TOXICITY OF MICROPLASTICS AND THEIR UPTAKE BY THE FISH

COLLECTED FROM RIVER CHENAB, PAKISTAN

BY

SADIA SAFDAR

2017-GCWUF-1163

Thesis submitted in partial fulfillment of requirements for the degree of

MASTER OF PHILOSOPHY

IN

ZOOLOGY

FACULTY OF SCIENCE AND TECHNOLOGY

GC WOMEN UNIVERSITY

FAISALABAD PAKISTAN

2022

1
2
DECLARATION

It is hereby declared that the contents of thesis, “Toxicity of microplastics and their uptake
by the fish collected from river Chenab, Pakistan ” are product of my own research and no
part has been copied from any published source (except the references). I further declare that
this work has not been submitted for award of any other diploma/degree. The university may
take action if the information provided is found inaccurate at any stage.

Sadia Safdar

2017-GCWUF-1163

3
The Controller of Examinations,

The members of the Supervisory Committee find the thesis submitted by Miss Sadia Safdar,
(2017-GCWUF-1163) satisfactory and recommend that it be processed for evaluation by the
External Examiner(s) for the award of degree.

Dr. SafinaKousar (Supervisor) -------------------------

Dr. Sumaira Aslam (Member) ------------------------

Dr. Sana Aslam (Member) --------------------------

Incharge Dean/ Coordinator

Department of Zoology, Faculty of Sci.& Tech.
G.C.W. University, Faisalabad GCWUF

Director Academics
GCWUF

4
 DEDICATION

This thesis is dedicated to

The ALLAH Almighty

My creator, my Strong pillar, My source of Inspiration,Wisdom, Knowledge and

Understanding

My Parents

Mr. and Mrs. Safdar

For whom, educated children are better than hefty bank deposits

My Supportive Siblings

Rana Aamir, Imran, Faisal, Nadia Zahid, Sonia Ali

For their unwavering support, protection, Faith and encouragement

My mentors

Rana Ghulam Mustafa, Madam Nasreen, Mrs. Rehana Talat, Nemrah Ahmad Niazi

And to me

Without whom the completion of this work would not possible.

5
 ACKNOWLEDGEMENTS

All acclamation and appreciation are for Allah the Almighty, the most merciful, the most
beneficent. All respect for his Holy Prophet (PBUH).
I wish to acknowledge my deep sense of profound gratitude to Prof. Dr. Robina Farooq,
Worthy Vice Chancellor GC Women University Faisalabad. I also acknowledge Dr.
Tamseela Mumtaz, Chairperson, Department of Zoology, GC Women University Faisalabad
for providing us research facilities.

The work presented in this manuscript was accomplished under the sympathetic attitude,
animate directions, observant pursuit, scholarly criticism, cheering perspective and
enlightened supervision of Dr. Safina Kousar. I offer deep sense of obligations to her to
encourage me to work independently in every field of life. I shall always remember her
admirable supervision, great support and marvelous knowledge. The words are inadequate to
acknowledge the sincere efforts of members of supervisory committee, Dr. Sumaira Aslam
Department of Zoology, Govt. College for Women University Faisalabad, Dr. Sana Aslam
Department of Chemistry, Govt. College for Women University Faisalabad. I have no words
to explain my obligation to Mrs. Zainab Tariq for her help during all the research work

No acknowledgement could ever adequately express my obligation to my supporting brother

Rana Imran and respected parents Mr. and Mrs. Safdar whose hands always rose in
prayers for me and without their moral and financial support the present destination would
merely be a dream. They always acted as a light house for me in the dark oceans of life path.
The name of my parents will always be in front of my eyes, as I will look on the cover of my
thesis, even though my name will be printed on it. I would like to extend my zealous thanks
to my loving and caring sisters Nadia Zahid and Sonia Ali and my little princess Enaya
Zahid and my Grand Mother Mukhtara Bibi (Late).

Last but not the least I offer my special and sincere thanks to my big brother Dr. Umair
Hassan and my friends Sadia sarwat, Sanam shehzadi and Nisha who gave me courage
all the time.

Sadia Safdar

vi
 TABLE OF CONTENTS
CHAPTER NO. CONTENTS PAGE

ACKNOWLEDGEMENTS Iv

TABLE OF CONTENTS V

LIST OF TABLES Vi

LIST OF FIGURES Vii

ABSTRACT X

CHAPTER-1 INTRODUCTION 1-5

CHAPTER-2 REVIEW OF LITERATURE 6-25

MATERIALS AND METHODS 26-40

Study area and experimental plan 26 -27

FTIR for microplastics detection in fish 27-28

CHAPTER-3
Water analysis 28-29

Comet assay 30-33

Histopathological alteration 34-40

RESULTS AND DISCUSSION 41-55

CHAPTER-4 Results 41-50

Discussion 51-55

CHAPTER-5 SUMMARY 56-57

REFERENCES 58-75

vii
 List of Tables
[Link]. Title Page

1. Physio-Chemical parameters of test system at 2 (mg) level …………………………… 39

2: Physio-Chemical parameters of test system at 4 (mg) level ……………..……......... 40

3: Physio-Chemical parameters of test system used for control group …………………. 41

4: DNA damage in peripheral erythrocytes of microplastically exposed fish…………. 46

5: Mean DNA damage in peripheral erythrocytes of microplastically exposed …….. 47

viii
 List of Figures
S. No Title Page

1. pH meter, NHᴣ kit, DO meter, Fish feed ....................................................................24

2. Acquaria containing experimental group……………………………….…………....24

2.3 Fourier Transform Infrared spectroscopy ...…………….………........27

3. Seives, magnetic stir, water sample….…………………………………………..…. 29

4. Blood sample of fish……………….……………………………………………...... 32

5. Microtome………………………….…………………………………..………...…. 37

6. FTIR spectra and microscopic view of polypropylene.…………………………….. 43

7. Microscopic view of MPs in water….…………….………………………..,……..…44

7.1 FTIR spectra of water…………………………………….………….…44

8. Damaged erythrocytes………………………………………………………………..45

8.1 Highly damaged erythrocytes …………………….……………….….45

8.2 Undamaged erythrocytes ……………………..……………………..…45

9. Photomicrography of undamaged and damaged and damaged muscles……………….

ix
ABSTRACT:

Plastics are widely considered to be a major threat particularly to the aqua-culture. Extensive
use of plastics on worldwide scale pose a great threat to contamination of waterbodies. By
passing through the process of bio and photodegradation, the plastic breakdown into small
particles. The particles below 5mm in diameter is known as microplastics. The potential
hazardous effects of Microplastics (MPs) on aquatic animals must be determined urgently.
For this purpose, an experiment was planned to determine presence of MPs in River Chenab
as well as its possible effects on fish. FTIR (Fourier Transform Infrared Spectroscopy)
technique was used to identify the microplastic content in fish organ and water sample taken
from River Chenab. Moreover, DNA damage and histopathological alteration in fish due to
MPs exposure were determined under laboratory conditions. Fish (n=8) were exposed to MPs
for 30 days and samples were collected fortnightly for determination of DNA damage and
histopathological alteration. DNA damage in fish blood was evaluated by using single cell gel
electrophoresis technique. Fish tissues were dried using a gradients of ethyl alcohol
concentrations, embedded in paraffin wax, sectioned, stained and seen under a microscope
for histology examinations. Computer software SPSS-28.0.1 was used to statistically analyse
the gathered data. Fish has been used as a bioindicator of pollution in aquatic ecosystem. This
study helped to evaluate the current status of MPs pollution in River Chenab and its ultimate
effects on aquatic fauna and human health.

x
CHAPTER. 1

INTRODUCTION

The word "plastic," which meaning "moldable," is derived from the Greek word plastikos
and refers to synthetic organic polymers like coal, oil and gas (Ivleva et al., 2017). Plastics
key distinguishing characteristics are its affordability, indestructibility and low cost (Van
Eygen et al., 2017). These charateristics have made the vast use of plastics in unending
applications (Andrady, 2011). Due to the increase in population and industrial advancement,
the need of plastic products has also increased. Plastics production has expanded
considerably during the last few decades, reaching upto 350 million tones (Plastics Europe,
2018). Due to its primacy, it is designated as the most widely manufactured and used material
on this planet (Andrady and Neal, 2009). Packaging, construction, automobiles, electronics
and electrical appliances, textiles, and agriculture are all the examples where plastics are used
in modern life (Plastics Europe, 2018).

Microplastics are particles with a diameter of less than 5mm (Eckert et al., 2018). However,
the MPs with 500 μm or less are more common in aquatic environment (Sharma and Chatter
jee, 2017) and siza quite easily engulfed by aquatic organisms (Lusher et al., 2013). Both
primary and secondary sources of MP can be found in nature. Plastic pallets, seeds, films,
fibres and powder are examples of primary microplastics utilised in the manufacturing of
cosmetics such as cleanser, facial scrub and sunscreen, as well as items created during ship
breaking (Fendall and Sewell, 2009; Guzzetti et al., 2018). Large plastic products (primary
MPs) which undergo biological, chemical and physical degradation results in secondary MPs
(Browne et al., 2008; Ryan et al., 2009). When larger plastics particles break through
chemical weathering, such as tidal currents, wind damage, and solar exposure, secondary
microplastics emerge (Andrady, 2011).

Over 5 trillion plastic trash particles were predicted to be floating in the sea, with >90
percent of these being secondary microplastics generated during breakdown (Eriksen et al.,
2014). Microplastics can be found in almost any sort of aquatic habitat, making them
accessible to a range of aquatic animals, such as fish (Lusher et al., 2016; Baldwin et al.,
2016; Dai et al., 2018).

According to reports, 192 coastal regions produced over 275 million metric tonnes of plastic
trash in 2010 (Jambeck et al., 2015). Annually, round about 20 million metric tonnes of
plastic thrown into the marine water (Vannela, 2012). In the time of COVID-19, the most pop
up source of plastic pollution to aquaculture was disposable facial masks (Fadare and
Okoffo., 2020). Plastic particles have been discovered on beaches throughout the world, not
simply in surface water (Eriksen et al., 2013; Fernandino et al., 2015; Fok and Cheung,
2015). The industries that generate plastics on the other hand, use tonnes of pellets, which
implies that during production, collection and transporting, basic micro-sized granules can
be set to release into waterbodies as well as the sea life (Lebreton et al., 2017). Living things
might devour these pellets, or they can be deposited in sediments over time (Karlsson et al.,
2018; Rochman, 2018). Additionally, spilling granules increase the probability of plastic
ingredients getting into the water, potentially endangering surrounding environment (Borges
xi
Ramirez et al., 2019). As a result, investigating the existence of microplastics in these kind of
critical microplastic particles point sources is critical. Plastic manufacturing plants have been
observed to leak micro-sized pellets into the environment in only one investigation (Karlsson
et al., 2018).

Large amount of microplastics are ingested by aquatic fauna including invertebrates,

mammals as well as fish (Blessling et al., 2015; Rummel et al., 2016; Scherer et al., 2017).
Microplastic particles are quickly ingested by fish due to their tiny size and similarity to feed
stuffs. In both unintentional and intentional way (Crawford and Quinn, 2017). Studies shown
the consumption of MPs by aquatic animals like fish and invertebrates (Rochmann et al.,
2013) that exert detrimental effects on various tissues, organs i.e gills, gut, circulatory system
and digestive tract (Sekirov et al., 2010; Moos et al., 2012). In aquatic environment, the most
commonly used bioindicator animal is fish (Wesch et al., 2016).

Human activities in surrounding populations and industrial entities regularly damage inland
rivers and water bodies in developing and underdeveloped Countries (Qadir and Malik,
2011). The pollution of water bodies by industrial effluents containing dangerous chemicals
has a significant impact on the physical, chemical and biological imperatives of the
surrounding water (Eqani et al., 2012). Although Pakistan is a member to the Stockholm
Convention, there is currently no effective regulatory framework in place to monitor
polychlorinated biphenyles (PCB) levels in various ecological matrices. Manufacturing and
use of PCBs are not regulated in this country, and these chemicals are not classified as
chemicals of serious concern in the National Environmental Quality Standard (Eqani et al.,
2012).

Pakistan's province, Punjab is the home to five rivers. Among all these rivers, the Chenab is
Punjab's lifeline. The Chenab River originates in the Himachal Pradesh districts of Kulu and
Kangra. This river enters in Pakistan, near the town of Diawara in Sialkot. The River Chenab
has a catchment area of 13469 square miles. The Chenab River has twelve significant
tributaries, two of which are in Pakistan: Nallah Palkhu and Nallah Aik. The river Chenab in,
four headworks of Punjab (Khanki, Marala, Trimmu and Qadirabad) and
six canals that connect (Marala Ravi link canal, Lower Chenab canal, upper Chenab canal,
Rasul Qadirabadd link, Qadirabad Balloki link, Trimmu Sidhnai link). Contaminated water
from Faisalabad's industrial and metropolitan regions is released into the Chenab River,
causing the extinction or population decline of major carps (Latif et al., 2016). The
Chakbandi Drain, which runs through the Faisalabad's eastern and southern districts carries a
large amount of industrial and sewage waste into the Chenab River. Hazardous chemicals
from a variety of industries, including fabric, preservative, and drug companies, along with
tanneries and refineries, have been identified in these industrial effluents (Latif et al., 2016).
Regardless of the fact that the advanced treatment plants are capable of removing huge
amounts of plastic particles from textile wastewaters, these particles proceed to exit and
pollute the waterways (Carr et al., 2016; Murphy et al., 2016). In Pakistan, there are 6634
recognised enterprises, 1228 of which are considered severely polluting. Through many
drains, the Chenab River collects pollutants in the form of sewage and industrial waste

xii
discharge from various districts and the surrounding area. Through Chakbandi Main Drains,
Faisalabad dumps sewage and industrial trash into the Chenab River (Ullah et al., 2014).

Because of their varying densities, microplastics can inhabit the surrounding water and
bottom sediments. Many aquatic organisms unintentionally or deliberately consume oceanic
waste due to contact of microplastic particles with plankton and sand particles. However, the
toxic effects of microplastic on the exposed organisms are expected to vary over the
microplastic particle size spectrum encountered, which varies according to the size of the
microplastic confronted pieces of plastic at the top range of the size (1-5 mm) may cause
nutritional status and digestive health issues (Wright et al., 2013). Primary microplastics,
that included synthetic fibres ejected from microbeads and washing machine runoff and used
as scrubbing agent or for other uses and found in skincare products and cosmetics are divided
into two types based on their source (Andrady, 2011).

Recent research has shown how MPs affect a variety of freshwater and saltwater fish species,
including zebrafish, which were used as samples to determine whether MPs pose a threat to
higher organisms. The results of the model are likely to provide insight into the dangers of
polymer toxicity, with a focus on long-term exposure in a variety of human organs and
tissues, including the brain, at different levels of biological complexity. Plastic toxicity is
regulated by a number of elements, such as particle size, form, density, time of exposure and
type of material, as well as co-factors, making this matter extremely difficult (Guerrera et al.,
2021). Because fish are one of the most essential feedstuffs for humans, they are commonly
used in evaluations of the existence, quantity, and dispersion of microplastic pollution
(Dehaut et al., 2019). Furthermore, multiple exposure studies have shown that high
microplastic concentrations might have detrimental effects on fish in the laboratory, including
decreased hatchability of sea bass, biological oedema and histopathologic abnormalities such
as gradual and incendiary modifications (Peda et al., 2016; Choi et al., 2018; Jabeen et al.,
2018).

Polystyrene microplastic exposure to fish show the physiological effects which lead to the
induction of oxidative stress (Lu et al., 2016). From the last two decads, the great attention
have been made on the process of MPs for inducing toxicity and death in aqua-system
varied from fish motality (Peda et al., 2016) to the internal injuries like obstruction in
digestive system, delayed enzyme synthesis, slowness of growth, genotoxicity, neurotoxic
effects and disruption based on reproductive system (Kaposi et al., 2014; Alimba and Faggio,
2019). The effects of microplastics on various fish depend on a number of factors, such as
particle size, the kind of microplastic, the availability or exclusion of other chemical
pollutants and other organisms (Rainieri et al., 2018).

Toxicological consequences in organisms can result from chemical and biological harm,
therefore, concerns about the effects of MP consumption go far beyond physical harm (De Sa
et al., 2015). This may results in distruction of habitat, retarded growth, genotoxicity
(Richards et al., 2000). The damage of DNA can be identified and repaired through Comet
assay which is the most advanced technique. It is inexpensive, non-invasive, rapid, sensitive
and easy to handle (Hussain et al., 2018).

xiii
 Histopathological alteration is used for the assessment of environmental stress and toxicity
of MPs to the fish (Yancheva et al., 2016; Santos and Martinez, 2014). Many studies have
looked structural alterations in fish of various species. The muscle, liver and kidney all
showed significant inflammatory alterations as well as tissue damage (Usman et al., 2021).
As a result when it comes to identifying toxicological consequences histopathology is the
gold standard. So that evaluating the impacts of MPs on the entire body, histopathological
examination is still an essential element (Reddy and Rwat, 2013).

NEED OF PROJECT:

Due to global administration and its correlated environmental impacts, plastic waste has
become a worldwide concerned matter. Fish are frequently used to assess the water quality
and it acts as a bioindicator of ecological ecosystem. Microplastic damage assessment has
been found to be an excellent method for biomonitoring in aquatic ecosystems. Microplastic
causes a variety of diseases in fish, which has a negative impact on the health and ecological
stability of organisms. The goal of this research is to determine the toxicity of microplastics
ingested by fish in the river Chenab.

AIMS AND OBJECTIVE:

The aims and objective of this research is the

Determination of MPs in fish organ and water samples taken from River Chenab.

Determination of DNA damage in MPs exposed fish under laboratory conditions.

Determination of the histopathological effects of MPs in fish

xiv
CHAPTER. 2

LITERATURE REVIEW

In the aquatic environment, microplastics (particles smaller than 5mm) are composed of
particulate that vary in size, particular specific gravity, chemical properties and morphology
(Duis and Coors, 2016). Plastic is lightweight, flexible, affordable, and corrosion-resistant,
among other features. Its elasticity is one of them (Thompson et al., 2009). In the last five
years, numerous studies have shown that MPs are present in the environment, especially in
water bodies such oceans, rivers, estuaries, lakes and polar water (Bellasi et al., 2020; Li et
al., 2020a; Xu et al., 2020). Initial researches were developing in the world, offering
validation of this hypothesis, but because of the inevitable lack of uniformity in
methodologies, they are difficult to use as a baseline. Microplastics have also been subjected
to organisms in lab trials to test whether they can spread disease (Browne et al., 2008, 2013;
Rochman et al., 2013; Wright et al., 2013). The biggest concern nowadays is
its consumption, which might result in severe harm (Wright 2014) in addition to toxic
potential on organisms (Teuten et al., 2007; Browne et al., 2008). It has been demonstrated
that plastics can remove metals and persistent organic contaminants from saltwater (Mato et
al., 2001; Ogata et al., 2009; Teuten et al., 2009) and organic contaminants can accumulate in
concentrations that are considerably greater on the substratum of plastic than those in the
surrounding water (Holmes et al., 2012; Mato et al., 2001; Ogata et al., 2009; Teuten et al.,
2009). According to laboratory research, these chemicals can be consumed by living things
and transported from plastics to them (Teuten et al., 2009), which might cause distress them
(Browne et al., 2013; Rochmanet et al., 2013; Wright et al., 2013). It causes harm to the
organisms who come into contact with it in the wild. The possibility of transmission differs
depending on plastic and pollutant combination, with some polymeric materials, such as
plastic polymer (polyethylene), which has a high likelihood of being transported (Bakir et al.,
2012). With some polymeric materials, like polyethylene, having a high capacity for
transport, the likelihood for translocation varies depending on the combination of plastic and
pollutant (Teuten et al., 2007; Bakir et al., 2012).

Since the mid-twentieth century, there has been an increase in overall production of plastic
polymers has also been followed by a deposit in the water ecosystems of plastic pollution
(Barnes et al., 2009; Thompson et al., 2004). It was determined in the 1990s that hand
sanitizer, which are hardly ever used by most consumers, are a negligible contributor to
microplastic contamination. However, because the majority of cleansers now
comprise of polyethylene microplastics, which are not accumulated by wastewater treatment
plants and result in ocean ecosystem, the common customer is more likely to use
microplastic-containing products on a daily basis in 2009. Four face cleansers that include
microplastic that are sold inthe surface area of the polythylene particles was measured using
New Zealand shops. Planktonic organisms at the base of the food chain could immediately
consume 75% of the brands since their median size was less than 100 microns. In the long
run, plastic particles will become smaller and more hazardous due to UV degradation and the
consumption of hydrophobic substances like PCBs. Marine biologists must inform the public
of the risks associated with utilising products that jeopardise the condition of the water

xv
bodies and the food we consume today and in the future (Fendall and Sewell, 2009).
Permanent plastics are often destroyed and disintegrate over time when distributed by
currents and winds, whether they are lost accidentally or on purpose (Thompson 2015). The
amount of microscopic plastic pieces, or microplastics, in the water has increased as a result
of these dissolved secondary micro-fragments and micro-sized primary plastic waste from
consumer products (Andrady, 2011). The density of microplastics has a significant impact on
their dispersal in the marine environment. The mass of a virgin polymer substance is changed
on a regular basis during production (for instance, by inorganic fillers increasing density or
by polymer foaming decreasing density), in addition to corrosion and photocatalytic
degradation mechanisms (Gregory 1983; Moret-Ferguson et al. 2010; Harrison et al. 2011).

Due to the lower density of most synthetic polymers than seawater, microplastic particles are
trapped in the intertidal zone to a lesser extent (0.014–12.51 items mᶾ ) than they are drifting
far above sea level (0.022-8,654 products mᶾ). Microplastics seem to collect in sediment
(18,000-125,000 items in subtidal sediments) neutrally buoyant, suspended polymers (185-
80,000 mᶾ items) subsiding polymers build up on beaches, which serve as intermediary
settings (Hidalgo-Ruz et al., 2012).

To identify locations of high and low concern for Micro Plastics in the freshwater
environment, it is essential to define the sources of MPs in the freshwater environment. Every
year, 1.15-2.41 million tones of plastic reach the seas via rivers and other waterways
(Lebreton et al., 2018). The most significant source of Micro Plastics in rivers is ascribed to
effluent from wastewater treatment plants (WWTP), which is likely due to the fact that MPs
may pass through treated effluent (Cole et al., 2011). Increased plastic loads in rivers are
associated with a combination of ineffective waste management and high population density,
which are positively linked with one another (Baldwin et al., 2016). Polyethylene
terephthalate (PET) is the most common kind of plastic in wastewater treatment plant
effluent, which means that PET concentrations are greater in the proximity of wastewater
treatment plants (Rodrigues et al., 2018).

Cok et al. (2011) aquatic environmental contamination with varying levels of contaminants
poses unanticipated risks to wildlife and human. The result of pollutants and xenobiotics
metabolism and exposure particularly mutagens and carcinogens adequately examined by
looking into sever events like DNA damage by using single cell gel electrophoresis. The fish
species Cyprinus carpio was used in this study to determined contamination in lake Morgan
of Turkey near Ankara, where the main source of pollution are agricultural pesticides and the
city’s waste water. The tail intensity, tail moment and tail length values of fish collected
Mogan Lake were determined to be 7.774.51, 1.501.48, 31.1010.39 respectively. They were
determined to be 0.40±0.51, 3.47±1.59, 22.80±1.08 for clean reference sites. The differences
in the values are significant statistically p= 0.0013, p 0.0001 and p 0.0001. These conclusion
indicate that Lake Mogan may be contaminated with genotoxic chemicals, and they serve as
an early warning for the lake system.

Kemp et al. (2011) stated that as a result of anthropogenic activities the increased in fine
sediment intake from aquatic and terrestrial sources has a detrimental influence on aquatic

xvi
ecosystem. This study has been concluded that fine sediments have an influence at all phases
of all fish species lives in fresh water. This study uncover the causative processes that
explain fish populations' apparent negative reaction to increased fine sediment loads, as well
as the heterogeneity among fish species.

Andrady (2011) studied that Plastic trash and microplastics will become more prevalent in
the ocean ecosystem as a consequence of shifting production trends, demography and usage
patterns. Microplastics appear to be primarily produced through weathering-related plastic
disintegration and surface cracks in coastal environments. When exposed to maritime
conditions, micro- and nanoplastics are stubborn materials. Plastics' proven ability to absorb
and concentrate Persistant Organic Pollutants (POP) is a severe problem, even though they
make up a relatively minor percentage of micro- and nanoparticles in marine water. Because
POP-laden fragments are ingestible by aquatic life, including micro- and nano plankton
species, this technique is very likely to transfer poisons throughout the trophic levels. The
effectiveness of such transmission will be determined by the availability of POPs and the
length of time that microplastics remain in species. Hazardous side effects may result the
consumption of plastic nanoparticle by micro- or nanofauna. Any danger to aquatic biota
which laid the framework of the aquatic food webs, could be dangerous and far-reaching
effects on the oceans across the world. It is vitally necessary to quantify the scope of these
possible consequences and predict the long-term effects of rising microplastics concentrations
on the world's oceans.

Claessens et al. (2013) Microplastics had been extracted from environmental materials
(sediment and biota) using two different procedures. Sediment samples are subjected to the
upward flow of water in an elutriation tube in order to isolate the lighter plastic items from
the heavier sand particles. A sodium iodide solution is then used to treat the product that
contains the microplastics. In this greater solution, plastic particles will float while heavier
particles will be spun out. The microplastic-containing supernatants are filtered using a 5 m
filter. Microplastics are recovered from biota by boiling them for two hours after killing the
organism in HNO3 at room temperature overnight. The mixture is then filtered through a 5 m
filter and diluted with hot filtered deionized water. Because both approaches produced high
distillation efficiency (from 93 to 98 percent) for a variety of MPs, they can aid in the
development of a more thorough understanding of the prevalence of microplastics, including
biota.

Lusher et al. (2013) worked on plastic in fish and compareds pelagic and demersal species.
Microplastic's potential negative consequences were not investigated. The fish's age and
gender were not taken into account, which could be useful in future research to identify
whether plastic accumulates over time or is naturally transported through the digestive
system. To determine the potential repercussions of ingestion, data on residence time as well
as potential plastic particle translocation is needed. To properly comprehend the potential
effects of plastic particles, greater study encompassing a larger variety of species and
ecosystems is required given the widespread distribution and consumption of this material.

xvii
Avio et al. (2015) evaluated the effectiveness of numerous existing techniques and developed
a new methodology that allows for a microplastic extraction yield of 78 percent to 98 percent
from fish tissues, depending on the polymer size. The extraction process had no effect on the
particle properties, according to FT-IR analysis. The approach was then tried on two fish
species which was subjected to polyethylene and polystyrene under laboratory settings; the
particles were organised and measured in the intestinal lumen and liver cell organ, and
histological tests confirmed their appearance in the hepatic [Link] investigation
reported the existence and dispersion of many aquatic organisms collected all around Aegean
Sea contained microplastics.. Polyethylene was shown to be the most prevalent polymer
(65%) in the stomachs of fish, according to FT-IR analysis. Overall, the findings proved that
the newly proposed method is a valid method for dete.

Phillips and Bonner (2015) determined the frequency and types of microplastics consumed
by fish in four Gulf of Mexico freshwater drainages and a Gulf of Mexico estuary.
Microplastics were found in the digestive tracts of 8% of freshwater fish and 10% of marine
fish in this study, which included 535 species. Especially in comparison to one of the
urbanised streams, the percentage of microplastics fish ingested in the proportion of
rural waterways (5%) was lower (Neches River; 29 percent). Regarding environment (For
example, planktonic, demersal) and food chain community (i.e. carnivore,
herbivore/omnivore, invertivore), microplastics were discovered in comparable numbers.
Although the effects on fish health are currently unknown, plastic contamination is common
in Gulf of Mexico watersheds, with low but extensive frequencies among drainage channels,
habitat guilds, and trophic guilds. Identifying and measuring MPs in aquatic biota.

Bellas et al. (2016) worked on the commercially significantthe small mottled carp
Scyliorhinus canicula, the European fish Merluccius merluccius, and the red mullet Mullus
barbatus have been studied as benthic fish from the Spanish Continental and Middle east
coasts. for microplastic ingestion. 212 fish in all, comprising 128 red mullets, 12 hake, and 72
dogfish, were examined. Microplastics were found in 17 percent of the fish (15.3 % dogfish,
18.8 % red mullets, and 16.7 % hakes), with an average of 1.56 pieces per fish varying in size
from 0.38 to 3.1 mm. These fish species are being used as bioindicator to pinpoint ocean
pollution as representatives of the Spanish Marine Pollution Monitoring Programme (SMP)
and they may be suitable for identifying temporal and geographic trends. The Spanish
Maritime Pollution Monitoring Programme (SMP) uses these fish species as biomonitors to
measure marine pollution, and they would be suitable for observing the temporal and
geographic trends of ingested litter.

Peda et al. (2016) examined the histopathological changes in freshwater fish Dicentrarchus
labrax that had ingested microplastics over a long period of time. The fish (n = 162) received
Control, natural polyvinyl chloride (PVC), or toxic polyvinyl chloride (PVC) pellets for 90
days. Standard processes were employed for histological investigation. A score value was
used to look at histopathological changes (from 0 to 4). The distal section of the intestine was
the most impacted by pathological changes in all samples with a progressive change in terms
of exposure time, they range from mild to severe. Both groups' histological image, reveal that

xviii
gastrointestinal functions may occasionally be completely disrupted, especially after 90 days
of treatment.

Lu et al. (2016) investigated (PS-MPs) Polystyrene microplastics absorption and tissue

accumulation in zebra fish and its toxic effects in the liver. The findings revealed that 5 m
diameter MPs accumulated in the gills of fish, glandular organ, and gestrointestinal tract after
7 days of exposure, but MPs with a diameter of 20 m accumulated only in the gut and gills of
fish. PS-MPs are available at both 5 m and 70 nm wavelengths in live fish, this caused
inflammation and lipid accumulation, according to histopathological examination.

Cincinelli et al. (2017) examined the presence and level of microplastic (MPs) pollution in
subsurface waters gathered from the Ross Sea coast's near-shore and offshore areas
(Antarctica). Additionally, a non-invasive technique for analysing MPs was proposed,
consisting of filtering after water sample and analysing the dried filter using FTIR 2D
imaging. The analytical setup's non-intrusiveness minimizes potential bias and enables
further investigation of the filtration sample to distinguish other types of pollutants MPs
demonstrates quantities lesser than those reported in the world ocean, with a mean value of
0.17 0.34 mᶾ. MPs varied from 0.0032 to 1.18 particle per cubic meters of saltwater. MPs
included particles (mean 71.9 21.6 %), fibres (mean 12.7 14.3 %) and others (mean 15.4 12.8
%). The existence of numerous MP types was determined using spectroscopy, with
polypropylene and polyethylene being the more abundant. There was also evidence of the
possible environmental impact of scientific research, such as marine research activities and
sewage treatment plant operations.

El-Moghazy and El-Morsy (2017) conducted a study to examine the effect of water change
on tilapia. Different time selected to change different fish tank water was after 1 day (control
group), 2 days, 6 days and 8 days. Fish was fed 2 days a day according to their body weight.
Biological dissolved oxygen level was also checked. It was concluded that fish growth was
maximum in trail in which water is changed after every two days than other trails.
Experiment group in which water was changed after 4 days shown better growth than the
experiment group in which water was changed after 8 days. BOD was checked by using
digital dissolved oxygen (DO) meter. Firstly, the DO of the freshwater sample was measured
after which the water sample was sited in dark room at 25°C temperature for 5 days. After
which oxygen was measure by digital DO meter.

Hermsen et al. (2017) explored the effects of eating more than the 400 specific fish from 4
distinct North Sea species were exposed to 20 micron-sized plastic: Sprat, Continental Hake,
Common Dollop, and Grouper. To prevent contamination during the trial, strict quality
control standards were adhered to. Only one (a Sprat) out of 400 animals (or 0.25 percent,
with a 95 percent confidence interval of 0.09-1.1 percent) had two plastic particles in their
bodies. FTIR spectroscopy was used to determine that the particles were made of
polymethylmethacrylate (PMMA). The fact that there was no contamination during the trial
demonstrates the suitability of the technology used for biota microplastic ingestion studies.
He compared the smaller plastic particle count for fish from the North Sea to that of studies
conducted elsewhere and proposed a link between the observed particle count and the level of

xix
quality assurance utilised. Given the low prevalence seen in this study and other studies that
adhered to high quality assurance standards, plastic particles consumption by fish would be
less than once believed.

Shafi et al., (2018) evaluated the effects of industrial and urban pollutants discharged in water
of river Ravi without treatment 11 sites of river Ravi were selected to gather the water
samples. These included polluted and relatively unpolluted sites. Physio-chemical parameters
and heavy metals concentration was examined in these samples. In water flowing from
Lahore up to Balloki head works the level of dissolved oxygen fall below the suggested
limited for the aquatic organisms. At various polluted sites, the levels of lead and manganese
were significantly greater than the recommended levels for aquatic populations. About 200
µg/l, 70.0µg/l, 190 that are 0µg/l, 2.0µg/l and 72.0µgl concentrations were detected in water
samples. It was concluded that due to low levels of dissolved oxygen, water present near the
drains carrying waste water was not fit for aquatic organisms.

Jabeen et al. (2018) explained how goldfish were fed three different types of virgin MPs,
including fibres, pieces, and granules (Carassius auratus). Numerous impacts were found
after six weeks of exposure, however no mortality was recorded. When compared to the
control group, fish exposed to plastic lost a substantial amount of weight. Fibers were found
in the gills and the digestive system (GIT), and faeces didn't seem to gather there. Fish
subjected to fibres had livers that underwent significant, negative alterations. The most
numerous progressive and inflammatory changes in the livers and intestines were caused by
fibre ingestion. The reason behind that these organs have a greater organ index than other
texa. Fragments and pellets, on the other hand, were chewed and ejected rather than
swallowed. Injury to the jaws resulted from the eating process, ranging from minor
exfoliation to extensive wounds. The upper and lower jaws, as well as the lower jaw and
liver, respectively, of fish exposed to fragments showed the maximum incidence of
regression and cardiovascular (e.g., dilated sinusoids) abnormalities. These studies
demonstrate that chewing and ingesting MPs injure numerous tissues and digestive organs,
and they also demonstrate that various materials can affect fish in vastly diverse ways.

Barboza et al. (2018) analysed how Acetylcholnesterase (AchE) brain activity significantly
decreased and lipid peroxidation increased in Dicentrarchus labrax following exposure to
MP alone or in conjunction with mercury (LPO). Alarm is growing over the amount of
floating plastic trash in the open ocean. The extent and effects of this contamination are still
unknown, though. Using findings from the Malaspina 2010 circumnavigation, local surveys,
and previously published findings, we depict a widespread distribution of plastic on the open
ocean's surface, mostly gathering in the converging zones of each of the five ocean gyres
with comparable density. On the other hand, it was predicted that the amount of plastic
littering the open ocean's surface would be far lower than expected, likely in the hundred of
thousands of tonns. Our research on the size distribution of floating plastic waste points to the
need for size-selective sinks that can effectively and widely remove floating plastic particles
as small as mm. A combination of fast nano-fragmentation of microplastic into micron-sized
particles, transit to the interior of the ocean via food webs and ballasting mechanisms and as
yet unidentified processes could make up this sink. Finding out what happened to the missing

xx
plastic waste is crucial in identifying the kind and scope of the effects of marine plastic
pollution (Cozar et al., 2014).

Halstead et al. (2018) High amounts of microplastics and fibres are found along metropolitan
beaches, but further research is needed to determine whether fish in these locations are
ingesting microplastics. In this study, the amount of debris consumed by three benthic-
feeding fish species: Mugil cephalus (sea mullet), Acanthopagrus australis (yellowfin
bream) and Gerres subfasciatus (silverbiddy) in Sydney harbour, Australia, has been
quantified. Vibrational spectroscopy has also been used to chemically speciate the debris and
determine the type of polymer. Raman microspectroscopies, Fourier transform infrared
(FTIR) and principal component analysis were used to quantify and identify the ingested
debris (PCA). Ingestion of debris at the time of survey varied between 21 and 64 percent for
the 3 varieties, and the average number of pieces per fish varied between 0.2 and 4.6, with
microplastic making up about 53 percent of the debris. When particle counts were centralised
to fish mass or gut content weight, there was no distinction between species, suggesting that
these organisms consume a comparable low concentration of particles relative to their
absorption rate of other substance. However, there had been a substantial distinction of
fragments ingested among species.72% of the debris was successfully identified using ATR-
FTIR microspectroscopy. An extra 1 percent of successful identification was due to Raman
spectroscopy. Additionally, the ATR-FTIR spectra were objectively classified using PCA,
which allowed for the detection of an extra 9% of the debris. Polyester (PET), acrylic-
polyester blends and rayon (semi-synthetic) fibres were the most often discovered
microplastics. Raman microspectroscopy was explored for its potential to be used for debris
identification and offered further details on the debris's nature and the presence of particular
dyes.

Yin et al. (2018) worked on Sebastes schlegelii to check microplastic effects in term of
histopathology. Polystyrene microplastics were exposed for 14 days. Histopathological
abnormalities in the gallbladder, liver and gut of fish were seen.

Baalkhuyur et al. (2018) sampled 26 fish included both commercial and non commercial
from four different habitats along the Saudi Arabian Red Sea coast to determine the amount
of microplastic litter present in their gastrointestinal tracts. 178 people in all had their bodies
checked for microplastics. A total of 26 pieces of microplastic were discovered. 16 of them
(61.5%) were flicks, and 10 were fishing thread (38.5 percent). Polypropylene and
polyethylene were found to be the most prevalent polymers, according to FTIR analyses. The
species of Parascolosps eriomma sampled in Jazan had the highest concentration of
microplastics in their stomachs. Benthic in nature, this fish species consumes benthic
invertebrates. Even while there were no statistically significant differences in the amount of
microplastics consumed by different species, there was a significant difference between
habitats in the amount of microplastics consumed. The existence of microplastic trash along
the seabed and fish habitats may be associated to the higher quantity of microplastic particles.
The findings of this study provide the first proof that microplastic contamination poses a
growing threat to fish in the Red Sea, their food chain, and human consumers.

xxi
Hussain et al. (2018) stated Labeo rohita has a significant commercial value, but in Chenab
River its population has declined as a result of pollution. Results of Comet assay from three
polluted river sites showed considerable (p.05) fragmentation of DNA in Labeo rohita with
42.21±2.06 percent, 21.84±2.21percent, 31.26±2.41 percent, DNA in comet tail, olive
moments of 8.10± 1.04, 13.58±1.306 and 5.88±0.06 and tail moments 7.81±1.56, 17.71±1.79
and 10.30±1.78, respectively. Single micronucleus induction was found to be 50.00±6.30, a
nuclear abnormality was found to be 150.00±2.92 and double MN was 14.40±2.56 in the
micronucleus assay. In an experimental region of the river Chenab, greater frequencies of NA
and MN induction were shown to be the cause of a ninety six percent decline in the
population of Labeo rohita. Labeo rohita has been discovered near extinction along Chenab
River.

Klein et al., (2018) analyzed that micro plastics deposited in rapidly moving and dynamic
freshwater systems such as rivers may ultimately reach the ocean, a more urgent issue is
when MPs come to rest in static freshwater systems (e.g. lakes or ponds) since they can more
readily collect there

Pannetier et al. (2019) described the work on Comet assay to check the DNA damage in fish.
In alkaline circumstances, the formamidopyrimidine glycosylase (Fpg)-modified comet assay
was done. The use of the Fpg enzyme increased the number of lesions observed, including
oxidised guanine, as well as the comet assay's sensitivity. The percentage of tail DNA (DNA
break (percent), which is the proportion of DNA which has relocated from forefront, was
used to measure DNA damage.

Xu et al. (2019) studied that oceans and the coastlines of all five continents have been
recorded to have MPs (5 mm). Due to the methodological difficulties mentioned above,
standardised methodologies are still lacking despite the fact that there are a rising number of
researches being conducted to detect and measure the presence of MPs in various sample
matrices (such as wastewater and canned fish). The scarcity of ideal approach, which results
in poor result comparability or, worse still, ambiguous conclusions, is being criticised by an
increasing number of academics. A promising method for autonomous MPs analysis is
chemical imaging, such as FTIR and Raman. To speed up identification and improve
accuracy, it is essential to create, test, and improve automated analytic algorithms. The
development of an effective automated pipeline will increase our knowledge of the existence,
persistence, and effects of MPs in the community and develop us to estimate their risk in a
way that is reasonable, comparable and accurate.

Xu et al. (2019) studied that oceans and the coastlines of all five continents have been
recorded to have MPs (5 mm). Due to the methodological difficulties mentioned above,
standardised methodologies are still lacking despite the fact that there are a rising number of
researches being conducted to detect and measure the presence of MPs in various sample
matrices (such as wastewater and canned fish). The scarcity of ideal approach, which results
in poor result comparability or, worse still, ambiguous conclusions, is being criticised by an
increasing number of academics. A promising method for autonomous MPs analysis is
chemical imaging, such as FTIR and Raman. To speed up identification and improve

xxii
accuracy, it is essential to create, test, and improve automated analytic algorithms. The
development of an effective automated pipeline will increase our knowledge of the existence,
persistence, and effects of MPs in the community and develop us to estimate their risk in a
way that is reasonable, comparable, and accurate.

Xu et al. (2019) studied that oceans and the coastlines of all five continents have been
recorded to have MPs (5 mm). Due to the methodological difficulties mentioned above,
standardised methodologies are still lacking despite the fact that there are a rising number of
researches being conducted to detect and measure the presence of MPs in various sample
matrices (such as wastewater and canned fish). The scarcity of ideal approach, which results
in poor result comparability or, worse still, ambiguous conclusions, is being criticised by an
increasing number of academics. A promising method for autonomous MPs analysis is
chemical imaging, such as FTIR and Raman. To speed up identification and improve
accuracy, it is essential to create, test, and improve automated analytic algorithms. The
development of an effective automated pipeline will increase our knowledge of the existence,
persistence, and effects of MPs in the community and develop us to estimate their risk in a
way that is reasonable, comparable, and accurate.

Xu et al. (2019) studied that oceans and the coastlines of all five continents have been
recorded to have MPs (5 mm). Due to the methodological difficulties mentioned above,
standardised methodologies are still lacking despite the fact that there are a rising number of
researches being conducted to detect and measure the presence of MPs in various sample
matrices (such as wastewater and canned fish). The scarcity of ideal approach, which results
in poor result comparability or, worse still, ambiguous conclusions, is being criticised by an
increasing number of academics. A promising method for autonomous MPs analysis is
chemical imaging, such as FTIR and Raman. To speed up identification and improve
accuracy, it is essential to create, test, and improve automated analytic algorithms. The
development of an effective automated pipeline will increase our knowledge of the existence,
persistence, and effects of MPs in the community and develop us to estimate their risk in a
way that is reasonable, comparable, and accurate.

Pozo et al. (2019) investegated and analysed the microplastic particles (MPs) that were
present in six commercially significant fish species in central Chile. The fish species came
from oceanic and coastal settings and are of various trophic levels. A microscope with
Fourier-transform infrared spectroscopy (FTIR) was used to extract, examine, and describe
the fish gastrointestinal content in order to study MPs (FT-IR). The majority of the MPs
discovered in fish samples (70–100%) were made up of red microfibers, which ranged in size
from 176 to 2842 m. The most common polymers found were polyester, polyethylene (PE),
and polyethylene terephthalate (PET). In comparison to marine species (29 percent), coastal
species shows the existence of microbeads with higher concentration (71 percent), indicating
a greater potential exposure. These results agree with those of previous global research that
has been conducted. To accurately determine the potential exposure danger for the general
public devouring these fish and the influence of MPs in Chilean fishery activities, more
research is still required.

xxiii
Lefebvre et al. (2019) studied to characterise and quantify the distribution of microplastics
(MP) in the surrounding water of the NW Mediterranean Sea and even the MP intake by the
two primary planktivorous species in the region, the sardine and the anchovy. In all samples
of the water column and all but two of the fish guts, debris of comparable sizes was
discovered (out of 169). 93% of water columns samples contained MP, with an average
density of 0.23 0.20 MPm3, whereas only 12 percent of sardines and 11 percent of anchovies
contained MP (0.20 0.69 MPind1, respectively). The sole type of MP found was in the form
of fibres and polyethylene terephthalate was perhaps the most prevalent polymer found in
water columns (61 percent), sardines (71 percent) and anchovies (89 percent). This study
provides modest evidence of MP in fish digestive systems while attesting to MP's prevalence
in the Mediterranean Sea.

Lorenz et al. (2019) explained that the global issue of microplastic pollution in the marine
ecosystems is urgent. Therefore, identifying sources and entrance points may be made
possible with the aid of geographical tracking of microplastic concentrations, composition,
and size distribution, which would then enable targeted mitigation to be started. In two
segments of the southern North Sea, the spatial variability features of microplastics were
examined through the collection of surface water and sub littoral sediment samples from 24
stations. Visually detecting and identifying large microplastics (500–5000 m) was
done by Fourier transform infrared (FTIR) spectroscopy. The residual material was digested
enzymatically, condensed onto filters, and examined FTIR imaging for tiny microplastics
(11500 m). In all samples, microplastics were found in amounts between 0.1 and 245.4
particles m3 for surface waters and 2.8 to 1188.8 particles kg1 for sediment. In sediments, 98
percent of microplastics were on average 100 m or smaller, while 86 percent were found in
surface waters. Acrylates, Polypropylene, and polyamide were the three polymer types that
were most frequently found in both compartments. However, there were considerable
differences in the composition of the polymers between samples of sediment and surface
water as well as between sites in the Frisian Islands and the English Channel. These findings
demonstrate that pieces of plastic are not uniformly distributed in terms of size or location,
which has important implications for the design of monitoring procedures.

Qiang and Cheng (2019) studied the impact of microplastics on zebrafish larvae and
embryos, with a focus on how well they can swim. The zebrafish embryos were subjected to
microplastics beginning four hours after fertilisation. Plastic particles initially adhered to the
embryonic chorion before entering the digestive tract of the larvae. Mostly in local swimming
test, zebrafish juveniles exposed to 1000 g/L (or around 1.91 107 particles/L) of microplastics
experienced declines in swimming distance and speed of 3.2% and 3.5%, respectively, when
swimming in the dark. In the alternate light-to-dark photoperiod stimulation experiment,
exposure to microplastics at concentrations of 100 and 1000 g/l (or roughly 1.91 106 and 1.91
107 particles/l) reduced swimming distance by 4.6 percent and 2.6 percent, respectively. At
the molecular level, as well as a 4.9 percent and 2.8 percent reduction in active speed,
possibly due to hindered dark avoidance in handled zebrafish larvae. MPs elevated the
expression of genes linked to oxidative stress (cat) and inflammatory disease (il1b).
According to this study, exposure to microplastics decreases larvae's capacity for swimming,

xxiv
which could have detrimental impacts on population fitness in aquatic environments. MPs
increased the expression of inflammation (il1b) and oxidative stress (cat) associated genes at
the molecular level. This research shows that exposure to microplastics reduces larvae
swimming ability, which could have serious implications for population fitness in the aquatic
environment and other ecological effects.

Baalkhuyuret et al. (2020) identified MPs, albeit at an extremely lower incidence of about 1
fish per 20 fish analysed, in a number of economical and environmentally significant fish
species collected from the Western Arabian Gulf. Each extracted and confirmed particle was
assigned to one of two shape categories, threads (fibres from fishing lines, ropes and nets), or
fragments (thick pieces from broken things), based on the characteristics of microplastic-like
particles that were found in the gastrointestinal tract (Devriese et al., 2015). Polyethylene and
polypropylene were the most prevalent polymers identified in MPs isolated from fish
gastrointestinal tract. Because MPs have the potential to cause acute poisoning in humans,
their presence in fish digestive systems is problematic.

Dane and sisman. (2020) examined the digestive tract of three fresh water fish species i.e.
Squalius cephalus , Alburnus mossulensis and Capoeta capoeta with the help of morpo-
histopathological technique. Between June 2015 and September 2016, sample of sediments
and fish were accumulated from four stations selected in Karasu River. The concentration of
same plastic particles were indicated in sediment and water samples. Histopathological
alteration index (HAI) was used to determine the histopathological changes in the digestive
tracts of these fish. There was calculation of the commonmarkers for introduction to
environmental stress i.e. intestinal coefficient (IC) and condition factor (CF) for each fish.
The site having the least contamination was observes to have highest condition factor (CF).
Among the three species, the highest value of intestinal co-efficient was observed in C.
capoeta. The fish at the polluted site were detected have abnormalities like hyperplasia,
infiltration, fibrosis, swelling, fusion, gastric degeneration, epithelial degenerations,
vacuolization and congestion. There was significant variation in the intestinal coefficient (IC)
and histopathological alterations index (HAI) values from site to the site. It was concluded
that the health condition of the fish species was affected badly due to heavy metals in the
sediments and water of river

Li et al. (2020) explored the microplastic contamination in physical and biological

compartments at three sites, two of which were in the vicinity of an industrial colony
producing plastic and one of which served as a reference site. 0.4-20.5 items/L in
groundwater, 44.4-124.7 items/kg (ww) in sediment, and 1.9-6.1 items/individual in the
gastrointestinal tract of Hemiculter leucisculus from the factories area were the
microplastic extensive. Of comparison, the abundances in the reference site's H. leucisculus
were substantially lower at 0.1 0.1 items/L in surface water, 0.5 0.2 items/kg (ww) in
sediment, and 0.2 0.01 items/individual. The area surrounding the plastic factory, there were
a lot of raw pellets to be found. Pieces (67 percent) and pellets (second-most common shape
of microplastics discovered in sediment) were the most prevalent morphologies (18 percent ).
Surprising, no fish included shards or pellets (greater than 1 mm). In Hemiculter leucisculus,
the liver organ indices included four different types of histopathological alterations, and it

xxv
ranged from 5.5 to 9.9 in the plastic manufacturing area while it was only 1.6 in the reference
site. The histopathological alteration in fish muscle and the results significantly support the
notion that microplastic toxicity was at a high level, as well as the ecological reaction of the
plastic producing area. Our findings further suggest that, despite significant levels of
microplastics present in their natural surroundings, the feeding habits of native fish species
may be to blame for the absence of uncooked pellets or pieces in fish.

Hamed et al. (2020) worked on microplastics exposure resulted in changes in antioxidant

biomarkers catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAC),
DNA damage, which causes a change in the muscle protein profile of tilapia early juveniles,
which could be a cause of fish death. The MPs have a dose-response relationship.

Ahrendt et al. (2020) Compared to control animals, benthic fish subjected to PS micro
particles had a higher frequency of intestinal plastic particle occurrence. This rise was linked
to the existence and degree of intestinal histological damage, demonstrating a dynamic series
of tissue modifications. This supports the assertion of how plastic abrasion affects the
structure of fish intestines, perhaps having an impact on their permeability, functionality, and
immune response. The impact of greater exposure to microplastic, particularly when
contrasted to environmental levels, was demonstrated in the current work. The results, in our
opinion, are pertinent since it is anticipated that there will be an increase in the amount of
microplastic pollution due to the breakdown of drifting macro and mesoplastics as well as the
direct creation and release of microplastics into the environment. Clarifying the effect of
plastic use on marine biota will take more research.

Xia et al. (2020) examined fish's exposure to microplastics and its toxicological effects.
The results of the experiments demonstrated that PVC microplastics hindered carp growth ca
used oxidative damage to the liver, intestine, gills and caused carp mortality.
Histopathological examination demonstrated cytoplasmic vacuolation in the liver. To better
understand and ascertain the effects of PVC microplastic particles on freshwater organisms,
our study gathered fundamental toxicological data.

Tang et al. (2020) worked on the influent, effluent, and sludge MP characteristics of two
wastewater treatment plants (WWTPs) in Wuhan, Central China. The extraction rate of MPs
in both WWTPs' wastewater were 62.7 and 66.1 %, respectively, which can be attributed to
the WWTP's wastewater sources and treatment procedures. Microbeads were identified in
large quantities only in frosted cleansers, but not in toothpaste. Furthermore, the majority of
MPs in domestic wastewater come from fibres in laundry waste. MPs in home wastewater
and WWTPs have similar shapes and basic components, according to our findings.
Furthermore, the major MPs in residential wastewater contributed mostly to the MPs in
WWTP wastewater. Finally, the daily discharge of MPs from home wastewater to WWTPs in
China was estimated to be 9.1 × 1010.

Bihanic et al. (2020) described comet assay. It was used to examine toxicity 13-day-old yolk
sac larvae that had earlier been sedated with Benzocaine 0.1 mg/L. The larvae were squashed
for 20 seconds in a tissue grinder. After 10 minutes of centrifugation at 1000 rpm and at room

xxvi
temp, the essential medium is ready to use. The assay employed was the Trypan blue
exclusion assay to determine cell viability in all samples, and it was consistently above 95%.

Yang et al., (2022) provided an overview of the existing literature on micro plastics (MPs) in
the marine environment to get a better understanding of micro plastics contamination. In
recent years, widespread public awareness has been raised about the severe worldwide micro
plastic contamination problem. Despite the fact that micro plastics are extensively dispersed
in a variety of settings and that their contamination is already pervasive in the ocean system,
there seems to be an exponential increase in public awareness over the last decade and in a
variety of study fields. Tiny size of micro plastics makes easier access in the stomach of
marine creatures and can put adverse effect. Moreover, Micro plastics in the coastal habitats
pose a significant danger to the health of marine creatures, and therefore to human life. So it
has gotten a lot of attention from society and academics. The issue of marine micro plastic
contamination should be taken into account while designing techniques for decomposing
plastics

Uurasjarvi et al. (2021) worked on Perch (Perca fluviatilis) and vendace, two popular wild
freshwater fish species, were investigated for their consumption of microplastic (Coregonus
albula). The development and validation of an imaging Fourier-transform infrared
spectroscopy method for quantifying ingested microplastics was another goal of ours.
Enzymatically digested materials were assessed for this purpose using an infrared microscope
with a focal plane array (FPA). Software called simple was used to evaluate the data and
provide counts, mass calculations, shapes and substances for the observed particles. Ten
procedure blanks and recovery assays were used to validate the method and the results
showed survival rates during pretreatment and infrared imaging was 75% and 77%,
respectively. The main cause of contamination during pretreatment was tiny microplastics
(100 m). The findings revealed that 25% of vendace and 17% of perch had consumed plastic.
Plenty of the fish had little to no plastic in them, but some of them had a lot of little pieces or
a limited number of big ones.

Guimaraes et al. (2021) evaluated DNA damage by using alkaline method in the comet test.
Little amount of Polystyrene nanoplastics (PS NPs) can cause DNA damage in
Ctenopharyngodon idella juveniles. The Blood samples (10 L) were obtained and diluted
1:19 in bovine foetal sera. Diluting 15 litres of cell solution yielded two slides (one for each
sample) in Low melting point (0.5%) agarose was dissolved in lysis solution (Triton X-100,
DMSO, stock lysis solution) at 6 ᴼC for 24 hours while being kept in the dark. Slides were
electrophoresed for 25 minutes at 300 mA and 25 V (0.90 V/cm) in complete darkness.
Results from a single cell gel electrophoresis investigation indicated that C. idella treated to
PS NPs exhibited induced DNA damage, even at low doses (comet assay).

Abarghouei et al. (2021) explained the histopathological effect of microplastic exposure to

goldfish Carassius auratus. A fluorescent microscope (Labo MedLx 400) was used to image
gill, liver and intestinal specimens for 168 hours. In comparison to the control, histological
lesions in the liver, gut and gill were identified in polystyrene-exposed fish. When the dose of

xxvii
exposure rose, the severity of the lesions increased dramatically. The buildup of polystyrene
in tissue samples over time was confirmed by fluorescent microscopy studies.

Bertoldiet et al. (2021) reported the standard data on MPs contamination in Lake Guaba in
southern Brazil. We investigated the quantity, composition and dispersion of microplastics on
the substratum of this freshwater body, and we found relationships between these parameters
and land use, population density, wind speed and geohydrologic processes. A manta net
(mesh size 60 m) was used to gather the samples. Microplastics were identified in all of the
samples, ranging from 11.9 0.6 to 61.2 6.1 items per mᵌ, indicating that the lake is heavily
contaminated with plastic particles. The most prevalent shades were white/transparent and
red, while the particle type was the most prevalent microplastic shape in the range of sizes of
100 to 250 m. Black-colored microplastics are more likely to be miscounted, leading to an
under or overestimation of the total amount of microplastics, according to visual microplastic
counts with measurement errors. The majority of the different types of polymers were
polypropylene and polyethylene (98 percent). 58 percent of the polymers tested were highly
oxidised, according to micro-Fourier transform infrared (micro-FTIR) spectroscopic
examination, demonstrating that the particles had been in the water for a considerable amount
of time. The geohydrological properties of the lake have a significant impact on the spread of
microplastics. This study may give information for further microplastic distribution studies in
Lake Guaba, as well as it serves as a starting point for bettering waste management practises
in order to successfully reduce pollution problems in freshwater ecosystems. Black
microplastics are more susceptible to measurement alterations, according to the measurement
uncertainty, it was also discovered.

Azevedo Santos et al. (2021) stated that all around the world, plastics are major contaminants
in fresh water ecosystem. In seminatural or natural environments, they discovered plastic
ingestion evidence by 206 species of fresh water. In addition, also describe synthetic
polymers effects in freshwater environment such as entanglement of creatures from various
groups. This study has been concluded that the situation in freshwater may be just as harmful
as pollution in the ocean, despite that it is often undervalued.

Nalbone et al. (2021) worked on three types of mussels that were purchased as fresh or
processed food from different Italian stores were examined for the presence of microplastics.
The possible weight of plastic that could be consumed with a piece of mussels was used to
empirically quantify the vulnerability to polycyclic aromatic hydrocarbons (PAHs)
and polychlorinated biphenyls (PCBs). After they were digested with a 30% hydrogen
peroxide solution, a strong saline solution was added to perform a density separation. The
filtered samples were examined under a stereomicroscope and FT-IR spectroscopy was used
to determine the chemical makeup of the items that had been visually sorted. Compared to
processed mussels, which contained significantly less things (0.9 0.10) and significantly more
(0.4 0.19), fresh mussels contained 0.20 0.24 items/g and 0.40 0.47 items/individual. By
using FT-IR, various plastic polymers were discovered, with polyethylene being the most
common one. The use of fresh and processed mussels could expose European shellfish
consumers to up to 585 MP per year and 253 MP per year, respectively. A piece of MP-
contaminated fresh or cooked mussels would have a negligible impact on the amount of

xxviii
PCBs and PAHs that people are exposed to on a daily basis. Store-bought mussels may
contain microplastics from a variety of sources and these sources may be connected to
contamination across the food supply chain.

Feld et al. (2021) studied to examine the incidence of MPs in tap water from 17 various
Danish homes and workplaces. To reduce airborne contamination, tap water samples were
taken using a closed sampling apparatus, and QA/QC tests were run to determine the level of
background contamination. Stereomicroscopy and spectroscopy analysis (-FTIR) were used
to visually examine particles larger than 100 m in order to determine their morphology and
chemical make-up. Hyperspectral image analysis was used to evaluate MP particles as small
as 10 m in water samples from three different locations. The findings point to a low
concentration of Mp in Danish drinking water, with MP particles: Polyethylenen, poly
poylene and poly styrene found in all samples. The most prevalent type of MP-like particles
in drinking water and blanks were microfibers, however their concentration in all samples
was below the level at which they could be detected, making it impossible to distinguish them
from background contamination. A large portion of the particles examined by -FTIR were
found to be cellulose fibres, whereas a lesser portion was found to be protein. The position of
MP in drinking water, difficulties and restrictions with regard to the assessment of MP in
drinking water based on this study.

Da Costa Araujo et al. (2022) explained the idea that after 2 weeks of exposure, mature
zebrafish (Danio rerio) exposed to a mixture of developing contaminants will have
increasingly harmful mutagenic, genotoxic and redox imbalance effects. Although MP
accumulation was higher in animals exposed to PE-MPs alone, the overall picture of the
current study shows that erythrocyte Genotoxicity (comet assay) and the intensity of
erythrocytic nuclear abnormalities (ENAs) in zebrafish exposed to PE-MPs alone were as
severe as those seen in animals exposed to the combination of pollutants (either alone or in
combination with MPs). Furthermore, we found that these effects were linked to an
imbalance between pro and antioxidant biotransformation in animals, whose catalase (CAT)
and superoxide dismutase (SOD) and activity was evaluated in various organs but was
insufficient to manage the generation of reactive oxygen species [nitrogen [nitric oxide (NO)
and hydrogen peroxide (H2O2)]. The principal component factor analysis (PCA) also showed
that although the animal's brain and liver displayed greater antioxidant activity, the animal's
gills and muscles produced the most (H 2O2), indicating that the animal's biochemical reaction
was organ-dependent. As a result, the interaction between PE-MPs and a mixture of toxins in
the zebrafish was not shown to have any antagonistic, mutually reinforcing, or additive
effects on the animals exposed to them in the current study. This supports the idea that the
relationships among both pollutants in marine environments as complicated as their impacts
on freshwater ichthyo fauna.

xxix
CHAPTER 3.

MATERIALS AND METHODS

Phase I:

Sample Collection

Fish samples were collected from river Ravi from Bahadur Shah bridge, Sahiwal road,
Faisalabad, Punjab sampling sites during February, April and June, 2022. The specimen of
each specie was captured alive and brought to laboratory through containers then water was
preserved separately for experiment and the fish was put in freezer for furthur experiment.
Experiment (Determination of toxicity and uptake of microplastics by the fish inhabiting river
ravi, Pakistan) was performed in the Microbiology and Aquatic Toxicology Laboratory in
Department of Zoology, Government College Women University Faisalabad.

Contamination Control

Before starting the experiment, it was necessary to control the possible ways of
contamination. For this purpose, all the glassware that were used in the experiment was
washed with distilled water. The chemicals were filtered by using filter paper. The lab coats
were wore during all the experiment. The treatment before the FTIR was done in laminar
flow cabinet and after FTIR treatment the samples were treated in the fume hood to contol the
sample contamination.

Identification and Characterization of MPs from water

The first step was the collection of river water, which was done through the net with the
attachment of bottle. The water was collected in the water bottle and stained to separate out
too much bigger or extra material from the water.

For that purpose, sieves having different size such as 0.3mm and 5mm used for filteration
[Link] 5mm seives was placed at the top and 0.3mm was placed at the bottom.

The water go through these seives ,the 5mm sieve material was discarded and material was
collected on 0.3mm that was kept separate in a beaker. KOH and magnetic stirr bar was
added into the beaker having filterate. The beaker was placed on the hot plate for 24hours.

After 24hours, the magnetic bar was removed and the solution was filtered. The filterate was
kept in the petri dish and it was analyzed in the microscope.

xxx
 Figure 3. a) seivevs b) magnetic stirr c) water sample

Identification and Characterization of MPs from fish gut

The fish was washed with distilled water before examination. The total length (cm) and
weight (g) was measured for each fish. The length was measured from the tip of the snout to
longer lobe of caudal fin.

Fish was dissected by using the dissecting scissors. The forceps was carefully used to
separate out the gastrointestinal tract (GIT) of fish. To minimize the contamination, the
gastrointestinal tract was placed in 250 ml laboratory beaker and covered with glass lids until
analyed. The gastrointestinal tract of different fish were kept separate to get the accurate
results. Weight and length of gastrointestinal tract (GIT) was measured using weighing
balance and measurement scale respectively.

xxxi
Materials and chemicals required for FTIR Analysis

Microscope, slides, filter paper, beakers, dissection box, Whatsmann No. 1004, 10%
potassium hydroxide (KOH), filtration assembly, distilled water and 70% ethanol were
required for FTIR analysis.

Analysis of fish gut for presence of MPs through FTIR

For this purpose, one individual from control and 1 individuals from each experimental
aquaria (with 2mg, 4mg) were taken. The whole gastrointestinal tract, gills and muscles were
separated. Then the gut of each individual was exposed to three folds of its volume of KOH.
For digestion purpose, the gills were incubated at 40°C for 72 hour, while the gastrointestinal
tract and the muscles were incubated at 60°C for 24 hours. During the incubation period, the
samples were shaken after 1min to digest the organic material completely. Stainless steel
sieve (200µm pore size) was used for filteration of samples. The left over residues were
washed with distilled water and placed in a petri dish. The results were observed under the
light microscope (OMAX 40X- 2000X biological compound microscope) and FTIR (JASCO
6100 system) with Specac Golden Gate MKII Diamond Window ATR that was connected
with the LED.

Figure 3. Fourier Transform infrared spectroscopy (FTIR)

xxxii
PHASE II:

Experimental Conditions

Through FTIR, three types of microplastics were detected from the fish sample collected
from river Ravi. During Phase II, fish [Link] was exposed to three types of plastics viz.
Polyethylene (PE), polypropylene (PP) and polystyrene (PS). For this purpose, [Link]
fingerlings were procurer from fish seed hatchery, Faisalabad. Healthy, disease free
specimens were selected and stocked in glass aquaria. Products containing these
microplastics were detergent bottles, plastic bottle caps and plastic bags etc. The plastic was
chopped into smaller particles of approximately 1–2 mm size with the help of a scalpel.

Fish (n=8) were exposed to each type of three selected MPs at a concentration rate of 2mg
and 4mg, separately in the labortory to determine the toxic effects of microplastic on growth
and antioxidant enzyme activity of fish. To maintain required level of dissolved oxygen each
aquarium was equipped with bubbler/air pump. (Figure )

Fish was fed to safiation on commercial fish feed throughout the experimental period @ of
3% of their body weight. Exposure was continued for one month. The experimental tanks
were cleaned and water quality parameters were dialy monitored by using different probes
before conducting experiment. All the possible measures were taken to minimize any type of
stress to the fish during experiment. Water quality was monitored and maintained to the best
level during [Link] experimental period physio-chemical parameters viz.
temperature, pH, hardness, dissolved oxygen and NH3 were measured daily while growth
responses in terms of weight gain, length increment, SGR and condition factor were
determined weekly. Fish survival rate was also continuously monitored. Fish length and
weight were measured weekly for estimation of its growth responses.

a) b) c) d)

Figure 1. a) pH meter b) NHᴣ kit c) DO meter d) Fish feed

xxxiii
Figure 2: Aquaria containing experimental fish

xxxiv
Comet assay

Solution preparation

Following solutions were made for the test:

Lysis buffer: pH 10

Ingredients per 200ml

0.24g Na2EDTA

29.22g of NaCl

1.6g NaOH

7.44g Tris-HCl

An Ingredient was added in 200ml distilled water, and mixed them. NaOH, about 8g, was
also added to the mixture, and it was left to dissolve for 20 minutes. HCl and concentrated
NaOH were used to keep the pH of the solution constant. Stock of the buffer solution was
stored at room temperature.

Electrophoresis Buffer: pH>13

NaOH (27g)

Na2EDTA (0.8g)

The pH was maintained at 13 by adding EDTA and NaOH and mixing them thoroughly. The
whole volume was then stored at room temperature after being adjusted for the gel box's
capacity.

Neutralization buffer

Tris HCl (pH 7.5)

Tris HCl (12.6g) +Distilled water (180ml) = buffer

In distilled water tris was added and maintained the 7.5 pH of solution by using HCl. Buffer
was kept at room temperature.

Procedure

Sample of fish species were collected from experimental group. The blood sample was taken
from caudal vein of fish by using syringe.

xxxv
Fig 4. Blood samples of fish

Slide preparation:

For the purpose of precoating, grease-free microscopic slides were utilised. By submerging
them in alcohol and wiping them dry with clean tissue, these slides were cleaned.

On frosted slides, 0.2µl fresh blood sample was mixed with low melting point agarose at a
temperature of 37ºC. Using tissue, the back of the slide was cleaned. Low melting point
agarose gel was warmed in a microwave before being cooled in a 37ºC water bath. Phosphate
buffer solution was then added after the supernatant was removed. Then, a cover slip was
placed over the precoated slides with the cell suspension in agarose. 10 minutes incubated for
solidification in a refrigerator.

Unwinding:

After solidification, the slides were immersed for an hour at 4ºC in a lysing solution buffer
made up of 100m M, Na₂EDTA, 10m M Tris HCl, 2.5 M NaCl, 1% Sodium Sarcosinate
(C3H6NNaO₂), 1% Triton X-100, and 10% dimethyl sulphoxide. All slides were placed in
buffer (pH 10, Na₂EDTA 1mM + NaOH 0.3M) for 20 minutes to unwind DNA.

Electrophoresis:

After unwinding the slides were put into electrophoresis tank. This tank was filled with
prepared buffer and run the [Link] was carried out by positioning the
electrodes in various directions and connecting them to an electric instrument. Slides were
positioned for 30 minutes during electrophoresis (20 V and 300 Ma).

Neutralization:

The slides were neutralised with Tris Hydrochloride buffer at 25ºC for two minutes
following electrophoresis.

xxxvi
Staining:

Slides were stained with ethidium bromide solution prior to DNA damage analysis.

Damage Analysis:

Slides were examined by using fluorescent microscope.

Histopathological alteration
Organ collection

Muscle and liver sample were collected from fish by disecting it.

Fixation

Organ tissues (muscle, liver) were fixed with 10% naturally buffered formalin for 2 days.
The volum of fixative agent was 10 times higher than that of tissues. The muscle and liver
were fixed.

Dehydration

Dehydrating the tissues after fixation allowed the water to be removed from them.
Ethyl alcohol was employed to dehydrate the tissues. Tissue deformity would result from the
fast dehydration. Therefore, tissues were not rapidly dehydrated; rather, dehydration was
performed gradually and step-by-step by passing the tissues through a sequence of increasing
ethyl alcohol solution concentrations from 70% to 100% grade. Thus, all of the water is
eliminated and replaced with ethyl alcohol.

Infilteration and embadding process

The tissue was infiltrated using a material that served as a support during sectioning before
being cut into sections. Alcohol was initially occupying all of the space in the tissue during
the infiltration phase as paraffin in the tissue block was equilibrating. The tissue was then
allowed to harden following the infiltration process in a mould contained within a tiny
paraffin cube.

Infiltration

After the final stage of dehydration, 100% alcohol is removed, and melted paraffin is used to
fill the vial to about [Link] gave tissue one hour to settle.

xxxvii
The container labeled for paraffin disposal poured the paraffin into this container.
By using fresh melted paraffin repeated the step1

Embedding

Base portions of two embedding moulds were put in a plastic petri dish. Paraffin from the
second step of infiltration poured into the waste container.
The paraffin allowed solidifying at room temperature.
Sectioning

Sections of tissue were cut after embedding. A cutting tool called a microtome was used to
cut sections. The microtome produced a succession of thin sections with extremely accurate
thickness by cutting across the surface of the paraffin cubes. The goal is to create a ribbon-
like pattern of parts that adhere to one another at their trailing and leading edges. The
thickness of the sections was predetermined to be 0.5µl, which is ideal for light microscope
viewing. On spotless microscope slides, the slices were mounted. The embedded tissue
block's preparation and mounting on the microtome are crucial for proper section cutting.
After trimming the paraffin around the tissue block, it was protected to a holder and poured
onto the microtome.

Clearing and staining

The paraffin was cleared by the clearing process before the stain. Only the tissue adhering
remained on the slides following the cleaning process. By putting the parts that had been
poured through the solvent that melted the paraffin, clearing was accomplished. Hematoxylen
and eosine were used to stain the slides.

Slides analysis

Under a microscope, slides were checked for histopathological changes.

xxxviii
Figure.5. Microtome

xxxix
Water quality parameters

The water quality parameters i.e Temperature, pH, DO and NHᴣ were monitored on weekly
basis. Water was needed to be changed on weekly basis or when the water parameters were
not in normal range.

Temperature

A Mercury thermometer is used for temperature determination. The thermometer was

checked [Link] thermometer was dipped directly in controlled and experimental tank.

After this, the point of mercury within the thermometer is noticed to check the temperature
The reading was shown on screen and noted.

pH

A digital pH meter was used to monitor pH of both controlled and experimental group.

 For this purpose, 50ml of water was taken in a beaker from both systems.
 The sensor of the pH meter is dipped in water.
 The reading was shown on screen and noted.

Dissolved oxygen (DO)

DO was determined by using bante 810 DO meter. Unit of dissolved oxygen was ppm.

 50ml of water was taken in a beaker from both controlled and experimental
group.
 The sensor of the DO meter was dipped in water.
 The reading was shown on screen and noted.

xl
Ammonia (NHᴣ)

For the determination of ammonia, a HANNA medium-range ammonia meter was used. Unit
of ammonia was ppm.

 Firstly, 10ml of distilled water was poured into the tank (given with ammonia
checker kit) and placed in the kit sensor to get zero reading. It is done to check
the reliability of the meter.
 Then 10ml of water sample was poured in another tank (given with ammonia
checker kit).
 Reagents given with the kit were poured into a tank (containing a water
sample). Firstly 4 drops of reagent A. Then 4 drops of reagent B.
 Mixed well and placed in sensor area of kit and timer started.

After the timer was completed, reading was shown on the screen.

xli
CHAPTER. 4

Results and
Discussion

Figure 6. Image of fiber microplastics found in fish guts by using microscope and
FTIR spectroscopy

xlii
Figure 7. FTIR spectra and microscopic view of polypropylene microplastics in fish gut

xliii
Figure 8. Microscopic view of MPs in water

xliv
Figure 8.1: FTIR spectra of Water

Figure 9. Damaged erythrocytes

Fig 9.1: Highly damaged erythrocytes

xlv
Figure 9.2: Undamaged erythrocytes

xlvi
Table 1. DNA damage in peripheral erythrocytes of microplastics exposed fish at 2 mg/l
concentration.

Treatment Replicat Un- Percentage of Damaged Nuclei (%) PDC * GDI

e damaged (II+III+IV)
# Nuclei
(%)
Type I Type II Type III Type
Type 0 IV

1 92.00 4.00 4.00 0.00 0.00 4.00 0.12

Initial 2 92.00 4.00 4.00 0.00 0.00
(control) 4.00 0.12
3 94.00 4.00 2.00 0.00 0.00 2.00 0.08

1 38.00 22.00 20.00 18.00 2.00 40.00 1.24

2 34.00 20.00 22.00 12.00 12.00 46.00 1.48
1st fortnight 3 38.00 22.00 20.00 18.00 2.00 40.00 1.24

1 48.00 36.00 12.00 2.00 2.00 16.00 0.74

2nd fortnight 2 52.00 38.00 10.00 0.00 0.00 10.00 0.58
3 38.00 22.00 20.00 18.00 2.00 40.00 1.24

**GDI = Genetic Damage Index

43
Table 2. Mean DNA damage in peripheral erythrocytes of microplastics exposed fish at 2 mg/l
concentration.

Fortnight Un- Percentage of Damaged Nuclei (%) %age of *Genetic

damaged Damaged Damage
Nuclei Cells Index
(%) (GDI)
(II+III+IV)
Type 0 Type I Type II Type III Type IV

Initial
(control) 92.67± 4.00± 3.33± 0.00± 0.00± 3.33± 0.11± 92.67±

1st fortnight 21.33 20.67 42.00

36.67± ± ± 16.00± 5.33± ± 1.32± 36.67±

2nd fortnight 32.00 14.00 22.00

46.00± ± ± 6.67± 1.33± ± 0.85± 46.00±

*PDC= %age of Damaged Cells

**GDI = Genetic Damage Index

44
Table 3. DNA damage in peripheral erythrocytes of microplastics exposed fish at 4 mg/l
concentration.

Treatment Replicat Un- Percentage of Damaged Nuclei (%) PDC * GDI

e damaged (II+III+IV)
# Nuclei
(%)
Type I Type II Type III Type
Type 0 IV

1 92.00 4.00 4.00 0.00 0.00 4.00 0.12

Initial 2 92.00 4.00 4.00 0.00 0.00
(control) 4.00 0.12
3 94.00 4.00 2.00 0.00 0.00 2.00 0.08

1 38.00 22.00 20.00 18.00 2.00 40.00 1.24

2 34.00 20.00 22.00 12.00 12.00 46.00 1.48
1st fortnight 3 38.00 22.00 20.00 18.00 2.00 40.00 1.24

1 48.00 36.00 12.00 2.00 2.00 16.00 0.74

2nd fortnight 2 52.00 38.00 10.00 0.00 0.00 10.00 0.58
3 38.00 22.00 20.00 18.00 2.00 40.00 1.24

**GDI = Genetic Damage Index

45
Table 2. Mean DNA damage in peripheral erythrocytes of microplastics exposed fish at 4mg/l
concentration.

Fortnight Un- Percentage of Damaged Nuclei (%) %age of *Genetic

damaged Damaged Damage
Nuclei Cells Index
(%) (GDI)
(II+III+IV)
Type 0 Type I Type II Type III Type IV

Initial
(control) 92.67± 4.00± 3.33± 0.00± 0.00± 3.33± 0.11± 92.67±

1st fortnight 21.33 20.67 42.00

36.67± ± ± 16.00± 5.33± ± 1.32± 36.67±

2nd fortnight 32.00 14.00 22.00

46.00± ± ± 6.67± 1.33± ± 0.85± 46.00±

*PDC= %age of Damaged Cells

**GDI = Genetic Damage Index

46
Fig 10. Photomicrography of undamaged (A) and damaged (B) muscle tissue

47
Table 1: Physio-Chemical Parameters of test system used for MPs exposed fish at 2mg
level

Exposure Week Temperature pH DO NHᴣ

conc. (ºC) (ppm) (ppm)

2 mg 1st 22.12± 0.01 7.12±0.02 6.81± 0.02 0.65 ± 0.03

2nd 24.28± 0.02 7.00± 0.01 8.53± 0.02 0.45 ± 0.03

3rd 24.26± 0.02 7.11 ± 0.01 8.42± 0.02 0.99 ± 0.02

4th 26.21± 0.03 7.15± 0.01 8.31± 0.02 0.56 ± 0.03

48
Table 2: Physio-Chemical Parameters of test system used for MPs exposed fish at 4mg
level

Exposure Week Temperature pH DO NHᴣ

conc. (ºC) (ppm) (ppm)
4 mg 1st 22.76± 0.02 7.34± 0.39 6.33 ± 0.58 0.99±0.02
2nd 24.22± 0.03 7.14± 0.02 8. 40 ± 0.02 0.78±0.03
3rd 23.04± 0.02 7.26± 0.02 6.74± 0.69 0.65±0.03

4th 24.12± 0.02 7.18± 0.02 7.01 ± 0.65 0.45±0.03

49
Table 3: Physio-Chemical Parameters of test system used for controlled group.

Week Temperature pH DO NHᴣ

(ºC) (ppm) (ppm)
1st 22.38 ± 0.02 7.10 ± 0.19 6.61 ± 0.69 0.66±0.03
2nd 22.50 ± 0.02 7.06 ± 0.34 7.20 ± 0.33 0.56±0.03
3rd 22.00 ± 0.01 7.14 ± 0.21 7.01 ± 0.65 0.97±0.02
4th 22.42 ± 0.02 7.12 ± 0.20 6.33 ± 0.58 0.96 ±0.02

50
 Discussion

FTIR analysis in fish from river Chenab

In this research study the microplastics found in water and fish gastrointestinal tract was
detected by using Fourier transform infrared spectroscopy (FTIR). It is well known that FTIR
(Fourier transform infrared) spectroscopy is a highly successful method for identifying
microplastics (Mecozzi et al., 2016). FTIR microspectroscopy, which collects data utilising
transmission, reflectance, or micro-ATR sampling modes, has been used in more recent
investigations (Song et al., 2014, Kappler et al., 2016).

Majority of microplastics particles in the form of fibers were found in GI. Polyethylene and
polypropylene were the main type of microplastics consumed by fish. The riverine fish has
the great exposure to the waste materials. Similarly in river Chenab fish, the waste from
domestic as well as industries carried a huge amount of plastics along with other pollutants
and toxic matels. FTIR not only help to identify microplastics but also helped to confirm the
polymer type.

All stages of plastic decomposition have been associated with the release of harmful
chemicals (Lithner et al., 2011). Fish are often accustomed to eating a variety of indigestible
particles, including sand, fish scales, and the shells of smaller invertebrates. As a result, their
guts have evolved to deal with these unwanted and unnecessary particles. There are numerous
studies that describe the toxicological effects of MPs on various fish species (Fonte et al.,
2016; Nadal et al., 2016; Mizraji et al., 2017)

The results showed that polyethylene and polypropylene microplastics and FTIR spectra also
showed its abundance.

Water analysis

Water sample was taken from river Chenab to detect microplastics by using advanced
technique FTIR. In order to detect the very fine microplastic particles, the large amount of
water was utilized. It was about one plastic particle in 1000ml of water. Centrifugation helped
to get the concentrated form of particles.

Comet Assay

51
50 cells were chosen at random from the sample, which consisted of the slides. The mean tail
length and mean tail moment served as the markers for the 400x fluorescent microscope
observation of the cells. It was discovered that fish exposed to microplastics had DNA
damage in their erythrocytes.

DNA Damage in Erythrocytes of microscopically exposed fish

Results showed significantly maximum DNA damage in peripheral erythrocytes of fish

collected from experimental group having microplastics exposure. However, proportion of
damage was variable in the erythrocytes of fish which shows fluctuation in the amount of
microplastics. Type 0 of level damage was maximum in the erythrocytes. While type 3 and
Type 4 level damage was not observed in the erythrocytes of microplastically exposed fish.

Fish has been used as the most imperative bio-indicators in aquatic ecosystems (Boran et al.,
2012).Pollution in water influence not only integrity of DNA structure but also affects its
expression and cause genotoxicity (Yu, 2000). DNA damage is used as an indicator of
pollution level in aquatic species like fish. Due to rapid growth and greater market demand
major carps are most cultivable fish. Comet assay is used for the measurement of damage of
DNA. Comet assay also known as SCGE is a form of micro gel electrophoresis used to
observe damage in [Link] assay has importance over other assay because it can detect
DNA damage at low level (Ali et al., 2014; Bucker et al., 2012).
Histopathological alterations in the Muscles and liver
Histopathological examinations of fish muscles and liver was performed to check the effects
of MPs. It was done by tissue sectioning on microtomy and staining by hematoxylin and
eosin staining method. For histopathological analysis a number of parts of 5micron thick
section were studied and observed underneath the microscope.
Signs of degeneration (cytoplasmic and nuclear degeneration and nuclear vacuolation) and
the focal necrosis in the liver parenchyma of fishes exposed to the industrial effluent. These
alterations have been reported as more acute changes, which are more commonly associated
with the exposure of the fishes to contamination by various metals.

Histopathological examination of muscles of Labeo rohita

Muscle histology of experimental group of L. rohita of control group showed the normal
structure of muscle showed in figure (A) but the muscle of L. rohita polluted group showed
breaking of the fiber muscle. The changes include the vacuolar destruction as well as

52
degenerative changes were also observed that are showed in figure (B), (C) and (D) (Figure
4.1).

Histopathological examination of liver of Labeo rohita

Liver histology of experimental group of L. rohita showed the presence of normal structure
of the liver (control group), while (B), (C), and(D) showed morphological changes due to
pollution including hepatic necrosis (HN), fatty infiltration (FI), hemorrhage vacuolation
(HV), glycogen vacuolation (GV), and congestion (C) (Figure 4.2) .

Liver

The liver of control fish revealed a normal homogeneous mass of hepatocytes with no
abnormalities when examined histopathologically. The sinusoids and central vein were
placed in a methodical order. Fish from a contaminated location had necrotic lesions,
hydrophobic vaculation, and megatocytic heptoses, according to histopathology. The same
observation sited by Hadi and Alwan (2012).

The toxicity of chemical is determined by the histopathology of liver of fish. Pollutants cause
alteration of hepatic tissue in the current study, including nuclear degeneration, vaculation,
cellular edema as well as pyknotic nucleus. Our finding also confirm by Sing and shrivastava
1998. In the fish species exposed to carbaryl, Boran et al 2012 that exposed fish to captan
Mishra and Mohanty 2008 in fish species channa punctatus exposed to chromium hexavalent.

Fatty infilteration, glycogen vaculation, hepatic necrosis, haemorrhage vacoulation and

cellular edema as well as congestion were detected in fish from polluted river. The same
result was given by (Ullah et al., 2018) found that oxidative stress cause biochemical damage
as well as the histological damage in the rohu liver, at an acute dose exposed to malathion.

The present findings are consistent with previous findings of research revealing various
alteration of histopathological in the various species of fish liver induced by the pesticide, the
fish species including Cyprinus carpio (Banaee et al.,2011), Cirrhinus Mrigala (Velmurugan
et al., 2009b), Salmo salar (Glover et al.,2007),Clarias gariepinus, Oreochromis niloticus
(Benli and Ozkul, 2010),Tor putitora (Ullah et al.,2015), etc.

The current findings of changes of histopathological are well supported by the work of (Saini
et al., 2013) found acute histopathological lesions in [Link] liver caught in natural

53
freshwaters of Punjab, including hepatocytes deformation, lympho-nuclear cells as well as
hepatic parenchyma degeneration, all caused by heavy metals.

The current investigation in fish from contaminated region revealed histological changes
including degradation of hepatic parenchyma, melano-macrophage centres and deformation
of hepatocytes, acute necrosis and leukocyte infiltration. This conclusion is consistent with
that of (Kaur et al., 2018), who found degradation of hepatic parenchyma, leukocytes
infiltration, acute necrosis and histological alterations [Link] liver owing to heavy metals
exposure when compared to the reference location.

(Chavan and Muley, 2014) subjected [Link] to the sub-lethal concentrations of Pb acetate
in the laboratory looked for congestion in venules and sinusoids localized necrosis and
cytoplasmic vacoulation in the liver tissue. These results are correlated with the present
study.

The changes in histopathology in the livers of both fish species might be a direct result of
sewage, salt, and fertilizers entering the Ravi river with drainage water, as reported by (Tayel
et al., 2014) who observed similar histology changes in the livers of Mugil species residing in
the lake. After cadmium sulphate exposure, the current study of histopathological changes in
liver of fish supported by (Jalaludeen et al., 2012) discovered numerous prominent changes
in O. mossambicus hepatic tissue, including ducted cells marked proliferation, acute damage,
liver tissue conversion into large vacuoles and sponge mass.

Muscles

To evaluate the impact of microplastics on the fish from experimental group. The
histopathological condition of the muscle was examined. Muscles were histopathologically
examined by tissue sectioning under a microscope and stained with the hematoxylin and
eosin procedure. A few pieces of a 5 micron thick segment were examined and evaluated
under the microscope for histopathological investigation.

Fish exposed to microplastics showed signs of cytoplasmic and nuclear degeneration and
localised necrosis in the muscle parenchyma. These abnormalities, which have been
described as more severe changes, have more frequently been linked to fish exposure to
microplastics.

A histopathological analysis of fish muscles:

54
Fish muscles that had been subjected to microplastics displayed fibre muscle breakdown.
Figure (A) showed the normal muscles while (B) depicted the degenerative alterations of
fish muscles exposed to microplastics.

Muscle tissue

The fish that does not consume microplastics displayed a variety of muscular layers, such as
dermis, myoepithelium, epidermis, and myotomes with evenly spaced muscle bundles,
showing that the fish were not under stress as depicted in the image. On the other hand,
polluted locations' muscle tissue shows severe modifications like edoema, necrosis, and
shortening of the muscular bundle. Muscle bundles were discovered to be elongated as well.
The muscle of the fish is the most delicious part, but it is also the tissue that is in contact with
toxins that are dissolved in the water. According to the findings, accumulation of
microplastics in fish muscle tissue at significantly high concentrations may have caused
alterations in the tissue.

Microplastics were found in the stomachs of almost 37% of the fish tested from 504 fish
evaluated from the English Channel (Lusher et al., 2013).Microplastics in fish have the
potential to change histological function and to obstruct the gut (Jovanovic. 2017). The very
small microplastic burden may be absorbed by the intestinal lining as it travels through the
digestive tract, after which it reaches the bloodstream, translocates, and poses a threat to life.
In lab tests, zebrafish (Danio rerio) gills (5 and 20 m), stomach (5 and 20 m), and liver (5 m)
were found to accumulate microplastic in several interior organs. Additionally, oxidative
stress, inflammatory reactions, and fat buildup in the liver were discovered by histological
investigation (Scherer et al., 2018).

55
Physio-chemical parameters of water

The experiment was designed between two groups i.e controlled and experiment group. The
fish sample (n=8) were kept in 3 different tanks. The controlled group fish was given the
normal feed. In order to determine the effect of microplastics on fish the experimental group
was fed along with microplastics. The source of microplastics was water bottles and bottles
cap. They were crushed and given the experimental group with 2mg and 4mg concentration.

CHAPTER 5.

Summery

A broad range of microplastics from physical sources contribute to water contamination.

Due to intense human activities, pollution level has greatly increased MPs in aquatic
ecosystems which exert extremly toxic effects on aquatic fauna espacialy fish. Industrial and
agriculture effluents contaminate the water bodies which in turn affect the fish health both
directly and indirectly. In addition to organic residues there are large amount of plastics and
their mixtures present in municipal and industrial wastewaters. Fish are mostly used for the
determination of quality of aquatic environment and has importance as a bioindicators of
environmental contamination. The microplastics are given to the experimental fish along
with the feed . That is why water quality should be checked regularly to keep the water clean
and in good condition. Any change in water cab cause fish stress, stop growth or even cause
death. Water in good condition means that all quality parameters of water for example pH,
temperature, dissolved oxygen should be in an acceptable range. Various factors including
water temperature, dissolved oxygen (DO), ammonia (NH 3) and pH. FTIR spectroscopy is
applied to detect MPs in water and fish sample taken from river Chenab. Fish gut was used
to examine MPs particles. Comet assay also called single cell gel electrophoresis used to
observe damage in DNA. Histopathological alteration is the best indicator for the assessment
of environmental stress. Histopathological evaluation is a common and sensitive technique
used for the assessment of toxicity caused by MPs to the fish.

The experiment ‘’Toxicity of microplastics and their uptake by the fish collected from river
Chenab, Pakistan ” was performed in Microbiology and Aquatic Toxicology laboratory in
Department of Zoology, Government College Women University Faisalabad. Fish and water
sample were taken from Ravi Chenab for the detection of microplastics experiment. Fish (n-
56
8) were kept in two different tanks labelled as controlled and experimental group. Constant
air was provided to the aquarium by using air pump.

The water and fish sample from river Chenab were analysed to detect MPs by FTIR. The fish
gut was removed and digested with 10% KOH for 24 hours. Solution was filtered, observed
under microscope and FTIR spectra of polypropylene and polyethylene was obtained.

For Comet assay blood sample was taken from fish caudal vein. Blood sample was coated on
the frosted slides after mixing with agarose. Slides were rinsed with lysing solution then
placed in unwinding solution. After electrophoresis, these sides were neutralized and then
stained with ethidium bromide. slides observed under fluorescent microscope.
For histopathological alteration muscle Organs was fixed with 10% buffered natural
formaline solution for 48 hours. Alcohol was used for dehydration with a series of ascending
concentrations from 70 to 100% grade. Xylene was used for cleared purpose. Then tissue was
allowed to solidify in a mold embedded within paraffin small cube. Tissues were cut in to
section by using microtome. The slides were stained by using eosine and hematoxylene.

Following conclusions were made for the above mentioned experiment.

 Polypropylene and polyethylene were found in fish gut.
 Polypropylene in the form of fibers was analysed in water .
 Results showed significantly maximum DNA damage in peripheral erythrocytes of fish
collected from experimental group while there is no DNA damage observed from controlled
group.
 No changes were observed in the fish muscles from control group.
 The muscles tissues of control group revealed different layers including
myoepithelium , normal myotomes and epidermis with evenly muscle bundle spaced
indicating that the fish was not stressed.
 The muscles tissue of experimental fish showed significant alteration such as edema
and necrosis.

57
REFERENCES:

Abarghouei, S., A. Hedayati, M. Raeisi, B. S. Hadavand, H. Rezaei and A. Abed-Elmdoust.

2021. Size-dependent effects of microplastic on uptake, immune system, related gene
expression and histopathology of goldfish (Carassius auratus). Chemosphere, 276:
129-977.

Ahrendt, C., D. J. Perez-Venegas, M. Urbina, C. Gonzalez, P. Echeveste, M. Aldana and

C. Galban-Malagon. 2020. Microplastic ingestion cause intestinal lesions in the
intertidal fish Girella laevifrons. Mar. Pollut. Bull., 151: 110-795.

Ali, A. S., S. A. US and R. Ahmad. 2014. Effect of different heavy metal pollution on
fish. Res. J. Chem. Env. Sci., 2(1): 74-79.

Alimba, C. G. and C. Faggi. 2019. Microplastics in the marine environment: current trends in
environmental pollution and mechanisms of toxicological
profile. Environ. Toxicol. Pharmacol., 68: 61-74.

Andrady, A. L. 2011. Microplastics in the marine environment. [Link]. Bull., 62 (8):

1596–1605.

Andrady, A. L. and M. A. Neal. 2009. Applications and societal benefits of plastics.

philosophical transactions of the Royal Society. B. Biol. Sci., 364 (1526) : 1977-1984.

Avio, C. G., S. Gorbi and F. Regoli. 2015. Experimental development of a new protocol for
extraction and characterization of microplastics in fish tissues: first observations in
commercial species from Adriatic Sea. Mar. Environ. Res., 111: 18-26.

Azevedo-Santos, V. M., M.F. Brito, P.S. Manoel, J.F. Perroca, J.L. Rodrigues-Filho, L.R.
Paschoal and F.M. Pelicice. 2021. Plastic pollution: A focus on freshwater
biodiversity. Ambio., 1-12.

58
Baldwin A. K, S.R. Corsi and Mason. 2016. Plastic Debris in 29 Great Lakes Tributaries:
Relations to Watershed Attributes and Hydrology. Environmental Science &
Technology, 50(19), 10377–10385.

Baalkhuyur, F. M., E. J. A. B. Dohaish, M. E. Elhalwagy, N. M. Alikunhi, A. M.

AlSuwailem, A. Rostad and C. M. Duarte. 2018. Microplastic in the gastrointestinal
tract of fishes along the Saudi Arabian Red Sea coast. Mar. Pollut. Bull., 131: 407-
415.

Baalkhuyur, F. M., E. J. A. B. Dohaish, M. E. Elhalwagy, N. M. Alikunhi, A. M.

AlSuwailem, A. Rostad and C. M. Duarte. 2018. Microplastic in the gastrointestinal
tract of fishes along the Saudi Arabian Red Sea coast. Mar. Pollut. Bull., 131: 407-
415.

Baalkhuyur, F. M., M. A. Qurban, P. Panickan and C. M. Duarte. 2020. Microplastics in

fishes of commercial and ecological importance from the Western Arabian
Gulf. . Mar. Pollut. Bull., 152: 110-920.

Bakir, A., S. J. Rowland and R. C. Thompson. 2012. Competitive sorption of persistent

organic pollutants onto microplastics in the marine environment. Mar. Pollut. Bull.,
64: 2782–2789.

Baldwin, A. K., S. R. Corsi and S. A. Mason. 2016. Plastic debris in 29 Great Lakes
tributaries: relations to watershed attributes and hydrology. [Link]
technol., 50(19): 10377-10385.

Barboza, L. G. A., L. R. Vieira, V. Branco, N. Figueiredo, F. Carvalho, C. Carvalho and L.

Guilhermino. 2018. Microplastics cause neurotoxicity, oxidative damage and energy-
related changes and interact with the bioaccumulation of mercury in the European
seabass, Dicentrarchus labrax (Linnaeus, 1758). Aquat. Toxicol. 195: 49-57.

Barnes, D. K., F. Galgani, R. C. Thompson, M. Barlaz. 2009. Accumulation and

fragmentation of plastic debris in global environments. Philosophical Transactions of
the Royal Society of London. Mar. Biol. Technol., 364(1526): 1985–1998..

Bellas, J., J. Martinez-Armental, A. Martinez-Camara, V. Besada, C. Martinez-Gomez.

2016. Ingestion of microplastics by demersal fish from the Spanish Atlantic and
Mediterranean coasts. Mar. Pollut. Bull., 109(1): 55-60.

59
Bellasi, A., G. Binda, A. Pozzi, S. Galafassi, P. Volta and R. Bettinetti. 2020. Microplastic
contamination in freshwater environments: A review, focusing on interactions with
sediments and benthic organisms. Env., 7(4): 1-30.

Bertoldi, C., L. Z. Lara, A. D. L. Fernanda, F. C. Martins, M. A. Battisti, R. Hinrichs, A.

N. Fernandes. 2021. First evidence of microplastic contamination in the freshwater
of Lake Guaíba, Porto Alegre, Brazil. Sci. Total Environ., 759: 143-503.

Blessing, E. M., M. M. Steenkamp, J. Manzanares and C. [Link]. 2015. Cannabidiol as a

potential treatment for anxiety disorders. Expert Rev. Neurother., 12(4): 825-836.

Boran, H., E. Capkin, I. Altinok and E. Terzi. 2012. Assessment of acute toxicity and
histopathology of the fungicide captan in rainbow trout. . Exp. Toxicol. Pathol., 64(3):
175-179

Browne, M. A., A. Dissanayake, T. S. Galloway, D. M. Lowe and R. M. Thompson. 2008.

Ingested microscopic plastic translocates to the circulatory system of the mussel,
Mytilus edulis (L.). Environ. Sci. Technol., 42: 5026–5031

Browne, M. A., S. J. Niven, T. S. Galloway, S. J. Rowland and R. C. Thompson. 2013.

Microplastic moves pollutants and additives to worms, reducing functions linked to
health and biodiversity. Curr. Biol., 23: 2388–2392.

Bucker, A., M. Carvalho, M. Conceicaoand J. Alves-Gomes. 2012. Micronucleus test and

comet assay in erythrocytes of the Amazonian electric fish
Apteronotusbonapartiiexposed to benzene. Ecotoxicol. Environ. Contam.,7(1). 341-
412.

C.B. Crawford and B. Quinn. 2017. Impacts of microplastics contamination. Biol., 22: 241-
286.

Canniff, P. M. and T. C. Hoang. 2018. Microplastic ingestion by Daphnia magna and its
enhancement on algal growth. Sci. Total Environ., 633: 500-507.

Carr, S. A., J. Liu and A. [Link]. 2016. Transport and fate of microplastic particles in
wastewater treatment plants. Water Res., 91: 174-182.

60
Chen, P., A. Nirula, B. Heller, R. L. Gottlieb, J. Boscia, J. Morris and D. M. Skovronsky.
2021. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with Covid-
19. NEJM. 384(3): 229-237.

Chen, W., R. Zheng, P. [Link], S. Zhang, H. Zeng, F. Bray and J. He. 2015. Cancer
statistics in China. CA, 66 (2): 115-132.

Choi, J. S., Y. J. Jung, N. H. Hong, S. H. Hong and J. [Link]. 2018. Toxicological

effects of irregularly shaped and spherical microplastics in a marine teleost, the
sheepshead minnow (Cyprinodon variegatus). Mar. pollut. Bull., 129 (1): 231-240.

Cincinelli, A., C. Scopetani, D. Chelazzi, E. Lombardini, T. Martellini, A. Katsoyiannis

and [Link]. 2017. Microplastic in the surface waters of the Ross Sea (Antarctica):
occurrence, distribution and characterization by FTIR. Chemo., 175: 391-400.

Claessens, M., L. Van Cauwenberghe, M. B. Vandegehuchte and C. R. Janssen. 2013.

New techniques for the detection of microplastics in sediments and field collected
organisms. [Link]. Bull., 70 (1-2): 227-233.

Cok, I., O. K. Ulutas , O. Okusluk, E. Durmaz and N. Demir. 2011. Evaluation of DNA
damage in common carp (Cyprinus carpio ) by comet assay for determination of
possible pollution in lake Morgan (Ankara). Sci. world J., 11: 1455-1461.

Cole, M., P. Lindeque, P. Halsband and T. S. Galloway. 2011. Microplastics as contaminants

in the marine environment: a review. Mar. Pollut. Bull., 62(12): 2588-2597.

Cole, M., [Link], E. Fileman, C. Halsband, R. Goodhead, J. Moger and T. S.

Galloway. 2013. Microplastic ingestion by zooplankton. Environ. Sci. Technol.,
47(12): 6646-6655.

Cozar, A., F. Echevarria, J. I. Gonzalez-Gordillo, X. Irigoien, B. Ubeda, S. Hernandez-

Leon and C. M. Duarte. 2014. Plastic debris in the open ocean. PNAS., 111(28):
10239-10244.

Da Costa Araujo, A. P., T. M. da Luz, T. L. Rocha, M. A. I. Ahmed, D. D. M. Silva, M.

M. Rahman and G. Malafaia. 2022. Toxicity evaluation of the combination of
emerging pollutants with polyethylene microplastics in zebrafish: Perspective study of
genotoxicity, mutagenicity, and redox unbalance. J. Hazard. Mater., 432: 128-691.

61
Dai, Z., H. Zhang, Q. Zhou, Y. Tian, T. Chen, C. Tu and Y. Luo. 2018. Occurrence of
microplastics in the water column and sediment in an inland sea affected by intensive
anthropogenic activities. Environ. Pollut., 242: 1557-156.

Dias Dane, H. and T. Şi̇ şman. 2020. A morpho-histopathological study in the digestive tract
of three fish species influenced with heavy metal pollution. Chemosphere, 242, 125-
212.

De Sa, L. C., L. G. Luis and L. Guilhermino. 2015. Effects of microplastics on juveniles of

the common goby (Pomatoschistus microps): confusion with prey, reduction of the
predatory performance and efficiency, and possible influence of developmental
conditions. Environ. pollut., 196: 359-362.

Dehaut, A., L. Hermabessiere and G. Duflos. 2019. Current frontiers and recommendations
for the study of microplastics in seafood. Trends Analyt Chem., 116: 346-359.

Devriese, L. I., M. D. Van der Meulen, T. Maes, K. kaert, I. Paul-Pont, L. Frere and A. D.
Vethaak. 2015. Microplastic contamination in brown shrimp (Crangon crangon,
Linnaeus 1758) from coastal waters of the Southern North Sea and Channel
area. Mar. Pollut. Bull., 98(1-2): 179-187.

Duis, K. and A. Coors. 2016. Microplastics in the aquatic and terrestrial environment:
sources (with a specific focus on personal care products), fate and effects. Environ.
Sci. Eur., 28(1): 1-25.

Eckert, E. M., A. Di Cesare, M. T. Kettner, M. Arias-Andres, D. Fontaneto, H. [Link]

and G. Corno. 2018. Microplastics increase impact of treated wastewater on
freshwater microbial community. Environ. Pollut., 234: 495-502.

Eerkes-Medrano, D., R. C. Thompson and D. C. Aldridge. 2015. Microplastics in freshwater

systems: a review of the emerging threats, identification of knowledge gaps and
prioritisation of research needs. Water Res., 75: 63-82.

El-Moghazy and El-Morsy. 2017. Food production and water conservation in a recirculating
aquaponic system in Saudi Arabia at different ratios of fish feed to plants. J. of the
world aquacul. society, 39(4): 5

62
Eqani, S. A. M. A. S., R. N. Malik, A. Katsoyiannis, G. Zhang, P. Chakraborty, A.
Mohammad and K. C. Jones. 2012. Distribution and risk assessment of
organochlorine contaminants in surface water from River Chenab,
Pakistan. Environ. Monit. Assess., 14(6): 1645-1654.

Eriksen, M., L. C. Lebreton, H. S. Carson, M. Thiel, C. J. Moore, J. C. Borerro and J.

Reisser. 2014. Plastic pollution in the world's oceans: more than 5 trillion plastic
pieces weighing over 250,000 tons afloat at sea. PloS on ., 9(12): 111-913.

Eriksen, M., N. Maximenko, M. Thiel, A. Cummins, G. Lattin, S. Wilsonand S. Rifman.

2013. Plastic pollution in the South Pacific subtropical gyre. Mar. Pollut. Bull., 68 (1-
2): 71-76.

Fadare, O. O. and E. D. Okoffo. 2020. Covid-19 face masks: A potential source of

microplastic fibers in the environment. Sci. Total Environ.,737: 140-279.

Feld, L., V. H. D. Silva, F. Murphy, N. B. Hartmann and [Link].2021. A Study of

Microplastic Particles in Danish Tap Water. Water., 13(15): 20-97.

Fendall, L. S. and M. A. Sewell. 2009. Contributing to marine pollution by washing your

face: microplastics in facial cleansers. [Link]. Bull., 58(8): 1225-1228.

Fernandino, G., C. I. Elliff, I. R. Silva and A. C. Bittencourt. 2015. How many pellets are
too many? The pellet pollution index as a tool to assess beach pollution by plastic
resin pellets in Salvador, Bahia, Brazil. Revista de Gestao Costeira
Integrada.J. Integr. Coast. Zone Manag., 15(3): 325-332.

Fok, L. and P. K. Cheung. 2015. Hong Kong at the Pearl River Estuary: A hotspot of
microplastic pollution Mar. Pollut. Bull., 99 (1-2): 112-118.

Fonte, E., P. Ferreira and L. Guilhermino. 2016. Temperature rise and microplastics interact
with the toxicity of the antibiotic cefalexin to juveniles of the common goby
(Pomatoschistus microps): post-exposure predatory behaviour, acetylcholinesterase
activity and lipid peroxidation. [Link]., 180: 173-185.

Gregory, M. R. 1983. Virgin plastic granules on some beaches of Eastern Canada and
Bermuda. Mar. Environ. Res., 10 (2): 73–92.

63
Guerrera, M. C., M. Aragona, C. Porcino, F. Fazio, [Link], M. Levanti and A. Germand.
2021. Micro and nano plastics distribution in fish as model organisms:
histopathology, blood response and bioaccumulation in different organs. Appl.
Sci., 11(13): 57-68.

Guimaraes, A. T. B., F.N. Estrela, P. S. Pereira, J. [Link] Andrade Vieira, A. S. de Lima

Rodrigues, F. G. Silva and G. Malafaia. 2021. Toxicity of polystyrene nanoplastics
in Ctenopharyngodon idella juveniles: a genotoxic, mutagenic and cytotoxic
perspectiv. Sci. Total Environ., 752: 141-937.

Guzzetti, E., A. Sureda, S. Tejada and C. Faggio. 2018. Microplastic in marine organism:
Environmental and toxicological effects. Environ. Toxicol. Pharmacol., 64: 164-171.

Halstead, J. E., J. A. Smith, E. A. Carter, P. A. Lay, E. L. Johnston. 2018. Assessment tools

for microplastics and natural fibres ingested by fish in an urbanised estuary. Environ.
Pollut., 234: 552-561.

Halstead, J. E., J. A. Smith, E. A. Carter, P. A. Lay, E. L. Johnston. 2018. Assessment tools

for microplastics and natural fibres ingested by fish in an urbanised estuary. Environ.
Pollut., 234: 552-561.

Harrison, J. P., M. Sapp, M. Schratzberger and A. M. Osborn. 2011. Interactions between

microorganisms and marine microplastics: A call for research. J. Mar. Sci. Technol.,
45(2): 12–20.

Hermsen, E., R. Pompe, E. Besseling and A. [Link]. 2017. Detection of low numbers
of microplastics in North Sea fish using strict quality assurance criteria. [Link].
Bull., 122(1-2): 253-258.

Hermsen, E., R. Pompe, E. Besseling and A. [Link]. 2017. Detection of low numbers
of microplastics in North Sea fish using strict quality assurance criteria. [Link].
Bull., 122(1-2): 253-258.

Hidalgo-Ruz, V., L. Gutow, R. C. Thompson, M. Thiel. 2012. Microplastics in the

marine environment: A review of the methods used for identification and
quantification. Environ. Sci. Technol., 46 (6): 3060–3075

64
Holmes, L. A., A. Turner and R. C. Thompson. 2012. Adsorption of trace metals to plastic
resin pellets in the marine environment. Environ. Pollut., 160: 42–48.

Horton, A. A., A. Walton, D. J. Spurgeon, E. Lahive and C. Svendsen. 2017. Microplastics

in freshwater and terrestrial environments: evaluating the current understanding to
identify the knowledge gaps and future research priorities. Sci. Total Environ., 586:
127-141.

Hussain, B., T. Sultana S. Sultana, M. S. Masoud, Z. Ahmed and S. Mahboob. 2018. Fish
eco-genotoxicology: Comet and micronucleus assay in fish erythrocytes as in situ
biomarker of freshwater pollution. Saudi. J. Biol. Sci., 25(2): 393-398.

Hussain, Z., M. Arslan, M. H. Malik, M. Mohsin, [Link] and M. Afzal. 2018. Integrated
perspectives on the use of bacterial endophytes in horizontal flow constructed
wetlands for the treatment of liquid textile effluent: phytoremediation advances in the
field. J. Environ. Manage., 224: 387-395.

Ivleva, N. P., A. C. Wiesheu and R. Niessner. 2017. Microplastic in aquatic

ecosystems. Angew. Chem. Int. Ed., 56(7): 1720-1739.

Jabeen, K., B. Li, Q. Chen, L. Su, C. Wu, H. Hollert and H. Shi. 2018. Effects of virgin
microplastics on goldfish (Carassius auratus). Chemo., 213: 323-332.

Jambeck, J. R., R. Geyer, C. Wilcox, T. R. Siegler, M. Perryman, [Link] and K. L.

Law.2015. Plastic waste inputs from land into the ocean. Sci., 347 (6223): 768-771.

Jambeck, J. R., R. Geyer, C. Wilcox, T. R. Siegler, M. Perryman, A. Andrady and K. L.

Law. 2015. Plastic waste inputs from land into the ocean. Sci., 347(6223): 768-771.

Jovanovic, B. [Link] of microplastics by fish and its potential consequences from a

physical [Link]., 13(3): 510-515.

Kaposi, K. L., B. Mos, B. P. Kelaher and S. A. Dworjanyn. 2014. Ingestion of microplastic

has limited impact on a marine larva. Environ. Sci. Technol., 48(3): 1638-1645.

Kappler, A., D. Fischer, S. Oberbeckmann, G. Schernewski, M. Labrenz, K. J. Eichhorn

and B. Voit. 2016. Analysis of environmental microplastics by vibrational
microspectroscopy: FTIR, Raman or both?. Anal. Bioanal. Chem., 408(29): 8377-
8391.

65
Karlsson, T. M., L. Arneborg, G. Brostrom, B. C. Almroth, L. Gipperth and M. Hassellov.
2018. The unaccountability case of plastic pellet pollution. Mar. Pollut. Bull., 129(1):
52-60.

Kemp, P., D. Sear, A. Collins, P. Naden and I. Jones. 2011. The impacts of fine sediment on
riverine fish. [Link]., 25(11): 1800-1821.

Klein S, I.K. Dimzon, J. Eubeler and [Link]. 2018. Analysis, Occurrence, and
Degradation of Microplastics in the Aqueous Environment. In M. Wagner & S.
Lambert (Eds.), Freshwater Microplastics: Emerging Environmental Contaminants?
Springer International Publishing., 31: 61-74.

Latif-Hernandez, A., D, Shah, T. Ahmed, A. C. Lo, Z. Callaerts-Vegh, A. Van der Linden

and R. D’Hooge. 2016. Quinolinic acid injection in mouse medial prefrontal cortex
affects reversal learning abilities, cortical connectivity and hippocampal synaptic
plasticity. Sci. Rep., 6(1): 1-14.

Le Bihanic, F., C. Clérandeau, B. Cormier, J. C. Crebassa, S. H. Keiter, R. Beiras and J.

Cachot. 2020. Organic contaminants sorbed to microplastics affect marine medaka
fish early life stages development. Mar. Pollut. Bull., 154: 11-1059.

Lebreton, L., J. Van Der Zwet, J. W. Damsteeg, B. Slat, A. Andrady and J. Reisser. 2017.
River plastic emissions to the world’s oceans. Nat. Commun., 8 (1): 1-10.

Lebreton L , B. Slat, F. Ferrari, B. Sainte-Rose, J. Aitken, R. Marthouse, S. Hajbane, S.

Cunsolo, A. Schwarz, A. Levivier, K. Noble, P. Debeljak, H. Maral, R. Schoeneich-
Argent, R.R. Brambini and J. Reisser. 2018. Evidence that the Great Pacific Garbage
Patch is rapidly accumulating plastic. Scientific Reports, 8(1), 1–15.

Lechner, M., M. Johannes, A. Kuppert and M. Merklein. 2014. Influence of pre-straining

and heat treatment on the yield surface of precipitation hardenable aluminum
alloys. Phys. Procedia., 56: 1400-1409.

Lefebvre, C., C. Saraux, O. Heitz, A. Nowaczyk and D. Bonnet. 2019. Microplastics FTIR
characterisation and distribution in the water column and digestive tracts of small
pelagic fish in the Gulf of Lions. Mar. Pollut. Bull., 142: 510-519.

66
Lefebvre, C., C. Saraux, O. Heitz, A. Nowaczyk and D. Bonnet. 2019. Microplastics FTIR
characterisation and distribution in the water column and digestive tracts of small
pelagic fish in the Gulf of Lions. Mar. Pollut. Bull., 142: 510-519.

Li, B., L. Su, H. Zhang, H. Deng, Q. Chen H. Shi. 2020. Microplastics in fishes and their
living environments surrounding a plastic production area. Sci. Total Environ., 727:
138-662.

Li, B., L. Su, H. Zhang, H. Deng, Q. Chen H. Shi. 2020. Microplastics in fishes and their
living environments surrounding a plastic production area. Sci. Total Environ., 727:
138-662.

Li, C., R. Busquets and L. C. Campos. 2020. Assessment of microplastics in freshwater

systems: A review. Sci. Total Environ., 707: 135-578.

Lithner, D., A. Larsson and G. Dave. 2011. Environmental and health hazard ranking and
assessment of plastic polymers based on chemical composition. Sci. Total Environ.,
409(18): 3309-3324.

Lorenz, C., L. Roscher, M. S. Meyer, L. Hildebrandt, J. Prume, M. G. Loder and G.

Gerdts. 2019. Spatial distribution of microplastics in sediments and surface waters of
the southern North Sea. Environ. Pollut., 252: 1719-1729.

Lu, Z., Q. C. Zhang, B. Lee, R. A. Flynn, M. A. Smith, J. T. Robinson and H. Y. Chang.

2016. RNA duplex map in living cells reveals higher-order transcriptome
structure. Cell, 165(5): 1267-1279.

Lusher, A. L., M. Mchugh and R. C. Thompson. 2013. Occurrence of microplastics in the

gastrointestinal tract of pelagic and demersal fish from the English
Channel. [Link]. Bull., 67(1-2): 94-99.

Lusher, A. L., M. Mchugh and R. C. Thompson. 2013. Occurrence of microplastics in the

gastrointestinal tract of pelagic and demersal fish from the English
Channel. [Link]. Bull., 67(1-2): 94-99.

Lusher, A. L., C. O'Donnell, R. Officer and I. O'Connor. 2016. Microplastic interactions

with North Atlantic mesopelagic fish. ICES Int. J. Mar. Sci., 73(4): 1214-1225.

67
Lusher, A. L., M. Mchugh and R. C. Thompson. 2013. Occurrence of microplastics in the
gastrointestinal tract of pelagic and demersal fish from the English Channel. Mar.
Pollut. Bull., 67(1-2): 94-99.

Lusher, A. L., M. Mchugh and R. C. Thompson. 2013. Occurrence of microplastics in the

gastrointestinal tract of pelagic and demersal fish from the English Channel. Mar.
Pollut. Bull., 67(1-2): 94-99.

Lusher, A. L., N. A. Welden, P. Sobral and M. Cole. 2020. Sampling, isolating and
identifying microplastics ingested by fish and invertebrates. Analysis of Nanoplastics
and Microplastics in Food, 119-148.

Mato, Y., T. Isobe, H. Takada, H. Kanehiro, C. Ohtake, T. Kaminuma. 2001. Plastic

resin pellets as a transport medium for toxic chemicals in the marine environment.
Environ. Sci. Technol., 35: 318–324.

Mecozzi, M., M. Pietroletti and Y. B. Monakhova. 2016. FTIR spectroscopy supported by

statistical techniques for the structural characterization of plastic debris in the marine
environment: application to monitoring studies. Mar. Pollut. Bull., 106(1-2): 155-161.

Mizraji, R., C. Ahrendt, D. Perez-Venegas, J. Vargas, J. Pulgar, M. Aldana and C. Galban-

Malagon. 2017. Is the feeding type related with the content of microplastics in
intertidal fish gut?. Mar. Pollut. Bull., 116(1-2): 498-500.

Moore, C. J. 2008. Synthetic polymers in the marine environment: a rapidly increasing, long-
term threat. Environ. Res., 108(2): 131-139.

Moret-Ferguson, S., K. L. Law, G. Proskurowski, E. K. Murphy, E. E. Peacock C. M.

Reddy. 2010. The size, mass, and composition of plastic debris in the Western North
Atlantic Ocean. [Link]. Bull., 60 (10): 1873–1878.

Moret-Ferguson, S., K. L. Law, G. Proskurowski, E. K. Murphy, E. E. Peacock C. M.

Reddy. 2010. The size, mass, and composition of plastic debris in the Western North
Atlantic Ocean. [Link]. Bull., 60 (10): 1873–1878.

Morritt, D., P. V. Stefanoudis, D. Pearce, O. A. Crimmen and P. F. Clark. 2014. Plastic in

the Thames: a river runs through it. Mar. Pollut. Bull., 78(1-2): 196-200.

68
Murphy, E. and E. A. King, E. A. 2016. Smartphone-based noise mapping: Integrating sound
level meter app data into the strategic noise mapping process. Sci. Total Environ. 562:
852-859.

Nadal, M. A., C. Alomar and S. Deudero. 2016. High levels of microplastic ingestion by the
semipelagic fish bogue Boops boops (L.) around the Balearic Islands. Environ.
Pollut., 214: 517-523.

Nalbone, L., F. Cincotta, F. Giarratana, G. Ziino and A. Panebianco. 2021. Microplastics in

fresh and processed mussels sampled from fish shops and large retail chains in
Italy. Food Control., 125: 108-1003.

Nalbone, L., F. Cincotta, F. Giarratana, G. Ziino and A. Panebianco. 2021. Microplastics in

fresh and processed mussels sampled from fish shops and large retail chains in
Italy. Food Control., 125: 108-1003.

Nizzetto, L., G. Bussi, M. N. Futter, [Link] and P. [Link]. 2016. A theoretical

assessment of microplastic transport in river catchments and their retention by soils
and river sediments. Environ Sci Process Impacts, 18(8): 1050-1059.

Ogata, Y., H. Takada, M. K. Master, H. Hirai, S. Iwasa, Endo. 2009. International pellet
watch: Global monitoring of persistent organic pollutants (POPs) in coastal waters. 1.
Initial phase data on PCBs, DDTs, and HCHs. Mar. Pollut. Bull., 58: 1437–1466.

Pan, Z., C. Zhang, C. Wang, S. Sun, S. D., A. Zhou, S. Xie and J. Zou. 2021. Occurrence
of Microplastics in the Gastrointestinal Tract and Gills of Fish from Guangdong,
South China. J. Mar. Sci., 9(9): 981.

Pan, Z., C. Zhang, C. Wang, S. Sun, S. D., A. Zhou, S. Xie and J. Zou. 2021. Occurrence
of Microplastics in the Gastrointestinal Tract and Gills of Fish from Guangdong,
South China. J. Mar. Sci., 9(9): 981.

Pannetier, P., J. Cachot, C. Clerandeau, F. Faure, K. Van Arkel, L. [Link] Alencastro and B.
Morin. 2019. Toxicity assessment of pollutants sorbed on environmental sample
microplastics collected on beaches: Part I-adverse effects on fish cell [Link].
Pollut., 248: 1088-1097

69
Peda, C., L. Caccamo, M. C. Fossi, F. Gai, F. Andaloro, L. Genovese and G.
Maricchiolo. 2016. Intestinal alterations in European sea bass Dicentrarchus labrax
(Linnaeus, 1758) exposed to microplastics: preliminary results. Environ. Pollut., 212:
251-256.

Peda, C., L. Caccamo, M. C. Fossi, F. Gai, F. Andaloro, L. Genovesa and G. Maricchiolo.

2016. Intestinal alterations in European sea bass Dicentrarchus labrax (Linnaeus,
1758) exposed to microplastics: preliminary results. Environ. Pollut., 212: 251-256.

Phillips, M. B. and T. [Link]. 2015. Occurrence and amount of microplastic ingested by

fishes in watersheds of the Gulf of Mexico. [Link]. Bull. 100(1): 264-269.

PlasticsEurope, E. P. R. O. 2018. Plastics—The Facts 2019. An Analysis of European

Plastics Production, Demand and Waste Data. Plast. Eur., 20: 12-55.

Pozo, K., V. Gomez, M. Torres, L. Vera, D. Nunez, P. Oyarzun and J. Klanova. 2019.
Presence and characterization of microplastics in fish of commercial importance from
the Biobio region in central Chile. Mar. Pollut. Bull. 140: 315-319.

Qadir, A. and R. [Link]. 2011. Heavy metals in eight edible fish species from two polluted
tributaries (Aik and Palkhu) of the River Chenab, Pakistan. Biol. Trace
Elem. Res., 143(3): 1524-1540.

Qiang, L. and [Link]. 2019. Exposure to microplastics decreases swimming competence in

larval zebrafish (Danio rerio). Ecotoxicol. Environ. Saf., 176: 226-233.

Rainieri, S., N. Conlledo, B. K. Larsen, K. Granby and A. Barranco. 2018. Combined

effects of microplastics and chemical contaminants on the organ toxicity of zebrafish
(Danio rerio). Environ. Res., 162: 135-143.

Ramirez, M. M. B., R. D. Caamal and J. R. Von Osten. 2019. Occurrence and seasonal
distribution of microplastics and phthalates in sediments from the urban channel of
the Ria and coast of Campeche, Mexico. Sci. Total Environ., 672: 97-105.

Reddy, P. B. and S. S. Rawat. 2013. Assessment of aquatic pollution using histopathology in

fish as a protocol. Int. Res. J. Environ. Sci., 2(8): 79-82.

Richards, M. J., J. R. Edwards, D. H. Culver, R. P. Gaynes and National Nosocomial

Infections Surveillance System. 2000. Nosocomial infections in combined medical-

70
 surgical intensive care units in the United States. Control Hosp Epidemiol,21(8): 510-
515.

Rochman, C. M. 2018. Microplastics research—from sink to source. Sci., 361 (6384): 28-29.

Rochman, C. M., E. Hoh, T. Kurobe and S. J. The. 2013. Ingested plastic transfers
hazardous chemicals to fish and induces hepatic stress. Nature Sci. Rep., 3: 32-63.

Rochman, C. M., E. Hoh, T. Kurobe and S. [Link]. 2013. Ingested plastic transfers hazardous
chemicals to fish and induces hepatic stress. Sci. Rep., 3 (1): 1-7.

Rodrigues M. O, N. Abrantes, F.J.M. Goncalves, H. Nogueira, J.C. Marquesand, A.M.M

Goncalves. 2018. Spatial and temporal distribution of microplastics in water and
sediments of a freshwater system (Antuã River, Portugal). The Science of the Total
Environment, 633, 1549–1559.

Rummel, C. D., M. G. Loder, N. F. Fricke, T. Lang, E. M. Griebeler, M. Janke and G.

Gerdts. 2016. Plastic ingestion by pelagic and demersal fish from the North Sea and
Baltic Sea. Mar. Pollut. Bull., 102 (1): 134-141.

Ryan, C., D. Huebner, R. M. Diaz and J. Sanchez. 2009. Family rejection as a predictor of
negative health outcomes in white and Latino lesbian, gay, and bisexual young
adults. 123(1): 346-352.

Santos, K. C., and C. B. Martinez. 2014. Genotoxic and biochemical effects of atrazine and
Roundup, alone and in combination, on the Asian clam Corbicula fluminea.
Ecotoxicol. Environ. Saf., 100: 7-14.

Scherer, C., [Link], S. Lambert and M. Wagner. 2018. Interactions of microplastics with
freshwater biota. Freshwater microplastics., 212: 153-180.

Scherer, C., N. Brennholt, G. Reifferscheid and M. Wagner. 2017. Feeding type and
development drive the ingestion of microplastics by freshwater
invertebrates. Sci. Rep., 7 (1): 1-9.

Sekirov, I., S. L. Russell, L. C. M. Antunes and B. B. Finlay. 2010. Gut microbiota in health
and disease. Physiol. Rev., 90: 30-104.

71
Shafi, H. A., J. I. Qazi and A. S. Chaudhry. 2018. Monitoring the impact of urban effluents
on mineral contents of water and sediments of four sites of the river Ravi,
Lahore. Environ. Monit. Assess. 185(12):9705-9715.

Sharma, N. and R. Chaterjee. 2017. A Retrospective Study of 6 Years to evaluate Clinico-

Histopathological Correlation in Leprosy. J Adv Med Dent Scie Res, 7(11): 216-311.

Song, Y. K., S. H. Hong, M. Jang, J. H. Kang, O. Y. Kwon, G. M. Han and W. [Link].

2014. Large accumulation of micro-sized synthetic polymer particles in the sea
surface microlayer. Environ. Sci. Technol., 48(16): 9014-9021.

Tang, N., X. Liu and W. Xing. 2020. Microplastics in wastewater treatment plants of
Wuhan, Central China: Abundance, removal, and potential source in household
wastewater. [Link] Environ., 745: 14-1026.

Teuten, E. L., J. M. Saquing, D. R. U. Knappe, M. A. Barlaz, S. Jonsson, A. Bjorn. 2009.

Transport and release of chemicals from plastics to the environment and to wildlife.
Philos. Trans. R. Soc. 364: 2027–2045.

Teuten, E. L.,S. J. Rowland, T. S. Galloway and R. C. Thompson. 2007. Potential for

plastics to transport hydrophobic contaminants. Environ. Sci. Technol., 41: 7759–
7764.

Thompson, R. C. andA. [Link]. 2015. Microplastics in the marine environment: Sources,

consequences and solutions. Environ. Sci. Eur., 22(3): 552-554.

Thompson, R. C., S. H. Swan, C. J. Moore and F. S. Vom Saal. 2009. Our plastic
age. Philosophical Transactions of the Royal [Link] Sci., 364(1526): 1973-
1976.

Thompson, R. C., Y. Olsen, R. P. Mitchell, A. Davis, S. J. Rowland, A. W. G. John. 2004.

Lost at sea: Where is all the plastic? Sci., 304 (5672): 1- 838.

Thompson, R. C., Y. Olsen, R. P. Mitchell, A. Davis, S. J. Rowland, A. W. G. John. 2004.

Lost at sea: Where is all the plastic? Sci., 304 (5672): 1- 838.

Ullah, H. M., S. Zaman, F. Juhara, L. Akter, S. M. Tareq, E. H. Masum and R.

Bhattacharjee. 2014. Evaluation of antinociceptive, in-vivo & in-vitro anti-

72
 inflammatory activity of ethanolic extract of Curcuma zedoaria rhizome. BMC, 14(1):
1-12.

Usman, S., A. F. Abdull Razis, K. Shaari, M. N. A. Amal, M. Z. Saad, N. Mat Isa and M. F.
Nazarudin. 2021. Polystyrene Microplastics Exposure: An Insight into Multiple
Organ Histological Alterations, Oxidative Stress and Neurotoxicity in Javanese
Medaka Fish (Oryzias javanicus Bleeker, 1854). Int. J. Environ. Health Res. 18(18):
94-149.

Uurasjarvi, E., E. Sainio, O. Setala, M. Lehtiniemi, A. Koistinen. 2021. Validation of an

imaging FTIR spectroscopic method for analyzing microplastics ingestion by Finnish
lake fish (Perca fluviatilis and Coregonus albula). Environ. Pollut., 288: 117- 780.

Van Eygen, E., J. Feketitsch, D. Laner, H. Rechberger and J. Fellner. 2017. Comprehensive
analysis and quantification of national plastic flows: The case of Austria. Resources,
Conservation and Recycling, 117: 183-194.

Van Sebille, E., C. Wilcox, L. Lebreton, N. Maximenko, B. D. Hardesty, J. A. Van Franeker

and K. L. Law. 2015. A global inventory of small floating plastic debris. Environ.
Res. Lett., 10 (12): 12-4006.

Vannela, R. 2012. Are we “digging our own grave” under the oceans? Biosphere-level effects
and global policy challenge from plastic (s) in oceans. Environ. Sci. Technol., 46:
7932–7933

Von Moos, N., P. Burkhardt-Holm and A. Koohler. 2012. Uptake and effects of
microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an
experimental exposure. Environ. Sci. Technol., 46 (20): 11327-11335.

Wesch, C., K. Bredimus, M. Paulus and R. Klein. 2016. Towards the suitable monitoring of
ingestion of microplastics by marine biota: A review. Environ. Pollut.,218: 1200-
1208.

Wright, S. L., D. Rowe, R. C. Thompson and T. S. Galloway. 2014. Microplastic ingestion

decreases energy reserves in marine worms. Curr. Biol., 23: 1031–1033.

Wright, S. L., R. C. Thompson and T. S. Galloway . 2013. The physical impacts of

microplastics on marine organisms. Environ. Pollut., 178: 483–492.

73
Xia, X., M. Sun, M. Zhou, Z. Chang and L. Li. 2020. Polyvinyl chloride microplastics induce
growth inhibition and oxidative stress in Cyprinus carpio var. [Link]. Total
Environ., 716: 136-479.

Xu, G., L. Strathearn, B. Liu and W. Bao. 2014. Prevalence of autism spectrum disorder
among US children and adolescents. Jama, 319 (1): 81-82.

Xu, J. L., K. V. Thomas, Z. Luo and A. A. Gowen. 2019. FTIR and Raman imaging for
microplastics analysis: State of the art, challenges and prospects. TRAC-TREND
ANAL CHEM., 119: 115-629.

Xu, S., J. Ma, R. Ji, K. Pan and A. J. Miao. 2020. Microplastics in aquatic environments:
occurrence, accumulation, and biological effects. Sci. Total Environ., 703: 134-699.

Yin, L., B. Chen, B. Xia, X. Shi and K. Qu. 2018. Polystyrene microplastics alter the
behavior, energy reserve and nutritional composition of marine jacopever (Sebastes
schlegelii). J. Hazard. Matter, 360: 97-105.

Yu, R. A. 2000. Cadmium and DNA damage, oncogene expression as well as cell
apoptosis. Overseas Medicine Hygienics Fascicule, 27(6): 359-363.

Yang H, [Link], K. Mi, W. Xue, Y. Zhang. 2020.. Toxicity comparison of nano-sized and
micron-sized microplastics to gold fish Carassius auratus larvae. J. Hazard. Mater.,
388: 122-1058.

Yancheva, V., I. Velcheva, S. Stoyanova and E. Georgieva. 2016. Histological biomarkers in

fish as a tool in ecological risk assessment and monitoring programs: a review Appl.
Ecol. Environ. Res., 14(1): 47-75.

74