Alterations in Haematological Indices of Clarias Garipinus Exposed To Sub Lethal Concentrations of Kola-Pod Mediated Silver-Gold Alloy Nanoparticles


Bachelor Thesis, 2017
45 Pages, Grade: 2.50

Excerpt

Table of Contents

CHAPTER ONE
1.0 INTRODUCTION
1.1 NANOTECHNOLOGY
1.2 NANOPARTICLES
1.2.1 PHYSICAL METHODS OF SYNTHESIZING NANOPARTICLES
1.2.2 CHEMICAL METHOD
1.2.3 BIOLOGICAL METHOD
1.3 NANOPARTICLES TOXICITY ON FISH
1.4 AIMS AND OBJECTIVES

CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 CHARACTERIZATION
2.2 HAEMATOLOGICAL INDICES OF THE AFRICAN CAT FISH, Clarias gariepinus

CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 BIOGENIC SYNTHESIS OF SILVER-GOLD ALLOY NANOPARTICLES
3.1.1 PREPARATION OF SILVER SALT SOLUTION
3.1.2 PREPARATION OF GOLD SALT SOLUTION
3.1.3 PREPARATION OF GOLD-SILVER ALLOY NANOPARTICLES
3.2 COLLECTION OF TEST ORGANISM
3.3 ACCLIMATIZATION OF FISH
3.4 BLOOD SAMPLE COLLECTION FROM FISH
3.4.1 PACKED CELL VOLUME (PCV)
3.4.2 HEAMOGLOBIN (HB)
3.4.3 RED BLOOD CELL (RBC)
3.4.4 WHITE BLOOD CELL
3.4.5 MEAN CELL VOLUME (MCV)
3.4.6 MEAN CORPUSCULAR HEAMOGLOBIN (MCH)
3.5 STATISTICAL ANALYSIS

CHAPTER FOUR
4.0 RESULTS

CHAPTER FIVE
5.0 DISCUSSION AND CONCLUSION
5.1 DISCUSSION
5.2 CONCLUSION

REFERENCES

APPENDIX
APPENDIX I
APPENDIX II
APPENDIX III

CHAPTER ONE

1.0 INTRODUCTION

Nanotechnology is an anticipated manufacturing technology that allows the long-established trend toward smaller, faster, cheaper materials and devices.

Silver nanoparticles have been found many applications in therapeutics, antimicrobial drugs, microelectronics and biosensing devices because of their unique properties (Sundaramoorthi et al., 2011).

Gold nanoparticles (GNPs) are the most compatible nanomaterial for preparation of engineered nanoplatforms in smart sensing devices. Surface Plasmon resonance property of GNP makes them most suitable engineered nanomaterial for bioimaging, biomedical therapeutics and biodiagnostic tools (Jain et al., 2006). GNPs, also named as gold colloids, have attracted attracted increasing attention due to their unique properties in multi-disciplinary research fields (Daniel and Astruc, 2004).

This nanometer sized particles was derived from nanotechnology down to nanomaterials and nanoparticles which has different classifications, dimensions and applications in many fields like nanomedicine, electronics and other nano-fields (Niemeyer and Mirkin, 2004).

In this study, evaluation of haematological indices of Clarias garipinus exposed to sub lethal concentrations of silver salt, gold salt and silver-gold alloy nanoparticles will be analysed.

Haematological indices are important parameters for the evaluation of fish physiological status. Their changes depend on the fish species, age, the cycle of sexual maturity and health condition (Blaxhall, 1972). Haematological parameters are closely related to the response of the animal to the environment, an indication that the environment where fishes live could exert some influence on the haematological characteristics (Gabriel et al., 2004). These indices have been employed in effectively monitoring the responses of fishes to the stressors and thus their health status under such adverse conditions. They can provide substantial diagnostic information once reference values are established under standardized conditions. Evaluation of the haematological indices involves the determination of the total Red Blood Cell (RBC), total white blood cell count (WBC), hematocrit (PCV), haemoglobin concentration (Hb), erythrocyte indices (MCV, MCH, MCHC) (Campbell, 2004).

1.1 NANOTECHNOLOGY

Nanotechnology is the creation of objects or surfaces whose unique functions are a direct result of their nanoscale dimensions and organisation. "Nanoscale" generally refers to objects that ranges from 1-100 nm in one or more dimensions (Klaine et al., 2008).

Nanotechnology can therefore be defined as the design, synthesis and application of materials and devices whose size and shape have been engineered at the nanoscale". It exploits unique properties which may be chemical, physical, electrical and mechanical properties that emerge when matter is structured at the nanoscale (Klaine et al., 2008).

1.2 NANOPARTICLES

Nanoparticles are particles with at least one dimension smaller than 1 micrometer and potentially as small as atomic and molecular length scales (~0.2 nm). Nanoparticles can have amorphous or crystalline (nanocrystals) form and their surfaces can act as carriers for liquid droplets or gases (Hoet et al., 2004).

Before nanomaterials is broken down to nanoparticles, synthyesis of materials must be done which helps to engineer the sizes and shapes of nanomaterials at nanoscale. This can be done by different methods such as Physical methods, Chemical methods and Biological methods.

Nanoparticles can be synthesized physically, chemically and biologically. Many adverse effects have been associated with chemical synthesis methods due to the presence of some toxic chemical absorbed on the surface. Eco friendly alternatives to Chemical and Physical methods are Biological ways of nanoparticles synthesis using microorganisms (Konishi and Uruga, 2007).

1.2.1 PHYSICAL METHODS OF SYNTHESIZING NANOPARTICLES

Different researchers have mentioned a wide range of the physical methods for preparation of nanoparticles like, evaporation-condensation, which might be carried out using a tube furnace at atmospheric pressure (Ingale and Chaudhari, 2013), evaporation-condensation and laser ablation together, electric arc discharge, chemical vapor deposition, gas phase synthesis and ball milling-annealing methods ,physical vapor deposition (Rajendran et al., 2013), etc. Ball mill is used to grind materials into extremely fine powder of nano size for use in livestock feeding. High energy ball milling (HEBM) can be used for synthesizing the nano minerals, which can provide 1000 times high impact grinding than traditional ball mills. However, a longer milling time is generally required for HEBM to activate and complete the structural changes. While using the HEBM, controlled milling atmosphere and temperature are to be taken care carefully. The demerit in the gas phase synthesis of nanomaterials is that it usually results in deposits of particles with larger size distributions, in some cases ranging from 10 nm to 200 nm. The absence of solvent contamination and maximum recovery of nanominerals are the advantages of physical methods of nanomineral synthesis (Iravani et al., 2014).

1.2.2 CHEMICAL METHOD

The chemical methods are having an upper hand over physical method with respect to the stabilization of nanomineral particles from agglomeration and extraction of nanomineral particles from solvent, surface modification, processing control and mass production. In the physical method, the produced nanoparticles are having a wider range of particle size. But in chemical method, a uniform nanosized particle can be produced (Lane et al., 2002). Hence, effective and controlled bulk production can be achieved by using the chemical methods. Reduction of mineral salts by chemical methods is the most convenient way to reduce the size of the particles (Rajendran et al., 2013). However, in chemical method there is always a chance of toxicity as hazardous chemicals are used during synthesis. Hence, attempts are made to produce nanomineral particles by using eco-friendly chemicals, plant and fungal origin as reducing agents, called as green chemistry method of nanoparticle synthesis. Eco-friendly as well as nontoxic chemicals such as glucose, starch, amino acids and plant extracts are used to synthesize metal nanomineral particles. (Rajendran et al., 2013).

1.2.3 BIOLOGICAL METHOD

The conventional methods are usually hazardous and energy consuming. This leads to focus on “green synthesis” of nano mineral particles which seems to be an easy, efficient and eco-friendly approach (Cardenas et al., 2007) and also minimize the toxicity (Narayanan and Sakthive 2010). Biosynthetic methods using either biological microorganisms, plant extracts or animal materials have emerged as a simple alternative to physical and chemical methods (Sri-Sindhura et al., 2014). Over the past several years, plants, animal parts, algae, fungi, bacteria and viruses have been used for the production of low cost, energy-efficient and non-toxic nano mineral particles (Sri-Sindhura et al., 2014). Various metal nanoparticles such as silver, gold, cadmium, selenium, palladium, barium titanate, and titanium has been successfully synthesized by biological methods (Narayanan et al., 2010) using different plant materials. Use of plant materials for the synthesis of nano minerals is easier and advantageous as this method is simple and is having upper hand as it follows single step synthesis procedures, one pot synthesis, easy product recovery from the final solutions, ecofriendly, compatible to pharmaceutical and biomedical applications, cost effective, economic viability, nontoxic, less time consuming, and no need to maintain selected culture (Rajendran et al., 2013).

Eco friendly alternatives to Physical and Chemical methods are Biological ways of nanoparticles synthesis using microorganisms (Konishi and Uruga, 2007; Klaus et al 1999), enzymes (Willner et al., 2006), fungus and plants or plant extracts (Ahmad et al., 2011). The development of these eco-friendly methods for the synthesis of nanoparticles is evolving into an important branch of nanotechnology especially silver nanoparticles, which have many applications.

1.3 NANOPARTICLES TOXICITY ON FISH

Given the increasing production of Nanoparticles of all types, however, the potential for their release in the environment can be transported to aquatic ecosystems and may induce malignant impacts on aquatic biota. Therefore, the aquatic ecotoxicology of engineered nanoparticles (aquatic nano-ecotoxicology) is a relatively new and evolving field. In recent years, silver nanoparticles due to their antimicrobial properties have been produced a lot so that 56% of nanoparticles global production is dedicated to Silver nanoparticles. The limited information that is slowly emerging demonstrates that Silver nanoparticles cause cytotoxity, oxidative stress, and inflammatory responses in Clarias gariepinus and other aquatic organisms. It has been shown that silver ion toxicity for fish is significantly less toxic in salt water than in fresh water. This difference is due to the high ionic strength that creates links between free silver ions and anions (e.g. silver chloride) in the salt water. In freshwater, silver toxicity is induced by ionic Ag+ which targets specific sites in fish gills. (Bielmyer et al., 2008). Since the released nanoparticles eventually enter water ecosystems, their maximum toxicity effects could be magnified in aquatic ecosystems.

1.4 AIMS AND OBJECTIVES

Therefore, this study is aimed to evaluate the effects of kola nitida mediated alloy of Silver and Gold nanoparticles on the haematology of juvenile Clarias gariepinus over exposed period of 42 days based on the following objectives:-

1. To evaluate the potential toxic effects of the alloy nanoparticles on the PCV, RBC, HB and WBC of the exposed fish.
2. To evaluate the potential toxic effects of the alloy nanoparticles on the derived parameters (MCV, MCH and MCHC).

CHAPTER TWO

2.0 LITERATURE REVIEW

Nanotechnology is one of the most prominent actors of the scientific revolution marking the beginning of the new millennium in which nature has been using nanotechnology since the beginnings of biological evolution (Kasemo, 1998). As for biotechnology, nanotechnology is the outcome of an interdisciplinary, new approach to old technological issues ranging from device manufacturing to energy conversion, from sensing to signal amplification and transmission. The discovery of unexpected physical and chemical behavior of matter at the nanometer scale has paved the way to a number of exploitations (some current, most real but prospective).

Nanoparticles are being applied in different fields such as in energy cells, due to their optical properties, as catalytic compounds for their high surface activity and in biomedicine field due to their bio conjugation possibilities and capability to be detected easily and rapidly.

Nanoparticles are been used in many toxicological studies to know the toxic effects on target and non-target organisms, environments, aquatic organism (Fish, etc) and terrestrial (Rat etc) organism.

So therefore, the present study was carried out in other to evaluate the toxicity of these nanoparticles (Silver, Gold and Silver-Gold alloy nanoparticles) on the haematological indices of juvenile Clarias gariepinus which was used for the Nanotechnology research group Lautech.

Fish are very sensitive to anthropogenic pollution and some of them have been widely used in toxicological studies as models to evaluate the health of aquatic ecosystems (Law, 2003).

Clarias gariepinus are subjected to many environmental influences which alter the healthy hemogram (a wide-ranging blood test used to assess overall health conditions­­­) most especially when exposed to nanoparticles (Law, 2003).

Haematological parameters are widely used indicators of environmental stress in fish. A literature survey shows that most experiments studying the hematological parameters of several marine and fresh water fish have been conducted in different labs under controlled conditions. The history of applying hematological methods as diagnostic aids in episodes of noninfectious and infectious diseases in confined and free-living populations of fish is quite meager (Blaxhall, 1972).

Haematological studies furnishes an index of physiological changes in fish and the fish blood acts as an impressive tool for detection of alterations in the tested organism (Rambhaskar and Rao, 1987).

A limited number of nanoparticles studies have been done with fish as a model organism (Karthikeyeni et al., 2013). Recently, preliminary reports showed that iron oxide nanoparticles could boost bioavailability than other forms of iron nanoparticles in both humans and rats through dietary administration (Stephen 2007).

Particularly, the toxicity tests on fish have mainly focused on carbon-based Nanoparticles (Smith et al., 2007). Moreover, the studies about toxicity of Gold-Silver alloy nanoparticles on fishes have concentrated the early developmental stages (Zhu et al., 2008,) and enzymatic studies. This has led to a consensus view on the main effects of Gold-Silver alloy nanoparticles on fish, as well as the associated environmental hazards and risks (Campbell et al., 2006).

Effects of gold nanoparticles and other nanoparticles on zebra fish were investigated demonstrating that zebra fish is a good animal model for assessing nanoparticles impacts from different exposure routes, at various life stages and concentration pressures. Most studies investigating gold nanoparticles impacts on zebra fish used waterborne exposure and did not represent realistic environmental concentrations. In fact, contamination values tested reached levels up to 250mg/L in the water column, and the majority of gold nanoparticles used were functionalized with organic compounds (Asharani et al., 2010). Dietary exposure of zebrafish to gold nanoparticles demonstrated a toxic impact at much lower concentrations (Geffroy et al., 2012).

Despite increased production of silver nanoparticles, relatively little is known about their environmental fate and potential effects, particularly in terrestrial environments (Klaine et al., 2008). The toxicity of silver nanoparticles has thus far been mainly studied in the aquatic environment, with toxicity demonstrated for bacteria, yeast, paramecia, algae (Griffitt et al. 2008) and various zebrafish life stages (Asharani et al. 2008). In the terrestrial environment, silver nanoparticles have not been investigated to such an extent and silver toxicity itself is not well studied (Nahmani et al., 2007).

The soil nematode has been used to assess silver nanoparticles toxicity but these experiments were performed in K-medium (NaCl and KCl solution) rather than in soils (Roh et al., 2009).

2.1 CHARACTERIZATION

The biogenic synthesis of silver nanoparticles (AgNPs) using pod extract of Cola nitida, evaluation of antibacterial activities, antioxidant activities and application as antimicrobial additive in paint were studied. The AgNPs were characterized through UV-Vis spectroscopy, Fourier-Transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM, Figure 1). The AgNPs was dark brown with maximum absorbance occurring at 431.5 nm and were spherical with sizes ranging from 12-80 nm (Lateef et al., 2016).

Cell-free extracts of four strains of non-pathogenic species of food origin, were studied for the green synthesis of gold nanoparticles (AuNPs), and characterized by UV–Vis absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM, Figure 2). The AuNPs were evaluated for their Anopheles gambiae larvicidal, dye degradation, antioxidant and thrombolytic activities. The blue-black colloidal AuNPs which absorbed maximally at 549–552 nm were nearly spherical in shape, and crystalline in nature with size of 8–50 nm (Oladipo et al., 2017).

The use of leaf, seed, seed shell and pod extracts of Cola nitida (Figure 3) were studied for the green synthesis of silver-alloy nanoparticles (Ag–AuNPs) and characterized by UV–Vis absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM, Figure 4).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1: TEM micrograph of synthesized AgNPs

Source:- Lateef et al., 2016

Abbildung in dieser Leseprobe nicht enthalten

Figure 2.The TEM micrographs (A), EDX spectra (B) and SAED patterns (C) of the biosynthesized AuNPs

Source:- Oladipo et al., 2017

Abbildung in dieser Leseprobe nicht enthalten

Figure 3: The schematic view of the biosynthesis of Ag–AuNPs using different extracts of C. nitida.

Source: Lateef et al., ( 2016)

The Ag–AuNPs formed were dark brown with maxima absorbance in the range of 497–531 nm. FTIR peaks at 3290–3396 and 1635–1647 cm-[1]showed that proteins were the capping and stabilization molecules for the synthesis of Ag–AuNPs. While leaf, seed and seed shell extract-mediated Ag–AuNPs had near spherical morphology, anisotropic structures of sphere, rod, hexagon and triangle were formed by pod extract. The poly dispersed particles were 17–91 nm in size, with crystalline characteristics and prominent presence of Ag and Au in the EDX spectra (Lateef et al., 2016).

2.2 HAEMATOLOGICAL INDICES OF THE AFRICAN CAT FISH, Clarias gariepinus

Haematological Indices are progressively used in fish as indicators of the physiological or sub-lethal stress response to endogenous or exogenous alterations and are more quickly reflected in the poor condition of fish than in other commonly measured (cataldi et al., 1998). These indices have been employed in effectively monitoring the responses of fishes to the stressors and thus their health status under such adverse conditions when exposed to Nanoparticles.

They can provide substantial diagnostic information once reference values are established under standardized conditions.

Abbildung in dieser Leseprobe nicht enthalten

Figure 4. The transmission electron micrographs (a), selected area electron diffraction patterns (b) and energy dispersive X-ray signals (c) of the biosynthesized Ag–AuNPs

Source:- Lateef et al., 2016

Hesser (1960) reviewed the literature on the examination of fish blood and discussed the diagnostic value of such an examination. Hunn (1960) prepared a bibliography of the chemistry of fish blood. Snieszko (1960) described in detail a microhematocrit (PCV) prodedure and microhematocrit values as obtained with trout blood. Larsen and Snieszko (1961) reviewed and evaluated methods of hemoglobin determination and discussed modification of the microhematocrit procedure applied to fish blood. The purpose of this paper is to furnish additional data on hematocrit values in trout, in order to contribute to the establishment of normal values for brook, brown, and rainbow trout of different sexes and age groups. The technique employed here was the same as that described by Snieszko (1960).

The red blood cell (RBC) is a determination of the total number of red cells present in blood. Red blood cells take up oxygen in the lungs or gills and release it into tissues while squeezing through the body’s capillaries (vinay et al., 2007).Binukumari and Vasanthi (2013) reported that the amount of RBC in the blood of the fishes exposed to 0.398ppm dimethoate 30% Effective Concentration for 24, 48 and 72 hours was found to contain 1.52, 1.49, 1.41 and mean control was found to be 1.79X10[6]mm[3] while the amount of WBC in the blood of fishes exposed to 0.398ppm dimethoate 30% EC for 24, 48 and 72 hours values were found to contain 5600, 6100, 7800 and mean control was found to be 4200 mm[3].

Evaluation of the hematological indices involves the determination of total white blood cell count (WBC) and erythrocyte indices which are Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH) and Mean Cell Haemoglobin Concentration (MCHC) (Campbell, 2004).

Sethi et al., (2008) reported that a long-term exposure to nanoalumina (aluminium oxide nanoparticles) could impair spatial learning abilities and increases anxiety in rats. Winklhofer et al., (2000) reported that rats with iron deficiency had a larger intestinal uptake and higher concentrations of Al in liver, spleen and plasma than the control group, whereas iron overload decreased intestinal absorption and tissue concentrations of Al.

Several literature reports indicated that haematological profile of different animal species may be influenced adversely by different chemicals. Transient changes are reported in the haematology rainbow trout, Oncorhynchus mykiss, under the effect of copper nanoparticles (Shaw et al., 2012). Both content of haemoglobin (Hb) and value of haematocrit (PCV) of rats reduced significantly after intra-peritoneal injection with aluminium sulphate as compared to controls (Farina et al., 2002). The stainless steel implantation caused a slight increase in Hb, PCV and red blood cell (RBC) count associated with a significant decrease in total white blood cell (WBC) count of male Wistar rats (Fadl-allah et al., 2011). Therefore, assessment of haematological and biochemical parameters are highly useful and benefit as markers for the extent of the deleterious effect of foreign substances on the blood constituents of an animal.

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Title
Alterations in Haematological Indices of Clarias Garipinus Exposed To Sub Lethal Concentrations of Kola-Pod Mediated Silver-Gold Alloy Nanoparticles
College
Ladoke Akintola University of Technology
Grade
2.50
Author
Year
2017
Pages
45
Catalog Number
V475214
ISBN (eBook)
9783668939677
ISBN (Book)
9783668939684
Language
English
Tags
alterations, haematological, indices, clarias, garipinus, exposed, lethal, concentrations, kola-pod, mediated, silver-gold, alloy, nanoparticles
Quote paper
Adeyemi Phillips (Author), 2017, Alterations in Haematological Indices of Clarias Garipinus Exposed To Sub Lethal Concentrations of Kola-Pod Mediated Silver-Gold Alloy Nanoparticles, Munich, GRIN Verlag, https://www.grin.com/document/475214

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