University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA, LEGON, GHANA COLLEGE OF BASIC AND APPLIED SCIENCES SCHOOL OF PHYSICAL AND MATHEMATICAL SCIENCES DEPARTMENT OF CHEMISTRY. STUDIES ON THE CAUSES OF EMULSION PAINT DEGRADATION ON BUILDINGS IN PORT HARCOURT, NIGERIA AND PAINT FORMING PROPERTIES OF SOME AFRICAN SEED OILS. BY ORIJI, ONUOHA G. (10325670) B.TECH (HONS) INDUSTRIAL CHEMISTRY (FUTY, NIGERIA) M.Sc INDUSTRIAL CHEMISTRY (UNIPORT, NIGERIA). A THESIS PRESENTED TO THE BOARD OF GRADUATE STUDIES, UNIVERSITY OF GHANA, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF A MASTER OF PHILOSOPHY DEGREE. MAY, 2018 i University of Ghana http://ugspace.ug.edu.gh DECLARATION I declare that the thesis is my own original work, except the references to other people‘s work which have been duly acknowledged. Furthermore, I declare that the work has never been presented wholly or partially for a degree or diploma in the University of Ghana or in any other University. ……………………………………. ORIJI, ONUOHA G (STUDENT) …………………………………… ………………………………………. PROF. V.K. NARTEY DR. A.K. BRIMAH (SUPERVISOR) (CO-SUPERVISOR) …………………………………… …………………………………… PROF. A.I. SPIFF(Nigeria) DR. L.K. DOAMEKPOR (CO-SUPERVISOR) (HEAD) DEPARTMENT OF CHEMISTRY University of Ghana, Legon,Ghana. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I must thank God for His divine grace during the period of this work. I very much appreciate all the efforts, advice and sacrifice of my Supervisor, Prof. V K. Nartey, and indeed all co- supervisors: Dr. A.K. Brimah and Prof. A.I. Spiff for their great inspiration and encouragement. I owe immense gratitude to my wife, Ataisi, for her patience, love, care and understanding. I am also grateful to Prof. Mike Horsfall, Prof. Leo Osuji, Mr. G.Arueria, and my colleagues and friends at the University of Port Harcourt and the University of Ghana, respectively. I very much appreciate all the assistance from staff of the Department of Chemistry, University of Ghana, Legon-Accra and staff of the Department of Pure and Industrial chemistry, University of Port Harcourt, Nigeria. Furthermore, I must extend my gratitude to the staff of the Department of Microbiology, University of Port Harcourt for their assistance in the collection and storage of the samples. Finally, I am much grateful to the Tertiary Education Trust Fund of the Federal Government of Nigeria for providing the necessary fund for this research work. All your support added much ―flesh‖ to the ―skeleton‖ of this work. God bless all of you. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to the Almighty God; to my beloved wife, Atisi; to my children: Prosper, Christan, Blossom, Shalom, Victory, and Immanuel; and to the memory of my late father, Eze.E.J.A.Oriji. iv University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS AAS…………Atomic Absorption Spectrometry AP--------------Acid Production st APHA------------- American Public Health Association (21 Edition 2005) ASTM----------American Society for Testing and Materials (2005 Edition) BH--------------Brinel Hardness CAEM-------------Chemical Analysis of Ecological Materials CFU/G-------- Colony Forming Unit per gram DW----------------Drinking Water FID…………Flame Ionization Detector GC……………Gas Chromatography GP--------------Gas Production HM---------------Heavy Metal J------------- Jatropha N-------------Neem LCFA----------Long Chain Fatty Acid MC----------------Maximum Concentration MCT-------------Medium Chain Triglycerides MR--------------Methyl Red Test v University of Ghana http://ugspace.ug.edu.gh PAH--------------Polycyclic Aromatic Hydrocarbon PK……………Palm Kernel PVA-------------Polyvinyl Acetate R…………….Rubber SB………….Soya Bean SCFA----------Short Chain Fatty Acid THBC-----------Total Heterotrophic Bacteria Count THFC-----------Total Heterotrophic Fungi Count TPH--------------Total Petroleum Hydrocarbon TS……………Tensile Strength USEPA------------United States Environmental Protection Agency VB1…………….Vitamin B1 VB2………………Vitamin B2 VB6,,,,,,,,,,,,,,,,,,, Vitamin B6 VBcom----------Vitamin B- complex VE----------------- Vitamin E VK………………Vitamin K VA……………….Vitamin A VP---------------Voges Proskauer Test WSAL------------Water- Supporting Aquatic Life vi University of Ghana http://ugspace.ug.edu.gh SUMMARY In southern Nigeria, it is common to notice ―infected patches‖ on emulsion paintings, mostly on the walls of buildings and fences. These emulsion paintings contain a wide range of constituents and therefore provide ecological niches that may be exploited by a large variety of microbial species. Samples of these paintings were collected from three different locations in University of Port Harcourt, Nigeria. In order to identify the microbes in both degraded and undegraded walls, o some of the samples were cultured and incubated for 24hours at 37 C. The colonies that developed were subjected to different tests: motility, methyl-red, voges proskauer, oxidase, indole, citrate utilization and catalase tests. Under the microbes, algae, bacteria (bacillus, flavobacteria, microcollus, and anthrobacteria species), and fungi (rhizopus, aspergillus, fusarium species) were identified, but no algae was detected in the undegraded wall. The pH of the environment was also determined. The pH range of the degraded wall was found to be 6.59 – 7.04. The presence of sulfate, nitrate and carbonate was also determined in both walls. The concentrations of sulfate and nitrate were higher in the undegraded wall, but there was no marked difference in the concentration of carbonate in both walls. The heavy metal content of these samples was determined using Atomic Absorption Spectrometry. It was discovered that Fe, + 2+ Na , Cr, Ni and Zn dominated these areas, followed by Ca , Ti and Cd. But Hg and Pb were not identified. The polycyclic aromatic hydrocarbon content was analyzed using Gas Chromatography. In this case, acenaphthylene, fluorene and anthracene were dominantly present, while naphthalene, pyrene and acenaphthene were in minute quantities Six different seed oil extracts: coconut fruit, palm kernel seed, soya bean seed and rubber seed (from Nigeria), as well as jatropha seed and neem seed (from Ghana), were screened by determining their fatty acid vii University of Ghana http://ugspace.ug.edu.gh profile using a GC-FID. It was found that coconut oil and palm kernel oil were composed more of saturated fatty acid, while soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil were composed more of unsaturated fatty acid. Thus, coconut oil and palm kernel oil can be used in the production of emulsion paint while soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil can be used in the production of oil paint. Furthermore, the anti-oxidant vitamin content of these oils was also determined using a GC-FID. All the oils had anti-oxidant vitamins and so they can be used for industrial applications like soap, cosmetic and paint production. In the performance test, different paints were formulated using these oils. The best formulations came out from rubber seed oil and the coconut oil. Thus rubber seed oil and coconut oil would be best suited for use in the formulation of oil paint and emulsion paint respectively. viii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ...................................................................................................................... ii SUMMARY ............................................................................................................................ vii TABLE OF CONTENTS ......................................................................................................... ix CHAPTER ONE ........................................................................................................................1 1.0 INTRODUCTION ............................................................ Error! Bookmark not defined. 1.1 BACKGROUND OF STUDY .............................................................................................1 1.2 Problem Statement ...............................................................................................................5 1.3 Aim of Study ........................................................................................................................6 1.4 Specific Objective of Study .................................................................................................6 1.5 Potential Benefits of the Study ............................................................................................6 CHAPTER TWO .......................................................................................................................8 2.0 REVIEW OF LITERATURE ..............................................................................................8 2.1 Paints ....................................................................................................................................8 2.1.1: Defects in the liquid paint ..............................................................................................19 2.1.2: Defects During Application ...........................................................................................21 2.1.3: Defects during drying/curing: These include: ...............................................................21 2.2 SEED OILS ........................................................................ Error! Bookmark not defined. 2.2.1 COCONUT OIL..............................................................................................................25 2.2.2: Rubber Seed Oil (Hevea brasilliensis) ..........................................................................33 2.2.3: Palm Kernel Seed Oil ....................................................................................................34 2.2.4: Soybean Seed Oil ...........................................................................................................35 2.2.5: Neem Seed Oil ..............................................................................................................36 2.2.6: Jatropha (Curcas) seed oil ..............................................................................................37 2.3: Oils ...................................................................................................................................38 2.4: FATTY ACIDS.................................................................................................................39 2.4.1: LAURIC ACID ..............................................................................................................41 2.4.2: PALMITIC ACID ..........................................................................................................43 ix University of Ghana http://ugspace.ug.edu.gh 2.4.3: CAPRYLIC ACID .........................................................................................................44 2.4.4: CAPRIC ACID ..............................................................................................................44 2.4.5: CAPROIC ACID ...........................................................................................................45 2.4.6: STEARIC ACID ............................................................................................................46 2.4.7: ARACHIDIC ACID ......................................................................................................47 2.4.8: MYRISTIC ACID..........................................................................................................47 2.4.9: OLEIC ACID .................................................................................................................48 2.4.10: LINOLEIC ACID ........................................................................................................49 2.4.11: ALPHA-LINOLENIC ACID .......................................................................................52 2.5: ANTIOXIDANT VITAMINS ..........................................................................................53 2.5.1: VITAMIN E, VE ............................................................................................................54 2.5.2: VITAMNE A .................................................................................................................55 2.6: GAS CHROMATOGRAPHY .........................................................................................56 2.7: ATOMIC ABSORPTION SPECTROSCOPY .................................................................59 2.8. POLYCYCLIC AROMATIC HYDROCARBONS .........................................................60 2.8.1: ANTHRACENE ............................................................................................................61 2.8.2: ACENAPHTHENE .......................................................................................................63 2.8.3: FLUORENE...................................................................................................................64 2.8.4: NAPHTHALENE ..........................................................................................................65 2.8.5:FLUORANTHENE ........................................................................................................67 2.8.6: PYRENE ........................................................................................................................68 2.8.7: PHENANTHRENE .......................................................................................................68 2.9: HEAVY METALS ...........................................................................................................69 2.9.1: IRON ..............................................................................................................................71 2.9.2: SODIUM ........................................................................................................................76 2.9.3: NICKEL .........................................................................................................................77 2.9.4: CHROMIUM .................................................................................................................79 2.9.5: CALCIUM .....................................................................................................................82 2.9.6: CADMIUM ....................................................................................................................83 2.9. 7: ZINC .............................................................................................................................85 x University of Ghana http://ugspace.ug.edu.gh 2.9.8 LEAD ..............................................................................................................................87 2.9.9: ARSENIC ......................................................................................................................88 2.10: CARBONATES, SULPHATES AND NITRATES .......................................................90 2.10.1: CARBONATE .............................................................................................................90 2.10.2 Sulfate ...........................................................................................................................91 2.10.3 Nitrate ...........................................................................................................................91 2.11: MICROBES AND METALS .........................................................................................92 CHAPTER THREE .................................................................................................................98 3.0: MATERIALS AND METHODS......................................................................................98 3.1 LOCATION OF STUDY ...................................................................................................98 3.2 STUDY SITES.................................................................................................................101 3.3 STUDY DESIGN.............................................................................................................101 3.4 MICROBIAL ANALYSIS ..............................................................................................102 3.5 Determination of heavy metals in degraded and undegraded emulsion paintings by the APHA 3111B AAS method (American Public Health Association) .................................................110 3.6: Determination of Total Petroleum Hydrocarbon and Polycyclic Aromatic Hydrocarbon in degraded and undegraded emulsion paintings by GC-FID USEPA 8015D Method (United States Environmental Protection Agency) ........................................................................................115 3.7 Determination of pH of degraded and undegraeded emulsion paintings by the ASTM4972 Method (American Standard for Testing Materials) ..............................................................119 3.8: Determination of Total Inorganic Carbon/Carbonate in degraded and undegraded emulsion paintings by the CAEM method (Chemical Analysis of Ecological Matter) ........................120 3.9: Determination of Extractable Sulphate in degraded and undegraded emulsion paintings by 2- Turbidimetric Method-APHA 4500SO4 E (American Public Health Association) .............121 3.10 Determination of Extractable Nitrate in degraded and undegraded emulsion paintings by Colometric Method-USEPA352.1 (United States Environmental Protection Agency) ........123 3.11 Determination of the Fatty Acid Composition for Oils [Coconut Oil (Agric and Native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil]124 3.12: Determination of Antioxidant Vitamins of the seed oils: .............................................131 3.13 Paint Analysis ................................................................................................................131 3.13.1: Determination of the Polycyclic Aromatic Hydrocarbon and Total Petroleum Hydrocarbon for Glaxo and Demcork Emulsion Paint. .........................................................132 xi University of Ghana http://ugspace.ug.edu.gh 3.13.2 Determination of Heavy Metals in Glaxo and Demcork Emulsion Paints .................133 3.14 Performance Test ...........................................................................................................133 3.15 Statistical Analysis .........................................................................................................134 3.16 Quality Control ..............................................................................................................135 CHAPTER FOUR ..................................................................................................................137 4.0 RESULTS AND DISCUSSION ......................................................................................137 4.1 MICROBIAL ANALYSIS: .............................................................................................137 4.2 Analysis of Emulsion Paintings. ......................................................................................152 4.2.1 Analysis of Heavy Metals in Degraded and Undegraded Emulsion Paintings. ............152 4.2.2: Analysis of Total Petroleum Hydrocarbon in Degraded Sites:. ...................................159 4.3.3: Analysis of Polycyclic Aromatic Hydrocarbon in Degraded and Undegraded Sites. .163 4.2.3 Determination of Sulphate: Nitrate and Carbonate of good and infected walls. ..........169 4.2.4: Analysis of the Saturated and Unsaturated Fatty Acid composition of Oils (Coconut Oil (agric & native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil) ................................................................................................................................175 4.2.5: Analysis of Anti-Oxidant Vitamins in Coconut Oil (agric & native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil: .....................................183 4.3 Paint Analysis ..................................................................................................................186 4.2.1 Analysis of Polycyclic Aromatic Hydrocarbon in Glaxo(Nig) and Demcork(Nig) Emulsion Paints: .....................................................................................................................................186 4.2.2: Analysis of Total Petroleum Hydrocarbon in Glaxo and Demcork Paints. .................189 4.2.3: Analysis of Heavy Metals in Glaxo and Demcork Paints. ..........................................191 CHAPTER FIVE ...................................................................................................................196 5.0 CONCLUSION AND RECCOMENDATIONS .............................................................196 5.1 CONCLUSION ................................................................................................................196 5.2 RECOMMENDATIONS .................................................................................................197 REFERENCES ......................................................................................................................198 APPENDIX ............................................................................................................................237 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: The Formulation For a Typical Alkyd Oil Paint (Mohammed et al ,2005) ................................................................................................................. 12 Table 2: The formulation for Water-based Paint(Mohammed et al, 2005) .... 17 Table 3: Formulation for a typical primer ( Mohammed et al ,2005) ........... 18 Table 4: Properties of lauric acid (Lide, 2005) ............................................ 42 Table 5: The % Alpha-Linolenic Acid from some extracted oils (Li, Thomas, 1999) ........................................................................................................ 52 Table 6: TOLERABLE LIMITS OF SOME HEAVY METALS (Hutton and Symon, 1986) ........................................................................................................ 70 Table 7: Characteristic Values of Tensile Strength (TS) and Brinel Hardness (BH) of different forms of Iron (Kohl, 1995) ................................................ 72 Table 8: Monthly average values of rainfall and accumulated total in the Niger Delta (1937—1997) from IITA,Nigeria (Adejuwon,2012) .............................. 99 Table 9: Summary of operating conditions for AAS machine (APHA 3111B)111 Table 10: Causes of Interference and possible Suppressants ................... 113 Table 11: Microbial Density of Samples in Degraded Wall ........................ 137 Table 12: Identification of Bacteria in Degraded Wall ............................... 139 Table 13: Identification of Fungi on Degraded Wall .................................. 143 Table 14: Microbial Density of Samples in Undegraded Wall .................................. 143 Table 15: Identification of Bacteria in Undegraded Wall ........................... 147 xiii University of Ghana http://ugspace.ug.edu.gh Table 16: Identification of Bacteria in Undegraded Wall ........................... 149 Table 17: Mean values of concentrations of heavy metals in degraded sites with their standard deviations (mg/kg) ........................................................... 152 Table 18: Correlation of Degraded Sites ................................................... 152 Table 19: Mean values of concentrations of heavy metals in undegraded sites with their standard deviations (mg/kg). ................................................... 154 Table 20: Correlation of Undegraded Sites ............................................... 155 Table 21: Mean values of concentrations of TPHs in degraded sites with their standard deviations (mg/kg).................................................................... 159 Table 22: Correlation of Mean values of concentrations of TPHs in degraded sites ........................................................................................................ 159 Table 23: Mean values of concentrations of PAHs in degraded sites with their standard deviations (mg/kg).................................................................... 163 Table 24: Correlation of Mean values of concentrations of PAHs in degraded sites ........................................................................................................ 163 Table 25: Mean Values of Concentrations of PAHs in Undegraded Sites with Their Standard Deviations (mg/kg) .......................................................... 165 Table 26: Coefficient of Correlation for Mean Values of Concentrations of PAHs in Undegraded sites ................................................................................ 165 Table 27: Mean values of pH (H O) at 25.802 C in good and degraded sites with their standard deviations (AST MD 49721) .............................................. 168 xiv University of Ghana http://ugspace.ug.edu.gh Table 28: Mean values of concentrations of sulphate, nitrate and, carbonate in good sites with their standard deviations (mg/kg) ..................................................................... 169 Table 29: Correlation Coefficient for mean values of concentrations .......................... 169 Table 30: Mean values of concentrations of sulphate, nitrate and carbonate in degraded sites with their standard deviations (mg/kg). ............................ 170 Table 31: Corrrelation Coefficient for mean values of concentrations of sulphate, nitrate and carbonate in degraded sites ................................... 171 Table 32: Mean Values of Concentrations of Saturated Fatty Acids with their standard deviations (mg/kg).................................................................... 175 Table 33: Mean values of concentrations of unsaturated fatty acids with their standard deviations (mg/kg).................................................................... 179 Table 34: Mean values of concentrations of antioxidant vitamins in the oils with their standard deviations (mg/kg) ........................................................... 183 Table 35: Mean values of concentrations of PAHs in Paints with their standard deviations (mg/kg) .................................................................................. 186 Table 36: Coefficient of Correlation for Mean values of concentrations of PAHs in Paints ................................................................................................. 186 Table 37: Mean values of concentrations of TPHs with their standard deviations (mg/kg) ................................................................................................... 189 Table 38: Coefficient of Correlation for Mean values of concentrations of TPHs ............................................................................................................... 189 xv University of Ghana http://ugspace.ug.edu.gh Table 39: Mean values of concentrations of heavy metals with their standard deviations (mg/kg) .................................................................................. 178 Table 40: Coefficient of Correlation for Mean values of concentrations of heavy metals ..................................................................................................... 178 Table 41:Drying Times of the Different Paint Formulatios, -------------------------181 xvi University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Flow Chart for Paint Manufacture Error! 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Figure 2: Linoleic Acid Metabolism ............................................................ 51 Figure 3: Block Diagram of a Gas Chromatograph (Pavia, 2006) ................ 56 Figure 4: Block Diagram of an Atomic Absorption Spectroscopic Machine (Welz and Sperting, 1999) .................................................................................. 59 Figure 5: An Anthracene Molecule (Iglesia-Groth et al, 2010) ..................... 62 Figure 6: An Anthraquinone ...................................................................... 63 Figure 7: Anthrancene is converted to anthroquinone, a precursor to dyes (Gerd et al, 2006). ..................................................................................... 63 Figure 8: Acenaphthene Molecule (KarCriesbaun,et al, 2002) .................... 64 Figure 9: Fluorene Molecule (Gerkins et al, 1984) ...................................... 65 Figure 10: Naphthalene (Erlenmeyer, 1866) ............................................... 65 Figure 11: Fluoranthene (Amoore and Hautala, 1983) ............................... 67 Figure 12: Pyrene Molecule (Senkan and Castald, 2003) ............................ 68 Figure 13: Phenanthrene (Amoore and Hautala, 1983) .............................. 69 Figure 14: Map of Africa showing the position of Nigeria. ........................... 99 xvii University of Ghana http://ugspace.ug.edu.gh Figure 15: Map of Nigeria showing the position of Port Harcourt in Rivers State ............................................................................................................... 100 Figure 16: Map of University of Port Harcourt Showing the “Study Buildings” ............................................................................................................... 101 Figure 17: Plate 10 (A freshly degraded emulsion painting (infected wall) . 103 Figure 18: Plate 7 (An undegraded emulsion painting (good wall) ............. 103 Figure 19: Plate 9(The AAS Instrument) ................................................... 113 Figure 20: GC-FID Instrument ................................................................ 116 Figure 21: Plate 11: UV Spectrometer ...................................................... 122 Figure 22: Plate12: The electric Oven Containing a fresh endosperm of coconut fruit for drying ........................................................................................ 125 Figure 23: Plate13: The electric oven with dried coconut endosperm ........ 126 Figure 24: Plate 16 (A tyical Extraction set-up in the laboratory used in the extraction of oil from the samples) ........................................................... 127 Figure 25: Plate14 (A typical laboratory grinding machine used in the grinding of seed samples) ...................................................................................... 128 Figure 26: Extracted coconut oil .................... Error! Bookmark not defined. Figure 27: Plate15 (A dried and ground endosperm of coconut) ........... Error! Bookmark not defined. Figure 28: Graph of Correlation between Heavy Metal Concentrations in Degraded sites ........................................................................................ 147 xviii University of Ghana http://ugspace.ug.edu.gh Figure 29: Histogramam of Correlation between Heavy Metal Concentrations in Degraded sites ........................................................................................ 147 Figure 30: Graph of Correlation between Heavy Metal Concentrations in Undegraded sites .................................................................................... 149 Figure 31: Histogram of Correlation between Heavy Metal Concentrations in Undegraded sites .................................................................................... 149 Figure 32: Correlation Plot of Mean values of Concentrations of TPHs in Degraded Sites ........................................................................................ 153 Figure 33: Histogram of Mean values of Concentrations of TPHs in Degraded Sites ....................................................................................................... 153 Figure 34: Correlation Plot of Mean Values of Concentrations of PAHs in Degraded sites ........................................................................................ 156 Figure 35: Histogram of Mean Values of Concentrations of PAHs in Degraded sites ........................................................................................................ 156 Figure 36: Corrlation Plot of Mean Values of Concentrations of PAHs in Undegraded Sites .................................................................................... 158 Figure 37: Histogram of Mean Values of Concentrations of PAHs in Undegraded Sites ....................................................................................................... 158 Figure 38: Correlation Plot for mean values of sulfate, nitrate & nitrate good sites. ....................................................................................................... 162 Figure 39: Histogram for mean values of sulfate, nitrate & nitrate good site162 xix University of Ghana http://ugspace.ug.edu.gh Figure 40: Correlation Plot of Mean Values of sulfate, nitrate & carbonate in degraded sites ......................................................................................... 164 Figure 41: Correlation Plot for Mean values of concentrations of PAHs in Paint 174 Figure 42: Plot of Correlation for Mean values of concentrations of TPHs .................. 177 Figure 43: Plot of Correlation for Mean values of concentrations of heavy metals ............................................................................................................... 179 Figure 44: Concentration of Heavy Metals in the paints, and sites 1, 2 and 3. ............... 180 xx University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 BACKGROUND OF STUDY Paints are used to protect, preserve, decorate (such as colour) or add functionality to an object or surface. Examples of protection are the covering of metal to retard corrosion and the painting of a house to help protect it from elements of the environment. An example of decoration is to add a festive trim to a room (interior). Paint may be used to add functionality by modifying light reflection or heat radiation on a surface. Another example of functionality is the use of colour to identify hazards or to specify the function of equipment, such as pipelines or military ammunition (Bently and Turner, 1997). Painting is the application of paint. Someone who paints artistically is usually called a painter or an artist, while a person who paints commercially is often referred to as a painter and decorator, or a house painter. There are many types of artist paints, for example, oil, water colour, acrylic gouache etc.which can be applied to almost any kind of object. They are used, among many other things, in the production of art or driving aids, in industrial coating, for road surface marking or as a barrier to prevent corrosion or water damage. Paint is a semi-finished product, or an intermediate good, as the final product is the final painted article itself. Paint can also be mixed with glaze to create various textures and patterns. This process is referred to as faux finish and is quite popular with discerning homeowners, architects, and interior designers (Talbert, 2007). 1 University of Ghana http://ugspace.ug.edu.gh Furthermore, paints must have certain properties to ensure that they are applicable, will dry in a reasonable length of time, can adapt to the physical changes of the surface and will maintain their function for an acceptable period of time. These basic properties include: Consistency: This refers to the fluidity of paint. Consistency depends on certain factors, for example, temperature, type of film former, type of pigment etc. Opacity. This is the property of the paint to obliterate the surface to which it is applied. Although the pigment is the principal component responsible for opacity, a careful blending of appropriate film formers and pigments is also important. Adhesion: The resin in the paint gives it the ability to stick to a surface. The condition of the surface is vital to the maximum adhesion of the paint film. Flow: Flow is the quality of the paint that enables the painter to apply coating so that the brush or roller marks are not visible. Some paints (eg gloss paints) have good painting properties and help the painter achieve a texture free finish (Turner, 1967). Emulsion paints are a mixture of two substances that do not mix, one of which is dispersed in the other. Such paintss have a clouded look due to the various substance boundaries that scatter light rays passing through them. Emulsions are not formed naturally and are generally unstable. The paint pigments are suspended in a solvent that is also used to control its viscosity. Emulsion paints have fillers to thicken the film, support the paint structure and increase their volume. They also contain catalysts, stabilizers, emulsifiers, adhesion enhancers, texturizers and flatteners. Once emulsion paint is applied, it becomes tack free. On solidifying, a hardening of the paint may result because of curing, evaporation or even phase change, depending on the type of binder. Emulsion paints also contain a lot of additives to impact various properties like foam control, skin control, bacterial-growth control, antifreeze and pigment stability (Turner, 1967). 2 University of Ghana http://ugspace.ug.edu.gh Emulsion paints have water as base, along with vinyl or acrylic resins added to make them hard wearing. They give various degrees of sheen. The paint becomes harder wearing as the shine increases. Special water based paints are available for wood works (Bently and Turner, 1997). There are three main types of emulsion used for walls and ceilings, each giving a different finish: Vinyl Matt Emulsiongives a matt, non-shiny finish that is good for concealing small imperfections on the wall or ceiling. Vinyl Satin Emulsion gives a subtle soft-sheen finish and is a more durable surface than vinyl matt. It is suitable for areas that might need to be occasionally lightly washed or sponged. Vinyl Silk Emulsion gives a high sheen finish and is the most durable of all emulsion paints. It is good for rooms that are subject to a lot of moisture, e.g., bathrooms. MICROBIAL ATTACK ON PAINTING: Paintings contain a wide range of organic and inorganic constituents and provide different ecological niches that may be exploited by a large variety of microbial species. Besides, many of the components of a painting are biodegradable, and so are the additives (glues, emulsifiers, thickeners, etc.) that facilitate the drawing or application of paint layers or enhance the aesthetic quality of the finished product (Agrawal et al, 1988). In easel paintings, the support material (the cellulose of paper, wood and the proteins of parchment, silk and wool) may be easily degraded by micro-organisms, as may be the materials (animal or plant glues) used to ―size‖ the support and to prepare a ground lay (Bock and Sand,1993). Paintings on paper or silk are laid, in general, directly on the support, since a ground or underlay is lacking, but the pigments are kept in emulsion with organic binders. Thus, besides the organic nature of the support, easel paintings contain molecules that many microorganisms may utilize for growth, such as sugars, gums and 3 University of Ghana http://ugspace.ug.edu.gh other polysaccharides, proteins, linseed and other oils, waxes, but also less chemically defined mixtures of biomolecules such as egg yolk, bile and even urine. Pigments are suspended in water or oil, often in the presence of binders such as casein and milk, and applied on the damp lime plaster. The calcium carbonate formed on contact with air consolidates the pigments. For both types of paintings, the spectrum of compounds that may be present is further increased by those that are added at later times during retouching or relining, or when a fresco is detached and transferred to a canvas or a board. In one case at least, extensive fungal colonization was reported even with frescoes that, after cleaning and consolidation, were removed from walls and transferred to a fiber glass support (Gettens et al, 1941). Finally, dirt, soot and other environmental contaminants accumulating on the painted surface may represent a significant source of nutrients. The growth of microorganisms on paintings may cause aesthetic and structural damage. With aesthetic damage, one must consider pigment discoloration, stains and the formation of a biofilm on the painted surface, whereas with structural damage, one must consider the cracking and disintegration of paint layers, the formation of paint blisters, and the degradation of support polymers or of glues and binders resulting in the detachment of the paint layer from the support (Ross,1963).Of course, the two types of damage are strongly linked, and in the long run, structural damage greatly affects the aesthetic quality of a painting. In fungal colonization of mural paintings, Saiz-Jimenez and Samson (1991) have shown that at the beginning, the growth of fungi on the surface of a mural caused only aesthetic damage since there was little or no alteration of the painted surface. Later on, fungal growth in depth occurred. Hyphae penetrated the painted layer, degrading some of its components (especially glues and binders), which resulted in a decrease in the cohesion of the painted layers, thus giving rise to exfoliations, cracking and loss of the paint. Similarly, cyanobacteria and algae growing on 4 University of Ghana http://ugspace.ug.edu.gh paintings exposed to light may cause considerable damage. Besides the aesthetic damage caused by a green, black, brown, or yellow algae patina covering the painted portions, these organisms may causeweathering of the surface layers, accelerating the detachment of portions of the painted layer as well as the underlying plaster (Klens and Lang,1956). Gettens and coworkers were among the first to point out in1941 that paintings could be defaced or destroyed by the growth of those small, parasitical plants, commonly called ‖mould‖or―mildew‖. They reported that these organisms could cause changes to the painted surfaces through staining, discoloration or formation of patinas and efflorescence. Besides, they showed that many of such organisms, especially the fungi, could grow between the paint layers and the wall, causing a swelling of the paint film which could lead to the detachment of portions of the painted layer and their disaggregation from the wall. 1.2 Problem Statement Emulsion paintings are susceptible to attack by heavy metals and microbes such as algae, fungi and bacteria. Paintings contain a wide range of organic and inorganic constituents, and provide different ecological niches that may be exploited by a large variety of microbial species. The growth of microorganisms on paintings may cause aesthetic and structural damages. The uptake of trace metals and their subsequent utilization in enzyme activation occurs in all microbes (Wackett et al, 1989). Some microbes are able to use some metals as electron donors or acceptors in energy metabolism (Ehrlich, 1996). Prokaryotic and eukaryotic microbes are capable of accumulating metals by binding them as cations to the cell surface in a passive process (Beveridge and Doyle, 1989; Gadd, 1993). Even dead cells can bind metal ions. Under some circumstances, microbes may cause non-enzymatic corrosion of metals like iron or some 5 University of Ghana http://ugspace.ug.edu.gh alloys through the formation and release of corrosive metabolic products (Edywan, 1995). In nature, noticeable microbial interaction with metals frequently manifests itself through metal immobilization or mobilization (Ferrris et al, 1989; Ghiorse and Ehrlich, 1992; Ehrlich, 1996). The activities of microbes and metals between the paint structure and the painted surface will eventually cause a detachment of the paintings from the wall. In emulsion paint formulation, oil with a higher degree of saturated fatty acid profile and more anti-oxidant vitamins is used, as this quality of oil enhances its performance on a given surface. Kabara (1978) and others have reported that certain fatty acids and their derivatives can have adverse effects on various microorganisms. Besides, Akinson (2008) and others have also reported that vitamin E protects lipids and prevents the oxidation of polyunsaturated fatty acids. 1.3 Aim of Study The major aim of this study is to investigate the major causes of the degradation of emulsion painting on buildings in Port Harcourt, Nigeria. 1.4 Specific Objective of Study (1) To identify the microbes present in degraded sites through microbial analysis and determine the pH of the environment. (2) To identify the heavy metals in degraded sites using atomic absorption spectroscopy. (3) To investigate the paint forming properties of some seed oils. 1.5 Potential Benefits of the Study (a) The results from this work will open a fresh window of research study in the effort to find a lasting solution to the degradation of emulsion paintings on walls of buildings. 6 University of Ghana http://ugspace.ug.edu.gh (b) The outcome of this work will assist paint producers in improving the quality of their products as they gradually become aware of the causes of degradation and take steps to address them. (c) This work will also help painters and other paint users to improve on their working methods. (d) The outcome of this work will also enhance the understanding of potential toxic elements in the environment and help policy makers in government to adopt approaches to minimize exposure to these elements, (e) The results of this work will contribute to the data base of indigenous knowledge and uses of natural products like plant seeds. 7 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 REVIEW OF LITERATURE 2.1 Paints Generally, paints are made up of: pigment and extenders, film former or binder, solvents, additives etc. Pigment: Pigments are granular solids incorporated into the paint to contribute color and toughness. Alternatively, some paints contain dyes instead of, or in combination with pigments. Pigments can be classified as either natural or synthetic. Natural pigments include various clays, calcium carbonate, mica, silicas and talcs. Synthetic pigments would include engineered molecules, calcined clays, blanc fix, precipitated calcium carbonate and synthetic silicas. Hiding pigments, in making paint opaque, also protect the substrate from the harmful effects of ultraviolet light. Hiding pigments include titanium dioxide, phthalo blue and red iron oxide. Fillers are a special type of pigments that serve to thicken the film, support its structure and simply increase the volume of the paint. Fillers usually comprise cheap and inert materials, such as talc, lime, barite and clay. Floor paints that will be subjected to abrasion may even contain fine quartz sand as a filler. Not all paints contain fillers. On the other hand, some paints contain very large proportions of pigment/filler and binder. Some pigments are toxic, for example, lead pigments that are used in lead paints. The titanium dioxide (less toxic substitute) used in most paints today is often coated with silicon or aluminium oxides for various reasons such as better 8 University of Ghana http://ugspace.ug.edu.gh exterior durability or better hiding performance (opacity) through better efficiency promoted by more optimal spacing within the paint. Binder/vehicle: The binder, commonly referred to as the vehicle, is the actual film forming component of paint. It is the only component that must be present in every type of paint. Other components listed below are (added) optionally, depending on the desired properties of the cured film. The binder imparts adhesion, binds the pigments together, and strongly influences such properties as gloss potential, exterior durability, flexibility, and toughness. Binders include synthetic or natural resins such as acrylics, polyurethanes, polyesters, melamine resins, epoxy or oils. Binders can be categorized according to drying or curing mechanism. The four most common drying or curing mechanisms are simple solvent evaporation, oxidative cross linking, catalyzed polymerization and coalescence. Drying generally refers to evaporation of the liquid (called the vehicle) which carries the pigment, whereas curing refers to polymerization of the binder. Depending on the chemistry and composition, any particular paint may undergo either or both processes. Thus, there are paints that only dry, those that dry and then cure, and those that do not depend on drying for curing. Paints that dry by simple solvent evaporation contain a solid binder dissolved in a solvent. This forms a solid film when the solvent evaporates, and the film can redissolve in the solvent again. Classic nitrocellulose lacquers fall into this category, as do non-grain raising stains composed of dyes dissolved in the solvent. Latex paint is a water-based dispersion of sub-micrometre polymer particles. The term ―latex‖ in the context of paint simply means an aqueous dispersion. Latex rubber (the sap of the rubber tree that has historically been called latex), is not an ingredient. These dispersions are prepared by emulsion polymerization. Latex paints cure by a process called coalescence where first the water and then the trace or coalescing solvent evaporates and 9 University of Ghana http://ugspace.ug.edu.gh draws together and softens the latex binder particles and fuses them together into irreversibly bound networked structures, so that the paint will not redissolve in the solvent/water that originally carried it. The presence of residual surfactants in the paint as well as hydrolytic effects with some polymers causes the paint to remain susceptible to softening and over time, to degradation by water. Paints that cure by oxidative cross-linking are generally single package coatings that, when applied, the exposure to oxygen in the air starts a process that cross-links and polymerizes the binder component. Classic alkyd enamels would fall into this category. Paints that cure by catalyzed polymerization are generally two package coatings that polymerize by way of a chemical reaction initiated by mixing resin and hardener, and which cure by forming a hard plastic structure. Depending on their composition, they may need to dry first by evaporation of the solvent. Classic two package epoxies or polyurethanes would fall into this category. Still other films are formed by a cooling of the binder. For example, encaustic or wax paints are liquid when warm and harden upon cooling. In many cases they will liquidify if reheated. Recent environmental requirements restrict the use of volatile organic compounds (VOC), and alternative means of curing have been developed, particularly for industrial purposes. In UV curing of paints, the solvent is evaporated first, and hardening is then initiated by ultraviolet light. In powder coatings, there is little or no solvent, and flow and cure are produced by a heating of the substrate after the application of the dry powder. Solvent: The main purpose of the solvent is to adjust the curing properties and viscosity of the paint. It is volatile and does not become part of the paint film. It also controls flow and application properties, and affects the stability of the paint while in the liquid state. Its main function is to serve as the carrier for non volatile components. In order to spread heavier oils (i.e 10 University of Ghana http://ugspace.ug.edu.gh linseed) as in oil-based interior house paint, a thinner is required. These volatile substances impart their properties temporarily once the solvent has evaporated or disintegrated and the remaining paint is fixed to the surface. This component is optional. Some paints have no diluent. Water is the main diluent for water- based paints. Solvent-based paints, sometimes called oil-based paints, can have various combinations of solvents as diluents, and these include aliphatics, aromatics, alcohols and ketones. These diuents may also include organic solvents such as petroleum distillate and esters. Sometimes, volatile low-molecular weight synthetic resins also serve as diluents. Such solvents are used when grease resistance or similar properties are desired. Additives: Besides the three main categories of ingredients, paints can have a wide variety of miscellaneous additives, which are usually added in very small amounts and yet impact a very significant effect on the product. Some examples include additives to modify surface tension, improve flow properties, improve the finished appearance, increase wet edge, improve pigment stability, impact antifreeze properties and control foaming and skinning. Other types of additives include catalysts, thickeness-stabilizers, emulsifiers, texturizers, adhesion-promoters, UV stabilizer flatteners (de-glossing agents) and biocides to fight bacterial growth (Woodbridge, 1991). Types of Paint: Paint consists of pigments and an oil or water-based binder. The proportion of pigment to binder in any paint dictates the amount of gloss the finished product will have. The glossier the finish, the more hard wearing it will be. There are various categories of finish, mainly matt and gloss, and a range in between the two which vary accordingly and are designated in a number of different terms like silk, satin, semi-gloss, eggshell etc. Water-based paints dry purely by evaporation while oil-based paints have chemical drying agents added. 11 University of Ghana http://ugspace.ug.edu.gh Paints with a water base are not as hard or durable as those with an oil base, although they are being improved upon all the time (Bently et al, 1997). Gloss Paints: They are oil-based and contain resins to give them a hard wearing quality. They normally include the following types: Liquid gloss: It needs an undercoat but gives the more traditional high gloss finish and is extremely hard wearing and resistant to dirt. Satinwood: It is a durable gloss paint that gives a more subtle sheen than the conventional shiny gloss; however, it is not usually very hardwearing. Eggshell: It is a paint type that gives a flatter (but not entirely matt) finish. It is often used for smaller pieces of decoration such as architrave and skirting. Polyurethane gloss: It is an oil-based paint with added polyurethane resin, making it tougher, providing a really hard wearing surface that withstands greater abrasion than standard gloss. Silthane: It is a combination of silicone and polyurethane. This paint is purported to give a stronger surface than polyurethane alone as the silicone gives it extra protection, especially during the drying period when paint is most vulnerable. Table 1: The Formulation For a Typical Alkyd Oil Paint (Mohammed et al ,2005) Materials Weight (kg) Volume(L) Rutile titanium dioxide 350.0 86.0 Antisag gel (8% NV) 35.0 42.0 Lecithin solution (50%) 4.0 4.4 12 University of Ghana http://ugspace.ug.edu.gh Long oil alkyd resin(70% 75.0 78.9 NV) White spirit 62.7 79.0 Long oil alkyd resin (50% 620.0 645.8 NV) Cobalt drier 2.5 2.5 ( 6% Co) Lead drier (24% Pb) 14.0 11.7 Calcium drier 6.0 6.2 (6% Ca) Antiskin solution (25%) 5.0 6.0 White spirit 30.4 37.5 TOTAL 1204.6 1000.0 Paint Variations: Various types of painits exist and are classified according to their uses. These are as stated below: 13 University of Ghana http://ugspace.ug.edu.gh (i) A primer is a preparatory coating put on materials before painting. Primering ensures better adhesion of paint to the surface, increases paint durability, and provides additional protection for the material being painted. (ii) Emulsion paint is a water-based paint used for painting interior or exterior surfaces. (iii) Varnish and shellac provide a protective coating without changing the colour. They are paints without pigment. (iv) Wood stain is a type of paint that is very ―thin,‖ low in viscosity and formulated so that the pigment penetrates the surface, rather than remaining in a film on top of the surface. Stain is predominantly pigment or dye and solvent with little binder, designed primarily to add colour without providing a surface coating. (v) Lacquer is usually a fast-drying solvent-based paint or vanish that produces an especially hard, durable finish. Enamel paint is a paint type that dries to an especially hard, usually glossy finish. They contain either gloss powder or tiny metal flake fragments instead of the colour pigments found in standard oil-based paints. Enamel paints are also mixed with varnish to increase shine as well as assist their hardening process. A glaze is an additive used with paint to slow drying time and increase translucency, as in Faux painting and Art painting. A roof coating is a fluid applied membrane which has elastic properties that allow it to stretch and return to its original shape without damage. It provides UV protection to polyurethane foam and is widely used as part of a roof restoration system. (vi) Finger paint is a kind of paint intended to be applied with the fingers; it typically comes in pots and is used by small children, though it has very occasionally been used by adults either to teach art to children, or for their own independent use. Inks are similar 14 University of Ghana http://ugspace.ug.edu.gh to paints, except they are typically made using finely ground pigments or dyes, and are designed so as not to leave a thick film of binder. Titanium dioxide is extensively used for both house paint and artist‘s paints, because it is permanent and has a good covering power. (vii) Anti-Graffiti paints are used to defeat the marking of surfaces by graffiti artists. There are two categories and they include sacrificial and non-bonding. Sacrificial coatings are clear coatings that allow the removal of graffiti and the coating, usually by pressure washing the surface with high-pressure water (hence, sacrificed). Sacrificial coatings must be re-applied afterward for continued protection. This is most commonly used on natural-looking masonry surfaces, such as statuary and marble walls, and on rougher surfaces that are difficult to clean. Non-bonding coatings are clear, high-performance coatings and are usually catalyzed polyurethanes that allow the graffiti very little to bond to. After the graffiti is discovered, it can be removed with the use of a solvent wash, without damaging the underlying substrate or protective coating. Non-bonding coatings work best when used on smoother surfaces, and especially over other painted surfaces, including murals. (viii) Anti-climb paint is a non-drying paint that appears normal while still being extremely slippery. It is usually used on drain pipes and ledges to deter burglars and vandals from climbing them, and is found in many public places. When a person attempts to climb objects coated with the paint, it rubs off onto the climber, as well as making it hard for the person to climb. No-VOC paints, which are solvent-free paints that do not contain volatile organic compounds, have been available since the late 1980s. 15 University of Ghana http://ugspace.ug.edu.gh Low VOC paints, which typically contain very low percentage of VOCs as coalescent or coalescing solvent, have been available since the 1960s. (Woodbridge, 1991). (ix) Application: Paint can be applied as a solid, a gaseous suspension (aerosol) or a liquid. Techniques vary depending on the practical or artistic results desired. As a solid (usually used in industrial and automotive applications), the paint is applied as a very fine powder, then baked at high temperature. This melts the powder and causes it to adhere (stick) to the surface. The reasons for doing this involve the chemistry of the paints, the surface itself and perhaps even the chemistry of the substrate (the overall object being painted). This is commonly referred to as powder coating an object. As a gas or as a gaseous suspension, the paint is suspended in solid or liquid form in a gas that is sprayed on an object and the paint sticks to the object. This is commonly referred to as spray painting an object. The reasons for doing this include: a. The application mechanism is air and thus no solid object ever touches the object being painted. b. The distribution of the paint is very uniform, so there are no sharp lines. c. It is possible to deliver very small amounts of paint. d. A chemical (typically a solvent) can be sprayed along with the paint to dissolve together both the delivered paint and the chemicals on the surface of the object being painted. e. Some chemical reactions in paint involve the orientation of the paint molecules. f. In the liquid application, paint can be applied by direct application using brushes, paint rollers, blades, other instruments, or body parts. Examples of body parts include finger painting, where the paint is applied by hand. 16 University of Ghana http://ugspace.ug.edu.gh g. Paint application by spray is the most popular method in industry. In this, paint is atomized by the force of compressed air or by the action of high pressure compression of the paint itself, which results in the paint being turned into small droplets that travel to the article which is to be painted. Rollers, generally have a handle that allows for different lengths of poles which can be attached to allow for painting at different heights. Roller application takes two coats for even colour. A roller with a thicker nap is used to apply paint on uneven surfaces. Edges are often finished with an angled brush (Boxall et al, 1980). Defects in Paint: A defect in a paint can be the result of any one of a number of causes and may, therefore, have a corresponding number of remedies. The type of defects can be classified as:- defects in the liquid paint, defects during application, defects during drying/curing and defects in the dry film (Tabbi, 1967). Table 2: The formulation for Water-based Paint (Mohammed et al, 2005) Materials Weight ( kg) Volume ( L) Water 280.0 280.0 Cellulosic thickener 1.5 1.0 Anionic dispersant 3.0 2.2 Wetting agent 2.5 2.4 Antifoam 1.0 1.1 Ammonia solution 1.5 1.6 Preservative (mercurial 0.5 0.2 17 University of Ghana http://ugspace.ug.edu.gh type) Talc 200.0 73.5 Diatomaceous silica 75.0 32.0 Rutile titanium dioxide 300.0 81.0 (special grade for high PVC latex paints) Coalescing agent 10.0 10.5 PVA acrylic emulsion ( 308.0 280.3 55% NV) Antifoam 1.5 1.6 Cellulosic thickener 150.0 148.0 solution (3% NV) Cellulosic thickener 83.0 83.0 solution or water TOTAL 1419.0 1000.0 Table 3: Formulation for a typical primer (Mohammed et al, 2005) Component % by weight Resin 7.2 Polyvinyl butryal resin 18 University of Ghana http://ugspace.ug.edu.gh Zinc tetroxychromate 7.0 Talc 1.0 Isopropyl alcohol 50.0 Toluol 14.8 Etchant 3.6 85% phosphoric acid Water 3.2 Isopropyl alcohol 13.2 2.1.1: Defects in the liquid paint a) Can Corrosion: This may be caused by incorrect pH of latex paints or incorrect choice of ingredients, leading to acidic by-products on storage. The remedy is a careful selection of can bearing linning or the addition of anti-corrosive agents to the paint or improved formulation and adjustment. b) Coagulation: This refers to the pre-mature coalescing of emulsion resin particles in the paint. It is often referred to as ―breaking of emulsion‖. Excessive stirring, solvent addition or addition of coalescing agents may be the cause. There is no truly effective remedy for coagulated paints. c) Gassing: This is aeration due to a chemical reaction within the liquid paint during storage. It can result in the explosion of cans. The action of water on aluminum or zinc based paints or acid on calcium carbonate will give this defect. The addition of water or acid scavenger in the paint could help to arrest this defect. 19 University of Ghana http://ugspace.ug.edu.gh d) Gelling: Deterioration of paint by the partial or complete changing of the medium into a jelly-like condition. This may be a result of chemical reaction between certain pigments and vehicles such as zinc oxide and acidic vehicle or between atmospheric oxygen and oxidisable or polymerisable oils in the vehicle. e) Settling: Separation of paint in a container in which the pigment and other dense insoluble materials accumulate and aggregate at the bottom. An increase in consistency will help, as will a thioxotropic rheology. Various additives are available to do this work. f) Skinning: This is the formation of a tough skin-like covering on liquid paints when exposed to air. If the skin is continuous and easily removed, it is not as troublesome as a slight, discontinuous skin which may easily become mixed with the rest of the paint. The formation of skin is due to oxidation and polymerization of the medium of the air-liquid interface. Anti-skinning agents, usually volatile antioxidants, are generally added to the paint to prevent skinning. g) Viscosity decrease: Many cases of reported low viscosity are the result of failure to stir thoroughly, or over-reduction. This may be checked by determining the density or solids content on a sample and comparing it with the original figure recorded for the bath. Other causes of viscosity drop are: (i.) Enzyme attack on cellulose thickeners, because of insufficient preservatives. (ii.) Failure of gallant or soap in solvent system to maintain gel, due to chemical or physical causes. (iii.) Change in pigment orientation on storage, such as dispersion of a partially flocculated system 20 University of Ghana http://ugspace.ug.edu.gh 2.1.2: Defects during Application Cobwebbing: This involves the formation of fine filaments of partly dried paint during the spray application of fast-drying paint. This can be caused by incorrect solvent blend in the coating or by spraying too far from the article. In order to reduce this defect the solvent should be properly mixed and spraying should be done closer to the article. Foaming: This is the formation of a stable gas-in-liquid dispersion in which the bubbles do not coalesce with each other or with the continuous gas phase. It is usually encountered in latex paints. The remedy is the addition of an anti-foam agent in the formulation of the paint. Drag or Sticky Application: Sticky application of paint may be caused by a number of factors which may be: the choice of formulation of ingredients, excessive rapid loss of solvent by evaporation during application, excessive viscosity, possibly because of bodying of the paint on storage,the use of a highly absorbent substrate or a poor quality brush. Poor application characteristics can be controlled to a certain extent during production by viscosity adjustment and by the choice of a suitable solvent balance, provided the basic formulation is correct. 2.1.3: Defects during drying/curing: These include: Bleaching: This is discolouration caused by migration of components from the underlying films. Substrates that can cause problems are timber stains that contain soluble dyes, and paints made from certain red and yellow organic pigments. 21 University of Ghana http://ugspace.ug.edu.gh Cissing: This is the recession of a wet paint film from a surface bearing small areas uncoated. It is a consequence of improper wetting of the substrate by the paint; surfactants can be used to remedy the situation. Floating: Refers to separation of pigment which occurs during drying, curing or storage that gives rise to patches in the surface of the film and produces a variegated effect. Thioxotropic paints will minimize this defect. Defects in the Dry Film: This defect is normally due to the following factors: Ageing: Gives rise to degeneration occurring in a coating with the passage of time and/or heating. Blistering: This is a deformation of a paint film in the form of blisters arising from the detachment of one or more of the coats. This is often the consequence of faulty surface preparation, leading to poor primer-substrate adhesion. Chalking: It refers to a change which involves the release of one or more of the constituents of the film in the form of loosely adherent fine powder. This is a result of the gradual breakdown of the binder because of the action of the weather. The use of a more durable binder type will reduce the chalking. Cracking: This is the formation of breaks in the paint film that expose the underlying surface. This is the most severe of a class of defects which include checking, crocodiling and embrittlement. The following should be avoided if cracking is to be reduced: applying paint 22 University of Ghana http://ugspace.ug.edu.gh before the previous coat is sufficiently hardened and applying a hard-drying finish over two elastic undercoats. Dirt Collection: The presence of matter adhering to the surface or embedded in a film but not derived from the film. This generally refers to atmospheric dust deposited on the film. The degree to which this can be removed by washing is related to the gloss and hardness of the film. Basic Process in Paint Formulation: Paint formulation involves several stages which include the following: Mill Charge/Dispersion Stage: This is the stage where specific quantifies of pigments and extenders are introduced to predetermine quantities of medium and solvent which are then ground by the use of a suitable machine. The aim of the dispersion is to reduce the particle size of pigments and extenders from different shapes to fine form/powder. The fineness of dispersion is usually tested with a Hedgeman gauge. However, the fineness/degree of grinding depends on the type of paint required. The finest dispersion is required on finishes with the highest gloss such as enamels, whereas a lower dispersion is adequate for a primer on under coat and flat finishes. The following factors can affect dispersion: particle size, particle shape, particle size distribution, agglomeration of dry pigment, adsorption and sedimentation behaviour (Boxall et al, 1980). Stabilization Stage: At this stage, more solvent is added to the dispersion product to increase the intermolecular distance between particles, thus preventing fine particles from aggregating together and thereby increasing stability. Let Down Stage: 23 University of Ghana http://ugspace.ug.edu.gh This is the stage where calculated amounts of additives are added to the paints, so as to ensure its good quality. A paint is obtained and then made to undergo series of quality control requirements such as: opacity measurement, gloss measurement, viscosity and drying test (Tubbi, 1967). 24 University of Ghana http://ugspace.ug.edu.gh Fig 1: Flow Chat for Paint Manufacture 2.2: SEED OILS Different seed oils play very important roles in the formulation of paints. They are as stated below: 2.2.1 COCONUT OIL Cocos nucifera is the scientific name of the common coconut fruit. The plant is one of the most valuable to man. It is a primary source of food, drink and shelter. Early Spanish explorers called it coco which means ‗monkey face‘ because the three indentations (eyes) on the nut resemble the 25 University of Ghana http://ugspace.ug.edu.gh head and face of a monkey. Nucifera means nut bearing. Man can use every part of the coconut. The white nut (meat) can be eaten raw or shredded and dried and used in most cooking recipes. A single coconut has as much protein as a quarter pound of beefsteak. Copra, the dried meat of the Kernels, when crushed is the source of coconut oil. The husks, known as coir, are short, coarse, elastic fibers used to make excellent thatch roofing material for houses. This very diverse plant is also an excellent source of charcoal which is produced from the shells, and which serves as fuel, not only in cooking but also in the production of gas masks and air filters (Woodruff, 1970). The outer part of the trunk of the coconut palm furnishes a construction lumber, known as porcupine wood, for houses and furniture. The swollen base of the trunk, when hollowed, can be turned into a hula drum that the Hawaiians use for entertainment. These are just a few examples of how extraordinary the coconut palm is. Palmae, the palm family to which the coconut belongs, is one of the oldest and most diverse of the family (Child, 1974). Palms have many botanical characteristics such as a woody trunk in many species, perennial growth leaves which are folded like a fan and the production of a single seed leaf which, qualifies them to be classified, along with grasses, lilies and other families, as monocotyledons. There have been sixty other species under the genus cocos, but the coconut palm stands by itself and is monotypic, meaning that within the genus cocos, only one species, nucifera, is recognized. Consequently, every coconut palm in the world is taxonomically the bane species, which probably makes it the most abundant single food tree in existence. Two major classes of coconuts are recognized on the basis of stature: tall and dwarf. The ones most commonly planted for commercial purposes are the tall varieties which are slow to mature and first flower six to ten years after planting. They produce medium-to-large size nuts and have 26 University of Ghana http://ugspace.ug.edu.gh a life span of sixty to seventy years. Within producing countries, it is noted that the wide and varied utilization of the coconut will always be important in their economy. Row copra used to be the major export form, but as coconut oil is becoming more widely used, its export as coconut oil is increasing. Another change is from the export of coconuts in the shell to the export of dessicated coconut. Both of these changes have benefited the countries of origin and the plant is still showing great potential by creating more employment in the tropics. The coconut has been a growing success since the time it was first discovered and to this day is still very diverse. In its pure state, coconut oil has no taste, odour or colour. It is liquid at ordinary temperatures and has low volatility. It is a non-drying oil and regarded to be one of the most consistent oils and quite stable at ordinary temperatures. It therefore serves a lot of purposes in both edible products and inedible ones (Style, 2008). Coconut oil can be emulsified with water in the presence of a suitable emulsifying agent to form a system in which the water is continuous or is dispersed as small droplets. The water can thus be used as diluents to reduce greasiness of the skin or hair (Griffin, 1949). It will undergo hydrolysis with acids, alkalis and high pressure steam to produce glycerine, a carbonyl acid, or its salts, depending upon experimental conditions. On continued exposure to moist air, coconut oil undergoes hydrolysis or oxidation in a process known as rancidification. In this case, the process takes place slowly and makes the oil lose its taste and develop an offensive smell. Slow hydrolysis by atmospheric moisture forms free fatty acids. Oxidation of C=C position takes place by the air trapped in the container to form peroxides. These decompose to form a number of carbonyl compounds which are responsible for bad taste and offensive odour. Rancidity may be 27 University of Ghana http://ugspace.ug.edu.gh regarded as a deterioration of odour and flavour in oils and fats. It can also be judged by chemical tests. Coconut oil, as a non-drying oil, does not form a hard surface in contact with air. In contact with 0 nickel catalyst at 120-150 C, and under 50-250psi pressure, coconut oil can undergo hydrogenation at C=C position to produce vanaspati Ghere (solid fat). The oil reacts with ICl or IBr in aqueous alcoholic solution at the points of unsaturation, giving rise to the formation of iodo-bromo derivatives. The process is known as mixed halogenation and the amount of iodine absorbed is proportional to the number of C=C present per mole. The amount of I-Cl absorbed by a known amount of fat or oil is expressed as an iodine number which is a direct measure of the unsaturation in the glycerides Coconut oil is a very important oil because it is a lauric oil. The lauric fats possess unique characteristics for food industry uses and also for the use of the soaps and cosmetic industries. This oil is unique because it is composed predominantly of medium chain triglycerides. It is the presence of these triglycerides in coconut oil that make it different from all other fats and for the most part gives it its unique character. Almost all of the medium-chain triglycerides used in research, medicine and food products come from coconut oil (Thampan, 1994). Kabara (1978) and others have reported that certain fatty acids (e.g. medium chain saturates) and their derivatives (e.g. monoglycerides) can have adverse effects on various microorganisms; those microorganisms that are inactivated include bacteria, yeast and enveloped viruses of the saturated fatty acids. Lauric acid has greater antiviral and antibacterial activity than either caprylic--C10 or myristic acid --C14 (Kabara, 1985). 28 University of Ghana http://ugspace.ug.edu.gh All three monoesters of lauric acid are shown to be active antimicrobials, e.g., alpha-alpha and beta-MG. Additionally, it is reported that the antimicrobial effects of the fatty acids and monoglycerides are additive and total concentration is critical for inactivating viruses (Isaacs and Thormar, 1990). The potentially pathogenic bacteria inactivated by monolaurin include listeria monocytogenes, staphylococcus aureus, streptococcus agalactiae, Groups AF and G streptococci, gram-positive organisms, and some gram-negative organisms, if pretreated with a chelator (Bodolie and Nickerson,1992; Kabara 1978, Kabara,1985, Isaacs et al, 1990, Isaacs and Schneidman ,1991, Isaacs and Thormar,1986; Isaacs and Thormar,1990, Isaac and Thormar ,1991, Thormar et al, 1987, Wang and Johnson ,1992). Decreased growth of staphylococcus aureus and decreased production of toxic shock syndrome toxin-1 was shown with 150g monolaurin per liter (Holland et al, 1994). Monolamin was 5000 times more inhibitory against listeria monocytogenes than ethanol (Oh and Marshal, 1993). Helicobacter pyloric is rapidly inactivated by medium-chain monoglycerides and lauric acid, and there appears to be very little development of resistance of the organisms of these natural antimicrobials (Petschow et al, 1996). A number of fungi, yeast and protozoa are inactivated or killed by lauric acid or monolaurin. The fungi include several species of ringworm (Isaacs et al, 1991). The yeast reported included candida albicans (Isaacs et al, 1991). The protozoan parasite Giadia is killed by free fatty acids and monoglycerides from hydrolysed human milk (Hernell et al, 1986; Reineret al, 1986; Crouchet al, 1991; Isaacset al, 1991). The medium-chain saturated fatty acids and their derivatives act by disrupting the lipid membranes of the organisms (Isaacs and Thormar, 1991; Isaacs et al, 1992). 29 University of Ghana http://ugspace.ug.edu.gh Coconut oil and palm kernel oil are the only two oils in existence that are made up predominantly of medium chain triglycerides (MCT). The medium chain fatty acids (MCFA) are six to twelve carbon chains in length and are saturated (6.0, 8.0, 10.0, 12.0); they comprise two- thirds of coconut oils fatty acids the saturated long chain fatty acids or LCTs (14.0, 16.0, 18.0) and less than a third (28 – 30%) and the unsaturates (18.0, 18.2) less than a fourth of coconut‘s fatty acids (Blackborn et al, 1990) Ohler (1984) had reported the chemical composition and fatty acid profile of coconut oil as follows:- Fats, %of part, dry basis 65 – 72 Acid value 1 – 10 Saponification value 251 – 364 Iodine value 7 – 10 Thiocyanogen value 61 – 70 R-m value 6 – 8 Polenske value 12 – 18 Unsaponifiable (%) 0.15 – 0.6 0 Refractive Index mp 40 C 1.448 – 1.450 0 0 Sp. Gravity, 40 /25 0.908 – 0.913 0 M.P. ( C) 25 – 26 0 Titer ( C) 20 – 24 Saturated acids, Capric 0 – 0.8 Caprylic 5.5 – 9.5 30 University of Ghana http://ugspace.ug.edu.gh Lauric 44 – 52 Myristic 13 – 19 Palmitic 7.5 – 10.5 Stearic 1 – 3 Arachidic 0 – 0.4 Unsaturated acids, Henadecanoic 0 – 1.3 Oleic 5 – 8 Lineleic 1.5 – 2.5 Composition of coconut shell, cocos minifera (dry basis) Ligmin – 36% Cellulose 53% Ash – 0.6% Approximately 50% of the fatty acids in coconut fat are lauric acid, and lauric acid is a medium chain fatty acid. Also, approximately 6 – 7% of the fatty acids in coconut fat are capric acid, and capric acid is another medium chain fatty acid. (Enigi, 1998). The molecule of coconut oil consists of one unit glycerine and a number of fatty acid components of medium carbon chain. Other plant oils consist of one unit of glycerine and typically three units of fatty acids of long carbon chain. The glycerine component of plant oil has a high boiling point which prevents the plant oil from volatizing. This is what makes it an excellent cooking oil. However, in biofuel application, what is used is the fatty acid component of the plant oil that is converted to another compound called ester. Esters volatize just like glycerine and so fatty acids can be separated from each other by esterification. The plant oil is reacted with an alcohol with the aid of a 31 University of Ghana http://ugspace.ug.edu.gh catalyst. If the plant oil is coconut and methanol is the alcohol reactant, the resulting element is coco methyl ester. Coco methyl ester is the chemical name of coco Biodiesel (Diaz, 1999). Modern medical science is confirming the use of coconut oil in treating many health conditions. Published studies in medical journals show that coconut oil in one form or another may provide a wide range of health benefits. Some of these are stated as follows: kills viruses that cause influenza, herpes, measles, hepatitis C, 1) Kills bacteria that cause ulcers, throat infections, urinary tract infections, gum disease and cavities, pneumonia and gonorrhea etc. 2) Kills fungi and yeasts that cause candidiasis, ringworm, athlete‘s foot, diaper rash etc. 3) Expels or kills tapeworms, lice, giardia, etc. 4) Provides a nutritional source of quick energy. 5) Improves digestion and absorption of other nutrients including vitamins, minerals, and amino acids. 6) Improves insulin secretion and utilization of blood glucose. 7) Relieves stress on pancreas and enzyme systems of the body. 8) Promotes healthy looking hair and complexion. 9) Prevents wrinkles, sagging skin, and age spots. 10) Softens skin and helps relieve dryness and flaking. 11) Supports the natural chemical balance of the skin. 12) Helps prevent obesity and over weight problems. 13) Is utilized by the body to produce energy in preference to being stored as body fat like other dietary fats. 32 University of Ghana http://ugspace.ug.edu.gh 14) Dissolves kidney stones. 15) Reduces epileptic seizure. 16) Helps prevent liver disease. 17) Helps to protect the body from harmful free radicals that promote premature aging and degenerative disease. 18) Improves calcium and magnesium absorption and supports the development of strong bones and teeth (Ellis, 1997). Lauric acid, the major fatty acid from the fat of the coconut, has long been recognized for the unique properties that it lends to non food uses in the soaps and cosmetics industry. More recently, lauric acid has been recognized for its unique properties in food use, which are related to its antiviral, antibacterial and antiprotozoal functions. Now, capric acid, another coconut fatty acid, has been added to the list of coconut‘s antimicrobial components. These fatty acids are found in the largest amounts only in traditional lauric fats, especially from coconut (Enigi, 1998). 2.2.2: Rubber Seed Oil (Hevea brasilliensis) It is a perennial plantation crop indigenous to South America and cultivated as an industrial crop (Abdullah et al, 2009). Its seeds are composed of about 48% oil (Nwokolo et al, 1988). The seed is a semi-dried substance (Aigbodin and Pillai, 2000) that does not contain any unusual fatty acids, but is a rich source of polyunsaturated fatty acids that make up 52% of its total fatty acid composition (Gandhi et al, 1990). It has been shown to have many industrial applications including the manufacture of fatty acids (Chin et al, 1977), paint, soap (Chin et al, 1977, Gandhi et al, 1990) and surface coatings (Aigbodin and Pillai, 2000). 33 University of Ghana http://ugspace.ug.edu.gh The rubber seed oil has potential applications in the manufacture of some consumer goods (Iyayi etal, 2007). However, the incidence of high free fatty acids in the oil may be a limiting factor in its application in the synthesis of some compounds such as vulcanized oil (Fernando. 1971). Attempts to deacidify the oil by using the alkali refining resulted in high refining losses (UNIDO, 1989). But studies carried out by Ebiwele and others in 2010 showed that rubber seed oil could be de-acidified by chemical means - a reesterification process using glycerol in the presence of a catalyst and under a vacuum. Furthermore, studies have also shown that rubber seed oil could be used in industries, especially the paint-, lubricating, cosmetic, as well as in the putty industries (Njoku et al, 1995a, 1995b, and 1996). Research has also shown that rubber seed oil may not be a good source of anti-oxidants, but it can provide adequate essential metals in nutrition (Ononogbu et al, 2001). 2.2.3: Palm Kernel Seed Oil The kernel nut of oil palm (Elaes guineesis) is cracked and milled before extraction of the palm kernel oil. The palm kernel oil is one of the most widely used of the lauric acid oils, which contain a high level of saturated fatty acids such as lauric acid and myristic.acid. And because it has a low degree of unsaturation, the oil possesses a high oxidative stability (Young, 1983). In the southern part of Nigeria, the oil is used for cooking, frying and even as cream in some families. The palm is similar to coconut palm. It grows straight and is able to reach 30m in maturity. It has no branches, but a trunk and sheets (Vandenput, 1981). The fruits are fleshy and elongated, egg-shaped, and of reddish colour. The nut has a hard shell which surrounds a kernel. It is from these kernels that palm kernel oil is extracted (Detheux, 2004). In the food industry, it 34 University of Ghana http://ugspace.ug.edu.gh is used in the preparation of certain traditional dishes and in the constitution of food fats (Dosumno and Ochu, 1995, Alonso etal, 2000). In the non-food industry, its higher lauric acid proportion gives this oil an important characteristic used in the preparation of soap and other beauty care products. This property also characterizes its strong use in the traditional pharmacopoeia (Salmich et al, 1998). 2.2.4: Soybean Seed Oil It is a valuable commercial source of edible oil and protein meal and contains about 42% protein and 25% oil at maturity (Bils and Howell, 1963). Studies have shown that soybean is widely used as the raw material for oil milling and the residue (Soy meal) is mainly used as feedstuff for domestic animals (Liu, 1997). According to this research, dry soybean contains 13% protein, 19% carbohydrate (17.5% of which is dietary fiber), 5% minerals and several other components including vitamins. Research has also shown that soybean oil contains tocopherols which are excellent natural anti-oxidants (Liu, 1997, Sugano, 2006). Soybean curd refuse (okara) contains soluble polysaccharides which are used to modify the physical properties of various foods (Espinosa-Martos, 2006). Soybean oil is characterized by large amounts of polyunsaturated fatty acids, i.e., linoleic acid and linolenic acid (Liu, 1997). However, due to the presence of lipoxygenates in soybean, linoleic acid renders the soybean oil prone to rancidification (Sugano, 2006). 35 University of Ghana http://ugspace.ug.edu.gh 2.2.5: Neem Seed Oil It is a vegetable oil pressed from the fruits and seeds of the neem (Azadirachia indica) tree which is an evergreen tree common in the tropics. The neem oil, much like other vegetable oils, is composed of triglycerides of oleic, stearic, linoleic and palmitic acids. The ―cold pressed oil‖ is mainly used in lamps, soaps and other non edible products. It is generally dark, bitter and smelly. Unlike most vegetable oils, it contains sulphur compounds whose pungent odor is reminiscent of garlic. The solvent extracted oil is not of as high a quality as the cold pressed oil. Components of neem oil can be found in many products which include toothpaste, cosmetics and soaps. Azadirachtin is the most active component for repelling pests and can be extracted from neem oil. The portion left over is called clarified hydrophobic neem oil which is made of fatty acids and glycerides. Research has shown that neem oil causes ‗solitarization of gregarious locust nymphs (Schmutterer and Freres, 1990). Also in a ‗‖taste test‖‖, American cockroach adults preferred neem treated pellets to untreated ones, but neem-treated cartons repelled them (Adler and Uchel, 1988). Neem cake (the residue left after oil has been removed from the kernel) has proved so successful that Philippi farmers are using it on a trial basis against the brown planthopper and other rice pests (Saxena et al, 1984, von der Hyde et al, 1984). In experiments in Virginia, neem seed extracts at relatively low concentrations were tested in potato fields both with and without the synergist piperonyl butoxide (PBO). All treatments significantly lowered the potato beetle populations and raised potato yields. However, the extracts containing PBO were the most effective. The sprayings were most effective when the larvae were young and were best when conducted as soon as the eggs hatched (Langs and Feuerhake, 1984). Certain limonoid fractions extracted from neem kennels 36 University of Ghana http://ugspace.ug.edu.gh are proving active against root-knot nematodes, the type most devastating to plants. They inhibit the larvae from emerging and the eggs from hatching, and in at least one test, they have done so at concentrations in the parts per million range (Devakomar et al, 1985). In a test in a green house and in the field in Germany, tomato plants were improved by neem products, but there was no significant difference in the numbers of some nematode in the soil. However, among treated and untreated soils the majority was extracted from the roots of plants in untreated soil. (Rossner and Zebitz, 1987) 2.2.6: Jatropha (Curcas) seed oil The jatropha tree is a multipurpose tree belonging to the family of Euphorbideae. The plant can be used to prevent or control erosion or to reclaim land; grown as a live-fence, especially to contain or exclude farm animals; or planted as a commercial crop. It is a native of tropical America, but now thrives in many parts of the tropics and sub-tropics in Africa (Gubitz et al, 1993; Kumar and Sharma, 2008; OpenShaw, 2000; Martinez H‘eriera et al, 2006). Studies have revealed that jatropha grows in tropical and subtropical climates across the developing world (OpenShaw, 2000). The fact that jatropha oil cannot be used for nutritional purposes without detoxification makes its use for energy or fuel source very attractive as biodiesel. In Madagascar, Cape Verde and Benin, jatropha oil was used as mineral diesel substitute during the Second World War (Agarwal, 2007). The wood and fruit of jatropha can be used for numerous processes including fuel. The seeds of jatropha contain viscous oil which can be used for the manufacture of candles and soap in the cosmetics industry, or as extender in a diesel/paraffin substitute. This latter use has 37 University of Ghana http://ugspace.ug.edu.gh important implications for meeting the demand for rural energy services and also exploring practical substitutes for fossil fuels to counter green house gas accumulation in the atmosphere. These characteristics, along with its versatility, make it of vital importance to developing countries (Kumar and Sharma, 2008). The high content of unsaturation in the fatty acid profile such as oleic and linoleic acids in jatropha places the oil in the drying oil group, and hence the oil can be used in the production of alkyd resin, shoe polish, varnishes etc. (Akintayo,2004, Eromosele et al ,1997). 2.3: Oils Oils are liquids and nonvolatile. They are readily soluble in benzene, petroleum, carbon disulphide, carbon tetrachloride, etc. Oils gradually decompose in light, air and moisture, i.e., they become rancid due to microbial action. Some oils do have natural odour, but this is due to some minor constituents (Sharma, 2006). There are two broad classifications for oils: edible and non-edible. The various kinds of edible oil include soya bean, corn and olive oil. These oils are employed for salad dressing, other table use and cooking purposes. The hydrogenated fats for cooking and baking include various vegetable oils such as cotton seed, soya bean and peanut. This is due to the fact that hydrogenation improves the colour, flavour and odour of the crude oil. Non-edible oils include tallow oil, coconut oil, palm oil and certain greases that are used for making soaps. The drying oil industry, including paints and varnishes, consume about 10% of non-edible oils (Martens, 1969). Castor oil, linseed oil, soya bean oil, rapeseed oil and cottonseed oil are used as plasticizers for lacquers and polymers. Oils, in general, have also been used for many other 38 University of Ghana http://ugspace.ug.edu.gh purposes such as the manufacture of lubricants and greases, various polishes, creams, and emulsions. In another classification of oils, depending on the nature of the carbon chain, vegetable oils can also be classified as non-drying, semi-drying and drying oils. In other words, glycerides occurring in nature are conveniently divided into non-drying, semi-drying and drying oils. These terms refer to the behaviour of the oil in contact with moist air, usually in diffused or strong day light. The drying oils thicken and form an elastic surface film. The non-drying oils do not change much in consistency. In non-drying oils, the carbon chain is saturated and these oils are quite stable at ordinary temperatures, but become viscouse at higher temperatures, e.g., castor oil and olive oil. Semi-drying oils have some degree of unsaturation in the carbon chain. They have a tendency of becoming thick in air without actually undergoing drying at ordinary temperature. This is probably due to slow oxidation at the point of unsaturation in the molecule, for example, rapeseed oil and cottonseed oil. Drying oils are spread in thin layers over a smooth surface. When exposed to light and air for a few hours, they set as a hard and firm film. This process is known as drying and may be regarded as rapid oxidation taking place at C=C positions involving various chemical reactions (Sharma, 2006). 2.4: FATTY ACIDS All fats and oils are composed of molecules called fatty acids. There are two methods of classifying fatty acids. One is based on saturation and the other is based on the molecular size or the length of the carbon chain in the fatty acid. For saturated fatty acids, some are mono- saturated while others are poly-saturated. For size or length classification, there are short chain 39 University of Ghana http://ugspace.ug.edu.gh fatty acids (SCFA), medium chain fatty acids (MCFA), and long chain fatty acids (LCFA). Another term most often used in this regard is triglyceride.hence there are short-chain triglycerides, medium-chain triglycerides or long-chain triglycerides. There are a dozen or so fatty acids that are common in our food. What makes corn oil different from soybean, olive or coconut oil is the combination and types of fatty acids each contains. The fatty acids that make up the various dietary oils are in the form of triglycerides.So, dietary fats, including coconut oil, consist of fatty acids that are in the form of triglycerides. The melting point of coconut and other oils is determined by the fatty acid content. The triglycerides in coconut oil consist of a mixture of ten different fatty acids. Each fatty acid has its own melting point. Saturated fatty acids have a higher melting point than mono-saturated fatty acids, and mono-saturated fatty acids have a higher melting point than polyunsaturated fatty acids. This is why animal fat, which is highly saturated, is solid at room temperature and why olive oil (mono- saturated fat) and corn oil (polyunsaturated fat) are liquid at the same temperature. When one leaves olive oil in the refrigerator, however, it will become solid, but corn oil will remain liquid. In addition to the degree of saturation, the size of the fatty acid also influences the melting point. Fatty acids are composed of predominantly a chain of carbon atoms. The longer the carbon chain, the larger the fatty acid and the higher the melting point. Consequently, long chain fatty acids have higher melting points than medium or short chain fatty acids. Therefore, each of the ten fatty acids in coconut has its unique melting point. To cloud the picture even more, triglycerides can be composed of any combination of three of the ten fatty acids and each combination (or each triglyceride) will have a unique melting point. Because of the various melting points of the fatty acids and triglycerides, oils normally do not have a sharp or precise 40 University of Ghana http://ugspace.ug.edu.gh 0 melting point. Unlike ice that melts at exactly 18 C, oils change from solid to liquid over a range of temperature. For this reason, the melting point is determined by the temperature at which only 3-5 % of solids are present. Because coconut oil is composed mainly of medium chain fatty acids which have similar melting points, the melting point of coconut oil is more precise than 0 that of other dietary oils. While 42 C is given as the official melting point, in reality portions of it begin to melt (or freeze) a few degrees lower or higher. Therefore, some of the oil may become 0 solid or start to crystallize at 40-43 C. If the change in temperature is rapid, the melting point appears to be more precise and if the change in temperature is slow, an oil with both liquid and solid components will result. 0 Many homes maintain a constant temperature of 40-42 C. This is precisely the range in which components of coconut oil begin to melt as well as freeze. When liquid coconut oil is stored in such an environment the transformation from liquid to solid is very slow. This allows portions of the oil that have the highest melting point to solidify first. If the change in temperature is very gradual it allows crystals to develop. These are the crystals that are usually found in coconut oil. 2.4.1: LAURIC ACID Lauric acid is a white, powdery solid with a faint odor of bay oil or soap. It is a component of triglycerides and comprises about half of the fatty acid content in coconut oil and palm kernel oil (Beare-Rogers et al, 2001; David et al, 2006). It is also found in human breast milk, cow milk and goat milk. Lauric acid has a long shelf-life, and is non-toxic and safe to handle. It is mainly used for the production of soaps and cosmetics. For these purposes, lauric acid is neutralized with sodium hydroxide to give sodium laurate, which is soap. Ordinarily, sodium laurate is 41 University of Ghana http://ugspace.ug.edu.gh obtained by saponification of various oils, such as coconut oil. These precursors are mixtures of sodium laurate and other soaps (David et al, 2006). In the laboratory, lauric acid is often used to investigate the molar mass of an unknown substance via the freezing point depression. Lauric acid is convenient because its melting point when pure 0 is relatively high (43.2 C). Its cryoscopic constant is 3.9k.kg/mol. By melting lauric acid with the unknown substance, allowing it to cool, and recording the temperature at which the mixture freezes, the molar mass of the unknown compound may be determined (Freezing Point Determination Method, 2010). Lauric acid has been claimed to have antimicrobial properties (Hoffman et al, 2001; Ouattaret al, 2001, Dawson et al ,2002; Alexey et al, 2000). It has been found to increase total cholesterol in almost all the fatty acids.But most of the increase is attributable to an increase in high-density lipoprotein (HDL), a good cholesterol. As a result, lauric acid has a more favourable effect on total HDL cholesterol than any other fatty acid, either saturated or unsaturated (Mensink et al, 2003). A lower total HDL cholesterol ratio suggests a decrease in atherosclerotic risk (Thijssen et al, 2005). Table 4: Properties of lauric acid (Lide, 2005) Molecular Formula C12H24O2 MolarMass 200.318 Appearance White powder Odor Slight odor of bay oil 3 Density 0.880g/cm 42 University of Ghana http://ugspace.ug.edu.gh 0 Melting Point 298.9 C 0 Solubility in Water 0.006g/100ml/20 C Refractive Index 1.423 Viscosity 7.30mPars at 323k 0 Flash Point 110 C 2.4.2: PALMITIC ACID Palmitic (hexadecanoic) acid is the most common fatty acid (saturated) found in animals, plants and microorganisms (Gunstone et al, 2007). Its molecular formula is CH3(CH2)14CO2H. Palmitate is a term for the salts and esters of palmitic acid. It occurs mainly as the ester in triglycerides (fats), especially palm oil. The cetyl ester of palmitic acid (cetyl palmitate) occurs in spermacell. It was discovered by Edmond Freny in 1840. Palmitic acid is mainly used to produce soaps, cosmetics, and release agents. These applications utilize sodium palmitate which is commonly obtained by saponification of palm oil. Hydrogenation of palmitic acid yields cetyl alcohol which is used to produce detergents and cosmetics. Palmitic acid is prepared by treating fats and oils with water at 0 a high pressure and temperature (above 200 C) leading to the hydrolysis of triglycerides. The resulting mixture is then distilled (Anneken et al, 2006). Excess carbohydrates in the body are 43 University of Ghana http://ugspace.ug.edu.gh converted to palmitic acid. It is the first fatty acid produced during fatty acid synthesis and the precursor to longer fatty acids. Hence palmitic acid is a major body component of animals. In humans, one analysis found it to comprise 21-30% (molar) of human depot fat and it is a major but highly variable, lipid component of human breast milk (Kingsburry et al, 1961). 2.4.3: CAPRYLIC ACID Caprylic (octanoic) acid is found naturally in the milk of various mammals and is a minor constituent of coconut oil and palm kernel oil. It is an oily liquid that is minimally soluble in water with a slightly unpleasant rancid-like smell and taste (Beare-Rogers et al, 2001). It is used in the production of esters which are used in the perfumery and also in the manufacture of dyes. Caprylic acid is also used in the treatment of some bacterial infections. Due to its relatively short chain length, it has no difficulty in penetrating fatty cell wall membranes, hence its effectiveness in containing certain lipid coated bacteria such as staphloccocus aureus (Nair et al, 2001). It is an antimicrobial pesticide used as food contact sanitizer in commercial food handling establishments, dairy equipment, and breweries etc. It is also used as a disinfectant, algaecide, bactericide, and fungicide in nurseries, green centres etc. 2.4.4: CAPRIC ACID Capric (decanoic) acid is a saturated fatty acid and its formula is CH(CH2)8COOH. Salts and esters of capric (decanoic) acid are called caprates or decanoates. 44 University of Ghana http://ugspace.ug.edu.gh It occurs naturally in coconut oil and palm kernel oil and is uncommon in typical seed oils (David et al, 2006). Capric acid can be prepared from oxidation of the primary alcohol, decanol, by using chromium trioxide (CrO3) oxidant under acidic conditions (John, 2008). Neutralization of decanoic acid or saponification of its esters, typically triglycerides with sodium hydroxide, will give sodium decanoate. It is used in the manufacturing of esters for artificial fruit flavours and perfumes. It also serves as an intermediate in many chemical syntheses. 2.4.5: CAPROIC ACID Caproic (hexanoic) acid is the carboxylic acid derived from hexane with the general formula C5H11COOH. It is a colourless oily liquid with an odor like that of goats. It is a fatty acid, found naturally in various animal fats and oils. It is also one of the chemicals that give the decomposing fleshy seed coat of the ginkgo its characteristic unpleasant odor. It is also one of the components of vanilla and is primarily used for the manufacture of its esters for artificial flavors, and in the manufacture of hexyl derivatives such as hexlphenols. The salts and esters of this acid are known as hexanoates or caproates. Caproic caprylic and capric acids are not only used for the formation of esters, but also commonly used in butter, milk, cream, strawbeery, bread, beer, nut, and other flavors. Capric acid is a crystal or wax-like substance, whereas caproic and caprylic acids are mobile liquids. 45 University of Ghana http://ugspace.ug.edu.gh 2.4.6: STEARIC ACID Stearic (octadecanoic) acid is a saturated fatty acid with a chemical formula CH3(CH2)11CO2H. The salts and esters of stearic acid are called stearates. It is one of the most common saturated fatty acids found in nature, following palmitic acid (Gunstone et al, 2007). It occurs in many animal and vegetable fats. The important ones are cocoa butter and Shea butter, where the stearic acid content (as triglyceride) is 28-45% (Beare- Rogers et al, 2001). Stearic acid is prepared by treating these fats and oils with a high pressure 0 and temperature (above 200 C), leading to the hydrolysis of triglycerides. The resulting mixture is then distilled. Commercial stearic acid is often a mixture of stearic and palmitic acids, although purified stearic acid is available (David et al, 2006}. Stearic acid is mainly used in the production of detergents, soaps and cosmetics, such as shampoos, and shaving cream products. Lithium stearate is an important compound of grease and the stearate salts of zinc, calcium, cadmium and lead are often used to soften polyvinyl chloride (a polymer). Stearic acid is also used along with castor oil for preparing softeners in textile sizing. They are heated and mixed with caustic potash or caustic soda. Fatty acids are common components of candle making and stearic acid is used along with simple sugar or corn syrup as a hardener in candles. In fireworks, stearic acid is often used to coat metal powders such as aluminium and iron. This prevents oxidation, allowing compositions to be stored for a longer period of time. Stearic acid is a common lubricant used for injection molding and pressing of ceramic powders (Tsenga et al, 1999). 46 University of Ghana http://ugspace.ug.edu.gh 2.4.7: ARACHIDIC ACID Arachidic (eicosanoid) acid is a saturated fatty acid with a 20-carbon chain. It is a minor constituent of peanut oil and corn oil (Beare-Rogers et al, 2001). It is formed by the hydrogenation of arachidonic acid. Reduction of arachidic acid yields arachidyl alcohol. Arachidic acid is used for the production of detergents, photographic materials and lubricants. 2.4.8: MYRISTIC ACID Myristic (tetradecanoic) acid is a common saturated fatty acid with the molecular formula CH3(CH2)12COOH. A myristate is a salt or ester of myristic acid. It is found in nutmeg, palm kernel oil, coconut oil, butter fat and is a minor component of many other animal fats. It is also found in spermacell, the crystallized fraction of oil from the sperm whale. The ester isopropyl myristate is used in cosmetics and tropical medicinal preparations where good absorption through the skin is desired. Reduction of myristic acid yields myristyl aldehyde and myristyl alcohol (Gunstone.et al, 2007). 47 University of Ghana http://ugspace.ug.edu.gh 2.4.9: OLEIC ACID Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats and oils. It is an odourless, colourless oil, although commercial samples may be yellowish. It has the formula: CH3(CH2)7CH═CH(CH2)7COOH (Thomas, 2000). Oleic acid occurs as its esters, commonly the triglycerides, which are the greasy materials in many natural oils. Generally, through the process of saponification, oleic acid can be obtained. Triglycerides of oleic acid compose the majority of olive oil. Oleic acid also makes up 59-75% of pecan oil (Villarreal-Lozoya et al 2007). It is abundantly present in many animal fats, constituting 37-56% of chicken and turkey fat (Nutter, 1943). It is the most abundant fatty acid in human adipose tissue (Kokatnur et al, 1979). Oleic acid is emitted by the decaying corpses of a number of insects, including bees and pogononymex ants, and triggers the instincts of living workers to remove the dead bodies from the hive. If a live bee ant is daubed with oleic acid, it is dragged off for disposal as if it were dead (Pumamadjaja and Russel, 2005; Ayasse, 2002). The oleic acid smell may also indicate danger to living insects, prompting them to avoid others who have succumbed to disease or places where predators lurk (Walker, 2009). Oleic acid undergoes the reactions of carboxylic acids and alkenes. It is soluble in an aqueous base to give soaps called oleates. Hydrogenation of the double bond yields the saturated derivative stearic acid. Oxidation at the double bond occurs slowly in air and is known as rancidification in foodstuffs or drying in coatings. Reduction of the carboxylic acid group yields oleyl alcohol. Ozonolysis of oleic acid is an important route to azelaic acid; the coproduct is nonanoic acid (Cornils and Lappe, 2000): H17C8CH═CHC7H14CO2H + 2O2 → H17C8CO2H + HO2CC7H14CO2H 48 University of Ghana http://ugspace.ug.edu.gh Esters of azelaic acid find applications in lubrication and plasticizers. In chemical analysis, fatty acids are separated by gas chromatography of methyl esters; additionally, a separation of unsaturated isomers is possible by argentation thin-layer chromatography (Breue et al, 1987). Oleic acid, as its sodium is a major component of soap, is an emulsifying agent. It is also used as an emollient (Carrasco, 2009). Small amounts of oleic acid are used as an excipient in pharmaceuticals; oleic acid is used as a solubilizing agent in aerosol products (Smolinske, 1992). The intravenous administration of oleic acid causes acute lung injury with corresponding pulmonary adena (Julien et al, 1992). Oleic acid may hinder the progression of adrenoleukodystrophy (ALD), a fatal disease that affects the brain and adrenal glands (Rizzo et al, 1986). Oleic acid may be responsible for the hypotensive (blood pressure reducing) effects of olive oil (Teres et al, 2008). Adverse effects, also, have been documented, however, since both oleic acid and monounsaturated fatty acid levels in the membranes of red blood cells have been associated with increased risk of breast cancer (Papa et al, 2001). 2.4.10: LINOLEIC ACID This is an unsaturated omega-6 fatty acid. It is a colorless liquid at room temperature. It is a carboxylic acid with an 18-carbon chain and two double bonds. The first double bond is located at the sixth carbon from the methyl end (David et al, 2006). Linoleic acid belongs to one of the two families of essential fatty acids; the body cannot synthesize linoleic acid from other food components (Burr et al, 1930). Linoleic acid is found in the lipids of cell membranes. It is abundant in many vegetable oils, comprising over half (by weight) of poppy seed, sunflower and corn oils (Cunname and Anderson, 1997). Linoleic acid is an essential fatty acid that must be 49 University of Ghana http://ugspace.ug.edu.gh consumed for proper health. A diet only deficient in linoleate causes mild skin scaling, hair loss, and poor wound healing in rats (Cunnane and Anderson, 1997; Ruthig and Meckling-Gill, 1999). Along with oleic acid, linoleic acid is released by cockroaches upon death, which has the effect of preventing other cockroaches from entering the area. This is similar to the mechanism found in ants and bees in which oleic acid is released upon death. In its metabolism, linoleic acid is first converted into gamma-linoleic acid. This is later converted into a group of metabolites called eicosanoids, a class of paracine hormones (David 1993; Piomelli, 2000). 50 University of Ghana http://ugspace.ug.edu.gh Figure 1: Linoleic Acid Metabolism It is used in making quick drying oils which are useful in oil paints and varnishes. These applications exploit the easy reaction of linoleic acid with oxygen in air which leads to crosslinking and formation of a stable film. Reduction of linoleic acid yields linoleyh alcohol. Linoleic acid is a surfactant, and has become increasingly popular in the beauty products industry because of its beneficial properties on the skin which include: anti-inflammatory and acne reductive qualities as well as its ability to retain moisture on the skin (Letawe et al, 1998; 51 University of Ghana http://ugspace.ug.edu.gh Darmastardt et al, 2002). Linoleic acid may be linked to obesity by promoting overeating and damaging the arcuate nucleus in the hypothatamus of the brain (Ralof, 2012). 2.4.11: ALPHA-LINOLENIC ACID Alpha-linolenic acid is an essential omega 3 fatty acid and organic compound found in seeds and many common vegetable oils. It is relatively more susceptible to oxidation and will become rancid more quickly than many other oils. The oxidative instability of alpha-linolenic acid is one reason why producers choose to partially hydrogenate oils containing the acid such as soybean oil (Kinney, 2007). Seed oils are the oldest sources of alpha-linolenic acid, for example, chia, perilia, linseed, rapseed and soybeans as shown in the table below. It is also obtained from the thylakoid membranes in the leaves of pisum sativum pea leaves (Chapman et al, 1983). It is not suitable for baking, as it will polymerize with itself, a feature exploited in paint with transition metal catalysts. Some of the acid will also oxidize at baking temperature. Preliminary research has found evidence that it is related to a lower risk of cardiovascular disease (Burdge and Cadder, 2005; Penny et al, 2002). A 2005 study found that daily administration of the acid greatly reduced both self-reported anxiety, stress levels and objective measured cortisol levels in college age students (Yehuda et al, 2005). Table 5: The % Alpha-Linolenic Acid from some extracted oils (Li, Thomas, 1999) † Common name Alternate name Linnaean name % ALA Chia chia sage Salvia hispanica 64% 52 University of Ghana http://ugspace.ug.edu.gh Kiwifruit seeds Chinese gooseberry Actinidia chinensis 62% Perilla Shiso Perilla frutescens 58% Flax Linseed Linum usitatissimum 55% Lingonberry Cowberry Vaccinium vitis-idaea 49% Camelina Camelina Camelina sativa 35-45% Purslane Portulaca Portulaca oleracea 35% Sea buckthorn Seaberry Hippophae rhamnoides L. 32% Hemp Cannabis Cannabis sativa 20% Rapeseed Canola Brassica napus 10% Soybean Soya Glycine max 8% 2.5: ANTIOXIDANT VITAMINS An antioxidant is a molecule that inhibits the oxidation of other molecules. And oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which in turn can start chain reactions. When a chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates and by inhibiting other oxidation reactions. They do this by being oxidized themselves; so antioxidants are often reducing agents, for example, ascorbic acids. Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipid (lipophilic). The water-soluble antioxidants react with oxidants in 53 University of Ghana http://ugspace.ug.edu.gh the cell cytosol and the blood plasma, while lipid=soluble antioxidants protect cell membranes from lipid peroxidation. These compounds may be synthesized in the body or obtained from the diet (Vertuani et al, 2004). 2.5.1: VITAMIN E, VE Vitamin E refers to a group of eight fat-soluble compounds that include both tocopherols and tocotrienols (Brigelius-Flohe and Traber, 1999). The eight forms of vitamin E are divided into two groups: four are tocopherols and four are tocotrienols. Alpha-tocopherol is the most biologically active form of vitamin E. It is the second most common form of vitamin E in diet and can be found most abundantly in wheat germ oil, sunflower and soybean oils (Reboul et al, 2006). As a fat-soluble antioxidant, it stops the production of reactive oxygen species formed when fat undergoes oxidation (Herera, 2001; Packer et al, 2001). However, the importance of the antioxidant properties of this molecule at the concentration present in the body are not clear and the reason vitamin E is required in the diet is possibly unrelated to its ability to act as an antioxidant (Brigelius-flohe, 2009). Vitamin E has many biological functions, the antioxidant function being the most important and best known (Bell, 1987). Other functions include enzymatic activities, gene expression and neurological functions (Azzi, 2007; Zingg-Azzi, 2004). Vitamin E also protects lipids and prevents the oxidation of polyunsaturated fatty acids (Akinson et al, 2008). The deficiency of vitamin E can cause spinocerebellar ataxia (Traber, 2007), red blood destruction (Whitney et al, 2011), 54 University of Ghana http://ugspace.ug.edu.gh peripheral neuropathy, skeletal myopathy, retinopathy and impairment of the immune response (Kowdley et al, 1992). 2.5.2: VITAMINE A Vitamin A is a group of unsaturated nutritional organic compounds that includes retinol, retinal, retinoic acid and several provitamin A carotenoids, among which beta-carotene is the most important (Fennema, 2000). Vitamin A has multiple functions; it is important for growth and development and for the maintenance of the immune system and poor vision (Tanumihardjo, 2011). Vitamin A is needed by the retina of the eye in the form of retinal which combines with protein, opsin, to form rhodopsin, the light-absorbing molecule that is necessary for both low- light (sotopic vision) and color vision. Vitamin A plays a role in a variety of functions throughout the body, such as vision, gene transcription, embryonic development,skin health and antioxidant activity. The conversion of retinol from provitamin carotenoids by the human body is actively regulated by the amount of retinol available to the body. The absorption of provitamins depends greatly on the amount of lipids ingested with the provitamin and lipids increase the uptake of the provitamin (Solomons, 2003). Conversion of carotene to retinol varies from person to person and bioavailability of carotene in food also varies (Borel et al, 2005; Tang et al, 2005). Vitamin A and more specifically retinoic acid, appears to maintain normal skin health by switching on genes and differentiating keratinocytes (immature skin cells) (Moore and Holmes, 1971). Vitamin A deficiency can occur as either a primary or secondary deficiency. A primary vitamin A deficiency occurs among children and adults who do not consume an adequate intake of provitamin A, carotenoids from fruits and vegetables or preformed vitamin A from animal and 55 University of Ghana http://ugspace.ug.edu.gh dietary products. Early weaning from breast milk can also increase the risk of vitamin A deficiency. Zinc deficiency can also impair absorption, transport and metabolism of vitamin A and zinc increases the severity of vitamin A deficiency (Combs, 2008). Night blindness is one of the first signs of vitamin A deficiency. Besides, vitamin A deficiency also diminishes the ability of the body to fight infections. Excessive dietary intake of beta-carotene can lead to carotenoderma which causes orange-yellow discoloration of the skin (Nishimura et al, 1998, Takita, 2006). Smokers and chronic alcohol consumers have been observed to have increased risk of mortality due to lung cancer, esophageal cancer, gastrointestinal cancer and colon cancer (Crabb et al, 2001). 2.6: GAS CHROMATOGRAPHY Gas Chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vapourized without decomposition. Typical uses of GC include testing the purity of a particular substance or separating the different components of a mixture; and the relative amounts of such components can also be determined. Figure 2: Block Diagram of a Gas Chromatograph (Pavia, 2006) 56 University of Ghana http://ugspace.ug.edu.gh Figure 3: Block Diagram of a Gas Chromatograph(Pavia,2006) In some cases, GC may help in identifying a compound in preparative chromatography (Pavia, 2006). In gas chromatography, the mobile phase (or ―moving phase‖) is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column. The instrument used to perform a gas chromatography is called a gas chromatograph. The gaseous compounds being analyzed interact with the walls of the column which is coated with a stationary phase. This causes each compound to elude at a different time, known as the retention time of the compound. The comparison of retention time is what gives GC its usefulness. In a typical GC analysis, a known volume of gaseous or liquid analyte is injected into the ―entrance‖ (head) of the column with the aid of a microsyringe. As the carrier gas sweeps the analyte molecules through the column, the motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the 57 University of Ghana http://ugspace.ug.edu.gh analyte mixture are separated as they progress along the column at different times (retention time). A detector is used to monitor the outlet stream from the column, thus the time at which each component reaches the outlet and the amount of that component can be determined. Thus, substances are identified (qualitatively) by the order in which they emerge (elude) from the column and by the retention time of the analyte in the column. In a qualitative analysis, chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis) which is called a chromatogram. This provides a spectrum of peaks for a sample representing the analytes present in a sample eluding from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant conditions and can be used to identify complex mixtures of analytes. In most modern applications, a GC is connected to a mass spectrometer. For a quantitative analysis, the area under a peak is proportional to the amount of the analyte present in the chromatogram. By calculating the area of the peak using the mathematical function of integration, the concentration can be calculated using a calibration curve created by finding the response for a series of concentrations of analyte. The relative response factor is the expected ratio of an analyte to an internal standard and is calculated by finding the response of a known amount of analyte and a constant amount of internal standard. An internal standard is a chemical added to the sample at a constant concentration, with a distinct retention time to the analyte (Robert and Eugene, 2004). The conditions which can be varied to accommodate a required analysis include inlet temperature, detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates, the column stationary phase, diameter and length, inlet type and flow rates, sample size and injection technique. 58 University of Ghana http://ugspace.ug.edu.gh 2.7: ATOMIC ABSORPTION SPECTROSCOPY Atomic Absorption Spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements employing the absorption of optical radiation (light) by free atoms in the gaseous state. In analytical chemistry, the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. Figure 3: Block Diagram of an Atomic Absorption Spectroscopic Machine (Welz and Sperting, 1999) It can be used to determine over seventy different elements in solution or directly in solid samples (Welz and Sperting, 1999). The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration, and relies therefore on the Beer-Lambert law. In the process, the electrons of the atom in the 59 University of Ghana http://ugspace.ug.edu.gh atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, that is wavelength, is specific to a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert law. In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used are flame and electrothermal (graphite tube) atomizers (Walsh, 1955). The atoms should then be irradiated by optical radiation, and the radiation source could be an element specific line radiation source or a continuum radiation source. The radiation then passes through a monochrometer in order to separate the element specific radiation from any other radiation emitted by the radiation source which is finally measured by a detector. Studies have revealed that a number of factors are responsible for the degradation of emulsion paintings. They include polycyclic aromatic hydrocarbons, heavy metals, microbes, inorganic carbonates, sulphates and nitrates. 2.8. POLYCYCLIC AROMATIC HYDROCARBONS Polycyclic Aromatic Hydrocarbons (PAHs) are chemicals that are often found together in groups of two or more. They are found naturally in the environment, but they can also be man-made. They are solid and range in appearance from colourless to white or pale yellow. PAHs are 60 University of Ghana http://ugspace.ug.edu.gh created when products like coal, oil, gas and garbage are burnt (ATSDR, 1990). PAHs are a concern because they are persistent, they do not burn easily. They can stay in the environment for long periods of time. Individual PAHs vary in behavior. Some can turn into a vapor in the air very easily. Most do not break down easily in the water. PAHs can enter the body through breathing contaminated air. Another way this happens is when one eats or drinks food and water contaminated with PAHs. Besides, exposure to PAHs can also occur if the skin contacts PAHs contaminated soil or products like heavy oils, coal tar, roofing tar or creosote. Creosote is an oily liquid found in coal tar and is used to preserve wood. Once in the body, PAHs target the kidney and liver and they leave the body through urine and faeces in a matter of days (USEPA, 2001). 2.8.1: ANTHRACENE Anthracene is a solid polycyclic aromatic hydrocarbon of formula C14H10 and consists of three fused benzene rings. It is a component of coal tar. It is used in the production of the red dye alizarin and other dyes. Anthracene is colourless but exhibits a blue fluorescence under ultraviolet light. In 2010, a strong absorption band of anthrancene was observed along a line of Sigh to a star in the open cluster, and this may be associated with an intervening molecular cloud (Iglesias-Groth et al, 2010). 61 University of Ghana http://ugspace.ug.edu.gh Figure 4: An Anthracene Molecule (Iglesia-Groth et al, 2010) Commercial anthracene is obtained from coal tar. A laboratory method for the preparation of anthracene is by cyclodehydration of alpha-methyl or alpha-methylene substituted diaryketones in the Elbs reaction. Anthrancene photodimerizes by the action of UV-light, as seen below: The reaction is affected by the presence of oxygen. Oxidation occurs readily, giving anthraquinone, C14H8O2, as seen below, for example, using hydrogen peroxide and vanadyl acetylacetonate (Kimberly et al, 2011). 62 University of Ghana http://ugspace.ug.edu.gh Figure 5: An Anthraquinone Anthrancene will also react with the denophile singlet oxygen in a (4+2) cycloaddition Diels- Alder reaction as seen below: Figure 6: Anthrancene is converted to anthroquinone, a precursor to dyes (Gerd et al, 2006). It is used as a scintillator for detectors of high energy photons, electrons and alpha particles. Plastics, such as polyvinyltoluene, can be doped with anthrancene to produce a plastic scintillator that is approximately water-equivalent for use in radiation therapy dosimetry. It can also be used in wood preservative, insecticides and coating materials (Gerd et al, 2006). 2.8.2: ACENAPHTHENE It is a polycyclic aromatic hydrocarbon consisting of naphthalene with an ethylene bridge connecting positions 1 and 8. Acenaphthene is a colourless solid. Coal tar consists of about 0.3% of this compound (KarGriesbaun et al, 2002). 63 University of Ghana http://ugspace.ug.edu.gh Figure 7: Acenaphthene Molecule (KarCriesbaun,et al, 2002) Acenaphthene was first prepared from coal tar by Marcelline Berthelot. Later Berthelot and Bardy synthesized the compound by cyclization of alpha-ethylnaphthalene. Industrially, it is still obtained from coal tar together with its derivative acnaphthylene. Like other arenes, acenaphthene forms complexes with low valent metal centres (Kim et al, 2010). It is used on a large scale to prepare naphthalic anhydride, which is a precursor to dyes and optical brighteners. Chemical reduction affords the radical anion sodium acenaphylenide, which is used as a strong reductant (Connelly et al, 1996). 2.8.3: FLUORENE It forms white crystals that exhibit a characteristic aromatic odour similar to that of naphthalene. Fluorene is combustible and has a fluorescence, hence, its name. It is insoluble in water and soluble in benzene and ether. It is obtained from coal tar but it can also be prepared by diphenylmethane (Burns et al, 1954) or by the reduction of diphenylene with zinc. The fluorene molecule is planar, although each of the two benzene rings is coplanar with the central carbon (Gerkin et al, 1984). The C9-A sites of the fluorene ring are weakly acidic. 64 University of Ghana http://ugspace.ug.edu.gh Figure 8: Fluorene Molecule (Gerkins et al, 1984) - Deprotonation gives the stable fluorenyl anion C13H 10 which is aromatic and has an intense orange colour. The anion is a nucleophile, and most electrophiles react with it by adding to the 9- position. The purification of fluorene exploits its acidity and the low solubility of its sodium derivative in hydrocarbon solvents. Both protons can be removed from C9, for example 9, 9- fluorenyldipotassium can be obtained by treating fluorene with potassium metal in boiling dioxin (Schief et al, 1960). 2.8.4: NAPHTHALENE Naphthalene is an organic compound with the formula C10H8. It is the simplest polycyclic aromatic hydrocarbon and is a white crystalline solid with a characteristic odor that is detectable at even very low concentrations (Amooreand Hautala,1983). Figure 9: Naphthalene (Erlenmeyer, 1866) As an aromatic hydrocarbn, naphthalene‘s structure consists of a fused pair of benzene rings. It is best known as the main ingredient of traditional mothballs. John Kidd proposed the name naphthalene, as it had derived from a kind of naphtha (Kidd, 1821). Naphthalene‘s chemical formula was determined by Michael Faraday in 1826. The structure of two fused benzene rings 65 University of Ghana http://ugspace.ug.edu.gh was proposed by Emil Erlenmeyer in 1866 and confirmed by Cawl Grabe three years later (Erllenmeyer, 1866). Naphthalene is classified as a benzenoid polycyclic aromatic hydrocarbon. There are two sets of equivalent hydrogen atoms, the alpha positions are positions 1,4,5 and 8 on the drawing above, and the beta positions are positions 2,3,6 and 7. Like benzene, naphthalene can undergo electrophilic aromatic substitution. For example, whereas both benzene and naphthalene react with chlorine in the presence of ferric chloride or aluminium chloride catalyst, naphthalene and chlorine can react to form 1-chloronaphthalene even without a catalyst. Naphthalene is the most abundant single component of coal tar. Petroleum derived naphthalene is usually purer than that derived from coal tar. Crude naphthalene can be purified by recrystallization from any of a variety of solvents (Collin et al, 2003). Naphthalene is used in the synthesis of 2-naphthol, a precursor for various dyestuffs, pigments, rubber processing chemicals and other miscellaneous chemicals and pharmaceuticals. Naphthalene sulfonate polymers are produced by reacting naphthalene with sulphuric acid and then polymerizing with formaldehyde, followed by neutralization with sodium hydroxide or calcium hydroxide. (1) H2SO4 + C10H8 → C10H7SO3H + H2O Sulfonation step: Sulfuric acid plus naphthalene 66 University of Ghana http://ugspace.ug.edu.gh (2) C10H7SO3H-(C10H7SO3H)n + CH2═O → SO3H-C10H7-(-CH2-C10H7SO3H)n + H2SO4 Polymerization step: naphthalene sulfonic acid plus formaldehyde. (3) C10H7SO3H-(C10H7SO3H)n + NaOH → C10H7SO3Na-(C10H7SO3)n + NaSO4 Neutralization step: naphthalene sulfonic acid condensate plus sodium hydroxide. Exposure to naphthalene is harmful to people and may cause hemolytic anaemia even at lower doses (Santucci.and Shah 2000). 2.8.5: FLUORANTHENE It is a polycyclic aromatic hydrocarbon that consists of a naphthalene and a benzene unit connected by a five membered ring. Figure 10: Fluoranthene (Amoore and Hautala, 1983) It is a member of the class of polycyclic aromatic hydrocarbons known as non-alternant polycyclic aromatic hydrocarbon because it has rings other than those with six carbon atoms. Fluoranthene is a structural isomer of the alternate pyrene. It is not as thermodynamically stable as pyrene because its electrons cannot resonate throughout the complete structure like the corresponding ones in pyrene. It is found in many combustion products along with other polycyclic aromatic hydrocarbons. Its presence is an indicator of less efficient combustion. It has 67 University of Ghana http://ugspace.ug.edu.gh been isolated from coal tar pitch, and its name is derived from its fluorescence under UV light (Amoore and Hautala, 1983). 2.8.6: PYRENE It is a polycyclic aromatic hydrocarbon consisting of four fused benzene rings, resulting in a flat aromatic system. The chemical formula is C18H10. Pyrene is a colourless solid and is the smallest pen fused polycyclic aromatic hydrocarbon, that is, one in which the rings are fused through more than one face. It forms during incomplete combustion of organic matter. Figure 11: Pyrene Molecule (Senkan and Castald, 2003) As a pen fused polycyclic aromatic hydrocarbon, pyrene is much more resonance-stabilized than its isomer, fluoranthene. Therefore, it is produced in a wide range of combustion conditions (Selim and Marco, 2003). It undergoes a series of hydrogenation reactions and it is susceptible to halogenations, Diels-Alder additions and nitration, all with varying degrees of selectivity. Pyrene and its derivatives are used commercially to make dyes. 2.8.7: PHENANTHRENE It is composed of three fused benzene rings. The name phenanthrene is a composite of phenyl and anthrancene. 68 University of Ghana http://ugspace.ug.edu.gh Figure 12: Phenanthrene (Amoore and Hautala, 1983) In its pure form, it is found in cigarette smoke and is a known irritant. Phenanthrene appears as a white powder which has a blue fluorescence. The compound with a phenanthrene skeleton and nitrogens at the 4 and 5 positions is known as phenanthroline. Phenanthrene is the backbone of morphinan. It is nearly insoluble in water but is soluble in most low polarity organic solvents such as toluene, carbon tetrachloride, ether, chloroform, acetic acid and benzene (Amoore and Hautala, 1983). 2.9: HEAVY METALS Literature sources point to the fact that these metals are released into the environment by both natural and anthropogenic sources, especially industrial activities and automobile exhausts, for example lead (Hutton and Symon, 1986; Battarbee et al, 1988; Niragu,1989). When ingested into the body, they combine with the body‘s biomolecules, like proteins and enzymes, to form stable biotoxic compounds, thereby mutilating their structures and hindering them from the bioreactions of their functions (Lenntech, 2004). ‗Heavy Metals‘ is a general collective term 3 which applies to the group of metals and metalloids with atomic density greater than 4g/cm or 5 times or more great than water (Hutton and Symon, 1986; Battarbee et al, 1988; Niragu and Pacyna, 1988; Niragu, 1989; Garbarino et al, 1995; Hawkes, 1997). Heavy metals include lead(Pb), cadmium(Cd), zinc(Zn), mercury (Hg), arsenic(As), silver(Ag), chromium(Cr), 69 University of Ghana http://ugspace.ug.edu.gh copper(Cu) and iron(Fe). An environment is defined as the totality of circumstances surrounding an organism or group of organisms, especially the combination of external physical conditions that affect and influence the growth, development and survival of organisms (Farlex, 2005). A pollutant is any substance in the environment which causes objectionable effects, impairing the welfare of the environment, reducing the quality of life and perhaps eventually causing death. Such a substance has to be present in the environment beyond a set or tolerable limit, which could be either a desirable or acceptable limit. Hence, environmental pollution is the presence of a pollutant in the environment, air, water and soil which may be poisonous or toxic and will cause harm to living things in the polluted environment (Hutton and Symon, 1986). The tolerance limits of some heavy metals are shown in the table below: Table 6: TOLERABLE LIMITS OF SOME HEAVY METALS (Hutton and Symon, 1986) HM MC,Air MC,Soil MC,DW MC,WSAL mg/l) 3 (mg/m ) (mg/l) (ppm) Cd 0.1-0.2 85 0.005 0.008 Pb 420 0.0 0.0058 Zn 1.5 7500 5.00 0.0766 70 University of Ghana http://ugspace.ug.edu.gh Hg <1 0.002 0.05 Ca 5 Tolerable 50 Tolerable>50 Ag 0,01 0.0 0.1 As 0.01 HM=Heavy Metal MC=Maximum Concentration DW=drinking Water 2.9.1: IRON Iron has the symbol Fe and the atomic number 26. It is a metal in the first transition series. Like other group 8 elements, iron exists in a wide range of oxidation states,-2, to +6, although -2 and +3 are the most common. Iron chemical compounds, which include ferrous and ferric compounds, have many uses. Iron oxide mixed with aluminium powder can be ignited to create a thermite reaction, used in welding and purifying ores. Among its organometallic compounds is ferrocene. Steels and low carbon alloys, along with other metals-alloy steels- are by far the most common metals in industrial use due to their range of desirable properties and the abundance of iron (Dernazeau et al, 1982). Iron plays an important role in biology, forming complexes with molecular oxygen in haemoglobin and myoglobin. These two compounds are common oxygen 71 University of Ghana http://ugspace.ug.edu.gh transport proteins in vertebrates. Iron is also the metal at the active site of many important redox enzymes dealing with cellular respiration and oxidation-reduction in plants and animals (Neilands, 1981). The mechanical properties of iron and its alloys can be evaluated using a variety of tests, including the Brinel test, the Rockwell test and the Vickers hardness test. Data on iron is so consistent that it is often used to calibrate measurements or to compare tests (Kuhn and Meddin, 2000). However, the mechanical properties of iron are affected by the sample‘s purity; pure research-purpose single crystals of iron are actually softer than alumunium (Walter, 1995) and the purest industrially produced iron (99%) has a hardness of 29-30 brinel (Takaji and Toshikatsu, 1964). An increase in the carbon content of the iron will initially cause a significant corresponding increase in the iron‘s hardness and tensile strength. Maximum hardness is achieved with 0.6% carbon content, although this produces a metal with a low tensile strength (Raghavan, 2004). Table 7: Characteristic Values of Tensile Strength (TS) and Brinel Hardness (BH) of different forms of Iron (Kohl, 1995) Material TS BH (Mpa) (Brine) Iron wiskers 1100 ---- Ausformed(hardened) Iron 2930 850 – 1200 Martensitic steel 2070 600 72 University of Ghana http://ugspace.ug.edu.gh Bainitic Steel 1380 400 Peartitic Steel 1200 350 Cold-worked Iron 690 200 Small grain Iron 340 100 Carbon-containing Iron 140 40 Pure Single crystal Iron 10 3 Iron is the sixth most abundant element in the universe (McDonald, et al, 2010). It is formed as the final exothermic stage of the stellar nucleosynthesis by silicon fusion in massive stars. Metallic iron is rarely found on the surface of the earth because it tends to oxidize, but its oxides are pervasive and represent the primary ores. Most of the iron in the crust is found combined with oxygen as iron oxide minerals such as hermatite (Fe2O3) and magnetite (Fe3O4) (Morgan and Anders, 1980). Iron forms compounds mainly in the +2 and +3 oxidation states. Iron (1) compounds are called ferrous and iron (2) compounds ferric. Iron also occurs in higher oxidation states, an example being the purple potassium ferrate (K2FeO4), which contains iron in its +6 oxidation state. Iron (V) is a common intermediate in many biochemical oxidation reactions (Nam Wonwoo, 2007). Numerous organometallic compounds contain formal oxidation states of +1, 0 or even -2. There are also many mixed valence compounds that contain both iron (1) and iron (11) centres, such as magnetite and Prussian blue Fe4(Fe⌠CN⌡5)3 which is used as the traditional ―blue‖ in 73 University of Ghana http://ugspace.ug.edu.gh blueprints(Ware, Mike,1999). Antoine Lavosier used the reaction of water steam with metallic iron inside an incandescent iron tube to produce hydrogen in the experiment for the demonstration of the mass conservation. Fe + H2O→ FeO + H2 2Fe + 3H2O → Fe2O3 + 3H2 2Fe + 4H2O → Fe2O4 + 4H2 Industrially, iron production involves iron ores, mainly hermatite (Fe2O3) and magnetite (Fe3O4) in a carbothermic reaction (reduction with carbon) in a blast furnace at temperatures of about 0 2000 C. In a given blast furnace, iron ore, carbon in the form of coke and a flex such as limestone (which is used to remove silicon dioxide impurities in the ore which would otherwise clog the furnace with solid material) are fed into the top of the furnace while a massive blast of heated air is forced into the furnace at the bottom (Wagner, 1993). 2C + O2 → 2CO-----------------------------(1) Fe2O3 + 3CO → 2Fe + 3CO2-------------------- (2) (Hermatite) (Molten iron) 2Fe2O3 + 3C → 4Fe + 3CO2------------------(3) CaCO3 → CaO + CO2 -------------------- (4) CaO + SiO2 → CaSiO3 --------------------------- (5) (Slag) 74 University of Ghana http://ugspace.ug.edu.gh In the furnace, the coke reacts with oxygen in the air blast to produce carbon monoxide as in eqn.1. In eqn.2, the carbon monoxide reduces the iron ore to molten iron, becoming carbon dioxide in the process. Some iron in the high temperature lower region of the furnace reacts with the coke, as in eqn (3). The flux, calcium carbonate is present to melt impurities in the ore like silicone dioxide. Then calcium oxide combines with silicone dioxide to form a liquid slag which melts in the heat of the furnace and floats away at the bottom of the furnace (eqns4&5). Iron is used in the construction of machinery and machine tools, automobiles, the hulls of large ships and structural components of buildings. Pure iron is commonly combined with alloying elements to make steel. Iron acquisition poses a problem to aerobic organisms because ferric iron is poorly soluble near neutral pH. Thus, bacteria have evolved high affinity sequestering agents called sideophores (Neilands, 1995; Neilands, 1981; and Boukhalfa, 2002). Iron accumulates in the hippocampus of the brains of people with Aizheeimer‘s disease and in the substantia nigra of those with Parkinson‘s disease (Brar et al, 2009). Iron eating bacteria live in the hulls of sunken ships such as the Titanic (Ward, 2012). Large amounts of ingested iron can cause excessive levels of iron in the blood. High blood levels of free ferrous iron react with peroxides to produce free radicals which are highly reactive and can damage DNA proteins, lipids and other cellular components (Cheney et al, 1995). The medical management of iron toxicity is complicated and can include the use of a specific chelating agent called deferoxamine to bind and expel excess iron from the body (Tenenbein, 1996). 75 University of Ghana http://ugspace.ug.edu.gh 2.9.2: SODIUM It has the symbol Na and the atomic number 11. Sodium is soft, silvery and highly reactive, and 23 is a member of the alkali metals. Its only stable isotope is Na. The free mental does not occur in, but instead must be prepared from its compounds. It was first isolated by Humphry Davy in 1807 by the electrolysis of sodium hydroxide. Many sodium compounds are useful, such as sodium hydroxide for soap making and sodium chloride for use as a nutrient (edible salt). Sodium is an essential element for all animals and some plants (Banks, 1990). Sodium, at saturated temperature and pressure, is a soft metal that can be readily cut with a knife and is a good conductor of electricity. Freshly exposed sodium has a bright, silvery lustre that is rapidly tarnished, forming a white coating of sodium hydroxide and sodium carbonate (Gatti et al, 2010). When sodium or its compounds are introduced into a flame, they turn it yellow (Schumann, 2008). Sodium is generally less reactive than potassium and more reactive than lithium (De Leon, 2007). Sodium is created in the carbon burning process in stars by fusing two carbon atoms together; this requires temperatures above 600megakelvins and a star of at least three solar masses (Denisenkov and Ivanov, 1987). The earth‘s crust contains 2.6% sodium by weight, making it the sixth most abundant element on earth (Lide, 2005). Because of its high reactivity, it is never found as a pure element. Rather, it is found in many different minerals such as cryollite and feldspar. Sodium compounds are of immense commercial importance, being particularly central to industries producing glass, paper, soap and textiles (Alfred etal, 2005). The sodium compounds that are the most important include table salt (Nacl), soda ash (Na2CO3), baking soda (NaHCO3), caustic soda (NaOH), sodium nitrate (NaNO3), di- and tri-sodium phosphates sodium thiosulphate (Na2S2O3.5H2O) and borax 76 University of Ghana http://ugspace.ug.edu.gh (Na2B4O7.10H2O)(Hollemann et al,1985). In its compounds, sodium is usually ionically bonded to water and anions and is viewed as a hard Lewis acid (Cowa, 1997). Metallic sodium was first 0 produced commercially in 1855 by carbothermal reduction of sodium carbonate at 1100 C in what is known as the Deville process (Cespar and Lemay, 1950). In humans, sodium is an essential nutrient that regulates blood volume, blood pressure, osmotic equilibrium and pH. Sodium chloride is the principal sodium in the diet, and is used as seasoning and preservative as in processed food (Geleijnse et al 2004). In C4 plants, sodium is a micronutrient that acts in metabolism, specifically in the regeneration of phophoenolpruvate and the synthesis of chlorophyll (Kering, 2008). In others, it substitutes for potassium in several roles such as maintaining tugor pressure and in the opening and closing of stomata (Subbarao et al, 2003). Excess sodium in the soil limits the uptake of water due to decreased water potential which may result in wilting; similar concentrations in the cytoplasm can lead to enzyme inhibition, which in turn causes necrosis and chlorosis. To avoid these problems, plants develop mechanisms that limit sodium uptake by roots, and control them over long distances (Zhu, 2001). 2.9.3: NICKEL It is a chemical element with the chemical symbol Ni and the atomic number 28. Nickel is a transition metal, and is hard and ductile. An iron-nickel mixture is thought to compose the earth‘s inner core (Lars Sibxude et al, 1997). The most common oxidation state of nickel is +2, 77 University of Ghana http://ugspace.ug.edu.gh + but other exotic oxidation states of do exist. Tetracarbonyl nickel,, discovered by Ludwig Mond, is a volatile, highly toxic liquid at room temperature. On heating, the complex decomposes back to nickel and carbon monoxide. Ni (CO)4 Ni + 4CO Purification of nickel oxides to obtain the purest metal is performed via the Mond process which increases the nickel concentration to greater than 99.9% purity (Mond et al, 1890). In this 0 process nickel is reacted with carbon monoxide at around 40-80 C to form nickel carbonyl in the presence of a sulfur catalyst. Iron gives iron pentacarboxyl too, but the reaction is slow. Dicobalt octacarbonyl is also formed in this process, but it decomposes to tetracobalt dodecacarbonyl at the reaction temperature to give a non-volatile solid (Derek, 2005). Nickel is re-obtained from the nickel carbonyl by one of two processes. It may be passed through a large chamber at high temperatures in which tens of thousands of nickel spheres called pellets are constantly stirred. It decomposes, depositing pure nickel onto the nickel spheres. Alternatively, the nickel carbonyl 0 may be decomposed in a smaller chamber at 230 C to create the nickel powder. The resultant carbon monoxide is re-circulated and re-used through the process. The highly pure nickel produced by this process is known as carbonyl nickel (Neikov et al, 2009). Nickel is used in many industrial and consumer products like stainless steel, almico magnets, coinage, rechargeable batteries, electric quitar strings, and in nickel cast irons, for example, nickel brasses and bronzes, and alloys with copper, chromium, aluminium, lead, cobalt, silver and gold (Davis, 2000). Nickel foam or nickel mesh is used in gas diffusion electrodes for alkaline fuel cells (Kharton, 2011). Nickel is used as a binder in the cemented tungsten carbide or hard metal industry. It can make the tungsten carbide magnetic and adds corrosion-resistant properties to the 78 University of Ghana http://ugspace.ug.edu.gh cemented tungsten carbide parts, although the hardness is lower than those of parts made with cobalt binder (Cheburaeva et al, 1992). The plant enzyme urease-- an enzyme that assists in the hydrolysis of urea--contains nickel (Sydor et al, 2013). Other nickel-containing enzymes include a rare bacterial class of superoxide dismutase and glyoxatase (Szilagyi et al, 2004). Sensitized individuals may show an allergy to nickel, which may affect their skin (dermatitis). Sensitivity to nickel may also be present in patients with pompholyx. Nickel is an important cause of contact allergy, partly due to its use in jewellery intended for pierced ears (Thyssen et al, 2007). Nickel allergies affecting pierced ears are often marked by itchy, red skin. Hence many earrings are now made nickel-free due to this problem. 2.9.4: CHROMIUM It has the symbol Cr and atomic number 24. It is the first element in group 6. It is a steely-grey, lustrous, hard and brittle metal which takes a high polish, resists tarnishing and has a high melting point (Brandes et al, 1956). Chromium is the only elemental solid which shows 0 antiferromagnetic ordering at room temperature (and below). Above 38 C it transforms into a paramagnetic state (Fawcett, 1988). Chromium exhibits a wide range of possible oxidation states where the +3 state is most stable energetically; the +3 and +6 states are most commonly observed in chromium compounds, whereas the +1, +4 and +5 states are rare. Chromium (111) can be obtained by dissolving elemental chromium in acids like hydrochloric acid or sulfuric acid. Chromium (V1) compounds are powerful oxidants at low or neutral pH. Most important are 2- 2- chromate anion (CrO4 ) and dichromate anion (Cr2O7 ) which exist in equilibrium: 2- + 2- 2(CrO4) + 2H (Cr2O7) + H2O 79 University of Ghana http://ugspace.ug.edu.gh Sodium chromate is produced industrially by the oxidative roasting of chromite ore with calcium or sodium carbonate. The dominant species is therefore, by the law of mass action, determined by the pH of the solution. The change in equilibrium is visible by a change from yellow (chromate) to orange (dichromate), such as when an acid is added to a neutral solution of potassium chromate. Both the chromate and dichromate anions are strong oxidizing reagents at low pH (Holleman et al, 1985). 2- + - 3+ Cr2O7 + 4H3O + 6e → 2Cr + 6H2O They are, however, only moderately oxidizing at high pH. The two main products of chromium ore refining are ferrochromium and metallic chromium (Papp et al, 2006). For the production of pure chromium, the iron has to be separated from the chromium in a two-step roasting and leaching process. The chromite ore is heated with a mixture of calcium carbonate in the presence of air. The chromium is oxidized to the hexavalent form while the iron forms the stable Fe2O3. The chromate is converted by sulfuric acid into the dichromate (Papp etal, 2006). 4FeCr2O4 + 8Na2CO3 + 7O2 → 8Na2CrO4 + 2Fe2O3 + 8CO2 2Na2CrO4 + H2SO4 → Na2Cr2O7 + Na2SO4 + H2O The dichromate is converted to the chromate (111) oxide by reduction with carbon and then reduced in an aluminothermic reaction to chromium; Na2Cr2O7 + 2C → Cr2O3 + Na2CO3 + CO Cr2O3 + 2Al → Al2O3 + 2Cr 80 University of Ghana http://ugspace.ug.edu.gh Chrome green is a mixture of Prussian blue and chrome yellow, while the chrome oxide green is chromium (111) oxide (Gettrns, 1966). Chromium oxides are also used as a green colour in glass making and as a glaze in ceramics (Gerd et al, 2005). Green chromium oxide is extremely light- fast and as such is used in cladding coatings. It is also the main ingredient in IR reflecting paints used by the armed forces to paint vehicles, to give them the same IR reflectance as green leaves (Marrion, 2004). Natural rubies are corundum (aluminium oxide) crystals that are coloured red (the rarest type) due to chromium (111) ions. Other colours of corundum gems are termed sapphires. A red coloured artificial ruby may also be achieved by doping chromium (111) into artificial corundum crystals, thus making chromium a requirement for making synthetic rubies (Moss and Newnham, 1964). Chromium (VI) salts are used for the preservation of wood, because of their toxicity. For example, chromate copper arsenate (CCA) is used in timber treatment to protect wood from decay fungi and wood attacking insects, including termites and marine borers (Hingston et al, 2001). Chromium (III) salts, especially chrome alum and chromium (III) sulfate, are used in the tanning of leather. The chromium (III) stabilizes the collagen fibres (Brown, 1997). Several chromium compounds are used as catalysts for processing hydrocarbons. For example, Phillips catalysts for the production of polyethylene are mixtures of chromium and aluminium oxide 3+ (Weckhuysen, and, Schoonheydt, 1999). Trivalent chromium (Cr (III) or Cr ) occurs in trace amounts in food and water, and appears to be benign (Mertz, 1993). In contrast, hexavalent 6+) chromium (Cr (VI) or Cr is very toxic and magnetic when inhaled. 81 University of Ghana http://ugspace.ug.edu.gh 2.9.5: CALCIUM It has the symbol Ca and the atomic number 20. Calcium is a soft grey alkaline earth metal and is the fifth most abundant element by mass in the earth‘s crust (Dickson and Goyet, 1994). It is a silvery metallic element that must be extracted by electrolysis from a fused salt-like calcium chloride. Calcium is reactive; it reacts with water, generating hydrogen gas at a rate rapid enough to be noticeable, but not fast enough at room temperature to generate much heat, making it useful for generating hydrogen (Pauling, 1970). Calcium is not naturally found in the elemental state, but it occurs mostly in sedimentary rocks like calcite, dolomite and gypsum. Calcium is used as a reducing agent in the extraction of other metals such as uranium and zirconium. It is also used as a deoxidizer for various ferrous and nonferrous alloys. Calcium is used as an alloying agent in the production of aluminium, beryllium, copper lead and magnesium alloys. It is also used in making cements and mortars to be used in construction. Calcium is essential to living organisms, 2+ in particular in cell physiology where movement of calcium ion Ca into and out of the cytoplasm functions as a signal to many cellular processes. As a major material used in mineralization of bone, teeth and shells, Calcium is the most abundant metal by mass in many animals (Curhan et al, 1998). Calcium is used in making cheese, where calcium ions influence the activity of rennin in bringing about the coagulation of milk (Lide, 2005). Calcium is an important component of a heathy diet and a mineral necessary for life. Dairy products such as milk and cheese are a well known source of calcium (Hall et al, 2001). Many good vegetable sources of calcium include seaweeds, nuts and seeds. An overlooked source of calcium is eggshell which can be ground into a powder and mixed into food or a glass of water (Schaafsma and Beelen, 1999; Schaafsma et al, 2002). Compared with other metals, the calcium ion and 82 University of Ghana http://ugspace.ug.edu.gh most calcium compounds have low toxicity. Calcium poses few serious environmental problems, with kidney stones being the most common side effects in clinical studies. Acute calcium poisoning is difficult to achieve unless calcium compounds are administered intravenously (Lewis, 1996). 2.9.6: CADMIUM Cadmium is an element with atomic number of 48. It is a soft, malleable, ductile, blush-white divalent metal. It is similar in many respects to zinc but forms complex compounds (Holleman et al, 1985). Unlike other metals, cadmium is resistant to corrosion and as a result it is used as a protective layer when deposited on other metals. It is insoluble in water and is not flammable. However, in its powdered form it may burn and release toxic fumes. Cadmium has an oxidation state of +2, but it also exists in the +1 state. Cadmium is not always considered a transition metal, in that it does not have partly filled d or f electron shells in the elemental or common oxidation states (Cotton, 1999). It burns in air to form brown amorphous cadmium oxide (CdO); the crystalline form of this compound is a dark red which changes colour when heated, similar to zinc oxide. Hydrochloric acid, sulfuric acid and nitric acid dissolve cadmium by forming cadmium chloride (CdCl2), cadmium sulfate(CdSO4), or cadmium nitrate(Cd(NO3)2). 2HCl + Cd → CdCl2 + H2 H2SO4 + Cd → CdSO4 + H2 2HNO3 + Cd → Cd(NO3)2 + H2 83 University of Ghana http://ugspace.ug.edu.gh The oxidation state of +1 can be reached by dissolving cadmium in a mixture of cadmium 2+ 2+ chloride, forming the Cd2 cation which is similar to the Hg2 cation in mercury (1) chloride: Cd + CdCl2 + 2AlCl2 → Cd2(AlCl2)2 The structures of many nucleobases, amino acids and vitamins have been determined (Carballo et al, 2013). Cadmium is a common impurity in zinc ores, and it is most often isolated during the production of zinc. Some zinc ores concentrated from sulfidic zinc ores contain up to 14% of cadmium (Golberg et al, 1969). Zinc sulfide ores are roasted in the presence of oxygen, converting the zinc sulfide to the oxide. Zinc metal is produced either by smelting the oxide with carbon or by electrolysis in sulfuric acid. Cadmium is isolated from the zinc metal by vacuum distillation if the zinc is smelted, or cadmium sulfate is precipitated out of the electrolytic solution (Scoullos, 2001). Cadmium has many common industrial uses as it is a key component in battery production, is present in cadmium pigments (Scoullos, 2001) and coatings (Smith et al, 1999), and is commonly used in electroplating (Scoullos et al, 2001). Cadmium oxide is used in black and white TV phosphors and in blue and green phosphors for colour TV picture tubes (Les Ching-Hwa, 2002). Cadmium sulfide (CdS) is used as a photoconductive surface coating for photo copier drums (Miller and Mullin, 1991). In PVC, cadmium was used as heat, light and weathering stabilizers (Jennings, 2005). Cadmium is used in many kinds of solder and bearing alloys due to a low coefficient of friction and fatique resistance. It is also found in some of the lowest-melting alloys such as wood‘s metal (Brady et al, 2002). Cadmium is used as a barrier to control neutrons in nuclear fission. The pressurized water reactor designed by Westinghouse Electric Company uses an alloy which consists of 80% silver, 15% indium and 5% cadmium (Scullos, 2001). Helium-cadmium 84 University of Ghana http://ugspace.ug.edu.gh lasers are a common source of blue ultraviolet laser light. They operate at either 325 or 422nm and are used in fluorescence microscopes and various laboratory experiments (Nambiar, 2006). In molecular biology, cadmium is used to block voltage dependent cadmium channels in chicken neutrons (Klotz et al, 2013). Analytical methods for the determination of cadmium in biological samples have been reviewed (Cullen and Maldonado, 2013). The most dangerous form of occupational exposure to cadmium is inhalation of fine dust and fumes of highly soluble cadmium compounds (Morrow, 2010). Inhalation of cadmium- containing fumes can result initially in metal fume fever but may progress to chemical pneumonitis, pulmonary edema and death (Hayes, 2007). 2.9. 7: ZINC Zinc is a chemical element with the symbol Zn and the atomic number of 30. It is the first element of group 12 of the periodic table. Zinc metal is produced by using extractive metallurgy (Bodsworth, 1994): 2ZnS + 3O2 → 2ZnO + 2SO2 2ZnO + C → 2Zn + CO2 ZnO +CO → Zn + CO2 Eletrowinning processing can leach zinc from the ore concentrate by sulfuric acid (Gupta and Mukherjee, 1990). ZnO +H2SO4→ ZnSO4 + H2O After this step, electrolysis is used to produce zinc metal: 85 University of Ghana http://ugspace.ug.edu.gh 2ZnSO4+ 2H2O→ 2Zn + 2H2SO4 + O2 The sulfuric acid regenerated is recycled to the leaching step. The metal is mostly used as an anti-corrosion agent. Galvanization, which is the coating of iron steel to protect the metals against corrosion, is the most familiar form of using zinc in this way. Zinc is more reactive than iron or steel and thus will attract almost all local oxidation until it completely corrodes away. Zinc is also used to cathodically protect metals from corrosion (Bounoughaz et al, 2003). Excessive absorption of zinc suppresses copper and iron absorption. The free zinc ion in solution is highly toxic to plants, invertebrate and even vertebrate fish. A recent example showed 6 micromolar killing 93% of all Daphnia in water (Muyssen et al, 2006). The free zinc ion is a powerful lewis acid up to the point of being corrosive. Stomach acid contains hydrochloric acid in which metallic zinc dissolves readily to give corrosive zinc chloride (Bothwell et al, 2003). Many alloys contain zinc, including brass, an alloy of copper and zinc. Other metals long known to form binary alloys with zinc are aluminium, antimony, gold, iron, lead, mercury, silver and tin (Ingalls, 1902). Zinc is an essential mineral of exceptional biologic and health importance. Zinc deficiency affects about two billion people in the developing world and is associated with many diseases (Prasad, 2003). Enzymes with a zinc atom in the reactive centre are widespread in biochemistry, such as alcohol dehydrogenase in humans. Consumption of excess zinc can cause ataxia, lethargy and copper deficiency (Maret, 2013). 86 University of Ghana http://ugspace.ug.edu.gh 2.9.8 LEAD It is a chemical element in the carbon group with the symbol Pb and the atomic number 82. Lead is a bright and silvery metal with a very slight shade of blue in a dry atmosphere. Upon contact with air, it begins to tarnish by forming a complex mixture of compounds depending on the conditions (Thurmer et al, 2002). Its characteristic properties include high density, softness, ductility and malleability, poor electrical conductivity compared to the corrosion and ability to react with organic chemicals (Tetreaulf et al, 1998). Lead compounds exist mainly in two oxidation states, +2 and +4. Three oxides are known: lead monoxide (PbO), lead tetroxide (Pb3O4) and lead dioxide. Upon heating under high pressure with sulfur, it gives the disulfide. In the compound, the lead atoms are linked with sulfur atoms (Silverman, 1966). It is also a semiconductor. A mixture of the monoxide and the monosulfide when heated forms the metal (Cava and Hor, 2011): 2PbO + PbS → 3Pb + SO2 Lead is used in applications where the low melting point, ductility and high density are advantageous. The low melting point makes casting of lead easy, and therefore small arms ammunition and short-gun pellets can be cast with minimal technical equipment. Lead is also used for automobiles, mostly as electrodes in the lead-acid battery, extensively used as a car battery. Cathode (reduction) + 2- - PbO2 + 4H + SO4 + 2e → PbSO4 + 2H2O Anode (oxidation) 87 University of Ghana http://ugspace.ug.edu.gh 2- - Pb + SO4 → PbSO4 + 2e It is also used as a shielding from radiation and molten lead is used as a coolant (lead cooled reactors) (Tucek et al, 2006). Lead compounds are used as a coloring element in ceramic glazes, notably in the colours of red and yellow (Leonard and Lynch, 1958). Lead is frequently used in polyvinyl chloride (PVC) plastic, which coats electrical cords (Wilkes et al, 2005). Lead is a highly poisonous metal. The main target for lead toxicity is the nervous system (Golub, 2005). Lead poisoning typically results from the ingestion of food or water contaminated with lead, but may also occur after accidential ingestion of contaminated soil or lead based paint. It is rapidly absorbed into the blood stream and is believed to have adverse effects on the central nervous system, the cardiovascular system, the kidneys and the immune system (Bergeson, 2008). 2.9.9: ARSENIC It is a chemical element with the symbol As and the atomic number 33. Arsenic occurs in many minerals, usually in conjunction with sulfur and metals, and also as a pure elemental crystal. It is a metalloid and was first documented by Albertus Magnus in 1250(Ernsley, 2001). The three most common arsenic allotropes are metallic gray, yellow and black arsenic, with gray being the most common (Norman, 1998). Naturally occurring arsenic is composed of one stable isotope, 75 As, which makes it a monoisotopic element (Georges et al, 2003). When heated in air, arsenic oxidizes to arsenic trioxide; the fumes for this reaction have an odor resembling garlic. Arsenic sublimes upon heating at atmospheric pressure, converting directly to a gaseous form without an intervening liquid. Arsenic makes arsenic acid with concentrated nitric acid, arsenious acid with 88 University of Ghana http://ugspace.ug.edu.gh dilute nitric acid, and arsenic trioxide with concentrated sulfuric acid (Chrisholm, Hugh ed, 1911). Arsenic is used as the group 5 element in the III-V semiconductors: gallium arsenide, indium arsenide and aluminium arsenide (Tanaka, 2004). The valence electron count of GaAs is the same as a pair of Si atoms, but the band structure is completely different, which results in distinct bulk properties; other arsenic alloys include the II-IV semiconductor, cadmium arsenide (Din and Gould, 1998). The toxicity of arsenic to insects, bacteria and fungi led to its use as a wood preservative (Rahman, 2004). Arsenic is used as a feed additive in poultry and swine production to increase weight gain, improve feed efficiency and to prevent disease. An example is roxarsone, which had been used as a broiler starter by about 70% of US broiler growers (Jones, 2007). In subtoxic doses, soluble arsenic compounds act as stimulants and were once popular in small doses as th medicine by people in the mid-18 century (Holieman et al, 1985). The main use of metallic arsenic is for alloying with lead. Lead components in car batteries are strengthened by the presence of some percentage of metallic arsenic (Sabina et al, 2005; Bagshaw, 1995). Denzincification can be strongly reduced by adding arsenic to brass, a copper-zinc alloy (Joseph and kundig, 1999). Some species of bacteria obtain their energy by oxidizing various fuels while reducing arsenite. Under oxidative environmental conditions, some bacteria use arsenite as fuel for their metabolism (Stolz etal, 2006). The enzymes involved are knowm as arsenate reductases (Mukhopadhyay et al, 2002). Inorganic arsenic and its compounds, upon entering the food chain, are progressively metabolized through a process of methylation (Sakurai, 2003; Reimer, 2010). 89 University of Ghana http://ugspace.ug.edu.gh For example, the mold scopulariopsis brevicaulis produces significant amounts of trimethylarsine if inorganic arsenic is present (Bentley and Chasteen, 2002). 2.10: CARBONATES, SULPHATES AND NITRATES 2.10.1: CARBONATE 2+ A carbonate is a salt of carbonic acid, characterized by the presence of the carbonate ion, CO . The carbonate ion is the simplest oxo carbon atom and consists of one carbon atom surrounded by three oxygen atoms in a trigonal planar arrangement. It is a conjugate base of the hydrogen carbonate (bicarbonate) ion, HCO3, which is the conjugate base of carbonic acid, H2CO3. It has a _1 molecular mass of 60.01gmol . (Kemper et al, 2002). Metal carbonates decompose on heating, liberating carbon dioxide from the long term carbon cycle and leaving behind an oxide of the + 2_ metals. This process is called calcination. 2M + CO3 → 2+ 2_ M2CO3 M + CO3 → 3+ 2 MCO3 2M + 3CO3 - → M2(CO3)3 Most carbonate salts are insoluble in water at standard temperature and pressure. In an aqueous solution, carbonate, bicarbonate, carbon dioxide and carbonic acid exist together in a dynamic equilibrium. Carbonated water is formed by dissolving CO2 in water under pressure. When the partial pressure of CO2 is reduced, for example, when a can of soda is opened, the equilibrium for each of the forms of carbonate (carbonate, bicarbonate, carbon dioxide and carbonic acid) 90 University of Ghana http://ugspace.ug.edu.gh shifts until the concentration of CO2 in the solution is equal to the solubility of CO2 at that temperature and pressure. 2.10.2 Sulfate A sulfate is a salt of sulfuric acid and is prepared from that acid. The anion consists of a central sulfur atom surrounded by four equivalent oxygen atoms in a tetrahedral arrangement. The sulfur atom is in the +6 oxidation state while the four oxygen atoms are each in the -2 state. The sulfate ion carries a negative two charge and is the conjugate base of the bisulfate (hydrogen sulfate) - ion, HSO4 which is the conjugate base of sulfuric acid (H2SO4). Many examples of ionic sulfates are known which are highly soluble in water. Sulfate reducing bacteria which are anaerobic microorganisms use the reduction of sulfates coupled with the oxidation of organic compounds or hydrogen as an energy source for chemosythesis (Cotton et al, 1966). 2.10.3 Nitrate _ A nitrate is a polyatomic ion with the molecular formular NO3 and a molecular mass of 62.0049g/mol. The anion is the conjugate base of nitric acid, consisting of one central nitrogen atom and surrounded by three identically bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a formal charge of -1. Almost all inorganic nitrate salts are soluble in water at standard temperature and pressure. A common example of an inorganic nitrate salt is potassium nitrate (Salt peuter). Nitrates are produced by a number of species of nitrifying bacteria. They are also found in man-made fertilizers. Nitrates are mainly produced for use as fertilizers in agriculture because of their high solubility and biodegradability. The second major 91 University of Ghana http://ugspace.ug.edu.gh application of nitrates is as oxidizing agents, most notably in explosives where the rapid oxidation of carbon compounds liberates large volumes of gases. Sodium nitrate is used to remove air bubbles from molten glass and some ceramics. Mixtures of the molten salt are used to harden some metals. Explosives and table tennis balls are made from celluloid, an organic nitrate (Wolfgang et al, 2006). 2.11: MICROBES AND METALS Beveridge and Doyle (1989), Ehrlich and Brierley (1990), Gadd (1993), Macaskie (1987), and so many others carried out extensive work on the interactions of microbes and metals and made several reports. The uptake of trace metals and their subsequent utilization in enzyme activation occur in all microbes (Wackett et al, 1989). Since ferric iron in the environment at around neutral pH exists mainly in a water-insoluble form, its uptake under aerobic conditions requires the microbial formation of ligands called siderophores, to render the ferric iron soluble (Neilands 1974). A number of microbes are able to use some metals as electron donors or acceptors in energy metabolism (Ehrlich, 1996). Depending on the element, the metal species may be in the simple ionic form or in the form of oxyanions. As energy sources, oxidizable metals may satisfy the entire energy demand of an organism (Chemolithotrophs). For example, the eubacteria Thiobacillus ferrooxidans, and the archaea Acidianus brierleyi, and sulfolobus acidocaldarius are able to obtain all their energy for growth from the oxidation Fe(11) to Fe(111) (Ehrlich,1996); stibiobacter senarmontii from the oxidation of Sb2O3 to Sb2O5 (Lyalikova et 2- 3- al,1976), and Pseudomonas asenitoxidans from the oxidation of AsO to AsO4 (IIyaletdinov and Abrashitova,1981). Some anaerobic hydrogen-oxidizing autotrophic bacteria also use oxidized metal species as terminal electron acceptors in their respiration. 92 University of Ghana http://ugspace.ug.edu.gh Examples of anaerobic respiration in which an oxidized metal specie serves as terminal electron 2+ acceptor include Fe (III) reduction to Fe , Fe3O4 or FeCO3 (Lovley and Philips 1988; Coleman 2+ et al 1993); MnO2 reduction to Mn or MnCO3 with acetate by the eubacterium, Geobacter 2- 2- o metallireducens (Lovely and Phillips 1988) and SeO4 and SeO3 reduction to Se by the eubacterium, Thauera selenatis, in the presence of nitrate (Rech and Macy, 1992; DeMoll- Decker and Macy, 1993). 2- The archeon Sulfolobus sp has been shown to reduce MoO4 to a lower oxidation state (Brierley 2- and Brierley 1982). Aerobic reduction of MnO2 to Mn by a few marine eubacteria and of 2- CrO4 to Cr (III) by the eubacterium Pseudomonas fluorescens LB300 as part of respiration has also been observed (Ehrlich 1996, Wang and Shen 1995). Enzymatic microbial detoxification of harmful metals is another type of microbe/metal interactiom. In this process, a toxic metal species may be converted to a less toxic or non-toxic entity by enzymatic oxidation or reduction. -- 3— The bacterial oxidation of AsO2 to AsO4 by a strain of Alcaligenes faecalis, and the 2- reduction of CrO4 to Cr(OH)3 by P. fluorescens are examples of such redox reduction (Ehrlich,1996, Wang and Shen,1995). The detoxification in the foregoing example is part of the respiratory process of the organisms. In some other cases, detoxification may be by enzymatic reduction that is not part of the respiratory process, as in mercury detoxification (Robinson and Tuovinen, 1984). In general, mercury-resistant bacteria produce the enzyme mercuric reductase, 2+ 0. which catalyses the conversion of Hg to volatile Hg Mecurric reductase formation is induced 2+ by Hg in all organisms tested except in Thiobacillus ferrooxidans, in which the enzyme is constitutive (Robinson and Tuovinen, 1984). Still other detoxification processes involve enzymatic or non-enzymatic methylation of metals and metalloids such as Sn, Hg, Pb, As, and Se (Chau et al, 1976; Frankenberger and Karlson, 1992; Guard et al,1981; Hallas et al, 1982; 93 University of Ghana http://ugspace.ug.edu.gh Summers and Silver, 1978; Trevors,1992; Wong et al, 1975). When microbes cannot detoxify harmful metals, they often have other genetically determined defenses against them (Ji and Silver 1995; Silver 1992). These defenses include modification or elimination of membrane transport systems into the cell for the harmful metal species, or efflux systems (molecular pumps) for their removal from the cell into the interior. Anaerobic enzymatically catalysed biocorrosion of metal/microbe interaction: In the original concept, as formulated by von Wolzogen Kuhr and van der Vlugt (1934), sulphate reducing bacteria promote biocorrosion of cast-iron metal surfaces anaerobically through cathodic depolarization. In this model, an iron surface exposed to aqueous moisture undergoes the spontaneous reation: 0 2+ -- Fe + 2H2O ---- Fe + 2OH + H2 ---------(1) With the reaction 0 2+ Fe --------------- Fe +…………. 2e ----------------- (2) At anodic regions, and the reaction - + 2H2O + 2e ----- 2OH H2---- (3) at cathodic regions. The H2 generated in a cathodic region was thought to accumulate at the iron surface where it was generated: its build-up causing passivation (polarization) of the surface, i.e., its build-up having stopped further corrosion. Sulfate-reducing bacteria, when using this hydrogen in their reduction of sulfate, as illustrated by this reaction: 2- + - 4H2 + SO4 + H  HS + 4H2O --------- (4) were thought to depolarize the surface, thereby promoting a continuation of the corrosion process. 94 University of Ghana http://ugspace.ug.edu.gh 2+ The sulfide they generate could react with Fe produced at anodic area which would also help to promote corrosion if iron sulfide did not precipitate on the surface as a uniform film that would passivate the iron surface as long as the film was undisturbed. Although some past experiments seemed to lend support to this model, the general view now is that anaerobic biocorrosion is the result of several different microbiological reactions. Metal surfaces are often colonized by biofilms and their activity has to be taken into account. These biofilms consist of a consortium of different types of bacteria often including aerobic, facultative and anaerobic bacteria with specific locations in the biofilm. Metalbolic products released by one consortium member in the biofilm and not consumed by any of the other members may be corrosive to the metal or act as chemical cathodic depolarizer beside the H2 consuming activity of the surface-reducing bacteria at the bottom of the biofilm (Videla 1995). Prokaryotic and eukaryotic microbes are capable of accumulating metals by binding them as cations to the cell surface in a passive process (Beveridge and Doyle 1989, Gadd 1993). Even dead cells can bind metal ions. Depending on conditions, such binding may be selective or non-selective. In some cases, if the cell surface becomes saturated by a metal species, the cell may subsequently act as a nucleus in the formation of a mineral containing the metal (Macaskie et al, 1967, 1992; Schultze-Lam et al, 1996). Some bacteria and fungi can promote selective and non-selective leaching of one or more metal constituents from an ore or other rock with metabolic product such as acid and/or ligands produced by them (Ehrlich,1996). The acid may be organic or inorganic; Groudev and Groudeva (1986) were able to leach aluminium from clays with oxalic and citric acids from Aspergillus niger. Alibhai et al (1991) were able to leach nickel selectively from low grade Greek laterite with citric acid produced by various species of Aspergillus and Penicillium. The process discriminated against iron, probably 95 University of Ghana http://ugspace.ug.edu.gh because of a higher affinity of citric acid for nickel than for iron. Microbes may excrete inorganic metabolic products such as sulfide, carbonate or phosphate irons in their respiratory metabolism and will then precipitate toxic metal ions as a form of non-enzymatic detoxification (Macaskie et al, 1987; Ehrlich, 1996). To be effective, the precipitation must decrease the concentration of the dissolved metal species below their inhibitory level. Under some circumstances, microbes may cause non-enzymatic corrosion of metals like aluminium or iron or some metal alloys through formation and release of corrosive metabolic products (Edyvan, 1995). These products are mainly organic and inorganic acids. In nature, noticeable microbial interaction with metals frequently manifests itself through metal immobilization or mobilization (Ferris et al, 1989, Ghiorse and Ehrlich 1992; Ehrlich 1996). Metal immobilization may be through extra-cellular precipitation. Metal mobilization results from dissolution of insoluble metal containing phases. Bioleaching of metals from ores is a practical example (Ehrlich and Brierley 1990). These processes are central to controlling biological availability of metals in soils, sediments, and water. Extra-cellular metal accumulations that result from microbial respiratory metabolism or through metal binding to microbial cell surfaces include some sedimentary iron and manganese oxide deposits, some iron and manganese carbonate deposits (Beveridge and kova 1981; Beveridge et al,1982; Doyle 1991; Ferris,1991;Ghiorse and Ehrlich,1992; Schutze-Lam et al,1996). With the exception of some rare natural occurrences, iron and copper in their metallic state are found in the environment only because human beings have placed them there. Even though artificially introduced, such metals and others are subject to biocorrosion by some members of the microbial flora indigenous at the site of emplacement. The ease and extent to 96 University of Ghana http://ugspace.ug.edu.gh which these metals corrode depends, in part, on whether they are pure or alloyed (Pope et al, 1994). 97 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0: MATERIALS AND METHODS This chapter describes the location of the study sites and the research methodology which includes research design, sample collection and sampling procedure, sample treatment analysis and experimental methods employed in this work. 3.1 LOCATION OF STUDY The location of the study is the city of Port Harcourt in Rivers State of Nigeria as seen in figures 14 and 15 which show the locations of Nigeria and Port Harcourt. Nigeria lies in the west of Africa and it consists of a federal capital territory and 36 states. Abuja is the capital city of nd Nigeria. Nigeria is considered to be the 32 largest country in the world. The largest ethnic groups are Yoruba, Igbo and Hausa. The Benue and the Niger are two main rivers of Nigeria and empty into the largest river delta, the Niger Delta. Port Harcourt is in the Niger Delta area of southern Nigeria, with the Niger Delta located at the Atlantic coast. The region spans over 20,000 square kilometers and it has been described as the largest wetland in Africa and among the three largest in the world (Ogunkoya and Efi, 2003). About 2,370 square kilometers of the Niger Delta area consist of rivers, creeks, and estuaries, while stagnant swamps cover about 86,000 square kilometers. The delta has the largest mangrove swamps in Africa (Awosika, 1995) 98 University of Ghana http://ugspace.ug.edu.gh . Figure 13: Map of Africa showing the position of Nigeria. The Niger Delta lies mainly within the wet equatorial climatic region, but in its northern extremities. The climate is the tropical wet and dry climate. Between February and November, the climate of the coastal zone is dominated by the tropical maritime air mass, while the remaining period from December to January is under the influence of the dry tropical continual air mass. These two air masses are separated by the inter-tropical discontinuity (ITD), which is a zone of relatively low pressure system. 99 University of Ghana http://ugspace.ug.edu.gh Figure 14: Map of Nigeria showing the position of Port Harcourt in Rivers State This zone controls the north-south movement of rainfall in the tropics (Lamb, 1983, Bello, 2003). The mean annual rainfall decreases from about 4500mm around the coastal margin to about 2000mm around the northern fringe of the Niger Delta. The wind tends to be omni- directional in the dry season but is concentrated in the south, southwest and west directions in the rainy season (Ogunkoya and Efi, 2003). Rainfall starts in February/March and terminates in November/December. Over 95% of the rainfall is received in the wet season while less than 5% is received in the dry season (Adejuwon, 100 University of Ghana http://ugspace.ug.edu.gh 2012). Table 8 shows the average monthly rainfall in this region, and in the case of Port Harcourt, for a period of 25 years. 3.2 STUDY SITES For this study, three buildings were selected within the community of University of Port Harcourt. Each building on each campus was marked as ―research site‖ and prevented from being renovated for the two years of the study. All the samples of the emulsion paintings were collected from these buildings. Figure 16 shows the map of University of Port Harcourt indicating various ‗study buildings‘ marked as red blocks. While figures 17 and 18 show a typical degraded wall and undegraded wall respectively. 101 University of Ghana http://ugspace.ug.edu.gh Table 8: Monthly average values of rainfall and accumulated total in the Niger Delta (1937—1997) from IITA, Nigeria (Adejuwon, 2012) Accumulated No. of Station Jan Feb March April May June July Aug Sept Oct Nov Dec total years Onset End Sapele 67 20.6 44.0 102.6 185.4 216.7 328.7 441.9 282.7 395.1 268.4 79.6 23.2 64.7 23.2 Warri 67 25.2 58 131 225.2 263.1 368.9 474.5 343.1 450.0 323.3 104.7 39.8 26.2 39.8 Forcados 67 42.2 42.8 137.2 191.4 320.6 551.7 691 341 540.6 401.5 123.5 58.8 84.5 42.2 Yenagoa 37 42.7 69.1 167.6 263.9 310.2 375.9 424.8 444.6 562.3 356.5 143.1 51.1 42.7 42.7 Ahoada 67 25.6 57.6 129.6 207.1 259.5 291.9 322.4 277.5 350.5 275.1 133.9 40.3 25.7 40.3 Port 25 31.1 60.4 168.1 177.8 213.0 270.5 393.0 352.5 367.1 264.4 76.2 19.3 31.3 19.3 Harcourt Degema 67 29.3 61.1 135.7 183.3 243.3 290.1 341.2 29.5 377.5 261 112.6 35 29.3 35 Onne 21 25.4 47.5 144.6 157.7 271.6 305.2 361.6 388.6 335.2 250.9 123.3 26.2 72.9 26.2 Opobo 67 47.3 81.8 139.8 238.8 354.6 534.6 697.4 483.3 511.2 437.4 192.1 49..4 47.3 49.4 99 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Figure 15: Map of University of Port Harcourt Showing the ―Study Buildings‖ 3.3 STUDY DESIGN In order to achieve the primary aim of this study, a laboratory in the research unit of the Nigerian National Petroleum Coporation in Port Harcourt was selected for the analysis of all the samples. A laboratory in the department of Microbiology of the University of Port Harcourt was also selected for the microbial analysis and a laboratory in the Department of Pure and Industrial Chemistry was selected for the preparation of the samples and subsequent extraction of all the oils. 101 University of Ghana http://ugspace.ug.edu.gh 3.4 MICROBIAL ANALYSIS Sample site description: [ The sample sites are located in Choba Park, Delta Park and Abuja Park of the University of Port Harcourt, Nigeria (see fig 16 showing the map of University of Port Harcourt). In Choba Park, the engineering workshop building was used as site 1, while in Delta Park, a hostel building was used as site 2, but in Abuja Park, the ofrima building was used as site 3. These buildings were clearly marked as Research Sites for the period of work. [ Sampling Procedure: Both degraded and undegraded emulsion paintings were scraped from the walls of these research sites in the University of Port Harcourt, Nigeria, using a plasticc spatula, into clearly labelled plastic bags. They were later transferred into preconditioned glass containers and stored in a refrigerator. Sample Treatment and Analysis: With the aid of a spatula, the sample was transferred into a sterile agar plate. Using a sterile pipette, a 0.1ml of physiological saline solution was transferred into the plate. The inocula were inoculated by spread plate technique in triplicates using a bent glass rod. The cultured plates 0 were all inoculated for 24 hrs at 37 C. After the incubation period, the colonies that developed were counted and the average mean value calculated and used for the calculation of colony forming units per gram (Cfu/g). 102 University of Ghana http://ugspace.ug.edu.gh Figure 16: Plate 10 (A freshly degraded emulsion painting (infected wall) Figure 17: Plate 7 (An undegraded emulsion painting (clean wall) Enumeration of Total Fungi: 103 University of Ghana http://ugspace.ug.edu.gh With a spatula the sample was transferred into a potato dextrose agar plate. The medium was aseptically dispensed into sterile petri dishes and allowed to solidify at room temperature. The solidified dry medium was then inoculated after performing ten fold serial dilutions. The cultured plates were all incubated for 72 hours at room temperature. After incubation, growth was observed and colonies counted in the plates. The colony forming units per gram were calculated from the mean value of the plates. Purification of Bacteria Isolates: Once the total heterotrophic bacteria were counted, culture plates from each sample were selected for purification. Based on cultural characteristics, the colonies were selected, labeled and transferred to fresh sterile nutrient agar plates and incubated for 24 hours at room temperature. After incubation, the isolates were all gram stained and viewed under the oil immersion objective (x 100) of a compound microscope. Mixed cultures were further sub- cultured to obtain a pure culture. Gram staining: To confirm the purity of the isolates, the gram staining procedure was performed as outlined, herewith using clean labeled and grease-free slides. A smear of each isolate was prepared, fixed and flooded with 0.5% crystal violet (stain) and stained for 60 seconds. It was then rinsed under slow running tap water and gram iodine was introduced into the smears for 30 seconds. It was rinsed off the same way and immediately decolorized using 95% ethanol within the period of 10- 15 seconds, keeping the slides in a slanting position. The slides were rinsed under slow running water. The decolorized smears were all stained for 60 seconds with 1% safarine and finally 104 University of Ghana http://ugspace.ug.edu.gh blotted dry for microscopy using the immersion objective lens of a microscope. The gram reaction of the isolates was observed and noted. Spore staining: The technique was performed by using Malachite green as the initial stain, while safarine was used for counter staining. A smear of each isolate was made to the grease–free labeled clean slide and allowed to fix by air drying. The fixed smear was placed in a beaker of water that was previously heated. The slide was flooded with 5% malachite green and boiled using a bunsen flame for 2 minutes, not allowing the smear to dry. The steaming allows heat to permeate the spore coat in order to ensure full penetration of the dye into the spore. Upon washing out the initial stain, 1% safarine was used to counter stain. The smear was rinsed under slow running tap water and air-dried. The slide was viewed under a microscope (x 100 objective lens). Vegetable cells took up the counter stain, thus appearing red, while the endospores retained green (Malachite Test). Motility Test: The test was performed to determine the presence of flagella in the bacterial isolates with the use of a semi solid agar medium. The medium is commonly referred to as motility medium. It was prepared by weighing its components, dissolving them in deionised water and boiling the solution to dissolve the agar completely. The medium was dispensed in 10ml aliquots into test tubes plugged with cotton wool and finally sterilized by autoclaving for 15 minutes at a 2- 0 1.2kg/cm pressure and a temperature of 121 C. It was then allowed to cool and solidify in an upright position. The incocula was labeled and inoculated aseptically using a straight wire. The 0 process was carried out for 72 hours at 37 C. 105 University of Ghana http://ugspace.ug.edu.gh Motility was observed as hazy growth that spreads throughout the stab line rendering the medium slightly opaque. Non motile ones grew only along the stab line. Biochemical Test for identification of bacteria isolates: All the tests were carried out in duplicates and controls were set up alongside the tests. They were, generally, not inoculated unless otherwise stated. Each isolate was examined for its ability to ferment some sugar (Carbohydrate), and then tested for the production of indole, the production of hydrogen sulphide from triple sugar iron agar, citrate utilization, catalase production, starch hydrolysis, methyl red and vogues proskaur (MRVP) and oxidase test. Sugar Fermentation: The ability of the isolates to ferment some sugars, such as glucose, lactose, sucrose, maltose, manitol and xylose was tested. Sugar solutions were made by weighting 10g of each, dissolving it in distilled water and adding the appropriate weight of peptone water powder. This medium was also incorporated with bromocresol propel which served as an indicator. Each fermentation medium was clearly labeled and finally dispensed into test tubes plugged with cotton wool. Each tube contained an inverted Durham tube to detect gas production. The preparations were all 0 -2 sterilized in the autoclave at 126 C for eleven minutes at the pressure of 1.2kgcm . After autoclaving, the medium was allowed to cool at room temperature, labeled according to the isolate code number to be characterized and finally inoculated. The inoculated medium was 0 incubated at 37 C for 72 hours. After incubation, the culture was observed for both acid and gas production or either. Acid production was indicated by a change of colour from purple to yellow while gas production was indicated by a displacement of fluid in the Durham tube. 106 University of Ghana http://ugspace.ug.edu.gh Methyl Red—Voges Proskauer Test: The test was carried out to determine the ability of the test organisms to produce acid (MR) or acetyl methyl carbinol (VP) as the end product of glucose metabolism. The test also aids in the identification of Enterobacteriacae. About 10ml aliquots were dispensed in test tubes plugged with cotton wool labeled clearly and auto cleaved. After sterilization, the medium was lightly inoculated and incubated for 72 hours 0 at 37 C. After incubation, the tubes were divided into two parts to test for methyl red and the vogues Proskauer test. Methyl Red Test: A few drops of methyl red reagent were introduced into a culture tube. The production of a red colour immediately indicates a positive (+) reaction. Vogues Proskauer Test: After production of acid from glucose some of the organisms are able to convert the acid to acetyl 1-2-carbinel or 2, 3-butane diol which are natural substances. Aeration in the presence of alkali then converts the products to di-acetyl which in turn reacts with peptone. Barit‘s method was used. To the 75 hours old culture 1ml of 40% potasion hydroxide and 3ml of 5% alpha- naphthol in absolute alcohol (ethanol) were added. A positive test was shown by the development of a pink or brick red colour within 5 to 15 minutes. Oxidase Test: The method used involved the use of paper soaked with the test reagent, N, N, N, N tetra methy 1-p- phenylene diamine dihydrochloride, and allowed to dry. A smear of each isolate was made 107 University of Ghana http://ugspace.ug.edu.gh on the filter paper. The development of purple colour within 3-10 seconds depicts a positive test due to the possession of cytochrome oxidase by the test organism. To confirm a positive or negative test, controls were equally made by placing a drop of the reagent on a paper without making a smear or film of the isolate. Indole Test: This test demonstrates the ability of some bacteria to decompose the amino acid-tryptophan to indole which accumulates in the medium. The medium used for this purpose is bacteriological peptone water dispensed in 10ml aliquots into tubes and labeled clearly. The tubes were 0 2 sterilized by autoclaving at 126 C for 11 minutes at the pressure of 1.2kg/cm . It was allowed to 0 cool, inoculated with the test organisms and incubated for 48 hours at 37 C. The culture was then tested with Kovac‘s reagent. To each culture, 0.3ml of the reagent was added and shaken gently. A positive reaction was indicated by the development of red colour in the alcohol layer of the culture medium. Citrate Utilization Test: This test is based on the ability of an organism to use citrate as its sole source of carbon and the ammonium salt as the sole source of nitrogen. Simmon‘s citrate agar was used for the test. The 0 medium was prepared with the screw caped bottles and autoclaved at 126 C for 11 minutes at the 2 pressure of 1.2kg/cm . After sterilization, the medium was allowed to cool at room temperature and solidify in a slanted position before they were inoculated. The cultures were incubated at 0 37 C for 72 hours. Positive tests are shown by a change in colour from green to blue, while in a negative test the original colour (green) of the medium is retained. 108 University of Ghana http://ugspace.ug.edu.gh Test for Hydrogen sulphide production Hydrogen sulphide is produced when certain bacteria are present following the decomposition of organic sulphur compounds such as cysteine methionine or through the reduction of inorganic sulphur compounds such as sulphate, sulphite and thiosulphate. The resulting hydrogen sulphide (H2S) is normally detected by incorporating a heavy metal salt in which the gas reacts to form metal sulphides. The medium used for the identification of nitrobacteria is based on the sugar fermentation and hydrogen sulphide production. It was prepared and dispended into test tubes in 5ml volumes and sterilized. The sterile medium was allowed to solidify as slants and inoculated by streaking and stabbing. 0 The culture was incubated for 48 hours at 37 C. Hydrogen Sulphide production was indicated by the presence of black mucoid growth on the medium black. Starch Hydrolysis Test A 0.4% soluble starch (BDH) was added to the nutrient agar. The agar was prepared by weighing and dissolving in de-ionized water and dispensed into sterile petri dishes, allowed to solidify at room temperature and dried prior to inoculation. The plates were labeled and inoculated by 0 streaking a strength line at their centre. The culture was incubated for 48 hours at 37 C. To test for amylase, the plates were flooded with Lugol‘s iodine solution. Starch hydrolysis was indicated by a clearing around the isolate. A dark blue coloration showed that starch was not hydrolyzed. Catalase Test This test demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from peroxide (H2O2). The glass slide technique was used. A drop of hydrogen peroxide (3%) was placed on a clean slide, and with the aid of a sterile loop, a lapful of isolate was collected 109 University of Ghana http://ugspace.ug.edu.gh from fresh plate culture and mixed with the drop of the test solution placed on the slide. The production of gas bubbles from the surface of the mixture indicates a positive test. Fungal Identification Once both yeast and mould were isolated, they were all transferred to a freshly prepared secondary potato dextrose agar plates. The plates were incubated at room temperature for three days. The isolates were further subcultured into McCartney bottles for preservation as stock cultures in the refrigerator. Characterization and Identification of Mould Isolates The moulds were subcultured aseptically in plates of potato destrose agar (PDA) and incubated for 72 hours at ambient temperature. Once there is growth and pigmentation, the cultural characters of the organisms were observed. The colour of the surface and that of the reverse were critically observed for colour and pattern of growth. Microscopic Examination of Mould Isolates To observe the organisms in the microscope, they were stained with lactophenol cotton blue as the stain. The wet mount preparation was made, using lactophenol cotton blue as the stain. This was observed with a lose power objective (X10) of the compound microscope. 3.5: Determination of heavy metals in degraded and undegraded emulsion paintings by the APHA 3111B AAS method (American Public Health Association) Sample treatment and analysis (APHA 3111D (V)): 110 University of Ghana http://ugspace.ug.edu.gh Each sample was thoroughly washed with deionised water by shaking it for 10 minutes. Then it 0 was air dried for 15 hours and was oven dried to a constant weight at 55 C. Exactly 0.1g of the sample was dissolved by using HNO3/HCLO4. The residue was filtered and diluted to desired volume with distilled water. The blank (distilled water) was first aspirated, then the quality control standard solution, and finally the sample solution according to the guildelines in table 9 below: Table 9: Summary of operating conditions for AAS machine (APHA 3111B) Cd 8.00 228.80 0.50 Norma Air - 1.20 10.0 l acetylen e Co 15.0 240.70 0.20 Norma Air - 1.20 10.0 0 l acetylen e Cr 15.0 357.90 0.30 Norma Air - 1.60 10.0 0 l acetylen e Cu 15.0 324.7 0.50 Norma Air - 1.00 10.0 0 l acetylen e 111 Metal Lamp current (mA) Wavelength (nm) Slit width (nm) Silt Height Flame type Fuel Flow(L/MIN) Air Flow(L/min) University of Ghana http://ugspace.ug.edu.gh Pb 8.00 217.0 1.00 Norma Air - 1.10 10.0 l acetylen e Zn 10.0 213.9 1.00 Norma Air - 1.10 10.0 0 l acetylen e Ni 15.0 232.0 0.20 Norma Air - 1.00 10.0 0 l acetylen e Fe 15.0 248.30 0.20 Norma Air - 1.20 10.0 0 l acetylen e Mn 15.0 279.50 0.20 Norma Air - 1.00 10.0 0 l acetylen e V 18.0 310.30 0.20 Norma N2O- 6.00 10.0 0 l acetylen e B 15.0 55.3 0.5 Norma N2O- 6.00 10.0 a 0 6 0 l acetylene 112 University of Ghana http://ugspace.ug.edu.gh Figure 18: Plate 9(The AAS Instrument) Data Processing: [ The instrument calculates the results automatically, provided the correct information is, properly entered into it. Figure 19 shows a typical AAS instrument. Metal concentration in mg/L = Instrument reading (mg/L) – blank result (if not) Chemical interference is a major cause of error in the operations of the AAS machine. And this occurs when atoms are bound in molecular combination in the flame and as such can not absorb light. In order to eliminate or reduce this interference, certain specific elements or compounds are added to the sample solutions. Some of the causes of interference are shown in table 10. Table 10: Causes of Interference and possible Suppressants Metal Chemical interference Ionization interference Suppressant 113 University of Ghana http://ugspace.ug.edu.gh Cd Ca concentrations Metal ionizes in very high 5ml lanthanum solution above 100mg/1 flame (fuel rich) to 100ml standard and sample before aspirating. Use background correction. Mn Mg concentrations above Metal ionizes in very high 25ml Ca solution to 100mg/l, Sio2 above 100mg/l flame (fuel rich) 100ml standards and samples before aspiring Use background correction. Cu - - Use background correction. Ni - - Pb - - Zn - - Cr Fe, Ni and Co concentrations‘ High flame (fuel rich) 5ml 10% lanthanum of 100mg/l and Mg chloride to 100ml concentrations of 30mg/l sample and standard depress absorption before aspirating Use background correction Fe - - Use background 114 University of Ghana http://ugspace.ug.edu.gh correction Ba High dissolved salts, matrix High flame (fuel rich) 5ml HCl 10ml potassium type solution, 7.5ml calcium solution to standard and sample before aspirating. V High dissolved salts, matrix High flame (rich) 2ml Al(NO3)3 9H2O to type 100ml sample and standard b/4 aspiring 3.6: Determination of Total Petroleum Hydrocarbon and Polycyclic Aromatic Hydrocarbon in degraded and undegraded emulsion paintings by GC-FID USEPA 8015D Method (United States Environmental Protection Agency) Sample Treatment and Analysis (USEPA 3550C): About 10g of wet sample was weighed out and transferred into a clean solvent -rinsed extraction bottle. This was dried using anhydrous sodium sulphate. Exactly 20ml of methylene chloride was added to the sample and shaken and shaken for 1hour on a water bath. The extract was allowed to settle for at least 20mins and carefully filtered through a glass funnel fitted with glass wool and sodium sulfate into a clean beaker and rinsed with methylene chloride. The residue was washed with 20ml ofextracting solvent and filtered through the funnel. Figure 20 shows a typical GC-FID instrument. 115 University of Ghana http://ugspace.ug.edu.gh Sample Clean-Up and Sample Concentration (USEPA 8440): A silica gel was washed with methylene chloride and pre-activated by heating overnight at 0 130 C. About 2g of pre-activated silica gel was added to the filtered sample extract and allowed to stand for about 10mins. The supernatant was decanted into a clean beaker. The sample extract was concentrated by evaporating to 1ml at room temperature in a fume cupboard. The concentrated extract underwent GC analysis with an FID detector. The operating conditions are listed in the table below: Figure 19: GC-FID Instrument Chromatographic Conditions for GC/FID (USEPA 8015D) Injector (Auto sampler): Use Front and Back injectors 116 University of Ghana http://ugspace.ug.edu.gh Injection volume 3µL Washes Pre-injection Post injection Sample 2 0 Solvent A 3 3 Solvent B 0 0 Pumps 3 0 Mode Split Heater (0C) 250 Pressure (psi) 18 Total flow (ml/min) 26.555 Split ratio 84.1 Split flow 10.5 Column: Mode Ramp flow Pressure (psi) 18 Flow (ml/min) 1.708 Average velocity (cm/sec) 37.343 Oven: Oven On 0 Set point 500 C Temperature Programme 0 Initial Temperature 500 C hold for 2mins 0 0 0 Program. 500 C to 3100 C @ 180 C/min. 117 University of Ghana http://ugspace.ug.edu.gh 0 Final Temperature 3100 C hold for15mins FID Detector 0 Heater ON 3400 C H2 Flow 46ml/min Air Flow 450ml/min Make-up Flow (N2) 34.43ml/min Flame On Electrometer On Signal 1: Data Rate 20Hz Minimum peak width 0.01min Integration Slope sensitivity 10 Peak width 0.04 Area reject 0.01 Height reject 0.01 Shoulders Off Data Processing The GC automatically calculates the result if entries are properly done in the sequence as shown below: 118 University of Ghana http://ugspace.ug.edu.gh Where, = Calculated mass of the analyte in µg = Total volume of the concentrated extract in µL = Dilution factor if extract is diluted, if not, D = 1 = Volume of the extract injected in µL = Weight of sample in g 3.7 Determination of pH of degraded and undegraeded emulsion paintings by the ASTM4972 Method (American Standard for Testing Materials) Sample Treatment and Analysis: With the aid of a glass rod, the sample was mixed to minimize stratification effect due to differential rate of settling and also to obtain a representative sample. Thereafter, a composite sample was formed as a result of the removal of smaller portions. The composite sample was 0 dried in the oven at 105 C. Then the dried sample was disaggregated by gently crushing in a mortar. The crushed sample was sieved through a 0.50-2mm stainless steel sieve. About 20g of sieved dried sample was weighed into a 100ml beaker and 20ml of distilled water was added. This content was stirred thoroughly with a glass rod and allowed to stand for 30mins. The partly settled suspension was decanted into a 50ml beaker. The electrodes were inserted into the suspension. The pH and temperature were recorded after 3mins. 119 University of Ghana http://ugspace.ug.edu.gh 3.8: Determination of Total Inorganic Carbon/Carbonate in degraded and undegraded emulsion paintings by the CAEM method (Chemical Analysis of Ecological Matter) Sample Treatment and Analysis: The sample was thoroughly mixed using a glass rod, and any foreign material was discarded. 0 Small portions of the sample were randomly removed and dried at 105 C to a costant weight. It was later cooled and desegregated by gently crushing any lumps in a mortar and sieved through a 0.5mm sieve. Blank Analysis: About 40ml of 0.5M HCl solution was placed in a 250ml wide mouth Erlemeyer flask and left standing for at least 1hour. Then 2-3 drops of phenolphthalein indicator solution was added to it and the solution was titrated with 0.5M NaOH to a persistent pink colour. Sample Analysis: About 2g of the sieved sample was put into a 250ml Erlenmeyer flask and 40ml of 0.5M HCl solution was added to it. The flask was gently swirled to disperse the sample in solution and left to stand for at least 1 hour. Then 2-3 drops of phenolphthalein indicator solution was added to the sample and it was titrated with 0.5M NaOH to a persistent pink colour. Data Processing: 120 University of Ghana http://ugspace.ug.edu.gh 3.9: Determination of Extractable Sulphate in degraded and undegraded emulsion 2- paintings by Turbidimetric Method-APHA 4500SO4 E (American Public Health Association) Sample Treatment and Analysis: About 5g of the sample was weighed into an extraction bottle. Then 25ml of the extracting solution (0.0164M potassium dihydrogen phosphate, KH2 PO4) was added into the bottle. The bottle was placed in a shaking water bath for 30 minutes and then allowed to settle. The suspension was filtered through a whatman No.1 qualitative filter paper into a beaker. Distilled water was used to dilute the sdlution to about half of the container. Then 5ml of 50% of acetic acid and 1ml of H3PO4 were added to the beaker and swirled gently to mix. This was later diluted to about ¾ of the beaker with water and mixed again. A measured spoonful of BaCl2.2H2O crystals was added without mixing and left to stand for 10 minutes. The beaker was stoppered and inverted 10 times and allowed to stand for 10 minutes. Then 1ml of 0.5% gum acacia solution was added and made up to the 50ml mark with distilled water. It was inverted several times more and left to stand for 1 hour. First a blank run was done before analyzing sample with the UV-Spectrometer. 121 University of Ghana http://ugspace.ug.edu.gh Figure 20: Plate 11: UV Spectrometer Data P rocessing: Where, All results were reported in mg/kg 122 University of Ghana http://ugspace.ug.edu.gh 3.10 Determination of Extractable Nitrate in degraded and undegraded emulsion paintings by Colometric Method-USEPA352.1 (United States Environmental Protection Agency) [ Sample Treatment and Analysis: The sample was mixed with a glass rod and a number of small portions were removed to obtain a 0 composite sample. This was dried in the oven at 105 C for 2 hours. The dried material was disaggregated by crushing any lumps in a mortar and sieved through a 2mm stainless steel sieve. About 1g of the sample was weighed into an extraction bottle, and then 25ml of distilled water was added into the bottle. It was placed on a rotary shaker for 10 minutes and filtered. About 5ml of the extract was pipetted into a 10ml test tube and 10ml of 30% NaCl was added to it. The 0 contents were mixed by swirling and the rack was placed in a cold bath (10-15 C). Then 5ml of H2SO4 was pipetted into each tube and mixed by swirling and allowed to cool. About 0.25ml brucine – sulphanic acid solution was added to the tubes and mixed by swirling. The rack of 0 tubes was placed in the 100 C water bath for 25 minutes. Then they were removed from the hot water and immersed in the cold water bath and allowed to reach a thermal equilibrium 0 temperature of 20-25 C. First a blank was run, before the sample was analysed using a UV- Spectrometer. Data Processing: 123 University of Ghana http://ugspace.ug.edu.gh 3.11 Determination of the Fatty Acid Composition for Oils [Coconut Oil (Agric and Native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil] Sample Site Description: Coconut oil: Ten top grade coconut fruits were obtained from Elele-Alimini and tagged native coconut (A) and another ten top grade coconut fruits were also obtained from Emohua and tagged agric coconut (B), (Elele-Alimini and Emohua are towns close to the city of Port Harcourt in Nigeria). 0 The endosperms of the collected fruits were cut into tiny pieces and dried in an oven at 70 C for eight hours. Thereafter, it was further ground into more tiny bits for extraction. The coconut oil was extracted using hexane as solvent with the soxhlet extraction method. Figure 22 shows fresh endosperm of coconut for drying in the oven.Whereas figure 23 shows the dried coconut endosperm in the oven and figure 26 shows the extracted coconut oil. But figure 24 shows a typical extraction set-up in the laboratory and figure 25 shows a typical grinding machine. 124 University of Ghana http://ugspace.ug.edu.gh Figure 21: Plate12: The electric Oven Containing a fresh endosperm of coconut fruit for drying 1. Soya Bean Oil: Fifty cups of soya bean were bought from Choba market, close to the University of Port 0 Harcourt, Nigeria. The endosperms of the collected seeds were dried in an oven at 70 C for eight hours. Then, it was ground into more tiny pieces for extraction. The soya bean oil was extracted using hexane as solvent with the soxhlet extraction method. 125 University of Ghana http://ugspace.ug.edu.gh Figure 22: Plate13: The electric oven with dried coconut endosperm 2. Palm Kernel Oil: The palm kernel seeds were obtained from Elele-Alimini market, which is close to the city of 0 Port Harcourt, Nigeria. The endosperms of the collected seeds were dried in an oven at 80 C for forty-eight hours. Later, it was ground into more tiny bits for extraction. The palm kernel oil was extracted using hexane as solvent with the soxhlet extraction method. 126 University of Ghana http://ugspace.ug.edu.gh Figure 23: Plate 16 (A tyical Extraction set-up in the laboratory used in the extraction of oil from the samples) 3. Rubber Seed Oil: The rubber seeds were obtained from Delta Rubber Estate in Etche, which is close to the city of o Port Harcourt. The endosperms of these seeds were dried in an oven at 80 C for forty-eight hours. Later they were ground into tiny pieces for extraction. The Rubber seed oil was extracted using hexane as solvent with the soxhlet extraction method. 127 University of Ghana http://ugspace.ug.edu.gh Figure 24: Plate14 (A typical laboratory grinding machine used for grinding seed samples) Jatropha Seed Oil: These seeds were obtained from the trees on the Legon Campus of the University of Ghana, Accra. The thick top skin was removed by hand to obtain the endosperm. The endosperms of the 0 seeds were dried in the oven at 70 C for 8 hours. They were ground into tiny pieces with a grinding machine for extraction. The oil was also extracted using hexane as solvent with the soxhlet extraction method. Neem Seed Oil: The neem seeds were also obtained from trees on the Legon campus of the University of Ghana, Accra. The slimy top cover was removed by hand, the seeds were dried under the sun for 10 hours, and the thick skin was removed. The seeds were dried in the oven at 700 C for 8 hours. With the aid of 128 University of Ghana http://ugspace.ug.edu.gh the grinding machine, the seeds were ground into very tiny pieces for extraction. The etraction was done with hexane. Figure 25: Extracted coconut oil 129 University of Ghana http://ugspace.ug.edu.gh Figure 26: Plate15 (A dried and ground endosperm of coconut) Analysis of the Fatty Acids The fatty acid composition of coconut oil, soya bean oil, palm kernel oil, rubber seed oil, Jatropha seed oil and neem seed oil was investigated using only the Gas chromatography technique. The GC-FID System Chloroform/methanol containing 0.005% butylated hydroxytoluene, to act as antioxidant, was 0 added to 100ml of sample and mixed vigorously for one minute, then left at 4 C overnight. Then 1ml of 0.9% NaCI was added and the sample was mixed again. The chloroform phase containing lipids was collected, pooled and dried under nitrogen, and then subjected to methylation. Peaks were identified by comparisons with fatty acid internal standards 130 University of Ghana http://ugspace.ug.edu.gh area and its percentage for each resolved peak was analysed using an atlas software integrator. 0 0 The temperature was programmed at 60 C and held for 6 minutes, then increased to 110 C at 0 0 0 1 C/min for 5 minutes. The injector and detector temperatures were set at 90 C and 110 C respectively. 3.12: Determination of Antioxidant Vitamins of the seed oils: The vitamins of these oils were analyzed with a GC-FID System. The following equipment parameters were used: Column pressure 20psi Split Ratio 50:1 0 0 Column Temperature 250 C-320 C GC Carrier Gas He, H2 and Air An aliquot of the extract was applied to the hexane preconditioned solid phase of the extraction column. The internal standard used was dihydro-vitamins of the vitamins, of which 1ml was injected into the GC, and since not all the vitamins have the same absorbance at a particular wavelength, the instrument was programmed to change wavelength in the course of a run. 3.13 Paint Analysis Sample Site Description: The paint samples were collected from Glaxo (Nig) ltd and Demcork (Nig) Ltd in Port Harcourt, Nigeria. 131 University of Ghana http://ugspace.ug.edu.gh 3.13.1: Determination of the Polycyclic Aromatic Hydrocarbon and Total Petroleum Hydrocarbon for Glaxo and Demcork Emulsion Paint. The analysis of Polycyclic Aromatic Hydrocarbon was done using GC-MS (Gas Chromatography-Mass Spectrometry) which has the ability to both identify and quantify all polycyclic aromatic hydrocarbons and total petroleum hydrocarbons. Sample Treatment and Analysis: 0 The sample was first charred in a furnace at 105 C for two 2 hours. About 10g of dried emulsion paint was thoroughly mixed with anhydrous sodium sulphate (10g) and was soxhlet extracted with dichloromethane (200ml) for 6 hours. The extract was concentrated to 5ml in a rotary evaporator under reduced pressure. Exactly 0.5M potassium hydroxide (100ml) in methanol was 0 added and the mixture was refluxed for 4 hours in a water bath at 80 C. After cooling 20ml deionsied water was added and extraction was performed with hexane. The combined organic extract was dried over 0.5g anhydrous sodium sulphate. The decanted extract was evaporated under pressure to near dryness and dissolved in 1ml iso-octane for silica clean up. The glass column was slurry-packed with 10g silica gel in dichloromethane with a top layer of 0.5g anhydrous sodium suplhate. The column was initially rinsed with 40ml hexane. The extract was transferred to a column and subsequently eluted with 60:40 hexane and hexane-dichloromethane mixture which gave fractions enriched with alkanes and polycyclic aromatic hydrocarbons respectively. After cleaning on a silica and column to prevent the alkanes from interfering, the sample was injected into the GC-FID column. The analysis was done as described in section 3.7. 132 University of Ghana http://ugspace.ug.edu.gh 3.13.2: Determination of Heavy Metals in Glaxo and Demcork Emulsion Paints Sample Treatment and Analysis 0 The sample was charred in a furnace at 105 C for 2 hours. About 1g of sample was dissolved by using HF/HCl/HClO4in a Teflon crucible. The sample was quantitatively transferred into a 100ml volumetric flask. The crucible was rinsed several times with deionised water into the volumetric flask and the solution was made up to the mark with deionised water. A known standard solution was taken through the same process to determine the recovery factor which is usually higher than 98%. The different levels of heavy metals were determined by the AAS methodas in section 3.8. 3.14: Performance Test: Paint formulations were made using the different seed oils. Best results were obtained with the rubber seed oil (oil paint) and coconut oil (emulsion paint). A given quantity of emulsion paint (100ml) was collected from Glaxo Paint and divided into four portions of 25ml each. Another quantity of this paint (100ml) which did not contain the normal oil used in the production of the paint was also collected and divided into four portions of 25ml each. Different volumes of coconut oil were made (10ml, 8ml, 5ml, and 2ml). Another different set of volumes of rubber seed oil was made (15ml, 10ml.5ml, and 2ml). Lastly, another 5ml of the original paint containing company oil was collected. Thus: Sample A: This is the original emulsion paint from the company with its oil (5ml). Sample B: This is the paint from the company but without the oil used in its production. This particular one was mixed with different volumes of coconut oil (10ml, 8ml, 5ml, and 2ml). 133 University of Ghana http://ugspace.ug.edu.gh Sample C: This is paint from the company but without the oil in its production. This paint is then mixed with 15ml, 10ml.5ml and 2ml of the rubber seed oil. A building wall was chosen as the site for the performance test, which is situated in Abuja Park of the University of Port Harcourt. Different portions of the wall were painted with the separate paint concentrations and observed for twelve months. At the end of this period, it was observed that the portion painted with sample C developed chalks, cracks due to poor adhesion and cohesion.There was a little loss of colour in the portion painted with sample A but the adhesion was intact. However, in the portion painted with sample B, both the adhesion and colour were intact and there was no form of cracking. Furthermore, these oils were used in different paint formulations to determine their performing characteristics using 3.5cm x 10cm aluminium panels. The touch method was used to determine their drying performances. 3.15 Statistical Analysis The correlation analysis of the data collated was done with the aid of excel‘s correlation data analysis tool, the pairwise correlation coefficient. All the calculations of mean and standard deviation of the results were done using Microsoft excel software version 2007. The same software was used in the preparation of the graphs arising from the results. 134 University of Ghana http://ugspace.ug.edu.gh 3.16 Quality Control The accuracy of the methods used for these measurements was evaluated through the analysis of the appropriate quality control standards. Blank reagents and standard solutions were analysed along with the samples in each set of measurements. In the case of the GC measurements, verification was done by running a solvent (Dichloromethane) blank, mid-concentration standard -Terphenyl Surrogate Quality Control standard (760m/L) every day as a mini-requirement before start of work. In the case of the AAS measurements, a blank (distilled water) was first aspirated, then a Quality Control standard solution before the sample solution. But in the case of the UV measurements, sample temperature is very important for reproducibility of results. Therefore, Quality Control solutions and Sample solutions were 0 adjusted to the same temperature range of 20 to 25 C before the analysis. In the case of the pH measurements, the following buffers were used: buffer pH4.00, buffer pH7.00 and buffer pH10.00. All these certified reference materials were purchased from INORGANIC VENTURES USA while the quality control standard materials were purchased from ACCU STD EUROPE. 135 University of Ghana http://ugspace.ug.edu.gh Oil Yield: The seed meals were weighed before extraction. After extraction, the oils were weighed. The % yield was determined using the formula: Weight of oil ⁄ weight of sample x100 Paint Preparation: Prepared alkyd resins were formulated into white gloss paints without the use of driers. This was to accurately determine the drying rate of the oils. The alkyd resins and part of the solvent were premixed in a clean vessel. The pigment, TiO2 was then added and mixed to uniform consistency. The emulsion paint was also prepared but with water as the base and polyvinyl acetate as the resin. Evaluation of Paint Films Preparation of Test Panels:Aluminium panels measuring 3.5cm x 10cm were wiped with a clean cotton cloth dipped in ethanol and allowed to dry in air. Paint samples were applied on the panels with a paint brush to obtain uniform coats. The panels were then left to air-dry. Drying Time:The touch method was used to determine the drying performances of the paints. The films were monitored to determine the extent of drying. 136 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS AND DISCUSSION 4.1 MICROBIAL ANALYSIS: RESULT: Table 11: Microbial Density of Samples in Degraded Wall S/N THBC THFC ALGAE 5 3 2 2.6 10 cfu/g 1.8 cfu/g 1.4 cfu/g Site 1 5 3 3.12 cfu/g 2.4 cfu/g Site 2 Nil 5 3 2 2.14 cfu/g 1.3 cfu/g 2.26 cfu/g Site 3 Sample Code: THBC→Total heterotrophic Bacteria Count THFC→Total heterotrophic fungi count CFU/G→Colony forming units per gram of sample 137 University of Ghana http://ugspace.ug.edu.gh 138 University of Ghana http://ugspace.ug.edu.gh Table 12: Identification of Bacteria in Degraded Wall M V SUGAR FERMENTATION R P Probabl e Generae 1A + Ro + - + + + + + - - A - A A Bacillus ds Sp 1B - Ro + - - - + - - + - A A - - Flavoba ds cterium Sp 2A + Ro + - + - - + - - - A/ A/ A A/ Arthrob ds G G G acter Sp 2B + Co + - - - + - - - - A/ A A A Microco cci G lus Sp 2C Ro + - - - ds 2D + Ro + - + + + + + - - A - A A Bacillus ds Sp. 3A + Ro + - + + + + - + - A A A A Bacillus ds Sp. 3B - Ro + - - - ds 3C + Ro + - + + + - - + - A A - A A Bacillus ds Sp. 139 Isolate lab no Gram reactions Cell Morphology Catalase test Oxidase test Moltility Spore test Citrate Starch hydrolysis Indole Glucose Sucrose Maltose Lactose University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Sample Code: + : Positive - : Negative A : Acid production G : Gas production A/G : Both acid and gas production MR : Methyl red test VP : Voges proskauer test. 141 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 13: Identification of Fungi on Degraded Wall Sample Number Name 1A Rhizopus SP 2A Aspergillus SP. (niger) 2B Aspergillus Flavus 2C Fusarium sp 3A Aspergillus sp SAMPLE CODE: 1A ---------------------- Sample A from site 1 2A ---------------------- Sample A from site 2 2B --------------------- Sample B from site 2 2C -------------------- Sample C from site 2 3A ------------------- Sample A from site 3 Table 14: Microbial Density of Samples in Undegraded Wall 143 University of Ghana http://ugspace.ug.edu.gh S/NO. THBC THFC ALGAE 3 3 Site 1 1.65x 10 cfu/g 1.7 x10 cfu/g Nil 4 2 Site 2 1.03x 10 cfu/g 3.6x 10 cfu/g Nil 3 2 Site 3 5.4x 10 cfu/g 8.0x10 cfu/g Nil 144 University of Ghana http://ugspace.ug.edu.gh Sample Code: THBC ------ Total heterotrophic bacteria Count THFC ------ Total heterotrophic fungi count CFU/G ---- Colony forming unit per gram of sample 145 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 15: Identification of Bacteria in Undegraded Wall MR VP SUGAR FERMENTATION Probable Generae 1A + Rods + - + + + + - + - A/G A A - Bacillus Sp 1B + Rods + + - + + + - + - A/G A - - Bacillus Sp 2A + Rods + - + + + + - + - A/G - A - Bacillus Sp 2B + Rods + - + + + + - + - A/G - A - Bacillus Sp 2C Rods + + - + + - + A/G A - Bacillus Sp 2D + Rods + - + + + - - + - A/G A A A Bacillus Sp. 3A + Rods + - - + - - - + - A/G - A - Bacillus Sp. 3B + Rods + + + - A/G - - Bacillus Sp 3C + Rods + - + + - - - + - AA/G - A A Bacillus Sp. 147 Isolate lab no Gram reactions Cell Morphology Catalase test Oxidase test Moltility Spore test Citrate Starch hydrolysis Indole Glucose Sucrose Maltose Lactose University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Sample Code: + : Positive - : Negative A : Acid production G : Gas production A/G : Both acid and gas production MR : Methyl red test VP : Voges proskauer test. Sample Number Name 1A Aspergillus sp 2A Rhizopus sp 2B Aspergillus sp 2C Aspergillus sp 3A Aspergillus sp Table 16: Identification of Fungi in Undegraded Wall 149 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh DISCUSSION: The result in table 11 shows that at site 2, bacteria and fungi were most abundant, hence causing much aesthetic and structural damage, so much so that there were no algae growing on it. Furthermore, at site 1, the population of bacteria and fungi was such that it allowed the growth of some algae with some damage on the painted wall. At site 3, it was observed that the bacteria and fungi were least abundant, hence causing little damage on the painted wall. It, therefore, follows that an increase in the population of the bacteria and fungi causes more damage to the painted wall and decreases the population of the algae. The result in table 12 shows the identification of bacteria in which it was observed that the bacillus species were most abundant. Whereas in table 13 which shows the identification of fungi, much of the aspergillus species were identified. The result in table 14 shows the density of microbes in the undegraded wall in which it was observed that no algae were identified. This explains why the naked eyes can not see any degradation. But site 3 in that result has a higher amount of bacteria and fungi than sites 1 and 2. Furthermore, in all these results, the bacillus species of the bacteria, the aspergillus species of the fungi dorminated the environment. The least of the microbes identified were the algae, but at site 2, no algae were present. However, this observation is supported by the work of Gettens and coworkers in 1941 in which they showed that microbes, especially the fungi, could grow between the paint layers and the wall, causing a swelling of the paint film which could lead to the detachment of portions of the painted layer and their disaggregation from the wall. Besides, work by Agrawal and others in 1988 has also shown that paintings contain a wide range of organic and inorganic constituents, which provide different ecological niches that may be exploited by a large variety of microbial species, and that many of the components of painting 151 University of Ghana http://ugspace.ug.edu.gh and additives are biodegradable. Thus, microbial activities contribute greatly to the degradation of emulsion paintings. 4.2 Analysis of Emulsion Paintings. 4.2.1 Analysis of Heavy Metals in Degraded and Undegraded Emulsion Paintings. RESULTS: Table 17: Mean values of concentrations of heavy metals in degraded sites with their standard deviations (mg/kg) HEAVY META LS S ITE 1 SITE 2 SITE 3 Titanium 0.97±0.02 0.97±0.02 0.94±0.02 Nickel 10.75±0.16 10.75±0.16 10.56±0.16 Iron 78.35±0.17 78.35±0.17 77.46±0.06 Zinc 4.47±0.20 4.47±0.20 4.48±0.17 Chromium 11.34±0.09 11.34±0.09 11.30±0.02 Calcium 2.70±0.24 2.70±0.24 2.59±0.01 Sodium 35.26±0.25 35.26±0.25 34.97±0.06 Cadmium 0.05±0.02 0.05±0.02 0.02±0.01 Table 18: Correlation of Degraded Sites SITE 1 SITE 2 SITE 3 SITE 1 1 152 University of Ghana http://ugspace.ug.edu.gh SITE 2 1 1 SITE 3 1 1 1 153 University of Ghana http://ugspace.ug.edu.gh Figure 27: Graph of Correlation between Heavy Metal Concentrations in Degraded sites Figure 28: Histogram of Correlation between Heavy Metal Concentrations in Degraded sites Table 19: Mean values of concentrations of heavy metals in undegraded sites with their standard deviations (mg/kg). 154 University of Ghana http://ugspace.ug.edu.gh HEAVY METALS SITE 1 SITE 2 SITE 3 TITANIUM 0.65±0.04 0.65±0.05 0.66 ±0.07 NICKEL 6.91±0.61 7.45±0.13 7.41±0.35 IRON 50.34±0.76 54.49±0.41 52.91±0.23 ZINC 2.50±0.44 2.85±0.10 2.42±0.14 CHROMIUM 6.62±0.24 7.45±0.27 6.74±0.23 CALCIUM 1.44±0.21 1.57±0.26 1.43±0.02 SODIUM 34.12±0.51 32.39±0.44 31.59±0.44 CADMIUM 0.02±0.00 0.01±0.00 0.02±0.01 Table 20: Correlation of Undegraded Sites SITE 1 SITE 2 SITE 3 SITE 1 1 SITE 2 0.997309074 1 SITE 3 0.996718624 0.999136327 1 155 University of Ghana http://ugspace.ug.edu.gh Figure 29: Graph of Correlation between Heavy Metal Concentrations in Undegraded sites Figure 30: Histogram of Correlation between Heavy Metal Concentrations in Undegraded sites DISCUSSION: 156 University of Ghana http://ugspace.ug.edu.gh The results in tables 17 and 19 are the concentrations of heavy metals in both degraded and undegraded sites. It is observed that there is high deposit of iron, sodium, chromium and nickel in both sites, although this is higher in the degraded sites. Perhaps the only reason is that there is no microbe-metal interaction yet in the undegraded sites. But in table 17, there is a microbe- metal interaction and other microbial activities giving rise to degradation. The city of Port Harcourt is home to many oil and gas companies and other production companies. Hence, it experiences a high level of industrial activities. A comparison of results in tables 39, 17 and 19 reveals a higher amount of metal content in the last two tables than in the first one. This is clearly illustrated as shown in figure 38. This difference in amount of metal content can only be explained by the fact that there is a gradual build up of these heavy metals from the environment. Work by Battarbee, Niragu and others in 1988 and 1989 showed that these metals are released into the environment by both natural and anthropogenic sources, especially from industrial activities and automobile exhausts. Gadd, Ehrlich, Macaskie, Beveridge, Brierley and so many others carried out extensive work on the interactions of microbes and metals and made several reports. The uptake of trace metals and their subsequent utilization in enzyme activation occur in all microbes (Wackett et al 1989). Enzymatic microbial detoxification of harmful metals is another type of microbe-metal interaction. In this process, a toxic metal specie may be converted to a less toxic or non-toxic entity by enzymatic oxidation or reduction. The bacterial oxidation of 3- 2- AsO2 to AsO4 by a strain of Alcaligenes faecalis, and the reduction of CrO4 to Cr(OH)3 by P. fluorescens are examoles of such redox reduction (Ehrlich, 1996; Wang and Shen, 1995). Besides, prockaryotic and eukaryotic microbes are capable of accumulating metals by binding them as cations to the cell surface in a passive process (Beverridge and Doyle, 1989, Gadd 1993). These results support our observations that there is usually a gradual build-up of metals 157 University of Ghana http://ugspace.ug.edu.gh from the environment on the painted surface and that they contribute extensively to the degradation of this surface. 2 The results in table 18 and figure 28 indicate that as R=1 and R =1, for each correlation, there is a perfect linear correlation of the concentrations of heavy metals for the three sites in the degraded area. This means that as the concentrations of heavy metals increase in site1 there is a perfectly corresponding increase of the concentrations of heavy metals in site 2 as well as in site 3. An interesting factor also is that the total variation in the concentrations in all the three sites is predictable by the regression line for both site 1 and site 2 and between site 2 and site 3 as well as between sites 1 and 3. Just as it is in the degraded sites, there is a perfect correlation of metallic concentration in the undegraded sites as well as shown in table 20 and figure 30. In this case, 99% of the total fluctuation is also predictable by the regression lines as indicated by the 2 value R =0.99. These results are further represented in the forms of histogram as shown in figures 29 and 31. 158 University of Ghana http://ugspace.ug.edu.gh 4.2.2: Analysis of Total Petroleum Hydrocarbon in Degraded Sites: RESULTS: Table 21: Mean values of concentrations of TPHs in degraded sites with their standard deviations (mg/kg) TPH SITE 1 SITE 2 SITE 3 C10 0.52±0.01 0.33±0.02 0.44±0.02 C11 0.64±0.02 0.45±0.03 0.53±0.02 C12 1.15±0.02 1.05±0.02 1.14±0.02 C13 0.94±0.02 0.75±0.02 0.37±0.04 C14 0.75±0.03 0.64±0.02 0.65±0.02 C15 0.49±0.02 0.42±0.01 0.48±0.02 C16 0.46±0.02 0.43±0.01 0.46±0.02 C17 ---------- ----------- ----------- C18 0.34±0.02 0.16±0.02 0.48±0.02 C19 ----------- 0.05±0.02 0.05±0.03 C20 0.14±0.02 0.14±0.02 0.06±0.01 Table 22: Correlation of Mean values of concentrations of TPHs in degraded sites SITE 1 SITE 2 SITE 3 SITE 1 1 159 University of Ghana http://ugspace.ug.edu.gh SITE 2 0.97127569 1 SITE 3 0.78584941 0.8343205 1 160 University of Ghana http://ugspace.ug.edu.gh Figure 31: Correlation Plot of Mean values of Concentrations of TPHs in Degraded Sites Figure 32: Histogram of Mean values of Concentrations of TPHs in Degraded Sites 161 University of Ghana http://ugspace.ug.edu.gh DISCUSSION: The results in tables 21 and 37 show the composition of total petroleum hydrocarbon in degraded sites and the raw paint. A close comparison of these results shows that there is no marked difference in the amount of TPHs present in these environments. The implication is that the presence of TPHs only indicates the type of oil used in the paint production, but they do not contribute to the degradation of the emulsion paintings. The coefficients of correlation R for the mean values of concentration of TPH in the degraded sites are computed as shown in table 22 and figure 32 above. And as indicated, the figures were R=0.97 between sites 1 and 2, showing an almost perfect linear correlation in the concentrations of TPH in both sites, R=0.79 between sites 1 and 3, indicating a relatively strong correlation, and R=0.83 between the concentrations in sites 2 and 3. From the results there is a stronger correlation between the concentrations of TPH between sites 2 and 3 than there is in the concentration of TPH between sites 1 and 3. However, sites 1 and 2 show the strongest correlation in concentration of TPH in all the three sites. The scatter plot shows the linear relationship between sites 1 and 2 (series 1) and between sites 2 and 2 3 (series 2). The coefficient of determination, R =0.9 (series 1), indicates a very strong linear association between sites 1 and 2, such that the regression line can be used to predict the concentration of TPH in both sites 94% of the time, while the regression line for series 2 (between sites 2 and 3) can only represent the data about 62% of the time. The histogram representation is as shown in figure 33. 162 University of Ghana http://ugspace.ug.edu.gh 4.3.3: Analysis of Polycyclic Aromatic Hydrocarbon in Degraded and Undegraded Sites. RESULTS Table 23: Mean values of concentrations of PAHs in degraded sites with their standard deviations (mg/kg) P. A. H. SITE 1 SITE 2 SITE 3 Anthracene 2.63±0.09 2.53±0.02 2.78±0.08 Fluorene 4.18±0.02 3.83±0.07 4.34±0.12 Pyrene 0.71±0.16 0.65±0.02 0.77±0.05 Acenaphthene 0.71±0.09 0.76±0.04 0.65±0.08 Acenaphthylene 9.03±0.02 8.70±0.01 9.04±0.02 Table 24: Naphthalene 1.26±0.02 1.15±0.02 1.36±0.04 Correlatio n of Mean concentrations of PAHs at degraded sites SITE 1 SITE 2 SITE 3 SITE 1 1 SITE 2 0.999568097 1 SITE 3 0.999649548 0.99861795 1 163 University of Ghana http://ugspace.ug.edu.gh Figure 33: Correlation Plot of Mean Values of Concentrations of PAHs at Degraded sites Figure 34: Histogram of Mean Values of Concentrations of PAHs at Degraded sites 164 University of Ghana http://ugspace.ug.edu.gh Table 25: Mean Values of Concentrations of PAHs at Undegraded Sites with Their Standard Deviations (mg/kg) P. A. H. SITE 1 SITE 2 SITE 3 Anthracene ------- 0.02±0.00 ------- Fluorene 2.60±0.08 2.47±0.17 2.13±0.13 Pyrene 0.47±0.05 1.52±0.08 0.27±0.03 Acenaphthene 0.42±0.07 0.49±0.23 0.28±0.07 Acenaphthylene 0.44±0.13 0.28±0.02 0.16±0.01 Naphthalene 3.71±0.15 2.75±0.13 2.39±0.02 Table 26: Coefficient of Correlation for Mean Values of Concentrations of PAHs at Undegraded sites SITE 1 SITE 2 SITE 3 SITE 1 1 SITE 2 0.900214 1 SITE 3 0.9837 0.917094 1 165 University of Ghana http://ugspace.ug.edu.gh Figure 35: Corrlation Plot of Mean Values of Concentrations of PAHs at Undegraded Sites Figure 36: Histogram of Mean Values of Concentrations of PAHs at Undegraded Sites DISCUSSION: 166 University of Ghana http://ugspace.ug.edu.gh The results in tables 23 and 25 show the composition of polycyclic aromatic hydrocarbons at both the degraded and the undegraded sites. It is expected that 48 hours after a surface is painted, the PAHs ought to have evaporated into the air because they are only used for the preservation of the paint. But the results show the heavy presence of PAHs, and that the PAHs are more abundant in the degraded sites than in the undegraded sites. This implies that there must have been some deposits of these chemicals from the environment. And the fact that they are more in the degraded sites shows that they are contributing to the degradation of the environment. In table 23, anthrancene, fluorene and acenaphthylene appear to occur in greater amounts than the others. The coefficient of correlation between the concentrations of PAH at all the three degraded sites(table 24 and figure 34) is almost uniform, indicating an almost perfect correlation which shows that the values of concentration of PAH in the degraded sites increases symmetrically, and 2 the value of coefficient of determination/adjusted correlation R =0.999 for the relationship between sites 1 and 2 and between sites 2 and 3 shows that both regression lines can explain all of the variations and make predictions at any time. In the undegraded area(table 26 and figure 36),sites 1 and 3 have the strongest correlation in terms of concentration of PAH as indicated by the almost perfect value of coefficient of correlation, R=0.98, followed by that between sites 2 and 3 (R=0.917) and finally that between sites 1 and 2 (R=0.90). Nonetheless, all the three undegraded sites show very strong correlation in their values of concentration of PAH, meaning that as the concentration of PAH increases in one site it also increases proportionally in the other 2 sites. This assertion is also supported by the scatter plot and the values of R for both series. 2 Series 1 (between sites 1 and 3), with R =0.97 gives better predictions when compared to Series 167 University of Ghana http://ugspace.ug.edu.gh 2 2 (Regression line for sites 1 and 2) with R =0.81 for the total variations of PAH concentration in the degraded sites.These results are represented in a histogram as shown in figure 37. Determination of the pH of clean wall and infected wall 0 Table 27: Mean values of pH (H2O) at 25.8 C in nondegraded and degraded sites with their standard deviations (AST MD 49721) Parameter Site 1 Site 2 Site 3 PH (H20) 7.79 + 0.32 6.78+0.34 7.46+0.31 (Nondegraded Wall) Degraded Wall 6.59+0.13 6.99+0.17 7.04+0.15 Discussion: From the results in table 27, the pH range of the nondegraded wall is 6.78 – 7.79 and that of the degraded wall is 6.59 -7.04. Studies have shown that one way of affecting the gowth of bacteria in a particular environment is by changing its pH level and that bacteria are sensitive to the hydrogen ion concentration in their environment. In one of his reports, Trudy Wassenaar of Argonne National Laboratory, USA, stated that all bacteria require a certain pH level, which can vary widely in many different types of bacteria. Besides, he also reported that most bacteria grow best around a neutral pH value (6.5 – 7.0). This is in support of the results as shown in table 27 above. 168 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Determination of Sulphate: Nitrate and Carbonate of clean and infected walls. Results: Table 28: Mean values of concentrations of sulphate, nitrate and, carbonate in clean sites with their standard deviations (mg/kg) Parameter Site 1 Site 2 Site 3 Sulphates 512 0.16 860 0.19 161 0.17 (CAEM/apha4500 2- S04 E) Nitrates 8.84 89.21 0.02 0.08 (EPA 352.1) Carbonates 33.01 65.10 65.12 (CAEM) Table 29: Correlation Coefficient for mean values of concentrations Of sulphate, nitrate and carbonate in good sites. Site 1 Site 2 Site 3 Site 1 1 Site 2 0.997602195 1 Site 3 0.932037656 0.904724 1 169 University of Ghana http://ugspace.ug.edu.gh Figure 37:Correlation Plot for mean values of sulfate, nitrate & carbonate in clean sites. Figure 38: Histogram for mean values of sulfate, nitrate & carbonate in clean sites Table 30: Mean values of concentrations of sulphate, nitrate and carbonate in infected sites with their standard deviations (mg/kg). Parameter Site 1 Site 2 Site 3 170 University of Ghana http://ugspace.ug.edu.gh Sulphates 163.32 134.73 159.46 0.25 CAEM/APHA 2- (4500 S04 E) Nitrates 0.04 0.04 0.04 (EPA 35.21) Carbonates 77.60 56.01 57.0 3 (CEAM) Table 31: Corrrelation Coefficient for mean values of concentrations of sulphate, nitrate and carbonate in infected sites Site 1 Site 2 Site 3 Site 1 1 Site 2 0.997664 1 Site 3 0.990973 0.997816 1 171 University of Ghana http://ugspace.ug.edu.gh Figure 39: Correlation Plot of Mean Values of sulfate, nitrate & carbonate in infected sites 172 University of Ghana http://ugspace.ug.edu.gh Discussion: The results in tables 28 and 30 indicate that there are greater concentrations of sulfates and nitrates on the clean wall than on the infected wall. This may be as a result of the presence of sulfate reducing bacteria and nitrifying bacteria in the degraded wall. The difference in concentration of the carbonates in the two different environments is not much, indicating that there are no bacteria that actually affect their concentrations. In the clean sites (table 29 and figure 38), correlation between the sites is almost perfect. However, Sites 1 and 2 have the strongest correlation (R =0.997602195), followed by sites 1 and 3 (R=0.932037656), and then finally, Sites 2 and 3 (R=0.904724). This result is also depicted by the values of the Adjusted 2 Correlation (R ) for Site 1, 2 and for Site 1, 3 which are 0.9952 and 0.8687 respectively, showing that the regression line for undegraded Site 1, 2 gives better predictions of the variations of Carbonates, Nitrates and Sulphates when compared to that of Site 1, 3. In the infected sites, correlation between the sites is almost perfect as seen in the table 31 and figure 40. However, unlike in the clean sites, Sites 2 and 3 have the strongest correlation (R =0.997816) in the infected sites, while sites 1 and 2 (R=0.997664) have a relatively stronger correlation when compared to Sites 1 and 3 (R=0.990973). The values of the Adjusted 2 Correlation (R ) for Site 1, 2 shows better predictability of the variations of Carbonates, Nitrates and Sulphates in the degraded sites with the regression line when compared to those of Site 1, 3, also confirming the results of the Coefficient of correlation. Both results are represented in forms of histogram as shown in figures 39 and 41 for clean and infected walls respectively. 173 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 4.2.4: Analysis of the Saturated and Unsaturated Fatty Acid composition of Oils (Coconut Oil (agric & native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil) RESULTS: Table 32: Mean Values of Concentrations of Saturated Fatty Acids with their standard deviations (mg/kg) Coconut 0il/ Coconut oil/ Fatty Acid P.K.Oil S. B. Oil R.Seed Oil Agric Native J.Seed Oil N.Seed Oil Caproic 0.57±0.05 0.47±0.09 -------------- ----------- ------------ ------------ -------------- Caprylic 8.30±0.16 7.30±0.16 3.23±0.02 ----------- ------------ ------------- -------------- Capric 7.37±0.21 9.17±0.09 3.06±0.01 ----------- ------------- ------------- ------------- Lauric 44.80±0.16 43.73±0.17 45.81±0.02 ------------ ------------- ------------- ------------- Myristic 17.97±0.12 19.57±0.09 18.23±0.03 0.08±0.00 0.60±0.08 0.70±0.16 1.27±0.25 Palmitic 9.40±0.24 8.57±0.21 6.94±0.02 14.36±0.11 16.57±0.25 16.40±0.29 14.50±0.24 Stearic 3.23±0.12 3.47±0.21 1.89±0.03 5.23±0.01 9.53±0.25 9.33±0.21 17.47±0.25 Arachidic ---------- ------------ 0.18±0.01 1.91±0.01 ------------- 0.27±0.05 1.40±0.16 175 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Sample Code P.K. ----Palm Kernel S.B……..Soya Bean R……….Rubber J………..Jatropha N………Neem 177 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 33: Mean values of concentrations of unsaturated fatty acids with their standard deviations (mg/kg) Coconut oil/ Coconut oil/ Fatty Acid P.K.Oil S.B.Oil R.Seed OIL J.Seed Oil N.Seed Oil Agric Native Oleic 5.00±0.08 3.50±0.02 3.51±0.02 24.06±0.02 18.38±0.21 48.57±0.21 52.60±0.16 Linoleic 2.20±0.08 2.30±0.16 ---------- 49.80±0.07 22.50±0.17 12.57±0.29 8.33±0.12 Linolenic -------- --------- 0.13±0.02 4.13±0.02 36.30±0.16 12.40±0.21 4.37±0.25 179 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh DISCUSSION: The results in tables 20 and 21 indicate that there is a higher level of saturation in the fatty acids of coconut oil and palm kernel oil, whereas there is a higher level of unsaturation in the fatty acids of soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil. Therefore, coconut oil and palm kernel oil can be described as semi-drying oils. A closer look at the results shows that these oils also have some level of unsaturation in their fatty acids. But the soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil can be described as drying oils because of the high level of unsaturation in their fatty acids. In the formulation of paint, generally, oils with a high level of saturation (like coconut and palm kernel oils) are used in the production of emulsion paint while the soya bean, rubber seed, jatropha seed and neem seed oils can be used in the production oil paint. Beare-Rogers and others, in their work in 2001 and 2002 showed that lauric acid comprises about half of the fatty acid content in coconut oil and palm kernel oil. This is in support of the results as shown in table 20. Besides, work by Hoffman, Quattar and others in 2001 and 2002 has shown that lauric acid has some anti-microbial properties. 181 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 4.2.5: Analysis of Anti-Oxidant Vitamins in Coconut Oil (agric & native), Palm Kernel Oil, Soya Bean Oil, Rubber Seed Oil, Jatropha Seed Oil and Neem Seed Oil: RESULTS: Table 34: Mean values of concentrations of antioxidant vitamins in the oils with their standard deviations (mg/kg) Vitamins Coconut Coconutoil/Nat P.K.Oil S.B.Oil R.Seed Oil J.Seed N.Seed Oil Oil Agric Oil VE 0.61±0.02 0.91±0.01 1.22±0.02 2.05±0.02 6.55±0.0 15.45±0.20 0.40±0.01 4 VK 0.05±0.01 1.22±0.02 0.72±0.01 1.66±0.13 ---- ---- --- VA 0.01±0.00 0.31±0.00 0.02±0.00 0.08±0.01 ------- ----- --- VB6 0.02±0.00 0.01±0.00 0.05±0.01 ---- ---- ---- VB1 0.01±0.00 0.01±0.00 0.02±0.00 0.04±0.03 0.01±0.00 ------- 0.23±0.02 VB- 1.38±0.0 COMP 0.62±0.02 0.73±0.09 0.10±0.01 0.93±0.01 0.27±0.05 2 2.12±0.01 VB2 ------- ------ ------ ------- ------- --------- 0.36±0,03 Sample Code: 183 University of Ghana http://ugspace.ug.edu.gh VE----Vitamin E; VK----Vitamin K; VA---Vitamin A; VB6----Vitamin B6 VB1---Vitamin B1; VBcom-----Vitamin Bcomplex; VB2----Vitamin B2 184 University of Ghana http://ugspace.ug.edu.gh DISCUSSION: From the results, it can be seen that vitamin E is the most active and abundant antioxidamt vitamin in all these oils. In the case of the coconut oil, the native coconut oil has a higher amount of vitamin E. All the other vitamins are in trace amounts. Research by Reboul and others in 2006 has revealed that vitamin E is the most abundant in these oils and that alpha-tocopherol is the most active form of vitamin E. Akinson and others in 2008 have found that vitamin E prevents the oxidation of polyunsaturated fatty acids. Herera, Packer and others in 2001 also found that vitamin E stops the production of reactive oxygen. According to results of research carried out by Chin, Ghandi and Aigbodion and others in 1977,1990, and 2000 respectively, it has been shown that rubber seed oil has many applications for industrial purposes, including the manufacture of paints, soap and other surface coating, because of its vitamin E content. Besides, the work of Kumar and Sharma in 2008 has shown that jatropha seed oil can be used in the manufacture of candles and soap as well as a diesel substitute because they have a high content of vitamin E. All these results lend support to our assertion that these oils have many industrial applications because they contain vitamin E. 185 University of Ghana http://ugspace.ug.edu.gh 4.3 Paint Analysis 4.2.1 Analysis of Polycyclic Aromatic Hydrocarbon in Glaxo (Nig) and Demcork (Nig) Emulsion Paints: RESULTS: Table 35: Mean values of concentrations of PAHs in Paints with their standard deviations (mg/kg) P .A.H GLAXO DEMCORK Anfhracene 67.30±0.27 67.27±0.26 Fluorene 53.41±0.25 42.54±0.15 Acenaphthene 37.51±0.15 41.42±0.45 Fluoranthene 36.31±0.42 52.36±0.34 Pyrene 38.86±3.57 36.49±0.26 Phenanthrene ----------- 38.30±0.26 Table 36: Coefficient of Correlation for Mean values of concentrations of PAHs in Paints GLAXO DEMCORK GLAXO 1 DEMCORK 0.709407816 1 186 University of Ghana http://ugspace.ug.edu.gh Figure 40: Correlation Plot for Mean values of concentrations of PAHs in Paint DISCUSSION: Results from table 35 indicate the abundance of polycyclic aromatic hydrocarbons. This is as a result of the large amount of these chemicals that are contained in paint preservatives. According 187 University of Ghana http://ugspace.ug.edu.gh to a report by the United States Environmental Protection Agency in 2001, these chemicals are harmful to the body. Thus the painter at any point in time is expected to wear a mask while painting a given surface. It is expected that these chemicals should evaporate into the air within 48 hours after a surface is painted. It is therefore advisable that people should keep away from a newly painted surface in order to avoid inhaling these chemicals. There is a strong positive correlation (R=0.7) between the mean values of concentrations of PAH for Glaxo and Demcork, meaning that as the concentration of PAH for Glaxo increases, that of Demcork also increases reasonably. As the values of the concentration becomes smaller, a more 2 unbiased estimate is given by the Adjusted Correlation Coefficient, R which gives the proportion of variance of one variable that is predictable from the other variable, or how well the 2 regression line represents the data and is able to explain it. In this case, R is 0.5, indicating that approximately 50% of the total variation in the concentration in Demcork can be explained by the linear relationship between Glaxo and Demcork, as described by the regression equation, while the other 50% of the total variation in the concentration of PAH in Demcork remains unexplained. 188 University of Ghana http://ugspace.ug.edu.gh 4.2.2: Analysis of Total Petroleum Hydrocarbon in Glaxo and Demcork Paints. RESULTS: Table 37: Mean values of concentrations of TPHs with their standard deviations (mg/kg) TPH GLAXO DEMCORK C11 0.01±0.00 0.24±0.02 C12 0.15±0.02 0.41±0.02 C13 0.01±0.00 1.14±0.02 C14 0.01±0.00 0.75±0.03 C15 0.13±0.01 0.55±0.03 C16 0.24±0.02 0.23±0.02 C17 0.67±0.03 0.48±0.21 C18 0.70±0.02 ----------- C19 1.32±0.07 0.15±0.02 C20 1.73±0.02 0.07±0.02 Table 38: Coefficient of Correlation for Mean values of concentrations of TPHs GLAXO DEMCORK GLAXO 1 DEMCORK 0.724885689 1 189 University of Ghana http://ugspace.ug.edu.gh Figure 41: Plot of Correlation for Mean values of concentrations of TPHs 190 University of Ghana http://ugspace.ug.edu.gh Discussion: The values of the TPHs show a medium range C-atom present in the oil which shows that the oil used in the production of the paint is light oil. Here, there is a negative linear correlation between the mean values of concentration of TPH in Glaxo and Demcork with R=0.72, indicating that as the concentration of TPH in Glaxo is increasing, that of Demcork is decreasing. However, the 2 coefficient of Determination, R =0.379, shows that this variation can be explained by the regression line only 38% of the time, the other 62% of the total variation remaining unexplained. 4.2.3: Analysis of Heavy Metals in Glaxo and Demcork Paints. RESULTS: Table 39: Mean values of concentrations of heavy metals with their standard deviations (mg/kg) HEAVY METALS GLAXO DEMCORK Titanium 1.09±0.01 0.50±0.01 Nickel 0.55±0.02 0.35±0.02 Arsenic 0.39±0.02 0.75±0.03 Zinc 1.45±0.03 1.51±0.02 Lead 0.02±0.00 0.00±0.00 Cadmium 0.07±0.02 0.01±0.01 Glaxo Demcork Tabl Glaxo 1 e 40: Demcork 0.944491 1 Coefficie nt of Correlation for mean values of concentrations of heavy metals. 191 University of Ghana http://ugspace.ug.edu.gh Figure 42: Plot of Correlation for Mean values of concentrations of heavy metals 192 University of Ghana http://ugspace.ug.edu.gh DISCUSSION: The results in table 39 show the composition of heavy metals in the paint samples. The heavy metals in these paints are less than 2mg/kg, i.e., within tolerable limits. The work by Hutton and Symon in 1986 showed that a heavy metal is harmful only when it is above the tolerable limit of 2mg/kg.However, figure 44 shows the concentrations of the heavy metals in the degraded sites, undegraded sites and the paints. It clearly shows the concentrations of these metals as indicated in tables 17, 19, and 39 and as earlier discussed. Here, R=0.94, implies an almost perfect positive correlation between the mean values of concentration of metals in Glaxo and Demcork such that as the metallic concentration in Glaxo increases, one is sure that the concentration in Demcork will also increase. The regression line on the other hand can only be used to explain, at any given instance, 72% of the total variation as 2 indicated by the value R =0.718. 193 University of Ghana http://ugspace.ug.edu.gh Figure 43: Concentration of Heavy Metals in the paints, and sites 1, 2 and 3. RESULTS: Table 41:Drying Times of the Different Paint Formulations Time(hrs) Coconut Palm Soybean Jatropha Rubber oil Neem oil oil Kernel oil oil oil Set-To- 120 125 216 123 126 210 Touch(STT) Dry-To- 124 129 ------ 127 130 ----- Touch(DTT) Dry-Hard- 128 134 ------ 129 134 ------- 194 University of Ghana http://ugspace.ug.edu.gh Touch(DHT) DISCUSSION:The results in table 41 indicate that paint formulations were possible with the various seed oils without any driers. But the extent of the drying abilities differ from one seed to another. The best formulations came out of the coconut oil and the jatropha oil because they had the lowest set-to-touch time. It is believed that if driers were used in their paint formulations, the set-to-touch time would decrease. 195 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 CONCLUSION AND RECOMMENDATIONS 5.1 CONCLUSION It is obvious from all the results that algae, bacteria, fungi and heavy metals play a major role in the degradation of emulsion paintings on the walls of buildings and fences in Port Harcourt, Nigeria. This degradation in the emulsion paintings is primarily caused by the growth of fungi between the painting and the wall and the various microbe/metal interactions.Other contributors to this degradation are polycyclic aromatic hydrocarbons. All these activities weaken the paint structure and the adhesion of the paint to the wall, causing it to break away from the wall eventually. Furthermore, total petroleum hydrocarbons do not play much role in a degraded emulsion painting. On the other hand, only coconut oil and palm kernel oil can be used in the production of emulsion paint. But soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil can also be used in the production of oil paint. The oils of coconut fruit, palm kernel seed, jatropha seed and neem seed have anti-oxidant vitamins and so can be used for various industrial applications. Finally, coconut oil, especially native coconut oil, is the best suited in the production of emulsion paint because of its high content of lauric acid. 196 University of Ghana http://ugspace.ug.edu.gh 5.2 RECOMMENDATIONS (1) This work has opened a fresh window of research in an effort to find a lasting solution to this problem of degradation of emulsion paintings in Port Harcourt, Nigeria.More effort and resources should be committed to further work in this research. (2) Coconut oil and palm kernel oil should be used in the production of emulsion paint. (3) Soya bean oil, rubber seed oil, jatropha seed oil and neem seed oil should be used in the production of oil paint. (4) All the oils have anti-oxidant vitamins, hence they should be used in many industrial applications like soap, cosmetics, paint production, biodiesel etc. (5) The results obtained from this work will be of immense assistance to the paint industries in improving the quality of their products.These results should be made available to them. 197 University of Ghana http://ugspace.ug.edu.gh REFERENCES (1) ―Agency for Toxic Substancces and Disease Registry‖. (1980) ATSDR. Department of Health and Human Services Atlanta, GA. (2) Abdullah, B.M.and Saliman, J. (2009): Physicochemical Characteristics of Malaysian Rubber Seed Oil, EuropeanJournal of Scientific Research 31(3), 437-445. (3) Adejuwon, J.O. (2012) Rainfall Seasonality in the Niger Delta, Nigeria. Journal of Geography and Regional Planning5(2), 51-60. (4) Adler, V.E. and Uchel, E.C. (1985): Effects of a formulation of neem extract on six species of cockroaches. Phytoparasitica13(1), 3-8. (5) Afred, K.; Gabriele, H.Ludwig, L; (2005): Sodium and Sodium Alloys in Ullmann’s Encyclopedia of Industrial Chemistry Wiley-VCH, Weinheim. (6) Agarwal, D., Agarwal, A.K. (2007): Performance and emission characteristics of Jatropha Oil(preheated and blends) in a direct injection compression ignition engine. Applied Thermal Engineering27, 2314-2323. (7) Agrawal,O.P;Dhawan,S;Garg,K.L;Shaheen,F;Pathak,N;Misra;A.(1988):Studyof biodeterioration of the Ajanta wall paintings. Int. Biodeterior, 24, 121-129. (8) Aigbodion, A.I. and Pillai, C.K.S (2000): Preparation, analysis, and applications of rubber seed oil and its derivatives in Surface Coatings Progress in organic coatings 38,187-192. (9) Akinson, E.R.F.and Epand, R.W. (2008): Tocopherols and tocotrienols in membranes: a critical review. Free radical biology and medicine44(5), 739-64. 198 University of Ghana http://ugspace.ug.edu.gh (10)Akintayo, E.T. (2004): Characteristics and Composition of biglobbossa and Jatropha curcas oils and cakes. Bioresource Technology92, 307—310. (11) Alexey, R.and Novick, R.P. (2000): Equivalence of Lauric Acid and Glyceroll Monolaurate as Inhibitors of Signal Transduction in Staphylococcus aureus. J.Bacteriol182(9), 2668— 2671. (12) Alibhai, K; Leak, D.J; Dudeney, A. W. L., Agatzini, S., Tzeferis, P. (1991): Microbial leaching of nickel from low grade Greek laterites. In Smith, R. W., Misra, M. (eds): Mineral bioprocessing. The Minerals, Metals and Materials Society. Warrendak Pa, 191- 205. (13) Amekon,D.J.;Both,S.;Christotoph,R.;Fieg,G.;Steinberner,U.Westfechtel,(2006) ―Fatty Acids‖ in Ullmann‘s Encyclopedia of Industrial Chemistry, Wiley—VCH, Weinheim. (14) Amoore, J.E.and Hautala, E. (1983): Odor, an aid to chemical safety. J. Appl Toxicology. 3(6), 272-290. (15)Anneken,D.J.,Both,S;Christoph,R;Fieng,G;teinberner,U;Westfechel,A(2006):Fatty Acids. Ullmann”s Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinhein. (16) Awosika, L.F. (1995): Impacts of global climate change and sea level rise on coastal resources and energy developmemt in Nigeria InUmolu, J.C. (Ed) Global Climate Change: Impact on Energy Development DAMTECH Nigeria Limited, Nigeria. (17) Ayassa, M; Paxton, R. (2002): Brood Protection in social insects. Berlin Blackwell. 117- 48. 199 University of Ghana http://ugspace.ug.edu.gh (18) Azzi, A. (2007): Molecular mechanism of alpha-tocopherol action. Free radical biology and medicine. 43(1), 16-21. (19) Bagshaw, N.E. (1995): Lead alloys: Past, present and future. Journal of Power Sources .53, 25. (20) Banks, A (1990): Sodium. Journal of Chemical Education .67(12) 1046. (21) Battarbee R., Anderson, N., Appleby, P., Flower, R. J., Fritz, S., Haworth, E., Higgit, S., Jones, V., Kreiser, A., Muro, M.A., Naikanski J; Okifield F; Patrick ST; Richardson N; Rippey B; Stevenson AC; (1988). ‗Lake Acidification in the United Kingdom, ENSIS, London. (22) Beare—Rogers, J;Dieffenbacher,A;Holm,J.V.(2001):―Lexicon of Lipid Nutrition(IUPAC Technical Report)‖ Pure and Applied Chemistry 73(4), 685—744. (23) Bell, E.F (1987):‗‘History of vitamin E in infant nutrition‖ American Journal of Clinical Nutrition.46, 183-186. (24) Bello, N.J (2008): Perturbation in the Plant Environment: The threads and Agro- Climatological implications for food security. An inaugural lecture delivered in June 18, 2008 at the University of Agriculture, Abeokuta (25) Bentley,R.;Chasteen,T.G,(2002):Microbial Methylation of Metalloids: Arsenic,Antimony and Bismuth. Microbiology and Molecular Biology Reviews66(2), 256-271. 200 University of Ghana http://ugspace.ug.edu.gh (26) Bently,J. and Turner,G.R.A.(1997):Introduction to Paint Chemistry and Principles of Paint technology. Unk ISBN 0412723204. (27) Bergesou, Lynn L. (2008):The proposed lead NAACS: consideration of cost in the clean air act‘s future? Environmental Quality Management 18, 79. (28) Beveridge, T.J.; Doyle, R. (1989): Metal ions and bacteria.Wiley.NewYork (29) Beveridge, T.J., Forsberg, C.W., Doyle, R.C. (1982): Major sites of metal binding in Bacillus licheniformis Walls.J.Bacteriol150, 1438-1448. (30) Beveridge, T.J; Kova, I. (1981): Binding of metals to cell envelopes of Escherichia coli K- 12. Appl Environ Microbiol42,325-335. (31) Bils, R.F.and Howell, R. (1963): Crop Sci Chemistry/Food science 3. 304. (32)Blackborn,G.L.;Kater,G.E.A.;Mascioli,M;Kowalchuk,V.K;Babayan;and Bistrian,B.R.:(1990): A Re-evaluation of coconut oils effect on serum chobaterol and atherogenesis. Coconuts Today, 24 – 31. (33) Bock,E;Sand,W,(1993):The Microbiology of Mason Biodeterioration. J. App Bacteriol. , 74,503-514. (34) Bodsworth,C. (1994):The Extraction and Refining of Metals. CRC Press 148. (35) Borel,P;Drai,J;Faure,H.(2005): Recent knowledge about intestinal absorption and cleavage of carotenoids. Ann Biol Clin (Paris) (In French) 63(2), 165-77. (36) Bothwell,D.N;Mair,E.A;Cable,B.B,(2003):Chronic Ingestion of a Zinc Based Penny. Peditrics 111(3), 689-91. 201 University of Ghana http://ugspace.ug.edu.gh (37) Boukhalfa,H;Grumbliss,A.L,(2002):―Chemical aspects of Siderophore mediated iron transport‖ Biometals15(4), 325-39. (38) Bounoughas,M;Salhi,E;Benzine,K;Ghali,E;Dalard,,F,(2003): A Comparative Study of the electrochemical behavior of Algerian zinc and a zinc from a commercial Sacrificial anode. Journal of Material Science,3896, 1139. (39)Boxall,J.and,Von.Fraunhofer,J.A,(1980):―Paint.Formulation,Principles,and Practice‖,CDNS 469,32. (40)Brady,G.S;Clauser,H.R;Vaccan,J.A.(2002):Materials handbook.McGrawHill Professional 425. (41)Brandes,E.A;Greenaway,H.T;Stone,H.E.N(1956):‖Ductility in Chromium‖ Nature 178(587), 587. (42)Brar,S;Henderson,D;Schenck,J;Zimmerman,E.A,,(2009):Iron accumulation in the Substantia nigra of patients with Allzheimer disease and parkinsonism. Archives of neurology 66(3), 371-4. (43)Breuer,B;Stuhifauth,T;Fock,H.P,(1987):Separation of fatty acids or methyl esters including positional and geometric isomers by alumina argentation thin-layer chromatography. J. of Chromatogr. Science25(5), 302-306. (44) Brierley, C.L; Brierley, J.A (1982): Anaerobic reduction of molybdenum by sulfolobus species.Zentral bi Baktariol Mikrobiol Hyg 1Abt orig C: 289-294 202 University of Ghana http://ugspace.ug.edu.gh (45) Brigelius-Flohe (2009): ―Vitamin E, the shrew waiting to be tamed‖ Free radical biology and medicine. 46(5), 543-54. (46)Brigelius-Flohe, T.B (1999): ―Vitamin E function and Metabolism‖ FASEB, J. 13(10), 1145- 1155. (47) Brown, E.M, (1997):A Conformational Study of Collagen as Affected by Tanning Journal of the American Leather Chemists Association92, 225-233.Procedures. (48) Buddie, R.L.and, Nickerson, S.C. (1992): Evaluationof Post-Milking teat germicides containing lauricidin, saturated fatty acids, and lauric acid. Journal of DairyScience75: 1725 – 1730. (49) Burdge,G.C;Calder,P.C,(2005):―Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults.‖ Reproduction nutrition development. 45(5), 581-97. (50) Burns, D.M; John, B, l (1954): Molecular Structure of Fluorene, Nature.173,635. (51) Burr, G.O; Burr, M.M; and Miller, E, (1930): ―On the nature and role of fatty acids essential in nutrition‖ J.Biol. Chem 86(587), 1-9. (52) Carballo, R; Casliieras, A; Dominiquez –M.A; Garcia-Santos,I; Niclos-Guttiarrez,J (2013): Chapter 7 Solid State Structures of Cd complexes with relevance to biological system.Metal Ions in life sciences.11 Springer pp147-189. th (53) Carrasco, F, (2009):‖Ingredientes Cosmeticos.‖ Diccionario de Ingredientes (4 ed) 248. 203 University of Ghana http://ugspace.ug.edu.gh (54) Cava, R.J; Hor, Y.S. (2011):―Pressure Stabilized Se-Se Dimer Formation in PbSe2‖ Solid State Sciences, 13(2011), 38-41. (55) Ceesper, R.E; Lemay, P (1950): ―Henri Sainte-Claire Deville 1818-1881‖ Chapman 3 , 205- 221. (56) Chau,Y.K;Wong,P.T.S;Silverberg,B.A;Luxon,P.L;Bengert,G.A,(1976):Methylation of selenium in the aquatic environment. Science, 1130-1131. (57) Cheburaeva,R.F;Chaporova,I.N;Krasina,T,(1992):―Structure and Propertties of Tungstein Carbide Hard Alloys with an alloyed nickel binder” Soviet Powder Metallurgy and Metal ceramics31(5), 423. (57) Cheney,K;Gumbiner,C;Benson,B;Tenenbein,M;(1995):―Survival after a severe iron poisoning treated with intermittent infusions of deferoxamine‖ J.Toxicol clin Toxicol33(1) ,61-6. nd (58) Child, R. (1974) ―Coconuts‖ 2 edition. Longman Group Ltd. (59) Chin, H.F., Enoch, I.C.and Rajaharun,R.M.(1977):Seed technology in the tropics‖ Faculty of Agriculture, Universiti Putra Malaysia Publishing, Kuala Lumpur, Malaysia. (60) Ching-Hwa, Lee; His, C.S, (2002):‖Recycling of Scrap Cathode Ray Tubes‖ Environmental Science and Technology, 36 (1), 69-75. th (61) Chrisholm,Hugh,ed,(1911):Arsenic,EncyclopediaBritannica(11 ed),Camb. University Press. 204 University of Ghana http://ugspace.ug.edu.gh (62) Coleman,M.L;Hedrick,D.B;Lovley,D.R;White,D.C;Pye,K,(1993);Reduction of Fe(111) in sediments by sulfate-reducing bacteria. Nature (Lond) 361,:436-438. th (63) Combs, G.F, (2008):The Vitamins Fundamental Aspects in Nutrition and Health (3 ed) Burlington Elsevier Academic Press. (64) Conelly, N.G; and Geiger, W.E, (1996): Chemical Redox Agents for Organometallic Chemistry, Chem. Rev. 96, 877-910. (65) Cornils, B; Lappe, P (2000):‖Dicarboxylic Acids: Aliphatics‖ Ullmann”s Encyclopedia of Industrial Chemistry.Wiey-VCH,08, 524 (66) Cotton,F.A.(1999):‖Survey of Transition Mstal Chemistry” Advanced Inorganic Chemistry th (6 ed), John Wiley and Sons p 633. nd (67) Cotton, F.A; Wilkinson,G,(1966):Advanced Inorganic Chemistry (2 Edition).Wiley,New York. Cowan, James A.(1997): Inorganic Biochemistry, An Introduction. Wiley-Vch 7 (68) Crabb,D.W;(2001):Alcohol and Retinoids.Clinical and Experimental Research 25(5), 2075- 2175. (69) Cullen,J.T;Maldonado,M.T.(2003);‖Biogeochemistry of Cadmium and its release to the environment‖ Metal Ions in Life Sciences 11 Springer pp 31-62. (70) Cunham,G.C;Willet,W.C;Rim,E.B; Stampler,M.J,(1993) :A Prospective Study of dietary calcium and other nutrients and the risk of symptomatic kidney stones: The new England Journal of Medicine328(12), 833-8. 205 University of Ghana http://ugspace.ug.edu.gh (71) Cunnane,S;Anderson,M,(1997);―Pure linoleate deficiency in the rat influence on growth,accumulation of n-6 polyunsaturates and linoleate oxidation” J.Lipid Res38(4), 807-12. (72)Darmstadt,GL;Mao-Qlang,M;Chi,E;Saha,SK;Zihol,V.A;Black,R.E;Santosham,W; Elias,P.M,(2002): ―Impact of topical oils on the skin barrier: possible implications for neonatal health in developing countries‖ Acta Paediatrica,91(5), 546-554. (73) Davies,J.R.(2000):‖Uses of Nickel‖ ASM Special Handbook: Nickel,Cobalt and Their Alloys.ASM International 7-13. (74) De leon N. (2007): ―Reactivity of Alkali Metals‖ Indiana University Northwest (75) DeMoll-Decker,H;Macy,J.M,(1993);The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium.Arch Microbiol 160,241-247. (76) Denisenkov,P.A;Ivanor,V.V,(1987):‖Sodium Synthesis in hydrogen Burning stars‖ Soviet Astronomy letters13, 214. (77) Derek,G.E;Kerfoot.(2005):‖Nickel‖ Ullmann”s Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim. (78) Dernazeen,G;Buffat,B;Pouchard,M;Hagemmuller,P,(1982):‖Recent developments in the field of high oxidation states of transition elements in the oxides stabilization of six- coodinated iron(V)‖ Zeifschift fu anorganische and allgemeine Chemie 491 60. 206 University of Ghana http://ugspace.ug.edu.gh (79) Detheux,B.(2004):Utilization de l‘‘huile de palme comme combustible dans les moteurs diesels. Travaux de fin d’etude, 31-33. (80) Diaz,R. S.(1999): Coconut for Clean Air,22(3) (81) Dickson,A.G and Goyet C,(1994):Handbook of method for the analysis of the various parameters of the carbon dioxide system in seawater, version 2. (82) Din,M.B;Gould,R.D.(1998):―High field conduction mechanism of the evaporated cadmium arsenide thin films. International Conference of semiconductor Electronics proceedings, 168. (83) Dosumno,M.T.and Ochu,C. (1995.):Physiochemical properties and Fatty acid composition of lipids extracted from some Nigerian fruits and seeds. Global J. Pure and applied Sci. 1(1/2). (84) Doyle,R.J,(1991):How cell walls of gram-positive bacteria interact with metal ions.In;Beveridge,TJ;Doyle,RJ(eds).Metal ions and bacteria.Wiley.New York, 275-293. (85) Ebiwele,R.O;Iyayi;A.F,Hymore,F.K,(2010): ―Deacidification of high acidic rubber seed oil‖5(6),841-846. (86) Edyvean,R.G.J,(1995);The influence of marine macrofouling on corrosion. In; Gaylarde,C.C;Videla,HA(eds).Bioextraction and biodeterioration of metals.Cambridge University Press. Cambridge, 169-196 (87) Ehrlich,H.L,(1996);Geomicrobiology, Dekker. New York. 207 University of Ghana http://ugspace.ug.edu.gh (88) Ehrlich,H.L;Brierley,C.L,(1990):Microbial mineral recovery.McGraw.New York. (89)Ellis,R.W.(1997):―Infection and Coronary heart disease‖. Journal of Medical Microbiology,46(197),535-539. Ellis,R.W.(1997):―Infection and Coronary heart disease‖.Emil Erlemmeyer(1866), Journal of Medical Microbiology,333,269-275 st (90) Enigi,M.G:(1998) ―Coconut: In Support of Good Health in the 21 Century‖ (RR Watson,ed), CRC Press Boca Raton 81-97 (91) Ernsley,J,(2001):Nature‘s building blocks: An A-Z Guide to the Elements. Oxford University Press 513, 529, 43. (92)Eromosele,I.C.,Eromosele,C.O.,Innazo,P.,Njerim,P.,(1997):Short Communication Studies on some seeds and seed oils Bioresour.Technol. 64, 254-247. (93) Espinosa-Martos, I. Ruperez. P. Nutr. Hosp.( 2006,): 21, 92-96. (94)Farlex Incorporated (2005); ‗Definition, Environment‘ The Free Dictionary, Farlex Inc. Publishing, U.S.A. (95)Fawcett, E. (1988):‖Spin density wave antiferromagnetism in Chromium‖ Reviews of modern Physics 60, 209. (96) Fennema, O. (2008): Fennema‘s Food Chemistry CRC Press Taylor and Francis, pp 454- 455. (97) Fernando M.R.N (1971): Manufacture of dark facice from rubber seed oil. J. Rubb Res. Inst. Caylon47, 59 – 64 208 University of Ghana http://ugspace.ug.edu.gh st (98) Ferris,F.G,(1991): Enigi, M. G.:(1998) ―Coconut: In Support of Good Health in the 21 Century Metallic ion interactions with the outer membrane of gram-negative bacteria.In;Beveridge,TJ;Doyle,RJ(eds): Metal ions and bacteria. Wiley New York,295- 323. (99) Ferris,F.G;Schultze,S;Witten,T.C;Fyfe,W.S;Beveridge,TJ,(1989).Metal interactions with microbial biofilms in acidic and neutral Ph environments.Appl Environ Microbiol55, 1249-1257. (100) Frankenberger,W.T.,Jr.,Karson,U.(1992):Dissipation of soil selenium by microbial volatilization. In: Adriano DC(ed).Biogeochemistry of trace metals. Lewis, Boca Raton,Fla, 365-381. (101) Freny,E,(1842): ―Memoire sue les produits de la saponification de l‖hulle de palme ― Journal de Pharmacie et de Chime XII 757. (102) Gadd, G.M, (1993): Interactions of fungi with toxic metals:New Phytol.124, 25-60. (103)Gandhi,V.M;Cheriank,M.and,Mulky,M.J,(1990):Nutritional,and toxicological evaluation of rubber seed oil, Journal of American Oil Chemical Society67,883-886. (104) Garborino, J.R., Hayes,H; Roth,D; Antwelder, R; Brinton,TI; Taylor,H,;(1995); ‗Contaminants in the Mississippi River, U,S. Geological Survey Circular 1133, Virginia, U.S.A. (105) Garkin,R.E; Lundisled,A.P; Reppart,W.J,(1984): Structure of fluorene C13H10 at K Acta Crystallographica, Vol.C40 pp1892-1894. 209 University of Ghana http://ugspace.ug.edu.gh (106) Gatti,M;Tokatly,J; Rubio,A,(2010):‖Sodium: A charge-Transfer insulator at High Pressures‖ Eu Spine J. 10(21), 216-404. (107)Geleijnse,J.M; Kok,FJ; Grobbee,D.E,(2004): ―Impact of dietary and lifestyle factors on the prevalence of hypertension in Western populations‖ European Journal of Public Health14(3),235-239. (108) Georges,A; Bersillon,O; Blachol,J; Wapstra,A.H(2003):―The NUBASE Evaluation of Nuclear and decay properties‖ Nuclear Physics A Review.729, 3-128. (109)Gerd,A(2005):―Chromium Compounds‖ Ullmann‖s Encyclopedia of Industrial Chemistry. Wiley-VCH,Weinheim. (110) Gerd C; Hartnut H;and Jorg T,(2006): ―Anthracene in Ullmann‖s Encyclopedia of Industrial Chemistry Wiley-VCH Weinheim. (111) Gerd C; Hatmut H; Helmut G,(2003): ―Naphthalene and Hydronaphthalenes‖ in Ullman‖s Encyclopedia of Industrial Chemistry.Wiley-VCH Weinheim. (112) Gettens, Rutherford,J,(1966): ―Chrome Yellow‖ Painting Materials. A short Encyclopaedia Courier Dover Publications. Pp 105-106. (113)Gettens,R.J,Pease,M,Stout,G.I,(1941):‖The Problem of Mold Growth in Paintings‖.Techn Stud Fine Arts.,9: 127-143. (114) Ghiorse,W.C;Ehrlich,H.L,(1992):Microbial biomineralization of iron and manganese.In;Skinner,HCW;Fitzpatrick,RW(eds) Biomineralization Processes of iron 210 University of Ghana http://ugspace.ug.edu.gh and manganese. Modern and ancient environments.Catena Supplement Z1.Catena Cremlingen-Destedt.75-99 (115) Golberg,D..C,,(1969):Trends in Usage of Cadmium:Report pp 1-3. (116) Golub,Mari S ed (2005):―Summary‖ Metals, fertility and reproductive toxicity, Boca Raton, Fla. Taylor and Francis,p,153. (117) Griffin,W.(1949):―Cassification of Surface Active Agents by Hydrophilic-Lipophilic Balance,‖ Journal of Society of Cosmetic Chemist.1(5)362-367. (118) Grilli,C.,M,Fornic,C;Albertano,P,(1987):‖Characterization of the Algal Flora Growing on Ancient Roman Frescoes‖, Phycologia, ,26,387-390. (119)Groudev, S.N; Groudeva, V.1, (1986): Biological leaching of aluminium from clays. Workshop on biotecnol Bioeng Symp.16: 91-99. (120) Guard, H.E; Cobet, A.B; Coleman, W.M, (1981): Methylation of trimethyltin compounds by estuarine sediments.Science 213:770-771. (121) Gubitz, G.M., Mittelbach, M., Trabi, M. (1993); Exploitation, the tropical oilseed plant, Jatropha curcas L.Bioresource Technology67, 73-82. th (122)Gunstone, F.D; John, L.H; and Albert, J.D, (2007): The Lipid Handbook with CD Rom 3 Boca Raton CRC Press. (123) Gupta, C.K; Mukherjee, J.K, (1990): Hydrometallurgy in Extraction Processes CRC Press 62. 211 University of Ghana http://ugspace.ug.edu.gh (124)Hall,W.D;Pettinger,M;Oberman,A,(2001):‖Risk factors for kidney stones in older women in the Southern United States. Am J.Med. Sci322 (1) 12-18. (125Hallas,L.E;Means,J.C;Cooney,J.J,(1982):Methylation of tin by estuarine microorganisms.Science 215:1505-1507. (126) Hawkes,J.S,(1997);‗Heavy Metals‘ J. Chem. Educ. 74(11), 1374. (127) Hayes, A.W, (2007): Principles and Methods of Toxicology. Philadelphia. CRC Press 858- 861. (128) Hernell,O;Ward,H.;Blackberg,L;Pereira,M.E.(1986):Killing of Giafdia Lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids: Journal of Infections diseases153: 715 – 720. (129) Herrera, B.C, (2001):―Vitamin E action, metabolism and perspectives‖ Journal of Physiology and Biochemistry57(2). 43-56. (130) Hingston, J (2001):‖Leaching of Chromated Copper Arsenate Wood Preservatives:a review‖ Environmental Pollution 11(1),53-66. (131) Hoffman,K.L;Han,I.Y;Dawson,P.L.(2001):Antimicrobial Effects of Corn Zein Films Impregnated with Nisin, Lauric Acid and EDTA‖ J.Food Prot.64(6), 885—889. (132) Holieman,A.F;Wiberg,F;Wiberg,N.(1985):―Arsenic‖ Lehrbuch der Anorganischen Chemie(in Gernan) (91-100ed) Walter de Gruyter pp 675-681. 212 University of Ghana http://ugspace.ug.edu.gh (133) Holland,K.T;Taylor,D;Farrell,A. M.(1994): The Effects of glycerol monolaurate on growth of and production of toxic shock syndrome toxin-I and lipase by staphylococcus aureus. Journal of anti-microbial chemotherapy33, 41 – 55. (134) Holleman,A.F;Wiberg,E;Wiberg,Ni,(1985):‖Chromium‖ Lehrbuch der Anorganischen Chemie, Walter de Gruyter,1081-1095. (135)Holleman,A;Wiberg,E;Wiberg,E;Wiberge,N,(1985):‖Natrium‖ Lehibuch der Anorganischien Chemie(91-100) Walter de Gruyter,931-943. (136) (Holleman,A.F;Wiberg,E;Wiberg,N,(1985):‖Cadmium‖Lehrbuch,der Anorganischen Chemie,91-100 Walter de Gruyter, 1056-1057(German). (137) Horrobin, D.F (1993): ―Fatty acid metabolism in health and disease: the role of 6- desaturase‖ American Journal of Clinical Nutrition, 57, 7325-75. (138) Hutton, M; Symon C (1986).‘The quantities of Cadmium, Lead, Mercury and Arsenic Entering the U.K. Environment from Human Activities‘ Sci Total Environ. 57 129—150. (139)IIyaletdinov, A.N.; Abdrashitova, S.A, (1981); Autotrophic oxidation of arsenic by a culture of Pseudomonas arsenitoxidans.Microbiogiya50, 197-204(Microbiology NY50:135-140). (140) Ingalls, W.B,(1902): Production and Properties of zinc: The Engineering and Mining Journal., La Bibioleca Pubica de Nueva York 213 University of Ghana http://ugspace.ug.edu.gh (141) Inglesias-G,S;Manciado,A;Rebolo,R;GonzalezHernandez,J.J;Garcia-Hernandez, D.A.;Lambert,D.L.,(May 2011): A search for interstellar anthracene toward the Perseus anomalous microwave emission report. (142) Isaacs C. E., Litor R. E., Marie, P., Thormar H.(1992): Addition of Lipases to Infant Formulas Produces Antiviral and Antibacterial Activity.Journal of Nutritional Biochemistry 3:304 – 308. (143) Isaacs, C. E. Thormar, H. (1986): Membrane disruptive effect of Human Milk: Inactivation of enveloped viruses. Journal of Infectious Diseases 154, 966 – 971. (144) Isaacs, C. E., Kashyap, S., Heird, W. C., Thormar, H (1990).Antiviral and antibacterial lipids in human milk and infant formula feeds. Archives of Disease in Childhood 65, 861 – 864. (145)Isaacs, C. E., Kim, K. S., Thormar, H.(1994): Inactivation of Enveloped viruses in human bodily fluids by Purified lips. Annals of the New York. Academy of Sciences 124, 457 – 464. (146) Isaacs, C. E., Schneidman, K.(1991). Enveloped viruses in Human and Bavine Milk are inactivated by Added Fatty Acids (FAs) and Monoglycerides (MGs) FASEB Journal 5: abstract5325, A 1288. (147) Isaacs, C. E., Thormar ,H.(1991): The role of Milk-derived antimicrobial lipids as anti- viral and antibacterial agents in immunology of milk and the Neonate (Mastecky J. et al eds) Pienom Press, New York. 214 University of Ghana http://ugspace.ug.edu.gh (148) Isaacs, C. E., Thormor, H. (1990). ―Human Milk Lipids Inactivated enveloped viruses, Breast feeding, Nutrition, infection and infant Growth in Developed and Emerging countries (Atkinson SA, Hanson LA, Chandra RK, Eds) Arts. Biomedical Publishers and Distributors, St. John‘s NF, Canada. (149) Iyayi, A.F, Akpaka, P.O, Ukpeoyibo, U, Momodi, I.O, (2007) ―‖Rubber Seed Oil. An oil with great potentials. Chem. Tech. J. 3, 507 – 516. (150) Ji,G;Silver,S(1995):Bacterial resistance mechanisms for heavy metals of environmental concern. J.Ind Microbiol14, 61-75. (151) Jones, F.T. (2007): ―A broad view of Arsenic‖ Poultry Sciences 86(1), 2-14. (152)Joseph,G;Kundig,Konrad;J.A(1999):Association International Copper: ―Dealloying‖ 123- 124. (153) Julien, M; Hoeffel, J.M;: Flick,M.R,(1986):‖Oleic acid lung injury in sheep‖ Journal of Applied Physiology. 60(2), 433-40. (154) Kabara,J.J,.(1985):―Inhibition of Staphylococcus Aureus in the Pharmacological Effect of Lipids II (JJ Kabara, ed) American oil Chemists Society, Champaign IL, , 71 – 75. Kabara,J.J.(1978):Fatty acids and Derivatives as antimicrobial agents. A review in the Pharmacological Effect of Lipids (JJ Kabara, ed) American Oil Chemists‘ Society, Champaign IL, 215 University of Ghana http://ugspace.ug.edu.gh (155)Kakatnur,M.G;Oalmann,M.C;Johnson,W.D;Malcom,G.T;Strong,J.P;(1979): ‖Fatty acid composition of human adipose tissue from two anatomical sites in a biracial community‖ The American Journal of Clinical Nutrition,32 (11), 2198-2205. (156) Karl,G;Biedenkapp,A.D.;Heinz-Werner,V;Dorothea G;Paetz;C;Gerd,C;Diete,M.Hatmurt,H,(2002):―Hydrocarbons‖ in Ullmann‖s (157)Encyclopedia of Industrial Chemistry Wiley-VCH. Weiheim. Kemper, F; Moister, FJ; Jager, C, and, Waters, LBFM (2002): The mineral composition and spatial distribution of the dust ejecta of NGC 6302. Astronomy and Astrophysics.394, 679-690. Kering, M.K, (2008): ―Manganese Nutrition and Photosynthesis in NAD –malic enzyme C4 plants, Ph.D Dissertation‖ University of Missouri- Columbia. (158) Kharton, V.V, (2011): Solid State Electrochemistry I: Electrodes Interface and Ceramic Membranes. Wiley-VCH 166— (159) Kid, J ;( 1821): ―Observations on Naphthalene‖ Philosophical Transactions 11, 209-221. (160) Kim,S.B;Cotz,S; Sun,S; Chung, Y.K; Pike, R.D; Sweigart, D.A,(2010): Manganese Tricarbonyl(Transfer(MTT) Agents) Inorganic Synthesis.35,109-128. (161)Kimberly,DMC;Ernest,M;Prokopchuk,(2011):CoordinationComplexes as catalyst,Journal of Chemical Education88(8).1155-1157 (162) Kinny, T, (2007): ―Metabolism in plants to produce healthier food oils‖ (163) Kinsbury, K.J; Paul, S; Crosley, A; Morgan, O.M (1961):‖The fatty acid composition of human depot fat‖ Biochemical Journal. 18, 54-550. 216 University of Ghana http://ugspace.ug.edu.gh (164) Klens,P.F,and Lang,J.R,(1956):‖Microbiological Factors in Paint Preservation‖ J.Oil Colour Chemists” Association,,38,887-899. (165) Klotz,K;Weistenhofer,W;Drexler,H,(2013):‖Chapter 4 Determination of Cadmium in Biological Samples‖ Metal Ions in Life Sciences.11 Springer 85-98. (166) Kohl,W. H,(1995): Handbook of materials and techniques for vacuum devices Springer 164-165. (167) Kowdley,K.V;Mason,J.B; Meydani,S.N; Cornwall,S; Grand,R.J,(1992): ―Vitamin E deficiency and impaired cellular immunity related to intestinal fat malabsorption‖ Gastroerheology. 102(6), 2139-42. (168) Kuck,P. H,(2006): Mineral Yearbook,Nickel, United States Geological Survey. (169) Kuhn,H and Medlin,D,(2000) ASM Handbook-Mechanical Testing and Evaluation p275. (170) Kumar,A,Sharma,S.(2008): An evaluation of multipurpose oil seed crop for industrial uses (jatropha curcas). A review: Industrial crops and products.doi 10101 blindcrop 2008 01,001. (171) Kumar,D;Goswani,C.B.K;andMukerjee,S.K.(1985):Nematicidal principles from Neem Chitwood,Indian Journal of Nematolog15(1), 121-124. (172) Lamb,PJ (1983): Sub Sahara Rainfall Uptake for 1982 Continued drought. J. Climatol, 3, 419-422. 217 University of Ghana http://ugspace.ug.edu.gh (173) Lange,W and K.Feuerhake,(1984): Increased activity of enriched neem seed extracts with Synergist piperonyl butoxide under laboratory conditions ,. Zeitschrift fur Angewandte Entomologie98.368. (174) Lars,S;.Eigeny,W. and Ronald,C,(1997):―Composition and Temperature of Eatth‖s inner core‖ Journal of Geophysical Research 102(BII), 24729-24740. (175) Lenntech Water Treatment and Air Purification (2004). ‗Water Treatment‘ Lenntech,Rotterdamseweg Netherlands. (176) Leonard,A.R; Lynch,G(1958): ―Disware as a Possible Source for Lead Poisoning‖ Calif.Med,89(6), 414-416. (177) Letawe,Letawe C; Boone,M; Pierard,GE,(1998): ―Digital image analysis of the effect of topically applied linoleic acid on acne microcone dones‖ Clinical and Experimental Dermatology 23,(2), 56-58. th (178) Lewis,RJ,(1996):Sax‖s Dangerous Properties of Industrial Materials (9 ed) New York,NY Van Nostrand Reinhold, p 635. (179) Li,Thomas,SC,(1999): Sea bukthom:New crop opportunity.Perspectives on new crops and new uses. Alexandria, VA ASHS Press. Pp335-557. th (180) Lide,DR,(2005): CRC Handbook of Chemistry and Physics(86 ed), Boca Raton, CRC Press. (181) Lide,DR,(2005): CRC Handbook of Chemistry and Physics, Boca Raton (FL),CRC Press. 218 University of Ghana http://ugspace.ug.edu.gh (182) Liu, KS,(1997);―Chemistry and nutritional value of soyabean components.In soyabean: chemistry, Technology, and utilization‖, Chapman and Hall, New York, 25,-113. (183) Lovley,DR,Phillips,EJP,(1988);Novel mode of microbial energy metabolism.Organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl Environ Microbiol54, 1472-1480. (184) Lyalikova, NN; Vedenina, IYa; Romanova, AK, (1976); Assimilation of carbon dioxide by a culture of stibiobacter senarmontii. Mikrobiologiya 45:552-554(Microbiology NY45 476- 477). (185) Macaskie,LE;Dean,ACR;Cheetham,AK;Jakeman,RJB;Skarnulis,AJ (1987). Cadmium accumulation by a citrobacter sp, the chemical nature of the accumulated metal precipitate and its location on the bacterial cells.J.Gen Microbiol133:,539-544. (186) Macaskie,LE;Empson,RM;Cheetham,AK;Grey,CP;Skarnulis,AJ (1992):Uranium bioaccumulation by a citrobacteria sp as a result of enzymically-mediated growth of polycrystalline.HUO2PO4.Science.257,782-784. (187) Maret Wolfgang,(2013): Chapter 14 Zinc and the zinc proteome:In Band Lucia(Ed) 12 Springer. (188) Marrion A,(2004): ―The Chemistry and Puysics of Coatings , Royal society of chemistry. 287--- (189) Martens,C.R.(1969): ―Technology of Paints, Varnishes and Lacquers‖. Colombus Ohio.3, 111-131. 219 University of Ghana http://ugspace.ug.edu.gh (190)Martinez-Herrera,J.,Sidehuraju,P,Francis,G.,Da‘vila-Ortiz,G.Becker,K.(,2006): Chemical Composition,toxic/antimetabolic constituents and effects of different treatments on their levels in four provenances of jatropha curcas L 96,80-89. (191) Martz, Walter,(1993):‖Chromium in Human Nutrition: A Review” Journal of Nutrition 123 (4), 625-33. (192)McDonald,I;Sloan,GC;Zijstra,AA;Matsunaga,N;Matsuura,M;Kraemer,KE;Bernard- Salas,J;Markwick,AJ,(2010): ―Rusty Old Stars: A source of the missing Interstellar Iron‖ The Astrophysical journal Letts717(2), L92-L97. (193) Mensink, R.P, Zock, P.L, Kester, A.D.M, katan, M.B, (2003): ―Effects of Dietary Fatty Acids and Carbohydrates of 60 controlled Trials‖ American Journal of Clinical Nutrition77(5), pp1146—1155. (194) Miller,IS; Mullin,JB,(1991):‖Crystalline Cadmium sulfide‖ Electronic materials; from silicon to organics. Springer 273. (195) Mohammad,FA;Basam,MEA:James,GS,:(2005),Handbook of Industrial Chemistry (organic chemicals), McGraw-Hill, New York. ISBN 007-141037-6. (196) Mond,L; Langer,K; Quinoke,F,(1890): ―Action of Carbon Monoxide on Nickel‖ Journal of the Chemical Society.57,749-753. (197) Moore,T; Holmes,PD,(1971): ―The production of experimental vitamin A deficiency in rats and mice‖ Laboratory Animals,5(2), 239-50. 220 University of Ghana http://ugspace.ug.edu.gh (198) Morgan,J.W.and Anders,E;(1980): ―Chemical Composition of Earth, Venus and Mercury‖ Proc Nat. Acad Sci 77(12), 6973-6977. (199) Morrow,H,(2010):‖Cadmium and Cadmium alloys‖ Kirk-Othomer Ecyclopedia of Chemical Technology. John Wiley and Sons pp 1-36. (200) Moss,SC and Newnham,RE,(1964): ―The Chromium position in ruby‖ Kristallographie, 120 (4-5), 359—363. (201) Mukhopadhyay,R;Rosen,BP;Phung;LT;Silver,Simon,(2002):‖Microbial Arsenic:From geocycles to genes and enzymes‖ FEMS Microbiology Reviews 26(3),311-25. (202) Muyssen,Brita,TA;De,Schamphielaere,Karel,AC;,anssen,Colin R,(2006):‖Mechanisms of chronic water borne Zn toxicity in Daphnia magna‖ Aquatic Toxicology, 77(4) 393-401. (203)Nair,MK;Joy,J;Vasudevan,P;Hinckley,L;Hoagland,TA;Venkitanarayanan,KS,(2005): ‖Anti-bacterial effect of caprylic acid and monocaprylin on major bacterial mastitis pathogens‖ J. Diary Sci. 88(10) 3488-95. (204) Nam,Wonwoo,(2007): ―Hig-valent Iron(V)-OXO Complexes oh Heme and Non-Heme Ligands in Oxygenation Reactions‖ Accounts of Chemical Research 40(7),522-531. (205)Namibiar,KR,,(2006):‖Helium-Cadmium Laser‖ Lasers principles,Types and Applications. (206) National Research Council, Neem: a tree for Solving global problems, National Research Council, National Academy Press, Washington DC,1992,p141. 221 University of Ghana http://ugspace.ug.edu.gh (207) Neem Oil: Tolerance Exemptions. Fed. Regist.,December 13 1998, 60, 1995,60(239), 63950-63953. (208) Neikov,O.D;Naboychenko,S.;Gopienko,V.G;and Frishberg,I.V.(2009): Handbook of Non- Ferrous Metal Powders, Technologies and applications. Elsevier 371— (209) Neilands,JB(ed,)(1974); Microbial iron metabolism.Academic Press New York. (210)Neilands,JB,(1981): ―Microbial Iron Compounds‖ Annual Review of Biochemistry 50, 715- 31. (211) Neilands,JB,(1995): ―Siderophores: Structure and function of microbial iron transport compounds‖ The journal of Biological Chemistry50 715-31. (212) Nishimura,Y;Ishi,N;Sugita,Y;Nakajima,H,(1998):―A case of carotenodernia caused by a diet of the dried seaweed‖ J. Dermatol25(10), 6. (213) Njoku, O.U; Ononogbu, I.C: and Muorieke, SMC,(1996): Wood finish from rubber seed alkyd Res Indian J. Nat. RubberRes.9(2), 137. (214) Njoku,O.U, and Ononogbu, I.C,(1995b): Preliminary studies of preparation of lubricating grease from bleached rubber seed oil. Indian J. Nat. Rubber Res.8(2), 140-141. (215) Njoku, O.U; Ononogbu I.C,(1995a)); Alkyd resins from rubber seed oil. Indian J. Nat. Rubber Res,8(1), 63-65. (216) Norman, N.C, (1998): Chemistry of Arsenic, Antimony and Bismuth.Springer. p 50. 222 University of Ghana http://ugspace.ug.edu.gh (217) Nriagu, J.O,(1989); ‗A Global Assessment of Natural Sources of Atmospheric Trace Metals‘, Nature 338.47-49. (218) Nriagu,J.O; Pacyna J;(1988); ―Quantitative Assessment of Worldwide Contamination of Air, Water and Soil by Trace Metalas”, Nature333 134-139. (219) Nutter,M.K; Lockhart,E.E; Harris,RS,(1943): ‖The Chemical Composition of depot fats in chickens and turkeys‖ Oil and Soap.20(11), 231-4. (220) Nwokol, E;Kitts,D.D. and Ken hail,J.,(1998:) ―Serum and Liver Lipids of rats fed rubber seed oil, Plant Foods for Human Nutrition38, 145-153. (221) Ogunkoya,O.O;Efi,EJ,(2003): Rainfall Quality and Sources of Rainwater Acidity in Warri Delta, Nigeria. J. Minina, Geol,39(2), 125-130. (222) Oh,D.Hand Marshall,D.L,(1993):Antimicrobial activity of ethanol, glycerol monolaurate or lactic acid against listeria monocytogenes. International Journal of food and Microbiology20,239- 246. (223) Ohler, L(1984):―Characteristics of Coconut oil from Copra‖, 65-72. (224) Ononogbu, I.C;Uzoamaka, C.I;and Njoku, O.U(2001): Antioxidants and inorganic elements in Rubber seed oil, Actes du Colloque de Sainte-Foy (Quebec) 7 an 9 ‗ (225) OpenShaw,K,(2000): A review of Jatropha curcas oil, an oil plant of unfulfilled promise. Biomass and Bioenergy19, 1-15. 223 University of Ghana http://ugspace.ug.edu.gh (226)Ouattar,B;Simard,R.E;Prett,G,Begin,A,Holley,R,A.(2000).‖Inhibition of Surface Spoilage Bacteria in Processed Meats by Application of Antimicrobial Films Prepared with Chitosan‖ Int. J. Food Microbiol. 62(1-2), 139-148. (227) Dawson, P.L, Carl, G.D, Acton, J.C and Han, I.Y, (2002):―Effect of Lauric Acid and Nisin-Impregnated Soy-based films on the growth of Listeria monocytogenes on Turkey bologna‖. Poultry Sci. 79(Suppl.1).54. (228)Packer,L;Weber,S.U;Rimbach,G,(2001);―Molecular aspects of alpha-tocotrienol antioxidant action and cell signaling‖ Journal of Nutrition.131,3698-3738. (229) Pala,V; Krogh,V; Muti,P; Chajes,V; Riboli,E; Mitchell,A; Saadatrian,M; Sieri,S; et al,(2001): ―Erythrocyte Membrane Fatty Acids and Subsequent Breast Cancer. Journal of the National Cancer Institute,JNCI,93(14), 1088-95. (230) Papp,J.F, and, Lipin, B.R, (2006):Chromite Industrial Minerals and Rocks:Commodities, th Markets and Uses(7 ed) SME 233-8 (231) Pauling L,(1970): General Chemistry,Dover Publications 627. (232) Pavia,D.L;Gary,M.L;George,S.K;Randall,G.F,(2006):Introduction to Organic Laboratory th Techniques(4 ed),pp 797-817. (233) Penny,Kris-Etherton;William,S.H;andLawrence,J.A,(2002):―Fish consumption,Fish oil omega-3 fatty acids and cardiovascular disease‖ Circulation.106(21), 2747-2757. 224 University of Ghana http://ugspace.ug.edu.gh (234) Petschow,B.W;Batema,R.P;Ford,L.L,(1996):Susceptibility of Helicobacter Pylori to bactericidal properties of medium – chain monoglycerides and free fatty acids antimicrobial agents and chemotherapy 145: 876 – 887. (235) Piomelli,D.(,2000): ―Arachidonic Acid‖ Neuropsychopharmacology. (236)Pope,D.H;Duquette,D.J;Johannes,A.H;Wayner,P.C,(1984):Microbially-influenced corrosion of industrial alloys.Mater Perf 23:14-18. (237) Prasad,A.S,(2003):‖ Zinc deficiency..‖ British Medical Journal326(7386) 409-10. (238) Pumamadjaja,A.H; Russell,RW,(2005):‖Pheromone Communication tn a robot swarm Necrophoric bee behavior and its replication‖ Robotica 23(6), 731-42. (239) Raghavan,V (2004): Materials Science and Enginering, PHI learning Prt Ltd p218. (240)Rahman,F.A;Allan,D.L;Rose,C.J;Sadowsky,M.J,(2004):―Arsenic,availability from Chromated Copper arsenate treated wood‖ Journal of Enviromental Quality33(1), 173-80. (241) Ralof,J,(2012): ―Tricks Food Play‖ Science News Magazine. 182(7), 25-28. (242)Reboul,E;Richielle,M;Perrot,E;DesmoulinsMatezet,C;Pirisi,V;Borel,P,(2006): ―Bioaccessibility of carotenoids and vitamin E from their main dietary sources‖ Journal of Agricultural and Food Chemistry 54(23), 8749-8755. (243) Rech,S;Macy,J.M,(1992);The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes. J.Bacteriol174 7361-73120. 225 University of Ghana http://ugspace.ug.edu.gh (244)Reimer,K.J;Koch,J;Cullen,W.R,(2010):‖Organoarsenicals:Distribution,and transformation in the environment‖ Metal ions in the Sciences 7, 165-229. (245)Reiner,D.S;Wang,CS;Gillin,F.D.(1986):Human Milk kills Giardia Lamblia by generating toxic lipolytic products. Journal of infectious Diseases104:55 – 56. (246)Rizzo,W.B;Watkins,P.A;Phillips,M.W;Cranin,D;Campbell;B;Avigan,J(1986): ―Adrenoleukodystrophy: Oleic acid lowers fibroblast saturated C22-25 fatty acids” Neurology36(3), 357-61. th (247)Robert,L.G;Eugene,FB:(2004):Modern Practice,Gas Chromatography(4 ed).John Wiley and Sons. (248)Robinson,J.B;Tuovinen,O.H,(1984):Mechanism of microbial resistance and detoxification of mercury and organomercury compounds,physiological,biochemical,and genetic analysis:Microbiol Rev 48;95-124. (249) Ross, R.T, (1963):‖The Microbiology of Paint Films”, Adv. Appl. Microbiol,5, 217-234. (250) Rossner, J and,Zebitz,C.P.W. (1987): Effect of neem products on nematodes and growth of tomato plant(lycopersicon esculentum), Schmutterer and Ascher(GTZ) 611-621. (251) Ruthig,D.J;and Meckling-Gill,K.A,(1999):―Both(n-3) and(n-6) fatty acids stimulate wound healing in the rat intestinal epithelial cell line EC-6‖ Journal of Nutrition129,(10), 1791-8. (252) Sabina,C.G;Kumbert,H;Hans Uwe Wolf (2005):―Arsenic and Arsenic Compounds‖ Ullmann‘s Encyclopedia of Industrial Chemistry. Wiley-VCH, Weinheim. 226 University of Ghana http://ugspace.ug.edu.gh (253) Sahmial, A; Zaharial, I; and Mohtar, Y (1998): Olechemicals for soaps and detergents. International seminar and exposition. World scenario in oils, oleochemicals and surfactant industries, OTAI, LUKNOW, India. (254) Saiz-Jimenez, C, Samson,R.A,(1991): ―Microorganisms and Environmental Pollution as Deteriorating Agents of the Frescoes of the Monastery‖, Santa Maria de la Rabida, Huelva,Spain. (255) Sakurai,T.S,(2003):―Biomethylation of Arsenic is Essentially Detoxicating Event‖ Journal of Health Science49(3),171-178. (256)Santucci K; Shah, B;(2000): Association of naphthalene with acute hemolytic anaemia. Acad Emerg Med 7(1) 42-47. (257) Sauren Annalender Chemie and Pharmacie 137(3), 327-359. (258) Saxena,R.C;Epino,P.B;Cheng-Wen,TU and Buma, B.C,(1984):Neem,Chinaberry and Custard apple: antifeedant and insecticidal effects on leafhopper and planthopper pests of rice, Schmutterer and Ascher 403-412. (259) Schaafsma A,van Doormaal J.J; Muskiet, F.A; Hofstede,G.J; Pakan,I; van der veer E(2002):―Positive effects of a chicken eggshell powder-enriched vitamin-mineral supplement on femoral neck bone mineral density in healthy late post menopausal Dutch women‖ Br.J.Nutr. 87(3)276-78. 227 University of Ghana http://ugspace.ug.edu.gh (260) Schaafsma, A and Beelen G.M, (1999):‖Eggshell powder, a comparable or better source of calcium than purified calcium carbonate piglet studies‖ Journal of the science of Food and Agriculture 79(2), 1596-1600. (261) Schmutterer,H; and.Freres,T,(1990): Influence of neem-seed oil on metamorphosis, color behavior of the desert locust,Zeitschrift fur pflanzenkrankeiten and pflanzenschut,97(4), 431-438. nd (262) Schumann,W,(2008): Minerals of the world(2 ed) Sterling 28.Amazon Publications (263) Scoullos, M.I,(2001): Mercury, Cadmium and Lead: Handbook for sustainable Heavy Metals Policy and Regulation. Springer 104-116. (264) Scoullos,M.J; Vonke man,Gerrit H; Thomton,Lain; Makuch, Zen,(2001): Mercury,Lead: Nercury and Lead, Handbook of sustainable Heavy Metals policy and regulation.Springer. (265) Selim,S. and Marco C;(2003): ―Combustion‖ in Ullmann‖s Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim. th (266)Sharma, B.K. (2006):―Industrial Chemistry (Including Chemical Engineering)‖ 15 Edition Goel Publishing House, Meerut. (267) Shierf, G.W, and Brown, R.K, (1960): Derivatives of Fluorene as intermediates in the preparation of fluorene and some C9-substituted Fluorene. Canadian Journal of Chemistry, 38, 697. 228 University of Ghana http://ugspace.ug.edu.gh (268) Silver,S,(1992):Bacterial heavy metal detoxification and resistance systems.In;Mongkolsuk,S;Lovett,PS;Trempy J(eds) Biotechnology and environmental Science:Molecular approaches. Plenum, New York 109-129. (269) Silverman,M.S,(1966):―High Pressure(70kilobar)Synthesis of New Crystalline Lead Dichalcogenides‖Inorganic Chemistry,5(11),2067-9. (270) Smith,C.J.E;Higgs,M.S; Baldwin,K.R:(1999): Advances to Protective coatings and their applications to Ageing Aircraft.RTO MP-25. (271) Smolinske,S.C,(1992): Handbook of Food,Drug and Cosmetic Excipients,pp 247-8. (272) Solomons,N.W; Orozco,M,(2003): ―Alleviation of Vitamin A deficiency with palm fruit and its product‖ Asia Pacific Journal of Clinical Nutrition12(3), 373-84. (273) Stolz,J.F;Basu,SJM;Oremland,RS,(2006):―Arsenic and Selenium in Microbial Metabolism‖ Annual Review of Microbiology60, 107-30. (274)Style, M.L.A,(2008)―Coconut palm‖ encyclopedia Britannica ultimate reference suite, Chicago. (275)Subbarao,G.V;Ito,O;Berry,W.L;Wheder;R.W,(2003):―Sodium-:A Functional Plant Nutrient‖ Critical Reviews on plant sciences 22(6),391-416. (276) Sugano, M, ED.(2006) Soy in health and Disease Prevention, CRC Press, FI, USA. (277) Summers,A.P;Silver,S,(1978):Microbial transformations of metals.Annu Rev Microbiol 32, 637-672. 229 University of Ghana http://ugspace.ug.edu.gh (278) Sydor,A.M;Zambie,D.B,(2013):Chapter11,Nickel Metallomics:General Themes Guiding Nickel Homeostasis‖ Springer 12. (279)Szilagyi,R.K;Bryngeson,P.A;Maroney,M.J;Hedman,B;Hodgsuk,C;Solomon,E.I,(2004):‖S. K-Edge x-ray absorption spectroscopic investigation of of the Ni—containing superoxide Dismutase.Journal of the American Chemical society 126(10), 3018-3019. (280)Takaji,K;Toshikatsu,O,(1964):‖Properties of various Pure Irons‖ Study on pure iron Tetsu- to-Hagane 50(1), 42-47. (281) Takita,Y; Ichimiya,M; Hamamoto,Y; Muto,M,(2006):―A case of caotenemia associated with ingestion of nutrient supplements‖ J. Dermatol33(2), 132-4. (282) Talbert R,(2007):―Paint Technology Handbook‖, Grand Rapids, Michigan, U. S. A., : ISBN 1574447033. (283) Tanaka,A (2004): ―Toxicity of indium arsenide, gallium arsenide and aluminium gallium arsenide‖ Toxicology and Applied Pharmacology198(3), 405-11. (284) Tang,G; Qin,J; Dohikowski,GG; Russell,RM; Grusak,MA,(2005): ―Spinach or Carrots can supply significant amounts of Vitamin A as assessed by feeding with intrinsically deuterated vegetables‖ Am. J. Clin. Nutr82(4), 821-8. (285)Tanumihardjo,S.A,(2011):―Vitamin A biomarkers of nutrition for development‖ American Journal of Clinical Nutrition,99(2), 6588-6658. 230 University of Ghana http://ugspace.ug.edu.gh (286) Tenenbein,M(1996)―Benefits of parenteral deferoxamine for acute iron poisoning ― J.Toxicol clin Toxicol34(5), 485-489. (287)Teres,S;Barcelo-Coblijn,G;Benet,M;Alvarez,R;Bressani,R;Halver,J.E; Escriba,P.V,(2008): Oleic acid content is responsible for the reduction in blood pressure induced by olive oil‖ Proceedings of the National Academy of Sciences 105(37), 13811-6. (288) Tetreauit,J; Siros,J; Stamatopoykou,E.(1998):‖Studies of Lead Corrosion in Acetic Acid Environments‖ Studies in Conservation.43(1),17-32. (289) Thampan, P.K.(1994):―Facts and Fallacies About Coconut oil‖. Asian and Pacific Coconut Community books and Proceedings, 8. (290) Thijssen,M.A;and,Mensink,RP,(2005):―Fatty,Acids,andAtherosclerotic Risk‖ pp171— 172. ISBN 9783540225690. (291) Thomas, J.C, (2005):‖Cadmium Environmental Concerns‘ PVC hamdbook Hanser Verlag p 149. (292) Thomas, A, (2000):‖Fats and Fatty Oils‖ Ullmann‖s Encyclopedia of Industrial Chemistry, 10, 173. (293) Thormar,H,Isaacs,E.C.,Brown,H.R.,Barshatzky,M.R.,Pessolano, T.(1989): Inactivation of enveloped Viruses and Killing of cells by fatty acids and monoglycerides. Antimicrobial agents and Chemotherapy 31, 27-31. 231 University of Ghana http://ugspace.ug.edu.gh (294) Thurmer,K;Williams,E;Reutt-Robey,J(2002): ―Autocatalytic oxidation of lead crystallite surfaces” Science 287(5589),2033-5. (295) Thyssen,J.P;Linneberg,A; Menne,T; Johansen,J.D,(2007):‖The epidemiology of contact allergy in the general population-prevalence and main findings‖ Contact Dermatitis 57(5), 287-99. (296) Traber, A.J,(2007):‖Vitamin E,Antioxidant and Nothing More‖ Free radical biology and medicine,43(1), 4-15. (297) Trevors,J.T,(1992);Mercury methylation by bacteria. J Basic Microbiol 32:637-672. (298) Tsenga Wenjea J; Mo Liua, Dean; Hsub,Chung-king,(1999):‖Influence of stearic acid on suspension structure and green microstructure of injection-molded Zirconia Ceramics‖ Ceramics International25(2) 191-195. nd (299) Tubbi, L, (1967): Painting and Decorating, 2 ed., Macmillan Pub. (300) Tucek,K;Carlson,J;Wider,H,(2006):‖Comparison of Sodium and Lead-cooled fast reactors regarding reactor physics aspects,severe safety and economical issues”.Nuclear Engineering and Design236(14-16),1589. (301) Turner, G. R. A,(1967): Introduction to Paint Chemistry, Chapman and Hall Ltd, London, (302) UNIDO Report (1989): The development of a rubber seed processing technology for the production of vegetable oil and animal feed. Report No US/GLO/81/03. 232 University of Ghana http://ugspace.ug.edu.gh (303) United States Environmental Protection Agency. (2001): Emergency Planning and Community Right-to-Know Act, Section 313. Washington D.C. (304) Using Freezing Point Depression to find Molecular Weight. University of California, Irvine, (2010): Pp04—12. (305) Vandenput,R,(1981): Les Principles Cultures En Afrique Centrale In Techargo,T.J.(Ed). Contribution a L‘etude du Vin De palme D‘elaeis Guineensis Au Cameroun Production, utilization et Quelques Analysis Nationale Superieure agoromi que, Centre Universitaire de Dscharq, Dschang, pp 52. (306) Vertuani,S;August,A;Mantredini,S,(2004):‖The Antioxidants and Pro-Antioxidants Network: An overview” Current Pharmaceutical Design 10, 1677-94. (307) Videla,H..A.(1995);Electrochemical aspects of biocorrosion. In; Gaylarde, CC; Videla HA(eds):Bioextraction and biodeterioration of metals.Cambridge University Press, Cambridge,85-127. (308)Villarreal-Lozoya,J.E;Lombardini,L;Cisneros-Zevallos,L,(2007):‖Phytochemical Consttuents and Antioxidant capacity of different pecan carya illinoinensis cultivavars‖ Food Chemistry102(4) 1241. (309) Von der Hyde,J;Saxena, R.C.and Schmutterer,H(1984):Neem oil and neem extracts as potential insecticides for control of hemipterous rice pests,Schrifteureiche GTZ161377. 233 University of Ghana http://ugspace.ug.edu.gh (310)Wackett,L.P;Orme-Johnson,W.H,Walsh,CT,(1989);Transition,metal enzymes in bacterial metabolism. In: Beveridge,T.J; Doyle,RJ(EDS),Metal ions and bacteria. Wiley,New York 165-206. (311) Wagner, D.B, (1993): Iron and Steel in Ancient China BRILL, 335-340. (312) Walker,M,(2009):‖Ancient smell of death revealed‖ BBC Earth News Retrieved 13 sept 2009. (313)Walsh,A, (1955): The application of atomic absorption spectra to chemical analysis, Spectrochim Acta7, 108-117. (324)Wang Y-T; Shen, H, (1995); Bacterial reduction of hexavalent chromium.J Ind Microbiol 14, 159-163. (325) Wang, L.L.and Johnson E. A, (1992): Inhibition of Listeria Monocytogenes by fatty acids and monoglycerides Applied and Environmental Microbiol58, 624 – 629. (326) Ward, G, (2012): The Rough Guide to the Titanic, London Rough Guides Ltd p 171. (327) Ware,M,(1999):‖An introduction in monochrome‖ Cyanotype the history, science and art of photographic printing in Prussian blue NIMSI Trading Ltd, pp 11-18. (328) Weckhuysen,B.M;Schoonheydt,R.A,(1999):‖Olefin Polymerization over Supported Chromium Oxide Catalysts‖ Catalysis Today 51, (2) 215-221. (329) Welz,B; Sperting, M, (1999): Atomic Absorption Spectrometry. Wiley-VCH, Weinheim, Germany. 234 University of Ghana http://ugspace.ug.edu.gh (330)Whitney,E;Sharon.R.R,(2011):Peggy,Williams,ed.Understanding Nutrition(Twelfith ed), Clifornia Wadsworth,Cengage Learning. (331)Wilkes,C.E;Sumers,J.W;Daniels,C.A;Benard,M.T,(2005):PVC,handbook,Munchen Hanser,p 106. (332)Wolfgang,L; Michael;T; Erich,S; Karl;W.W(2006):‘Nitrates and Nitrites‘ in Ulmann‘s Encyclopedia of Imdustrial Chemistry Wiley-VCH, Weinheim.Wolzogen KÜhr CAH von;Vlugt LS van der,(1934);Graphitization of cast iron as an electro-biochemical process in anaerobic soils.Water 18:147-165. (333) Wong, P.T.S; Chau, Y.K; Luxon, P.L, (1975): Methylation of lead in the environment. Nature253: 263-264. (334) Woodbridge, P, R, (1991):―Principles of Paint Formulation‖ UnK. ISBN 0412029510. (335) Woodruff, J G, (1970): Coconuts: Production, Processing, Products The Avi Publishing Co. Inc. (336) Yehuda; S; Rabinovtz, S; Mostolsky; D.I, (2005): ―Mixture of essential fatty acids lowers test anxiety‖. Nutritional Neuroscience 8(4)265-267. (337)Young, F.V.K, (1983): Palm Kernel and Coconut Oil: Analytical Characteristics, Process Technology and Uses. J.Am.Oil Chem.Soc.60(2), 374-379. (338) Zhu,J.K,(2001):‖Plant Salt Tolerance Trends in Plant Sciences‖ 6(2) 66-71. 235 University of Ghana http://ugspace.ug.edu.gh (339) Zing-g; Azzi,A,(2004): ―Non-antioxidantal activities of vitamin E‖ Current medicinal chemistry.11(9), 1113-33. 236 University of Ghana http://ugspace.ug.edu.gh APPENDIX 237 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 42: Monthly average values of rainfall and accumulated total in the Niger Delta(1937=1997);from IITA,Nigeria. (Adejuwon,2012) No. Accumul Statio of Ja Fe Mar Apr Ma Jun Jul Au Sep No De ated total Oct n yea n b ch il y e y g t v c Ons En rs et d Sapel 20 44 102. 185 216 328 441 282 395 268 79. 23. 64. 23. 67 e .6 .0 6 .4 .7 .7 .9 .7 .1 .4 6 2 7 2 25 225 263 368 474 343 450 323 104 39. 26. 39. Warri 67 58 131 .2 .2 .1 .9 .5 .1 .0 .3 .7 8 2 8 Forca 42 42 137. 191 320 551 540 401 123 58. 84. 42. 67 691 341 dos .2 .8 2 .4 .6 .7 .6 .5 .5 8 5 2 Yena 42 69 167. 263 310 375 424 444 562 356 143 51. 42. 42. 37 goa .7 .1 6 .9 .2 .9 .8 .6 .3 .5 .1 1 7 7 Ahoa 25 57 129. 207 259 291 322 277 350 275 133 40. 25. 40. 67 da .6 .6 6 .1 .5 .9 .4 .5 .5 .1 .9 3 7 3 Port 31 60 168. 177 213 270 393 352 367 264 76. 19. 31. 19. Harco 25 .1 .4 1 .8 .0 .5 .0 .5 .1 .4 2 3 3 3 urt Dege 29 61 135. 183 243 290 341 29. 377 112 29. 67 261 35 35 ma .3 .1 7 .3 .3 .1 .2 5 .5 .6 3 25 47 144. 157 271 305 361 388 335 250 123 26. 72. 26. Onne 21 .4 .5 6 .7 .6 .2 .6 .6 .2 .9 .3 2 9 2 Opob 47 81 139. 238 354 534 697 483 511 437 192 49. 47. 49. 67 o .3 .8 8 .8 .6 .6 .4 .3 .2 .4 .1 .4 3 4 239 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 43: Summary of operating conditions for AAS machine (APHA 3111B) Cd 8.00 228.80 0.50 Normal Air - 1.20 10.0 acetylene Co 15.00 240.70 0.20 Normal Air - 1.20 10.0 acetylene Cr 15.00 357.90 0.30 Normal Air - 1.60 10.0 acetylene Cu 15.00 324.7 0.50 Normal Air - 1.00 10.0 acetylene Pb 8.00 217.0 1.00 Normal Air - 1.10 10.0 acetylene Zn 10.00 213.9 1.00 Normal Air - 1.10 10.0 acetylene Ni 15.00 232.0 0.20 Normal Air - 1.00 10.0 acetylene Fe 15.00 248.30 0.20 Normal Air - 1.20 10.0 acetylene Mn 15.00 279.50 0.20 Normal Air - 1.00 10.0 acetylene 241 Metal Lamp current (mA) Wavelength (nm) Slit width (nm) Silt Height Flame type Fuel Flow(L/MIN) Air Flow(L/min) University of Ghana http://ugspace.ug.edu.gh V 18.00 310.30 0.20 Normal N2O- 6.00 10.0 acetylene Ba 15.00 55.36 0.50 Normal N2O- 6.00 10.0 acetylene Table 44: Causes of Interference and possible Suppressants Metal Chemical interference Ionization interference Suppressant Cd Ca concentrations Metal ionizes in very high 5ml lanthanum solution above 100mg/1 flame (fuel rich) to 100ml standard and sample before aspirating. Use background correction. Mn Mg concentrations above Metal ionizes in very high 25ml Ca solution to 100mg/l, Sio2 above 100mg/l flame (fuel rich) 100ml standards and samples before aspiring Use background correction. Cu - - Use background correction. Ni - - Pb - - 242 University of Ghana http://ugspace.ug.edu.gh Zn - - Cr Fe, Ni and Co concentrations‘ High flame (fuel rich) 5ml 10% lanthanum of 100mg/l and Mg chloride to 100ml concentrations of 30mg/l sample and standard depress absorption before aspirating Use background correction Fe - - Use background correction Ba High dissolved salts, matrix High flame (fuel rich) 5ml HCl 10ml potassium type solution, 7.5ml calcium solution to standard and sample before aspirating. V High dissolved salts, matrix High flame (rich) 2ml Al(NO3)3 9H2O to type 100ml sample and standard b/4 aspiring 243 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh Table 45:Guide to the Preparation of Intermediate Standard Solution Metal Concentration Volume of stock Volume of Volume of Concentration of Of stock Standard concentration solution intermediate standard Solution(m1) acid added (ml) made up to standard solution solution (mg/1) (ml) (mg/1) C d, 1000 1.0 1ml HNO3 100 10.0 Zn Mn 1000 10.0 1ml HNO3 100 100 Cu 1000 10.0 1ml HNO3 100 100 Fe, 1000 10.0 1ml HNO3 100 100 Cr, Pb 1000 10.0 1ml HNO3 100 100 Ba 1000 10.0 5ml HNC 100 100 245 University of Ghana http://ugspace.ug.edu.gh V 1000 10.0 1ml HNO3 100 100 Ni 1000 10.0 1ml HNO3 100 100 Co 1000 10.0 1ml HNO3 100 100 Table 46:Guide to Preparation of Working Standards (PerkinElmer AA200) Metal Concentration of Volume of Volume of Concentration intermediate standard intermediate solution made up of work standard solution (mg/1) standard solution to (mg/1) solution (mg/1) (mg/1) Cd 10.0 2.5 100 0.25 Zn 5.0 0.50 10.0 1.00 20.0 2.00 Mn 10.0 1.0 100 1.0 3.0 3.0 5.0 5.0 Cu 10.0 2.0 100 2.0 4.0 4.0 6.0 6.0 246 University of Ghana http://ugspace.ug.edu.gh Fe,Cr,Ni 10.0 2.0 100 2.0 Co 4.0 4.0 6.0 6.0 Pb 10.0 1.0 100 1.0 2.0 2.0 5.0100 5.0 Ba 10.0 5.0 100 10.0 10.0 20.0 15.0 30.0 V 10.0 10.0 100.0 10.0 20.0 20.0 30.0 30.0 Table 47:Guide to Preparation of Q.C. Standards (GBC AVANTA AAS) Metal Concentrations of Volume of Volume of Concentration of intermediate Q.C solution intermediate Q.C solution made up Q.C standard (mg/1) solution (mg/1) to (mg1) solution (mg/1) Cd, Zn 10.0 5.00 100 0.50 Mn 100 4.00 100 4.00 Cu, Fe 100 5.00 100 5.00 247 University of Ghana http://ugspace.ug.edu.gh Cr, Ni 100 3.00 100 3.00 Pb 100 3.00 100 3.00 Ba 100 3.00 100 15.0 V 10 15.0 100 15.0 Co 100 15.0 100 5.00 Table 48: % Yield Values of the Seed Oils: OIL %YIELD Coconut 40 Palm Kernel 38 Jatropha 45 Rubber 42 Neem 5 Soya bean 2 Figure 51: An old degraded emulsion painting 248 University of Ghana http://ugspace.ug.edu.gh Figure 52: Concentration of PAHs in the paints,and sites 1,2 and 3. 249 University of Ghana http://ugspace.ug.edu.gh Figure 54:Concentration of TPS in undegraded wall(Lichen) Figure 55: Concentrations of Polycyclic Aromatic Hydrocarbons in An Ungraded Wall (Site 1) 250 University of Ghana http://ugspace.ug.edu.gh Figure 56: CONCENTRATION OF TOTAL PETROLEUM HYDROCARBONS IN AN UNDEGRADED WALL (SITE 3) 251 University of Ghana http://ugspace.ug.edu.gh Figure 45: JATROPHA SEED OIL CHROMATOGRAM 252 University of Ghana http://ugspace.ug.edu.gh Figure 46: RUBBER SEED OIL CHROMATOGRAM Figure 47: NEEM SEED OIL CHROMATOGRAM Figure 48: CHROMATOGRAM FOR FATTY ACID IN NATIVE COCONUT OIL SAMPLE 253 University of Ghana http://ugspace.ug.edu.gh Component Retention Area External Area% Solvent 0.946 3452.52 0.0000 84.4210 C6 0.410 321.134 0.0000 0.2210 C8 0.452 336.521 0.0000 5.3210 C10 0.328 946.523 0.0000 2.5561 C12 0.863 109.059 0.0000 1.3521 C14 0.655 321.011 0.0000 1.8631 C16 0.531 586.321 0.0000 1.8421 C18 0.104 869.911 0.0000 1.3697 C18 0.086 763.421 0.0000 1.0539 Figure 49 CHROMATOGRAM FOR FATTY ACID IN AGRIC COCONUT OIL SAMPLE Component Retention Area External Area% Solvent 0.546 4452.52 0.0000 83.4210 C6 0.110 351.134 0.0000 1.2810 C8 0.472 736.521 0.0000 4.1210 C10 0.128 146.523 0.0000 3.5661 C12 0.893 179.059 0.0000 1.3521 C14 0.655 421.011 0.0000 2.8031 C16 0.231 186.321 0.0000 1.2421 254 University of Ghana http://ugspace.ug.edu.gh C18 0.194 879.911 0.0000 1.3678 C18 0.286 963.421 0.0000 1.0339 Component Retention Area External Area% Solvent 0.936 6252.52 0.0000 86.3210 VA 0.210 428.134 0.0000 5.2455 VE 0.672 126.521 0.0000 4.3210 VK 0.421 746.523 0.0000 2.5561 CHOLINE 0.661 199.050 0.0000 0.3621 THIAMIN 0.350 321.011 0.0000 1.1943 255 University of Ghana http://ugspace.ug.edu.gh Figure 50: CHROMATOGRAM FOR VITAMINS IN AGRIC COCONUT OIL SAMPLE 256