Amna Thesis Black Print 5 Set

MS DISSERTATION
I, Amna Nawaz D/O Shah Nawaz student of MS Zoology, Government College

Women University, Sialkot, Pakistan, hereby declare that the data quoted in this thesis
entitled “PHTHALATES INDUCED TOXICITY ASSESSMENT BY
HISTOMORPHOMETRIC AND SEMEN ANALYSIS IN Oreochromis
mossambicus (TILAPIA)” Is based on my original work and has 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.
Amna Nawaz
2021-2023
ii
PHTHALATES INDUCED TOXICITY ASSESSMENT BY
HISTOMORPHOMETRIC AND SEMEN ANALYSIS IN
Oreochromis mossambicus (TILAPIA)
A DISSERTATION SUBMITTED TO GOVERNMENT COLLEGE WOMEN

UNIVERSITY SIALKOT
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD

OF DEGREE OF MS IN ZOOLOGY
Amna Nawaz
2021-GCWUS-1885
DEPARTMENT OF ZOOLOGY
GOVERNMENT COLLEGE WOMEN UNIVERSITY
SIALKOT
PAKISTAN
2021-2023
iii
GOVERNMENT COLLEGE WOMEN UNIVERSITY
SIALKOT DEPARTMENT OF ZOOLOGY
Dated: ____________
FINAL APPROVAL
This is to certify that we have evaluated the thesis submitted by Ms. Amna Nawaz
D/O Shah Nawaz on “Phthalates induced toxicity assessment by histomorphometric
and semen analysis in Oreochromis mossambicus (Tilapia)” 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 my whole work to Allah Almighty, thank you for the guidance, strength,
power of mind, protection, skills and for giving me a healthy life. I wholeheartedly
dedicated this research to my beloved parents, Bushra Nawaz and Shah Nawaz, 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.
Amna Nawaz
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.
Amna Nawaz
vi
ACKNOWLEDGEMENTS
First and foremost praise to Allah, the Almighty, and the greatest of all. On whom
ultimately we depend for sustenance and guidance. I would like to thank Almighty
Allah for giving me courage, strength and determination to perform my research in a
successful manner. His continuous grace and mercy was with me throughout my life,
and ever more during the tenure of my research. I also offer my humblest and sincere
words of thanks with special Durood and Salam to my beloved Holy Prophet
Muhammad (PBUH) who is the foremost source of knowledge for all mankind.
My profound gratitude goes to my respectable supervisor Dr. Qurat-ul-Ain for her

countless hours of reflecting, reading, encouraging and most of all patience
throughout the entire process. I would like to thank Dr. Moazama Batool for her
guidance and attention.
I would also like to express my gratitude to my head of department Dr. Asma

Waheed Qureshi for all the things that facilitated smooth work of my research.
I would like to dedicate this work to my brave mother Bushra Nawaz and my sincere
and generous father Shah Nawaz, for providing their moral, spiritual, emotional and
financial support.
My humble thanks to my sisters, Aisha Nawaz, Maryam Nawaz and my brother and
bhabi, Tahir Nawaz and Nazish Tahir who share their words of advice and
encouragement to finish this research and helped me at every stage of academic life.
Moreover, my special regard goes to my seniors for guidance, discussion, and

criticism. I would like to thanks to my fellows Asma Aziz, Fatima Arshad, Farwa
Shehzadi, Zunaira Khalid, Hina Nabi and Tehniat Tahir who assisted me in my
research. Their excitement and willingness to provide feedback made the completion
of this research an enjoyable experience.
Amna Nawaz
vii
CONTENTS
LIST OF TABLES X
LIST OF FIGURES xi-xii
LIST OF APPENDICES xiii
LIST OF ABBREVATIONS xiv
ABSTRACT 1
Chapter 1 INTRODUCTION 2-9
1.1 Aims and Objectives 10
Chapter 2 REVIEW OF LITERATURE 11-17
Chapter 3 MATERIALS AND METHODS 18-32
Experimental design 18
Ethical considerations 19
Acute toxicity (96-hr LC50) determination 21
Physico-chemical parameters determination 21
Determination of sub-lethal toxicity of phthalates 24
Semen Analysis 25
Sperm parameters 25
Sperm motility 25
Sperm viability 25
DNA Damage 26
Histomorphometric Analysis 28
Statistical Analysis 32
Chapter 4 RESULTS 33-52
Determination of Acute Toxicity (LC50) of DBP, 33
DINP and Combination (DBP+DINP) in Oreochromis
mossambicus during exposure period of 96 hours
Determination of Physico-chemical parameters of 40
water during 96 hours of exposure
Determination of sub lethal (1/3rd of LC50) toxicity 40
Determination of Physico-chemical parameters of 41
water under sub lethal (1/3rd of LC50) toxicity
exposure
viii
Determination of Semen Analysis 42
Motility 42
Viability 43
Determination of DNA Damage 46
Histomorphometric Analysis 52
Chapter 5 DISCUSSION 54-59
CONCLUSION 60
REFERENCES 61-74
APPENDICES 75-81
ix
LIST OF TABLES
Sr. No. Title Page No.
Table 1 Determination of Acute Toxicity (LC50) of di-butyl 34
phthalate in O. mossambicus during the exposure of
96 hour through Probit analysis
Table 2 Determination of Acute Toxicity (LC50) of di- 36

isononyl phthalate in O. mossambicus during the
exposure of 96 hour through Probit analysis
Table 3 Determination of Acute Toxicity (LC50) of mixture 38

(DBP+DINP) in O. mossambicus during the
exposure of 96 hour through Probit analysis
Table 4 Motility parameters in all experimental groups 42

Table 5 Mean±SEM Head length (µm), Tail length(µm), 46
%DNA in head, %DNA in tail and Tail moment
(µm) in sperms of O. mossambicus measured
against different groups Control, DINP, DBP and
their Mixture (DBP+DINP) groups after 30 days of
sublethal exposure.
x
LIST OF FIGURES
Fig: 1.1 Structural formula of Di-butyl phthalate (DBP). 5
Fig: 1.2 Structural formula of Diisononyl phthalate (DINP). 6
Fig: 1.3 Oreochromis Mossambicus (Mozambique Tilapia). 8
Fig: 3.1 Head Marala hatchery of Sialkot. 18
Fig: 3.2 Control and Treatments Groups of Fish. 18
Fig: 3.3 Fish housing and management. 19
Fig: 3.4 Dissection of Oreochromis mossambicus. 20
Fig: 3.5 Water hardness was checked against the EDTA. 22
Fig: 3.6 Slide Preparation for Comet 26
Fig: 3.7 Staining the slides. 27
Fig: 3.8 Slides were observed under flourescence 28
microscope.
Fig: 3.9 Embedding process. 30
Fig: 3.10 Microtomy. 30
Fig: 3.11 Staining the slides. 32

Fig: 4.1 The Probit analysis of mortality (%) of di-butyl 35
phthalate in O.mossambicus.
Fig: 4.2 The Probit analysis of mortality (%) of di-isononyl 37
phthalate in O.mossambicus.
Fig: 4.3 The Probit analysis of mortality (%) of mixture 49
(DBP+DINP) in O.mossambicus.
Fig: 4.4 a. represented the sperms in control group which 43
was significantly high in numbers b. represented
sperms of di-isononyl phthalates which shows the
non-significant difference c and d figure represent
the sperms of DBP and DBP+DINP respectively
which showed a significant reduction in sperm
numbers.
Fig: 4.5 Percentage sperm motility in Control and the 44
exposure groups (DBP, DINP and DBP+DINP) of
xi
O. mossambicus.
Fig: 4.6 Progressive movement (min/s) of sperms in 44
Control, and the exposure groups (DBP, DINP and
DBP+DINP) of O. mossambicus.
Fig: 4.7 Moderate movement (min/s) of sperms in Control, 45
and the exposure groups (DBP, DINP and
DBP+DINP) of O. mossambicus.
Fig: 4.8 Percentage sperm viability in Control and the 45
exposure groups (DBP, DINP and DBP+DINP) of
O. mossambicus.
Fig: 4.9 Chromatin material scattered in sperms of O. 48
mossambicus at 40X in different groups as (a)
Control, (b) DINP, (c) DBP and (d) Mixture
(DBP+DINP)
Fig: 4.10 Head length (µm) in sperms of O. mossambicus 49
measured against Control, DBP, DINP and Mixture
(DBP+DINP) after 30 days of sublethal exposure.
Fig: 4.11 Tail length (µm) in sperms of O. mossambicus 49
Fig: 4.12 % DNA in Head in sperms of O. mossambicus 50
Fig: 4.13 % DNA in Tail in sperms of O. mossambicus 50
Fig: 4.14 Tail moment (µm) in sperms of O. mossambicus 51
Fig: 4.15 Histological changes showed in testicular tissues of 53
O. mossambicus exposed in DINP, DBP and
Mixture (DBP+DINP) for 30 days.
xii
LIST OF APPENDICES
Appendix-1 Mortality of Oreochromis mossambicus in different 75
concentrations of Di-butyl phathalate at 96h
exposure period
concentrations of Di-isononyl phathalates at 96h
exposure period
concentrations of DBP+DINP at 96h exposure
period
Appendix-4 Physiochemistry of Control group on daily basis in 78
Oreochromis mossambicus
Appendix-5 Physicochemistry of Sub-lethal toxicity (1/3rd 79
(LC50) of DBP on daily basis in Oreochromis
mossambicus
(LC50) of DINP on daily basis in Oreochromis
mossambicus
(LC50) of DBP+DINP on daily basis in
Oreochromis mossambicus
xiii
LIST OF ABBREVIATIONS
Acronyms Abbreviations
PEs Phthalate esters
DBP Dibutyl phthalate
DnBP Di-n-butyl phthalate
DIDP Diisododecyle phthalate
BBP Benzyl butyl phthalate
DEP Diethyl phthalate
DINP Diisononyl phthalate
DEHP Diethylhexyl phthalate
HMW High molecular weight
SVHC Substance of very high concern
HPG Hypothalamo- pitutary gonadal

axis
FSH Follicle stimulating hormones
LH Leutinizing harmones
ECS Endocannabonoid system
GSI Gonadosomatic index
EDCs Endocrice distrupting chemicals
IUPAC International Union of Pure and

Applied Chemistry
EDTA Ethylene diaminetetra acetic acid
DNA Deoxyribonucleic acid
xiv
Abstract
Phthalates are plasticizers that are commonly used to enhance the properties of
polyvinyl chloride products. When they are released into the environment, they pose a
serious risk to human health. The primary concern with phthalate plasticizers is their
potential toxicity to aquatic organisms and their effects on reproductive systems.
Therefore, the present research was conducted to examine the reproductive toxicity of
phthalates i.e. di-butyl phthalate (DBP) and di-isononyl phthalate (DINP) by finding
out their LC50 for Oreochromis mossambicus (tilapia) and to check their sublethal
effects on reproductive parameters. After acclimatization, adult fish were divided into
three experimental groups and one control group. Mortality data of all the
experimental groups was collected to find the LC50 of DBP and DINP and their
mixture. These LC50 were obtained by using the Probit analysis method. The 96hr
LC50 for O. mossambicus was found to be 12.17 mgL-1 of DBP, and 11.75 mgL-1 in
case of mixture (DBP+DINP) group. The LC50 of DINP was not found at its highest
dissolving concentration i.e 300 mgL-1. The sub-lethal dose was calculated as 4 mgL-1
of DBP, 300 mgL-1 of DINP, and 3.91 mgL-1 of DBP+ DINP for O. mossambicus. All
physico-chemical parameters of water were checked thoroughly during acute and sub-
lethal doses of DBP, DINP, and their mixture. Results of sperm parameters showed a
significant decline in the sperm numbers, sperm motility and viability in the DBP and
mixture groups. Comet assay of sperm also showed the highly significant increase of
tail length (µm), percent tail DNA (%), and the tail moment (µm) in the exposure
groups. In Histological analysis of testes, DBP-treated fishes show irregular
seminiferous tubules with disintegration of spermatozoa, and reduced spermatocytes
numbers while DINP-treated groups show elongated seminiferous tubules, and
reduced sperm count. A mixture group of DBP+DINP shows the highest variation
which includes interstitial spaces, disintegration of spermatozoa and the empty lumen.
Thus results of histomorphometric and semen analysis of fish showed that phthalates
induced the potential toxic effects on the male reproduction of fish.
Key words: Tilapia, histomorphometric analysis, semen analysis, and male

reproductive toxicity
1
Chapter 1
Introduction
Plasticizer, a man made chemical, is an integratable material that is used to

increase the flexibility, dilateability and workability in a plastic or other material like
elastomer, specified through the IUPAC - International Union of Pure and Applied
chemistry (Godwin, 2011; Yan et al., 2021). Globally, Plasticizers are used to make
the plastic products and it is anticipated that by 2026, their demand will have
increased from 7.45 million tons to 10.5 million tons annually (Ceresana, 2019).
Plasticizers comprises upto 40% of total weight of plastic but are not covalently
attached with the products and may leach out from the products that cause the
environmental pollution (Bauer and Herrmann, 1997). It is thought that commonly
used plasticizers may have endocrine-disrupting qualities, which raises widespread
public health concerns (Ghisari, 2009; Swan, 2008). Plasticizers are classified into
two types on the base of original polymer i.e. internal plasticization and external
plasticization but the common classification depends upon the composition of
chemicals. Therefore, Esters of phthalates, diabasic acid esters, epoxy plasticizers,
glycol connate, citrates, and phosphate esters are among the crucial forms of
plasticizers (Langer et al., 2020).
Due to incorrect discharge of effluents and contaminated runoff from

increased consumption of consumer goods like plasticizers, xenobiotics substances
such as phthalic acid esters has caused a dramatic increase in freshwater and marine
habitats (Khalil et al., 2016). Aquatic ecosystems are eventually contaminated as a
result of environmental contaminants like xenobiotics substances that are released as a
by-product of human activities (Khalil et al., 2016). Acute and chronic exposures of
these chemicals contain a significant proportion of environmental mishaps and have
adverse effects on ecosystem (Johnson and Yund, 2007). Endocrine disruptions are a
concern due to the risks associated with the usage of synthetic drugs causing a drastic
decline in both the quantity and quality of the aquatic population which may impede
the growth, developmental, reproductive and immune functions of many organisms
(Reynolds et al., 2005). A persistent impact on reproduction is listed as one of the
2
main risks to environmental organisms among the many endocrine disruption
mechanisms (Guillette et al., 1994; Steinmetz et al., 1996). Endocrine disruptors are
those substances or molecules that prevent the endocrine system from operating
normally, which would otherwise allow an organism to coexist peacefully with its
environment (Mohan et al., 2022). The health of people and animals is impacted as a
result of this endocrine disruptors, particularly when consumption or usage of fish as a
nutritious source, because fish have the greatest potential to expose people to these
residues because they are known to be bioaccumulators of pollutants (Dorea, 2006).
One of the greatest examples of xenobiotics is phthalate esters (PEs), an endocrine
disruptive molecule that can affect a variety of biological systems, including those of
invertebrates, fish, reptiles, birds, and mammals (Moder et al., 2007).
An ester of phthalic acid is called phthalate. Phthalate is a category of

aromatic compounds consisting of two extended and linked acetate groups bonded to
a phenyl ring (Hu et al., 2020). Phthalates, which are categorized as endocrine
disruptors and were originally developed for commercial usage in the 1920s, are a
large set of additives, or plasticizers, used in the production of synthetic materials
(Huang et al., 2008). The abundantly used plasticizers are phthalates because this
chemical compound is used to impart the softness in normally rigid plastics i.e they
are useful to provide the flexibility, distensibility and elasticity in the polymeric
products (Wan et al., 2013). Widely, phthalates are used to make the polyvinyl
chloride products i.e. in building materials pipes, flooring, sheeting, in cosmetics,
food packaging, pharmaceutical and items for personal hygiene (Zhang et al., 2019).
Also used in many solvents, lubricating oils, fixatives and as a detergent (Zhang et al.,
2016; Chen et al., 2018). Cuisine, water, human blood plasma, urine, and other
substances have been reported to contain phthalates, with an estimated 6.0 million
tons produced annually (Liu et al., 2011; Schecter et al., 2013). Phthalates, which
make approximately 10 to 40% of a toy's weight overall, have been banned in several
countries because they may have negative health effects on people (Duty et al., 2003).
Soft toys in the local market contained more phthalates than hard toys (Johnson et al.,
2011). Because the phthalate esters and plastic do not chemically interact so the direct
discharge, draining, evaporation, erosion, and migration are easy ways for them to
cause pollution into the ecosystem (Staples et al., 1997; Wittassek et al., 2011).
3
Phthalates are also released into the atmosphere and surface water through burning
and plastic manufacturing, respectively (Jobling et al., 1995). Phthalates are not
durable in aquatic environments due to their easy microbial and abiotic breakdown,
but they are quite widespread and frequently present at high concentrations due to
their continuous release in a huge volume (Staples et al., 1997).
Diester phthalates are found in nearly 25 different types of commercial

products, the most common of which are di-butyl phthalates (DBP), benzyl butyl
phthalates (BBP), di-ethyl hexyl phthalates (DEHP), di-isononyl phthalates (DINP),
and di-ethyl phthalates (DEP) (Jarmolowicz et al., 2010). Phthalates are categorized
according to their molecular weight and carbon numbers that present in their ester
chain. Number of carbon atoms shows inverse relationship with the solubility of water
and volatility and therefore, the toxicity of phthalates depends upon these relationship
(KEMI et al., 2015). Phthalates induced toxic effects on human health are known
since 1950, and these toxic effects include endocrine toxicity, cardiotoxicity,
hepatotoxicity, reprotoxicity, nephrotoxicity and carcinogenesis (Seyoum and
Pradhan, 2019). Humans are exposed to phthalates on a large scale via ingesting foods
and beverages that have come into contact with phthalate-containing items and
containers. People are exposed to phthalates by two main ways, one through the
dermal absorption and second through the inhalation, in addition to food sources
(Silvia et al., 2003). In the past two decades, numerous studies have connected
phthalates to adverse health effects on people, including asthma, obesity, diabetes,
breast cancer, ADHD, autism, neurodisorder, behavioural change, aberrant
reproductive processes, and infertility (Stojanoska et al., 2017; Wang et al., 2019).
Di-butyl phthalate (DBP), a liquid that is oily, colourless to very faintly

yellow, and has a hazy pleasant odour, is used to manufacture pliable polymers and is
present in an assortment of goods for consumption (ATSDR, 2001). DBP belongs to a
unique catagory of plasticizer that have a low molecular weight, more volatile and
more soluble in water as compared to any other high molecular weight that's why it is
often used as a combination with HMW phthalates (Sun et al., 2015). N-butanol and
phthalic anhydride, both of these are the substrates of DBP. This commonly used
plasticizer is formed as a component in a prepration of paints, resins, adhesives and
other various types of polymers (Xu et al., 2015). A vast assortment of consumer
4
goods, such as floor tiles, food wrapping, raincoats, shower curtains, vinyl textiles,
and car interiors, use DBP (ATSDR, 2001).
Figure 1.1: Structural formula of Di-butyl phthalate (DBP).
Due to its close proximity to DEHP in terms of global manufacturing, DBP is

widely present in the atmosphere, water, soil, and living things (Tiwari et al., 2019).
Air, surface water and soil are the non- persistive source for DBP at some extent. In
soil, DBP has a short half-life of just a few days (WHO, 1997). It is known that DBP
from the atmosphere can wash out into the aquatic environments through precipitation
and dry deposition. While the airborne photodegradation half-life of DBP is between
7.4 hours and 3.1 days, its estimated 144-day biodegradation half-life in water spans
from one to fourteen days in naturally occurring surface water, in ground water and
soil, it ranges from two to twenty- three days (Staples et al., 1997).
The main source of food-borne di-butyl phthalate exposure is probably fish

and other seafood, as exposure levels in fish have been found to range between 78 and
200 parts per billion (US, 1999). Aquatic species are extremely susceptible to DBP
due to its lipophilic nature and high persistence in aquatic sediments. Moreover, DBP
was found in several drinking water supplies at concentrations between 0.1 and 5ppb
(US, 1999). DBP increases its solubility relative to the parent chemical by forming
complexes in freshwater with fulvic acid and humic compounds/ substances (Tiwari
et al., 2019). Di-butyl phthalates are known since 1991, of inducing toxic effects in
the reproductive system. Therefore, numbers of research have led to the classification
of di-butyl phthalate as an endocrine disruptor (Jergenson et al., 2019). According to
published data released by the European Chemical Agency, di-butyl phthalate is
5
categorized as a chemical of extreme concern as SVHC because of its massive
amount, multiple widespread applications, and hazardous characteristics (REACH,
2018).
DINP, a colourless and odourless oily viscous liquid of high molecular weight
phthalates is composed of more than six carbons in their backbone which give more
durability and permanency. Without DINP, vinyl would lose the ability to become
flexible enough to be used for synthetic leather and electrical wires (CPSC, 2010).
PVC applications account for about 95% of its manufacture, with the remaining 5%
going into non-PVC goods (ECHA, 2010). DINP is a less soluble phthalate and shows
the slower rate of migration in the aquatic environment and was first introduced for
the replacement of more toxic diethyl hexyl phthalates (Zhang et al., 2019; Vieira et
al., 2011).
Figure 1.2: Structural formula of Di-isononyl phthalate (DINP).
In aquatic environment, DINP released from multiple sources, including air

deposition, external surface water runoff, and direct or indirect sewage discharge. Due
to its strong hydrophobicity, DINP is adsorbs into sediments and is also linked to
surface water. Thus, sediments act as a reservoir and provide a grave danger to the life
of organisms that are present in the aquatic environments (Adeniyi et al., 2011). In the
air, surface water, soil, and sediments, the half-life of DINP is 0.7 days, 50 days, 300
days, and 3000 days, respectively (Oehlmann et al., 2008). The human cord, urine,
blood plasma, and mother's milk have all been reported to contain DINP and its
metabolites (Lin et al., 2011). DINP is also very toxic and hazardous for the aquatic
6
life and if inhaled then it also cause damage for humans because it is considered that it
induced the fertility damage (Saad, 2021). Since DINP has been found to have
endocrine disruptive qualities, it is known to imitate or oppose the actions of
endogenous hormones, having detrimental consequences on growth, development,
and reproduction (Fisher, 2004). It is becoming more and more concerning that DINP
is frequently found in aquatic ecosystems because of its constant discharge into the
environment and cause potential damage to aquatic organisms (Liu et al., 2014). As
soon as DINP enters the aquatic environment, it may find its way up the food chain to
higher creatures, including humans (Chen et al., 2014).
Fish are among the vertebrate animals that have a great deal in common with
mammals, including humans, in terms of their functional and sequence similarities.
Fish are an excellent bioindicator of the contamination of aquatic environment
because of their diversity of species. Therefore, fish were utilized as a model
organism in the majority of ecotoxicological investigations, which were conducted
both in the lab and in the field. Fish are preferred as an appropriate animal model due
to their relatively modest size, straight forward manner of life in the natural world,
high fertility rate, affordability, ease of handling, and up keep in lab settings (Sajla et
al., 2019).
Fish have well-developed endocrine, neurological, immunological, and

osmoregulatory systems (Song et al., 2012). Fish are more receptive to and sensitive
to many contaminants in water as compared to other vertebrate life forms. The fish's
body can easily absorb toxicants that have been discharged into the aquatic ecosystem
through skin contact, gill and mouth breathing, and consumption along the food chain.
As a result, any tiny variation from the aquatic environment, typical biological
condition instantly manifest as changes in the physiology and behaviour of exposed
fish (Solman and McNeil, 2012).
In jawed vertebrates, the hypothalamo-pituitary-gonadal (HPG) axis is a

highly conserved area that controls the production and release of the hypothalamic
pituitary gonadotropin hormones i.e. FSH- follicle stimulating hormone and LH-
luteinizing hormone , which in turn govern gonadal growth and development (Sower
et al., 2009). Additionally, gonad-derived activin and inhibin's endocrine feedback on
7
the control of gonadotropins performs an essential function in the synchronisation of
the HPG axis (Kretser et al., 2002). Majority of male and female reproductive issues
in fish populations are linked to exposure to substances having endocrine disrupting
characteristics. The evaluation of a number of reproductive endpoints in fish,
including reproductive behaviour, delayed sexual maturity, gonado-somatic indices,
vitellogenin levels, sperm parameters, activities of steroidogenic enzymes, and
changes in hormonal parameters, reveals the negative effects of endocrine disrupting
chemicals (EDCs) (Scholz et al., 2009).
Oreochromis mossambicus is a rare fish in Pakistan due to its African origin,

Adults consume trash, while its juveniles consume a variety of foods. It has an
accelerated maturation rate and breeds all year round. Tilapia is an excellent food
source for humans and has very high biological properties. It is therefore seen as
having tremendous significance on a global scale (Reynoldz et al., 2005; Russell et
al., 2012). Furthermore, the local inhabitants love to prefer this fish because of its
flavor (Naeem et al., 2011). O. mossambicus is frequently chosen as an experimental
model due to its hardiness, tolerance, and extreme environmental adaptability.
Figure 1.3: Oreochromis Mossambicus (Mozambique Tilapia).
The river dominions must regularly monitor the fish habitat and, if needed,
perform biological remediation in fish dwelling areas to preserve its well-being,
reproduction, and flesh quality (Jabeen and Chaudhary, 2010). In order to ascertain
whether phthalates—that are, DBP, DINP and their mixture—are detrimental to
8
reproduction, the current study selected an adult male O. mossambicus as an
experimental model and performed semen and histomorphometric analysis on that
animal.
9
Aims and Objectives
The primary goals and objectives of the current study are as follows:
 To find out/ ascertain the acute toxicity (96- hr LC50) of phthalates (di-butyl
phthalate and di-isononyl phthalate) to the fish, O. mossambicus.
 To evaluate the sublethal effects of phthalates by performing histomorphometric

analysis in O. mossambicus.
 To assess the reproductive toxicity in phthalates exposed fish, O. mossambicus by

performing semen analysis.
10
Chapter 2
Review of Literature
Revathy and Chitra (2018) assessed that the Mozambique tilapia (freshwater
fish) inhibited the reproduction process due to the exposure of di-isononyl phthalate
(DINP). DINP was exposed to fish at 300 ppm (mgL-1) for lengths ranging from
seven to sixty days, as well as short term (24, 48, 72, and 96 hours). Results
demonstrated a discernible drop in each antioxidant enzyme's activity accompanied by
a rise in lipid peroxidation in the testis and ovary. Steroidogenic enzyme activity
dramatically dropped, indicating that DINP changed the normal steroidogenesis of the
testicles and ovaries, which hinders fish reproduction.
Santangeli et al. (2017) studied the di-isononyl phthalate (DINP) and its
impact on Danio rerio reproduction. Five distinct sublethal DINP concentrations were
given to fish i.e. 0ugL-1, 0.42ugL-1, 4.2 ugL-1, 420ugL-1, 4200ugL-1 for a period of 21
days. Findings from the research illustrated that DINP has an adverse consequence on
the reproduction and the development of Zebrafish, which causes aberrant alterations
in oocyte growth and maturation.
Sruthi et al. (2021) assessed the reproductive toxicity of di-butyl phthalate

(DBP) on the Pseudetroplus maculates, fresh water fish. Sublethal exposure of DBP
with concentration 0.2mgL-1 to fish for 1, 3, 4, 7 and 15 days and as a result, together
with a concurrent rise in the quantity of estradiol and a decline in testosterone, a
significant rise in the amount of alkali-labile phosphates and whole protein in
testis and plasma was noted, demonstrating how male reproductive functions are
negatively impacted by DBP. By confirming the absence of mature sperms ,
unorganized spermatogonia, and sperm cells, histological analysis revealed that DBP
is toxic to the testicles, which may cause infertility and a drop in the numbers of fish.
Chen et al. (2014) assessed that phthalates have the ability to disrupt
estrogenic hormones in the body and induced toxicity. Using a 72h toxicity test on a
Zebrafish embryo and a 24-hour test on a ChgH-EGFP (A receptive to estrogen)
transgenic medaka (Oryzias melastigma) eleutheroembryos with different
11
concentrations and mixtures of phthalates. The results showed that BBP and DBP
were toxic to the developing organisms, and that BBP, DBP, DEHP, and DINP all had
estrogenic endocrine disrupting properties on whole organisms. These findings
suggested that the prolonged exposure to these phthalates may present adverse health
effects.
Revathy and Chitra (2019) conducted an experiment to determine the role of

di-ethyl hexyl phthalates (DEHP) on the antioxidant status in the ovary and testes of
Oreochromis mossambicus. The efficacy of antioxidant enzymes in the ovary and
testes was assessed after fish were exposed to 60 ppm of DEHP for varying lengths of
time. Results of this experiment indicated that the levels of lipid peroxidation in the
testes and ovaries increased simultaneously with a decrease in antioxidant enzyme
activity following exposure to DEHP. Results suggested that the exposure of DEHP
disturbed the pro-oxidant and antioxidant balance that preferentially affected the
normal reproductive functions of fish.
Bhatia et al. (2014) studied the antiestrogenic impacts of di-n-butyl phthalate

on adult male Murray Rainbow fish, Melanotaenia flauviatilis. In his study, fish were
exposed to the high concentration of 125, 250, 500, and 1000 ugL-1 of DnBP.
Testicular degeneration caused by high DnBP concentrations included cell
vacuolation, dying cell bodies, intestitial fibrosis, and delayed development with a
marked decrease in the percentage of mature sperms.
Hu et al. (2020) studied the reproductive toxicity of DnBP in Zebrafish. In his

experiment, embryos of Zebrafish were exposed to low concentrations of DnBP (4.9,
13.6, and 43.8 ugL-1) for two hours after fertilization until they reached sexual
maturity. The chronic exposure of DnBP (43.8 ugL-1) demonstrated the impairs
reproductive functions, as evidenced by decreased egg production, altered steroid
hormone levels, and a notable change in HPG axis gene expression. Level of
testosterone was also increased. Prenatal exposure to DnBP also illustrated that DnBP
lowered the rate of hatching, survival, and F1 generation growth.
Bhaisare et al. (2022) conducted an investigation to clarify the impact of

benzyl butyl phthalate (BBP) on the gonads i.e testes and ovaries of Clarias
gariepinus (African catfish). BBP was administered to fish at a low concentration i.e
12
2mgL-1 for 5 days and at high concentration i.e 6mgL-1 for 10 days. As a result, severe
disruption was observed in the gonads of fish. The anomalous fish reproductive
system was observed because of vacuolized seminiferous tubules, damaged germ
cells, and enlargement of sertoli cells in the testes.
Forner-Piquer et al. (2018) carried out an investigation to examine the impacts

of di-isononyl phthalate on Zebrafish's gonads for the study of the endocannabinoid
system (that controls the reproductive and metabolic processes). Three
environmentally relevant DINP concentrations (42ugL-1, 4.2ugL-1, 0.42ugL-1) were
chronically exposed to adult Zebrafish via water for a duration of 21 days. Results of
this experiment demonstrated that the ECS was disrupted and reproduction was
impacted in Zebrafish due to the ecological suitable concentrations of DINP. This was
due to the alterations in the genes coding enzymes for ECS receptors, as well as a
gender-specific higher activation of fatty acid amide hydrolase.
Kim et al. (2002) aimed this investigation to highlight the consequences of

phthalate i.e. DEHP's effect on the inhibtion of oocytes development in Japanese
medaka (Oryzias latipes). For this purpose, fish were exposed to the minimal
concentartion of DEHP i.e. 1, 10 and 50ugL-1 for a specific period, starts from
hatching to 3 months of age. A notable decreased were observed in blood vitellogenin
level, GSI and matured oocytes in DEHP treated groups of fish because this phthalate
prevents fish reproductive organs from developing.
Mohan et al. (2022) conducted an experiment to elucidate the sub-lethal

consequences of DEP plasticizer on reproduction of fresh water fish, Murrel, Channa
striatus. Ova diameter, fecundity, and gonadosomatic index significantly decreased in
Channa striatus as a result of the endocrine disrupting action of DEP, that harmfuly
affects the reproductive system of fish.
Ghisari (2009) performed an in vitro experiment to investigate the

implications of plasticizers and their combination on thyroid and estrogen receptor
activity. By using rat pitutary GH3 cells for T-secreening and MVLN cells for
assessing the estrogenic activities of these plasticizers. Results of this experiment
demonstrated that plasticizers induced endocrine disprupting potential that further
13
enhanced with the interferences of estrogen receptors and the TH (thyroid) system
that creates bad impacts on health.
Patyna et al. (2006) evaluated the hazardous consequences of di-isododecyle

and di-sononyl phthalates on the Japanese medaka (Oryzias latipes). Fish were fed a
diet that included both chemicals at a concentration of 20ugg-1. According to the
results, DINP exposed male fish showed huge metabolites of testosterone while DIDP
exposed female fish showed increased testosterone hydroxylase activity.
Godai et al. (2021) aimed this study to illustrate the di-isononyl phthalate's
effects on zebrafish ovaries either this phthalates cause atresial follicles or not. DINP
was given to adult female zebrafish for a period of 21 days with specific
concentrations i.e. 0.42ugL-1, 4.2ugL-1 and 42ugL-1. Results of this study
demonstrated that the atresial follicles occured due to exposure of DINP that leads to
the abnormality in the reproductive system of female Zebra fish.
Muhammad et al. (2018) investigated the long-term exposure effects of

plasticizers (DEHP and ATBC) on Danio rerio reproduction and on the gonads
development. Zebrafish were exposed to the plasticizers for a long period. i.e. 6
months at 0.5ugL-1 that was an environmental appropriate concentration. As a results,
significant adverse effects were observed in the inhibtion of body length, weight, GSI
with impaired fecundity which indicates that plasticizers creates bad impacts on fish
development and reproduction. While Suzuki et al. (2011) demonstrated that the
prenatal exposure of DEHP di (2- ethylhexyl) phthalate elicit bad affects on the
reproduction and development.
Yuen et al. (2020) assessed that short-term exposure of DEHP impaired early
survival (larval reproduction) success in Japanese medaka. Embryos of Japanese
medaka were exposed to DEHP at 0.001ppb, 0.1 ppb and 10 ppb concentrations for a
transient period of 21 days. Significant increased mortality was observed in medaka
embryos before hatching. Young hatchling showed poor progress in egg production
and fertilization. In histological examination, significant reduction of mature sperm
was also observed in male medaka. Results indicated that DEHP, the phthalates
impaired the reproductive progress in fish.
14
Park et al. (2020) conducted an experiment to examined the reproductive
toxicity of phthalates i.e. mono (2-ethylhexyl) phthalates on endocrine activity of
Zebrafish. For this study, fish were exposed with MEHP at their effective
concentration of 0.2, 10, 50 ug/ml for the duration of 21 days. A significant reduction
in the egg numbers was observed in MEHP exposed fish that disturb the normal
function of endocrine in zebrafish and cause reprotoxocity.
Adeogun et al. (2018) assessed how the Clarias gariepinus- African catfish
reacted to endocrine disruptors i.e DEHP. For three to four days, juvenile of Clarias
gariepinus were exposed at 10, 100, 200, 400ugL-1 concentration of DEHP.
Histological analysis of gonads showed that atresial oocyte/oocyte atresia, Inter sex
were occured in female while in male distortion on degenation of seminiferous
tubules were observed in DEHP exposed fish.
Ma et al. (2017) studied the phthalates effects on the reproductive system

linked with the DNA methylation in male Zebrafish. For a period of 3 months male
Zebrafish were chronically exposed with DEHP at environmentally suitable
concentration i.e. 0, 10, 33 and 100ugL-1. Inhibited spermatogenesis, damaged
testicular ultrastructure observered in male Zebrafish that were revealed by
histological analysis. The exposure of male testes to DEHP resulted in a notable
increase in global DNA methylation.
Zhu et al. (2016) performed an experiment to study the dysfunctional effects

of reproduction in Zebrafish caused by phthalates exposure. For this purpose, fish
were exposed with minute concentration i.e (0ugL-1, 0.46ugL-1, 4.0ugL-1 and 37.5ugL-
1
) of MEHP for 81 days. Results of this experiment demonstrated that egg production
(with reduced egg diameter, egg shell and low content of egg proteins) and sperm
quality were reduced after the chronic exposure of phthalates i.e. MEHP, which
indicated that phthalates have potential to impair the reproduction of fish of both
sexes.
Guo et al. (2015) aimed this study to investigate the impacts of esters of
phthalic acid by its acute exposure on the reproduction of Gobiocypris rarus
(Chinese-rare minnow). The concentration of DEHP that fish larvae were exposed to
for six months, or nearly 180 days, were 0, 4.2, 13.3, and 40. 8ugL-1. In female fish,
15
plasma testosterone and 17B-estradiol levels decreased, while male fish showed the
opposite effects. Furthermore, it was noted that male spermiation was retarded and
female oocyte maturation was inhibited, along with decreased egg production.
Guo et al. (2017) assessed that combined exposure of DEHP, DBP and ATBC
(plasticizers) caused harmful effects on Zebrafish development and reproduction. All
three plasticizers at a concentration of 50ugL-1 were exposed to adult fish for 3
months. Results demonstrated that male Zebrafish were more sensitive to combined
exposure as comparison with the females. Significant decreased in GSI, disturbed the
process of oogensis and spermatogensis and impariment of fecundity were observed.
Golshan et al. (2015) assessed that DEHP disrupts pituitary and normal
functions of testicular hormones that reduced sperm quality in mature goldfish
(Carassius auratus). In this study, DEHP exposed to fish at 100ugL-1 concentration
for 30 days. As a result, a significant decline was observerd in sperm number, sperm
motility and sperm velocity in goldfish due to DEHP exposure. The amount of
luteinizing hormone was likewise lower in the exposed fish, suggesting that
phthalates, or DEHP, disrupt testicular and pituitary hormone function, which in turn
lowers the quality of sperm.
Wang et al. (2013) investigated how di-(2-ethylhexyl)-phthalate affected

Gobiocypris rarus, a Chinese rare minnow, in terms of endocrine disruption. For 21
days, fish were exposed with chemical at environmentally relatable concentration of
0ugL-1, 3.6ugL-1, 12.8ugL-1, 39.4ugL-1 and 117.6ugL-1. Exposure to phthalates
resulted in greater concentration of circulating testosterone and reduced concentration
of estradiol in female gonads while in male gondas T/E2 ratio was decreased. DEHP
altered plasma sex hormones.
Bhatia et al. (2013) investigated that the antiestrogenic effects of DBP in

female Murray rainbow fish (Melanotaenia fluviatilis). For this investigation, 7 days
exposure of DnBP to adult female rainbow fish at 125ugL-1, 250ugL-1, 500ugL-1 and
1000ugL-1 concentrations were conducted and determined the changes in ovarian
histology. Consequently, the female fish gonads showed signs of granulomatous
inflammation, interstitial fibrosis, shurken ooplasm, impaired or reduced yolk
production, and chorion folding. Reduction of circulating level of vitellogenin was
16
also observed due to the exposure of DnBP which indicate that phthalates cause
antiestrogenic effects in female reproductive system.
Bhatia et al. (2015) assessed that juvenile Murray rainbow fish (Melanotaenia
fluviatilis) exhibit retarded body growth and disturbed gonad development when
exposed to DnBP (di-n-butyl phthalate) at chronic level. Over the course of 30 to 90
days, fish were exposed to DnBP at environmentally appropriate concentration of 5,
15 and 50ug-1. Results demonstrated that, after the environmentally exposure of
DnBP, gonadal development was strongly affected.
Corradetti et al. (2013) investigated the outcomes of Bis-(2-ethyexhyl)

phthalate on spermatogenesis that impaired in Danio rerio (Zebrafish) due to DEHP.
Adult male fish were exposed at 0.2 and 20ugL-1 for 7 to 21 days. The findings
showed that DEHP significantly decreased the production of embryos, increased
DNA fragmentation in sperm cells, and caused mitotic arrest during spermatogenesis
in Zebrafish, all of which had an adverse effect on reproduction.
Jarmolorwicz et al. (2013) demonstrated that during sex differentiation period

in European pikeperch (Sander luciperca), exposure of DnBP induced feminization
process. Observed reproductive effects indicated that DBP significantly affected the
sex ratio in european pikeperch (Sander luciperca). Forner-Piquer et al. (2019)
illustrated that after 21 days feeding with DINP at the dose of 15 and 1500ug/L/day
alters the histoarchitecture of testes in S. aurata.
Jarmolowicz et al. (2014) examined the benzyl butyl phthalate’s impacts on

pikeperch reproduction and evaluated that BBP induce adversed effects on the
reproductive system of fish associated with growth inhibition, feminisation (ovatestis)
process and delayed testicular development.
17
Chapter 3
Materials and Methods
3.1 Experimental Location: Zoology research laboratory at the GC Woman

University, Sialkot was served as the experimental site.
3.2 Collection of fish: Fish were procured from the Head Marala hatchery of Sialkot
and transported to Zoology research laboratory at GC Woman University, Sialkot.
Figure 3.1: Collection of fish from Head Marala hatchery of Sialkot.
3.3 Experimental Design: Adult male fish, O. mossambicus in good health were
acclimatized for ten days in a lab setting. There were three experimental groups and
one control group. Each group was contained 8 fish. The three experimental groups
which were contain di-butyl and di-isononyl phthalates and their mixture
(DBP+DINP) respectively and the control group without exposure.
Figure 3.2: Control and Treatments Groups of Fish.

18
Di-butyl phthalate with greater than 95% purity and Di-isononyl phthalate with 98%
purity were used for sublethal exposure according to 1/3rd value of lethal
concentration. Proper aeration was maintained throughout the experiment.
3.4 Ethical considerations: Following the National Institutes of Health's Guide for
the Care and Use of Laboratory Animals, all fish handling, aquatic system
management, and procedures were carried out (NIH publication no. 8523, revised
1996). Ethical approval was obtained from EIRB, GCWUS.
3.4.1 Fish housing and management: The adult male fish of freshwater, were
procured from the Head Marala hatchery of Sialkot and transported to Zoology
research laboratory at GC Woman University Sialkot. Healthy fish of almost similar
weight were acclimatized for ten days in a lab setting to overcome the stress caused
by transportation and other environmental changes. Small sample size of fish was
used to stable aquatic environment needed for physiological functions e.g feeding and
movement. Commercially accessible food prepared in accordance with standards and
manufacturer instructions was used.
Figure 3.3: Fish housing and management.
19
3.4.2 Aquatic system management: Fish were kept in glass tanks with fresh water
under ideal laboratory conditions and on a normal circadian cycle. By maintaining
optimal water quality standards, fish welfare was ensured i.e. temperature, pH,
hardness, CO2, dissolved oxygen, ammonia, calcium, and magnesium. For stable
husbandry, regular measurements of different water properties (water quality tests)
were carried out. For adequate aeration and gaseous exchange in an aquatic system, a
capillary system was used. To maintain the quality of the water, tank water was
replaced every week with fresh water.
3.4.3 Disinfection/ Decontamination: Before the experiment begins, fresh tap water
was used to wash all the tanks. Every day, hot water and a disinfectant spray were
used to clean and sterilize the experiment site. By regularly changing tank water to
remove waste or fecal material of fishes, the aquatic system sanitization was
maintained.
3.4.4 Dissections: Fish dissections were carried out under suitable instruction and
with personal training. Before the day of dissection, all the instruments were
autoclaved to prevent contamination.
Figure 3.4: Dissection of Oreochromis mossambicus.
Heat was used as a disinfectant during the whole dissection. After completion,
all the waste were tightly packed in polythene bags and properly disposed in separate
covered bins.
In case of allergic reactions to researchers, immediate medical care was followed.
20
PHASE I
3.5 Acute toxicity (96-hr LC50) Determination: The lethal toxicity test of phthalates
(di-butyl phthalate, di-isononyl phthalate and their mixture (DBP+DINP)) were
utilized to assess the LC50 and potentially fetal dose for 96 hrs for O. mossambicus.
For this purpose, adult male fish were exposed to varing doses of phthalates
separately, and data on fish mortality was collected. Probit analysis was used to
calculate the LC50 of phthalates for O. mossambicus, mainly based on fish mortality
rate with 95 percent confidence interval (Finney, 1971). Dead fish were evacuated
immediately. Feed was not provided to fish during lethal toxicity exposure.
3.6 Physico-chemical parameters determination:
During experiment, physico-chemical parameters including temperature, pH,

hardness, carbon dioxide, dissolved oxygen, ammonia, calcium and magnesium were
checked daily by following (APHA, 2005).
3.6.1 Water temperature and pH
Temperature and pH of the test media i.e experimental and control groups were
determined by using pH meter model 120 benchtop pH/mv meter.
3.6.2 Water Hardness:
For determination of water hardness following chemicals were used:
 Ammonia buffer
 Erichrome Black-T as an Indicator
 EDTA as a Titrant
Procedure:
Water hardness was determined by titration of the water sample of test and control
group with the standard solution of a strong mineral acid as EDTA. Water sample of
25 ml volume was taken in beaker from test media for measurement of water
hardness. Put in 25 ml of deionized water (distilled water). Then add 1ml of ammonia
buffer to raise the pH of water sample upto 7.5. The reaction mixture was mixed
21
thoroughly with the help of stirrer and then added 1 to 2 drops of indicator (EBT).
After mixing well it was titrated against EDTA until blue colour appeared.
Figure 3.5: Water hardness was checked against the EDTA.
The formula below was used to estimate total hardness:
(Where A=mg CaCO3 equivalent to 1.0 ml EDTA titrant at the Ca＋＋ indicator end
point)
3.6.3 Carbon dioxide:
Chemicals required for the determination of CO2 are given below:
 Phenolphtalein as an Indicator
 Titrant Sodium carbonate (IN)
22
Procedure:
To find out the carbon dioxide, water sample of known quantity was titrated against
sodium carbonate. As a result of reaction between free carbon dioxide and sodium
carbonate, sodium bicarbonate was formed and at a particular pH the reaction was
ended to mark the end point of titration. Phenolphthalein as an indicator was used due
to which colorless sample changed to pink colour.
The CO2 concentration was computed using the formula below:
3.6.4 Dissolved Oxygen:
Dissolved oxygen was determined using DO meter
3.6.5 Total Ammonia:
Following chemicals were used to determine ammonia:
 Sodium-potassium tartrate
 Ammonium chloride solution
 Nessler's reagent (ammonia indicator)
Procedure:
A 50 ml sample of water was taken from each test medium, and it was thoroughly
mixed in with one to two drops of the sodium-potassium tartrate solution. A solution
containing two to three drops of ammonium chloride was then added. After that, the
mixture was given 1 milliliter of Nessler's reagent and allowed to sit for 15 minutes.
Samples were run through one centimeter of light path in a spectrophotometer at a
420 nm wavelength after 15 minutes to measure the absorbance. By running standard
solution calibration curve was established at same reaction time and temperature used
for absorbance and samples reading were measured besides a blank reagent.
23
3.6.6 Calcium:
For determination of calcium following chemicals were used:
 Sodium hydroxide solution (1 N)

 Ammonium purpurate as an Indicator
 EDTA as a titrant
Procedure:
Water sample (25 ml) was taken from the test medium in a flask. After that, add 1
milliliter of NaOH to raise the pH to 12 or 13. Then one drop of indicator was added
to determine the end point of reaction and mixed it well. The end point was achieved
by titrating it against EDTA.
The calcium content of the samples was determined using the formula below:
3.6.7 Magnesium:
The following formula was used to calculate magnesium based on the values of
calcium and total hardness:
A-B equals C/4-Mg (mg L-1)
A = total hardness and B = calcium × 2.50
PHASE II
3.7 Determination of Sublethal toxicity of phthalates on fish:
Fish (O. mossambicus) were given sub-lethal phthalates dosages for 30 days after
acute toxicity experiment. Eight adult male O. mossambicus were given (1/3rd of
LC50) sub-lethal doses of DINP (300 mgL-1) and DBP (4 mgL-1) and DBP+DINP
(3.91 mgL-1). During the exposure time, fish were fed with rice bran twice a day.
Generally, the exposing media was changed every week. The normally required
phthalate dose had been maintained throughout the study, DBP was given to the fish
24
directly while DINP needs to dissolve in ethanol because of their low solubility. Each
tank was maintained at a steady air flow to protect the fish from being anxious and
stressed. Physicochemical parameters of water were periodically measured throughout
the second phase.
3.8 Semen Analysis:
Reproductive toxicity of phthalates i.e DBP, DINP and their mixture (DBP+DINP)
were determined by semen analysis. For the analysis of semen, testis were collected
after the dissection of adult male fish and placed in petridish where 0.9% saline
solution was present as an activator. By the longitudinal cut of testis leaking milt or
sperms were collected and further semen analysis was checked by performing the
following sperm parameters i.e sperm motility, their motile duration, viability and
DNA damage.
3.8.1 Sperm parameters:
Following sperm parameters were analyzed for semen analysis.
Sperm motiliy: Motility was perfomed by making the sperm suspension. For sperm
suspension, sperms were mixed with 0.9% saline solution with the ratio of 1:2. Sperm
suspension was mixed with tap water in a glass slide with the ratio of 1:2 and the
motility of the cells was then examined under a compound microscope at a 100X
magnification (Hayati et al., 2019). The measurement of sperm motility took into
account the time interval between the start of activation (using 0.9% saline) and the
complete cessation of all observed spermatozoa movements. Spermatozoa movements
were captured at 25 frames per second by using a digital camera (DOM300) from
Labomed Labo America, USA.
Sperm viability: For sperm viability, a mixture was prepared on a spot plate by
mixing 1% aqueous Eosin Y solution with the sperm suspension (few drops). After 15
seconds, 10% of an aqueous solution of Nigrosin (with two drops) was added, and
everything was mixed. This blend was applied to a slide in a droplet form and allowed
to air dry. A glass slide was examined at a 100X magnification using a compound
microscope. By counting the sperm with intact cell membranes, the percentage of live
25
sperm was ascertained. The non-viable sperms will absorb the stain, while the viable
sperms will remain clear. Dead sperms appeared pink, while viable sperms were
transparent (Hayati et al., 2019).
DNA Damage:
With the use of the comet assay/neutral single cell gel electrophoresis -SCGE method,
the spermatozoa DNA damage in tilapia was examined. Procedure followed by
Donnelly et al. (1999) and Boe hensen et al. (2007).
A simple technique for determining single and double strand DNA breaks and also the
ALSs (alkali-labile sites) within the DNA, is the comet assay. Fish sperms from testes
were separated after dissection and placed in cold normal saline. After
homogenization, the sperm suspension was centrifuged for 15 minutes at 1000 rpm,
and pellets were then diluted in phosphate-buffered saline.
Slide Preparation:
After being coated with 100 μl of 1% agarose in regular melting point (RMPA),
frozen microscope slides were immediately covered with cover slip.
Figure 3.6: Slide Preparation for Comet.
Slides were maintained at 4 °C to solidify agarose gel. After that, a 65-micro liter
solution of low melting point agarose (LMPA) and 20-micro liters of homogenate was
prepared and placed on the first slide. Slides were then covered to settle the gel.
26
Lysis
Slides were arranged in a tank (that contained the newly prepared lysis buffer in fresh
form) in a horizontal position. Lysis buffer was prepared by following this recipe i.e.
2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% (w/v) Triton X-100, pH=10.3
(Villani et al., 2012). The slides were removed from the jars following a 24-hour
incubation period at room temperature and washed three times in saline, each time
with a 20 minute interval, to remove any salt or chemical remains.
Neutral electrophoresis
Slides were stacked in a systematic manner in the tank of electrophoresis after adding
the electrophoretic buffer (54 g/L Tris base, 0.5 M EDTA, 27.5 g/L boric acid, pH
8.0). Twenty minutes of electrophoresis at 25V (0.714 V cm) was performed. Slides
were taken out of the tank when the treatment was finished, allowed to air dry, and
then covered in darkness to protect them from light. Draining the buffer and removing
the tray allowed the tank to be emptied. Slides were kept overnight at 5 °C.
Fluorescent microscopy and assessing of slides
For about sixty minutes, rehydrated the slides by using distilled water and then stained
with acridine orange (200-300 μl of 20ug/ml) before analysis.
Figure 3.7: Staining the slides.
27
A fluorescence microscope was at 40X was used to observe the slides and digital
photographs were captured. Various comet parameters i.e head length (µm), tail
length (µm), head DNA (%), tail DNA (%), tail moment (µm) were documented by
using software Tritek Casplab, V.12.3b2.
Figure 3.8: Slides were observed under flourescence microscope.
3.9 Histomorphometric Analysis:
To determine the possible toxicity of phthalates i.e DBP, DINP and their mixture,
testicular tissues were histologically examined using a previously described technique
(Sumon et al., 2019). After the reproductive organs from several experimental groups
had been dissected and collected, the subsequent operation was carried out.
3.9.1 Fixation:
Fish from both the experimental group i.e control and 3 exposure groups were
dissected at the end of the experiment. After dissection, Organ of testis were removed
and placed in petri-dish that contained 0.9% saline solution for washing purpose.
After rinsing with saline solution, organs were fixed in 10% formalin for 48 hours.
Fixation induces many chemical and physical alterations that protect the tissues
during processing.
28
3.9.2 Dehydration:
After fixation, the dehydration of preserved tissues was performed. At the ideal room
temperature, preserved tissues from the control and experiment groups were serially
dehydrated with progressively higher concentrations of alcohol until water-free
alcohol was attained.
Steps Reagents Time

1 70% Ethanol 120 mins
7 Xylene I 120 mins
8 Xylene II 120 mins
3.9.3 Embedding:
The method of tissue embedding was carried out after the dehydration step.
Embedding was performed to remove xylene completely from the tissues by using the
following steps. Testicular tissues were embedded in paraffin wax that was placed
inside a boat in melting form.
Tissues were emptied of bubbles and allowed to solidify. Using a knife or scalpel for
section cutting, tissue blocks made of paraffin wax were carved into desired shapes
and mounted on wooden blocks.
Steps Reagents Temperature Time
1 Xylene + Paraffin 62 °C 120 mins

(1:1)
2 Paraffin I 62 °C 4 hours
3 Paraffin II 62 °C 4 hours
29
Figure 3.9: Embedding process.
3.9.4 Microtomy:
A rotatory microtome was used to slice the tissues that mounted on blocks into thin
sections of 5µm in size after embedding. The paraffin sections were cut into long
ribbons and mounted on albuminized glass slides after being stretched in warm water.
Figure 3.10: Microtomy.
After placing the tissue section on albuminized glass slides, the tissue sections were
given time to drain and dry. These slides were first placed on slide warmer and then
placed in an incubator (60 °C) overnight for complete deparaffinization
30
3.9.5 Staining:
These sections-containing slides were first run through xylene, followed by a

decreasing concentration of alcohol, as shown below, before being stained with
hematoxylin and eosin. Following staining, the slices of tissue were once more
dehydrated and put in xylene in descending order as given below:
Steps Reagents Time

1 Xylene I 10 mins
2 Xylene II 10 mins
3 100% alcohol I 3 mins
4 100% alcohol II 3 mins
5 90% alcohol 3 mins
8 Water 3 mins
9 Hematoxylin 8 mins
10 Wash with tap water 5-10 mins
11 95% Acidified alcohol 2 mins
12 Wash with tap water 2 mins
13 Bluing solution 2 mins
16 Eosin 2 mins
20 Xylene I 5 mins
21 Xylene II 5 mins
Per-mount: After staining, the addition of 3 small drops/slides of DPX was

performed. A thin glass slide was placed upon tissue sections carefully before per-
mount dried.
31
Figure 3.11: Staining the slides.
3.9.6 Microscopy:
Glass slides that had been prepared were examined with a compound
microscope.Slides were analysed, and active regions were photographed using a
digital camera (DOM300) from Labomed Labo America, USA. At 40X, various
photos of the sections were taken, and image J2x was utilized in order to perform
morphology and histomorphometry.
3.10 Statistical Analysis:
Throughout the experiment, all parameter values were obtained and described
in Mean ± SEM. All the parameters in the experimental groups (control and three
exposures) were compared by using one-way ANOVA and Tukey's multiple
comparison test through SPSS. A significance level p<0.05 was utilized. The
mortality rate was calculated by using Probit analysis method (Finney, 1971).
32
Chapter 4
Results
The harmful and toxic effects of di- butyl phthalate, di-isononyl phthalate and their
mixture (DBP+DINP) were observed on adult male Oreochromis mossambicus in the
current study. The Acute toxicity (96 hours LC50) of DBP, DINP and DBP+ DINP
was determined and analyzed using Probit analysis method. The Physico-chemistry of
water was examined throughout the experiment. Under sub lethal (1/3rd of LC50)
exposure of di-butyl phthalate, di-isononyl phthalate and the mixture of these two
phthalates for 30 days, reproductive organ impairment was analyzed by semen and
histological analysis.
Determination of Acute Toxicity (LC50) of DBP, DINP and DBP+DINP in

Oreochromis mossambicus during exposure period of 96 hours
The acute toxicity assay was carried out to evaluate 96 hours LC50 of DBP, DINP and
DBP+DINP in the adult male O. mossambicus. The fish were not fed at that time. The
experiment was carried out in replicates. Control group fish showed no mortality
during the whole period. Aquariums were checked out on regular basis to remove
dead fish in order to avoid stress for other fish. Acute toxicity (96h LC50) of DBP for
O. mossambicus was found out to be 12.17 mgL-1 and the LC50 value of both the
phthalates in mixture form (DBP+DINP) was found to be 11.75 mgL-1 by using the
Probit analysis while acute toxicity of DINP was not observed at even 300 mgL-1
which one is the maximum concentration value of di-isononyl phthalate’s solubility.
The Probability plot showing the mortality rate of males of adult O. mossambicus in
accordance with the concentration is presented in fig. 4.1, 4.2, and 4.3. The detailed
analysis of mortality data was presented in table 1, 2 and 3.
33
Table 1: Determination of Acute Toxicity (LC50) of di-butyl phthalate in O.
mossambicus during the exposure of 96 hour through Probit analysis
Regression Table
Standard
Variable Coef Error Z P
Constant -1.63447 0.162020 -10.09 0.000
Conc. of DBP (mg/L) 0.134211 0.0118231 11.35 0.000
Natural
Response 0
Log-Likelihood = -184.499
Goodness-of-Fit Tests
Method Chi-Square DF P
Pearson 6.16811 11 0.862
Deviance 9.23344 11 0.600
Tolerance Distribution
Parameter Estimates
Standard 95.0% Normal CI

Parameter Estimate Error Lower Upper
Mean 12.1783 0.556230 11.0882 13.2685
StDev 7.45094 0.656379 6.26940 8.85516
Table of Percentiles
Standard 95.0% Fiducial CI
Percent Percentile Error Lower Upper
1 -5.15514 1.62379 -8.96188 -2.41264
2 -3.12402 1.45698 -6.53070 -0.656759
3 -1.83534 1.35275 -4.99136 0.460457
4 -0.865916 1.27538 -3.83544 1.30296
5 -0.0773648 1.21325 -2.89676 1.98985
6 0.593816 1.16102 -2.09910 2.57579
7 1.18231 1.11579 -1.40082 3.09067
8 1.70924 1.07580 -0.776602 3.55268
9 2.18846 1.03989 -0.209820 3.97379
10 2.62958 1.00728 0.311045 4.36228
20 5.90747 0.782944 4.14553 7.28503
30 8.27106 0.653373 6.84590 9.45710
40 10.2907 0.580150 9.07731 11.3890
50 12.1783 0.556230 11.0720 13.2856
60 14.0660 0.580960 12.9670 15.2820
70 16.0856 0.654860 14.8975 17.5148
80 18.4492 0.784935 17.0686 20.2162
90 21.7271 1.00963 19.9906 24.0514
91 22.1682 1.04228 20.3790 24.5723
92 22.6474 1.07822 20.8001 25.1392
93 23.1744 1.11824 21.2620 25.7635
94 23.7629 1.16350 21.7768 26.4618
95 24.4340 1.21576 22.3627 27.2595
96 25.2226 1.27793 23.0496 28.1983
97 26.1920 1.35532 23.8920 29.3542
98 27.4807 1.45959 25.0091 30.8937
99 29.5118 1.62645 26.7649 33.3249
34
Table 2: Determination of Acute Toxicity (LC50) of di-isononyl phthalate in O.
mossambicus during the exposure of 96 hour through Probit analysis
Regression Table
Standard
Constant -5.99530 1.31224 -4.57 0.000
Conc. of DINP mg/L 0.0178673 0.0048238 3.70 0.000
Natural
Response 0
Pearson 0.74160 11 1.000
Deviance 1.03485 11 1.000
Parameter Estimates

Mean 335.547 19.5770 297.176 373.917
StDev 55.9682 15.1103 32.9711 95.0056

1 205.345 19.4620 128.572 231.668
2 220.602 15.8577 159.911 242.709
3 230.282 13.7221 179.460 250.049
4 237.564 12.2383 193.880 255.857
5 243.487 11.1420 205.340 260.850
6 248.529 10.3129 214.828 265.367
7 252.949 9.68554 222.879 269.594
8 256.907 9.21914 229.820 273.648
9 260.507 8.88576 235.865 277.602
10 263.820 8.66432 241.169 281.503
20 288.442 9.81467 271.780 319.287
30 306.197 12.9033 287.265 353.119
40 321.367 16.2129 298.856 383.668
50 335.547 19.5770 309.086 412.825
60 349.726 23.0861 319.006 442.292
70 364.896 26.9369 329.420 474.019
80 382.651 31.5221 341.446 511.311
90 407.273 37.9661 357.951 563.200
91 410.586 38.8386 360.162 570.194
92 414.186 39.7875 362.562 577.793
93 418.144 40.8321 365.197 586.152
94 422.564 42.0002 368.138 595.490
95 427.606 43.3341 371.489 606.144
96 433.529 44.9034 375.422 618.665
97 440.811 46.8353 380.251 634.063
98 450.491 49.4077 386.662 654.541
99 465.748 53.4702 396.751 686.832
36
Table 3: Determination of Acute Toxicity (LC50) of mixture (DBP+DINP) in O.
mossambicus during the exposure of 96 hours through Probit analysis.
Regression Table
Standard
Constant -1.59381 0.160807 -9.91 0.000
Conc . of (DBP+DINP) mg/L 0.135556 0.0119174 11.37 0.000
Natural
Response 0
Pearson 7.9535 11 0.717
Deviance 10.8378 11 0.457
Parameter Estimates

Mean 11.7577 0.552480 10.6748 12.8405
StDev 7.37705 0.648557 6.20939 8.76429
1 -5.40394 1.62191 -9.20523 -2.66422
2 -3.39296 1.45692 -6.79881 -0.925611
3 -2.11706 1.35378 -5.27506 0.180529
4 -1.15725 1.27721 -4.13079 1.01462
5 -0.376517 1.21568 -3.20153 1.69459
6 0.288008 1.16393 -2.41181 2.27460
7 0.870666 1.11911 -1.72046 2.78422
8 1.39237 1.07945 -1.10239 3.24148
9 1.86683 1.04383 -0.541158 3.65822
10 2.30358 1.01145 -0.0253609 4.04265
20 5.54897 0.787963 3.77335 6.93333
30 7.88912 0.657222 6.45154 9.07866
40 9.88870 0.581002 8.66812 10.9836
50 11.7577 0.552480 10.6527 12.8513
60 13.6266 0.571753 12.5394 14.8169
70 15.6262 0.640211 14.4601 17.0177
80 17.9663 0.765157 16.6171 19.6842
90 21.2117 0.984425 19.5162 23.4745
91 21.6485 1.01644 19.9014 23.9896
92 22.1229 1.05170 20.3188 24.5501
93 22.6446 1.09100 20.7768 25.1675
94 23.2273 1.13548 21.2871 25.8581
95 23.8918 1.18686 21.8678 26.6471
96 24.6726 1.24803 22.5485 27.5757
97 25.6324 1.32423 23.3833 28.7192
98 26.9083 1.42694 24.4903 30.2421
99 28.9192 1.59144 26.2299 32.6476
38
Determination of Physico-chemical parameters of water during 96 hours of
exposure
The physicochemical parameters of water were properly measured throughout the

experiment. The temperature (28°C), pH (7.5) and water hardness (300 mgL-1) were
kept constant. No significant variations were detected in their computed values. The
levels of carbon dioxide, calcium and total ammonia were increased with the gradual
increase in concentration of DBP, DINP and DBP+ DINP. However, the levels of
dissolved oxygen and magnesium were observed to be decrease with the increase
concentration of exposed phthalates. Thus inverse relation exists between these
parameters and phthalates concentration. The carbon dioxide, calcium and ammonia
increased from (1.29 to 1.72mgL-1), (22.25 to 23.71 mgL-1) and (1.30 to 1.46 mgL-1)
respectively. The dissolved oxygen and magnesium decreased from (5.05 to 4.70
mgL-1) and (61.09 to 59.88 mgL-1) respectively. These alterations were observed due
to the toxic effects of the di-butyl phthalate. The carbon dioxide, calcium and
ammonia increased from (1.27 to 1.72 mgL-1), (21.25 to 23.75 mgL-1) and (1.30 to
1.37 mgL-1) respectively. The dissolved oxygen and magnesium decreased from (5.75
to 4.73 mgL-1) and (61.72 to 59.99 mgL-1) respectively. These alterations were
observed due to the toxic effects of the di-isononyl phthalate. The carbon dioxide,
calcium and ammonia increased from (1.29 to 1.60 mgL-1), (22.00 to 23.30 mgL-1)
and (1.31 to 1.34 mgL-1) respectively. The dissolved oxygen and magnesium
decreased from (5.65 to 4.91 mgL-1) and (61.25 to 59.98 mgL-1) respectively. These
alterations were observed due to the toxic effects of the DBP+ DINP
Determination of sub lethal (1/3rd of LC50) toxicity
After acute toxicity assay the experimental groups were exposed to sub-lethal (1/3rd
LC50) concentrations of DBP, DINP and their mixture (DBP +DINP) calculated for O.
mossambicus, It was calculated as 4 mgL-1, 300 mgL-1 and 3.9mgL-1 respectively. The
calculated amount was introduced into each of the three experimental groups. The
control group was left untreated.
40
Determination of Physico-chemical parameters of water under sub lethal (1/3rd
of LC50) exposure
The physico chemical parameters of water were measured on daily basis in all the
groups. The temperature (28°C), pH (7.5) and hardness (299 mgL-1) were kept at
constant level. In control group, the mean values with standard deviation were found
on daily basis to be 4.88±0.00 mgL-1, 1.06±0.09 mgL-1, 1.13±0.04 mgL-1, 21.97±1.76
mgL-1 and 61.06±1.15 mgL-1 of dissolved oxygen, carbon dioxide, total ammonia,
calcium and magnesium respectively. In DBP group, the mean values with standard
deviation calculated on daily basis were found to be 4.87±0.01 mgL-1, 1.22±0.22
mgL-1, 1.24±0.03 mgL-1, 23.94±1.26 mgL-1and 59.86±0.81 mgL-1 of dissolved
oxygen, carbon dioxide, total ammonia, calcium and magnesium respectively. While
in DINP group, the mean values with standard deviation calculated on daily basis
were found to be 4.88±0.00 mgL-1, 1.20±0.17 mgL-1, 1.18±0.03 mgL-1, 22.97±1.30
mgL-1 and 60.39±0.83 mgL-1 of dissolved oxygen, carbon dioxide, total ammonia,
calcium and magnesium respectively. Similarly in phthalates mixture (DBP+ DINP)
group, the mean values with standard deviation calculated on daily basis were found
to be 4.86±0.01 mgL-1, 1.23±0.05 mgL-1, 1.31±0.03 mgL-1, 24.95±1.59 mgL-1 and
59.16±0.97 mgL-1 of dissolved oxygen, carbon dioxide, total ammonia, calcium and
magnesium respectively.
41
Determination of Semen Analysis
A definite positive visual coexists between the viscosity and colour of fish semen
(milt). Fish semen was white in colour. In all groups of experiment, no difference was
observed in the colour of semen. The viscosity of the clear translucent semen of O.
mossambicus was higher in the control group as compared to the treated groups.
Motility
Exposure of phthalates in their specified concentrations affects the sperm motility of

fish. After activation, sperm motility declines and reaches an extremely low level
within a few minutes. In comparison to the other treatment groups i.e DBP, DINP and
DBP+DINP, the control group had significantly (p<0.05) the greatest duration
(23´17´´±0´29´´) according to the measurements that were taken for the sperm mass
duration. Phthalates exposure led to a decrease in individual motility duration
commensurate with the duration of mass motility. All treatment groups i.e DBP with
74% and DBP+DINP with 49% showed a significant decrease (p<0.05) in sperm
motility as compared to the control group with 89% motility, except for DINP (82%
sperm motility) exposure that did not differ significantly (table 4.4).
Table 4.4: Motility parameters in all experimental groups
sperm Control DBP DINP DBP+DINP

motility
parameters
Percentage 89.70±0.02%c 74.20±0.01%b 82.70±0.03%bc 49±0.02%a

motility (%)
Progressive
10´22´´±0´12´´ 1´55´´±0´26´áb 5´22´´±0´42´ć 1´59´´±0´17´á
movement
(min/s)
Moderate
12´55´´±0´17´ć 7´08´´±0´32´áb 8´58´´±0´14´´bc 6´56´´±0´2´á
movement
(min/s)
The values are given in mean ± SEM
P<0.05 are significant.

42
Motility parameter of sperm
100.0% c
bc
90.0%
b
80.0%
Sperm Motility (%)
70.0%
60.0% a Control
50.0%
DBP
40.0%
DINP
30.0%
DBP+DINP
20.0%
10.0%
0.0%
Control DBP DINP DBP+DINP
Groups
Figure 4.5: Percentage sperm motility in Control, and the exposure groups (DBP,
DINP and DBP+DINP) of O. mossambicus.

12
Progressive movement (min/s)
10
8
c Control
6
DBP
4 DINP
ab a DBP+DINP
2
0
Groups
Figure 4.6: Progressive movement (min/s) of sperms in Control, and the exposure
groups (DBP, DINP and DBP+DINP) of O. mossambicus.
44
14 c
Moderate movement (min/s) 12
10 bc
ab
8 a Control
6 DBP
DINP
4
DBP+DINP
2
0
Groups
Figure 4.7: Moderate movement (min/s) of sperms in Control, and the exposure
groups (DBP, DINP and DBP+DINP) of O. mossambicus.
Sperm Viability
120.0%
c
100.0% bc
ab
Sperm Viability (%)
80.0%
Control
60.0% a
DBP
40.0% DINP
DBP+DINP
20.0%
0.0%
Groups
Figure 4.8: Percentage Sperm Viability in Control, and the exposure groups (DBP,
DINP and DBP+DINP) of O. mossambicus.
45
Determination of DNA damage
DNA damage in the sperm of O. mossambicus exposed to DBP, DINP and their
mixture DBP+DINP was evaluated by comet assay. Changes in different DNA
parameters i.e. head length, tail length, percent DNA in head, percent DNA in tail and
tail moment were measured and presented in table 4.5 and figure 4.9.
Table 4.5 Mean±SEM Head length (µm), Tail length(µm), %DNA in head,
%DNA in tail and Tail moment (µm) in sperms of O. mossambicus measured
against different groups Control, DINP, DBP and their mixture (DBP+DINP)
groups after 30 days of sublethal exposure.
Control DINP DBP DBP+DINP p value
Head Length
102.33±2.91b 88.00±4.51b 86.00±3.00ab 74.33±3.28a <0.003
(µm)
Tail Length
7.33±0.33c 9.00±0.58bc 11.33±0.33ab 12.33±0.88a <0.001
(µm)
Head DNA
96.77±1.05c 90.65±1.48b 85.97±1.07b 78.99±1.31a <0.0001
(%)
Tail DNA
3.22±1.05c 9.35±1.48b 14.02±1.07b 21.00±1.31a <0.0001
(%)
Tail Moment
0.82±0.33c 1.88±0.32bc 2.79±0.20ab 3.78±0.39a <0.001
(µm)
Values are given in Mean±SEM.

P<0.05 are Significant
a = Values vs Control
b = Values vs DBP
c = Values vs DINP
46
Head length
In sperm, head length was non-significantly decreased in DINP group when compared
with a control group. Similarly, highly significant reduction in head length was
observed in DBP and in mixture (DBP+DINP) groups as compared to control group
with a significance value p<0.003. DINP group showed moderate changes as
compared to DBP group. Similarly, head length was significantly decreased in the
mixture (DBP+DINP) group with a non-significant difference against the moderate
changes in DINP and DBP groups, respectively (Figure 4.10).
Tail length
In sperms, increase in tail length was observed in all treatments group when compared
to the control group. But the high significant increased in tail length was observed in
both DBP and mixture (DBP+DINP) groups when compared with control group with
a significance value p<0.001 but non-significantly increased in DINP group. Tail
length was moderately changed in DINP group when compared with DBP. Similar
results were observed in mixture (DBP+DINP) group against both the DINP and DBP
groups (Figure 4.11).
Percent DNA in head
In sperms %DNA in head was significantly decreased in DINP, DBP and their
mixture (DBP+DINP) groups when compared to control group with significance
value p<0.0001. In the same way like head length, DINP group showed moderate
changes with the DBP with a non- significant results (p>0.05) while the mixture
(DBP+DINP) group showed significant results (p<0.05) when compared to the DINP
group. Significant reduction in the %DNA in head was also noticed in mixture
(DBP+DINP) group when compared to DBP group (Figure 4.12).
Percent DNA in tail
In sperms, a highly significant increased in %DNA of tail was observed in all groups
of treatments i.e DINP, DBP and their mixture (DBP+DINP) groups when compared
to control group with significance value p<0.0001. DINP group showed a non-
significant results (p>0.05) with the DBP group through some moderate alterations
47
Sperm DNA Damage
120
b
100 b
ab
Head Length (µm)
a
80
Control
60
DINP
40 DBP
DBP+DINP
20
0
Control DINP DBP DBP+DINP
Groups
Figure 4.10: Head length (µm) in sperms of O. mossambicus measured against

Control, DBP, DINP and Mixture (DBP+DINP) after 30 days of sublethal exposure.
Sperm DNA Damage

14 a
ab
12
bc
10
Tail length (µm)
c
8 Control
6 DINP
DBP
4
DBP+DINP
2
0
Groups
Figure 4.11: Tail length (µm) in sperms of O. mossambicus measured against

49
Sperm DNA Damage
120
c
100 b
b
a
Head DNA (%)
80
Control
60
DINP
40 DBP
DBP+DINP
20
0
Groups
Figure 4.12: % DNA in Head in sperms of O. mossambicus measured against

Sperm DNA Damage

25
a
20
b
Tail DNA (%)
15
Control
b
DINP
10
DBP
c DBP+DINP
5
0
Groups
Figure 4.13: % DNA in Tail in sperms of O. mossambicus measured against Control,

DBP, DINP and Mixture (DBP+DINP) after 30 days of sublethal exposure.
50
Sperm DNA Damage
4.5 a
4
3.5
Tail moment (µm)
ab
3
2.5 bc Control
2 DINP
1.5 c DBP
1 DBP+DINP
0.5
0
Groups
Figure 4.14: Tail moment (µm) in sperms of O. mossambicus measured against

51
Histological analysis
Histological alterations were seen in reproductive organ i.e Testes of Oreochromis

mossambicus in control and three treatments groups that were treated with the sub-
lethal concentrations (1/3rd of LC50) of DINP, DBP and their Mixture (DBP+DINP)
(figure 4.14).
Effect on Testicular tissues
Testes of control group showed regular pattern of seminiferous tubules which were
distinguished by oval, round or somewhat rectangular shape and regular shape of
sertoli cells and interstitial cell of Leydig. Testes of control group contained various
germ cells of spermatogenesis, such as, spermatocytes, spermatids and spermatozoa
(Figure 4.15 a).
Elongated seminiferous tubules, decreased spermatozoa, damaged sertoli cells and

interstitial spaces were observed in DINP exposed group (Figure 4.15 b).
In di-butyl phathalate treated group irregular shaped seminiferous tubules with

necrosis, reduced spermatocytes and decreased and disintegration of spermatozoa and
detachment of seminiferous tubules were observed (Figure 4.15 c).
Testes from the mixture (DBP+DINP) treated group showed breakdown of

seminiferous tubule, empty lumen, degeneration of interstitial cell of leydig and
reduction in spermatozoa. Disintegration of spermatozoa and increased in the amount
of interstitial tissues (Figure 4.15 d).
52
Chapter 5
Discussion
In current study, harmful and lethal effects of di-butyl phthalate (DBP), di-
isononyl phthalate (DINP) and their mixture (DBP+DINP) was assessed by
histomorphometric and semen analysis of Oreochromis mossambicus, fresh water fish
(Tilapia). The results had distinctly represented that DBP, DINP and their mixture
(DBP+DINP) were enormously toxic and deleterious to O. mossambicus. In present
study, the acute toxicity (96-h LC50) of di-butyl phthalate and the mixture of
phthalates (DBP+DINP) were found to be 12.17 mgL-1 and 11.75 mgL-1 respectively
by using the Probit method while acute toxicity of di-isononyl was not found.
Previously, LC50 values of DBP against Mozambique tilapia (Oreochromis
mossambicus) and Common carp (Cyprinus carpio) were observed to be 50 ppm and
35mgL-1 respectively which were much higher values in comparison to the current
study (Sepperumal and Saminathan, 2013; Poopal et al., 2017). In Pseudetroplus
maculatus, Struthi et al. (2020) determined that the 96-hours acute toxicity value of
DBP by using Probit analysis and found that it was 2 mgL-1, which was significantly
lower than our current research. LC50 value of di-butyl phthalate against Cyprinus
carpio observed to be 16.3 mgL-1 assessed by Zhao et al. (2014) which was near to
our results. Erkmen et al. (2015) reported 96-hr acute toxicity of DBP to Nile tilapia
at 10mgL-1, similarly, Khalil et al. (2016) also stated the LC50 value of DBP for the
juveniles of Oreochromis niloticus at 11.8 mgL-1 which was close to our present
results. According to Johnson and Finley's 1980 study, the 96-hours median lethal
toxicity (LC50 values) of di-butyl phthalate were reported as 1.4 mgL-1 in
Oncorhynchus mykiss (rainbow trout), 1.7 mgL-1 in Pimephales promelas (fathead
minnows), 2 mgL-1 in Ictaluras punctatus (channel catfish), and 2.2 mgL-1 in
Lepomis macrochirus (bluegill fish) respectively (Johnson and Finley, 1980).
In the present research, acute toxicity (96-hr LC50) of di-isononyl phthale was
not observed, no mortality related to the chemical was observed at even 300 mgL-1
which is the maximum concentration of di-isononyl phthalate’s solubility. In the same
way, Revathy and Chitra, (2015, 2018) reported no LC50 value of di-isononyl
54
phthalate and used 300mgL-1 DINP as a sub-lethal dose. Different researchers
reported different doses of DINP which was used as sublethal concentrations to
determine the chronic toxicity but not determined the acute toxicity (Patyna et al.,
2006; Forner-piquer et al, 2018; Godai et al., 2021). Chen et al. (2014) reported that
in the mixtures of phthalates i.e, DBP, BBP, DEHP, DIDP, DINP and DNOP, the
LC50 value was observed to be 0.50 ppm. In that study, the LC50 value of DINP as a
separate compound was not found similar to our results. The fish specie of this
experiment seemed to be highly sensitive in case of DBP and in mixture
(DBP+DINP) groups but not in DINP group. Some other studies were also reported
the toxicity of phthalates, both in a separate or in the mixture groups and
demonstrated that phthalate as a combined exposure have showed more synergistic or
additive effects on fish as compared to the single exposure (Zhao et al., 2014; Chen et
al., 2020).
The alteration in LC50 values may be because of the toxicity of the chemicals
or compounds depend upon the formulation of the phthalates, variation in genetic
makeup that can fluctuate the capability of the body to detoxify specific harmful
substances or chemicals, source of the fish, inertness of the chemical, distinct
behavior of the species, exposure condition and durations, the kind of the fish species,
size, health, sex and weight of the fish as well as the parameters of water.
In fish, motility parameters of sperms varied according to the species, mostly

or anywhere it lasts for a few seconds to several minutes (Cosson, 2010). Mature
sperms (spermatozoa) in Nile tilapia (Oreochromis niloticus) stay motile for four to
over forty minutes following activation (Chao et al., 1987; Mochida et al., 1999;
Pinheiro et al., 2003)). An analogous motility duration of more than half an hour have
also been reported in another relevant specie i.e, Mozabique tilapia (O. mossambicus)
(Morita et al., 2003). According to Gennotte et al. (2012), tilapia was distinguished
from other fish species by having long motility of sperm durations (24´52´´±10´40´´)
and low sperm concentrations (2.22 ± 1.11×109 cells mL-1). Following 20 seconds of
activation, Linhart et al. (1999) observed somewhat reduced motility level with 48%
of the spermatozoa (mature sperms) was motile and the velocity was also decreased of
approximately 42µm s-1. In our current study a significant reduction was observed in
the motility and viability of sperms of Oreochromis mossambicus in the exposure
55
groups of DBP, DINP and their mixture group (DBP+DINP) as compared to the
control group with the progressive movement of 1´59´´±0´17´´, 5´55´´±0´19´´,
1´55´´±0´26´´ and 10´22´´±0´12´´ respectively. Struthi et al. (2020) reported a
significant reduction in the sperm motility with the increase concentration of di-butyl
phthalate, similarly, the sperm viability was also decreased with upto 82, 63 and 40
percent respectively with the increase concentration of DBP in their study. The sperm
quantity and motility of the rainbow trout, Oncorhynchus mykiss, were shown to be
inhibited by the exposure of environmental estrogenic alkylphenolic chemicals,
corresponding to the study of Jobling et al. (2002). DINP induced testosterone
inhibition that caused a marked decrease in sperm number, motility, and viability
assessed by Revathy and Chitra, (2018). Golshan et al. (2015) reported a significant
reduced production of sperms and motility in DEHP exposed Carassius auratus
(golden fish) that showed the resemblance to our results.
In fish, successful reproduction has been linked to the motility and viability of
sperms (Marchand et al., 2008). Alteration in the production of hormones can have an
impact on sperm development. Numerous studies have demonstrated that xenobiotics
like phthalates or plasticizers can act through these mechanisms to slow down
the testosterone production and delay the development of spermatozoa (mature sperm)
(Kime and Nash, 1999). In addition to many other factors like the pH level,
temperature, osmotic concentration, storage duration, and the impacts of pollutants,
endocrine disruptors (phthalates) can also have an impact on both the amount and the
quality of sperms in production (Alvai and Cosson, 2005, 2006).
Phthalates possess lipophilic characteristics and has been demonstrated that,

among others xenobiotics and environmental pollutants, phthalates can induce the
oxidative stress. This stress ultimately affects the endocrinal system with the
reproductive system being the most efficiently targeted (Sedha et al., 2015). Reactive
oxygen species (ROS), a type of free radical, can be produced outside by radiation,
chemicals, or internally by activities like cellular metabolism. Research shows that
ROS behave like a sharp sword (2- ended): they can function as both in anatomical
processes as an important indicator as well as in morbific processes. Oxidative stress
is brought on by ROS because, at certain concentrations, they substantially exceed the
ability of the body’s natural antioxidant defence mechanism. The harm that results to
56
the organs and cells could cause or speed up the course of illness (Sharma and
Agarwal, 1996; Buhler and Miranda, 2000). Oxygen-dependent stress that induce by
phthalates is one of the main causes of male infertility. The attacks by reactive oxygen
species (ROS), have the potential to cause lipid per-oxidation and DNA damage
which can upset cell motility and impair spermatozoon development normally (Aitken
and Roman, 2008).
The fluorescent microscopy of comet slides in current research study showed

phthalates induced DNA damage in all treatment groups compared to untreated group.
Results of the present study showed that DNA tail length, % DNA in tail and tail
moment increased significantly while head length and % DNA in head decreased
significantly in all treatment groups following the 30 days of exposure. In our study,
sperm DNA damage was significantly observed in the mixture groups (DBP+DINP)
where tail length, % DNA in tail and tail moment were significantly increased with
specific values of 12.33± 0.88, 21.00±1.31, 3.78±0.39 respectively. Previous research
on Zebrafish treated with hydrogen peroxide revealed an increase in % DNA in tail
with approximate value of 88.7±3.9% (Reinardy et al., 2012). Another study also
revealed a sperm DNA damage in Cyprinus carpio due to the environmental
pollutants including xenobiotics compounds with 7.77±4.51 % tail DNA (Cok et al.,
2011), both of the above studies were non-significantly correlates with our results.
In line with the current study’s findings, previous research on Zebrafish

treated with DEHP revealed an increase in DNA fragments (Corradetti et al., 2013).
In contrast to our findings, a study by Webster et al. (2010) revealed that Zebrafish
sperm exposed to DEHP did not exhibit any appreciable DNA damage.
Exposure to elevated levels of phthalates results in distress, anxiety, disease,

and even death in affected animals. Kime and Nash (1999) have reported that
reproductive abnormalities at lower levels can be attributed to two possible
mechanisms: either by direct act on the gonads (testes and ovaries), or indirectly
acting through modification of the endocrine system to cause gamete development
during a hormonal imbalanced surroundings. The general effect is a change in the
abundance and attributes of gametes, nevertheless the activity occurs in the gonad,
hypothalamus, pituitary, or in the gemetes themselves. The reproductive system's
57
ultimate objective is to generate a sufficient quantity of viable gametes. In order to
monitor or determine the reproductive dysfunction, gamete (sperm) viability can be
examined (Kime 1995; Kime and Nash, 1999).
A thorough, useful and illuminating technique for examining changes in the

morphology of gonads caused by Endocrine Disrupting Chemicals (EDCs) is
Histology (Meyers and Hendricks, 1985; Pieterse, 2004). Histological parameter act
as a biomarker to study the reprotoxicity. Reproductive toxicity of phthalates was
investigated through histomorphometric analysis in Oreochromis mossambicus after
exposure to 1/3rd of LC50 of di-butyl phthalate, di-isononyl phthalate and the mixture
of both phthalates (DBP+DINP) in the current research. In the present study,
elongated seminiferous tubules, decreased spermatozoa, disintegration of
spermatozoa, detachment of seminiferous tubules from their basal membranes,
increasement of interstitial tissues between the tubules, reduction of spermatocytes,
irregular seminiferous tubules, empty lumen, necrosis, breakage of seminiferous
tubules, degeneration of interstitial cells of leydig, all were observed in phthalate
(DBP, DINP and (DBP+DINP)) exposure’s testes of O. mossambicus.
Previous research revealed that endocrine disrupting chemicals caused

epithelial detachment from the basal membrane, elongated sections of the testis,
penetration of blood cells, and interstitial spaces in the testes of O. mossambicus
(Mlambo et al., 2009). In the same way Marchand et al. (2008) reported the testicular
alterations in O. mossambicus which includes intersex, detachment of tubules and
growing of connective tissues in greater amount due to EDCs. Muhammad et al.
(2018) assessed that the phthalate exposure i.e DEHP and ATBC significantly alters
the male reproductive system of Zebrafish through the cellular disintegration of
sperms and by the breakage of spermatocyst.
Significant decreased in the number of spermatozoa, impaired

spermatogenesis and the empty lumen with greater size were observed in DEHP
exposed testes of Japanese medaka investigated by Yuen et al. (2020) which give
resemblance to our results. Another study revealed the toxic effects of DBP in the
histology of Pseudetroplus maculatus’s testes which includes significant decline in
numbers of mature sperms and the irregular pattern of spermatogonia and sperm’s
58
cells (Struthi et al., 2020). Adeogun et al. (2018) reported the vacuolation process
between the tubules, inflammation, aberrations, breakdown of tubular epithelium and
seminiferous tubules and the condensed formation of tubular cells in the DEHP
exposed testes of Clarias gariepinus. Guo et al. (2017) reported the breakage of
speminferous tubules , disintegration of spermatozoa, significant reduction in
numbers of spermatogenic cells and the spermatogoniain Danio rerio due to the
combined exposure of phthalates , these results was close to our current findings.
59
Conclusion
Present study concluded that sublethal doses (1/3rd of LC50) of di-butyl phthalate, di-
isononyl phthalate and their mixture (DBP+DINP) had toxic and deleterious effect on
the adult male fish of Oreochromis mossambicus. Furthermore, decline of percentage
sperm motility and viability and sperm DNA damage due to phthalate exposure,
caused abnormality in fish and also caused significant histological changes in
testicular organ. Results concluded that di-butyl, di-isononyl and their mixture
(DBP+DINP) caused reprotoxicity in fish and disturb the male reproductive system in
fish. For ecological development, we should utilize the minimum concentrations of
plasticizer that can be able to control moreover can protect the marine and terrestrial
habitat.
60
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74
APPENDICES
Phase # 01
Appendix-01: Mortality of Oreochromis mossambicus in different

concentrations of Di-butyl phathalate at 96h exposure period
Con. of
R1 R2 R3 Mean±SD
DBP
mgl-1 % % %
0.0 0 0 0 0.00±0.00
2.0 10 0 10 6.66±5.77
4.0 10 20 20 16.66±5.77
6.0 20 30 20 23.33±5.77
8.0 30 30 40 33.33±5.77
10.0 40 40 40 40.00±0.00
12.0 50 50 60 53.33±5.77
14.0 60 60 60 60.00±0.00
16.0 70 70 70 70.00±0.00
18.0 70 80 70 73.00±5.77
20.0 80 80 80 80.00±0.00
22.0 90 80 90 86.66±5.77
24.0 100 100 100 100.00±0.00
75
concentrations of Di-isononyl phathalates at 96h exposure period
Con. of
R1 R2 R3 Mean±SD
DINP
mgl-1 % % %
0.00 0 0 0 0.00±0.00
25.0 0 0 0 0.00±0.00
50.0 0 0 0 0.00±0.00
75.0 0 0 0 0.00±0.00
100.0 0 0 0 0.00±0.00
125.0 0 0 0 0.00±0.00
150.0 0 0 0 0.00±0.00
175.0 0 0 0 0.00±0.00
200.0 0 0 0 0.00±0.00
225.0 10 0 0 3.33±5.77
250.0 10 0 10 6.66±5.77
275.0 20 10 20 16.66±5.77
300.0 20 20 30 23.33±5.77
76
concentrations of DBP+DINP at 96h exposure period
Con. of DBP+DINP
where DINP= R1 R2 R3 Mean±SD
300mgl-1
mgl-1 % % %
0.0 0 0 0 0.00±0.00
2.0 10 0 10 6.66±5.77
4.0 20 20 10 16.66±5.77
6.0 20 20 30 23.33±5.77
8.0 30 40 40 36.66±5.77
10.0 50 50 50 50.00±0.00
12.0 50 60 60 56.66±5.77
14.0 60 60 50 56.66±5.77
16.0 70 70 70 70.00±0.00
18.0 70 80 70 73.00±5.77
20.0 80 90 90 86.66±5.77
22.0 90 80 90 86.66±5.77
24.0 100 100 100 100.00±0.00
77
Phase # 02
Appendix # 04 Physiochemistry of Control group on daily basis in Oreochromis mossambicus
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 27.00 299 7.49 1.09 4.89 1.01 24.05 59.72
2 27.00 299 7.50 1.05 4.89 1.00 22.44 60.72
3 27.50 299 7.50 1.07 4.89 1.02 20.24 62.10
4 28.00 300 7.50 1.12 4.88 1.00 22.44 60.97
5 28.00 298 7.49 1.19 4.88 1.00 24.05 59.47
6 28.00 298 7.50 1.12 4.89 1.00 24.02 59.49
7 27.00 299 7.50 1.13 4.88 1.21 22.44 60.72
8 28.00 300 7.51 1.18 4.87 1.22 22.44 60.97
9 27.50 300 7.52 1.19 4.89 1.23 20.25 62.34
10 28.00 299 7.53 1.17 4.89 1.00 22.44 60.72
11 29.00 298 7.50 1.12 4.88 1.00 20.23 61.86
12 29.50 299 7.50 1.15 4.88 1.00 22.44 60.72
13 28.00 299 7.51 1.07 4.88 1.00 20.24 62.10
14 27.00 300 7.50 1.05 4.87 1.08 19.24 62.98
15 28.00 299 7.50 1.09 4.87 1.07 22.44 60.72
16 27.00 299 7.49 1.12 4.89 1.00 24.05 59.72
17 28.00 300 7.50 1.19 4.89 1.21 23.21 60.49
18 27.00 298 7.52 1.17 4.88 1.02 19.24 62.48
19 28.00 299 7.50 1.08 4.89 1.00 24.05 59.72
20 28.00 300 7.50 1.16 4.89 1.00 19.24 62.98
21 29.00 299 7.47 1.17 4.88 1.00 19.24 62.73
22 28.00 300 7.50 1.14 4.88 1.00 20.21 62.37
23 27.50 298 7.53 1.15 4.89 1.00 24.05 59.47
24 29.00 299 7.50 1.13 4.89 1.21 22.44 60.72
25 28.50 300 7.50 1.17 4.88 1.22 19.24 62.98
26 28.50 300 7.52 1.19 4.88 1.22 22.44 60.97
27 29.00 299 7.50 1.18 4.87 1.19 24.05 59.72
28 29.50 300 7.51 1.17 4.87 1.00 24.05 59.97
29 29.00 299 7.50 1.12 4.89 1.00 22.23 60.86
30 29.00 300 7.52 1.09 4.89 1.00 22.21 61.12
Mean±S.D 28.08±0.77 299.2±0.71 7.50±0.01 1.13±0.04 4.88±0.00 1.06±0.09 21.97±1.76 61.06±1.15
78
Appendix # 05 Physicochemistry of Sub-lethal toxicity (1/3rd (LC50) of DINP on daily basis in Oreochromis mossambicus
Dissolved
oxygen
1 27.00 299 7.75 1.17 4.89 1.22 20.22 62.11
2 27.00 298 7.50 1.15 4.89 1.23 21.25 61.22
3 28.00 300 7.51 1.19 4.88 1.22 23.24 60.48
4 28.50 298 7.50 1.20 4.87 1.00 21.24 61.23
5 29.00 299 7.50 1.19 4.87 1.20 24.05 59.72
6 29.50 300 7.52 1.20 4.89 1.00 22.44 60.97
7 28.00 300 7.75 1.21 4.89 2.00 21.24 61.73
8 27.00 297 7.48 1.17 4.89 1.24 24.05 59.22
9 28.00 297 7.49 1.19 4.89 1.22 20.25 61.59
10 29.00 298 7.50 1.23 4.88 1.23 22.12 60.68
11 29.50 299 7.50 1.21 4.89 1.21 22.21 60.87
12 28.00 299 7.50 1.20 4.87 1.02 24.23 59.61
13 29.50 300 7.00 1.15 4.88 1.09 23.25 60.47
14 28.50 300 7.51 1.09 4.87 1.19 22.21 61.12
15 29.00 301 7.50 1.18 4.87 1.00 23.25 60.72
16 27.00 301 7.53 1.19 4.89 1.20 24.05 60.22
17 28.00 300 7.52 1.17 4.89 1.22 22.44 60.97
18 28.50 300 7.50 1.21 4.89 1.19 24.23 59.86
19 29.00 299 7.49 1.20 4.88 1.20 23.21 60.24
20 28.00 298 7.49 1.19 4.89 1.21 22.23 60.61
21 29.00 297 7.50 1.15 4.89 1.22 24.24 59.10
22 29.00 298 7.50 1.17 4.87 1.23 23.21 59.99
23 29.00 299 7.49 1.15 4.89 1.23 22.23 60.86
24 28.00 299 7.50 1.09 4.89 1.00 25.25 58.97
25 28.00 298 7.52 1.15 4.88 1.24 24.05 59.47
26 29.00 298 7.51 1.17 4.89 1.25 23.24 59.98
27 29.00 300 7.50 1.19 4.89 1.19 24.05 59.97
28 28.50 301 7.49 1.20 4.88 1.19 22.44 61.22
29 29.00 298 7.50 1.21 4.88 1.20 24.05 59.47
30 29.00 299 7.49 1.23 4.89 1.19 25.02 59.11
Mean±S.D 28.45±0.75 299±1.17 7.50±0.11 1.18±0.03 4.88±0.00 1.20±0.17 22.97±1.30 60.39±0.83
79
Appendix # 06 Physicochemistry of Sub-lethal toxicity (1/3rd (LC50) of DBP on daily basis in Oreochromis mossambicus
Dissolved
oxygen
1 27.00 299 7.49 1.23 4.89 1.21 24.23 59.61
2 27.00 298 7.50 1.22 4.89 1.22 23.25 59.97
3 28.00 300 7.49 1.25 4.88 1.23 22.21 61.12
4 28.50 301 7.52 1.27 4.87 1.00 23.12 60.80
5 29.00 300 7.49 1.26 4.86 1.02 23.21 60.49
6 29.50 299 7.50 1.28 4.85 1.19 24.25 59.59
7 28.00 299 7.75 1.29 4.85 1.21 24.19 59.63
8 27.00 298 7.50 1.30 4.86 1.02 25.21 58.74
9 29.00 300 7.48 1.25 4.89 2.00 25.22 59.24
10 29.00 301 7.49 1.28 4.88 1.19 24.23 60.11
11 29.50 300 7.49 1.27 4.89 1.22 24.23 59.86
12 28.00 299 7.50 1.29 4.87 1.23 26.23 58.36
13 29.50 299 7.53 1.30 4.88 1.22 20.21 62.12
14 28.00 298 7.50 1.33 4.86 1.19 22.12 60.68
15 29.00 298 7.53 1.24 4.85 1.19 22.21 60.62
16 27.00 299 7.49 1.25 4.89 1.00 23.24 60.23
17 28.50 300 7.50 1.21 4.89 1.00 23.21 60.49
18 29.00 300 7.52 1.22 4.89 2.00 24.22 59.86
19 29.50 301 7.53 1.23 4.88 1.00 25.12 59.55
20 30.00 300 7.47 1.20 4.87 1.19 24.25 59.84
21 30.00 299 7.51 1.19 4.88 1.21 25.12 59.05
22 29.00 299 7.50 1.21 4.87 1.22 25.21 58.99
23 29.00 299 7.75 1.23 4.89 1.20 23.23 60.23
24 30.50 298 7.50 1.22 4.89 1.19 25.22 58.74
25 29.00 299 7.51 1.25 4.88 1.23 25.24 58.98
26 29.50 299 7.52 1.24 4.87 1.24 24.25 59.59
27 30.00 300 7.50 1.23 4.84 1.23 24.21 59.87
28 29.00 301 7.53 1.22 4.85 1.22 23.24 60.73
29 29.00 298 7.75 1.21 4.88 1.22 23.21 59.99
30 29.00 299 7.51 1.20 4.89 1.20 25.22 58.99
Mean±S.D 28.8±0.94 299.33±0.95 7.52±0.07 1.24±0.03 4.87±0.01 1.22±0.22 23.94±1.26 59.86±0.81
80
Appendix # 07 Physicochemistry of Sub-lethal toxicity (1/3rd (LC50) of DBP+DINP on daily basis in Oreochromis mossambicus
Dissolved
oxygen
°C (mg/I) (mg/l) (mg/I) (mg/I) (mg/I) (mg/l)
1 27.00 299 7.49 1.30 4.85 1.19 24.05 59.72
2 27.00 298 7.50 1.31 4.85 1.20 26.25 58.09
3 28.00 297 7.52 1.25 4.84 1.22 25.15 58.53
4 28.50 296 7.51 1.29 4.85 1.21 24.05 58.97
5 29.00 299 7.52 1.33 4.86 1.22 24.05 59.72
6 29.50 298 7.50 1.34 4.86 1.23 25.23 58.73
7 28.00 298 7.75 1.35 4.87 1.23 23.22 59.99
8 28.00 299 7.75 1.32 4.89 1.24 24.05 59.72
9 29.00 301 7.52 1.34 4.85 1.25 24.05 60.22
10 28.00 300 7.51 1.29 4.88 1.20 28.21 57.37
11 27.00 300 7.48 1.28 4.89 1.20 24.22 59.86
12 28.00 299 7.51 1.34 4.89 1.21 24.05 59.72
13 29.50 300 7.49 1.33 4.88 1.19 24.05 59.97
14 28.50 300 7.50 1.25 4.89 1.23 24.22 59.86
15 29.00 298 7.50 1.23 4.89 1.19 25.23 58.73
16 29.50 301 7.53 1.25 4.85 1.24 24.05 60.22
17 30.00 299 7.75 1.32 4.86 1.19 24.05 59.72
18 30.00 300 7.51 1.34 4.88 1.20 26.25 58.59
19 29.00 299 7.49 1.32 4.88 1.20 29.06 56.59
20 29.00 298 7.50 1.31 4.89 1.21 24.05 59.47
21 30.00 297 7.50 1.30 4.89 1.22 24.05 59.22
22 28.00 299 7.50 1.29 4.89 1.23 25.22 58.99
23 29.00 300 7.51 1.34 4.86 1.21 23.24 60.48
24 29.50 300 7.52 1.33 4.86 1.20 24.05 59.97
25 28.50 301 7.48 1.37 4.88 1.20 29.06 57.09
26 29.50 299 7.49 1.39 4.86 1.21 24.05 59.72
27 29.00 298 7.50 1.33 4.89 1.21 24.05 59.47
28 28.50 299 7.00 1.30 4.86 1.20 26.21 58.37
29 29.00 300 7.50 1.29 4.87 1.20 27.12 58.05
30 28.00 299 7.51 1.31 4.86 1.21 24.05 59.72
Mean±S.D 28.68±0.85 299.03±1.21 7.51±0.12 1.31±0.03 4.86±0.01 1.23±0.05 24.95±1.59 59.16±0.97
81
SOPS
Students/researchers handling fishes and experimental chemicals were followed given

SOPS for reliable practices:
1. They were use face masks, lab coats, gloves and head covers to prevent inhalation
and direct contact to chemicals.
2. Later chemical treatment to fishes they were properly rinse and sanitize their hands.
3. They were not be allowed to eat, drink or use any cosmetics in research labs.
4. It was necessary for them to maintain good personal hygiene.
5. They were discarded the waste properly.
6. Suitable personal protection was implemented for dose preparation, administration

and dealing with waste disposal.
82

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I, Amna Nawaz D/O Shah Nawaz student of MS Zoology, Government College

A DISSERTATION SUBMITTED TO GOVERNMENT COLLEGE WOMEN

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD

GOVERNMENT COLLEGE WOMEN UNIVERSITY

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Supervisor Internal Examiner

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External Examiner Chairperson

My profound gratitude goes to my respectable supervisor Dr. Qurat-ul-Ain for her

I would also like to express my gratitude to my head of department Dr. Asma

Moreover, my special regard goes to my seniors for guidance, discussion, and

Table 2 Determination of Acute Toxicity (LC50) of di- 36

Table 3 Determination of Acute Toxicity (LC50) of mixture 38

Table 4 Motility parameters in all experimental groups 42

Fig: 3.11 Staining the slides. 32

PEs Phthalate esters

DBP Dibutyl phthalate

DnBP Di-n-butyl phthalate

DIDP Diisododecyle phthalate

BBP Benzyl butyl phthalate

DEP Diethyl phthalate

DINP Diisononyl phthalate

DEHP Diethylhexyl phthalate

HMW High molecular weight

SVHC Substance of very high concern

HPG Hypothalamo- pitutary gonadal

FSH Follicle stimulating hormones

ECS Endocannabonoid system

GSI Gonadosomatic index

EDCs Endocrice distrupting chemicals

IUPAC International Union of Pure and

EDTA Ethylene diaminetetra acetic acid

DNA Deoxyribonucleic acid

Key words: Tilapia, histomorphometric analysis, semen analysis, and male

Plasticizer, a man made chemical, is an integratable material that is used to

Due to incorrect discharge of effluents and contaminated runoff from

An ester of phthalic acid is called phthalate. Phthalate is a category of

Diester phthalates are found in nearly 25 different types of commercial

Di-butyl phthalate (DBP), a liquid that is oily, colourless to very faintly

Figure 1.1: Structural formula of Di-butyl phthalate (DBP).

Due to its close proximity to DEHP in terms of global manufacturing, DBP is

The main source of food-borne di-butyl phthalate exposure is probably fish

Figure 1.2: Structural formula of Di-isononyl phthalate (DINP).

In aquatic environment, DINP released from multiple sources, including air

Fish have well-developed endocrine, neurological, immunological, and

In jawed vertebrates, the hypothalamo-pituitary-gonadal (HPG) axis is a

Oreochromis mossambicus is a rare fish in Pakistan due to its African origin,

Figure 1.3: Oreochromis Mossambicus (Mozambique Tilapia).

 To evaluate the sublethal effects of phthalates by performing histomorphometric

 To assess the reproductive toxicity in phthalates exposed fish, O. mossambicus by

Sruthi et al. (2021) assessed the reproductive toxicity of di-butyl phthalate

Revathy and Chitra (2019) conducted an experiment to determine the role of

Bhatia et al. (2014) studied the antiestrogenic impacts of di-n-butyl phthalate

Hu et al. (2020) studied the reproductive toxicity of DnBP in Zebrafish. In his

Bhaisare et al. (2022) conducted an investigation to clarify the impact of

Forner-Piquer et al. (2018) carried out an investigation to examine the impacts

Kim et al. (2002) aimed this investigation to highlight the consequences of

Mohan et al. (2022) conducted an experiment to elucidate the sub-lethal

Ghisari (2009) performed an in vitro experiment to investigate the

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