DQE-R(1)

Research Proposal/Synopsis for M.S Thesis

Department of Biology

1. Name of the Student: Rehana balqees

2. VU ID: MS170401619 3. Session: 2017

4. Semester: 3rd 5. Field of Specialization: Zoology

6. Title of Research Proposal: Effect of nano-particles (FeO) on hematological

and serological profiles of Grass Carp

7. Date of Enrolment in Research: As per approval of synopsis

8. Duration of Proposed Research: 2 semesters

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Supervisor, Supervisory Committee (SC) Information

1. Name of Supervisor: Dr Aasma Noureen

Designation: Assistant professor Email ID: aasma.noureen@vu.edu.pk

Affiliation: Virtual University

2. Name of Supervisory Committee (SC) Member 1: Dr Mohammad Baber

Designation: Assistant Professor Email ID:

muhammadbabar@bzu.edu.pk

Affiliation: BZU, Multan

3. Name of Supervisory Committee (SC) Member 2: Ms.Aneela Kanwal

Designation: Tutor/Instructor Email ID: aneela.kanwal@vu.edu.pk

Affiliation: Virtual University

Topic

Effect of nano-particles (FeO) on hematological and serological profiles of Grass

Carp

Summary

Fish is an essential supply of meals and represents a key part of many natural
meals chains. Fish is especially nutritious due to its excessive protein content and
the presence of omega 3fattyacids, fat, amino acids and vitamins. incorporates
numerous minerals, such as Ca, Fe, Cd, Pb, Cu, and Zn.

The organisms which can be streamlined vertebrates own gills for respiration, fins
for locomotion and jawed oviparous and viviparous. The have variety in their
length range,morphology and habitat. Fishes are incorporated in the oldest
vertebrate which are developed at some point of devonian and Silurian duration
that are about 350 to 450 million years ago. Aquatic creature need healthy
surrounding to live and have sufficient nutrients for their development. Number of
home appliances and household products, in the manufacture of textiles and
electronics, in addition to medical merchandise and in bioremediation technology.
Actually all of those will create waste water or effluents, and there are issues about
the environmental dangers of nanotechnology which need to be balanced in
opposition to their undoubted blessings to human society
So this work is designed to check the effect of nanoparticles (Feo)on hematological
and serological profiles in grass carp . I will collect the fish sample of random size
from the fish seed hatchery Muzaffar Garh and transport it to fish laboratory at VU
lab ,here it will b acclimatized in laboratory condition for two weeks in fiberglass
container in recirculation aerated system. The initial length and weight of each fish
sample will b recorded. Precise temperature pH. and oxygen concentration will b
maintained throughout the experiment. At the end of experimental design I will
collect the fish by using fish net and will take the blood sample divide it into parts
one for hematological parameters and second for serum biochemical profiling.

Introduction

Nanoscience is the study of materials on the nanoscale level approximately

between 1 and 100 nm (Rotello,2003) and deals with manipulation of formation of
two and three-dimensional assemblies of molecular scale building blocks into well-
defined nanostructures or nanomaterials. Metal oxide nanoparticles are extensively
used in a considerable number of applications in food, material, chemical and
biological sciences (Aitken et al., 2006). It is well known that bulk materials based
on TiO2, SiO2, aluminium and iron oxides have been massively produced for
many years.

More recently, nano particulate versions of these metal oxides have been
manufactured and introduced in commercial products such as cosmetics and
sunscreens (TiO2, Fe2O3 and ZnO) (Nowack and Bucheli, 2007), fillers in dental
fillings (SiO2), in catalysis (TiO2) (Aitken et al., 2006), and as fuel additives
(CeO2) (Laosiripojana et al., 2005). However, due to the increasing interest of
these nanomaterials to be used as potential devices for biomedical applications,
water soluble iron oxide NPs is an active area of research. Iron is necessitated by
most of living organisms.

Iron oxide nanoparticles were synthesized biologically using potato as starch

template (FeSo4.7H2O as substrate). The X-ray diffraction (XRD) and Scanning
Electron Microscope (SEM) analysis revealed that the particles were spherical in
shape and the size ranges from 29-40 nm. Toxicity tests were performed to
investigate the possible harmful effects on fresh water fish Oreochromis
mossambicus exposed to as synthesized α-Fe2O3 NPs at exponential
concentrations (0.5, 5 and 50 μg/mL). The significant changes in haematological
(RBC, WBC, Hb, HCT) and biochemical parameters (SGOT, SGPT) were
observed after 96 h in treated groups than control, which may be a consequence of
iron oxide NPs in the blood cells of fish. Another factor worthy of mention is that
the increase in aquaculture production has been accompanied by the appearance of
emerging diseases.

The aquatic environment is a dynamic medium capable of transporting pathogens

over many kilometres (Munro & Gregory 2009; Murray 2013). Furthermore,
pathogens can be moved between farms, due to the substantial movement of
organisms and vehicles, creating a highly complex web of disease transmission
(Munro & Gregory 2009; Murray 2013). To control infectious diseases, large
quantities of veterinary drugs such as antibiotics are employed, in some cases
indiscriminately, resulting in the appearance of resistant pests (Gjedrem 2015;
Huang et al. 2015; Lafferty et al.

To address the challenges mentioned above, the UK Centre for Environment,

Fisheries and Aquaculture Science (CEFAS), and the UK and Asian governments,
are seeking to develop new aquaculture technology, such as DNA‐based diagnostic
techniques for the rapid detection of potentially devastating diseases. According to
CEFAS, these new technologies would reduce annual losses across the global
aquaculture industry by up to US$6 billion (new technology aims to combat
disease in aquaculture – Press releases – GOV.UK 2016). Another technology that
has attracted widespread interest among the scientific community for use in
aquaculture is nanotechnology. These new approaches in aquaculture will help to
address the challenges involved in increasing production, reducing environmental
degradation, controlling diseases and ensuring food safety (Bhattacharyya et al.
2015; Huang et al. 2015).

Maintaining fish resources Fish farming is the world’s fastest-developing area of

agricultural enterprise. Patron demand for fish merchandise is growing. At the
equal time, wild fish shares are unexpectedly declining, especially because of over-
fishing. Merchandise from capture fisheries and aquaculture supplied the arena
with about 106 million heaps in 2004. Aquaculture contributes to the arena food
supply with more than 45.five million tons (43%) of fish and shellfish
annually(Díaz-Almela, Marbà et al. 2008) Aquaculture is developing, expanding
and intensifying in almost all regions of the arena. Aquaculture seems to have the
capability to make a great contribution to this increasing demand for aquatic food
in most areas of the world; on the other hand, for its achievement, the region (and
aqua farmers) will have to face great challenges.. Within aquaculture's extensive
variety of species and developed practices, two different subsectors have appeared
throughout the past decade4. The rst institution includes business farms that mostly
use extensive and semi intensive in depth methods to provide medium- to
excessive-valued supplies for regional or worldwide markets and for serum
biochemical profiling.
Aquatic nanotoxicologists and ecotoxicologists have begun to identify the unique
properties of the nanomaterials (NMs) that potentially affect the health of wildlife.
The scientific aims are to discuss the main challenges nanotoxicologists currently
face in aquatic toxicity testing, including the transformations of NMs in aquatic
test media and modes of NM interference.

NPs in aquaculture are a challenging topic that must be developed in the near
future to assure human health and environmental safety.

Research Question(s)

1. How would you determine the effect of (Feo) nanoparticle in Grass carp?

2. What would be the effect of iron oxide nanoparticle on serological profile of

grass carp?

3. How would you determine the toxic effect of (Feo) nanoparticle in

Ctenopharyngodon idella (grass carp)?

4. What is the effect of nano materials (FeO) on the physiology of fish?

Research Objectives

1. To evaluate the effects of nano iron on blood biochemistry of grass carp

2. To determine the positive or negative effect of nano iron on grass carp

Socio-Economic Importance/Benefits

This research would help to know the effect of nanoparticles in blood serum of
grass carp
Literature review

Adeel, M., at all 2017 study the environmental impact of estrogens on fish, human,
and rat.

Hormones are used in fish farming to increase fish production when one sex of a
species has the capacity to grow bigger and faster than the other sex. The technique
to increase fish production based on sexual dimorphism mostly uses estrogens and
androgens. These chemicals must be handled carefully to ensure environmental,
biological and food safety, since they can contaminate the environment and
promote changes in the endocrine system inducing adverse effects on the consumer
health. Another important issue is the reliability of the analytical methods used to
identify and/or quantify hormone residues in the meat of treated fish. Therefore,
the aim of this review is to bring into view the use of hormones in fish farming, the
possible impacts of this practice on humans and on the environment and to discuss
the current methods of analysis for determining the hormone residues in food
matrices, especially in fish.

America and China Environmental sustainability of freshwater prawn farming and

recent technological developments in the culture methods has boosted
Macrobrachium production The fisheries and aquaculture industries can be
revolutionized by using nanotechnology with new tools, such as rapid diagnosis of
diseases which will enhance the ability of cultivable organisms to uptake drugs like
hormones, vaccines and external nutrients etc.,The metal nanoparticles (NPs), such
as Fe, FeO, Se, Zn, ZnO, Cu, and MgO etc., play a crucial role in aquaculture
operations. It has been reported that iron NPs when fed to young carp, Carassius
auratus and sturgeon, Acipenser gueldenstaedtii showed a faster growth rate, 30%
and 24% respectively . Different Se sources (nano-Se and Selenomethionine)
supplemented diets improved the growth, antioxidant status and muscle Se
concentration of Crucian carp, Carassius auratus gibelio]. In recent reports, dietary
supplementation of Zn, nano Zn and Cu have produced better survival and growth
in M. rosenbergii .Furthermore, iron-based nanoparticles (NPs) are also used for
soil and groundwater remediation and water treatment processes ]. Iron is one of
the most essential micron.(Muralisankar T and Bhavan PS et al.2015)

This study was performed to assess the growth promoting potential of dietary
Fe2O3 NPs in M.rosenbergi,Fe2O3 NPs was supplemented at 0, 10, 20, 30, 40 and
50 mg kg-1 with the basal diet (containing 5.45 mg Iron kg-1). The concentration
of Fe in Fe2O3 NPs supplemented diet was increased correspondingly. Fe2O3 NPs
supplemented diets were fed to M. rosenbergii PL (initial weight, 0.11±0.04 g) for
a period of 90 days. In the carcass of experimental prawns, the content of Fe was
found to be elevated significantly when compared with controll, simillarly, Cu, Zn,
Ca, Mg, Na and K levels were also elevated. Therefore, the supplemented Fe2O3
NPs were taken up and incorporated into the body of the prawns, which facilitates
absorption of other minerals as well. Significant (P < 0.05) improvements were
observed in survival, growth, activities of digestive enzymes (protease, amylase
and lipase), concentrations of basic biochemical constituents (total protein, amino
acid, carbohydrate and lipid, profiles of amino acids and fatty acids) and
population of haemocytes [total and differential (hyalinocytes, semigranulocytes
and granulocytes)] at 10 and 20 mg kg-1 Fe2O3 NPs supplemented feeds fed
prawns, whereas, 30-50 mg kg-1 Fe2O3 NPs supplementations showed negative
performance. The polynomial regression analysis revealed that a dietary
requirement of 24.56 mg kg-1 Fe2O3 NPs for optimal growth of prawns. SDS-
PAGE revealed separation of 14 polypeptide bands in the muscle of prawns.
Among these, the staining intensity of 116, 99, 50, 26, 20, 18, 15, 14 and 13 kDa
polypeptide bands were found to be higher in 20 mg kg-1 Fe2O3 NPs
supplemented feed fed prawn when compared with control. The lowest food
conversion ratio (FCR) recorded in 20 mg kg-1 of Fe2O3 NPs supplemented diet
fed PL reflects the superior quality of the diet prepared. Therefore, up to optimum
level Supplementation of Fe2O3 NPs, the general health of the experimental
prawns was improved due to its incorporation in the body. But beyond the
optimum level it turned towards toxic. Therefore significant (P<0.05) elevations in
activities of antioxidant enzymes (superoxide dismutase and and catalase), lipid
peroxidation, and metabolic enzymes (glutamic oxaloacetic transaminase and
glutamic pyruvic transaminase) were recorded in 30-50 mg kg-1 Fe2O3 NPs
supplemented diets fed prawns. The better profiles of protein, amino acids, fatty
acids, biochemical constituents and haemocytes helped to attain appreciable
survival and growth of M. rosenbergii PL. Thus, this study recommends Fe2O3
NPs supplementation up to 20 mg kg-1 for its sustainable maintenance. Rather MA
and Sharma et al.(2015)

Muralisankar T, Bhavan PS, Radhakrishnan S, Seenivasan C, Srinivasan V,

Santhanam P. Effects of dietary zinc on the growth, digestive enzyme activities,
muscle biochemical compositions, and antioxidant status of the giant freshwater
prawn Macrobrachium rosenbergii. Aquaculture 2015;

The effect of nanoparticles on the spermatogenesis is influenced by such factors as

dosage, administration routes, size of nanoparticles, and type of animal ].Recent
animal research has demonstrated that nanoparticles can deposit in various organs
throughout the body, and move from the initial absorption site, e.g., the lungs and
skin to secondary organs such as ovaries and testes . The aim of this study was to
examine the adverse effect of FeNP on the testicular tissue and sperms of adult
male mice.(Wang ECW AZ.et al.2015)

Kanipandian N, Thirumurugan R (2014) A feasible approach to phyto-mediated

synthesis of silver nanoparticles using industrial crop Gossypium hirsutum (cotton)
extract as stabilizing agent and assessment of its in vitro biomedical potential.

The increasing use of nano based-products induces the potential hazards from their
manufacture, transportation, waste disposal and management processes. In this
report, scientist emphasized the acute toxicity of silver nanoparticles (AgNPs)
using freshwater fish Labeo rohita as an aquatic animal model. The AgNPs were
synthesized using chemical reduction method and the formation of AgNPs was
monitored by UV–Visible spectroscopy analysis. The functional groups, crystaline
nature and morphological characterizations were carried out by fourier transform
infrared spectroscopy (FTIR), X-ray diffraction (XRD) and high resolution
transmission electron microscopy (HRTEM) analysis. UV-Vis range was observed
at 420 nm and XRD pattern showed that the particles are crystalline nature.
HRTEM analysis revealed that the morphology of particles was spherical and size
ranges between 50 and 100 nm. This investigation was extended to determine the
potential acute toxicity, L. rohita was treated orally with the lethal concentration
(LC50) of AgNPs. The antioxidative responses were studied in the three major
tissues such as gill, liver and muscle of L. rohita. The results of this investigation
showed that increasing the concentration of AgNPs led to bioaccumulation of
AgNPs in the major tissues. The haematological parameters showed significant
alterations in the treated fish. The histological changes caused by chemically
synthesized AgNPs demonstrated the damages in the tissues, primary lamella and
blood vessels of L. rohita. The histological study also displayed the formation of
vacuolation in liver and muscle when compared with untreated tissues(control) of
L. rohita ( Kanipandian N, Thirumurugan R (2014)

An experiment was conducted to compare the effects of iron oxide nanoparticles

(T1) and ferrous sulfate(T2) on Indian major carp, Labeo rohita H. There were
significant differences (P\0.05) in the final weight of T1and T2 compared with the
control. Survival rates were not affected by the dietary treatments. Fish fed a basal
diet(control) showed lower (P\0.05) iron content in muscle compared to T1 and T2.
Furthermore, the highest value (P\0.05) of iron content was observed in T1. In
addition,RBCs and hemoglobin levels were significantly higher in T1 as compared
to other treated groups. Different innate immune parameters such as respiratory
burst activity,bactericidal activity and myeloperoxidase activity were higher in
nano-Fe-treated diet (T1) as compared to other iron source (T2) and control in 30
days post-feeding.

Moreover, nano-Fe appeared to be more effective (P\0.05) than ferrous sulfate in

increasing muscle iron and hemoglobin contents. Dietary administration of nanoFe
did not cause any oxidative damage, but improved antioxidant enzymatic activities
(SOD and GSH level) irrespective of different iron sources in the basal
diet.Melodie A (2011)

Spherical superparamagnetic iron oxide nanoparticles have been developed as T2-

negative

contrast agents for magnetic resonance imaging in clinical use because of their
biocompatibility and ease of synthesis; however, they exhibit relatively low
transverse relaxivity Here scientist report a new strategy to achieve high transverse
relaxivity by controlling the morphology of iron oxide nanoparticles. We
successfully fabricate size-controllable octapod iron oxide nanoparticles by
introducing chloride anions. The octapod iron oxide nanoparticles (edgelength of
30 nm) exhibit an ultrahigh transverse relaxivity value (679.3±30 mM 1 s 1),

indicating that these octapod iron oxide nanoparticles are much more effective T2
contrast

agents for in vivo imaging and small tumour detection in comparison with
conventional iron

oxide nanoparticles, which holds great promise for highly sensitive, early stage and
accurate

detection of cancer in the clinic.(Terreno,E., et al.2010)

Methodology/Research Design

The sudy will be conducted at the research lab of Virtual

University of Pakistan I will collect the fish sample ofdifferent size
,randomly selected fingerlings of Grass carp(Ctenopharyngodonidella)by
purchasing them from the fish seed hatchery MuzaffarGarh and
transported to fish laboratory atanimal house of Virtual University,
Pakistan. Fish will be acclimatized to laboratory condition for two weeks
in fiber-glass container with recirculation aerated system.I will perfer
these experiment in semi static conditions with renewal of water after
every twenty four hours,temperature and oxygen conc. will be maintained
through out the experimental duration.then I will calculate the LC50
group of seven juvenile fishes were exposed to one of the three
concentrations 200.300.400, mg/kg of (FeO) nano material in feed in the
form of pellets in untreated control group will maintan in parallel.Then I
will observe the fish mortality after 1-7days and also repeated for 1-
15days.

I will design the experiment in short term phase for 1-7days and
long term phase for 1-15days 6 treatment groups were exposed to
different doses of nano materials of Feo. . I will use separate group for
each treatment.All fishes will be fed with ordinary fish diet used in fish
farm in the form of pellets. Nano materials of FeO are mixed with fish
feed to ensure the intake by fish. I will take the blood sample through
direct cardiac puncture and will use these sample for Hematological
parameters Serum biochemical profiling Determination of hematological
parameters.

In hematological parameters blood sample will use for estimation

of Haemoglobin(Hb).The quantitative determination of Hb will be carried
out by using MICRO-LAB 3000 (Merck, Germany). The wavelength of
the instrument will be adjusted at 540 nm. 5 ml Hb solution would be
taken in a tube and mixed with 20µl of whole blood. Mixture would be
left at room temperature for 5 minutes and Hb conentration would be
determined in gd/l.

Pack cell volume(PCV).

The PCV will be calculated by the following formula;

Red blood cells(RBC).

Total Red Blood Cell Count (TRBC) will be determined by using

improved Neubaur Ruling Hemacytometer.)

,Mean cell volum(MCV).

Mean Corpuscular Volume (MCV) will be calculated by using

conventional formula given below

Mean cell haemoglobin(MCH).

Mean Cell Haemoglobin (MCH) would be calculated by using

conventional formula

,Mean cell haemoglobin concentration (MCHC).

Mean Cell Hemoglobin Concentration (MCHC) would be determined by
conventional formula and expressed in g/dl.

Platlets counting.

To calculate the number of platelets, 10 square on either side of a

Neubaur haemocyto-meter would be counted and multiplied by a 10000.
The platelets would be measured in per µL.

Determination of Serological parameters:

Blood samples will be taken in eppendorf tubes and centrifuged at

maximum speed (13000 RPM) for 10 minutes to separate the serum from
the blood cells. The separated serum would be transferred into new
labeled tubes for the determination of glucose, total proteins, albumins,
triacylglycerols, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase
(ALP), calcium (C ), magnesium (M ), inorganic phosphate, uric
acid, cholesterol, urea, creatinine, total billirubin and lactate
dehydrogenase was determined in blood plasma for all treated and
untreated fish groups.

Risk Analysis and Management

 Fish handling is very difficult task and it is very sensitive to transport fish
from fish seed hatchery to Bio lab in sense that it may increase mortality
rate and there is chance to loss collected sample, so care should be taken
to handle fish samples

References.

The environmental impact of estrogens on fish, human, and rat.Adeel, M., at all
(2017)

New technology aims to combat disease in aquaculture – Press releases – GOV.UK

(2016)

Nanotechnology in aquaculture will help to address the challenges involved in

increasing production, reducing environmental degradation, controlling diseases
and ensuring food safety (Bhattacharyya et al. 2015; Huang et al. 2015).

To control infectious diseases, large quantities of veterinary drugs such as

antibiotics are employed, in some cases indiscriminately, resulting in the
appearance of resistant pests (Gjedrem 2015; Huang et al. 2015; )

Effects of dietary zinc on the growth, digestive enzyme activities, muscle

biochemical compositions, and antioxidant status of the giant freshwater prawn
Macrobrachium rosenbergii. Aquaculture . Muralisankar T, Bhavan PS ,et al.(2015)

Nanotechnology: Anoval tool for aquaculture and fisheries development. .Rather

MA, and Sharma et al.(2015).
A feasible approach to phyto-mediated synthesis of silver nanoparticles using
industrial crop Gossypium hirsutum (cotton) Kanipandian N, Thirumurugan R
(2014)

Fish additionally incorporates numerous minerals, such as Ca, Fe, Cd, Pb, Cu, and
Zn (Taweel, Shuhaimi-Othman et al. 2013)

The aquatic environment is a dynamic medium capable of transporting pathogens

over many kilometres (Munro & Gregory 2009; Murray 2013)

Effect of body size on elemental concentration in wild Wallago attu . Yousaf M,

Salam et al. (2012).

The most production is acquired while the bodily parameters are at the best degree
(Lawson 2011).

Iron supplements best increase ferritin.Melodie A (2011)

Spherical superparamagnetic iron oxide nanoparticles have been developed as T2-

negativecontrast agents for magnetic resonance imaging in clinical use.(Terreno,E.,
et al.2010)

Aquaculture contributes to the arena food supply with more than 45.five million
tons (43%) of fish and shellfish annually(Díaz-Almela, Marbà et al. 2008)

Nutritional necessities which might be reported about fish including some vitamin
materials consisting of diet C and vitamin D.)((((Nasi, Brown et al. 2008)

Environmental dangers of nanotechnology which need to be balanced in opposition

to their undoubted blessings to human society (Crane and reachable et al. 2007)
More recently, nanoparticulate versions of these metal oxides have been
manufactured and introduced in commercial products such as cosmetics and
sunscreens (TiO2, Fe2O3 and ZnO) (Nowack and Bucheli, 2007)

Nanoparticles (NPs) within the surroundings now seem inevitable given the rapidly
growing quantity of products containing these materials (Aitken, Chaudhry et al.
2006)

Nanoscience is the study of materials on the nanoscale level approximately

between 1 and 100 nm (Rotello,2003).

Gantt Chart

Provide activity Plan relevant to research/objectives.

Specific Semester-I Semester-II
Objectives
Activities Month-I Month-II Month-III Month-IV Month-I Month-II Month-III Month-IV
Literature’s
Survey
I Start study
Identify
research
area
Formulate
research
question
Strategic
plan for
research
II Write
synopsis
Data
collection
Preparation
of material
II Lab work
Lab work
Data
analysis
III Conclusion
and writing
Thesis writing
& Submission
Details of Funds/Expenditure (if applicable) [Maximum 1 page]

Provide detail of funds and expected expenditure on this research such as

Consumable/Chemicals/field Survey/Transport etc.

S. No. Details No(s) Unit Rate Total

1 Consumables & Chemicals
2 Surveys or Transport
3 Contingencies
4
5
Grand Total

Student Signature
Date: ______

DECLARATION
We hereby agree to supervise the research work as per above proposal/synopsis.

____________________
Signature of Supervisor

Signature of SC Member 1 Signature of SC Member 2

Date: _________ Date: _________
Note: Hard and soft copy of synopsis/research proposal must be submitted to secretary ASRB for final approval.

Profile of Supervisor

Name of Supervisor: _________________________________________________________

Designation: _______________________________________________________________
 Total No. of Impact Factor Research Publications during last 5 years: ____
  Total No. of Publications without Impact Factor during last 5 years: _______________

Ongoing
Research students

Number of MS/M.Phil. Number of PhD students

students

Signature of Supervisor

Endst. No. ___________ Dated:______________

The Proposal entitled “ ”duly

recommended by the Graduate Research Committee (GRC) in its meeting held on __________ is
forwarded to ASRB through the Dean of the Faculty for approval and allocation of funds (if
requested).

Signature / Seal
Chairperson of the Department
Date: ___________

Signature / Seal Signature / Seal

Dean of the Faculty Secretary ASRB
Date: ___________ Date: ___________