SPECIATION OF MERCURY IN DIFFERENT 
ENVIRONMENTAL COMPARTMENTS. 
DESIGN, DEVELOPMENT AND OPTIMIZATION OF 
ANALYTICAL METHODS AND PROCEDURES. 
 
 
 
Julien Lusilao Makiese 
 
 
 
 
A dissertation submitted to the faculty of science, University of Witwatersrand, in 
fulfilment of the requirements for the degree of Master of Science. 
 
Johannesburg 2008 
 ii 
Declaration 
I declare that this dissertation is my own, unaided work. It is being submitted for the 
degree of Master of Science in the University of the Witwatersrand, Johannesburg. It has 
not been submitted before for any degree or examination in any other University. 
 
 
(Signature of Candidate) 
 
Day of 2009 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 iii 
Abstract 
 
 
The widespread use of organometallic compounds and their subsequent release 
into the environment has created a great environmental concern about the toxicity 
and effects of these pollutants. Mercury pollution is a growing concern worldwide 
because its can reach high concentrations in various environmental media and thus 
adversely affect humans, wildlife and ecosystem functioning. 
 
Mercury is present in the environment in different molecular forms with specific 
biogeochemical transformation and ecotoxicity. Inorganic Hg2+ is the main form in 
water and sediment samples. Concentration levels of organomercury species is 
very low (usually ng L-1) in aquatic environments but the toxic effect of these 
compounds can be significant due to their tendency for bioaccumulation and 
biomagnification in the food chain. 
 
The development of a sensitive, reliable, simple, and cost effective procedure for 
speciation analysis of mercury in different environmental compartments is 
currently one of the principal research challenges in environmental analytical 
chemistry. To this end, this study aimed to develop and optimize analytical 
methods and procedures for the determination of total mercury and the speciation 
of inorganic and organic forms of mercury. The hyphenation of gas 
chromatography and inductively coupled plasma mass spectrometry (GC-ICP-MS) 
was achieved and used successfully.  
Rapid and efficient sample preparation procedures based on microwave-assisted 
extraction for solid samples were developed. The optimized analytical methods 
and procedures were validated by the analysis of environmental certified reference 
materials (CRM 015-050 sediment for HgTOT and CRM 463 tuna fish for HgTOT 
and MeHg). 
 
 iv 
The developed methodologies were finally applied to real environmental samples, 
namely soil, sediment, water, fish and human hair, collected in some South 
African regions affected by environmental pollution due to reprocessing of old 
tailings dumps and chlor-alakali facilities. The study included collection of 
ancillary data (pH, redox potential) which are critically important for mercury 
monitoring program. Predictive models of mercury speciation in water samples 
based on thermodynamic solution equilibria were also established. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 v 
Dedication 
 
 
 
 
 
 
 
To Mickel-Ange Lusilao  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 vi 
Acknowledgements 
 
 
I am indebted to my supervisor, Prof.  Ewa M. Cukrowska and thank her for the 
advice, guidance, support and patience throughout this work. I would also like to 
thank Dr Hermogene Nsengimana for his valuable critique and contribution to the 
development of analytical methodologies.  
 
Special thanks to the National Research Foundation (NRF) and the University of 
the Witwatersrand for the financial support during my studies. These studies were 
supported in part by NRF Grantholders through Prof Ewa M. Cukrowska and Wits 
University Postgraduate Merit Award. 
 
Many thanks to the following individuals and organization: 
 
Dr Hlanganani Tutu for his advice, his help with the modeling and assistance with 
sampling; Mr Mvuyisi Ngqola for the technical assistance and Dr Agata Bartyzel 
for her help with diagrams; South Africa Mercury Assessment Program (SAMA) 
for organizing relevant discussions through workshops on mercury assessment in 
SA. 
Misses Lise Etumba, Elysee Bakatula, Bakholise Mabuyane, Mr Robson 
Lokothwayo and all my colleagues in the Environmental Analytical Chemistry 
research Group for their help, support and encouragement.  
 
 
 
 
 
 
 
 
 
 vii 
TABLE OF CONTENTS  
 
CONTENTS             Page 
 
DECLARATION ???????????????.. ?????????.??.ii 
ABSTRACT ?..?????????????????????..??????iii 
DEDICATION????????????????????????????..v 
ACKNOWLEDGEMENTS???????????????????????.vi 
TABLE OF CONTENTS ???????????????????.???......vii 
LIST OF FIGURES ?????????????????????...???? xi 
LIST OF TABLES ??????????????????????????xiii 
LIST OF ABBREVIATIONS ?????????????????????. ..xv 
 
 
CHAPTER ONE ? INTRODUCTION 
 
1.1 General background ??????????????????????? 1 
1.2 Problem statement ??????????????????????...? 2 
 
CHAPTER TWO  ? LITERATURE REVIEW 
 
2.1 Chemistry of mercury ??????????????????????. 6 
2.2 Mineralogy ??????????????????????????.. 8 
2.3 Abundance and distribution in the environment ????????????. 8 
 2.3.1 Rocks, soils and sediments ???????????????.?? 8 
2.3.2 Minable deposits ???????????????????...? 11 
2.3.3 Fossil fuels ??????????????????????? 11 
2.3.4 Natural water ??????????????????????. 12  
2.4 Sources ???????????????????????????.. 14 
 2.4.1 Natural mercury emissions ?????????????............... 14 
 2.4.2 Anthropogenic sources ????????????????...?... 14 
 2.4.3 Mineral working ????????????????????? 17 
2.5 Pathways and behaviour of mercury in the environment ???????...... 18 
 2.5.1 Mercury in the atmosphere ??.??????????????.. 19 
 2.5.2 Terrestrial components ??????????????????.. 22 
 2.5.3 Aquatic components ??????????????????.?. 24 
 2.5.4 Transport and exchange processes ?????????????? 27 
2.6 Organomercury compounds in the environment ???????????... 28 
 2.6.1 Methylmercury in water ?????????????????? 29 
 2.6.2 Methylated mercury in the atmosphere ???????????.? 32 
 2.6.3 Methylmercury production and decomposition ????????? 33 
 2.6.4 Bioaccumulation ????????????????.......??? 36 
2.7 Health effects ????????????????????????.... 40 
2.8 Risk assessment and exposure pathways ??????????????.. 42 
 2.8.1 Exposure assessment ??????????????????..... 42 
 2.8.2 Risk characterization ??????????????????..... 42 
 viii 
 2.8.3 Risk management ????????????????????.. 43 
 2.8.4 Exposure pathways ???????????????????... 43 
2.9 Legislation and guidelines ???????????????????.... 45 
 2.9.1 Water ????????????????????????..... 45 
 2.9.2 Food ?????????????????????????.. 46 
 2.9.3 Air ??????????????????????????. 46 
 
 
CHAPTER THREE ? AN OVERVIEW OF ANALYTICAL METHODS USED IN 
   MERCURY SPECIATION 
 
3.1       Sampling and storage ?????????????????????? 49 
3.1.1    Water samples ?????????????????????... 50 
3.1.2    Solid sample ??????????????????????.. 51 
3.1.3    Biological samples ???????????????????? 51 
3.2       Preparation of solid samples ???????????????????. 52 
3.3       Methods for mercury species determination ?????????????. 54 
3.3.1    Comparison of modification techniques applied in CE, LC and GC ? 55 
3.3.2    LC and GC in mercury speciation ???????????.......?. 58 
3.3.3    Detection of mercury species ???????????????? 60 
3.3.4    Coupling of GC with ICP-MS ?????????????...?... 60 
3.4       Methods validation ?????????????????????.?... 60 
 
 
CHAPTER FOUR ? OBJECTIVES OF THE STUDY??????????? 68  
 
 
CHAPTER FIVE ? DEVELOPMENT OF INSTRUMENTAL METHODS FOR 
   MERCURY SPECIES ANALYSIS 
 
5.1 Optimization of ICP-MS ???????????????????....... 70 
5.1.1 Chemicals ?????????????????.????...?.. 70 
5.1.2 Sample preparation ?????????????....????...? 71 
5.1.3 ICP-MS analysis ?????????????.?...?..????. 72 
5.1.4 Results and discussion ??????????????????.. 73 
5.1.5 Conclusion ??????????????.?...??????? 76 
5.2 Coupling of GC with ICP-MS ??????????????????.. 76 
5.2.1 Design, development and optimization of hyphenated GC-ICP-MS ?77 
5.2.1.1 Instrumental design ????...??????????...?. 77 
5.2.1.2 Method development and optimization ???????........ 79 
5.2.2 Material and reagents ??????????????????? 81 
5.2.3 Sample preparation ?????????????????...?.... 82 
5.2.4 Analysis ??????????????????????.?? 83 
5.2.5 Results and discussion ??????????????????.. 84 
5.2.5.1 Analytical instrument ???????????????? 84 
5.2.5.2 Linearity ????????????????????? 85 
 ix 
5.2.5.3 Repeatability and detection limit ??????????...... 87 
5.2.5.4 Analysis of the certified reference material ???????.. 88 
5.2.6 Conclusion ?????????????????????...?. 90 
5.2.7 Recommendations ????????????????.???? 91 
 
 
 
CHAPTER SIX ?  APPLICATION OF DEVELOPED METHODS  
 
6.1 Introduction ?????????????????.????????.. 92 
6.2 Mercury determination in a dismantled chlor-alkali plant ???????? 93 
6.2.1 Material and reagents ??????????????????? 96 
6.2.2 Sampling and sample preparation ??????????????. 96 
6.2.2.1 Soil samples ?????????????...?????... 96 
6.2.2.2 Water samples ??????????????????... 97 
6.2.2.3 Sample preparation ??????...?????????? 98 
6.2.3 Analysis ???????????????????????.?100 
6.2.3.1 Total mercury ???????????????????100 
6.2.3.2 Mercury speciation ???????????..?????. 101 
6.2.4 Results and discussion ????????????...?????. .101 
6.2.4.1 Standard calibration ????...???????????. .101 
6.2.4.2 Soil samples ??????????????????? .105 
6.2.4.3 Water samples ??????????????????. .119 
6.2.5 Conclusion ??????????????...???????... .122 
6.3 Ecotoxicology assessment in three villages affected by mercury pollution?..123 
6.3.1 Sample preparation ???????????????????.. 125 
6.3.1.1 Hair samples ??????????????????.... 125 
6.3.1.2 Fish samples ?????...?????????????. 126 
6.3.2 Results and discussion ???????????????...??.. 127 
6.3.2.1 Speciation calibration ???????????????.. 128 
6.3.2.2 Hair samples ??????????????????? 128 
6.3.2.3 Fish samples ????????????...??????. 136 
6.3.3 Conclusion ??????????????...???????... 140 
6.4 Mercury from gold tailings ????????????????..??... 140 
6.4.1 Sampling area ?????????????????????.. 141 
6.4.2 Sampling and sample preparation ?????????????... 144 
6.4.3 Results and discussion ?????????????????? 145 
6.4.3.1 Mercury in water samples ?????????????... 150 
6.4.3.2 Mercury in sediment samples ????????????. 151 
6.4.4 Conclusion?????????????? ????????...155 
 
 
CHAPTER SEVEN ? GENERAL CONCLUSION ????????????157 
 
REFERENCES ........................................................................................................... 161 
 
 x 
APPENDIX 1ICP-MS Calibration Graph.....???????????????.. 191 
 
APPENDIX 2   GC-ICP-MS Chromatograms?..??????????.???...192 
 
APPENDIX 3  Photographs of features in the study area (Klip River, Johannesburg). 
?????????????????????????...????????194 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xi 
List of Figures  
 
Figure 2.1: A comparison of the mercury concentrations measured in the 
South Atlantic with those measured in 2002 in the North 
Pacific ????...?????.???????????? 
 
 
13 
Figure 2.2: Spatially distributed inventory of global anthropogenic 
emissions of mercury to the atmosphere, 2000??????. 
 
17 
Figure 2.3: TGM in the atmosphere at several locations???????. 21 
Figure 2.4: Summary of some of the important physical and chemical 
transformations of mercury in the atmosphere ??????. 
 
21 
Figure 2.5: Proposed mechanism for elemental oxidation in the marine 
boundary layer, or in other regimes where there is the 
presence of halogen-containing aerosol ????????? 
 
 
22 
Figure 2.6: Generalised view of mercury biogeochemistry in the aquatic 
environment???????????????????? 26 
Figure 3.1: Schematic representation of an ICP-MS instrument????.. 61 
Figure 3.2: GC-ICP-MS hyphenated technique??????????? 64 
Figure 5.1: ICP-MS calibration for different mercury isotopes????? 74 
Figure 5.2: schematic representation of the used hyphenated technique?. 77 
Figure 5.3: GC-ICP-MS coupled with the transfer line on top of the GC? 78 
Figure 5.4: ?T-piece? glass used for the connection of the transfer line 
and the nebuliser to the plasma torch??????????. 78 
Figure 5.5: Example of Chromatogram of inorganic and organic mercury 
Standard?????????????????????. 84 
Figure 5.6: Example of Chromatogram of Hg isotopes 202 and 199 
without baseline correction?????????????? 85 
Figure 5.7: Chromatogram of a blank??????????????.. 85 
Figure 5.8a, 
b and c: 
 
Chromatograms of calibration standards????????? 86 
Figure 5.9: Calibration for inorganic and organic mercury species???. 87 
Figure 5.10a 
and b: 
 
Chromatograms of CRM 463 at different extraction conditions 89 
Figure 6.1: Sketch of the sampling site with indications where soil and 
water samples were collected?????????????. 94 
Figure 6.2: Sample preparation chart for mercury determination in soil?.. 99 
Figure 6.3: Sample preparation chart for mercury determination in water.. 100 
Figure 6.4: ICP-MS calibration for 199Hg isotope?????????? 102 
Figure 6.5: ICP-MS calibration for 202Hg isotope?????????? 103 
Figure 6.6: Chromatograms of Hg standards???????????? 103 
Figure 6.7: GC-ICP-MS calibration for 202Hg isotope????????. 104 
Figure 6.8: Example of soil chromatogram obtained with GC-ICP-MS?. 105 
Figure 6.9: Example of water chromatogram obtained with GC-ICP-MS... 105 
Figure 6.10: Eh-pH diagram for some of the most important chloride and 
sulphur mercury species???????????????. 107 
 xii 
Figure 6.11: Predominant Hg(II) species with pH??????????.. 108 
 
Figure 6.12: 
 
Variation of pH with depth for the different profiles???? 
 
109 
Figure 6.13: Variation of redox potential in soil wit the depth?????.. 109 
Figure 6.14: Variation of the conductivity with the depth???????. 110 
Figure 6.15: Examples of metal concentrations with depth??????? 113 
Figure 6.16: Concentration of inorganic mercury with depth?????? 115 
Figure 6.17: Mercury concentrations in the sampling site???????. 116 
Figure 6.18: MeHg concentration at different profiles????????.. 117 
Figure 6.19: Chromatogram of sample 9C60-80??????????? 117 
Figure 6.20: Variation of IHg, MeHg concentrations and redox potential 
with depth????????????????????... 118 
Figure 6.21a: Water speciation by Geochemist?s Workbench (-Log a Cl- = 
0.143)??????????????????????.. 121 
Figure 6.21b: Water speciation if Cl- concentration is increased to - Log a = 
0.8???????????????????????... 121 
Figure 6.22: Map of the sampling site??????????????? 124 
Figure 6.23: Sample preparation for the determination of mercury in hair? 126 
Figure 6.24: Total mercury concentration in hair for each village????. 132 
Figure 6.25: Example of chromatogram of hair sample (H74)?????... 133 
Figure 6.26: Example of chromatogram of fish sample (carp fish)???? 137 
Figure 6.27: Eventual pathway of mercury contamination of vegetarians 
living in Madimeni?????????????????. 139 
Figure 6.28: Map of the sampling site??????????????? 144 
Figure 6.29: Redox potential, pH and conductivity of sediment profiles?.. 148 
Figure 6.30: Examples of metals and anions concentrations with depth for 
the sediment profile B???????????????? 149 
Figure 6.31: Example of chromatogram obtained on a sediment sample?.. 151 
Figure 6.32: Speciation of mercury compounds in the sediment profile B? 154 
Figure 6.33: % carbon in the sediment profile B??????????? 155 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xiii 
List of tables  
 
Table 2.1: Physical/ Chemical properties of mercury and some of its 
compounds???????????????????.. 7  
Table 2.2: Mercury-bearing minerals?????????????.. 9  
Table 2.3: Distribution of mercury in the Earth?s crust??????.. 10  
Table 2.4: Statistical data of analytical results from FOREGS???? 10  
Table 2.5: Range of mean concentrations of Hg and MeHg in 
subsurface water at different stations of the world ocean?.. 13  
Table 2.6: Major classes of anthropogenic emissions of mercury to the 
atmosphere in 1995???????????????? 15  
Table 2.7: Percent of mercury present as MeHg in tissues of 
invertebrae Aquatic...???????????????. 39  
Table 2.8: Levels of Total Mercury in Seafood?????????.. 41  
Table 2.9: Pathways of exposure to various species of mercury???. 44  
Table 2.10: Absorption of mercury species by routes???????? 45 
Table 3.1: Comparison of the speciation analysis using analyte 
modification by CE, LC and GC??????????? 56  
Table 3.2: Most frequently used methods or quantification of Hg and 
their relative detection limit????????????? 65 
Table 5.1: Microwave programme for sample extraction?????? 72  
Table 5.2: ICP-MS parameters????????????????. 73 
Table 5.3: ICP-MS calibration parameters???????????... 74  
Table 5.4: Total Hg concentrations on CRM015-050 measured with 
ICP-MS and % recovery determined from the certified value 75 
Table 5.5: ICP-MS and GC operating conditions????????? 80 
Table 5.6: Optimized digestion parameter for the determination of 
HgTOT in CRM 463 tuna fish????????????.. 82 
Table 5.7: RSD (%) of 0.5 ?g ml-1 IHg and 0.2 ?g ml-1 MeHg???? 88 
Table 5.8: MeHg concentration determined in CRM 463 Tuna fish by 
GC-ICP-MS???????????????????. 90 
Table 6.1: Description of collected samples???????????. 95 
Table 6.2: Microwave program for soil digestion????????? 98 
Table 6.3: ICP-MS Standard calibration parameters???????? 102 
 
  
 xiv 
Table 6.4: Samples measurement results obtained with ICP-MS 
(HgTOT) and GC- ICP-MS (IHg and MeHg)??????? 
 
106 
Table 6.5: Mercury and total ions concentrations in water and soil 
samples????????????????????? 112 
Table 6.6: Optimized digestion parameters for the determination of 
HgTOT in hair??????????????????? 125 
Tables 6.7.: Determination of HgTOT in hair samples collected from a 
non- exposed area ??????????????? 129 
Table 6.8: HgTOT in samples collected in Mshazi ???????? 129 
Table 6.9: HgTOT in samples collected in Nqetho ??????.?.. 130 
Table 6.10: HgTOT in samples collected in Madimeni ???????? 131 
Table 6.11: Inorganic and mono-methylmercury concentrations in hair 
samples ????????????????????. 133 
Table 6.12: comparison between total analysis and speciation result on 3 
hair samples ??????????????????. 134 
Table 6.13: Summary of total Hg concentration in hair from inhabitants 
of the 3 villages.????????????????? 134 
Table 6.14: Fish mercury levels, Inanda dam, KwaZulu-Natal, South 
Africa?????????????????????. 136  
Table 6.15:      Sampling GPS data????????????????.     145 
Table 6.16:      Field parameters and mercury concentrations in water samples   
                         and sediment profiles????????????????   147       
 
Table 6.17:      Ions concentrations and % carbon on the sediment profile B.    147 
 
 
 
 
 
 
 
 
 
 xv 
 
ABBREVIATIONS 
 
AAS: atomic absorption spectrometry 
 
AFS: atomic fluorescence spectrometry  
 
BCR: Community Bureau of Reference  
 
CE: Capillary electrophoresis  
 
CNRS: Centre national de recherch? scientifique 
 
CRM: certified reference material  
 
CV: cold vapor  
 
CVG: chemical vapor generation  
 
CVT: cold vapor technique  
 
CZE: capillary zone electrophoresis  
 
Eth: Ethylation 
 
FOREGS: Forum of the European Geological Surveys 
 
GC: gas chromatography  
 
GC-ICP-MS: gas chromatography- inductively coupled plasma-mass  
                        Spectrometry 
 
GPS: Global Positioning System 
 
HG: hydride generation  
 
HgEt2: Diethylmercury 
 
Hg-P: particulate-bound mercury 
 
HPLC: high-performance liquid chromatography  
 
ICP-MS: inductively coupled plasma-mass spectrometry 
 xvi 
 
IDMS: isotope dilution mass spectrometry  
 
IHg: inorganic mercury 
 
IPCS: International Programme on Chemical Safety 
 
LC: liquid chromatography  
 
LCABIE: Laboratoire de chimie analytique bio-inorganique et environment 
 
LOAEL: lowest-adverse-affect-effect-level 
 
LOD: Limit of detection  
 
LOQ: Limit of quantitation  
 
MAE: microwave-assisted extraction  
 
MCL: maximum contaminant level 
 
MeHg: monomethylmercury 
 
MeHgEt: Methylethylmercury  
 
MIP-AES: microwave-induced plasma atomic emission spectrometry 
 
MRC SA: Medical Research Council South Africa  
 
MS: mass spectrometry 
 
NaBEt4: sodium tetraethylborate 
 
NOAEL: no-adverse-affect-effect-level 
 
QC: quality control  
 
RfD: reference dose  
 
RGHg: reactive gaseous mercury 
 
RSD: Relative standard deviation 
 
 xvii 
SA: South Africa 
 
SABS: South African Bureau of Standards 
 
SAWQG: South African Water Quality Guidelines 
 
SEM: secondary electron multiplier 
 
SFE: supercritical fluid chromatography  
 
TDI: tolerable daily intake 
 
TMAH: Tetramethylammonium hydroxide  
 
USEPA: United States Environmental Protection Agency 
 
WHO: World Health Organization 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xviii