QUINALPHOS INDUCED BIOCHEMICAL
AND PATHOPHYSIOLOGICAL CHANGES
IN FRESHWATER EXOTIC CARP,
CYPRINUS CARPIO (LINNAEUS)
By
Mr....
Introduction

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Water resources have been the most
exploited natural system since man stepped
the earth.
On the one...
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The pollution of rivers, canals, and lakes with
chemical substances of anthropogenic origin
may have adverse consequ...
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More than 50% of India’s economy is
dependent upon agriculture and India’s
agriculture has to go a long way to match...
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PESTICIDE POLLUTION
Pesticides are toxic and designed to repel or
kill unwanted organisms, and when applied to
the...
Many of the more than 1000 pesticides
currently used in most of the countries of the
world inadvertently reach aquatic
eco...
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Many pesticides can be grouped into
chemical families. Prominent insecticide
families
include
organochlorines,
organ...
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Organochlorine toxicities vary greatly, but they
have been phased out because of their persistence
and potential ...
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Organophosphate pesticides are irreversibly
inactivate acetylcholinesterase, which is
essential to nerve function...
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It is estimated that only 0.1% of the insecticide
reaches the specific target (Aguiar, 2002) or often
less, leavi...
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SCOPE FOR THE PRESENT STUDY:
Fish is a vital source of food for people. It is man’s
most important single source o...
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Fish species are most sensitive to aquatic
pollutants during their early life stages
(Jiraungkoorskul et al., 2002)....
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The evaluation and assessment of the
ecotoxicological risks caused by pesticides
are based on the toxicity and effec...
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Quinalphos
(O,
O-diethyl-O-quinoxalin-2-yl
phosphorothioate) is one of the most widely used
organophosphorus inse...
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Among exotic carps Cyprinus carpio, is
widely cultured in ponds and lakes of this
region. This fish is largely prefe...
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Hence, present study focus on the
toxicological impact of organophosphate
pesticide, quinalphos on the freshwater
ex...
Chapter 1: Studies on Toxicity Evaluation
 Chapter 2: Studies on Behavioral changes
 Chapter 3: Studies on Respiratory d...
Aim of Study
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL
CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LIN...
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Toxicant:
The commercial grade organophosphate
insecticide, quinalphos (25% emulsified
concentration) is procured fro...
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Quinalphos
(O,
O-diethyl-O-quinoxalin-2-yl
phosphorothioate) is one of the most widely used
organophosphorus inse...
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Some chemical and physical properties of quinalphos.
Chemical Structure:
Molecular formula:...
The consumption of quinalphos during the year 2007 – 2010 in
Dharwad district (Karnataka) is presented below in terms of l...
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Test Toxicant: Quinalphos
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Biology of fish Cyprinus carpio:
Body elongated and somewhat compressed. Lips
thick. Two pairs of barbels at...
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Test Animal : Cyprinus carpio
Classification: Systematic position of Cyprinus carpio:
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Phylum
Sub-Phylum
Division
Super Class
Class
...
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Procurement and maintenance of fish:
Fish, Cyprinus carpio weighing 4 ± 2 g and
measuring an average length of...
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Experimental Design:

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Five groups of ten fish each were exposed to
lethal and sub lethal concentrations (1/10th
of t...
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Fixation of Exposure Periods:
The effect of the lethal and sub lethal
concentrations of quinalphos on fishes were
...
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Fixation of Lethal and Sub Lethal
Concentrations:
The LC50 96 h of quinalphos concentration
was taken is fixed as le...
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Tissue selected:

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Biochemical Parameters: Gill, kidney and
liver

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Histopathology: Gill, kidney and liver
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Methods followed
Parameters
Methods
Toxicity evaluation : Finney, 1971 and Carpente...
Chapter 1: Studies on Toxicity Evaluation
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The toxicity tests have been historically played an
important role in a...
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The LC50 96 hr value for the fish, Cyprinus carpio
was determined after conducting static renewal
bioassay test.
...
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Toxicity evaluation (RESULT)
Hence the present study is initiated with the determination
of 96 h LC50 value ...
Table 2. Mortality of Cyprinus carpio in different concentrations of
Quinalphos at 96-hour exposure period.

Concentration...
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Toxicity evaluation
LC50 studies are believed to provide
information on the relative lethality of a
toxicant to an or...
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LC50 studies are highly useful in determining the
sublethal concentrations of a particular compound.
Most of th...
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Toxicity of quinalphos to C. carpio is
relatively lower when compared with other
species of fishes.
The 96 hour LC50...
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The results obtained form the present
toxicity study shows that the quinalphos EC
25% was more toxic to the aquatic org...
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Chapter 2: Studies on Behavioral changes

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The pesticide brings tremendous changes in
the organism. Changes in regula...
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Behavioural study (RESULT)
The behavioural tests are useful in
evaluating the toxicant induced effects on
whole po...
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The fishes were exposed to the lethal
concentration of quinalphos exhibited the
behavioural changes like excitability, ...
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Whereas in sub lethal concentration such
behavioural changes were negligible.
Suffocation caused by the mucus film o...
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Behavioural characteristics are obviously sensitive
indicators of toxicant effect.
It is necessary, however, t...
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Chapter 3: Studies on Respiratory distress
A change in respiration rate is one of the
common
physiological
respon...
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A reduction in oxygen consumption is observed
when the fish is exposed to the toxicant and the
mortality is du...
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Oxygen consumption (RESULT)
Generally when a toxicant gains entry
through food chain or respiratory surfaces,
the ...
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The study of respiration at whole animal
level needs emphasis to ascertain the oxygen
requirements
of
fish
under
tox...
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In the present study, the oxygen consumption was
gradually decreasing with increasing exposure periods as
observe...
Table 3: Whole animal oxygen consumption (ml/gm wet wt/h) of the fish, Cyprinus carpio
on exposure to the lethal and sub l...
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Chapter 4: Studies on Quinalphos
Accumulation (residue analysis)
In environmental studies, certain organisms
provi...
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Persistent hydrophobic chemicals may
accumulate in aquatic organisms through
different mechanisms: via the direct up...
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In the present study an attempt has been
made to determine the residue levels of
quinalphos in different tissues viz., ...
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Bio-accumulation (RESULT)
A very important biological property of
pesticide
is
their
tendency
to
bioaccumulate. A ...
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However this increase is predominantly
more in lethal concentration and sub lethal
concentration. Further, it appear...
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Several authors have reported that, pesticide
residue can cause cellular damage to the gill tissue,
as it is the fir...
Table 4: Accumulation of quinalphos (µg/g wet wt.) in the organs of fish, Cyprinus carpio on exposure to the lethal
and su...
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Chapter 5: Ions and associated ATPases
Ions play a vital role in several body functions, viz.
the monovalent ions ...
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In freshwater fishes, blood and electrolyte
concentration are regulated by interacting
processes, such as absorption...
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Sodium (Na+) ion plays an important role in the
osmotic regulation of body fluids and also serves
as an essential...
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Adenosine triphosphatase (ATPase) is a membrane bound
enzyme group for regulating oxidative phosphorylation,...
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Results
In this investigation, it is evident that Na+ loss is higher in
the case of gill indicating the dera...
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In the present study, the decrease in the levels of
Na+- K+, Ca2+ ions in the gill, kidney and liver
exposed to leth...
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However this change increased over time of
exposure in lethal but decrease in sub lethal
concentration. The results ...
Table 5:
Sodium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the lethal
and sublethal...
Table 6: Potassium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the
lethal and sublet...
Table 7:
Calcium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the lethal
and subletha...
Table 8: Na+-K+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on
exposure to t...
Table 9: Mg2+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on
exposure to the...
Table 10: Ca2+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on exposure
to th...
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Chapter 6: Studies on Histopathology
Histopathology is mainly directed to study the effect
of chemicals and ...
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Histopathological study (RESULT)
Gill
Gills are the vital organs for respiration in
fish which establish a direc...
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Fish on exposure to the lethal concentration on day 1, the
enlargement of the base of primary gill lamellae wa...
H&E:X 100 and
X200

24 hr exposed gill to lethal
dose of quinalphos

Control Fish Gill

48 hr exposed gill to lethal
dose ...
H&E:X 100 and
X200

72 hr exposed gill to lethal
dose of quinalphos

Control Fish Gill

96 hr exposed gill to lethal
dose ...
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In the sublethal concentration of quinalphos a mild
degree of degenerative changes and sign of shrinkage in
the p...
H&E:X 100 and
X200

Day 1st exposed gill to
lethal dose of quinalphos

Control Fish Gill

Day 5th exposed gill to
lethal d...
H&E:X 100 and
X200

Day 10 exposed gill to
lethal dose of quinalphos
th

Control Fish Gill

Day 15th exposed gill to
letha...
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Liver
Fish liver is regarded as a major site of
storage, biotransformation and excretion of
pesticides. These orga...
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On day 1 of exposure to the lethal concentration of
quinalphos, the liver of fish exhibited enlarged nuclei an...
H&E:X 200

Control Fish

and X400

Liver

24 hr exposed fish liver to
lethal dose of quinalphos

48 hr exposed fish liver ...
H&E:X 200

Control Fish

and X400

Liver

72 hr exposed fish liver to
lethal dose of quinalphos

96 hr exposed fish liver ...
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Compared to the structure of the liver of control fish,
exposed to sublethal concentration of quinalphos
initiall...
H&E:X 200

Control Fish

and X400

Liver

Day 1st exposed fish liver to
lethal dose of quinalphos

Day 5th exposed fish li...
H&E:X 200

Control Fish

and X400

Liver

Day 10th exposed fish liver to lethal
dose of quinalphos

Day 15th exposed fish ...
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Kidney
In fish, the kidney performs an important
function related to electrolyte and water
balance and the mainten...
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In lethal concentration of quinalphos the kidney showed
reduction in renal cell number in the proximal and dis...
H&E:X 200

Control Fish

and X400

Kidney

24 hr exposed kidney to lethal
dose of quinalphos

48 hr exposed kidney to leth...
H&E:X 200

Control Fish

and X400

Kidney

72 hr exposed kidney to lethal
dose of quinalphos

96 hr exposed kidney to leth...
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In sublethal concentration of quinalphos the kidney of the fish
exhibited a mild degree of changes.
1st, 5th ...
H&E:X 200

Control Fish

and X400

Kidney

Day 1 exposed kidney to lethal dose
of quinalphos
st

Day 5th exposed kidney to...
H&E:X 200

Control Fish

and X400

Kidney

Day 10th exposed kidney to lethal
dose of quinalphos

Day 15th exposed kidney t...
Ultrastructure
Control Fish liver

In control fish the hepatocytes are characterized by a system of
parallelly stacked cis...
Ultrastructure of
Fish liver exposed
to sublethal dose
of quinalphos

In comparison to the controls, hepatocytes of Cyprin...
Ultrastructure of
Fish liver exposed
to sublethal dose
of quinalphos

Hepatocytes of common carp exposed to the
sublethal ...
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Chapter 7: Study on Metabolic and
Enzymological aspects
To
understand
quinalphos
induced
oxidative damage on chan...
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Biochemical Studies (RESULT)
Organophosphates are neuro toxins that disrupt the
central nervous system of an...
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Depression of acetyl cholinesterase activity
suggests decreased cholinergic transmission
and
consequent
accumulation...
Table 11: Ach level (µM/g wet wt.) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal
concentr...
Table 12: AChE activity (nM of acetylthiocholine iodide hydrolyzed/mg protein/min) in the organs of fish, Cyprinus
carpio ...
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The assessment of the protein content can be
considered as a diagnostic tool to determine the
physiological proce...
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Among the exposure periods, in lethal
concentrations, the levels of soluble, structural and
total proteins sign...
Table 13: Soluble protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio on
exposure ...
Table 14: Structural protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio
on exposu...
Table 15: Total protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio on
exposure to...
Table 16: Free amino acid levels (µmol of tyrosine equivalents/g wet wt.) in the organs of fish, Cyprinus
carpio on exposu...
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Oxidative stress
Among the most commonly used biomarkers,
those related to oxidative stress assume an
important...
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Results

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The increase in catalase activity and H2O2
level observed in all the organs of fish at all
the exposure per...
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In the present study, lethal and sublethal
concentrations of quinalphos were resulted in the
significant decrease...
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The most widely used assay for lipid peroxidation is
the malondialdehyde (MDA) formation, which
represents the se...
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Increase in the protease activity as evidenced from the
present study suggests that damage to proteins thus
...
Table 17: Catalase activity (mmol of hydrogen peroxide decomposed/mg protein/min) in the organs of fish,
Cyprinus carpio o...
Table 18: Hydrogen peroxide levels (nmol of hydrogen peroxide/mg protein) in the organs of fish, Cyprinus carpio
on exposu...
Table 19: MDA levels (nmol of TBARS formed/mg of protein) in the organs of fish, Cyprinus carpio on exposure to
the lethal...
Table 20: Protein carbonyls (nmol of DNPH incorporated/mg protein) in the organs of fish, Cyprinus carpio
on exposure to t...
Table 21: Protease activity (µmol of tyrosine equivalents/mg protein/h) in the organs of fish, Cyprinus
carpio on exposure...
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Chapter 8: Studies on Haematology
The study of blood parameters in fishes has been
widely used for the detectio...
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Blood is a vehicle for quickly mobilizing defense
against trauma and diseases. Since, fishes differ
considerably in ...
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Hematological Study (RESULT)
There was decrease in RBC, Hb, and
values of MCH, MCHC, PCV and MCV on
the fish expos...
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Measurement of haematological parameters are important
in diagnosing the structural and functional status of
anim...
Table 22: RBC count (x106/mm3) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal
concentration...
Table 23: WBC count (x103/mm3) in the blood of the fish, Cyprinus carpio on exposure to lethal and
sublethal concentration...
Table 24: Haemoglobin level (g/100 ml) in the blood of the fish, Cyprinus carpio on exposure to lethal
and sublethal conce...
Table 25: PCV level (%) in the blood of the fish, Cyprinus carpio on exposure to lethal and
sublethal concentrations of qu...
Table 26: MCV level (cu mm) in the blood of the fish, Cyprinus carpio on exposure to lethal
and sublethal concentrations o...
Table 27: MCH level (pg) in the blood of the fish, Cyprinus carpio on exposure to
lethal and sublethal concentrations of q...
Table 28: MCHC level (%) in the blood of the fish, Cyprinus carpio on
exposure to lethal and sublethal concentrations of q...
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In the present investigation, fish Cyprinus carpio
treated to the lethal and sublethal concentrations of
quinalph...
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Conclusion
As food source, fish interferes on man’s life quality
and so more detailed analysis of the action of wh...
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From the above assessment pertinent to
physiological, behavioural and biochemical
response of freshwater fish, C. ca...
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Under low concentration, i.e., sublethal
concentration stress in fish was observed
only for short period (1 to 5 day...
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Therefore, the above statement suggests that the
fish can adapt to low concentration of quinalphos
toxicity durin...
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It is hoped that based on some of the significant
differences observed in all the aspects of the
present study, useful ...
Thank you
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Acknowledgement
Dr. M. David sir and family
My Parents and In-laws'
My Family, Wife and Daughter
Univers...
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)
QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)
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QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS)

  1. 1. QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS) By Mr. Sameer Gopal Chebbi Under the Guidance of Lt. Dr. M. David Professor Department of Zoology Karnatak University Dharwad
  2. 2. Introduction    Water resources have been the most exploited natural system since man stepped the earth. On the one hand rapid population growth, increasing living standards, wide spheres of human activities and industrialization have resulted in greater demand of good quality water while on the other, pollution of water resources is increasing steadily. Water pollution is one of the main concerns of the world today.
  3. 3.   The pollution of rivers, canals, and lakes with chemical substances of anthropogenic origin may have adverse consequences, the waters become unsuitable for drinking and other household purposes, irrigation, and fish cultivation, and also the animal communities living in them may suffer seriously (Koprucu and Aydln, 2004; Ural and Saglam, 2005). The increasing use of synthetic pesticides is intensifying worldwide pollution risks.
  4. 4.   More than 50% of India’s economy is dependent upon agriculture and India’s agriculture has to go a long way to match the yield of the advanced countries. Indiscriminate discharge of waste by agriculture has variety of problems including those owing to pesticide and other exotic chemicals that are unfavorable to all life forms (Nagaraju, et al., 2011).
  5. 5.    PESTICIDE POLLUTION Pesticides are toxic and designed to repel or kill unwanted organisms, and when applied to the land they may be washed into surface waters and kill or, at least adversely influence, the life of aquatic organisms (El-Sayed et al., 2009). Dependence on pesticides for pest control has been increasing since the onset of the green revolution.
  6. 6. Many of the more than 1000 pesticides currently used in most of the countries of the world inadvertently reach aquatic ecosystems.  All pesticides are potentially toxic to living organisms.  Pesticide use, therefore, is one of the many factors contributing to the decline of fish and other aquatic species in tropical areas (Helfrich et al., 1996). 
  7. 7.   Many pesticides can be grouped into chemical families. Prominent insecticide families include organochlorines, organophosphates, and carbamates. Among different classes of pesticides, organophosphates are more frequently used, because of their high insecticidal property, low mammalian toxicity, less persistence and rapid biodegradability in the environment (Singh et al., 2010).
  8. 8.    Organochlorine toxicities vary greatly, but they have been phased out because of their persistence and potential to bioaccumulate. Organophosphate and carbamates largely replaced organochlorines. Both operate through inhibiting the enzyme acetylcholinesterase, allowing acetylcholine to transfer nerve impulses indefinitely and causing a variety of symptoms such as weakness or paralysis. Organophosphates are quite toxic to vertebrates, and have in some cases been replaced by less toxic carbamates (Kamrin, 1997).
  9. 9.    Organophosphate pesticides are irreversibly inactivate acetylcholinesterase, which is essential to nerve function in insects, humans, and many other animals. Organophosphate pesticides affect this enzyme in varied ways, and thus in their potential for poisoning. Although organophosphates degrade faster than the organochlorides, they have greater acute toxicity, posing risks to people who may be exposed to large amounts.
  10. 10.    It is estimated that only 0.1% of the insecticide reaches the specific target (Aguiar, 2002) or often less, leaving 99.9% as an unintended pollutant in the environment, including in the soil, air, and water, or on nearby vegetation (Pimentel, 1995). The aquatic environment is the ultimate sink for all anthropogenic chemicals and global pollutants. Any compound that has been used in large quantities ultimately reaches the aquatic ecosystem (Zitko, 1974). The size and nature of the water body and the extent of possible dilution influence the level of accumulation of residue by organisms.
  11. 11.    SCOPE FOR THE PRESENT STUDY: Fish is a vital source of food for people. It is man’s most important single source of high-quality protein, providing ~16% of the animal protein consumed by the world’s population, according to the Food and Agriculture Organisation of the United Nations (2000). The FAO estimates that about one billion people world-wide rely on fish as their primary source of animal protein (FAO, 2000). Fish is a food of excellent nutritional value, providing high quality protein and a wide variety of vitamins and minerals.
  12. 12.   Fish species are most sensitive to aquatic pollutants during their early life stages (Jiraungkoorskul et al., 2002). The indiscriminate use of pesticides, careless handling, accidental spillage or discharge of untreated effluents into natural waterways have harmful effects on fish population and other forms of aquatic life and may contribute long term effects in the environment. Fish communities are often used as an indicator of high water quality areas, as these individuals have extremely specific water quality requirements (Bauer and Ralph, 2001).
  13. 13.   The evaluation and assessment of the ecotoxicological risks caused by pesticides are based on the toxicity and effects of pesticide preparations to non-target organisms like fish, on which exert a wide range of effects (Velisek, et al., 2008). With increased interest from public and scientific communities in man’s use, abuse and misuse of the aquatic environment, a diverse array of bioassay methods have evolved..
  14. 14.    Quinalphos (O, O-diethyl-O-quinoxalin-2-yl phosphorothioate) is one of the most widely used organophosphorus insecticides in agriculture, due to its acaricidal and an insecticidal property is in large scale use in this country. Quinalphos formulations are widely used as contact and systematic insecticide against broad range of insects. Quinalphos effectively controls caterpillars on fruit trees, cotton, vegetables and peanuts; scale insect on fruit trees and pest complex on rice. Quinalphos also controls aphids, bollworms, borers, leafhoppers, mites, thrips, etc. on vines, ornamentals, potatoes, soya beans, rice, tea, coffee, cocoa, and other crops.
  15. 15.   Among exotic carps Cyprinus carpio, is widely cultured in ponds and lakes of this region. This fish is largely preferred for table purpose by the people because of its low cost proteins and vitamins. This fish being specifically a bottom feeder in habit is widely considered for composite fish culture. As the water bodies are the ultimate recipient of all the toxic chemicals and wastes emitted from industries, agricultural practices, household application, forest spraying, etc aquatic organisms especially the fishes becomes the target.
  16. 16.   Hence, present study focus on the toxicological impact of organophosphate pesticide, quinalphos on the freshwater exotic carp, Cyprinus carpio. The objective of the present investigation is to understand the toxic effects lethal and sublethal concentration of quinalphos (EC 25%) on freshwater exotic carp Cyprinus carpio with following objectives.
  17. 17. Chapter 1: Studies on Toxicity Evaluation  Chapter 2: Studies on Behavioral changes  Chapter 3: Studies on Respiratory distress  Chapter 4: Studies on Quinalphos Accumulation  Chapter 5: Ions and associated ATPases  Chapter 6: Studies on Histopathology  Chapter 7: Study on Metabolic and Enzymological aspects  Chapter 8: Studies on Haematology 
  18. 18. Aim of Study QUINALPHOS INDUCED BIOCHEMICAL AND PATHOPHYSIOLOGICAL CHANGES IN FRESHWATER EXOTIC CARP, CYPRINUS CARPIO (LINNAEUS) Test Animal: CYPRINUS CARPIO Test Toxicant: QUINALPHOS (OP) Dose: Toxicity test LC50 Subacute Dose: 0.75 μl/l Exposure Periods: 1, 5, 10 and 15 days Acute Dose: 7.5 μl/l Exposure Periods: 1, 2, 3 and 4 days Study on Biochemical Parameters: Proteins, Oxidative stress and AchE, Ions and ATPase Tissue selected: Gill, Kidney and Liver Studies on Respiratory distress Studies on Accumulation Result Discussion Conclusion Study on Pathological Parameters: Histology and Haematology
  19. 19.   Toxicant: The commercial grade organophosphate insecticide, quinalphos (25% emulsified concentration) is procured from local market as Quinalphos 25% EC (VAZRA-25).
  20. 20.    Quinalphos (O, O-diethyl-O-quinoxalin-2-yl phosphorothioate) is one of the most widely used organophosphorus insecticides in agriculture, due to its acaricidal and an insecticidal property is in large scale use in the country. Quinalphos effectively controls caterpillars on fruit trees, cotton, vegetables and peanuts; scale insect on fruit trees and pest complex on rice. Quinalphos also controls aphids, bollworms, borers, leafhoppers, mites, thrips, etc. potatoes, soya beans, rice, tea, coffee, cocoa, and other crops.
  21. 21.                Some chemical and physical properties of quinalphos. Chemical Structure: Molecular formula: C12H15N2O3PS Molecular Weight: 298.3 Common Name: Quinalphos Common trade names: Vazra IUPAC name: O,O-diethyl O-quinoxalin-2-yl phosphorothioate Formulation in solvent: 2-methoxy and 2-ethoxyethnol CAS chemical name: O,O-diethyl O-2-quinoxalinyl phosphorothioate Melting point: 31 – 32oC Boiling point: 107 oC at 0.05mmHg, 86 oC at 0.01 mmHg Solubility in water (21 oC) up to 17.8 gm/L Half- Life in aqueous media, at pH 2-7, relatively stable. Relative molecular mass: 298.25 Volatility: 1.235 mg/m3
  22. 22. The consumption of quinalphos during the year 2007 – 2010 in Dharwad district (Karnataka) is presented below in terms of liters used to control different pests. Name of the Pesticides Year Quantity (in liters) Quinalphos 2007 1,380 Quinalphos 2008 1,650 Quinalphos 2009 2,220 Quinalphos 2010 4,310 Source: Joint Director of Agriculture, Dharwad, Karnataka, India.
  23. 23.  Test Toxicant: Quinalphos
  24. 24.      Biology of fish Cyprinus carpio: Body elongated and somewhat compressed. Lips thick. Two pairs of barbels at angle of mouth, shorter ones on the upper lip.  Dorsal fin base long with 17-22 branched rays and a strong, toothed spine in front; dorsal fin outline concave anteriorly. Anal fin with 6-7 soft rays; posterior edge of 3rd dorsal and anal fin spines with sharp spinules.  Lateral line with 32 to 38 scales.  Pharyngeal teeth 5:5, teeth with flattened crowns.  Colour variable, wild carp are brownish-green on the back and upper sides, shading to golden yellow ventrally. The fins are dusky, ventrally with a reddish tinge. Golden carp are bred for ornamental purposes. 
  25. 25.  Test Animal : Cyprinus carpio
  26. 26. Classification: Systematic position of Cyprinus carpio:           Phylum Sub-Phylum Division Super Class Class Sub Class Super order Order Genus Species : : : : : : : : : : Chordata Vertebrata Gnathostomata Pisces Osteichthyes Actinopterygii Teleostei Cypriniformes Cyprinus carpio
  27. 27.     Procurement and maintenance of fish: Fish, Cyprinus carpio weighing 4 ± 2 g and measuring an average length of 5 ± 2 cm were collected from the State Fisheries Department, Dharwad, and maintained in large cement tank previously washed with potassium permanganate solution. The water was aerated twice a day so as to provide sufficient oxygen. The fish were fed daily with commercial fish pellets (40% protein content) procured from market. They were acclimated to laboratory conditions for fifteen days. The tanks were cleaned periodically to avoid infection to fish. The temperature of the water in the aquaria was 29 ± 1oC and the same was maintained throughout the course of investigation.
  28. 28.  Experimental Design:  Five groups of ten fish each were exposed to lethal and sub lethal concentrations (1/10th of the LC50) of quinalphos.  Similarly one such group of thirty fish was taken as control.
  29. 29.    Fixation of Exposure Periods: The effect of the lethal and sub lethal concentrations of quinalphos on fishes were studied at different periods of exposure in order to understand the influence of time over toxicity. Thus in lethal 1, 2, 3 and 4 days and in the sub lethal 1, 5, 10 and 15 days were chosen to observe the short term and long term acute and chronic effects of quinalphos on fish, Cyprinus carpio, respectively.
  30. 30.   Fixation of Lethal and Sub Lethal Concentrations: The LC50 96 h of quinalphos concentration was taken is fixed as lethal concentration (7.5 μl/l) and sub lethal concentration (1/10th of the LC50 i.e., 0.75 μl/l) to study the physiological, biochemical, haematological and histological responses of fish.
  31. 31.  Tissue selected:  Biochemical Parameters: Gill, kidney and liver  Histopathology: Gill, kidney and liver
  32. 32.                    Methods followed Parameters Methods Toxicity evaluation : Finney, 1971 and Carpenter, 1975 Behavioural studies : David et al., 2008 Estimation of AChE activity: Ellman et al., 1961 Studies on Whole animal oxygen consumption: Welsh and Smith, 1953 Quinalphos bioaccumulation : Rao et al., 2003 Catalase activity: Luck et al., 1974 Protease activity: Davis and Smith, 1955 Malondialdehyde (MDA) Placer et al., 1966, Free amino acids Moore and Stein, 1954 Protein Lowry et al., 1951 Estimation of Ca2+, K+, Mg2+ and Na+ ions Dall, 1967 Ca2+, Mg2+ and Na+/K+ ATPases activities Watson and Beamish, 1981 RBC count Donald and Henry, 1969 WBC count Donald Hunter and Bomford, 1963 Haemoglobin Dacie and Lewis, 1961 Histopathology Humason, 1972 Statistical analysis of data Duncan, 1955
  33. 33. Chapter 1: Studies on Toxicity Evaluation    The toxicity tests have been historically played an important role in assessing the effect of human activities on animals. These tests have wide applicability in evaluating the acute toxicity of pesticides to fish. Teleost fish have proved to be good models to evaluate the toxicity and effects of contaminants on animals, since their biochemical responses are similar to those of mammals and of other vertebrates (Sancho et al., 2000).
  34. 34.    The LC50 96 hr value for the fish, Cyprinus carpio was determined after conducting static renewal bioassay test. In the present test, acute toxicity is expressed as the median lethal concentration (LC50) that is the concentration in water which kills 50% of a test batch of fish within a continuous period of exposure (96 h). One tenth of the LC50 was selected as sub lethal concentration for sub acute studies (1, 5, 10 and 15 days) to find out quinalphos induced metabolic changes.
  35. 35.      Toxicity evaluation (RESULT) Hence the present study is initiated with the determination of 96 h LC50 value on exposure on Cyprinus carpio to different concentrations of quinalphos as described by Finney, 1971 and Carpenter (1975). 96 h exposure is preferred with a view that the effects of the toxicant on these animals become consistent within this period (Eisler, 1977). The dosage response studies conducted for 96 h reveled that the LC50 value was found to be 7.4 μl/l and 7.6 μl/l (sigmoid curve/linear curve, Finney method) and 7.7 μl/l according Dragstedt-Behrens’s method. Thus, the average 96 h LC50 was found to be 7.5 μl/l.
  36. 36. Table 2. Mortality of Cyprinus carpio in different concentrations of Quinalphos at 96-hour exposure period. Concentration of Quinalphos 25 % EC (µl/L) 5.0 5.5 6.4 6.5 7.0 7.1 7.3 7.5 8.5 Log concentrati No. of fish No. of on of exposed fish alive quinalphos 0.6989 0.7403 0.8016 0.8129 0.8450 0.8526 0.8678 0.8772 0.9294 10 10 10 10 10 10 10 10 10 10 9 8 7 5 4 2 1 0 No. Fish dead 0 1 2 3 5 6 8 9 10 Perce Probit nt kill kill 0 10 20 30 50 60 80 90 100 ----3.72 4.16 4.48 5.00 5.52 5.84 6.28 8.09
  37. 37.   Toxicity evaluation LC50 studies are believed to provide information on the relative lethality of a toxicant to an organism and establishing the tolerance limits and safe levels of toxic agents for the biota of aquatic environment and also for the evaluation of lethal and sub lethal concentrations.
  38. 38.     LC50 studies are highly useful in determining the sublethal concentrations of a particular compound. Most of the information on the effects of pesticides in aquatic animals is on short-term experiments carried out at the lethal concentrations (Tilak and Swarna Kumari, 2009). Hence, there is need for the sublethal toxicity studies, which prove to be of great worth in evaluating the sequence of events involving in response of the test animal to the sublethal concentrations (Sprague, 1971). So, to derive such sublethal concentrations and to compare responses of animal at the sublethal concentration with those of the lethal, LC50s are prime requisites (Das and Mukherjee, 2000).
  39. 39.   Toxicity of quinalphos to C. carpio is relatively lower when compared with other species of fishes. The 96 hour LC50 value (7.5µ l/L) obtained in the present study is lower than the values reported in literature for other species of fish (Das and Mukherjee, 2000; Tilak and Swarna Kumari, 2009; Nikam, et al., 2011).
  40. 40.  The results obtained form the present toxicity study shows that the quinalphos EC 25% was more toxic to the aquatic organism because of its liquid formulation of technical quinalphos in the solvent, 2-methoxy and 2ethoxyethnol (Das and Mukherjee, 2000).
  41. 41.  Chapter 2: Studies on Behavioral changes  The pesticide brings tremendous changes in the organism. Changes in regular behavioural pattern of the organism link it with the disturbed physiology. In the laboratory fish behaviour can be a sensitive marker of toxicant induced stress. Hence an attempt has been made to study physical, morphological and behavioural changes in quinalphos exposed fish. 
  42. 42.    Behavioural study (RESULT) The behavioural tests are useful in evaluating the toxicant induced effects on whole population, small change in learning, dominance, parental behaviour, food selection, migration etc. Further they are excellent tool for quick screening and for understanding the toxic effects of the organophosphate pesticide such as quinalphos.
  43. 43.  The fishes were exposed to the lethal concentration of quinalphos exhibited the behavioural changes like excitability, erratic swimming, jumping, respiratory disruptions like discomfort movement (S jerks, partial jerk, fin flicker, burst swimming), mucus secretion all over the body, precipitation of mucus on the gills and caudal bending etc. were observed.
  44. 44.   Whereas in sub lethal concentration such behavioural changes were negligible. Suffocation caused by the mucus film on gills could be one of the reasons for the death of fish in the lethal concentration of quinalphos. Hence an approximate concentration of pesticide in the ambient medium can be predicted based on the behavioural response of the inhabitatiting animals.
  45. 45.     Behavioural characteristics are obviously sensitive indicators of toxicant effect. It is necessary, however, to select behavioural indices for monitoring that relation to the organism’s behaviour in the field in order to derive a more accurate assessment of the hazards. In the present study as evidenced by the results the abnormal changes in the fish exposed to lethal concentration of quinalphos are time dependent. However, the normal behaviour of the fish at 10 and 15 days on exposed to sub lethal concentrations indicates its adaptability to the sub lethal concentration due to long term exposure of quinalphos.
  46. 46.    Chapter 3: Studies on Respiratory distress A change in respiration rate is one of the common physiological responses to toxicants and is easily detectable through changes in oxygen consumption rate, which is frequently used to evaluate the changes in metabolism under environmental deterioration. Respiration may be defined as an oxidative process during which food materials undergo oxidation and get converted into carbon dioxide, water and energy.
  47. 47.     A reduction in oxygen consumption is observed when the fish is exposed to the toxicant and the mortality is due to effect of metabolism of energy synthesis (Tilak and Swarna Kumari, 2009). Determination of oxygen consumption of aquatic animals will undoubtedly provide information on the effects of interactions of toxicants on the physiology of aquatic life (Sarkar, 1999). Oxygen consumption is an important parameter to assess the toxicological stress, since it serves as index of energy expanded and speaks of physiological and metabolic state of an organism. Generally when toxicants gain entry through food chain or respiratory surfaces, the physiological function to be affected is oxygen consumption.
  48. 48.    Oxygen consumption (RESULT) Generally when a toxicant gains entry through food chain or respiratory surfaces, the first physiological function to be affected is whole animal oxygen consumption. In view of the vital role ascribed to the enzymes involved in the energy pathways through respiratory process by utilizing oxygen.
  49. 49.   The study of respiration at whole animal level needs emphasis to ascertain the oxygen requirements of fish under toxic environmental condition. In the present investigation the whole animal oxygen consumption was decreased under lethal and sub lethal concentrations of quinalphos indicating prevalence of respiratory distress due to interference of quinalphos in oxidative metabolism.
  50. 50.    In the present study, the oxygen consumption was gradually decreasing with increasing exposure periods as observed by Mathivanan (2004) in Oreochromis mossambicus exposed to sublethal concentrations of quinalphos. Oxygen consumption is widely considered to be a critical factor for evaluating the physiological response and a useful variable for an early warning for monitoring aquatic organisms (Chinni et al., 2000). Like most fish, common carp (Cyprinus carpio) are oxygen regulators, i.e., they maintain their oxygen consumption at a constant level along a gradient of environmental oxygen concentrations, until critical oxygen concentration is reached, and below which oxygen consumption begins to fall. Under conditions of stress, this critical oxygen concentration is likely to increase, reflecting the decreased capacity of the fish to cope with environmental perturbations.
  51. 51. Table 3: Whole animal oxygen consumption (ml/gm wet wt/h) of the fish, Cyprinus carpio on exposure to the lethal and sub lethal concentrations of quinalphos. Exposure period in days Estim ations Cont rol Mean 0.83 A Lethal 1 2 Sub lethal 3 4 1 0.0823 0. 0333 0.1646 0.2597 0.1391 E H C B D 0.001 0.011 0.002 0.003 SD ± 0.004 0.002 % Chang e ----- -90.08 -95.98 5 10 15 0.075 0.0590 0.0288 I G 8F 0.013 0.012 0.003 -80.16 -68.71 -83.24 -90.86 -96.53 -92.89 Values are Means ± SD (n=6) for oxygen consumption in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  52. 52.    Chapter 4: Studies on Quinalphos Accumulation (residue analysis) In environmental studies, certain organisms provide valuable information about chemical states of their environment not through their absence or presence but ability to concentrate environmental toxins within their tissues. Since fish spend their entire lives in the aquatic environment and are often found at higher feeding levels of the aquatic food chain, they incorporate chemicals from the environment into their body tissues through feeding relationships.
  53. 53.   Persistent hydrophobic chemicals may accumulate in aquatic organisms through different mechanisms: via the direct uptake from water by gills or skin (bioconcentration), via uptake of suspended particles (ingestion) and via the consumption of contaminated food (biomagnification). Parameters affecting these processes include lipid and water solubility, degree of ionization, chemical stability and molecular size.
  54. 54.  In the present study an attempt has been made to determine the residue levels of quinalphos in different tissues viz., gill, muscle, and liver of fish Cyprinus carpio at different exposure periods under median lethal and sublethal concentrations employing High Performance Liquid Chromatographic (HPLC) technique.
  55. 55.    Bio-accumulation (RESULT) A very important biological property of pesticide is their tendency to bioaccumulate. A significant amount of quinalphos is accumulated in the organs of the fish in both lethal and sub lethal concentrations of exposure. Relative to control a significant increase in the concentration of quinalphos is observed in the organs of the fish.
  56. 56.   However this increase is predominantly more in lethal concentration and sub lethal concentration. Further, it appeared that the amount of quinalphos accumulated in the fish is time dependent, as it increased with the increase in the exposure period in both lethal and sub lethal concentrations. The rate of accumulation of quinalphos in the fish exposed to sub lethal concentration could be due to the activation of the toxicant bioconcentration and biomagnification processes.
  57. 57.   Several authors have reported that, pesticide residue can cause cellular damage to the gill tissue, as it is the first organ to face pesticide medium (Kalavathy et al., 2001; Kanabur and Sannadurgappa, 2001, Chanchal et al., 1990 Bashamohideen et al., 1989), which offers support to the present findings. Simultaneously, the residues are absorbed by the aquatic organisms, bioaccumulated in the tropic chain and deposit in the tissues. Earlier studies revealed that, the accumulation of pesticides in fish tissues (David and Philip, 2005; .Tilak. et al., 2001; Gupta et al., 2001; Sharma, 1994) shows similar result of present study.
  58. 58. Table 4: Accumulation of quinalphos (µg/g wet wt.) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Lethal 1 Gill 98.21 H SD ± 0.013 Kidney 78.54 H SD ± 0.002 Liver 65.91 H SD ± 0.001 Sub lethal 2 3 4 155.34 312.54 F D 443.78 C 0.001 0.002 0.011 133.67 198.23 G E 218.99 C 0.001 0.002 112.23 163.91 F 0.013 1 5 10 15 148.23 257.73 G E 489.87 B 502.33 A 0.012 0.001 0.003 0.012 136.85 215.33 F D 281.34 B 376.54 A 0.001 0.001 0.003 0.001 0.011 D 197.92 C 98.79 G E 203.73 B 261.81 A 0.003 0.004 0.002 0.001 0.001 0.003 156.82 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  59. 59.    Chapter 5: Ions and associated ATPases Ions play a vital role in several body functions, viz. the monovalent ions sodium, potassium and chloride are involved in neuromuscular excitability, acid base balance and osmotic pressure (Verma et al., 1981), whereas divalent cations, calcium and magnesium facilitate neuromuscular excitability, enzymatic reactions and retention of membrane permeability. Alteration in osmotic regulatory mechanism under toxic conditions may cause severe imbalance in biochemical composition of the tissue fluids followed by undesirable metabolic consequences
  60. 60.   In freshwater fishes, blood and electrolyte concentration are regulated by interacting processes, such as absorption of electrolytes from surrounding medium through active mechanisms predominantly at the gill, control of water permeability and selective re-absorption of electrolytes from urine. Any alteration in one or more of these processes results in a change in the plasma electrolyte composition.
  61. 61.    Sodium (Na+) ion plays an important role in the osmotic regulation of body fluids and also serves as an essential activating ion for specific enzyme system. Potassium ion (K+) is the prominent intracellular cation of animals. It is an important co-factor in the regulation of osmotic pressure and acid-base balance (Sexena, 1957). Calcium ion (Ca2+) is also important osmotic effectors and plays significant role in the regulation of cellular metabolism and is involved in conferring stability to the cell membrane.
  62. 62.      Adenosine triphosphatase (ATPase) is a membrane bound enzyme group for regulating oxidative phosphorylation, ionic transport, muscle function and several other membrane transport dependent phenomena. ATPase have the central role in physiological function of cells as energy transducers by coupling the chemical reactions of ATP hydrolysis (Laugher, 1987). Membrane bound Na+- K+ ATPase is the enzymatic machinery for the active transport of sodium and potassium across the cell membrane. Magnesium translocation is dependent on membrane bound Mg2+ ATPase (Beeler, 1983). The implication of ATP inhibition as the mechanism of toxic action by insecticides has been proposed from studies on the inhibition of ATPases activities by the pesticides (Henery et al., 1996).
  63. 63.      Results In this investigation, it is evident that Na+ loss is higher in the case of gill indicating the derangement in Na+ transport and rupture in the respiratory epithelium of gill tissue (David et al., 2003). The decreases in K+ ion content in the tissues of Cyprinus carpio exposed to quinalphos might attribute to the derangement in whole animal oxygen consumption and ionic content at tissue levels as observed in the present investigation. The decrease in Ca2+ ion level indicates increased decalcification. Ca2+ is concerned with neuromuscular excitability, cell membrane permeability and regulation of protein binding capacity (Walser, 1960). In the present study, the restlessness in fish during organophosphate stress corresponds to structural change in mitochondrial integrity.
  64. 64.   In the present study, the decrease in the levels of Na+- K+, Ca2+ ions in the gill, kidney and liver exposed to lethal and sublethal concentrations of quinalphos indicates changes in the permeable properties of the cell membrane of these organs and of deranged Na+-K+ and Ca2+ ionic pumps due to the probable consequences of tissue damage. The imperative reason for the diminish of sodium, potassium and calcium ion levels in the organs of fish, exposed to quinalphos could be attributed to the suppressed activities of Na+- K+, Mg2+ and Ca2+ ATPase (Renfro et al., 1974), since ATPases have been described as prominent energy linked enzymes in fishes (Desaiah et al., 1975).
  65. 65.   However this change increased over time of exposure in lethal but decrease in sub lethal concentration. The results indicted the changes in the permeable properties of the cell membrane on exposure to quinalphos. These decreases in the ionic levels by high quinalphos concentration could be due to the consequences of tissue damage and severe disruption in cellular ionic regulation of the fish leading the osmo-regulatory failure.
  66. 66. Table 5: Sodium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure periods in days Tissue Gill ± SD % Change Kidney ± SD % Change Liver ± SD % Change Contro l 0.5443 Lethal 1 Sublethal 2 3 4 1 5 10 15 0.3324 0.3112 0.3064 0.4343 0.3422 0.3321 0.3395 F H I B D G E A 0.4211 C 0.002 0.001 0.012 0.013 0.003 0.004 0.012 0.011 0.013 ---- -22.63 -38.93 -42.82 -43.70 -20.20 -37.13 -38.98 -37.62 0.4227 0.3835 0.3338 0.2748 0.2443 0.3974 0.3468 0.2948 0.2887 A C E H I B D F G 0.003 0.011 0.002 0.003 0.013 0.004 0.011 0.003 0.013 ---- -9.27 -21.03 -34.98 -42.21 -5.98 -17.95 -30.25 -31.70 0.5033 0.4960 0.4128 0.3765 0.3284 0.4995 0.4674 0.3877 0.3855 A C E H I B D G F 0.002 0.001 0.013 0.004 0.011 0.004 0.012 0.004 0.012 ---- -1.45 -17.98 -25.19 -34.75 -3.63 -7.13 -22.96 -23.41 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  67. 67. Table 6: Potassium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure periods in days Tissue Contro l Lethal Sublethal 1 2 3 4 1 5 10 15 0.9937 0.9025 0.8737 0.8152 0.7430 0.9832 0.9453 0.8743 0.7998 A D F G I B C E H ± SD 0.011 0.001 0.002 0.003 0.013 0.011 0.012 0.001 0.011 % Change ---- -15.12 -23.46 -41.58 -45.71 -7.85 -13.75 -20.55 -11.95 1.0743 0.9833 0.9023 0.8764 0.7289 0.9673 0.9567 0.8346 0.7321 A B E F I C D G H ± SD 0.002 0.011 0.002 0.001 0.004 0.001 0.002 0.004 0.012 % Change ---- -8.47 -16.01 -18.42 -32.15 -9.95 -10.94 -22.31 -31.85 0.8837 0.8027 0.7655 0.7121 0.6525 0.8665 0.7964 0.7343 0.6678 A C E G I B D F H ± SD 0.003 0.012 0.002 0.004 0.003 0.011 0.003 0.004 0.013 % Change ---- -9.16 -13.37 -19.41 -26.16 -1.94 -9.87 -16.91 -24.43 Gill Kidney Liver Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  68. 68. Table 7: Calcium ion content (µg/g wet wt.) in the tissues of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure periods in days Tissue Gill ± SD % Change Kidney ± SD % Change Liver ± SD % Change Contro l Lethal Sublethal 1 2 3 4 1 5 10 15 0.8627 0.7932 0.7457 0.7217 A D E H 0.7035 I 0.8112 B 0.7976 C 0.7523 0.7398 F G 0.001 0.003 0.011 0.002 0.001 0.012 0.004 0.003 0.013 ---- -8.05 -13.56 -16.34 -18.45 -5.96 -7.54 -12.79 -14.24 1.2232 1.0538 0.9425 0.9228 1.1123 B 0.9758 D 0.9566 0.8985 G 0.8847 I A C F E H 0.011 0.001 0.003 0.002 0.013 0.004 0.002 0.003 0.012 ---- -13.84 -22.94 -24.55 -27.67 -9.06 -20.22 -21.79 -26.54 0.9128 0.8835 0.8663 0.7968 E G 0.8967 B 0.8856 C 0.7769 D 0.7062 I 0.8563 A F H 0.004 0.003 0.011 0.002 0.003 0.004 0.013 0.012 0.001 ---- -3.21 -5.09 -12.71 -22.63 -1.76 -2.97 -6.18 -14.88 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  69. 69. Table 8: Na+-K+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on exposure to the lethal and sub lethal concentrations of quinalphos. Exposure period in days Organ Gill SD ± % Change Kidney SD ± % Change Liver SD ± % Change Contro l Lethal 1 3.7512 A 3.3112 C Sub lethal 2 3 4 1 5 10 15 3.0245 2.9137 2.0621 3.4185 3.2233 2.7823 3.0579 D G I B E H F 0.003 0.012 0.004 0.003 0.001 0.002 0.004 0.011 0.003 ------ -11.64 -19.34 -22.52 -44.88 -9.11 -19.43 -25.69 -18.63 3.2234 2.8544 2.0653 1.1647 0.9853 2.9785 2.6355 A C G H I B E 2.1866 F 0.002 0.001 0.003 0.002 0.004 0.011 0.012 0.004 0.002 ----- -11.44 -35.92 -63.86 -69.43 -7.59 -18.23 -32.16 -16.65 3.5495 3.0344 2.5755 2.2785 2.6507 2.1756 2.7864 B F G 1.5893 I 2.9709 A C E H D 0.001 0.002 0.003 0.004 0.011 0.013 0.012 0.002 0.004 ------ -14.51 -27.44 -35.81 -55.22 -16.30 -25.32 -38.71 -21.49 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test. 2.6866 D
  70. 70. Table 9: Mg2+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Gill SD ± % Change Kidney SD ± % Change Liver Contro l Lethal Sub lethal 1 2 3 1 5 10 15 3.2233 2.6553 2.0656 1.7785 A D G H 0.9365 I 2.8577 2.3754 2.4743 2.7873 B F E C 0.004 0.002 0.003 0.011 0.002 0.012 0.004 0.011 0.001 ------- -17.62 -35.91 -44.82 -70.94 -11.34 -26.31 -23.23 -13.52 3.5434 3.1112 D 2.5754 1.4865 1.2668 3.3843 2.6987 2.2378 3.3874 F G H C D F B 0.011 0.002 0.004 0.012 0.004 0.001 0.002 0.013 0.003 ------ -12.19 -27.31 -58.04 -64.24 -4.49 -23.83 -36.84 -4.41 3.8745 2.6873 2.4836 1.4884 3.6775 3.2862 1.8833 A D E H B C G A 4 0.1896 I 2.3885 F 0.003 0.002 0.004 0.011 0.012 0.003 0.002 0.001 0.003 SD ± % are Means ------30.64 -35.89 -61.58 -95.11 -5.08 -15.18 -51.39 Values Change ± SD (n=6) for a tissue in a column followed by the same letters are not significantly -38.35 different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  71. 71. Table 10: Ca2+ ATPase activity (µM of Pi formed / mg protein / h) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Gill SD ± % Change Kidney SD ± % Change Liver SD ± % Change Contro l Lethal Sub lethal 1 2 3 4 1 5 10 15 3.9687 3.5646 3.3881 2.0965 1.2793 3.6945 3.2899 2.0455 3.6235 A D E G I B F H C 0.012 0.013 0.001 0.003 0.014 0.013 0.014 0.013 0.011 ------ -10.18 -14.62 -47.17 -67.76 -6.91 -17.11 -48.45 -8.69 3.2676 2.2443 1.9677 1.7894 1.1673 2.8673 2.4879 2.2853 2.9878 A F G H I C D E B 0.022 0.011 0.012 0.015 0.023 0.014 0.013 0.014 0.012 ------ -31.31 -39.78 -45.23 -64.27 -12.25 -23.86 -30.06 -8.56 2.4784 2.2894 2.1984 1.7783 1.5835 2.2569 1.9963 1.8343 2.3785 A C E H I D F G B 0.001 0.013 0.011 0.014 0.012 0.014 0.011 0.002 0.014 ----- -7.62 -11.29 -28.24 -36.10 -8.93 -19.45 -25.98 -4.03 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  72. 72.      Chapter 6: Studies on Histopathology Histopathology is mainly directed to study the effect of chemicals and pesticides on the structural components of the living system and the ways in which cells and tissues respond to injury. As an indicator of exposure to pollutants, histology represents a useful tool to assess the degree of pollution, particularly for lethal and chronic effects. The severity of histological damages in any particular aquatic organism is directly proportional to the concentration of a pollutant in the medium. Gill, kidney and liver (Bucher and Hofer, 1993) are suitable organs for histological examination to determine the effect of pollution.
  73. 73.     Histopathological study (RESULT) Gill Gills are the vital organs for respiration in fish which establish a direct contact with the medium through which a pollutant largely enters into the body. The gills of fish are the main target organs for toxic action of chemical pollutants, as well as for detoxification process.
  74. 74.     Fish on exposure to the lethal concentration on day 1, the enlargement of the base of primary gill lamellae was observed. On day second lamellar oedema and secondary gill lamellae clubbing at the distal end was seen leading towards telangiectatic secondary lamellae. On day 3rd lamellar telangiectasis was observed, which continued up to day 4. Lamellar hypertrophy and lamellar hyperplasia was observed. Fusion of these lamellae was noticed all along their length on day 4. The cells seemed to have undergone a clear increased hyperplasia further desquamation of the hyperplasic epithelium and capillaries with loss of the lamellar structure of the gill were seen all along the gill filaments.
  75. 75. H&E:X 100 and X200 24 hr exposed gill to lethal dose of quinalphos Control Fish Gill 48 hr exposed gill to lethal dose of quinalphos
  76. 76. H&E:X 100 and X200 72 hr exposed gill to lethal dose of quinalphos Control Fish Gill 96 hr exposed gill to lethal dose of quinalphos
  77. 77.    In the sublethal concentration of quinalphos a mild degree of degenerative changes and sign of shrinkage in the primary gill lamellae was observed at day 1 exposure. The slight damage to the base of secondary lamellae and inter lamellar tissue was observed. On day 5 the changes were more marked when compared to day 1. The degeneration of epithelial cells encapsulating primary and secondary gill lamellae with necrosis. Fusion of secondary lamellar, bubbling of primary gill lamellae, atrophy is also observed. The excess secretion of mucus and necrosis of basal filament was observed. But on further exposures, for 10 and 15 days the gill structure was just similar to that of control fish except mild degree of precipitation of mucus over the gill lamellae.
  78. 78. H&E:X 100 and X200 Day 1st exposed gill to lethal dose of quinalphos Control Fish Gill Day 5th exposed gill to lethal dose of quinalphos
  79. 79. H&E:X 100 and X200 Day 10 exposed gill to lethal dose of quinalphos th Control Fish Gill Day 15th exposed gill to lethal dose of quinalphos
  80. 80.    Liver Fish liver is regarded as a major site of storage, biotransformation and excretion of pesticides. These organs have been proven to be indicative of pollution. The liver has the ability to degrade toxic compounds, but its regulating mechanisms can be overwhelmed by elevated concentrations of these compounds, and could subsequently result in structural damage.
  81. 81.     On day 1 of exposure to the lethal concentration of quinalphos, the liver of fish exhibited enlarged nuclei and vacuolization in hepatic cells. Liver cords were seen disarrayed. On day 2 of exposure, the parenchymatous nature of the liver was greatly disrupted with congested blood vessels. The hepatocyte cell membranes were ruptured and granular degeneration was evident in most of the hepatocytes. Nuclei became slightly hypertrophic. Further on day 3 severe degrees of atrophic changes were noticed in the liver cords. Hemorrhagic condition was prominent with heavy vacuolization in the liver tissue. At some regions exfoliation and congregation of hepatocytic nuclei and focal necrosis were seen. This was followed by the severe degree of vacuolization, shrinkage of hepatocytes, atrophy, cytoplasmic degeneration, rupture of blood vessels, diffused necrosis, dissolution of laminar structure and cytoplasmic disintegration in hepatocytes on day 4 of exposure.
  82. 82. H&E:X 200 Control Fish and X400 Liver 24 hr exposed fish liver to lethal dose of quinalphos 48 hr exposed fish liver l to lethal dose of quinalphos
  83. 83. H&E:X 200 Control Fish and X400 Liver 72 hr exposed fish liver to lethal dose of quinalphos 96 hr exposed fish liver l to lethal dose of quinalphos
  84. 84.    Compared to the structure of the liver of control fish, exposed to sublethal concentration of quinalphos initially exhibited few changes like slight disarray of liver lobes, mild degree of degeneration of cytoplasm, occasional blood clots and congregation of nuclei at day 1 and cloudy swelling of hepatocytes, granulization of cytoplasm, hypertrophic and pyknotic nuclei on day 5. However, on further exposure to day 10 certain degree of reorganization in the structure of liver cords was observed. The nuclei appeared normal, with a very little degree of cytoplasmic vacuolization. At 15 days of exposure, no significant changes were seen different from controls, except a slight degree of hyperchromatic condition of the nuclei.
  85. 85. H&E:X 200 Control Fish and X400 Liver Day 1st exposed fish liver to lethal dose of quinalphos Day 5th exposed fish liver to lethal dose of quinalphos
  86. 86. H&E:X 200 Control Fish and X400 Liver Day 10th exposed fish liver to lethal dose of quinalphos Day 15th exposed fish liver to lethal dose of quinalphos
  87. 87.    Kidney In fish, the kidney performs an important function related to electrolyte and water balance and the maintenance of a stable internal environment. The kidney of fish receives much the largest proportion of post-branchial blood, and therefore renal lesions might be expected to be good indicators of environmental pollution.
  88. 88.     In lethal concentration of quinalphos the kidney showed reduction in renal cell number in the proximal and distal collecting tubules, which have resulted in narrowness of lumen. On day 1 the tubular cells have undergone hypertrophy and some of the renal tubules have lost their normal shape. On day 2 Vacuolization due to degeneration of cytoplasm is quite obvious. The nuclei of epithelial cells have become quite dominant and are found infiltrating into the surrounding tissue. The perforation of kidney tubules is commonly observed. On day 3 the kidney demonstrated hyperplasia, vacuolization, degeneration and necrosis leading to the complete necrosis. Cubiodal epithelial cells lining the tubules showed complete vacuolization with degenerating cytoplasm and more nuclear division and their disorderly scattering nature. The hemopoietic tissue was fully studded with lymphatic cells at the highest rate of nuclear division. The lumen of the tubules was found to be dilated. Kidney tubules were also found to be perforated on day 4.
  89. 89. H&E:X 200 Control Fish and X400 Kidney 24 hr exposed kidney to lethal dose of quinalphos 48 hr exposed kidney to lethal dose of quinalphos
  90. 90. H&E:X 200 Control Fish and X400 Kidney 72 hr exposed kidney to lethal dose of quinalphos 96 hr exposed kidney to lethal dose of quinalphos
  91. 91.      In sublethal concentration of quinalphos the kidney of the fish exhibited a mild degree of changes. 1st, 5th and 10th day of shows more changes i.e., epithelial cells of the tubules which showed desquamation, irregular orientation of the nuclei in the cells, lumen of the tubules became wider as a result of flattening of epithelial cells. Ruptures of the tubules were quite prominent, cell fragments could be seen inside the lumen of some tubules. Vacuolar degeneration was seen in the few tubules. Hemopoeitic tissue was degenerated. But on 15th day kidney showed recovery tendency. Glomerular cells attained normalcy in structure. Cytoplasm appeared clear and vacuolization and karyolysis of cell was completely reduced. Necrotic changes in uriniferous tubules were reduced. Clumping of damaged blood cells was seen.
  92. 92. H&E:X 200 Control Fish and X400 Kidney Day 1 exposed kidney to lethal dose of quinalphos st Day 5th exposed kidney to lethal dose of quinalphos
  93. 93. H&E:X 200 Control Fish and X400 Kidney Day 10th exposed kidney to lethal dose of quinalphos Day 15th exposed kidney to lethal dose of quinalphos
  94. 94. Ultrastructure Control Fish liver In control fish the hepatocytes are characterized by a system of parallelly stacked cisternae of rough ER in the vicinity of the centrally located round nucleus, associated by a few mitochondria and a few other cell organelles such as lysosomes and peroxisomes. The cellular compartmentation is clearly separated into a central, organelle-rich area and a peripheral cell area with storage material, consisting mainly of glycogen.
  95. 95. Ultrastructure of Fish liver exposed to sublethal dose of quinalphos In comparison to the controls, hepatocytes of Cyprinus carpio exposed to quinalphos, demonstrated a loss of cellular compartmentation. The organelles were distributed through the entire cytoplasm and the amount of glycogen was decreased compared to controls. The rough ER showed numerous structural alterations including proliferation, fragmentation and vesiculation of cisternae and little dilation and degranulation. Lysosomes, peroxisomes, macrophages and myelin-like bodies increased in number in quinalphos exposed fish compared to controls. Generally, ultrastructural responses in liver of carp were significantly different between the control.
  96. 96. Ultrastructure of Fish liver exposed to sublethal dose of quinalphos Hepatocytes of common carp exposed to the sublethal concentration of quinalphos. Cells with a disturbed cellular compartmentation. The RER is dilated and vesiculated, the amount of glycogen is reduced. ga: Golgi apparatus, RER: rough endoplasmic reticulum.
  97. 97.    Chapter 7: Study on Metabolic and Enzymological aspects To understand quinalphos induced oxidative damage on changes in the protein, malondialdehyde and protein carbonyls levels in the tissue of control and experimental fish tissues. Enzymes associated also studied i.e., AChE activity, and catalase activity in the control and exposed fish.
  98. 98.      Biochemical Studies (RESULT) Organophosphates are neuro toxins that disrupt the central nervous system of animals by inhibiting the enzyme acetylcholinesterase (Rangarsdottir, 2000). Inhibition of acetylcholinesterase activity results in changes in behaviour and morphological alterations seen in the test animal. The enzyme AChE and its closely related ones are responsible for the toxicity of organophosphates (OP) compounds to vertebrates. In the present study, the level of AChE activity in gill, kidney and liver tissues of fish, C. carpio exposed to quinalphos were decreased suggesting the increase in the level of ACh
  99. 99.   Depression of acetyl cholinesterase activity suggests decreased cholinergic transmission and consequent accumulation of acetylcholine in the tissues leading to cessation of nerve impulses. This has lead to behavioral and morphological changes due to impaired neurophysiology of the fish.
  100. 100. Table 11: Ach level (µM/g wet wt.) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Contr ol Lethal 1 2 133.15 G 147.73 D Sub lethal 3 156.22 4 1 5 10 154.55 C Gill 86.54 I SD ± % Change 0.001 0.001 0.002 0.012 0.003 0.002 0.013 0.011 0.002 ------ 53.86 70.71 80.52 88.21 41.97 58.48 66.55 78.59 Kidney 59.58 I 94.89 G 119.11 A 84.34 H 97.23 F 108.51 D 113.63 B SD ± % Change 0.003 0.012 0.013 0.011 0.003 0.001 0.012 0.003 0.002 ------ 59.26 71.75 89.06 99.92 41.56 63.19 82.12 90.72 Liver 66.98 I 118.72 F 123.91 C 132.24 A 95.45 H 119.93 E 121.23 D B 102.33 E 112.64 C 128.44 B 162.88 A 122.86 H 137.15 F 144.13 E 15 105.34 G 0.002 0.003 0.001 0.002 0.011 0.001 0.013 0.003 0.002 SD ± % Change 77.25 91.76 40.51 57.27 79.05 80.99 Values are Means ± -----SD (n=6) for a tissue 85.00 in a column followed97.43 same letters are not significantly different (P by the ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  101. 101. Table 12: AChE activity (nM of acetylthiocholine iodide hydrolyzed/mg protein/min) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Contro l Lethal Sub lethal 1 2 3 135.34 123.29 D E 110.87 G 4 102.67 1 5 10 166.47 Gill 197.21 A SD ± % Change 0.001 0.003 0.002 0.012 0.011 0.001 0.021 0.003 0.011 ------ -31.37 -54.61 -70.03 -85.27 -20.31 -48.94 -79.69 -30.33 112.23 152.86 133.51 169.99 H C 147.15 D F B Kidney 178.28 A 138.21 E 121.87 G 103.22 I H 157.15 C 120.29 F 101.34 I 15 B SD ± % Change 0.003 0.002 0.012 0.002 0.011 0.001 0.003 0.011 0.003 ------ -22.47 -40.81 -61.59 -63.98 -14.25 -20.36 -30.42 -6.20 Liver 186.44 A SD ± % Change 0.003 0.001 0.002 0.003 ----- -33.90 -54.81 -71.48 123.23 E 118.89 G 101.45 I 111.67 H 155.97 C 131.71 D 0.012 0.001 -73.70 -16.34 119.53 F 161.23 B 0.013 0.003 0.011 -35.09 -58.394 -35.88 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  102. 102.    The assessment of the protein content can be considered as a diagnostic tool to determine the physiological process of the cell (David et al., 2004). Proteins are involved in major physiological events. Therefore, the estimation of protein under toxic stress is useful to determine the physiological phases of organisms (Kapila and Ragothaman, 1999). The pesticides are found to alter the structural and soluble proteins by causing histopathological and biochemical changes in the cell (Shakoori et al., 1976).
  103. 103.     Among the exposure periods, in lethal concentrations, the levels of soluble, structural and total proteins significantly decreased in the gill, kidney and liver relative to its controls. Protein synthesis is an energetically expensive process. It appears that protein degradation is in active phase over synthesis in the gill, kidney and liver of fish, at day 1 and 5 of exposure to the sublethal concentration of quinalphos as evidenced from the decrease in soluble, structural and total proteins with the significant increase in amino acid levels. An increase in free amino acid content, which might have come as a result of tissue damage, is also suggestive finding of the present study.
  104. 104. Table 13: Soluble protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal Sub lethal 1 2 3 4 1 5 10 15 Gill 16.54 A 15.52 B 14.44 D 13.21 G 10.71 I 15.01 C 12.96 H 13.63 F 14.12 E SD ± 0.001 0.002 0.011 0.004 0.003 0.013 0.002 0.003 0.011 % Change ------ -6.16 -12.69 -20.13 -35.24 -9.25 -21.64 -17.59 -14.63 Kidney 15.92 A 14.97 B 13.88 C 13.72 D 9.64 I 13.65 E 11.12 H 12.91 G 13.36 F SD ± 0.011 0.001 0.003 0.013 0.014 0.002 0.003 0.004 0.012 % Change ------ -5.96 -12.81 -13.81 -39.44 -14.25 -30.15 -18.91 -16.08 Liver 22.92 A 20.86 B 19.05 C 17.25 F 10.88 I 18.81 D 16.87 H 17.18 G 17.97 E SD ± 0.002 0.001 0.003 0.002 0.011 0.004 0.013 0.012 0.004 % Change ----- -8.98 -16.88 -24.73 -52.53 -17.93 -26.39 -25.04 -21.59 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  105. 105. Table 14: Structural protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal Sub lethal 1 2 3 4 1 5 10 15 Gill 22.39 A 20.11 B 17.65 E 15.56 H 11.82 I 19.21 C 15.91 G 17.34 F 18.02 D SD ± % Change 0.001 0.003 0.012 0.001 0.021 0.003 0.004 0.003 0.013 ------ -10.18 -21.17 -30.51 -47.21 -14.20 -28.94 -22.55 -19.51 Kidney 19.88 A 17.45 B 15.66 E 12.52 H 10.61 I 16.88 C 14.22 G 15.35 F 16.54 D SD ± % Change 0.012 0.002 0.004 0.011 0.014 0.021 0.013 0.001 0.013 ------ -12.22 -21.22 -37.02 -46.62 -15.09 -28.47 -22.78 -16.81 Liver 29.77 A 25.92 B 22.13 D 19.04 G 15.64 I 22.73 C 18.23 H 19.22 F 20.71 E SD ± 0.003 0.001 0.021 0.004 0.011 0.023 0.013 0.004 0.002 % Change ----- -12.93 -25.66 -36.04 -47.46 -23.64 -38.76 -35.43 -30.43 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  106. 106. Table 15: Total protein contents (mg of protein/g wet wt. of tissue) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal Sub lethal 1 2 3 4 1 5 10 15 Gill 55.87 A 50.12 B 47.44 D 39.84 G 35.35 I 48.65 C 39.77 H 42.07 F 45.78 E SD ± 0.012 0.001 0.002 0.013 0.004 0.014 0.004 0.003 0.013 % Change ------ -10.29 -15.08 -28.69 -36.72 -12.92 -28.81 -24.71 -18.05 Kidney 47.93 A 42.67 B 39.03 E 33.67 H 28.32 I 40.65 C 35.99 G 37.45 F 39.11 D SD ± 0.003 0.002 0.013 0.021 0.003 0.013 0.002 0.001 0.002 % Change ------ -10.97 -18.56 -29.75 -40.91 -15.18 -24.91 -21.86 -18.41 Liver 95.32 A 91.34 B 88.11 D 82.78 G 75.99 I 89.98 C 82.06 H 85.27 F 87.66 E SD ± 0.013 0.003 0.001 0.012 0.021 0.013 0.004 0.002 0.001 % Change ----- -4.17 -7.56 -13.15 -20.27 -5.61 -13.91 -10.54 -8.03 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  107. 107. Table 16: Free amino acid levels (µmol of tyrosine equivalents/g wet wt.) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Contro l Lethal Sub lethal 1 2 3 4 1 5 10 15 Gill 7.04 I 7.55 H 8.22 F 9.33 D 9.88 C 7.87 G 8.99 E 10.51 B 10.92 A SD ± 0.011 0.002 0.003 0.004 0.001 0.013 0.012 0.014 0.003 % Change ------ 2.17 13.06 26.28 29.54 6.84 17.57 34.21 39.51 Kidney 5.33 I 5.82 H 6.68 F 7.76 D 7.92 C 6.05 G 6.96 E 7.97 B 8.29 A SD ± 0.003 0.013 0.021 0.002 0.001 0.004 0.023 0.002 0.003 % Change ------ 9.26 25.45 45.61 48.66 13.61 30.71 49.60 55.51 Liver 12.65 I 13.42 H SD ± % Change 0.021 0.002 0.011 0.013 0.004 0.003 0.001 0.023 0.014 ----- 6.08 21.50 32.96 38.65 13.75 32.56 49.17 57.86 15.37 F 16.82 D 17.54 C 14.39 G 16.77 E 18.87 B 19.97 A Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  108. 108.     Oxidative stress Among the most commonly used biomarkers, those related to oxidative stress assume an important position, being frequently used both in environmental monitoring and laboratory assays (Pandey, et al., 2003). Rates or amounts of reactive oxygen species (ROS) production can be increased by the presence of a wide range of natural and man-made xenobiotics (Livingstone, 2001). The stimulation of free radical production, induction of lipid peroxidation, and disturbance of the total antioxidant capability of the body are mechanisms of toxicity for most pesticides (Abdollahi, et al., 2004).
  109. 109.  Results  The increase in catalase activity and H2O2 level observed in all the organs of fish at all the exposure periods studied in the lethal concentration of quinalphos. The increase in MDA level in all the organs of fish at all the exposure periods studied. The steep increase in protein carbonyl observed in all the organs of fish at all the exposure periods studied.  
  110. 110.    In the present study, lethal and sublethal concentrations of quinalphos were resulted in the significant decrease of antioxidant enzymes with concomitant increase in the lipid peroxidation in time-dependent manner when compared with corresponding control groups. The cellular defense machinery against H2O2 is very efficient and involves both low molecular weight antioxidants and enzymes such as catalase and glutathione peroxidase. Catalase activity increased during experimental periods and is probably a response to toxicant stress and serves to neutralize the impact of increased ROS generation (John et al., 2001; Zaidi and Slotani, 2010)
  111. 111.    The most widely used assay for lipid peroxidation is the malondialdehyde (MDA) formation, which represents the secondary lipid peroxidation product with the thiobarbituric acid reactive substances test (Draper et al. 1993; Janero 1990). Malondialdehyde (MDA) is the final product of lipid peroxidation. The concentration of MDA is the direct evidence of toxic processes caused by free radicals (Sieja and Talerczyk 2004). The formation of carbonyl proteins is non-reversible, causing conformational changes, decreased catalytic activity in enzymes and ultimately resulting, owing to increased susceptibility to protease action, in breakdown of proteins by proteases (Zhang et al., 2008).
  112. 112.      Increase in the protease activity as evidenced from the present study suggests that damage to proteins thus releasing their monomers due to oxidative damage and chopping by protease. Protein degradation is in active phase over synthesis in the kidney, gill and liver of fish during experimental periods in both the lethal and sublethal concentration of quinalphos. Moreover our results recommended that oxidative stress may, in part, be contributing to quinalphos induced hepatic, renal and gill damage. It may provide an indication of quinalphos is the affected carp C. carpio. Also our results indicated that the adverse effects of quinalphos on most of biochemical parameters, lipid peroxidation and enzymatic activities if used in low concentration. Also, produced oxidative stress in fish gill more than liver and kidney both at catalase activity and MDA levels.
  113. 113. Table 17: Catalase activity (mmol of hydrogen peroxide decomposed/mg protein/min) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Contro l Lethal 1 2 Sub lethal 3 4 1 5 10 15 Gill 6.56 I 7.12 H 7.99 E 9.23 B 7.33 G 7.38 F 8.16 D 9.47 A 8.56 C SD ± 0.014 0.002 0.004 0.013 0.011 0.023 0.001 0.002 0.012 % Change ------ 8.53 21.79 40.71 11.73 12.51 24.39 44.35 30.48 Kidney 3.44 I 3.96 H 4.57 E 4.96 B 4.11 G 4.23 F 4.78 D 5.23 A 4.83 C SD ± 0.011 0.001 0.002 0.003 0.004 0.002 0.021 0.013 0.001 % Change ------ 15.11 32.84 44.18 19.47 22.96 38.95 52.03 40.41 Liver 4.96 I 5.42 H 6.11 E 7.12 B 5.72 G 5.88 F 6.54 D 7.32 A 6.77 C SD ± 0.001 0.003 0.002 0.004 0.011 0.013 0.021 0.012 0.002 % Change ----- 9.27 23.18 43.54 15.32 18.54 31.85 47.58 36.49 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  114. 114. Table 18: Hydrogen peroxide levels (nmol of hydrogen peroxide/mg protein) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal Sub lethal 1 2 3 4 1 5 10 15 Gill 6.45 I 6.56 H 6.67 F 6.92 C 6.76 E 6.72 G 6.83 D 7.24 B 7.37 A SD ± 0.002 0.011 0.021 0.004 0.013 0.003 0.003 0.014 0.003 % Change ------ 1.71 3.41 7.28 4.81 4.18 5.89 12.24 14.26 Kidney 2.83 I 2.96 H 3.12 F 3.22 C 3.15 E 3.07 G 3.18 D 3.31 A 3.27 B SD ± 0.001 0.004 0.001 0.023 0.024 0.013 0.004 0.012 0.004 % Change ------ 4.59 10.24 13.78 11.30 8.48 12.36 16.96 15.54 Liver 4.53 I 4.66 G 4.81 E 5.02 C 4.64 H 4.78 F 4.96 D 5.44 B 5.54 A SD ± 0.004 0.011 0.023 0.001 0.004 0.003 0.002 0.021 0.012 % Change ----- 2.86 6.18 10.81 2.42 5.51 9.49 20.08 22.29 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  115. 115. Table 19: MDA levels (nmol of TBARS formed/mg of protein) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal 1 2 Sub lethal 3 4 1 5 10 15 F 3.031 D 2.656 E 2.198 G 3.075 C 3.262 A 3.11 B 2.522 Gill 1.423 I 1.835 H SD ± 0.001 0.011 0.013 0.003 0.021 0.014 0.002 0.001 0.004 % Change ------ 28.87 77.46 113.38 86.61 54.22 116.19 129.57 119.01 Kidney 0.298 I 0.323 H F 0.407 B 0.393 D 0.335 G 0.383 E 0.425 A SD ± 0.003 0.003 0.011 0.013 0.001 0.031 0.013 0.004 0.002 % Change ------ 8.38 21.81 36.57 31.87 12.41 28.52 42.61 36.24 Liver 0.402 I 0.468 H F 0.633 C 0.597 E 0.501 G 0.612 D 0.696 A 0.636 B SD ± 0.011 0.002 0.004 0.0013 0.021 0.015 0.001 0.014 0.013 % Change ----- 16.41 35.57 57.46 48.51 24.62 52.23 73.13 58.20 0.363 0.545 0.406 C Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  116. 116. Table 20: Protein carbonyls (nmol of DNPH incorporated/mg protein) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Lethal Contr ol Sub lethal 1 2 3 4 1 5 10 15 0.331 0.348 0.402 0.422 0.345 0.395 0.442 0.468 H F D C G E B A Gill 0.323 I SD ± 0.013 0.001 0.012 0.011 0.003 0.002 0.021 0.022 0.004 % Change ------ 2.47 7.73 24.45 30.65 6.81 22.29 36.84 44.89 Kidney 0.847 I 0.896 0.977 1.112 1.218 0.932 1.011 1.233 1.341 H F D C G E B A SD ± 0.004 0.002 0.001 0.003 0.004 0.001 0.021 0.013 0.022 % Change ------ 5.78 15.34 31.05 42.85 10.03 19.36 45.21 58.20 Liver 0.511 I 0.553 0.591 0.644 0.674 0.577 0.624 0.702 0.737 H F D C G E B A SD ± 0.021 0.002 0.001 0.004 0.014 0.011 0.003 0.021 0.001 % Change ----- 8.21 15.65 26.02 31.89 12.91 22.11 37.37 44.22 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  117. 117. Table 21: Protease activity (µmol of tyrosine equivalents/mg protein/h) in the organs of fish, Cyprinus carpio on exposure to the lethal and sublethal concentrations of quinalphos. Exposure period in days Organ Control Lethal 1 Gill 0.634 I SD ± 0.665 2 Sub lethal 3 4 1 5 0.822 0.847 0.696 D C G 0.764 E H 0.735 F 0.002 0.001 0.021 0.013 0.021 0.004 % Change ------ 4.88 15.93 29.65 33.59 9.77 Kidney 0.523 I 0.628 F 0.654 E 0.675 0.595 H C G 0.657 D SD ± 0.011 0.033 0.003 0.001 0.014 0.003 % Change ------ 10.32 20.07 25.04 29.06 Liver 0.753 I 0.783 0.837 0.948 0.958 H G D SD ± 0.003 0.002 0.001 % Change ----- 3.98 11.15 0.577 10 0.874 15 B 0.902 A 0.003 0.011 0.001 20.50 37.85 42.27 0.711 B 0.733 A 0.021 0.013 0.022 13.76 25.62 35.94 40.15 C 0.838 F 0.933 E 0.014 0.004 0.002 25.89 27.22 11.28 1.112 B 1.231 A 0.011 0.001 0.021 23.91 47.67 63.47 Values are Means ± SD (n=6) for a tissue in a column followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  118. 118.     Chapter 8: Studies on Haematology The study of blood parameters in fishes has been widely used for the detection of physiopathological alterations in different conditions of stress (Nussey et al., 1995). Moreover, haematological parameters are closely associated to the response of the fish to the environment (Tiwari and Sing, 2006). Accordingly, haematology can be used as clinical tool for the investigations of physiological and metabolic alterations in fish caused by pollution of the aquatic environment (Anand Kumar, 1994).
  119. 119.   Blood is a vehicle for quickly mobilizing defense against trauma and diseases. Since, fishes differ considerably in their activity patterns and respond to the pollutant. The blood parameters like Red blood corpuscle (RBC), white blood corpuscle (WBC), Haemoglobin (Hb), Packed cell volume (PCV), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH) and Mean corpuscular haemoglobin concentration (MCHC) are commonly studied in fishes to assess the impact of pesticides in aquatic biota.
  120. 120.    Hematological Study (RESULT) There was decrease in RBC, Hb, and values of MCH, MCHC, PCV and MCV on the fish exposed to both lethal and sub lethal concentrations of quinalphos. Whereas WBC recorded an elevation. The effect is conspicuous under quinalphos toxicity suggesting augmentation of additive effect on RBC and haemoglobin synthesis and leucocytosis.
  121. 121.    Measurement of haematological parameters are important in diagnosing the structural and functional status of animals exposed to the toxicant because blood parameters are highly sensitive to environmental or physiological changes and health conditions. Pesticides are known to alter the blood parameters of fishes. A significant decrease in RBC, Hb content and PCV has been observed earlier in fishes exposed to different pesticides. The findings of the present investigation also reveal a similar decreasing trend in all the parameters such as RBC, Hb content and PCV suggesting that the Organiphosphorous pesticides also induce changes which give evidence for decrease haematopoiesis followed by anemia induction in test fishes.
  122. 122. Table 22: RBC count (x106/mm3) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Parame ter Lethal Control Sublethal 1 2 3 4 1 5 10 15 1.53 A 1.42 B 1.36 D 1.11 H 0.95 I 1.29 E 1.17 G 1.21 F 1.39 C SD ± 0.003 0.025 0.013 0.012 0.015 0.014 0.012 0.016 0.012 % Change ---- -7.18 -11.11 -27.45 -37.90 -15.68 -23.52 -20.91 -9.15 RBC Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  123. 123. Table 23: WBC count (x103/mm3) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Paramete Contro r l Lethal Sublethal 1 2 3 4 10.45 F 10.67 E 9.78 G 8.59 H 6.49 I SD ± 0.022 0.011 0.013 0.011 % Change ---- 2.11 -6.41 -17.79 WBC 1 5 10 D 12.31 B 12.87 A 0.0123 0.013 0.012 0.013 0.012 -37.89 2.67 17.79 23.15 14.16 10.73 15 11.93 C Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  124. 124. Table 24: Haemoglobin level (g/100 ml) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Paramet er Haemogl obin Lethal Control Sublethal 1 2 3 4 1 5 10 15 7.64 A 6.55 D 5.94 G 3.87 H 3.53 I 6.88 B 6.12 F 6.34 E 6.62 C SD ± 0.011 0.012 0.013 0.012 0.014 0.003 0.015 0.012 0.011 % Change ---- -14.26 -22.25 -49.34 -53.79 -9.94 -19.89 -17.01 -13.35 Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  125. 125. Table 25: PCV level (%) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Param Control eter 25.22 A PCV Lethal Sublethal 1 2 3 4 23.21 20.55 16.73 C D H 10.49 I 1 23.58 B 5 10 15 19.96 E 18.64 G 19.66 F SD ± 0.012 0.001 0.013 0.012 0.014 0.015 0.013 0.002 0.021 % Chang e ---- -7.96 -18.51 -33.66 -58.41 -6.50 -20.85 -26.09 -22.04 Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  126. 126. Table 26: MCV level (cu mm) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Param eter Contr ol Lethal Sublethal 1 2 3 70.32 75.33 82.21 92.83 H G C A 87.37 B SD ± 0.013 0.001 0.014 0.032 % Chang e ---- 7.12 16.91 32.01 MCV 4 1 5 78.73 79.26 10 15 75.65 F 72.81 I E D 0.002 0.021 0.003 0.013 0.012 24.24 11.95 12.71 7.57 3.52 Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  127. 127. Table 27: MCH level (pg) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Parame ter MCH SD ± % Change Cont rol Lethal 1 2 3 Sublethal 4 1 5 10 15 22.38 23.84 24.92 26.79 28.66 24.87 27.23 27.48 25.12 I H F D 0.022 0.003 0.023 0.012 ---- 6.52 11.34 A 0.011 19.71 28.06 G C B E 0.014 0.001 0.015 0.013 11.12 21.67 22.78 12.24 Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  128. 128. Table 28: MCHC level (%) in the blood of the fish, Cyprinus carpio on exposure to lethal and sublethal concentrations of quinalphos. Exposure periods in days Paramet Contr er ol Lethal Sublethal 1 2 3 4 1 22.67 20.54 20.94 18.84 18.55 19.88 A E D G H F SD ± 0.011 0.001 0.013 0.021 0.013 0.002 0.012 0.014 0.023 % Change ---- -9.39 -7.63 -16.89 -18.17 -12.31 -18.79 -7.41 -5.51 MCHC 5 10 15 18.41 I 20.99 C 21.42 B Values are Means ± SD (n=6) for a parameter in a row followed by the same letters are not significantly different (P ≤ 0.05) from each other according to Duncan’s multiple range (DMR) test.
  129. 129.    In the present investigation, fish Cyprinus carpio treated to the lethal and sublethal concentrations of quinalphos, showed considerable alteration in the level of different blood parameters. Hematological parameters in fish can significantly change in response towards chemical stressors; however, these alterations are non-specific to a wide range of substances. Some of these changes may be the result of the activation of protective mechanisms (Cazenave et al., 2005) such as the results of the blood parameters observe in the present work. The anaemia produced due to quinalphos treatment might be the reasons for the decrease in the RBC count, haemoglobin percentage and haematocrit value at lethal concentration, which therefore clearly suggests the species response to pesticide and other toxicants.
  130. 130.    Conclusion As food source, fish interferes on man’s life quality and so more detailed analysis of the action of what may lethal and sub lethal concentrations of pesticide and insecticide substances provoke in these organisms is necessary. The over all study inferred that, the death of fish under lethal concentration might be due to the collective interference of the pesticide with all the systems at physiological, biochemical and histopathological levels.
  131. 131.   From the above assessment pertinent to physiological, behavioural and biochemical response of freshwater fish, C. carpio to quinalphos (EC 25%), the conclusion could be drawn that the changes arrived at are dependent on concentration of pesticide and the duration of exposure. Irreparable damage was caused to the physiological, histological, biochemical and behavioural activities of the fish at higher concentration. The damage increased and prevailed over time of exposure.
  132. 132.   Under low concentration, i.e., sublethal concentration stress in fish was observed only for short period (1 to 5 days) and on later days of exposure the stress appeared to lessen and the fish seemed to adapt the toxic environment. The recovery tendency shown by the fish, perhaps, could be due to physiological resistance developed by the animal, which also be reasoned as possible enhancement of detoxification mechanism and quinalphos elimination processes.
  133. 133.    Therefore, the above statement suggests that the fish can adapt to low concentration of quinalphos toxicity during long-term exposure periods. The over all study inferred that, the death of fish under lethal concentration might be due to the collective interference of the pesticide with all the systems at physiological, biochemical and histopathological levels. But in sub lethal concentration the fish can survive to a grater extent with significant metabolic compensation to overcome the chronic stress. The results are processed with statistical treatment and discussed in the light of available literature.
  134. 134.  It is hoped that based on some of the significant differences observed in all the aspects of the present study, useful in determining the safe concentration of quinalphos to ensure protection of worthy fishery resources and provide base line information for future monitoring of pesticides in the aquatic environment.
  135. 135. Thank you
  136. 136.         Acknowledgement Dr. M. David sir and family My Parents and In-laws' My Family, Wife and Daughter University authorities My College Karnatak Science College, Dharwad All Teaching and Non-teaching staff Friends

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