Developmental toxicity of PAHs in zebrafish embryos

DEVELOPMENTAL TOXICITY OF SELECTED PAHS IN
ZEBRAFISH (Danio rerio) EMBRYOS
Dissertation submitted in partial fulfillment for the degree of
B.Tech & M. Tech Dual Degree in Biotechnology
Submitted By
Arunima Pandey
School of Biotechnology
KIIT University
Bhubaneswar, Odisha, India
Under the Supervision of

Dr. Anbumani Sadasivam
(Senior Scientist)
Ecotoxicology Laboratory
Regulatory Toxicology Group
CSIR-INDIAN INSTITUTE OF TOXICOLOGY RESEARCH

VISHVIGYAN BHAWAN, 31,
MAHATMA GANDHI MARG, P.O. BOX NO. 80
LUCKNOW - 226 001, UTTAR PRADESH, INDIA
ii
DECLARATION
I hereby declare that the dissertation entitled 'Developmental toxicity of selected PAHs in zebrafish
(Danio rerio) embryos ' submitted by me, for the fulfillment of the degree of B. Tech & M. Tech, Dual
Degree Biotechnology, KIIT University is a record of bona fide work carried by me under the
supervision and guidance of Dr. Anbumani Sadasivam, Senior Scientist, Ecotoxicology Laboratory,
Regulatory Toxicology Group, CSIR-IITR, Lucknow.
Date: 24/ 05/ 2022

Place: Lucknow Arunima Pandey
iii
Acknowledgements
In our life, parents hold a position above the almighty God, so first of all, I would like to thank
them, my mother and father, Mrs. Archana Pandey, and Mr. Ravindra Nath Pandey.
I want to express my sincere gratitude to my supervisor, Dr. Anbumani Sadasivam, Senior Scientist,
Ecotoxicology LaboratoryRegulatory Toxicology Group, CSIR-IITR, for not only providing us a
quality research laboratory facility during this research but rather also for providing me the
opportunity to join his lab along with his constant guidance, support, and encouragement.
Also, I am very much thankful to my senior mentors, Mr. Ved Prakash and Mrs. Kavita for their all-time
support and proper guidance. Lastly, I would also like to express my gratitude towards my lab mate,
M/s. Dolly Dhapola who always encouraged me to work and helped me to complete the dissertation. My
special thanks to Mr. Sanjay Yadav, lab assistant for his kind consideration and cooperation.
Date: 24/ 05/ 2022

Place :Lucknow Arunima Pandey
iv
Table of Contents
Acknowledgements…………………………............................................................. ………………………………….v
Table of Contents................................................................................ ………………………..v-vii
List of Figures...................................................................................... ………………………..viii
List of Abbreviation..................................................................................................................... ix
Abstract......................................................................................................................................... x
Chapter 1- Introduction................................................................................................................. 11-14
1.1 Current Research................................................................................................................... 14
1.2 Scope and Objectives..................................................................... ……………………….………..14
Chapter 2- Review of Literature.........................................................……………………………………15-30
2.1 Phenanthrene................................................................................................................... 15-18
2.2 Pyrene.............................................................................................................................. 19-22
2.3 Zebrafish Testing Standarization..........................................................................................23
2.4 Summary Table.................................................................................................................24-30
Chapter 3- Materials and Methods................................................................................................ 31-45
3.1 Materials.......................................................................................................................... 32-35
3.1.1 Phenanthrene..................................................................................................................... 32
3.1.2 Pyrene................................................................................................................................33
3.1.3. Instruments........................................................................................................................34
3.1.4 Softwares............................................................................................................................35
3.2 Methods...........................................................................................................................36- 45
v
3.2.1 System Maintenance..................................................................................................... 36-37
3.2.2 Zebrafish and embryo Maintenance......................................…………………...………………..….38
3.2.3 Feeding............................................................................................................................... 38
3.2.4 Zebrafish Developmental Profile.............................................................................. ...39-40
3.2.5 Zebrafish Development: An overview......................................................................... …40-41
3.2.6 PAHs Exposure..................................................................................................................42
3.2.6.1 Phenanthrene Exposure................................................................................................... 42
3.2.6.2 Pyrene Exposure..............................................................................................................42
3.2.7 Embryo-larval toxicity test................................................................................................. 42
3.2.8 Determination of LC50..........................................................................................................................................42
3.2.8.1 Determination of LC50 Of Phenanthrene in zebrafish embryos..................................... 43
3.2.8.1 Determination fo LC50 of Pyrene in zebrafish embryos................................................. 43
3.2.9 Analysis of developmental defects.................................................................................... 44
3.2.10 Isolation of total protein....................................................................................................44
3.2.11 Antioxidant defense enzymes activity..............................................................................44-45
Chapter 4 - Results....................................................................................................................46-55
4.1 Phenanthrene........................................................................................................ 47-49
4.1.1 LC50 of Phenathrene in zebrafish embryos at 96 hpf.......................................47
4.1.2 Developmental defects induced by PAHs exposure in zebrafish embryos at
environment relevant concentration.....................................................................48
4.1.3 Oxidative stress profiling in zebrafish larvae at 4df............................................ 49-51
4.2. Pyrene......................................................................................................................51-55
4.2.1 LC50 of Phenathrene in zebrafish embryos at 96 hpf...........................................51-52
vi
4.2.2. Developmental defects induced by PAHs exposure in zebrafish embryos at
environment relevant concentration.....................................................................52
4.2.3 Oxidative stress profiling in zebrafish larvae at 4df..........................................52-55
4.3 Morphological abnormalities................................................................................56-57.
4.3.1 Phenanthrene...................................................................................................... 56
4.3.2 Pyrene..................................................................................................................57
Chapter 5 - Discussion............................................................................................................58-61
Chapter 6 - Conclusion............................................................................................................62-63
6.1 - Future Direction..................................................................................................63
Chapter 7 - References............................................................................................................. 64-77
vii
List of figures
Figure 1: Structure of Phenanthrene

Figure 2: Structure of Pyrene
Table 1: Summary Table
Figure 3: Phenanthrene from Sigma- Aldrich (St. Lois, MO, USA) at CSIR-IITR
Figure 4A: Pyrene from Sigma Aldrich (St. Lois, MO, USA) at CSIR-IITR,
B)DMSO ( Dimethyl sulfoxide)’, solvent used to dissolve Pyrene
Figure 5: Ocean salt
Figure 6: Nanodrop spectrophotometer ( BioTek Synergy H1Microplate Reader)
Figure 7: Fluoroscence Microscope
Figure 8: Zebrafish Aquaneering system (Italy) zebrafish reasearch facility,
CSIR-IITR, Lucknow
Figure 9: Breeding tanks with seperator with Male and female (2:1) A) Front
view B) Side view C) Zebrafish embryos
Figure 10: Fertilization cycle of Zebrafish Embryo
Figure 11: Graphical representation of dose response curve of phenanthrene
Figure 12: Graphical representation of the heart rate of phenanthrene exposed
zebrafish embryos
Figure 13: Represents the catalse activity of phenanthrene exposed zebrafish
embryos
Figure 14:Showing the GST activity of phenanthrene exposed zebrafish
embryos
Table 2: Cumulative mortality table of pyrene
Figure 15: Represents the catalase activity of Pyrene exposed zebrafish embryos
Figure 16: Showing the GST activity of pyrene exposed zebrafish embyos
Figure 17: Represents the total protein content in pyrene exposed zebrafish
embryos
Figure 18: Environment relevant exposure concentration of phenanthrene
Figure 19: Environment relevant exposure concentration of phenanthrene
viii
List of Abbreviation
 μg/g Micrograms per gram

 μg Microgram
 μg/L Micrograms per litre
 μm Micrometre
 ºC Degrees centigrade
 ANOVA Analysis of variance
 BMR Basal metabolic rate
 BPM Beats per minute
 CAT Catalase
 cm Centimetre
 CO Cardiac output
 dpf Day post fertilization
 D.Rerio Danio rerio
 DMSO Dimethyl sulfoxide
 DNA Deoxyribonucleic acid
 EDV End diastolic volume
 EF Ejection fraction
 ESV End systolic volume
 g Gram
 fh Heart Rate
 GST Glutathione-S-transferase
 Hpf Hours post fertilization
 K Condition factor
 Kg Kilogram
 L Litre
 LC50 Lethal concentration required to kill 50% of population
 m Metre
 mg Milligram
 mg/kg Milligrams per kilogram
 MtOH Methanol
 PAH Polycyclic hydrocarbon
 Phe Phenanthrene
 Pyr Pyrene
 TLM Median
ix
Abstract
Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread organic toxins released into the
environment from a variety of anthropogenic and natural sources. Concern continues to grow for the
increasing concentration of PAHs found in marine and fresh water environments and their toxic effects
on the organisms therein. In the aquatic environment PAHs tend to get distributed in the fresh water
ecosystem this raises concern over the toxic effects of PAHs to freshwater communities. Despite PAHs
have been the subject of several reviews, their toxicity to freshwater species has not been
comprehensively discussed. This review aimed to provide an overview on PAHs toxicity to freshwater
fish (Danio rerio). Phenanthrene and Pyrene have been reported to cause numerous cardiotoxic,
teratogenic, embryotoxic, metabolic toxic effects in a number of developing organisms including
zebrafish. Because of their unique characteristics, such as larval stage transparency, cost-effectiveness,
and high production zebrafish ( Danio rerio) are now used on a large scale. Another intriguing trait is
that it has a genetic structure that is ~70% similar to that of Human (Homo sapiens). Reliable
experimental early life stage acute toxicity data for fish are limited and further data are needed for
polyaromatic hydrocarbons to establish environmental quality objectives. Using a suite of biological
endpoints,we exposed zebrafish embryos to environmentally relevant concentrations of Phenanthrene
(10, 100, 500, 1000µg/L) and Pyrene (1, 10, 100, 1000µg/l) for 96 hours and then investigated its
developmental defect, oxidative stress at 96 hpf. Acute results indicated that spinal curvature and
pericardial edema the most sensitive sub-lethal effects observed and in many cases preceded observed
mortality. The LC50 for Phe and Pyr was 655µg/L and 1000µg/L respectively. The environmental
relevant concentrations were found to induce the CAT activity and GST activity at significant level
compared to solvent control group.So , results indicated that both phenanthrene and pyrene can induce
developmental deformities mediated by oxidative stress in developmental stage of zebrafish (Danio
rerio).
x
CHAPTER 1
INTRODUCTION
11
1.1 Introduction
Environmental toxins are known to have a deleterious impact on embryonic development and overall
health of organisms. Polycyclic aromatic hydrocarbons (PAHs) are a class of over 100 common
environmental contaminants made up of two or more benzene rings that are entirely made up of carbon
and hydrogen. Petroleum products, oil spills, forest fires, and insufficient combustion of organic
materials such as fossil fuels and wood are among the natural and anthropogenic causes of these
pollutants[1]. Industrial uses of PAHs comprise pesticides, pharmaceuticals, plastics, roofing tar, and
asphalt. Increased vehicle use in metropolitan areas has been identified as a common source of PAH
buildup in the environment [2]. The persistence and abundance of these contaminants in the air, soil, and
water have raised health concerns, with various kinds of cancer in humans being included [3-5]. PAHs
are usually found as a mixture, although examining the harmful consequences of a single PAH is simple.
The EPA's Urban Air Toxic Monitoring Program considers phenanthrene (C14H10), one of the simplest
PAHs with three benzene rings (Figure 1), to be one of 16 "priority pollutants" [6]. It has been shown
that phenanthrene alone is capable of causing developmental defects similar to those observed in
zebrafish embryos exposed to a combination of PAHs [7,8]. In the industrial world, phenanthrene has
been used to make insecticides and resins, such as coal-to-tar and wood-tar creosote, which is commonly
used as a wood preservative [9]. In addition, phenanthrene is found in tobacco products and is released
into the environment through the combustion of gasoline, diesel, wood, and coal [10]. Humans are
regularly exposed to phenanthrene by inhalation, which has been linked to an increased risk of lung
cancer [11]. PAHs are easily transported into aquatic environments of land-based runoff or atmospheric
deposition. Because PAHs are insoluble in water, they bind to solid particles when they settle to the
bottom of rivers, most commonly in aquatic species' breeding grounds [12,13]. Phenanthrene is
stimulated to permeate membranes and accumulate in exposed organisms' cells due to its hydrophobic
characteristics [14]. It has been shown that once within the cell, phenanthrene has the ability to cause
teratogenic consequences in a number of developing species [15-20]. Bioaccumulation is problematic
because the concentration of PAHs found within the organism is substantially higher than the levels
found in the surrounding environment[21]. PAHs exist as both gases and fine particulate materials,
making cleanup a difficult task[22]. For example, phenanthrene concentrations in crude oil can reach
400 mg/kg and up to 32 g/L in generated water, a byproduct of oil drilling [23], whereas phenanthrene
12
levels in ambient air can reach 299 ng/m3 in urban areas[24]. Out of a mixture of PAHs totaling an
average of 5502 g/kg in urban soil samples, phenanthrene alone adds up to 1985 g/kg [25]. According to
a recent study 3, the average concentration of PAHs in soils from urban areas in Florida ranged from
4562 g/kg to 10,031 g/kg, with the highest concentration reaching 58,640 g/kg [26]. Exogastrulation in
sea urchins [28], spinal curvature and craniofacial anomalies in rainbow trout [27], and abnormal heart
development and diminished locomotion in pacific herring [29] are just some of the teratogenic effects
of PAH exposure in aquatic species [30]. Several studies have also discovered morphological defects in
zebrafish embryos exposed to sublethal phenanthrene concentrations, including abnormal heart, eye,
yolk sac, and tail development [31-39]. After exposure to specific PAH components, delayed hatching
and developmental arrest leading to embryo death have been observed in these species [40,41]. The
smallest of the peri-fused PAHs, pyrene, is made up of four fused benzene rings in a colourless solid. In
addition to occurring ubiquitously as a product of incomplete combustion, anthropogenic sources of
pyrene in the environment include automotive care products, laundry and dishwashing products,
personal care products, water treatment products, and relatively high quantities in coal tar [42]. In
aquatic habitats, petroleum spills, atmospheric deposition, wastewaters, and surface-land runoff are all
key sources of pyrene [43]. Cardiac conduction, on the other hand, appears to be unaffected [44]. Pyrene
induced developmental abnormalities are thought to cause toxicity via altering the activity of the hepatic
antioxidant enzymes superoxide dismutase (SOD), glutathione-S-transferase (GST), and catalase [45,46].
With continued aqueous pyrene exposure in larval Carassius auratus, all three enzyme activities reached
maximum levels within 48 hours and subsequently steadily dropped until day 21 (end of the experiment)
[47] The production of GSSG followed the levels of antioxidant sensitive genes, but GSH levels were
inversely related, with the maximal increase and drop occurring 48 hours after exposure,
respectively[48]. Throughout the rest of the exposure period, things gradually returned to normal. These
findings suggest that pyrene bioaccumulation in the liver leads to redox cycling and the generation of
free radicals, both of which are significant mechanisms of pyrene toxicity in goldfish liver. These
deficits are strikingly comparable to those characterised by TCDD, a strong ligand for the AhR. While
PAHs are historically of concern for potential carcinogenicity [49] they are emerging contaminants of
concern for their developmental toxicity [50-52] and are characterized by malformations such as
skeletal defects, pericardial and yolk sac edema and cardiac dysfunction [53-57].
13
The zebrafish (Danio rerio) is a well-known model organism for toxicity assessment in early life stages
[58-61]. They have a fast reproductive rate, are transparent during development, and have a fully
sequenced genome [62]. Because it can be performed in multi-well microtiter plates, needs relatively
modest sample quantities, and most of the embryo and liquid handling may be automated, the zebrafish
embryo test (ZFET) is well suited as a bioanalytical screen [63,64]. Many endpoints related to diverse
mechanisms of action can be examined in one exposure using an in vivo test. Toxicology can be
detected and mechanistic ideas generated simply by monitoring morphological alterations in transparent
embryos (ETC).
1.2 Current Research work

Research focused on the effect of phenanthrene and pyrene, well-studied components of PAHs, on early
development in zebrafish. Documentation of the extent of morphological abnormalities occurring during
somitogenesis when zebrafish embryos are exposed to environment relevant concentrations of
phenanthrene, pyrene, throughout early development, with particular attention paid to spinal curvature,
incomplete somitogenesis, abnormal somite structure, pericardial edema and yolk sac edema.
1.3 Scope and Objectives
Objectives of the study:
1. Toxicity evaluation of Phenanthrene in zebrafish embryo at 96 hpf

a. LC50 Determination at 96 hpf
b. Developmental deformities
c. Oxidative stress
d. Gene expression
2. Toxicity evaluation of Pyrene in zebrafish embryo at 96 hpf

a. L50 Dertermination at 96 hpf
b. Developmental deformities
c. Oxidative stress
14
CHAPTER 2
REVIEW OF LITERATURE
15
2.1 Phenanthrene
Phenanthrene is a tricyclic polycyclic aromatic hydrocarbon that is widely diffused in the aquatic
environment. However, understanding of its effect on fish reproductive is still limited, especially in the
case of chronic exposure. Phenanthrene causes morphological defects in zebrafish embryos, such as tail
curvature, yolk-sac edema, and pericardial edema [60]. A recent study in which Peng et al., exposed
zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 μg/L) of
phenanthrene for 4 months and assessed the impact on reproduction. The results showed that fish
exposed to phenanthrene had lower egg production, with a substantial drop at 5.0 g/L. Female fish had
lower circulating levels of estradiol (E2) and testosterone (T), while male fish had lower levels of E2.
Furthermore, after phenanthrene exposure, plasma vitellogenin levels in female fish were significantly
reduced. The transcription of genes associated to the hypothalamic-pituitary-gonadal (HPG) axis
(GnRH2, FSH, LH, 17-HSD, CYP11A1, and CYP19a) was changed in a sex-specific manner.
Furthermore, in the F1 generation, embryos produced from exposed parents showed higher deformity
and lower hatching success[66]. There are few reliable experimental early life stage chronic toxicity data
for fish, and more data for polyaromatic hydrocarbons is needed to set environmental quality goals and
compare with toxicity models predictions. To decrease, refine, and replace the use of vertebrates in
animal testing, efforts are underway to produce a zebrafish embryo toxicity test guideline. Exposing
zebrafish embryos to environmentally relevant concentrations (35, 122, 145 g/L) of phenanthrene for
embryo lethal and sub-lethal developmental endpoints after a 5-day exposure, as well as larval survival
and growth endpoints during a subsequent 25-day test period (30 days) yielded mortality during the
chronic phase (6-30 days) for the former endpoint, and reduction in both the pericardial and yolk sac
elongation for the latter endpoint [67]. Furthermore, in addition to developmental toxicity, it is
commonly acknowledged that phenanthrene has cardiotoxic effects [ 68]. Cardiac dysfunction is known
as a subset of consequences in fish after exposure to PAHs [69-79]. To determine if or not exposure to
ecologically relevant Phe concentrations can cause heart abnormalities. Zhang et al., exposed zebrafish
embryos to environmentally relevant concentrations of phenanthrene ( (0.05–50 nmol L-1 ) and then
investigated its cardiotoxic effects and the mechanism(s) involved. The phenanthrene treated groups
showed certain cardiac morphogenetic abnormalities, such as an abnormally looping and enlarged heart,
dilated and thinner ventricular wall, and enhanced interstitial fibrosis. Similarly, when rockfish
16
(Sebastiscus marmoratus) embryos were exposed to phenanthrene, they showed similar abnormalities
(Li et al., 2011). According to Incardona et al. (2004, 2005), Phenanthrene exposure causes cardiac
dysfunction during cardiac morphogenesis via an aryl hydrocarbon receptor-independent mechanism,
which could be mediated by alternative cardiac conduction system targets. In contrast to findings that
phenanthrene exposure at 24hpf causes bradycardia in embryos [71], Zhang et al. found a quicker heart
beat after phenanthrene therapy at 72hpf which might be attributed to the different level of phenanthrene
exposure. Phenanthrene has long-term effects on fish development, reproduction, and behaviour [72].
Phenanthrene endangers the long-term viability of fisheries [73]. In fish embryos [74, 75], larvae [76],
and adults [77], phenanthrene exposure produces pathological alterations. Mai et al., by exposing adult
zebrafish to 300g/L for 15 days (96-hr LC50 3.12 mg/l), researchers were able to explore the
physiological and molecular responses associated with the toxic effects of phenanthrene on zebrafish.
and Histological investigation revealed that in phenanthrene exposed zebrafish, liver shape decreased
and cellular damage increased. Phenanthrene caused considerable modifications in glutathione S-
transferases (GST) and superoxide dismutase (SOD) activity, according to biological research. Similarly,
the toxicity of Phe was assessed in adult zebrafish (Danio rerio) at concentrations of 100, 500, 750, and
1000 g/l, with a 96-hour LC50 of 922.81 g/L [78]. In addition to the studies conducted on embryonal
and adult stages, Cypher et al. investigated the interaction of phenanthrene with hypoxia and its effect on
the cardiovascular system of larval zebrafish (Danio rerio) by exposing zebrafish larvae to 0, 1, 100, and
1000 g/L of phenanthrene in combination with normoxia and hypoxia. The results showed that the
highest concentration of phenanthrene caused a 58, 80, and 84% decrease in ƒH, Q, and arterial red
blood cell velocity in normoxia and an 88, 98, and 99 % decrease in hypoxia, respectively. Co-exposed
larvae also experienced higher rates of edema and lordosis in addition to a 33% increase in mortality rate
with co-exposure to hypoxia at the 1000 µg/L concentration of phenanthrene[79]. Furthermore,
Hooftman and Evers-de ruiter et al., worked on Brachydanio rerio (zebrafish) and Daphnia magna
(embryonal and larval stage) with the exposure concentration (18, 32, 56, 100, 180, 320 and 560 µg/L)
for 28 days and summarized for the former that the hatching rate was not afftected by the exposure
concentration, mortality was not significantly different from the control group, whereas a noticeable
decrease in length of the zebrafish was observed at 56µg/L, in contrast, the latter showed 21d NOEC at
18µg/L and LOEC at 32µg/L[80]. Moreover, other freshwater fishes showed similar pattern when
exposed with environmentally relevant concentration of phenanthrene, For eg. Carassius auratus (adult
17
stage) when exposed with 50 µg/L of Phe for 21 days yielded results in terms of bioaccumulation,
depuration and oxidative stress, where on one hand the increment of phenanathrene in tissues was
noticeably observed, just within the span of 3 days the decrease in the phenathrene level cannot be
neglected, the generation of free radical (hydroxyl radicle) was indicated, whereas upsurge in the
induction of hepatic SOD after 4 days was also marked. While, CAT levels increased during the early
stage and latter decreased, GST showed contrasting results by decreasing during the early stage and
increasing latter [81]
Fig 1: Structure of Phenanthrene ( C14H10 )
18
2.2 Pyrene
Pyrene is a polycyclic aromatic hydrocarbon (PAH) made up of four fused benzene rings. Pyrene and its
derivatives are used to manufacture dyes and dye precursors in the industry. Pyrene levels in surface
water in the Jiulong River Estuary and Western Xiamen Sea, China, range from 0.22 to 2.66 g/L (about
1–13 nmol/L) [82]. Pyrene (5 mol/L) exposure causes apparent abnormalities in zebrafish during
development, such as loss of peripheral circulation, peripheral edoema, anaemia, and neural tube cell
death [83]. In reaction to pyrene treatment (60 g/L, 150 g/L),. Yeung,(2019)., mentioned in his thesis
that he worked on adult zebrafish with pyrene concentrations of (0, 0.25, 2.5, 25g/L) and evaluated
certain end points such as cardiorspiratory toxicity, mortality rate, and metabolic toxicity, and found no
mortality, an increase in O2 consumption (MO2), a significantly altered critical swimmimg speed
(Ucrit), no change in Standard Metabolic Rate (SMR), and an increase in Active Metabolic Rate (AMR)
in 0.25µg/L, no significant effect on cardiac output, elevation in GSR expression [84]. Additionally,
certain studies were conducted on Danio rerio (l.arval stage) in terms of photo induced toxicity
parameter ( UV and No- UV enhanced), where former LC50 came out to be 1.56µg/L and latter one was
68µg/L with exposure concentration (5, 10, 12, 20, 30, 40, 50 µg/L) of pyrene the observed results
indicated acute toxicity to larvae in the presence of UV, photo induced toxicity, mortality rate was
73.3% [85]. Zhang et al., assesed the cardiotoxicity of pyrene in the zebrafish embryo for 72 hours with
exposure concentration (0, 0.0390, 0.3905, and 39.475 µg/L ) the results indicated Larger pericardial
edema and cardiac looping defects, increase in cardiac abnormalities including elongated ventricle,
string-like morphology and edema increment in malformation ratio (by 4.5-fold) was detected in the 50
nmol/L, rapid increase in heart rate, whereas, no change in cardiac output and QPCR, AhR1a, AhR1b,
AhR2 and Cyp1A, decrease in the expression levels of NKx2.5. However, it did not lead to a
remarkable abnormality of some cardiac function related genes such as actin, alpha cardiac muscle 1-
like (actc1l), myosin light polypeptide (myl7) and troponin T2, cardiac (Tnnt2) Gata5 had an equal
expression as control [86]. Other than cardiotoxicity, metabolic toxicity, reproductive toxicity a study
evaluated teratogenicity of Danio rerio (embryonal stage ) for 72 hr with the exposure concentration
((2.5, 25 and 50 μM) and summarized by stating the induction of the lowest levels of oxidative lesions in
DNA, stunted growth, Severe yolk-sac edema, increase in mortality rate, whereas , decrease in hatching
19
rate. Sogbanmu et al., (2018) employed Clarias gariepinus (adult stage) to assess the physiological,
biochemical and histological indices, teratogenic indices by exposing 150 µg/L of pyrene for 96 hr (96-
h LC50 is 1.53mg/l) and the findings demonstrated that pyrene induced increment in the hepatosomatic
index (HSI) values and no significant differences were observed in the condition factor, gill somatic
index and cardiosomatic index, however, fecundity was lowered by factors of 2.4x, whereas no
statistically significant differences between the exposed and control groups in the values of MDA, SOD,
CAT, GSH, and GST, gill alterations observed include inflammatory cells in pyrene-treated males and
oedema, epithelial hyperplasia and lifting in pyrene-treated females severe vacuolation in male pyrene-
treated catfish [87].
Fig 2: Structure of Pyrene (C16H10)
20
The significance of toxicology studies [88] -
i. To construct a dose-response curve.
ii. To guarantee that novel compounds intended for use as insecticides, medications, or food
additives are safe before they are approved for use in industry or doctor's offices.
iii. To determine the mechanism or mode of action for a harmful impact that has been
observed in earlier investigations.
iv. To produce epidemiological studies to explain population observations, such as the
protracted inquiry of the link between smoking and lung cancer.
v. Validation of new testing or investigative procedures, particularly those carried out in
vitro rather than on animals.
The 3Rs, or Replacement, Reduction, and Refinement of animal studies in research have
developed throughout time, as having the philosophies that underpin their usage, legislative
directives, and new technology, the latter of which can benefit both science and animal welfare.
The zebrafish has emerged as a prominent alternative animal model among innovative
techniques. Zebrafish offer several natural benefits for drug screening: tiny, low-maintenance,
and easily reproduced in large numbers (one spawning generates 100–200 eggs). Zebrafish are 3-
cm long when fully grown. Larvae, which are only 1–4 mm long, can survive in a single well of
a typical 96- or 386-well microtiter plate for seven days, thanks to nutrients stored in the yolk
sac.
21
The zebrafish larvae absorb small chemicals diluted in the surrounding water through their skin
and gills, making drug administration simple. Because zebrafish begin to swallow 72 hours after
fertilization, drugs can be administered orally for experiments performed after this stage (hpf). In
the yolk sac, the sinus venosus, or the circulation, very hydrophobic substances, big molecules,
and proteins can be injected. Oral intubation can also be used to give medications to adult
zebrafish. Compared to other animal models, zebrafish can be employed in large numbers for
each assay, and only tiny amounts of medication (mg) are required.
Furthermore, zebrafish's transparency allows in vivo study of life or the whole mount fixed
specimens, including the imaging of vital dyes, fluorescent tracers, antibodies, and riboprobes.
Zebrafish develop distinct organs and tissues by 120 hpf, including the brain, heart, liver,
pancreas, kidney, intestines, bone, muscles, nervous systems, and sensory organs. These organs
and tissues are anatomically, physiologically, and molecularly comparable to their mammalian
counterparts.
22
2.3 Zebrafish Testing Standardization:
The Organization for Economic Cooperation and Development has created test standards based
on the effectiveness of utilizing zebrafish and other fish species for toxicity investigations
(OECD). The Fish Embryo Toxicity (FET) test, the Fish: Juvenile Growth Test, and the adult-
based Fish: Acute Toxicity Test are among them. The FET test has been proven to be a reliable
and repeatable process through independent laboratory testing.
It's also been demonstrated to have a good connection with the Acute Toxicity Test in adults, and
it's been suggested as a good range-finder before more intensive testing. The FET test was
recently employed to assess the toxicity of methylxanthine medications. The results showed a
good connection between TC50 measures of mortality, morphological abnormalities, and
teratogenicity in zebrafish embryos and reported mammalian LD50 values.
23
Model Exposure Duration End points Observation References
PAHs organism concentrat of
ion exposure
Paralichthys 0.5, 1 and 4 weeks Bioindex ↓Average weight gain (WG) Jee et al
olivaceus 2 µm paramaters No significant changes in Hepatosomatic (2003)
(Adult stage) Hematological index (HSI) and
parameter Condition factor (CF)
Lysozyme ↓Erythrocyte count
activity ↓Hemocrit value (Ht)
↓Hemoglobin content (Hb)
RBC hemolysis and/or hemorrhaging from
damaged gill filament
↓Mean corpuscular hemoglobin ( MCH)
↓ Mean corpuscular hemoglobin
concentration (MCHC)
↑Mean corpuscular volume
↑Plasma bilirubin concentration
No significant Plasma total cholesterol
concentration
↑Mean plasma lysozome activity
↑Kidney lysozome activity
Danio rerio 0.2, 1.0, 4 months Hatching rate F0 Gen F1 Gen Peng et al.
(Embryo 5.0 µg/L Malformation No significant changes ↓Fecundity (2019)
stage) rate in hatching, No significant
Survival rate malformation, survival changes in
Phenanthre Reproductive rates and sex ratio malformation
ne parameters No significant change rates
Sex hormones in body length,body ↓Hatchability
Gene weight or condition rate
transcription factor (K- factor) ↓Survival rate
profile ↑HSI in male fish ↓Plasma E2 and
Inhibition of Plasma T of
Gonadsomatic Index female
(GSI) in male fish ↓Plasma E2 of
No significant change males
in GSI of female fish No significant
changes in
plasma T of
males
No significant
difference in
VTG
concentration in
males
↓VTG
concentration in
females
↑GnRH2 and
FSHß in the
females
↓Transcription
of FSHß and
LHß in the
males
Inhibition of
24
17ß-HSD
gene
Transcription
and
downregulation
of CYP19 in the
ovaries
↑CYP11A1
gene
transcription
and inhibition
of CYP19a in
the testes
No significant
differences in
hepatic ERα and
ERß gene
transcription
Danio rerio 35, 122, 30 days Lethal effects Mortality observed during chronic phase Butler et
(embryo 145 µg/L (6-30 days) al. (2013)
stage) Sub - Lethal ↓Pericardial edema
effects ↓Yolk sac edema
Spinal curvature observed at 120 h
affected 58% embryos
Carassius 50 µg/L 0.25, 0.5, Bioaccumulatio ↑Phenanthrene concentration in the tissues Sun et al.
auratus 1, 1.5, 2, n (2006)
(Adult stage) 3, 4, 7,
14, and Depuration ↓Phenanthrene concentration in whole
21 days fish tissue within 3 days
25
Oxidative stress Free radical (Hydroxyl radical) generation
ROS generation
No significant difference in SOD at early
exposure
↑Induction of hepatic SOD after 4 days
O
↑CAT activity at 6h
↓CAT activity gradually
Activation of CAT after 14 days
↓GST activity during early days
↑GST activity to the control value
Pacifastacus 500 1000 15 days Mortality rate 20, 30, 40% Mortality for 0.5, 1.0, 1.5 Ainerua et
leniusculus 1500 mg/L al. (2021)
(Embryo µg/L
stage)
Cardiac ↓Heart rate (HR) and prolonged recovery
function phase
Electrical disruption in the heart
Antioxidant Tissue and dise specific increment and
capacity decrement of SOD activity
Danio rerio 300µg/L 15 days Mortality rate Mortality reached 100% within 96 hr Mai et al
(Adult stage) 96-hr Biochemical Severe hepatocyte hypertropy (2019)
LC50 alterations ↓GST levels
(3.12mg/ ↑SOD activity
L)
Danio rerio 0.039, 72 h Cardiac toxicity Abnormally looped and enlarged heart Zhang et
(Embryo 0.39, Histomorpholog Dilated and thinner ventricle walls al. (2013)
stage) 39.047 ical alterations ↑Interstitial fibrosis
µg/L ↑EDV and ESV of ventricles
↓SO and CO
Abnormal heart contraction
Danio rerio 100, 500, 96 h ↑vtg, esr1 and CYP19A1resulting in Kim et al.
(Adult stage) 750, 1000 LC50 endrogen production in males (2017)
µg/L (922.81µ ↑CYP11A1 and CYP17A1 resulting in
g/L) adrenal stereoidogenesis
26
Danio rerio 0, 1, 100, 96 h Cardiotoxicity 58% decrease in heart rate (fH) Cypher et
(Larval stage) 1000µg/L LC50 Normoxia 80% decrease in Cardiac output(q) al. (2017)
( Hypoxia 84% aterial red blood cell velocity
induction 88% decrease in heart rate (fH)
Mortality rate 98% decrease in cardiac output (Q)
99% decrease in arterial red blood cell
volume
Eye area was smaller in hypoxia
treatments and decreased further with co-
exposure to 1000 µg/L phenanthrene in
late hatching larvae (72-96 hours)
Yolk width was smaller with normoxia but
larger with hypoxia at 1000 µg/L
phenanthrene in late hatching larvae (72-
96 hours)
33% mortality rate with co- exposure
Brachidanio 18, 32, 28 days Hatching rate Hatching rate is not affected at any Hooftman
rerio 56, 100 , Mortality rate concentration and Evers-
(embryo and 180, 320 Growth rate Mortality was not significantly different de Ruiter
larvae stage) and 560 from control et al
µg/L ↓ Length at 0.56mg/L (1992)
↓Wet weight at 0.10 mg/L
Daphnia 18, 32, 28 days Reproduction 21d NOEC at 0.018 mg/L Hooftman
magma 56, 100 , rate 21d LOEC at 0.032mg/L and Evers-
180, 320 Mortality de Ruiter
and 560 Morphological et al
µg/L or behavioral (1992)
criterion
Oryzias 12.5, 18 days Hatching PHEN exposure correlated significantly Rhodes et
latipes 25, 50, Length with concentration. Neither PHEN al. (2004)
(Embryo 100 and Hatching time congener had a significant impact on
larval stage) 200 Hatching Length (HL) or Time to hatch
µg/L , (TTH).
At 200µg/L hatching rate was equivalent
to the control (100%)
Oncorhynchs 60 days Mortality rate Test 1: Dora et al.
mykiss Test1: LC50 Test Growth rate ↑ mortality at all concentrations except 44 (1995)
(Fry fish) 44µg/L - 1 - µg/L.
700µg/L 1000µg/ ↓ survival time at the highest
L concentration (700 µg/L).
88, 180, and 350 µg/L treatments were not
significantly different in length and
weight, but this collective group differed
from both the control and the 0.044 mgIL
treatments
The few fry that did survive to 60 days in
350 and 700 µg/L were small
Test 2: ↓Mortality
Test 2: Since no mortalities >5O% occurred in
19µg/L- any treatment, an LC50 was not calculated
300µg/L General decline in length and weight over
the range tested
27
Clarias 150 µg/L 96-Hr Physiological, ↑Hepatosomatic index (HSI) values Sogbanmu
gariepinus LC50 Biochemical There were no significant differences in et al.,
(adult stage) (1.53 mg and Histological the condition factor, gill somatic index and (2018)
/L) indices cardiosomatic index
Teratogenic Fecundity was lower by factors of 2.4x
indices No statistically significant differences
Pyrene between the exposed and control groups in
the values of MDA, SOD, CAT, GSH,
and GST
Gill alterations observed include
inflammatory cells in pyrene-treated males
and oedema, Epithelial hyperplasia and
lifting in pyrene-treated females
Severe vacuola tion in male pyrene-treated
catfish
Sinusoidal congestion in female pyrene-
treated catfish
Revealed no histological alterations
Danio rerio 5,10,12,2 96 h Photoxicity Acute toxic to larvae in the presence of Alission
(Larval stage) 0,30,40,5 No-UV Mortality rate UV Wills
0µg/L enhanced Bioconcentratio Photo-induced toxicity observed (2013)
(LC50 n factor Mortality rate was 73.3%
68µg/L) ↑Uptake and elimination rate
UV
enhanced
LC50
( 1.56µg/
L)
28
Danio rerio (0, 0.25, 48 h Cardiorespitory Body burdens that ranged from 8.89 ng/g Yeung et
(Adult stage) 2.5 and toxicity in control to 6.84 ng/g in exposed fish al.,(2019)
25 µg/L Mortality rate No mortality observed
Metabolic ↑Oxygen consumption (MO2)
toxicity Significantly altered Critical swimming
speed (Ucrit)
Standard Metabolic Rate (SMR) remained
unchanged
↑Active Metabolic Rate (AMR) exposed
to 0.25µg/L
↓ESVand without changes in EDV or SV
leading to a greater ejection fraction
↓Artrial and ventricular contractile rates
No significant effect on cardiac output
↑glycogen content liver from
zebrafish exposed to 2.5 µg
↑mRNA level of CS in heart, liver and
skeletal muscle from zebrafish exposed to
0.25 and 25 µg/L Unchanged triglyceride
levels in heart tissue
↓ HOAD mRNA levels in liver
↑GSR expression
↑ cardiac and hepatic SOD mRNA
transcript levels was observed in the same
0.25 µg PYR/L group, as well as heart
from the 25 µg PYR/L-exposed zebrafish
no significant changes were observed for
SOD expression at any PYR concentration
in skeletal muscle
29
Danio rerio 0, 0.0390, 72h Cardiac toxicity Larger pericardial edema and cardiac Zhang et
(Embryonal 0.3905, looping defects al.,(2012)
stage) and ↑Cardiac abnormalities including
39.475 elongated ventricle, string-like
µg/L morphology and edema among
↑ Malformation ratio (by
4.5-fold) was detected in the 50 nmol/L
group
↑Heart rate
Smaller ventricular EDV and ESV of the
heart than that of the age-matched vehicle-
treatment,
No change in cardiac output
No change in QPCR, AhR1a, AhR1b,
AhR2 and Cyp1A
↓Expression level of NKx2.5
did not lead to a remarkable abnormality
of some cardiac function related
genes such as actin, alpha cardiac muscle
1-like (actc1l), myosin
light polypeptide (myl7) and troponin T2,
cardiac (Tnnt2)
Gata5 had an equal expression as control
↓ Bmp2b
Danio rerio (2.5, 25 72 h Embryotoxicity Induced the lowest levels of oxidative Sogbanmu
(Embryonal and 50 Teratogenicity lesions in DNA et al.,
stage) μM) Stunted growth (2016).
Severe yolk-sac oedema
↑Mortality rate
↓Hatching rate
Asellus 0.70µg/L 7 days Bioconcentratio Maximum isopod concentrations occurred Hattum et

Benzo(g,h, aquaticus n Toxicokinetic after 3 day al.,(1999)
i)perylene (Adult stage) rate constants Slow elimination rate
The rate constant-based BCFs are more
than 1 order of magnitude higher than the
apparent BCFs.
Table 1: Summary Table
30
CHAPTER 3
MATERIALS AND METHODS
31
3.1 Materials
3.1.1 Phenanthrene
Test compound:
Phenanthrene, purchased from Sigma–Aldrich (St. Louis, MO, USA). The purity was 98%. [CAS
No.: 85-01-8] C14H10, MW: 178.23 g/mol, mp: 98-100 ℃ (lit), bp: 340℃(lit), fp: 171℃, d:1 063
g/ml at 25℃ (lit), form: crystalline powder, was dissolved in Methanol( 0.001%), Potassium
phosphate buffer (pH 7.4), post mitochondrial supernatant (PMS), Bradford reagent, H2O2,
potassium dichromate, CDNB assay, stopping reagent, distilled water.
Fig 3: Phenathrene from Sigma–Aldrich (St. Louis, MO, USA) compound at CSIR- IITR
32
3.1.2 Pyrene
Test compound:
Pyrene, purchased from Sigma–Aldrich (St. Louis, MO, USA),was procured from HiMedia Laboratories Pvt.
Ltd., Mumbai, India. The purity was 98%. CAS Number: 129-00-0, C16H10, MW: 202.25 g/mol, DMSO
(solvent control), ocean salt, Potassium phosphate buffer (pH 7.4), post mitochondrial supernatant
(PMS), Bradford reagent, H2O2 , potassium dichromate, CDNB assay, stopping reagent, distilled
water,
A B
Fig 4A&B: Pyrene and DMSO (Dimethyl sulfoxide), solvent used to dissolve Pyrene
Fig 5: Ocean salt
33
3.2 Instruments
1. Light microscope
2. Nanodrop Spectrophotometer (BioTek Synergy H1 Microplate Reader)
3. Weighing machine
4. Breeding tank
5. Fluoroscence Microscope Nikon DS-Ri2 (made in Japan)
6. Centrifuge
7. Hot water plate
Fig 6: Nanodrop Spectrophotometer (BioTek Synergy H1 Microplate Reader )
34
Fig 7: Fluoroscence Microscope Nikon DS-Ri2 (made in Japan)
3.3 Softwares
1. Graphpad Prism 8.4.0

2. Gen next
35
3.4 Methods
3.4.1 System Maintenance
Zebrafish are housed in a moving structure that filters and circulates air through thesystem water to
maintain the water quality necessary for a safe aquatic environment. Excess food and fish waste are
also channeled into the circulation mechanism. Different organizations provide Zebrafish systems,
but CSIR,IITR has Aquaneering's scheme from Italy. Maintained and cultured wild type zebrafish
(Danio rerio) zebrafish embryos were collected as described by (Cheng SH et al., 2000) [89]. Wild
type zebrafish was maintained at 26-27 C0 temperature, pH 7-7.5 and conductivity was 500 µs/m, in
photoperiod of 14:10 h light: dark. After spawning,collection of the embryos was done within one
hour of fertilization from light/dark photoperiod as 14/10 hours. After collection of the embryos,
washed them with E3 medium four times to prevent them from fungal infection. Post-washing, we
kept the embryos in the embryo medium (60 mg of ocean salt in 1L of distilled water) and incubated
at 28 °C until chemical exposure. The selection of healthy and proper developing embryos has been
done under Nikon DS-Ri2 (made in Japan) at 16 – 64 celled stage (1.5 to 2 hours post fertilization;
hpf) for the subsequent experiment.
36
Fig 8 : Zebrafish Aquaneering system (Italy), Zebrafish Research Facility, CSIR-IITR, Lucknow
37
3.4.2 Zebrafish and embryo Maintenance
ASWT (Assam wild-type) strain zebrafish embryos from CSIRInstitute of Genomics and
Integrative Biology (IGIB) on gratis were maintained in Zebrafish facility of Ecotoxicology
Laboratory since 2017 in CSIR-IITR, Lucknow. Following physicochemical parameters
Zebrafish were maintained in a flow-through standalone water system (ZebTEC, Techniplast,
Italy): 14 h/10 h (light/dark) photoperiod, temperature: 27–28 °C; pH: 7–7.5 and conductivity:
500 μs/m
3.4.3 Feeding:
Different diets are available for different stages of zebrafish larvae and adults. Adult zebrafish
can be given dried blood worms, brime shrimps, or other zooplanktons (size 300-400 microns),
while larvae can be fed live blood worms, brime shrimps, or other zooplanktons (size 300-400
microns) (size from 100 microns). In present study, a modified feeding protocol as described by
Khan et al. (2018) [90] is followed, fish were fed twice a day (morning at 10 am; evening at 5
pm) with live cultured brine shrimps; Artemia nauplii and commercial feed (ZM-400 once daily
in noon) from Zebrafish Management Limited, the UK. Protocols of the Institutional Animal
Ethics Committee were followed for fish husbandry with prior approval (IITR/IAEC/41/20).
Overfeeding zebrafish can raise nitrate levels in the water, compromising the fish's health,
reproduction, and survivability. Hence, the tanks were cleaned on the daily basis.
38
3.4.4 Zebrafish Developmental Profile
Breeding:
The day before embryo collection, male and female fish at a ratio of 2:1
were kept in a breeding chamber and at the beginning of a light cycle of
spawning day, the separator in the breeding chamber was removed to allow
natural mating. After 30 min, embryos were collected, washed four times
with embryo (E3) medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2,
0.33 mM MgSO4, and 5% methylene blue) to prevent fungal infection.
Then the embryos were incubated at 28.5 °C until 4-MBC exposure in a
cooling incubator (REMI). Healthy and fertilized embryos at 16–64 celled
stage (1.5 to 2 hour post-fertilization; hpf) visualized under a microscope
(Nikon EclipseNi-U; Tokyo, Japan) were selected for PAHs exposure.
39
C
Fig 9. Breeding tanks with and Male and female (2:1) respectively A) Front view;
B) Side view
3.4.5 Zebrafish development: An Overview:
Fig10: Fertilization cycle of Zebrafish Embryo
40
Within 24 hours of fertilization, the animal's basic body plan emerges. This corresponds to
around nine days in the mouse's life. Newly hatched 'early' larvae (3 days post-fertilization) are
mostly inactive and negatively buoyant, lying immobile on the bottom, except for a few tail
flicks. The larvae inflate their gas bladders by swimming up and gulping air at the surface or just
before day 5 (usually related to water temperature). After this stage, they are neutrally buoyant
and can swim indefinitely while holding their place in the water column [91].
41
3.4.6 PAHs Exposure
3.4.6.1 Phenanthrene Exposure
In order to evaluate the toxicity in zebrafish embryos in accordance with ‘OECD

guideline, 236’determined the LC50 of phenantrene compound in zebrafish embryos at
96 hour post fertilization (hpf) and Methanol (MtOH) used as the final concentration
treatment; this concentration did not showed observable effects in zebrafish embryos in
the development stage.
3.4.6.2 Pyrene Exposure
In order to evaluate the toxicity in zebrafish embryos in accordance with ‘OECD

guideline, 236’determined the LC50 of pyrene compound in zebrafish embryos at 96
hour post fertilization (hpf) and DMSO. used as the final concentration treatment; this
concentration showed observable effects in zebrafish embryos in the development stage.
3.4.7 Embyo - larval toxicity
Toxic effects were assessed in zebrafish embryos exposed to phenanthrene (10, 100, 500, 1000
µg/ml) and for pyrene ( 1, 10, 100, 1000 µg/ml) for 24–96 hpf. Under a stereo microscope, the
hatching rate and survival of the embryos in each well were assessed every 24 hours. Embryonic
malformations were photographed and examined under fluoroscence microscope during the
exposure. The frequency of the malformations was measured at various doses.
3.4.8 Determination of LC50:

For determination of LC50, the apical endpoints recommended by OECD guideline are as follows :
1. Coagulated embryos
2. Lack of somite formation
3. Lack of tail detachment
4. Lack of heartbeat
42
Any of the above four endpoints was positively considered as the mortality of Zebrafish embryo. Based
on this observation, LC50 of phenanthrene was determined.
3.4.8.1 Determination of LC50 of Phenanthrene in zebrafish embryos
Zebrafish embryos were exposed to the chemical test compound at different concentrations of 10µg/L,
100µg/L, 500µg/L, and 1000 µg/L along with control and solvent control to find out the LC50 at 96 hpf.
Embryos were exposeed in duplicates in 24 well plates with each replicate having 10 embryos that is 20
embryos in each concentration. The embryos were exposed to phenanthrene until 96 hpf. LC50
determination was done based on the 4 apical endpoints (mentioned above) including coagulation of
embryo, the lack of heart beat, the lack of tail detachment and the lack of somites formation. Based on
the observation, the LC50 results were determined . Based on the LC50, we chose the environmentally
relevant concentrations for further experimental studies. Zebrafish embryos were exposed to
environmental relevant concentration to determine the developmental defects (Heart rate, hatching rate,
and Mortality), oxidative stress marker level (CATand GST) in embryos.
3.4.8.2 Determination of LC50 of Pyrene in zebrafish embryos
Zebrafish embryos were exposed to the chemical test compound at different concentrations of 1µg/L,
10µg/L, 100µg/L, and 1000 µg/L along with control and solvent control to find out the LC50 at 96 hpf.
Embryos were exposed in duplicates in 24 well plates with each replicate having 10 embryos that is 20
embryos in each concentration. The embryos were exposed to pyrene until 96 hpf. LC50 determination
was done based on the 4 apical endpoints (mentioned above) including coagulation of embryo, the lack
of heart beat, the lack of tail detachment and the lack of somites formation. Based on the observation,
the LC50 results were determined . Based on the LC50, we chose the environmentally relevant
concentrations for further experimental studies. Zebrafish embryos were exposed to environmental
relevant concentration to determine the developmental defects (Heart rate, hatching rate, and Mortality),
oxidative stress marker level (CATand GST) in embryos.
43
3.4.9 Analysis of developmental defects in exposed zebrafish embryos:
A.Heart rate From each concentration, 10 embryos at 72 hours post fertilization (hpf) were
selected and analyzed the heart rate per minute with control and solvent control by Nikon DS-
Ri2 microscope (Japan). For analysis hatching rate, 20 embryos were exposed per
concentration with control and solvent control in triplicate in 24 well plates and incubate the
embryos for 72 hpf at 280C and analyzed the hatching rate, in developing zebrafish embryos as
well under microscope.
B.Axial curvature: 10 embryos were selected from each sub-lethal concentration, observed the
axial curvature at 96 hpf in zebrafish larvae with respect to solvent control by Nikon DS-Ri2
microscope (Japan)
3.4.10 Isolation of total protein from Phe exposed zebrafish embryos:
For protein isolation, 20 zebrafish larvae/500µL of 0.1 Molar potassium phosphate buffer (pH 7.4) at 4
day post fertilization (dpf) time points were homogenized followed by centrifugation 10000 g during 10
min at 4°C and collected the post mitochondrial supernatant (PMS). The PMS stored in - 200C deep
freezer for further experimental analysis. Total protein concentration in zebrafish larvae were measured
by Bradford method (Bradford, 1976) using 20µL of enzyme supernatant and the Bovine serum albumin
(BSA) as a standard by Biorad's Bradford micro-assay and the absorbance was read at 595 nm without
any prior incubation.
3.4.11 Antioxidant defense enzymes activity:
Oxidative stress markers including, CAT and GST activities analyzed by the methods Aebi et al., 1974,
Okhawa et al., 1979 and Habig et al., 1974 respectively [91-93].
44
A) Catalase activity analyzed using 100µL of enzyme supernatant, H2O2 and 0.1M Phosphate
Buffer (pH- 7.4) by measuring the consumption of hydrogen peroxide (H2O2) at 240 nm [94] and the
enzyme activity determined as µmol/min/µg protein content.
B) Glutathione-S-transferase (GST) activity was determined from the 100µL of supernatant using
the L-glutathione and 1-chloro-2-4-dinitrobenzene and noted the activity at 340 nm[95] . The enzymatic
activity concluded in nmole/min/mg of protein.
45
CHAPTER 4
RESULTS
46
4.1 Phenanthrene
We analyzed the mortality data in GraphPad prism software. The LC50 was 655 µg/L of Phenanthrene
at 96 hours post fertilization (hpf).
4.1.1 LC50 of Phe in Zebrafish embryos at 96 hour post fertilization (hpf):
Primarily, investigation of the LC50 of the chemical compound phenanthrene in zebrafish embryos at
96 hours post fertilization (hpf) in static exposure was done. Zebrafish embryos were selected and
exposed to different concentrations of phenanthrene ( 10,100, 500, 1000µg/l) at 4 dpf and were scored
for mortality after 96 h of exposure. The results indicated that mortality increased with increasing
phenanthrene concentration in a dose-dependent manner. The LC50 of Phenanthrene is 655µg/L with
in zebrafish embryos at 96 hpf.
Figure 11: Graphical representation of dose response curve of Phenanthrene. Briefly the zebrafish
embryos were exposed to different concentration of phenanthrene at 10,100, 500, 1000 µg/L. The
exposure was given from the stage of 8 to 16 celled stages. Mortality of zebrafish embryos were
observed following OECD guideline, 236 [101].
47
4.1.2 Developmental defects induced by Phenanthrene exposure in Zebrafish
embryos at environmental relevant concentrations.
A. Heart rate at 72 hpf Phenanthrene exposed zebrafish embryos:
In Phe exposed zebrafish embryos, the heart rate per minute increased with increasing concentrations at
72 hpf. In result, the highest concentration of phenanthrene (1000 µg/l ) elevated the heart rate at a
significant level of 162 beat per minute.
Figure 12: Graphical representation of the heart rate . Shows significant increment in heart rate
per minute with increased concentration (1000 µg/l )
48
4.1.3 Oxidative stress profiling in Zebrafish larvae 4 dpf:
1. Catalase activity:
Most of the studies suggested that, phenanthrene; three fused benzene rings PAHs results in to the
oxidative stressby the production of reactive oxygen species (ROS), which leads to the activation of
antioxidant defenseor detoxification system to prevent the cell from the oxidative damage [102].
Activity of anti-oxidative stress enzyme could be induced in slight oxidative stress, however excessive
oxidative stress alleviate enzyme activity because of the impairment of compensatory mechanisms [103].
Catalase is an enzyme involve in the scavenging the H2O2 from the cellular environment and catalyses
the breakdown of H2O2 into O2 and H2O. Increased CAT activity suggests the higher production of H2O2
inside the cell means facing oxidative stress[104] . In our results, phenanthrene induced the CAT
activity in zebrafish larvae at 4dpf butfrom both developmental stages the responses were different
according to the concentrations. In the present study, the induction and inhibition of CAT activity
depends on the lower and higher concentration of Phe in zebrafish developmental stages. In 4 dpf
exposed zebrafish embryos, significant difference of CAT activity observed at 4 dpf between
phenathrene concentration and solvent control. At the concentration, of 10µg/l, a decrement in CAT
activity observed and with increasing concentrations like500 µg/l and 1000 µg/l of phenanthrene, the
CAT activity inhibited at significant level with respect to the solvent control.
49
Figure 13: Represents the catalase activity. At 4 dpf, the induction of CAT at 100µg/l and inhibition
at 500, and 1000 µg/l. At 100µg/L at significant level (* p<0.05).
2. Glutathione-S-transferase:
GST is a enzyme involve in the phase II detoxification metabolism and it helps to interact the
lipophilic compound to glutathione to make compound water soluble and this water soluble compound
readily excreted from the body. at 96 hpf, the activity of GST was observed at significant level between
treatment and solvent control groups after phenanthrene exposure. At 500 µg/l and 1000 µg/l of
concentration, the GST activity was increased as compared to solvent control. Phenanthrene was found
to have elevated GST activity in zebrafish embryo at 96 hpf. Present results suggested that, the GST
induction is involve in the activation of phase II detoxification system mediated by oxidative stress.
50
Figure 14: Showing the GST activity. At 4 dpf, the concentration of 500µg/l induced the GST activity
with a significant level (* p <0.05)
4.2 Pyrene
4.2.1 LC50 of Pyrene in Zebrafish embryos at 96 hour post fertilization (hpf):
Primarily investigation of the LC50 of the chemical compound pyrene in zebrafish embryos at 96 hours
post fertilization (hpf) in static exposure was done. Zebrafish embryos were selected and exposed to
different concentrations of pyrene ( 1,10, 100, 1000µg/l) at 4 dpf and were scored for mortalityafter 96
h of exposure. The results indicated that mortality increased with increasing Phe concentration in a
dose-dependent manner. The LC50 of pyrene is 1000 µg/L with in zebrafish embryos at 96 hpf.
51
Table 2: Table showing the LC50 of pyrene at 96 hpf
4.2.2 Developmental defects induced by Pyrene exposure in Zebrafish embryos at
environment relevant concentrations.
A. Spinal or axial curvature:

Pyrene exposure induced the spinal/axial curvature in zebrafish embryos with higher concentrations
including 100 and 1000 µg/l, other concentrations did not significantly induce the axial curvature. The
overall percentage of 4-MBC-exposed embryos with altered axial curvature at 4 dpf increased with Pyr
concentration. Pyrene exposed embryos presented trunk malformation characterized by a distorted body
axis with a curled tail as the most prominent deformity whereas untreated control embryos exhibited a
straight trunk. In the 100 µg/l concentration of pyrene, the percentage of axial curvature was observed is
40 % in zebrafish embryos at 4 dpf, while at 1000µg/l 20% increment was observed. In terms of
embryonic development, pyrene caused teratogenic effect in higher concentration of was observed by
Sogbanmu et al.,(2016) .
52
4.2.3 Oxidative stress profiling in Zebrafish larvae 4 dpf:
1. Catalase activity:
Most of the studies suggested that, pyrene; four fused benzene rings PAHs results in to the oxidative
stress by the production of reactive oxygen species (ROS), which leads to the activation of antioxidant
defense or detoxification system to prevent the cell from the oxidative damage. Activity of anti-
oxidative stress enzyme could be induced in slight oxidative stress, however excessive oxidative stress
alleviate enzyme activity because of the impairment of compensatory mechanisms [105]. Catalase is an
enzyme involve in the scavenging the H2O2 from the cellular environment and catalyses the breakdown
of H2O2 into O2 and H2O. Increased CAT activity suggests the higher production of H2O2 inside the cell
means facing oxidative stress[106]. In our results, pyrene induced the CAT activity in zebrafish larvae at
4dpf but the activity differed according to the concentrations. In the present study, the induction and
inhibition of CAT activity depends on the lower and higher concentration of Phe in zebrafish
developmental stages. In 4 dpf exposed zebrafish embryos, significant difference of CAT activity
observed at 4 dpf between pyrene concentration and solvent control. At the concentration, of 10µg/l, a
decrement in CAT activity observed and with increasing concentrations like 100 µg/l and 1000 µg/l of
pyrene, the CAT activity inhibited at significant level with respect to the solvent control.
Fig 17: Represents the catalase activity. At 4 dpf, the induction of CAT at 10µg/l , 100 µg/l
and 1000µg/l with a significant level (*, ** p<0.05)
53
2. Glutathione-S-transferase activity:
GST is a enzyme involve in the phase II detoxification metabolism and it helps to interact the
lipophilic compound to glutathione to make compound water soluble and this water soluble
compound readily excreted from the body. At 96 hpf, the activity of GST was observed at
significant level between treatment and solvent control groups after pyrene exposure. At 100 µg/l
and 1000 µg/l of concentration, the GST activity was increased as compared to solvent control.
Pyrene was found to elevated GST activity in zebrafish embryo at 96 hpf. Present results suggested
that, the GST induction is involve in the activation of phase II detoxification system mediated by
oxidative stress.
Fig 16: Showing the GST activity. At 4 dpf, the concentration of 10 µg/l and 1000µg/l
induced the GST activity at significant level activity (* represent the p<0.05)
54
3. Total protein content:
Fig 17: At 4 dpf, the concentration of 1µg/l and 10 µg/l induced the GST activity at significant
level activity (** represent the p<0.05)
55
4.3 Morphological abnormalities:
4.3.1 Phenanthrene
After treating embryos from phenanthrene there was no significant deformation in any of the
concentration.. The prepared concentration of phenanthrene was 10, 100, 500, 1000 µg/l.
24 hpf 48 hpf 72 hpf 96 hpf
SC
10µg/l
100µg/l
500µg/l
1000µg/l
Fig 18: Environment relevant exposure concentrations of phenanthrene did not induce axial
curvature in zebrafish embryos. Scale bar: 500 µm
56
4.5.2 Pyrene
After treating embryos from pyrene there were significant deformation in higher concentrations ..
the prepared concentration of pyrene 1, 10, 100, 1000 µg/l. In least concentration i.e. 1, 10 µg/l
there was no change in morphology of Zebrafish embryo at 48 hrs. With the increase in
concentration i.e. 100, 1000 µg/l there was morphological deformation (pericardial edema, yolk-
sac edema and axial curvature)
24 hpf 48 hpf 72 hpf 96 hpf
SC
1 µg/l
10 µg/l
100 µg/l
CHAPTER 5
1000 µg/l
Fig 19: Environment relevant exposure concentration of pyrene concentrations of pyrene

induced axial curvature and pericardial edema in zebrafish embryos from 48 hpf at 1000 µg/l, 72
hpf and 96 hpf for both 100 and 1000 µg/l concentrations. Scale bar: 500 µm
57
CHAPTER 5
DISCUSSION
58
Zebrafish is the most widely used vertebrate model in biomedical and ecotoxicological research in
studying the adverse effects of emerging environmental contaminants in aquatic ecosystems. Embryo-
larval stages are of particular interest to ecotoxicologists in screening the toxicity potential at early
developing stages and also serve the purpose of 3R's (reduction, refinement and replacement) approach.
5.1. Phe and Pyr induced embryotoxicity and developmental deformities
In the present study, induced embryotoxicity was carried out as per the OECD test guideline 236 and
deduced the 96 h LC50 in D. rerio embryos. Exposure of Danio rerio embryos to sub-lethal
concentrations of phenanthrene and pyrene induced developmental abnormalities like axial curvature,
reduced hatching rate, larval length, and heart rate. However, the at low concentrations of pyrene (1 and
10 μg/L) and all the phe exposure concentrations did not induce the aforementioned developmental
anomalies. Similar to the present study, PAHs induced teratogenicity has been noted in aquatic
organisms [107, 108]. Significant increase in axial curvature and pericardial edema with an increasing
concentration of pyrene, whereas no such observation could be noted for phenanthrene exposed
zebrafish embryos even at the higher concentrations (500 and 1000µg/l) at 72 hpf . Interestingly, at 3 dpf
the heart rate per minute elevated with increasing concentrations of at 72 hpf. In result, the highest
concentration of phenanthrene, 1000 µg/l elevated the heart rate at a significant level of 162 beat per
minute. Additionally, acute exposure of environmental relevant concentrations of phenanthrene resulted
in 33% larval mortality. The LC50 of phenanthrene and pyrene is 665 and 1000µg/L, respectively.
Whereas, Mai et al., calculated the LC50 of phenanthrene exposed adult zebrafish (Danio rerio), which
came about to be 3.12mg/l (3120µg/l) which clearly shows that at embyronic stage the zebrafish are
sensitive to the exposure concentrations of phenanthrene [109]. Similarly, Kim et al, calculated
922.8µg/l for phenanthrene exposed adult zebrafish [110]. Butler et al., mentioned that the LC50 of
phenanthrene exposed zebrafish should be more than 450µg/l [111]. Nevertheless, TLM (Median
Tolerance Limit), methodolgy which is capable of predicting both acute and chronic toxcity of MAHs
and PAHs in single exposures and mixtures. In consonance with the previously reported studies, the
LC50 in the present study lies within the reported values. However, in the present work, embryo/larval
stages exposure to near environmental concentration of phenanthrene did not induce any notable effects
59
at the organism level but the toxic effects at molecular level remains unclear. In the case of pyrene, the
LC50 calculated in the present study in agreement with the previously reported LC50 of pyrene exposed
zebrafish.. Hence, molecular studies on the exposures are crucial to decipher the response of signaling
molecules in zebrafish early developing stages towards PAHs exposure. Axial curvature is a well-
observed developmental deformity in developing zebrafish upon toxicants exposure [112]. In the present
study, only the larvae exposed to higher environmental relevant concentrations showed developmental
deformities. Deformed spine or axial curvature is usually appeared as a curled tail owing to the
impairment in the sonic hedgehog pathway leading to abnormal somitogenesis with deformed
musculature [113,114]. Zebrafish larvae exposed to pyrene showed different defects at different
developmental stages, such as anemia, mild pericardial edema, a minor dorsal curvature, peripheral
vascular defects and neuronal cell death . In the present study pericardial edema and axial curvature has
been noticed and corroborated with other studies after pyrene exposure [115,116]. The visible signs of
pyrene toxicity included dorsal curvature of the body axis, reduced peripheral circulation, anemia,
pericardial edema that evolves into yolk sac edema, and cell death beginning in the brain and later
involving the spinal cord. Although the hatching rate is not an important apical endpoint to assess the
teratogenic potential of xenobiotics [117], yet we have not noticed any significant inhibition in the
hatching rate after phenanthrene and pyrene exposure. Nonetheless, the decrease in larval length was
observed in both the PAHs. The observed hatching defect might be due to the reduced enzyme activity
and embryonic movement within the chorion. Xenobiotics induced hatching defect by affecting the
hatching gland via yolk-sac abnormality has been noticed in zebrafish embryos Nevertheless, in the
present study, we were to find yolk-sac abnormalities such as yolk-sac edema after pyr exposure.
5.2. Oxidative stress as a major mechanism in zebrafish embryo-larval stages after Pyrene and
Phenanthrene exposure that leads to developmental defects
It has been widely thought that oxidative stress plays a major role in developmental toxicity during
zebrafish embryogenesis, resulting in abnormal embryos [118, 119, 120]. In the present study, varying
levels of oxidative stress enzymes was noticed in zebrafish larvae sampled at 72 hpf intervals show
differential response towards phenanthrene and pyrene exposure. Increased CAT activity suggests the
higher production of H2O2 inside the cell means facing oxidative stress [121]. In our results, pyrene
60
induced the CAT activity in zebrafish larvae at 4dpf but the activity differed according to the
concentrations. In the present study, the induction and inhibition of CAT activity depends on the lower
and higher concentration of pyrene in zebrafish developmental stages. In 4 dpf exposed zebrafish
embryos, significant difference of CAT activity observed at 4 dpf between pyrene concentration and
solvent control. At the concentration, of 10µg/l, a decrement in CAT activity observed and with
increasing concentrations (100 µg/l and 1000 µg/l) of pyrene, the CAT activity inhibited at significant
level with respect to the solvent control. Under stressful conditions, ROS levels increased dramatically
leading to intracellular damage of macro molecules through oxidation. Perturbations between oxidation
and endogenous antioxidant systems that eventually resulted in excess accumulation of ROS is
considered to be the leading cause of oxidative stress [122, 123]. It has been reported that owing to
limited antioxidant capacity, vertebrate early developing stages are highly susceptible to oxidative stress
On the other hand, At 96 hpf, the activity of GST was observed at significant level between treatment
and solvent control groups after pyrene exposure. At 100 µg/l and 1000 µg/l of concentration, the GST
activity was increased as compared to solvent control. Pyrene was found to elevated GST activity in
zebrafish embryo at 96 hpf. Present results suggested that, the GST induction is involve in the activation
of phase II detoxification system mediated by oxidative stress. In the case of the phenanthrene, the
exposure concentration did not induce CAT activity at the significant level at 96 hpf zebrafish embryos,
while at the same concentration of Phe wasobserved to induce ST activity at 500 µg/l (p<0.05) as
compared to the SC group. The result of phenanthrene indicated that in the oxidative stress condition
the GST enzyme was able to metabolize the phenanthrene or organic compound and eliminated from
the body. So, the overall oxidative stress have been overcomed as demostrated by significant induction
of CAT activity. In accordance with the LC50 of phenanthrene determined was 655 µg/l. It maybe due to
the phenanthrene have the role to induce the mortality followed by different toxicological pathways at
96 hpf developmental lethality.
61
CHAPTER 6
CONCLUSION
62
The present work was carried out to explore the developmental toxicity potential of Phe and PYR using
D. rerio model system. Based on the research findings from the present study, exposure of D. rerio
embryos acutely to phenanthrene and pyrene induced embryo lethality and the 96 h LC50 value
whereas, resulting in larvae mortality and oxidative stress. The overall results show that, phenanthrene e
at all the concentrations (10, 100, 500, 1000 μg/L) did not induce any developmental defects in zebrafish
larvae except a significant increase in heart rate at sub-lethal concentrations (1000 μg/L). On the
contrary, pyrene exposure results in development defects including axial curvature, curling of tail,
pericardial edema and yolk sac edema. With varying levels of CAT and GST content in pyrene and
phenanthrene representing oxidative stress. Incardona et al., (2006) found that pyrene produced
developmental toxicity in zebrafish, in accordance with the current study provides satisfactory results
however, zebrafish toxicity to phenanthrene differs [120]. While the exact mechanism of toxicity was
not explained by this study alone, it is clear that oxidative stress as a major mechanism in zebrafish
embryo-larval stages is more complex than expected.
6.1 Future Direction

This study answered questions regarding phenanthrene and pyrene acute toxicity in zebrafish embryonic
stage, but there are many questions yet to be answered. The exact mechanism of toxicity of
phenanthrene and pyrene are still unknown. Although, based on OCED guidelines, the four apical
endpoints, the determination of LC50 was possible, but failed to determine the exact pathway of
phenanthrene and pyrene toxicity. Moreover, most of the studies concerning phenanthrene and pyrene
focus on high dose exposure, and there are few reports concerning the symptoms and mechanisms of
exposure of fish embryos to environmentally relevant concentrations of phenanthrene and pyrene, so
more research is needed
63
CHAPTER 7
REFERENCES
64
References
1. Abbas, I., Badran, G., Verdin, A., Ledoux, F., Roumié, M., Courcot, D., & Garçon, G.
(2018). Polycyclic aromatic hydrocarbon derivatives in airborne particulate matter:
sources, analysis and toxicity. Environmental chemistry letters, 16(2), 439-475.
2. Agudelo-Castañeda, D. M., Teixeira, E. C., Schneider, I. L., Lara, S. R., & Silva, L. F.
(2017). Exposure to polycyclic aromatic hydrocarbons in atmospheric PM1. 0 of urban
environments: Carcinogenic and mutagenic respiratory health risk by age groups.
Environmental pollution, 224, 158-170.
3. Petit, P., Maître, A., Persoons, R., & Bicout, D. J. (2019). Lung cancer risk assessment
for workers exposed to polycyclic aromatic hydrocarbons in various industries.
Environment international, 124, 109-120.
4. Rengarajan, T., Rajendran, P., Nandakumar, N., Lokeshkumar, B., Rajendran, P., &
Nishigaki, I. (2015). Exposure to polycyclic aromatic hydrocarbons with special focus
on cancer. Asian Pacific Journal of Tropical Biomedicine, 5(3), 182-189.
5. Stec, A. A., Dickens, K. E., Salden, M., Hewitt, F. E., Watts, D. P., Houldsworth, P. E.,
& Martin, F. L. (2018). Occupational exposure to polycyclic aromatic hydrocarbons and
elevated cancer incidence in firefighters. Scientific reports, 8(1), 2476.
6. Liu, B., Xue, Z., Zhu, X., & Jia, C. (2017). Long-term trends (1990–2014), health risks,
and sources of atmospheric polycyclic aromatic hydrocarbons (PAHs) in the US.
7. Incardona, J. P., Collier, T. K., & Scholz, N. L. (2004). Defects in cardiac function
precede morphological abnormalities in fish embryos exposed to polycyclic aromatic
hydrocarbons. Toxicology and applied pharmacology, 196(2), 191-205.
65
8. Sogbanmu, T. O., Nagy, E., Phillips, D. H., Arlt, V. M., Otitoloju, A. A., & Bury, N. R.
(2016). Lagos lagoon sediment organic extracts and polycyclic aromatic hydrocarbons
induce embryotoxic, teratogenic and genotoxic effects in Danio rerio (zebrafish)
embryos. Environmental Science and Pollution Research, 23(14), 14489-14501
9. Abdel-Shafy, H. I., & Mansour, M. S. (2016). A review on polycyclic aromatic

hydrocarbons: source, environmental impact, effect on human health and remediation.
Egyptian Journal of Petroleum, 25(1), 107-123.
10. Teixeira, E. C., Agudelo-Castañeda, D. M., & Mattiuzi, C. D. P. (2015). Contribution of

polycyclic aromatic hydrocarbon (PAH) sources to the urban environment: a comparison
of receptor models. Science of the Total Environment, 538, 212-219.
11. Carotenuto, M., De Antonellis, P., Liguori, L., Benvenuto, G., Magliulo, D., Alonzi, A.,
Turino, C., Attanasio, C., Damiani, V., Bello, A.M. and Vitiello, F. (2014). H-Prune
through GSK-3β interaction sustains canonical WNT/β-catenin signaling enhancing
cancer progression in NSCLC. Oncotarget, 5(14), 5736.
12. Cabrerizo, A., Dachs, ., Moeckel, C., O eda, M. ., Caballero, G., Barcel , D., & Jones, K.
C. (2011). Ubiquitous net volatilization of polycyclic aromatic hydrocarbons from soils
and parameters influencing their soil− air partitioning. Environmental Science &
Technology, 45(11), 4740-4747.
13. Pampanin, D. M., & Sydnes, M. O. (2013). Polycyclic aromatic hydrocarbons a

constituent of petroleum: presence and influence in the aquatic environment. In
Hydrocarbon. IntechOpen. 83-118.
14. Samanta, S. K., Singh, O. V., & Jain, R. K. (2002). Polycyclic aromatic hydrocarbons:
environmental pollution and bioremediation. TRENDS in Biotechnology, 20(6), 243-248
66
15. Charry, M. P., Northcott, G. L., Gaw, S., Keesing, V., Costello, M. J., & Tremblay, L. A.
(2019). Development of acute and chronic toxicity bioassays using the pelagic copepod
Gladioferens pectinatus. Ecotoxicology and environmental safety, 174, 611-617.
16. Fairbairn, E. A., Bonthius, J., & Cherr, G. N. (2012). Polycyclic aromatic hydrocarbons
and dibutyl phthalate disrupt dorsal–ventral axis determination via the Wnt/β-catenin
signaling pathway in zebrafish embryos. Aquatic toxicology, 124, 188-196
18. Mu, J., Wang, J., Jin, F., Wang, X., & Hong, H. (2014). Comparative embryotoxicity of
phenanthrene and alkyl-phenanthrene to marine medaka (Oryzias melastigma). Marine
pollution bulletin, 85(2), 505-515.
19. Sestak, M. C., Pinette, J. A., Lamoureux, C. M., & Whittemore, S. L. (2018). Early
Exposure to Polycyclic Aromatic Hydrocarbons (PAHs) and Cardiac Toxicity in a
Species (Xenopus laevis) with Low Aryl Hydrocarbon Receptor (AHR) Responsiveness.
bioRxiv, 301846.
20. Abdel-Shafy, H. I., & Mansour, M. S. (2016). A review on polycyclic aromatic

hydrocarbons: source, environmental impact, effect on human health and remediation.
Egyptian Journal of Petroleum, 25(1), 107-123
21. Manzetti, S. (2013). Polycyclic aromatic hydrocarbons in the environment:

environmental fate and transformation. Polycyclic Aromatic Compounds, 33(4), 311-330.
22. Pampanin, D. M., & Sydnes, M. O. (2013). Polycyclic aromatic hydrocarbons a

constituent of petroleum: presence and influence in the aquatic environment. In
Hydrocarbon. IntechOpen. 83-118.
67
23. Liu, B., Xue, Z., Zhu, X., & Jia, C. (2017). Long-term trends (1990–2014), health risks,
and sources of atmospheric polycyclic aromatic hydrocarbons (PAHs) in the US.
24. Wang, D., Ma, J., Li, H., & Zhang, X. (2018). Concentration and Potential Ecological
Risk of PAHs in Different Layers of Soil in the Petroleum-Contaminated Areas of the
Loess Plateau, China. International journal of environmental research and public health,
15(8), 1785.
25. Liu, Y., Gao, P., Su, J., da Silva, E. B., de Oliveira, L. M., Townsend, T., Xiang, P., &
Ma, L. Q. (2019). PAHs in urban soils of two Florida cities: Background concentrations,
distribution, and sources. Chemosphere, 214, 220-227.
26. Pillai, M. C., Vines, C. A., Wikramanayake, A. H., & Cherr, G. N. (2003). Polycyclic
aromatic hydrocarbons disrupt axial development in sea urchin embryos through a β-
catenin dependent pathway. Toxicology, 186(1-2), 93-108.
27. Le Bihanic, F., Morin, B., Cousin, X., Le Menach, K., Budzinski, H., & Cachot, J. (2014).
Developmental toxicity of PAH mixtures in fish early life stages. Part I: adverse effects
in rainbow trout. Environmental Science and Pollution Research, 21(24), 13720- 13731.
28. Le Bihanic, F., Morin, B., Cousin, X., Le Menach, K., Budzinski, H., & Cachot, J. (2014).
Developmental toxicity of PAH mixtures in fish early life stages. Part I: adverse effects
in rainbow trout. Environmental Science and Pollution Research, 21(24), 13720- 13731.
29. Vines, C. A., Robbins, T., Griffin, F. J., & Cherr, G. N. (2000). The effects of diffusible
creosote-derived compounds on development in Pacific herring (Clupea pallasi). Aquatic
oxicology, 51(2), 225-239.
68
30. Huang, L., Wang, C., Zhang, Y., Wu, M., & Zuo, Z. (2013). Phenanthrene causes ocular
developmental toxicity in zebrafish embryos and the possible mechanisms involved.
Journal of hazardous materials, 261, 172-180
32. Kamelia, L., Brugman, S., de Haan, L., Ketelslegers, H. B., Rietjens, I. M., & Boogaard,
P. J. (2019). Prenatal developmental toxicity testing of petroleum substances using the
zebrafish embryotoxicity test. ALTEX-Alternatives to animal experimentation, 36(2),
245-260.
33. Massei, R., Hollert, H., Krauss, M., von Tümpling, W., Weidauer, C., Haglund, P.,
Küster, E., Gallampois, C., Tysklind, M., & Brack, W. (2019). Toxicity and
neurotoxicity profiling of contaminated sediments from Gulf of Bothnia (Sweden): a
multi-endpoint assay with Zebrafish embryos. Environmental Sciences Europe, 31(1), 8.
34. Wolińska, L., Brzuzan, P., Woźny, M., G ra, M., Łuczyński, M. K., Podlasz, P., Kolwicz,
S., & Piasecka, A. (2011). Preliminary study on adverse effects of phenanthrene and its
methyl and phenyl derivatives in larval zebrafish, Danio rerio. Environmental
Biotechnology, 7, 26-33.
35. Zhang, Y., Huang, L., Wang, C., Gao, D., & Zuo, Z. (2013). Phenanthrene exposure
produces cardiac defects during embryo development of zebrafish (Danio rerio) through
activation of MMP-9. Chemosphere, 93(6), 1168-1175.
36. He, Y., Sun, C., Zhang, Y., Folkerts, E. J., Martin, J. W., & Goss, G. G. (2018).
Developmental toxicity of the organic fraction from hydraulic fracturing flowback and
69
produced waters to early life stages of zebrafish (Danio rerio). Environmental science &
technology, 52(6), 3820-3830.
37. McIntyre, J. K., Davis, J. W., Incardona, J. P., Stark, J. D., Anulacion, B. F., & Scholz, N. L.
(2014). Zebrafish and clean water technology: assessing soil bioretention as a protective
treatment for toxic urban runoff. Science of the Total Environment, 500, 173- 180.
38. Douben, P. E. T. (n.d.). PAHs: An Ecotoxicological Perspective Edited by. Retrieved from
http://dl.mozh.org/up/PAHs An_Ecotoxicological_Perspective Ecological amp Envir
onmental_Toxicology_Series_.pdf#page=23
39. Eisler, R. (1987). Biological Report 85(1.11) Contaminant Hazard Reviews POLYCYCLIC
AROMATIC HYDROCARBON HAZARDS TO FISH, WILDLIFE, AND INVERTEBRATES: A
SYNOPTIC REVIEW. Retrieved from
https://pdfs.semanticscholar.org/dca7/88a0a2ba218fe4b3fbf77c72f2df50950401.pdf
40. Incardona, J. P., Collier, T. K., & Scholz, N. L. (2004). Defects in cardiac function precede
morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons.
Toxicology and Applied Pharmacology, 196(2), 191–205.
https://doi.org/10.1016/j.taap.2003.11.026
41. Incardona, J. P., Carls, M. G., Teraoka, H., Sloan, C. A., Collier, T. K., & Scholz, N. L.
(2005). Aryl Hydrocarbon Receptor-Independent Toxicity of Weathered Crude Oil during
Fish. Health Perspectives (Vol. 113). Retrieved from
https://www.jstor.org/stable/pdf/3436747.pdf?refreqid=excelsior%3A9559e2e470f
6f94ab8fbff85751fafcd
42. Incardona, J. P., Collier, T. K., & Scholz, N. L. (2004). Defects in cardiac function precede
Toxicology and Applied Pharmacology, 196(2), 191–205.
https://doi.org/10.1016/j.taap.2003.11.026
70
43. Yin, Y., Jia, J., Guo, H. Y., Yang, L. Y., Wang, X. R., & Sun, Y. Y. (2014). Pyrene-
stimulated reactive oxygen species generation and oxidative damage in Carassius auratus.
Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and
Environmental Engineering, 49(2), 162–170.
https://doi.org/10.1080/10934529.2013.838846
44. Yin, Y., Jia, J., Guo, H. Y., Yang, L. Y., Wang, X. R., & Sun, Y. Y. (2014). Pyrene-
stimulated reactive oxygen species generation and oxidative damage in Carassius auratus.
Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and
Environmental Engineering, 49(2), 162–170. https://doi.org/10.1080/10934529.2013.838846
45. Verma SK, Nisha K, Panda PK, et al. Green synthesized MgO nanoparticles infer
biocompatibility by reducing in vivo molecular nanotoxicity in embryonic zebrafish
through arginine interaction elicited apoptosis. Sci TotalEnviron. 2020;713:136521.
doi:10.1016/j.scitotenv.2020.136521
46. Sheel R, Kumari P, Panda PK, et al. Molecular intrinsic proximal interaction infer
oxidative stress and apoptosis modulated in vivo biocompatibility of P.niruri contrived
antibacterial iron oxide nanoparticles with zebrafish. Environ Pollut. 2020;267:115482.
doi:10.1016/j.envpol.2020.115482
47. Kumari S, Kumari P, Panda PK, et al. Biocompatible biogenic silver nanoparticles
interact with caspases on an atomic level to elicit apoptosis. Nanomedicine.
2020;15(22):2119-2132. doi:10.2217/nnm-2020-0138
48. Begum NA, Mondal S, Basu S, Laskar RA, Mandal D. Biogenic synthesis of Au and Ag
nanoparticles using aqueous solutions of Black Tea leaf extracts. Colloids Surfaces B
Biointerfaces. 2009;71(1):113-118. doi:10.1016/j.colsurfb.2009.01.012
71
49. Heintz, R.A., Rice, S.D., Wertheimer, A.C., Bradshaw, R.F., Thrower, F.P., Joyce, J.E.,
Short, J.W., 2000. Delayed effects on growth and marine survival of pink salmon
Oncorhynchus gorbuscha after exposure to crude oil during embryonic development.
Mar. Ecol. Prog. Ser. 208, 205–216.
50. Incardona, J.P., Collier, T.K., Scholz, N.L., 2004. Defects in cardiac function precede
morphological abnormalities in fish embryos exposed to polycyclic aromatic
hydrocarbons. Toxicol. Appl. Pharmacol. 196, 191–20
51. Carls, M.G., Heintz, R.A., Marty, G.D., Rice, S.D., 2005. Cytochrome P4501A induction
in oil-exposed pink salmon Oncorhynchus gorbuscha embryos predicts reduced survival
potential. Mar. Ecol. Prog. Ser. 301, 253–265
52. Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of
4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH
receptor isoforms and hepatic cytochrome P450 1A metabolism. Toxicol. Appl.
Pharmacol. 217, 308–321.
53. Incardona, J.P., Carls, M.G., Day, H.L., Sloan, C.A., Bolton, J.L., Collier, T.K., Scholz,
N.L., 2009. Cardiac arrhythmia is the primary response of embryonic Pacific herring
(Clupea pallasi) exposed to crude oil during weathering. Environ. Sci. Technol. 43, 201–
207.
54. D’Costa A, Shepherd IT. Zebrafish development and genetics: Introducing

undergraduates to developmental biology and genetics in a large introductorylaboratory
class. Zebrafish. 2009;6(2):169-177. doi:10.1089/zeb.2008.0562
55. Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N. Identification
of 315 genes essential for early zebrafish development. Proc Natl Acad Sci
U S A. 2004;101(35):12792-12797.doi:10.1073/pnas.0403929101
72
56. Kim MS, Louis KM, Pedersen JA, Hamers RJ, Peterson RE, Heideman W. Using
citrate-functionalized TiO2 nanoparticles to study the effect of particle size on
zebrafish embryo toxicity. Analyst. 2014;139(5):964-972. doi:10.1039/c3an01966g
57. Iravani S. Green synthesis of metal nanoparticles using plants. Green Chem.
2011;13(10):2638-2650. doi:10.1039/c1gc15386b
58. Verma SK, Jha E, Panda PK, et al. Mechanistic insight into ROS and neutral lipid
alteration induced toxicity in the human model with fins (Danio rerio) by
industrially synthesized titanium dioxide nanoparticles. Toxicol Res (Camb).
2018;7(2):244-257. doi:10.1039/c7tx00300e
59. Kumari P, Panda PK, Jha E, et al. Mechanistic insight to ROS and Apoptosis
regulated cytotoxicity inferred by Green synthesized CuO nanoparticles from
Calotropis gigantea to Embryonic Zebrafish. Sci Rep. 2017;7(1):1-17.
doi:10.1038/s41598-017-16581-1
60. Ellis, T.R., Crawford, B.D., 2016. Experimental dissection of metalloproteinase

inhibitionmediated and toxic effects of phenanthroline on zebrafish development. Int J Mol Sci 17,
1503
61. Peng X, Sun X, Yu M, Fu W, Chen H, Chen J. Chronic exposure to environmental concentrations

of phenanthrene impairs zebrafish reproduction. Ecotoxicol Environ Saf. 2019 Oct
30;182:109376. doi: 10.1016/j.ecoenv.2019.109376. Epub 2019 Jun 26. PMID: 31254851.
62. Butler JD, Parkerton TF, Letinski DJ, Bragin GE, Lampi MA, Cooper KR. A novel passive
dosing system for determining the toxicity of phenanthrene to early life stages of zebrafish. Sci
Total Environ. 2013 Oct 1;463-464:952-8. doi: 10.1016/j.scitotenv.2013.06.079. Epub 2013 Jul
18. PMID: 23872248.
63. Heintz, R.A., Rice, S.D., Wertheimer, A.C., Bradshaw, R.F., Thrower, F.P., Joyce, J.E., Short,
J.W., 2000. Delayed effects on growth and marine survival of pink salmon Oncorhynchus
73
gorbuscha after exposure to crude oil during embryonic development. Mar. Ecol. Prog. Ser. 208,
205–216
Toxicol. Appl. Pharmacol. 196, 191–205.
65. Carls, M.G., Heintz, R.A., Marty, G.D., Rice, S.D., 2005. Cytochrome P4501A induction in oil-
exposed pink salmon Oncorhynchus gorbuscha embryos predicts reduced survival potential. Mar.
Ecol. Prog. Ser. 301, 253–265.
66. Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of 4-ring
polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor
isoforms and hepatic cytochrome P450 1A metabolism. Toxicol. Appl. Pharmacol. 217, 308–321
67. Incardona, J.P., Carls, M.G., Day, H.L., Sloan, C.A., Bolton, J.L., Collier, T.K., Scholz,
N.L.,2009. Cardiac arrhythmia is the primary response of embryonic Pacifific herring (Clupea
pallasi) exposed to crude oil during weathering. Environ. Sci. Technol. 43, 201–207.
69. Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of 4-ring
polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor
isoforms and hepatic cytochrome P450 1A metabolism. Toxicol. Appl. Pharmacol. 217, 308–321
70. Billiard, S.M., Timme-Laragy, A.R., Wassenberg, D.M., Cockman, C., Di Giulio, R.T., 2006.
The role of the aryl hydrocarbon receptor pathway in mediating synergistic developmental
toxicity of polycyclic aromatic hydrocarbons to zebrafish. Toxicol. Sci. 92, 526–536.
74
71. Hendon, L.A., Carlson, E.A., Manning, S., Brouwe, M., 2008. Molecular and developmental
effects of exposure to pyrene in the early life-stages of Cyprinodon variegates. Comp. Biochem.
Physiol. C: Pharmacol. Toxicol. 147, 205–215
morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol.
Appl. Pharmacol. 196, 191–205.
74. Vignet C, Devier MH, Le Menach K, Lyphout L, Potier J, Cachot J, Budzinski H, Bégout ML,
Cousin X. Long-term disruption of growth, reproduction, and behavior after embryonic exposure of
zebrafish to PAH-spiked sediment. Environ Sci Pollut Res Int. 2014 Dec;21(24):13877-87. doi:
10.1007/s11356-014-2585-5. Epub 2014 Mar 23. PMID: 24659400.
75. Sogbanmu TO, Nagy E, Phillips DH, Arlt VM, Otitoloju AA, Bury NR. Lagos lagoon sediment
organic extracts and polycyclic aromatic hydrocarbons induce embryotoxic, teratogenic and
genotoxic effects in Danio rerio (zebrafish) embryos. Environ Sci Pollut Res Int. 2016
Jul;23(14):14489-501. doi: 10.1007/s11356-016-6490-y. Epub 2016 Apr 11. PMID: 27068906;
PMCID: PMC4943991.
76. Zhang Y, Wang C, Huang L, Chen R, Chen Y, Zuo Z. Low-level pyrene exposure causes cardiac
toxicity in zebrafish (Danio rerio) embryos. Aquat Toxicol. 2012 Jun 15;114-115:119-24. doi:
10.1016/j.aquatox.2012.02.022. Epub 2012 Mar 3. PMID: 22446823.
77. Victoria A. Sleight, Adil Bakir, Richard C. Thompson, Theodore B. Henry,Assessment of

microplastic-sorbed contaminant bioavailability through analysis of biomarker gene expression in
larval zebrafish,Marine Pollution Bulletin,Volume 116, Issues 1–2,2017,Pages 291-297,ISSN 0025-
75
326X,https://doi.org/10.1016/j.marpolbul.2016.12.055.(https://www.sciencedirect.com/science/artic
le/pii/S0025326X16310591)
78. Kim, K., Jeon, H.J., Choi, S.D., Tsang, D.C.W., Oleszczuk, P., Ok, Y.S., Lee, H.S., Lee, S.E., 2017.
Combined toxicity of endosulfan and phenanthrene mixtures and induced molecular changes in
adult zebrafish (Danio rerio). Chemosphere 194, 30–41.
79. Alysha D. Cypher, Joanna Consiglio, Brian Bagatto,Hypoxia exacerbates the cardiotoxic effect of
the polycyclic aromatic hydrocarbon, phenanthrene in Danio rerio,Chemosphere Volume
183 ,2017,Pages 574-581,ISSN 0045-
6535,https://doi.org/10.1016/j.chemosphere.2017.05.109.(https://www.sciencedirect.com/science/ar
ticle/pii/S0045653517308135)
80. Hooftman , R.N., Evers-de Ruiter, A , Investigations into the aquatic toxicity of
phenanthrene.:cover report for reproduction tests with the waterflea Daphnia magna and an Early
Life Stage (ELS) test with the zebrafish Brachydanio rerio, 1992
81. Sun Y, Yu H, Zhang J, Yin Y, Shi H, Wang X. Bioaccumulation, depuration and oxidative stress in
fish Carassius auratus under phenanthrene exposure. Chemosphere. 2006 May;63(8):1319-27. doi:
10.1016/j.chemosphere.2005.09.032. Epub 2005 Nov 15. PMID: 16293290.
82. K Maskaoui, J.L Zhou, H.S Hong, Z.L Zhang,Contamination by polycyclic aromatic hydrocarbons
in the Jiulong River Estuary and Western Xiamen Sea, China,Environmental PollutionVolume 118,
Issue 1 2002,Pages 109-122,ISSN 0269-7491, https://doi.org/10.1016/S0269-7491(01)00208-1.
(https://www.sciencedirect.com/science/article/pii/S0269749101002081)
morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol.
Appl. Pharmacol. 196, 191–205.
76
84. Yeung, Chanel 1993-The Cardiorespiratory and metabolic toxicity of acute naphthalene and pyrene
exposure in adult zebrafish (Danio rerio): Relationship to aryl hydrocarbon receptor activation
http://hdl.handle.net/10388/12080
85. Pecquet (nee Willis), Alison & Oris, James. (2014). Acute photo-induced toxicity and toxicokinetics
of single compounds and mixtures of polycyclic aromatic hydrocarbons in zebrafish. Environmental
Toxicology and Chemistry. in press.
86. Zhang, Youyu & Wang, Chonggang & Huang, Lixing & Chen, Rong & Chen, Yixin & Zuo,
Zhenghong. (2012). Low-level pyrene exposure causes cardiac toxicity in zebrafish (Danio rerio)
embryos. Aquatic toxicology (Amsterdam, Netherlands). 114-115. 119-24.
10.1016/j.aquatox.2012.02.022.
87. Sogbanmu, T.O., Nagy, E., Phillips, D.H. et al. Lagos lagoon sediment organic extracts and
polycyclic aromatic hydrocarbons induce embryotoxic, teratogenic and genotoxic effects in Danio
rerio (zebrafish) embryos. Environ Sci Pollut Res 23, 14489–14501 (2016).
https://doi.org/10.1007/s11356-016-6490-y
88. McGrath P, Li CQ. Zebrafish: a predictive model for assessing drug-induced toxicity.
Drug Discov Today. 2008;13(9-10):394-401.
doi:10.1016/j.drudis.2008.03.002
89. Cheng SH, Wai AWK, So CH, Wu RSS (2000) Cellular and molecular basis of cadmium-induced
deformities in zebrafish embryos. Environ Toxicol Chem 19(12):3024–3031.
90. Khan, Z.A., Labala, R.K., Yumnamcha, T., Devi, S.D., Mondal, G., Devi, H.S., Rajiv, C., Bharali,
R., Chattoraj, A., 2018. Artificial light at night (ALAN), an alarm to ovarian physiology: a study of
possible chronodisruption on zebrafish (Danio rerio). Sci. Total Environ. 628, 1407–1421.
91. Mukherjee P, Ahmad A, Mandal D, et al. Fungus-Mediated Synthesis of Silver

Nanoparticles and Their Immobilization in the Mycelial Matrix: A Novel Biological Approach
to Nanoparticle Synthesis. Nano Lett. 2001;1(10):515-519. doi:10.1021/nl0155274
77
92. Aebi et al., (1974) catalase. In H.U. Bergmeyer, Editor, methods of enzymeatic analysis,
Academic press, London.
93. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction. Anal. Biochem. 95, 351–358.
94. Habig, W.; Pabst, M.J.; Jacoby, W.B. Glutathione-S-transferase: the first step
inmercapturic acid formation. J. Biol. Chem., 1974, 249, 1730-1739.
95. OECD, 2013. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Publishing.
96. Liu, H., Sun, P., Liu, H., Yang, S., Wang, L., Wang, Z., 2015. Hepatic oxidative stress
biomarker responses in freshwater fish Carassius auratus exposed to four benzophenone
UV filters. Ecotoxicology and Environmental Safety 119, 116-122.
97. Gao, L., Yuan, T., Zhou, C., Cheng, P., Bai, Q., Ao, J., Wang, W., Zhang, H., 2013.
Effects of four commonly used UV filters on the growth, cell viability and oxidative
stress responses of the Tetrahymena thermophila. Chemosphere 93, 2507-2513.
98. Barata, C., Varo, I., Navarro, J.C., Arun, S., Porte, C., 2005. Antioxidant enzyme
activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to
redox cycling compounds. Comparative Biochemistry and Physiology Part C:
Toxicology & Pharmacology 140, 175-186.
99. Gao, L., Yuan, T., Zhou, C., Cheng, P., Bai, Q., Ao, J., Wang, W., Zhang, H., 2013.
Effects of four commonly used UV filters on the growth, cell viability and oxidative
stress responses of the Tetrahymena thermophila. Chemosphere 93, 2507-2513.
106. Barata, C., Varo, I., Navarro, J.C., Arun, S., Porte, C., 2005. Antioxidant enzyme activities
and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox
cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology &
78
Pharmacology 140, 175-186.
107. Butler JD, Parkerton TF, Letinski DJ, Bragin GE, Lampi MA, Cooper KR. A novel passive
dosing system for determining the toxicity of phenanthrene to early life stages of zebrafish.
Sci Total Environ. 2013 Oct 1;463-464:952-8. doi: 10.1016/j.scitotenv.2013.06.079. Epub
2013 Jul 18. PMID: 23872248.
108. Sogbanmu, T.O., Nagy, E., Phillips, D.H. et al. Lagos lagoon sediment organic extracts and
polycyclic aromatic hydrocarbons induce embryotoxic, teratogenic and genotoxic effects
in Danio rerio (zebrafish) embryos. Environ Sci Pollut Res 23, 14489–14501 (2016).
https://doi.org/10.1007/s11356-016-6490-y
109. Mai Y, Peng S, Li H, Lai Z. Histological, biochemical and transcriptomic analyses reveal
liver damage in zebrafish (Danio rerio) exposed to phenanthrene. Comp Biochem Physiol
C Toxicol Pharmacol. 2019 Nov;225:108582. doi: 10.1016/j.cbpc.2019.108582. Epub
2019 Jul 31. PMID: 31374294.
110. Kim K, Jeon HJ, Choi SD, Tsang DCW, Oleszczuk P, Ok YS, Lee HS, Lee SE. Combined
toxicity of endosulfan and phenanthrene mixtures and induced molecular changes in adult
Zebrafish (Danio rerio). Chemosphere. 2018 Mar;194:30-41. doi:
10.1016/j.chemosphere.2017.11.128. Epub 2017 Nov 23. PMID: 29197246.
111. A novel passive dosing system for determining the toxicity of phenanthrene to early life
stages of zebrafish
112. Chen TH, Wang YH, Wu YH (2011) Developmental exposures to ethanol or

dimethylsulfoxide at low concentrations alter locomotor activity in larval zebrafish:
implications for behavioral toxicity bioassays. Aquat Toxicol 102:162–166
113. Li VW, Tsui MP, Chen X, Hui MN, Jin L, Lam RH, Yu RM, Murphy MB, Cheng J, Lam
PK, Cheng SH. Effects of 4-methylbenzylidene camphor (4-MBC) on neuronal and
79
muscular development in zebrafish (Danio rerio) embryos. Environ Sci Pollut Res Int.
2016 May;23 (9):8275-85.
114. Hen Chow, E.S., Cheng, S.H., 2003. Cadmium affects muscle type development and axon
growth in zebrafish embryonic somitogenesis. Toxicol. Sci. 73, 149-159.
Toxicol. Appl. Pharmacol. 196, 191–205
116. Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of 4-
ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH
receptor isoforms and hepatic cytochrome P450 1A metabolism. Toxicol. Appl. Pharmacol.
217, 308–321
117. Ved Prakash, Veena Jain, Shweta Singh Chauhan, Ramakrishnan Parthasarathi, Somendu K.
Roy, Sadasivam Anbumani,Developmental toxicity assessment of 4-MBC in Danio rerio
embryo-larval stages,Science of The Total Environment,Volume 804, 2022,149920,ISSN
0048-9697,https://doi.org/10.1016/j.scitotenv.2021.149920.
(https://www.sciencedirect.com/science/article/pii/S0048969721049950)
118. Nerbonne, J.M., Kass, R.S., 2005. Molecular Physiology of Cardiac Repolarization. Physiol.
Rev. 85, 1205-1253.
119. Ren, F., Ji, C., Huang, Y., Aniagu, S., Jiang, Y., Chen, T., 2020. AHR-mediated ROS
production contributes to the cardiac developmental toxicity of PM2. 5 in zebrafish
embryos. Sci. Total Environ. 719, 135097.
120. Jin, H., Ji, C., Ren, F., Aniagu, S., Tong, J., Jiang, Y., Chen, T., 2020. AHR-mediated
oxidative stress contributes to the cardiac developmental toxicity of trichloroethylene in
zebrafish embryos. J. Hazard. Mater. 385, 121521
80
121. Barata, C., Varo, I., Navarro, J.C., Arun, S., Porte, C., 2005. Antioxidant enzyme activities
and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox
cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology &
Pharmacology 140, 175-186
122. Xing, J., Wang, G., Zhao, J., Wang, E., Yin, B., Fang, D., Zhao, J., Zhang, H., Chen, Y.Q.,
Chen, W., 2016. Toxicity assessment of perfluorooctane sulfonate using acute and
subchronic male C57BL/6J mouse models. Environ. Pollut. 210, 388–396
123. Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of 4-
ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH
receptor isoforms and hepatic cytochrome P450 1A metabolism. Toxicol. Appl. Pharmacol.
217, 308–321
124. Xu, D., Li, C.,Wen, Y., Liu,W., 2013.Antioxidant defense system responses and DNA
damage of earthworms exposed to perfluorooctane sulfonate (PFOS). Environ. Pollut. 174,
121–127.
81

Developmental toxicity of PAHs in zebrafish embryos

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Developmental toxicity of PAHs in zebrafish embryos

Uploaded by

Copyright:

Available Formats

DEVELOPMENTAL TOXICITY OF SELECTED PAHS IN

ZEBRAFISH (Danio rerio) EMBRYOS

Dissertation submitted in partial fulfillment for the degree of

B.Tech & M. Tech Dual Degree in Biotechnology

Under the Supervision of

CSIR-INDIAN INSTITUTE OF TOXICOLOGY RESEARCH

Date: 24/ 05/ 2022

Date: 24/ 05/ 2022

Table of Contents................................................................................ ………………………..v-vii

List of Figures...................................................................................... ………………………..viii

Chapter 1- Introduction................................................................................................................. 11-14

1.1 Current Research................................................................................................................... 14

1.2 Scope and Objectives..................................................................... ……………………….………..14

Chapter 2- Review of Literature.........................................................……………………………………15-30

2.1 Phenanthrene................................................................................................................... 15-18

2.2 Pyrene.............................................................................................................................. 19-22

2.3 Zebrafish Testing Standarization..........................................................................................23

2.4 Summary Table.................................................................................................................24-30

Chapter 3- Materials and Methods................................................................................................ 31-45

3.1 Materials.......................................................................................................................... 32-35

3.2.2 Zebrafish and embryo Maintenance......................................…………………...………………..….38

3.2.4 Zebrafish Developmental Profile.............................................................................. ...39-40

3.2.5 Zebrafish Development: An overview......................................................................... …40-41

3.2.6 PAHs Exposure..................................................................................................................42

3.2.6.1 Phenanthrene Exposure................................................................................................... 42

3.2.6.2 Pyrene Exposure..............................................................................................................42

3.2.7 Embryo-larval toxicity test................................................................................................. 42

3.2.8 Determination of LC50..........................................................................................................................................42

3.2.8.1 Determination of LC50 Of Phenanthrene in zebrafish embryos..................................... 43

3.2.8.1 Determination fo LC50 of Pyrene in zebrafish embryos................................................. 43

3.2.9 Analysis of developmental defects.................................................................................... 44

3.2.10 Isolation of total protein....................................................................................................44

3.2.11 Antioxidant defense enzymes activity..............................................................................44-45

4.1 Phenanthrene........................................................................................................ 47-49

4.1.1 LC50 of Phenathrene in zebrafish embryos at 96 hpf.......................................47

4.1.2 Developmental defects induced by PAHs exposure in zebrafish embryos at

environment relevant concentration.....................................................................48

4.1.3 Oxidative stress profiling in zebrafish larvae at 4df............................................ 49-51

4.2.1 LC50 of Phenathrene in zebrafish embryos at 96 hpf...........................................51-52

environment relevant concentration.....................................................................52

4.2.3 Oxidative stress profiling in zebrafish larvae at 4df..........................................52-55

4.3 Morphological abnormalities................................................................................56-57.

6.1 - Future Direction..................................................................................................63

Chapter 7 - References............................................................................................................. 64-77

Figure 1: Structure of Phenanthrene

 μg/g Micrograms per gram

1.2 Current Research work

1.3 Scope and Objectives

Objectives of the study:

1. Toxicity evaluation of Phenanthrene in zebrafish embryo at 96 hpf

2. Toxicity evaluation of Pyrene in zebrafish embryo at 96 hpf

Fig 1: Structure of Phenanthrene ( C14H10 )

Fig 2: Structure of Pyrene (C16H10)

i. To construct a dose-response curve.

observed in earlier investigations.

iv. To produce epidemiological studies to explain population observations, such as the

protracted inquiry of the link between smoking and lung cancer.

v. Validation of new testing or investigative procedures, particularly those carried out in

vitro rather than on animals.

and repeatable process through independent laboratory testing.

good connection between TC50 measures of mortality, morphological abnormalities, and

teratogenicity in zebrafish embryos and reported mammalian LD50 values.

Asellus 0.70µg/L 7 days Bioconcentratio Maximum isopod concentrations occurred Hattum et