UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES SPECIATION OF INORGANIC ANTIMONY IN POLYETHYLENE TEREPHTHALATE (PET) BOTTLED WATER USING HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROPHOTOMETRY (HG-AAS) MARKWO ALI DEPARTMENT OF CHEMISTRY JULY 2015 University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES SPECIATION OF INORGANIC ANTIMONY IN POLYETHYLENE TEREPHTHALATE (PET) BOTTLED WATER USING HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROPHOTOMETRY (HG-AAS) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON BY MARKWO ALI (10274409) IN PARTIAL FULFILLMENT FOR THE AWARD OF DEGREE OF MASTER OF PHILOSOPHY IN CHEMISTRY DEPARTMENT OF CHEMISTRY JULY 2015 University of Ghana http://ugspace.ug.edu.gh i DECLARATION This is to certify that, this thesis is the outcome of a research undertaken by Markwo Ali towards the award of MPhil chemistry degree in the Department of Chemistry, University of Ghana and has neither in part nor in whole been presented for another degree elsewhere. Markwo Ali ……………………. ……………………… (Student) Signature Date Professor Derick Carboo ……………………… …………………….. (Principal Supervisor) Signature Date Professor Robert Kingsford-Adaboh ……………………... ………………........... (Co-Supervisor) Signature Date University of Ghana http://ugspace.ug.edu.gh ii DEDICATION To the Almighty God and to the Ali Kansake family, Buipe, Central Gonja District. …… M. Ali University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS I thank the Lord almighty for the wisdom and strength he has bestowed on me. I bless his name all my lifetime. I am extremely grateful to my supervisor, Professor Derick Carboo for his patience, guidance and invaluable contributions to make this work a success. My sincere thanks to Dr. Walter Affo for seeing the potential in me and giving me the chance to soar further in education. Without your guidance and advice, this MPhil degree will not have started in the first place. You are an inspiration to me. God richly bless you. To my Co-Supervisor, Professor Robert Kingsford-Adaboh, I say thank you for your time and resources to make this labour an achievement. Gratitude to my family for their support in all my ways, especially my elderly sister, Mrs Yakubu Joyce. Her advice, prayers and financial assistance throughout my education has been wonderful. Sister, I say God almighty richly bless you and the seed you have sown in me, you will surely reap. Many thanks to my fiancée, Ms. Sandra Ahadjie for your support and prayers throughout the working period. Sunny! I love you, and may God almighty, bless you all the days of your life. I will also thank Mr. Ofori and Mr Michael Doleku of the Water Research Institute (Council for Scientific and Industrial Research (CSIR)) and Mr. Essien of the chemistry department for their support in measurements and chemicals plus glassware respectively. My appreciation to Mr. Afful of the chemistry department (Ghana Atomic Energy Commission (GAEC)) for helping me with the determination of the physicochemical properties of the bottled water samples. Gratitude to the Foods and Drugs Authority (FDA) for providing the ultrapure milli-Q water used in the study. University of Ghana http://ugspace.ug.edu.gh iv Finally, I am grateful to all my friends and MPhil mates who have been with me and supported me to the end. Mr. Richmond Darko, Mr. Edem Dinku, Mr. Justice F. Awuku, Mr. Majeed S. Bakari and Mr. Gideon Akolgo, God richly bless you all. University of Ghana http://ugspace.ug.edu.gh v ABSTRACT Antimony (Sb) is a regulated drinking water contaminant that has been found to leach from polyethylene terephthalate (PET) plastic containers into the waters stored in them. The common inorganic species of antimony in water are Sb(III) and Sb(V), with the former being more toxic and the latter being more soluble. In order to assess the extent to which waters stored in PET bottles are contaminated with inorganic Sb and to further examine the effect of typical storage conditions on migration rates, speciation analysis of inorganic Sb using hydride generation atomic absorption spectrophotometry (HG-AAS) was undertaken on selected PET plastic bottled waters marketed in the Greater Accra Region of Ghana. Six brands of PET plastic bottled waters were obtained at source on the day of packaging, and analyses undertaken on samples of the waters stored in the plastic containers at intervals of four weeks for twelve weeks, under three carefully chosen storage conditions distinctive of bottled water usage. Selected physicochemical properties of samples of the waters stored in the plastic containers and total Sb of samples of the plastic containers were also determined to discover the effect of some physical properties and certain major ions, and the influence of the different quality PET plastic types on Sb migration respectively. The study revealed amounts of total Sb in the PET plastic containers of the 6 brands ranging from 123.46 mg/kg to 146.45 mg/kg. The selected physicochemical properties of the waters stored in the PET plastic containers considered were pH (6.78 – 7.43), Ca2+ (1.61 – 12.39 mg/L), Mg2+ (1.00 – 4.96 mg/L), HCO3 − (6.18 – 55.41 mg/L) and TDS (8.70 – 70.40 mg/L)). PET bottled waters of 5 out of the 6 brands contained Sb (initial total Sb ranging from 1.11 – 14.65 µg/L) before storage. Total Sb concentrations of the waters stored in the plastic containers were observed to increase with storage time under all the three storage conditions for all the brands of PET plastic bottled waters. Sb(III) and Sb(V) of the waters stored in the University of Ghana http://ugspace.ug.edu.gh vi plastic containers were observed to increase with storage time, with the latter in higher amounts in solution under all the three storage conditions. Waters stored in plastic containers exposed to harsh conditions of the weather like high air temperatures (average temperature for 12 weeks: 23.0ºC in the morning and 39.5ºC in the afternoon) and sunlight outdoors registered the highest Sb concentrations. In conclusion, PET bottled waters marketed in the Greater Accra region were not found to be contaminated with Sb leaching from their packaging PET plastic containers within the one year limited time of expiry using WHO MCL. Notwithstanding that, harsh weather conditions outdoors is most likely to contribute to the rapid migration of the metalloid (Sb) from the plastic containers, making the bottled water unwholesome before its due expiry time is reached. University of Ghana http://ugspace.ug.edu.gh vii TABLE OF CONTENTS DECLARATION ............................................................................................................... i DEDICATION .................................................................................................................. ii ACKNOWLEDGEMENTS .............................................................................................. iii ABSTRACT ..................................................................................................................... v TABLE OF CONTENTS ................................................................................................ vii LIST OF TABLES ........................................................................................................... xi LIST OF FIGURES......................................................................................................... xii LIST OF ABBREVIATIONS ......................................................................................... xiv CHAPTER ONE 1.0 GENERAL INTRODUCTION............................................................................. 1 1.1 Background............................................................................................................. 1 1.2 Problem statement ................................................................................................... 3 1.3 Justification............................................................................................................. 5 1.4 Hypotheses ............................................................................................................. 6 1.5 Objectives of the research ....................................................................................... 7 1.5.1 Aim ................................................................................................................. 7 1.5.2 Specific objectives ........................................................................................... 7 CHAPTER TWO 2.0 LITERATURE REVIEW ..................................................................................... 8 2.1 Antimony occurrence and applications .................................................................... 8 University of Ghana http://ugspace.ug.edu.gh viii 2.2 Antimony species in speciation analysis ................................................................ 10 2.3 Synthesis of polyethylene terephthalate (PET) ...................................................... 11 2.4 Effects of antimony toxicity .................................................................................. 12 2.5 Antimony migration from PET bottles .................................................................. 14 2.5.1 Effect of sunlight and temperature on antimony migration ............................. 14 2.5.2 Effect of storage condition and duration on antimony migration ..................... 15 2.5.3 Effect of physicochemical properties of water on antimony migration ............ 16 2.5.4 Effect of different plastic types on antimony migration .................................. 17 2.6 Analytical method employed in antimony speciation ............................................. 18 CHAPTER THREE 3.0 MATERIALS AND METHODS ........................................................................ 21 3.1 Introduction .......................................................................................................... 21 3.2 Sample selection and collection procedure ............................................................ 21 3.3 Reagents and standards ......................................................................................... 24 3.4 Instrumentation ..................................................................................................... 25 3.5 Sample preparation ............................................................................................... 26 3.6 Containers and cleaning process ............................................................................ 26 3.7 Migration experiment ............................................................................................ 26 3.8 Analysis of water samples ..................................................................................... 27 3.8.1 Physicochemical properties of the water samples ........................................... 27 3.8.2 Total antimony determination ........................................................................ 28 University of Ghana http://ugspace.ug.edu.gh ix 3.8.3 Antimony (III) determination ......................................................................... 29 3.8.4 Antimony (V) determination .......................................................................... 29 3.9 Analysis of PET plastic containers ........................................................................ 29 3.10 Data analysis ..................................................................................................... 30 CHAPTER FOUR 4.0 RESULTS ............................................................................................................ 32 4.1 Introduction .......................................................................................................... 32 4.2 Validation of analytical procedures used in inorganic antimony determination ...... 33 4.3 Relationship between physicochemical properties and inorganic antimony species 34 4.3.1 pH versus antimony species ........................................................................... 34 4.3.2 Bicarbonate content versus antimony species ................................................. 35 4.3.3 Calcium content versus antimony species ....................................................... 36 4.3.4 Magnesium content versus antimony species.................................................. 36 4.3.5 Total dissolved solids content versus antimony species .................................. 37 4.4 Effect of different plastics on antimony migration ................................................. 37 4.5 Effect of storage conditions on inorganic antimony species ................................... 38 4.5.1 Effect of storage conditions on total antimony ................................................ 38 4.5.2 Effect of storage conditions on antimony (III) species .................................... 50 4.5.3 Effect of storage conditions antimony (V) species .......................................... 53 4.6 Effect of storage time on total antimony concentrations ........................................ 57 University of Ghana http://ugspace.ug.edu.gh x CHAPTER FIVE 5.0 DISCUSSION ...................................................................................................... 63 CHAPTER SIX 6.0 CONCLUSIONS AND RECOMMENDATIONS .............................................. 73 6.1 Conclusions .......................................................................................................... 73 6.2 Recommendations ................................................................................................. 75 REFERENCES ............................................................................................................... 76 APPENDICES ................................................................................................................ 87 APPENDIX A ................................................................................................................. 87 APPENDIX B ................................................................................................................. 88 APPENDIX C ................................................................................................................. 90 APPENDIX D ................................................................................................................. 91 APPENDIX E ................................................................................................................. 93 APPENDIX F ................................................................................................................. 96 APPENDIX G ................................................................................................................. 97 APPENDIX H ............................................................................................................... 106 APPENDIX I ................................................................................................................ 115 University of Ghana http://ugspace.ug.edu.gh xi LIST OF TABLES Table 3.2.1: Bottling plant sites in the Greater Accra Region ............................................... 23 Table 3.4.1: Operating conditions of the spectrophotometer ................................................ 25 Table 4.2.1: Quality control methods for the procedures used in inorganic Sb analysis ........ 33 Table 4.3.1: Physicochemical properties of the water samples ............................................. 36 Table 4.5.1: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined on first day and after four weeks in refrigerator ...................................................................... 40 Table 4.5.2: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after eight weeks and twelve weeks in refrigerator ...................................................................... 41 Table 4.5.3: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after four and eight weeks indoors ............................................................................................. 42 Table 4.5.4: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after twelve weeks indoors and four weeks outdoor ............................................................ 43 Table 4.5.5: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after eight and twelve weeks outdoor .......................................................................................... 44 Table 4.5.6: Difference in mean total antimony of water samples under storage conditions . 49 Table 4.5.7: Migration rates of antimony under the three storage conditions ........................ 49 University of Ghana http://ugspace.ug.edu.gh xii LIST OF FIGURES Figure 3.2.1: Geographical map showing the locations of the bottled water companies in Greater Accra. ............................................................................................................ 22 Figure 3.2.2: The six brands of bottled water samples displayed on a clean laboratory bench. .................................................................................................................................. 23 Figure 4.3.1: Selected ionic mineral content of bottled water samples. ................................ 35 Figure 4.4.1: Total antimony concentrations of the plastic material used in making the bottles. .................................................................................................................................. 38 Figure 4.5.1: Total antimony concentrations of bottled water samples stored in a refrigerator for twelve weeks. ....................................................................................................... 45 Figure 4.5.2: Total antimony concentrations of bottled water samples stored indoor for twelve weeks. ........................................................................................................................ 46 Figure 4.5.3: Total antimony concentrations of bottled water samples stored outdoor for twelve weeks. ........................................................................................................................ 47 Figure 4.5.4: Antimony (III) concentrations of bottled water samples stored in a refrigerator for twelve weeks. ....................................................................................................... 50 Figure 4.5.5: Antimony (III) concentrations of bottled water samples stored indoor for twelve weeks. ........................................................................................................................ 51 Figure 4.5.6: Antimony (III) concentrations of bottled water samples stored outdoor for twelve weeks. ........................................................................................................................ 52 Figure 4.5.7: Antimony (V) concentrations of bottled water samples stored in a refrigerator for twelve weeks. ............................................................................................................. 54 Figure 4.5.8: Antimony (V) concentrations of bottled water samples stored indoor for twelve weeks. ........................................................................................................................ 55 University of Ghana http://ugspace.ug.edu.gh xiii Figure 4.5.9: Antimony (V) concentrations of bottled water samples stored outdoor for twelve weeks. ........................................................................................................................ 56 University of Ghana http://ugspace.ug.edu.gh xiv LIST OF ABBREVIATIONS AAS Atomic Absorption Spectrophotometry AFS Atomic Fluorescence Spectrometry CI Confidence Interval CoV Coefficient of Variation CPE Cloud Point Extraction CRM Certified Reference Material CSIR Council for Scientific and Industrial Research EU European Union FDA Foods and Drugs Authority GEMS Global Environment Monitoring System HPLC High Performance Liquid Chromatography IC Ion Chromatography LOD Limit Of Detection LOQ Limit Of Quantification MCL Maximum Contaminant Level PET Polyethylene Terephthalate SPE Solid-Phase Extraction TDS Total Dissolved Solids WEEE Waste Electrical and Electronic Equipment WHO World Health Organisation University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 GENERAL INTRODUCTION 1.1 Background Access to safe drinking water is vital to human health, a basic human right and an element of effective policy for health protection (Graham, 1999). Germs and contaminants such as bacteria, viruses, heavy metals and pesticides when present in water, reduces its quality and as such can cause toxic effects (Fleeger, Carman, & Nisbet, 2003). This has influenced growing concerns, particularly with regards to municipal tap water, as contaminants such as lead (Pb), copper (Cu) and other toxic heavy metals through plumbings and fittings enter the water lowering its wholesomeness, and thus, making bottled water the most preferred and popular choice worldwide (Krachler & Shotyk, 2009; Osei, Newman, Mingle, Ayeh-Kumi, & Kwasi, 2013; Schmid, Kohler, Meierhofer, Luzi, & Wegelin, 2008). For the past fifteen years, global bottled water market has witnessed tremendous annual growth, reaching a total value of about €66 billion in the year 2010. China became the largest market in the consumption of bottled water at 40 million tons in the year 2011. (Y. Y. Fan et al., 2014; Rani, Maheshwari, Garg, & Prasad, 2012). The United Kingdom observed an increase in consumption of bottled water from 1415 to 2275 million litres between 2000 and 2006, spending about £1 billion. (Ward et al., 2009). Italy tops as the greatest annual producer of bottled water, manufacturing about 10 billion litres per year with a consumption rate of 151 litres per capita per year annually (Krachler & Shotyk, 2009). There is, therefore, no doubt that, bottled water in few years to come will replace tap water completely. In Ghana, the majority of the public consume drinking water that is stored in plastic container bottles or ‘sachets’. These bottled- and “sachet” waters are generally perceived to be clean, convenient, healthy and safer compared to tap water (Y. Y. Fan et al., 2014; Grant & University of Ghana http://ugspace.ug.edu.gh 2 Yankson, 2003; Osei et al., 2013; Ward et al., 2009; Westerhoff, Prapaipong, Shock, & Hillaireau, 2008). Therefore, to satisfy domestic Ghanaian need for bottled- and “sachet” water, many manufacturers have proliferated the market, most of whom are suspected not to have permits. This leads to difficulty in estimating the exact number of bottled- and ‘sachet’ water companies in Ghana. Bottled- and “sachet”-water companies to begin production will require three basic things: a water source, systems to purify the water and a packaging material for the finished product ― purified water. Depending on the factory’s location, some companies use water from aquifers and streams. Others depend on rivers, harvested rain or tap water supplied by urban or municipal water companies. Two water works are involved in the supply of pipe borne water to the city of Accra: Weija dam off the river Densu and Kpong water works off the Volta river (Machdar, van der Steen, Raschid-Sally, & Lens, 2013). Waters from these various sources are then passed through purification systems, which are usually in stages, some up to fourteen depending on the type of manufacturing plant. Some of the purification steps involved are activated carbon filtration, reverse osmosis, ultra violet sterilisation and ozonation post carbon filtration (Belaqua.com.gh, 2015). Hence, most of the germs and particulate dirt are removed or reduced to a level that is harmless to the consumer. Nonetheless, most of the purification processes used are not directly involved in the removal of dissolved trace metallic contaminants. After purification, the waters are usually packaged in plastics or in glass containers. The plastic material for drinking water packaging differ from country to country, but the common package material used is polyethylene terephthalate (PET) (Keresztes et al., 2009). PET plastics have become the most preferred packaging material for drinking water and other beverages. This is due to its excellent material properties like unbreakability, good barrier University of Ghana http://ugspace.ug.edu.gh 3 properties towards moisture, high clarity, low migration trends for residuary constituents and very low weight of the bottles compared with glass bottles of the same filling capacity (Y. Y. Fan et al., 2014; Sánchez-Martínez, Pérez-Corona, Cámara, & Madrid, 2013; Welle, 2011; Westerhoff et al., 2008). In Ghana, most of the “sachet”-water companies use polyethylene plastics whilst the bottled water companies use PET plastics in packaging their treated waters. PET is made from the polymerisation of monomers of terephthalic acid and ethylene glycol using antimony-, titanium-, or germanium-based catalysts (Westerhoff et al., 2008). Over ninety percent of globally manufactured PET utilizes antimony-based catalysts, predominantly antimony trioxide (Sb2O3) with beneficial qualities like high catalytic activity, low tendency to catalyse side reactions, creates no colour in the final product and has low-cost price (Carneado et al., 2014; Shotyk et al., 2006; Welle, 2011; Westerhoff et al., 2008). An estimated 150 billion plastic bottles are produced from PET resins annually (Shotyk, Krachler, & Chen, 2006). Commercialised PET resins produced from the use of antimony trioxide catalysts ordinarily have residual antimony concentrations ranging between 150 mg/kg – 300 mg/kg (Carneado, Hernández-Nataren, López-Sánchez, & Sahuquillo, 2015; Hureiki & Mouneimne, 2012; Keresztes et al., 2009). These residual antimony concentrations are very high and raise concerns, considering earlier findings that, antimony can migrate into the waters and beverages stored in PET plastic containers (Carneado et al., 2015; Y. Y. Fan et al., 2014; Keresztes et al., 2009). 1.2 Problem statement Bottled water sales volume, popularity, and consumption have increased considerably in recent times. This raises worries about the quality of the stored water and the packaging container, as previous studies have suggested that, heavy metalloids like antimony leach into juice, drinking water and other beverages packaged with PET plastics over time (Carneado et al., 2015; Y. Y. Fan et al., 2014; Hureiki & Mouneimne, 2012; Keresztes et al., 2009; Tostar, University of Ghana http://ugspace.ug.edu.gh 4 Stenvall, Boldizar, & Foreman, 2013). Critical public health concerns are therefore evoked, as antimony has been established as a regularised drinking water contaminant by monitoring organizations like United States Environmental Protection Agency (US EPA), European Union (EU), and World Health Organization (WHO) (Carneado et al., 2015; Hureiki & Mouneimne, 2012; Keresztes et al., 2009; Sánchez-Martínez et al., 2013; Westerhoff et al., 2008). Antimony is a non-essential element for plants and animals which has no known biological or physiological function and on a long-term exposure has been suspected to be carcinogenic (Roberts & Orisakwe, 2011; Sayago, Beltrán, & Gómez-Ariza, 2000; Shotyk et al., 2006; Tostar et al., 2013; Westerhoff et al., 2008). In cases of acute intoxication, conditions such stomach and muscle aches, diarrhoea, desiccation, shocks, anaemia, and uraemia may arise. These lead to serious myocardial inflammation, shivering, necrosis and finally death (Keresztes et al., 2009). Other detrimental ailments associated with exposure to this metalloid include pneumonitis, fibrosis, bone marrow damage and carcinomas (Sayago et al., 2000). Within the last two decades, scientists have come to the realisation that, total concentrations of elements do not provide the requisite information about mobility, bioavailability and how they affect ecological systems or biological organisms. Knowledge about the chemical forms or species of these elements is paramount to understanding the chemical and biochemical reactions involving these species. Thus, providing information about toxicity, innocuousness or essentiality of the element (Caruso & Montes-Bayon, 2003; Michalke, 2003). In water, the common antimony species reported are antimony (III) and antimony (V), with the former being more soluble as the pentavalent oxo-anion Sb(OH) 6 - under oxic conditions and the latter as Sb(OH) 3 , which is ten times more toxic probably because it has higher reactivity in living systems (Cornelis, Crews, Caruso, & Heumann, 2005; Krupka & University of Ghana http://ugspace.ug.edu.gh 5 Serne, 2002; Okkenhaug et al., 2011; Quiroz et al., 2013; World Health Organization, 2003). Total antimony concentrations only, will therefore not be sufficient in determining toxicity and subsequently contamination. Several environmental factors affect the migration of antimony from the plastic container into the water stored inside. Effects of factors like temperature, sunlight, duration and physicochemical properties amongst others on migration have been studied in other parts of the world using total antimony concentrations (Bach et al., 2013, 2014; Carneado et al., 2015; Hureiki & Mouneimne, 2012; Keresztes et al., 2009; Shotyk et al., 2006; Westerhoff et al., 2008). Climatic conditions or factors in our part of the world are very different and more severe with variations in seasons. Various ways in which consumers store plastic bottled water therefore becomes critical. Furthermore, the fate of the individual species vis-à-vis the effects of the factors studied based on the literature reviewed, has not been explored. Thus, it becomes imperative antimony speciation under typical storage conditions be conducted on PET plastic bottled waters to determine the levels of inorganic antimony species and to ascertain whether migration rates are high leading to contamination. 1.3 Justification Data obtained from antimony speciation in PET plastic bottled waters will be invaluable, as it will inform manufacturers, consumers and regulatory authorities on the extent to which the bottled waters are contaminated and how storage conditions typical of consumers may influence the release of antimony from the residual catalysts in the plastic containers into the waters stored inside. Information on the residual antimony in the plastic containers and migration rates will advise PET plastics manufacturers on the quality of the plastics they are producing. This can University of Ghana http://ugspace.ug.edu.gh 6 be used to decide whether to use PET plastic bottles manufactured using catalyst other than antimony or to switch to other types of plastics entirely. Typical conditions in which consumers store plastic bottled water affect antimony release under prevailing climatic conditions. Findings of this study will advise consumers on proper ways of storing plastic bottled water to avoid getting them contaminated before their due expiry date. Lastly, plastic bottled water manufacturers will be informed on some the factors to take into consideration when specifying the duration period before expiry. This should be done in order to protect the consumer. 1.4 Hypotheses H0: Waters stored in PET plastic containers marketed in Greater Accra Region are contaminated with antimony leaching from the plastic containers within the limited time of expiry. H1: Waters stored in PET plastic containers marketed in Greater Accra Region are not contaminated with antimony leaching from the plastic containers within the limited time of expiry. University of Ghana http://ugspace.ug.edu.gh 7 1.5 Objectives of the research 1.5.1 Aim The aim of this study is to assess the extent to which waters stored in PET plastic containers are contaminated with inorganic antimony. 1.5.2 Specific objectives The specific objectives comprise the following: 1. To determine total inorganic antimony in the PET plastic containers. 2. To determine inorganic antimony species in the source waters (within 12 hours after filling) 3. To determine inorganic antimony species of waters stored in PET plastic containers for twelve weeks (at intervals of four weeks). 4. To determine the impact of typical storage conditions on the migration rate of antimony from the PET plastic containers into the waters stored inside. 5. To establish the relationship between physicochemical properties and inorganic antimony species of the waters stored in PET plastic containers. University of Ghana http://ugspace.ug.edu.gh 8 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Antimony occurrence and applications Antimony (atomic number 51) is universally present in the environment as a result of natural developments and human activities (Filella, Belzile, & Chen, 2002). It belongs to group VA (symbol Sb from the latin word stibium) of the periodic table of elements and according to the Gold-Schmidt classical classification, is a strong chalcophile element that exists predominantly in nature as Sb2S3 1 and Sb2O3 2 (Ferreira et al., 2014; Filella et al., 2002). In the Earth’s crust, antimony and its compounds have a mean crustal abundance of ca 0.20 to 0.3 mg/kg, and are released into the environment via natural discharges like windblown dust, volcanic eruptions, sea sprays, forest fires, and biogenic sources (Filella et al., 2002; Kubota, Kawakami, Sagara, Ookubo, & Okutani, 2001; Sundar & Chakravarty, 2010). Anthropogenic sources of antimony in the environment include fossil fuel combustion, mining and smelting activities, waste incineration of plastic materials and vehicular emissions (Amereih, Meisel, Kahr, & Wegscheider, 2005). The uses of antimony were already known from ancient times as it could dissolve precious metals. This attribute was used in the purification process of gold (Au) from copper (Cu) and silver (Ag) up to the 18th century (Filella et al., 2002). It is very mobile in the environment and as such, has been used as a prospecting guide for ore deposits containing antimony-bearing minerals like gold (Au), silver (Ag), and some other metals (Krupka & Serne, 2002). In elemental form, the metalloid is sturdy, indissoluble in water and forms very hard and technically interesting alloys with tin (Sn), Cu and lead (Pb). Therefore, its presence 1 Stibnite (Antimony trisulphide) – Sometimes known in Geology as antimonite. 2 Valentinite (Antimony trioxide) – Transformation product of Stibnite University of Ghana http://ugspace.ug.edu.gh 9 in metals like Pb greatly improves hardness and mechanical strength. This successively has resulted in its use in batteries, antifriction alloys, type-metal, small arms, tracer bullets, cable sheathing and in semiconductors for making infrared detectors, diodes and Hall-effect devices (Dessuy, Kratzer, Vale, Welz, & Dědina, 2011; Filella et al., 2002; Kubota et al., 2001; Sayago et al., 2000; Sundar & Chakravarty, 2010; World Health Organization, 2003). Various antimony compounds are used in industry for the manufacture of several products. Antimony trioxide (Sb2O3) is utilised in fire retardant preparations for plastics, rubbers, textiles, papers, and adhesives. It is correspondingly used as, a paint pigment (a turbidifier in white enamel paint), ceramic opacifier, catalyst (initiator or additive in the production of polyethylene terephthalate), mordant, and glass decolouriser (Cavallo et al., 2002; dos Santos et al., 2013; Filella et al., 2002; Sayago et al., 2000; Sundar & Chakravarty, 2010; World Health Organization, 2003). Other antimony compounds like Sb4O8 3 and Sb2S3 are also widely known, with the former being used as an oxidation catalyst in the oxidative dehydrogenation of olefins and the latter in break linings, vulcanisation process of rubber, production of explosives, antimony salts and ruby glass (Filella et al., 2002; Fujiwara, Rebagliati, Marrero, Gómez, & Smichowski, 2011; Sundar & Chakravarty, 2010). The pentavalent antimony compounds (stibosamine, sodium stibogluconate, pentostam, glucantime) are used in the biomedical field as therapeutic agents against parasitic tropical protozoan diseases like leishmaniasis, schistosomiasis, ascariasis, trypanosomiasis and bilharziasis (Filella et al., 2002; Sundar & Chakravarty, 2010; World Health Organization, 2003). Certain compounds of antimony were also formally used to treat syphilis (Filella et al., 2002). 3 Antimony tetra-oxide University of Ghana http://ugspace.ug.edu.gh 10 In conclusion, antimony is ubiquitously present in the environment and has many industrial applications for the production of goods for man’s use. 2.2 Antimony species in speciation analysis The speciation analysis of antimony takes into account two groups of species: inorganic antimonials which are evaluated using their oxidation states (trivalent (III) and pentavalent (V)) and the organic antimonials in environmental and biological matrices (mainly methylated species comprising monomethylated methylstibonic acid [MeSbO(OH)2], dimethylated dimethylstibinic acid [Me2Sb(OH)], monomethylstibine [MeSbH2] and dimethylstibine [Me2SbH]) (Cornelis et al., 2005; Hernández-Nataren, Sahuquillo, Rubio, & López-Sánchez, 2011; Miravet, Hernández-Nataren, Sahuquillo, Rubio, & López-Sánchez, 2010). In the urine of occupationally exposed humans, methylated forms like [(CH3)3SbCl2] have also been reported (Quiroz et al., 2011). Antimony is further observed to associate strongly with soil organic matter, oxyhydroxides of Iron (Fe), Manganese (Mn), Aluminium (Al) and clay minerals. These types of associations have resulted in subjects for several studies as different antimony species can be formed with organic chelating macromolecules (Thanabalasingam & Pickering, 1990; Tserenpil & Liu, 2011; Van Vleek, Amarasiriwardena, & Xing, 2011; Wilson, Lockwood, Ashley, & Tighe, 2010). In some rivers and marine waters, methylated forms have been discovered and usually constitute about 10% or less of the total dissolved antimony (Krupka & Serne, 2002). Based on thermodynamic equilibrium considerations, the inorganic trivalent (III) and pentavalent (V) forms are the stable states under reducing and oxidising conditions respectively. Nonetheless, these two inorganic forms have been found to co-exist in natural aqueous systems (Krupka & Serne, 2002). In water, both species are strongly hydrolysed with antimony (V) existing as Sb(OH) 6 - and antimony (III) as SbO2 - (Serafimovska, Arpadjan, & University of Ghana http://ugspace.ug.edu.gh 11 Stafilov, 2011). Under oxic to slightly reducing conditions (pH ˃ ~ 2.5), the hydrolytic pentavalent oxo anion Sb(OH) 6 - exist. At averagely reducing conditions (pH 2 – 12), the dominant species is Sb(OH) 3 . At pH ˂ 2, the dominant species is Sb(OH) 2 + and at pH ˃ 12, the dominant species is Sb(OH) 4 - (Krupka & Serne, 2002; World Health Organization, 2003). The focus of this study will be on inorganic antimonials because of the major draw back of the lack of standard substances for calibration for organic antimonials (Ferreira et al., 2014). 2.3 Synthesis of polyethylene terephthalate (PET) Polyethylene terephthalate (PET) is a thermoplastic material that is made for a wide variety of applications, particularly food and beverage packaging, including drinking water bottles (Carneado et al., 2015). It is synthesised from the polymerization of the petroleum-derived monomers of terephthalic acid and ethylene glycol using Sb-, Ti-, or Ge-based catalyst. The use of Ti-based catalysts causes the formation of the PET resins to occur at higher temperatures whereas Ge- based catalyst are expensive compared to Sb-based catalyst. This makes the latter the best choice worldwide (over 90% manufactured PET) (Westerhoff et al., 2008). Furthermore, Sb- based catalysts (specifically antimony trioxide (Sb2O3)) are widely used because, they are efficient, present minimal tendency to produce side effects and do not produce undesirable colour in the polymer material (Aharoni, 1998; Duh, 2002). The first industrial step in the synthesis of PET involves a pre-polymerization reaction that generates low-weight oligomers and bis(hydroxyethyl) terephthalate (BHET) as an intermediate compound. A second poly-condensation reaction uses various catalysts such as the Sb-based ones to transform BHET to PET. Below is a scheme, showing the polycondensation reaction process adopted from Habaue et al. (2010). University of Ghana http://ugspace.ug.edu.gh 12 2.4 Effects of antimony toxicity The toxicity of antimony and its compounds raises global health concerns (Amarasiriwardena & Wu, 2011). They are regarded as pollutants of priority by international statutory bodies like the United States Environmental Protection Agency (US EPA), European union (EU) and the World Health Organisation (WHO). This is because, antimony can accumulate in living organisms and stimulate health effects like nausea, vomiting, and diarrhoea. These occur when maximum contaminant levels (MCLs) are surpassed over comparatively short periods (Carneado et al., 2015; Filella et al., 2002; Kubota et al., 2001; Mendil, Bardak, Tuzen, & Soylak, 2013; Miravet, López-Sánchez, Rubio, Smichowski, & Polla, 2007; Sánchez-Martínez et al., 2013; Westerhoff et al., 2008; Yousefi, Shemirani, & Jamali, 2010). Antimony also has biological properties similar to arsenic (As) and as such, may possess some carcinogenic tendencies (Tostar et al., 2013). Therefore, to protect consumers of bottled water and other beverages store in PET plastic containers, these international bodies and governmental organisations provide guideline values derived from various experimental data. The maximum permissible concentration of antimony in drinking water signed by the WHO, EU and US EPA are 20 µg/L, 5 µg/L and 6 µg/L respectively (Carneado et al., 2015; COOCH 2 CH 2 OHn HOH 2 CH 2 COOC BHET Catalyst COOCH 2 CH 2 OOC n + n HOCH 2 CH 2 OH PET University of Ghana http://ugspace.ug.edu.gh 13 Hureiki & Mouneimne, 2012; Sánchez-Martínez et al., 2013; Tostar et al., 2013; Westerhoff et al., 2008; World Health Organization, 2003). There is no guideline value for the maximum allowable concentration of antimony in drinking water in Ghana. Antimony toxicity chiefly comes into existence due to occupational exposure, domestic use or during therapy. Furthermore, the chemical form, oxidation state and water solubility of the species under consideration is a key function of toxicity (Amereih et al., 2005; Hagarová, Kubová, Matúš, & Bujdoš, 2008; Sánchez-Martínez et al., 2013; Sundar & Chakravarty, 2010; World Health Organization, 2003). Antimony concentrations in air ordinarily range from nanogram per cubic meter to approximately 170 ng m-3. In rivers and lakes, dissolved concentrations usually range less than 5 parts of the metalloid in 1 billion parts of water and are mostly found attached to dirt particles (Sundar & Chakravarty, 2010). Stibine (SbH3), a lipophilic gas, when inhaled is the most toxic of the inorganic forms of the metalloid. Some of the diseases associated with inhalation exposure to antimony are pneumonitis, fibrosis, bone-marrow damage and carcinomas (Amereih et al., 2005). The elemental form (Sb0) of the metalloid is more toxic than its inorganic salts. Its inorganic salts are in turn more toxic than the organic forms (Sundar & Chakravarty, 2010; World Health Organization, 2003). The inorganic Sb(III) and Sb(V) states constitute the predominant forms in inorganic environmental matrices with the former being more toxic because it has higher responsiveness in living systems (i.e, it expresses high affinity for red cells and thiol groups of cell components and has physicochemical properties similar to As (III) of known toxicity) (Amereih et al., 2005; Cornelis et al., 2005; Hagarová et al., 2008; Kubota et al., 2001; Quiroz et al., 2013; Sánchez- Martínez et al., 2013). Conditions like optical nerve destruction, uveitis, and retinal bleeding are linked to repeated oral exposure to therapeutic doses of antimony (III) with specific University of Ghana http://ugspace.ug.edu.gh 14 symptoms of intoxication generally followed by coughing, anorexia, troubled sleep and vertigo. Soluble antimony (III) salts have also been found to exert genotoxic effects in vitro and in vivo (World Health Organization, 2003). Thus, antimony can pose serious health risks to humans and as such, must be regulated and monitored frequently in the environment. 2.5 Antimony migration from PET bottles 2.5.1 Effect of sunlight and temperature on antimony migration Few studies have investigated the impact of sunlight and surrounding temperature on the migration of antimony from the PET plastic container into the water stored in it. Bach et al. (2014) discovered that bottled waters on exposure to sunlight for 2, 4 and 10 days had migration dependent on the type of water (carbonated or non-carbonated) and the migration rates for formaldehyde, acetaldehyde and antimony increased. Bach et al. (2013) also recognised that, increase in temperature impelled the release of plastic constituents like antimony, formaldehyde and acetaldehyde. Carneado et al. (2014) further examined the influence of temperature and storage time on antimony migration from PET bottles into the mineral water in a short term and long term. Their study showed that waters stored in the temperature range of 4ºC to 20ºC were not subject to antimony migration whilst, at temperatures above 40ºC and 60ºC, there was a significant increase in migration. Hureiki and Mouneimne, in their study too realised that in outdoor conditions under sunlight at temperatures below 45ºC, there was no significant effect on antimony release from PET bottles into natural water (Hureiki & Mouneimne, 2012). Fan et al. (2014) considered the effect of storage temperature and duration on sixteen brands of PET bottled water. They discovered that, an increase in temperature from 4ºC to 70ºC increased antimony release from the PET plastic container for up to four weeks and then decreased. This indicates that, under long-term storage, University of Ghana http://ugspace.ug.edu.gh 15 release rates may probably stabilise. Westerhoff et al. (2008) investigated how sunlight affects the migration of antimony from PET plastics. Natural sunlight experiments were conducted with controls held at the same temperature, but wrapped in aluminium foils. Samples were exposed to natural sunlight for seven days. From their results, it was realised that the effect of sunlight irradiation on antimony migration was minimal (5 – 10% higher than the control). It was also discovered that, increasing storage temperatures from 22ºC to 80ºC led to higher rates of antimony migration. Thus, exposure of plastic bottled water to sunlight and higher air temperatures tends to accelerate the migration process of antimony from the PET plastic container into the water stored in it. 2.5.2 Effect of storage condition and duration on antimony migration The state in which PET plastic bottled waters are stored over time influences the migration of constituents like antimony from the packaging material into the water stored inside. Carneado et al. (2014) storing bottled waters under conditions similar to refrigeratory and indoor settings (4ºC and 20ºC respectively) observed no antimony migration whilst at high- temperature conditions (above 40ºC) such as in outdoor exposed to high air temperatures and sunlight, significant increase in antimony concentration was witnessed. Hureiki and Mouneimne (2012) assessed eight Lebanese brands of bottled waters in the dark indoors (at 22ºC) and outdoor under sunlight (at a maximum temperature of 45ºC) for antimony migration. It was observed that the migrated antimony concentration of six of the brands increased reaching a concentration of 5.5 µg/L after 544 days of contact time with the PET packaging material. A study by Fan et al. (2014) revealed that storage duration affects the release rates of antimony from the PET plastic container. It was realised that release rates increased for a short University of Ghana http://ugspace.ug.edu.gh 16 period and then stabilise on a long-term. Modelling improper storage conditions by bottled water usage, Keresztes et al. (2009) stored purchased Hungarian mineral water for the duration of 10 to 950 days under various conditions. It was observed that the leaching rate increased with storage time and sparkling waters had higher migration rates than still mineral waters. Thus, longer storage periods are likely to result in higher antimony concentrations in the stored water. 2.5.3 Effect of physicochemical properties of water on antimony migration The presence of various degrees of ions in the stored water can affect the release of antimony from the PET plastic container. A small number of studies have considered the effect of physicochemical properties of the stored water on antimony release. Hureiki and Mouneimne (2012) studied four physicochemical properties (pH, calcium, magnesium, and bicarbonate) on antimony release. It was realised that only calcium had a significant effect on antimony release. Keresztes et al. (2009) in their study realised that carbonated water had higher antimony leaching rates compared to non-carbonated water. This observation was attributed to the lower pH values (4.94 – 5.27) of sparkling mineral water, indicating that lower pH may accelerate the release of antimony from the packaging material. Bach et al. (2013, 2014) discovered that antimony migration was higher in carbonated compared with non-carbonated waters. Carbonated waters have lower pH values by virtue of the dissolution carbon dioxide (CO2) to form carbonic acid. The carbonic acid then dissociates and contributes mores hydrogen ions in solution, thereby lowering the pH. A study by Westerhoff et al. (2007) showed that pH had no effect on antimony migration. They suggested that the typical pH range of 6 to 8 for drinking waters, regardless of the location would not influence antimony migration. University of Ghana http://ugspace.ug.edu.gh 17 In effect, lower pH (< 6) and few ions like calcium are likely to affect the release of antimony from the PET plastic container. 2.5.4 Effect of different plastic types on antimony migration The quality of the PET plastic containers used in packaging differs from country to country. Information regarding the effect of the different types of plastics on antimony migration is scanty. Nonetheless, few workers as part of their studies have tried to assess the effect of the different plastic types on antimony release or migration. Westerhoff et al. (2007) examined antimony migration between two types of PET plastic bottles (based on the colour of packaging material – clear and blue). Samples were incubated at 80ºC over a ten-day period. The results indicated that clear PET plastics released about four times more antimony than blue-coloured PET plastics. In another study by Carneado et al. (2014) based on colour and the residual antimony content (191 – 268 mg/kg) in the PET plastic containers, dark blue plastics contained slightly higher antimony levels than clear and light blue plastics. It was realised that leaching of antimony (III) into water samples stored in dark blue, clear and light blue bottles at 60ºC for 50 – 220 days had waters held in dark blue bottles having higher antimony levels in the stored water than clear and light blue bottles. The concentrations of stored waters (in dark blue plastics) increased from 5.61 at day 50, peaked at 7.12 at day 78 and decreased to 3.34 at day 220. Fan et al. (2014) determine total antimony in sixteen brands of PET drinking bottled waters in China. Comparing the maximum antimony concentrations in the stored waters if all the antimony in PET plastic containers were to be released into the stored waters showed that, the higher the total antimony in the PET packaging material, the higher the maximum antimony concentration released into the stored water. In summary, based on the literature reviewed, the colour and the residual amount of antimony in the plastic material tends to affect the levels of antimony in the stored water. University of Ghana http://ugspace.ug.edu.gh 18 2.6 Analytical method employed in antimony speciation Elemental speciation is outlined ‘…as the analyses that lead to determining the distribution of an element’s particular chemical species in a sample’ (Caruso & Montes- Bayon, 2003). Antimony speciation in plastic bottled water has been the topic of focus for the past decades. Nonetheless, few published works have been centred on identification, quantification and the fate of the species in the stored water and in the PET plastic container (Carneado et al., 2015). Furthermore, the extremely low concentrations of the inorganic forms (Sb(III) and Sb(V)) in aqueous environments makes direct speciation more difficult (Z. Fan, 2005; Hagarová et al., 2008). To understand the fate of antimony in the aquatic environment, low detection limits are usually required. This is accomplished by using speciation analysis methods which generally comprise a selective separation or pre-concentration technique and a sensitive detection method capable of determining analyte concentrations naturally in the range of 1 – 300 ng/L (Amereih et al., 2005; Apte & Howard, 1986; Hagarová et al., 2008; Krachler & Emons, 2001). Some of the analytical methods employed are: high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP MS), HPLC coupled online to hydride generation AAS or to ICP-MS, ion chromatography (IC) coupled to hydride generation inductively coupled plasma optical emission spectrometry (HG-ICP OES), cloud point extraction (CPE)/ atomic absorption spectrophotometry (AAS) and hydride generation coupled to atomic fluorescence spectrometry (AFS). Other techniques applied are anodic striping voltammetry, cathodic stripping voltammetry, X-ray fluorescence, microwave induced plasma atomic emission spectrometry (Ferreira et al., 2014; Krachler & Emons, 2001). Most of these procedures are largely complicated, time-consuming and bear high operation costs. Hence, the need for a simple, express, efficient and cost effective analytical method. University of Ghana http://ugspace.ug.edu.gh 19 Hydride generation atomic absorption spectrophotometry (HG-AAS) is the analytical technique frequently used in the determination of trace amounts of antimony typically in the µg/L concentration range (Apte & Howard, 1986; Ferreira et al., 2014). The hydride generation method has the benefit of being simple, rapid, is comparatively free from interference and by careful control of the reduction process, information of analyte oxidation state may be reached (Apte & Howard, 1986). The hydride generation process basically involves reacting antimony compounds with tetrahydroborate (III) in acidic medium to produce stibine (SbH3). Antimony (V) cannot be completely reduced to stibine with tetrahydroborate (III) because it demonstrates poor sensitivity compared to antimony (III). Thus, to achieve total amounts of the metalloid, antimony (V) should be pre-reduced to antimony (III) first and then further reduced with the tetrahydroborate to generate the stibine. The reaction chemical equations adopted from Feng et al. (1999) are presented below: 4H3SbO4 + BH4 − +H+→ 4H3SbO3 + H3BO3 + H2O (Slow) H3SbO3+ 3BH4 − + 3H+ → 4SbH3 + 3H3BO3 + H2O (Fast) The electrochemical equation of the reduction of antimony (V) to antimony (III) in acidic medium shows an important feature. SbO4 3- + 2H+ + 2e- ⇌ SbO3 3- + H2O ESb(+5)/Sb(+3) o = + 0.69 volts. The reduction process is highly favourable in acidic medium (Ferreira et al., 2014). However, not all acids will favour this reduction process. The choice of the acid, therefore, becomes critical in speciation analysis of antimony, as acids like oxidising ones would not be suitable for the reduction process to proceed. Hydrochloric acid is the principal acidifying agent routinely used for extracting antimony in aqueous matrices (Ferreira et al., 2014). Also from the reaction, the use of a reducing agent can be supportive in the reduction process. A variety of reducing agents have been utilised, but the most favoured is L-cysteine, as its solutions are University of Ghana http://ugspace.ug.edu.gh 20 usually more stable compared to other reducing agents (Z. Fan, 2005, 2007; Ferreira et al., 2014). When in solution with antimony (V), L-cysteine reduces it to antimony (III) as follows: 2R–SH + H3SbO4 → R–S–S–R + H3SbO3 + H2O (Slow) (Feng, Narasaki, Chen, & Tian, 1999) The antimony (III) is further reduced to stibine using tetrahydroborate (III) in acidic medium. A neutral inert gas then carries it for atomisation (by AAS or AFS) or excitation (using ICP or MIP). AAS is a technique in which free gaseous atoms absorbs light of characteristic wavelength (monochromatic radiation) in a flame or furnace resulting in the quantification of the atoms. To measure the concentration of an element in the sample, a liquid sample of the element is first aspirated into a nebulizer system. The fine mist produced by the nebulizer system is mixed with an oxidant gas that is drawn under pressure, thus producing aerosols. These aerosols are then carried into a flame that is commonly produced by a mixture of air and acetylene (2400ºC) or nitrous oxide and acetylene (2800ºC). In the flame, the solvated aerosols lose their solvent (desolvation) and the gaseous metallic element undergoes excitation. At the same, a light beam from a lamp (usually a hollow cathode lamp whose cathode is made of the element being determined) is passed through the flame. A photomultiplier tube is then used to detect the reduction of light intensity due to absorption by the atoms of the analyte in the flame. Using the Beer-Lambert law, the absorbance is proportional to the concentration of the metal ions (Skoog, Holler, & Nieman, 1998). In summary, inorganic speciation of antimony in the water samples can be undertaken by carefully controlling the reduction processes to determine total antimony and then antimony (III). Antimony (V) is the obtained from the difference between total antimony and antimony (III). University of Ghana http://ugspace.ug.edu.gh 21 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Introduction This chapter describes the sample selection and collection procedure, sample preparation, sample treatment and analyses carried out to speciate inorganic antimony in waters contained in PET plastic bottles stored under three distinct conditions. 3.2 Sample selection and collection procedure PET plastic bottled water samples of six popular brands (shown in Figure 3.2.2) in Greater Accra were selected and purchased at source (bottling plant locations are shown in Table 3.2.1 and geographically mapped out in Figure 3.2.1). Samples were obtained on the day of bottling in the month of January 2015. For each brand of plastic bottled water, forty samples were required. Thus for the six brands, two-hundred and forty bottled water samples were procured. All samples of a particular brand originate from the same batch of bottled waters bottled at that time of the day. All brands of bottled waters had a transparent layer of PET material used in packaging and the waters stored in them had different degrees of mineral composition. The container capacity (volume) chosen was 0.5 L for all the six brands of plastic bottled water samples. Bottled water samples were not of the same bottling date but were all packaged in the period of two weeks in the month of January 2015. All bottled water samples remained sealed in their original containers until analysis and all antimony analysis (total antimony and antimony (III)) for each brand (water samples and PET plastic containers) were carried out in quadruplicate (n = 4). Thus, the obtained results will correspond to the mean of four independent measurements for both water samples and plastic material originating from bottled water samples of the same batch. University of Ghana http://ugspace.ug.edu.gh 22 Figure 3.2.1: Geographical map showing the locations of the bottled water companies in Greater Accra. University of Ghana http://ugspace.ug.edu.gh 23 Table 3.2.1: Bottling plant sites in the Greater Accra Region Figure 3.2.2: The six brands of bottled water samples displayed on a clean laboratory bench. From left, bottled water coded names are VOL, AQF, VER, ICP, BQA, and SPI respectively. Bottled water establishment Location (Off) Latitude Longitude Voltic Ghana Limited Nsawam Road 5º45’29.81”N 0º19’40.40”W Special Ice water facility Oyarifa Road 5º46’23.47”N 0º10’49.75”W Bel-Aqua Ghana Limited Tema heavy industrial Area 5º43’4.71”N 0º1’5.72”E Verna water company Nsawan Road 5º45’3.09”N 0º20’30.04”W Ice Pak water company Adenta-Dodowa Road 5º45’39.55”N 0º8’30.03”W Aqua Fill water company North industrial area 5º35’2.52”N 0º13’25.94”W University of Ghana http://ugspace.ug.edu.gh 24 3.3 Reagents and standards The standards and reagents used in this study were prepared with ultrapure water (8 – 10 MΩ·cm-1 at 24.5ºC) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). All chemicals were analytical-reagent grade unless otherwise stated. The 1 mg/mL stock standard solutions of antimony (III) and antimony (V) were prepared by dissolving 0.274 g of dried (at 105ºC for 2 hours) antimony potassium tartrate hemihydrate (Sigma-Aldrich, 99%) in 0.1 L of 6 M HCl (Springer Shenstone Lichfield, 36% – 38%) and 0.216 g of potassium hexahydroxyantimonate (Sigma-Aldrich, 99%) in 0.1 L of 2.4 M HCl, respectively. Antimony stock solutions were stored in polyethylene plastic bottles in refrigerator at 4ºC and working solutions were prepared daily by dilution. Sodium borohydride solution was prepared from dissolving 0.700 g granular NaBH4 (to a 0.7% (w/v) concentration, KEM Light laboratory purpose reagent, >97%) in 0.4% (w/v) sodium hydroxide (prepared from 0.400 g NaOH pellets, Merck, 99%). In the microwave digestion of the plastic samples, concentrated HNO3 (Panreac, hyper- pure reagent grade, 69%) and H2O2 (Merck, 96%) were used. The 1 M citrate solution used was prepared by dissolving 2.940 g sodium citrate dihydrate (≥ 99%, Sigma-Aldrich) in 10 mL ultrapure water. The 1 M L-Cysteine solution used to reduce antimony (V) in total antimony determination was prepared by dissolving 1.212 g of L-Cysteine (97%, Sigma-Aldrich) in 10 mL of HCl (2.4 M). The 0.5 M aqueous sulphuric acid (Sigma-Aldrich, 99.99%) used in the titration determination of bicarbonate ion concentration was prepared by slowly adding 2.81 mL of the stock acid to 25 mL double distilled deionised water. The final volume of the mixture was then adjusted to 100 mL to obtain the required concentration. University of Ghana http://ugspace.ug.edu.gh 25 The 1 M potassium chloride used in the validation of the pH determination procedure was prepared by dissolving 0.750 g of KCl (Sigma-Aldrich, ˃ 99%) in 10 mL of double distilled deionised water. The ionisation suppression agent (1 M lanthanum chloride solution) used in the spectrophotometric determination of magnesium and calcium was prepared by dissolving 18.570 g of LaCl3·7H2O (Sigma-Aldrich, 99.99%) in 50 ml of 1.5 M HCl solution. 3.4 Instrumentation A 240 FS AA spectrophotometer (New York, US) with an antimony hollow cathode lamp (Photron Super Lamp) was used in the determination of trace inorganic antimony, calcium and magnesium. Background absorption was corrected using a deuterium lamp. The operating conditions of the spectrophotometer during the determinations are shown in Table 3.4.1. Table 3.4.1: Operating conditions of the spectrophotometer Element Wavelength, λ (nm) Current (mA) Spectral resolution (nm) Gas Antimony (Sb) 217.6 14 0.7 C2H2/air Calcium (Ca) 422.7 10 0.5 C2H2/N2O Magnesium (Mg) 285.2 4 0.5 C2H2/air Temperature measurements were done using a mercury (Hg) bulb thermometer (US Coater). The Hg-filled thermometer had a precision of ± 0.1ºC. All pH measurements were carried out using an HI 2210 basic Bench top pH meter. Total dissolved solids (TDS) for the bottled water samples were measured using HANNA HI 4321 conductivity/TDS meter. University of Ghana http://ugspace.ug.edu.gh 26 Microwave digestion of the PET plastic container samples was performed using a Milestone Ethos Touch control digester instrument, with a temperature controller and a power of 1000W. For this procedure, eight Teflon pressure vessels were used simultaneously. 3.5 Sample preparation Before analysis, water samples were allowed to stand an hour before treatment starts. This is to enable all the water samples held under the different storage conditions, re-adapt to the same experimental settings before treatment and subsequent analysis. PET plastic containers were cut into approximately 5 × 5 mm2 pieces using a ceramic blade. Each cut-out replicate of the PET plastic container weighed approximately 0.250 g. 3.6 Containers and cleaning process Glassware and polyethylene containers were cleansed by soaking in 1.5 M HCl overnight and rinsing with double distilled de-ionized water. They were then placed in a clean oven overnight at 35ºC. The moisture-free containers were then placed in a clean glass cabinet ready for use. The ceramic blade used in cutting the plastic containers was pre-cleansed with 1.5 M HCl 3.7 Migration experiment To simulate the effect of storage conditions typical of bottled water usage on the migration of antimony from the plastic containers into the waters stored inside, bottled water samples were assigned into three storage groupings, with each group comprising bottled waters from the six different brands. Each group (excluding samples for analysis on the day of acquiring samples – day one) was further divided into three subgroups. The three subgroupings (containing four bottled water samples for each brand) will represent four weeks, eight weeks and twelve weeks under a specified storage condition. One group will be stored in a refrigerator at 4ºC; the second group University of Ghana http://ugspace.ug.edu.gh 27 exposed to high air temperatures and sunlight light outdoor, and the third group in a closed cabinet away from sunlight indoor. The samples were held under these conditions from January to March 2015. During the study, temperatures of the bottled waters kept outside and indoors were monitored at regular intervals using the mercury bulb thermometer and those in the refrigerator maintained at 4ºC. To establish the concentrations of the inorganic antimony species in the source waters, determinations were done on some of the bottled water samples for each brand on the day of acquiring the samples (within 12 hours after filling). The rest of the bottled water samples were then stored according to the group they were assigned into for the rest of the period of study. 3.8 Analysis of water samples 3.8.1 Physicochemical properties of the water samples 3.8.1.1 pH The pH of the water samples of the six brands of bottled waters were taken using the basic Benchtop pH meter on the day the bottled water samples were acquired. The meter was first calibrated with buffers at pH 4.70 and 10.01 at ambient temperature (24ºC). The probe of the meter was then inserted into specific volumes (10 mL) of the water samples and readings taken from the screen. For validation purposes, the pH of 1 M KCl solution was similarly determined (APHA, 1998). 3.8.1.2 Total dissolved solids (TDS) The total dissolved solids of the water samples were measured using the conductivity/TDS meter that was calibrated at ambient temperature (24ºC) using HANNA standards. The probe of the meter was inserted into specific volumes (20 mL) of the water samples. Total dissolved solids readings were then taken from the screen (HANNA, 2012). University of Ghana http://ugspace.ug.edu.gh 28 3.8.1.3 Calcium The calcium content of the water samples was determined spectrophotometrically at 422.7 nm by atomic absorption. Aliquots of the water samples were first mixed with lanthanum chloride (LaCl3) solution and aspirated into nitrous oxide-acetylene flame. The absorbance was then measured and compared to identically-prepared standard and blank solutions from which concentrations were obtained (Environment Canada, 1979a). 3.8.1.4 Magnesium The magnesium concentration was determined by atomic absorption spectrophotometry (AAS) – direct aspiration. Aliquots (10 mL) of water samples were mixed with 5 mL of 1 M lanthanum chloride (LaCl3) solution and aspirated into an air-acetylene reducing flame. The absorbance was measured at 285.2 nm and compared to prepared standards and blank solutions. From the absorbance, concentration is then obtained (Environment Canada, 1979b). 3.8.1.5 Bicarbonate Bicarbonate ion concentration was determined by titration. Aliquots (10 mL) of bottled water samples were titrated with 0.5 M sulphuric acid using phenolphthalein and mixed bromocresol green and methyl red indicators. The endpoints reached was used to evaluate the amount of hydrogen carbonate present in the water samples (APHA, 1967). 3.8.2 Total antimony determination For total inorganic antimony determination, the pH of water samples (10 mL) were adjusted to 2 by adding 10 mL of 8 M HCl, after which 5 mL of 1 M L-cysteine was added to reduce all the antimony (V) to antimony (III). Solutions were then allowed to stand for 15 University of Ghana http://ugspace.ug.edu.gh 29 minutes after which they were place in the auto sampler section of the atomic absorption spectrophotometer for analysis to begin. Prior to the commencement of total antimony analysis, a calibration curve was generated using pre-reduced (by 1 M L-cysteine) working standards of antimony (V) prepared by serial dilutions of the stock antimony (V) standard. Blanks were also prepared and used to correct interferences originating from the matrices of the solutions. A global environment monitoring system (GEMS) water standard reference material having the same matrix as the water samples was equally determine using the same procedure. The result obtained will be used to check the accuracy of the method. 3.8.3 Antimony (III) determination In antimony (III) determination, 1 M citrate solution in HCl is used instead of L-cysteine. Thus, there was no pre-reduction. The rest of the procedure remains the same as in total antimony determination. Calibration was done using working standards obtained by serially diluting the antimony (III) stock standard solution. For quality control purposes, ultrapure water was spiked with 20 µg/L antimony (III) and determined using the same procedure. 3.8.4 Antimony (V) determination Antimony (V) concentrations of the waters stored in the PET plastic containers were evaluated by subtracting antimony (III) concentrations from total antimony concentrations. 3.9 Analysis of PET plastic containers Each cut-out replicate of the PET plastic containers was mixed with 10 mL of concentrated HNO3 and 2 mL of concentrated H2O2 and digested in Teflon closed vessels using University of Ghana http://ugspace.ug.edu.gh 30 the Milestone Ethos Touch control digester instrument. The maximum temperature and pressure was 180ºC and 250 psi respectively for 15 minutes using 50% of the digestion system’s maximum power of 1000 W. After digestion, 5 mL of 1 M L-cysteine was added to the digestates and solutions allowed to stand for 15 minutes to reduce all the antimony (V) to antimony (III). Solutions were finally topped to 25 mL with ultrapure water and analysed for antimony using the atomic absorption spectrophotometer. Calibration of the spectrophotometer was done using working standards of antimony (V) that were prepared from serial dilutions of the stock antimony (V) standard. Concentrated HNO3 and H2O2 were added to the antimony (V) standards and digested as in the determination of the unknown. Antimony (V) in these solutions were then pre-reduced using L-cysteine for the same time as in the unknown. Blanks were also prepared and used to eliminate interferences that may be coming from the matrices of the solutions. For quality control purposes, cut samples of polyethylene plastic containers were spiked with 20 µg/L antimony (V) and determined the same way as in the total antimony determination in the PET container samples. 3.10 Data analysis SPSS (IBM Corporation, 2012) and Excel (Microsoft Corporation, 2012) software tools were used to analyse data obtained in the study. Means, standard deviations, the coefficient of variations and confidence intervals were carried out using Excel whilst, Pearson’s correlations, Paired-Samples T Test, linear regressions, line and bar graphs were generated using SPSS. To determine how total antimony concentrations of the water samples change with time under the typical storage conditions, linear regressions were used. The results will reveal how migration is occurring over time. University of Ghana http://ugspace.ug.edu.gh 31 Pearson’s correlation analysis was used to highlight the relationship between the five selected physicochemical properties (pH, total dissolved solids, calcium, magnesium, and bicarbonate) and antimony content (total antimony, antimony (III) and antimony (V)). University of Ghana http://ugspace.ug.edu.gh 32 CHAPTER FOUR 4.0 RESULTS 4.1 Introduction This chapter presents the results obtained in the speciation analysis of inorganic antimony in selected PET plastic bottled waters marketed in the Greater Accra Region. Quality control methods used in validating the procedures used in the determination of the inorganic species of antimony are presented in Table 4.2.1. The selected physicochemical properties of the waters stored in the PET plastic containers for the six brands of PET bottled waters are presented in Table 4.3.1 and illustrated in Figure 4.3.1. Total inorganic antimony concentrations of the PET plastic containers of the six brands of PET plastic bottled waters are displayed in a table in Appendix F and illustrated in Figure 4.4.1. Inorganic antimony species of the waters stored in PET plastic containers exposed outdoor in the sun at high temperatures (average temperature: 23.0ºC in the morning and 39.5ºC in the afternoon – Appendix D ), indoor in a closed cabinet at room temperature (average temperature: 20.4ºC in the morning and 25.3ºC in the afternoon – Appendix D) and in a refrigerator (at 4ºC) for twelve weeks at intervals of four weeks are presented in Tables 4.5.1 – 4.5.5 and illustrated in Figures 4.5.1 – 4.5.9. The results of correlation analysis depicting the relationships between the selected physicochemical properties and inorganic antimony species of the waters stores in PET plastic containers are presented in Appendix A. University of Ghana http://ugspace.ug.edu.gh 33 Linear regressions showing how total inorganic antimony concentrations of the waters stored in PET plastic containers vary with time under the selected unique storage conditions for the six brands of PET plastic bottled waters are presented in Appendix C. 4.2 Validation of analytical procedures used in inorganic antimony determination Table 4.2.1: Quality control methods for the procedures used in inorganic Sb analysis The certified total antimony content in the GEMS material used in validating the total antimony determination procedure was 100 µg Sb/kg ± 2%. This standard reference material was analysed in quadruplicate (n = 4), in order to establish the precision of the measurement. The quantified total antimony concentration matched the certified value considering the associated uncertainties, achieving a value of 99.49 µg Sb/kg ± 2% (Table 4.2.1). Spiked ultrapure water samples and cut polyethylene container samples in the determination of antimony (III) in the waters stored in the PET plastic containers and total Procedure Reference material Certified amount (µg/kg) Measured amount (µg/kg) Recovery (%) Total Sb determination in water samples GEMS water standard 100 99.49 99.5 Total Sb determination in PET plastic container samples Spiked polyethylene plastic 20 19.98 99.9 Sb(III) determination in water samples Spiked ultrapure water 20 19.97 99.9 University of Ghana http://ugspace.ug.edu.gh 34 antimony in PET plastic containers respectively, had uncertainties within acceptable limits (Appendix B) and recoveries > 95% (Table 4.2.1). 4.3 Relationship between physicochemical properties and inorganic antimony species The results for physicochemical properties of the bottled water samples are presented in Table 4.3.1. The properties considered are pH, bicarbonate ion (HCO3 − ) concentration, calcium ion (Ca2+) concentration, magnesium ion (Mg2+) concentration and total dissolved solids (TDS). These were measured on the day bottled water samples were acquired (day one – week 0). Pearson correlation was used to highlight the relationship between the water physicochemical properties on one hand and total antimony, antimony (III) and antimony (V) concentrations on another hand. Appendix A shows the results for correlation analysis undertaken for each brand of bottled water. 4.3.1 pH versus antimony species From Table 4.3.1, the pH of the six brands of bottled water ranged 6.78 – 7.43 with a mean and median value of 7.12 and 7.11 respectively. The range obtained was similar to those obtained in literature (Hureiki & Mouneimne, 2012; Westerhoff et al., 2008).VOL recorded the highest pH value (7.43) and AQF the least (6.78). The rest of the brands had pH values varying between the figures 6.78 and 7.43. pH statistically registered an insignificant positive correlation (P = 0.085, 0.091 > 0.05, 0.01) to total antimony and antimony (V) (Appendix A). This suggests that pH does not statistically affect total antimony and antimony (V) at the 0.05 and 0.01 significance level. On another hand, pH significantly correlated antimony (III) positively (P = 0.046 < 0.05). This suggests that pH statistically affects antimony (III) at 0.05 level. University of Ghana http://ugspace.ug.edu.gh 35 Figure 4.3.1: Selected ionic mineral content of bottled water samples. Bars represent mean concentration values (n = 4). 4.3.2 Bicarbonate content versus antimony species The bicarbonate ion (HCO3 − ) concentrations of the waters stored in the PET containers are shown in Table 4.3.1 and in Figure 4.1. Concentrations ranged from 6.18 – 55.41 mg/L. These range of concentrations are lower compared with those obtain by (Hureiki & Mouneimne, 2012; Keresztes et al., 2009). The bicarbonate ion concentration is positively correlated to total antimony, antimony (III) and antimony (V) concentrations (P = 0.344, 0.307, 0.351 > 0.05, 0.01 – Appendix A). The correlation is, however, insignificant. This suggests that the bicarbonate ion concentration statistically has no relationship with antimony content in the stored water. Hureiki and Mouneimne (2012) obtained similar results. University of Ghana http://ugspace.ug.edu.gh 36 Table 4.3.1: Physicochemical properties of the water samples 4.3.3 Calcium content versus antimony species The calcium ion (Ca2+) content of the six brands of bottled waters are shown in Table 4.3.1. The concentrations ranged from 1.61 – 12.39 mg/L with a mean and median value of 4.53 and 3.36 mg/L respectively. These are lower compared with those obtained by Hureiki and Mouneimne (2012) and Keresztes et al. (2009). The calcium content registered statistically insignificant positive correlation to total antimony, antimony (III) and antimony (V) concentrations (P = 0.193, 0.064, 0.205 > 0.05, 0.01) at 0.05 and 0.01 significance level (Appendix A). Thus, the calcium content does not appear to affect the changing levels of the antimony content in the stored water. 4.3.4 Magnesium content versus antimony species Figure 4.3.1 displays the magnesium ion concentration for the six brands of bottled waters. The concentrations ranged from 1.00 – 4.96 mg/L with a mean and median value of 2.87 and 2.78 mg/L respectively. This range of concentrations are lower compared to those obtained in a study by Hureiki and Mouneimne, (2012) and Keresztes et al., (2009). Bottled water pH TDS (mg/L) Ca2+ (mg/L) Mg2+ (mg/L) HCO3 − (mg/L) AQF 6.78 ± 0.06 41.22 ± 0.19 3.49 ± 0.03 2.96 ± 0.04 34.77 ± 0.16 BQA 7.21 ± 0.04 39.82 ± 0.37 4.06 ± 0.05 4.96 ± 0.05 30.80 ± 0.30 ICP 7.02 ± 0.04 32.39 ± 1.29 2.40 ± 0.21 3.26 ± 0.07 26.73 ± 1.29 SPI 6.91 ± 0.07 27.05 ± 1.02 3.24 ± 0.06 2.43 ± 0.06 21.38 ± 0.93 VER 7.38 ± 0.08 8.78 ± 0.12 1.61 ± 0.07 1.00 ± 0.08 6.18 ± 0.06 VOL 7.43 ± 0.15 70.40 ± 0.33 12.39 ± 0.06 2.60 ± 0.06 55.41 ± 0.39 University of Ghana http://ugspace.ug.edu.gh 37 The magnesium ion concentration is statistically correlated positively to total antimony and antimony (V) (P = 0.453, 0.429 > 0.05, 0.01 – Appendix A). These correlations are however insignificant. The magnesium concentration is also negatively correlated to antimony (III) concentration. The correlation is statistically insignificant (P = 0.708 > 0.05, 0.01). Therefore, the magnesium ion concentration does not statistically affect inorganic antimony species in the waters at the respective significance levels. 4.3.5 Total dissolved solids content versus antimony species Results for the total dissolved solids for the six brands of bottled water are presented in Table 4.3.1. This range of concentration was lower compared to those obtained by Hureiki and Mouneimne, (2012) and Keresztes et al., (2009). TDS was found to be positively correlated to total antimony, antimony (III) and antimony (V) concentrations (Appendix A). This correlation was however found to be insignificant (P = 0.288, 0.264, 0.295> 0.05, 0.01). This suggests that the TDS content does not statistically affect the inorganic antimony species in the waters at the respective significance levels. 4.4 Effect of different plastics on antimony migration Figure 4.4.1 displays the total antimony concentrations in the plastic containers the waters were stored in for the six brands of bottled water. Differences were clearly shown in the residual total antimony concentrations. The concentrations ranged from 123.5 mg/kg to 146.5 mg/kg. VER had the highest total antimony concentration (146.5 mg/kg) and AQF had the least (123.5 mg/kg). These concentrations were lower compared to concentrations of PET bottles obtained in some previous studies (Carneado et al., 2015; Keresztes et al., 2009; Tukur, Sharp, Stern, Tizaoui, & Benkreira, 2012). However, the concentrations obtained were within the range of concentrations ((104 – 166 mg/kg) obtained in a study by Fan et al. (2014). Sanchez-Martinez University of Ghana http://ugspace.ug.edu.gh 38 Figure 4.4.1: Total antimony concentrations of the plastic material used in making the bottles. AQF – VOL represents the branded PET bottles used in packaging the waters. I-beams represent the mean total antimony concentrations of the plastic materials (n = 4). et al. (2013) recorded higher total antimony concentrations in the PET plastic containers used in their study. ICP-MS and HG-AFS were used in the determinations of antimony in PET plastic containers. 4.5 Effect of storage conditions on inorganic antimony species 4.5.1 Effect of storage conditions on total antimony The Figures 4.5.1 – 4.5.9 display the total antimony concentrations of water samples determined for twelve weeks at intervals of four weeks under the three selected storage conditions for the six brands of bottled waters. The three selected storage conditions (in a refrigerator at 4ºC, indoor at room temperature and outdoor exposed to direct sunlight and high air temperatures) were specific and characteristic of bottled water usage in Ghana. University of Ghana http://ugspace.ug.edu.gh 39 Five (AQF, BQA, ICP, VER and VOL) out the six brands of bottled water samples already had some amounts of antimony present in them on the day their samples were acquired – day one (week 0). The total antimony concentrations ranged below the detection limit (0.05 µg/L) for SPI to 13.77 µg/L for VOL (Table 4.5.1). The detected concentrations on the day of acquiring bottled water samples are about 1000 times higher compared to the analysis of twelve brands of PET bottled water from Canada (112 – 375 ng/L Sb) (Shotyk et al., 2006). In another study by Westerhoff et al., (2008), antimony concentrations of PET bottled waters ranged 0.095 – 0.521 µg/L. These concentrations were far lower compared to those obtained in this study. In Lebanese natural water bottled in PET, determined antimony concentrations ranged from 0.2 µg/L to about 5.12 µg/L after 544 days (Hureiki & Mouneimne, 2012). Concentrations ranging between 0.03 and 6.61 µg/L have also been obtained for 47 freshly purchased British bottled contents (Tukur et al., 2012).These concentrations are similar to concentrations determined on the day the bottled water samples were acquired in this study. In the study of the influence of storage time and temperature on antimony migration from PET bottles into mineral water, Carneado et al. (2014) detected antimony in all tested water samples before storage (day 0). Detected total antimony concentrations ranged from 0.3 to 0.7 µg/L. These concentrations compared with detected antimony concentrations in this study for bottled waters on the first day (week 0), were far lower. BQA, VER, and VOL registered total antimony concentrations greater than the maximum contaminant level specified by US EPA and EU but lower than the maximum contaminant level specified by WHO for day one. University of Ghana http://ugspace.ug.edu.gh 40 Table 4.5.1: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined on first day and after four weeks in refrigerator SbTot: Total antimony, SbV: antimony (V), SbIII: antimony (III), SbV = SbTot - SbIII, Concentrations shown are expressed as mean ± standard deviation, n = 4. LD: Limit of detection. Week 0 (First day): day bottled water samples were acquired. Bottled water brands Bottling date (2015) Week 0 4 SbTot ʶ(µg/L) SbIII (µg/L) SbV (µg/L) SbTot (µg/L) SbIII (µg/L) SbV (µg/L) AQF 5th January 1.76 ± 0.09 0.14 ± 0.01 1.62 ± 0.09 1.89 ± 0.03 0.22 ± 0.01 1.67 ± 0.02 BQA 7th January 14.65 ± 0.14 0.24 ± 0.01 14.42 ± 0.14 15.51 ± 0.39 0.35 ± 0.02 15.16 ± 0.39 ICP 8th January 1.11 ± 0.04 < LD 1.11 ± 0.04 1.77 ± 0.09 0.38 ± 0.03 1.40 ± 0.09 SPI 9th January < LD < LD < LD 1.33 ± 0.09 0.95 ± 0.06 0.38 ± 0.14 VER 10th January 6.12 ± 0.47 0.35 ± 0.02 5.78 ± 0.47 8.27 ± 0.37 0.86 ± 0.06 7.41 ± 0.42 VOL 12th January 13.77 ± 0.13 0.65 ± 0.07 13.12 ± 0.15 14.86 ± 0.37 1.21 ± 0.08 13.65 ± 0.38 University of Ghana http://ugspace.ug.edu.gh 41 Table 4.5.2: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after eight weeks and twelve weeks in refrigerator SbTot: Total antimony, SbV: antimony (V), SbIII: antimony (III), SbV = SbTot - SbIII, Concentrations shown are expressed as mean ± standard deviation n = 4. Bottled Water brands Week 8 12 SbTot (µg/L) SbIII (µg/L) SbV (µg/L) SbTot (µg/L) SbIII (µg/L) SbV (µg/L) AQF 2.02 ± 0.06 0.57 ± 0.04 1.45 ± 0.06 2.92 ± 0.08 1.09 ± 0.06 1.83 ± 0.08 BQA 16.67 ± 0.34 0.84 ± 0.08 15.84 ± 0.32 17.32 ± 0.10 1.50 ± 0.04 15.82 ± 014 ICP 1.96 ± 0.03 0.69 ± 0.06 1.27 ± 0.07 2.55 ± 0.20 1.05 ± 0.06 1.50 ± 0.22 SPI 1.43 ± 0.04 1.09 ± 0.06 0.34 ± 0.10 1.94 ± 0.04 1.76 ± 0.06 0.19 ± 0.09 VER 9.03 ± 0.05 1.07 ± 0.04 7.96 ± 0.06 10.16 ± 0.05 1.90 ± 0.05 8.26 ± 0.08 VOL 15.75 ± 0.21 1.37 ± 0.04 14.37 ± 0.18 16.79 ± 0.06 2.20 ± 0.05 14.60 ± 0.09 University of Ghana http://ugspace.ug.edu.gh 42 Table 4.5.3: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after four and eight weeks indoors SbTot: Total antimony, SbV: antimony (V), SbIII: antimony (III), SbV = SbTot - SbIII, Concentrations shown are expressed as mean ± standard deviation, n = 4. Bottled Water brands Week 4 8 SbTot (µg/L) SbIII (µg/L) SbV (µg/L) SbTot (µg/L) SbIII (µg/L) SbV (µg/L) AQF 2.86 ± 0.13 0.56 ± 0.06 2.30 ± 0.10 3.31 ± 0.11 0.96 ± 0.07 2.36 ± 0.13 BQA 15.11 ± 0.08 0.80 ± 0.06 14.32 ± 0.03 15.93 ± 0.09 1.27 ± 0.04 14.66 ± 0.11 ICP 2.14 ± 0.06 0.27 ± 0.04 1.87 ± 0.07 2.95 ±0.07 0.59 ± 0.03 2.36 ± 0.07 SPI 1.85 ± 0.06 1.17 ±0.05 0.68 ± 0.10 2.68 ± 0.10 1.82 ± 0.04 0.85 ± 0.07 VER 6.56 ± 0.07 0.77 ± 0.05 5.78 ± 0.08 6.90 ± 0.07 1.12 ± 0.03 5.78 ± 0.09 VOL 14.69 ± 0.08 1.08 ± 0.04 13.60 ± 0.07 15.70 ± 0.11 1.62 ±0.05 14.07 ± 0.07 University of Ghana http://ugspace.ug.edu.gh 43 Table 4.5.4: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after twelve weeks indoors and four weeks outdoor SbTot: Total antimony, SbV: antimony (V), SbIII: antimony (III), SbV = SbTot - SbIII, Concentrations shown are expressed as mean ± standard deviation, n = 4. Bottled Water brands Week 12 4 SbTot (mg L-1) SbIII (mg L-1) SbV (mg L-1) SbTot (mg L-1) SbIII (mg L-1) SbV (mg L-1) AQF 3.91 ± 0.07 1.53 ± 0.04 2.38 ± 0.08 3.51 ± 0.22 1.67 ± 0.05 1.84 ± 0.17 BQA 16.84 ± 0.06 1.79 ± 0.04 15.05 ± 0.06 17.56 ± 0.23 1.62 ± 0.06 15.94 ± 0.17 ICP 3.68 ± 0.13 1.01 ± 0.07 2.68 ± 0.17 3.47 ± 0.20 1.00 ± 0.05 2.47 ± 0.24 SPI 3.78 ± 0.11 2.30 ± 0.03 1.48 ± 0.12 3.57 ± 0.25 2.90 ± 0.06 0.68 ± 0.23 VER 7.94 ± 0.06 1.72 ± 0.05 6.22 ± 0.09 8.86 ± 0.17 1.81 ± 0.05 7.05 ± 0.16 VOL 16.86 ± 0.06 2.08 ± 0.05 14.78 ± 0.06 15.69 ± 0.23 1.91 ± 0.05 13.78 ± 0.21 University of Ghana http://ugspace.ug.edu.gh 44 Table 4.5.5: Total Sb, Sb(III) and Sb(V) concentrations of water samples determined after eight and twelve weeks outdoor SbTot: Total antimony, SbV: antimony (V), SbIII: antimony (III), SbV = SbTot - SbIII, Concentrations shown are expressed as mean ± standard deviation, n = 4. Bottled Water brands Time (weeks) 8 12 SbTot (mg L-1) SbIII (mg L-1) SbV (mg L-1) SbTot (mg L-1) SbIII (mg L-1) SbV (mg L-1) AQF 6.69 ± 0.18 2.70 ± 0.05 3.99 ± 0.14 8.63 ± 0.19 3.83 ± 0.04 4.79 ± 0.19 BQA 19.48 ± 0.27 2.25 ± 0.05 17.23 ±0.28 22.62 ± 0.13 3.48 ± 0.04 19.15 ± 0.17 ICP 6.83 ± 0.26 1.88 ± 0.04 4.96 ± 0.30 10.05 ± 0.14 2.81 ± 0.05 7.24 ± 0.10 SPI 7.63 ± 0.24 4.72 ± 0.06 2.92 ±0.24 11.99 ± 0.19 7.06 ± 0.06 4.93 ± 0.15 VER 11.69 ± 0.29 2.97 ± 0.07 8.72 ± 0.32 14.65 ± 0.22 4.06 ± 0.06 10.60 ± 0.20 VOL 19.57 ± 0.34 3.08 ± 0.09 16.49 ± 0.29 23.88 ± 0.16 4.18 ± 0.04 19.70 ± 0.18 University of Ghana http://ugspace.ug.edu.gh 45 Figure 4.5.1: Total antimony concentrations of bottled water samples stored in a refrigerator for twelve weeks. Horizontal reference lines represent maximum contaminant levels (MCL). AQF – VOL represents coded names of the brands of bottled water purchased. Water samples stored in the refrigerator witnessed an increase in total antimony concentrations from day one to the end of the twelve weeks for all brands of bottled water (Figure 4.5.1). Increments observed from week 0 – 4 for BQA, ICP, SPI, VER and VOL had a mean difference of total antimony concentration greater than 0.5 µg/L. Only AQF had a mean difference of total antimony between week 0 and 4 to be less than 0.5 µg/L. The mean difference between week 1 and 4 for BQA, VER and VOL exceeded 0.5 µg/L. The mean difference between week 1 and 4 for AQF, ICP and SPI were below 0.5 µg/L. For the mean difference between week 4 and 8, all the brands of bottled water exceeded the 0.5 µg/L value. BQA recorded the highest total antimony concentration at the end of week 12 under refrigeratory conditions. This was reached because of the major contribution from total University of Ghana http://ugspace.ug.edu.gh 46 antimony determined on day one. On the other hand, looking at the difference in mean total antimony concentrations between week 0 and 12 for the six brands suggested an interesting effect. VER recorded the highest difference (4.04) followed by VOL (3.02) (Table 4.2). BQA, Figure 4.5.2: Total antimony concentrations of bottled water samples stored indoor for twelve weeks. Horizontal reference lines represent maximum contaminant levels (MCL). AQF – VOL represents coded names of the brands of bottled water. SPI, ICP and AQF then followed in decreasing order of difference in mean total antimony concentrations. Despite the increments observed, none of the brands of bottled water had total antimony concentrations exceeding the MCL specified by WHO at the end of the twelve weeks under refrigeratory conditions. Water samples stored indoor saw increase in total antimony concentrations from week 0 to 12 for all the six brands of bottled water (Figure 4.5.2). Increments observed from week 0 to 4 for AQF, ICP, SPI and VOL had a mean difference in total antimony concentrations University of Ghana http://ugspace.ug.edu.gh 47 exceeding 0.5 µg/L. The mean difference of total antimony for BQA and VER were below 0.5 µg/L from week 0 to 4. The difference in mean total antimony concentrations between week 4 and 8 for BQA, ICP, SPI and VOL were greater than 0.5 µg/L. On the other hand, AQF and VER had their mean difference in total antimony concentration between week 4 and 8 to be Figure 4.5.3: Total antimony concentrations of bottled water samples stored outdoor for twelve weeks. Horizontal reference lines represent maximum contaminant levels (MCL). AQF – VOL represents coded names of the brands of bottled water. below 0.5 µg/L. For the mean difference in total antimony concentrations between week 8 and 12, all brands of bottled water samples had a difference in mean total antimony concentrations exceeding 0.5 µg/L. VOL recorded the highest total antimony concentration (16.86 µg/L) indoors at the end of week 12. The major contribution of total antimony was from concentration determined one the first day (day one). Considering the difference in mean total antimony University of Ghana http://ugspace.ug.edu.gh 48 concentrations between week 0 and 12, resulted in SPI registering the highest with a value of 3.78 difference. This was followed in decreasing order of difference in mean total antimony concentrations by VOL, ICP, BQA, AQF and VER. Bottled water brands BQA, VER and VOL had concentrations greater than the MCL defined by EU and US EPA but less than WHO MCL at the end of week 12 indoor. Total antimony concentrations of water samples stored outdoor exposed to sunlight and high air temperatures increased from week 0 to 12 for all brands of bottled water (Figure 4.5.3). For the period from week 0 to 4, all bottled water brands recorded a difference in mean total antimony concentrations greater than 0.5 µg/L. The difference in mean total antimony concentration from week 4 to 8 and 8 to 12 were all found to be greater than 0.5 µg/L. At the end of week 12, VOL recorded the highest total antimony concentration (23.88 µg/L) followed by BQA. AQF recorded the least total antimony concentration (8.63 µg/L) at the end of week 12. Total antimony concentration obtained at day one were taken into considerations to achieve such “high” concentrations. However, the difference in mean total antimony concentrations from week 0 to 12 for the six brands had SPI register the highest difference (11.99) (Table 4.5.6). This was followed in decreasing order of difference in mean total antimony concentrations by VOL, ICP, VER, BQA and AQF. Bottled water brands VER, AQF, SPI and ICP had total antimony concentrations exceeding MCL Specified by EU and US EPA but below WHO MCL at the end of week 12. VOL and BQA however, had their total antimony concentrations exceeding WHO MCL at the end of week 12. Comparing the difference in mean total antimony concentrations from week 0 to 12 for each brand of bottled water under the three selected storage conditions (Table 4.5.6) generated various migration rates (Table 4.5.7). Bottled water samples exposed to high air