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A stepwise reduction approach reveals mercury competitive binding and exchange reactions within natural organic matter and mixed organic ligands Xujun Liang, Xia Lu, Jiating Zhao, Liyuan Liang, Eddy Y. Zeng, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02586 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019
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A stepwise reduction approach reveals mercury competitive binding
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and exchange reactions within natural organic matter
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and mixed organic ligands
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Xujun Liang†,‡,¶, Xia Lu§,‡,¶, Jiating Zhao‡, Liyuan Liang‡, Eddy Y. Zeng†,
6
Baohua Gu‡,£,*
7 8
†
9
and Health, Jinan University, Guangzhou 511443, China
School of Environment and Guangdong Key Laboratory of Environmental Pollution
10
‡
11
37831, USA
12
§
School of Environmental Sciences, Lanzhou University, Lanzhou 730000, China
13
£
Department of Biosystems Engineering and Soil Science, University of Tennessee,
14
Knoxville, TN 37996, USA
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN
15 16
¶ These
authors contributed equally.
17 18 19 20 21
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ABSTRACT
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The kinetics of mercuric ion (Hg2+) binding with heterogeneous naturally
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dissolved organic matter (DOM) has been hypothesized to result from competitive
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interactions among different organic ligands and functional groups of DOM for Hg2+.
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However, experimental protocol is lacking to determine Hg2+ binding with various
27
competitive ligands and DOM, their binding strengths, and their dynamic exchange
28
reactions. In this study, a stepwise reduction approach using ascorbic acid (AA) and
29
stannous tin [Sn(II)] was devised to differentiate Hg(II) species in the presence of two
30
major functional groups in DOM: the carboxylate-bound Hg(II) is reducible by both
31
AA and Sn(II), whereas the thiolate-bound Hg(II) is reducible only by Sn(II). Using
32
this operational approach, the relative binding strength of Hg2+ with selected organic
33
ligands was found in order: dimercaptopropanesulfonate (DMPS) > glutathione (GSH)
34
> penicillamine (PEN) > cysteine (CYS) > ethylenediaminetetraacetate (EDTA) >
35
citrate, acetate, and glycine at the ligand-to-Hg molar ratio < 2. Dynamic, competitive
36
ligand exchanges for Hg2+ from weak carboxylate to strong thiolate functional groups
37
were observed among these ligands and within DOM, and the reaction depended on the
38
relative binding strength and abundance of thiols and carboxylates, as well as reaction
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time. These results provide additional insights into dynamic exchange reactions of Hg2+
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within multicompositional DOM in controlling the transformation and bioavailability
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of Hg2+ in natural aquatic environments.
42 43
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INTRODUCTION
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Natural organic matter consists of thousands of mixed heterogeneous organic
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compounds,1-3 and is known to play an important role in controlling the chemical
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speciation and biogeochemical cycling and toxicity of mercury (Hg) in natural aquatic
48
ecosystems.4-11 This effect is in part due to the formation of strong complexes between
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mercuric ion (Hg2+) and a suite of organic ligands, particularly the thiol functional
50
groups present in dissolved organic matter (DOM).6,8,12-15 Many low-molecular-weight
51
(LMW) organic ligands have been identified in DOM, such as acetate, citrate, cysteine,
52
glutathione, phenol, and glutamate,16-19 although their stability constants (log β) for
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Hg2+ binding vary by many orders of magnitude.8,14,20,21 In general, Hg2+ forms weaker
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complexes with carboxylate (log β ranging from 3.7 to 23.2) and much stronger
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complexes with thiolate ligands with log β ranging from 26 to 53.1 (Supporting
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Information (SI), Table S1). However, large discrepancies have been reported for the
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measured log β values for Hg(II)-DOM species (ranging from 10 to 40),4,8,15,22-28 and
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these differences have often been attributed to different origins and properties of DOM
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(e.g., its sulfur or thiol contents), as well as to methodologies and environmental
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conditions used in measurements. Large discrepancies also exist in the reported log β
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values for thiolate binding with Hg2+, such as Hg(II)-cysteine (log β =14.2–43.4) and
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Hg(II)-glutathione (log β =26–41.6) (Table S1).14,20,21,29 However, a recent study using
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Hg LIII-edge EXAFS spectroscopy indicates that the thermodynamic stabilities of
64
Hg(NOM-RS)2, Hg(Cys)(NOM-RS) and Hg(Cys)2 in a mixed Hg2+-NOM-Cys system
65
are very similar at pH < 7.15 These discrepancies in reported log β values of DOM and
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organic ligands make it difficult to assess their relative binding strengths for Hg2+, how
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they may competitively interact with Hg2+, and what are the dominant Hg(II) species 3
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in order to assess its availability for biological uptake and methylation under realistic
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environmental conditions.
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Unlike bonding between Hg2+ and a single LMW organic ligand,14,15 Hg2+
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complexation with DOM is also known to be kinetically controlled, usually requiring >
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9 h to reach an equilibrium.4, 5, 30 An even longer equilibrium time (> 24 h) was reported
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for Hg(II)-DOM complexation in a contaminated creek water,5,30 as indicated by
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decreasing stannous [Sn(II)]-reducible Hg(II) over time. Similarly, Hg(II) species were
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found to be more bioavailable under non-equilibrium conditions than when Hg2+ and
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DOM had reached pseudo-equilibrium (24 h) prior to exposure to bacteria in a Hg(II)
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uptake study.9 It is therefore hypothesized that newly deposited Hg2+ binds rapidly to
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biologically labile ligands present in the DOM, but over time Hg(II) species becomes
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much less bioavailable, due to a series of ligand exchange reactions between Hg2+ and
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different hypothetical DOM pools within the DOM.4,9 Likewise, competitive ligand
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exchanges for Hg2+ among many different functional groups in DOM, such as weak
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carboxylates and strong thiolates, are thought to be the main cause of the kinetic effect,5,
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30
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species. For example, the Sn(II) reduction technique used in previous studies cannot
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differentiate Hg2+ bound to carboxylate from thiolate. No direct experimental evidence
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has been presented to support the kinetic exchange behavior of Hg(II)-DOM species,
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partly due to technical difficulties in separating or detecting unknown Hg(II)-DOM
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species, particularly at nanomolar Hg(II) concentrations.2, 14
although so far few techniques have been developed to differentiate these Hg(II)
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This study was therefore aimed at devising a technique to differentiate Hg(II)
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species that are bound to carboxylates and thiolates, two typical weak and strong
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binding groups/domains in DOM. This is based on Hg(II) reducibility by a weaker 4
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reducing agent, ascorbic acid (AA), and a stronger reductant, Sn(II) chloride. Using this
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operationally defined, stepwise AA- and Sn(II)-reducible Hg(II) approach as proxies
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for carboxylate- and thiolate-bound Hg(II), we compared relative binding strengths of
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Hg2+ with a suite of LMW organic ligands and their competitive exchange reactions
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among themselves and with DOM. We also investigated the dynamic, competitive
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ligand exchange reactions for Hg2+ from the weak carboxylate to the strong thiolate
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ligands, and then to the strongest non-reducible fractions within DOM over time.
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MATERIALS AND METHODS
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Materials and sample preparation. L-cysteine (CYS), L-penicillamine (PEN),
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glutathione (GSH), glycine (GLY), and 2,3-dimercaptopropanesulfonate monohydrate
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(DMPS) were purchased from Sigma-Aldrich. Disodium ethylenediaminetetraacetate
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(EDTA), citric acid (CTA), and sodium acetate (ACE) were obtained from Fisher
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Scientific. These organic compounds were selected to represent a suite of thiolate and
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carboxylate organic ligands commonly used in Hg(II) biogeochemical investigations.8,
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14, 21, 31, 32
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Hg2+ are summarized in Table S1. Ascorbic acid and SnCl2 were purchased from Fisher
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Scientific and used as reductants for Hg(II). All chemicals and organic ligands are
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certified analytical reagents and were used without further purification.
Their molecular structures, molecular weights, and stability constants with
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Two DOM isolates from terrestrial and aquatic origins were used in this study.
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Elliott soil humic acid (SHA) was obtained from the International Humic Substances
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Society, and EFPC-DOM was isolated from the East Fork Poplar Creek (EFPC) water
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in Oak Ridge, Tennessee, as previously described.2,11 The basic chemical
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characteristics of these DOM isolates are given in Table S2. All DOM stock solutions 5
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(containing ~ 24 mM C) were prepared in a deoxygenated phosphate-buffered saline
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(PBS) solution at pH 7.4, as used in biological and biochemical investigations.11,33-35
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The PBS consisted of 0.14 M NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4,
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as previously described.11, 33-35 The DOM stock solution was filtered through a 0.2-µm
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filter (Acrodisc® with Supor® membrane) before use. Dissolved organic carbon (DOC)
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concentrations were determined using a Shimazu TOC-5000A total organic carbon
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analyzer.28
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Stepwise reduction of Hg(II)-ligand complexes. Complexes between Hg2+ and
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selected organic ligands (ACE, CTA, EDTA, GLY, CYS, PEN, GSH, DMPS) were
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prepared in 4-mL glass vials by mixing solutions of HgCl2 and organic ligands at a
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fixed Hg(II) concentration (25 nM), but varying ligand concentrations from 0 to 25 M
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to yield ligand-to-Hg molar ratios of 0–1000 in PBS. All sample preparations were
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performed in an anoxic chamber under N2 (~98%) and H2 (~2%) (Coy Lab Products,
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Grass Lake, MI), and all vials were sealed with PTFE-lined silicone screw caps and
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then placed on a rotary shaker (90 rpm). Samples were equilibrated for about 1 h, which
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was sufficient to reach an equilibrium based on our preliminary results and previous
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studies,14,15 and were then removed from the anoxic chamber for analysis. An aliquot
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(100 µL) was immediately transferred to a clean 4-mL vial prefilled with 800 µL
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deionized water. This was followed by adding an excess amount of AA (100 µL of 1
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mM AA stock) to reduce Hg(II), and subsequent detection of the purgeable gaseous
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Hg(0) directly using a Hg(0) analyzer (Lumex RA-915+, Ohio Lumex Co.).6,33,36,37
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After completion of the AA reduction (or when Hg(0) was non-detectable), 100 µL
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SnCl2 (20% SnCl2 in 10% HCl) was immediately added to the remaining sample and
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again analyzed for Hg(0) by Lumex RA-915+. Herein, the AA-reducible Hg(II) and 6
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Sn(II)-reducible Hg(II) are respectively denoted as HgR-AA and HgR-Sn throughout
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the text. Separately, another aliquot (100 µL) of the sample was transferred to a 4 mL
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clean vial prefilled with 850 µL deionized water and 50 µL BrCl (5%, v/v), and samples
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were equilibrated overnight at 4°C and analyzed for total Hg(II) (HgT).5,33,38
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Similar experiments were performed for DOM bound Hg(II) species, except
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that samples were pre-equilibrated for either 1 or 24 h before measurement of HgR-AA
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and HgR-Sn, since Hg(II)-DOM complexation is known to be time dependent.4, 5, 30
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While the Hg(II) concentration was kept constant (25 nM), DOM concentrations varied
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from 0 to 1 mM C (equivalent to DOC:Hg molar ratios of 0 to 40,000).23-25 Additionally,
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since DOM is known to reduce a small amount of Hg(II),6, 38 all samples were purged
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with ultrapure N2 to remove purgeable gaseous Hg(0), HgP, before measuring HgR-
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AA and HgR-Sn. For clarity of presentation and comparisons, these data were
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normalized to the total non-purgeable Hg(II) (HgNP) remaining in the system since
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HgP varied slightly with different concentrations of DOM. All samples were prepared
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in duplicate in batch experiments and, in some cases, batch experiments were repeated
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2–3 times for quality assurance. A good mass balance was obtained during sample
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preparation and complexation experiments (Figures S1 and S2). Average
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concentrations are reported, and error bars in figures represent one standard deviation
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of all replicate samples. In the case of duplicate measurements, error bars represent the
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deviation between the two replicates (X1 and X2), defined as the absolute value of (X1–
160
X2)/2.39
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Competitive ligand exchange reactions with Hg2+. Ligand exchange reactions were
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subsequently performed to evaluate relative binding strengths of selected organic
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ligands and their competitive interactions with Hg2+. To study if carboxylate can 7
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exchange thiolate ligands for Hg2+, solutions of Hg2+-thiolate complexes were first
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prepared with molar ratios of DMPS:Hg and GSH:Hg at 1:1 and CYS:Hg at 2:1 in PBS,
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and equilibrated for about 1 h in a series of 4-mL glass vials under anoxic conditions.
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Different thiol:Hg ratios were used to ensure that at the selected minimal thiol
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concentrations, the complexed Hg(II) was mostly non-reducible by AA. Therefore,
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when weaker-binding ligands such as CTA and EDTA were added, the exchanged
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Hg(II), if any, was expected to be reducible or detectable by AA reduction. CTA and
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EDTA were added at varying concentrations (up to a ligand:Hg ratio of 1000), and the
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amounts of Hg(II) exchanged by CTA or EDTA were determined after either 1 or 24 h
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equilibration, using the stepwise reduction technique. Similarly, CYS and GSH at
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varying concentrations (up to a ligand:Hg ratio of 4) were added to the pre-prepared
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DMPS:Hg solutions to study if CYS and GSH could exchange DMPS for Hg2+. Here
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DMPS is the strongest ligand among all the organic ligands studied. The amounts of
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Hg(II) exchanged by CYS or GSH were determined after 1 or 24 h equilibration to
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observe whether the exchange reactions were time-dependent.
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Exchange reactions between LMW ligands and the two DOM isolates were also
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conducted to improve our understanding of competitive interactions within DOM over
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time. In this case, Hg(II)-DOM complexes were first prepared in solutions with a
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DOC:Hg molar ratio of ~19,000, typical of the natural aquatic environment.10, 40 The
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solution was equilibrated for either 1 or 24 h in an anoxic chamber. Before the addition
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of competing organic ligands, DOM reducible Hg(II) was determined by purging with
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ultrapure N2 to remove gaseous Hg(0) (Figure S3), as noted above. After purging,
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samples were placed back in the anoxic chamber and spiked with varying
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concentrations of competing ligands, including CYS, GSH, DMPS, CTA and EDTA, 8
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at varying ligand:Hg ratios, based on their relative binding strengths. After an additional
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1 h equilibration, samples were taken out of the chamber and analyzed for the
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exchanged Hg(II) by the stepwise reduction technique. All results were again
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normalized to the total HgNP (i.e., Hg(II) remaining in solution).
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RESULTS AND DISCUSSION
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Differentiating Hg(II)-carboxylate and thiolate complexes by stepwise reduction
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To determine interactions between Hg2+ and various organic ligands, we first developed
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a reduction scheme to differentiate Hg(II)-carboxylate (–COO-) and thiolate (–S-)
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complexes, using a suite of natural and synthetic organic ligands commonly used in
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Hg(II) biogeochemical investigations.8,14,21,31,32 Carboxylate/amine and thiolate
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compounds were selected to represent the most abundant and important functional
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groups of DOM for binding with Hg2+ in the natural environment.5,8,10,28 Results
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indicate that at ligand:Hg molar ratios < 5, more than 90% of the Hg(II)-carboxylate
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species (ACE, CTA, EDTA and GLY) were reduced by AA (Figure 1a), whereas
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negligible amounts of Hg(II) ( 2 (Figure 1a), indicating that Hg2+ forms
206
stronger bonds with thiolate than with carboxylate ligands, as expected.5,8,10,28 However,
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all Hg(II)-thiolate species, except Hg(II)-DMPS, were completely reducible when Sn(II)
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was used as a reductant (Figure 1b). For Hg(II)-DMPS, ~75% of the Hg(II) was non-
209
reducible at the DMPS:Hg ratio of 1, but this fraction decreased to ~60% at DMPS:Hg
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ratios ≥3. This result is explained by the formation of binuclear bidentate Hg2(DMPS)2
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species at the DMPS:Hg molar ratio of 1, but formation of mixed Hg2(DMPS)2 and 9
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mono-nuclear tetra-dentate, Hg(DMPS)4 complexes at higher DMPS:Hg ratios.41 The
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Hg2(DMPS)2 complex forms a ring-structure and is among the most stable since its Hg-
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S bond distance is shorter than those of Hg(DMPS)4 species.41 Regardless of its
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structure, DMPS formed the strongest Hg(II)-thiolate species, as they were the most
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resistant to reduction by both Sn(II) and AA. Figure 1a also shows that, at the thiol:Hg
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ratio of 1, < 5% of the Hg(II) species in the presence of GSH were reducible by AA,
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but ~35% and 50% of the Hg(II) species in the presence of PEN and CYS were AA-
219
reducible. The result indicates that the amounts of Hg2+ bound to thiol ligands that are
220
non-AA-reducible are in order: GSH > PEN > CYS.
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While all Hg(II)-carboxylate complexes are reducible by AA at the ligand:Hg
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ratio < 5 (Figure 1a), increasing EDTA:Hg ratios decreased HgR-AA (Figure 1c). At
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the EDTA:Hg ratio of 1000, HgR-AA was essentially non-detectable. However, ACE,
224
GLY or CTA bound Hg2+ was completely reducible by AA, even at the ligand:Hg ratio
225
of 1000 (Figure 1c), indicating that Hg2+ forms stronger complexes with EDTA than
226
with ACE, CTA, and GLY. This finding is supported by the formation of multidentate
227
Hg(II)-EDTA complexes via both carboxyl and amine functional groups, since EDTA
228
is a known metal chelator.42 However, when Sn(II) was used as a reducing agent, all
229
carboxylate-bound Hg2+, including Hg-EDTA, were completely reducible, irrespective
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of the ligand:Hg ratio (up to 1000) (Figure 1b, d). Together, based on the amounts of
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AA- or Sn(II)-reducible Hg(II) bound to each ligand at the thiol:Hg ratio PEN > CYS > EDTA >
234
CTA, ACE, GLY. These results suggest that the stepwise AA and Sn(II) reduction
235
approach could offer a means to qualitatively discern relative binding strengths of 10
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ligands for Hg2+, particularly the two major functional groups of carboxyls and thiols
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observed in natural organic matter.
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Competitive ligand-exchange reactions
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To demonstrate the application of the stepwise AA and Sn(II) reduction approach, we
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subsequently investigated Hg(II) competitive ligand exchange reactions between
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selected carboxylate (CTA and EDTA) and thiolate (CYS and GSH) ligands, and
242
between these and DMPS, the strongest Hg(II)-binding ligand observed in this study.
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Results show that, when CTA was added to CYS:Hg (2:1) or GSH:Hg (1:1) pre-
244
equilibrated solutions, HgR-AA remained low or unchanged (Figure 2a). No ligand
245
exchange occurred even with the addition of 500–1000 times more CTA than CYS or
246
GSH, indicating that Hg(II)-thiol complexes are strong and the thiolates cannot be
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exchanged by CTA, as expected. Similarly, at the EDTA:Hg ratio of 3, EDTA could
248
not replace CYS or GSH. However, at EDTA:Hg ratios of 500 and 1000, HgR-AA
249
decreased to near zero (Figure 2a), whereas HgR-Sn remained unchanged (Figure 2b).
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In these cases, neither AA nor Sn(II) reduction could differentiate whether the Hg(II)
251
is complexed with thiols or EDTA since all Hg(II) complexes with CYS, GSH or EDTA
252
(at high EDTA:Hg ratios) were non-reducible by AA (Figure 1a,c) but completely
253
reducible by Sn(II) (Figure 1b,d).
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We then examined ligand exchange reactions between the strongest Hg(II)-
255
binding ligand DMPS and CTA, EDTA, CYS, or GSH at varying ligand:Hg ratios.
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Again, no ligand exchange was observed with CTA (up to a CTA:Hg ratio of 1000) or
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with EDTA (at the EDTA:Hg ratio of < 3) after 1 h or 24 h reactions (Figure 2c). With
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the addition of CYS or GSH (at ligand:Hg ratios of 1–4) or excess amounts of EDTA
259
(at EDTA:Hg ratios of 20–1000), HgR-AA remained low to non-detect since AA 11
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cannot reduce these Hg(II) species (Figure 2), as noted above. But using Sn(II)
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reduction, a substantially increased HgR-Sn was observed following the addition of
262
CYS, GSH, or EDTA (at EDTA:Hg ratios > 20; Figure 2d), indicating that Hg(II) bound
263
to DMPS was exchanged by CYS, GSH, or EDTA. With the addition of only 50 nM
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CYS (or CYS:Hg ratio = 2), HgR-Sn increased from ~23% to 55%, and to 90% at the
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CYS:Hg ratio of 4. Similarly, HgR-Sn increased to about 63% and 87% with the
266
addition of 25 and 100 nM GSH (or GSH:Hg ratios of 1 and 4), respectively. EDTA
267
was also effective in exchanging DMPS at relatively high EDTA:Hg ratios (Figure 2d).
268
HgR-Sn remained at ~23% at the EDTA:Hg ratio of 3, but increased to 40%, 55%, and
269
82% when EDTA:Hg ratios increased to 20, 200, and 1000, respectively. These
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findings indicate that, despite DMPS forms strong complexes with Hg2+(Figure 1b),
271
CYS, GSH, and EDTA at relatively high concentrations can competitively displace
272
DMPS, likely by breaking the ring structure of Hg2(DMPS)2 formed at the DMPS:Hg
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molar ratio of 1.41 The resulting product is a mixed DMPS-Hg(II)-ligand species (i.e.,
274
CYS, GSH and EDTA), which is less stable than Hg2(DMPS)2 and thus readily reduced
275
by Sn(II). However, the amounts of Hg(II) exchanged by EDTA were unexpectedly
276
higher than those predicted by the thermodynamic speciation model (Table S3). The
277
model predicts that Hg(II) is completely associated with DMPS even at the EDTA:Hg
278
ratio of 1000, because the reported log β values for Hg(II)-DMPS species are about 19
279
orders of magnitude higher than that of Hg(II)-EDTA (Table S1). Similarly, model
280
shows that Hg(II) is predominantly bound by DMPS in the presence of CYS or GSH
281
both at 4 times of the DMPS concentration (Table S3), which differs from our
282
experimental observations. These results therefore question the validity of some of the
283
reported log β values. Additional work is needed to improve log β measurements for 12
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Hg2+ binding with various organic ligands (Table S1), as noted earlier.14,20,21,29 For
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example, the reported log β value for Hg(II)-CYS was similar or even greater than
286
Hg(II)-GSH (Table S1), but our data (Figures 1 and 2) indicate that more Hg2+ is
287
associated with GSH (that is non-AA-reducible) than with CYS at the ligand:Hg ratio
288
< 2. These results again illustrate the novel application of the stepwise reduction
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approach for comparing relative binding strengths of organic ligands and determining
290
their competitive exchange reactions with Hg2+.
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We observed no significant differences in exchange reactions when the reaction
292
time increased from 1 to 24 h (Figure 2c,d), indicating that these ligand exchange
293
reactions are rapid, and generally complete < 1 h. This result is consistent with previous
294
13C
295
between free thiols and the thiolate bound to Hg(II) are usually within seconds.43, 44
296
These reactions are also reversible, depending on the binding strength and relative
297
concentrations of the ligand in solution. Similarly, a recent study of Hg(II) ligand
298
exchange between CYS and thiol groups in Suwanee River DOM showed that chemical
299
equilibrium between Hg-CYS and Hg-DOM species could be achieved within minutes
300
of reaction.15
301
Hg(II)-DOM complexation dynamics
302
Using the same approach, we also investigated the dynamics of Hg2+ binding with two
303
major functional groups (i.e., –COO- and –S-) in two DOM isolates: The SHA was used
304
to represent a high molecular weight, high aromaticity terrestrially derived DOM,
305
whereas EFPC-DOM was used to represent aquatic DOM with a relatively low
306
molecular weight and aromaticity.2,
307
semiquinone moieties acting as weak reducing agents, a small amount of Hg(II) was
and 1H NMR spectroscopic studies, which showed that Hg(II) exchange reactions
6, 38
Since all DOM contains some levels of
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reduced to elemental Hg(0) by DOM itself upon mixing (Figures S2), as previously
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reported.6, 30, 38 Slightly more Hg(II) was reduced by SHA than EFPC-DOM, especially
310
after 24 h reaction, since EFPC-DOM contains a higher S or thiol content (Table S2)
311
and thus forms more Hg(II)-thiol complexes which could not be reduced by the
312
semiquinone compounds in DOM.6,13,15,31,45 Therefore, lower Hg(II) reduction was
313
observed with increasing EFPC-DOM concentrations or DOM:Hg ratios and with
314
increasing reaction time, as more Hg(II) became complexed with thiol functional
315
groups in EFPC-DOM than SHA (Figure S2).
316
Following the removal of Hg(0) by purging, HgR-AA, HgR-Sn, and non-
317
reducible Hg(II) (HgNR) were analyzed in sequence, and the dynamics of Hg(II)
318
complexation and competitive ligand exchange among different DOM functional
319
groups were determined with time and varying DOM:Hg ratios (Figure 3). Since the
320
amount of purgeable Hg(0) varied slightly with the addition of different amounts of
321
DOM (Figure S2), data in Figure 3 were normalized to total non-purgeable Hg(II)
322
(HgNP) in the system for comparison. Results indicate that HgR-AA decreased
323
continually with increasing DOM concentrations, and lower amounts of HgR-AA were
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produced in the presence of EFPC-DOM than SHA due to a higher thiol content of
325
EFPC-DOM (Table S2). Additionally, sharp decrease in HgR-AA was observed when
326
Hg(II) was pre-equilibrated with DOM for a longer time (24 h). For example, with 1-h
327
pre-equilibration, HgR-AA in EFPC-DOM (at 0.1 mM C) was ~27%, but decreased to
328
zero when EFPC-DOM-Hg pre-equilibrated for 24-h (Figure 3a,b). For SHA under
329
same conditions, HgR-AA (at 0.1 mM C) decreased from ~65% to 45%, and further
330
decreased to zero at the DOM concentration above 0.5 mM C (Figure 3c,d). Coincident
331
with decreasing HgR-AA, HgR-Sn increased with DOM concentration, reached a 14
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maximum and then decreased with increasing DOM concentration or DOM:Hg ratio.
333
This is especially evident for samples that were pre-equilibrated for 24 h, although the
334
HgR-Sn maxima varied with DOM isolates and the reaction time during pre-
335
equilibration (Figure 3b,d). In the same experiments, a consistently increased non-
336
reducible HgNR was observed with increasing DOM concentrations, as HgNR is the
337
most stable Hg(II)-DOM species that can neither be reduced by Sn(II) nor AA.
338
The dynamic changes in HgR-AA, HgR-Sn, and HgNR with time and DOM
339
concentrations (Figure 3) indicate competitive binding and exchange reactions between
340
different pools or functional groups of DOM for Hg2+, as previously hypothesized,5, 6,
341
46
342
concentration (e.g., < 0.2 mM C), most Hg(II) may be bound to carboxylate initially in
343
SHA (at 1 h), and could therefore be reduced by AA (Figure 3c). However, with
344
increasing reaction time (24 h; Figure 3d), HgR-AA decreased, whereas HgR-Sn and
345
HgNR increased, suggesting that some weakly-bound Hg(II)-carboxylate species were
346
exchanged with thiol ligands, or formed strong Hg(II)-thiolate complexes or other non-
347
reducible Hg(II) species. In the case of EFPC-DOM, a shorter reaction time (pre-
348
equilibrated for 1 h) resulted in the production of ~ 25% HgR-AA, but most Hg(II)
349
(~75%) was bound to thiolates when the DOM concentration is at or greater than 0.2
350
mM C (Figure 3a). However, no HgR-AA could be detected for samples pre-
351
equilibrated for 24 h even at a low DOM concentration (0.1 mM C) (Figure 3b). The
352
result suggests that all weakly-bound Hg(II) had been replaced by strongly-bound
353
thiolate or non-reducible Hg(II) species within 24 h. However, HgR-Sn increased and
354
reached a maximum of ~65% (at 0.1 mM C). It then decreased, while HgNR increased
355
with increasing DOM concentrations. These observations again indicate time-
but have not been experimentally validated. Our results suggest that, at a low DOM
15
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dependent Hg(II) exchange from the carboxyl to the thiol functional groups in DOM,
357
and then to the non-reducible pool of DOM. Similar trends were observed with SHA
358
(Figure 3c,d), although the HgR-Sn maximum occurred at a higher DOM concentration
359
(~ 0.35 mM C), which can be attributed to a lower S or thiol content in SHA than EFPC-
360
DOM, as discussed above (Table S2). Therefore, the HgR-Sn maxima depend not only
361
on the reaction time and the DOM:Hg ratio, but also on the type of DOM and its thiol
362
content.
363
Competitive ligand exchange reactions with DOM
364
Although DOM forms exceptionally strong complexes with Hg2+,6,10,13,15 it remains
365
unclear to what extent the DOM-bound Hg(II) may be exchanged by competing organic
366
ligands, such as CTA, EDTA, CYS, GSH, and DMPS. We therefore further
367
investigated the dynamics of competitive exchange reactions between various
368
carboxylate or thiolate ligands and DOM that had been pre-equilibrated with Hg(II) for
369
either 1 h or 24 h. Based on their relative binding strengths (Figure 1), a low thiol:Hg
370
ratio (either 1 or 2) but a high CTA:Hg or EDTA:Hg ratio (1000) was used in these
371
studies. The measured HgR-AA, HgR-Sn, and HgNR (Figure 4) were also normalized
372
to the total HgNP after removing Hg(0) following its pre-equilibration with DOM
373
(Figure S3), as described above. No HgR-AA was observed for Hg2+ complexed with
374
EFPC-DOM 1 h after the addition of EDTA, CTA, CYS, GSH, and DMPS (Figure 4a).
375
However, about 25% HgR-AA was observed for Hg(II) complexed with SHA (pre-
376
equilibrated for 1 h) either with or without addition of CTA (Figure 4b), again
377
indicating that CTA is ineffective in competitively binding or exchanging Hg2+ that had
378
been bound with DOM. A slightly lower amount of HgR-AA (~9%) was observed for
16
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Hg(II) complexed with SHA after 24 h pre-equilibration, as more Hg2+ became
380
complexed with thiol functional groups or shifted to the HgNR pool over time.
381
However, HgR-Sn increased 10-15% with the addition of CYS, GSH, and
382
EDTA but decreased (up to 25%) with the addition of DMPS when Hg2+ was pre-
383
equilibrated with DOM for 1 h (Figure 4c,d). The increase in HgR-Sn suggests that a
384
fraction of HgNR was exchanged by CYS, GSH, or EDTA and thus reduced by Sn(II).
385
Both CYS and GSH appeared equally effective in exchanging DOM functional groups
386
for Hg2+ although a lower amount of GSH (GSH:Hg = 1:1) was used than CYS
387
(CYS:Hg = 2:1). A slightly lower amount of HgR-Sn was observed with the addition
388
of EDTA at a high EDTA:Hg ratio of 1000, in line with the binding strength of these
389
ligands established above in the order GSH > CYS > EDTA (Figure 1). However, the
390
addition of DMPS substantially decreased HgR-Sn but increased HgNR, especially for
391
SHA (Figure 4c-f), indicating DMPS replacement of Hg2+ that had been bound to DOM
392
thiolates. The result implies that the HgNR pool could, at least partially, be attributed
393
to the formation of stable chelated Hg(II) species such as Hg(II)-DMPS. Such exchange
394
reactions appeared lower (< 15%) on EFPC-DOM than SHA, particularly after samples
395
were pre-equilibrated for a longer time (Figure 4c,d). When Hg(II) was pre-equilibrated
396
with EFPC-DOM for 24 h, no differences in HgR-Sn were observed (Figure 4c),
397
regardless of whether or not there was addition of CTA, EDTA, CYS, GSH or DMPS.
398
It suggests that all Hg(II) had been complexed with strong binding sites in EFPC-DOM,
399
and no Hg2+ could be exchanged or replaced by the competing ligands. These results
400
are indicative of the transfer of Hg2+ from weak to strong, and to stronger, nonreducible
401
binding sites within DOM macromolecules, making Hg2+ less or non-exchangeable
402
over time. 17
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ENVIRONMENTAL IMPLICATIONS
404
The present study reveals that stepwise AA and Sn(II) reduction could offer a means to
405
assess relative binding strengths of Hg2+ with a suite of organic ligands, such as DMPS
406
> GSH > PEN > CYS, based on the amounts of Hg(II) bound to each ligand that are
407
non-AA-reducible at the ligand:Hg molar ratio < 2. This order of Hg2+ binding strengths
408
with thiol ligands differs from that calculated based on reported log values, which vary
409
by many orders of magnitude,20,21,47-49 and also deviates from recently determined
410
stability constants for HgL2 species with the following ligands (L): GSH (log = 38.8),
411
PEN (log = 36.9), and CYS (log = 37.5).14 However, the order is consistent with
412
the effects of these thiols on Hg(II) methylation by certain anaerobic bacteria, such as
413
G. sulfurreducens PCA and D. desulfuricans ND132, assuming that bacteria take up
414
Hg(II) via ligand exchange with thiols in solution.11, 50 While many biochemical factors
415
may influence the rate and extent of Hg(II) methylation, our results offer a reasonable
416
explanation as bacterial cells have to compete with thiols for Hg2+ binding and uptake
417
in solution.50, 51 Among the thiol ligands examined, CYS forms the weakest Hg(II)
418
complexes and shows to enhance Hg(II) methylation by G. sulfurreducens PCA,32, 34,
419
52, 53
420
are shown to enhance Hg(II) methylation by D. desulfuricans ND132, since ND132
421
cells carry more thiol functional groups and are more competitive in Hg(II) sorption
422
and uptake than PCA cells.32, 36 Similarly, since EFPC-DOM binds strongly with Hg2+,
423
it inhibits Hg(II) uptake and methylation by G. sulfurreducens PCA cells but not by D.
424
desulfuricans ND132.11 DMPS forms the strongest Hg(II) complexes and thus inhibits
425
Hg(II) uptake and methylation by both PCA and ND132 cells.11, 36
whereas all other thiols inhibit its methylation. However, all thiols except DMPS
18
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Importantly, EDTA at relatively high concentrations (or high EDTA:Hg ratios)
427
is capable of competing with thiols, such as DMPS, for Hg2+ binding (Figure 2d),
428
despite that the reported stability constant of Hg(II)-DMPS is 19 orders of magnitude
429
higher than that of Hg(II)-EDTA (Table S1).7,13,19 Speciation calculations indicate that
430
EDTA, even at the concentration of 1000× greater than DMPS, is unable to replace
431
DMPS for Hg(II) binding (Table S3). These results suggest that Hg2+ interaction with
432
DOM may be more complicated than previously thought, as DOM contains abundant
433
carboxyl and amine functional groups (in addition to thiols). DOM compounds
434
containing both carboxylates and amines with certain configurations (like EDTA) at
435
relatively high concentrations could form strong complexes with Hg2+, although
436
experimental studies and speciation calculations so far have shown that thiolates are the
437
dominant functional groups in DOM responsible for Hg2+ binding.6,10,13,15,24 Similarly,
438
GSH can form stronger complexes with Hg(II) than CYS and PEN (Figure 1a),
439
resulting from multi-coordination involving cysteinyl sulfur and amine in GSH at a low
440
thiol:Hg ratio (< 2).29 Although additional investigations are required to determine
441
whether such a relationship holds at higher thiol:Hg ratios (> 2), these exchange
442
reactions could occur in natural water and DOM where many individual thiol ligands
443
likely exist at very low concentrations (or at the thiol:Hg ratio < 2), as DOM consists
444
of thousands of individual molecules.1, 2, 3 Therefore, the chemical structure and relative
445
abundance of thiol, carboxyl, and amine functional groups in DOM may ultimately
446
dictate Hg(II)-ligand binding and its chemical speciation in natural aquatic
447
environments.
448
Time-dependent competitive ligand exchange reactions for Hg2+ in DOM were
449
illustrated by dynamic changes in HgR-AA, HgR-Sn, and HgNR pools within 19
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heterogeneous DOM macromolecules (Figure 3). At the DOC:Hg molar ratios of 4,000
451
to 40,000,10, 24 HgR-AA was found to generally decrease whereas HgNR increase with
452
time, as Hg2+ is likely exchanged from carboxyl to thiol and finally to stronger binding
453
functional groups/domains, making Hg(II) non-reducible by Sn(II). The shorter the
454
reaction time between Hg2+ and DOM, the more Hg2+ may be complexed with
455
carboxylates and thus more readily reduced by AA or Sn(II). However, increasing
456
reaction time results in increase in the HgNR pool in DOM, supporting the hypothesis
457
that the reducibility of Hg(II) bound to DOM decreases with time as a result of Hg(II)
458
rearrangement or exchange reactions from abundant weak binding sites (e.g., carboxyls
459
and amines) to strong thiolate binding sites in DOM.5, 30, 46, 54 These observations are
460
consistent with the observed reduction rate of Hg(II)-thiolates nearly 2 orders of
461
magnitude slower than that of Hg(II)-carboxylate complexes.54 The results also agree
462
with findings that Hg(II) is more bioavailable for microbial uptake under
463
nonequilibrium conditions (a short reaction time) than when Hg2+ and DOM have
464
reached pseudoequilibrium (24 h) prior to cell exposure.9
465
The stepwise reduction approach enables experimental determination of
466
competitive ligand-exchange reactions, and it shows that the complexed Hg(II) is
467
exchangeable among LMW organic ligands and between DOM and LMW ligands.
468
Increase in thiol concentrations such as CYS and GSH could release Hg(II) that had
469
been bound to DMPS or DOM although their binding strengths with Hg2+ may be
470
higher than those of CYS and GSH. These results support that Hg(II) chemical
471
speciation in natural aquatic systems may change constantly with input of new organic
472
ligands and could thus make Hg(II) species more or less available for biological uptake
473
and methylation. For example, enhanced Hg(II) methylation has been observed in 20
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benthic biofilms and green alga periphyton, likely due to increased levels of LMW thiol
475
ligands.19, 55 Similarly, a positive correlation is found between LMW thiols and Hg(II)
476
in lake periphytic biofilms.56 In line with these studies, high resolution mass
477
spectrometry examination reported compositional changes in organic ligands and in
478
Hg(II) speciation during freshwater algal growth under different lighting conditions.19
479
Phytoplankton-derived organic ligands are also shown to significantly enhance Hg(II)
480
methylation during planktonic blooms, although the authors attributed this effect to an
481
overall increased bacterial activity.18 Our results, as well as others,19, 55, 57 suggest that
482
changes in Hg(II) speciation due to input of new organic ligands during algal blooms
483
could be partially responsible. Molecular compositional changes of DOM and its
484
dynamic equilibrium with Hg(II) could thus be critically important in controlling the
485
transformation and bioavailability of Hg(II) in natural aquatic ecosystems.
486 487
ASSOCIATED CONTENT
488
Supporting Information
489
The Supporting Information is available free of charge on the ACS Publications website
490
at DOI: XXXX.
491
Molecular structure of organic ligands (L) used in this study and their binding
492
stability constants (log β) with Hg2+ (Table S1); Elemental composition and
493
characteristics of dissolved organic matter (DOM) isolates, soil humic acid (SHA)
494
and EFPC-DOM, used in this study (Table S2); Speciation calculations of Hg(II) (25
495
nM) reactions with DMPS (25 nM) with or without added competing ligands
496
(EDTA, CYS, and GSH) (Table S3); Total Hg (HgT) determination during stepwise
497
reduction of Hg(II)-ligand complexes (Figure S1); Analyses of purgeable Hg(II) 21
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(HgP), nonpurgeable Hg(II) (HgNP), and total Hg (HgT) (Figure S2); Mass balance
499
analyses of purgeable Hg (HgP) and nonpurgeable Hg(II) (HgNP) (Figure S3);
500
Determination of Hg(II) reduction or purgeable Hg(0) (HgP) in PBS by citric acid
501
(CTA) (Figure S4).
502
AUTHOR INFORMATION
503
Corresponding Author
504
* Telephone: (865)-574-7286; E-mail:
[email protected] 505
Notes
506
The authors declare no competing financial interest.
507 508
ACKNOWLEDGMENTS
509
We thank X. Yin, L. Zhao, L. Wang, and X. Yin for technical assistance in laboratory
510
preparations and sample analyses. This research was sponsored in part by the Office of
511
Biological and Environmental Research within the Office of Science of the U.S.
512
Department of Energy (DOE), as part of the Mercury Science Focus Area project at the
513
Oak Ridge National Laboratory (ORNL) and by China Postdoctoral Science program
514
(2017M622906). The Department of Energy will provide public access to these results
515
of federally sponsored research in accordance with the DOE Public Access Plan
516
(http://energy.gov/downloads/doe-public-access-plan). ORNL is managed by UT-
517
Battelle, LLC under Contract No. DE-AC05-00OR22725 with DOE.
518 519 520 521 522
REFERENCES 1. Zark, M.; Dittmar, T., Universal molecular structures in natural dissolved organic matter. Nat. Commun. 2018, 9, (1), 3178. 22
ACS Paragon Plus Environment
Environmental Science & Technology
523 524 525
2. Chen, H.; Johnston, R. C.; Mann, B. F.; Chu, R. K.; Tolic, N.; Parks, J. M.; Gu, B., Identification of mercury and dissolved organic matter complexes using ultrahigh resolution mass spectrometry. Environ. Sci. Technol. Lett. 2017, 4, (2), 59-65.
526 527 528
3. Chen, H.; Yang, Z.; Chu, R. K.; Tolic, N.; Liang, L.; Graham, D. E.; Wullschleger, S. D.; Gu, B., Molecular insights into arctic soil organic matter degradation under warming. Environ. Sci. Technol. 2018, 52, (8), 4555-4564.
529 530 531 532
4. Lamborg, C. H.; Tseng, C.-M.; Fitzgerald, W. F.; Balcom, P. H.; Hammerschmidt, C. R., Determination of the mercury complexation characteristics of dissolved organic matter in natural waters with “reducible Hg” titrations. Environ. Sci. Technol. 2003, 37, (15), 3316-3322.
533 534 535
5. Miller, C. L.; Southworth, G.; Brooks, S.; Liang, L.; Gu, B., Kinetic controls on the complexation between mercury and dissolved organic matter in a contaminated environment. Environ. Sci. Technol. 2009, 43, (22), 8548-8553.
536 537 538
6. Gu, B.; Bian, Y.; Miller, C. L.; Dong, W.; Jiang, X.; Liang, L., Mercury reduction and complexation by natural organic matter in anoxic environments. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1479-1483.
539 540
7. Fitzgerald, W. F.; Lamborg, C. H.; Hammerschmidt, C. R., Marine biogeochemical cycling of mercury. Chem. Rev. 2007, 107, (2), 641-662.
541 542 543 544
8. Skyllberg, U., Competition among thiols and inorganic sulfides and polysulfides for Hg and MeHg in wetland soils and sediments under suboxic conditions: illumination of controversies and implications for MeHg net production. J. Geophys. Res. 113, G00C03, doi:10.1029/2008JG000745.
545 546 547
9. Chiasson-Gould, S. A.; Blais, J. M.; Poulain, A. J., Dissolved organic matter kinetically controls mercury bioavailability to bacteria. Environ. Sci. Technol. 2014, 48, (6), 3153-3161.
548 549 550
10. Dong, W.; Liang, L.; Brooks, S.; Southworth, G.; Gu, B., Roles of dissolved organic matter in the speciation of mercury and methylmercury in a contaminated ecosystem in Oak Ridge, Tennessee. Environ. Chem. 2010, 7, (1), 94-102.
551 552 553 554
11. Zhao, L.; Chen, H.; Lu, X.; Lin, H.; Christensen, G. A.; Pierce, E. M.; Gu, B., Contrasting effects of dissolved organic matter on mercury methylation by Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132. Environ. Sci. Technol. 2017, 51, (18), 10468-10475.
555 556 557
12. Aiken, G. R.; Hsu-Kim, H.; Ryan, J. N., Influence of dissolved organic matter on the environmental fate of metals, nanoparticles, and colloids. Environ. Sci. Technol. 2011, 45, (8), 3196-3201.
558 559 560
13. Skyllberg, U.; Bloom, P. R.; Qian, J.; Lin, C.-M.; Bleam, W. F., Complexation of mercury(II) in soil organic matter: EXAFS evidence for linear two-coordination with reduced sulfur groups. Environ. Sci. Technol. 2006, 40, (13), 4174-4180. 23
ACS Paragon Plus Environment
Page 24 of 32
Page 25 of 32
Environmental Science & Technology
561 562 563 564
14. Liem-Nguyen, V.; Skyllberg, U.; Nam, K.; Björn, E., Thermodynamic stability of mercury(II) complexes formed with environmentally relevant low-molecularmass thiols studied by competing ligand exchange and density functional theory. Environ. Chem. 2017, 14, (4), 243-253.
565 566 567 568
15. Song, Y.; Jiang, T.; Liem-Nguyen, V.; Sparrman, T.; Björn, E.; Skyllberg, U., Thermodynamics of Hg(II) bonding to thiol Groups in Suwannee River natural organic matter resolved by competitive ligand exchange, Hg LIII-edge EXAFS and 1H NMR spectroscopy. Environ. Sci. Technol. 2018, 52, (15), 8292-8301.
569 570
16. Leenheer, J. A.; Croué, J.-P., Peer reviewed: characterizing aquatic dissolved organic matter. Environ. Sci. Technol. 2003, 37, (1), 18A-26A.
571 572
17. Ogawa, H.; Amagai, Y.; Koike, I.; Kaiser, K.; Benner, R., Production of refractory dissolved organic matter by bacteria. Science 2001, 292, 917-920.
573 574 575
18. Bravo, A. G.; Bouchet, S.; Tolu, J.; Björn, E.; Mateos-Rivera, A.; Bertilsson, S., Molecular composition of organic matter controls methylmercury formation in boreal lakes. Nat. Commun. 2017, 8, 14255.
576 577 578
19. Mangal, V.; Stenzler, B. R.; Poulain, A. J.; Guéguen, C., Aerobic and anaerobic bacterial mercury uptake is driven by algal organic matter composition and molecular weight. Environ. Sci. Technol. 2019, 53, (1), 157-165.
579 580 581
20. Foti, C.; Giuffrè, O.; Lando, G.; Sammartano, S., Interaction of inorganic mercury(II) with polyamines, polycarboxylates, and amino acids. J. Chem. Eng. Data 2009, 54, (3), 893-903.
582 583 584
21. Cardiano, P.; Cucinotta, D.; Foti, C.; Giuffrè, O.; Sammartano, S., Potentiometric, calorimetric, and 1H NMR investigation on Hg2+-mercaptocarboxylate interaction in aqueous solution. J. Chem. Eng. Data 2011, 56, (5), 1995-2004.
585 586 587
22. Gasper, J. D.; Aiken, G. R.; Ryan, J. N., A critical review of three methods used for the measurement of mercury (Hg2+)-dissolved organic matter stability constants. Appl. Geochem. 2007, 22, (8), 1583-1597.
588 589 590
23. Benoit, J. M.; Mason, R. P.; Gilmour, C. C.; Aiken, G. R., Constants for mercury binding by dissolved organic matter isolates from the Florida Everglades. Geochim. Cosmochim. Acta 2001, 65, (24), 4445-4451.
591 592 593
24. Skyllberg, U.; Xia, K.; Bloom, P. R.; Nater, E. A.; Bleam, W. F., Binding of mercury(II) to reduced sulfur in soil organic matter along upland-peat soil transects. J. Environ. Qual. 2000, 29, (3), 855-865.
594 595 596 597
25. Drexel, R. T.; Haitzer, M.; Ryan, J. N.; Aiken, G. R.; Nagy, K. L., Mercury(II) sorption to two florida everglades peats: evidence for strong and weak binding and competition by dissolved organic matter released from the peat. Environ. Sci. Technol. 2002, 36, (19), 4058-4064. 24
ACS Paragon Plus Environment
Environmental Science & Technology
598 599 600 601
26. Black, F. J.; Bruland, K. W.; Flegal, A. R., Competing ligand exchange-solid phase extraction method for the determination of the complexation of dissolved inorganic mercury (II) in natural waters. Anal. Chim. Acta 2007, 598, (2), 318333.
602 603
27. Hsu, H.; Sedlak, D. L., Strong Hg(II) Complexation in municipal wastewater effluent and surface waters. Environ. Sci. Technol. 2003, 37, (12), 2743-2749.
604 605 606
28. Dong, W.; Bian, Y.; Liang, L.; Gu, B., Binding constants of mercury and dissolved organic matter determined by a modified ion exchange technique. Environ. Sci. Technol. 2011, 45, (8), 3576-3583.
607 608 609
29. Cardiano, P.; Falcone, G.; Foti, C.; Sammartano, S., Sequestration of Hg2+ by some biologically important thiols. J. Chem. Eng. Data 2011, 56, (12), 47414750.
610 611 612
30. Miller, C. L.; Liang, L.; Gu, B., Competitive ligand exchange reveals time dependant changes in the reactivity of Hg–dissolved organic matter complexes. Environ. Chem. 2012, 9, (6), 495-501.
613 614 615
31. Zheng, W.; Lin, H.; Mann, B. F.; Liang, L.; Gu, B., Oxidation of dissolved elemental mercury by thiol compounds under anoxic conditions. Environ. Sci. Technol. 2013, 47, (22), 12827-12834.
616 617 618
32. Schaefer, J. K.; Rocks, S. S.; Zheng, W.; Liang, L.; Gu, B.; Morel, F. M. M., Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 8714-8719.
619 620 621
33. Hu, H.; Lin, H.; Zheng, W.; Tomanicek, S. J.; Johs, A.; Feng, X.; Elias, D. A.; Liang, L.; Gu, B., Oxidation and methylation of dissolved elemental mercury by anaerobic bacteria. Nat. Geosci. 2013, 6, (9), 751-754.
622 623 624 625
34. Lin, H.; Morrell-Falvey, J. L.; Rao, B.; Liang, L.; Gu, B., Coupled mercury-cell sorption, reduction, and oxidation affecting methylmercury production by Geobacter sulfurreducens PCA. Environ. Sci. Technol. 2014, 48, (20), 1196911976.
626 627 628
35. Lu, X.; Johs, A.; Zhao, L.; Wang, L.; Pierce, E. M.; Gu, B., Nanomolar copper enhances mercury methylation by Desulfovibrio desulfuricans ND132. Environ. Sci. Technol. Lett. 2018, 5, (6), 372-376.
629 630 631 632
36. Liu, Y.; Lu, X.; Zhao, L.; An, J.; He, J. Z.; Pierce, E. M.; Johs, A.; Gu, B., Effects of cellular sorption on mercury bioavailability and methylmercury production by Desulfovibrio desulfuricans ND132. Environ. Sci. Technol. 2016, 50, 1333513341.
633 634 635
37. Lu, X.; Gu, W. Y.; Zhao, L.; Ul Haque, M. F.; DiSpirito, A. A.; Semrau, J. D.; Gu, B., Methylmercury uptake and degradation by methanotrophs. Science Adv. 2017, 3, e1700041. 25
ACS Paragon Plus Environment
Page 26 of 32
Page 27 of 32
Environmental Science & Technology
636 637 638
38. Zheng, W.; Liang, L.; Gu, B., Mercury reduction and oxidation by reduced natural organic matter in anoxic environments. Environ. Sci. Technol. 2012, 46, (1), 292299.
639 640 641 642
39. Lu, X.; Zhao, J.; Liang, X.; Zhang, L.; Liu, Y.; Yin, X.; Li, X.; Gu, B., The application and potential artifacts of Zeeman cold vapor atomic absorption spectrometry in mercury stable isotope analysis. Environ. Sci. Technol. Lett. 2019, 6, (3), 165-170.
643 644 645 646
40. Southworth, G. R.; Turner, R. R.; Peterson, M. J.; Bogle, M. A.; Ryon, M. G., Response of mercury contamination in fish to decreased aqueous concentrations and loading of inorganic mercury in a small stream. Environ. Monit. Assess. 2000, 63, (3), 481-494.
647 648 649 650
41. George, G. N.; Prince, R. C.; Gailer, J.; Buttigieg, G. A.; Denton, M. B.; Harris, H. H.; Pickering, I. J., Mercury binding to the chelation therapy agents DMSA and DMPS and the rational design of custom chelators for mercury. Chem. Res. Toxicol. 2004, 17, (8), 999-1006.
651 652 653
42. Thomas, S. A.; Gaillard, J.-F., The molecular structure of aqueous Hg(II)-EDTA as determined by X-ray absorption spectroscopy. J. Phys. Chem. A 2015, 119, (12), 2878-2884.
654 655 656 657
43. Cheesman, B. V.; Arnold, A. P.; Rabenstein, D. L., Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 25. Hg(thiol)3 complexes and Hg(II)-thiol ligand exchange kinetics. J. Am. Chem. Soc. 1988, 110, (19), 6359-6364.
658 659 660
44. Rabenstein, D. L.; Isab, A. A., A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes. BBA-Mol. Cell Res. 1982, 721, (4), 374-384.
661 662 663 664
45. Zheng, W.; Demers, J. D.; Lu, X.; Bergquist, B. A.; Anbar, A. D.; Blum, J. D.; Gu, B., Mercury stable isotope fractionation during abiotic dark oxidation in the presence of thiols and natural organic matter. Environ. Sci. Technol. 2019, 53, (4), 1853-1862.
665 666 667 668
46. Lamborg, C. H.; Von Damm, K. L.; Fitzgerald, W. F.; Hammerschmidt, C. R.; Zierenberg, R., Mercury and monomethylmercury in fluids from Sea Cliff submarine hydrothermal field, Gorda Ridge. Geophys. Res. Lett. 2006, 33, (L17606), 1-4.
669 670 671
47. Starý, J.; Kratzer, K. J. J. o. R.; Chemistry, N., Radiometric determination of stability constants of mercury species complexes with L-cysteine. J. Radioanal. Nucl. Chem. 1988, 126, (1), 69-75.
672 673 674
48. Casas, J. S.; Jones, M. M., Mercury(II) complexes with sulfhydryl containing chelating agents: stability constant inconsistencies and their resolution. J. Inorg. Nucl. Chem. 1980, 42, (1), 99-102. 26
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Environmental Science & Technology
675 676 677
49. Oram, P. D.; Fang, X.; Fernando, Q.; Letkeman, P.; Letkeman, D., The formation constants of mercury(II)−glutathione complexes. Chem. Res. Toxicol. 1996, 9, (4), 709-712.
678 679 680 681
50. Thomas, S. A.; Tong, T.; Gaillard, J.-F., Hg(II) bacterial biouptake: the role of anthropogenic and biogenic ligands present in solution and spectroscopic evidence of ligand exchange reactions at the cell surface. Metallomics 2014, 6, (12), 2213-2222.
682 683 684
51. Dunham-Cheatham, S.; Mishra, B.; Myneni, S.; Fein, J. B., The effect of natural organic matter on the adsorption of mercury to bacterial cells. Geochim. Cosmochim. Acta 2015, 150, 1-10.
685 686
52. Schaefer, J. K.; Morel, F. M. M., High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens. Nat. Geosci. 2009, 2, 123.
687 688 689
53. Lin, H.; Lu, X.; Liang, L.; Gu, B., Cysteine inhibits mercury methylation by Geobacter Sulfurreducens PCA mutant ΔomcBESTZ. Environ. Sci. Technol. Lett. 2015, 2, 144–148.
690 691 692 693 694
54. Jiang, T.; Skyllberg, U.; Wei, S.; Wang, D.; Lu, S.; Jiang, Z.; Flanagan, D. C., Modeling of the structure-specific kinetics of abiotic, dark reduction of Hg(II) complexed by O/N and S functional groups in humic acids while accounting for time-dependent structural rearrangement. Geochim. Cosmochim. Acta 2015, 154, 151-167.
695 696 697 698 699
55. Bouchet, S.; Goñi-Urriza, M.; Monperrus, M.; Guyoneaud, R.; Fernandez, P.; Heredia, C.; Tessier, E.; Gassie, C.; Point, D.; Guédron, S.; Achá, D.; Amouroux, D., Linking microbial activities and low-molecular-weight thiols to Hg methylation in biofilms and periphyton from high-altitude tropical lakes in the Bolivian Altiplano. Environ. Sci. Technol. 2018, 52, (17), 9758-9767.
700 701 702
56. Leclerc, M.; Planas, D.; Amyot, M., Relationship between extracellular lowmolecular-weight thiols and mercury species in natural lake periphytic biofilms. Environ. Sci. Technol. 2015, 49, (13), 7709-7716.
703 704 705
57. Mangal, V.; Stenzler, B. R.; Poulain, A. J.; Guéguen, C., Aerobic and anaerobic bacterial mercury uptake is driven by algal organic matter composition and molecular weight. Environ. Sci. Technol. 2019, 53, (1), 157-165.
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707
100
80 DMPS GSH CYS PEN ACE CTA EDTA GLY
60 40 20 0
20
1
2
(c)
3
ACE
CTA
(b)
4
5
0
EDTA
GLY
(d)
80
100
HgR-Sn (% of HgT)
HgR-AA (% of HgT)
40
0
60 40 20
1
2
3
4
5
80 60 40 20
0
0 20
708
60
(a) 0
100
80
HgR-Sn (% of HgT)
HgR-AA (% of HgT)
100
50
200
500
1000
20
Ligand:Hg(II) molar ratio
50
200
500
1000
Ligand:Hg(II) molar ratio
709 710 711
Figure 1. Determination of ascorbic acid-reducible Hg(II) (HgR-AA) (a, c) and Sn(II)-
712
reducible Hg(II) (HgR-Sn) (b, d) that are bound to various natural and synthetic organic
713
ligands, including acetate (ACE), citric acid (CTA), ethylenediaminetetraacetate
714
(EDTA), glycine (GLY), L-cysteine (CYS), L-penicillamine (PEN), glutathione (GSH),
715
and 2,3-dimercaptopropanesulfonate (DMPS) at varying ligand:Hg(II) molar ratios in
716
PBS (pH = 7.4).
717 718
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(a) HgR-AA No EDTA/CTA +CTA/Hg(II)=3 +CTA/Hg(II)=500 +CTA/Hg(II)=1000 +EDTA/Hg(II)=3 +EDTA/Hg(II)=500 +EDTA/Hg(II)=1000
80
60
10
100
HgR-Sn (% of HgT)
HgR-AA (% of HgT)
100
CYS/Hg(II)=2 (c) HgR-AA
DMPS/Hg(II)=1 +CYS/Hg(II)=2 +CYS/Hg(II)=4 +GSH/Hg(II)=1 +GSH/Hg(II)=4 +CTA/Hg(II)=3 +CTA/Hg(II)=500 +CTA/Hg(II)=1000 +EDTA/Hg(II)=3 +EDTA/Hg(II)=20 +EDTA/Hg(II)=200 +EDTA/Hg(II)=1000
80
60
10
719
60
40
100
CYS/Hg(II)=2
GSH/Hg(II)=1
(d) HgR-Sn
80
60
40
20
5 0
80
0
GSH/Hg(II)=1
HgR-Sn (% of HgT)
HgR-AA (% of HgT)
100
(b) HgR-Sn
20
5 0
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1
Exchange time (h)
0
24
1
Exchange time (h)
24
720 721 722
Figure 2. Competitive ligand exchange for Hg2+ in (a, b) Hg(II)-thiol complexes (CYS
723
and GSH) by CTA or EDTA, and (c, d) Hg(II)-DMPS by CTA, EDTA, CYS, or GSH
724
at varying ligand concentrations by stepwise measurements of HgR-AA and HgR-Sn.
725
Complexes of Hg-CYS, Hg-GSH, and Hg-DMPS were formed by pre-equilibrating
726
corresponding Hg-Ligand solutions for ~1 h, followed by the addition of competing
727
ligands. Exchange reaction times were set at 1 h for Hg-CYS and Hg-GSH complexes
728
(a, b) and 1 and 24 h for the Hg-DMPS complex (c, d). All reactions were performed
729
in PBS (pH = 7.4). See main text for additional details.
730
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731 732 733 734
Figure 3. Hg(II) species distributions, normalized to the total non-purgeable Hg(II)
735
(HgNP), during stepwise AA and Sn(II) reduction of Hg(II) that has been pre-
736
equilibrated with varying concentrations of EFPC-DOM (a, b) or SHA (c, d) in PBS
737
(pH = 7.4) for either 1 h or 24 h. HgR-AA represents AA-reducible Hg(II), and HgR-
738
Sn represents Sn(II)-reducible Hg(II). Non-reducible Hg (HgNR) denotes Hg(II) that
739
cannot be reduced by either AA or Sn(II).
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100 (b) HgR-AA_SHA
HgR-AA (% of HgNP)
100 (a) HgR-AA_EFPC-DOM DOC only +CYS/Hg(II)=2 +GSH/Hg(II)=1 +DMPS/Hg(II)=1 +CTA/Hg(II)=1000 +EDTA/Hg(II)=1000
80 60
80 60 40
4 2
20
0
0 100 (d) HgR-Sn_SHA
HgR-Sn (% of HgNP)
100 (c) HgR-Sn_EFPC-DOM 80
80
60
60
40
40
20
20
0
0 100 (f) HgNR_SHA
HgNR (% of HgNP)
100 (e) HgNR_EFPC-DOM 80
80
60
60
40
40
20
20
0
1
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0
24
EFPC_DOM-Hg(II) binding time (h)
1
SHA-Hg(II) binding time (h)
24
740 741 742 743
Figure 4. Evaluation of competitive ligand exchange for Hg2+ in Hg(II)-DOM
744
complexes by CYS, GSH, DMPS, CTA, and EDTA, by stepwise determination of
745
HgR-AA, HgR-Sn, and HgNR. Complexes of EFPC-DOM-Hg(II) (a, c, e) and SHA-
746
Hg(II) (b, d, f) were formed by pre-equilibrating the DOM solutions with Hg2+ for either
747
1 or 24 h before the addition of competing ligands. The exchange reaction time was set
748
at 1 h after the addition of competing ligands. The initial Hg2+ concentration was 25
749
nM, and DOC:Hg molar ratios were 18400 and 19200 for EFPC-DOM and SHA,
750
respectively. All experiments were conducted in PBS (pH = 7.4). All data were
751
normalized to the total non-purgeable Hg(II) (HgNP).
752 753
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