 63.4788

± 2.42

63.2318

± 1.26

62.9123

± 1.32

47.8271**

± 2.80 (-24.66)

43.2162**

± 3.71 (-31.65)

38.1732**

± 2.50 (-39.32)

53.6312***

± 1.19 (-15.51)

47.9823***

± 2.11 (-24.12)

43.8720***

± 3.26 (-30.11)

3 Gills 54.1568

± 2.48

53.7018

± 1.65

53.2013

± 2.67

29.8322**

± 3.06 (-44.92)

25.9337**

± 4.86 (-51.71)

22.8513***

± 5.04 (-57.05)

36.9365***

± 2.95 (-31.80)

32.0520***

± 3.45 (-40.31)

28.8203***

±3.94 (-45.83)

4 Digestive

glands 50.7416 ± 1.25

51.1671 ± 2.68

50.8570 ± 2.55

25.8718*

± 4.01

(-49.01)

17.9168**

± 4.98

(-64.98)

17.1020***

± 6.37

(-66.37)

41.9512*

± 1.32

(-17.32)

36.8710*

± 2.94

(-27.94)

26.8993**

± 3.24

(-47.11)

5 Gonad 48.5821 ± 2.88

48.2130 ± 1.62

47.1236 ± 1.93

38.3589*

± 3.14

(-20.44)

32.5621**

± 2.31

(-32.46)

24.6588**

± 2.20

(-47.67)

38.8657***

± 1.90

(-24.12)

38.1204***

± 1.30

(-20.93)

27.9861***

± 2.87

(-40.61)

6 Whole soft

body

61.6578

± 3.04

61.1216

± 1.58

60.7217

± 1.90

43.7419** ± 2.05

(-29.06)

34.9772** ± 4.41

(-42.77)

31.0788*** ± 5.62

(-48.82)

46.2712*** ± 2.95

(-24.95)

39.1817*** ± 1.13

(-35.90)

35.0618*** ± 3.08

(-42.26)

109

Table No. 3.1.b. Total protein content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol and its

subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr. No.

Tissue Dicofol

Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 21.2190

(-50.82)

24.4712NS

±9.13

(+15.33)

25.812** ± 1.07

(+21.65)

29.9515** ± 2.92

(+41.15)

26.1415* ± 2.56

(+23.20)

31.4751** ± 3.36

(+48.33)

36.7851*** ± 4.14

(+73.36)

2 Foot 33.1732

(-39.32)

36.7051NS

± 2.63 (+10.65)

39.1751NS

± 5.06 (+18.09)

41.5700 *

± 1.41 (+25.31)

39.914NS

± 5.33 (+20.32)

45.194*

± 1.58 (+36.24)

53.2123***

± 2.18 (+60.41)

3 Gills 22.8513

(-57.05)

26.021NS

± 4.80

(+13.87)

27.8121*

± 1.27

(+21.71)

33.3521**

± 3.28

(+45.95)

28.2151**

± 1.92

(+23.47)

34.2157**

± 3.16

(+49.73)

42.311***

± 6.93

(+85.16)

4 Digestive

glands

17.1020

(-66.37)

19.842*

± 9.13 (+16.02

22.112*

± 1.07 (+29.29)

25.3182**

± 2.92 (+48.04)

22.4257**

± 1.23 (+31.13)

26.2030**

± 2.83 (+53.22)

32.671***

± 6.28 (+91.04)

5 Gonad 24.6588

(-47.67)

27.6531* ± 2.38

(+12.14)

29.7242* ± 1.32

(+20.54)

34.4578** ± 3.52

(+39.74)

30.1511** ± 2.11

(+22.27)

35.3515** ± 3.79

(+43.36)

42.1302*** ± 3.8

(+70.85)

6 Whole soft

body

31.0788

(-48.82)

35.5420NS

± 4.01 (+14.36)

38.3510*

± 2.79 (+23.40)

45.5412**

± 338 (+46.53)

39.1188*

± 1.23 (+25.87)

46.2128**

± 2.83 (+48.70)

55.9028***

± 3.28 (+79.87)

110

Table No. 3.2.a. Total protein content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos without

and with ascorbic acid.

Sr. No.

Tissue

Control

(A)

Dichlorovos

(B)

Dichlorovos + A.A.(50 mg/lit)

(C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 44.8718

±1.32

43.6821 ±1.58

43.1478

±1.97

30.5605** ± 1.00

(-31.89)

26.6721*** ± 3.04

(-38.90)

24.5012*** ± 2.17

(-43.22)

35.9816** ± 2.17

(-19.81)

32.4241** ± 2.49

(-25.77)

26.8941*** ± 1.34

(-37.67)

2 Foot 63.4788

±2.38

63.2318

± 1.20

62.9123

± 1.28

52.5271* ± 2.80

(-17.25)

47.9162*** ± 3.71

(-24.22)

42.0732*** ± 2.50

(-33.12)

57.6312** ± 1.19

(-9.21)

51.5823** ± 2.11

(-18.42)

47.8720*** ± 3.26

(-23.91)

3 Gills 54.1568

±2.43

53.7018

± 1.60

53.2013

± 0.58

33.8322**

± 3.06 (-37.53)

29.9337***

± 1.86 (-44.26)

24.8513***

±1.04 (-53.29)

39.9722**

± 2.95 (-26.19)

35.052***

±3.45 (-34.73)

32.4203***

± 3.94 (-39.06)

4 Digestive

glands

50.7416

± 1.21

51.1671

± 0.64

50.8570

± 0.50

28.8718** ± 1.01

(-43.10)

21.9168*** ± 4.98

(-57.17)

19.1020*** ±1.37

(-62.44)

43.2512* ± 1.32

(-14.76)

36.5710** ± 2.94

(-28.53)

29.8993*** ± 3.24

(-41.21)

5 Gonad 48.5821

± 0.82

48.2130

± 1.56

47.1236

± 0.85

34.4189* ± 3.14

(-29.15)

30.5621* ± 2.31

(-36.61)

27.6588** ± 2.20

(-41.31)

37.9657* ± 2 .81

(-21.85)

34.1204* ±1.30

(-29.23)

31.9861** ± 2.87

(-32.12)

6 Whole

soft body

61.6578

± 2.93

61.1216

± 1.52

60.7217

± 1.85

47.7419* ± 2.05

(-22.57)

37.9772** ± 4.41

(-37.87)

33.9788*** ± 1.62

(-44.04)

49.8712* ± 2.95

(-19.12)

43.9817 ± 1.13

(-28.04)

39.5618** ± 3.08

(-34.85)

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

111

Table No. 3.2.b. Total protein content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos

and its subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation 4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr.

No. Tissue

Dichlorovos Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 24.5012

(-43.22)

27.5618NS

± 9.13

(+12.49)

28.9618* ± 1.07

(+18.21)

32.5016** ± 2.92

(+32.65)

29.5102** ± 2.56

(+20.44)

35.5812*** ± 3.36

(+45.22)

41.9701*** ± 4.14

(+71.30)

2 Foot 42.0732

(-33.12)

44.7341NS

± 2.63 (+6.32 )

47.5281*

± 5.06 (+12.97)

51.7651**

± 1.41 (+23.04)

48.0365**

± 5.33 (+14.17)

56.4665**

± 1.58 (+34.21)

63.4665***

± 1.18 (+50.85)

3 Gills 24.8513 (-53.29)

27.6381NS

± 4.80

(+11.21)

30.4983*

± 1.27

(+22.72)

35.6911*

± 3.28

(+43.62)

31.1912**

± 1.92

(+25.51)

36.8211**

± 3.16

(+48.17)

45.3211***

± 6.93

(+82.37)

4 Digestive

glands 19.1020 (-62.44)

16.1411*

± 9.13

(+15.50)

24.2811**

± 1.07

(+27.11)

27.9811***

± 2.92

(+46.48)

24.8985*

± 1.23

(+30.34)

28.5285***

± 2.83

(+49.35)

36.1285***

± 6.28

(+89.13)

5 Gonad 27.6588

(-41.31)

30.8912* + 2.38

(+11.69)

32.5112* ± 1.32

(+17.54)

35.1912** ± 3.52

(+27.23)

33.6137** ± 2.11

(+21.53)

38.9194** ± 3.79

(+40.71)

46.5158*** ± 3.8

(+68.18)

6 Whole soft

body

33.9788

(-44.04)

38.5272 NS

± 4.01

(+13.39)

41.3532* ± 2.79

(+21.70)

48.9422** ± 3.38

(+44.04)

42.0423* ± 1.23

(+23.73)

47.9649** ± 2.83

(+41.16)

60.8642*** ± 6.28

(+79.12)

112

Table No. 3.3.a. Total Ascorbic acid content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol without

and with ascorbic acid.

Sr.

No. Tissue

Control (A)

Dicofol (B)

Dicofol + A.A.(50 mg/lit) (C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.9556

± 1.87

0.9465

± 1.32

0.9411

± 1.56

0.6703* ± 2.62

(-29.86)

0.6310** ± 2.95

(-33.33)

0.4978*** ± 3.43

(-47.10)

0.7325* ± 1.89

(-23.35)

0.6681** ± 2.94

(-29.41)

0.5913*** ± 3.93

(-37.17)

2 Foot 0.4846 ± 1.42

0.4690 ± 1.78

0.4610 ± 1.41

0.3573*

± 2.11

(-26.27)

0.3385*

± 2.82

(-32.71)

0.2715***

± 3.59

(-41.11)

0.3953*

± 1.91

(-18.43)

0.3510**

± 2.15

(-25.90)

0.2981***

± 2.19

(-35.34)

3 Gills 1.1565

± 1.71

1.1320

± 1.89

1.1185

± 1.32

0.8188*

± 2.55 (-29.20)

0.6738**

± 2.99 (-40.48)

0.5321***

± 3.74 (-52.43)

0.8623*

± 2.25 (-25.44)

0.7952**

± 2.56 (-29.75)

0.6552***

± 3.37 (-41.42)

4 Digestive

glands 1.5840 ± 1.65

1.4112 ± 1.98

1.4368 ± 1.62

1.0642**

± 3.81

(-32.82)

0.7422***

± 5.17

(-47.41)

0.5682***

± 4.15

(-60.56)

1.1341*

± 2.82

(-28.40)

0.8636**

± 3.71

(-38.80)

0.7751***

± 4.77

(-46.05)

5 Gonad 1.4728 ± 1.33

1.4611 ± 1.68

1.4598 ± 1.82

0.9989*

± 3.78

(-33.18)

0.8411**

± 4.49

(-42.43)

0.7032***

± 5.82

(-45.78)

1.0698*

± 2.44

(-27.36)

0.9611**

± 2.61

(-34.22)

0.9212***

± 4.33

(-36.90)

6 Whole soft

body

1.0820

± 1.27

1.0910

± 1.21

1.0906

± 1.67

0.7490* ± 3.77

(-26.45)

0.6741** ± 3.21

(-37.80)

0.5187*** ± 4.55

(-52.44)

0.8814* ± 1.53

(-21.78)

0.7624** ± 3.11

(-33.00)

0.6453** ± 3.09

(-40.83)

1. Values expressed as mg/100mg dry wt. of tissue 2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

113

Table No. 3.3.b. Total Ascorbic acid content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol

and its subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation 4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr.

No. Tissue

Dicofol Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.4978

(-47.10)

0.5455NS

± 5.46

(+9.58)

0.5607* ± 9.64

(+12.64)

0.6147** ± 2.69

(+23.48)

0.6078* ± 1.69

(+22.10)

0.7178** ± 3.64

(+44.19)

0.8671*** ± 4.91

(+74.19)

2 Foot 0.2715

(-41.11)

0.3017*

± 9.85 (+11.12)

0.3114*

± 1.80 (+14.70)

0.3654***

± 3.92 (+34.59)

0.3690*

± 1.84 (+35.91)

0.4159**

± 4.13 (+53.19)

0.4759***

± 2.90 (+75.29)

3 Gills 0.5321

(-52.43)

0.6205*

± 1.67

(+16.61)

0.6525*

± 2.84

(+22.63)

0.7255**

± 3.27

(+36.35)

0.7135**

± 2.06

(+34.09)

0.7982***

± 3.26

(+50.01)

1.0132***

± 4.45

(+90.42)

4 Digestive

glands 0.5682

(-60.56)

0.6329*

± 5.56

(+11.39)

0.6695NS

± 8.14

(+17.83)

0.7345**

± 1.63

(+29.27)

0.7182NS

± 14.00

(+26.40)

0.8205**

± 2.39

(+44.40)

1.0415***

± 3.73

(+83.30)

5 Gonad 0.7032

(-45.78)

0.8026* ± 1.06

(+14.14)

0.8407** ± 1.40

(+19.55)

0.9808*** ± 2.86

(+39.48)

0.9903** ± 1.31

(+40.83)

1.0974** ± 2.04

(+56.06)

1.1974*** ± 5.78

(+70.28)

6 Whole soft

body

0.5187

(-52.44)

0.5832*

± 8.36 (+12.43)

0.6418 NS

± 1.50 (+23.73)

0.7320**

± 3.76 (+41.12)

0.7201**

± 1.41 (+38.83)

0.7655**

± 3.21 (+47.58)

0.9205***

± 4.13 (+77.46)

114

Table No. 3.4.a. Total Ascorbic acid content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos

without and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Dichlorovos

(B)

Dichlorovos + A.A.(50 mg/lit)

(C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.9823 ± 0.26

0.9631 ± 0.59

0.9590 ± 0.45

0.7285*

± 0.23

(-25.84)

0.6721**

± 1.02

(- 30.21)

0.5523***

± 1.12

(- 42.41)

0.7910*

± 1.22

(- 19.47)

0.6921**

± 0.71

(- 28.14)

0.6430***

± 0.48

(- 32.95)

2 Foot 0.4880

± 0.01

0.4711

± 0.78

0.4698

± 0.33

0.3730*

± 0.89

(- 23.57)

0.3456**

± 1.11

(- 27.56)

0.2972**

± 1.18

(- 36.74)

0.4206**

± 1.38

(- 13.81)

0.3673**

± 0.05

(- 22.03)

0.3271***

± 1.70

(- 30.37)

3 Gills 1.1778

± 0.49

1.1538

± 0.88

1.1387

± 0.18

0.8932** ± 1.52

(- 24.16)

0.7432** ± 1.31

(- 35.59)

0.5941*** ± 0.86

(- 47.83)

0.9140* ± 1.41

(- 22.40)

0.8330** ± 1.81

(- 27.80)

0.7255*** ± 1.92

(- 36.29)

4 Digestive

glands

1.6538

± 0.13

1.5310

± 0.68

1.5572

± 0.05

1.1573**

± 0.78 (- 30.02)

0.8612**

± 1.37 (- 43.75)

0.6256***

± 1.16 (- 59.83)

1.2845*

± 1.38 (- 22.33)

0.9940**

± 1.82 (- 35.08)

0.8956***

± 1.87 (- 42.49)

5 Gonad 1.4718

± 0.32

1.4632

± 0.52

1.4512

± 0.71

1.0121*

± 1.29 (- 31.23)

0.8665**

± 1.61 (- 40.78)

0.7893**

± 0.82 (- 45.61)

1.1752*

± 1.12 (- 20.15)

1.0485**

± 1.31 (- 28.34)

0.9985***

± 1.34 (- 31.19)

6 Whole soft

body 1.0780 ± 0.37

1.0891 ± 0.91

1.0895 ± 0.61

0.8326*

± 0.77

(-22.76)

0.6997**

± 1.18

(-35.75)

0.5259***

± 1.71

(-51.73)

0.8942*

± 1.78

(- 17.35)

0.7845**

± 1.87

(- 28.09)

0.7089***

± 1.43

(- 34.93)

1. Values expressed as mg/100mg dry wt. of tissue 2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05 5. NS (Not significant)

115

Table No. 3.4.b. Total Ascorbic acid content in different soft body tissues of Parreysia cylindrica after chronic exposure to

Dichlorovos and its subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr.

No. Tissue

Dichlorovos Recovery in normal water

(i) Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.5523

(- 42.41)

0.6084*

± 6.32 (+10.16)

0.6312*

± 1.11 (+14.29)

0.6895**

± 2.11 (+24.84)

0.6803*

± 1.58 (+23.18)

0.8104**

± 3.08 (+46.73)

0.9712***

± 4.60 (+75.85)

2 Foot 0.2972

(- 36.74)

0.3384 NS

± 1.79

(+13.86)

0.3484*

± 1.61

(+17.23)

0.4022***

± 3.53

(+35.33)

0.3974*

± 1.42

(+33.71)

0.4504**

± 4.28

(+51.55)

0.5244***

± 1.43

(+76.45)

3 Gills 0.5941

(- 47.83)

0.6914*

± 2.28

(+16.38)

0.7277**

± 2.83

(+22.49)

0.8215***

± 3.36

(+38.28)

0.8161**

± 2.97

(+37.37)

0.9108***

± 3.76

(+53.31)

1.1548***

± 3.04

(+94.38)

4 Digestive

glands

0.6256

(- 59.83)

0.7188NS

± 6.01

(+14.90)

0.7503* ± 9.41

(+19.93)

0.8225** ± 1.70

(+31.47)

0.7798* ± 1.47

(+24.65)

0.9218*** ± 2.45

(+47.35)

1.1618*** ± 3.40

(+85.71)

5 Gonad 0.7893

(- 45.61)

0.8765* ± 1.23

(+11.05)

0.9755** ± 1.49

(+23.59)

1.0985*** ± 2.69

(+39.17)

1.0895** ± 1.55

(+38.03)

1.2295*** ± 2.06

(+55.77)

1.4571*** ± 3.50

(+84.61)

6 Whole soft

body

0.5259

(-51.73)

0.5866NS

± 7.86 (+11.54)

0.6553**

± 1.82 (+24.61)

0.7533***

± 3.90 (+43.24)

0.7213**

± 1.51 (+37.16)

0.7819***

± 3.60 (+48.68)

0.9462***

± 4.04 (+79.92)

116

Table No. 3.5.a. Total DNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol without and

with ascorbic acid.

Sr.

No. Tissue

Control (A)

Dicofol (B)

Dicofol + A.A.(50 mg/lit) (C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 2.463 ± 1.32

2.421 ± 1.38

2.382 ± 176

1.821*

± 2.29

(-26.07)

1.521**

± 3.48

(-37.17)

1.218***

± 4.35

(-48.87)

2.113*

± 1.11

(-14.21)

1.709**

± 2.08

(-29.41)

1.378***

± 3.10

(-42.15)

2 Foot 3.643

± 0.62

3.570

± 1.71

3.532

± 1.10

2.938*

± 1.17 (-19.35)

2.588**

± 2.02 (-27.51)

2.113***

± 3.86 (-40.18)

3.298*

± 9.96 (-9.47)

2.918**

± 1.48 (-18.26)

2.358***

± 3.63 (-33.24)

3 Gills 2.371 ± 1.72

2.322 ± 1.56

2.281 ± 1.63

1.705*

± 2.72

(-28.09)

1.315**

± 4.00

(-43.37)

1.105**

± 1.55

(-52.41)

1.872**

± 2.79

(-19.38)

1.592**

± 2.80

(-31.44)

1.177***

± 4.05

(-48.40)

4 Digestive

glands 3.398 ± 1.21

3.365 ± 1.53

3.316 ± 1.82

1.978**

± 3.14

(-41.79)

1.657***

± 4.75

(-50.76)

1.137***

± 2.57

(-65.71)

2.571*

± 2.13

(-24.34)

2.217**

± 3.43

(-34.12)

1.667***

± 4.08

(-49.73)

5 Gonad 3.520

± 1.23

3.481

± 1.74

3.422

± 1.05

2.217** ± 3.21

(-37.02)

1.941** ± 4.20

(-43.28)

1.901*** ± 4.38

(-45.68)

2.610*** ± 2.97

(-25.85)

2.461*** ± 3.70

(-29.30)

2.211*** ± 2.14

(-35.39)

6 Whole soft

body

3.430

± 1.49

3.412

± 1.58

3.383

± 1.16

2.653**

± 1.65 (-22.65)

2.323**

± 2.16 (-31.92)

1.798***

± 1.68 (-46.85)

2.993*

± 1.94 (-12.74)

2.371**

± 2.50 (-30.51)

1.997***

± 3.72 (-40.97)

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

117

Table No. 3.5.b. Total DNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol and its

subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05 5. NS (Not significant)

Sr. No.

Tissue Dicofol

Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 1.218

(-48.87)

1.308*

± 8.30 (+7.43)

1.397*

± 2.51 (+14.70)

1.560**

± 1.92 (+28.08)

1.524**

± 2.26 (+25.12)

1.797**

± 1.68 (+47.54)

2.159***

± 4.00 (+77.26)

2 Foot 2.113

(-40.18)

2.224 NS

± 5.97

(+5.25)

2.374*

± 1.24

(+12.35)

2.588***

± 1.98

(+22.48)

2.545*

± 1.39

(+20.44)

2.952**

± 2.52

(+39.71)

3.520***

± 1.76

(+66.59)

3 Gills 1.105

(-52.41)

1.247*

± 1.80

(+12.85)

1.391**

± 2.45

(+25.88)

1.501***

± 4.46

(+35.84)

1.475**

± 2.67

(+33.48)

1.811***

± 4.03

(+63.89)

2.108***

± 3.60

(+90.77)

4 Digestive

glands

1.137

(-65.71)

1.254NS

± 1.05

(+10.30)

1.298* ± 2.99

(+14.18)

1.468** ± 3.79

(+29.09)

1.457** ± 3.22

(+28.17)

1.684** ± 3.91

(+48.09)

2.077*** ± 2.15

(+82.67)

5 Gonad 1.941

(-43.28)

2.118 NS

± 3.80 (+9.12)

2.274*

± 1.83 (+17.16)

2.560**

± 3.00 (+31.89)

2.591***

± 2.71 (+33.49)

2.812***

± 4.58 (+44.87)

3.347***

± 4.06 (+72.44)

6 Whole soft

body 1.798

(-46.85)

1.951*

± 9.78

(+8.51)

2.051**

± 1.23

(+14.07)

2.211***

± 2.71

(+22.97)

2.164**

± 1.75

(+20.36)

2.578**

± 2.78

(+43.38)

3.142***

± 4.54

(+74.75)

118

Table No. 3.6.a. Total DNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos without

and with ascorbic acid.

Sr.

No. Tissue

Control (A)

Dichlorovos (B)

Dichlorovos + A.A.(50 mg/lit) (C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 2.463

± 1.70

2.421

± 1.51

2.382

± 1.22

1.963**

± 2.29 (-20.30)

1.795**

± 3.48 (-27.10)

1.293***

± 4.35 (-45.72)

2.185*

± 1.11 (-11.29)

1.815**

± 2.08 (-25.03)

1.422***

± 3.10 (-40.30)

2 Foot 3.643 ± 1.34

3.570 ± 1.15

3.532 ± 1.72

3.182*

± 1.17

(-12.65)

2.798**

± 2.02

(-21.62)

2.193***

± 3.86

(-37.91)

3.392*

± 2.96

(-6.89)

2.985**

± 1.48

(-16.39)

2.451***

± 3.63

(-30.61)

3 Gills 2.371 ± 1.57

2.322 ± 1.77

2.281 ± 0.76

1.756*

± 2.72

(-25.94)

1.482**

± 4.00

(-36.18)

1.220***

± 1.55

(-46.51)

1.995*

± 2.79

(-15.86)

1.645**

± 2.80

(-29.16)

1.251**

± 4.05

(-45.16)

4 Digestive

glands

3.398

± 1.37

3.365

± 1.53

3.316

± 1.81

2.102** ± 3.14

(-32.09)

1.758*** ± 4.75

(-42.00)

1.241*** ± 2.57

(-54.07)

2.712** ± 2.13

(-20.19)

2.241** ± 3.43

(-33.40)

1.754*** ± 4.08

(-47.10)

5 Gonad 3.520

± 1.58

3.481

± 1.14

3.422

± 1.88

2.891* ± 3.21

(-17.87)

2.501* ± 2.20

(-28.15)

2.071** ± 3.38

(-39.48)

3.071* ± 2.97

(-12.76)

2.631** ± 3.70

(-24.42)

2.321*** ± 2.14

(-32.17)

6 Whole soft

body

3.430

± 1.25

3.412

± 1.51

3.283

± 1.69

2.871*

± 1.65 (-16.30)

2.481**

± 2.16 (-27.29)

1.961***

± 1.68 (-40.27)

3.057*

± 1.94 (-10.87)

2.671**

± 2.50 (-21.72)

2.028***

± 3.72 (-38.23)

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05 5. NS (Not significant)

119

Table No. 3.6.b. Total DNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos and

its subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05 5. NS (Not significant)

Sr.

No. Tissue

Dichlorovos Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 1.423

(-40.26)

1.332NS

± 1.30

(+6.39)

1.594* ± 2.51

(+12.02)

1.780** ± 1.92

(+25.09)

1.755* ± 2.26

(+23.33)

2.085** ± 1.68

(+46.52)

2.488*** ± 4.00

(+74.84)

2 Foot 2.193

(-37.91)

2.288NS

± 2.97 (+4.33)

2.397*

± 1.24 (+9.30)

2.637**

± 2.98 (+20.25)

2.597*

± 1.39 (+18.42)

2.923**

± 2.52 (+33.29)

3.602***

± 1.76 (+64.25)

3 Gills 1.220

(-46.51)

1.345*

± 1.80

(+10.25)

1.482**

± 2.45

(+21.48)

1.593***

± 2.46

(+30.57)

1.556*

± 2.67

(+27.54)

1.952**

± 2.03

(+60.00)

2.313***

± 3.60

(+89.59)

4 Digestive

glands 1.241

(-54.07)

1.342NS

± 1.05

(+8.14)

1.392*

± 2.99

(+12.17)

1.635**

± 3.79

(+31.75)

1.585**

± 3.22

(+27.72)

1.815**

± 3.91

(+46.25)

2.112***

± 2.15

(+70.19)

5 Gonad 2.071

(-39.48)

2.242*

± 3.80

(+8.26)

2.412*

± 1.83

(+16.47)

2.705**

± 3.00

(+30.61)

2.685**

± 2.71

(+29.65)

2.902**

± 1.58

(+40.13)

3.515***

± 2.06

(+69.72)

6 Whole soft

body

1.961

(-40.27)

2.082NS

± 3.78

(+6.17)

2.208* ± 1.23

(+12.60)

2.360** ± 2.71

(+20.35)

2.325** ± 1.75

(+18.56)

2.718*** ± 2.78

(+38.60)

3.358*** ± 3.54

(+71.24)

120

Table No. 3.7.a. Total RNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol without and

with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Dicofol

(B)

Dicofol + A.A.(50 mg/lit)

(C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 5.628

± 1.41

5.587

± 1.23

5.510

± 1.57

4.572*

± 3.32 (-18.76)

4.112**

± 2.20 (-26.40)

2.978***

± 3.39 (-45.95)

4.832*

± 2.78 (-14.14)

4.317**

± 2.69 (-22.73)

3.238***

± 3.91 (-41.23)

2 Foot 6.342

± 1.63

6.340

± 1.82

6.339

± 1.53

4.892**

± 1.88 (-22.86)

4.385**

± 2.98 (-30.84)

3.976***

± 3.09 (-37.28)

5.622*

± 1.80 (-11.35)

4.724**

± 2.30 (-25.49)

4.327***

± 3.11 (-31.65)

3 Gills 7.038 ± 0.85

7.107 ± 0.73

7.210 ± 1.22

4.998**

± 1.76

(-28.99)

4.488**

± 2.94

(-36.85)

3.461***

± 3.64

(-52.00)

5.778*

± 1.74

(-17.90)

4.821**

± 2.87

(-32.17)

4.021***

± 3.47

(-44.23)

4 Digestive

glands

9.258

± 1.88

9.187

± 1.47

9.122

± 1.71

6.421** ± 2.74

(-30.64)

5.118** ± 3.26

(-44.29)

3.788*** ± 3.58

(-58.47)

7.481** ± 1.49

(-19.19)

5.978** ± 3.32

(-34.93)

4.868*** ± 3.92

(-46.63)

5 Gonad 5.183

± 0.92

5.127

± 1.23

5.103

± 0.61

4.128** ± 1.73

(-20.36)

3.528*** ± 2.78

(-31.19)

2.971*** ± 3.42

(-41.78)

4.242** ± 2.59

(-18.16)

3.778** ± 3.49

(-26.31)

3.258*** ± 3.69

(-36.16)

6 Whole soft

body

7.524

± 2.11

7.514

± 1.63

7.424

± 1.91

6.122*

± 1.62 (-18.63)

5.382**

± 1.47 (-28.37)

4.232***

± 2.78 (-43.00)

6.732*

± 1.83 (-10.53)

5.792**

± 2.86 (-22.92)

4.585**

± 3.08 (-38.24)

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

121

Table No. 3.7.b. Total RNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dicofol and its

subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr.

No. Tissue

Dicofol Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 2.978

(-45.95)

3.230NS

± 3.47

(+8.46)

3.627*

± 1.90

(+21.79)

3.995**

± 2.18

(+34.15)

3.681*

± 3.91

(+23.61)

4.243**

± 1.14

(+42.48)

5.317***

± 2.09

(+78.54)

2 Foot 3.976

(-37.28)

4.208NS

± 3.27

(+5.84)

4.465*

± 2.78

(+12.30)

5.004*

± 2.74

(+25.86)

4.662*

± 3.11

(+17.25)

5.307**

± 1.29

(+33.48)

6.647***

± 2.24

(+67.18)

3 Gills 3.461

(-52.00)

3.825* ± 2.38

(+10.52)

4.051** ± 1.58

(+17.05)

4.821*** ± 2.70

(+39.30)

4.218** ± 3.47

(+21.87)

5.088*** ± 3.66

(+47.01)

6.449*** ± 1.27

(+86.33)

4 Digestive

glands

3.788

(-58.47)

4.078* ± 3.92

(+7.66)

4.328** ± 1.29

(+14.26)

5.132*** ± 2.25

(+35.48)

4.697* ± 3.92

(+24.00)

5.498** ± 1.07

(+45.14)

6.915*** ± 3.47

(+82.55)

5 Gonad 2.971

(-41.78)

3.154NS

± 2.52 (+6.16)

3.350*

± 3.51 (+12.76)

3.824**

± 3.29 (+28.71)

3.587**

± 2.69 (+20.73)

4.075***

± 1.73 (+37.16)

5.072***

± 3.23 (+70.72)

6 Whole soft

body 4.232

(-43.00)

4.478NS

± 3.42

(+5.81)

4.728*

± 1.22

(+11.72)

5.546**

± 1.31

(+31.05)

4.926**

± 3.08

(+16.40)

5.952***

± 3.82

(+40.64)

7.372***

± 2.44

(+74.20)

122

Table No. 3.8.a. Total RNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos without

and with ascorbic acid.

Sr. No.

Tissue

Control

(A)

Dichlorovos

(B)

Dichlorovos + A.A.(50 mg/lit)

(C)

7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 5.876

± 1.06

5.816

± 1.13

5.801

± 0.87

5.253* ± 2.36

(-10.60)

4.572** ± 3.81

(-21.39)

3.372*** ± 3.87

(-41.87)

5.422* ± 1.03

(-7.73)

4.745** ± 2.07

(-18.41)

3.958*** ± 3.13

(-31.77)

2 Foot 6.635

± 1.22

6.602

± 1.43

6.582

± 1.02

5.672**

± 2.11 (-14.51)

4.923**

± 3.49 (-25.43)

4.357***

± 2.08 (-33.80)

5.997*

± 1.17 (-9.62)

5.287**

± 2.00 (-19.92)

4.917***

± 3.43 (-25.30)

3 Gills 7.214

± 1.54

7.144

± 1.81

7.103

± 1.35

5.917**

± 2.97 (-17.98)

5.127**

± 3.38 (-28.23)

3.696***

± 1.34 (-47.97)

6.417*

± 1.54 (-11.05)

5.396**

± 2.24 (-24.47)

4.525***

± 3.16 (-36.29)

4 Digestive

glands 9.162 ± 0.60

9.103 ± 0.89

9.087 ± 1.15

6.675**

± 3.34

(-27.14)

5.713***

± 2.50

(-37.24)

4.415***

± 3.62

(-51.41)

7.515*

± 2.36

(-17.98)

6.516**

± 2.73

(-28.42)

5.522***

± 3.61

(-39.23)

5 Gonad 5.492 ± 1.75

5.411 ± 1.32

5.376 ± 1.78

4.485*

± 2.42

(-18.34)

3.931**

± 3.78

(-27.35)

3.490***

± 2.27

(-35.08)

4.783*

± 2.32

(-12.91)

4.259**

± 2.97

(-21.29)

3.811***

± 3.27

(-29.11)

6 Whole soft

body

7.806

± 1.00.

7.758

± 0.71

7.685

± 0.57

6.724* ± 1.88

(-13.86)

6.118* ± 2.22

(-21.14)

4.722** ± 3.57

(-38.56)

7.117NS

± 1.70

(-8.83)

6.418* ± 2.87

(-17.27)

5.236** ± 2.91

(-31.87)

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control 3. ± indicate S.D. of three observation

4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

123

Table No. 3.8.b. Total RNA content in different soft body tissues of Parreysia cylindrica after chronic exposure to Dichlorovos and

its subsequent recovery.

1. Values expressed as mg/100mg dry wt. of tissue

2. (+) or (-) indicate percent variation over control

3. ± indicate S.D. of three observation 4. Values are significant at *P<0.001, **P<0.01, ***P<0.05

5. NS (Not significant)

Sr. No.

Tissue Dichlorovos

Recovery in normal water

(i)

Recovery in A.A.(50 mg/lit)

(ii)

21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 3.372

(-41.87)

3.633**

± 3.35

(+7.74)

3.983**

± 1.03

(+18.12)

4.512***

± 2.20

(+33.81)

4.112*

± 3.13

(+21.95)

4.732**

± 1.90

(+40.33)

5.892***

± 2.58

(+74.73)

2 Foot 4.357

(-33.80)

4.652*

± 3.37

(+6.77)

4.954**

± 1.26

(+13.70)

5.384***

± 2.03

(+23.57)

5.062**

± 3.43

(+16.18)

5.724**

± 1.39

(+31.37)

7.053***

± 2.32

(+61.88)

3 Gills 3.696

(-47.97)

3.995*

± 2.45

(+8.09)

4.263**

± 3.98

(+15.34)

5.125**

± 2.07

(+38.66)

4.518**

± 3.16

(+22.24)

5.325**

± 2.36

(+44.07)

6.759***

± 1.78

(+82.87)

4 Digestive

glands

4.415

(-51.41)

4.682NS

± 1.89

(+6.05)

5.042*

± 2.88

(+14.20)

6.017**

± 2.77

(+36.29)

5.420**

± 3.61

(+22.76)

6.258***

± 1.08

(+41.74)

7.938***

± 1.26

(+79.80)

5 Gonad 3.490

(-35.08)

3.810NS

± 3.18

(+9.17)

3.908* ± 1.76

(+11.98)

4.375** ± 2.21

(+25.36)

4.144* ± 2.27

(+18.74)

4.705** ± 1.39

(+34.81)

5.764*** ± 2.17

(+65.16)

6 Whole soft

body

4.722

(-38.56)

4.912* ± 3.14

(+4.02)

5.188** ± 1.59

(+9.87)

6.122*** ± 2.41

(+29.65)

5.427* ± 2.91

(+14.95)

6.557** ± 1.13

(+38.86)

7.990*** ± 2.45

(+69.21)

124

Fig. 3.1.1 Profiles of proteins (mg/100mg of dry tissue) in mantle of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

Fig. 3.1.2 Profiles of proteins (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

0

5

10

15

20

25

30

35

40

45

07 days

14 days

21 days

07 days

14 days

21 days

Prote

in c

on

ten

t in

mg/1

00m

g

of

dry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

70

07 days

14 days

21 days

07 days

14 days

21 days

Prote

in c

on

ten

t in

mg

/10

0m

g

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

125

Fig. 3.1.3 Profiles of proteins (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.1.4 Profiles of proteins (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and

with ascorbic acid and during recovery.

0

10

20

30

40

50

60

07 days

14 days

21 days

07 days

14 days

21 days

Prote

in c

on

ten

t in

mg/1

00m

g

of

dry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

126

Fig. 3.1.5 Profiles of proteins (mg/100mg of dry tissue) in gonad of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

Fig. 3.1.6 Profiles of proteins (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and

with ascorbic acid and during recovery.

0

5

10

15

20

25

30

35

40

45

50

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

70

07 days

14 days

21 days

07 days

14 days

21 days

Prote

in c

on

ten

t in

mg

/10

0m

g

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

127

Fig. 3.1.7 Profiles of proteins (mg/100mg of dry tissue) in mantle of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.1.8 Profiles of proteins (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

0

5

10

15

20

25

30

35

40

45

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

70

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

128

Fig. 3.1.9 Profiles of proteins (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.1.10 Profiles of proteins (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

10

20

30

40

50

60

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

129

Fig. 3.1.11 Profiles of proteins (mg/100mg of dry tissue) in gonad of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.1.12 Profiles of proteins (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

5

10

15

20

25

30

35

40

45

50

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

10

20

30

40

50

60

70

07 days

14 days

21 days

07 days

14 days

21 days

Pro

tein

co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

130

Fig. 3.2.1 Profiles of ascorbic acid (mg/100mg of dry tissue) in mantle of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

Fig. 3.2.2 Profiles of ascorbic acid (mg/100mg of dry tissue) in foot of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

131

Fig. 3.2.3 Profiles of ascorbic acid (mg/100mg of dry tissue) in gills of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

Fig. 3.2.4 Profiles of ascorbic acid (mg/100mg of dry tissue) in digestive glands of

fresh water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without

and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

132

Fig. 3.2.5 Profiles of ascorbic acid (mg/100mg of dry tissue) in gonad of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

Fig. 3.2.6 Profiles of ascorbic acid (mg/100mg of dry tissue) in whole soft body of

fresh water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without

and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.2

0.4

0.6

0.8

1

1.2

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

133

Fig. 3.2.7 Profiles of ascorbic acid (mg/100mg of dry tissue) in mantle of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.2.8 Profiles of ascorbic acid (mg/100mg of dry tissue) in foot of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.1

0.2

0.3

0.4

0.5

0.6

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

134

Fig. 3.2.9 Profiles of ascorbic acid (mg/100mg of dry tissue) in gills of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.2.10 Profiles of ascorbic acid (mg/100mg of dry tissue) in digestive glands of

fresh water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos

without and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

gof

dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

135

Fig. 3.2.11 Profiles of ascorbic acid (mg/100mg of dry tissue) in gonad of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.2.12 Profiles of ascorbic acid (mg/100mg of dry tissue) in whole soft body of

fresh water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos

without and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.2

0.4

0.6

0.8

1

1.2

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt i

n m

g/1

00m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

136

Fig. 3.3.1 Profiles of DNA (mg/100mg of dry tissue) in mantle of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.3.2 Profiles of DNA (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

0

0.5

1

1.5

2

2.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

137

Fig. 3.3.3 Profiles of DNA (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.3.4 Profiles of DNA (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and

with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

138

Fig. 3.3.5 Profiles of DNA (mg/100mg of dry tissue) in gonad of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.3.6 Profiles of DNA (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dicofol without and

with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f dry

tis

sue

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

139

Fig. 3.3.7 Profiles of DNA (mg/100mg of dry tissue) in mantle of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.3.8 Profiles of DNA (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

0

0.5

1

1.5

2

2.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

140

Fig. 3.3.9 Profiles of DNA (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia Cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.3.10 Profiles of DNA (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

141

Fig. 3.3.11 Profiles of DNA (mg/100mg of dry tissue) in gonad of fresh water

bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and with

ascorbic acid and during recovery.

Fig. 3.3.12 Profiles of DNA (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia Cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

100

mg

of

dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

3.5

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f dry

tis

sue

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

142

Fig. 3.4.1 Profiles of RNA (mg/100mg of dry tissue) in mantle of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.4.2 Profiles of RNA (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

0

1

2

3

4

5

6

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

143

Fig. 3.4.3 Profiles of RNA (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.4.4 Profiles of RNA (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

0

1

2

3

4

5

6

7

8

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

9

10

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0mg

of d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

144

Fig. 3.4.5 Profiles of RNA (mg/100mg of dry tissue) in gonad of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dicofol without and with ascorbic acid

and during recovery.

Fig. 3.4.6 Profiles of RNA (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia cylindrica after chronic exposure to dicofol without and with

ascorbic acid and during recovery.

0

1

2

3

4

5

6

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dicofol

Dicofol +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

145

Fig. 3.4.7 Profiles of RNA (mg/100mg of dry tissue) in mantle of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.4.8 Profiles of RNA (mg/100mg of dry tissue) in foot of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

0

1

2

3

4

5

6

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

146

Fig. 3.4.9 Profiles of RNA (mg/100mg of dry tissue) in gills of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.4.10 Profiles of RNA (mg/100mg of dry tissue) in digestive glands of fresh

water bivalve, Parreysia cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

1

2

3

4

5

6

7

8

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

9

10

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0mg

of d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

147

Fig. 3.4.11 Profiles of RNA (mg/100mg of dry tissue) in gonad of fresh water bivalve,

Parreysia cylindrica after chronic exposure to dichlorovos without and with ascorbic

acid and during recovery.

Fig. 3.4.12 Profiles of RNA (mg/100mg of dry tissue) in whole soft body of fresh

water bivalve, Parreysia cylindrica after chronic exposure to dichlorovos without and

with ascorbic acid and during recovery.

0

1

2

3

4

5

6

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

100m

go

f d

ry t

issu

e

Time of exposure

control

Dichlorovos

Dichlorovos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

148

DISCUSSION

The use of synthetic chemicals in agriculture, forestry and public

health has become so inevitable and common that the environment is

continuously being contaminated by these toxic substances (Patil and

Saidapur, 1989). Complex composition and cumulative action of

synthetic pesticides causes enormous amount of stress in the recipient

ecosystem (Madhyastha, 1996). Pollution is recognized as one of the

most significant factors causing major declines in populations of

freshwater species in many parts of the world (Winfield, 1992; Lawton

and May, 1995; Maitland, 1995; Mason and Jenkins 1995). Nearly 70%

of the freshwater mussel (Bivalvia: Unionidae) species in the United

States are considered as endangered, threatened, or of special concern

(Williams et al., 1993). Pesticides due to their potential toxicity produce

biochemical changes in the tissues and organs of exposed animals

(Shastry and Sharma, 1979; Kumar and Singh, 2000; Tilak et al., 2003;

Mathivanan, 2004; Shrivastava and Singh, 2004). Acute poisoning by

pesticides certainly represents stress (Matton and La Ham, 1969).

Biochemical constituents like glycogen, proteins and lipids considered to

be sensitive indicator of stress and act as key substrates for metabolism

(Peter, 1973). In order to investigate the physiological changes after

pesticide treatment, the study of changes in the biochemical constituents

is the most fundamental tool.

Ascorbic acid is an antioxidant vitamin. It plays vital role as an

antioxidant that serves protective function against oxidative damage in

tissues. Antioxidant property of ascorbic acid helps to prevent free radical

formation from water soluble molecules, which may causes cellular

injuries and diseases. Vitamin-C has been shown to play an important

role in the process of hydroxylation, oxygenation and oxidation of

corticosteroids (Chatrjeee, 1967). The role of ascorbic acid in disease and

149

tissue repair is well known (Halver, 1972). It is confirmed that the free

radical scavenging property of L-ascorbate is responsible for reducing

genotoxic damage (Edgar, 1974). So that, for the detoxification of

pesticides from animal body ascorbic acid can be ideally useful.

Present investigation is concerned with changes in biochemical

composition in different tissues of Parreysia cylindrica exposed to

chronic dose of pesticide dicofol and dichlorovos and its subsequent

recovery after withdrawal of pesticide and its subsequent recovery by

exogenous administration of L-ascorbic acid. Taking in consideration the

vital role of protein, ascorbic acid, DNA and RNA in structure and

functions of cells, the changing pattern in these biochemical constituents

have been studied.

Changes in Protein content:-

In the present study obtained results demonstrated that, after

chronic exposure to pesticides dicofol and dichlorovos a marked

depletion in the protein contents in the mantle, foot, gills, gonad,

digestive glands and whole soft body tissues of the experimental

freshwater bivalve Parreysia cylindrica was observed as compared to

bivalves maintained as control. The obtained results are presented in the

table nos. 3.1.a; 3.2.b and 3.2.a; 3.2.b and figures 3.1.1 to3.1.12. The

result clearly indicated that dicofol causes more protein depletion in all

soft body tissues of the experimental bivalve as compared to dichlorovos.

The results showed that, there was progressive decrease in the

protein content as exposure period was increased. The depletion in

protein contents with increased exposure period suggest high protein

hydrolysis that could be due to pesticide interfering and impairment as

well as lowering of protein synthesis (Ghosh and Chatterjee, 1985). The

results recorded in the present study are in harmony with the results of

previous investigators (Lomte et al., 2000; Mahajan and Zambare, 2005;

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Gulbhile, 2006; Satyaparameshwar et al., 2006; Nawale, 2008; Pardeshi

and Gapat, 2012).

In the present study the decrease in amount of protein content in all

tissues after exposure to pesticides dicofol and dichlorovos was attributed

due to oxidative stress generated by these pesticides. Pesticides are

inducers of reactive oxygen species (ROS) (Abdullah et al., 2004). In the

presence of reactive oxygen species (ROS), proteins can be damaged by

direct oxidation of their amino acid residues and cofactors or by

secondary attack via lipid peroxidation (Grune, 2000; Requena et al.,

2003). Oxidative attack on proteins results in site-specific amino acid

modifications, fragmentation of the peptide chain, aggregation of cross-

linked reaction products, altered electrical charge and increased

susceptibility to proteolysis. The amino acids in a peptide differ in their

susceptibility to attack, and the various forms of activated oxygen differ

in their potential reactivity. Primary, secondary, and tertiary protein

structures alter the relative susceptibility of certain amino acids.

Oxidative modification of specific amino acids is one mechanism of

marking a protein for proteolysis (Stadtman, 1986). In mollusk the

proteases enzyme degrade oxidised proteins (Farr and Kogoma, 1991).

The decrease in amount of protein content in all tissues after

exposure to dicofol and dichlorovos was attributed to the impairment of

protein synthesis and increase in the rate of their degradation to amino

acid which may be fed to TCA cycle through aminotransferase probably

to cope up with the high energy demands and stress conditions (Waykar

and Lomte, 2001). These results are in agreement with the earlier findings

(Parthasarathy and Joseph, 2011) which indicated that the decreased

protein content might also be attributed to the destruction/necrosis of cells

and consequent impairment in protein synthetic machinery (Umminger,

1977; Bradbury et al., 1987). Catabolism of proteins and amino acids

151

make a major contribution to the total energy production. It is known that

structural proteins are used as energy source under stressful conditions

(Claybrook, 1983). Jha (1988) and Waykar and Lomte (2001) supported

the idea of consumption of amino acid for metabolic processes as energy

source. According to Sivaprasad and Raman Rao (1980) depletion in

protein content in pollutant treated animals might be due to either

enhanced proteolytic activity or decreased protein synthesis. Increase in

protease activity also supported depletion of protein content (Srinivas and

Purushotam Rao, 1987). They observed increased protease activity in

Bombyx mori. Waykar and Lomte (2002, 2004) observed increased

protease activity in experimental bivalves after exposure to pesticides.

Reddy and Bashamohideen (1998) reported that increased protease

activity can increase protein breakdown in the tissue of the animal

exposed to the pesticide. Parate and Kulkarni (2003) suggested that

depletion of protein may be due to utilization of protein for the

production of energy and to prevent from fatigue due to the effect of

pesticide. The decrease in the protein level recorded during study was an

indicative of increased proteolysis resulting to shift in nitrogen

metabolism. Inhibition of ribosomal activity result in protein degradation

was also one of the possible reasons for protein decrease (Shelke, 2010).

The decrease in amount of protein content in different tissues after

chronic exposure to pesticides indicate that, pesticides inhibits the

synthesis of protein which ultimately results in increase in the free amino

acid pool in the cell or due to enhancement of proteolysis to cope with the

high energy demands under toxic stress (Vincent et al., 1995; Waykar

and Lomte, 2001). A marked fall in the protein level in all the tissues

indicates a rapid initiation of breakdown of protein. To meet energy

demands during toxic stress mobilization of protein might have taken

place. The depletion of protein tissue was due to diversification of

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energy, to meet the impending energy demand under toxic stress (Vincent

et al., 1995) and to prevent fatigue due to pesticide toxicity (Parate and

Kulkarni, 2003). At high pollution stress however, protein synthesis can

be suppressed indicating disturbance of normal metabolic processes

(Pottinger et al., 2002).

The results of total protein contents in all tissues clearly indicate

that digestive glands was the most affected organ followed by gill, whole

body, mantle, gonads and foot. The higher depletion of protein in the

digestive gland might be due to high metabolic potency and efficiency of

the gland when compared to other tissues like mantle, foot, gills, gonads

and whole soft body of the bivalve. The digestive gland is the site of

action of pollutants in the body of bivalves or digestive gland seems to be

the main site of degradation and detoxification of pesticides and hence

has the largest demand of energy for the metabolic processes resulting

into increasing utilization of protein in digestive gland provides better

indication of the extent of toxicity. Mule and Lomte (1992, 1993, and

1995), Waykar and Lomte (2001) supported the most alteration of protein

contents in digestive glands of freshwater bivalves. Patil (2011) observed

maximum depletion of protein content in digestive glands than in mantle,

gills, foot and whole body tissue.

Large body of literature reported that, pesticide stress caused

depletion of protein content. According to Lynch et al., (1969) decreased

level of protein could be due to the reduction in protein synthesis because

of liver cirrhosis. Yagana Bano et al., (1981) reported that the depletion

in the protein content from the muscles of fish, Clarias batrachus after

pesticide treatment. Lomte and Alam (1982) observed the decline in

protein level in Bellamya (V) bengalensis after pesticide stress. Swami et

al., (1983) noted decrease in total protein content in foot, mantle and

hepatopancreas of the fresh water mussel, Lamellidens marginalis due to

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metacid and flodit exposure. Vijayalakshmi and Rao (1985) reported the

decrease in protein content in the muscles of prawn Metapenaeus

monoceros due to phosphomidon exposure. Observations were made

about the decrease in protein content in the muscles of prawn, Penaeus

indicus after exposure to pesticide phosphomidon and methyl parathion

by Reddy et al., (1988). The effect of endosulfan in various tissues of the

freshwater field crab, Barytelphusa gureini were studied and noted a

remarkable decrease in protein content in the muscle tissues by Reddy et

al., (1989). Krishnamoorthy and Subramanian (1995) noted decrease in

protein level was recorded in muscle, gill and hepatopancrease of

Macrobrachium lamarri when exposed to copper. Singh and Agarwal

(1996) reported significant decrease in levels of protein in foot tissue in

Lymnea accuminata on exposure to deltamethrin. Decrease in protein

content in gills, liver, muscles and brain of Labio rohita, Mystus vittatus

and Channa punctatus on exposure to monocrotophos was reported by

Rao and Ramaneshwari (2000). Mahajan and Zambare (2001) reported

decrease in the protein contents of freshwater bivalve, Corbicula

striatella after heavy metal stress in most of the tissues. Valarmathi and

Azariah (2002) reported maximum decrease in protein content in the gill

tissues of a crab, Sesarma quadratum when treated with 9.3 ppm copper

chloride solution for 21 days. Tilak et al., (2003) reported significant

decrease in the protein content in almost all tissues in Channa punctatus

when exposed to sub lethal and lethal concentration of fenvalerate.

Parate and Kulkarni (2003) were of the opinion that, depletion of the

protein may be due to its utilization for the production of energy to

alleviate the pesticide stress and to prevent fatigue which may occur due

to the effect of pesticides. Amsath et al., (2003) noted decrease in total

protein content of L. maginalis exposed to monocrotophos. Same effect

was observed by Shukla et al. (2005) in liver and kidney of cat fish

154

Clarias batracus during exposure to organophosphorous pesticide, nuvan.

Abdul Naveed et al. (2005) reported that, serum biochemical parameters

of fish Channa punctatus could be altered on treatment with sub lethal

concentration of triazophos and found decreased level of protein. They

concluded that the level of the protein decreased due to increased

proteolytic activity which might be the cause of increased amino acid

pool during the pesticide exposure period. Ghanbahadur et al. (2005)

found variations in protein content of gills and liver of fish, Rasbora

daniconius when was exposed to three sub lethal concentrations of

organophosphate nuvan. Jagatheeswari (2005) found significant depletion

in protein content in different tissues of Cyprinus carpio at a sub acute

period of exposure to pesticide phosalone. Kulkarni et al. (2005) found

significant decrease in total protein content in foot, hepatopancreas and

gills of the fresh water mussel, Lamellidens corrianus on exposure to the

sub lethal concentration of organochlorine insecticide, hildan. They

further concluded that, decline in protein content indicates intensive

proteolysis which is followed by corresponding decrease in total free

amino acids. Total rotein content in muscles, hepatopancreas and gills of

crab, Barytelphusa guerini was decreased due to sub lethal dose of hildan

(Keshavan et al., 2005). The acute and chronic exposure to tetracycline

and chloramphenicol, L. corrianus showed decrease in protein levels, in

proportion with the period of exposure (Nagpure and Zambare, 2005).

Bhide et al., (2006) studied the morphology and biochemistry of different

developmental stages of fresh water snail, Lymnea stagnalis after the

treatment of baygon and nuvan and found decrease in protein fractions in

most of the developmental stages. Satyaparameshwar et al., (2006)

observed decrease in total protein content on exposure to chromium in

three different tissue viz. adductor muscles, gills and mantle of fresh

water mussel, Lamellidens marginalis. Agrahari et al., (2006) reported

155

decrease in total protein content in liver, muscle, brain and gills of

Channa punctatus after monocrotophos exposure for 30 days.

Venkataramana et al., (2006) found decreasing trend in protein content in

the heart of Glossogobius giuris on exposure to malathion. Singh et al.

(2006) noted decrease in total protein in ovaries of fresh water fish,

Channa punctatus treated with organophosphorous insecticide nuvan.

Senthilkumar et al., (2007) showed that when Spirolotelphusa

hydrodroma treated with chloropyriphos, the protein content decreased in

gills. Siddiqui et al., (2010) reported depletion of protein in the gills of

freshwater crab, Barytelphusa gureini when exposed to sub lethal

concentration of copper sulphate solution. Anilkumar et al., (2010) noted

decrease in protein content of male albino rats after exposure to toxicant.

Patil (2010) observed decrease in protein content in different tissues like

foot, mantle, gills, digestive gland and whole body of freshwater bivalve,

Parreysia cylindrica after exposure to thiamethoxam and indoxacarb.

Andhale and Zambare (2011) studied the nickel induced biochemical

alterations in freshwater bivalve, Lammellidens marginalis and reported

that the protein contents were decreased in treated animals than the

control. Kamble and Rao (2011) reported decrease in protein contents of

freshwater mollusc, Lamellidens corrianus after chronic exposure to

thiodan. Waykar and Pulate (2012) reported decreased protein contents in

different soft tissues of freshwater bivalve, Lamellidens marginals (L)

after chronic exposure to profenofos and reported highest decrease in

protein contents in digestive gland. Pardeshi and Gapat (2012) reported a

marked decrease in protein content in different soft tissues of the

freshwater bivalve, Lamellidens corrianus after chronic exposure to

nickel chloride. Tripathi et al., (2012) reported significant reduction in

protein, glycogen and nucleic acid (DNA and RNA) content in freshwater

fish, Colisa fasciatus after exposure to sub-lethal dose of cadmium

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sulphate for 30 days. Mahajan and Zambare (2012) observed decrease in

protein content of hepatopacreas and gonad of Bellamya bengalensis due

to heavy metal toxicity and concluded that decrease in protein contents

was due to severe disturbances of the metabolism in the animal.

In combined exposure to dicofol and dichlorovos with 50mg/l of L-

ascorbic acid the severity of protein depletion was much reduced.

Ascorbic acid is capable of counteracting the damage. Antioxidants block

the process of oxidation by neutralizing free radical. Pesticides are known

to enhance the formation of reactive oxygen species. The ROS have the

capacity to cause damage to biomolecules such as proteins, nucleic acids

etc. The toxicity of ROS can be mitigated by free radical scavengers such

as ascorbic acid. Ascorbic acid usually acts as an antioxidant. It typically

reacts with oxidants of the reactive oxygen species, such as the hydroxyl

radical formed from hydrogen peroxide. Such radicals are damaging to

animals at the molecular level due to their possible interaction with

proteins, and nucleic acids. Sometimes these radicals initiate chain

reactions. Ascorbic acid can terminate these chain radical reactions by

electron transfer. Ascorbic acid functions as a reductant for many free

radicals, thereby minimizing the damage caused by oxidative stress. This

study indicates that the use of L-ascorbic acid protect the tissues from

oxidative damage caused by pesticide.

Many investigators reported the similar results. Mahajan and

Zambare, (2001) reported the protection by ascorbic acid against the

heavy metal induced alterations in protein levels in fresh water bivalve,

Corbicula striatella. The bioregulatory role of ascorbic acid to protect

extracellular protein function through gene expression is already

highlighted (Griffiths and Lunec, 2001). Agarwal et al., (2003) reported

that the stimulatory action of ascorbic acid is indicated by increase in cell

population, protein content and level of lysosomal enzymes, antioxidants

157

and enhanced capacity for phagocytosis. Waykar and Pulate (2011)

reported the role of ascorbic acid in amelioration of protein alteration

induced by pesticide. Pardeshi and Gapat (2012) reported the effect of

ascorbic acid on protein content during nickel intoxication in the

freshwater bivalve, Lamellidens corrianus. The present results showed

that ascorbic acid, one of the most important antioxidant, spares the other

oxidants by forming the first line defense against free radicals and

peroxides that are generated during cellular metabolism (May, 2000).

Changes in ascorbic acid content:-

In the present study depletion of ascorbic acid levels in the mantle,

foot, gill, gonad, digestive glands and whole soft body tissues of the

experimental freshwater bivalve Parreysia cylindrica was observed after

chronic exposure to pesticides dicofol and dichlorovos. Obtained results

were presented in the table nos. 3.3.a; 3.3.b and 3.4.a; 3.4.b and figures

3.2.1 to 3.2.12. The result clearly indicates that highest depletion in

ascorbic acid content was reported in bivalves exposed to dicofol as

compared to dichlorovos. Results obtained during the present study are in

harmony with the findings of Jadhav et al., (1996), Padmaja and Reddy

(1998), Waykar and Lomte (2001 and 2004), Borane (2006), Gulbhile

(2006), Waykar (2006 and 2007), Phirke (2008), Nawale (2008).

In the present study, the observed decreased content of ascorbic

acid in different soft body tissues of experimental bivalve, might be due

to its contribution in detoxification or due to impairment in its synthesis

(Waykar et al., 2001), repairing of injuries in tissues and to cope up

against the toxic stress caused by pesticides. This also suggests the

increased demand of energy being provided by utilization of ascorbic acid

in responses to pesticide stress.

Stress caused alterations in the normal physiology of animal

leading to enhanced utilization and mobilization of ascorbic acid (Chinoy

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and Kamalakumari, 1976) as ascorbic acid is recognized as antistress

factor (Kutsky, 1973). Kachole et al., (1977) concluded that, the ascorbic

acid might have induced hepatic mixed function of oxidase system and

played important role of bio-transformation of toxic substances into non

toxic. Same observations were reported by Ali and Ilyas, (1981) and Ali

et al., (1983). Ascorbic acid is known to participate in several

biosynthesis reactions as source of electron energy (Gorbunova, 1966;

Chinoy, 1972 a, b). At stressful condition on exposure to toxicants

ascorbic acid indicates positive role in detoxification (Mahajan and

Zambare, 2001) and also perform therapeutic role against pollutant

toxicity in mollusc (Waykar, 2006; Waykar and Pulate, 2012). Parihar

and Dubey, (1995) and Lackner, (1998) reported that during acute

response to different stressors ascorbic acid was depleted. Decrease in

ascorbic acid content indicated its involvement in counteracting oxidative

damage.

The antioxidant role of ascorbic acid is a well-known phenomenon,

which protects the tissues from the superoxide radical generated due to

different toxicological effects. Changes in the environment cause

alteration in the ascorbic acid content. The varied functions of the

ascorbic acid make it dynamic. Any alteration in the surrounding water

due to the contamination of water also alters ascorbic acid contents.

Different pollutant stresses has its impact on the concentration of ascorbic

acid (Bhusari, 1987).

Number of researchers reported that due to toxicant stress ascorbic

acid content was decreased. Dedemeyer (1969) reported a decrease in the

ascorbic acid in the kidney of salmonids on exposure to stressful

situation. Bannerjee and Basu (1975) reported that the depletion of

ascorbic acid content might be due to decreased biosynthesis and

increased catabolism. Chitra and Ramana Rao (1977) suggested variety of

159

changes in blood glycogen and ascorbic acid levels at low temperature.

Bhusari (1987) reported alteration in the ascorbic acid content in the

tissues of fresh water fish, Barbus ticto on exposure of endosulfan and

ekalux. Jadhav et al., (1996) observed decrease in ascorbic acid content

on acute and on chronic exposure of fresh water bivalve, Corbicula

striatella to carbaryl. Kulkarni et al., (1988) estimated the effect of

temperature and pH on ascorbic acid content of Indonaia caeruleus.

Waykar et al., (2001) reported depletion in the ascorbic acid contents in

mantle, gills, digestive glands and whole soft body tissues of the

freshwater bivalve, Parreysia cylindrica after acute and chronic exposure

to cypermethrin. Waykar and Lomte (2004) reported decreased in

ascorbic acid contents in different soft tissues of freshwater bivalve after

exposure to carbaryl. They suggested that decline in ascorbic acid might

be due to the impairment in its synthesis due to pesticide stress and

possible utilization of ascorbic acid to overcome the stressful condition.

Gulbhile (2006) reported a decrease in the ascorbic acid content after

acute exposure to mercuric chloride and sodium arsenate in freshwater

bivalve, Lamellidens corrianus. Nawale (2008) reported a decrease in

ascorbic acid content in freshwater bivalve, Lamellidens corrianus after

chronic exposure to lead nitrate and sodium arsenate.

As for as present work is concerned, decrease in ascorbic acid

content in different tissues of Parreysia cylindrica might be due to its

involvement in detoxification and repairing of injuries in tissues which

occurred due to pesticide stress.

In the present study it was observed that the ascorbic acid contents

were more in combined exposure to pesticides with 50mg/l of L-ascorbic

acid as compared to those exposed to only pesticides. This study indicates

that the use of L- ascorbic acid protect the tissues from oxidative damage

caused by pesticides. Several other workers studied the protective role of

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ascorbic acid against pesticide induced depletion in ascorbic acid

contents. Gulbhile, (2006) reported that the ascorbic acid content was

decreased after acute exposure to mercuric chloride and sodium arsenate,

while less depletion was observed on exposure with caffeine in

freshwater bivalve, Lamellidens corrianus. Mahajan and Zambare (2006)

reported the protection by ascorbic acid against the arsenic induced

alterations in ascorbic acid levels in freshwater bivalve, Lamellidens

marginalis. Mahajan (2007) reported depletion in ascorbic acid content in

various tissues of bivalve Lamellidens marginalis after exposure to heavy

metals. He noted less depletion in bivalves exposed to heavy metals with

ascorbic acid. Nawale (2008) reported that the ascorbic acid content was

decreased after exposure to heavy metals while ascorbic acid showed less

decrease with caffeine and ascorbic acid in freshwater bivalve,

Lamellidens corrianus. Shinde (2008) recorded significant decrease in

ascorbic acid content in various tissues of fish Channa orientalis after

chronic treatment by pesticides and reduction of impact of pesticides

during simultaneous exposure with ascorbic acid. Padmaja and Reddy

(1998) found decrease in ascorbic acid content in heavy metals exposed

Anabas testudinus, probably for detoxification and restoration to normal

level during recovery. During chromium-induced toxicity, ascorbic acid

may maintain osmo-regulation by altering ATPase activity in the gills for

the synthesis of metallothiones (Padmaja and Reddy, 1998).

Changes in the DNA content:

In the present study it was observed that after chronic exposure of

pesticides dicofol and dichlorovos there was significant decrease in the

DNA content in mantle, foot, gill, gonad, digestive glands and whole soft

body of experimental bivalves as compared to those of control bivalves.

Obtained results were presented in the table nos. 3.5.a; 3.5.b and 3.6.a;

3.6.b and figure nos. 3.3.1 to 3.3.12. The result clearly indicates that

161

highest depletion in DNA content was reported in bivalves exposed to

cdicofol as compared to dichlorovos. Results obtained during the present

study are in harmony with the findings of Borane (2006), Gulbhile

(2006), Phirke (2008), Nawale (2008). The depletion of DNA content in

mantle, foot, gills, digestive glands, gonads and whole body of

experimental bivalves was due to pesticide stress.

DNA content, the index of capacity of an organism for protein

synthesis in the different stress conditions was affected by heavy metals

or any toxic metals or pesticides. Patiashvili et al., (1989) reported that

copper ions introduced into asides tumors penetrate the nucleic acid

(DNA) and damage it, causing incardinating of the chromatin structure,

copper associates with DNA at higher copper concentrations.

Pesticides generate oxidative stress that induces numerous lesions

in DNA that leads to deletions, mutations and other lethal genetic effects.

Characterization of this damage to DNA has indicated that both the sugar

and the base moieties are susceptible to oxidation, causing base

degradation, single strand breakage, and cross-linking to protein (Imlay

and Linn, 1986). The principle cause of single strand breaks is oxidation

of the sugar moiety by the hydroxyl radical. Cross-linking of DNA to

protein is another consequence of hydroxyl radical attack on either DNA

or its associated proteins (Oleinick et al., 1986). Treatment with ionising

radiation or other hydroxyl radical generating agents causes covalent

leakages such as thymine-cysteine addicts, between DNA and protein.

When these cross-linkages exist, separation of protein from DNA by

various extraction methods is ineffective. Although DNA-protein cross-

links are about an order of magnitude less abundant than single strand

breaks, they are not as readily repaired, and may be lethal if replication or

transcription precedes repair. Black et al., (1996) observed significant

DNA strand breakage in the foot tissue from Anodonta grandis exposed

162

to toxicants. The pesticides may be carcinogenic because of their ability

to generate reactive oxygen species and other reactive intermediates or

react directly with DNA (Brien et al., 2003).

The decreased levels of DNA and RNA were observed by various

investigators, Asifa Parveen and Vasantha (1986) in Clarius batrachus,

Patil and Lomte (1989) in Mythima seperata, Choudhari et al., (1993) in

Thiara lineata under various different toxic stresses. Rao et al., (1998)

studied the effect of Fluoride toxicity on the nucleic acid contents of

freshwater crab, Barytelphusa cunicularis. They observed that the level

of DNA in muscles and hepatopancreas were found to be elevated

initially and then a gradual decrease was noted in gills, testes and ovaries.

Pawar and Kulkarni (2000) reported the decrease in DNA levels of

Paratelphusa jacquemonti exposed to cythion at different periods. Tiwari

and Singh (2003) reported that DNA level was decreased to 46 % and 30

% of controls after treatment with sublethal doses of methanol extract of

Euphorbia Royleana latex in the liver and muscle tissues Channa

punctatus. Zahran et al., (2005) reported decrease in DNA and RNA

contents in rat after exposure to sub acute dose of organphosphorus

pesticide, Nuvacron. Nwani et al., (2010) demonstrated DNA damage

after treatment with carbosulfan in freshwater fish, Channa punctatus.

Patil (2010) observed decrease in DNA content in different tissues like

foot, mantle, gills, digestive gland and whole body of freshwater bivalve,

Parreysia cylindrica after exposure to thiamethoxam and indoxacarb.

Bhosale et al., (2011) reported that DNA content of gill and gonad of

Corbicula striatella was decreased due to 5- fluorouracil after 15 and 30

days. Profenofos induced DNA damage in freshwater fish, Channa

punctatus was reported by Pandey et al., (2011). Thenmozi et al., (2011)

showed significant decrease in nucleic acid content in the liver, muscle

and gill of freshwater fish, Labeo rohita after treatment of malathion.

163

Andhale and Zambare (2011) observed decrease in DNA content in gills,

foot, digestive gland and whole body of Lamellidens marginalis and

concluded that the toxicant interact with the DNA and disturbs its normal

double helical structure and this disturbed structure is vulnerable to the

attacks of the DNase enzyme.

As compared and supported by above literature, the present

investigation of the chronic exposure of dicofol and dichlorovos to

bivalve P. cylindrica, showed decreased DNA contents as compared with

control bivalves, and those exposed to pesticides with ascorbic acid. The

bivalves showed faster recovery due to ascorbic acid, as compared with

those recovered in natural water.

In combined exposure to dicofol and dichlorovos with 50 mg/l of

ascorbic acid the severity of DNA depletion was much reduced. Fraga et

al., (1991) concluded that vitamin C supplementation may minimize

endogenous oxidative DNA damage, thereby decreasing the risk of

genetic defects, particularly in populations with low vitamin C levels.

Ascorbic acid increases the therapeutic effect of different drugs and

medicines by making them more effective. L-ascorbate possesses

substantial nucleophilic property, attack on cellular DNA by intercepting

reactive agent for ascorbyl anion radical with high extent of unpaired

electron. Delocalization accelerates the scavenging of free radicals. L-

ascorbic acid reduces genotoxic damage caused by CMA (chlormadione

acetate), which also increases the production of reactive oxygen species

by CMA. Therefore it is confirmed that the free radical scavenging

property of L-ascorbate is responsible for reducing genotoxic damage

(Edgar 1974). So, for the detoxification of toxicants from animal body

ascorbic acid can be ideally useful. Vitamin C has been shown to play an

important role in the process of hydroxylation, oxygenation, and

oxidation of corticosteroids (Chaterjee 1967). The role of ascorbic acid in

164

disease and tissue repair is well known (Halver 1972). Surjyo and Anisur

(2004) reported the protective action of L-ascorbic acid against

genotoxicity and cytotoxicity in mice during p-DAB induced

hepatocarcinogenesis. Greco et al., (2005) demonstrated that combined

supplementation of vitamin C and E significantly reduced the percentage

of DNA-fragmented sperm. Sohini and Rana (2007) reported that co-

treatments with ascorbic acid reduced the DNA damage and increased the

amount of total DNA in liver and kidney of rat after arsenic toxicity.

Nawale (2008) studied the protective effect of caffeine and ascorbic acid

on heavy metal induced depletion in DNA content. Preventive effect of

vitamin C on renal DNA damage of mice exposed to arsenic was reported

by Zongyuan et al., (2009).

Changes in the RNA contents: -

In the present study depletion of RNA levels in the mantle, foot,

gills, digestive glands, gonad and whole soft body tissues of the

experimental freshwater bivalve was observed after chronic exposure to

pesticides dicofol and dichlorovos as compared with bivalves maintained

as control. Obtained results were presented in the table nos. 3.7.a; 3.7.b

and 3.8.a; 3.8.b and figures 3.4.1 to 3.4.12. The result clearly indicates

that highest depletion in RNA content was reported in bivalves exposed

to dicofol as compared to dichlorovos. Results obtained during the

present study are in harmony with the findings of Borane (2006),

Gulbhile (2006), Phirke (2008), Nawale (2008). The decrease in RNA on

exposure to pesticides may be due to damage in DNA, poor rate of

synthesis of enzymes necessary for transcription or increased catabolism

of RNA due to their abnormalities on binding to pesticides or abnormal.

The cellular degradation, rapid histolysis and decreased rate of protein

synthesis are the possible reasons.

165

Several reports are available on the reduction in RNA levels on

exposure to different pesticide (Tarig et al., 1977; Nordenskjold et al.,

1979). The decreased amount of RNA levels was observed by Patil and

Lomte (1989) in Mythimna (Pseudoletia) seperata under different toxic

stress. Asifa Parveen and Vasantha (1986) in Clarias batrachus,

Chaudhari et al., (1993) in Thiara lineata and Rao et al., (1998) in B.

cunicularis observed decreased level of RNA on pollutant stress. Pawar

and Kulkarni (2000) reported the decrease in RNA levels of Paratelphusa

jacquemonti exposed to cythion at diffrerent periods. Ester Saball et al.,

(2000) observed the total tissue m-RNA of liver and kidneys of control

and HgCl2 treated rats. Tong Lu et al., (2001) observed that 10% genes,

mostly related to cell cycle regulation, apoptosis, DNA damage response

etc were differentially expressed in the form of RNA and such abnormal

RNA are vulnerable to RNA are attack. These have been referred to as

reliable tools for evaluating the extent of hazard of any chemicals much

before any gross signs become apparent (Johnson and Heijnen, 2001).

Tiwari and Singh (2003) reported that RNA level was decreased to 33%

and 38 % of controls after treatment with sublethal doses of methanol

extract of Euphorbia Royleana latex in the liver and muscle tissues

Channa punctatus. Singh et al., (2010) reported a significant decline in

RNA levels in various tissues of Labeo rohita after cypermethrin

intoxication. Patil (2010) observed decrease in RNA content in different

tissues like foot, mantle, gills, digestive gland and whole body of

freshwater bivalve, Parreysia cylindrica after exposure to thiamethoxam

and indoxacarb. Andhale and Zambare (2011) observed decrease in RNA

content in gills, foot, mantle, digestive gland and whole body of

Lamellidens marginalis and concluded that the decrease in RNA content

was due to damage in DNA, poor rate of synthesis of enzymes necessary

166

for transcription or increased catabolism due to the abnormalities in

binding to the toxicant.

In combined exposure to dicofol and dichlorovos with 50 mg/l of

L-ascorbic acid the severity of RNA depletion was much reduced. This

study indicates that the use of L-ascorbic acid protect the tissues from

oxidative damage caused by pesticide. Gulbhile (2006) showed that the

heavy metal exposure reduces the RNA contents in various tissues of

Lamellidens corrianus and the RNA contents were less decreased in

exposure along with ascorbic acid. Nawale (2008) reported significant

decline in RNA contents after lead and arsenic exposure in various tissues

of Lamellidens corrianus and it was less during exposure with ascorbic

acid.

Recovery studies:

In present study, the bivalves pre-exposed to chronic concentration

to pesticides dicofol and dichlorovos showed fast recovery in biochemical

constituents like protein, ascorbic acid, DNA and RNA level in presence

of 50 mg/l L-ascorbic acid than those allowed curing naturally. The

results recorded in the present study are in harmony with the results of

previous investigators (Bhagyalakshmi et al., 1980; Mukhopadhaya et al.,

1982; Tulasi 1992; Holmberg et al., 1992; Singh and Srivastva’,1995;

Joshi et al., 2002; Maruthanyagam and Sharmila, 2004; Mahajan and

Zambare, 2006; Gulbhile, 2006; Mahajan, 2007; Shinde, 2008; Nawale,

2008; Pardeshi and Gapat, 2012).

Ascorbic acid has promising antioxidant property. L-Ascorbic acid

play curative role against pesticide induced biochemical alteration and

cures structural damages caused by pesticides in the animal body. Many

studies have demonstrated that vitamin C, can readily scavenge ROS,

reactive nitrogen species and prevent oxidative damage to many

important biological macromolecules such as DNA, lipids and proteins

167

(Carr and Frei, 1999; Konopacka, 2004). Hence the preventive effect of

vitamin C on tissue damage induced by pesticides may be associated with

its antioxidation capacity or as free radical scavenger that inhibits lipid

peroxidation (Frei, 1999; Carr and Frei, 2000). In addition to scavenging

of ROS and reactive nitrogen species, ascorbic acid can regenerate other

small molecule antioxidants, such as a-tocopherol, GSH, urate, and β-

carotene, from their respective radical species (Englard and Seifter,

1985). These properties of ascorbic acid make it a suitable antidote for

pollutant toxicity in rodents and possibly in human subjects (Sohini and

Rana, 2007). Blankenship et al., (1997) showed that vitamin C protected

cells from undergoing apoptosis.

At cellular level, ascorbic acid has been reported to mitigate the

deleterious effect of ROS directly by increasing antioxidant enzyme

activities of cells and indirectly by reducing oxidized form of vitamin E

and GSH (Neuzil et al., 1997; Wu et al., 2004). Antioxidant and free

radical scavenger properties of ascorbic acid possibly prevent the effects

of oxidative stress (Carnes et al., 2001). Ascorbic acid protects dox-

induced biochemical changes in the cardiac tissue of rats either by

restoring endogenous antioxidant activity or as antioxidant or both

(Viswanatha Swamy et al., 2011).

Many studies were carried out to evaluate the potential role of

antioxidant vitamins, such as vitamin C, vitamin E and $-carotene

(Yousef et al., 1999; Salem et al., 2001). Vitamin C (ascorbic acid) is an

essential micronutrient required for normal metabolic functioning of the

body. Many biochemicals, clinical and epidemiologic studies have

indicated that vitamin C may be of benefit in chronic diseases such as

cardiovascular disease, cancer and cataract, probably through antioxidant

mechanisms (Carr and Frei, 1999). Vitamin C is a cofactor for several

enzymes involved in the biosynthesis of collagen, carnitine and

168

neurotransmitters (Burri and Jacob, 1997; Tsao, 1997). In addition,

vitamin C is used as a cofactor for catecholamine biosynthesis, in

particular the conversion of dopamine to norepinephrine catalyzed by

dopamine -monooxygenase (Burri and Jacob, 1997). Vitamin C prevents

free radical damage in the lungs and may even help to protect the central

nervous system from such damage. It acts against the toxic, mutagenic

and carcinogenic effects of environmental pollutants by stimulating liver

detoxifying enzymes (Kronhausen, 1989).

Mahajan and Zambare (2006) showed faster recovery by ascorbic

acid against the arsenic induced alterations in protein, ascorbic acid, DNA

and RNA levels in freshwater bivalve, Lamellidens marginalis. Mahajan

(2007) reported the decrease in collagen, ascorbic acid, protein content of

the various tissues of freshwater bivalve Lamellidens marginalis, on

exposure to heavy metal cadmium and arsenic and lead and fast recovery

of tissue protein, ascorbic acid, DNA and RNA level in presence of

ascorbic acid than those cured naturally during recovery. Nawale (2008)

reported that protein, ascorbic acid, DNA and RNA content was

decreased after exposure to heavy metals, while ascorbic acid showed

faster recovery on exposure with caffeine and ascorbic acid in freshwater

bivalve, Lamellidens corrianus. Shinde (2008) recorded significant

decrease in ascorbic acid content in various tissues of fish Channa

orientalis after chronic treatment by pesticides also showed fast recovery

in presence of ascorbic acid. Gapat (2011) reported L-ascorbic mediated

protection against the pesticide induced biochemical changes in fresh

water bivalve, Lamellidens corrianus.

From the obtained results it may be concluded that the

physiological disturbances arising in animals after exposure to pesticides

exhibits trends towards normalization and this rate of recovery from

pesticide induced damage was faster on exposure to L-ascorbic acid

169

indicating the preventive and curative property of the L-ascorbic acid

against the pesticide induced damage. Thus it is evident that vitamin C

not only confirm protection against pesticide toxicity but can also

perform therapeutic role against pesticide toxicity in mollusc.

170

SUMMARY

The present investigation showed the role of ascorbic acid in dicofol

and dichlorovos induced biochemical alterations in an experiment

model, the freshwater bivalve, Parreysia cylindrica.

The biochemical contents such as protein, ascorbic acid, DNA and

RNA in various tissues like gills, gonad, digestive glands, foot,

mantle and whole soft body tissues of freshwater bivalves, Parreysia

cylindrica were studied after chronic exposures to dicofol and

dichlorovos with and without ascorbic acid and during recovery.

The protein, ascorbic acid, DNA and RNA content in gills, gonad,

digestive glands, foot, mantle and whole soft body tissues were found

to be significantly decreased after chronic treatment of dicofol and

dichlorovos. The depletion was maximum in digestive glands than

other studied tissues. Pesticide stress might have increased the

proteolysis activities in the cells.

The protein, ascorbic acid, DNA and RNA contents were more in

gills, gonad, digestive glands, foot, mantle and whole soft body

tissues of freshwater bivalves, Parreysia cylindrica when exposed to

dicofol and dichlorovos with ascorbic acid as compared to those

exposed to only pesticides.

After 21 days exposure to pesticides, the bivalves showed fast

recovery of tissue biochemical contents in presence of 50mg/l of L-

ascorbic acid than those allowed to cure naturally.

The results indicate the detoxifying effect of ascorbic acid on

pesticide induced alterations.

171

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