Asma Thesis Black Print 5 Set

Effects of di-butyl and di-isononyl phthalate on antioxidant
enzyme status and DNA damage in Cyprinus carpio (Common

carp)
Submitted by
Asma Aziz
Supervised by
Dr. Moazama Batool
DEPARTMENT OF ZOOLOGY
GOVERNMENT COLLEGE WOMEN UNIVERSITY
SIALKOT
PAKISTAN
2021-2023
i
MS DISSERTATION
I Author Asma Aziz D/O Ghulam Mustafa student of MS Zoology, Government College
Women University, Sialkot, Pakistan, hereby declare that the data quoted in this thesis
entitled “EFFECTS OF DI-BUTYL AND DI-ISONONYL PHTHALATE ON
ANTIOXIDANT ENZYME STATUS AND DNA DAMAGE IN Cyprinus carpio
(Common carp)” not yet been submitted or published elsewhere. I also solemnly declare
that the entire thesis is free of plagiarism and I shall not use this thesis for obtaining any
other degree from this or any other university or institution.
Asma Aziz
2021-2023
ii
Effects of di-butyl and di-isononyl phthalate on antioxidant
enzyme status and DNA damage in Cyprinus carpio (Common
carp)
A DISSERTATION SUBMITTED TO GOVERNMENT COLLEGE WOMEN

UNIVERSITY SIALKOT IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF DEGREE OF MS IN ZOOLOGY
Asma Aziz
2021-GCWUS-1877
DEPARTMENT OF ZOOLOGY
SIALKOT
PAKISTAN
2021-2023
iii
SIALKOT DEPARTMENT OF ZOOLOGY
Dated: ____________
FINAL APPROVAL
This is to certify that we have evaluated the thesis submitted by Ms. Asma Aziz D/O
Ghulam Mustafa on “Effects of di-butyl and di-isononyl phthalate on antioxidant enzyme
status and DNA damage in Cyprinus carpio (Common carp)” and it is our judgement that
this is sufficient standard to warrant its acceptance by Government College Women
University, Sialkot for the degree of MS Zoology.
Name: ______________________ Name: ______________________
Signature: ____________________ Signature: ____________________
Supervisor Internal Examiner
Name: ______________________ Name: ______________________
Signature: ____________________ Signature: ____________________
External Examiner Chairperson
iv
DEDICATION
I dedicated this research to Allah Almighty thank you for the guidance, strength, power of
mind, protection, skills and for giving me a healthy life and wholeheartedly dedicated this
research is dedicated to my beloved parents, Ghulam Mustafa and Shagufta Khanum,
who gave me strength when I thought of giving up, whose words of encouragement and
push for tenacity ring in my ears, whose unceasing support and prayers cherished a way to
my success.
This work is also dedicated to my sisters, brothers, friends and mentor who given me the
drive and discipline to tackle the task with enthusiast and determination. Without their love
and support this research would not have been made possible.
Asma Aziz
v
DECLARATION
I hereby certify that this thesis has been written entirely by myself and that it has not
previously been approved in whole part in any prior application for a higher degree. This
thesis is a record of my own effort; any collaborative work as well as all sources of
information has been clearly acknowledged.
Asma Aziz
vi
ACKNOWLEDGEMENT
First, I thankful to Allah Almighty, who bless me sound health, abilities and gives courage
me to perform and complete my work in a successful manner and without help of ALLAH
was not able to do my work completely.
My profound gratitude goes to my respectable supervisor Dr. Moazama Batool for her
ongoing mentorship and never-ending supply of fascinating tasks.
I would also like to express my gratitude to my Head of Department Dr. Asma Waheed
Qureshi for all the thing that facilitated smooth work for my research purpose. She guided
me with her knowledge and empower me during whole research work. I would like thanks
to Dr. Qurat Ul Ain for her countless hours of reflecting, reading, encouraging and most
of all patience throughout the entire process. She proves a succour in my hours of need.
I would like to express my sincere gratitude to my advisor Dr. Shiza Bano for her patience,
motivation, enthusiasm and immense knowledge. Her guidance helped me a lot in writing
of this thesis. I could not imagine having a better advisor for my MS thesis study. Beside
my mentor and adivisors I would like to thanks my research colleagues Zunaira, Amna
Nawaz, Fatima Arshad, Tehniat Tahir, Farwa, Hina Nabi Ahmad who assisted me in
my research their excitement. They always work in a team and support me in whole work.
Their excitement and willingness to provide feedback made the completion of this research
an enjoyable experience. I would like to thanks to my parents, Ghulam Mustafa and
Shagufta Khanum, for providing their moral, spiritual, emotional and financial support.
My humble thanks to my sisters Maryam Aziz, Samra Komal and Fakhra Khanum and
my brother, Abdul Rehman who share their words of advice and encouragement to finish
this research and helped me at every stage of academic life. Thank you for always being
so supportive and helping me in every step of the way.
Moreover, my special regard goes to my seniors Hafsa Sultan for guidance, discussion,
and criticism. I would like to thanks to my fellows Arooba, Saja, Rabia Nawaz, Urfa,
Arjamand and Sehrish who assisted me in my research.
vii
CONTENTS
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF APPENDICES xvii
LIST OF ABBREVATIONS xix
ABSTRACT 1
Chapter 1 INTRODUCTION 2-6
1.1 Aims and Objectives 6
Chapter 2 Review of Literature 7-13
Chapter 3 Materials and Methods 14-26
3.1 Experimental Location 14
3.2 Collection of Fish 14
3.4 Ethical Consideration 15
3.4.2 Aquatic system management 15-16
3.4.4 Dissections 16
3.5 Acute toxicity (LC50) 96 hours 17
3.6 Sub-Lethal Toxicity of di-butyl and di-isononyl
phthalate on fish 17
3.7 Determination of fish growth performance 17-18
3.8 Determination of Physio-chemical Variants 18-21
3.8.1 Water pH and Temperature 18
3.8.2 Hardness of water 18-19
3.8.3 Estimation of Carbon Dioxide (CO2) 19-20
3.8.4 Dissolved Oxygen 20
3.8.5 Total Ammonia 20
3.8.6 Calcium 21
3.8.7 Magnesium 21
3.9 Antioxidant Enzymes Activity 22-24
3.9.1 Estimation of Reactive Oxygen Specie (ROS) 22
3.9.2 Estimation of Superoxidase dismutase assay (SOD) 22
viii
3.9.3 Estimation of (Peroxidase) POD 22-23
3.9.4 Thiobarbituric acid reactive substance assay (TBARS) 23
3.9.5 Estimation of Catalase (CAT) 23
3.10 Calculating DNA damage 24-26
3.10.1 Slides preparation 24
3.10.2 Lysis 25
3.10.3 Neutral electrophoresis 25
3.10.4 Examination of the prepared slides 26
3.11 Statistical Analysis 26
Chapter 4 RESULTS 27
4.1 Determination of acute toxicity of di-butyl phthalate, di-
isononyl phthalate and their mixture in Cyprinus carpio 27
during 96-hour exposure period
4.2 Determination of physicochemical parameters of water
during 96 hours exposure 34
4.3 Determination of sublethal (1/3rd of LC50) toxicity 34
4.4 Determination of physicochemical parameters of water
after sub lethal exposure 34-35
4.5 Antioxidant Enzymes activity in gills 36
4.5.1 Catalase (CAT) 36
4.5.2 Superoxide Dismutase (SOD) 36
4.5.3 Peroxide Dismutase (POD) 36
4.5.4 Thiobarbituric acid reactive substance (TBARS) 36
4.5.5 Reactive Oxygen Species (ROS) 37
4.6 Antioxidant Enzymes activity in Kidney 41
ix
4.7 Antioxidant Enzymes activity in Muscles 46
4.8 Antioxidant Enzymes activity in Liver 51
4.9 Comet Assay 56
4.9.1 Liver 56
Head length 56
Tail Length 56
% DNA in Head 56
% DNA in Tail 56
Tail Moment 57
4.9.2 Kidney 62
Head length 62
Tail Length 62
% DNA in Head 62
% DNA in Tail 62
Tail Moment 62
Chapter 4 Discussion 67-71
Conclusion 72
SOPs 73
x
List of Tables
Sr. No. Title Page No.
Table 1 Determination of Acute Toxicity (LC50) of di-butyl
phthalate (DBP) in common carp (C. carpio) during the 28
exposure of 96 hour through Probit analysis
Table 2 Determination of Acute Toxicity (LC50) of di-isononyl

phthalate (DINP) in common carp (C. carpio) during the
exposure of 96 hour through Probit analysis 30
Table 3 Determination of Acute Toxicity (LC50) of Mixture

(DBP+DINP) in C. carpio during the exposure of 96
hour through Probit analysis 32
Table 4 The activity of catalase (CAT), superoxide Dismutase

(SOD), peroxidase (POD), thiobarbituric acid reactive
substances (TBARS) and Reactive oxygen species
(ROS)in the gills of Cyprinus carpio in control, DBP, 38
DINP and mixture (DBP+DINP) treated groups.

substances (TBARS) and Reactive oxygen species 43
(ROS)in the kidney of Cyprinus carpio in control, DBP,
DINP and mixture (DBP+DINP) treated
Table 6
The activity of catalase (CAT), superoxide Dismutase
47
xi
(ROS) in the muscles of Cyprinus carpio in control,
DBP, DINP and mixture (DBP+DINP) treated groups.

(ROS)in the liver of Cyprinus carpio in control, DBP,
DINP and mixture (DBP+DINP) treated groups 51
Table 8 Mean ± SEM head length (µm), tail length (µm), DNA
in head (%), DNA in tail (%) and Tail moment (µm)in
the DNA in liver cells of Cyprinus carpio in control,
DBP, DINP and mixture (DBP+DINP) after 30 days 56
exposure.
Table 9 Mean ± SEM head length (µm), tail length (µm), DNA
in head (%), DNA in tail (%) and Tail moment (µm)in
the DNA in liver cells of Cyprinus carpio in control,
DBP, DINP and mixture (DBP+DINP) after 30 days 61
exposure
xii
LIST OF FIGURES
Fig:1 Collection of adult fish from fish hatchery 14
Fig: 2 Experimental setup for research purpose 15
Fig: 2 Fish Dissection 16
Fig: 3 Determining the growth performance of fish during 18

experiment
Fig: 4 Determining hardness of water 19
Fig: 5 Observing the antioxidant activity on 24

Spectrophotometer
Fig: 6 Preparation of comet slides 25
Fig: 7 Observation of Comet slides under fluorescent 26

microscope
Fig: 8 The probit analysis of mortality (%) of Di-butyl 29

phthalate exposed Cyprinus carpio
Fig: 9 The probit analysis of mortality (%) of Di-isononyl 31

phthalate exposed C. carpio
Fig: `10 The probit analysis of mortality (%) of mixture 33
(DBP+DINP) exposed common carp (Cyprinus carpio)
Fig: 11 CAT (U/mg protein) in gills of Cyprinus carpio exposed
to DBP (5.43 mg/L), DINP (300 mg/L) and mixture for
30 days. Each value represents the mean ±S.E.M of 10 39
individuals.
Fig: 12 SOD (U/mg protein) in gills of Cyprinus carpio exposed
individuals
xiii
Fig: 13 POD (nmole) in gills of Cyprinus carpio exposed to
DBP (5.43 mg/L), DINP (300 mg/L) and mixture for 30 39
days. Each value represents the mean ±S.E.M of 10
individuals.
Fig: 14 TBARS (nM/mg) in gills of Cyprinus carpio exposed to
DBP (5.43 mg/L), DINP (300 mg/L) and mixture for 30
days. Each value represents the mean ±S.E.M of 10 40
individuals
Fig: 15 ROS (U/g tissue) in gills of Cyprinus carpio exposed to
individuals
Fig: 16 CAT (U/mg protein) in kidney tissue of Cyprinus carpio
exposed to DBP (5.43 mg/L), DINP (300 mg/L) and
mixture for 30 days. Each value represents the mean 44
±S.E.M of 10 individuals.
Fig: 17 SOD (U/mg protein) in kidney tissue of Cyprinus carpio
Fig: 18 POD (nmole) in kidney tissue of Cyprinus carpio
Fig: 19 T-BARS (nM/mg) in kidney tissue of Cyprinus carpio
±S.E.M of 10 individuals
Fig: 20 ROS (U/g tissue) in kidney of Cyprinus carpio exposed
45
xiv
30 days. Each value represents the mean ±S.E.M of 10
individuals.
Fig: 21 CAT (U/mg protein) in muscle tissue of Cyprinus
carpio exposed to DBP (5.43 mg/L), DINP (300 mg/L) 49
and mixture for 30 days. Each value represents the mean
48±S.E.M of 10 individuals.
Fig: 22 SOD (U/mg protein) in muscles tissue of Cyprinus
carpio exposed to DBP (5.43 mg/L), DINP (300 mg/L)
and mixture for 30 days. Each value represents the mean 49
Fig: 23 POD (nmole) in muscles tissue of Cyprinus carpio
Fig: 24 T-BARS (nM/mg) in muscles tissue of Cyprinus carpio
Fig: 25 ROS (U/g tissue) in muscles of Cyprinus carpio
Fig: 26 CAT (U/mg protein) in liver of Cyprinus carpio
Fig: 27 SOD (U/mg protein) in liver tissue of Cyprinus carpio
xv
Fig: 28 POD (nmole) in liver tissue of Cyprinus carpio exposed
individuals
Fig: 29 T-BARS (nM/mg) in liver of Cyprinus carpio exposed
individuals
Fig: 30 ROS (U/g tissue) in liver of Cyprinus carpio exposed to
individuals
Fig: 31 Head length (µm) measured in control, DBP, DINP and
their mixture exposed in Cyprinus carpio liver tissue 59
after 30 days exposure
Fig: 32 Tail length (µm) measured in control, DBP, DINP and
Fig: 33 DNA in head (%) measured in control, DBP, DINP and
Fig: 34 DNA in Tail (%) measured in control, DBP, DINP and
Fig: 35 Tail Moment (µm)) measured in control, DBP, DINP
and their mixture exposed in Cyprinus carpio liver 60
tissue after 30 days exposure
Fig: 36 Diagram of Chromatin material scattered in Liver cells
of Cyprinus carpio at 40 X exposed with A) control B) 61
DBP C) DINP D) DBP+DINP group after 30 days of
treatment
xvi
Fig: 37 Head Length (µm)) measured in control, DBP, DINP
and their mixture exposed in Cyprinus carpio kidney 64
Fig: 38 Tail Length (µm)) measured in control, DBP, DINP and
their mixture exposed in Cyprinus carpio kidney tissue 64
Fig: 39 DNA in Head (%)) measured in control, DBP, DINP
and their mixture exposed in Cyprinus carpio kidney 64
Fig: 40 DNA in Tail (%)) measured in control, DBP, DINP and
Fig: 41 Tail Moment (µm) measured in control, DBP, DINP and
after 30 days exposure.
Fig: 42 Diagram of Chromatin material scattered in kidney cells
of Cyprinus carpio at 40 X exposed with A) control B) 66
DBP C) DINP) DBP+DINP group after 30 days of
treatment
xvii
LIST OF APPENDICES
Appendix-1 Physiochemical parameters of water in control group 85
of Cyprinus carpio.
Appendix-2 Physiochemical parameters of water on daily basis
in di-isononyl phthalate (DINP) exposed group of 86
Cyprinus carpio
Appendix-3 Physiochemical parameters of water of di-butyl 87
Phthalate (DBP) treated group in Cyprinus carpio
Appendix-4 Physiochemical parameters of water in mixture of di-
butyl and di-isononyl phthalate (DBP+DINP) treated 88
group of Cyprinus carpio.
Appendix-5 Mortality of Cyprinus carpio in different
concentration of di-butyl phthalate at 96 hours 89
exposure period
concentration of di-isononyl phthalate at 96 hours 90
exposure period
concentration of Mixture (DBP+DINP) at 96 hours 91
exposure period
LIST OF ABBREVIATIONS
xviii
Abbreviations Acronyms
DBP Di-butyl Phthalate
DINP Di-isononyl Phthalate
DBP+DINP Di-butyl and Di-isononyl Phthalate
LC50 Lethal Concentration
DNA Deoxyribonucleic Acid
ROS Reactive Oxygen Species
SOD Superoxide Dismutase
CAT Catalase
POD Peroxide Dismutase
T-BARS Thiobarbituric acid reactive

substances
LPO Lipid peroxidation
RBCs Red blood cells
WBCs White blood Cells
EDTA Ethylenediaminetetraacetic acid
GR Glutathione reductase
DEP Di-ethyl phthalate
MDA Melanodialdehyde
SCGE Single cell gel electrophoresis
H2O2 Hydrogen Peroxide
xix
CO2 Carbon dioxide
CH3COONa Sodium Acetate Buffer
pH Potential hydrogen
DO Dissolve Oxygen
EBT Erichrome black Tea
PBS Phosphate Buffer Saline
NaOH Sodium hydroxide
NaCl Sodium chloride
FAO Food and Agriculture Organization
GST Glutathione S-transferase
ANOVA Analysis of Variance
SPSS Statistical package of social scince
DEPPD N-N, Diethyl Paraphenyldamine
RMPA Regular Melting point Agarose
LMPA Low Melting point Agarose
RPM Rotation per minute
TL Tail length
HL Head length
xx
Abstract
Phthalates released from plastic industries and these phthalates when leach into the
water bodies they seriously affect the aquatic life causing genotoxicity and disturbance
to antioxidant enzymes status in different organs of an organism. The current study was
conducted to check the acute toxicity of di-butyl phthalate (DBP) and di-isononyl
phthalate (DINP) and their mixture (DBP+DINP) in Cyprinus carpio (common carp).
The status of antioxidant enzymes in liver, kidney, gills and muscles and DNA damage
in kidney and liver of Cyprinus carpio was determined. The experiment was performed
in glass aquaria. Fish were divided into four groups one control group and three
experimental groups. Mortality data was collected to find LC50 of di-butyl and di-
isononyl by using Probit analysis. Experimental groups were exposed to DBP, DINP
and mixture. The 96 hours LC50 of DBP and mixture was 15.90 mg/L and 13.13 mg/L
while, LC50 of DINP was not found at its highest dissolving concentration 300 mg/L
determined by probit analysis. The sub lethal concentration (1/3rd of LC50) of DBP (5.3
mg/L), DINP (300 mg/L) and mixture (4.37 mg/L) were administered to fish. Physico
chemical parameters were checked on daily basis. Water temperature, pH, and water
hardness were remained constant throughout the experiment. Concentration of CO2,
ammonia and calcium increased while magnesium and dissolved oxygen were
decreased. Antioxidant enzymes activity of catalase (CAT), superoxide dismutase
(SOD) and peroxidase (POD) was decreased with the increase in activity of reactive
oxygen species (ROS) and thiobarbituric acid reactive substance (T-BARS) in different
organs, exposed to DBP, DINP and their mixture. The obtained result showed that
prominent changes were recorded in mixture group and DBP treated group. DNA
damage in kidney and liver cells was also assessed by comet assay, results indicated
that with the increase in ROS activity damage in DNA increased in treatment groups
with more prominent change in mixture group. DNA in tail (%), tail length and tail
moment of liver and kidney cells was increased with the increase in toxicity in mixture
group showing more prominent changes. Whereas, decrease in head length and head
DNA (%) was noted in DBP and mixture group. Therefore, it is concluded that DBP
and DINP has the potential to cause organ impairment through disturbing antioxidant
enzymes status and altering DNA integrity in various organs of C. Carpio.
Key words: Di-butyl phthalate, Di-isononyl phthalate, Cyprinus carpio, Antioxidant
enzymes, DNA damage
1
Chapter I
Introduction
Water is the most important resource that humans use for survival, more than any other
resource. There is huge amount of water present on Earth in the form of polar ice caps
and oceans. In polluted water quality and composition of water changes sometimes by
human activities and sometimes naturally. This contaminated water is not suitable for
domestic, agricultural, industrial, recreational, wildlife and other uses. Water pollutants
are chemical, physical and biological factors that have harmful effects on aquatic
organisms and water consumers. Water pollution mainly manifests itself in the form of
chemicals that remain undissolved in water and cause harmful reactions in the
environment (Goel., 2006).
Nowadays, disposable plastics productions increase day by day so plastic pollution has
become trending environmental issue. Waste is also transported to the sea by large
rivers, which act like a conveyor belt and absorb more and more waste as they sink.
With development of social economy, the demands of plastic products increase rapidly
and simultaneously the manufacturing speed of plastic products is becoming faster
(Hopewell et al., 2009). Widespread use of plastic products leads to increase the
accumulation of Phthalates in environment (Li et al., 2020). Infiltered waste water from
many industries and commercial cites enter in freshwater sources is the main cause of
aqueous Phthalates ester (PAE) pollution. When this polluted water enters in water
bodies these PAEs cause many drastic effects such as carcinogenicity, metabolic
toxicity, hormonal toxicity, developmental toxicity, and immunodeficient and many
more (Yu et al., 2019).
Large scale production of Phthalates by plastic industries puts aquatic system and
human lives at risk. Plastic is non-biodegradable, it is buoyant so it flows over the
surface of oceans (Weiss et al., 2006). Plastic buoyancy means that they can be easily
carried by water currents and transported the depth of water bodies (Pichel et al., 2007).
Phthalates are divided into two main classes based on their molecular weight. High
molecular phthalates are with carbon atom more than 7-13 in their backbone, so they
have more durability. Low molecular weight phthalates have carbon atom
approximately 3-6 in their backbone (Ye et al., 2014). To improve plastics products at
2
industrial level polymer is a chemical agent uses (sear et al., 1982) these plastic
products used in our daily life such as for toys, paints, medical devices, personal care
items etc. (Horn et al., 2004; Shea, 2003). Although these plasticizing agents have very
beneficial impacts on plastic products but they do not have property to bind with
polymer by a covalent linkage and they leach from the forge (Fromme et al., 2013).
This phthalic acid if release in environment they will travel a long distance and ultimate
enter in food chain (FAO 2013).
The aquatic environment contaminated with different types of toxicants which are
having different levels of toxicity. Phthalates found in large amount in water sources. It
is reported that these phthalates accumulated in the body of fish via gill respiration,
dermal exposure and food ingestion these accumulations led to genotoxicity and other
abnormalities (Ohkawa et al., 1979). Exposure to phthalates not only result to damage
in cellular lipid but also DNA damage by increased production of MDA
(malondialdehyde) (by-product of oxidative stress) (Kleinsasser et al., 2001).
Phthalates are responsible for disturbing antioxidant enzyme status in organisms who
consume the phthalates in any way. High concentration of phthalates can cause
oxidative stress in fish (Martinze-Alvarez et al., 2003). Studies show that phthalates
exposure disturb CAT, GST and SOD gene expression level (Kim et al., 2017).
Chemical contamination in an environment responsible for production of free radicals
such as hydrogen peroxide, hydroxyl radical and superoxide radical in an organism
(Martinze-Alvarez et al., 2003). An organism will be oxidatively damaged when free
radical generation rate is faster than eliminating rate it will include enzyme inactivation,
DNA and cholesterol damage in a fish body or any exposed organism (Sies et al., 1986).
Di-butyl phthalate (DBP) is an oily, colorless to very slightly yellow liquid. DBP is a
major pollutant according to the Environmental Monitoring Center. Dibutyl phthalate
(DBP) is a plasticizer used in vast range of products such as paints, inks, plastics and
cosmetics (Biedermann et al., 2008). Dibutyl phthalate is more soluble in fat and
slightly soluble in water because it is an oily liquid. It cannot be vaporized readily in
environment because it is not volatile. According to European union in 1998 about
26,000 tones DBP were produced annually (Sun et al., 2016).
Each year approximately 150 million tons of plastic products are consumed globally
(Net et al., 2015) and PAE annual production is 6-8 million tons worldwide (Wittassek
3
et al., 2011). According to a global study, one of the largest producers and consumers
of PAE is China, which weighs nearly 4.50 x 107 tons and consumes 2.20x107 tons per
year. Most important PAEs in the aquatic environment is DBP and requires greater
attention. Many countries have banned the use of PAE in various industries. For
example, China banned DBP and DEHP because they affected water quality standards,
but PAEs continued to be detected in both aquatic terrestrial and organisms such as fish,
algae, zooplankton and mice (Mankidy et al., 2013).
Janjua et al., 2007 reported that DBP impose very harmful effects on aquatic organisms
such as the increase in unusual growth and also leads to the destruction of both the
developmental system and the reproductive system (Xu et al., 2015). DBP leads to cell
death, where it can accumulate in body tissues, break down, and alter other enzymatic
processes (Zhao et al., 2014). Furthermore, level of reactive oxygen species increases
by use of DBP which stimulate oxidative damage in aquatic organisms (Bie et al.,
2012). Homeostasis of body maintains due to antioxidant defense system and it also
prevent further oxidative damage (Carnevali et al., 2019). Reactive oxygen species
(ROS) are terminated by some enzymatic and non-enzymatic scavengers such as
catalase (CAT), superoxide dismutase (SOD) (Xing et al., 2012).
Di-isononyl phthalate (DINP) is a mixture of branched-chain alkyl phthalate isomers

with nine carbon atoms. Di-ethylhexyl phthalate (DEHP) is replaced by DINP as major
phthalate for PVC polymer (Kavlock et al., 2002). The di-isononyl phthalate (DINP)
has taken the place of the extensively used chemical DEHP, which is notorious for
having negative effects on aquatic biodiversity (Maradonna et al., 2013). DINP is a
high molecular weight phthalate. It has also been found to release into the aquatic
environment through direct and indirect sewage discharge, surface water runoff, and
air. Di-isononyl phthalate (DINP) cause bad hormone activity, disturb metabolism, and
impair reproductive system in fish (Godoi et al., 2021). Di-isononyl (DINP) is an
endocrine disruptor in freshwater fish. Studies revealed that its exposure can cause
disruption in reproductive endocrine system and disturb the production and release of
follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Hauser et al., 2005).
Common carp (Cyprinus carpio) is a fish that occurs naturally in the wild in many parts
of the world and is currently found in 91 of the 120 countries in the world (Casal, 2006).
In temperate regions of Asia, particularly China, common carp is the most widely
4
cultivated and refined carp species (Reddy et al., 2002; Miah et al., 1997). Common
carp is a seafloor omnivore that survives by feeding on bottom fauna and decaying plant
material. Common carp belongs to the class Osteichthyes, the order Cyprinidae and the
family Cyprinidae. It has different body colors, from gray to silver to brown, with a
yellowish or reddish belly. The mouth is large and opens like an accordion. There are
two pairs of whisker-like whiskers, one pair located on the upper lip and the other pair
at the corner of the mouth. Its belly is yellowish and its lower fins are orange-red.
Common carp has a single backbone and the cheeks and gills are partly scaly. Carp
survives in a temperature range between 3 and 35 °C (Froese and Pauly, 2011). For
common carp’s optimal growth and reproduction, the optimal temperature is between
20 and 25 °C. It grows quickly and becomes moderately large, reaching a weight of
around 80 pounds. The maximum life expectancy is 15 years; carp breed mainly in
spring and summer.
Common carp is bred in a complex fish farm together with the Indian carp and the
Chinese carp, which is a great advantage. It also feeds directly on the broken material
of the grass carp. In polyculture it can grow by 1 kg per year. When growing in a tropical
climate, spawning occurs all year round, while in a pond climate there are two main
seasons, one from January to March and the other from July to August. Carp eggs are
naturally small and sticky and reach maturity in12 months (Aikhuni, 1996). Common
carp (C. carpio) is the most widespread exotic species in the world. Due to its unique
characteristics, this species preferred the breeding waters of Asia, the Middle East and
the Far East, together with other fish and sometimes alone. Special characteristics
include excellent growth speed, omnivorous behavior, reproduction in closed waters
and easy adaptation to artificial nutrition. Studies have shown that the Indian carp
(Cirrhinus mirgala) has a slow growth rate compared to common carp (Cyprinus
carpio) (Parameswaran et al., 1971). Common carp (Cyprinus carpio) acquires high
economic value in terms of growth rate, high meat yield, tasty meat, length, weight non-
selective habitat and production availability in fish farming (Demirkalp, 1992). In
cyprinid species common carp (Cyprinus carpio) is most common specie which is
introduce into inland water system such as dams, streams and lakes and also account
for a significant portion of inland freshwater fish production.
Common carp can modify ecological characteristics of aquatic system so it is frequently

called as “ecological engineers” (Matsuzaki et al., 2009, Bajer and Sorensen 2015).
5
According to FAO ranking (C. carpio) is 3rd most commercial and cultivated freshwater
fish (FAO, 2013). The aquatic environment nutrient turnover is highly affected by
planktivorous and benthivores fish population (Rahman and Verdegem, 2007).
Acute toxicity is generally determined by short-term exposure of fish to a range of

chemical concentrations. The lethal concentration for 50% of the fish tested is
calculated and expressed as LC50. This phthalate dosages which kills 50% of total
population under study in a certain time frame is generally calculated between 24-96
hours (Shen et al., 2019).
Fish have proven to be very useful experimental models for assessing the health of
aquatic ecosystem exposed to environmental pollution and the biological changes that
occur as a result. Common carp was selected for this investigation, because it quickly
feeds on artificial feed, has a rapid rate of growth, and is adaptable to lab conditions
(Tasneem et al., 2018). Previously, no study has done on effects of di butyl and di-
isononyl phthalates on common carp (C. carpio) up to this point. Phthalates are
adversely affecting the fish source, so the current study check the hazardous effects of
lethal and sub lethal exposure of di-isononyl and di-butyl phthalates (individually and
their mixtures) induced toxicity in different organs of C. carpio.
1.1 Objectives:
The aims and objectives of present study are following;
• To determine the acute toxicity (96h LC50) of di-butyl and di-isononyl phthalate
to the fish, Cyprinus carpio
• To evaluate sublethal effects of phthalates on antioxidant enzyme status in
different organs of C. carpio
• To investigate DNA damage in C. carpio exposed with phthalate
6
Chapter II
Review of literature
Ogunwole et al. (2021) examined the catfish (Clarias gariepinus) for its antioxidant
enzymes for 30 days expose with dibutyl phthalate (DBP). Exposure to catfish at 0.2
mg/L DBP showed a significant decrease in GSH activity. A significant decrease was
observed in GSH activity after 30 days in whole organs of fish which were exposed to
DBP, however, a surge increase in GSH activity was noted after 15 days of exposure.
The level of TBARS was decrease significantly in liver, gill, kidney and muscles after
15 days of experiment. The findings of this study suggested that a very small amount
of DBP can cause stress (oxidative stress) by changing in oxidative markers.
Bisaï et al. (2022) treated common carp (Cyprinus carpio) with DEHP 3 at different
concentrations of 10, 100 and 1000 μgL-1 for 30 days. Hematological studies showed a
significant lessen (p < 0.05) in RBCs count, while a significant increase in WBCs count
in the treatment groups with increasing DEHP concentration. Furthermore, DEHP
treatment resulted in concentration-dependent deformations of the erythrocytes, while
the leukocytes remained intact. Total plasma protein levels decreased significantly at
lower and higher exposure concentrations (p < 0.05). This study concluded a decrease
in erythrocytes while leukocytes remained unchanged. Histological examinations
showed damage to gills and liver tissue.
Sajla et al. (2019) investigated the sublethal effects of (DBP) di-butyl phthalates in
ovary of freshwater fish orange chromide (Pseudotroplus maculatus). DBP exposed at
0.2 mg/L for 96h which showed significant changes, body weight reduces, increase in
the mucous deposition. DBP disturb the whole cycle of hormones, all the hypothalamo-
pituitary-gonadal hormones disrupts demonstrated by the increasing level of luteinizing
hormone and testosterone histological examination revealed that DBP cause very severe
damage in empty follicle and atretic oocytes, vacuolization and broken theca granulosa
membrane. This study’s finding suggests that exposure of DBP cause the ovarian
toxicity in freshwater fish orange chromide (P. maculatus).
Poople et al. (2017) examined the toxic effects of di-butyl and di-ethyl phthalate on
freshwater fish common carp (Cyprinus carpio). The LC50 for DBP was 35 mgL-1 and
DEP was 53 mgL-1 for 96h. Exposure of DBP was resulted decrease in the blood cells
variables at the same time it was noticed that leukocytes number was increase. Exposure
of both toxins (DBP, DEP) cause a decrease in chloride, sodium-Potassium levels in the
7
brain and gills of fish. Plasma glucose level was also decreased in all treatment groups.
The activity of superoxide dismutase and catalase decrease in gills and liver of fish
treated with DBP and DEP.
Agus et al. (2015) evaluated the toxicological. Stress on common carp (Cyprinus
carpio) at a sublethal concentration of DBP. The exposed sublethal concentration of
DBP. The exposed DBP sub-lethal concentration was 1ppm for 96 hours. During study
it was observed that CYP1A (Cytochrome P4501A) messenger RNA level immediately
increases. In liver the level of GPX and SOD increase moderately.
Faheem et al. (2021) studied the toxic effect of DBP in term of oxidative stress. For this
study 21days exposure was conducted by exposing grass carp with graded
concentration of DBP (1,10,100,1000 μg/L). The activity level of glutathione S
transferase (GST), catalase (CAT), and TBARS checked in gills, liver and kidney after
21 days of exposure. It is concluded that DBP exposure resulted in oxidative stress in
grass carp as its reduction in antioxidant enzymes and increase in (LPO) lipid
peroxidation level. The outcomes showed that most effected organs were kidney
followed by liver and gills.
Jaffar et al. (2019) studied the effect of DBP low concentration (10 microgram per liter
and 100 micrograms per liter) on freshwater fish rohu (Labeo rohita) for 7 days. In this
study oxidative stress in vital organs of fish exposed to 10 μgL-1 of DBP evaluated by
change in activity of catalase (CAT), glutathione-S-transferase, glutathione (GPX) by
measuring lipid peroxidation activity elevated significantly. This study concluded that
low concentration of DBP can lead to stress in vital organs of exposed organism (fish)
that can induce lethal effects.
Xu et al. (2020) studied the direct causal relationship and mechanism between phthalate
with neurotoxicity and neurodevelopment. It was found that phthalate (DBP, DINP,
BBP) disrupts the expression of estrogen receptor and impaired neurogenesis in zebra
fish (Danio rerio). The phthalate exposure also causes estrogen disturbance and
estrogen receptor antagonism.
Poople et al. (2017) investigated that both groups of toxins showed a significant change
in SOD activity in the gills at all doses compared to non-treated animals. At last 7th,
14th, 21st, 28th and 35th treatment days I, the (DBP) groups showed a low percentage
(range of 0.076%, 0.063%, 0.00%.059%, 0.054% and 0.049%). 0.155% was the highest
8
decrease in percentage observed at the end of the 7th day of treatment II, while the
minimum percentage decrease was 0. At last, on 35th day, 0.078% was observed.
Throughout the study, a remarkable reduction in superoxide dismutase (SOD) level in
liver was observed in fish exposed to both phthalates DEP and DBP (treatments I and
II).
Boran and Terzi (2019) examined the zebrafish (Danio rerio) larvae when sublethal
dose of DEHP expose which induce small amounts of breaks in DNA strand and
alternations in transcriptional processes in genes related to oxidative stress such as p53,
rad51 and xrcc5. DEHP is also toxic to early developing zebrafish larvae and has some
external body defects. Diethyl hexyl phthalate has been proven to have a genotoxic
effect. A dose dependent damage increase in DNA observed in larval cells of fish.
Xu et al. (2013) studies zebra fish have shown that DINP, DBP and DEHP can cause
genetic toxicity in aquatic animals through DNA methylation, DNA strand breaks and
increased micronuclei abundance. It is known that increased ROS levels in cells can
lead to a direct attack on bases, the deoxynucleotide structure of deoxyribonucleic acid
(DNA) and other cellular components, which can lead to DNA strand breaks, DNA
cross-links, proteins and others. oxidative phenomena. DNA damage. All of this leads
to the toxicity at genetic level.
Xu et al. (2015) examined zebra fish embryo when exposed to dibutyl phthalate (DBP)
for 96 hours shows the immunotoxic effects on the liver at various concentrations.
Exposure to DBP inhibits the formation of neutrophils and macrophages. It was
observed during study that activity of macrophage phagocytosis decreases when cells
were exposed to DBP, this led to immunotoxicity. This study resulted that DBP cause
immunotoxicity in aquatic environment greatly the immune system of fish.
Cui et al. (2021) studied the DBP effect on oxidative stress and apoptosis in hepatocytes
of grass carp. For this 300 μM exposed to fish for 24h when DBP exposed stress level
and the inflammation in hepatocytes increased. This study resulted that DBP cause
toxicity and oxidative stress in hepatocytes.
Revathy et al. (2018) investigated the role of DINP in reproductive disorders in tilapia
(Oreochromis mossambicus). DINP at a concentration of 300 ppm was given to fish for
96 hours. After treatment, the activity of different antioxidant enzymes in the ovaries
9
and testes of fish was evaluated. Antioxidant enzymes indicated a significant (p<0.05)
decrease in the activity with an elevation in TBARS was observed in both the ovary
and testis. DINP has a hindrance effect on antioxidant enzymes that increases with time,
indicating stress in the gonads. This research resulted that the activity of steroidogenic
enzymes significant decreased (p < 0.05), indicating that DINP change normal ovarian
dysfunction.
Aoki et al. (2011) observed the hazardous effects of DBP on fish by exposing different
concentration of DBP. During study 3 spined sticklebacks (Gasterosteus aculetaus)
adult male exposed to DBP concentrations (0, 15, 35 μg/L) for 22 days. The noted
changes were in nesting behaviour, spiggin concentration, steroidogenic gene
expression. The observed changes were significant increased concentration of plasma
testosterone from 35 μg/L. There was no significant change in 3B hydroxysteroid
dehydrogenase and steroid acute regulatory protein level. In 35 μg/L expression of
spiggin were significantly lower. Nesting behaviour also become slow in some adult
fish. This study resulted that DBP has androgen- antagonists effect in sticklebacks.
Sruthi et al. (2021) evaluated the male conceptive toxicity on freshwater fish
Pseudotroplus maculatus exposed to DBP. 2 mgL-1 was the median lethal concentration
and sublethal was 0.2 mgL-1 for dibutyl phthalate. This concentration showed
significant changes in reproductive parameter. The observed changes were impairment
testicular.
Jee et al. (2009) conducted this study to find out the detrimental effects of di-ethylhexyl
phthalate (DEHP) and di-butyl phthalate (DBP). DBP and DEHP exposed to fish at
100, 500 and 1000 mgkg-1 dose to evaluate the acetylcholinesterase effect on liver, gills,
heart, kidney, muscles and eye. It was observed that acetylcholinesterase activity
inhibited significantly in brain and muscles after exposing to DBP and DEHP. Less
restriction was observed in liver and kidney. This resulted its first report as that the
amount of AChE work as important biomarker of DBP and DEHP exposure. This study
also resulted that DBP and DEHP exerts neurological toxicity by inhibiting AChE
activity in fish.
Sepperumal et al. (2014) evaluated the DBP effects on tilapia Oreochromis

mossambicus. The observed changes were in the oxidative enzymes such as ACP, ALP,
10
SDH. This study reports that exposure of DBP at sublethal concentration (2.5 and 5
ppm) caused the modulation in the enzyme’s concentration of gill, liver and muscles of
Oreochromis mossambicus.
Forner-Piquer et al. (2017) conducted study to check effects of DINP on liver, brain
and hepatic lipid storage of zebrafish. For this study exposed adult female zebrafish
with the varying concentration of DINP (0.42, 4.2 and 42 micrograms per liter) for 3
weeks. This study resulted that DINP upregulated the appetite stimulates signals and
simultaneously cause hepatosteatosis with degradation of peripheral ECS and lipid
metabolism. Finding of this research suggested that DINP has very mild to severe
effects on aquatic organisms.
MacDougall (2014) examined the effect of DIHeP (di-isoheptyl phthalate) and DINP
(di-isononyl phthalate) on the Asian fish Oryzias latipes. Fish were exposed to DIHeP
and DINP 1 day post hatching with concentration DIHeP (0.037-0.3 microliter per liter)
and DINP (0.012-0.1 3 microliter per liter) for 7 days. The treatments did not induce
any significant mortality or malformation. This study resulted that the tested
concentration of DINP and DIHeP did not induce any significant effect on gene
expression, mortality, endocrine disruption or malformation in Oryzias latipes.
Sun and Li (2019) investigated the toxic effect DBP on cardiac development in
zebrafish embryo. During study embryos at 4h post fertilization was treated to 3
different concentrations of DBP (0.036, 1.8 and 3.6 μM). These concentrations resulted
in morphological abnormalities in fish. At 1.8 μM DBP exposure observed changes
were upset cardiac functioning rate, cardiovascular looping, growth malformation rate.
3.6 μM DBP affected all organs at remarkable point. It was observed that DBP exposure
significantly reduced reflection of two master cardiac transcription factor NKX2.5 and
TBX5. This study resulted that in zebrafish DBP exposure cause developmental
toxicity, cardiac structure deformities, pericardial edema and altered the expression
NKX2.5 and TBX5 (master cardiac transcription factor).
Rodrigues et al. (2020) conducted a study and in This study inquire the impacts DBP
exposure to the mechanism of action on Ca+ influx in gills of zebra fish (Danio rerio).
In this invitro study 1 pM, 1 nM, and 1 μM concentration of DBP given to gills of
treated fish for 60 minutes. Disorders in gill tissues of fish were observed when compare
11
high environmental Ca+ to normal Ca2+ and also with low Ca2. 1pM DBP exposure
disturbed the Ca2+ homeostasis. This study resulted that short term exposure of DBP to
gills of fish cause histopathological changes in Ca2+ influx and disturb the
Ca2+homeostasis.
Khalil et al. (2016) examined the sublethal toxicity in juvenile Nile tilapia
(Oreochromis niloticus) exposed of dibutyl phthalate (DBP) to determine the biological
effect of exposure to ½ and 1/3 of LC50 at 96 hours. The LC50 in this research measured
was 11.8 mg/l. In this study, indices of oxidative potential melanodialdehyde (MDA)),
antioxidant parameters (decreased activity of reduced glutathione (GSH) and SOD
(superoxide dismutase) and comet assay was performed to estimate the DNA damage.
In addition, lethal changes at tissue level were observed in the gills, liver and in hepatic
tissue. Comparative results between 1/3 LC50 at 96 hours and ½ LC50 at 96 hours
indicated a remarkable elevation in MDA levels and a remarkable increase in DNA
damage. Increased liver markers and cortisol levels were also observed. Histology
examination of the gills, liver and kidneys revealed malformation alternations.
Qu et al. (2015) examined the goldfish (Carassius auratus) liver to check the
antioxidant activity exposed to 9 phthalates (PAEs). According to body weight 10
mg/kg injections were given to fish of each phthalate for interval of days 1, 4, 8, and
15 days. After long-term treatment, the antioxidant enzymes such as malondialdehyde
(MDA), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase
(SOD) showed different directions, suggesting that metabolism produces fewer or more
dangerous chemicals. The degree of enzyme inhibition, the amount of MDA and the
calculated IBR (integrated biomarker response) were applied to determine order of
toxicity. Dialyl phthalate (DAP), Di-butyl phthalate (DBP) and Dimethyl phthalate
(DMP) is followed by di-isodecyl phthalate (DIDP), di-ethyl phthalate (DEP), Diphenyl
phthalate (DPP), butyl benzyl phthalate (BBP), and di(2-ethylhexyl) phthalate (DEHP).
Specifically, Dibutyl phthalate may be a very potentially dangerous pollutant as it
significantly inhibited activity of enzyme and caused the remarkable decrease in MDA
level. This study resulted that integrated biomarker response (IBR) is a general marker
of pollution.
Shen et al. (2019) conducted a study to measure parameters such as antioxidant enzyme,
toxicity and oxidative stress and in newborn and fully grown water fleas (Daphnia
12
magna) contact to dibutyl phthalates (DBP). The data obtained showed that DBP like
toxic reactions are observed in infants and adults. The mean lethal DBP concentration
in newborn treated for 24 hours is 3.48 mg/l and for 48 hours was 2.83 mg/l. High levels
melanodialdehyde and hydrogen peroxide were observed in infants and fully grown at
1st day and 2nd day, while total capacity of antioxidant was reduced. After exposure to
DBP 0.5 mg/L, superoxide dismutase activity significantly increases in infants and
adults. In adults and newborn glutathione S-transferase and Catalase activity were
reduced, and these changes were concentration and time dependent. The results showed
that the DBP effect is more common in newborns than in adults. The overall results of
this study indicate an important relationship between Thiobarbituric Acid Reactive
Substances (TBARS) and antioxidant enzyme levels in the responses of water flea
(Daphnia magna) to DBP exposure.
13
Chapter III
Methodology
3.1 Experimental Location
Research was conducted in the Zoology Research Lab of GC Women University

Sialkot, Pakistan.
3.2 Collection of Fish
Common carp (Cyprinus carpio) were purchased from a fish hatchery Head marala
Sialkot and delivered to the study site in properly aerated plastic tanks.
Figure 3.1: Collection of adult fish from fish

hatchery
3.3 Acclimatization and Experimental procedure
Fish of the same weight were acclimatized to laboratory conditions in a glass aquarium
for nearly few days prior to the experiment. Fish were provided with rice bran feed
twice daily during the acclimatization period, at least one hour after water refill and
before leaving lab in evening. To avoid stress in tank, environment remains of feed and
excretory material were removed out on daily basis. Fish were classified into four
groups each having 10 fish. One control group and three treated groups. Using the
14
A.P.H.A. (2005) approach, all of the water physio-chemical characteristics (dissolve
oxygen, temperature, pH, total hardness, CO2, total calcium, and magnesium) were
measured daily during the experiment.
Figure 3.2: Experimental setup for research purpose
3.4 Ethical consideration
All the fish handling, aquatic system management and procedure was conducted in
accordance with the national institute of health guide for the care and use of laboratory
animals (NIH publication no.23 revised 1996) (Ethical approval was obtained from
EIRB/23/578, GCWUS).
3.4.1 Fish housing and management
Fish were collected from Head Marala Sialkot and transported to research lab of GC
Women University Sialkot. Healthy fish of almost similar weight acclimatized for ten
days under laboratory conditions to overcome stress caused by transportation and
environmental changes. Small sample size was used to stable the aquatic environment
needed for physiological functions e.g., feeding and movement.
3.4.2 Aquatic system management
Fish were kept in glass tanks having freshwater with optimal laboratory conditions and
normal circadian cycle. Water quality for the wellbeing of fish maintained by
optimizing water quality parameters i.e., temperature, pH, hardness, CO2, dissolved
oxygen ammonia, calcium and magnesium. Routine measurement of various water
characteristics (water quality testing) was done for stable husbandry. Capillary system
was used for proper aeration and gaseous exchange in aquatic system. Water in tanks
15
was weekly changed with freshwater to maintain quality. Researcher dealing with
experiment and managing aquatic system were trained in biologically relevant aspects
of water chemistry and how water quality parameters may affect fish. Students were
trained to handle fish gently and follow SOPs for safe practices throughout experiment.
3.4.3 Disinfection
All the tanks were washed with fresh tap water before starting experiment. Area of
experiment was cleaned and sanitized on daily basis by disinfectant spray. Sanitation
of aquatic media was maintained by changing water in tanks routinely for removal of
debris and other waste material.
3.4.4 Dissection
Fish dissection was performed with proper planning and personal training. All the
instrument were autoclaved before a day of dissection to avoid any contamination.
When dissection completed, all the waste was tightly packed in polythene bags and
properly disposed in separate covered bins.
Figure 3.3: Fish Dissection
16
3.5 Acute Toxicity (96h-LC50) Determination:
The acute toxicity of phthalates (di-butyl, di-isononyl and their mixture) for common
carp was utilized to assess the LC50 and potentially fatal dose for 96 hours. For this
research, fish were exposed to varying dose of di-isononyl phthalate and di-butyl
phthalate individually and the mixture (DBP+DINP), and data on fish mortality was
collected. Probit analysis (Finney, 1971) was used to calculate the LC50 of di-butyl, di-
isononyl phthalate and mixture for common carp (Cyprinus carpio). The dead fish was
removed immediately. Feed was not provided to fish during lethal toxicity exposure.
3. 6 Sub-Lethal Toxicity of di-butyl and di-isononyl phthalate on fish
Fish common carp (Cyprinus carpio) were given sub-lethal dose of di-butyl phthalate,
di-isononyl phthalate and their mixtures dosages for 30 days after acute toxicity
experiment. Common carp (C. carpio) were given (1/3rd of LC50) sub-lethal doses of
DBP, DINP and (DBP+DINP). During the exposure time, fish fed twice a day. Mostly,
the exposing media was changed every week. The normally required di-butyl phthalate,
di-isononyl phthalate and mixture dose has been maintained throughout the study.
Steady airflow in tank was maintained in every tank during study to protect fish from
being stressed and anxious.
3.7 Determination of fish growth performance
The total body length and fork length of fish was noted at the start of the research and
at the finale of each week of experiment. Body weight of each fish was determined
using an electronic balance (Model KERN 572), and its overall length and fork length
were determined using a scale. According to these observed values, the given below
formulas were used to evaluate the measure of total fork length (cm), feed intake (FI),
total length increase (cm), gain in weight (grammes), feed efficiency ratio (FER), feed
conversion ratio (FCR), and rate of survival.
Total Weight (g) = Final weight – initial weight
Body Length (cm) = Final total length – initial total length
Fork length (cm) = Final Fork length- initial fork length
Feed Intake (g/fish) = Dry feed intake ÷ No. of fish
Rate of Survival (%) = (No. of fish survived ÷ initial No. of fish) x100
17
Figure 3.4: determining the growth performance of fish during experiment
3.8 Determination of Physio-chemical Variants
Physio-chemical properties of water were evaluated on daily basis till the study was
completed (Appendix I, II, III).
3.8.1 Water pH and Temperature:
pH meter model 120 benchtop pH/mv meter was used to determine the temperature and
pH of test medium.
3.8.2 Hardness of water:
The following compounds were used to determine the hardness of water:
• Titrant [Ethylenediaminetetraacetic acid (EDTA)]

• An Ammonia buffers
• Eri-chrome Black-T (indicator)
Procedure
Hardness of water was evaluated by the titration of collected water sample with known
concentration of a potent mineral acid. 25 ml amount of water sample was collected
from the test medium and placed in a beaker for the analysis of water hardness. Add
25 ml of distilled water in this water sample. To increase the pH of water sample up to
7.5 it was necessary to add a suitable amount of ammonia buffer in it. Properly mix the
18
sample solution by stirrer and 2-3 drops of indicator (EBT) were added. After mixing
reaction mixture was titrated against EDTA until sample color turned to blue color. The
following general formula was used to evaluate the total hardness of water
Total water hardness (mg/L) = Vol. of titrant (EDTA) used x 1000 x A
Sample Volume (ml)
Where A= mg CaCO3 which is equivalent to 1.0ml of titrant (EDTA) at the end point
of Ca++ indicator.
Figure 3.5: Determining of Hardness of water
3.8.3 Carbon dioxide
The following chemical are used for the measure amount of carbon dioxide in water:
• An acid-base indicator Phenolphthalein

• A titrant 1N sodium carbonate
Procedure:
By titrating a known amount of water with sodium carbonate the concentration of

carbon dioxide (CO2) was determined. Sodium bicarbonate was produced as a reaction
19
product of sodium carbonate and free CO2. In order to show the completion of the
titration, reaction was stopped at a certain pH. Colorless sample turned to pink when an
indicator phenolphthalein was added.
According to formula given below, CO2 concentration was determined;
CO2 (mg/L) = Volume of sodium carbonate used x 1000
Volume of water sample used in ml
3.8.4 Dissolve Oxygen:
A dissolve oxygen meter (DO meter) was used to measure the amount of oxygen in
gaseous form that had been present in water sample. The electrode of DO meter was
put in sample water and the amount of soluble oxygen in mgL-1 was displayed on the
LCD of the DO meter.
3.8.5 Total Ammonia:
In order to determine ammonia, following compounds were use:
• Sodium Potassium tartrate

• Nessler’s reagent (K2 [HgI4])
• Ammonium chloride (Buffer solution)
Procedure:
50 ml of water sample was obtained from each testing media to which 1-2 drops of
solution of sodium potassium tartrate was added and the mixture thoroughly mixed.
Ammonium chloride (buffer) solution was then added in little amounts (about 2-3
droplets). The mixture was mixed with one ml of Nessler’s reagent then allowed to sit
for 15 min. Samples were run in a spectrophotometer for 1 min at 420 nm of wavelength
range for 1cm light path to analyze absorbance. At around the same reaction time,
calibration curves were generated by running standard concentration and temperature
utilized for sample reading and absorbance were determined along blank reagent.
3.8.6 Calcium:
The following compounds were used to measure calcium levels:
• 1N solution of sodium hydroxide
20
• Titrant EDTA
• An indicator Ammonium purpurate
Procedure:
50ml of water was collected from test media in glass beaker. The pH was then raised to
12-13 by adding a known amount of sodium hydroxide in glass beaker. In order to
identify the end point of reaction, 1 drop of indicator was introduced and thoroughly
mixed. By titrating it with EDTA, the end point was obtained.
Content of calcium in water sample was determined by the formula:
Calcium (Ca) in m/L = Used volume of EDTA titrant x 400.8
Sample volume in ml
3.8.7 Magnesium:
By knowing the values of calcium (Ca) and total hardness content of magnesium was
calculated.
The following formula determines the magnesium values of test medium.
C = A-B
Mg (mgL-1) = C/4
Where,
B = Ca x 2.50 and A = total hardness
21
3.9 Antioxidant enzyme Activity
For determination of antioxidant enzymes activity sample tissues were centrifuge and
their supernatant used.
3.9.1 Catalase (CAT)
Activity of catalase in liver, gills, muscles and kidney of fish status check by following
procedure designed by Chance and Maehly (1955).
Procedure
2.5 ml of phosphate buffer (50 mM, pH 5.0), supernatant (0.1 ml) and 0.4 ml of H2O2
(5.9 mM) was used for preparation of reaction mixture in cuvette. Spectrophotometer
was used for the recording the absorbance at 240 nm after one minute. One unit of CAT
activity considered as 0.01 unit/minute.
3.9.2 Estimation of Superoxidase dismutase assay (SOD)
Kakkar et al. 1984 procedure follow to evaluated the activity of superoxide dismutase
Procedure
0.3 ml of homogenate, 0.1 ml of phenazinemethosulphate (186 µM) and 1.2 ml of

sodium pyrophosphate (0.052 mM, pH 7.0) was used to make reaction mixture. 0.2 ml
NADH (780 µM) was introduced to start a reaction then 1 ml of glacial acetic acid was
added to stop reaction mixture after 60 seconds. Chromogen intensity of color
formation was associated with absorbance of reaction mixture. On spectrophotometer
560 nm wavelength set to note absorbance of reaction mixture. SOD values were in
units/mg protein.
3.9.3 Estimation of POD
The activity of Peroxidase (POD) is evaluated by Chance and Maehly (1955) procedure.
Procedure
0.3 ml of H2O2 (40 mM), 100 µl homogenate, 2.5 ml of phosphate buffer (50 mM, pH
5.0) and 0.1 ml of glycol (20 mM). At wavelength of 470 nm absorbance was observed
after one minute. One unit of POD activity is actually change in 0.01 units/ minutes of
absorbance. mU/mg was unit for the value of POD.
22
3.9.4 Thiobarbituric acid reactive substance assay (TBARS)
Homogenate of liver, kidney, gills and muscles tissues was prepared to evaluate the
activity of lipid peroxidation. Procedure followed given by Wright et al. 1981 as
modified by Iqbal et al. 1996.
Procedure
0.58 ml phosphate buffer (0.1M pH 7.4) was distributed along with 0.2 ml ascorbic acid
(100mM) and 0.02 ml ferric chloride (100mM) in the test tube. In the reaction mixture
0.2 ml homogenate was added and for 1 hour it was incubated in water bath at 37 ºC.
1.0 ml of trichloroacetic acid (10%) was incorporated to cease the reaction after
incubation. All test tubes were dispensed with 1.0 ml of thiobarbituric acid (0.67%) and
then incubated for 30 minutes in boiling water and then placed in crushed ice.
Centrifugation at 2500 × g was conducted for all Samples for 10 minutes. Wavelength
of 535nm was kept for investigation of absorbance of mixture. The values were
specified as nM TBARS/min/mg tissue using molar extinction coefficient of 1.56 × 105
/M cm.
3.9.5 Estimation of ROS
Hayashi et al. (2007) reported procedure was used to estimate the activity of reactive
oxygen species (ROS) in tissue mixture of gills, liver, kidney and muscles.
Procedure
To evaluate the activity of the activity of ROS enzyme 2 reagents were prepared, for
reagent I 10 mg N, N-diethyl-para-phenylenediamine (DEPPD) added in 10ml sodium
acetate buffer (0.1 M, pH 4.8) and to prepare reagent II 50 µL of ferrous sulphate added
in 100 ml of sodium acetate buffer (0.1 M, pH= 4.8). Mixed both reagent I and reagent
II (1:25) and incubated in dark for 20 minutes. Dispense of 5 µL of H2O2 and 5 µL
homogenate in each test tube. 140 µL of reagent mixture added in each test tube. Then
incubated at 37 ºC for 1 minute. In spectrophotometer absorbance was noted at 505nm
for 60 seconds. Hydrogen peroxide concentration in sample equal to one unit of ROS.
23
Figure 3.4: observing the antioxidant activity on
Spectrophotometer
3.10 Calculating DNA damage
Comet assay known as single cell gel electrophoresis use to determine the damage in
DNA followed procedure was by Boe hensen et al. (2007); Donnelly et al. (1999). It
is a simple protocol to find out single- stranded and double-stranded DNA breaks and
base-labile sites in DNA. At 1000 rpm for 15 minutes tissue samples were centrifuged
and the pellet was thinner with phosphate-buffer saline solution.
3.10.1 Slides preparation
0.1 ml of 1% regular melting point agarose gel (RMPA) was coated over frosted
microscopic slides and immediately covered with large size (22 x 50 mm) coverslips.
To solidify the agarose gel placed slides at 4 ºC for few minutes. When gel solidify over
slides added mixture of 20 µl homogenate and 65 µl low melting agarose gel (LMPA)
(1%). Slides enfolded and let the agarose gel to dry.
24
Figure 3.5: Preparation of comet slides
3.10.2 Lysis
Lysis buffer is prepared by adding 2.5 M NaCl, 1% (w/v) Triton X- 100, 100 mM EDTA
and 10 mM Tris base pH maintain at 10.3. When this lysis buffer prepared slides were
placed in clean tray and added lysis buffer slowly and with care (Villani et al., 2012).
Kept the slides at 37oC for incubation overnight. After incubation slides were takeoff
from tray rinsed with saline solution 3 times for 20 minutes with gaps, so if there is any
salt or traces of chemical wash out.
3.10.3 Neutral electrophoresis
Electrophoresis buffer prepared by adding 54000 mg/L Tris base, 500 mM EDTA, 27.5
g/L boric acid and maintain pH at 8. When electrophoresis buffer prepared added in
electrophoresis tank and slides were arranged according to tag. Electrophoresis process
was conducted for 20 minutes at 25 V (0.714 V/cm). When electrophoresis completed,
slides were brought out from tank, let them dried in air and covered to avoid light. Drain
out whole buffer from tank and remove the tray from electrophoresis tank. Slides kept
at 5°C for 24 hours.
3.10.4 Examination of the prepared slides
Slides rinsed with distilled water for 30 minutes and used acridine orange (200-300 µl
of 20 µg/ml) for staining before analyzing. Fluorescent microscope (AFX-1 Optiphot,
25
Nikon, Tokyo, Japan) with 40 X lens was used to examined the slides and for analysis
photographs were captured. For more analysis a comet software name as Casplab,
version (V.1.2.3bs). In Capslab observed parameters were the number of comet/100
cells counted and head DNA (%), Tail DNA (%), tail movement (µm), Tail length (TL,
µm), and head length (HL, µm) observed.
Figure 3.6: Observation of Comet slides under fluorescent microscope
3.11 Statistical Analysis
Probit analysis (Finney, 1971) method was applied on fish mortality % data to find LC50
and total concentration. To determine the statistical differences among various variables
under study one way analysis of variance (ANOVA) using SPSS software was applied
(Steel et al., 1996).
26
CHAPTER-IV
Results
The hazardous effects of di-butyl phthalate (DBP) and di-isononyl phthalate (DINP)
and their mixtures (DBP+DINP) were observed on common carp (Cyprinus carpio).
By using probit analysis, the 96 hours LC50 values of di-butyl and di-isononyl phthalate
and their mixtures were determined in present study. Throughout experiment all
physico-chemical parameters of water were determined by use of different chemicals
and equipment. The change in antioxidant activities and damage in DNA in cells of
liver, gill, muscles and kidney were analyzed when fish exposed to di-butyl and di-
isononyl phthalate and their mixtures. All results were analyzed by one way Analysis
of variance (ANOVA) in SPSS software.
4.1 Determination of acute toxicity of di-butyl phthalate, di-isononyl phthalate and

their mixture in Cyprinus carpio during 96-hour exposure period
The 96hr LC50 of di-butyl and di-isononyl phthalate and their mixture in C. carpio was
determined by performing acute toxicity experiment. Different and gradually increasing
concentrations of DBP, DINP and DBP+DINP were exposed to fish during 96hr time
period to find out LC50. There were 4 different groups of fish 3 treated groups and 1
was control group each group contain 10 fish. Tanks were cleaned on daily basis any
expired fish removed immediately from tanks to avoid stress in remaining fish. For
Cyprinus carpio LC50 96hr of di-butyl phthalate found was 15.90 mg/l and for di-
isononyl LC50 was not found even at its highest dissolving concentration 300 mg/L and
LC50 of mixtures was 13.13 mg/l by using probit analysis (Finney, 1971). By using
probit analysis method at different concentration of DBP, DINP and DBP+DINP
mortality rate was found show in figures 4.1, 4.2, 4.3, respectively (Appendix
V,VI,VII).
27
Table 4.1: Determination of Acute Toxicity (LC50) of dibutyl phthalate (DBP) in common carp (C.
carpio) during the exposure of 96 hour through Probit analysis
Regression Table
Standard
Variable Coef Error Z P
Constant -2.25761 0.197025 -11.46 0.000
Conc. of DBP mg/L 0.141911 0.0116656 12.16 0.000
Natural
Response 0
Log-Likelihood = -181.950
Goodness-of-Fit Tests
Method Chi-Square DF P
Pearson 7.42073 12 0.829
Deviance 9.42623 12 0.666
Tolerance Distribution
Parameter Estimates
Standard 95.0% Normal CI

Parameter Estimate Error Lower Upper
Mean 15.9086 0.528643 14.8725 16.9447

StDev 7.04665 0.579261 5.99806 8.27856
Table of Percentiles
Standard 95.0% Fiducial CI
Percent Percentile Error Lower Upper
1 -0.484340 1.42527 -3.77633 1.94929
2 1.43657 1.27994 -1.51121 3.62838
3 2.65533 1.18940 -0.0773521 4.69699
4 3.57215 1.12237 0.999150 5.50300
5 4.31791 1.06866 1.87318 6.16025
6 4.95268 1.02362 2.61579 6.72100
7 5.50924 0.984715 3.26576 7.21382
8 6.00757 0.950394 3.84671 7.65611
9 6.46079 0.919652 4.37412 8.05929
10 6.87798 0.891793 4.85873 8.43128
20 9.97801 0.702840 8.42381 11.2315
30 12.2134 0.597754 10.9320 13.3131
40 14.1234 0.542246 13.0059 15.1611
50 15.9086 0.528643 14.8665 16.9662
60 17.6939 0.554951 16.6457 18.8526
70 19.6039 0.621421 18.4714 20.9487
80 21.8392 0.735046 20.5359 23.4741
90 24.9393 0.930489 23.3232 27.0521
91 25.3565 0.958922 23.6940 27.5379
92 25.8097 0.990231 24.0961 28.0664
93 26.3080 1.02512 24.5372 28.6485
94 26.8646 1.06459 25.0289 29.2996
95 27.4993 1.11020 25.5886 30.0433
96 28.2451 1.16449 26.2447 30.9185
97 29.1619 1.23213 27.0495 31.9962
98 30.3807 1.32335 28.1167 33.4314
99 32.3016 1.46949 29.7942 35.6981
28
Table 4.2: Determination of Acute Toxicity (LC50) of di-isononyl phthalate (DINP) in common carp
(C. carpio) during the exposure of 96 hour through Probit analysis
Regression Table
Standard
Constant -3.87701 0.593944 -6.53 0.000
Conc. of DINP (mg/L) 0.0110474 0.0023723 4.66 0.000
Natural
Response 0
Pearson 4.80132 11 0.940
Deviance 5.75231 11 0.889
Parameter Estimates

Mean 350.943 25.1616 301.628 400.259
StDev 90.5191 19.4383 59.4225 137.889

1 140.364 24.9916 59.2655 176.318
2 165.040 20.3480 100.548 195.011
3 180.696 17.5949 126.331 207.280
4 192.473 15.6810 145.385 216.852
5 202.053 14.2663 160.566 224.955
6 210.207 13.1960 173.179 232.161
7 217.356 12.3857 183.933 238.784
8 223.758 11.7834 193.263 245.014
9 229.579 11.3530 201.454 250.973
10 234.938 11.0674 208.711 256.741
20 274.761 12.5647 253.238 309.004
30 303.475 16.5572 278.108 353.927
40 328.011 20.8263 297.435 394.235
50 350.943 25.1616 314.767 432.643
60 373.876 29.6813 331.717 471.432
70 398.412 34.6393 349.604 513.181
80 427.126 40.5415 370.336 562.241
90 466.948 48.8350 398.872 630.495
91 472.307 49.9577 402.699 639.693
92 478.129 51.1788 406.854 649.689
93 484.531 52.5231 411.419 660.683
94 491.680 54.0262 416.514 672.965
95 499.834 55.7427 422.321 686.977
96 509.414 57.7620 429.138 703.445
97 521.191 60.2479 437.511 723.697
98 536.847 63.5578 448.632 750.628
99 561.522 68.7849 466.139 793.096
30
Table 4.3: Determination of Acute Toxicity (LC50) of Mixture (DBP+DINP) in C. carpio during the
exposure of 96 hour through Probit analysis
Regression Table
Standard
Constant -1.92582 0.186870 -10.31 0.000
Conc. of (DBP+DINP) mg/L 0.146643 0.0125931 11.64 0.000
Natural
Response 0
Pearson 5.80044 11 0.886
Deviance 8.07483 11 0.707
Parameter Estimates

Mean 13.1327 0.524630 12.1044 14.1609
StDev 6.81927 0.585611 5.76289 8.06930
1 -2.73131 1.49128 -6.20820 -0.202569
2 -0.872381 1.34297 -3.99534 1.41082
3 0.307048 1.25032 -2.59421 2.43732
4 1.19429 1.18156 -1.54204 3.21136
5 1.91599 1.12634 -0.687573 3.84238
6 2.53027 1.07991 0.0385691 4.38062
7 3.06887 1.03969 0.674271 4.85353
8 3.55112 1.00413 1.24259 5.27784
9 3.98972 0.972189 1.75866 5.66453
10 4.39344 0.943159 2.23296 6.02122
20 7.39344 0.742761 5.72691 8.70221
30 9.55666 0.624855 8.19304 10.6886
40 11.4050 0.554388 10.2391 12.4472
50 13.1327 0.524630 12.0779 14.1643
60 14.8603 0.535865 13.8330 15.9653
70 16.7087 0.590461 15.6237 17.9791
80 18.8719 0.696138 17.6348 20.4206
90 21.8719 0.887354 20.3341 23.8962
91 22.2757 0.915576 20.6924 24.3689
92 22.7142 0.946720 21.0807 24.8834
93 23.1965 0.981493 21.5066 25.4502
94 23.7351 1.02092 21.9811 26.0843
95 24.3494 1.06655 22.5209 26.8088
96 25.0711 1.12096 23.1535 27.6617
97 25.9583 1.18886 23.9292 28.7122
98 27.1378 1.28057 24.9576 30.1115
99 28.9967 1.42775 26.5732 32.3221
32
4.2 Determination of physicochemical parameters of water during 96 hours
exposure
On regular basis physicochemical parameters were checked during experiment. The pH

was 7.5 water hardness level 299 mgL-1 and temperature 28oC remain constant. There
was no change observed in their computed values. A significant increase was observed
in level of total ammonia, carbon dioxide and level of calcium, however there was
observed a decrease in level of magnesium and dissolved oxygen with gradual increase
in concentration of di-butyl phthalate, di-isononyl phthalate and their mixtures
(DBP+DINP). The calcium (15.56 mgL-1 to 17.13 mgL-1), total ammonia (0.16 mgL-1
to 0.29 mgL-1) and carbon dioxide (1.21 mgL-1 to 1.22 mgL-1) increased respectively
and the dissolved oxygen (4.87 mgL-1 to 4.74 mgL-1) and magnesium (65.06 mgL-1 to
64.4 mgL-1) decreased respectively. Whereas, during the exposure of mixture
(DBP+DINP) the physicochemical parameters of water included calcium (17.10 mg/L-
1
to 16.73 mgL-1), total ammonia (0.28 mgL-1 0.29 mgL-1) and carbon dioxide (1.21
mgL-1 to 1.22 mgL-1) increased respectively and dissolved oxygen (4.86 mgL-1 to 4.80
mgL-1) and magnesium (64.21 mgL-1 to 65.5 mgL-1) decreased respectively. Theses
variations in the level of different parameters were observed due to the toxic effects of
DBP, DINP and mixture (DBP+DINP).
4.3 Determination of sublethal (1/3rd of LC50) toxicity
When acute toxicity protocols performed then experimental groups were treated to
sublethal concentration 1/3rd of LC50 of di-butyl, di-isononyl phthalate and their
mixtures calculated for Cyprinus carpio. Sub lethal of phthalates were calculated as 5.3
mgL-1 (DBP), DINP (300 mgL-1) and for mixture was 4.37 mgL-1. The calculated
amounts were introduced in different experimental groups. The control group
throughout the experiment was left untreated. Feed was given to fish during sublethal
period.
4.4 Determination of physicochemical parameters of water after sub lethal

exposure
The physicochemical parameters of water in all treated and control groups were
determined on daily basis for 30 days. The temperature (28.04±0.039 oC), pH
(7.5±0.01), and total hardness (299±0.59) were remained constant. In control group, the
34
mean values with standard deviation were determined on weekly basis to be of total
ammonia (0.12±0.17), dissolved oxygen (4.87±0.01), carbon dioxide (1.21±0.01),
calcium (15.56±2.34) and magnesium (65.15±1.45) (Appendix I). In DBP exposed
group treated group, the mean values with standard deviation were evaluated on weekly
basis to be dissolved oxygen (4.78±0.08), carbon dioxide (1.24±0.02), ammonia
(0.23±0.012), calcium (17.16±5.27) and magnesium (64.12±3.30) (Appendix II). While
in DINP treated group the mean values of physicochemical parameters with standard
deviation were found to be dissolved oxygen (4.80±0.07), carbon dioxide (1.23±0.02),
ammonia (1.202±0.09), calcium (13.26±2.11) and magnesium (66.46±1.31). However,
in groups treated by mixture (DBP+DINP) the mean value of different parameters of
water with standard deviation was investigated to be dissolved oxygen (4.77±0.6),
carbon dioxide (1.25±0.02), ammonia (0.24±0.10), calcium (17.13±4.89) and
magnesium (64.17±3.03) (Appendix III).
35
4.5 Antioxidant enzymes in Gills
In table 4.1 all the calculated values of antioxidant enzymes in gill tissues of common
carp are mentioned.
The antioxidant enzymes activity of common carp gills was determined after 30 days
exposure with di-butyl, di-isononyl and their mixture. In gills catalase activity was
significantly decreased (p<0.05) in mixture (DBP+DINP), DBP and DINP when
compare to control. DBP, DINP and mixture group showed non-significant results in
gills of Cyprinus carpio (Figure 4.5.1).
4.5.2 Superoxide Dismutase (SOD)
The activity of SOD in gills was significantly reduced (p<0.05) in mixture

(DBP+DINP) groups in contrast to control group. While DBP and DINP group showed
non-significant differences when compare to control group. Overall, the activity of
SOD decreases in gills. (Figure 4.5.2).
4.5.3 Peroxidase (POD)
In gills tissue level of peroxidase (POD) significantly decreased (p<0.05) in mixture

group (DBP+DINP) in comparison to control group. DBP and DINP group showed a
non-significant alteration when compare to control. Moreover, a significant decrease
(p<0.05) was observed in mixture group as it was compared with DBP (Figure 4.5.3).
4.5.4 Thiobarbituric acid reactive substance (TBARS)
In gills tissue of Cyprinus carpio thiobarbituric acid reactive substances level was
significantly increased (p<0.05) in mixture (DBP+DINP) and DBP treated groups when
compare to control. A non-significant variation was seen within DBP, DINP and
mixture group when compare to control group (Figure 4.5.4).
36
4.5.5 Reactive Oxygen Species (ROS)
The reactive oxygen species (ROS) activity in gill tissues of common carp significantly
increased (p<0.05) in all groups control, DBP, DINP and mixture when compare to
control. A non-significant variation was seen within DBP, DINP and mixture group
when compare to control group (Figure 4.5.5)
37
Table 4.1: The activity of catalase (CAT), superoxide Dismutase (SOD), peroxidase (POD), thiobarbituric acid reactive substances
(TBARS) and Reactive oxygen species (ROS)in the gills of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP) treated
groups.
Parameters Groups
Control DBP DINP DBP+DINP P-value
CAT (U/mg Protein) 12.74±0.44a 10.96±0.64ab 11.29±0.64ab 10.10±0.33c 0.045

SOD ((U/mg Protein) 26.65±1.23a 20.54±0.32bc 23.69±1.22ab 19.09±0.43c 0.002
POD (nmole) 30.47±4.86a 26.82±0.99ab 21.95±0.75ab 15.43±1.65b 0.019
TBARS (nM/mg tissue) 5.11±0.57a 5.28±0.72a 5.12±0.81a 7.22±0.74a 0.222
ROS (U/g tissue) 0.72±0.43a 0.94±0.03a 0.86±0.04a 0.94±0.01a 0.393
Note: Data are presented as mean value with ± S.E.M Different superscripts on the same row indicate there is
a significant difference (p<0.05)
38
GILLS
14 a
ab ab
12
CAT (U/mg Protein)

c
10
8 CONTROL
6 DBP
4
DINP
2
DBP+DINP
0
CONTROL DBP DINP DBP+DINP
GROUPS
Figure 4.5.1: CAT (U/mg protein) in gills of Cyprinus carpio exposed to DBP (5.3 mg/L),
DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean
GILLS
30 a
ab
SOD (U/mg Protein)
25
bc
20 c
CONTROL
15
DBP
10
DINP
5
DBP+DINP
0
Groups
Figure 4.5.2: SOD (U/mg protein) in gills of Cyprinus carpio exposed to DBP (5.3 mg/L), DINP
(300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean ±S.E.M of 10
individuals.
GILLS
35 a
30 ab
ab
POD (nmole)
25
20 b CONTROL
15 DBP
10 DINP
5
DBP+DINP
0
GROUPS
Figure 4.5.3: POD (nmole) in gills of Cyprinus carpio exposed to DBP (5.43 mg/L),
DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the
mean ±S.E.M of 10 individuals.
39
GILLS
9
8
a
7 a
a
T-BARS (nM/mg)
6 a
CONTROL
5
4 DBP
3 DINP
2 DBP+DINP
1
0
GROUPS
Figure 4.5.4: TBARS (nM/mg) in gills of Cyprinus carpio exposed to DBP (5.43 mg/L),
GILLS
1.4 a a
a
1.2
ROS (U/g tissue)
1 a
0.8 CONTROL
0.6 DBP
0.4 DINP
0.2 DBP+DINP
0
GROUPS
Figure 4.5.5: ROS (U/g tissue) in gills of Cyprinus carpio exposed to DBP (5.43 mg/L),
40
4.6 Antioxidant enzymes of Kidney
In table 4.2 all the calculated values of antioxidant enzymes in kidney tissues of
common carp are mentioned
In comparison to control group DBP and DIP showed no significant variation whereas,
mixture group show a significant decrease (p<0.05) in catalase activity when compared
to control group. Overall, the activity of CAT decreased in all treated groups when
compare among themselves (4.6.1).
In comparison with control group mixture group exhibited a significant decrease

(p<0.05) while DBP and DINP indicated no significant alteration. Mixture group
showed a significant decline (p<0.05) in superoxide dismutase activity when compare
to DINP. As a result, the activity of SOD decreased in DBP, DINP and mixture group
when compare with control (Figure 4.6.2).
In kidney peroxidase activity is significantly reduced (p<0.05) in DBP and mixture

while DINP expressed no significant alteration when compare to control. DINP and
mixture exhibited no significant changes when compare to DBP. Overall, the activity
of POD decreased in all groups when compare among themselves (figure 4.6.3).
A significant elevation (p<0.05) was noticed in the level of thiobarbituric acid reactive
substance in DBP group, when compared with control group while DINP and mixture
show non-significant changes in comparison to control group. Thiobarbituric
acid reactive substance were significantly increased (p<0.05) within mixture
(DBP+DINP) and DBP when compare with control group (Figure 4.6.4).
41
4.6.5 Reactive Oxygen specie (ROS)
A significant increase (p<0.05) was observed in the ROS level in DBP, DINP and
mixture group when compare with control group. ROS level significantly increase
(p<0.05) within mixture (DBP+DINP) and DBP group while non-significant increase
was observed in DINP when compare to control group (Figure 4.6.5).
42
(TBARS) and Reactive oxygen species (ROS)in the kidney of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP)
treated
Parameters Groups
CAT (U/mg Protein) 28.37±0.43 a 23.85±2.27ab 26.14±1.78 a 19.27±0.67b 0.014
SOD ((U/mg Protein) 23.80±0.96 a 19.97±0.53ab 23.40±1.12 a 18.84±0.88b 0.010
POD (nmole) 16.63±1.17 a 10.78±0.48b 13.89±1.84ab 9.66±0.47b 0.010
TBARS (nM/mg tissue) 9.12±2.15 a 9.28±1.88 a 9.20±0.34 a 9.87±2.09 a 0.094
ROS (U/g tissue) 0.21±0.33 a 1.25±0.33 a 0.73±0.36 a 2.34±0.68 a 0.161
Note: Data are presented as mean value with ± S.E.M Different superscripts on the same row indicate there is a significant
difference (p<0.05)
43
35 KIDNEY
a
CAT (U/mg Protein)

30 a
a
25
b
20
15 CONTROL
10 DBP
5
DINP
0
CONTROL DBP DINP DBP+DINP DBP+DINP
GROUPS
Figure4.6.1: CAT (U/mg protein) in kidney tissue of Cyprinus carpio exposed to DBP (5.43
mg/L), DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the
KIDNEY
30
a a
SOD (U/mg Protein)
25
ab b
20
15 CONTROL
10 DBP
5 DINP
0 DBP+DINP
GROUPS
Figure 4.6.2: SOD (U/mg protein) in kidney tissue of Cyprinus carpio exposed to DBP (5.43 mg/L),
DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean ±S.E.M of
10 individuals.
KIDNEY
20
18 a
16 ab
14
POD (nmle)
12 b CONTROL
b
10 DBP
8
6 DINP
4 DBP+DINP
2
0
GROUPS
Figure 4.6.3: POD (nmole) in kidney tissue of Cyprinus carpio exposed to DBP (5.43
mg/L), DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents
the mean ±S.E.M of 10 individuals.
44
KIDNEY
14
a a
12 a
T-BARS (nM/mg) 10
8 CONTROL
6 DBP
a DINP
4
DBP+DINP
2
0
GROUPS
Figure 4.6.4: T-BARS (nM/mg) in kidney tissue of Cyprinus carpio exposed to DBP (5.43
KIDNEY
3.5
3 a
ROS (U/g tissue)
2.5
2 CONTROL
a a
1.5 DBP
a
1 DINP
0.5 DBP+DINP
0
GROUPS
Figure 4.6.5: ROS (U/g tissue) in kidney of Cyprinus carpio exposed to DBP (5.43 mg/L),
45
4.7 Antioxidant enzymes in Muscles
In table 4.3 all the calculated values of antioxidant enzymes in muscle tissues of
common carp are mentioned
The catalase activity in muscle tissue of Cyprinus carpio show a significant change
(p<0.05) in DBP, DINP and mixture group when compare to control group. Whereas,
DINP and mixture group did not show significant alteration when compare with DBP.
Similarly, DBP and mixture group exhibited the non-significant change when compare
with DINP. Overall, the activity of CAT decreased in DBP, DINP and mixture when
compare among themselves (Figure 4.7.1).
SOD activity in muscles of Cyprinus carpio showed a significant decrease (p<0.05) in

mixture (DBP+DINP) and exhibited non-significant results in DINP and DBP when
compare with control. Moreover, mixture indicated significant results while DINP show
no significant alteration when compare with DBP. As a result, DBP, DINP and mixture
showed a significant decline (p<0.05) when compare with control group (Figure 4.7.2).
Peroxidase (POD) activity in Cyprinus carpio muscles showed a significant decrease

(p<0.05) in mixture group while DINP and DBP exhibited when compare with control.
It was observed that DINP and mixture did not show significant changes when compare
with DBP. Similarly, DBP and mixture (DBP+DINP) indicated non-significant changes
when compare with DINP treated group (Figure 4.7.3).
The activity of TBARS in muscles of C. carpio showed a significant increase (p<0.05)

in DBP, DINP and mixture group when compared with control. Similarly, DINP and
mixture group indicated non-significant change when compare with DBP. The overall
activity of TBARS significantly increases (p<0.05) in all groups when compare among
themselves.
46
Level of reactive oxygen species in muscles of common carp significantly increased

(p< 0.05) in DINP, DBP and mixture group when compare with control group.
Similarly, all treated group showed non-significant alteration when compared with each
other (Figure 4.7.5).
47
Table 4.3: The activity of catalase (CAT), superoxide Dismutase (SOD), peroxidase (POD), thiobarbituric acid reactive substances (TBARS)
and Reactive oxygen species (ROS) in the muscles of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP) treated groups.
Parameters Groups
CAT (U/mg Protein) 17.35±3.04 a 8.67±2.06b 15.88±2.21ab 8.11±1.19b 0.42

SOD ((U/mg Protein) 20.57±1.84 a 19.93±1.00ab 20.50±0.29 a 14.10±1.46b 0.020
POD (nmole) 14.90±1.96 a 9.86±1.04ab 11.89±1.00ab 6.81±1.03b 0.015
a a a a
TBARS (nM/mg tissue) 6.55±1.29 9.52±0.73 6.93±1.17 10.48±1.29 0.108
ROS (U/g tissue) 1.94±0.02a 1.33±0.32a 1.24±0.34a 1.59±0.33a 0.373
Note: Data are presented as mean value with ± S.E.M Different superscripts on the same row indicate there is a
significant difference (p<0.05)
48
MUSCLES
20
a
ab
CAT (U/mg Protein)

15
CONTROL
10 b b DBP
DINP
5
DBP+DINP
0
DBP DINP CONTROL
DBP+DINP
GROUPS
Figure 4.7.1: CAT (U/mg protein) in muscle tissue of Cyprinus carpio exposed to DBP (5.43
25 MUSCLES
a ab a
SOD (U/mg Protein)
20
b
15 CONTROL
10 DBP
DINP
5
DBP+DINP
0
GROUPS
Figure 4.7.2: SOD (U/mg protein) in muscles tissue of Cyprinus carpio exposed to DBP (5.43
mg/L), DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean
MUSCLES
18 a
16
14 ab
POD (nmle)
12 ab
10 CONTROL
8 b DBP
6 DINP
4
DBP+DINP
2
0
GROUP
Figure 4.7.3: POD (nmole) in muscles tissue of Cyprinus carpio exposed to DBP (5.43
49
MUSCLE
14
12 a
T-BARS (nM/mg)
a
10
a a
8 CONTROL
6 DBP
4 DINP
2 DBP+DINP
0
GROUPS
Figure 4.7.4: T-BARS (nM/mg) in muscles tissue of Cyprinus carpio exposed to DBP (5.43
mg/L), DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean
MUSCLE
2.5
2 a a
ROS (U/g tissue)
a a
1.5 CONTROL
DBP
1
DINP
0.5 DBP+DINP
0
GROUPS
Figure 4.7.5: ROS (U/g tissue) in muscles of Cyprinus carpio exposed to DBP (5.43 mg/L), DINP
individuals.
50
4.8 Antioxidant enzymes in Liver
In table 4.4 all the calculated values of antioxidant enzymes in liver tissues of common
carp are mentioned
The catalase activity in liver tissue of Cyprinus carpio showed a significant decrease
(p<0.05) in mixture group while non-significant results were observed in DBP and
DINP when compare with control group. The level of CAT showed a non-significant
decrease (p<0.05) in all treated groups (Figure 4.8.1).
In liver of C. carpio SOD activity significantly decrease (p<0.05) in DBP and DINP
and mixture group when compare with control group. All groups indicated non-
significant changes when compare with DBP. Similarly, when compare the DINP and
mixture group all treatments indicated the non-significant increase in their antioxidant
activity (Figure 4.8.2).
Activity of peroxidase in liver tissues of common carp significantly reduced in DBP,

DINP and in mixture group (p<0.05) when compare with control group. On comparison
with DBP control group showed significant results while other indicated non-
significant changes. Whereas, comparison with DINP exhibited no significant changes
(p<0.05) with all groups (Figure 4.8.3)
In liver tissues of Cyprinus carpio significant increase (p<0.05) was observed in DBP,
DINP and mixture group when compare with control group. While a non-significant
change (p<0.05) indicated in DINP and mixture group when compare with DBP.
Similarly, mixture group showed a significant increase (p<0.05) compare along with
DBP and DINP (Figure 4.8.4).
51
The activity of ROS in liver tissues of Cyprinus carpio significantly increased (p<0.05).
The level of ROS in all treated groups and control groups indicated a significant
increase (Figure 4.8.5).
52
(TBARS) and Reactive oxygen species (ROS) in the liver of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP)
treated groups
Parameters Groups
CAT (U/mg Protein) 12.54±0.29 a 11.16±0.86ab 11.18±0.74ab 9.29±0.32b 0.034

SOD ((U/mg Protein) 25.89±1.56 a 19.09±0.17b 21.33±0.55b 18.07±0.60b 0.001
a ab ab b
POD (nmole) 15.65±1.56 8.98±2.64 10.26±0.68 7.62±0.28 0.031
TBARS (nM/mg tissue) 5.33±0.23 a 7.09±1.14 a 5.23±0.34 a 7.69±0.80 a 0.102
ROS (U/g tissue) 1.22±0.32 a 1.57±0.33 a 0.93±0.03 a 1.60±0.34 a 0.373
Note: Data are presented as mean value with ± S.E.M Different superscripts on the same row indicate there is a significant
difference (p<0.05)
53
LIVER
14 a
12
ab ab
10 b
CAT (U/mg Protein) 8 CONTROL
6 DBP
4 DINP
2 DBP+DINP
0
GROUPS
Figure 4.8.1: CAT (U/mg protein) in liver of Cyprinus carpio exposed to DBP (5.43 mg/L), DINP (300
mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean ±S.E.M of 10
individuals.
LIVER
30 a
SOD (U/mg Protein)
25 b
b b
20 CONTROL
15 DBP
10 DINP
5 DBP+DINP
0
GROUPS
Figure 4.8.2: SOD (U/mg protein) in liver tissue of Cyprinus carpio exposed to DBP (5.43 mg/L),
DINP (300 mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean ±S.E.M
of 10 individuals.
LIVER
20
18 a
16
14
POD (nmle)
12 ab ab
CONTROL
10
8 b DBP
6 DINP
4
2 DBP+DINP
0
GROUPS
Figure 4.8.3: POD (nmole) in liver tissue of Cyprinus carpio exposed to DBP (5.43 mg/L), DINP
individuals.
54
LIVER
9 a a
8
7
T-BARS (nM/mg)
6 a a
5 CONTROL
4 DBP
3 DINP
2 DBP+DINP
1
0
GROUPS
Figure 4.8.4: T-BARS (nM/mg) in liver of Cyprinus carpio exposed to DBP (5.43 mg/L), DINP
individuals.
LIVER
2.5
2 a a
ROS (U/g tissue)
a
1.5
CONTROL
1 a DBP
DINP
0.5 DBP+DINP
0
GROUPS
Figure 4.8.5: ROS (U/g tissue) in liver of Cyprinus carpio exposed to DBP (5.43 mg/L), DINP (300
mg/L) and mixture (4.37 mg/L) for 30 days. Each value represents the mean ±S.E.M of 10
individuals.
55
4.9 Comet Assay
4.9.1 Liver
DNA damage in the liver of common carp (Cyprinus carpio) was observed by following
comet assay. In table 4 explanation is given relating to different parameters which were
attended in comet assay.
Head length
In common carp (C. carpio) liver head length exhibited a significant decreased (p<0.05)
in DINP, DBP and mixture (DBP+DINP) when compare with control group. Despite
the fact DINP and DBP show a non-significant change while mixture showed a
significant decrease (p<0.05) in head length when compare with control group. Overall,
the head length of DNA significantly decreased in mixture and DBP treated groups
(Figure 4.9.1).
Tail Length
In liver highly significant decreased (p<0.05) was noticed in tail length in all treated
groups DBP, DINP and mixture when compare with control group. DBP and mixture
group indicated a highly significant decrease (p<0.001) on comparison with control
group while DINP showed a non-significant change (p<0.05). Mixture group exhibited
a highly significant (p<0.05) results when compare with control group. Overall, tail
length of DNA increased in all treated groups (Figure 4.9.2).
% DNA in Head
% DNA in head of common carp liver cells showed a significant decrease (p<0.05) in
DBP, DINP and mixture treated groups on comparison with control group. Mixture
group showed a significant decreased in % DNA in head when compare with control
group. On contrary all groups exhibited a non-significant change (p<0.05) when
compared among themselves (Figure 4.9.3).
% DNA in Tail
% DNA in tail of common carp liver cells manifest the highly significant results
(p<0.05) in treated groups. Therefore, DBP and mixture group showed a significant
56
result whereas DINP display the non-significant changes on comparison with control.
DBP and DINP showed a not significant results in comparison with mixture as well as
among themselves. % Tail DNA significantly increase (p<0.05) in mixture when
compare to both DBP and DINP (Figure 4.9.4).
Tail Moment
In liver cells of Cyprinus carpio tail moment revealed the significant increase (p<0.05)
when comparison was made with control group. However, no significant alterations
were observed in DBP group as compared to control. In contrast to DBP a highly non-
significant (p<0.05) change was observed in mixture (DBP+DINP) group and no
significant change was observed in DINP group. When compare with control group
significant increase was also detected in DBP+DINP in comparison with DINP (Figure
4.9.5).
57
Table 5: Mean ± S.E.M head length (µm), tail length (µm), DNA in head (%), DNA in tail (%) and Tail moment (µm)in the DNA in
liver cells of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP) after 30 days exposure.
Parameters Groups
Head Length (µm) 119.66±2.72 a 111.66±2.18 a 116.77±1.88 a 110.70±1.19 a 0.047
Tail Length (µm) 25.04±1.70b 35.23±2.55a 27.30±1.81ab 35.39±1.08a 0.008
DNA in head (%) 87.82±0.52a 78.04±4.32ab 79.43±0.67ab 75.76±2.57b 0.045
DNA in tail (%) 12.17±0.52b 21.95±4.32ab 20.56±0.67ab 24.23±2.57a 0.045
Tail Moment (µm) 2.33±0.63a 3.11±0.82a 2.41±0.86a 3.71±0.64a 0.552
Note: Data are presented as mean value with ± S.E.M. Different superscripts on the same row indicate there is a significant difference (p<0.05)
58
Liver
125 a
120 a
115 a Control
Head Length (µm)
a
110 DBP
DINP
105
DBP+DINP
100
Control DBP DINP DBP+DINP
Groups
Figure 4.9.1: Head length (µm) measured in control, DBP, DINP and their mixture exposed
in Cyprinus carpio liver tissue after 30 days exposure
Liver
40 a a
35
30 b ab
Tail Length (µm)
25 Control
20
DBP
15
DINP
10
5 DBP+DINP
0
Groups
Figure 4.9.2: Tail length (µm) measured in control, DBP, DINP and their mixture exposed
in Cyprinus carpio liver tissue after 30 days exposure
Liver
100 a
90 ab ab
80 b
DNA in Head (%)
70
60 Control
50
DBP
40
30 DINP
20 DBP+DINP
10
0
DINP Control
DBP+DINP DBP
Groups
Figure 4.9.3: DNA in head (%) measured in control, DBP, DINP and their mixture
exposed in Cyprinus carpio liver tissue after 30 days exposure
59
Liver
30
ab a
25
DNA in Tail (%) ab
20
Control
15 b DBP
DINP
10
DBP+DINP
5
0
Control
DINP DBP
DBP+DINP
Groups
Figure 4.9.4: DNA in Tail (%) measured in control, DBP, DINP and their mixture exposed in
Cyprinus carpio liver tissue after 30 days exposure
Liver
6 a
5
a
Tail Moment (µm)
a
4
a Control
3 DBP
2 DINP
DBP+DINP
1
0
Groups
Figure 4.9.5: Tail Moment (µm)) measured in control, DBP, DINP and their mixture
exposed in Cyprinus carpio liver tissue after 30 days exposure
60
4.9.2 Kidney
In common carp (Cyprinus carpio) DNA damage caused by exposure of di-butyl and
di-isononyl phthalate and their mixture (DBP+DINP) was assessed by comet assay.
Description about different parameters is given in table 4.5.
Head Length
In kidney cells of common carp head length exhibited significant (p<0.05) change in
mixture group while DBP and DINP showed a non-significant change upon comparison
with control. A significant change was observed when compare the all treated groups
among themselves (Figure 4.9.2.1).
Tail Length
Tail length of DNA showed a significant result (p<0.01) in mixture and DBP when
compare with control group. DINP showed a significant result when compare with DBP
treated group. Similarly, DBP and mixture group exhibited the significant (p<0.01)
decrease when compare with the DINP (Figure 4.9.2.2)
% DNA in Head
% Head DNA indicated the significant decrease (p<0.01) in DBP and mixture group on
comparison with control group. However, all other treated groups shown non-
significant alteration on comparison among themselves.
% DNA in Tail
% Tail DNA in C. carpio kidney cells manifest the significant decrease (p<0.01) in DBP
and DBP+DINP when compare with control group. Whereas, remaining all treated
groups did not show significant alteration when compare with each other.
Tail Moment
In kidney non-significant changes were demonstrated in tail moment by DBP. DINP

and mixture (DBP+DINP) groups in contrary to control group as well as among
themselves.
62
Table 4.5: Mean ± S.E.M head length (µm), tail length (µm), DNA in head (%), DNA in tail (%) and Tail moment (µm)in the DNA
in liver cells of Cyprinus carpio in control, DBP, DINP and mixture (DBP+DINP) after 30 days exposure.
Parameters Groups
Head Length (µm) 94.89±0.53a 85.82±1.86 b 88.51±1.71ab 81.13±2.03b 0.002
Tail Length (µm) 17.46±0.32 b 24.14±2.66ab 23.22±1.57ab 26.54±2.16a 0.048
DNA in head (%) 87.56±0.72 a 78.18±0.71 b 79.36±1.84 b 76.75±1.84 b 0.003
DNA in tail (%) 11.84±1.15 b 21.81±0.71 a 20.63±1.84a 23.24±1.84 a 0.002
Tail Moment (µm) 4.64±1.20 a 5.44±0.68 a 4.86±0.50 a 6.85±0.61 a 0.276
Note: Data are presented as mean value with ± S.E.M Different superscripts on the same row indicate there is a significant difference (p<0.05)
63
Kidney
120
100 a
b ab
Head Lenght (µm)

b
80 Control
60 DBP
DINP
40
DBP+DINP
20
0
Groups
Figure 4.9.2.1: Head Length (µm)) measured in control, DBP, DINP and their mixture exposed
in Cyprinus carpio kidney tissue after 30 days exposure
Kidney
35
30 a
ab
Tail Length (µm)
ab
25
20 b Control
15 DBP
10 DINP
5 DBP+DINP
0
Groups
Figure 4.9.2.2: Tail Length (µm)) measured in control, DBP, DINP and their mixture exposed
in Cyprinus carpio kidney tissue after 30 days exposure
Kidney
90 a
85
DNA in head (%)
b
80 b b Control
DBP
75
DINP
70 DBP+DINP
65
Groups
Figure 4.9.2.3: DNA in Head (%)) measured in control, DBP, DINP and their mixture exposed in
Cyprinus carpio kidney tissue after 30 days exposure
64
Kidney
30
25 a
a a
DNA in Tail (%)

20
Control
15 b DBP
DINP
10
DBP+DINP
5
0
Groups
Figure 4.9.2.4: DNA in Tail (%)) measured in control, DBP, DINP and their mixture exposed in
Cyprinus carpio kidney tissue after 30 days exposure
Kideny
8 a
7
a a
6 a
Tail oment(µm)
5
Control
4
DBP
3
DINP
2
DBP+DINP
1
0
Groups
Figure 4.9.2.5: Tail Moment (µm) measured in control, DBP, DINP and their mixture exposed in Cyprinus
carpio kidney tissue after 30 days exposure.
65
Chapter # V
Discussion
Fishes not only provides food to community but is also a source of livelihood for
individuals involved in commercial fishing especially in Inland water-logged areas
(Sheikh et al., 2017). Fish are frequently utilized as sentinel organism for
ecotoxicological studies since they play a number of parts within the trophic web,
accumulate harmful substances and react to low concentration of mutagens (Cavas and
Ergene, 2005). In this manner, the use of fish biomarkers as indices of the impacts of
contamination, are of increasing significance and can allow early detection of aquatic
environmental issues (Van Der Oost et al., 2003). Phthalates are colorless, low
instability, ineffectively water-soluble synthetic natural compounds (Paluselli et al.,
2020). In most commercial items, PAEs are utilized as additives and are not combined
with plastics within the shape of covalent bonds. They can effectively move from those
items to the environment through dissipation, filtering, and scraped area (Dutta et al.,
2020).
Lethal concentration 50 (LC50), describes the amount of a chemical induced to

laboratory animals that results in the death of 50% of the laboratory animals used in a
toxicity study. The LC50 values are then used to determine which concentration is
necessary to show toxic effects in organisms (Braunbeck et al., 2005). In current study,
lethal and detrimental effects of di-isononyl phthalate (DINP) and di-butyl phthalate
(DBP) and their mixture (DBP+DINP) on antioxidant enzymes status and DNA
integrity in Cyprinus carpio were observed. The results had distinctly represented that
DBP, DINP and mixture (DBP+DINP) tremendously lethal and deleterious to C. carpio.
The value of 96 hours LC50 (acute toxicity) of DBP and their mixture (DBP+DINP) in
Cyprinus carpio was found for DBP was 15.90 mg/L-1 and for mixture 13.13 mgL-1 the
LC50 of DINP did not find even at its highest dissolving concentration 300 mgL-1 which
agrees well with Zhao et al. (2014) who observed LC50 value for 96 hours of DBP for
Cyprinus carpio found 16.30 mgL-1. LC50 value of di-n-butyl phthalate (DBP) against
Oreochromis niloticus (Juvenile Nile tilapia) observed to be 11.8 mg/L for 96 hours by
Khalil et al. (2016) which inlined the present study. Grass carp (Ctenopharyngodon
Idella) was exposed to DBP for 21 days with grading concentrations 1, 10, 100 and
1000 µg/L (Faheem et al., 2021). In a 72-hour zebrafish embryo toxicity test, the LC50
67
value for BBP, DBP and the six-phthalate mixture were found to be 0.72, 0.63 and 0.50
ppm (Chen et al., 2014). Similarly, DINP was exposed to tilapia (Oreochromis
mossambicus) at a concentration of 300 ppm for short term (24, 48, 72 and 96 hours)
and long term (7, 14, 30 and 60 days) (Revathy et al., 2018) and in this experiment
author also did not find LC50 value of DINP at its maximum concentration 300 mgL-1.
Antioxidant enzymes are proteins involved in the catalytic conversion of reactive

oxygen species and their byproducts into stable, nontoxic molecules (Fridovich et al.,
1999). Therefore, represent the most important defense mechanism against cell damage
caused by oxidative stress. The role of antioxidants is to balance the cellular production
of ROS, maintaining the intracellular redox balance by preventing the cellular damage
caused by ROS (Gargallo et al., 2018). In an organism antioxidant defense system helps
combat oxidative stress and includes several enzymes and vitamins (Wilhelm et al.,
1993). Oxidative stress is basically an imbalance between reactive metabolites and
production of free radicals, and their eradication by defensive mechanism known as
antioxidants (Yonar, 2018). CAT is highly sensitive and provides the first line of
defense against ROS thereby protecting the organism from oxidative stress (Gauvin et
al., 2017). Since lipid peroxidation (LPO) is one of the primary signs of oxidative
damage brought on by a variety of substances, including metals (Ercal et al., 2001) it
has been employed as a pollution biomarker (Almroth et al., 2005). The enzyme
superoxide dismutase (SOD) catalyses the dismutation of the superoxide anion into O2
and H2O2, while catalase (CAT) combines with H2O2 to produce oxygen molecules and
water (Kohen et al., 2002). In aerobic organisms, the SOD group of metalloenzymes
serves as the main line of defense against the harmful effects of superoxide radical and
plays a critical antioxidant role (Pandey et al., 2001). Saglam et al. 2014 proposed that
diminished SOD activity could be an indicator of damage within the antioxidant
mechanisms caused by toxicants. Within the display ponder, the decline in Sod activity
moreover demonstrates harm in Grass protein due to the overproduction of ROS from
DBP and DEP stress
Fish are of special interest to study oxidative stress because of the properties of the
water environment and its relationship with organisms (Francour et al., 1995). In
current study the activity of CAT (catalase) significantly decreased in gill, liver, kidney
and muscles tissue which indicate the increase production of ROS. Higher level of
TBARS was observed in kidney, liver, muscles and gills tissue in exposure with DBP
68
and DINP and their mixture. Level of SOD and POD was significant low in vital organ
(kidney, liver, muscles, gills) tissues of common carp (Cyprinus carpio). A significant
increase was observed in activity of ROS in kidney tissues followed by liver, gills and
muscles. The obtained results were not unexpected as gills are the first organ to be in
contact with toxicant an after the biotransformation in liver should affect in a lesser
level the kidney. Similarly in previous study Latif et al. 2020 observed rohu antioxidant
defense system when exposed to DEP concentration 0.01 µgL-1. The activity of CAT
decrease significantly in all exposed tissues. Higher LPO levels were observed for gills,
followed by liver and kidneys. Catalase showed significant inhibition of its activity only
at the kidney level at all exposure concentrations. Same results as compare with present
study shown when an important culture fish Olive founder (Paralichthys olivaceous)
was treated with Di-ethyl phthalate (DEP) for 3 days with dose (0, 100, 300, 900 mg/kg)
body weight. After 24 hours final dose the biochemical effect on fish body was detected
in liver, kidney and blood serum. The parameters measured mostly were restricted to
oxidative stress and toxicity of organ. In hepatic tissue there was a significant increase
in TBARS at 100 mg/kg while decrease in catalase activity was also observed (Kang et
al., 2010). The occurrence of DINP in fish has been documented in various literatures
with the levels ranging from undetectable to 11,576μg/Kg (Munshi et al., 2013). The
water solubility of DINP is less than 0.001mg/ L (Staples et al., 1997) and the highest
soluble concentration of DINP dissolved in propylene glycol as organic solvent was
found to be 300 ppm, where no mortality was noticed for 96 h (Revathy and Chitra,
2015). According to the maximum solubility limit 300 mg/L was selected as the test
concentration in the present study. Prediction of toxicity of any chemicals on single
species provides basic information to improve the quality of life and protect the
environment from the adverse effects of the toxicant. Thus the present study adopted
short-term exposure of DINP for 96 h using single test species, Cyprinus carpio.
Similarly, Revathty et al. 2018 examined the Oreochromis mossambicus when exposed
with DINP 300 mg/L for long- and short-term duration. There was a significant change
in antioxidant enzymes activity in gills, liver and muscles. A significant decrease
(p<0.05) was observed in CAT (catalase) and SOD (superoxide Dismutase) and
induction in ROS activity.
Alkaline single-cell gel electrophoresis (SCGE), often known as the comet assay, is a
highly simple, fast, and sensitive method for measuring the amount of DNA damage
69
produced in individual cells in order to determine genotoxicity. It is a vital instrument
for monitoring the environment and evaluating the health of aquatic life since it can
identify DNA damage in fish, clams, shellfish, and mussels (Andrade et al., 2004). DNA
strand breaks are an additional sensitive sign of genetic damage that can be found using
the comet assay. This test has been used in aquatic settings to evaluate and track the
genetic health of both vertebrate and invertebrate species (Kleinjans et al., 2002).
Usually, cellular digestion system is well set up as the source of endogenous reactive
oxygen species (ROS), and it is these (normally non-pathogenic) cellular forms that
account for the foundation levels of oxidative DNA damage detected in normal tissue
(Cooke et al., 2003). Within the present study, the noteworthy increment in DNA
damage compared to the controls was due to oxidative stress, which may be induced by
the ROS collection. Also, the ROS collection induced by phthalates moreover
specifically caused DNA damage and the activation of DNA repair instruments. A
number of studies have appeared that ROS is the major source of DNA damage by
causing strand breaks, expulsion of nucleotides, and different alterations of the
nucleotide bases (Cooke et al., 2003). In spite of the fact that cells have created repair
components to adjust normally happening changes in DNA, excessive ROS can lead to
damage of DNA. In current study di-butyl, di-isononyl phthalates and their mixture
(DBP+DINP) exposure for 30 days resulted in DNA damage which was investigated in
different organs of Cyprinus carpio included liver and kidney by comet assay, which is
an extremely sensitive, accurate, easily adaptable, simple, fast and give definitive
results (Ullah et al., 2022).`This study showed DBP, DINP and DBP+DINP exposure
induced decline in the head length and % DNA in head whereas significant increase in
% DNA in tail, tail length and tail moment in treated groups as compare to control
group. Similarly, Khalil et al. (2016) did a study to find the genotoxic impact of di-
butyl phthalate on juvenile nile tilapia (Oreochromis niloticus) by using alkaline comet
assay. Data obtained from this study demonstrated the similar result with current study,
that there was a significant alteration (p<0.05) in values of all observed parameters such
as tail length, tail moment and %DNA in tail. Afshan et al. 2018 exposed the crucian
carp (Carassius carassius) with di-n-butyl phthalate (3.88 ppm) for 3 weeks. The
obtained results indicated that there was a significant increase in DNA damage in
exposed cells as compared to control group.
70
It is known that the increment of ROS substance in cells can cause it to attack bases
specifically, deoxyribonucleic Acid (DNA) deoxynucleotide backbone and other
cellular components, causing DNA strand disturbance, base adjustment, DNA-DNA
cross-linking, DNA-protein crosslinking and other oxidative DNA harm, all of which
lead to genotoxicity (Chen et al., 2008).
Previous studies shown that increase in ROS content synchronized with DNA damage
(Ray et al., 2012). Therefore, the DBP and DINP induced cellular toxicity could be
mainly related to the ROS content generated by cellular oxidative which indicated that
ROS play an important role in inducing genotoxicity (Shao et al., 2018). In particular,
the overproduction of ROS will attack the DNA structure when oxidants and
antioxidants are not balanced, which will result in more serious DNA damage and cell
dysfunction. If DNA damage is not repaired in a timely manner, it may also cause
additional disruptions and negative effects on cell homeostasis (Song et al., 2022).
The current study showed that observed amount of DNA damage is more in tissues of
kidney as compare to liver. The kidney plays an important role in protecting the animal
from potentially toxic substances by excrete excess water while retaining most of the filtered
solutes (Latif and Faheem, 2020).
71
Conclusion
Current study concluded that sublethal dose (1/3rd) of di-butyl phthalate and di-isononyl
phthalate and their mixture (DBP+DINP) has toxic and lethal effects on the adult
common carp (Cyprinus carpio). Moreover, suppressed activities of antioxidant
enzymes due to plasticizers caused oxidative stress and also significant change in
different organs along with DNA damage. It can be concluded that di-butyl and di-
isononyl phthalate and their mixture (DBP+DINP) are genotoxic plasticizers and
genotoxic possibility of pollutants in biomonitoring analyses can be determined by
comet assay using common carp (Cyprinus carpio) as a model organism. For ecological
development it is suggested to discard the waste products of plastic industries in a safe
way, moreover it can protect the aquatic and terrestrial habitat.
72
SOPs
Students/researcher handling fishes and experimental chemicals will follow given

SOPs for reliable practice:
1. Students use face mask, lab coats, gloves and head cover to prevent inhalation
and direct contact to chemicals.
2. Later chemical treatment to finish they will properly rinse and sanitize their
hands.
3. They will not be allowed to eat, drink or use any cosmetics in research lab.
4. It will be necessary for them to maintain a good personal hygiene.
5. They should discard the waste properly
6. Suitable personal protection will also be implemented for dose preparation,
administration and dealing with waste disposal.
73
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(2019). Di (2-ethyl hexyl) phthalate (DEHP)-induced spleen toxicity in quail
(Coturnix japonica) via disturbing Nrf2-mediated defense response. Environmental
pollution, 251, 984-989.
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(PAEs) exposure: A review of aquatic animal toxicology studies. Science of the
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111. Zhao, X., Gao, Y., & Qi, M. (2014). Toxicity of phthalate esters exposure to
carp (Cyprinus carpio) and antioxidant response by biomarker. Ecotoxicology, 23,
626-632.
84
Appendix-I Physicochemical parameters of water in control group of Cyprinus carpio
Dissolved
No. of days Temperature Hardness pH Ammonia Carbon dioxide Calcium Magnesium
oxygen
°C (mg/I) (mg/I) (mg/I) (mg/I) (mg/I) (mg/I)
1 28.01 300 7.51 0.023 4.89 1.19 11.222 67.986
2 28.09 300 7.50 0.031 4.88 1.21 11.984 67.510
3 28.50 299 7.49 0.035 4.87 1.22 12.124 67.172
4 28.89 299 7.50 0.022 4.89 1.20 12.545 66.910
5 27.89 299 7.51 0.049 4.88 1.21 12.984 66.635
6 28.00 299 7.49 0.050 4.89 1.21 13.124 66.547
7 28.03 299 7.52 0.051 4.88 1.20 13.346 66.409
8 28.00 299 7.51 0.051 4.87 1.19 13.679 66.201
9 28.71 300 7.49 0.045 4.87 1.21 13.998 66.251
10 27.97 300 7.50 0.055 4.89 1.21 14.134 66.166
11 27.94 300 7.51 0.056 4.88 1.22 14.346 66.034
12 27.99 300 7.52 0.061 4.89 1.23 14.785 65.760
13 28.03 299 7.50 0.055 4.87 1.21 14.990 65.382
14 28.02 299 7.49 0.071 4.86 1.22 15.232 65.230
15 28.98 300 7.50 0.071 4.89 1.23 15.460 65.338
16 27.94 300 7.48 0.078 4.87 1.20 15.790 65.131
17 28.09 300 7.50 0.081 4.88 1.22 15.900 65.063
18 27.88 299 7.51 0.099 4.87 1.21 16.033 64.729
19 27.03 299 7.52 0.091 4.89 1.22 16.347 64.533
20 28.09 300 7.51 0.990 4.88 1.23 16.568 64.645
21 27.93 300 7.50 0.110 4.86 1.22 17.010 64.369
22 28.01 300 7.48 0.137 4.86 1.21 17.456 64.090
23 27.05 299 7.50 0.133 4.86 1.22 17.679 63.701
24 27.91 299 7.49 0.159 4.86 1.23 17.999 63.501
25 28.05 300 7.50 0.158 4.86 1.22 18.011 63.743
26 28.09 300 7.50 0.161 4.86 1.23 18.346 63.534
27 27.94 300 7.52 0.171 4.87 1.20 18.679 63.326
28 28.01 300 7.51 0.197 4.87 1.21 18.999 63.126
29 28.04 299 7.52 0.196 4.88 1.21 19.032 62.855
30 28.00 299 7.50 0.280 4.88 1.23 19.238 62.726
Mean±S.D 28.04±0.39 299.53±0.59 7.5±0.01 0.12±0.17 4.87±0.01 1.21±0.01 15.56±2.34 65.15±1.45
85
Appendix -II Physiochemical parameters of water of Di-butyl Phthalate (DBP) treated group in Cyprinus carpio
Dissolved
Temperature Hardness Ammonia Carbon dioxide Calcium Magnesium
No. of days pH oxygen
°C (mg/I) (mg/I) (mg/I) (mg/I) (mg/I)
(mg/I)
1 28.00 300 7.50 0.007 4.89 1.19 10.013 68.741875

2 28.09 299 7.50 0.021 4.88 1.22 11.964 67.27225
3 28.00 299 7.52 0.027 4.87 1.22 12.154 67.1535
4 28.89 300 7.51 0.017 4.89 1.20 12.535 67.1658125
5 27.89 300 7.49 0.019 4.88 1.22 12.884 66.94725
6 28.00 299 7.51 0.019 4.89 1.22 13.110 66.556
7 28.03 299 7.52 0.122 4.88 1.23 13.347 66.4084375
8 28.00 299 7.48 0.124 4.87 1.23 13.658 66.21375
9 28.71 299 7.50 0.127 4.87 1.23 13.898 66.0635
10 27.97 299 7.52 0.242 4.85 1.23 14.034 65.9785
11 27.94 299 7.51 0.256 4.86 1.24 14.366 65.7715625
12 27.99 300 7.50 0.278 4.86 1.24 14.790 65.7565625
13 28.03 300 7.48 0.281 4.86 1.24 14.890 65.6940625
14 28.02 300 7.53 0.292 4.83 1.25 15.272 65.4551875
15 29.98 299 7.52 0.310 4.8 1.25 15.460 65.087625
16 27.94 299 7.50 0.322 4.79 1.26 15.789 64.8819375
17 28.09 299 7.49 0.328 4.78 1.26 15.891 64.8181875
18 27.88 299 7.50 0.299 4.76 1.26 16.033 64.729125
19 27.03 299 7.51 0.300 4.74 1.24 16.347 64.5334375
20 28.09 299 7.52 0.329 4.71 1.26 17.661 63.7120625
21 27.93 300 7.48 0.341 4.7 1.25 17.010 64.368875
22 28.01 300 7.49 0.309 4.71 1.24 18.135 63.665625
23 27.05 300 7.52 0.339 4.69 1.26 19.679 62.7006875
24 27.91 300 7.51 0.333 4.69 1.25 20.999 61.8758125
25 28.05 300 7.52 0.344 4.68 1.27 21.011 61.868
26 28.09 300 7.51 0.351 4.68 1.27 21.346 61.6590625
27 27.94 300 7.52 0.311 4.68 1.27 24.679 59.57575
28 28.01 299 7.50 0.311 4.7 1.24 26.999 57.8758125
29 28.04 299 7.51 0.319 4.68 1.26 29.032 56.60475
30 28.00 299 7.51 0.322 4.67 1.26 32.064 54.71
Mean±S.D 28.05±0.49 299.43±0.50 7.5±0.01 0.23±0.12 4.78±0.08 1.24±0.02 17.16±5.27 64.12±3.30
86
Appendix-III Physiochemical parameters of water on daily basis in Di-isononyl phthalate (DINP) exposed group of Cyprinus
carpio
No. of days Temperature Hardness pH Ammonia Dissolved oxygen Carbon dioxide Calcium Magnesium
°C (mg/I) (mg/I) (mg/I) (mg/I) (mg/I) (mg/I)

1 27.90 299 7.48 0.008 4.89 1.190 8.016 69.74
2 27.02 300 7.50 0.011 4.88 1.210 8.016 69.99
3 27.92 299 7.51 0.025 4.87 1.220 10.032 68.48
4 27.02 299 7.51 0.015 4.86 1.200 11.024 67.86
5 28.90 299 7.52 0.112 4.88 1.210 11.214 67.74125
6 27.93 299 7.49 0.168 4.86 1.210 11.341 67.661875
7 28.03 298 7.52 0.141 4.88 1.200 11.459 67.338125
8 28.05 299 7.50 0.198 4.87 1.190 12.568 66.895
9 28.06 298 7.51 0.204 4.85 1.210 12.726 66.54625
10 28.07 298 7.52 0.232 4.85 1.210 12.902 66.43625
11 27.80 299 7.52 0.158 4.88 1.220 12.888 66.695
12 28.23 299 7.50 0.167 4.86 1.230 12.898 66.68875
13 28.98 299 7.48 0.225 4.87 1.210 13.360 66.4
14 28.23 300 7.52 0.236 4.84 1.220 13.479 66.575625
15 28.90 300 7.50 0.279 4.84 1.230 13.692 66.4425
16 28.02 298 7.51 0.281 4.87 1.240 13.786 65.88375
17 27.90 298 7.50 0.257 4.84 1.240 13.989 65.756875
18 27.99 298 7.52 0.276 4.84 1.250 13.815 65.865625
19 27.12 299 7.50 0.209 4.76 1.250 13.230 66.48125
20 28.02 300 7.50 0.229 4.77 1.255 14.347 66.033125
21 28.02 300 7.51 0.281 4.75 1.256 14.653 65.841875
22 28.05 299 7.50 0.239 4.75 1.259 14.801 65.499375
23 28.06 299 7.48 0.227 4.74 1.259 14.942 65.41125
24 28.00 299 7.52 0.235 4.73 1.261 15.001 65.374375
25 27.98 299 7.50 0.284 4.72 1.266 15.022 65.36125
26 28.00 300 7.49 0.261 4.7 1.260 15.234 65.47875
27 28.03 299 7.48 0.311 4.73 1.270 15.546 65.03375
28 27.89 299 7.51 0.323 4.69 1.271 15.892 64.8175
29 27.90 299 7.50 0.318 4.7 1.272 16.010 64.74375
30 28.99 300 7.52 0.339 4.68 1.272 16.032 64.98
Mean±S.D 28.03±0.47 299.03±0.66 7.5±0.01 0.208±0.09 4.80±0.07 1.23±0.02 13.26±2.11 66.46±1.31
87
Appendix-IV Physiochemical parameters of water in mixture of Di-butyl and Di-isononyl phthalate (DBP+DINP) treated group
of Cyprinus carpio.
No. of Dissolved Carbon
Temperature Hardness pH Ammonia Calcium Magnesium
days oxygen dioxide
°C (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
1 28.03 300 7.50 0.035 4.89 1.190 8.016 69.99
2 28.02 299 7.50 0.049 4.88 1.210 8.016 69.74
3 28.06 299 7.52 0.134 4.82 1.220 10.052 68.4675
4 28.00 299 7.49 0.142 4.85 1.230 11.028 67.8575
5 28.00 299 7.50 0.158 4.8 1.230 12.214 67.11625
6 27.90 300 7.52 0.157 4.81 1.240 12.341 67.286875
7 28.02 300 7.48 0.161 4.8 1.240 13.556 66.5275
8 28.09 300 7.49 0.167 4.82 1.250 13.877 66.327
9 28.00 299 7.50 0.172 4.81 1.250 14.245 65.846875
10 28.09 299 7.51 0.177 4.84 1.250 14.653 65.591875
11 28.03 300 7.50 0.183 4.84 1.260 14.920 65.675
12 28.09 300 7.50 0.189 4.85 1.260 15.345 65.409375
13 27.98 300 7.50 0.190 4.79 1.240 15.657 65.214375
14 27.90 300 7.51 0.195 4.78 1.270 16.450 64.71875
15 27.98 299 7.51 0.203 4.83 1.230 16.782 64.26125
16 27.99 299 7.50 0.233 4.78 1.230 16.986 64.13375
17 28.09 299 7.50 0.256 4.8 1.250 17.224 63.985
18 28.01 299 7.48 0.273 4.75 1.260 17.982 63.51125
19 28.04 300 7.50 0.281 4.79 1.280 18.678 63.32625
20 28.09 300 7.49 0.289 4.76 1.230 19.356 62.9025
21 28.02 299 7.51 0.296 4.79 1.270 19.872 62.33
22 28.03 300 7.50 0.301 4.75 1.260 20.780 62.0124375
23 27.98 300 7.50 0.327 4.71 1.250 21.450 61.59375
24 27.90 299 7.50 0.345 4.68 1.280 21.890 61.06875
25 28.00 299 7.51 0.360 4.71 1.250 22.286 60.82125
26 28.03 299 7.50 0.371 4.72 1.270 22.580 60.6375
27 28.09 300 7.50 0.379 4.69 1.270 23.460 60.3375
28 28.09 300 7.52 0.382 4.67 1.280 24.379 59.763125
29 28.99 300 7.50 0.391 4.67 1.290 24.420 59.7375
30 29.03 300 7.50 0.423 4.64 1.290 25.651 58.968125
Mean±S.D 28.09±0.25 299.53±0.50 7.50±0.009 0.24±0.10 4.77±0.06 1.25±0.02 17.13±4.89 64.17±3.03
88
Appendix- V Mortality of Cyprinus carpio in different concentration of di-butyl phthalate at 96 hours exposure period
Con. of Dibutyl phthalate R1 R2 R3

Mean±SD
mgL-1 % % %
2.0 0 0 0 0.00±0.00
4.0 0 10 0 3.33±5.77
6.0 0 10 10 6.66±5.77
8.0 20 10 20 16.66±5.77
10.0 30 30 20 26.66±5.77
12.0 30 40 30 33.33±5.77
14.0 40 50 40 43.33±5.77
16.0 50 60 60 56.66±5.77
18.0 50 60 60 56.66±5.77
20.0 60 60 70 63.33±5.77
22.0 70 70 80 73.33±5.77
24.0 80 80 90 83.33±5.77
26.0 90 100 100 96.66±5.77
28.0 100 100 100 100±0.00
89
Appendix-VI Mortality of Cyprinus carpio in different concentration of di-isononyl phthalate at 96 hours exposure period
Con. of Di-isononyl phthalate R1 R2 R3

Mean±SD
mgl-1 % % %
0.00 0 0 0 0.00±0.00
25.00 0 0 0 0.00±0.00
50.00 0 0 0 0.00±0.00
75.00 0 0 0 0.00±0.00
100.00 0 0 0 0.00±0.00
125.00 0 0 0 0.00±0.00
150.00 0 0 0 0.00±0.00
175.00 0 0 0 0.00±0.00
200.00 10 10 10 10±0.00
225.00 10 10 20 13.33±5.77
250.00 10 10 20 13.33±5.77
275.00 20 20 20 20.00±0.00
300.00 20 20 30 23.33±5.77
90
Appendix-VII Mortality of Cyprinus carpio in different concentration of Mixture (DBP+DINP) at 96 hours exposure period
Con. of DBP+DINP where DINP =

R1 R2 R3
300mg/L Mean±SD
% % %
mgl-1
2.0 0 0 0 0.00±0.00
4.0 0 10 10 6.66±5.77
6.0 10 10 20 13.33±5.77
8.0 20 30 40 30.00±10.00
10.0 30 40 50 40.00±10.00
12.0 40 50 50 46.00±0.00
14.0 50 60 60 56.66±5.77
16.0 60 70 70 66.66±5.77
18.0 70 70 80 73.33±5.77
20.0 80 80 80 80.00±0.00
22.0 90 80 90 86.66±5.77
24.0 90 100 90 93.33±5.77
26.0 100 100 100 100.0±0.00
91

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Effects of di-butyl and di-isononyl phthalate on antioxidant

enzyme status and DNA damage in Cyprinus carpio (Common

Dr. Moazama Batool

GOVERNMENT COLLEGE WOMEN UNIVERSITY

A DISSERTATION SUBMITTED TO GOVERNMENT COLLEGE WOMEN

GOVERNMENT COLLEGE WOMEN UNIVERSITY

Name: ______________________ Name: ______________________

Signature: ____________________ Signature: ____________________

Supervisor Internal Examiner

Name: ______________________ Name: ______________________

Signature: ____________________ Signature: ____________________

External Examiner Chairperson

Table 2 Determination of Acute Toxicity (LC50) of di-isononyl

Table 3 Determination of Acute Toxicity (LC50) of Mixture

Table 4 The activity of catalase (CAT), superoxide Dismutase

Table 5 The activity of catalase (CAT), superoxide Dismutase

Table 7 The activity of catalase (CAT), superoxide Dismutase

Fig: 2 Fish Dissection 16

Fig: 3 Determining the growth performance of fish during 18

Fig: 4 Determining hardness of water 19

Fig: 5 Observing the antioxidant activity on 24

Fig: 6 Preparation of comet slides 25

Fig: 7 Observation of Comet slides under fluorescent 26

Fig: 8 The probit analysis of mortality (%) of Di-butyl 29

Fig: 9 The probit analysis of mortality (%) of Di-isononyl 31

DBP Di-butyl Phthalate

DINP Di-isononyl Phthalate

DBP+DINP Di-butyl and Di-isononyl Phthalate

LC50 Lethal Concentration

DNA Deoxyribonucleic Acid

ROS Reactive Oxygen Species

SOD Superoxide Dismutase

POD Peroxide Dismutase

T-BARS Thiobarbituric acid reactive

LPO Lipid peroxidation

RBCs Red blood cells

WBCs White blood Cells

EDTA Ethylenediaminetetraacetic acid

DEP Di-ethyl phthalate

SCGE Single cell gel electrophoresis

H2O2 Hydrogen Peroxide

CH3COONa Sodium Acetate Buffer

EBT Erichrome black Tea

PBS Phosphate Buffer Saline

NaOH Sodium hydroxide

NaCl Sodium chloride

FAO Food and Agriculture Organization

GST Glutathione S-transferase

ANOVA Analysis of Variance

SPSS Statistical package of social scince

DEPPD N-N, Diethyl Paraphenyldamine

RMPA Regular Melting point Agarose

LMPA Low Melting point Agarose

RPM Rotation per minute

Di-isononyl phthalate (DINP) is a mixture of branched-chain alkyl phthalate isomers

Common carp can modify ecological characteristics of aquatic system so it is frequently

Acute toxicity is generally determined by short-term exposure of fish to a range of

The aims and objectives of present study are following;

Sepperumal et al. (2014) evaluated the DBP effects on tilapia Oreochromis

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