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INTRODUCTION

Since introduction of the synthetic insecticides in the 1940’s there

has been rapid increase in the use of chemicals of high biological activity

for pest control. Pesticides have brought tremendous benefits to mankind

by increasing food production and controlling the vectors of man and

animal diseases. At the same time use of these pesticides has posed

potential health hazards to the life of aquatic animals. Pesticides are

major cause of concern for aquatic environment because of their toxicity,

persistency and tendency to accumulate in the organisms (Olea and

Fernandez, 2007; Joseph and Raj, 2010). The toxic effect of pesticide to

several aquatic organisms even in minute concentrations is recorded

(Charjan et al., 2008). Many pesticides are known inducers of oxidative

stress by directly producing reactive oxygen species (ROS) and impede

the natural antioxidants or oxygen free radical scavenging enzyme system

(Geter et al., 2008; El-Gendy et al., 2010). The ROS have a strong

reactivity and can potentially interact with all other cellular components

(lipids, proteins, DNA) and disable them (Manduzio et al., 2005;

Gwozdzinski et al., 2010). All aerobic organisms have a network of

antioxidants and enzymes which limit the effects of ROS to prevent

oxidative damage. The production and the destruction of the radicals of

oxygen coexist in a weak balance. If this balance is broken in favour of

the ROS, an oxidative stress is generated. Xenobiotics could influence

this balance by catalysing production of ROS. ROS create new radical

species thus, causing oxidations in chain. Such an effect may be at

cellular or molecular level but ultimately it would lead to physiological,

pathological and biochemical disorders that may prove fatal to the

organism (Jain and Kulshrstha, 2000).

Severe alterations in the tissue biochemistry of bivalves caused by

pesticide exposure were studied by different workers (Waykar and

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Lomte, 2001, 2004; Kulkarni et al., 2005; Nagpure and Zambare, 2005;

Mohanty et al., 2005; Satyaparameshwar et al., 2006; Pawar et al., 2009

Nandurkar and Zambare, 2011).

Organisms have developed many defense mechanisms to protect

themselves from injuries by ROS. The small molecule antioxidants, such

as vitamin E (α-tocopherol) and vitamin C (ascorbic acid) are able to

interact with oxidizing radicals directly (Agarwal, 2003), whereas vitamin

C scavenges aqueous-phase ROS by very rapid electron transfer and thus

inhibits lipid peroxidation (Halliwell et al., 1987; Halliwell and

Gutteridge, 1999), as well as reduces the oxidized tocopheroxyl radicals.

Therefore, vitamin C and vitamin E function together to protect

membrane lipids from damage (Frei, 1991). Kannan and Flora (2004)

also reported that co-administration of vitamin E or vitamin C may be

useful in the restoration of altered biochemical variables. Ascorbic acid

serves important function in tissue synthesis and growth processes. It also

indirectly protects the fat-soluble vitamins A, D and E, also some of the B

vitamins like riboflavin, folic acid, thiamine, and pantothenic acid from

oxidants. Ascorbic acid also prevents free radical induced DNA damages

(Dawson et al., 1990). However, still there is limited study on

effectiveness of ascorbic acid against pesticides toxicity. We have

proposed to use the ascorbic acid; commonly called vitamin “C” to study

its efficacy to protect the body from harmful effects of pesticides.

The interaction between pollutants and organisms can be

understood properly if various biochemical changes occurring within the

body of an organism are known (Geetha and Govindan, 1992).

Qualitative and quantitative study of changes in major biochemical

components of organisms such as proteins, ascorbic acid, DNA and RNA

and vital enzymes is useful to know different toxicants and defensive

mechanism of the body against toxic effects of pesticides. These

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biochemical components are indices of pollution as they determine

nutritional status, health and vigor of an organism. Investigation

regarding the physiological and biochemical changes in non target

aquatic species such as molluscs and its subsequent recovery by

supplementation of ascorbic acid is insufficient.

The present study investigates the propensity of carbosulfan and

profenofos induced variation in protein, ascorbic acid, DNA and RNA

level and its possible attenuation by vitamin C in a convenient model, the

fresh water bivalve, Lamellidens marginalis after chronic exposure. 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. gills, gonads, digestive glands, mantle, foot and whole body

mass are used to determine the changes in protein, ascorbic acid, DNA

and RNA content of their tissues on exposure of profenofos and

carbosulfan and its subsequent recovery in presence of ascorbic acid.

Proteins -

Proteins are essential constituents of protoplasm. Protein acts as

growth material for organism. Proteins are the known biological

compounds which regulate and integrate several physiological and

metabolic processes in the body through hormones, enzymes and

nucleoproteins. As a constituent of cell membrane, proteins regulate the

process of interaction between intra and extra cellular media. The protein

plays a major role in the synthesis of microsomal detoxifying enzymes

and helps to detoxify the toxicants when entering into the animals

(Wilkinson, 1976). Protein play important role in metabolism of

organisms. Herper et al., (1978) reported that, the proteins are among the

most abundant biological macromolecules and are extremely versatile in

their function and interaction during metabolism in protein, amino acids,

enzymes and co-enzymes. Proteins play an important role in energy

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production. Normally, tissue proteins in aquatic animals under toxic

stress are known to play a role in the activation of compensatory

mechanism (Wigglesworth, 1972 and Downer, 1982). Protein metabolism

is considered the most sensitive physiological responding to

environmental stress.

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. Any undesirable change in the

environment affects the protein level by changing the physiology of

organism. Reports are available regarding the toxic effects of pesticides

on protein content of some aquatic animals. Kabeer et al., (1971) studied

Malathion induced alterations in total protein content of the pelecypod,

Lamellidens marginalis. The biochemical variations in protein content of

Pila globosa after exposing to pesticide were studied by Ramanna Rao

and Ramamurthi (1978). Sivaprasad et al., (1981) evaluated the impact of

methyl parathion on protein content in tissues of Pila globosa. Lomte and

Alam (1982) studied the biochemical composition of Bellamya

(Viviparous) bengalensis after treatment with the pesticides. Mane and

Muley (1989) reported alteration in protein content in Lamellidens

marginalis on exposure to endoslfan. Jadhav (1993) observed impact of

pesticides on some physiological aspects of freshwater bivalve, Corbicula

striatella and reported decrease in protein content. Mahajan (2005)

studied the biochemical changes induced by heavy metals, lead, mercury

and arsenic in the protein content on the gastropod, Bellamya

(Viviparous) bengalensis. Gupta and Bhide (2001, 2004) reported gradual

decline in a number of protein fractions in Lymnaea stagnalis when

exposed to nuvan. Tripathi and Singh (2002; 2003) reported that

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organophosphorus and carbamate pesticides caused disruptive effects on

carbohydrate and protein metabolism of the fresh water snail, Lymnaea

acuminata. Valarmathi and Azariah (2002) studied protein changes in

tissues of crab Sesarma quadratum exposed to Cucl2. Ghanbahadur et al.,

(2005) studied the effect of organophosphate (Nuvan) on protein contents

of gills and liver in, Rasbora daniconius. Keshvan et al., (2005) studied

total protein content in fresh water crab, Barytelphusa guerini on

exposure to Hildan. Jagatheeswari (2005) reported 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.

Suryavanshi et al., (2009) studied changes in the protein content in the

penacid shrimp, Metapneus monoceros after exposure to sublethal does of

organochlorine pesticides. Kamble et al., (2010) studied the biochemical

changes in the protein, glycogen, lactic acid and cholesterol, in the tissues

like gills, hepatopancreas, gonads, muscle, mantle and foot fresh water

bivalve. Parthasarthy and Joseph (2011) studied on the changes in the

levels of protein metabolism in cyclothrin induced heaptotoxicity in fresh

water Tilapia (Oreochromis massambicus).

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 -

L-ascorbic acid (C6H8O6) is one of the important water soluble

vitamins and essential for collagen, carnitine and neurotransmitters

biosynthesis. It is synthesized in the body of most of the animals

endogenously. Man, primates and guinea pigs cannot synthesize this

vitamin. It has much significance in the body of animals. Many health

benefits have been attributed to ascorbic acid such as antioxidant,

antiatherogenic, anti-carcinogenic, immunomodulator and prevents cold

etc (Naidu, 2003). Antioxidants from our diet appear to be of great

importance in controlling damage by free radicals. Each nutrient is

unique in terms of its structure and antioxidant function. Vitamin C is an

important dietary antioxidant; it significantly decreases the adverse

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effects of reactive species such as reactive oxygen and nitrogen species

that can cause oxidative damage to macromolecules such as lipids, DNA

and proteins which are implicated in chronic diseases (Halliwell and

Gutteridge, 1999).

Ascorbic acid has a few therapeutic characters. It predominantly

works as a radical chain terminator. One ascorbate molecule reacts with a

peroxyl radical to yield a hydroperoxide and ascorbyl radical,

subsequently the ascorbyl radical can react with another peroxyradical

and produce the oxidized ascorbic acid i.e., dihydroscorbic acid (Combs

and Gray, 1998). Thus one molecule of ascorbate can trap two molecules

of peroxyl radicals. Another hypothesis suggests that ascorbic acid may

be involved in the regeneration or restoration of antioxidant properties of

a-tocopherol. The tocopherol is converted to a-tocopherol quinone

(Chow, 1985).

Ascorbic acid takes part in the synthesis of collagen and bone

formation and in wound healing (Gould, 1963). Chinoy and

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

steriodogenesis in molluscs. Halver (1972) states that the ascorbic acid

plays a major role in tissue synthesis and growth processes and obviously

mediates rapid tissue repair in trauma or diseased condition. Ascorbic

acid is necessary in the formation and maintenance of collagen, the basis

of connective tissue, which is formed in skin, ligaments, cartilage,

vertebral discs, joint linings, capillary walls, bones and teeth. It acts as

radio protective agent in several tissues including reproductive organs by

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

an important role in the process of hydroxylation, oxygenation and

oxidation of corticosteroids (Chatterjee, 1967). It takes part in synthesis

of collagen and maturation of red blood corpuscles (Talwar, 1980).

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Many pollutants are known to enhance the formation of reactive

oxygen species (ROS) (Gomez-Mendikute, Cajarville, 2003). ROS create

new radical species thus causing oxidations in chain. All the

biomolecules of the cell (proteins, lipids, nucleic acids, polysaccharides)

are potential substrates of ROS. Both antioxidant enzymes, such as

superoxide dismutase, catalase and glutathione peroxidase and low

molecular weight antioxidants, such as glutathione, ascorbate and α-

tocopherol, belong to the cellular antioxidant system that counteracts the

toxicity of reactive oxygen species (Santovito et al., 2005).The toxicity of

ROS can be mitigated by free radical scavengers, such as glutathione,

ascorbic acid, α-tocopherol and by antioxidant enzymes (Gwozdzinski et

al., 2010). These antioxidant systems make it possible to stop the chain

reactions and destroy the free radicals.

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. Bhusari (1987) reported

alteration in the ascorbic acid content in the tissues of fresh water fish,

Barbus ticto on exposure of endosulfan and ekalux. Kulkarni et al.,

(1988) estimated the effect of temperature and pH on ascorbic acid

content of Indonaia caeruleus. 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

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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 profenofos and carbosulfan toxicity in fresh water

bivalve, Lamellidens marginalis. 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 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 –

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.

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.

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Nucleic acid contents are considered as an index of capacity of an

organism for protein synthesis. Different hormones and stress conditions

may exert control over synthesis, activity and break down of nucleic

acids. The nucleic acid contents can cause alterations in genetic

information and genome functioning so it is important to investigate the

levels of DNA and RNA periodically in different tissues of the organisms

undergoing stress conditions (Khanuja, 1981). Structural changes in the

DNA can be monitored using biochemical methods and usually low

quantitative changes are observed on pesticide 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 (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).

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

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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 others. Fraga et al., (1991) studied the protective action of ascorbic

acid against endogenous oxidative damage in human sperm. Suriyo and

Anisur (2004) suited 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

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

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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

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.

The role of RNA is to help protein synthesis in the cytoplasm

hence depletion of RNA level also resulted decreased rate of protein

synthesis (Rao et al., 1990). Similar decreased amount of RNA levels

was observed by Asfia and Vasantha (1986) in Clarius batracus, by Patil

and Lomte (1989) in Pseudoletia seperata and by Choudhari et al.,

(1993) in Thiara lineata under different toxic stress. The cellular

degradation, rapid histolysis and decreased rate of protein synthesis are

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the possible reasons. 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.

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 and arsenic induced

DNA 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 DNA damage of mice

exposed to arsenic

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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 is made to

investigate the effect of chronic treatment of pesticides profenofos and

carbosulfan on biochemical contents of different tissues and its

subsequent recovery by exogenous administration of L-ascorbic acid in

fresh water bivalve, Lamellidens marginalis.

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, Lamellidens marginalis is used as test

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

profenofos and carbosulfan. The biochemical contents such as protein,

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

tissues. Reduction of toxicant reduces the stress and hence reduces level

of stress effect. Protective and curative role of ascorbic acid was observed

after pesticide treatment and during recovery in experimental model

Lamellidens marginalis.

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MATERIALS AND METHODS

Medium sized, healthy, fresh water bivalve, Lamellidens

marginalis were collected from Girna dam, 48 km away from Chalisgaon

Dist. Jalgaon (M.S.). Animals were brought in laboratory and were

acclimatized for a week to dechlorinated tap water. The medium sized

animals were selected for experiment.

Experimental design:

Set – I

For experimental studies the animals were divided into three

groups–

a) Group ‘A’ was maintained as control.

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

(0.6191ppm, LC50/10 of 96 hours) and carbosulfan (0.5564 ppm,

LC50/10 of 96 hours) upto 21 days.

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

(0.6191 ppm, LC50/10 of 96 hours) and carbosulfan (0.5564 ppm,

LC50/10 of 96 hours) along with 50 mg/l of L-ascorbic acid.

Experimental design for recovery studies:

Set – II

Group ‘B’ animals from set-I after 21 days exposure to profenofos

were divided into two groups for recovery studies.

i) Animals pre-exposed to chronic dose of profenofos (0.6191 ppm) and

carbosulfan (0.5564 ppm) were allowed to self cure in normal fresh

water upto 21 days.

ii) Animals pre-exposed to chronic dose of profenofos (0.6191 ppm) and

carbosulfan (0.5564 ppm) were allowed to cure in 50 mg/l of L-

ascorbic acid added fresh water upto 21 days.

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During experimentation animals were fed on fresh water algae.

After every 7th

, 14th and 21

st days interval, animals from set-I and set-II

were, dissected and tissues such as digestive glands, gills, gonads, foot,

mantle were separated and whole body mass was dried at 800

C in an

oven till constant weights were obtained and blended into dry powder.

These powders were used for the estimation of various biochemical

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

The methods of estimation were 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 in 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

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 and

0.9 ml distilled water were 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

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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.

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).

10 mg of dry tissue powder was homogenized by adding 10 ml

distilled water. Then it was centrifuged at 3000 rpm for 10 minutes. The

supernatant containing DNA was removed. After that 1ml supernatant

was taken and 3ml diphenylamine reagent was added. Then the solution

in the test tube was placed in boiling 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 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 containing RNA was removed.

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

Then the solution in the test tube was placed 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 nm filter.

Each observation was confirmed by taking at least three replicates.

The differences in control and experimental animal group was tested for

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significance by using student ‘t’ test (Bailey, 1965) and the percentage of

decrease or increase over control was calculated for each value.

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OBSERVATIONS AND RESULTS

Biochemical estimation of protein, ascorbic acid, DNA and RNA

contents were determined from the different tissues i.e. mantle, foot, gills,

digestive glands, gonads and whole soft body tissues of experimental

model, the freshwater bivalve Lamellidens marginalis from control and

experimental groups and obtained results are presented in table nos. 3.1

to 3.8 and figure nos. 3.1.1 to 3.4.12.

Protein profile:

Table No. 3.1.a,b and 3.2.a,b indicates changes in protein levels of

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

of Lamellidens marginalis on chronic exposure of profenofos (0.6191

ppm) and carbosulfan (0.5564 ppm) without and with ascorbic acid and

during recovery. It was noticed that protein contents were significantly

reduced after profenofos and carbosulfan exposure in all tissues of the

bivalves as compared to control. Bivalves exposed to profenofos or

carbosulfan with ascorbic acid showed fewer alterations in the protein

contents showing the protective role of the ascorbic acid.

The results clearly indicate that carbosulfan causes more protein

depletion in all soft body tissues of the experimental bivalve as compared

to profenofos. The results also demonstrate that there was progressive

decrease in the protein contents as exposure period was increased.

Compared to other tissues the higher depletion in protein contents was

observed in digestive glands.

When the bivalves exposed for 21 days to profenofos or

carbosulfan was allowed to recover, protein recovery was at a very slow

rate in naturally curing bivalves. Protein contents recovered faster during

21 days in all tissues in ascorbic acid. Rate of recovery was better in

ascorbic acid than in normal water recovery.

149

Ascorbic acid profile:

Table No. 3.3.a,b and 3.4.a,b indicates changes in ascorbic acid

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

of Lamellidens marginalis on chronic exposure of profenofos (0.6191

ppm) and carbosulfan (0.5564 ppm) without and with ascorbic acid and

during recovery. It is noticed that ascorbic acid contents were

significantly reduced after profenofos and carbosulfan exposure in all

tissues of the bivalves as compared to control. Bivalves exposed to

profenofos or carbosulfan with ascorbic acid showed fewer alterations in

the ascorbic acid contents showing the protective role of the ascorbic

acid.

The results clearly indicate that carbosulfan causes more depletion

in ascorbic acid contents in all soft body tissues of the experimental

bivalve as compared to profenofos. The results also demonstrate that

there was progressive decrease in the ascorbic acid contents as exposure

period was increased.

When the bivalves exposed for 21 days to profenofos or

carbosulfan were allowed to recover, ascorbic acid recovery was at a very

slow rate in naturally curing bivalves. Ascorbic acid contents recovered

faster during 21 days in all tissues in ascorbic acid and the comparative

rate of recovery was better in ascorbic acid.

DNA Profile:

Table No. 3.5.a,b and 3.6.a,b indicates changes in DNA levels of

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

Lamellidens marginalis on chronic exposure of profenofos (0.6191 ppm)

and carbosulfan (0.5564 ppm) without and with ascorbic acid and during

recovery. It is noticed that DNA contents were significantly reduced after

profenofos and carbosulfan exposure in all tissues of the bivalves as

compared to control. Bivalves exposed to profenofos or carbosulfan with

150

ascorbic acid showed fewer alterations in the DNA contents showing the

protective role of the ascorbic acid.

The results clearly indicate that carbosulfan causes more depletion

in DNA contents in all soft body tissues of the experimental bivalve as

compared to profenofos. The results also demonstrate that there was

progressive decrease in the DNA contents as exposure period was

increased.

When the bivalves exposed for 21 days to profenofos or

carbosulfan were allowed to recover, DNA recovery was at a very slow

rate in naturally curing bivalves. DNA contents recovered faster during

21 days in all tissues in ascorbic acid and the comparative rate of

recovery was better in ascorbic acid.

RNA Profile:

Table No. 3.7.a,b and 3.8.a,b indicates changes in RNA levels of

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

Lamellidens marginalis on chronic exposure of profenofos (0.6191 ppm)

and carbosulfan (0.5564 ppm) without and with ascorbic acid and during

recovery. It is noticed that RNA contents was significantly reduced after

profenofos and carbosulfan exposure in all tissues of the bivalves as

compared to control. Bivalves exposed to profenofos or carbosulfan with

ascorbic acid showed fewer alterations in the RNA contents showing the

protective role of the ascorbic acid.

The results clearly indicate that carbosulfan causes more depletion

in RNA contents in all soft body tissues of the experimental bivalve as

compared to profenofos. The results also demonstrate that there was

progressive decrease in the RNA contents as exposure period was

increased.

When the bivalves exposed for 21 days to profenofos or

carbosulfan were allowed to recover, RNA recovery was at a very slow

151

rate in naturally curing bivalves. RNA contents recovered faster during

21 days in all tissues in ascorbic acid and the comparative rate of

recovery was better in ascorbic acid.

The comparative tabulated results represent increased or decreased

levels of different biochemicals in respective tissues during treatment and

recovery period. Results of “t” test shown are at significant levels, P <

0.001, P < 0.01 or P < 0.05 for chronic exposure of profenofos and

carbosulfan with and without ascorbic acid as well as in combination and

during the recovery. Two “t” tests are applied; one for simultaneous

exposure where variations in the levels of the biochemicals are compared

with respective levels of the control, while second is applied for recovery

where the levels in the variations are compared with the 21 days exposure

values of the respective pesticide.

152

Table No. 3.1.a. Total protein content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos

without and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Profenofos

(B)

Profenofos + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 43.9560

±1.40

42.8559

±1.61

41.7589

±1.50

27.7892*

±1.95

(-36.78)

25.0736*

±1.31

(-41.49)

21.9780*

±2.54

(-47.36)

34.5604**

±1.88

(-21.37)

31.0436*

±1.30

(-27.56)

25.4092*

±2.04

(-39.15)

2 Foot 61.5384

±2.40

61.2280

±1.24

60.4851

±1.30

49.8168**

±2.03

(-19.05)

45.2346**

±1.88

(-26.12)

39.2132**

±1.87

(-35.17)

54.2124**

±1.68

(-11.90)

48.4416**

±2.06

(-20.88)

45.2413**

±1.45

(-25.20)

3 Gills 52.7472

±2.45

51.6240

±1.63

51.5242

±0.62

31.9228**

±1.56

(-39.48)

27.3624*

±1.61

(-47.00)

22.7802**

±2.04

(-55.79)

38.3516*

±2.08

(-27.29)

32.0460*

±1.42

(-37.92)

30.4560*

±1.25

(-40.89)

4 Digestive

glands 48.3516

±1.23

47.5163

±0.65

47.5152

±0.53

26.3736***

±1.03

(-45.46)

18.5824*

±2.03

(-60.89)

16.6812*

±1.03

(-64.89)

43.8560**

±1.24

(-9.30)

38.1635**

±0.89

(-19.68)

26.9344***

±1.52

(-43.31)

5 Gonad 47.8072

±0.85

47.6345

±1.59

47.1895

±0.91

32.0465**

±1.36

(-32.97)

28.8427*

±2.14

(-39.45)

26.8167*

±1.75

(-43.17)

36.2486**

±1.77

(-24.18)

32.3745**

±1.25

(-32.03)

30.5286**

±1.80

(-35.31)

6 Whole soft

body 60.8142

±3.01

58.1452

±1.56

57.1428

±1.88

45.4212***

±2.03

(-25.31)

35.1648***

±1.29

(-39.52)

30.7692**

±2.09

(-46.15)

48.3516**

±0.58

(-20.49)

38.0952*

±2.01

(-34.48)

35.5604**

±1.94

(-37.77)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

153

Table No. 3.1.b. Total protein content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos

and its subsequent recovery.

Sr.

No. Tissue

Profenofos Recovery in normal water

(i)

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

(ii) 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 21.9780

(-47.36)

26.4736**

±0.45

(+20.46)

27.9692*

±0.74

(+27.26)

30.3648*

±0.50

(+38.16)

29.7852**

±1.85

(+35.52)

31.4632*

±2.04

(+43.16)

37.6315**

±1.82

(+71.22)

2 Foot 39.2132

(-35.17)

42.5232***

±0.40

(+8.44)

45.1359***

±0.46

(+15.10)

47.3674**

±0.23

(+20.79)

44.7692**

±1.76

(+14.24)

49.6291**

±1.05

(+26.56)

60.8984**

±2.24

(+55.30)

3 Gills 22.7802

(-55.79)

25.3784**

±0.45

(+11.41)

27.6524*

±0.78

(+21.39)

32.1658*

±0.45

(+41.20)

28.3760**

±2.06

(+24.56)

33.1631*

±0.29

(+45.58)

41.7166**

±0.46

(+83.13)

4 Digestive

glands 16.6812

(-64.89)

19.6812**

±0.46 (+17.78)

21.5872***

±0.79

(+29.41)

24.8692*

±0.24

(+49.09)

23.3382*

±0.94

(+39.91)

25.2328*

±1.02

(+51.26)

31.7263***

±0.79

(+90.19)

5 Gonad 26.8167

(-43.17)

30.6597***

±1.37

(+14.33)

31.4897***

±1.76

(+17.43)

34.3684**

±0.93

(+28.16)

33.0925**

±1.54

(+23.40)

35.2685***

±1.43

(+31.52)

45.1456**

±1.28

(+68.35)

6 Whole soft

body 30.7692

(-46.15)

35.5692**

±1.23

(+15.60)

42.5604*

±1.08

(+38.32)

46.9560*

±2.05

(+52.61)

40.6952***

±1.05

(+32..26)

46.0138**

±1.56

(+49.54)

55.0436*

±0.45

(+78.89)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

154

Table No. 3.2.a. Total protein content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan

without and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Carbosulfan

(B)

Carbosulfan + A.A.

(50 mg/l)

(C)

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

1 Mantle 49.8168

±1.48

48.7195

±1.68

48.1244

±1.37

33.4289**

±1.25

(-32.90)

27.7692***

±1.56

(-43.00)

24.8388**

±1.77

(-48.39)

38.4212*

±1.42

(-22.87)

34.9560***

±1.54

(-28.25)

28.3648**

±1.48

(-41.06)

2 Foot 64.4688

±1.55

64.2589

±1.84

63.8341

±1.72

47.8864*

±1.58

(-25.72)

43.9560**

±1.39

(-31.59)

38.0852**

±1.78

(-40.34)

55.6080***

±1.98

(-13.74)

49.7472**

±1.43

(-22.56)

45.5268**

±1.37

(-28.68)

3 Gills 57.1428

±1.29

56.6335

±1.18

56.2148

±1.63

32.1692***

±0.89

(-43.70)

28.3736*

±1.33

(-49.90)

24.3225***

±2.01

(-56.73)

39.8560**

±1.29

(-30.25)

34.0952***

±1.36

(-39.80)

31.8604*

±1.86

(-43.32)

4 Digestive

glands 52.7472

±1.59

51.9780

±1.81

50.6845

±1.42

27.2344**

±1.59

(-48.37)

18.9675***

±1.49

(-63.51)

17.5824**

±1.35

(-65.31)

44.9158*

±2.12

(-14.85)

39.2908**

±1.88

(-24.41)

27.2648***

±1.62

(-46.21)

5 Gonad 51.9103

±0.98

50.2492

±0.74

50.1165

±1.50

36.1268*

±1.12

(-30.41)

31.5246**

±1.90

(-37.26)

27.4233*

±1.35

(-45.28)

38.8452**

±1.30

(-25.17)

32.6812*

±0.87

(-34.96)

30.6124**

±1.78

(-38.92)

6 Whole soft

body 61.5384

±2.06

61.1644

±1.97

60.2482

±1.78

44.3516**

±1.73

(-27.93)

34.9560*

±2.09

(-42.85)

30.6300***

±1.44

(-49.16)

48.4772***

±1.93

(-21.22)

37.8864*

±1.73

(-38.06)

35.5616**

±1.91

(-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 obsevation

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

5. NS (Not significant)

155

Table No. 3.2.b. Total protein content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan

and its subsequent recovery.

Sr.

No. Tissue

Carbosulfan Recovery in normal water

(i)

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

(ii) 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 24.8388

(-48.39)

28.8647**

±1.45

(+16.21)

30.0952**

±1.65

(+21.16)

32.6212**

±1.21

(+31.33)

31.6645***

±1.50

(+27.48)

33.9612**

±2.04

(+36.73)

41.0656***

±1.30

(+65.33)

2 Foot 38.0852

(-40.34)

41.1863*

±1.80

(+8.14)

42.9812***

±1.44

(+12.86)

46.0948**

±1.90

(+21.03)

43.2256**

±1.88

(+13.50)

47.0237***

±1.24

(+23.47)

57.9384**

±1.49

(+52.13)

3 Gills 24.3225

(-56.73)

26.6288*

±1.17

(+9.48)

29.3289*

±1.40

(+20.58)

33.4604*

±2.53

(+37.57)

30.3504*

±1.37

(+24.78)

33.6513**

±1.73

(+38.35)

43.8472**

±2.13

(+80.27)

4 Digestive

glands 17.5824

(-65.31)

20.2432**

±1.25

(+15.13)

21.9422**

±1.70

(+24.80)

26.1300**

±1.77

(+48.61)

23.7692***

±1.53

(+35.19)

27.1326*

±1.26

(+54.32)

32.1557***

±1.69

(+82..89)

5 Gonad 27.4233

(-45.28)

27.0236**

±0.85

(+13.13)

32.9013*

±1.45

(+19.98)

35.4435**

±1.66

(+29.25)

34.3715*

±0.79

(+25.34)

36.5715***

±1.06

(+33.36)

44.7508**

±0.95

(+63.19)

6 Whole soft

body 30.6300

(-49.16)

34.1952*

±1.35

(+11.64)

41.5660***

±1.83

(+35.70)

44.5164**

±1.65

(+45.34)

39.9560**

±1.36

(+30.45)

44.2864***

±1.95

(+44.59)

53.4080***

±1.87

(+74.37)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

156

Table No. 3.3.a. Total ascorbic acid content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos

without and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Profenofos

(B)

Profenofos + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.9652

±0.28

0.9492

±0.63

0.9451

±0.47

0.7054*

±0.27

(-26.92)

0.6589**

±1.06

(-30.58)

0.5668**

±1.15

(-40.03)

0.7835**±1.

27

(-18.82)

0.7062**

±0.79

(-25.60)

0.6396**

±0.55

(-32.37)

2 Foot 0.4818

±0.03

0.4718

±0.81

0.4723

±0.36

0.3842***

±0.95

(-20.26)

0.3497**

±1.14

(-25.88)

0.3056*

±1.21

(-35.30)

0.4055**

±1.41

(-15.84)

0.3692*

±0.09

(-21.75)

0.3568**

±1.75

(-24.45)

3 Gills 1.2641

±0.52

1.2564

±0.92

1.2494

±0.21

0.9425**

±1.57

(-25.44)

0.7985***

±1.36

(-36.44)

0.6385**

±0.91

(-48.90)

0.9934**

±1.18

(-21.41)

0.9388**

±1.91

(-25.28)

0.8012**

±1.98

(-35.87)

4 Digestive

glands 1.5137

±0.16

1.4328

±0.72

1.4416

±0.09

1.0532*

±0.84

(-30.42)

0.7916*

±1.43

(-44.75)

0.6215**

±1.19

(-56.89)

1.2365**

±1.45

(-18.31)

0.9426**

±1.87

(-34.21)

0.8245***

±2.04

(-42.81)

5 Gonad 1.4623

±0.37

1.4568

±0.59

1.4512

±0.76

1.1626**

±1.33

(-20.50)

1.0722***

±1.65

(-26.40)

0.9012**

±0.87

(-37.90)

1.2343*

±1.17

(-15.59)

1.1373**

±1.35

(-21.93)

1.0085**

±1.39

(-30.51)

6 Whole soft

body 1.0185

±0.41

1.0134

±0.97

1.0086

±0.68

0.7845*

±0.81

(-22.97)

0.6835*

±1.22

(-32.55)

0.5416**

±1.75

(-46.30)

0.8534**

±1.83

(-16.21)

0.7234***

±1.94

(-28.62)

0.6515**

±1.49

(-35.40)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

157

Table No. 3.3.b. Total ascorbic acid content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos

and its subsequent recovery.

Sr.

No. Tissue

Profenofos Recovery in normal water

(i)

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

(ii) 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.5668

(-40.03)

0.6217**

±1.23

(+9.69)

0.6486**

±0.68

(+14.43)

0.7122*

±0.67

(+25.65)

0.6978*

±1.04

(+23.11)

0.8023***

±1.15

(+41.55)

0.9645**

±1.22

(+70.17)

2 Foot 0.3056

(-35.30)

0.3096***

±1.92

(+12.34)

0.3249*

±1.13

(+17.89)

0.3725**

±1.44

(+35.16)

0.3675**

±1.66

(+33.35)

0.4197**

±1.26

(+52.29)

0.4805***

±1.51

(+74.35)

3 Gills 0.6385

(-48.90)

0.7542**

±1.74

(+18.12)

0.7956***

±1.78

(+24.60)

0.8955**

±1.59

(+40.25)

0.8891***

±1.73

(+39.25)

0.9617*

±0.85

(+50.62)

1.2553*

±0.91

(+96.60)

4 Digestive

glands 0.6215

(-56.89)

0.6873*

±1.25

(+10.59)

0.7425***

±1.07

(+19.47)

0.7983**

±0.98

(+28.45)

0.7885*

±1.33

(+26.87)

0.9276**

±1.39

(+49.25)

1.1542**

±1.87

(+85.71)

5 Gonad 0.9012

(-37.90)

0.8429**

±1.51

(+6.47)

1.0098**

±1.39

(+12.05)

1.0884*

±1.41

(+20.77)

1.0774***

±1.55

(+19.55)

1.2774*

±1.49

(+41.74)

1.4878***

±1.78

(+65.09)

6 Whole soft

body 0.5416

(-46.30)

0.6032**

±2.04

(+11.37)

0.6728**

±2.07

(+24.22)

0.7675**

±1.76

(+41.71)

0.7564**

±1.77

(+39.66)

0.8074**

±1.65

(+49.08)

0.9685**

±1.83

(+78.82)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

158

Table No. 3.4.a. Total ascorbic acid content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan without and

with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Carbosulfan

(B)

Carbosulfan + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.9245

±1.84

0.9228

±1.28

0.9180

±1.52

0.6675**

±0.89

(-27.80)

0.6327*

±1.15

(-31.44)

0.5042**

±1.27

(-45.08)

0.7289**

±1.47

(-21.16)

0.6735*

±0.94

(-27.01)

0.5916**

±1.55

(-35.56)

2 Foot 0.4654

±1.39

0.4598

±1.71

0.4474

±1.37

0.3508**

±1.75

(-24.62)

0.3176**

±1.73

(-30.93)

0.2654***

±1.63

(-40.68)

0.3878**

±1.56

(-16.67)

0.3504***

±1.29

(-23.79)

0.3017***

±1.56

(-32.56)

3 Gills 1.1686

±1.65

1.1536

±1.82

1.1477

±1.25

0.8477*

±1.16

(-27.46)

0.7124**

±1.51

(-38.24)

0.5645***

±1.31

(- 50.81)

0.8944***

±1.21

(-23.46)

0.8377*

±1.83

(-27.38)

0.7722**

±1.66

(-37.94)

4 Digestive

glands 1.4817

±1.59

1.4284

±1.93

1.4268

±1.55

0.9841* *

±1.44

(-33.58)

0.7816***

±1.84

(-45.28)

0.5833***

±1.42

(-59.12)

1.0925**

±1.75

(-26.27)

0.9122***

±1.78

(-36.14)

0.7911*

±1.04

(-44.55)

5 Gonad 1.3256

±1.27

1.3245

±1.61

1.3159

±1.76

0.9477*

±1.79

(-28.51)

0.7864**

±1.92

(-40.63)

0.7582**

±1.13

(-42.38)

0.9843***

±1.25

(-25.75)

0.8915**

±1.63

(-32.69)

0.8651***

±1.17

(-34.26)

6 Whole soft

body 0.9688

±1.07

0.9450

±1.18

0.9385

±1.46

0.7324***

±1.08

(-24.40)

0.6125*

±1.77

(-35.18)

0.4865***

±1.31

(-48.16)

0.7763*

±1.98

(-19.87)

0.6458**

±1.74

(-31.66)

0.5766**

±1.65

(-38.56)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

159

Table No. 3.4.b. Total ascorbic acid content in different soft body tissues of Lamellidens marginalis after chronic exposure to Carbosulfan and its

subsequent recovery.

Sr.

No. Tissue

Carbosulfan Recovery in normal water

(i) Recovery in A.A. (50 mg/l) (ii)

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

1 Mantle 0.5042

(-45.08)

0.5407*

±1.73

(+7.24)

0.5579**

±1.48

(+10.65)

0.6145**

±1.61

(+21.88)

0.6078***

±1.13

(+20.55)

0.7195**

±1.51

(+42.70)

0.8512***

±1.55

(+68.82)

2 Foot 0.2654

(-40.68)

0.2896**

±1.86

(+9.12)

0.3010***

±1.36

(+13.41)

0.3545*

±1.64

(+33.57)

0.3495*

±1.76

(+31.69)

0.3987***

±1.76

(+50.23)

0.4601**

±1.47

(+73.36)

3 Gills 0.5645

(-50.81)

0.6495**

±1.35

(+15.06)

0.6854**

±1.87

(+21.42)

0.7659***

±1.91

(+35.68)

0.7579**

±1.53

(+34.26)

0.8419***

±1.81

(+49.14)

1.0945***

±1.41

(+93.89)

4 Digestive

glands 0.5833

(-59.12)

0.6494*

±1.29

(+11.33)

0.6835**

±1.19

(+17.18)

0.7412*

±1.68

(+27.07)

0.7385**

±1.35

(+26.61)

0.8365**

±1.73

(+43.41)

1.0687***

±1.99

(+83.22)

5 Gonad 0.7582

(-42.38)

0.8016**

±1.21

(+5.72)

0.8456**

±1.88

(+11.53)

0.8973***

±1.72

(+18.35)

0.8879***

±1.78

(+17.11)

1.0574***

±1.95

(+39.46)

1.2454*

±0.96

(+64.26)

6 Whole soft

body 0.4865

(-48.16)

0.5274*

±2.01

(+8.41)

0.5913*

±1.12

(+21.54)

0.6773**

±1.43

(+39.22)

0.6671***

±1.88

(+37.12)

0.7125**

±1.59

(+46.45)

0.8526*

±1.17

(+75.25)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

160

Table No. 3.5.a. Total DNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos without

and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Profenofos

(B)

Profenofos + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 1.392

±1.72

1.354

±1.54

1.287

±1.25

1.075**

±1.61

(-22.77)

0.952*

±0.76

(-29.69)

0.705***

±1.21

(-45.22)

1.245***

±0.59

(-10.56)

1.095*

±1.98

(-19.13)

0.866**

±1.72

(-32.71)

2 Foot 2.584

±1.39

2.496

±1.18

2.418

±1.76

2.207***

±1.71

(-14.59)

2.017**

±1.64

(-19.19)

1.464**

±1.82

(-39.45)

2.452**

±1.62

(-5.11)

2.165***

±1.33

(-13.26)

1.794**

±0.78

(-25.81)

3 Gills 1.243

±1.63

1.212

±1.80

1.183

±1.47

0.894*

±0.81

(-28.08)

0.782***

±1.33

(-35.48)

0.610*

±0.96

(-48.44)

1.075***

±1.75

(-13.51)

0.948**

±1.51

(-21.78)

0.762***

±1.45

(-35.59)

4 Digestive

glands 2.232

±1.41

2.219

±1.59

2.197

±1.88

1.451***

±1.22

(-34.99)

1.215***

±1.58

(-45.24)

0.958**

±1.49

(-56.39)

1.815*

±1.12

(-18.68)

1.557***

±1.29

(-29.83)

1.275*

±1.66

(-41.97)

5 Gonad 2.427

±1.67

2.378

±1.18

2.356

±1.92

1.921**

±1.61

(-20.85)

1.728**

±1.65

(-27.33)

1.395*

±1.17

(-41.34)

2.162**

±1.37

(-9.08)

1.952*

±1.31

(-17.91)

1.697***

±0.84

(-28.64)

6 Whole soft

body 2.365

±1.29

2.352

±1.55

2.329

±1.73

1.917*

±1.35

(-18.94)

1.803***

±1.83

(-23.34)

1.336***

±1.53

(-42.64)

2.174***

±0.75

(-8.08)

1.984***

±1.48

(-15.65)

1.590**

±1.93

(-31.73)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

161

Table No. 3.5.b. Total DNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos and

its subsequent recovery.

Sr.

No. Tissue

Profenofos Recovery in normal water

(i)

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

(ii) 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 0.705

(-45.22)

0.754**

±1.77

(+6.95)

0.796***

±1.43

(+12.91)

0.897**

±1.87

(+27.23)

0.886***

±1.08

(+25.67)

1.048*

±1.44

(+48.65)

1.245***

±1.54

(+76.60)

2 Foot 1.464

(-39.45)

1.549***

±1.27

(+5.81)

1.619**

±1.54

(+10.59)

1.782*

±1.71

(+21.72)

1.748*

±1.17

(+19.40)

1.988***

±1.21

(+35.79)

2.423**

±1.09

(+65.51)

3 Gills 0.610

(-48.44)

0.682*

±1.92

(+11.80)

0.752**

±1.68

(+23.28)

0.807***

±0.83

(+32.30)

0.796**

±1.88

(+30.49)

0.993**

±1.89

(+62.79)

1.168***

±0.62

(+91.48)

4 Digestive

glands 0.958

(-56.39)

1.049**

±1.15

(+9.50)

1.087**

±1.23

(+13.47)

1.255***

±1.06

(+31.00)

1.242***

±1.22

(+29.65)

1.378*

±1.05

(+43.84)

1.742***

±1.13

(+81.84)

5 Gonad 1.395

(-41.34)

1.488***

±1.49

(+6.67)

1.557**

±0.94

(+11.61)

1.774**

±1.35

(+27.17)

1.768*

±1.38

(+26.74)

1.980***

±1.03

(+41.94)

2.375**

±1.53

(+70.25)

6 Whole soft

body 1.336

(-42.64)

1.452**

±1.18

(+8.68)

1.528*

±1.75

(+14.37)

1.637**

±1.96

(+22.53)

1.615***

±1.62

(+20.88)

1.943**

±1.82

(+45.43)

2.315***

±1.79

(+73.28)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

162

Table No. 3.6.a. Total DNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan without

and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Carbosulfan

(B)

Carbosulfan + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 1.452

±1.29

1.415

±1.34

1.396

±1.38

1.095**

±1.71

(-24.59)

0.917**

±1.39

(-35.19)

0.732*

±1.86

(-47.56)

1.265***

±1.16

(-12.89)

1.024*

±1.40

(-27.63)

0.925***

±1.48

(-33.74)

2 Foot 2.638

±0.56

2.590

±1.12

2.568

±0.84

2.184*

±0.87

(-17.21)

1.953**

±1.55

(-24.59)

1.494**

±1.17

(-41.82)

2.417**

±1.50

(-8.38)

2.155**

±1.61

(-16.79)

1.776***

±1.59

(-30.84)

3 Gills 1.365

±1.66

1.327

±1.47

1.289

±1.70

0.997**

±1.19

(-26.96)

0.772***

±1.53

(-41.82)

0.634*

±1.26

(-50.81)

1.123*

±0.91

(-17.73)

0.963***

±1.05

(-27.43)

0.745**

±1.20

(-42.20)

4 Digestive

glands 2.396

±1.26

2.375

±1.61

2.321

±1.75

1.457**

±1.60

(-39.19)

1.212*

±1.43

(-48.97)

0.847***

±1.35

(-63.51)

1.862***

±1.77

(-22.29)

1.614**

±1.19

(-32.04)

1.210***

±1.62

(-47.87)

5 Gonad 2.518

±1.17

2.492

±1.68

2.438

±0.92

2.018**

±0.95

(-19.86)

1.785**

±0.57

(-28.37)

1.382*

±1.04

(-43.31)

2.251**

±0.67

(-10.60)

1.983***

±1.14

(-21.25)

1.693**

±1.06

(-32.76)

6 Whole soft

body 2.443

±1.44

2.414

±1.57

2.385

±1.63

1.946*

±1.22

(-20.34)

1.731**

±1.25

(-28.29)

1.314**

±1.07

(-44.91)

2.175*

±1.80

(-10.97)

1.887**

±0.75

(-21.83)

1.461**

±1.13

(-38.74)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

163

Table No. 3.6.b. Total DNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan and its

subsequent recovery.

Sr.

No. Tissue

Carbosulfan Recovery in normal water

(i) Recovery in A.A. (50 mg/l) (ii)

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

1 Mantle 0.732

(-47.56)

0.773**

±1.46

(+5.60)

0.829*

±1.51

(+13.25)

0.917**

±1.49

(+25.27)

0.897**

±1.52

(+22.54)

1.035*

±1.35

(+41.39)

1.274***

±1.41

(+74.04)

2 Foot 1.494

(-41.82)

1.559***

±1.08

(+4.35)

1.668**

±1.13

(+11.65)

1.782*

±1.25

(+19.28)

1.759*

±1.13

(+17.74)

2.064***

±1.06

(+38.15)

2.514*

±1.37

(+68.27)

3 Gills 0.634

(-50.81)

0.693**

±0.94

(+9.31)

0.733***

±1.04

(+15.62)

0.835***

±1.28

(+31.70)

0.823**

±1.32

(+29.81)

1.006*

±0.52

(+58.68)

1.204***

±1.14

(+89.91)

4 Digestive

glands 0.847

(-63.51)

0.920*

±1.31

(+8.62)

0.947*

±1.43

(+11.81)

1.087**

±1.60

(+28.34)

1.079*

±1.84

(+27.39)

1.194**

±1.63

(+40.97)

1.525*

±1.24

(+80.05)

5 Gonad 1.382

(-43.31)

1.455**

±1.19

(+5.28)

1.517***

±1.27

(+9.77)

1.718**

±1.33

(+24.31)

1.664***

±1.78

(+20.41)

1.975***

±1.07

(+42.91)

2.345**

±1.29

(+69.68)

6 Whole soft

body 1.314

(-44.91)

1.417***

±0.85

(+7.84)

1.479*

±1.02

(+12.56)

1.595***

±1.65

(+21.39)

1.567**

±0.46

(+19.25)

1.872**

±1.15

(+42.47)

2.243***

±1.34

(+70.70)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

164

Table No. 3.7.a. Total RNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos without

and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Profenofos

(B)

Profenofos + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 4.743

±1.08

4.687

±1.15

4.615

±0.89

3.804***

±1.47

(-19.80)

3.357*

±1.38

(-28.38)

2.804***

±1.55

(-39.24)

4.312*

±0.53

(-9.09)

3.542***

±1.30

(-24.43)

3.074**

±1.22

(-33.38)

2 Foot 5.562

±1.25

5.472

±1.45

5.426

±1.05

4.637**

±1.40

(-16.63)

4.087***

±0.74

(-25.31)

3.387**

±1.18

(-37.58)

4.981***

±1.77

(-10.44)

4.284**

±1.41

(-21.71)

3.937**

±1.57

(-27.44)

3 Gills 6.125

±1.57

6.118

±1.84

6.084

±1.39

4.892***

±1.63

(-20.13)

4.164**

±1.28

(-31.94)

3.334***

±1.72

(-45.20)

5.421**

±0.71

(-11.49)

4.525*

±1.19

(-26.04)

3.907*

±0.93

(-35.78)

4 Digestive

glands 8.488

±0.63

8.391

±0.92

8.346

±1.18

6.032*

±1.96

(-28.93)

5.025***

±1.07

(-40.11)

3.915*

±1.49

(-53.09)

6.865***

±1.89

(-19.12)

5.793***

±1.36

(-30.96)

4.851**

±1.53

(-41.88)

5 Gonad 4.278

±1.78

4.209

±1.35

4.182

±1.81

3.517**

±1.86

(-17.79)

2.992**

±1.61

(-28.91)

2.582***

±1.52

(-38.26)

3.922*

±1.09

(-8.32)

3.323**

±0.44

(-22.32)

2.867**

±1.85

(-31.44)

6 Whole soft

body 6.764

±1.03

6.728

±0.75

6.683

±0.59

5.713***

±0.48

(-15.54)

5.128*

±1.12

(-23.78)

4.315*

±1.23

(-35.43)

6.012**

±0.86

(-11.12)

5.572*

±0.92

(-17.18)

4.918***

±1.35

(-26.41)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

165

Table No. 3.7.b. Total RNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to profenofos and its

subsequent recovery.

Sr.

No. Tissue

Profenofos Recovery in normal water

(i)

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

(ii) 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 2.804

(-39.24)

3.049**

±1.76

(+8.74)

3.248***

±1.88

(+15.83)

3.546**

±2.07

(+26.46)

3.465*

±1.77

(+23.57)

4.023***

±0.98

(+43.47)

4.792**

±1.06

(+70.90)

2 Foot 3.387

(-37.58)

3.612***

±0.56

(+6.64)

3.772*

±1.38

(+11.37)

4.084*

±0.84

(+20.58)

3.984**

±1.35

(+17.63)

4.538*

±1.69

(+33.98)

5.423***

±1.77

(+60.11)

3 Gills 3.334

(-45.20)

3.647*

±1.43

(+9.39)

3.824***

±1.54

(+14.70)

4.206**

±1.22

(+26.15)

4.156***

±0.49

(+24.66)

4.982**

±1.23

(+49.43)

6.144***

±0.95

(+84.28)

4 Digestive

glands 3.915

(-53.09)

4.218**

±1.67

(+7.74)

4.538**

±1.90

(+15.91)

4.807***

±1.29

(+23.30)

4.762**

±1.30

(+21.63)

5.464*

±1.65

(+39.57)

7.035*

±1.83

(+79.69)

5 Gonad 2.582

(-38.26)

2.754***

±2.12

(+6.66)

2.927*

±1.94

(+13.36)

3.057**

±1.65

(+18.40)

3.024***

±0.88

(+17.12)

3.472**

±1.27

(+34.47)

4.167***

±1.42

(+61.39)

6 Whole soft

body 4.315

(-35.43)

4.102**

±1.33

(+4.94)

4.814**

±1.25

(+11.56)

5.113*

±1.71

(+18.49)

5.024*

±1.64

(+16.43)

5.795***

±1.85

(+34.30)

6.737**

±1.90

(+56.13)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

166

Table No. 3.8.a. Total RNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan without

and with ascorbic acid.

Sr.

No. Tissue

Control

(A)

Carbosulfan

(B)

Carbosulfan + A.A.

(50 mg/l)

(C) 7 days 14 days 21 days 7 days 14 days 21 days 7 days 14 days 21 days

1 Mantle 4.924

±1.34

4.893

±1.08

4.865

±1.21

3.872**

±1.36

(-23.19)

3.317***

±1.51

(-32.21)

2.736**

±1.72

(-43.76)

4.316**

±1.13

(-12.35)

3.564**

±1.53

(-27.16)

3.341***

±1.33

(-31.32)

2 Foot 5.737

±1.59

5.703

±1.77

5.681

±1.48

4.645*

±1.24

(-19.03)

4.102**

±0.75

(-28.07)

3.373*

±1.05

(-40.63)

5.197***

±0.63

(-9.41)

4.379*

±0.95

(-23.21)

4.015**

±1.27

(-29.32)

3 Gills 6.297

±0.76

6.284

±0.67

6.228

±1.17

4.586***

±1.18

(-27.17)

4.136***

±1.35

(-34.18)

3.075**

±1.44

(-50.62)

5.342**

±1.85

(-15.16)

4.408**

±1.40

(-29.85)

3.847***

±1.52

(-38.23)

4 Digestive

glands 8.612

±1.83

8.573

±1.42

8.542

±1.67

5.768**

±1.67

(-33.02)

4.873*

±0.92

(-43.16)

3.735***

±1.25

(-56.27)

6.437*

±1.79

(-25.25)

5.749***

±1.46

(-32.94)

4.684*

±1.17

(-45.16)

5 Gonad 4.543

±0.87

4.516

±1.19

4.495

±0.55

3.597***

±0.79

(-20.82)

3.204**

±1.04

(-29.05)

2.644**

±1.49

(-41.18)

4.084***

±0.93

(-10.10)

3.424**

±1.60

(-24.63)

3.144***

±0.81

(-30.79)

6 Whole soft

body 6.906

±1.98

6.887

±1.50

6.854

±1.81

5.719*

±1.57

(-17.19)

5.074***

±1.43

(-26.32)

4.216*

±1.88

(-38.49)

6.343**

±1.76

(-8.15)

5.485***

±1.98

(-20.36)

4.893**

±1.90

(-28.61)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

167

Table No. 3.8.b. Total RNA content in different soft body tissues of Lamellidens marginalis after chronic exposure to carbosulfan and its

subsequent recovery.

Sr.

No. Tissue

Carbosulfan Recovery in normal water

(i) Recovery in A.A. (50 mg/l) (ii)

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

1 Mantle 2.736

(-43.76)

2.916*

±1.28

(+6.58)

3.106**

±1.46

(+13.52)

3.374**

±1.68

(+23.32)

3.327**

±0.68

(+21.60)

3.835**

±1.32

(+40.17)

4.614**

±0.59

(+68.64)

2 Foot 3.373

(-40.63)

3.535*

±1.54

(+4.80)

3.775*

±1.18

(+11.92)

4.024**

±1.73

(+19.30)

3.894**

±1.94

(+15.45)

4.436**

±1.70

(+31.51)

5.312**

±1.55

(+57.49)

3 Gills 3.075

(-50.62)

3.294*

±0.87

(+7.12)

3.476***

±1.53

(+13.04)

3.863*

±1.75

(+25.63)

3.788*

±1.49

(+23.19)

4.473**

±1.06

(+45.46)

5.546***

±0.86

(+80.36)

4 Digestive

glands 3.735

(-56.27)

3.977*

±1.98

(+6.48)

4.218**

±1.22

(+12.93)

4.507**

±1.25

(+20.67)

4.378**

±0.94

(+17.22)

5.063*

±1.14

(+35.56)

6.615***

±1.51

(+77.11)

5 Gonad 2.644

(-41.18)

2.784*

±1.38

(+5.30)

2.955**

±0.89

(+11.76)

3.157**

±0.48

(+19.40)

3.065*

±1.10

(+15.92)

3.495**

±1.65

(+32.19)

4.253***

±0.88

(+60.85)

6 Whole soft

body 4.216

(-38.49)

4.381*

±1.05

(+3.91)

4.632*

±1.16

(+9.87)

4.926*

±1.43

(+16.84)

4.837*

±1.28

(+14.73)

5.487**

±0.68

(+30.15)

6.817***

±1.84

(+61.69)

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

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

3. ± indicate S.D. of three obsevation

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

5. NS (Not significant)

168

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

bivalve, L. marginalis after chronic exposure to profenofos 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,

L. marginalis after chronic exposure to profenofos 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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

169

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

L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos 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

Pro

tein

co

nte

nt

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

170

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

bivalve, L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos 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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

171

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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,

L. marginalis after chronic exposure to carbosulfan 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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

172

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

L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan 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

in m

g/1

00

mg

off

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

173

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

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

Pro

tein

co

nte

nt

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

174

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

bivalve, L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos 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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

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

. c

on

ten

t in

mg

/10

0m

g

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

175

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

bivalve, L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos

without and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. c

on

ten

t in

mg

/10

0m

g

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

176

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

bivalve, L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

177

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan 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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

178

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

179

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

without and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

07 days

14 days

21 days

07 days

14 days

21 days

A.A

. co

nte

nt

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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

in m

g/1

00

mg

of

dry

tis

sue

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

180

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

L. marginalis after chronic exposure to profenofos 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, L.

marginalis after chronic exposure to profenofos without and with ascorbic

acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

181

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

marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos without

and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

182

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

L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos 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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

183

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

L. marginalis after chronic exposure to carbosulfan 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, L.

marginalis after chronic exposure to carbosulfan 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

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

0.5

1

1.5

2

2.5

3

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

184

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

marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

without and with ascorbic acid and during recovery.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

185

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

bivalve, L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

without and with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

3

07 days

14 days

21 days

07 days

14 days

21 days

DN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

186

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

L. marginalis after chronic exposure to profenofos 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, L.

marginalis after chronic exposure to profenofos without and with ascorbic

acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

187

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

marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos without

and with ascorbic acid and during 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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

9

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

188

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

L. marginalis after chronic exposure to profenofos 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, L. marginalis after chronic exposure to profenofos without

and with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Profenofos

Profenofos +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

189

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

L. marginalis after chronic exposure to carbosulfan 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, L.

marginalis after chronic exposure to carbosulfan without and with ascorbic

acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

190

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

marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

without and with ascorbic acid and during 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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

0

1

2

3

4

5

6

7

8

9

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

191

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

L. marginalis after chronic exposure to carbosulfan 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, L. marginalis after chronic exposure to carbosulfan

without and with ascorbic acid and during recovery.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

07 days

14 days

21 days

07 days

14 days

21 days

RN

A c

on

ten

t in

mg/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

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/

10

0m

go

f d

ry t

issu

e

Time of exposure

control

Carbosulfan

Carbosulfan +

Ascorbic acid

Normal water

(Recovery)

Ascorbic acid

(Recovery)

192

DISCUSSION

The degradation of aquatic system is a worldwide phenomenon

originated from the intense population and from the corresponding

increase in agriculture practices as well as industrial and domestic

activities. Pesticides are major cause of concern for degradation of

aquatic environment because of their toxicity, persistency and tendency to

concentrate in organisms as they move up the food chain, increase their

toxicity to fish, birds and other wildlife and, in turn to man (Rand, 1995).

While the pesticides are instrumental in achieving significant increase in

crop productivity, it has to be considered that they cause serious

ecological hazards to the non-target animals especially the bivalves,

fishes which form an important part of food chain for various animals

including human beings. The exposure of aquatic organisms to even very

low levels of pesticides causing alterations in the nutritional value of

aquatic animals as well as their biochemical constituents, physiological

and histological functions has been widely documented (Reddy et al.,

1991; Bhavan and Geraldine, 1997; 2001; 2002). Most of the pesticides

act as metabolic depressors and generally affect the activity of

biologically active molecules such as proteins, carbohydrates and lipids

(Agrahari and Gopal, 2009). For this reason, there is an increasing need

to develop the methods for identification, estimation, comparative

assessment and management of risk posed by chemical pollutants

discharge into the aquatic environment. Therefore, the measuring

biological effects of pollutants in relation to body organic constituents are

essential for assessing the quality of the freshwater environment (Kharat

et al., 2009). In order to investigate the physiological changes after

pesticide treatment, the study of changes in the biochemical constituents

is the most fundamental tool. Biochemical studies are good parameters

193

which help to see the effect of pesticide on biochemical composition of

vital tissue of bivalve.

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

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.

Changes in protein contents:

Protein is one of the important biochemical components and plays

an important role in metabolic pathways and biochemical reactions.

Under extreme stress conditions, protein supply energy in metabolic

pathways and biochemical reactions. The proteins are indeed of primary

and paramount importance in living world not only because of their

peculiar chemicals and physicochemical properties but also because of

fact that they appear to confer their biological specificity among various

type of cells (Bhushan et al., 2002). Therefore, an assessment of the total

protein content in different tissues could be used as a diagnostic tool for

determining the physiological status of an organism (Prasath and Arivoli,

2008).

Present investigation clearly showed that, after chronic exposure to

pesticides profenofos and carbosulfan a marked depletion in the protein

contents in the mantle, gills, gonad, digestive glands, foot and whole soft

body tissues of the experimental freshwater bivalve was observed as

194

compared to bivalves maintained as control. The obtained results are

presented in the table nos. 3.1 to 3.2 and figure nos. 3.1.1 to 3.1.12. The

result clearly indicated that carbosulfan causes more protein depletion in

all soft body tissues of the experimental bivalve as compared to

profenofos. The results recorded in the present study are in harmony with

the results of previous investigators (Lomte et al., 2000; Mahajan and

Zambare, 2005; Gulbhile, 2006; Satyaparameshwar et al., 2006; Nawale,

2008; Pardeshi and Gapat, 2012).

The results also demonstrated that, there was progressive decrease

in the protein content as exposure period was increased. Toxicity

response generally depends upon the toxicant concentration and the

duration of exposure in the tissue. 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).

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

tissues after exposure to pesticides profenofos and carbosulfan is

attributed to oxidative stress generated by these pesticides. Pesticides are

inducers of reactive oxygen species (ROS). 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). Regarding protein

cofactors, the oxidation of 4Fe-4S clusters by superoxide radicals in

enzymes such as the mitochondrial aconitase has major detrimental

effects, due to the lost of activity that impairs respiratory function.

Furthermore, the free iron released promotes the formation of highly

reactive hydroxyl radicals that propagate molecular damages (Costa and

Moradas, 2001). Protein oxidation can lead to conformational changes

associated with the decrease in activity, when target amino acid residues

195

are at or close to active sites. Oxidation of amino acid side chains results

in the formation of carbonyl derivatives which are non-reversible, causing

conformational changes, decreased catalytic activity of enzymes and,

eventually, resulting in a breakdown of proteins by proteases due to

increased susceptibility (Almroth et al., 2005). In mollusk the proteases

enzyme degrade oxidised proteins (Farr and Kogoma, 1991). It is well

documented that the various peptide components of photosystem II

turnover at different frequencies; the D1 protein specifically is noted for

its high rate of turnover, and it is assumed that this is a consequence of

oxidative attack at specific sites on the protein (Barber and Anderson,

1992). Waykar and Lomte (2002, 2004) and Mahajan (2005) reported

increase in protease activity in fresh water bivalve, 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.

According to Sivaprasad and Rao (1980) depletion in protein

content in pesticide treated animals may be due to either enhanced

proteolytic activity or decreased protein synthesis. 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; Parate

and Kulkarni 2003). This would involve interfering with protein

metabolism or the modification of sulphydryl groups and other ligands of

the cell membrane, thus altering its structure and function (Webb, 1966),

rather than by the specific inhibition of certain enzymes. It is known that

structural proteins are used as energy source under stressful conditions

(Claybrook, 1983). The decreased protein concentrations might also be

196

attributed to the destruction or necrosis of cells and consequent

impairment in protein synthesis machinery (Bradbury et al., 1987;

Parthasarathy and Joseph, 2011). At high pollution stress however,

protein synthesis can be suppressed indicating disturbance of normal

metabolic processes (Pottinger et al., 2002). The synthesis of protein in

any of a tissue can be affected in two ways by a chemical; (1) it either

affects the RNA synthesis at the transcription stage, or (2) it somehow

affects the uptake of amino acids in the polypetide chain. Both these

possibilities may account for the lower protein content in the affected

tissue. In the first case, the RNA synthesis would be inhibited resulting in

reduced RNA as well protein content. In the second case, only the protein

content would be affected (Tariq et al., 1977).

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 body of the bivalve. The 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 to meet energy demand. The higher

degradation of protein in digestive glands provided better indication of

the extent of toxicity. Singaraju et al., (1991), Mule and Lomte (1992,

1993, and 1995), Waykar and Lomte (2001) supported the most alteration

of protein contents in digestive glands of freshwater bivalves.

Large body of literature reported that, pesticide stress caused

depletion of protein content. Singh and Agarwal (1996) reported

significant decrease in endogenous levels of protein in foot tissues in

Lymnea accuminata on exposure to pesticides. Gupta and Bhide (2001,

197

2004) reported gradual decline in a number of protein fractions as well as

in the intensities of some of the protein fractions in Lymnaea stagnalis

when exposed to pesticides. Waykar and Lomte (2001) reported

decreased levels of total protein in different soft tissues of freshwater

bivalve, Parreysia cylindrica after acute and chronic exposure to

cypermethrin and also reported highest decrease in protein contents in

digestive glands. Pottinger et al., (2002) reported that at high pollution

stress however, protein synthesis can be suppressed indicating

disturbance of normal metabolic processes. Mahajan and Zambare,

(2005) reported that the protein contents in different soft tissues of the

Bellamya bengalensis were decreased after exposure to chronic

concentration of pollutants. Kulkarni et al., (2005) reported significant

decrease in total protein content in foot, hepatopancreas and gills of the

freshwater mussel, Lamellidens corrianus on exposure to pesticide. Kaur

and Dhaniu (2005) reported decreased protein content after exposure to

organophosphorus pesticides. Zahran et al., (2005) reported decrease in

protein contents in rat after exposure to sub acute dose of

organphosphorus pesticide, Nuvacron. 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. 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. Waykar and Pulate (2012)

reported decreased in protein contents in different soft tissues of

freshwater bivalve, Lamellidens marginals (L) after 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

198

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 sulphate for 30 days.

In combined exposure to profenofos and carbosulfan with 50 mg/l

of L-ascorbic acid the severity of protein depletion was much reduced.

Antioxidants (ascorbic acid) are capable of counteracting the damage.

Oxidative stress occurs when the production of harmful molecules called

free radicals is beyond the protective capability of the antioxidants

defenses. Antioxidants block the process of oxidation by neutralizing free

radical. In doing so, the antioxidants themselves become oxidized.

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. 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

199

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

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.

Changes in ascorbic acid content:

Ascorbic acid performs antioxidative functions in vivo by serving

as a hydrogen ion donor at various metabolic sites. The ascorbic acid has

potential role to reduce the activity of free radical-induced reactions

(Holloway, 1984). L- Ascorbic acid is a powerful antioxidant which

plays an important role in intracellular oxidation-reduction system and in

binding of free radicals produced endogenously (Laurence et al., 1977).

L- Ascorbic acid reduced the clastogenic effects generated by certain

chemical agents in the vivo and vitro assays (Amare-Mokrane et al.,

1996, Khan., 1996). According to Edgar (1974), L-Ascorbic acid

possesses substantial nucleophilic property and ascorbate might protect

against elecrophilic attack on cellular DNA by intercepting reactive

agents or ascorbic anion radical with a high extent of unpaired electron

delocalization which is the responsible for the scavenging of free radicals

(Bieski,1982).

In the present study depletion of ascorbic acid levels in the mantle,

foot, gills, digestive glands, gonads and whole soft body tissues of the

experimental freshwater bivalve was observed after chronic exposure to

pesticides profenofos and carbosulfan as compared to bivalves

maintained as control. Obtained results were presented in the table nos.

3.3 to 3.4 and figure nos. 3.2.1 to 3.2.12. The result clearly indicates that

200

highest depletion in ascorbic acid content was reported in bivalves

exposed to carbosulfan as compared to profenofos. 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), Phirke (2008), Nawale (2008). The

ascorbic acid content decreased in mantle, foot, gill, digestive glands,

gonad and whole body due to pesticide stress.

In the present study, it was observed decreased in content of

ascorbic acid in different soft body tissues of the experimental bivalve

species, 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 pesticides stress.

Stress caused alterations in the normal physiology of animal

leading to enhanced utilization and mobilization of ascorbic acid (Chinoy

and Kamalakumari, 1976) as ascorbic acid is recognized as antistress

factor (Kutsky, 1973). 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). During

acute response to different stressors such as metals and heat shock etc

ascorbic acid is depleted (Parihar and Dubey, 1995 and Lackner, 1998).

Decrease in ascorbic acid content indicated its involvement in

counteracting oxidative damage.

Number of researchers reported that due to toxicant stress ascorbic

acid content was decreased. 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

201

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 far as present work is concerned, decrease in ascorbic acid

content in different tissues of Lamellidens marginalis 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

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

202

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.

Changes in the DNA content:

In the present study depletion of DNA levels in the mantle, gills,

digestive glands, gonads and whole soft body tissues of the experimental

freshwater bivalve was observed after chronic exposure to pesticides

profenofos and carbosulfan compared to bivalves maintained as control.

Obtained results were presented in the table nos. 3.5 to 3.6 and figure nos

3.3.1 to 3.3.12. The result clearly indicates that highest depletion in DNA

content was reported in bivalves exposed to carbosulfan as compared to

profenofos. 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.

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

203

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 to toxicants. Tong Lu et

al., (2001) observed that approximately 60 genes (10%) were

differentially expressed in arsenic exposed human livers compared to

controls. The differentially expressed genes induced those involved in

cell cycle regulation, apoptosis, DNA damage response, and intermediate

filaments. The observed gene alterations appear to be reflective of hepatic

degenerative lesions seen in the arsenic exposed patients. This array

analysis revealed important patterns of aberrant gene expression

occurring with arsenic exposure in human liver. Aberrant expressions of

several genes were consistent with the results of array analysis of chronic

arsenic exposed mouse livers and chronic arsenic - transformed rat liver

cells. They suggested that clearly a variety of gene expression changes

might play an integral role in arsenic hepatotoxicity and possibly

carcinogenesis. 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).

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

204

gills, testes and ovaries. 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. The cellular degradation rapid histolysis and decreased rate of

protein synthesis are possible reasons behind this.

As compared and supported by above literature, the present

investigation of the chronic exposure of profenofos and carbosulfan to

bivalve Lamellidens marginalis showed decreased DNA contents

compared with control bivalves, and those exposed to pesticides with

ascorbic acid.

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.

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.

In combined exposure to profenofos and carbosulfan 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

205

endogenous oxidative DNA damage, thereby decreasing the risk of

genetic defects, particularly in populations with low vitamin C levels.

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 profenofos and carbosulfan as compared with bivalves

maintained as control. Obtained results were presented in the tables no

3.7 to 3.8 and figure 3.4.1 to 3.4.12. The result clearly indicates that

highest depletion in RNA content was reported in bivalves exposed to

carbosulfan as compared to profenofos. 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, gill, digestive gland and whole body of

experimental bivalves was due to pesticide stress. 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.

206

Several reports are available on the reduction in RNA levels on

exposure to different pesticide (Tarig et al., 1977; Nordenskjold et al.,

1979). 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. 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.

In combined exposure to profenofos and carbosulfan 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.

Changes in Protein, Ascorbic acid, DNA and RNA contents during

the recovery due to the ascorbic acid exposure:

Detoxification can be used as a beneficial curative measure and as

a tool to increase overall health and vitality. Detoxification treatment has

become one of the cornerstones of alternative medicine. Detoxification

therapies are having increasing importance and popularity.

207

In present study, the bivalves pre-exposed to chronic concentration

to pesticides profenofos and carbosulfan 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 (Mahajan and Zambare, 2006;

Gulbhile, 2006; Mahajan, 2007; Shinde, 2008 Gulbhile, 2006; Nawale,

2008; Pardeshi and Gapat, 2012).

The obtained results indicate that the physiological disturbances

arising in animals after exposure to pesticides exhibits trends towards

normalization and this rate of recovery from pesticide induced damage is

faster on exposure to L-ascorbic acid 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.

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, as a water soluble antioxidant,

can readily scavenge ROS, reactive nitrogen species and prevent

oxidative damage to many important biological macromolecules such as

DNA, lipids and proteins (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. L-ascorbic acid is a

primary defensive nutrient by virtue of its function as a free radical

scavenger (Frei, 1999; Carr and Frei, 2000). These properties of ascorbic

acid make it a suitable antidote for pollutant toxicity in rodents and

possibly in human subjects (Sohini and Rana, 2007). The preventive

208

activity of vitamin C may be related to its antioxidant efficacy that

inhibits lipid peroxidation enhanced by lead. Blankenship et al., (1997)

showed that vitamin C protected cells from undergoing apoptosis.

The possible action of ascorbic acid is mediated through

scavenging physiologically relevant reactive oxygen and nitrogen species.

These include free radicals such as hydroxyl radicals, aqueous radicals,

superoxide anion, and nitrogen dioxide, as well as nonradical species

such as hypochlorus acid, ozone, singlet oxygen, nitrosating species

(N2O3/N2O4), nitroxide, and peroxynitrite. 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).

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). Preservation of intracellular

ascorbic acid levels minimizes the peroxynitrite-mediated injury which is

attributable to the beneficial effect of ascorbic acid (Mihm 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

209

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

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 levels 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.

210

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 is faster on exposure to L-ascorbic acid

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.

211

SUMMARY

The present investigation showed the role of ascorbic acid in

profenofos and carbosulfan induced biochemical alterations

in an experiment model, the freshwater bivalve, Lamellidens

marginalis.

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 of freshwater bivalves,

Lamellidens marginalis were studied after chronic exposures

to profenofos (0.6191 ppm) and carbosulfan (0.5564 ppm)

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 were

found to be significantly decreased after chronic treatment of

profenofos and carbosulfan.

The protein, ascorbic acid, DNA and RNA contents were more

in gills, gonad, digestive glands, foot, mantle and whole soft

body of freshwater bivalves, Lamellidens marginalis when

exposed to profenofos and carbosulfan 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.

212

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