Mercury species transformations in marine and

biological systems studied by isotope dilution

mass spectrometry and stable isotope tracers

by

Lars Lambertsson

b

AKADEMISK AVHANDLING

Som med vederbörligt tillstånd av Rektorsämbetet vid Umeå Universitet för erhållande av filosofie doktorsexamen framlägges till offentlig granskning vid Kemiska Instutitionen, Sal

KB3A9 (Lilla hörsalen) KBC-huset, fredagen den 1 April, Klockan 10.00.

Fakultetsopponent: Dr. John Munthe, IVL Svenska Miljöinstitutet AB, Göteborg

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Titel: Mercury species transformations in marine and biological systems studied by isotope dilution mass spectrometry and stable isotope tracers

Author: Lars Lambertsson

Department of Chemistry, Analytical Chemistry, Umeå University, S-90187 Umeå, Sweden

Abstract: This thesis focuses on the implementation of species-specific isotope dilution

(SSID) methodology and stable isotope tracers to determine mercury species occurrence and transformation processes in-situ and during sample treatment. Isotope enriched tracers of methyl-, ethyl- and inorganic mercury were synthesised and applied in different combinations to marine and biological samples. Experimental results were obtained using gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS).

Mercury methylation and methylmercury demethylation processes in surface sediments were studied in the brackish Öre River estuary, Bothnian Bay. Uni- and multivariate data evaluation identified the organic material content and mercury methylation potential in the sediments as important factors controlling incipient methylmercury levels. Mercury species distribution in mice treated with the pharmaceutical preservative Thimerosal (ethylmercurithiosalicylate) was studied. The ethylmercury moiety of Thimerosal was observed to rapidly convert to inorganic mercury in the mice during the treatment period as well as during sample treatment, hence necessitating SSID methodology for accurate ethylmercury determinations in biological samples.

To facilitate the introduction of SSID as a routine quantitative method in mercury speciation, a methylmercury isotopic certified reference material (ICRM) was produced. Prior to certification, the stability of the material was examined in conventional and isochronous stability studies spanning 12 months, which permitted uncertainty estimation of the methylmercury amount content for two years of shelf-life.

Finally, a field-adapted SSID method for methylmercury determinations in natural water samples was developed. The proposed analytical protocol significantly simplified sample storage- and treatment procedures without sacrifices in analytical accuracy.

Keywords: Mercury, methylmercury, ethylmercury, Thimerosal, speciation, methylation,

demethylation, brackish water sediment, natural waters, GC-ICP-MS, species-specific isotope dilution, isotope enriched tracers

ISBN: 91-7305-838-6

© Copyright, Lars Lambertsson, 2005. All rights reserved Printed by Solfjädern Offset AB, Umeå

iii

This thesis includes the following papers, which are referred to in the text by Roman numerals I L. Lambertsson, E. Lundberg, M. Nilsson and W. Frech

Applications of enriched stable isotope tracers in combination with isotope dilution GC-ICP-MS to study mercury species transformation in sea sediments during in-situ ethylation and determination Journal of Analytical Atomic Spectrometry, 2001, 16, 1296.

II L. Lambertsson and M. Nilsson Organic material as the primary control on net mercury methylation in estuarine sediments Environmental Science and Technology, submitted.

III J. Qvarnström, L. Lambertsson, S. Havarinasab, P. Hultman and W. Frech Determination of methylmercury, ethylmercury and inorganic mercury in mouse tissues, following administration of Thimerosal, by species-specific isotope dilution GC-inductively coupled plasma MS Analytical Chemistry, 2003, 75, 4120.

IV J. P. Snell, C. R. Quetél, L. Lambertsson and J. Qvarnström Preparation and certification of ERM-AE670, a 202Hg enriched methylmercury isotopic reference material Journal of Analytical Atomic Spectrometry, 2004, 19, 1315.

V L. Lambertsson and E. Björn Validation of a simplified field-adapted procedure for routine determinations of methylmercury at trace levels in natural water samples using species-specific isotope dilution mass spectrometry Analytical & Bioanalytical Chemistry, 2004, 380, 871.

Paper I and IV are reprinted from Journal of Analytical Atomic Spectrometry with permission from the Royal Society of Chemistry (copyright 2001 and 2004, respectively). Paper III is reprinted from Analytical Chemistry with permission from the American Chemical Society (copyright 2003) and paper V is reprinted from Analytical & Bioanalytical Chemistry with permission from Springer Verlag (copyright 2004).

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Also by the author

S. Havarinasab, L. Lambertsson, J. Qvarnström and P. Hultman Dose-response study of thimerosal-induced murine systemic autoimmunity Toxicology and Applied Pharmacology, 2004, 194, 169.

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Table of contents

1. Introduction 1

2. Mercury 1 2.1 Toxicology 2

2.1.1 Elemental mercury 2 2.1.2 Inorganic mercury salts 3 2.1.3 Organic mercury 3

2.2 The mercury cycle 3 2.2.1 Mercury methylation 6 2.2.2 Methylmercury demethylation 7

3. Analytical techniques and methodology for mercury speciation 8 3.1 Sample storage, preservation and preconcentration 9 3.2 Mercury species separation 10

3.2.1 Gas Chromatography 10 3.3 Mercury species detection 12

3.3.1 ICP-MS 12 3.3.2 GC-ICP-MS 13

3.4 Isotope dilution mass spectrometry (IDMS) 14 3.4.1 Isotopomer synthesis 16

3.5 Analytical methods for monitoring mercury species transformations in the environment 17

4. Results: Summary of published papers and manuscripts 20 4.1 Mercury methylation and methylmercury demethylation

in brackish water sediments: Improved methodology and geochemical controls, papers I and II 20

4.2 Mercury species transformations in Thimerosal treated mice monitored with multi labelled SSID, paper III 23

4.3 Towards a wider implementation of SSID methodology in mercury speciation, papers IV and V 26

5. Conclusions and prospects 27

6. Acknowledgements 29

7. References 30

1

1. Introduction

In nature, an element may exist in different chemical forms (species) that are characterised by oxidation state and/or molecular structure. The toxicity and bioavailability of some elements is often directly related to the chemical form, for example trivalent Chromium (Cr3+) is essential for glucose metabolism in animals whereas hexavalent Chromium (Cr6+) is carcinogenic. Many other elements, such as mercury, tin, arsenic, lead and selenium, display similar “inter-species” toxicity differences. Obviously, it is of concern to have available sensitive and accurate analytical methods for qualitative and quantitative element speciation in any given sample. During the last 40 years there has been a steadily increasing interest in the field of speciation science, which is the commonly used name for this branch of analytical chemistry. The term speciation was recently defined by IUPAC (International Union for Pure and Applied Chemistry) as “Determination of the exact chemical form or compound in which an element occurs in a sample, for instance determination of whether arsenic occurs in the form of trivalent or pentavalent ions or as part of an organic molecule, and the quantitative distribution of the different chemical forms that may coexist” [1]. Often, establishment of the total concentrations of many elements would not provide sufficient information in risk assessment regarding for instance food and environmental safety. In fact, present European legislation requires speciation of mercury, cadmium, lead, tin and nickel in certain environmental samples and biota [2].

This thesis describes development of analytical methodology based on species-specific isotope dilution mass spectrometry and isotope enriched tracers for determination of mercury species and their transformation processes in marine and biological matrices.

2. Mercury

Mercury (Hg) is a natural constituent element of the earth crust. It exists in three oxidation states: elemental mercury (Hg0), mercurous mercury (Hg+) and mercuric mercury (Hg2+). At room temperature, elemental mercury (Hg0) is a silvery liquid metal, hence the latin name “hydrargyrum” meaning water-silver. Given a high vapour pressure (0.17 Pa at 25 ° C), however, Hg0 is easily emitted to the atmosphere and is rarely encountered as the liquid metal in nature. A majority of the immobilised or “buried” mercury found in the earth crust are inorganic mercuric compounds (mercury salts), e.g. mercuric oxide (HgO), mercuric chloride (HgCl2) and in particular mercuric sulphide (HgS) or cinnabar. Cinnabar is mined to produce elemental mercury for a range of industrial applications, for instance as a catalyst in chemical processes and in small-scale gold mining operations. Elemental mercury is also used in various products such as electrical switches, thermometers and medical equipment, batteries and copper/silver amalgams tooth filling material to name a few. The recorded annual global primary production of mercury in year 2000 was 1800 metric tonnes and in addition an estimated 700-900 metric tonnes of recycled mercury has been marketed yearly since the mid 90’s [3].

Mercuric mercury (Hg2+), hereafter referred to as inorganic mercury, may also form mono- or di-substituted organometallic compounds through covalent bonding to phenyl or short-chained alkyl substituents, yielding for example dimethylmercury ((CH3)2Hg), ethylmercury (C2H5Hg+), phenylmercury (C6H5Hg+) and, of particular environmental concern, methylmercury (CH3Hg+).

The first documented human activity involving organic mercury compounds dates back to the 1860’s, when methylmercury compounds were first synthesised. Apparently, two of the

2

scientists involved in the experiment died from unsafe exposure to the products. As a result, the scientific community avoided organic mercury compounds until the beginning of the twentieth century when it was found that methyl- and other short-chained alkyl- or phenylmercury derivatives had excellent anti-fungal properties [4]. Organic mercury compounds were from then on used in numerous applications where such properties were desired. For example, methyl- and ethylmercury compounds were efficiently used globally for a long time as a cereal seed dressing and also in the paper pulp industry where phenylmercury acetate was used as a fungicidal additive in the pulp.

Adverse effects on human health associated with organic mercury compounds were brought to broad public attention in the late 1950s when it was discovered that the widespread occurrence of neurological disease among residents in Minamata Bay and along the Agano River, Japan was linked to methylmercury poisoning [5]. Local chemical industries producing acetaldehyde and vinyl products were eventually pinned down as pollution sources. In the manufacturing processes used, catalytic inorganic mercury compounds were unintentionally converted into methylmercury that was discharged with process wastewater to nearby fishing grounds. The methylmercury accumulated through the aquatic trophic levels and eventually the people in these areas, who depended heavily on a fish-rich diet, were exposed to considerable amounts of methylmercury. In 1999 nearly 3000 diagnosed cases of methylmercury poisoning (Minamata disease) had been recorded in the Minamata-Agano area [5]. Other severe cases of alkyl mercury poisonings have occurred, for example in Iraq during the early 1970’s when flour made from methyl- and ethylmercury treated seed-grain was used to prepare bread. In retrospect, some 40,000 cases of mercury poisonings were observed and of which approximately 500 were lethal [4].

2.1 Toxicology

The toxicity of mercury is explained by the strong attraction between mercury and sulphur. When mercury enters a cell it will interact with thiol moieties of amino acids, such as Cysteine. In this manner protein structure and function, which is largely provided by the sulphur bridging between thiol functionalised amino acids, is deteriorated. For example, the enzyme choline acetyl transferase that is involved in the final step of acetylcholine production is inhibited by mercury. This inhibition may lead to acetylcholine deficiency, contributing to signs and symptoms of motor dysfunction. The most affected organs for mercury poisonings are the central nervous system and kidneys. For a detailed summary of biological abnormalities in connection to mercury exposure see reference 6.

Mercury toxicity is highly dependent on the chemical form, or species, in which it is present and consequently there are also differences in symptoms developing from exposures to the three main mercury categories: elemental-, inorganic- and organic mercury. As a result of the markedly dissimilar chemical and physical properties between mercury categories, sources of exposure are different. The main exposure source for elemental mercury vapour nowadays is dental amalgam. For organic mercury compounds, particularly methylmercury, the major source of exposure stems from consumption of fish and other seafood. This also applies to inorganic mercury, although some parts of the global population may be subject to significant exposures through use of mercury in ritualistic/cultural purposes, for example body decoration with cinnabar based pigments or in traditional medicine.

2.1.1 Elemental mercury

Elemental mercury seldom causes acute toxic effects if orally ingested. The gastro-intestinal absorption is very low (less than 0.01 %) and the lethal oral dose corresponds to about 100 g [7]. However, if the vapours are inhaled absorption occurs with near 100 % efficiency. The

3

absorbed elemental mercury is rapidly distributed within the organism and can readily pass the blood-brain barrier. The elemental mercury is subsequently oxidised to inorganic mercury via the catalase-peroxide pathway for further interaction with proteins as outlined above. Chronic mercury vapour exposures affect the central nervous system and kidneys with symptoms such as tremors, fine motor difficulties and proteinuria and ultimately-death.

2.1.2 Inorganic mercury salts

Owing to the higher water solubility of inorganic mercury salts the lethal oral dose is a mere 0.5 g. However, since inorganic mercury is not lipid soluble it cannot pass the blood-brain barrier sufficiently enough to cause significant damages to the nervous system. In most cases of acute inorganic mercury poisonings affected organs include kidney and intestines. Although rare, mild forms of chronic inorganic mercury are exemplified by the occurrence of acrodynia or “Pink disease” during the middle of the last century among small children treated with teething powders containing inorganic mercury salts. Affected persons often displayed symptoms such as pink extremities and severe leg cramps.

2.1.3 Organic mercury

Organic mercury compounds and in particular methylmercury are nowadays treated as potent neurotoxins (lethal oral dose is about 0.2 g). Numerous toxicology studies have revealed that the main target organ for organic mercury compounds is the central nervous system due to high lipid solubilities that enable efficient penetration of the blood brain barrier as well as the placental barrier. For methylmercury, absorption occurs at 90-100 % efficiency if orally ingested. There are marked differences in the toxic actions of methylmercury between the mature and the developing nervous systems, the latter being most sensitive. In adults, the first symptom to appear, which can occur after a latency period as long as several month from the time of exposure, at low dose exposures is usually paresthesia (numbness or a pins and needles sensation). As the exposure increases, symptoms such as tremors, dysarthria, ataxia, deafness and constriction of the visual fields occur [8].

The severe toxic actions of methylmercury on the developing nervous system were first recognized during the Minamata incident outlined above. There it was observed that women with symptoms of mild methylmercury poisoning gave birth to children with severe neurological damages that was overall analogous to cerebral palsy. Facilitated by the capacity to cross the placental barrier methylmercury interferes teratogenically with normal neuronal development in the foetus and may also have an effect on cell division [9].

In 1999 attention was drawn towards another potential organic mercury poisoning source when the American Academy of Pediatrics and the US Public Health Service in a joint statement identified the use of Thimerosal (sodium ethylmercurithiosalicylate) for anti microbial purposes in medicinal preparations as an extensive source of organic mercury exposure (ethylmercury) in infants and small children. Eventually, it was hypothesized that the administration of Thimerosal containing vaccines was associated with neuro-developmental disorders such as autism and speech delay [6]. In 2001 the US Institute of Medicine (IOM) concluded that such a relationship was biologically plausible and recommended that Thimerosal containing vaccines should not be administered to infants, small children and pregnant women [10].

2.2 The mercury cycle

Around the beginning of the 1960’s, scientists in Sweden were witnessing abnormal neurological behaviour in predatory birds. As mentioned, alkyl mercury compounds were at

4

the time a widely used agricultural fungicide, and as a result birds on top of the food chain accumulated high amounts of mercury by preying on small seed-feeding birds and rodents. Surprisingly, when control groups of fish-feeding birds were analysed, researchers found high concentrations of mercury in them as well even though these birds had no direct contact with mercury treated grain seed [11].

Following studies revealed the occurrence of high mercury levels in fish from remote lakes isolated from point mercury pollution sources. Initially, it was hypothesized that the ever increasing anthropogenic input of mercury to the atmosphere gave rise to increased mercury deposition and combined with acid rain caused an elevated mobility and hence accumulation of mercury in higher organisms within the aquatic compartment [12]. However, since mercury accumulates through food webs primarily as methylmercury [13] the atmospherically deposited mercury has to undergo methylation at some stage prior to bioaccumulation.

In 1969 Jensen and Jernelöv [14] presented evidence that inorganic mercury was transformed microbiologically to methylmercury in aquatic sediments. This finding combined with the knowledge gained in Minamata ultimately crystallized into the revelation that people who were dependent on a seafood based diet could be subjected to methylmercury poisoning (Minamata disease) just about anywhere. Consequently, in many countries legislative regulations were imposed to control mercury exposure from ingestion through maximum allowed mercury concentrations in fish and other seafood. In the European union, the present limit for mercury in fish is set at 0.5 mg kg-1 for non-predatory species and 1 mg kg-1 in predatory fish to meet the recommendations issued by the JECFA (Joint FAO/WHO (Food and Agriculture Organisation of the United Nations and World Health Organisation) Expert Committee on Food Additives) on a provisional tolerable weekly intake of 1.6 µg methylmercury per kg body mass [15].

The findings by Westöö and Jensen et al [13, 14] initiated an ongoing effort to unravel the complex biogeochemical cycling of mercury in the environment as well its toxicology and involves numerous branches of natural science. The current understanding of the environmental “mercury-cycle” is displayed graphically in figure 1.

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Figure 1. Mercury emission sources and transformation mechanisms in the environment.

Elemental mercury is the dominant mercury species in the atmosphere and around 4400 metric tonnes a year is emitted to it from both natural and anthropogenic sources [16]. Natural emissions occur from soil and sea surfaces but also from volcanoes at significant amounts. Anthropogenic mercury emission sources include combustion of fossil fuels, waste incineration and mining activities, with the two former examples accounting for 70 % of the anthropogenic emissions. Being a single atom gas, elemental mercury is highly stable in the atmosphere with an average residence time of 1 year and can therefore be transported over very long distances. Mercury is therefore considered a global pollutant in the sense that a local release can have global effects.

Mercury is deposited to the aquatic and terrestrial compartments with precipitation following oxidation of elemental- to inorganic mercury. Once the deposited inorganic mercury enters an aquatic system it may complex with, for example, anions like chloride and hydroxide or to thiol sites on dissolved or particulate organic material. The various inorganic mercury species subsequently sediment at bottoms where they either are immobilised by formation of cinnabar (HgS) or undergo biological transformations into methyl-, dimethyl- or elemental mercury. Mercury species formed in sediments may thereafter transfer to the water column where methylmercury is available for further biomagnification in aquatic food webs while a majority of the dimethyl- and elemental mercury ultimately will emit to the atmosphere (figure 1).

Anthropogenic Hg emissions

Sediment

Water

Atmosphere Natural Hgemissions

Natural Hg runoff

Anthropogenic Hg runoff

Hg0 (part)

Hg0 (aq) Hg2+ (aq)

Hg2+ (part)

Hg0 (g) Hg2+ (g)

Wet deposition Dry deposition

Hg2+Hg0

Hg2+

CH3Hg+

Hg0

HgS

(CH3)2Hg

(CH3)2HgCH3Hg+

Hg0 Hg2+

(CH3)2HgCH3Hg+EvasionAnthropogenic Hg emissions

Sediment

Water

Atmosphere Natural Hgemissions

Natural Hg runoff

Anthropogenic Hg runoff

Hg0 (part)

Hg0 (aq) Hg2+ (aq)

Hg2+ (part)

Hg0 (g) Hg2+ (g)

Hg0 (part)

Hg0 (aq) Hg2+ (aq)

Hg2+ (part)

Hg0 (g) Hg2+ (g)

Wet deposition Dry deposition

Hg2+Hg0

Hg2+

CH3Hg+

Hg0

HgS

(CH3)2Hg

(CH3)2HgCH3Hg+

Hg0 Hg2+

(CH3)2HgCH3Hg+Evasion

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Table 1. Typical methyl- and total mercury concentrations in environmental and biological matrices, compiled from US EPA data [17]

Environmental media HgTot Methylmercury Air 1-170a 0-40a Precipitation 4-90b 0.04-0.6b Fresh water 0.2-15b 0.04-0.8b Sea water 0.3-15b 0.01-0.5b Soil 8-406c 0.3-23c Ocean sediments 2-2200c 0.06-70c Lake sediments 10-750c 0.3-30c Fresh water fish 30-330d 28-310d Marine fish 10-1300d 10-1240d

a. ng m-3

b. ng l-1 c. ng g-1 dry weight d. ng g-1 wet weight 2.2.1 Mercury methylation In 1985 Compeau and Bartha identified sulphate reducing bacteria (SRB) to be primary mercury methylators in aquatic sediments [18]. Through a series of inhibition-stimulation experiments of anoxic estuarine sediment they found that the strain Desulfovibrio desulfuricans LS displayed very high mercury methylating potential. In following studies [19], the same research group showed that mercury methylation in Desulfovibrio desulfuricans LS occurred via donation of a methyl group from methylcobalamin, or methylated vitamin B12, and further suggested that the reaction was mediated through enzymatic catalysis in the acetyl-CoA pathway [20]. However, the biochemical pathway for methylation of inorganic mercury in SRB is still somewhat unclear since methylcobalamine and acetyl-CoA mechanisms are not at all unique to SRB and it is likely that another, so far unidentified, component is most likely also involved in the process. Recently it was also shown that some SRB strains that do not utilize the acetyl-CoA pathway also are capable of mercury methylation and possibly even without the presence of methylcobalamin [21].

At present there seems to be no absolute answer to why SRB methylate mercury. In living organisms there are a number of biochemical pathways performing methylation on a variety of substrates, e.g protective methylation of DNA, and it is likely that mercury methylation is a coincidental result of inorganic mercury competing with intended substrates to be methylated. Nevertheless, it has been shown that the methylation of mercury provides resistance against the toxicity of inorganic mercury in the fungus Neurospora crassa [22].

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Figure 2. The biochemical pathway for mercury methylation as proposed by Choi et al [20].

Apart from pure culture mercury methylation experiments, the importance of SRB for

the production of methylmercury in different environmental compartments has been further verified in a number of studies [23-26]. In these, measurements of sediment microbial community composition and activity in combination with inorganic mercury radiotracers have shown correlation between the abundance and respiration of SRB populations and rates of mercury methylation.

SRB have also been found capable of producing dimethylmercury [27]. The rate at which this process occurs has been shown to be about 1000 times lower than the mono methylation rate in pure culture as well as in sediments and sediment slurries [28]. Whether or not the acetyl-CoA pathway is also involved in the mercury dimethylation process is not clearly established. For instance, Baldi et al [29] suggested that the formation of dimethylmercury in Desulfovibrio desulfuricans LS originated from the decomposition of insoluble di methylmercury sulphide ((CH3Hg)2S) formed by this bacteria. 2.2.2 Methylmercury demethylation A number of bacterial strains, both anaerobic and aerobic, are able to degrade or demethylate methylmercury (and other alkyl- or phenylmercury species) as a resistance mechanism against organic mercury compounds. This mechanism is mediated through two different biochemical

Acetate

Acetyl-CoA

CODH

CH3

B12-protein

CH3

CH3-THF

Formate

CO2

CO

CO2

Methyltransferase

2e-

CODH

4e-

FDH2e-

Hg2+

CH3Hg+

Acetate

Acetyl-CoA

CODH

CH3

B12-protein

CH3

CH3-THF

Formate

CO2

CO

CO2

Methyltransferase

2e-

CODH

4e-

FDH2e-

Hg2+

CH3Hg+

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pathways, reductive- and oxidative demethylation. Reductive demethylation is genetically controlled via the mercury resistance operon (mer), which is fairly common among microbes in the environment [30]. Transcription of the mer operon is induced in the presence of methylmercury resulting in synthesis of organomercurial lyase, an enzyme that cleaves the mercury-carbon bond in methylmercury yielding inorganic mercury and methane. The resulting inorganic mercury in turn induces transcription of the enzyme mercuric reductase, which reduces inorganic mercury to elemental mercury, which subsequently evaporates from the organism due to the high vapour pressure and low aqueous solubility. Attempts have been made to incorporate the mer-operon into plants, called transgenic plants, for phytoremediation of mercury-contaminated soils [31].

Figure 3. The reductive demethylation pathway as proposed by Robinson and Tuovinen [30].

The oxidative demethylation pathway was recently proposed based on observations of

carbon dioxide instead of the expected methane in the demethylation pathway [32, 33]. Oxidative demethylation is thought to occur within biochemical mechanisms that utilize single-carbon substrates to derive energy. Presumably, the oxidative demethylation mechanism yields inorganic mercury as an end product as opposed to elemental mercury in mer-mediated demethylation. It also appears that the oxidative demethylation pathway is more dominant in pristine ecosystems whereas the mer-operon is more abundant and hence reductive demethylation dominates in mercury-contaminated areas [34].

3. Analytical techniques and methodology for mercury speciation

Mercury speciation analysis can be performed with direct or indirect methods. The direct methods, involving techniques such as nuclear magnetic resonance (NMR), X-ray absorption spectroscopy (XAS) and X-ray absorption near-edge structure spectroscopy (XANES), are highly attractive in the way that samples can be analysed with minimal treatment prior to measurements. Unfortunately, the direct techniques display relatively poor detection limits, typically in the low mg kg-1 range, and are therefore not suitable for quantitative mercury measurements in most samples. Direct techniques are, however, important tools for the establishment of mercury species chemical ligands and bonding strengths in various matrices [35, 36].

RHg+ RH

NADPH NADP+

Hg2+

Hg0

NADPH

NADP+

Organomercurial lyase

Mercuric reductase

RHg+ RH

NADPH NADP+

Hg2+

Hg0

NADPH

NADP+

Organomercurial lyase

Mercuric reductase

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Indirect methods are typically based on hyphenated techniques, a combination between a separation (chromatographic) technique and a detection technique, for example gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-atomic fluorescence spectrometry (GC-AFS). For hyphenated techniques it is required that the sample is pre-treated to permit introduction on the instrumentation. Such sample pre-treatment often involve a number of operative steps but, on the other hand, it is possible to achieve very high analyte enrichment in the sample through optimisation of the treatment procedure. Indirect methods for mercury speciation usually display substantially lower detection limits (<µg kg-1) compared to direct methods and are hence well suited for quantitative analysis at trace level analyte concentrations.

3.1 Sample storage, preservation and preconcentration

Mercury species are normally present at very low concentrations in pristine environmental samples, see table 1, and especially in natural water samples in which, for example, methylmercury concentrations in the low to mid pg kg-1 range are quite common. Contamination of samples during sampling is a potential source of error at such low analyte levels, which requires that clean sampling- and laboratory protocols are employed [37].

In water samples it has been observed that the relative mercury species concentrations are unstable over extended periods of storage (< 1 month) prior to analysis as a result of biological transformation and/or adsorption to sample container surfaces [38, 39]. For short term storage (< 1 week) these effects can be subdued by storing samples in containers made of inert materials like polytetrafluoroethylene (PTFE) at + 4°C but obviously it is desirable to maintain the original species distribution more or less permanently. Some studies have been concerned with water sample preservation by means of acidification with mineral acids [40, 41] or complex binding [42] of methylmercury on additives such as dithiocarbamate functionalised resins followed by elution under acidic conditions. However, the subsequent treatment steps usually require less acidic conditions that must be restored with additional reagents, which potentially can increase blank levels. Freeze preservation of water samples at –80 ° C has been investigated with positive results [39, 43]. For biological and sediment samples the problem with analyte instability is less pronounced. Increased analyte stability in these matrices might be explained by the high content of reduced sulphur groups on which mercury species are immobilised. Storage in a regular household freezer dedicated to trace level samples at –20 ° C is normally a sufficient preservation strategy for these types of samples.

Sub ng kg-1 levels of mercury species found in natural waters necessitate extraction and preconcentration of the analytes regardless of detection technique used. To accomplish this various preconcentration techniques such as distillation [44, 45], solid phase extraction (SPE) [46, 47] and solid phase micro extraction (SPME) [48, 49] can be employed. In the author’s opinion though, purge-trap techniques based on aqueous phase derivatisation combined with adsorbent trapping are best suited for this purpose given that analyte derivatisation and extraction is carried out in a single step with very high preconcentration capacity [39, 50-52, I, II, V]. The required equipment is relatively simple and adaptable for field use if so desired. The technique also offers great sample volume flexibility, which can be an important feature in order to obtain satisfactory method detection limits when dealing with trace level analyte concentrations. Using the purge-trap methods for mercury species derivatisation and preconcentration in aqueous samples is not problem-free though. It has been observed [39, 52] that aqueous phase ethylation, using sodium tetraethylborate (NaB(C2H5)4), in aqueous samples containing high concentrations of halides and/or DOC can

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result in reduced analyte recoveries or transformations of methylmercury, deteriorating detection limits and increasing the risk for systematic errors. As a consequence, methylmercury determinations in sea- and mire waters with purge-trap methods based on aqueous phase ethylation usually require analyte-matrix separation by repeated liquid extractions under acidic conditions prior to derivatisation to boost analyte recoveries. Unfortunately, such extraction procedures require the use of several solvents and reagents, which will increase the blank levels and deteriorate detection limits.

For solid samples such as soils, sediments and biota a majority of current sample treatment strategies for mercury species extraction are in general variations on the original Westöö-method, developed by Swedish scientist Gunnel Westöö in 1966 [53]. These solid/liquid (or liquid/liquid) extraction methods involve either a complete dissolution or leaching of the samples under acidic or alkaline conditions to liberate mercury species attached to reduced sulphur groups present in the matrix. The analyte is thereafter separated from the matrix by liquid extraction using a suitable organic solvent. A variety of leaching media are used, for example diluted hydrochloric acid, mixtures of sulphuric acid/potassium bromide/copper sulphate or methanolic potassium hydroxide (KOH) solutions. Sample digestion methods used for total mercury concentration measurements, for example pressurised microwave digestion in concentrated mineral acids, are in most cases not well suited for speciation analysis since such treatment can cause substantial degradation of organic mercury species. Complete sample dissolution under the relatively mild treatment conditions required to maintain the species distribution integrity is typically only achievable with biological matrices. For this purpose, samples can be treated with aqueous solutions of tetramethyl ammonium hydroxide (TMAH) or KOH (20-25 % w/v) assisted with mechanical or ultrasonic shaking.

In addition to traditional solid-liquid or liquid-liquid extraction methods, more “up to date” sample preparation techniques like accelerated solvent extraction (ASE) [54], supercritical fluid extraction (SFE) [55, 56] and open focused microwave digestion [57] have been tested in a variety of environmental and biological matrices. However, the techniques mentioned have not enjoyed widespread acceptance either because analyte recoveries obtainable are insufficient or that instrument production has been cancelled.

3.2 Mercury species separation

To perform a speciation analysis using an indirect method the mercury species present in the processed sample need to be separated in time prior to detection. Initially in the 1960s, gas chromatography (GC) was the preferred separation technique in mercury speciation science. Today virtually all known chromatography techniques have been evaluated and applied for mercury speciation. All contemporary chromatographic techniques have their pros and cons but, in the author’s opinion, gas chromatography still stands out as the most attractive to apply in the field of mercury speciation on account of unparalleled separation efficiency and robustness.

3.2.1 Gas Chromatography

In gas chromatography (GC) the sample is normally introduced as an organic solution in an injector, where it is vaporized and transferred by the mobile phase (gas) through a chromatographic column situated inside a heated oven. The GC-oven can be either held at a constant temperature (isothermal) or programmed to increase the temperature at a given rate. Analytes are separated on basis of differences in volatility and, for some applications, polarity between analyte compounds and their interaction with the column stationary phase. Today, most GC separations are carried out on capillary columns, with inner diameters of 0.25 to

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0.53 mm, 15-60 m of length and stationary phase thickness between 0.25-3 µm. The stationary phase consists of a polysiloxane polymer that is characterized by a repeated siloxane backbone in which each silicon atom contains two functional groups. The amount and type of functional groups are varied to obtain different stationary phase properties, for instance 100 % dimethyl polysiloxane is a non-polar phase whereas the addition of 50 % cyanopropylmethyl polysiloxane or trifluoropropylmethyl polysiloxane gives a high polarity phase.

Figure 4. Polysiloxane polymers in capillary GC column stationary phases: Dimethyl polysiloxane, cyanopropylmethyl polysiloxane and trifluoropropylmethyl polysiloxane

For mercury speciation capillary column GC with non-polar or slightly polar stationary phases (e.g. 5 % diphenyl polysiloxane phases like DB-5 and other equivalent phases) offers fast analyses (3-5 minutes) with excellent chromatographic performance that helps obtaining low detection limits because chromatographic peaks can be highly compressed in narrow bandwidths, thereby resulting in improved signal to noise ratios. However, a disadvantage with GC compared to other chromatographic techniques, e.g. HPLC, is that ionic analytes, such as methylmercury and inorganic mercury, are difficult to separate regardless of stationary phase used (non-polar or polar). To circumvent this, ionic species are typically transformed into volatile and stable derivatives through nucleophilic alkyl substitution by adding a derivatisation reagent to the sample. Halogenated alkyl magnesium compounds, called Grignard reagents (after the inventor Victor Grignard), and tetra-alkylated borates like tetraethylborate or tetrapropylborate are the most popular derivatisation reagents in mercury speciation. Grignard reagents are very unstable in aqueous media and therefore the analytes have to be derivatised in a dry (water free) organic solvent, such as toluene or hexane. It is therefore necessary to extract mercury species present in aqueous sample solutions into such a solvent. This can be accomplished by adding a complexing agent to the sample, for example diethyldithiocarbamate (DDTC). Tetra-alkylated borate derivatisation reagents on the other hand are relatively stable in both aqueous and organic solutions, which means that these derivatisation reagents can be added directly to aqueous sample solutions. The alkylated non-polar mercury species can thereafter either be

Si

CH3

CH3

O

nSi

CH2

CH3

O

n

CH2

CH2

CN

Si

CH2

CH3

O

n

CH2

CF3

Si

CH3

CH3

O

nSi

CH2

CH3

O

n

CH2

CH2

CN

Si

CH2

CH3

O

n

CH2

CF3

12

extracted into an organic solvent, for traditional syringe injection on the GC, or be purged-trapped on adsorbent columns as discussed in section 3.1.

3.3 Mercury species detection

Traditionally, electron capture detectors (ECD) have been used extensively in combination with GC separations of mercury species. This type of detector is selective towards electronegative compounds, especially halogenated molecules, and therefore mercury species have to be derivatised towards such functionality when the ECD is used. Nowadays ECD detectors have been more or less abandoned in the field of mercury speciation mainly due to the risk that other halogenated (electronegative) compounds co-elute with the mercury species under study, thereby producing erroneous results. Instead spectrometric detectors that display high selectivity towards mercury are preferred, e.g. atomic absorbtion spectrometry (AAS), atomic fluorescence spectrometry (AFS), atomic emission spectrometry (AES) and mass spectrometry (MS), since these detection techniques greatly reduce the number of possible matrix interferences. The work presented in this thesis was carried out using inductively coupled plasma quadropole mass spectrometry (ICP-QMS) as a GC detector (GC-ICP-QMS). Several benefits can be gained by using mass spectrometric detection for mercury speciation work. The ability to discriminate between elemental isotopes is particularly useful as it permits unique experiments to be carried out.

3.3.1 ICP-MS

In mass spectrometric techniques, molecular or elemental ions produced in an ion source are sorted and counted in a mass analyser on basis of their mass to charge ratio (m/z). There are several different mass analyser designs, for example quadropole-, time-of-flight-, ion trap- and sector field mass analysers that display varying capacities to resolve different mass to charge ratios. For characterisation of inorganic/organometallic compounds mass spectrometry can be used with common ion sources, such as electron ionisation (EI) and electro spray ionisation (ESI). However, for quantitative elemental analysis of such compounds a complete decomposition of the sample is required to avoid severe spectral interferences (isobaric overlap). To accomplish this, “hard” ion sources that atomise and ionise the analytes with high efficiency, like thermal ionisation, glow discharge and inductively coupled plasma, are applied.

Among hard ion sources, the inductively coupled plasma is the most suitable to use for elemental speciation analysis since it is compatible with continuous sample introduction and therefore more easily coupled to chromatographic systems like GC and HPLC. It consists of three concentric quartz tubes, called a torch, through which streams of Argon are passed at a combined flow rate of typically 15 l min-1. The top of the torch is surrounded by an induction coil that is connected to a radio frequency generator operating at either 27 or 40 MHz with about 1500 W power capacity. The argon gas is initially ionised by introducing Tesla sparks into it and an Argon-plasma is formed at the top of the torch. The plasma is sustained by the collisions between Argon atoms and electrons in the oscillating inductive field. At the high frequencies used plasma temperatures up to 10000 ° C are obtained locally, which is sufficient to produce singly charged atomic ions at high efficiency.

13

Ions produced in the ICP ion source are extracted into the mass spectrometer through a source/spectrometer-interface that sequentially lowers the pressure from atmospheric to about 10-4 Pa by differential pumping. After passing the interface analyte ions are then focused and accelerated by a series of ion lenses into the quadrupole mass analyser or mass filter, which separates the ions based on their mass/charge ratio. The quadrupole type of mass analyser is built up of four parallel hyperbolic stainless steel rods to which a combination of RF and DC voltages are applied. For a specific combination of these voltages only ions of a specific mass/charge ratio will have a stable flight path through the mass analyser to the detector, enabling selective mass filtering. Filtered ions are subsequently counted in the detector (electron multiplier). The high ionisation efficiency of the ICP source and combined with a clean background spectrum generate very high signal to noise ratios and hence low detection limits for most elements.

Figure 5. Schematic presentation of ICP-MS (M+ signifies analyte ion)

3.3.2 GC-ICP-MS

Couplings between capillary column GC and ICP-MS were first described almost 15 years ago [58]. The GC-ICP-MS technique has quickly increased in popularity and is nowadays a frequently used technique for speciation of most metals and metalloids but also for halides or halogenated hydrocarbons as well as organic phosphor and sulphur compounds. The main benefit of using GC-ICP-MS for mercury speciation is a significant gain in sensitivity compared to other mass spectrometric techniques such as GC-MS. In general, detection limits 1-2 orders of magnitude lower than those of GC-MS can be obtained, which of course is highly valuable when dealing with trace analyte concentrations, e.g. natural water samples. A major drawback of the technique, in addition to high running costs (Argon consumption at ~15 l min-1), lies in the fact that no molecular weight or structure information for polyatomic analytes can be extracted as a result of the complete analyte decomposition that is effected in the ICP ion source. Identification of individual mercury species therefore has to rely upon retention time matching against high purity standard compounds.

DetectorM+

Ion lenses

Interface

Auxilary gas

Plasma gas

Torch

Quadropole

Sample

Plasma

Mass analyserIon source

Induction coilDetector

M+

Ion lenses

Interface

Auxilary gas

Plasma gas

Torch

Quadropole

Sample

Plasma

Mass analyserIon source

Induction coil

14

3.4 Isotope dilution mass spectrometry (IDMS)

The isotope selectivity of a mass spectrometric detector makes it possible to employ so called isotope dilution calibration to mercury speciation analysis. The general principle of isotope dilution methodology is that the sample is standardised by adding a known amount of a compound (commonly called “spiking”) that has identical chemical properties as the analyte apart from being labelled with an enriched isotope (isotopomer). This results in an experimental isotopic abundance ratio between isotopomer and incipient analyte (reference) that deviates from the naturally occurring ratio (figure 6).

Figure 6. Isotope dilution demonstrated by an analyte having two isotopes with m/z A and B in the sample, isotopomer standard and spiked sample

Based on the measured experimental isotope ratio in the mixture between reference and isotopomer in the sample as well as corresponding ratios in the unspiked sample and isotopomer standard, the analyte amount can be calculated according to equation 1.

−

−

=

xxm

smS

xx

sssx ABR

BRAMm

MmCC (1)

m/z

Inte

nsi

ty

m/z

Inte

nsi

ty

m/z

Inte

nsi

ty

Sample

Isotopomer standard

Mixture

A B

A

A

B

B

m/z

Inte

nsi

ty

m/z

Inte

nsi

ty

m/z

Inte

nsi

ty

Sample

Isotopomer standard

Mixture

A B

A

A

B

B

15

In this equation Cx and Cs denote the analyte concentration in the sample and the concentration of the isotopomer standard and ms and mx are the masses of the added spike and sample, respectively. Ms and Mx are the relative molar masses of the spike and analyte. Rm is the measured ratio between reference (A) and isotopomer (B) in the spiked sample. As and Bs are the atom fractions of reference and isotopomer in the isotopomer standard, while Ax and Bx are the equivalent for the original sample. As can be seen, the analyte concentration in the sample can be determined without the establishment of calibration curves, in one single measurement of Rm. All other parameters are given in the isotopomer standard specifications and from weighing the sample and spike. However, in the case of the ICP-MS technique used in this thesis, analyte ions with high m/z are detected at higher relative sensitivity compared to lower m/z analyte ions, which means that measured ratios are distorted. This so called mass bias effect is compensated for in the calculation of results by applying experimentally determined correction factors or abundance values, which requires additional analyses.

Since isotope dilution quantitation is based on isotope ratios that remain constant during the full analysis cycle (sample pre-treatment, pre-concentration, analysis), potential error sources such as analyte degradation and detector drift etc will not influence the final result since analyte and standard are affected to an exact equal extent. Provided that isotopomer and analyte extraction yields are equal, the accuracy and precision that can be obtained by using IDMS at optimum conditions is typically superior to external or regular internal calibration approaches. Equally important is the fact that, when using IDMS, non-quantitative analyte recoveries will not have an effect on the accuracy of results (within reasonable limits), which ultimately means that sample pre-treatment procedures can be significantly simplified and overall sample throughput improved.

IDMS has been an established quantitative method for quite some time in organic mass spectrometry, for example for the determination of PCBs and other persistent organic pollutants (POPs) in a wide range of matrices by using 2H or 13C labelled isotopomers. The application of IDMS in speciation analysis (usually referred to as “species-specific isotope dilution” (SSID)) was pioneered by Heumann some 15 years ago parallel to the initiation of the GC-ICP-MS technique [59, 60]. To date SSID based speciation methods for the determination of selenium, chromium, thallium, lead, iodine, tin and mercury have been published. Contrary to the isotopomers used in organic IDMS, the isotopic labelling of the tracer compounds used in speciation IDMS is usually incorporated in the parent trace element, for example CH3

200Hg+ rather than C2H3Hg+ or 13CH3Hg+, to maintain high sensitivity at higher atomic mass/charge ratios with ICP-MS detection and also to allow for SSID calibration of valency species, inorganic mercury (Hg2+) for example. However, since this approach for isotopic labelling of trace element species requires that the parent trace element holds at least two stable isotopes (polyisotopic), SSID speciation methods for monoisotopic elements such as Arsenic would have to be based on long lived radioisotopmers or deuterated/13C labelled isotopomers where applicable.

Table 2. IUPAC mercury isotopic abundances (atom %).

Isotope 196Hg 198Hg 199Hg 200Hg 201Hg 202Hg 204Hg

Abundance 0.15 9.97 16.87 23.10 13.18 29.86 6.87

16

No method is perfect and of course this also applies to isotope dilution calibration. The precision of ID-based measurements is dependent on the ratio between reference and spike isotopes, A and B in figure 6. For any given system of spike/reference isotopes there is an optimum ratio between A and B at which the optimal precision in measurements is obtained [61]. Ratios higher or lower than the optimum increases the uncertainty of the measurement to a proportion that is determined by a so-called error propagation factor (EPF), which in turn is derived from the isotopic abundances of the spike and reference isotopes in both the enriched standard and natural sample [62]. To achieve the optimal ratio when adding the isotopomer standard it is therefore necessary to know the concentration of the analyte present in the sample. In reality, however, or at least in the case of mercury, the detrimental effect caused by non-optimal isotope ratios on the overall measurement uncertainty is relatively small compared to contributions from other uncertainty sources [V]. An estimation of the incipient analyte concentration based on previous results or results presented in the literature for similar matrices can in many cases be adequate to obtain reasonably optimal ratios between spike and reference isotopes. Figure 7 describes the EPF as a function of 200Hg to 202Hg isotopomer/reference isotope ratios and as can be seen the ratio can be varied within a quite wide range without causing dramatic increase in the EPF.

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

0 5 10 15 20 25 30 35 40 45

200Hg/202Hg

Err

or p

ropa

gatio

n fa

ctor

Figure 7. Error propagation factor as a function of 200Hg/202Hg ratio.

3.4.1 Isotopomer synthesis

SSID calibration is a recently established methodology in speciation analysis and the number of laboratories that apply it are relatively few, therefore isotopomer standards or certified reference materials (CRMs) have, up until recently [IV], not been commercially available. As a result scientists in the field have had to synthesise their own isotope enriched standards. In the case of mercury speciation, aqueous isotope standards of inorganic mercury can easily be produced by dissolving the enriched mercury isotope (usually supplied as oxide or as the metal) in nitric acid followed by dilution with doubly distilled (or better) water to desired concentrations. For the preparation of methylmercury isotopomer standards three approaches for synthesis routes have developed. The most popular seems to be to use methylcobalamine [52, 63] or tetramethyltin ((CH3)4Sn) [64-66] to accomplish a mono methyl group transfer to

17

isotope enriched inorganic mercury in aqueous media with high synthesis yields. These two similar synthesis routes have the advantage that methylmercury can be synthesised without significant amounts of residual mercury species forming, like dimethylmercury or inorganic mercury. Consequently, subsequent product purification procedures are drastically simplified or in best case unnecessary. For the work presented in this thesis a different synthesis strategy based on a so called comproportionation reaction [67], was applied in the production of mercury species isotopomer standards. This synthesis route initially produces dimethylmercury quantitatively by reacting mercuric chloride with a Grignard reagent, CH3MgCl, in organic solution (toluene). Mono methylmercury chloride is subsequently produced by refluxing equimolar amounts of dimethylmercury and mercuric chloride. This so called comproportionation reaction yields mono methylmercury chloride at near 100 % efficiency. However, the handling of dimethylmercury can be extremely hazardous if dealing with concentrated solutions and appropriate safety precautions must always be taken [68]. The largest benefits of this method for isotopomer synthesis are that high yields are obtained and that it is equally applicable to synthesis of other alkylated or phenylated mercury species, such as ethylmercury chloride [III], simply by adjusting the type of Grignard reagent.

In cases when a speciation analysis concerns more than one element species, SSID calibration can be applied as a quantitative method in either “single labelled”- [67, 69, 70] or “multi labelled” mode [III, 71, 72]. In single labelled SSID isotopomers for all element species under study are labelled with the same isotope and correspondingly, in multi labelled mode, each species-specific isotopomer is labelled with an individual isotope. Multi-labelled SSID speciation analysis is highly useful since it facilitates detection and subsequent correction [III, 73] for possible species interconversion reactions, or transalkylation between species and matrix occurring during sample storage and pre-treatment that in some cases can introduce severe bias in the quantitative analysis. This can be exemplified by the unintentional formation of methylmercury during sample processing of sediment samples that occur when steam distillation techniques are used for analyte extraction/pre-concentration. The steam distillation technique became a subject of debate in the late 1990s when Hintelmann and co-workers found by spiking sediment samples with an inorganic mercury isotopomer followed by distillation enrichment and GC-ICP-MS analysis, that methylmercury was produced during the distillation processing [74]. The degree of such “artefact formation” of methylmercury was found to be around 0.03 % of the added inorganic mercury spike. The ratio between inorganic to methylmercury concentrations in marine sediments normally is about 100:1 [75] and therefore a small unintentional formation of methylmercury during sample processing would not influence the analysis result significantly. On the other hand, in contaminated sediments this ratio can sometimes be as high as 1500:1 and in such samples even a 0.03 % “artefact formation” of methylmercury would result in a substantial over estimation of the methylmercury content. Studies aimed at understanding the mechanisms leading to so-called “artifact formation” of methylmercury during distillation sample processing have showed that it is related to the inorganic mercury content of the sample and that it can be kept very low or insignificant if conventional solid/liquid extraction methods are employed instead of distillation in the processing of sediment samples [I, II, 74, 76-79].

3.5 Analytical methods for monitoring mercury species transformations in the environment

It is nowadays established that observed methylmercury concentrations in aquatic sediments is the net product between mercury methylating and methylmercury demethylating mechanisms effected by micro organisms. Consequently, it is of interest to gain knowledge about the rates at which these reactions occur as well as their environmental controls. Such

18

information can be valuable in, for example, risk assessment of mercury polluted industrial areas and the development of preventive measures to minimise mercury methylation and emission at such sites.

The methods by which mercury species transformation rates have been determined in sediments involve three different strategies. Jensen and Jernelöv [14] first studied mercury methylation in aquatic environments by spiking sediments with inorganic mercury of natural isotopic composition. In most cases when using this method the sediments have to be amended with very high concentrations of inorganic mercury, several µg g-1, to differentiate any significant methylation of the added inorganic mercury spike from the methylmercury already present in the sample. Unless the sediment under study originates from a heavily Hg-polluted site, the results obtained could be considered uncertain since large inorganic mercury additions will probably affect the incipient mercury species equilibria and microbial fauna drastically. More specific methods that utilize radiochemically labelled tracers, e.g. 203Hg2+ [80] and 14CH3Hg+ [81], have been used frequently for mercury methylation as well as methylmercury demethylation studies. In the radiotracer methods the amount of methylated radioactive 203Hg2+ in the sediment is typically quantified by selective methylmercury extraction followed by scintillation counting. The amount of demethylated 14CH3Hg+ can be estimated by collecting the headspace of the incubation vessel and measuring the radioactive methane (14CH4) or carbon dioxide (14CO2) formed in the reductive or oxidative demethylation pathways. Radiotracer methods are appealing because the required instrumentation is relatively simple and methods can easily be adopted for field use. However, the radioactive 203Hg2+ tracer is usually of low specific activity and therefore samples have had to be spiked to unrealistically high concentrations to detect the methylated tracer. Gilmour and Riedel [82] used a custom-made 203Hg2+ tracer with very high specific activity to circumvent this dilemma, which allowed them to determine mercury methylation rates in sediments with very small tracer additions (<10 % of incipient levels). Nevertheless, the selectivity of the methylmercury extractions used in radiotracer methods is questionable, meaning that there is a risk for residual 203Hg2+ to be co-extracted together with the CH3

203Hg+ and thereby methylation rates may be overestimated. Demethylation studies using the radiotracer methodology are also problematic since a given proportion of the end products in the demethylation pathway, 14CH4 and/or 14CO2, are most likely reincorporated into the microbiological biomass, leading to underestimated demethylation rates.

Hintelmann and co-workers significantly improved the methodology for mercury species transformation studies about ten years ago when they presented a method that made use of enriched stable inorganic mercury as a tracer to monitor mercury methylation in combination with GC-ICP-MS instrumentation [83]. By spiking sediment slurries with 199Hg2+ at sub-incipient concentrations, mercury methylation rates as well as changes in incipient methylmercury levels could be determined over a period of three weeks. They further developed their method to also include a methylmercury tracer, which enabled measurement of demethylation rates simultaneously in the same sample as methylation rates were measured [84, 85].

The general theory of using enriched stable mercury tracers for this type of studies resembles isotope dilution calibration methodology. If an isotope enriched inorganic mercury tracer is added to a sediment, for example 199Hg2+, the isotopic abundance ratio between 199Hg and a reference isotope (normally 202Hg) for that species will be shifted from the natural ratio to a higher value that is dependent on the amount of added tracer. Methylmercury produced from the inorganic mercury present in the sample will then also display a 199 to 202 ratio that is higher than the natural 199/202 ratio. The amount of produced CH3

199Hg+ is proportional to the degree that the measured methylmercury 199/202 ratio exceeds the natural 199/202 ratio and can usually be quantified with high accuracy and precision, especially if the tracer

19

experiment is combined with SSID calibration during sample treatment. The limit of detection for mercury methylation using the stable isotope methodology will be dependent on the precision of the isotope ratio measurements and the incipient concentration of methylmercury. To detect a significant methylation of the added 199Hg2+ tracer, the resulting increase in the 199/202-methylmercury ratio has to exceed the natural 199/202 ratio by an extent equivalent to three times the standard deviation interval at which the 199/202 ratio can be measured. In line with this, the incipient methylmercury concentration also determines the methylation limit of detection in each individual sample, as a higher incipient amount of methylmercury means that a higher amount of the tracer has to be methylated to produce a significant increase in the 199/202 methylmercury ratio and vice versa. In that respect, radiotracer methods are advantageous since the added 203Hg2+ tracer does not exist naturally and hence does not suffer from mercury related spectral interferences. The precision at which isotope ratios can be measured with a ICP quadropole mass spectrometer in continuous signal mode is normally in the neighbourhood of 0.1-0.2 % relative standard deviation (RSD) [86] but deteriorates if chromatography is involved, like in the case of GC-ICP-MS instrumentation, since measurements then are based on transient signals. Precision in transient signal measurements can be improved by using, for example, multi collector ICP-MS (MC-ICP-MS) instrumentation that is capable of measuring isotope ratios at parts per million level RSD [87]. The strategy for methylmercury demethylation rate measurements using stable isotopes is different compared to radiotracer methods. Instead of targeting the end products of the demethylation pathway demethylation rates are indirectly measured based on the decrease in concentration of the added methylmercury tracer during the incubation period. The reductive and oxidative demethylation mechanisms produce different end products, Hg0 and Hg2+ respectively, of which the former is lost to the head space/atmosphere and hence escapes detection. Furthermore, the fact that methylmercury is generally present at 1-5 % of inorganic mercury concentrations would render very high demethylation detection limits if measurements were based on an increase of the inorganic tracer/reference isotope ratio due to methylmercury tracer demethylation. For an in-depth description on the calculation scheme of methylation and demethylation detection limits using the stable isotope methodology, see reference 84. In summary, the stable isotope approach is a very convenient approach for measurements of mercury species transformations in sediments and other ecological or biological systems (see figure 8 for a schematic presentation of both radiotracer and enriched stable isotope tracer methodology for measurement of mercury species transformation rates in sediment). The greatest benefits gained in using the stable isotope tracer methodology is that methylation- and demethylation rates can be determined simultaneously in one individual sample with high specificity and with very low tracer additions, which enables the establishment of “true” net methylation rates for the system under study.

20

Figure 8. Comparison between radioisotope- and stable isotope methodologies for monitoring mercury methylation and methylmercury demethylation in sediments.

4. Results: Summary of published papers and manuscripts

4.1 Mercury methylation and methylmercury demethylation in brackish water sediments: Improved methodology and geochemical controls, papers I and II

In paper I a method for simultaneous monitoring of mercury methylation and methylmercury demethylation rates in brackish water sediment based on enriched mercury isotope tracers was developed. In addition, by adding enriched isotope standards of methyl- and inorganic mercury to the sediment prior to sample treatment, it was also possible to determine both the incipient methylmercury concentrations by SSID calibration with a precision of 2-3 % RSD as well as the amount of artefact methylmercury produced in the sample treatment. Methylmercury was acid leached from the sediments using a mixture of KBr/CuSO4/H2SO4 and extracted to an organic phase (CH2Cl2) followed by back-extraction of the organic phase to water and subsequent aqueous phase ethylation using sodium tetraethylborate. It was found that this sample treatment resulted in an average methylmercury “artefact formation” of about 0.05 %. It was also found, in line with observations made by Demuth et al [52], that methylmercury was partly degraded and reduced to elemental mercury during the sample

203Hg2+(aq)N2

Anaerobic incubation

14CH3Hg+(aq)

CH3203Hg-extraction

Headspace 14CO2, 14CH4

Scint.Counter

CH3198Hg+(aq) 201Hg2+(aq)

N2

Anaerobic incubation

CH3200Hg+(aq)

199Hg2+(aq)

ExtractionGC-ICP-MS

Radioisotope tracers Stable isotope tracers

- Low sensitivity - Selective MeHg extraction?- CO2 and CH4 can be incorporated intothe microbial biomass

- No incipient MeHg data extractable+ Simple instrumentation

+ High sensitivity + Incipient MeHg concentration,methylation-/de-methylation rates and“artifact MeHg” in one experiment.+ IDMS- Complex instrumentation

Sed

imen

t co

re

Sed

imen

t co

re

21

treatment cycle and/or analysis as the specific isotopic pattern displayed for methylmercury was also found in the peak corresponding to elemental mercury. Another peak that displayed a similar isotopic pattern eluted shortly after diethylmercury (inorganic mercury) but the compound could not be identified. However, when the solid/liquid extraction was performed in diluted HCl instead of the KBr/CuSO4/H2SO4 mixture this peak was not present and therefore it was suggested that the unidentified peak might have resulted from methylmercury bromide. Although the developed method obviously was prone to species transformations during sample treatment and analysis accurate results were still obtained as a result of the SSID calibration.

The developed method was applied to assess methylation and demethylation rate profiles in brackish water sediment from the Öre estuary located in the Gulf of Bothnia. Measurements were carried out by spiking 1 cm slices of the top 10 cm of a sediment core with methyl- and inorganic isotope enriched tracers (CH3

198Hg+ and 201Hg2+, respectively) to about 20 % of the incipient methyl- and inorganic mercury concentrations followed by anaerobic incubation in a nitrogen atmosphere for 10 days. From the obtained results a corresponding net methylation profile was derived (figure 9). The highest observed net methylation occurred in the 2-3 cm depth region of the sediment core in which a net methylmercury amount of about 3.5 ng per gram of dry sediment was produced during a 10-day anaerobic incubation. This observation was in line with other studies that also have described high methylation rates in the top section of lake- and sea sediments [88, 89]. Logically, the net methylation depth profile in sediments ought to reflect the vertical depth distribution of mercury methylating micro organisms (SRB). For instance, Devereux et al [90] were able to correlate high near-surface mercury methylation rates to SRB population density in estuarine sediment by using oligonucleotide molecular probes to determine SRB community depth distribution.

Incipient methylmercury concentrations varied as much as 5 times over the sampled 10 cm depth and given that the methylmercury levels could be determined with high precision, even small differences in concentration could be detected (figure 9). Similar to the net methylation profile, incipient methylmercury concentrations peaked in the 2-3 cm segment at around 5 ng g-1 but also displayed a second increase at 6-8 cm depth below the sediment surface with concentrations of about 5.5 ng g-1. The second methylmercury maximum coincided with a sharp increase in sediment pore water sulphide concentration, which was measured in the sediment core using a sulphide specific microelectrode, and therefore the increase in methylmercury concentration at the 6-8 cm depth was possibly due to immobilisation of methylmercury on reduced sulphur.

22

Figure 9. Depth profiles of sulphide-, oxygen-, total mercury and methylmercury concentrations in addition to corresponding net mercury methylation rates observed in a brackish water sediment

A number of factors have been identified to influence microbial mercury methylation processes in aquatic sediments, e.g. temperature [88], salinity [91], sediment sulphate- and sulphide concentrations [92-94] and sediment organic material content [88, 95, 96]. In contrast, basically nothing is known about how by these factors affect methylmercury demethylation processes.

In paper II the effects of organic material (OM) content and sulphide concentration on methylation and demethylation processes as well as incipient methylmercury concentrations were investigated in Öre River estuary sediments by using the method developed in paper I. Samples were collected from five different sites (erosion- or accumulation type bottoms) in October, May and June 2001-2002 to incorporate a broad spatial and temporal variation in sediment OM- and sulphide content into the experimental design (table 1 and figure 1 in paper II).

The measured incipient methylmercury concentrations and mercury methylation rates varied considerably with bottom type, sediment depth and sampling occasion (figure 2 in paper II). In general, sediments that contained higher levels of OM also displayed relatively high methylmercury concentrations compared to low OM containing sediments. In the accumulation bottoms, methylmercury concentrations comparable to those normally found in Hg-contaminated marine environments were observed, which was remarkable since the Öre estuary is considered a pristine area [97]. This finding indicated that process-controlling factors, other than the availability of substrate (Hg2+), are important for microbial synthesis of methylmercury in sediments unpolluted with mercury. The correlation between sediment organic material and methylmercury concentrations was found to be significantly correlated in an exponential regression model based on the total set of data (figure 3 in paper II). With respect to temporal variation in incipient methylmercury concentrations, the highest levels were observed in October and the lowest in May. Mercury methylation followed the same spatial pattern and varied by factor of ~10 between sites. Temporarily, the highest mercury methylation rates were recorded in June or October and the lowest in May in all sites. In most cases the depth of maximum methylation rates for individual sampling sites were similar between sampling occasions. Demethylation depth profiles were, however, surprisingly

0

1

2

3

4

5

6

7

8

9

10

0 20 40 60 80 100 120

Total Hg (ng/g); Total Sulfide (µM); O2 Saturation (%)

De

pth

(c

m)

0 1 2 3 4 5 6Ambient methyl mercury (ng/g)

Total Hg

Total Sulfide

O2 saturation

Ambient MeHg

0

1

2

3

4

5

6

7

8

9

10

0 0.5 1 1.5 2 2.5 3 3.5 4

Net methylation (ng/g dry weight)

Dep

th (

cm

)

23

constant spatially as well as temporarily. Consequently, the calculated net methylation depth profiles were to large degree determined by the mercury methylation potential at all sites except for a shallow, low OM, erosion bottom that almost exclusively displayed negative net methylation rates.

To further evaluate how the variables: OM-content, sulphide concentration, mercury methylation and methylmercury demethylation correlated with incipient methylmercury depth profiles, partial least squares regression modelling (PLS) was applied to each site and sampling occasion. In a majority of cases the PLS models explained >85 % of the variance in incipient methylmercury concentration although the distribution of significant regression coefficients between models was mixed (table 4 in paper II). However, a PLS model based on the total data set explained 69 % of the variance in incipient methylmercury with OM-content and mercury methylation as positive significantly correlated regression coefficients.

Combined, the results indicated that sulphide concentrations in the sediments were in most cases to low to inhibit mercury methylation, which has been reported by others to occur at >10 µM sulfide concentrations [92]. Furthermore, methylmercury demethylation processes in the sediments were not markedly affected by changes in OM- or sulphide concentrations. The observed spatial variation in incipient methylmercury concentrations as well as depth and rates of maximal methylation were controlled by the organic material content and differences in sediment redox conditions. Based on the results obtained in paper II and data reported by others (table 5 in paper II), it was concluded that non Hg-polluted brackish environments might constitute potential methylmercury “hotspots”, which may be activated if organic material loads increase.

4.2 Mercury species transformations in Thimerosal treated mice monitored with multi labelled SSID, paper III

Suspicion that Thimerosal (ethylmercurithiosalicylate) as a preservative in vaccines used in child immunisation programmes is linked to increasing numbers of children diagnosed with neuro developmental disorders (particularly autistic spectrum disorder, ASD) was raised by Bernard et al [6] in 2001. Since then a number of epidemiological research studies have either confirmed [98] the suspicion or have not found any correlation at all [99]. Even though conclusive evidence that confirms a Thimerosal-autism connection is still missing the “Thimerosal-controversy” has stimulated the filing of a number of lawsuits in the US by affected individuals that seek compensation from vaccine producing companies. But given the rather limited knowledgebase on the toxicity, distribution and metabolism of Thimerosal in higher organisms it certainly seems difficult to conclude whether or not Thimerosal containing vaccines might cause neuro developmental disorders. Systematic research studies aimed at these topics are therefore needed, as was concluded by the IOM in their 2001 report [10]. The toxicological effects of ethylmercury, which is the toxic moiety of Thimerosal, are understudied and most often researchers have assumed its toxicity to be analogous to that of methylmercury. It has been suggested that ethylmercury is rapidly released from the Thimerosal moiety upon its administration in the human body and therefore disposition pathways similar to those of methylmercury can be expected [4]. However, ethylmercury seems to be less stable than methylmercury within the body, which may result in toxilogical differences between the two compounds. For example, Matheson et al [100] reported five fold higher blood levels of inorganic mercury for a patient exposed to large amounts of Thimerosal compared to what is usually found in cases of methylmercury poisoning of similar degree.

In paper III a multi-labelled SSID GC-ICP-MS method was developed to study the disposition and metabolism/transformation of Thimerosal in mice. As mentioned the ethylmercury moiety of Thimerosal is rapidly dissociated from the thiosalicylic acid upon its

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introduction in biological systems and therefore an ethylmercury isotopomer was synthesised from enriched 199HgO to enable SSID quantitation of this metabolite. By using the comproportionation reaction previously applied for the synthesis of methylmercury isotopomers in paper I, C2H5

199HgCl could be produced with a yield of >90 %. The isotope labelled ethylmercury chloride was subsequently transferred from the reaction medium (toluene) to aqueous media by evaporating the organic solvent and redissolving the resulting dry white powder in 10 % aqueous ethanol solution. In this way the isotopomer could be added to samples in a medium that was more compatible with the biological tissues under study and, in addition, unwanted residual mercury by-products like diethylmercury were removed to a large extent facilitated by the moderate heat evaporation of the organic solvent. In addition to the ethylmercury isotope spike the samples were also standardised with CH3

200HgCl and 201Hg2+. Accurate results for the methyl- and inorganic mercury content of two certified reference materials (CRM) were obtained. Ethylmercury was added to the CRMs in varying concentrations and quantitative recoveries were obtained. Nevertheless, it was found from the isotope ratio measurements that almost one tenth of the ethylmercury isotopomer, and consequently also the incipient ethylmercury present in the sample, was degraded to inorganic mercury during sample pre-treatment and analysis. Given that such species conversions are very difficult to correct for when using non-isotope selective techniques, it could be concluded that SSID calibration is essential for achieving accurate results in ethylmercury determinations.

Species concentrations of methyl-, ethyl- and inorganic mercury were assessed in duplicates in kidney, liver and mesenterial lymph nodes following 0 (control), 1, 2.5, 6 and 14 days of Thimerosal treatment ad libitum (figure 10). A rapid absorption of the ethylmercury moiety of Thimerosal in the experimental animals was reflected in the ethylmercury concentrations observed in the analysed mouse tissues. However, it was clearly evident that ethylmercury displayed limited stability once absorbed as found concentrations of inorganic mercury in the organs increased with time, which was in line with the observations made by Matheson et al. Interestingly, methylmercury concentrations also increased with time in the experimental animals, but this observation could be linked to methylmercury impurities in the mouse food as well as the administered Thimerosal.

25

Figure 10. Determined (+) CH3Hg+, ( ) C2H5Hg+ and ( ) Hg2+concentrations in a) kidney b) liver and c) mesenterial lymph nodes as a function of days of Thimerosal exposure.

Days of Thimerosal exposure

0 2 4 6 8 10 12 14

C2H

5Hg+ a

nd H

g2+ c

onc.

/ µ g

g-1

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20

40

60

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3Hg+ c

onc.

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g-1

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C2H

5Hg+ a

nd H

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5

10

15

20

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30

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3Hg+ c

onc.

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0 2 4 6 8 10 12 14

C2H

5Hg+ a

nd H

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onc.

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2

4

6

8

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g-1

0.00

0.02

0.04

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4.3 Towards a wider implementation of SSID methodology in mercury speciation, papers IV and V

Species-specific isotope dilution facilitates considerably simpler and more flexible analytical protocols in terms of sample processing compared to most other methods for mercury speciation analysis. So far though, the methodology has mainly been used as a reference technique by a relatively few research laboratories. A wider implementation on routine basis at commercial laboratories has been hindered by the lack of available certified isotope standards and to some extent lack of certified reference materials [101], which are a requisite for quality assurance in terms of accuracy and traceability of results at accredited analytical laboratories.

Paper IV describes the preparation and certification of aqueous methylmercury isotopic certified reference material (ICRM) enriched to 98 % in the 202Hg isotope with a certified methylmercury amount content and isotopic composition that is traceable to SI-units. This ICRM will hopefully help launch SSID calibration as a standard method in mercury speciation analysis. The synthesis route that had previously been used successfully for the production of methyl- and ethylmercury isotopomers [I, III] was again applied here, although in a somewhat larger scale. However, special emphasis was in this case put into producing as pure a product as possible to minimise the risk of unwanted side reactions that potentially could influence the species integrity. This meant applying product purification procedures to quantitatively remove residual mercury species in the raw product, e.g. Hg2+, (CH3)2Hg and Hg0. Two independent yearlong stability tests were carried out to investigate the stability of the material. One conventional, in which the standard was stored at -20 ° C and the methylmercury amount content analysed every three months, and one isochronous stability test, where six sets of standard aliquots were stored at temperatures of -20 ° C, 4 ° C and 20 ° C and were then analysed all at the same occasion. The results clearly showed that the isotopomer standard was stable for at least one year independent of storage temperature. The resulting aqueous CH3

202Hg standard was certified with total CH3Hg as well as CH3202Hg

amount contents of 35.3 and 34.5 µg g-1, respectively. Inorganic mercury, which was the only detectable residual species, was found to be present at 2 % of the total mercury content. The CH3

202Hg ICRM, named ERM-AE670, is currently commercially available through European Reference Materials, ERM [102].

A potential application for the ICRM developed in paper IV is for routine determinations of methylmercury in water samples. Natural waters are perhaps the most difficult samples to deal with in mercury speciation analysis owing to the very low levels of mercury species present and their limited stability during sample storage, methylmercury in particular (section 3.1). Analyte instability should be efficiently corrected for if species-specific standardisation is applied during sampling since losses of analyte and added isotopomer will occur to equal extents, given that the isotopomer has been properly equilibrated with the sample. In that way, previous requirements associated with non-SSID methods of keeping sample storage time as short as possible to prevent erroneous results from analyte degradation/losses could be evaded. This would be very convenient during e.g. long-term studies since samples then could be accumulated batch wise and analysed accordingly at a suitable occasion instead of having to analyse samples continuously. Likewise, by applying SSID standardisation to water samples it should also be possible circumvent the need for analyte-matrix separation prior to derivatisation that is required with non-SSID methods (section 3.1) since non-quantitative analyte recoveries due to matrix related interferences will not effect the accuracy of results. Thereby sample preparation protocols could be kept extremely simple. The above hypotheses were tested in paper V in which a field-adapted

27

method for methylmercury determination in natural water samples based on SSID standardisation was developed and applied in the field. The possibility of direct analyte derivatisation through aqueous phase ethylation using sodium tetraethylborate without any kind of sample treatment (other than filtration of suspended particles) was tested against conventional liquid-liquid (quantitative) extractions of CH3

200HgCl standardised water samples of varying matrix composition with respect to salinity and DOC content (mire-, fresh- and seawater). Nearly identical results were obtained between the two methods and it could thus be concluded that SSID based methylmercury determinations can be conducted in a wide range of natural waters without any analyte-matrix separation prior to analyte derivatisation. Furthermore, it was observed that the applied liquid-liquid extraction treatment generated methylmercury blank levels, and accordingly detection limits, an order of magnitude higher than the direct ethylation approach. Although the application of SSID dramatically simplified sample handling and treatment protocols, it should be pointed out that analyte loss at trace level concentrations could turn out to be crucial in terms of detectability. It was thus desirable to combine SSID sample standardisation with some kind of sample preservation procedure to subdue analyte losses. Therefore sample storage at –20 º C was investigated and results were compared to those obtained from analysing the same samples as soon as possible after sampling. Again, no significant differences in either analyte concentrations or recoveries between freeze stored or immediately analysed samples could be detected in the tested matrices, which showed that samples could be safely stored in a regular household freezer.

Adapting the developed method for the field study meant that samples had to be standardised and frozen in conjunction to sampling. For simplicity, the CH3

200HgCl isotopomer was added volumetrically by a micropipette even though this procedure was likely to have a greater detrimental effect on the overall uncertainty of the measurements than if the standard had been added gravimetrically. However, total uncertainty budgets for the field application of the method revealed that the highest uncertainty contributions derived from, depending on the analyte concentration, either uncertainty of the concentration of the isotopomer standard or from variation in field blanks. Nevertheless, the relative expanded uncertainties derived from the uncertainty budgeting ranged between 5-13 % for the observed methylmercury concentration range in 50 analysed mire water samples, which was sufficient to allow for detection of small differences in analyte concentration at trace level.

5. Conclusions and prospects

The work presented in this thesis demonstrates the potential and usefulness of SSID methodology in mercury speciation, either if it is applied solely for quantification or if it is combined with isotope enriched tracers for studies of mercury species transformations in marine, or other, environments. In the latter case, benefits can be gained with respect to the number of simultaneously observable parameters, as well as the specificity and precision at which they can be measured. The method presented in paper I and similar SSID based approaches presented by others can, in the authors opinion, be considered as more or less perfect for the purpose of assessing mercury species transformation rates in sediments. With this methodology available the conditions to further resolve biological and geochemical mechanisms that control microbial mercury methylation are quite favourable. However, information regarding tracer versus incipient species availability for species transformations and its effect on observed transformation rates in sediments is scarce [85] and more research is needed on this topic. This knowledge would facilitate finer tuned stable isotope tracer

28

methods. In wider perspective, it is important to include other environmental compartments of the “mercury cycle” in mercury species transformation studies e.g. water, biota and atmosphere. In that way it should be possible to obtain good quality data on important parameters such as mercury species biomagnification and “inter-compartment” transfer rates. Such experiments, based on microcosm simulations of brackish water systems, are currently under way in our laboratory utilizing newly developed SSID-based technology for measurement of gaseous mercury species [103].

The developed SSID method for determination of ethylmercury evidently enables accurate determinations of this apparently unstable mercury species in biological samples. The next logical step to further develop the method should involve synthesis of isotope enriched Thimerosal that is administered to experimental animals followed by SSID calibration using an ethylmercury isotopomer labelled in a different isotope in the analytical stage. This would certainly make a “sharper” analytical tool at hand for Thimerosal toxicity studies in the way that traceability of in-situ species conversions would be much improved.

Paper V showed that SSID makes sample storage less critical and reduces sampling treatment procedures associated with natural water samples. At the same time, accuracy of the analytical results is maintained. These features should certainly appeal to commercial laboratories that handle large quantities samples. However, accurate in-field SSID standardisation of water samples (or other sample matrices) is still hampered by the requirement of background data on analyte concentration for the sample under study. A solution to this problem could be to standardise samples with an assortment of different methylmercury isotopomers that cover a wide concentration range. In that way the probability to obtain an optimal isotopomer/reference ratio, to minimise measurement uncertainty for an unknown sample, is increased.

Finally, from an analytical viewpoint it is desirable to develop mercury speciation methods in analogy to the IUPAC definition of speciation as being “determination of the exact chemical form or compound in which an element occurs in a sample” (see introduction). With respect to this, the GC-ICP-MS technique commonly used in mercury speciation, and throughout this thesis, can fulfil these criteria for elemental mercury but is unfortunately a blunt technique when other mercury species are concerned. Future efforts should therefore be aimed at developing complementary methods based on molecular mass spectrometric techniques, such as LC-MS(-MS) or GC-MS, as well as improvement of existing direct methods.

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6. Acknowledgements Wolfgang: Först och främst ska du ha tack för alla ovärderliga synpunkter på laborativa och skriftliga moment samt att du i de allra flesta fall ser till att allt “snack” resulterar i nånting konkret. Stort tack även för alla festliga sammankomster du arrangerar-höjdpunkter under min tid som doktorand. Erik L: Tack för alla tips och råd och för att du alltid förbehållslöst ställt upp och ordnat både det ena och det andra. Mats: Tack för din entusiasm och alla högintressanta projekt (gamla som nya). Dina kunskaper och idéer har haft avsevärd betydelse. Ett jättetack går ut till alla fantastiska medarbetare/vänner i AAS-gruppen, nuvarande och föredettingar, som på ett eller annat sett bidragit till att göra de här åren så roliga och stimulerande. Det har varit ett nöje att arbeta tillsammans, tack: Tom för att du låter mej ta hand om tjädrarna i Vargträsk och andra ställen-absoluta höjdpunkter! Ser fram emot 25/8!, Erik B för givande diskussioner, “laborationer” och kollaborationer rörande diverse vattenlösningar samt för alla klarsynta kommentarer, Johanna för givande samarbete, lycka till med familj och karriär, Solomon för din positiva attityd och stöd genom åren, James för all teknisk hjälp i början och för gemensamma projekt på senare tid, Dong and Daniel - good luck in the future!, Karin, Jin och Qiang. Tack också till övriga kollegor på analytisk kemi: Lasse för all hjälp med instrument, datorer och trevliga jaktturer på ditt natursköna hemman, Svante J för tekniska konstruktioner, Patrik, Tobias o Nina & Co., Petrus, William, Michael, Svante Å, Camilla, Anna S, Anders, Josefina, Knut, Anna N, Julien, Britta, Wen, Christer, Emil, Ahn Mai, Fredrik, Erika, Einar, Jufang och Gerd. De senaste året har jag varit relativt osynlig på min andra arbetsplats UMF av olika skäl (boken ni läser just nu t ex). De perioder som jag tillbringat på UMF har emellertid varit otroligt trevliga, vilket till stor del berott på personalen-tack allihop! Särskilt tack till Calle och Tommy för laborativ assistans samt Mikael och Jonas för assistans i jakten på svavelosande gegga. Tack även till SLU:are Ulf, Andreas, Torbjörn och Kevin. Per Hultman och Said Havarinasab i Linköping ska ha stort tack för ett givande samarbete. Vi kanske ses IRL nångång! Tack till klanmedlemmar som på ett eller annat sätt bidragit till att underlätta doktorandlivet: Morsan, Einar, Karin, Jörgen, Kalle, Elsa, Edvin, Farsan, Eva, Oskar, Lisa, Ulla, Börje och Kicki.

Slutligen vill jag ge min underbara familj ett gäng jättekramar: Lotta, Max, Greta och Elvira, NI ÄR BÄST!

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