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MASS-INDEPENDENT FRACTIONATION OF
MERCURY ISOTOPES IN FRESHWATER
SYSTEMS
by
Carla Hendrika Rose
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science
Graduate Department of Geology University of Toronto
© Copyright by Carla H. Rose (2010)
ii
Mass-Independent Fractionation of Mercury Isotopes in Freshwater Systems
Carla H. Rose
Master of Applied Science
Graduate Department of Geology
University of Toronto
2010
ABSTRACT Mass-independent fractionation (MIF) of Hg isotopes has the potential to track the
environmental transport and fate of Hg. Herein we demonstrate that reducing both the
frequency and intensity of light have a large effect on the expression and magnitude of
MIF. This strongly supports the magnetic isotope effect as the mechanism behind MIF
observed during aqueous photo-reduction of Hg(II) and MeHg. The ratios of MIF,
199Hg/201Hg, were 1.00 ± 0.04 (2SE) for Hg(II) and 1.35 ± 0.16 (2SE) for MeHg
respectively and did not change as incident radiation energy and magnitude of MIF
diminished, suggesting the respective MIF pathways remained constant regardless of
experimental conditions. Comparable amounts of total photo-reduction were shown to
coincide with different magnitudes of MIF depending the wavelength light available for
photo-reduction. This confirms there are multiple pathways for photo-reduction in
freshwater reservoirs and indicates that quantitatively relating photo-reduction and MIF
will be challenging.
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ACKNOWLEDGMENTS
I would like to thank my advisor, Prof. Bridget Bergquist, without whom this thesis would
have been impossible. I have learned a great deal from her over the past two years, and
deeply appreciate both her scientific insight and her personal concern for my success.
I would also like to thank Prof. Joel Blum, Marcus Johnson and the members of the U.M.
BEIGL laboratory for assistance and guidance in obtaining the isotope measurements
presented in this thesis and for their warm welcome and discussions when I was a guest in
their lab.
The members of my supervisory committee, Prof. Barbara Sherwood-Lollar and Prof. Grant
Ferris both welcomed me when I came to them with questions and broadened my perspective
of isotopes and biogeochemical cycling and I appreciate their help very much.
Dr. Sanghamitra Ghosh has been a wonderful colleague over the last couple of years and
offered help, discussions, moral support and generally a great attitude. Priyanka Chandan has
also been a great colleague and I have enjoyed working with her. Dr. Georges Lacrampe-
Couloume has been extremely helpful over the last two years and I am grateful for his
assistance and support.
Finally, Mom and Dad, I don’t know how I would have come this far without your
encouragement and support. Thank you a hundred times over and I love you very much.
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TABLE OF CONTENTS ABSTRACT……………………………………………………………………….… ii AKNOWLEDGEMENTS……………………………………………………………… iii TABLE OF CONTENTS……………………………………………………………… iv LIST OF TABLES…………………………………………………………………… vi LIST OF FIGURES……………………………………………………………………vii CHAPTER 1: INTRODUCTION INTRODUCTION…………………………………………………………………… 1 RESEARCH OBJECTIVE……………………………………………………………… 7 THESIS OUTLINE…………………………………………………………………… 8 STATEMENT OF AUTHORSHIP AND PUBLICATION STATUS………………………… 10 LITERATURE CITED………………………………………………………………… 11 CHAPTER 2: OVERVIEW OF STABLE ISOTOPE GEOCHEMISTRY, FRESHWATER MASS
TRANSFER PROCESSES AND SPECIES TRANSFORMATIONS 2.1. MASS-DEPENDENT FRACTIONATION (CLASSICAL EFFECT)…………………… 17
2.1.1. Mechanisms of mass-dependent fractionation………………………… 18 2.1.2. Mercury isotopes and mass-dependent fractionation………………… 19
2.2. MASS-INDEPENDENT FRACTIONATION………………………………………… 25 2.2.1. Mercury isotopes and mass-independent fractionation……………… 25 2.2.2. Mechanisms of mass-independent fractionation……………………… 28
2.3. FRESHWATER MERCURY MASS TRANSFER PROCESSES AND SPECIES
TRANSFORMATIONS………………………………………………………… 32 2.3.1. Concentrations………………………………………………………… 32 2.3.2. Sources………………………………………………………………… 33 2.3.3. Sinks…………………………………………………………………… 34
2.3.3.1. Outflow…………………………………………………………… 34 2.3.3.2. Sedimentation…………………………………………………… 35 2.3.3.3. Bioaccumulation………………………………………………… 35 2.3.3.4. Degradation of MeHg…………………………………………… 36 2.3.3.5. Volatilization………………………………………………………38
2.3.4. Light Penetration……………………………………………………… 43 2.3.5. Organic Matter Complexation………………………………………… 45
LITERATURE CITED………………………………………………………………… 47
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CHAPTER 3: EFFECTS OF ULTRAVIOLET RADIATION ON THE MAGNETIC ISOTOPE
EFFECT IN FRESHWATER PHOTO-REDUCTION OF ORGANIC AND INORGANIC
MERCURY
3.1. INTRODUCTION…………………………………………………………………58 3.2. METHODS …………………………………………………………………… 62
3.2.1. Cleaning equipment…………………………………………………… 62 3.2.2. Photo-reduction experiments…………………………………………… 62 3.2.3. Hg concentration analysis ……………………………………………… 65 3.2.4. Matrix transfer in preparation for isotope analysis…………………… 65 3.2.5. MC-ICP-MS analysis…………………………………………………… 67 3.2.6. Reporting analytical uncertainty ……………………………………… 68 3.2.7. Calculations…………………………………………………………… 69
3.3. RESULTS……………………………………………………………………… 71 3.3.1. Concentrations ………………………………………………………… 71 3.3.2. Mass-dependent fractionation………………………………………… 73 3.3.3. Mass-independent fractionation………………………………………… 75
3.4 DISCUSSION …………………………………………………………………… 78 3.4.1. Relationship between MIF and light available ………………………… 78 3.4.2. MIF and Hg(II) ………………………………………………………… 78 3.4.3. MIF and MeHg………………………………………………………… 83 3.4.4. Mass-dependent fractionation………………………………………… 87 3.4.5. Implications for the natural world ……………………………………… 87
3.5 CONCLUSIONS ………………………………………………………………… 92 FIGURES…………………………………………………………………………… 93 TABLES…………………………………………………………………………… 102 LITERATURE CITED ……………………………………………………………… 110 CHAPTER 4: MASS-INDEPENDENT FRACTIONATION DURING CHEMICAL REDUCTION
OF AQUEOUS INORGANIC MERCURY IN THE ABSENCE OF LIGHT 4.1. INTRODUCTION…………………………………………………………………116 4.2. METHODS…………………………………………………………………… 117 4.3. RESULTS……………………………………………………………………… 118 4.4. DISCUSSION…………………………………………………………………… 118 4.5. CONCLUSION………………………………………………………………… 121 FIGURES…………………………………………………………………………… 122 TABLE……………………………………………………………………………… 125 LITERATURE CITED ………………………………………………………………… 126 CHAPTER 5: CONCLUSIONS 4.1. CONCLUSIONS………………………………………………………………… 129 LITERATURE CITED ………………………………………………………………… 131 APPENDIX A.1. TABLE OF ALL PHOTOEXPERIMENTS AND ANALYTICAL STATUS…………… 133
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LIST OF TABLES Table 2.1. Isotopic abundances of mercury ………………………………………21 Table 2.2. Typical Hg concentrations in natural, unpolluted freshwaters………… 32 Table 2.3. Example depths of 99% attenuation of solar radiation at various
DOC concentrations………………………………………………… 44 Table 3.1. Summary of solar radiation measurements…………………………… 102 Table 3.2. Isotopic Data for Hg(II) aqueous photo-reduction with UV filtration…103 Table 3.3. Summary of subsrate losses through photo-reduction, isotopic
signatures and fractionation factors…………………………………… 105 Table 3.4. Isotopic Data for MeHg aqueous photo-reduction with UV filtration…106 Table 3.5. Summary of substrate losses through photo-reduction, isotopic
signatures and fractionation factors…………………………………… 108 Table 3.6. Sample recoveries after matrix transfer, all experiments…………… 109 Table 3.7. Calculated properties of CH3HgL complexes, from Tossell 1998…… 85 Table 4.1. Isotopic Data for Hg(II) dark reduction with SnCl2 in the presence
of organic matter……………………………………………………… 125
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LIST OF FIGURES Figure 3.1. Schematic of the photochemical reaction apparatus………………… 93 Figure 3.2. Schematic of the phase separator…………………………………… 94 Figure 3.3. Mercury concentration in the substrate reservoir over time for
Hg(II) and MeHg. …………………………………………………… 95 Figure 3.4. Mass-dependent isotopic fractionation (202Hg) of the substrate
reservoir for Hg(II) and MeHg……………………………………… 96 Figure 3.5. Mass-independent isotopic fractionation (MIF, 199Hg), observed
in odd isotopes only, for Hg(II) photo-reduction experiments……… 97 Figure 3.6. Mass-independent isotopic fractionation (MIF, 199Hgas a
function of Hg(II) remaining………………………………………… 98 Figure 3.7. Mass-independent isotopic fractionation (MIF, 199Hg), observed
in odd isotopes only, for MeHg photo-reduction experiments……… 99 Figure 3.8. Mass-independent isotopic fractionation (MIF, 199Hg), observed
in odd isotopes only, as a function of MeHg remaining…………… 100 Figure 3.9. 201Hg versus 199Hg for photochemical reduction of Hg(II)
and MeHg…………………………………………………………… 101 Figure 4.1. Mercury concentration over time for dark chemical reduction
of Hg(II) by SnCl2……………………………………………………122 Figure 4.2. Mass-dependent isotopic fractionation (202Hg) as a function of Hg
remaining for dark chemical reduction of Hg(II) by SnCl2………… 123 Figure 4.3. Mass-independent isotopic fractionation (199Hg), as a function of
Hg(II) remaining for dark chemical reduction of Hg(II) by SnCl2 … 124
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CHAPTER 1: INTRODUCTION
This thesis is principally concerned with understanding and quantifying the effects of
frequency and intensity of sunlight on the magnitude and signature of mass-independent
fractionation (MIF) that occurs through photo-reduction of mercury (Hg). MIF in Hg
isotopes has been observed in both laboratory demonstrations (Bergquist and Blum 2007,
Estrade et al. 2009, Zheng and Hintelmann 2009, 2010a, 2010b, Malinovsky et al. 2010;
Wiederhold et al., 2010; Sherman et al., 2010) and natural samples (Bergquist and Blum
2007, Bergquist and Blum 2009, Ghosh et al. 2008, Biswas et al. 2008, Jackson et al.
2008, Gantner et al. 2009, Laffont et al. 2009, Sherman et al. 2009). Mass-independent
effects, and stable isotope behavior of Hg in general, have great potential to improve our
understanding of biogeochemical cycling of Hg in nature.
Mercury has always circulated in the atmosphere, oceans and terrestrial surface. Natural
emissions originate from volcanic activity (as Hg(II)P and Hg(0)g, Patterson and Settle
1987, Zambardi et al. 2009), geothermal activity (Hg(0) from fumaroles and hot springs,
Gustin et al. 2008), halos of Hg enrichment in deposits surrounding hot springs and
sulfide ore bodies, (Varekamp and Buseck 1984) and emissions from land areas that are
naturally enriched in Hg (see review Fitzgerald 2007). The majority of surface Hg
deposits are in the form of red and black cinnabar (HgS), which is poorly soluble in water
under standard conditions and mobilized chiefly by erosion. Based on sediment cores
taken from remote seepage lakes (Engstrom and Swain 1997 and references therein), peat
deposits (Jensen and Jensen 1990, Martinez-Cortizas et al. 1999) and glacial ice cores
(Vandal et al. 1993, Schuster et al. 2002), anthropogenic emissions have increased the Hg
that circulates in the atmosphere, oceans and terrestrial surface by a factor of
approximately 3 since the industrial revolution. Mercury use in commercial products
(thermostats, batteries, fluorescent lighting) and processes (cement, metals, iron & steel
and chlor-alkali production) has peaked and is declining in much of the western world;
currently the largest source category of anthropogenic emissions worldwide is stationary
combustion of coal (and to a lesser extent fossil fuels) in which it is present as a trace
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element (Pacyna et al. 2010). Emissions in many of these categories have been growing
in Asia with China now being the largest global emitter due to its reliance on coal for
power and fuel (Jaffe and Strode 2008, Pacyna et al. 2010). Burial in sediments, in
particular deep-ocean sediments, is the ultimate sink for Hg but this process is far too
slow to counterbalance anthropogenic inputs (Fitzgerald and Lamborg 2007, Selin 2009).
The actively cycling lifetime of Hg in the atmosphere, terrestrial surface and ocean
surface is difficult to assess. Although the atmosphere contains less than one percent of
actively cycling Hg, it is the major avenue of global distribution. There is a great deal of
uncertainty around inputs to the atmosphere. Volcanic emissions, for example, are
inherently difficult to monitor and are spatially and temporally variable. They are
estimated by applying the few Hg/SO2 mass ratios available to SO2 concentrations
measured for a given eruption (Gustin et al. 2008). This leads to estimates that vary by
over an order of magnitude, and the portion of global emissions recently assigned to
volcanoes ranges from less than five to over 20 percent (Zambardi et al. 2009, Selin
2009). The most recent inventory of primary anthropogenic emissions is by Pacyna et al.
2010, for the year 2005. They cite an uncertainty in emissions by continent of at best
27% (North Amercia) and at worst 50% (Africa and South America). Mercury is emitted
to the atmosphere dominantly in three forms: volatile Hg(0), reactive gaseous Hg(II)
(RGM), and particulate Hg(II) (HgP). The atmospheric residence times of RGM and HgP
are days to weeks (Selin 2009), whereas the residence time for highly volatile Hg(0) is on
the order of 0.75 y (the uncertainty in this estimate is a factor of 2, Lindberg et al. 2007).
Most Hg in the lower atmosphere is therefore Hg(0) and well-mixed. Thus, a given
region may have important Hg contributions from distant sources as well as local ones.
For example, Sunderland et al. 2008 estimate that approximately half of the atmospheric
deposition to the Bay of Fundy area in the late 1990s was from global sources.
Deposition to terrestrial and ocean surfaces is thought to follow Hg(0) oxidation to Hg(II)
and scavenging by water droplets and/or particulate matter. Measurement networks exist
in some regions for wet deposition (snow and rain), but nowhere for dry deposition and
this is a significant gap in our understanding of Hg cycling (Lindberg et al. 2007, Selin
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2009). Vegetation and soils contain about 80% of actively cycling Hg. Some portion of
the Hg newly deposited to land is promptly revolatilized (“prompt recycling”, 5 to 60%
depending on the surface, Selin 2009 and references therein). The remainder is
incorporated into the soil and vegetation pools where it is released over longer timescales
(decades to centuries; “legacy mercury”) by chemical transformations or physical erosion
and runoff. It has been found that a portion of Hg newly deposited to water bodies
(which contain about 20% of actively cycling Hg) also revolatilizes promptly (Garcia, et
al. 2005, Poulain et al. 2006). To complicate the problem, revolatilization depends on
substrate concentration, matrix composition, light, temperature, precipitation, and for
soils, moisture content (Gustin et al. 2008). Assessing the relative importance of natural
versus anthropogenic sources of mercury in soils, water bodies and the atmosphere, and
also the fluxes between them, are therefore fundamental problems in understanding the
global Hg cycle. In addition, it is unlikely that Hg mobilized by natural biotic and abiotic
processes can be measured without interference of previously deposited Hg (“legacy
mercury”) of anthropogenic origin (Lindberg et al. 2007).
Although high or repeated exposure to different forms of Hg can have serious health
consequences, the most important toxicity risk that Hg poses to humans is as
methylmercury (MeHg) and exposure is mainly through consumption of fish. MeHg is a
neurotoxin and bioaccumulates, particularly in aquatic food webs. There is at least a
hundred-fold variation in Hg concentrations among species of fish and shellfish, but
concentrations in piscivorous fish can reach levels one million times greater than in the
surrounding water (Lindqvist et al. 1991, Mergler et al. 2007). The sites where MeHg
production is most intense in nature are wetlands, lake sediments and anoxic bottom
waters (Rudd 1995, Eckley and Hintelmann 2006). Since Hg is delivered to these areas
mainly as particulate and/or ionic species, biogeochemical cycling is a principle
modulator of mercury toxicity (Barkay and Wagner-Dobler 2005). Methylation appears
to be largely mediated by sulfur-reducing and iron-reducing bacteria (Compeau and
Bartha 1985, Gilmour et al. 1992, Selin 2009) and although Hg can be methylated
abiotically (Akagi and Takabatake 1973, Weber 1993), microbial production is sufficient
to account the amount of MeHg that is bioaccumulated (Fitzgerald and Lamborg 2007).
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Both the amount of bioavailable inorganic Hg and physiochemical factors that stimulate
microbial activity affect MeHg production (Barkay and Wagner-Dobler 2005 and
references therein). Characterizing the methylation and degradation capacities of natural
systems will clarify the relationship between continued anthropogenic loading and
contamination of food webs, including fish harvested for human consumption.
Stable isotope fractionation has been successfully used to trace biogeochemical processes
at both human and geologic timescales. The recent development of an analytical
technique using cold vapor multicollector inductively coupled plasma mass spectrometry
(CV-MC-ICP, Lauretta et al. 2001) now allows accurate measurements of Hg isotope
composition, and the reported range of isotopic signatures imparted by mass-dependent
fractionation (MDF) already exceeds 7‰ (typical uncertainty of ± 0.2 ‰, Bergquist and
Blum 2009, Sherman et al. 2010). In order to use the isotopes of Hg as a tool to track its
transport and fate in the environment, however, it is necessary to characterize source
signatures and, in particular, processes that induce fractionation.
Mass-independent fractionation (MIF) of Hg has been observed in some environmental
samples, such as snow (Sherman et al., 2010), fish and food webs (Bergquist and Blum
2007, Epov et al. 2008, Jackson et al. 2008, Gantner et al. 2009, Laffont et al. 2009),
mosses and lichens (Ghosh et al. 2008, Carignan et al. 2009, Bergquist and Blum 2009),
sediments, soils and peats (Foucher and Hintelmann 2006, Biswas al. 2008, Gehrke et al.
2009, Feng et al. 2010) and hydrothermal ores (Sherman et al. 2009; 0.13 ± 0.06‰) but
not in others, for example hydrothermal deposits and ores (Hintelmann and Lu 2003,
Smith et al. 2005, 2008, Sherman et al. 2009), chondrites (Lauretta et al. 2001), volcanic
emissions (Zambardi et al. 2009), and fly ash from waste incineration (Estrade et al.
2009). MIF has also been associated with some abiotic transformative processes, such as
photo-reduction of Hg(II) (Bergquist 2007, Zheng 2009, 2010b, Sherman 2010) and
photo-degradation of MeHg (Bergquist 2007, Malinovsky 2010) and volatilization from a
liquid phase (Estrade 2009). It has been observed not to occur in other processes, such as
hydrothermal transport (Smith 2005, 2008), volatilization from an aqueous phase (Zheng
et al. 2007), oxidative condensation of Hg(0) to Hg(II)P in volcanic plumes (Zambardi
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2009) and biological transformations such as demethylation and Hg(II) reduction (Kritee
et al. 2007, 2008, 2009). The Hg samples displaying large MIF to date (>1‰) are snow,
fish, food web components and moss and may be assumed to have undergone previous
photochemical transformation. For those sediments, hydrothermal deposits, soils and
peats that display small but significant MIF, previous photochemical transformation of
the Hg that is preserved in them is likely, although other MIF pathways might need to be
considered.
Reliable measurements of Hg isotopic composition are only a decade old with the first
report published in 2001 (Lauretta et al. 2001). Thus Hg isotope is a young field, and
there are many processes left to investigate and many natural archives that have yet to be
sampled. With the evidence available to date, the emerging picture is that MIF (greater
than approximately 0.2 ‰) is particular to photochemical transformations. MIF should
not be altered by mass-dependent fractionation (MDF). This is not to say that MIF
signatures are unsusceptible to alteration; they can be changed by other MIF processes or
diluted by the mixing of Hg pools bearing different MIF signatures (Bergquist and Blum
2009). Understanding the factors that control MIF will permit assessment of which
signatures are susceptible to alteration by successive transformations or mixing and
whether the various contributions to MIF can be deciphered. This is a prerequisite for
using MIF signatures to shed light on the transformations and fate of environmental Hg.
The geochemical processes investigated in this thesis are photochemical reduction of
inorganic Hg(II) and photochemical degradation of MeHg in freshwater aqueous
reservoirs. Two plausible mechanisms, the nuclear volume effect (NVE) and the
magnetic isotope effect (MIE), have been put forward to explain mass-independent
anomalies in Hg isotopes. The most probable mechanism behind the large MIF
signatures observed in Hg is the MIE, which can influence the rates of chemical reactions
that are electron spin selective. Photo-reduction and photo-degradation have been
demonstrated to produce relatively large MIF (> 2 ‰) in lab experiments, and the
photochemistry involved is characterized by radical intermediates. Radical pair reactions
are spin-selective and provide a venue for the manifestation of the MIE. Investigating the
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factors that affect MIF in photo-reduction and photo-degradation will thus shed light on
MIF processes in general. In addition, photo-reduction represents a significant pathway
by which the Hg burden of aqueous reservoirs is lost. Likewise, photo-degradation of
MeHg represents a significant portion of the demethylation capacity of ecosystems. If
MIF or some aspect of it is particular to these pathways among others, then it could
provide a method of identifying and quantifying them absolutely or relative to other
reduction and demethylation processes respectively. The extent to which MIF is
characterized by photo-reduction and photo-demethylation respectively will determine
how useful it is in quantifying these processes.
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RESEARCH OBJECTIVES:
While the MIE is the most probable cause of the large kinetic MIF observed in the
experimental results of Bergquist and Blum 2007, the only direct test of the mechanism
to date has been by Malinovsky et al. 2009. These investigators found that MIF during
photo-demethylation of MeHg was suppressed in solutions with ascorbic acid and with
ammonia, attributed to suppression or scavenging of the radical pair during
decomposition. Based on the explanation above, the efficiency of the MIE should be
affected by several aspects of the reaction pathway. The frequency of light required to
transform a molecule or complex into a radical pair will depend on the nature and energy
of the bond that needs to be broken. This means there will be an energy threshold below
which radiation cannot effect electronic transition to a radical pair. Bond energy is
affected by the nature of the ligand, and by solution properties such as pH and ionic
strength. The chemical composition of the solution can affect the formation and fate of
the radicals, as demonstrated by Malinovsky et al. Varying the intensity of radiation may
also affect the magnitude of MIF expression
One of the more straightforward tests of the mechanism to perform is elimination of the
photo-generated radical pair. If the higher energy wavelengths of the solar spectrum are
removed gradually, then at some point the incident radiation will not have enough energy
to excite an Hg complex to form a radical pair. This closes the avenue of expression for
the MIE.
Objectives:
To determine whether removing the UVA or the UVB portions of the electromagnetic
spectrum affects the expression and magnitude of the MIF observed during the aqueous
photo-reduction of MeHg and Hg2+ respectively.
Does the MIF allow us to distinguish and quantify photoreductive processes from other
process that reduce Hg in freshwater aqueous environments?
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THESIS OUTLINE
CHAPTER 1: INTRODUCTION. This chapter introduces some of the outstanding problems in mercury research and the
potential of stable isotope geochemistry to elucidate them. In particular, it introduces the
importance of a mechanistic understanding of mass-independent fractionation (MIF) as a
prerequisite for using mass-independent isotopic signatures to characterize fluxes and
transformations of mercury in the environment.
CHAPTER 2: OVERVIEW OF MERCURY ISOTOPES AND FRESHWATER MASS TRANSFER
PROCESSES AND PHOTOCHEMISTRY This chapter provides background information on mercury stable isotopes as well as on
processes of physical transport and species transformations of Hg in freshwaters.
CHAPTER 3: EFFECTS OF ULTRAVIOLET RADIATION ON THE MAGNETIC ISOTOPE
EFFECT IN FRESHWATER PHOTO-REDUCTION OF ORGANIC AND INORGANIC MERCURY This chapter addresses the objective of this thesis. It describes experiments in which
quartz reactors containing Hg(II) or MeHg in aqueous solutions with organic matter
were simultaneously exposed to three different light regimes to examine the effect of
photo-reduction on the isotopic fractionation of the respective Hg species. It demonstrates
that limiting the energy of incident radiation has a pronounced effect on MIF, supporting
the magnetic isotope effect (MIF) as the mechanism behind the MIF observed during
aqueous photo-reduction of Hg species. It also demonstrates that comparable amounts of
total photo-reduction may coincide with different amounts of MIF depending on the
wavelengths of light available for photo-reduction. This will make quantitatively relating
photo-reduction and MIF difficult.
CHAPTER 4: MASS-INDEPENDENT FRACTIONATION DURING CHEMICAL REDUCTION OF
AQUEOUS INORGANIC MERCURY IN THE ABSENCE OF LIGHT This chapter describes an experiment where Hg(II) is chemically reduced in the dark. It
is not directly related to the objectives of this thesis and experimental conditions were
significantly different. It demonstrates that during the process of dark chemical reduction
of aqueous Hg(II) by SnCl2 (in the presence of organic matter) and subsequent evasion of
the Hg(0), mass-independent anomalies in isotopic composition are produced.
9
Attributing these effects to a particular mechanism or combination of mechanisms is not
possible without further investigation.
10
STATEMENT OF AUTHORSHIP AND PUBLICATION STATUS
CHAPTER 1: INTRODUCTION Contributors: Carla Rose, Bridget Bergquist. Contributions: Research and writing was carried out by CR with guidance and editing by BB. Publication Status: Not submitted for publication. CHAPTER 2: OVERVIEW OF MERCURY ISOTOPES AND FRESHWATER
PHOTOCHEMISTRY CHAPTER 3: EFFECTS OF ULTRAVIOLET RADIATION ON THE MAGNETIC ISOTOPE
EFFECT IN FRESHWATER PHOTO-REDUCTION OF ORGANIC AND INORGANIC MERCURY Contributors (in alphabetical order other than first author): Carla Rose, Bridget Bergquist and Joel Blum. We thank Marcus Johnson, Sanghamitra Ghosh and Georges Lacrampe-Couloume for discussions and experimental assistance. Contributions: Photoexperiments were carried out by CR. Isotopic analysis were carried out both by MJ and by CR under close supervision of MJ. These analyses were funded by an NSF Grant to JB. Isotopic analysis of additional dark controls will be carried under supervision of SG. Data interpretation and writing was done by CR with input from BB. Publication Status: Not submitted for publication CHAPTER 4: MASS-INDEPENDENT FRACTIONATION DURING CHEMICAL REDUCTION OF
AQUEOUS INORGANIC MERCURY IN THE ABSENCE OF LIGHT Contributors (in alphabetical order other than first author): Carla Rose, Bridget Bergquist, Joel Blum and Marcus Johnson. We thank Sanghamitra Ghosh for discussions. Contributions: Photoexperiments were carried out by CR. Isotopic analysis were carried out by MJ and funded by an NSF Grant to JB. Data interpretation and writing was done by CR with input from BB. Publication Status: Not submitted for publication.
11
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Barkay, T. and Wagner-Dobler, I., 2005. Microbial transformations of mercury: Potentials, challenges, and achievements in controlling mercury toxicity in the environment, Advances in Applied Microbiology, Vol 57.
Bergquist, B. A. and Blum, J. D., 2007. Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems. Science 318, 417-420.
Bergquist, R. A. and Blum, J. D., 2009. The Odds and Evens of Mercury Isotopes: Applications of Mass-Dependent and Mass-Independent Isotope Fractionation. Elements 5, 353-357.
Biswas, A., Blum, J. D., Bergquist, B. A., Keeler, G. J., and Xie, Z. Q., 2008. Natural Mercury Isotope Variation in Coal Deposits and Organic Soils. Environmental Science & Technology 42, 8303-8309.
Carignan, J., Estrade, N., Sonke, J. E., and Donard, O. F. X., 2009. Odd Isotope Deficits in Atmospheric Hg Measured in Lichens. Environmental Science & Technology 43, 5660-5664.
Compeau, G. C. and Bartha, R., 1985. SULFATE-REDUCING BACTERIA - PRINCIPAL METHYLATORS OF MERCURY IN ANOXIC ESTUARINE SEDIMENT. Applied and Environmental Microbiology 50, 498-502.
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Engstrom, D. R. and Swain, E. B., 1997. Recent declines in atmospheric mercury deposition in the upper Midwest. Environmental Science & Technology 31, 960-967.
Epov, V. N., Rodriguez-Gonzalez, P., Sonke, J. E., Tessier, E., Amouroux, D., Bourgoin, L. M., and Donard, O. F. X., 2008. Simultaneous determination of species-specific isotopic composition of Hg by gas chromatography coupled to multicollector ICPMS. Analytical Chemistry 80, 3530-3538.
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Estrade, N., Carignan, J., Sonke, J. E., and Donard, O. F. X., 2009. Mercury isotope fractionation during liquid-vapor evaporation experiments. Geochimica Et Cosmochimica Acta 73, 2693-2711.
Feng, X. B., Foucher, D., Hintelmann, H., Yan, H. Y., He, T. R., and Qiu, G. L., 2010. Tracing Mercury Contamination Sources in Sediments Using Mercury Isotope Compositions. Environmental Science & Technology 44, 3363-3368.
Fitzgerald, W. F. and Lamborg, C. H., 2007. Geochemistry of Mercury in the Environment. In: Holland, H. D. and Turekian, K. K. Eds.)Treatise on Geochemistry. Elsevier.
Foucher, D. and Hintelmann, H., 2006. High-precision measurement of mercury isotope ratios in sediments using cold-vapor generation multi-collector inductively coupled plasma mass spectrometry. Analytical and Bioanalytical Chemistry 384, 1470-1478.
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17
CHAPTER 2: OVERVIEW OF MERCURY
ISOTOPE GEOCHEMISTRY, FRESHWATER
MASS TRANFER PROCESSES AND SPECIES
TRANSFORMATIONS
2.1. MASS-DEPENDENT FRACTIONATION (CLASSICAL
EFFECT) The discovery and early study of multiple stable isotopes of elements happened in the
first decades of the twentieth century and is reviewed by Urey 1947. Some of the earliest
demonstrations of variable isotopic compositions of elements were of differences in the
vapor pressures of neon and of hydrogen (Keesom and van Dijk 1931, Urey et al. 1932).
The theoretical laws that describe how much of isotopic fractionation happens (i.e., the
mass-dependent laws) were also developed early (Urey 1947, Biegleisen 1949) and gave
rise to the field of traditional stable isotope geochemistry. This early isotope work
focused on lighter isotopic systems where the relative mass differences, and thus the
expected fractionation of the isotopes, were large. It also focused on elements that were
amenable to gas source mass spectrometry (i.e., H, C, N, O and S). The varying isotopic
composition of different reservoirs provided insight into, for example, historical climates
(the oxygen isotope paleotemperature scale, Urey et al. 1948), metabolism and reaction
mechanisms (the relative abundance of carbon isotopes in plants, Wickman 1952) and
fluxes in the meteoric water cycle (Craig 1961). With recent analytical developments and
improvements in precision, the isotopic variations of heavier elements (including Mg, Ca,
Fe, Cu, Zn, Mo, and Hg) is now being explored and has led to the development of a new
field called non-traditional stable isotope geochemistry.
Isotopic compositions in stable isotope geochemistry are generally reported as the per mil
(‰) deviation of the ratio of two isotopes from the same ratio observed in a standard
18
reference material. This deviation is referred to as delta () notation. The lowest-mass
major isotope is usually used as the denominator:
otherX (‰) = (other/lowest majorRsampleother/lowest majorRstandard) – 1) x 1000
Relative differences in isotopic ratios can be determined with far greater precision than
absolute differences.
2.2.1. Mechanisms of mass dependent fractionation
Much of the observed variations in isotope ratios found in nature follow mass dependent
fractionation (MDF) laws. These were the first laws used to predict and describe isotope
fractionation (Urey 1947, Bigeleisen and Mayer 1947, Bigeleisen 1949). Mass-
dependent fractionation laws have also been derived in detail, as in Hulston and Thode
1965, Mariotti et al. 1981 and Young et al. 2002. Mass dependent fractionation occurs
in both equilibrium and kinetic reactions.
Equilibrium effects arise from the tendency of systems to adjust to minimize their energy.
The equilibrium constant of a chemical reaction is the difference in the standard free
energies of the reactants and product divided by RT. For an isotopic exchange reaction,
this is a small difference between large numbers that cannot be well-characterized by
classical models. The equilibrium constant for an isotopic exchange reaction can be
determined through a quantum mechanical consideration of two equilibrium constants:
those for each of the two isotopic molecules to dissociate into atoms. The magnitude of
these dissociation constants depends on the translational, rotational and vibrational
energies of the molecules. It is differences in the vibrational energy of the two molecules
at the quantum mechanical level that dominantly leads to equilibrium isotopic
fractionation, such that the heavy isotope is more stable in the molecule with stronger
bonds. The lighter isotope will preferentially be found as individual atoms and in weaker
bonds. The vibrational energy differences decrease as a function of temperature (1/T2) so
that at higher temperatures, isotope fractionation disappears (Bigeleisen and Mayer
1947). It is worth mentioning that chemical equilibrium does not guarantee isotopic
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equilibrium because the times required to achieve isotopic and chemical equilibria
respectively are not necessarily related. As an example, the chemical equilibrium
between CO2(g) and water is established more quickly that oxygen istotope equilibrium
between the two (Fritz and Fontes 1980).
Kinetic isotope effects are associated with processes that are fast, unidirectional or
incomplete (such as evaporation, diffusion and enzymatic reactions). Zero point energies
(the energy at which a molecule vibrates in its ground state) are lower for heavier
isotopes, so the energy gap between the ground state of a heavier isotope and its bond
dissociation energy is larger. Lighter isotopes require less energy to break chemical
bonds and frequently display slightly lower chemical stability. Lighter isotopes may also
have slightly greater translational velocities and diffusion coefficients. This also leads to
kinetic fractionation where transfer between compartments/reservoirs is incomplete or
unidirectional.
For both equilibrium and kinetic mass-dependent isotope effects, the relationship
between the mass difference of the isotopes (m) being studied and the magnitude of
fractionation is approximately linear (Hulston and Thode 1965, Bigeleisen 1949, Young
et al. 2002). For this reason, MDF effects are the largest for hydrogen and tend to
decrease. For the first few rows of the periodic table, m/m is generally 5 to 10% or more
but by row six (containing Hg), m/m is only 1 to 3%. The magnitude of isotopic
fractionation, however, does not decrease proportionately. Smith et al. 2005 note that all
elements from Fe to Hg showing unexpectedly large fractionation have multiple
oxidation states.
2.1.2. Mercury isotopes and mass-dependent fractionation.
Separation of Hg isotopes was achieved as early as 1920 by Bronsted and von Hevesy by
evaporation and subsequent condensation on a cold surface. In 1950, Alfred Nier
reported the relative isotopic abundances of a laboratory sample of Hg on a gas-source,
electron bombardment mass spectrometer using sample-standard bracketing and
calibration of machine mass discrimination using 36Ar and 40Ar. These values became
20
the isotopic composition of Hg accepted by IUPAC until Zadnik et al. repeated the
measurements in 1989.
In the late 20th century, there was considerable interest in the cosmochemical behavior of
Hg and several investigations of Hg in the Muchison and Allende carbonaceous
chondrites (for example) were undertaken between 1970 and 2001. However these
studies were conducted by neutron activation analysis (NAA) and the measured
variations have been shown to be analytical artifacts (Nier and Schlutter 1986). Nier and
Schlutter 1986 obtained samples of the Allende meteorite and measured all Hg isotopes
relative to 202Hg using an electron bombardment gas-source mass spectrometer. They
failed to find anomalous abundance ratios relative to a laboratory standard for 196/202Hg
(uncertainty of ~ 20‰) or any of the other isotopes (uncertainty of ~ 1‰).
Lauretta et al. 2001 undertook measurements of both the Allende and Murchison
chondrites on a multi-collector inductively coupled plasma mass spectrometer (MC-ICP-
MS). The samples were reduced to Hg(0) online and introduced through a gas-liquid
separator (cold vapor generation; this produces a steady signal for isotope measurement),
along with a Tl aerosol to correct for machine mass bias and bracketed with a reference
standard. They found no significant variations in isotopic abundances between the
chondrites within their precision of 0.2 – 0.5‰, with the exception of 200/202Hg in the
Murchison which fell only slightly outside the analytical uncertainty.
The CV-MC-ICP-MS technique of Lauretta 2001 has become the standard method of
determining Hg isotope ratios. Blum and Bergquist 2007 proposed NIST SRM 3133 as a
universal reference standard and the method described above for reporting ratios, XXXHg/198Hg where XXX is the isotope of interest. These have generally been adopted,
and investigation of the isotopic variation in terrestrial Hg samples has been underway
for the last few years. Mercury has seven stable isotopes which span a relative mass
difference of 4% as summarized in Table 2.1.
21
Table 2.1 Isotopic abundances of mercury
Atomic Mass Number
Relative Abundance (%)
196 0.16 198 10.0 199 16.9 magnetic 200 23.1 201 13.2 magnetic 202 29.7 204 6.8
The lowest-mass isotope (196Hg) has by far the lowest abundance (0.15 %) and is
generally not measured. Thus 198Hg is used as the denominator, and the accepted
reference standard has come to be NIST Standard Reference Material 3133 (Blum and
Bergquist 2007):
XXXHg (‰) = (XXX/198Rsample/XXX/198RSRM 3133) – 1) x 1000.
The other five isotopes can be measured conveniently by MC-ICP-MS. Isotopic shifts
are not as large as for lighter elements such as C, N and O, but with typical uncertainties
for 202Hg of 0.1 to 0.3 ‰ (2SD, Bergquist et al. 2009), isotopic differences in samples
can be readily resolved.
Mercury’s active redox chemistry, tendency to form covalent bonds, biological cycling,
phase changes and volatile form (Hg0) gives rise to many circumstances in which isotope
fractionation based on nuclear mass and diffusion coefficients may be expressed. A large
range of mass-dependent isotopic signatures, 202Hg, has been reported for hydrothermal
systems and ore (from -4 ‰ to nearly 3‰ , Hintelmann and Lu 2003, Smith et al. 2005,
2008, Sherman et al. 2009). Sherman et al. 2009 conclude that multiple processes
contribute to the variation in isotopic composition of such systems. Smith et al. 2005
identify the boiling of hydrothermal fluids as an important fractionating process. For
example, Hg mobilized from magma or country rock by epithermal systems travels
principally as Hg(0)aq in reduced, low sulfur fluids. Lighter isotopes of Hg(0) partition
preferentially into the vapor phase as the fluid ascends and boils. Isotopically heavy Hg
22
is deposited in solid solution at depth, whereas isotopically light Hg(0) can mix with and
be oxidized by groundwater at the surface and precipitate with reduced sulfur as
isotopically light cinnabar. The range in 202Hg signatures resulting from hydrothermal
fractionation is much wider (for example, -3.5 ± 0.1 ‰ to 2.1 ± 0.1 ‰, Smith et al.
2005) than the -0.6 ± 0.2 ‰ that is typical of crustal rocks investigated so far (Bergquist
and Blum 2009, Smith et al. 2008).
Phase changes have been demonstrated to produce MDF in Hg. Estrade et al. 2009
investigated both equilibrium and kinetic volatilization of pure liquid mercury. In the
equilibrium experiments, the Hg vapor was slightly depleted in the heavier isotopes
(202Hgvapor was 0.20 ‰ less negative that 202Hgliquid) and was independent of
temperature in the range 2 to 22 ºC. Kinetic volatilization under a 10-5 bar vacuum
showed an increase in residual liquid 202Hg of 7.23 ± 0.22‰ for a fraction of Hg
remaining (fR ) = 0.3, a dramatic enrichment in the heavier isotopes. Zheng et al. 2007
tested free volatilization of aqueous Hg(0) (unassisted by sparging) by reducing a
solution of inorganic Hg(II) SnCl2 in a reactor while flushing the headspace with N2.
They report that volatilization was subject to Rayleigh-type mass-dependent fractionation
only, resulting in enrichment of the unvolatilized substrate in 202Hg by 1.5 ± 0.1 ‰
when fR had dropped to 0.04.
Reductive process, both biological and abiotic, have been demonstrated to result in mass-
dependent fractionation of Hg. Biological reduction of Hg(II) (Kritee et al., 2007, 2008)
has been reported to enrich the heavier isotopes in the unreacted substrate. Kritee et al.
2008 observed that MDF during reduction of Hg(II) to Hg(0) by various strains of
bacteria initially followed a Rayleigh distillation model. As the reaction proceeded,
however, MDF was suppressed to different degrees according to the microbial strain
studied. The authors hypothesized that with unlimited bioavailable Hg(II), reduction of
Hg(II) by MerA is the rate-limiting and single fractionation-determining step. As cell
densities grow and the Hg(II) supply shrinks, non-fractionating process such as Hg(II)
diffusion and cell uptake become rate-limiting. Reduction by MerA is then quantitiative
and fractionation is suppressed. Biological demethylation and reduction of MeHg also
23
induces MDF. Kritee et al. 2009 observed preferential degradation of the lighter isotopes
of MeHg during the initial phases of mer-mediated microbial degradation. Complete
dampening of fractionation was observed as the reactions progressed, seemingly after cell
densities exceeded a certain level. The authors suggest that, as breakage of the organo-
Hg bond by MerB happens prior to and much more slowly than reduction of Hg(II) by
MerA, that catalysis by MerB was initially rate-limiting and fractionation determining but
that at higher cell concentrations, uptake of MeHg by the cell becomes rate-determining
and causes no significant fractionation. These examples demonstrate that the magnitude
of MDF during enzymatic reaction depends the reaction conditions. If the fractionating
step is rate-limiting, biological processes can produce significant changes in Hg isotopic
composition. If a previous step becomes rate-limiting, however, the fractionating step
will quantitatively transform all isotopes and no MDF will be observed.
Depending on reaction conditions, the product of reduction, Hg(0), may be isotopically
light and can be lost from the reaction medium through volatilization. As mentioned
above, light isotopes preferentially volatilize from an aqueous phase. Abiotic reduction
of aqueous Hg(II) and MeHg has also been shown to enrich the unreacted substrate in
202Hg. This includes photochemical processes in the presence of organic matter
(Bergquist and Blum 2007, Zheng and Hintelmann 2009, 2010b). Malinovsky et al. 2010
report MDF during photo-degradation of MeHg under UVC radiation in the absence of
organic matter, but it seems doubtful that this would occur in nature (see, for example,
Zhang and Hsu-Kim 2010). Abiotic reduction by organic matter occurs in the dark as
well, and has been shown to enrich the unreacted substrate in 202Hg (Bergquist and
Blum, 2007; Zheng and Hintelmann 2009). Chemical derivitization of Hg species (dark
chemical ethylation of Hg(II) by NaBEt4 , Yang and Sturgeon 2009) and biological
methylation (Rodriguez-Gonzalez et al. 2009) have both been shown to induce MDF.
Overall, it might be expected that Hg remaining in a cell, the reaction medium or the
aqueous phase generally will be isotopically heavy.
In contrast, oxidative condensation of Hg(0) as Hg(II) on particulates and acidic aerosols
in volcanic plumes shows the oxidized Hg(II)P is enriched in the heavy isotopes
24
(Zambardi et al. 2009). If this process is representative of atmospheric oxidation of
Hg(0) in general, it may be expected that the Hg(II) deposed as Hg(II)P and Hg(II)aq will
be isotopically heavy and Hg(0) remaining in the atmosphere will be isotopically light.
Assessing the relative importance of reductive and oxidative processes, as well as of
phase changes, on Hg isotopes in nature is difficult at this early stage of research.
Natural archives show both positive and negative signatures. Zambardi et al. 2009 report
202Hg from -1.74 to 0.01‰ for fumarolic gasses. Biswas et al. 2008 report a range of -
2.8 to 0‰ in 202Hg for 30 coal deposits from the United States, China and Russia-
Kazakhstan. Lichen and moss show negative MDF to about -2 ‰ (Ghosh et al. 2008,
Carignan et al. 2009, Bergquist and Blum 2009). Soils, peat and sediments show larger
negative range than coal (as low as -4 ‰) but also exhibit positive signatures (with
202Hg up to 1 ‰ or more; Foucher and Hintelmann 2006, Biswas et al. 2008, Ghosh et
al. 2008, Foucher et al. 2009, Gehrke et al. 2009.) Fish and food web 202Hg signatures
range from -3 ‰ to nearly 2 ‰ (Bergquist and Blum 2007 and 2009, Epov et al. 2008,
Laffont et al. 2009, Gantner et al. 2009). Sherman et al. 2010 report signatures in Arctic
snow ranging from -0.49 to 0.70 ± 0.16‰.
Differentiating between natural and anthropogenic Hg emissions based on isotopic
compositions is difficult at present because of the limited measurements, the likely source
variability and also fractionation induced by processes after the point source (Bergquist
and Blum 2009). It is worth mentioning, however, that the various coal deposits
sampled by Biswas et al. 2008 were generally isotopically distinguishable. In addition,
Foucher et al. 2009 demonstrated that there is a well-resolved difference between
background Hg isotopic composition of Adriatic sea sediment (depleted in 202Hg) and
Hg derived originally from the Idrija region (depleted in 202Hg), which drains into the
Adriatic. One of the largest Hg mines in the world is located in the Idrija region, and
isotopic compositions can be used to track, differentiate and quantify its contributions to
Hg in the Adriatic. This is a well-constrained system with one dominant Hg source that
did not undergo much fractionation during transport, however, and the general usefulness
of MDF to track the transport and fate of anthropogenic Hg in the environment requires
further investigation.
25
2.2. MASS-INDEPENDENT FRACTIONATION
Mass-independent fractionation (MIF) was first reported by Thiemens and Heidenreich in
1983, in the ozone produced by molecular oxygen in an electric plasma. It was
subsequently observed in other kinds of reactions, such as UV-photolysis (Thiemens &
Jackson 1987) and in ion-molecule encounters (Griffith & Gellene 1992). Mass-
independent isotopic signatures have since been helpful in problems such as deciphering
the reaction pathways of ozone (Schueler et a. 1990) and determining the historical
oxidative capacity of Earth (see review: Thiemens 2006). Small MIF (much less than 0.5
‰) has also been observed in several other elements, including Ti, Cr, Zn, Sr, Mo, Ru,
Cd, Sn, Te, Ba, Nd, Sm, Gd, Yb and U (see review: Fujii 2009).
MIF is the difference in the measured isotope ratio from the ratio predicted by mass-
dependent fractionation laws. It is reported in capital delta notation, and can be
approximated (for 5 ‰) by
XXXHg = XXXHg – (202Hg*XXX)
where is the kinetic or equilibrium factor applicable to the isotope of interest (see
Younge et al. 2002; see also the methods section of Chapter 3 of this document).
2.2.1. Mercury isotopes and mass-independent fractionation
MIF of Hg isotopes is observed in many natural samples and is only ever seen in the odd
isotopes. The most dramatic deviations are positive anomalies in food webs, with 199Hg
signatures in fish tissue reaching nearly 5‰ (Bergquist and Blum 2007, 2009, Epov et al.
2008, Jackson et al. 2008, Gantner et al. 2009, Laffont et al. 2009) and negative
anomalies, with 199Hg signatures lower than -5 ‰, in arctic snow (Sherman et al.
2010). When measuring MIF, typical analytical uncertainties are lower than 0.1 ‰,
2(Bergquist and Blum 2009) To date, O and S are the only other elements that have
displayed large degrees ( >1‰) of MIF in natural samples (Bergquist and Blum 2009).
26
Tin, however, has been reported to undergo MIF during laboratory experiments, with
117Sn reaching nearly 5 ‰ (Malinovsky et. al 2009).
Hydrothermal deposits, ore, host rock and volcanic emissions rarely display MIF
(Hintelmann and Lu 2003, Smith et al. 2005, 2008, Sherman et al. 2009, Zambardi et al.
2009). Where they do, it is quite small. For example, Sherman et al. 2009 report small
but significant MIF at a hydrothermal site in Yellowstone (199Hg = 0.13 0.06 ‰) that
likely had some exposure to sunlight. Whereas, hydrothermal samples from a deep sea
floor rift showed no MIF. The authors suggested that a portion of the Hg in the
hydrothermal fluids from Yellowstone had been photochemically reduced in its
geological history. Biswas et al. 2009 report 199Hg in coal from 0.3 to -0.6 ± 0.04 ‰. In
contrast to the samples above, natural samples that are part of the surface pools of Hg that
undergo active biogeochmical cycling including photochemistry do display MIF. Soils,
sediments and peats display small negative MIF (less than 1 ‰, Foucher and Hintelmann
2006, Biswas et al. 2008, Ghosh et al. 2008, Jackson et al. 2008, Foucher et al. 2009,
Gehrke et al. 2009). Moss and Lichen display similar small, negative MIF (Ghosh et al.
2008, Carignan et al. 2009, Bergquist and Blum 2009).
The first demonstration of a process that induced MIF was photochemical reduction of
methylmercury (MeHg) and Hg(II) in the presence of dissolved organic matter
(Bergquist and Blum, 2007). Odd isotopes were preferentially retained in the unreacted
substrate, resulting in mass-independent anomalies of over 2 ‰. Examination of
199Hg/201Hg revealed that the ratios of each process were distinct: 1.00 ± 0.02 (2SE)
for of Hg(II) and 1.36 ± 0.03 (2SE) for of MeHg. The authors suggested that the ratio
might be unique to the pathway, which raised the possibility of using MIF signatures to
identify and quantify photochemical transformations in nature. They suggested the
mechanism causing this MIF was the magnetic isotope effect (MIE), which will be
discussed hereafter. MIF during photochemical reduction of Hg has also been reported
by Yang and Sturgeon 2009, Zheng and Hintelmann 2009, 2010b. Malinovsky et al.
2010 report MIF during photochemical reduction of MeHg, with a 199Hg/201Hg ratio of
1.28 ± 0.03 (2SE).
27
Recently, Sherman et al. 2010 reported MIF during photochemical reduction of snow.
The direction of MIF is opposite aqueous photochemical reduction, with the odd isotopes
being depleted in the substrate snow. They note a 199Hg/201Hg ratio of 1.07 ± 0.04 ‰.
They are unsure about the mechanism, but suggest multi-step heterogeneous redox
reactions in the quasi-liquid layer on snow crystal surfaces as a starting point for
investigation.
While equilibrium process do not produce the large MIF signatures that kintetic photo-
processes do, there have been a few demonstrations of small (< 0.2 ‰) Hg MIF during
equilibrium processes. Equilbration of Hg metal with its vapor phase has been reported
to cause small MIF with 199Hg ranging from +0.10 to 0.14 ± 0.03 ‰ in the vapor phase
(Estrade et al. 2009) and from +0.13 to 0.31 ± 0. 05‰ (Ghosh et a., in prepartation). This
indicates the non-volatilized substrate was slightly depleted in the odd isotopes. Estrade
et al. obtained a ratio 199Hg/201Hg of 2.0 ± 0.6 and Ghosh et al. a ratio of 1.62 ±
0.06(2SE), which are distinct from the ratios obtained for photo-reductive processes by
Bergquist and Blum 2007 and Malinovsky et al., 2010. Equilibrium fractionation is
attributed to the nuclear volume effect (NVE), which will be discussed hereafter. Estrade
et al. also observed 199Hg ranging from -0.12 to -0.01 ± 0.03 ‰ in residual liquid
phase for kinetic volatilization of pure Hg. Weiderhold et al. 2010 report small MIF (<
0.1‰) during equilibration binding of aqueous Hg(II) with various thiols.
While volatilization of aqueous Hg(0) has been shown not to produce MIF in Hg (Zheng
and Hintelmann 2007), Zheng and Hintelmann 2010a report that dark chemical reduction
of Hg(II) with SnCl2 results in small but significant and consistent negative MIF in the
unreacted substrate. In contrast, dark chemical reduction of Hg(II) in the presence of
organic matter was not observed to induce MIF in the unreacted substrate, as reported by
Bergquist and Blum 2007 and Zheng and Hintelmann 2009. Zheng and Hintelmann 2009
do report a small, positive MIF anomaly in volatilized Hg trapped after dark abiotic
reduction, indicating the unreacted substrate was slightly depleted in the odd isotopes. It
should be noted that dark reduction in the presence of organic matter is a slow process,
28
and investigators were considering reactions with a maximum of 20% loss of initial Hg.
The SnCl2 reduction, on the other hand, could be pushed to completion.
Several of the processes demonstrated to cause MDF do not cause MIF in Hg. Smith et
al. 2005, 2008 and Sherman et al., 2009, conclude that hydrothermal processes do not
induce MIF. None of the microbially mediated reduction or degradation processes
investigated by Kritee et al. 2007, 2008 or 2009 show any MIF and Kritee et al. 2009
present a detailed discussion of why MIF is unlikely to be induced by dark enzymatic
pathways. Yang and Sturgeon 2009 report that dark ethylation of Hg(II) by NaBEt4
shows only strong MDF. Neither do Malinovsky et al. 2009 see MIF in dark methylation
of inorganic tin by methylcobalamin. They do see MIF if they expose the solution to UV
light, but only for acidic and semineutral pH. Oxidative condensation of Hg(0) as Hg(II)
on particulates and acidic aerosols in volcanic plumes shows no MIF (Zambardi et al.
2009). In general, MIF signatures larger than approximately 0.2‰ seem likely to be
associated with a small group of reaction pathways and the pathways seem to be
identifiable by their 199Hg/201Hg ratio (Bergquist and Blum 2009). MIF has the
potential to highlight these few pathways of Hg transformation among the many
pathways that result in MDF and this potential increases the utility of Hg in tracking the
transport and fate of Hg in the environment.
2.2.2. Mechanisms of mass-independent fractionation
There are two plausible mechanisms that have been proposed to account for the MIF
observed in Hg isotopes: The nuclear volume effect (NVE) and the magnetic isotope
effect (MIE). The NVE was postulated by Bigeleisen (1996) to explain the even-odd
MIF observed for uranium (Fujii et al. 1989a, 1989b) and is relevant for heavy elements
in general. Schauble 2007 gives an extensive theoretical discussion of its equilibrium
effects on the isotopes of mercury and thallium. As atoms get larger, nuclear volume and
nuclear charge radii do not increase linearly with mass. This effect becomes significant
for heavy elements such as U, Hg and Tl, but has been observed for lighter elements such
as Cr as well (see review: Fuji et al. 2009). It is predicted to have the largest deviations
29
from MDF for the odd isotopes resulting in an even-odd pattern for isotopic fractionation.
NV predicts that the volume of the odd isotopes (ex. 199Hg) is closer to that of the next
lower even nucleus rather than being intermediate between the volumes of its even
neighbours (ex. 198Hg, 200Hg). This effects the shielding of electrons and thus bond
strengths. Schauble 2007 calculated expected NV contributions to Hg MIF based on
mean squares of nuclear charge radii, which he retrieved from calculations by Angeli
2004, and predicted mass deviations for 196Hg, 199Hg, 201Hg and 204Hg. Mass deviations
are observed, however, only in 199Hg and 201Hg. Ghosh et al. 2008 pointed out that the
experimentally determined 204Hg charge radius (Hahn et al. 1979) differed from the
Angeli et al.’s calculations; after revising Schauble’s scaling factor they found no mass-
independent anomalies were expected for 204Hg.
The smaller relative size and larger relative charge density of the odd nuclei mean that s
orbitals, which have a higher electron density at the nucleus, will be more strongly bound
to the odd nuclei. P, d and f electronic orbitals have a smaller electron density at the
nucleus. When orbiting an odd nucleus, these three will be more effectively screened by
the s electrons and therefore more weakly bound to odd nuclei. The two major oxidation
states of mercury (Hg0 and Hg2+) have zero or two electrons in the 6s orbital respectively.
At equilibrium, therefore, the even isotopes will be more stable in a state where they have
a lower electron density around the nucleus (Hg2+) because their valence orbitals (5d)
will be more strongly bound. The odd isotopes will be more stable in a state with a
higher electron density around the nucleus (Hg0) because their valence orbitals (6s) will
be more strongly bound (Bigeleisen 1996 and Schauble 2007). If all isotopes are in the
form of elemental Hg, the odd isotopes will form weaker covalent bonds and be more
volatile.
The second mechanism that is likely to produce MIF in mercury is the magnetic isotope
effect, first observed for 13C in the photodecomposition of dibenzyl ketone (Buchachenko
et al. 1976) and proposed to apply to natural mercury isotopic fractionation by Bergquist
and Blum 2007. This effect is a result of interactions between the magnetic moments of
nuclei (only odd isotopes have non-zero nuclear magnetic moments) and the spin
30
magnetic moments of electrons (any paramagnetic species will have net spin angular
momentum and therefore a magnetic moment). The interactions are called hyperfine
coupling, and are so small that they have no effect on the equilibrium characteristics of
chemical systems (Turro1983, Bigeleisen 1996, Engel and Reid 2006). They can,
however, produce a kinetic effect by allowing isotopes with different nuclear spins to
react at different rates.
Consider as an example an aqueous photo-dissociation reaction that involves a pair of
radical intermediates. The two radical electrons could be spin-paired in a singlet state:
one electron has spin + ½ and the other - ½ such that their spins are precisely opposite in
three-dimensional space. Within the solvent cage formed by water molecules, such a
radical pair is highly reactive and likely to recombine or disproportionate (Turro 1983
and Engel and Reid 2006). The two radical electrons could also be in a triplet state: the
electrons are unpaired and have spins of + ½, + ½ or - ½,- ½ or + ½, - ½. In this last
case, the projections of the two spins on a reference axis add to zero, but the spins are not
opposite in three-dimensional space.
Recombination (or disproportionation) requires the radical electrons to finish in a spin-
paired singlet state, but this path is unavailable to the electrons in the triplet state because
it does not conserve their total spin angular momentum. Within a solvent cage, the triplet
radicals could undergo chemical rearrangement resulting in a structurally different radical
pair or they could diffuse out of the solvent cage and become free (uncorreleated)
radicals. A third possibility is triplet-singlet conversion, also called intersystem crossing
(ISC), to produce a set of singlet radicals. If circumstances are favorable, hyperfine
coupling can induce ISC through a simultaneous exchange of electron-nuclear spin (total
spin angular momentum is conserved). Re-phasing or reversal of an electron spin results.
A spin-selective process such as ISC can sort isotopes because the (odd) magnetic nuclei
provide a mechanism for electrons to undergo ISC that the (even) non-magnetic nuclei do
not. Radicals with magnetic nuclei will undergo triplet-singlet spin conversion more
quickly and accumulate preferentially in the recombination (or disproportionation)
products, whereas the non-magnetic nuclei will be more likely to diffuse out of the
31
solvent cage or undergo structural rearrangement (see reviews: Turro 1983, Buchachenko
2001).
In order for hyperfine coupling to effectively induce ISC, the radical fragments must
separate to a physical distance where the energy gap between the singlet and triplet states
is on the same order or smaller than the hyperfine interaction. At such a distance, the
original chemical bond will have completely dissociated and one or more solvent
molecules may separate the radicals. The probability of the newly-singlet radical
fragments re-encountering each other before separating completely is high because they
will “bounce” against the surrounding solvent molecules and each other several times
before finding avenues through the solvent to become fully separated (Turro et al. 2009).
Example reaction schemes for the photo-reduction of Hg2+ and the photo-degradation of
MeHg have been proposed (Bergquist and Blum 2007, 2009; Buchachenko 2009;
schematic courtesy of B. Bergquist) and are presented below for Hg2+:
X Hg L hv X Hg
L
T 198,200202,204 L
Hg
dissociation tofree radicals
199,201
X Hg
L
S
Hg0
and for MeHg:
CH3HgXodd
hv
X Hg
C
H3
even Hg0
T-S conversion kTS(odd) > kTS(even)
32
2.3. FRESHWATER MERCURY MASS TRANSFER
PROCESSES AND SPECIES TRANSFORMATIONS
2.3.1. Concentrations
Aqueous freshwater Hg species are usually grouped into three categories: total mercury
(HgT), dissolved gaseous mercury (DGM) and methyl mercury (MeHg). Representative
concentrations are displayed in Table 2.2 below.
Table 2.2. Typical Hg concentrations in natural, unpolluted freshwaters
Species low
concentration high
concentration Comment Reference a few hundred
pg/L
Arctic: 3 lakes and 1 wetland
Amyot et al. 1997a
same 100 high-altitude
U.S. lakes Krabbenhoft et al. 2002
a few ng/L 23 northern
Wisconsin lakes Watras et al. 1995a
total mercruy (HgT)
same 6 temperate
Canadian lakes Amyot et al. 1997b
HgT*10-3 or less high altitude lakes Krabbenhoft et al. 2002
0.1*HgT to 0.01*HgT
Wisconsin Lakes Watras et al. 1995a methyl
mercury (MeHg)
0.05 ng/L 3.0 ng/L Experimental Lake Area, NW Ontario
Sellers et al. 1996
HgT*10-5 HgT*10-2 Wisconsin lakes;
Great Lakes Vandal et al. 1991, Jeremiason et al. 2009
106-215 pg/L maximum summer
concentrations Garcia et al. 2005a
dissolved gaseous mercury (DGM)
17-126 pg/L maximum summer
concentrations Amyot et al. 1997b
Both DGM and MeHg can be measured directly. HgT is generally the portion in an
acidified sample that can be reduced by SnCl2, purged from the solution and collected on
a gold trap. Mercury remaining after subtraction of DGM and MeHg from the total is
assumed to be Hg(II) of some variety. Approximately a quarter of both Hg(II) and MeHg
is estimated to be particulate bound in freshwaters (Watras et al., 1995a, Rolfhus et al.
2003). These estimates are based on the difference in the total Hg (HgT and MeHg)
detected in unfiltered versus filtered water samples (Watras et al. 1995a) or multiplying
the particulate concentration by suspended particulate matter concentration (Rolfhus et al.
33
2003). There remain, however, substantial uncertainties in how Hg species partition
between the aqueous phase and particulate matter, as well as in the chemical lability of
particulate-bound Hg (Lindberg et al. 2007). Lamborg et al. 2003 conclude that over
99% of Hg in freshwaters is complexed to organic ligands. Some authors see no
significant seasonal variation in either MeHg or HgT when measuring in spring and fall
(ex. Watras et al. 1995a) but others report seasonal variation by a factor of two or three in
both HgT and MeHg (Krabbenhoft et al. 2002, Selvendiran et al. 2009),
2.3.2. Sources
2.3.2.1. Deposition
For precipitation-dominated lakes, atmospheric deposition (rain, snow and dry
deposition) is the dominant source of Hg (Watras et al. 1995a, Watras and Morrison
2008). Although ambient tropospheric Hg is principally Hg(0) (98% or more), the
majority of Hg in rain is derived from atmospheric scavenging of particulate Hg.
Fitzgerald et al. 1991 report that HgR at their Wisconson study site averaged only 19%
(2.0 0.3 ng/L) of the total Hg content in rain and 52% (3.1 2.2 ng/L) in snow. They
conclude that atmospheric deposition could easily account for the total mass of Hg
detected in fish, water and sediments in their study lake. MeHg in precipitation is
sometimes detectable in trace amounts (tens to hundreds of pg/L, Fitzgerald et al. 1991,
Hammerschmidt et al. 2007).
2.3.2.2. Runoff
In drainage lakes, runoff can be a large source of HgT. Runoff is a significant source of
MeHg in water bodies, in particular runoff from wetland areas (Watras et al. 1995a,
Sellers et al. 2001, Selvendiran et al. 2009). Terrestrial organic carbon is an important
transport vector for HgT and MeHg. Events causing an export of catchment soils
(agriculture, forestry, urbanization) to water bodies can contribute substantially to
waterborn Hg loads. Export to lakes from watersheds generally lags far behind
atmospheric deposition, but is expected to continue for decades or longer after
atmospheric deposition subsides (Harris et al. 2007, Watras and Morrison 2008, Mills et
al. 2009).
34
2.3.2.3. In-situ production of MeHg
Inorganic methylation of Hg has been both argued (Weber 1993) and demonstrated
(Akagi and Takabatake 1973, Gardfeldt et al., 2003). In general, however, most mercury
methylation is considered a biologically mediated process (Fitzgerald and Lamborg 2007,
Selin 2009). While sulfate reducing bacteria (SRB) are assumed to play the largest role,
iron-reducing bacteria have also been implicated (as in Kerin et al. 2006) and
methanogens have not been ruled out (Barkay and Wagner-Dobler 2005). For example,
mechanisms involving corrinoid-containing proteins have been proposed (Barkay and
Wagner-Dobler 2005 and references therein). Cinnabar may serve as a substrate,
although uncharged soluble forms of mercuric sulfide are the more likely source for
methylation. Mercury bound to organic ligands may also be methylated (Barkay and
Wagner-Dobler 2005), however, and Schaefer and Morel 2009 demonstrate that mercury
methylation is greatly enhanced by the formation of Hg-cysteine complexes.
Methylation of mercury can occur both in anoxic sediments (Gilmour et al. 1992) and
anoxic hypolimnia of lakes (Watras et al. 1995b, Eckley and Hintelmann 2006).
Methylation in the water column has been associated with bacterial communities
migrating to the oxic-anoxic interface that can move from the sediment surface to higher
in the water column during periods of stratification. While methylation rates are more
intense in the surficial sediments, methylation zones in the water column can represent a
larger volume (depending on the depth of the hypolimnion, which changes seasonally)
and also generate significant quantities MeHg. MeHg produced in the water column may
also account for more of the MeHg that is bioaccumulated.
2.3.3. Sinks
2.3.3.1. Outflow
Lake and watershed morphology, precipitation, groundwater discharge and recharge and
other variables that influence the hydraulic residence time of water in a lake affect
whether a given water body will be an overall source or sink for mercury. The relative
magnitude of inflow and outflow of a particular Hg species varies from case to case (for
35
example, seepage versus drainage lakes) and from season to season (Krabbenhoft et al.
2002, Selvendrian et al. 2009).
2.3.3.2. Sedimentation
Sediments represent both the largest repository of MeHg and HgT (Fitzgerald et al. 1991)
and also an important removal pathway. Mercury sequestration through sedimentation
can be calculated using annual sediment deposition rates and Hg concentration in
sediment material (Krabbenhoft et al. 1992, Mills et al. 2009, Rolfhus et al. 2003).
Unfortunately, estimates of sediment accumulation rates are lacking in many studies
(Jeremiason et al. 2009) and researchers frequently estimate sedimentation rates by the
discrepancies of other mass components (ex. Sellers et al. 1996, Watras and Morrison
2008, Selvendiran et al. 2009).
2.3.3.3. Bioaccumulation
The relationship between environmental Hg loading and bioaccumulation is the subject
of ongoing research. Both MeHg and and Hg(II) species can passively diffuse across a
lipid membrane in the form of small, uncharged complexes. Trophic transfer, however,
is more efficient for MeHg. MeHg accumulates in the cell cytoplasm of phytoplankton
and is assimilated more efficiently (a factor of 4) by zooplankton than Hg(II), which
remains bound to phytoplankton membranes and is more likely to be excreted by
zooplankton (Mason et al. 1996). Biota can respond quickly to alterations in deposition
rates. A recent and striking example of this has been noted during the METAALICUS1
project, involving the addition (or spike) of three different highly enriched stable isotopes
of Hg (198Hg, 200Hg and 202Hg) separately to a lake, its watershed and a large wetland that
drains into the lake. The methylated lake spike was detected in the anoxic bottom waters
three days after the first surface addition. In sediments, zooplankton and benthos it was
detected after one month and in several fish species after 2 months. Spiking increased the
Hg loading of the lake by approximately 120% annually. By the end of the third year of
consecutive spiking, MeHg in water and biota was 30 to 40% higher than they would
1 Mercury Experiment to Assess Atmospheric Loading in Canada and the United States, conducted at Lake 658 in the Experimental Lakes Area of northwestern Ontario.
36
have been if the spike had not been added (Harris et al. 2007). Concentrations of Hg in
the water column and in short-lived organisms such as periphyton, zooplankton and water
mites can respond quickly to reductions in atmospheric deposition and to MeHg
availability (Orihel et al. 2008). Hg concentrations in older individuals of long-lived
species, especially those participating in benthic food webs,are likely to be much slower
to respond (Orihel et al. 2008). Reductions in fish Hg concentration (50% or more) have
been reported, however, in both freshwater and marine environments subjected to point-
source contamination within ten years of remedial actions. Hg levels of fish in remote
waterbodies have likewise been observed to systematically decline when a decline in wet
or bulk Hg deposition is also documented (see review: Munthe et al. 2007). Total Hg
concentrations in sediments, however, are slow to respond to decreases in loading (ex.
Orihel et al. 2008). Recent experimental manipulations such as the labeled ecosystem
additions in the METAALICUS project are designed to increase our understanding of the
relationships between Hg loading and the response of Hg in organisms. Reports on these
manipulations will be forthcoming once enough time has passed for adequate observation
of the recovery phase.
2.3.3.4. Degradation of MeHg
Methyl mercury can be degraded by both biotic and abiotic processes. Two microbial
degradations mechanisms are known to exist. Oxidative demethylation (OD) results
principally in CO2 and in Hg(II), which becomes available for re-methylation. Small
amounts of CH4 are generated. Pathways of oxidative demethylation are not known and
pure cultures have not been characterized (Barkay and Wagner-Dobler 2005 and
references therein). Reductive demethylation (RD) produces only CH4 and Hg(0), which
may escape the aqueous reservoir by evasion. The best known path of RD is mediated by
microorganisms that carry Hg resistance (mer) operons, which include the enzymes
organomercury lyase and merucuric reductase. Two dominant factors that control which
of the two pathways is followed are redox conditions and levels of mercury
contamination. RD is favored when mercury concentrations exceed thousands of ng/g
sediment in anaerobic conditions (or hundreds of ng/g sediment in aerobic conditions),
whereas OD dominates at low mercury concentrations and anoxic conditions. Overall,
37
MeHg is degraded reductively in highly contaminated environments but in less
contaminated conditions, OD dominates.
It does not appear that dark abiotic processes result in significant degradation of MeHg.
Aqueous solutions containing MeHg and organic matter have been kept strictly in the
dark under laboratory conditions. Concentrations have been monitored for days to weeks
at a time, with no reported decrease (Bergquist and Blum 2007, Zheng and Hsu-Kim
2010). In sediments and bottom waters, microbial processes most likely dominate MeHg
degradation, whereas in light-exposed environments, photo-degradation may be the major
mechanism (Barkay and Wagner-Dobler 2005).
Photodemethylation was reported as an important sink for MeHg in an Ontario lake by
Sellers et al. 1996. The investigators estimated that it represented approximately twice
the sum of MeHg inputs, which necessitated significant in-situ MeHg production. In a
more detailed mass-balance analysis, Sellers et al. 2001 concluded that over the course of
a single year, photo-degradation represented ~78% of MeHg losses from the lake under
study. Other studies support the significance of MeHg photo-degradation. Krabbenhoft
et al. 2001 found photo-degradation to be active in the top meter of a high altitude lake
and to be a MeHg sink of comparable importance to sedimentation and stream flow.
Hammerschmidt and Fitzgerald 2006 concluded that photodecomposition could account
for as much as 80% of the MeHg mobilized annually from sediments in Toolik Lake,
Alaska.
UV radiation has been identified as the portion of naturally available radiation resulting
in the highest rates of photo-degradation (Sellers et al. 2001, Lehnherr and St. Louis
2009, Hammerschmidt and Fitzgerald 2006). Hammerschmidt and Fitzgerald 2006 found
that photodecomposition due to visible radiation was important in their Arctic study lake.
Photodecomposition of MeHg was a linear function of PAR (which decreased
exponentially with depth) with the exception of the top few cm of the water column.
Photo-decomposition in these top few centimeters is more intense (see also Sellers 1996),
but Hammerschmidt and Fitzgerald 2006 point out that the penetration depth of visible
38
radiation is much larger than of UV radiation, and the photodecomposition zone of
visible radiation represents a much larger volume. They did not identify a mechanism of
photodemethylation due to visible light (see Table 2.3 p. 44) but noted that
photosensitization of dissolved organic material (DOM) seemed to be a requisite.
When normalized for initial mercury concentration and incident radiation,
photodecomposition rates for a temperate Ontario lake (Sellers et al. 1996), for controlled
field experiments (Lehnherr and St. Louis 2009) and the Alaskan lake from
Hammerschmidt and Fitzgerald 2006 were similar (0.0018 ng m2 L-1 E-1).
Environmental factors such as pH and DOC were very different in these lakes, and it is
possible that this rate is approximately uniform among freshwater bodies (Lehnherr and
St. Louis 2009). In addition to light intensity and frequency, photo-degradation rates also
depend on the presence of humic substances. Specifically, the relative concentrations of
MeHg and humic acid with thiol-containing ligands has been shown to speed degradation
rates (Zhang and Hsu-Kim 2010), possibly by decreasing electron transition energies
sufficiently for low UV wavelengths to induce demethylation.
2.3.3.5. Volatilization
There are both biotic and abiotic process that carry out the reduction of Hg(II) to Hg(0) in
natural freshwaters. Dissolved gaseous mercury (Hg(0); DGM) is a volatile species that
can diffuse to the lake surface and escape to the atmosphere. DGM is also a product of
both the biotic and abiotic processes that degrade MeHg mentioned above.
Concentrations of MeHg, however, are typically one or more orders of magnitude lower
than those of HgT. (see Table 1.2). Contributions of MeHg to DGM, while representing
an important component of the MeHg aquatic cycle, do not comprise a very significant
portion of DGM produced in lakes. Experimentally, it is more convenient to monitor the
production of dissolved gaseous mercury than the reduction of either Hg(II) or MeHg.
The phrase “DGM production” is somewhat interchangeable with “Hg reduction”. While
DGM production represents the fate of reduced Hg(II) well, it is not indicative of MeHg
reduction in natural waters where a large excess of Hg(II) species are present. The
39
following discussion of DGM production mechanisms and conditions, therefore, applies
to Hg(II).
Inorganic mercury(II) can be reduced by biotic and abiotic pathways. Both direct and
indirect biotic pathways have been reported. As with reductive demethylation, the most
well-studied pathway of direct biological reduction of Hg involves the mer operon (and
MerA, mercuric reductase), which allows cells to avoid the toxicity of Hg(II) by reducing
it to Hg(0) and removing it from their environment. It is inducible by high environmental
concentrations of Hg(II) (50 pM or more, Siciliano et al. 2002). Actively growing
cultures (105 – 106 cells/mL) in a cell medium contaminated by as much as 20 ppm
Hg(II) can reduce 99% of the Hg in under an hour (Kritee et al. 2007). Between 1 and
10% of culturable microbes taken from natural, uncontaminated environments are likely
to have a functional MerA enzyme; this proportion increases among culturable microbes
that survive in Hg-contaminated sites (Kritee et al. 2007).
Mer-independent microbial reduction has also been observed. For example, it has been
reported for the species Shewanella oneidensis MR-1 under anoxic conditions, which
cannot live at the micromolar concentrations of mercury tolerated by mer-carrying
species (Wiatrowski et al. 2006). Despite not being able to survive at high concentrations
of Hg, it has been observed to reduce Hg(II) more effectively than its mer-containing
transconjugate at nM concentrations (Wiatrowski et al. 2006). Such mer-independent
pathways of microbial reduction may contribute to the removal of Hg(II) in anoxic
aqueous environments, which are important sites for Hg(II) methylation.
Thus there are microbes that do not carry an Hg-specific reductive pathway that
nevertheless appear to mediate the reduction of Hg species. Ben-Bassat and Mayer 1978
showed that illuminated algal suspensions of Chlorella pyrenoidosa volatilize Hg(0) at a
fairly constant rate, given a renewable supply of Hg(II). By alternating light and dark
conditions, they documented tight coupling between illumination and volatilization of
Hg(0) and suggested the reduction occurred in the cell medium as a result of leakage of
some reducing agent from the cells, possibility metabolites produced during
40
photosynthesis. Poulain et al. 2004 observed peaks in DGM concentration at the bottom
of the metalimnion in two lakes in the Experimental Lake Area (Ontario) at depths where
over 90% of incident radiation had been attenuated, which coincided with maxima in
chlorophyll and phytoplankton biomass. No transcripts of the merA gene could be
detected from RNA extracts.
The following discussion of abiotic reductive pathways will focus on photo-degradation,
but it must be noted that dark abiotic reduction does occur, although at much slower rates
than photo-induced processes. A small increase in DGM concentrations is sometimes
observed in field samples taken early in the morning and incubated, starting before dawn,
in dark bottles (for example Garcia et al. 2005a) . Such dark abiotic reduction is expected
to be a result of humic substances. In laboratory experiments, Alberts et al. 1974 showed
that purified (and sterilized) humic acid from pond sediment would slowly reduce Hg(II)
(added as HgCl2) at room temperature in a way that showed some dependence on pH.
Allard and Arsenie 1991 confirmed that reduction of Hg(II) by humic substances (in
filtered solutions, using sterile equipment) would take place aerobically in the dark. Low
rates of dark reduction relative to photo-reduction have been demonstrated in laboratory
experiments (17% compared to 90% HgT losses over 5 hours, Bergquist and Blum 2007)
and in-situ field experiments (up to 50% of DGM photo-production in a wetland but
negligible in lakes, Garcia et al. 2005a). By definition, dark reduction is not dependent
on sunlight, however, and can continue at all hours of the day.
The production of Hg(0) through the midday hours of summer can represent several
percent of the HgT burden in surface waters, for example up to ~8% over the course of a
day in lakes (Amyot et al. 1997a, Amyot et al. 1997b). Through multiple additions of
isotopically enriched Hg(II) to field mesocosms, Poulain et al. 2006 showed that 30 to
60% of the added mercury was lost to the atmosphere, and that loss was unrelated to the
rate of loading.
41
The link between solar radiation and in situ freshwater DGM production was
demonstrated by Amyot et al. 1994. Samples of lake water (at approximately 5 mg/L
DOC) incubated in transparent bottles yielded DGM concentrations 2 to 9 times higher
than those kept in black bottles. Blocking UVB radiation (280-320 nm) by wrapping
bottles in mylar film did not affect DGM production significantly. DGM concentrations
showed a clear diel pattern, with the lowest concentrations (~ 0.2 pM) before sunrise and
the highest at noon (0.6 to 0.7 pM). Comparison of midday concentrations from August,
September and November 1993 showed large seasonal differences as well (the November
maximum DGM was less than 0.2 pM compared with 0.7 pM in August).
UV radiation on the whole has been observed to contribute more to DGM production
than visible radiation. Investigators find that visible radiation alone generates far less
DGM (over 70% decrease, Garcia et al. 2005a) or none at all (O’Driscoll et al. 2006).
In some cases, it has been shown that UVB makes a significant contribution to DGM
production apart from UVA (Amyot et al. 1997a, 1997b, Garcia et al. 2005a). Other
studies have not shown that UVB is more important than UVA (Amyot et al. 1994,
1997b).
An additional important influence on photo-reduction is organic matter. Amyot 1997a
proposed that high DOC concentrations in some lakes would absorb most of the UV
radiation at the lake surface, making it unavailable for direct reduction of Hg complexes.
In contrast, increased penetration in clear lakes would allow UV radiation to affect DGM
production significantly. Garcia et al. 2005a also concluded that UVB contributions to
photo-reduction were more important in clearer lakes. They found a strong negative
correlation between DGM production and DOC concentration for samples exposed to
UVB radiation (r = 0.99) and a strong positive correlation between DGM production and
DOC concentration under UVA and visible range (400-700 nm) exposure (r=0.99). They
remark that incoming photons (presumably in the UVA range) could be photosensitizing
the chromophoric dissolved organic carbon. It should be mentioned that some lakes
show no DGM production at all, for example the arctic lake Amituk (Amyot et al. 1997a)
42
and Lake Eerie (Amyot et al. 1997b); in both cases the authors suggested low levels of
photoreducible complexes as an explanation.
In general, DGM levels incubated in temperate lake water samples under sunlight reach a
plateau after a few hours (about 3, Amyot et al. 1997b) and drop slightly thereafter.
DGM levels in samples incubated in black bottles show slow decay. By incubating pre-
purged samples (14 hours in quartz bottles under cloudless conditions followed by
flushing with Hg-free air in an effort to exhaust the photoreducible Hg), Garcia et al.
2005a showed that plateaus were not likely due to limited HgR because DGM production
resumed upon exposure to daylight the following day. The authors suggest that plateaus
are more likely a balance between oxidation and reduction.
Photo-oxidation was suggested as a simultaneous competitive process with photo-
reduction by Lalonde et al. 2001 to explain why lake water samples spiked with Hg(0)
(introduced by bubbling with a gas) and subjected to UVB radiation showed a decrease in
Hg(0) over time. Garcia et al. 2005b gathered evidence of competition between photo-
reduction and photo-induced oxidation in natural freshwater systems by collecting
surface water samples in batches and monitoring DGM concentration over time under
different incubation conditions. They noted initial DGM concentration, DGM after one
hour of dark and light incubation respectively and DGM after two hours of dark and light
incubation respectively. Samples incubated in the light showed an increase in DGM
while samples incubated in the dark showed a decrease. The greatest decrease was
observed in samples collected at 12:50 pm, when oxidation was equivalent to 77% of the
DGM production under sunlight exposure. The authors concluded that both processes
occur simultaneously in sunlight with the leading process depending on light intensity: at
high irradiance, photo-reduction dominates but under cloudy conditions and towards
sunset, photo-induced oxidation dominates.
To investigate photo-reduction alone, therefore, it is necessary to find a way to decouple
it from photo-oxidation. One way to do this is to remove the DGM as it is formed so that
photo-oxidation has no substrate (i.e., via sparging with Hg-free gas), as in Allard and
43
Arsenie 1991 and O’Driscoll et al. 2006. The photochemical reactions can be
generalized as follows (as in O’Driscoll et al. 2004):
Hgreducible + photo-reductants DGM + photo-oxidants
Thus removal of DGM pushes this equation toward reduction.
In order to estimate the losses of Hg via reduction to Hg(0) in an environmental system, it
is necessary to quantify the relative importance of all three (biotic, dark aboitic and
photochemical) reduction pathways. Distinguishing between these three processes can be
difficult in the environment. All three types of reduction are drawn on by Poulain et al.
2004 to explain the DGM depth profiles they obtained for two lakes in the Experimental
Lakes Area (sampled in summer, fall and under ice in the late winter). DGM
concentrations were always highest in the top two meters of the water column (~ 2 pM
L-1), but a second, smaller peak in concentration was frequently observed at the limit of
the euphotic zone, which was at the bottom of the metalimnion (at depths of 3.5 to 5.5 m,
depending on the lake and sampling occasion). A third, small peak (up to 0.3 pM L-1)
was observed at the sediment-water interface when the bottom of the hypolimnion was
anoxic. They proposed that DGM production at the surface was photo-chemically
mediated, since DGM production stopped in the dark and filtration to remove organisms
did not affect the production rates. DGM production in the metalimnion was associated
with phytoplankton biomass. As discussed above, and the authors drew on Ben-Bassat
and Mayer’s (year) observations of reduction of Hg(II) by illuminated algal suspensions
to explain it. No transcripts of the merA gene could be detected from RNA extracts. For
the peaks observed at the sediment-water interface, they propose anoxic reactions
involving humic substances or microbial activities. Similar rationales can be applied to
other DGM depth profiles, for example Vandal et al. 1991 who report that DGM
concentrations reach a maximum at a depth of 7 m before decreasing and then rising
slightly at the sediment-water interface.
2.3.4. Light penetration
44
When considering photo-reduction, light penetration is an important variable needed to
quantify and understand the amount of photo-reactions possible in a natural water body.
Because photo-reduction is a major source of DGM, high DGM production rates drop off
quickly with depth. There have been several studies that have attempted to quantify light
and UV penetration in freshwater systems. Scully and Lean 1994 undertook systematic
investigations of UV attenuation in temperate freshwater lakes (n = 20, DOC from 0.50 to
7.80 mg/L). They found that light attenuation coefficients, generally calculated as:
z
EK depth
depth
ln
Where E is downward irradiance (in W*area-1*nm-1) and
z is distance below the water surface
varied considerably across survey lakes. DOC was the principal attenuating substance of
UVB and UVA respectively, and from their data Scully and Lean derived integrated
(over wavebands) attenuation coefficients that were power functions of DOC
concentration. Morris et al. 1995 also investigated UV attenuation in freshwater lakes (n
= 59; 30 lakes with DOC below 2 mg/L DOC). They offer a modification to Scully and
Lean’s model for calculating attenuation coefficients in low DOC environments.
Examples depths of 99% attenuation of various radiation regions are given in Table 2.3:
Table 2.3. Example depths of 99% attenuation of solar radiation at various DOC concentrations. Depths were calculated from reported measured attenuation coefficients and not generalized equations.
Depth of 99% attenuation (m) Lake DOC (mg/L)
UVB UVA Visible Reference
Gutierrez 0.32 8.5 15 38 Morris et al. 1995 Giles 1.16 11.5 17.5 35.5 Morris et al. 1995 Hamilton Harbor 4.90 0.43 0.8 1.7 Scully and Lean 1994 Sharpes Bay, Ontario 5.60 0.48 0.76 2.1 Scully and Lean 1994 Toolik 5.07 n/a 0.83 10 Morris et al. 1995 Red Rock 10.11 0.15 0.19 1.9 Morris et al. 1995
45
Crump et al. 1999 find that the equations in Scully and Lean 1994 and Morris et al. 1995
were poor predictors for UV attenuation in high DOC environments (n = 11 ponds, DOC
from 8 to 16 mg/L). They found the most useful predictor for attenuation coefficients
with depth to be absorption coeffecients measured on filtered water using a laboratory
spectrophotometer (r2 = 0.94). Ninety-nine percent of the UVB was attenuated in 12-60
cm while the depth of UVA penetration was twice as large. They also noted that the
percentage of DOC that acted to absorb UVB radiation changed throughout the year, with
the highest attenuation per unit DOC occurring in May and the lowest in September.
Thus light penetration is important in predicting the amount of Hg photo-reduction that
occurs in nature, but is a difficult variable to use to quantify photo-reduction.
2.3.5. Organic matter complexation
Natural organic matter also plays an important role in determining the amount of Hg that
is reduced to DGM in the environment. Trace metals, including mercury, are usually
bound to the acid sites of natural organic matter (NOM). Carboxyllic acids and phenols
comprise over 90% of these sites. However Hg is expected to bind preferentially to
reduced sulfur sites, which are thought to play a role disproportionate to their abundance
in the complexation of “soft” trace metals in the environment due to their high binding
coefficients (Xia et al. 1998, 1999, Ravichandran 2004 and references therein).
Sulfur is a minor component of DOM, but Hg(II) stability constants for compounds
containing reduced sulfur are usually much stronger than stability compounds with other
ligands. Since the amount of reduced sulfur available for binding in natural freshwaters
(up to a few ppt) is a few orders of magnitude larger than Hg concentration in
uncontaminated sites, S-containing moieties are predicted to dominate complexation
(Hesterberg et al. 2001, Haitzer et al. 2002). It has been suggested that the reactivity of
thiol-complexed Hg differs from the reactivity of Hg complexed to oxygen species
(Zheng and Hsu-Kim 2010, Zhang and Hintelmann 2009).
There are large variations in Hg isotopic compositions in natural samples. Many
environmentally relevant transformations have been shown to cause MDF in Hg isotopes,
46
and distinguishing source MDF signatures from MDF imparted during transport and
transformation in the environment will be challenging. There are fewer processes that
induce MIF signatures in Hg samples, and large MIF ( > than 0.2‰) seem to be
associated with photochemical transformations. MIF therefore has the potential to
highlight and track photochemical transformations among the many processes that affect
the environmental fate of Hg. For example, using both mass-dependent and mass-
independent Hg isotope signatures, along with 15N and 13C signatures, Senn et al. 2010
build a strong argument that coastal and oceanic fish in the Gulf of Mexico derive their
MeHg from different sources. The strong possibility that benthic and pelagic food webs
incorporate MeHg from different sources has important implications for future toxicity
mitigation and remediation efforts. As discussed in the previous pages, photochemical
reduction is a significant pathway whereby water bodies release their burden of Hg back
to the atmosphere, and photo-degradation is an important sink (and detoxification
mechanism) for the MeHg in water bodies. These processes are strongly influenced by
the intensity and frequency of sunlight available, and the kind and quantity of dissolved
organic matter in the water column. If MIF is to be used as a tool to understand the
photochemical transformations of Hg, it is important to investigate whether and how the
frequency and intensity of light and the kind and quantity of organic matter affect MIF.
Chapter 3 of this thesis is devoted to the first of these questions: the manner in which
frequency and intensity of available sunlight affects the expression and magnitude of MIF
during photo-reduction of Hg(II) and MeHg respectively.
47
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58
CHAPTER 3: EFFECTS OF ULTRAVIOLET
RADIATION ON THE MAGNETIC ISOTOPE
EFFECT IN FRESHWATER PHOTO-REDUCTION OF ORGANIC AND INORGANIC
MERCURY 3.1. INTRODUCTION
The toxicity of various forms of mercury (Hg) is well established. The form that is of
most concern is monomethylmercury (MeHg), which is a potent neurotoxin that
bioaccumulates in aquatic food chains with concentrations in piscivorous fish reaching
levels one million times greater than in the surrounding water (Lindqvist et al., 1991;
Mergler et al., 2007). Humans have increased the amount of mercury that is actively
cycling in the environment by mining, industrial activities and combustion of fossil fuels
by a factor of approximately three since pre-industrial times. Once released to the
environment, Hg undergoes redox, biological, and phase transformations. Its elemental
form is volatile and is transported globally in the atmosphere. Uncertainty about the
origin and magnitude of primary emissions to the atmosphere, deposition to terrestrial
and ocean surfaces and re-emission of previously deposed Hg presents challenges to
scientists and policy-makers alike (Fitzgerald and Lamborg, 2007; Lindberg et al., 2007;
Selin, 2009).
Stable isotope fractionation has been successfully used to trace biogeochemical processes
at both human and geologic timescales. Mercury has seven stable isotopes: 196Hg
(0.16%) 198Hg (10.0%) 199Hg (16.9%), 200Hg (23.1%), 201Hg (13.2%), 202Hg (29.7%) and 204Hg(6.8%). The recent development of an analytical technique using cold vapor
multicollector inductively coupled plasma mass spectrometry (CV-MC-ICP, (Lauretta et
al., 2001) has reduced measurement uncertainty (typically 0.1 to 0.3 ‰, (Bergquist and
Blum, 2009)to a point where variations in the isotope compositions of natural samples are
readily resolved for Hg. The reported range of isotopic signatures imparted by mass-
59
dependent fractionation (MDF) already exceeds 7‰ (Bergquist and Blum, 2009;
Sherman et al., 2010) and MDF has recently been employed to track sources of Hg in the
environment (Feng et al., 2010; Foucher et al., 2009). Mass-independent fractionation
(MIF) of the odd isotopes of Hg has also been observed, with a reported range of
signatures spanning nearly 10 ‰ (Bergquist and Blum, 2009; Sherman et al., 2010). Such
large MIF is unusual in isotopic systems, with O (during ozone formation) and S (only in
the Archean) being the only other elements showing MIF of comparable magnitude in
natural samples.
Processes that induce MDF for Hg include hydrothermal transport (Sherman et al., 2009;
Smith et al., 2005), biological reduction (Kritee et al., 2009; 2008; 2007) , abiotic redox
transformations (Bergquist and Blum, 2007; Yang and Sturgeon, 2009; Zheng and
Hintelmann, 2009; Zheng and Hintelmann, 2010a; Zheng and Hintelmann, 2010b) and
evaporation (Estrade et al., 2009; Zheng et al., 2007). One challenge to using MDF as a
tool to elucidate the transformation and fate of Hg in the environment is distinguishing
source signatures from MDF imparted during transport and subsequent transformations.
Processes reported to induce MIF are fewer and large MIF (> 0.3‰) has only been
observed in photochemical reduction (Bergquist and Blum, 2007; Malinovsky et al.,
2010; Sherman et al., 2010; Zheng and Hintelmann, 2009; Zheng and Hintelmann,
2010b) and now photo-oxidation (Ghosh). While MIF is not affected by MDF processes,
it could be altered by other MIF processes or through mixing of Hg pools bearing
different MIF signatures (Bergquist and Blum, 2009). Understanding the factors that
control MIF will permit MIF to be utilized to track and quantify specific pathways in
nature. It will also permit assessment of which signatures are susceptible to alteration by
successive transformations or mixing and whether the various contributions to MIF can
be deciphered.
Of the two plausible mechanisms, the nuclear volume effect (NVE) and the magnetic
isotope effect (MIE), put forward to explain mass-independent anomalies in Hg isotopes,
the more probable cause of the large MIF observed in photochemical reactions is the MIE
(Bergquist and Blum, 2007; Bergquist and Blum, 2009; Malinovsky et al., 2010). Only
60
the odd isotopes are magnetic, which is to say, have non-zero nuclear spin and nuclear
magnetic moments that can interact with electron magnetic moments (hyperfine
coupling). This interaction can become important where the orientation of electron spin
affects the reaction pathway, as in the fate of a geminate excited radical pair. If the
unpaired electrons are in a triplet state (unpaired spins) with respect to each other, then
the forming of a covalent bond is spin-forbidden (total electron spin would not be
conserved) and the radicals are likely to drift apart. Hyperfine coupling can cause
electron spin conversion (intersystem crossing, ISC), such that the unpaired electrons
convert from a triplet state into a singlet state (spin paired). From a singlet state,
recombination of the radical pair in a covalent bond is spin-allowed. The difference in
the rates of spin conversion for the odd versus the even isotopes sorts the nuclei into
different products. Odd nuclei are more likely to be found in the recombination product
due to faster ISC and even nuclei are more likely to dissociate spatially and meet other
fates. Other forces that influence the electron spin orientation, such as coupling between
electron spin and orbital angular momenta (spin-orbital coupling (SOC)), can also affect
expression of the MIE. Another factor that is important in the expression of the MIE is
the lifetime of the radical pair. If the lifetime is very short relative to ISC, the MIE will
be suppressed. If it is very long relative to ISC, spin-conversion may take place by other
processes and nuclear sorting of the odd and even isotopes will not be consistent
(Bergquist and Blum, 2009; Buchachenko, 2001).
This mechanism was first proposed by Bergquist and Blum 2007 to explain MIF of over
2 ‰ observed during the photochemical reduction of aqueous Hg species in the presence
of dissolved organic matter and MIF of over 4 ‰ in Hg isolated from fish. While it has
been widely adopted by subsequent investigators to explain mass-independent anomalies,
the only direct test of the mechanism to date has been by Malinovsky et al. 2009. They
found that MIF during photodemethylation of MeHg was suppressed in solutions with
ascorbic acid and with ammonia, attributed to suppression or scavenging of the radical
pair during decomposition. Expression of the MIE is dependent on many factors, a few
of which are mentioned above. Another test of the mechanism is elimination of the
photo-generated radical pair. If the higher energy wavelengths of the solar spectrum are
61
removed gradually, the radiation available will eventually not carry enough energy to
effect the electronic transition to an anti-bonding orbital and radical pairs will not be
produced. This closes the avenue of expression for the MIE. The objective of this study
is the following: to determine whether removing the ultraviolet B (290-320 nm) or the
ultraviolet A (320-400 nm) portions of the electromagnetic spectrum affects the
expression and magnitude of MIF during the aqueous photo-reduction of MeHg and
Hg(II) respectively. Other goals were to determine how MIF is related to total photo-
reduction and to determine whether MIF wil allow us to distinguish photo-reduction from
other processes that reduce Hg in aqueous freshwater environment.
62
3.2. METHODS
3.2.1. Cleaning equipment
All sample containers and analytical vials were glass and cleaned according to the
following procedure:
(1) glass vials were rinsed five times with Milli-Q (18 M) water
(2) glass vials were soaked in cold 10% HCl for 48 hours
(3) glass vials were rinsed five times with Milli-Q water along with the teflon-lined
caps
(4) glass vials were then filled with 2-3% BrCl (acidic and a complexing agent for
Hg), capped and left to stand at least 12 hours right side up and 12 hours inverted to
clean the entire inner surface.
(5) five final Milli-Q water rinses (including caps)
(6) drying in a class 100 laminar workstation (which was equipped with a carbon
filtration unit prior to the HEPA filter to remove Hg(0) from air) followed by capping
and storage in clean, sealed plastic bags.
If the 48-hour cold HCl soak was not feasible, it was substituted with a preliminary BrCl
soak. The quartz photochemical reactors and associated teflon tubing and fittings were
rinsed five times with Milli-Q water, filled with 2-3% BrCl and left to soak for at least 12
hours and then rinsed copiously with Milli-Q water before use.
3.2.2. Photo-reduction experiments
Two sets of three photochemical reduction experiments each were carried out using a
Hg(NO3)2 standard (J.T. Baker lot E04632, hereafter called Hg(II)). Two sets of three
photochemical reduction experiments each were carried out using a CH3HgCl standard
(Alfa Aesar lot 82-082844A, hereafter called MeHg). Photo-reduction took place under
natural sunlight on the roof of the McLennan Physical Building, 60 St. George Street,
Toronto, Ontario. (43 N, 79 W). The initial set for each species was carried out in July,
2009 and the replicate set was carried out in September, 2009
Aqueous mercury solutions
63
Aqueous solutions of 2 mg/L Suwannee River Fulvic Acid Standard (1S101F) from the
International Humic Substance Society (http://ihss.gatech.edu/ihss2/) were made up in
glass bottles in simulation of natural fresh waters and sampled to verify negligible
mercury content. The fulvic acid solutions were then spiked to a final concentration of
approximately 30 g/L of the Hg species under study, and immediately subsampled to
measure the initial isotopic composition. These experimental Hg concentrations are four
to five orders of magnitude higher than in natural freshwaters, but were chosen as the
minimum concentrations at which isotopic composition could be measured with
confidence.
The Hg-fulvic acid solutions were left to equilibrate overnight in aluminum foil, which
was not removed until the moment of filling the quartz photochemical reactors. For UV-
filtration experiments, the filtration boxes were placed over the reactors immediately after
filling and the solution was sampled to monitor any change in concentration or isotopic
composition that occurred overnight. All subsamples for isotopic analysis were
immediately preserved by spiking with concentrated BrCl to achieve a sample
concentration of 2.5%.
Photoreactor setup
Photochemical reactors were 1 L quartz Erlenmeyer flasks with air input and sample
withdrawal lines running through a teflon cap and an arm for headspace purging, as
depicted in Figure 2.1. Before entering the reaction vessel, the air passed through a 0.2
m filter to remove particulate matter and a gold-covered sand trap to remove any
ambient Hg(0). Experiments were started between 9:30 and 10:30 a.m. and allowed to
run for several hours, through the solar noon (approximately 1 p.m.). Sub-samples were
removed from the aqueous reservoir with a syringe as the reaction progressed and
preserved immediately with Hg-clean 2.5% BrCl. The sum of volumes sampled never
exceeded half the reservoir volume.
Light filtration
64
Light filtration was accomplished by building boxes, closed on five sides, which stood
over the quartz reactors. Mylar (poly(ethylene terphthalate) sheets were used to
selectively screen the UVB portion of the electromagnetic spectrum (290-320 nm); five
sheets per side) were attached to a 0.5-inch acrylic frame. Mylar’s transmission of
ultraviolet light is negligible at 305 nm and less than 30% at 310 nm but rises quickly to
over 60% at 315 nm (Mitrofanov et al., 2009; Mizuno et al., 2008). It is not completely
transparent to the UVA spectrum, and in removing the majority of the UVB spectrum
(93-95%), some of the UVA spectrum was also lost (320 – 400 nm, 43-45%). A small
amount of the visible spectrum was also lost (400 - 700 nm, 19-22%). To selectively
screen both the UVB and the UVA portions of the spectrum, solid acrylic sheets covered
with a 3M Scotchshield (Ultra S150) window film were used. This consistently blocked
96% of UVB, 98% of the UVA, and a small amount the visible spectrum when the sun
was not directly overhead. Around mid-day, the intensity of visible light was higher
under the acrylic box (by 30% or more) than the intensity of visible light under direct
solar radiation. Measurements of the light reaching each reactor were taken four or five
times throughout the experiment, with the three readings for each electromagnetic region
taken in as rapid a sequence as possible (Table 3.1). Measurements were taken with a
Solar Light PMA2200 Radiometer fitted with detectors specific to the three spectral
regions of interest to us: UVB (PMA2106), UVA (PMA2110) and Visible (PMA2130).
Dark controls
Dark control experiments for Hg2+ and MeHg were performed in the laboratory with the
same reactor configuration except that the quartz reactor was covered with aluminum
foil. Initial dark controls (Number 1 for both species) showed no concentration loss
during 8 h of continuous sparging. Subsequent dark controls for Hg(II) left in a sealed,
foil-wrapped bottle with no sparging over a few weeks showed significant concentration
losses (ex. Hg(II) Dark Control 2, 40 % loss). Dark control experiments for both Hg(II)
and MeHg, with the quartz reactor wrapped in foil and continuous sparging, were
performed outdoors in Michigan by B. Bergquist, as published in Bergquist and Blum
2007.
65
3.2.3. Hg concentration analysis
Concentrations of Hg in the aqueous samples were measured by a Tekran 2600 Cold
Vapor Atomic Fluorescence Spectrometer (CVAFS). Samples were introduced through a
gas-liquid separator modified by B. Bergquist. The method was an adaptation of US
EPA Method 1631, Revision E. The BrCl (or KMnO4 for trap solutions, discussed
hereafter) preserving the solutions was reduced with hydroxylamine hydrochloride
(prepared fresh monthly) prior to analysis. This reduces the oxidizing matrix promptly,
but takes weeks to reduce the Hg. After uptake, mercury in solution was reduced on-line
by a Sn(II)Cl2 solution. The dissolved Hg(0) that was generated was stripped from the
solution by Hg-free argon through the gas-liquid separator and concentrated on a series of
two gold traps before being released to the detector. To maximize signal stability and
reproducibility, all tubing, connections and the phase-separating surface were routinely
cleaned and changed immediately if any sort of precipitate (including organic matter and
tin oxide) was suspected of accumulating on the interior. Ten minutes of apparatus
washing with a 7% (each) HNO3 and HCl aqueous solution at the end of each analytical
session was found to prolong the useful lifetime of tubing and connections, and to
improve signal stability in general. Instrument operation and maintenance was still being
optimized during the July analyses and typical precisions (± 4.9 %, Hg(II), 2SE and ±
3.76 %, MeHg, 2SE) are poorer than in September (± 2.8 % , Hg(II), 2SE and 2.8 %,
MeHg, 2SE). Calibration accuracy and instrument drift were monitored with a reference
mercury solution prepared from NIST 1641d. Reference standard accuracy varied from
5% to ±1%, 2SD. This accuracy was based on the first two hours of sample run time
only (n = 4 or more). This metric was chosen because the Tekran 2600 could (but did not
always) drift by 5% to 8% during an analytical session. This drift was monitored closely
with the reference standard so that concentrations could be corrected. Drift was not
significant during the first two hours of analysis. Errors shown on graphs are the greater
of average sample 2SE and standard 2SD for one analytical session. Method blanks were
run prior to calibration and periodically throughout the analysis.
3.2.4. Mercury purifcation and matrix transfer in preparation for isotope analysis
66
Any variability in instrumental mass bias introduced into isotopic analysis through the
matrix effects of natural samples is not consistent enough to be corrected for. To obtain
reliable isotope compositions, therefore, it was necessary to transfer the Hg from complex
sample matrices to an aqueous acidic matrix consisting of 10% (wt) H2SO4 and 1% (wt)
KMnO4. This was done by cold vapor generation in a manner similar to that described
for concentration analysis by reduction with SnCl2 and the use of a gas-liquid separator
(Figure 2.2). The Hg(0) generated was trapped by sparging into the acidic permanganate
solution. Through a process of recovery optimization, it was found that a gas flow rate
(ultrapure N2) of approximately 170 mL/min streaming into 35 mL of trapping solution
yielded the best results. The diameter of the trapping vials was approximately 22 mm.
If less trapping solution was desired in order to concentrate the sample, the gas flow rate
had to be reduced to maintain recoveries above 95%. Trap reagents were tested for
negligible Hg content (less than 0.05% of sample concentrations). The apparatus was
cleared of each successive sample with a minimum of 15 minutes of washout time with
10% (wt) HNO3 followed by 10% (wt) H2SO4. A heat gun was applied to the transfer
lines to desorb Hg(0) stuck to the interior and Teflon lines, and spargers were rinsed
between samples with dilute hydroxylamine hydrochloride, soaked in 2.5 % BrCl for 15-
30 minutes and finally rinsed in 10% H2SO4. Blanks during this matrix transfer were on
the order of 0.25 % of sample concentrations. Matrix transfer process standards using
NIST 3133 reference mercury solution (concentration- and matrix-matched to samples)
were run routinely at the beginning of each session and then throughout the day as
needed. Sample recoveries after matrix transfer were rarely lower than 95% and never
less than 90% (see Table 2.6). While trouble-shooting the apparatus, process standard
recoveries were sometimes lower than this. Process standards with recoveries ranging
from 86% to 100% were analyzed for isotopic composition to monitor how incomplete
recovery affected isotopic fractionation. The lowest recovery (86%) showed small
offsets (less than 0.2 ¥) in mass-dependent fractionation, but no offset in mass-
independent fractionation. Transfer replicates (indeed, overall process replicates) were
achieved by processing R0 from each of the three experiments from both sets. These
were all prepared from the same Hg(II) and MeHg stock respectively and sampled before
67
Hg-DOM equilibration and transfer to the quartz reactor. Thus they should represent the
experimental reproducibility in terms of isotopic composition.
3.2.5. MC-ICP-MS analysis
Isotopic compositions were measured on a Nu Instruments multiple collector inductively
coupled plasma mass spectrometer (MC-ICP-MS) at the Department of Geological
Sciences, University of Michigan. Samples were introduced to the plasma by the cold
vapor generation technique already described. Instrumental mass bias was corrected in
two ways as recommended in (Blum and Bergquist, 2007). First, the isotopic
composition of a thallium internal standard (NIST 997; reported 205Tl/203Tl ratio of
2.38714) added to the Hg vapor by a CETAC Aridus desolvating nebulizer was
monitored and used to determine the instrumental mass bias factor using an exponential
fractionation law. This factor was used to correct Hg isotope abundances. Second,
sample-standard bracketing (SSB) was carried out with a reference Hg solution of NIST
SRM 3133 that was matched to the samples both in concentration (5 ppb, usually to
within 5%) and in matrix (1% KMnO4 in 10% H2SO4). Prior to cold vapor generation,
the matrix was neutralized with hydroxylamine hydrochloride a minimum of one hour
(but usually much longer) before sampling. On-peak zero corrections were applied to all
masses by measuring a matrix matched blank solution prior to each sample after 10 to 12
minutes of washout. Isobaric interference of 204Pb on 204Hg was corrected by monitoring 206Pb. The Faraday cups were configured to collect masses of 196, 198, 199, 200, 201,
202, 203, 204, 205 and 206 atomic mass units.
Isotopic compositions are reported in delta notation as the permil (‰) deviation from the
NIST SRM 3133 standard. It is determined through SSB as follows, where the
(XXXHg/198Hg)NIST SRM 3133) ratio in the denominator is the average ratio of the two
bracketing standards run prior to and after the sample:
XXXHg = [(XXXHg/198Hg)sample/(XXXHg/198Hg)NIST SRM 3133) – 1] x 1000
68
where XXX is the mass the Hg isotope of interest from 198 to 204 amu. This convention
uses the lowest-mass major isotope (196Hg has an abundance of 0.15% and cannot be
measured as precisely as 198Hg, with an abundance of 10.0%) in the denominator. Thus
higher values (more positive) will indicate a sample is “isotopically heavier” than the
SRM, as is the convention in other stable isotope systems.
Mass-independent fractionation is reported using capital delta notation (XXXHg) as the
deviation in isotope ratios from the theoretical values predicted by mass-dependent
fractionation laws, also in units of permil (‰). The theoretical isotope ratios are
determined using the 202Hg/198Hg ratio (i.e. 202Hg) and applying the kinetic mass-
dependent fractionation law derived by Bigeleisen 1949 and reviewed by (Young et al.,
2002)as follows:
199Hg = 1000 x { ln[199Hg/1000 + 1] - 0.2520 x ln[202Hg/1000 +1] }
200Hg = 1000 x { ln[200Hg/1000 + 1] - 0.5024 x ln[202Hg/1000 +1] }
201Hg = 1000 x { ln[201Hg/1000 + 1] - 0.7520 x ln[202Hg/1000 +1] }
204Hg = 1000 x { ln[204Hg/1000 + 1] - 1.493 x ln[202Hg/1000 +1] }.
For values lower than 5 ‰, these may be approximated by:
199Hg = 199Hg - (0.2520 x 202Hg)
200Hg = 200Hg - (0.5024 x 202Hg)
201Hg = 201Hg - (0.7520 x 202Hg)
204Hg = 204Hg - (1.493 x 202Hg)
(all equations from (Blum and Bergquist, 2007).
3.2.6. Reporting of analytical uncertainty
External reproducibility of the method was monitored with a standard of different
composition than the bracketing standard and reported as 2SD (since it is the distribution
69
of measurements about a fixed value). At the University of Michigan Biogeochemistry
and Environmental Isotope Geochemistry Laboratory, this secondary standard solution
was made from metallic Hg mined from Almadén, Spain (UM-Almadén). The data in
this thesis was produced over 8 analytical sessions (October 2009 –July 2010). The mean
Almadén values for the October through January sessions were 202Hg = -0.58 ± 0.13 ‰
and 199Hg value of 0.00 ± 0.07 ‰ (2SD, n = 12). The mean Almadén values for the
June and July sessions were 202Hg = -0.58 ± 0.09 ‰ and 199Hg value of -0.02 ± 0.07 ‰
(2SD, n = 20). These values are within error of the values for UM-Almadén cited in
(Bergquist and Blum, 2007)for an aqueous BrCl sample matrix and for solutions with
higher Hg concentrations (30 ppb): 202HgAlmaden =-0.54 ± 0.08 ‰ (2SD, n = 25) and
199HgAlmaden of -0.01 ± 0.05 ‰ (2SD, n=25). Our uncertainties are likely larger due to
the different matrix and lower concentrations used for analyses (5 ppb).
The mean isotopic values for JTBaker Hg(II), taken from 16 samples of R0 over 5
analytical sessions were 202Hg = -0.69 ± 0.18 (2SD) and 199Hg = 0.03 ± 0.10 ‰
(2SD). The mean isotopic values for Alfa Aesar MeHg, taken from 12 samples of R0
over 4 analytical sessions were 202Hg = -0.17 ± 0.10 ‰ (2SD) and 199Hg = 0.10 ±
0.10‰ (2SD).
3.2.7. Calculations
Photo-reduction of both Hg(II) and MeHg in these experiments appears to follow a
Rayleigh distillation model well. Rayleigh models describe the isotopic composition of a
substrate-reactant system under equilibrium isotope separation or under kinetic isotope
separation where the product is removed instantaneously after its formation (Fritz and
Fontes, 1980). Kinetic fractionation factors were determined from the results of our
experiments using the form of the Rayleigh distillation equation outlined by (Mariotti et
al., 1981) Briefly, with 199Hg as an example, 199Hg is defined in reference to the
abundance of the isotope of interest:
70
199Hg
d199HgPr oduct
d198HgPr oduct199HgReac tan t198HgReac tan t
.
With as series of substitutions, it can be shown that 199Hg
d198HgR198HgR
d199HgR199HgR
.
Assuming is constant during the reaction and integrating both sides, one obtains
199Hg ln198HgR , f198HgR ,i
ln
199HgR , f199HgR ,i
where i and f refer to the initial abundance and the abundance after some time
respectively. Through another series of substitutions, it can be shown that
i
i
f
f
i
f
i
fHg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
198
199
198
199
198
198
198
198
199 lnlnln .
In delta notation and assuming that 198Hgf/198Hgi can be approximated by f, the fraction
of substrate remaining, this equation can be re-written for 199Hg (or any other isotope) as
1 ln f ln103substrate, f 1
103substrate,i 1
.
.
The fractionation factor, , can thus be obtained from the slope of a plot of ln(f) against
the right side of the previous equation. Conversely, a value of allows one to predict the
isotopic signature of (in this case) the substrate for a given fraction remaining:
e(-1)lnf
(i + 1000) – 1000 = f.
Fractionation factors reported in this thesis were obtained in this way from experimental
data.
71
3.3. RESULTS
Quartz reactors containing either Hg(II) or MeHg in aqueous solutions with organic
matter were simultaneously exposed to three different light regimes to examine the effect
of photo-reduction on the isotopic fractionation of the respective Hg species. Initial sets
of three were carried out on July 19th (MeHg) and July 30th (Hg(II)), 2009 and replicate
sets of three two months later on September 1st (MeHg) and September 3rd (HgII), 2009.
Dark controls, wrapped in aluminum foil throughout the experiment, were also
performed. Solar radiation intensity (W/m2) in each of three wavebands, UVB (290-320
nm), UVA (380-400 nm) and Visible (400-700 nm) was measured at intervals throughout
the experiments (see Table 3.1).
3.3.1. Concentrations
Photo-reduction of Hg(II) and MeHg resulted in highly volatile Hg(0), which was
continuously purged from the substrate reservoir by sparging with Hg-free air. The
amount of Hg(II) or MeHg lost via photo-reduction was estimated from the decreasing
concentration of Hg in the substrate reservoir over time (Figure 3.3). Blocking different
wavebands did not always have the same effect on September photo-reduction as it did
on July photo-reduction. Ultraviolet intensities in September were generally lower, for
example, the maximum UVB intensities (recorded near solar noon, which is
approximately 1 pm) are 15 and 30% lower than the corresponding July intensities and
the maximum September UVA intensities are 10 and 20% lower than in July. Visible
waveband intensities were not significantly lower in September than they were in July.
Hg(II)
In July, Hg(II) showed maximum similar losses of 80% in both the experiment exposed
to full sun and the experiment with UVB blocked (Figure 3.3a). Blocking both the UVB
and the UVA suppressed most of the photo-reduction (only 10% loss). In contrast,
blocking both the UVB and the UVA in September did not suppress photo-reduction
nearly as much (40% loss). Maximum September loss (70%) was in the full spectrum
experiment, and blocking of UVB resulted in approximately 40% loss. Dark control
experiments were carried out in the laboratory with the Hg(II)-organic matter solution
72
shielded with aluminum foil at all times. They showed no detectable losses after 8.5
hours of sparging with Hg-free air (for example, Dark Control 1, Figure 3.3a). Thus, we
conclude that Hg loss due to non-photochemical reduction during the 6-7 hour UV
filtration experiments was negligible.
In contrast, an Hg-organic matter solution kept in a foil-wrapped glass bottle with no
sparging showed 20% loss after 2 days and 40% loss after 8 days (Dark Control 2,
Figure 3.3a), indicating that while non-photochemical pathways can contribute to Hg
volatilization, the kinetics of these are far slower than the kinetics of photochemical
pathways. An additional dark control with Hg(II) in a foil-wrapped photochemical
reactor with continuous sparging was carried out by B. Bergquist (as published in
(Bergquist and Blum, 2007) that showed approximately 20% loss after 5.5 hours.
In the September experiments with UVB and UVA removed (Figure 2.3b), the lowest
reservoir Hg concentrations are recorded after approximately four hours and are followed
by small concentration increases during the last two hours of exposure. It is possible that
this is a result of reservoir evaporation, as the water vapor would have been pumped out
of the reactor headspace along with the Hg(0). It is likely that the UV filtering boxes
increased the ambient temperature as compared with the uncovered, full sun experiment.
No such Hg concentration increase is seen in the September full spectrum trial, which
may reflect a lower ambient temperature. Unfortunately, temperature under the boxes in
contrast to outside the boxes was not monitored.
MeHg
In the July MeHg photoexperiments, neither blocking the UVB waveband only nor
blocking all UV radiation had a significant effect on the total amount of MeHg lost as
Hg(0) (Figure 3.3c). All three experiments show losses of approximately 20% after
seven hours of exposure. Overall concentration losses in the September trials were
smaller, at approximately 10% loss in the full spectrum experiment and the UVB blocked
experiment (Figure 3.3d). The smaller losses observed in September may be due to the
lower intensities of light available in September (see Table 3.1). In contrast to the July
73
trial, the September trial with both UVB and UVA blocked underwent significantly less
photo-reduction (~ 4%) than its full spectrum counterpart (~ 10% loss). Our method of
concentration measurement, however, cannot be relied upon to resolve differences
smaller than 5% and so it is difficult to determine whether loss in the September no UV
trial is indeed significantly lower than losses in the other two trials.
The dark MeHg control experiment carried out in the laboratory showed no detectable
losses after 8.5 hours of sparging with Hg-free air. We therefore conclude that any losses
due to non-photochemical reduction during the 6-7 hour UV filtration experiments were
negligible. Based on literature reports (Bergquist and Blum, 2007; Zhang and Hsu-Kim,
2010) no loss was expected.
Apparent kinetic plateaus
Figure 3.3 displaying reservoir losses of both Hg(II) and MeHg against time suggests
that photo-experiments were allowed to proceed until a kinetic plateau was reached. This
is somewhat misleading in that the experimental hours were chosen to cover the brightest
six hours of daylight available (10 a.m. to 4 p.m.), and while the rates of reaction appear
to slow considerably after 2 to 3 hours of exposure, it is not clear whether the kinetic
plateaus are due to reaction exhaustion or to a decline in the radiation available. It is
likely that photo-reduction would resume in some or all of the reactors if bright daylight
was restored, as was observed in other photoexperiments left outside for 30 subsequent
hours.
3.3.2. Mass-dependent fractionation
When considering the mass-dependent fractionation (MDF) of Hg, it is sufficient to
examine one isotope ratio that does not display mass-independent anomalies. As
recommended in (Blum and Bergquist, 2007), MDF will be reported using 202Hg (see
Methods). If fractionation follows a Rayleigh distillation model, the fractionation factor
() can relate the change in isotope ratios to the amount of substrate that has reacted.
Photo-reduction of both Hg(II) and MeHg in continuously purged reactors appears to
follow a Rayleigh model well, and in the discussion that follows, comparisons will be
74
made in terms of the kinetic fractionation factor . An = 1.0000 indicates no
fractionation; fractionation becomes more pronounced as the absolute distance of from
1.0000 increases. Throughout this thesis is defined as Rproduct/Rreactant (see Methods)
so, for example, 202 = 0.9980 for a given process means the substrate is becoming
isotopically heavier as the reaction progresses.
Hg(II)
MDF was observed in all experiments where there was photo-reduction with the heavier
isotopes being preferentially retained in the reactor. Complete isotopic data are displayed
in Table 3.2. In the July experiments, the largest change in 202Hg was 1.88 0.17 ‰
(2SE) in the no UVB experiment. MDF was greater in the experiment with UVA
blocked (202 = 0.9988 0.0001, 2SE) than in the full spectrum experiment (202 =
0.9992 0.0002, 2SE; Figure 3.4a and Table 3.3). In the September experiments, the
largest change in 202Hg was 2.02 0.17 ‰ (2SE) in the full sun experiment. September
MDF was not significantly affected by blocking any of the wavebands. All fractionation
factors 202 fall within error of each other (Figure 3.4b and Table 3.3). The July
experiments do not show greater fractionation than the September experiments, despite
exposure to ultraviolet radiation of higher intensity.
MeHg
MDF was observed in all experiments where there was significant photochemical loss,
with the heavier isotopes being preferentially retained in the reactor. Complete isotopic
data are displayed in Table 3.4. In the July experiments, the largest change in 202Hg is
0.33 0.22 ‰ (2SE) in the no UV experiment. July MDF was not significantly affected
by blocking any of the wavebands; 202 for all three experiments fall within error of each
other (Figure 3.4c and Table 3.5). In the September experiments, the largest change in
202Hg is 0.28 0.13 ‰ (2SD) in the no UVB experiments. Differences in mass-
dependent fractionation between the September full sun experiment and the September
no UVB experiment were not significant; fractionation factors (202 values) were not
meaningful due to the small extent of reaction and are not included in Table 3.5 (see also
75
Figure 3.4d). The September, no UV experiment shows no significant MDF. This is to
be expected if very little photo-reduction occurred, as the concentration data suggests.
The July experiments do not show greater fractionation than the September experiments,
despite exposure to ultraviolet radiation of higher intensity. The dark control is not
shown in Figure 3.4 because, as no significant loss occurred, there could be no
fractionation.
3.3.3 Mass-independent fractionation
Mass-independent fractionation (MIF) occurred during photo-reduction of both Hg(II)
and MeHg in continuously purged reactors and appears to follow a Rayleigh model well.
Comparisons will be made in terms of the kinetic fractionation factor XXX, where XXX
is 199Hg or 201Hg. It is calculated in the same way as mass-dependent fractionation
factors (see Methods) except that a capital delta () value is used in place of a delta ()
value. An XXX = 1.0000 still indicates no fractionation. Throughout this thesis, is
defined as Rproduct/Rreactant (see Methods) so, for example, 199 = 0.9980 for a given
process means the substrate is becoming enriched in the odd isotopes as the reaction
progresses.
Hg (II)
MIF was observed with the odd isotopes, 199Hg and 201Hg, being preferentially retained in
the reactor as observed in (Bergquist and Blum, 2007). MIF occurred for the odd
isotopes only. An example is shown in Figure 3.5, using the July experiments. In
Figure 3.5a, 199Hg for all three experiments is plotted against 202Hg to demonstrate
odd-isotope deviation from mass-dependent behavior. In contrast, when even isotopes
are plotted against 202Hg (for example, 204Hg in Figure 2.5b), the progressive changes
in even follow the values predicted by kinetic mass-dependent fractionation laws (see
Methods).
In July, the largest MIF signature was 2.45 0.14 ‰ (2SE) in the experiment exposed to
full sun. Complete MIF data are displayed in Table 3.2. Blocking the UVB waveband
reduced MIF significantly (Figure 3.6a) with 199 going from 0.9986 0.0001 (full sun)
76
to 0.9993 0.0001 (no UVB). Blocking all UV radiation suppressed most of the photo-
reduction and isotopic fractionation, so it is not possible to determine what effect this had
on MIF. Rayleigh models based on estimated MIF fractionation factors for July
experiments are shown in Figure 3.6b. Given the insignificant MIF signature and the
small extent of reaction, the July no UV experiment was not plotted on a Rayleigh model.
In September, the largest MIF signature, 0.69 0.14 ‰ was once again in the experiment
exposed to full sun, and a small but significant decrease was observed in fractionation
when the UVB waveband was blocked (Figure 3.6b and Table 3.3). In contrast to July,
photo-reduction in September was not suppressed by blocking all UV radiation, and
fractionation observed in the no UV experiment was not significantly smaller than
fractionation with only the UVB waveband blocked. MIF in the July experiments was
greater than in the September experiments, corresponding with higher intensity of
ultraviolet radiation. No MIF was observed in Dark Control 2 (left for weeks in a sealed
bottle), despite a 40% substrate loss. Nor was MIF observed in the dark control
performed by B. Bergquist (a foil-wrapped reactor with continuous sparging), despite a
20% substrate loss, as reported by (Bergquist and Blum, 2007).
MeHg
Similar to the Hg(II) experiments, MIF was observed with the odd isotopes, 199Hg and 201Hg, being preferentially retained in the reactor. MIF occurred in the odd isotopes only.
An example is shown in Figure 3.7, using the September experiments. In Figure 3.7a,
199Hg for each of the three experiments is plotted against 202Hg to demonstrate mass-
independent anomalies in the odd isotopes. Even isotopes show no such anomalies:
when they are plotted against 202Hg (for example, 200Hg in Figure 3.7b), isotopic
signatures show mass-dependent behavior as predicted by kinetic mass-dependent
fractionation laws (see Methods).
In the July, the largest MIF signature was 0.55 0.07 ‰ (2SD) in the experiment
exposed to full sun. Complete MIF data are displayed in Table 3.4. Although blocking
the UVB and UVA wavebands did not have an appreciable effect on total losses of MeHg
through photo-reduction, MIF was suppressed in both the experiment with only UVB
77
blocked and the experiment with all UV blocked (Figure 3.8a and Table 3.5).
Unfortunately, a rather large amount of instrumental noise accompanied the production of
this data (2SE is at times half as large or larger than the measurement) and it is not clear
if MIF was suppressed completely or only partially.
In September, the full sun experiment also displayed the largest MIF signature, 0.42
0.14 ‰. Fractionation was significantly reduced by blocking the UVB waveband
(Figure 3.8b and Table 3.5). The September no UVB experiment shows an MIF
signature only half as large. The effect of blocking all UV on MIF in the September
experiments is not clear, since photo-reduction was almost completely suppressed in this
experiment. Uncertainty associated with the fractionation factors in the July full sun and
the September full sun experiments (Table 3.5) makes it difficult to determine if MIF in
July was greater than in September. No change in MeHg concentration was observed in
the dark control, providing no opportunity for MIF. Similarly, no drop in MeHg
concentration was reported for dark controls by (Bergquist and Blum, 2007).
Relationship between 199Hg and 201Hg
The average ratio 199Hg/201Hg was 1.00 0.04 (2SE) for the Hg(II) photo-reduction
experiments. Figure 3.9a shows this ratio for all samples where MIF was significant
(4SE or larger). Although not all Hg(II) experiments lost substrate Hg through photo-
reduction to the same extent, all samples showing significant MIF fall within error of a
line of slope 1.00 ± 0.04, 2SE. In contrast, the average ratio 199Hg/201Hg for MeHg
samples showing significant MIF (4SE or larger) was 1.35 0.16 (2SE), as shown in
Figure 3.9b. All points showing significant MIF but one fall within error of this line.
78
3.4. DISCUSSION
3.4.1. We see a relationship between the expression and magnitude of MIF and the
energy of light available. Removing the UVB waveband clearly reduces the magnitude
of MIF in Hg(II). In both the July and September experiments, the most pronounced MIF
is in the experiments exposed to full sun. The July experiments were exposed to more
intense ultraviolet radiation and show correspondingly larger fractionation factors. No
MIF was observed in a sealed dark control with 40% substrate loss, or in a dark control
with continuous sparging (carried out by B. Bergquist and reported in (Bergquist and
Blum, 2007) with a substrate loss of 20%.
Removing the UVB waveband also clearly reduces the magnitude of MIF in MeHg.
Fractionation is largest in the experiments exposed to full sun in both the July and the
September experiments. Uncertainty in the fractionation factors, 199 make it difficult to
asses whether exposure to more intense sunlight in July was accompanied by greater
MIF. No MeHg degradation was observed in the dark control, which agrees with other
published results (Bergquist and Blum, 2007; Zhang and Hsu-Kim, 2010). These results
strongly support the magnetic isotope effect (MIE), expressed through spin-selective
reactions such as geminate radical pair recombination, as the likely mechanism of
MIF in photo-reduction. Radical pair generation is a frequency-dependent process and
irradiation by higher frequencies increases the production of radical pairs, allowing
greater expression of the MIE.
3.4.2. For Hg(II), UVB is the larger contributor to MIF but both the UVA and the
visible portions of the spectrum contribute significantly to MIF. In the July
experiments, blocking the UVB spectrum reduces MIF by 50 ± 10%. In the September
experiments, blocking UVB radiation also reduces MIF significantly (by 30 ± 20%).
Blocking all UV radiation reduced MIF (in September) by a similar amount. In the case
of the July experiments, total photo-reduction in the absence of UVB is not significantly
reduced despite the decrease in MIF. This indicates that there are multiple pathways for
photo-reduction, and that not all of them express the MIF. Multiple pathways for
79
aqueous photo-reduction have been proposed in the literature and include direct
photolysis, indirect photolysis and also direct followed by indirect photolysis.
Direct photolysis of Hg halides, HgXn2-n in organic solvents under laboratory UVC
radiation has been well studied (Kunkely et al., 1997) and yields Hg(I) species and X·
radicals as primary products. There are far fewer studies of photolysis of Hg compounds
in water under environmentally relevant conditions. One such is (Zepp et al., 1973), who
report that photo-degradation of aqueous phenylmercury salts by broadband wavelengths
greater than 290 nm takes place through homolytic cleavage of the carbon-mercury bond:
L HgC6H5hv L Hg C6H5 (1)
if the ligand is another phenyl group, the organomercury radical will decompose to
Hg(0). The singlet-triplet excitation energy of the phenylmercurials was determined to
be approximately 3.47 eV (the energy of radiation at 357 nm), which allowed them to be
photosensitized by acetone (which has comparable singlet-triplet transition energy). This
is an interesting example because it illustrates photo-dissociation of organically-bound
Hg(II) to a radical pair over a range of environmentally available wavelengths. These are
conditions that would allow the expression of the MIE. In addition, it demonstrates that
in-situ photosensitization by a small organic compound can accelerate photo-
decomposition (for energy transfer to be effective unless, the excited state energy of the
sensitizer must be greater than that of the photoreactive compound, (Montalti, 2006; Zepp
et al., 1973). It is very likely that components of organic matter serve as photosensitizers
of Hg complexes in natural waters. More evidence of this comes from the lack of or very
slow photo-reduction of Hg(II) without organic matter.
If the mercury(I) radical (as in equation 1) does not recombine with the original ligand or
spontaneously decompose to Hg(0), indirect photo-reduction may reduce the radical to
Hg(0). (Han et al., 2007; Zheng et al., 2005) demonstrate that photo-generated reactive
intermediates (such as •H and CO•) produced from small organic molecules
(formaldehyde, methanol, acetic, oxalic and malonic acid) can reduce Hg(II) to Hg(0) in
aqueous solution. Alternatively, photo-generated oxidants such as 1O2 and •OH could
convert the radical back to Hg(II).
80
Two pathways of direct photolysis were proposed by (Lin and Ariya, 2008) to explain the
reduction of Hg(II) in the presence of oxalic, malonic or succinic acid under UV radiation
(290-400 nm). Three small dicarboxylic acids were chosen because carboxylic acid
functional groups are abundant in natural organic matter (NOM) and important sites of
trace metal complexation. One pathway will be discussed subsequently; the other is the
following:
HgCRCOOROOCHg hv222)( (2)
from where •Hg+ can be reduced by some electron donor (such as the photo-generated
reactive intermediates mentioned above) if it is not re-oxidized by (for example)
dissolved O2. Here also, initial photo-excitation of the complex by environmentally
available wavelengths produces a radical pair and provides an opportunity for expression
of the MIE. While carboxylic acids are ubiquitous in NOM, Hg is expected to be
primarily associated with reduced sulfur complexes. Sulfur is only a minor component of
NOM (usually less than 1%, based on the product analyses of the International Humic
Substance Society, n = 23) but its molar concentrations in natural freshwaters exceeds
those of Hg by several orders of magnitude and the stability constants of Hg-thiol
complexes are particularly high (Ravichandran, 2004) and references therein). Thus it is
predicted that most Hg will be bound to reduced S ligands. Based on ab initio
calculations, (Stromberg et al., 1991) expect the aqueous complexes Hg(SH)2 and
HgS(SH)- to be photolysed by sunlight. In addition, the aqueous photo-reduction of Hg-
cysteine complex has been demonstrated in sunlight (this author, unpublished data) and
under UV irradiation ( > 300 nm, (Zheng and Hintelmann, 2010b). Whether Hg in
freshwater is bound to thiolate or to carboxylate groups, therefore, it is plausible that
radical pairs generated by photo-excitation of organomercury complexes are significant
in nature, particularly if the photosensitizing potential of small organic compounds
associated with NOM is considered.
A second pathway of direct photolysis of Hg-organic complexes is two-electron donation
by the organic ligand to Hg(II). This was invoked by (Gardfeldt and Jonsson, 2003)to
81
explain aqueous photo-reduction ( > 290 nm) of HgC2O4 complexes after reduction by
HO2•/O2•- had been ruled out:
Hg(C2O4 )n (22n ) hv Hg0 2CO2 (n 1)C2O42 (3)
This two-electron transfer is the second pathway proposed by (Lin and Ariya, 2008) to
explain photo-reduction of Hg(II) by small dicarboxylic acids:
[Hg((OOC)2R)n](2-2n) h/H2O HORCO2H + Hg0 + CO2 + (n-1)R(CO2)2
2- (4)
Here is an avenue of photo-reduction that results in the direct production of Hg(0).
Although the lifetime of an excited molecule can range as widely as 10-14 to 100 seconds
(Turro et al. 2008), it is frequently orders of magnitude shorter than the time required for
triplet-singlet conversion (up to about 0.1 s). The MIE would not necessarily have time
to influence the rate of this reaction and MIF would not be expressed. When their entire
experimental reaction system was modeled, Lin and Ariya, 2008 found that the majority
of Hg(II) reduction took place through intramolecular 2-electron transfer (as in equation
4), but that processes starting with photo-generated radical pairs (as in equation 2) also
contributed.
The modeling results of (Lin and Ariya, 2008) suggest that a major pathway of Hg(II)
photo-reduction in the experiments of this study is by 2-electron transfer from an organic
ligand, which provides one avenue for mercury loss that would not allow expression of
the MIE. Another such mechanism is indirect photolysis, that is, two-step reduction by
the reactive intermediates generated through photolysis of organic molecules. (Zheng et
al., 2005), for example, show that Hg(II) could be reduced to Hg(0) by photolysis
products of aqueous formic acid under ambient laboratory light only (although the rate of
Hg(0) production was far higher under UV radiation). This is one example of an indirect
photolysis mechanism through which Hg(II) can be reduced by a small organic molecule
in the absence of ultraviolet radiation. Here is a second avenue for reservoir mercury to
be lost without expressing the MIE.
Radical pairs are produced through absorption of a photon when a photochemically
excited electron orbits at a higher energy than its usual ground state. Such electronic
82
promotion usually results in two half-filled orbitals, one of which is bonding and the
other non-bonding. If the two moieties no longer share orbitals of net bonding character,
they can drift apart spatially. The energy difference between the highest occupied
molecular orbital and the lowest unoccupied molecular orbital determines the longest
wavelength of light with sufficient energy to cause photoexcitation. If this difference is
small, then wavelengths in the visible region may suffice. As the difference gets larger,
wavelengths in the UVA, UVB, or higher will be required. It is to be expected that direct
photolysis of Hg-organic complexes resulting in radical pairs would be more effectively
stimulated by higher-energy radiation, and that blocking UVB radiation would limit these
reactions and MIF expression considerably.
Relationship between 199Hg and 201Hg for Hg(II)
In examining MIF during photo-reduction of Hg(II), (Bergquist and Blum, 2007) noted
that the ratio199Hg/201Hg was very consistent (1.00 ± 0.02, 2SE) and distinct from the
ratio199Hg/201Hg resulting from photo-degradation of MeHg. It is also distinct from
199Hg/201Hg ratios obtained from transformations of Hg attributed to the nuclear
volume effect (Estrade et al., 2009; Wiederhold et al., 2010). Bergquist and Blum 2007
suggested the ratio might be indicative of the reductive pathway. A ratio of
approximately 1 is observed in lichens and in coals (Bergquist and Blum, 2009; Biswas et
al., 2008; Carignan et al., 2009; Ghosh et al., 2008). Lichens, in particular, are presumed
to derive their Hg from the atmosphere (they are not reported to fractionate during
assimilation) and suggest that atmospheric mercury may also have a ratio 199Hg/201Hg
of 1.0. In contrast, 199Hg/201Hg from freshwater fish tissue samples (which is
approximately all MeHg) has been reported to be 1.3 ± ~0.1 (Bergquist and Blum, 2007;
Jackson et al., 2008; Laffont et al., 2009).
In all Hg(II) reduction experiments in this thesis, the ratio 199Hg/201Hg falls within
error of 1.0, even when photo-reduction is severely reduced. If the ratio is indeed
indicative of the pathway, then the mechanism of MIF is not changing even as the
magnitude is reduced by screening higher energy radiation. Based on the above
83
mechanistic explanation, it suggests that direct photolysis of mercury-organic compounds
takes place to some extent even in the range of visible radiation (as in the September, no
UVB experiment).
3.4.3. For methyl mercury, UVB is likely the major contributor to MIF during
photo-reduction. A small contribution is made by other wavelengths. When UVB is
blocked, we see at most a small MIF signature in MeHg during the July experiments, and
a small but significant signature in the September experiment exposed to UVA radiation.
Photo-reduction, however, can take place through processes that do not cause MIF. This
is particularly apparent in the July photo-experiments, where MIF was largely or
completely suppressed when the UVB and UVA wavebands were blocked, but the
overall amount of total photo-reduction was not significantly affected. A considerable
amount has been published on the photo-degradation of MeHg, and considering some of
this work provides insight into the relationship between photo-reduction and MIF.
Organomercury compounds have long been known to decompose photolytically (and
thermally) into radical components; evidence for both one-step and two step-mechanisms
R1HgR2 R1Hg R2 R2 (Hg R1) (6)
is reviewed by (Friswell and Gowenlock, 1965). (Janzen and Blackbur.Bj, 1969)note
that photolysed organomercury compounds (R2Hg, RHgX) tend to cleave on the organic
group rather than the halide or carboxylate group. (Inoko, 1981)invokes a mechanism of
this type to account for the aqueous photochemical decomposition of CH3HgCl:
CH3HgCl hv CH3 HgCl
CH3 HgCl CH3Cl Hg0 (7, 8)
where the second line is a reaction known to take place in solvent cages. This pathway of
direct photolysis involves a geminate radical pair whose fate (recombination or
dissociation to free radicals in solution) could be influenced by the MIE. (Malinovsky et
al., 2010)and co-workers have recently published a study examining isotopic
fractionation of aqueous MeHg during UV photolysis (2010). They observe large MIF
signatures (greater than 0.7‰) in acidic and acidic saline solutions. In contrast, MIF is
suppressed by the addition of a radical scavenger (ascorbic acid) and in alkaline
84
solutions, which inhibit radical formation. These findings are entirely compatible with a
mechanism where MIF is expressed through radical pair chemistry.
Direct photolysis, however, has been studied principally in laboratories using lamps that
produce radiation of much higher frequency than is available at the earth’s surface.
Malinovsky 2010 use a mercury vapor lamp emitting radiation at 254 nm as a light
source (in addition, Hg from the lamp may directly photo-excite Hg in solution), and
Inoko, 1981 presents data obtained with wavelengths of 206 nm. There has been debate
over whether a direct photolysis mechanism is applicable to natural systems. (Takizawa
et al., 1981), for example, find that MeHg was not degraded by a black light lamp
(maximum wavelength 366 nm). Since photo-degradation of MeHg in natural fresh
waters has been clearly demonstrated (Hammerschmidt and Fitzgerald, 2006; Lehnherr
and Louis, 2009; Sellers et al., 1996), other mechanisms have been proposed. (Chen et
al., 2003) investigate the degradation of aqueous MeHg by hydoxyl radicals (•OH)
generated at a steady state by photolysis of nitrate (absorption maximum at 302 nm) at
naturally relevant wavelengths. Degradation of MeHg followed pseudo-first-order
kinetics and among the mechanistic paths proposed were both stepwise and simultaneous
decomposition:
CH3HgCl OH CH3OH HgCl CH3OH Hg0 Cl (9)
In addition, methyl radicals (generated by natural organic matter, NOM) could react with
HgCl radicals to produce elemental Hg:
CH3 HgCl Hg0 CH3Cl (10)
Here is an encounter between a radical pair. Whether such an encounter could allow the
expression of the MIE requires an understanding of whether spin coupling between a
magnetic nucleus and its electrons affects random radical pair interactions, as well as
geminate radical pair reactions. The environmental significance of this reaction depends
on the steady state concentration of •OH in environmental systems and also on competing
photo-degradation reactions.
Field reports of MeHg photo-degradation in fresh water have been from water bodies
with dissolved natural organic matter (NOM), which Chen et al. 2003 do not add to their
85
experiments. (Zhang and Hsu-Kim, 2010) report that aqueous MeHg is readily degraded
by both sunlight and a UVA lamp, but only in the presence of NOM and only when there
was a molar excess of reduced sulfur relative to MeHg. Theoretical studies offer some
explanation of the increased photo-reactivity of MeHg with thiol complexes in particular.
The highest-energy radiation that reaches the surface of the earth can induce an electronic
transition of approximately 4.4 electron volts (eV; this is ~ 282 nm). Through ab initio
calculations,(Tossell, 1998) estimates singlet to triplet-state excitation energies (ES-T)
for various for various MeHg-ligand complexes (see Table 3.7).
Table 3.7. Calculated properties of CH3HgL complexes, from Tossell 1998
ESinglet-Triplet in eV ESinglet-Triplet in nm
CH3HL' Hartree-Frock
Moller-Plesset
Hartree-Frock
Moller-Plesset
changes upon S-T excitation
CH3HgOH2+ 3.97 4.97 312 249 CH3 leaves
CH3HgOH ~5.9 210 CH3 and OH leave CH3HgCl 5.41 6.04 229 205 CH3 leaves CH3HgSH 5.16 4.98 240 249 CH3 leaves CH3Hg(SH)2 5.47 4.26 227 291 one SH leaves
Most of these ES-T transitions are greater than the high-energy boundary for solar
radiation. Singlet-singlet transitions energies are always approximately 2-3 eV (400-600
nm) higher than S-T energies and lie well into the UVC range. Electronic excitation
through direct photon absorption, therefore, is most likely to take place through the
lowest energy triplet state. Depending on pH, the MeHg-thiol complexes appear to have
the lowest ES-T excitation energies, and are therefore the most likely to have absorption
tails that extend to the longer wavelengths reaching earth’s surface. Table 2.7 above
shows there is a strong tendency toward dissociation in the lowest energy triplet state of
the complex, with methyl radicals being generated. This tendency is because, while the
highest occupied molecular orbitals (HOMO) are bonding, the lowest unoccupied
molecular orbitals (LUMO) are anti-bonding. In a triplet state, the antibonding orbital
becomes half-occupied and the bonding orbital occupancy decreases to half. The Hg-C
bond strength is greatly reduced and •CH3 moiety dissociates during geometry
optimization.
86
The larger MIF signatures observed under exposure to full sun (and hence to higher
energy UVB and UVA) in both July and September could be due particularly to
dissociation of •CH3 and •Hg-thiol radical intermediates following singlet-triplet
excitation of a MeHg-thiol complexes. This would allow an opportunity for the MIE to
be expressed. Radical pairs generated from odd-isotope complexes would have a higher
probability of recombining to form the starting products before complete dissociation. If
the radical pair does dissociate, the fate of radical Hg(I) species (both odd and even) may
be re-oxidation (Zhang and Hsu-Kim 2010 present strong evidence for 1O2; •OH is also a
possibility) or reduction (by •H or CO• generated by irradiation of low molecular weight
organic compounds as in Zheng et al., 2005 and Han et al 2007). At lower wavelengths,
mechanisms such as the one involving •OH proposed by Chen et al. 2003 could induce
photo-reduction through pathways that do not involve radical pairs, reducing the
possibility for expression of the MIE.
Relationship between 199Hg and 201Hg for MeHg
Bergquist and Blum 2007 noted that the ratio199Hg/201Hg obtained during the photo-
reduction of MeHg was very consistent (1.36 ± 0.02, 2SE) and distinct from the
199Hg/201Hg ratio associated with photo-degradation of Hg(II). It is also distinct from
the ratio of 199Hg/201Hg ratios obtained from transformations of Hg attributed to the
nuclear volume effect (2.0 ± 0.6, Estrade et al., 2009, and 1.62 ± 0.06 (2SE), Ghosh et
al., in preparation and 1.54 ± 0.22 (2SE), Weiderhold et al., 2010). The average ratio
199Hg/201Hg for all MeHg experiments in this thesis was 1.35 ± 0.16 (2SE). The
consistency of this ratio among experiments of the same Hg species suggest that the
mechanism(s) of photo-reduction is/are the same for a given species regardless of
changes in the radiation available. The significant difference of this ratio between MeHg
and Hg(II) suggest that the pathway(s) of photo-reduction are not the same for the
organic Hg species as for the inorganic one.
Malinkovsky et al. 2010, report a ratio 199Hg/201Hg for experimental photo-reduction
of MeHg under UVC radiation (1.28 ± 0.03, 1SD) that is similar to that of Bergquist and
Blum 2007, which was obtained experimentally under natural sunlight. It is also within
87
error of the ratio obtained in this study under natural sun. This supports the possibility
that the mechanism leading to MIF under exposure to radiation of wavelength 254 nm
(leading to radical pairs through direct photolysis) is similar to the mechanism leading to
MIF under exposure to natural sunlight. The 199Hg/201Hg ratio obtained from samples
of temperate lake fish tissue (1.28 ± 0.03, 2SE , Bergquist and Blum 2007, 2009),
tropical lake fish tissue (1.28 ± 0.12, 2SE , Laffont et al. 2009) and arctic lake fish tissue
(1.32 ± 0.06, 2SE for 9 out of 10 lakes, (Gantner et al., 2009) support the possibility that
MIF signatures carried by MeHg in organic tissue was imparted by the same mechanism,
prior to entry into the food web.
3.4.4. Mass-Dependent Fractionation
It is interesting that MDF in the July Hg(II) experiments increases significantly as higher-
energy wavebands are blocked. This effect is not observed in September, however,
where the mass-dependent fractionation factors of the three experiments are
indistinguishable. As noted in Table 3.1, visible radiation in July at mid-day was more
intense under the UV-filtering box than it was under full solar exposure. It is likely that
the July no UVB and the no UV experiments underwent more evaporation than the July
full spectrum experiment. In this case, 202 will reflect a combination of reduction and
evaporation effects, and should not be over-interpreted. In September, the MeHg
experiment showing the largest MDF signature is the one shielded from UVB.
Generally, the effects of UV filtration on MDF during photo-reduction of Hg species are
not amenable to interpretation.
3.4.5. Implications for the natural world
In nature, photo-reductive processes operate in competition with photo-oxidative
processes, and the rates of photo-reduction measured during daylight in natural systems
usually reflect the net rather than the total photo-reduction (Garcia et al., 2005). In our
experiments, continuous sparging with Hg-free air removed most (if not all) of the Hg(0)
from the reaction vessel as it formed, likely eliminating the photo-oxidative reaction
pathway. For this reason, and also the fact that experimental concentrations of Hg were
88
several orders of magnitude higher than concentrations of Hg in natural fresh waters, the
rates of photo-reduction that we observe are not representative of natural rates.
Mass-dependent fractionation
The range of Hg(II) mass-dependent fractionation factors in this thesis, using 202Hg as the
example isotope, is from 0.9992 ± 0.0002 (July full sun) to 0.9983 ± 0.0003 (September
no UVB). Zheng and Hintelmann report on photoexperiments similar to the ones in this
thesis investigating the effects of the ratio of Hg to DOM as the variable of interest
(2009) and the effects of sulfur-less versus sulfur-containing ligands (2010b).
Fractionation factors range from 0.9986 ± 0.0001 to 0.9996 ± 0.0007 with the least
significant fractionation at the highest Hg/DOM ratio and most significant associated
with an intermediate ratio. Dark controls reached a maximum of 0.9981 ± 0.0004. Thus
intensity, frequency, and Hg/DOM ratio all affect MDF over the same range and one
would need to have additional information about the ecosystem of interest to interpret
MDF values. Additionaly, the variability of 202 with available radiation and with kind
and proportion of organic matter falls into the same range as dark abiotic reduction
fractionation factors and does not appear useful in distinguishing photochemical from
dark abiotic reduction.
MDF does not appear to be helpful in distinguishing photochemical from biological
reductive processes either. Kritee et al., 2007; 2008 investigated fractionation of Hg(II)
during dark reduction by microorganisms, using both aerobic and facultative anearobes
that reduce Hg by the mer pathway and one facultative anaerobe the employs an
undetermined, non-mer mechanism. They report fractionation factors ranging from
0.9982 ± 0.0003 to 0.9988 ± 0.0001. Kritee et al., 2009 report an MDF fractionation
factor of 0.0096 ± 0.0002 for the mer-mediated degradation of MeHg. The narrowness of
this range is interesting in view of the bacterial diversity, but falls squarely into the range
of MDF fractionation factors generated by abiotic photochemical and dark reduction.
Mass-independent fractionation: Quantifying Hg(II) photo-reduction
89
Large mass-independent fractionation of Hg(II) (greater than approximately 0.2 ‰)
remains associated with photochemical reduction. No MIF was reported by Bergquist
and Blum 2007 during reduction in dark abiotic (organic matter) controls or by Kritee et
al. 2008 for any of the strains of Hg-reducing bacteria. Zheng and Hintelmann 2010a
report 199Hg as large as -0.6‰ after nearly complete dark chemical reduction of Hg(II)
by SnCl2 with continuous removal of the product Hg(0), although it is not clear whether
this represents any natural process. Relating MIF to photo-reduction quantitatively,
however, will be difficult because, as this thesis has demonstrated, comparable amounts
of photo-reduction may coincide with different amounts of MIF depending on the
frequency of light available. In addition, it has been argued that there are multiple
simultaneous pathways for photo-reduction of Hg(II) in natural fresh waters, and that not
all of them permit the expression of the MIE.
Other factors influencing MIF during aqueous photo-reduction of Hg(II)
Of interest is the dramatic effect Hg/DOM ratios have on the magnitude of MIF. Zheng
and Hintelmann (2009) investigated this variable during aqueous photo-reduction and
reported maximum MIF in a trial with 10 gL-1 Hg/12 mgL-1 DOM exposed to sunlight
(199 = 0.9943 ± 0.0008). This max MIF occurred at an intermediate value of Hg/DOM
that they investigated (they varied Hg/DOM ratios from 34.6 to 8330). The observed
MIF at 10 gL-1 Hg/12 mgL-1 DOM is a much larger effect than is observed by blocking
portions of the electromagnetic spectrum and was obtained under conditions of poorer
light penetration of the reactor. As the Hg/DOM ratio continues to diminish, however,
the MIF effects become less pronounced and the lowest ratio experiment (2 ppb Hg in 60
mg DOM/L) has an 199 = 0.9973 ± 0.0006 which is not that different from the July, full
spectrum experiment of this thesis with an 199 of 0.9986 ± 0.0001. However, even the
smaller fractionation seen at this low Hg/DOM is larger than any of the fractionation
factors observed in this thesis. It should be noted that important variables such as the
kind of organic matter, the light intensity (also the light source) were not the same for this
thesis as for the work of Zheng and Hintelmann, 2009. Zheng and Hintelmann 2009
investigate Hg/DOM ratios over three orders of magnitude. It is not clear if variability
demonstrated in their experiments can be expected to be relevant to MIF signatures in
90
nature, where aqueous concentrations of reduced sulfur (to say nothing of organic matter
as a whole) exceed aqueous Hg concentrations by orders of magnitude and Hg/DOM
ratios are still considerable smaller than what they investigated.
Also of interest are photoexperiments conducted by Zheng and Hintelmann, 2010b on the
effects of a sulfur-containing ligand cysteine and its sulfur-less counterpart serine. Using
cysteine, they acheive a molar concentration of reduced sulfur three orders of magnitude
larger than that of Hg (natural ratios are 3 to 5 orders larger), which may create a more
realistic model of Hg-NOM bonds in freshwater environments. Large, consistently
negative MIF was observed in the reactant reservoir indicating that the odd isotopes were
preferentially reduced and purged from the reactant reservoir. This effect is opposite the
MIF associated with photo-reduction in the presence of organic matter reported in
Bergquist and Blum, 2007, Zheng and Hintelmann, 2009 and in this study. The
explanation was put forth as follows by the authors: the original radical pair produced
through photo-exitation of a complex could start in an excited singlet state. Photolysis
products from an excited singlet state would be inhibited by recombination to the starting
material, but hyperfine coupling between a magnetic (odd) nucleus and an electron could
invert or rephase the spin of the electron such that the pair becomes triplet.
Recombination would no longer be possible and the triplet pair would be likely to
dissociate spatially into independent radicals, resulting in a preferential depletion of the
odd isotopes in the starting material. Whether photo-excitation produces a singlet state or
a triplet state radical pair depends on the nature of the chemical bond broken and the
reaction conditions, such as temperature and exciting wavelength. This is an interesting
complication and raises the possibility that MIF signatures in nature (as Zheng and
Hintelmann 2010b argue) will be the net result of competing photo-processes.
Mass-independent fractionation: Quantifying MeHg photo-reduction
Mass-independent fractionation is associated with photochemical reduction of MeHg, but
it is not associated with dark biological reduction (Kritee et al., 2009). It is not clear
under what conditions MeHg undergoes dark abiotic reduction; none was observed in the
dark controls of this study. Relating MIF to photo-reduction quantitatively, however,
91
will be difficult because, as this thesis has demonstrated, comparable amounts of photo-
reduction may coincide with different amounts of MIF depending on the
wavelengths of light available for photo-reduction. In addition, it has been argued that
there are multiple simultaneous pathways for photo-reduction of MeHg in natural fresh
waters, and that not all of them permit the expression of the MIE.
Other factors influencing MIF during aqueous photo-reduction of MeHg
There is some evidence that Hg/DOM ratios may have an important effect on photo-
reduction of MeHg as well. Bergquist and Blum 2007 undertook photoexperiments
similar to the ones in this thesis, with aqueous MeHg in 2 and in 20 mg/L NOM
(Suwannee River, of the same origin as the NOM in this study). Their data show an
199 = 0.9967 ± 0.0006 for the 2 mg NOM/L experiment and an 199 = 0.9921 ± 0.0013
for the 20 mg NOM/L experiment with fR = 0.80 respectively. The magnitude of the
fractionation factor 199 obtained under full sun with 20 mg/L NOM is surprisingly large
compared to the changes in 202 effected by varying UV exposure in this experiment.
The increase in fractionation appears to be associated with decreasing the ratio of Hg to
DOM in solution. The mechanistic discussion presented earlier of MeHg-thiol
complexes enabling photo-generation of radical pairs at naturally available wavelengths
is compatible with these results. However Bergquist and Blum also conducted these
experiments on different days, and variations in solar radiation intensity and frequency
cannot be ruled out as a driver of the differences observed for the two experiments.
92
3.5. CONCLUSIONS
3.5.1. Blocking the UVB spectrum significantly reduces MIF during photochemical
reduction of both MeHg and Hg(II). This is evidence of a mechanism that is stimulated
by photo-excitation and has a certain energy threshold. The magnetic isotope effect,
expressed through photo-excitation of a solvated complex to a radical pair, is such a
mechanism and our experiments solidly support it as the cause of large MIF signatures
during photochemical reduction of Hg species.
3.5.2. UVA radiation can contribute to MIF during photochemical reduction of both
MeHg and Hg(II). Electronic excitation energies are influenced by many factors such as
the atoms in the complex, the chemical composition of the solution and the ambient
temperature. A mechanism where the primary step is electronic excitation of a solvated
complex is compatible with a range of excitation energies. Visible light can contribute
to MIF during photochemical reduction of Hg(II) as well.
3.5.3. There are multiple pathways for photo-reduction of both MeHg and Hg(II) in
freshwater reservoirs. Some of these are spin-selective (since they express the MIE) and
some are not. Pathways that express the MIE have spin-selective processes at or before
their rate-limiting steps. This knowledge can be used to elucidate the photochemistry of
Hg in natural waters.
3.5.4. Quantitatively relating total photo-reduction in a freshwater system and the
magnitude of MIF will not be straightforward for either Hg(II) or MeHg. For both
species, we have demonstrated cases where comparable amounts of total photo-reduction
are accompanied by significantly different MIF.
93
FIGURES
Figure 3.1. Taken from Bergquist 2007. Schematic of the photochemical
reaction apparatus with Hg(II) as an example species.
94
Figure 3.2. Courtesy of B. Bergquist. Phase separator. Hg(II) in solution is reduced online with
SnCl2 to produce Hg(0), a vapor. The solution carrying dissolved Hg vapor runs in a thin film
down a frosted tip. The Hg0 is stripped out by a clean gas flowing upwards and out an arm at
the top of the phase separator case. From there, the Hg can be either trapped in an acidic
solution or directed into an analytical machine.
95
Figure 3.3. Mercury concentration in the substrate reservoir over time for replicate photo-
reduction experiments on two Hg species, inorganic Hg(II) and organic MeHg, in the presence of
natural organic matter and under varying exposure to solar radiation. Error in time elapsed is 1
minute, which is smaller than the width of the symbols used. Maxima in solar irradiation were
recorded at approximately 1 pm, Eastern Standard Time, which was found to coincide with solar
noon in Toronto, Ontario. Effective blocking of the UVB spectrum necessitated partial blocking
of the UVA spectrum (by 30 to 52%). Complete radiation measurements are shown in Table
3.1. a) Dark Control 1 was carried out in a foil-wrapped reactor with continuous sparging.
Dark Control 2 was carried out in a sealed, foil-wrapped bottle with no sparging.
96
Figure 3.4. Mass-dependent isotopic fractionation (202Hg) of the substrate
reservoir as a function of Hg remaining for replicate photo-reduction experiments
on two Hg species, inorganic Hg(II) and organic MeHg with dissolved natural
organic matter under varying exposure to solar radiation. Error in fraction of
substrate Hg remaining is set at 0.05 or less (see Reporting analytical
uncertainty, p. 68), but not shown on plots (c) and (d) for clarity. Rayleigh
distillation models based on estimated fractionation factors (202) are shown
where there was significant loss in concentration. Solid black lines: full sun;
broken grey lines: UVB spectrum blocked; broken black lines: UVB and UVA
spectra blocked. Complete isotope data are displayed in Table 3.2 and Table 3.4.
Mass-dependent fractionation factors for all isotopes and associated uncertainty
are displayed in Table 3.3.
97
Figure 3.5. Mass-independent isotopic fractionation (MIF, 199Hg), observed in
odd isotopes only, for Hg(II) photo-reduction experiments with dissolved natural
organic matter under varying exposure to solar radiation. (a) Deviation from
theoretical mass-dependent 199Hg values (black line) based on measured 202Hg
and the kinetic mass-dependent fractionation law derived from transition state
theory (Young et al. 2002). (b) Adherence to theoretical mass-dependent 204Hg
values (black line). Complete isotope data are displayed in Table 3.2
98
Figure 3.6. Mass-independent isotopic fractionation (MIF, 199Hg), observed in
odd isotopes only, as a function of inorganic Hg(II) remaining for replicate photo-
reduction experiments with dissolved natural organic matter under varying
exposure to solar radiation. No MIF was observed in dark controls. Rayleigh
distillation models based on estimated mass-independent fractionation factors are
shown where MIF was significant. Solid black lines: full sun; broken grey
lines: UVB spectrum blocked; broken black lines: UVB and UVA spectra
blocked. Complete isotope data are displayed in Table 3.2 Mass-independent
fractionation factors for both odd isotopes and associated uncertainty are
displayed in Table 3.3.
99
Figure 3.7. Mass-independent isotopic fractionation (MIF, 199Hg), observed in
odd isotopes only, for MeHg photo-reduction experiments with dissolved natural
organic matter under varying exposure to solar radiation. (a) Deviation from
theoretical mass-dependent 199Hg values (black line) based on measured 202Hg
and the kinetic mass-dependent fractionation law derived from transition state
theory (Young et al. 2002). (b) Adherence to theoretical mass-dependent 200Hg
values (black line). Complete isotope data are displayed in Table 3.4. Mass-
independent fractionation factors for both odd isotopes and associated uncertainty
are displayed in Table 3.5.
100
Figure 3.8. Mass-independent isotopic fractionation (MIF, 199Hg), observed in
odd isotopes only, as a function of organic MeHg remaining for replicate photo-
reduction experiments with dissolved natural organic matter under varying
exposure to solar radiation. MeHg was not significantly reduced in dark controls.
A Rayleigh distillation model based on an estimated mass-independent
fractionation factor is shown where significant MIF took place: Solid black line:
full sun. Complete isotope data are displayed in Table 3.4. Mass-independent
fractionation factors for both odd isotopes and associated uncertainty are
displayed in Table 3.5.
101
Figure 3.9. 201Hg versus 199Hg for photochemical reduction of inorganic
Hg(II) and organic MeHg in the presence of natural organic matter under varying
exposure to solar radiation. Dark controls all plot at the origin. Only samples
with significant MIF were plotted, so R0 (pre-exposure) and R1 (taken
immediately after first exposure to solar radiation) do not appear for any
experiment. (a) For Hg(II), the average ratio 201Hg/199Hg is 1.00 0.04, 2SE,
n = 21 (black line). (b) for MeHg, the average ratio 201Hg/199Hg is 1.35 0.16,
2SE, n = 11 (black line).
102
TABLES
103
104
105
106
107
108
109
110
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CHAPTER 4: MASS-INDEPENDENT
FRACTIONATION DURING CHEMICAL
REDUCTION OF AQUEOUS INORGANIC
MERCURY IN THE ABSENCE OF LIGHT 4.1. INTRODUCTION Over the last few years, large mass-independent fractionation (MIF) of mercury (Hg)
isotopes has been reported for kinetic photo-reduction (Bergquist and Blum 2007, Zheng
and Hintelmann 2009, 2010a, 2010b, Sherman et al. 2010, this thesis) and now also
photo-oxidation (Ghosh et al., in preparation). Kinetic, reductive biological processes
have been investigated in the absence of light and found not to generate MIF in Hg
(Kritee et al. 2007, 2008, 2009). Bergquist and Blum 2007 investigated dark, kinetic
abiotic reduction of aqueous Hg(II) in the presence of dissolved organic matter (DOM)
and observed no MIF. Zheng and Hintelmann 2010a, however, report that small MIF
signatures are observed during the dark reduction of aqueous Hg(II) in the presence of
organic matter (actual -values are not reported). A significant difference in
experimental design is that Zheng and Hintelmann 2010a allowed their reaction to
continue for a few weeks, while Bergquist and Blum 2007 monitored their reaction for a
few hours. Zheng and Hintelmann 2010a went on to push the dark reductive reaction
forward by adding Sn(II)Cl2 (without DOM) to their aqueous mercury solutions. This
resulted in MIF with the odd isotopes being preferentially lost from the substrate
reservoir (199Hg reaching -0.6 ± 0.05 ‰, 2SD). This is opposite the MIF generated by
photochemical reduction (odd isotopes are preferentially retained in the substrate
reservoir). Apparently the process causing MIF is associated with reduction and not with
phase change, since volatilization of Hg from aqueous solution was shown not to induce
MIF by (Zheng et al. 2007). The objective of this investigation was to see if dark
chemical reduction of aqueous Hg(II) in the presence of organic matter produces mass-
independent isotopic anomalies. In addition, it provided a way to investigate non-
photochemical contributors to MIF in the experiments described in Chapter 2.
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4.2. METHODS Equipment, sample vials and analytical containers were cleaned according to the process
described in Chapter 2 of this document. An aqueous solution of 2 mg/L Suwannee
River Fulvic Acid Standard (1S101F) from the International Humic Substance Society
(http://ihss.gatech.edu/ihss2/) was made up in a glass bottle and then spiked to a final
concentration of approximately 31 g/L (ppb) of Hg(II) in the form of Hg(NO3)2 (J.T.
Baker lot E04632, hereafter called Hg(II)). This solution was quickly subsampled to
measure the initial isotopic composition and then wrapped in aluminum foil to protect it
from light. It was left to stand and equilibrate for 3.5 hours. It was then poured into the
photochemical reactor described in Chapter 2 and in Figure 3.1, except that the reactor
was completely wrapped in aluminum foil at all times. Continuous sparging with Hg-free
air removed the Hg(0) from the reaction vessel as it formed. Once inside the reactor, the
Hg-fulvic acid solution was initially subsampled again to verify that no change in
isotopic composition occurred on standing. Ten mL of (get moles) SnCl2 were then
injected into the reactor through the sample withdrawal line. Subsamples were removed
from the aqueous reservoir with a syringe as the reaction progressed. All subsamples for
isotopic analysis were immediately preserved by spiking with concentrated BrCl.
Concentrations were analysed as in Chapter 2. Overall uncertainty in concentration was
set at ± 5%, the 2SD of the reference standard for that analytical session. Matrix
separation and transfer for isotopic analysis was carried out as in Chapter 2. Sample
recoveries after matrix transfer were mostly 98% and 100% respectively (± 3%, 2SD).
However, sample recoveries for R2, R3 and R4 were only 83 ± 3%. Two attempts were
made to trap R3 with no improvement in recovery despite the fact that it was bracketed
by NIST 3133 processes standards at 97-100 % recovery. On the second attempt, an
effort was made to simultaneously trap sample Hg running down the drain. Nine percent
of the original sample Hg was caught from the drain, indicating incomplete reduction of
the sample. Isotopic analysis, carried out as in Chapter 2, included process standards
with recoveries as low as 86%. This lowest recovery (86%) showed small offsets
associated with this incomplete reduction (less than 0.2 ¥) in mass-dependent
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fractionation, but no offset in mass-independent fractionation was observed. Analytical
uncertainty is determined as in Chapter 2 and given in Table 4.1.
4.3. RESULTS An aqueous solution of Hg(II) with 2 mg/L fulvic acid was poured into a photo-reactor
covered with aluminum foil. SnCl2, a reducing agent, was added subsequently while the
solution was sparged with Hg-free air to remove any Hg(0) generated. The amount of
Hg(II) lost via dark chemical reduction was estimated from the decreasing concentration
of Hg(0) in the reactor over time (Figure 4.1). Sampling was discontinued after loss in
the reactor reached 78 ± 5%. Mass-dependent fractionation (MDF) was observed with
the heavier isotopes being preferentially retained in the reactor (Figure 4.2). Complete
isotopic data are displayed in Table 4.1. MDF followed a Rayleigh distillation model,
with 202 = 0.9997 ± 0.0002, R2 = 0.85.
Mass-independent fractionation (MIF) was observed (Figure 4.3) with the odd isotopes
being preferentially retained in the reactor during the earlier part of the reaction, similar
to photo-reduction, but opposite what Zheng et al., 2010 observed. Maximum MIF was
observed in the subsample taken soonest after SnCl2 addition (201Hg = 0.16 ± 0.05 ‰
and 199Hg = 0.24 ± 0.07 ‰, Table 4.1). MIF decreased in magnitude thereafter with the
final data point (fraction remaining = 0.22 ± 0.05) showing 201Hg = 0.01 ± 0.05 ‰ and
199Hg = 0.10 ± 0.07 ‰). The ratio 199Hg/201Hg for both data points showing
significant MIF (4SE or more) is 1.50 ± 0.86, 2SE. The point at fR = 0.22 did not show
MIF of 4SE or more, and so the 199Hg/201Hg ratio was not calculated.
4.4. DISCUSSION
During the process of dark chemical reduction of aqueous Hg(II) by SnCl2 (in the
presence of organic matter) and subsequent evasion of the Hg(0), mass-independent
anomalies in isotopic composition are produced, as displayed in Figure 4.3. The
mechanism of MIF in this process is not immediately clear. Two plausible mechanisms
have been put forward to explain the MIF observed in odd isotopes of Hg. The
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mechanism normally associated with photochemical reduction is likely the magnetic
isotope effect (MIE). It is caused by hyperfine coupling between electrons and the
nuclear magnetic moment present in odd nuclei, which can speed up triplet-singlet
conversion in a geminate radical pair. From a singlet state, the radical pair can
recombine to form the starting products anew. Triplet-singlet conversion of a non-
magnetic radical pair is slower, so non-magnetic pairs are more likely to dissociate and
form reaction products (Bergquist and Blum 2009, Buchachenko 2001). Since the
reaction took place in the dark, there could not have been photo-generated radical pairs.
Investigation of other possible radical reactions under these experimental conditions is
beyond the scope of this thesis.
The other mechanism suggested to produce MIF in odd isotopes of mercury is the nuclear
volume effect (NVE). The NVE arises because the nuclear volume and charge radius do
not scale linearly with mass. It produces even/odd staggering of mass-independent
anomalies because the odd isotopes have ground-state energies closer to those of the
adjacent lower-mass even isotope (Bigeleisen 1996, Schauble 2007). Estimates of the
relationship 199Hg/Hg produced by the NVE vary, depending on how the
(experimentally determined) values for nuclear charge radii are chosen. Theoretical
estimations range from 1.65 (Wiederhold et al. 2010) to 2.7 (Schauble 2007) for
equilibrium conditions. Experimentally determined relationships include 2.0 ± 0.6
(Estrade et al. 2009) and 1.62 ± 0.06, (2SE, Ghosh et al. in preparation) for equilibrium
volatilization of pure Hg metal, and 1.54 ± 0.44, 2SE for equilibrium binding of aqueous
Hg(II) to various thiol groups (Weiderhold et al., 2010) and 1.60 ± 0.06, 2SE for kinetic
reduction of Hg(II) by SnCl2 in the dark (attributed principally to the NVE, Zheng and
Hintelmmann 2010a).
Different 199Hg/Hg ratios have been associated with particular chemical pathways,
for example 1.28 ± ~0.12 (2SE) with photo-degradation of methylmercury (Bergquist
and Blum 2007, Malinovsky et al. 2010), and 1.0 ± ~0.1 (2SE) for photo-reduction of
Hg(II) (Bergquist and Blum 2007). These ratios have the potential to identify processes
that transform Hg in nature (Bergquist and Blum 2009). Of the ratios mentioned above,
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the 199Hg/Hg observed in this study is closer to those associated with the NVE than
with the ratios associated with the MIE if the more recent estimates (Weiderhold et al.,
2010; Ghosh et al., in prep, Zheng and Hintelmann 2010a) of NV 199Hg/Hg (1.6)
are considered.
In Hg however, the NVE causes a concentration of the odd isotopes in the chemical
species with higher electron density at the nucleus, which is to say, odd isotopes will
preferentially be found as Hg(0) and Hg(II) will be enriched in even isotopes. In the
experiment described above, Hg(0) was continuously removed from the substrate
reservoir through sparging with Hg-cleaned ambient air. If the odd isotopes were
reacting preferentially to form Hg(0) then the substrate reservoir should have been
depleted in the odd isotopes. This is the opposite of what was observed. In addition, our
observations are contrary to what is found by Zheng and Hintelmann 2010a, who use the
NVE to explain their data for SnCl2 reduction. One difference in experimental design is
that Zheng and Hintelmann 2010a do not use both SnCl2 and organic matter in the same
reactor. A second significant difference is that they add the reducing agent, SnCl2 in tiny
increments such that a molar excesss (by several orders of magnitude) of Hg is always
maintained. In contrast, the initial injection of SnCl2 in this experiment created a vast ( x
106) molar excess of Sn(II) with respect to Hg. SnCl2 reduction of Hg(II) is prompt (as
demonstrated by the matrix transfer process in Chapter 2: Methods). It is likely that the
entirety of Hg(II) was reduced immediately and loss of Hg(0) from the reactor was
initially limited by diffusion velocity. This would result in an abundance of Hg(0) in the
substrate reservoir and increased time for it to react further in the reservoir before exiting.
Initial quantitative reduction of Hg(II) entails complete mass transfer to Hg(0) and would
not induce isotopic fractionation of any kind during the reduction step. The pattern of
data points (Figure 4.3) suggests that a large positive MIF signature is imparted early in
the progress of the reaction and then slowly erased by a different processes that depletes
odd isotopes preferentially in the reservoir. The NVE might be invoked to explain the
decreasing MIF signature over time, but the question remains of how the positive MIF
signature was imparted in the first place. It is unclear what if any implications these
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findings have for non-photochemical contributors to MIF in the experiments described in
Chapter 2. Because of quite different experimental conditions, they are not likely
comparable.
4.5. CONCLUSION
Mass-independent anomalies in isotopic composition are observed during dark chemical
reduction of aqueous Hg(II) by SnCl2 in the presence of organic matter. Whether they
may be attributed to the MIE, the NVE, to some combination of the two or to another
mechanism entirely requires further investigation.
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FIGURES
Figure 4.1. Mercury concentrationin the substrate reservoir over time for dark chemical
reduction of Hg(II) by SnCl2 in the presence or organic matter. Error in time elapsed is ±
1 minute. Error in concentration is ± 0.05, which is 2SD of the reference standard for one
analytical session.
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Figure 4.2. Mass-dependent isotopic fractionation (202Hg) of the substrate reservoir as a
function of Hg remaining for dark chemical reduction of Hg(II) by SnCl2 in the presence
of organic matter. Line shown is a Rayleigh distillation model estimated from the data.
202 = 0.9997 ± 0.0001
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Figure 4.3. Mass-independent isotopic fractionation (199Hg), observed in the odd
isotopes only, as a function of inorganic Hg(II) remaining for dark chemical reduction of
Hg(II) by SnCl2 in the presence of organic matter.
125
126
127
LITERATURE CITED
Bergquist, B. A. and Blum, J. D., 2007. Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems. Science 318, 417-420.
Bergquist, R. A. and Blum, J. D., 2009. The Odds and Evens of Mercury Isotopes: Applications of Mass-Dependent and Mass-Independent Isotope Fractionation. Elements 5, 353-357.
Bigeleisen, J., 1996. Nuclear size and shape effects in chemical reactions. Isotope chemistry of the heavy elements. Journal of the American Chemical Society 118, 3676-3680.
Buchachenko, A. L., 2001. Magnetic isotope effect: Nuclear spin control of chemical reactions. Journal of Physical Chemistry A 105, 9995-10011.
Carignan, J., Estrade, N., Sonke, J. E., and Donard, O. F. X., 2009. Odd Isotope Deficits in Atmospheric Hg Measured in Lichens. Environmental Science & Technology 43, 5660-5664.
Estrade, N., Carignan, J., Sonke, J. E., and Donard, O. F. X., 2009. Mercury isotope fractionation during liquid-vapor evaporation experiments. Geochimica Et Cosmochimica Acta 73, 2693-2711.
Kritee, K., Barkay, T., and Blum, J. D., 2009. Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury. Geochimica Et Cosmochimica Acta 73, 1285-1296.
Kritee, K., Blum, J. D., and Barkay, T., 2008. Mercury Stable Isotope Fractionation during Reduction of Hg(II) by Different Microbial Pathways. Environmental Science & Technology 42, 9171-9177.
Kritee, K., Blum, J. D., Johnson, M. W., Bergquist, B. A., and Barkay, T., 2007. Mercury stable isotope fractionation during reduction of Hg(II) to Hg(0) by mercury resistant microorganisms. Environmental Science & Technology 41, 1889-1895.
Laffont, L., Sonke, J. E., Maurice, L., Hintelmann, H., Pouilly, M., Bacarreza, Y. S., Perez, T., and Behra, P., 2009. Anomalous Mercury Isotopic Compositions of Fish and Human Hair in the Bolivian Amazon. Environmental Science & Technology 43, 8985-8990.
Malinovsky, D., Latruwe, K., Moens, L., and Vanhaecke, F., 2010. Experimental study of mass-independence of Hg isotope fractionation during photodecomposition of
128
dissolved methylmercury. Journal of Analytical Atomic Spectrometry 25, 950-956.
Sherman, L. S., Blum, J. D., Johnson, K. P., Keeler, G. J., Barres, J. A., and Douglas, T. A., 2010. Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight. Nature Geoscience 3, 173-177.
Yang, L. and Sturgeon, R., 2009. Isotopic fractionation of mercury induced by reduction and ethylation. Analytical and Bioanalytical Chemistry 393, 377-385.
Zheng, W., Foucher, D., and Hintelmann, H., 2007. Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase. Journal of Analytical Atomic Spectrometry 22, 1097-1104.
Zheng, W. and Hintelmann, H., 2009. Mercury isotope fractionation during photoreduction in natural water is controlled by its Hg/DOC ratio. Geochimica Et Cosmochimica Acta 73, 6704-6715.
Zheng, W. and Hintelmann, H., 2010a. Isotope Fractionation of Mercury during Its Photochemical Reduction by Low-Molecular-Weight Organic Compounds. Journal of Physical Chemistry A 114, 4246-4253.
Zheng, W. and Hintelmann, H., 2010b. Nuclear Field Shift Effect in Isotope Fractionation of Mercury during Abiotic Reduction in the Absence of Light. Journal of Physical Chemistry A 114, 4238-4245.
129
CHAPTER 5: CONCLUSIONS Mass-independent fractionation (MIF) of Hg isotopes has the potential to highlight and
track photochemical transformations among the many processes that affect the
environmental fate of Hg. A solid mechanistic understanding of MIF in nature is
necessary, however, before it can be applied as a tool. Two plausible mechanisms have
been proposed to cause MIF in Hg isotopes: The magnetic isotope effect (MIE) and the
nuclear volume effect (NVE). The MIE is the most likely mechanism behind large (> 0.2
‰) MIF observed during photochemical reduction of Hg species (Bergquist and Blum,
2007; Malinovsky et al., 2010; Zheng and Hintelmann, 2009; 2010b). Photochemical
reduction is a significant pathway whereby water bodies release their burden of Hg back
to the atmosphere, and photo-degradation is an important sink (and detoxification
mechanism) for the MeHg in water bodies. These processes are strongly influenced by
the intensity and frequency of sunlight available. This thesis investigated whether and
how the frequency and intensity of sunlight available affected MIF in photo-reduction
and clarified the potential of MIF to document these processes. It also a tested the
hypothesis that the MIE was the mechanism behind the large MIF signatures observed
during photo-reduction of Hg species.
Experiments were conducted to determine whether removing the UVA or the UVB
portions of the electromagnetic spectrum affected the expression and magnitude of the
MIF observed during the aqueous photoreduction of MeHg and Hg(II) respectively.
Quartz reactors containing either Hg(II) or MeHg in aqueous solutions with organic
matter were simultaneously exposed to three different light regimes to examine the effect
on photo-reduction and on the isotopic fractionation of the respective Hg species.
Replicate sets of experiments were carried out for each species, with the first groups in
July and the second groups in September. Blocking UVB radiation had no effect on total
photoreduction of MeHg. It had some effect on total reduction of Hg(II) in the
September replicates (~70% loss under full spectrum and ~40% loss with UVB blocked)
but no effect in the July replicates. For both MeHg and Hg(II), however, blocking UVB
radiation suppressed MIF significantly (by ~30 to 50% for Hg(II); by ~70% or more for
130
MeHg). In addition, the lower light intensity in September was accompanied by a
significant decrease in the magnitude of MIF in Hg(II).
This is evidence of a mechanism that is stimulated by photo-excitation and has a certain
energy threshold. The magnetic isotope effect, expressed through photo-excitation of a
solvated complex to a radical pair, is such a mechanism and our experiments solidly
support it as the cause of large MIF signatures during photochemical reduction of Hg
species. Exposure to UVA radiation produced significant MIF in both Hg(II) and MeHg.
Electronic excitation energies are influenced by many factors such as the atoms in the
complex, the chemical composition of the solution and the ambient temperature. A
mechanism where the primary step is electronic excitation of a solvated complex is
compatible with a range of excitation energies. Visible light can contribute slightly to
MIF during photochemical reduction of Hg(II) as well.
For both species, we have demonstrated cases where comparable amounts of total photo-
reduction are accompanied by MIF of significantly different magnitude. This confirms
there are multiple pathways of photo-reduction for both MeHg and Hg(II) in freshwater
reservoirs. Some of these pathways are spin-selective (since they express the MIE) and
some are not. Pathways that express the MIE have spin-selective processes at or before
their rate-limiting steps. This knowledge can be used to elucidate the photochemistry of
Hg in natural waters. The observation that MIF in both Hg(II) and MeHg can be
significantly reduced while total photo-reduction remains constant means that
quantitatively relating MIF and photo-reduction in natural freshwater systems will be
challenging.
In all Hg(II) reduction experiments in this thesis, the ratio 199Hg/201Hg falls within
error of 1.0, even when photo-reduction and MIF are severely reduced. If this ratio is
indicative of the reductive pathway, then the mechanism of MIF is not changing even as
the magnitude is reduced by screening higher energy radiation. If the mechanism of
Hg(II) MIF was constant for all experimental conditions, this suggests that direct
photolysis of mercury(II)-organic compounds takes place to some extent even in the
131
range of visible radiation (as in the September, no UVB experiment). The average ratio
199Hg/201Hg for all MeHg experiments in this thesis was 1.35 ± 0.16 (2SE), regardless
of whether or not UVB radiation was blocked. The consistency of this ratio among
MeHg experiments suggests the mechanism of photo-reduction was the same regardless
of changes in the radiation available. The significant difference of this ratio between
MeHg and Hg(II) suggest that the pathway(s) of photo-reduction are not the same for the
organic Hg species as for the inorganic one.
The ratio 199Hg/201Hg for Hg(II) in this thesis is consistent with other experimental
results for aqueous photo-reduction of Hg(II) (1.00 ± 0.02, 2SE,(Bergquist and Blum,
2007). The ratio 199Hg/201Hg for MeHg in this thesis is consistent with other
experimental results for aqueous photoreduction of MeHg (1.36 ± 0.02, 2SE, Bergquist
and Blum, 2007, 1.28 ± 0.06, 2SD, Malinovsky et al., 2010). The ratios for aqueous
photoreduction of both species (in this thesis) are significantly different from
experimentally determined ratios 199Hg/201Hg for other transformative processes where
MIF has been attributed to the NVE. These process include the equilibrium
volatilization of pure Hg metal (2.0 ± 0.6, Estrade et al., 2009 and 1.62 ± 0.06 (2SE),
Ghosh et al., in preparation) and also equilibrium binding of aqueous Hg(II) to various
thiol groups (1.54 ± 0.22, 2SE, Weiderhold et al., 2010). This contributes to the evidence
suggesting that the ratios of the mass-independent isotopic signatures are unique to the
reaction pathways.
In a separate experiment not directly related to the main research objectives of this thesis,
and under different experimental conditions, it was observed that the process of dark
chemical reduction of aqueous Hg(II) by SnCl2 (in the presence of organic matter) and
subsequent evasion of the Hg(0), produced mass-independent anomalies in Hg isotopic
compositions. These anomalies are not immediately attributable to either the MIE or the
NVE and illustrate that much remains to be discovered about the processes that
fractionate Hg isotopes.
132
LITERATURE CITED Bergquist, B. A. and Blum, J. D., 2007. Mass-dependent and -independent fractionation
of Hg isotopes by photoreduction in aquatic systems. Science 318, 417-420.
Estrade, N., Carignan, J., Sonke, J. E., and Donard, O. F. X., 2009. Mercury isotope fractionation during liquid-vapor evaporation experiments. Geochimica Et Cosmochimica Acta 73, 2693-2711.
Ghosh, S., In preparation.
Malinovsky, D., Latruwe, K., Moens, L., and Vanhaecke, F., 2010. Experimental study of mass-independence of Hg isotope fractionation during photodecomposition of dissolved methylmercury. Journal of Analytical Atomic Spectrometry 25, 950-956.
Wiederhold, J. G., Cramer, C. J., Daniel, K., Infante, I., Bourdon, B., and Kretzschmar, R., 2010. Equilibrium Mercury Isotope Fractionation between Dissolved Hg(II) Species and Thiol-Bound Hg. Environmental Science & Technology 44, 4191-4197.
Zheng, W. and Hintelmann, H., 2009. Mercury isotope fractionation during photoreduction in natural water is controlled by its Hg/DOC ratio. Geochimica Et Cosmochimica Acta 73, 6704-6715.
Zheng, W. and Hintelmann, H., 2010b. Isotope Fractionation of Mercury during Its Photochemical Reduction by Low-Molecular-Weight Organic Compounds. Journal of Physical Chemistry A 114, 4246-4253.
133
134
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136