CSIRO PUBLISHINGResearch Paper

W. Dong et al., Environ. Chem. 2010, 7, 94–102. doi:10.1071/EN09091 www.publish.csiro.au/journals/env

Roles of dissolved organic matter in the speciationof mercury and methylmercury in a contaminatedecosystem in Oak Ridge, Tennessee

Wenming Dong,A Liyuan Liang,A Scott Brooks,A George SouthworthA

and Baohua GuA,B

AOak Ridge National Laboratory, Environmental Sciences Division, PO Box 2008,MS 6036, Oak Ridge, TN 37831-6036, USA.

BCorresponding author. Email: gub1@ornl.gov

Environmental context. Mercury (Hg) presents an environmental concern owing to its transformation to thepotent neurotoxin methylmercury (CH3Hg+). The environmental factors that control bacterial methylation ofmercury are poorly understood, but we know that methylmercury is bioaccumulated and biomagnified inaquatic food webs. We show that, even at low concentrations (∼3 mg L−1), natural dissolved organic matterstrongly complexes with ionic Hg2+ and CH3Hg+, thereby influencing biological uptake, and methylation ofHg in aquatic environments.

Abstract. Complexation of the mercuric ion (Hg2+) and methylmercury (CH3Hg+) with organic and inorganic ligandsinfluences mercury transformation and bioaccumulation in aquatic environments. Using aqueous geochemical modelling,we show that natural dissolved organic matter (DOM), even at low concentrations (∼3 mg L−1), controls the Hg speciationby forming strong Hg-DOM and CH3Hg-DOM complexes through the reactive sulfur or thiol-like functional groups inDOM in the contaminated East Fork Poplar Creek at Oak Ridge,Tennessee. Concentrations of neutral Hg(OH)2, Hg(OH)Cl,CH3HgCl, and CH3HgOH species are negligible. Of the coexisting metal ions, only Zn2+, at concentrations of 1.6–2.6 × 10−7 M, competes with Hg2+ for binding with DOM, causing decrease in Hg-DOM complexation but having littleimpact on CH3Hg-DOM complexation. DOM may thus play a dominant role in controlling the transformation, biologicaluptake, and methylation of Hg in this contaminated ecosystem.

Additional keywords: aquatic environments, complexation, geochemical model, methylation, reduced sulfur, thiols.

Introduction

Past industrial operations at the US Department of Energy’s(DOE) Y-12 National Security Complex (NSC) in Oak Ridge,Tennessee, resulted in the release of more than 200 t of mercury(Hg) into the nearby watershed, including the East Fork PoplarCreek (EFPC).[1–4] The EFPC originates within the Y-12 NSCfacility and represents a significant source of Hg contaminationin the sediments and water, leading to elevated methylmer-cury (CH3Hg+) concentration in biota. Past remedial actionshave reduced the total Hg loading to the local streams, andthe dissolved inorganic Hg concentration in the lower EFPCdecreased from ∼200–800 ng L−1 in 1998 to the current levels of12–85 ng L−1 (with a total Hg concentration ∼100–300 ng L−1

in water).[3,5] However, these corrective actions had little suc-cess in reducing Hg levels in biota; the concentration of toxicCH3Hg+ in fish tissue remains relatively constant and exceedsthe US Environmental Protection Agency (EPA) regulatory cri-terion of 0.3 µg g−1. Field monitoring data indicate that theCH3Hg+ concentration correlates to neither the total Hg, northe dissolved inorganic Hg concentrations in water, suggestingthat site-specific geochemical conditions significantly controlthe speciation, biological transformation, and accumulation ofmercury in the EFPC aquatic ecosystem.

As a soft metal ion, Hg2+ shows strong Lewis-acid charac-teristics and a high tendency to form complexes, particularly

with the reduced sulfur species such as inorganic S2−, HS−,and organic S−.[6–8] For example, dissolved organic matter(DOM) has been shown to form exceptionally strong complexeswith Hg2+ owing to interactions between Hg2+ and reactivefunctional groups of reduced sulfur or thiols (R–SH).[9,10]The conditional formation constants (log K) between Hg2+ andDOM were reported to vary from 5 to 47,[9,10] dependingon the methodologies and conditions, such as Hg2+ to DOMconcentration ratios, pH, and ionic composition used in the deter-mination of log Ks.[9–13] Studies have shown that, at relativelylow ratios of Hg to DOM, Hg2+ and CH3Hg+ are primar-ily associated with thiol groups,[8,10,11,14–19] as evidenced bythe extended X-ray absorption fine structure (EXAFS) spectro-scopic analyses.[8,16–19] However, at relatively high ratios of Hgto DOM, thiols will be saturated; O- and N-containing groupssuch as carboxylic (R–COOH), phenolic (R–OH), amino (R–NH–R, R–NH2), quinone, and hydroquinone (R–C=O) func-tional groups in DOM become dominant binding sites[8,9,20,21]and form relatively weaker complexes.[11,22]

An important implication of the formation of Hg-DOM com-plexes is the potential in controlling the forms or precursors ofbioavailable Hg species and thus affecting the rate and extentof CH3Hg+ formation by methylating bacteria.[23,24] DOM isconsidered to be among the most important complexing agentsthat affect the speciation, transformation, and bioavailability of

© CSIRO 2010 94 1448-2517/10/010094

Mercury and methylmercury speciation

Table 1. Aqueous geochemical properties of the East Fork Poplar Creek (EFPC) waterData collected between November 2007 and October 2008 unless noted otherwise. DOM, dissolved organic matter;

DOC, dissolved organic carbon

Aqueous composition Concentration range (M) Average concentration (M)

HCO−3 2.0 × 10−3–2.1 × 10−3 2.0 × 10−3

Cl− 2.2 × 10−4–6.5 × 10−4 3.1 × 10−4

SO2−4 3.0 × 10−4–4.1 × 10−4 3.4 × 10−4

F− 3.0 × 10−4–7.0 × 10−4 5.0 × 10−4

NO−3 1.0 × 10−4–2.8 × 10−4 1.3 × 10−4

PO3−4 2.5 × 10−6–1.2 × 10−5 5.0 × 10−6

Ca2+ 7.7 × 10−4–1.1 × 10−3 9.0 × 10−4

Mg2+ 4.5 × 10−4–4.8 × 10−4 4.6 × 10−4

Na+ 7.7 × 10−4–1.1 × 10−3 9.0 × 10−4

K+ 5.0 × 10−5–9.0 × 10−5 6.0 × 10−5

Li+ 7.0 × 10−7–1.0 × 10−6 9.0 × 10−7

Zn2+ 1.6 × 10−7–2.6 × 10−7 2.1 × 10−7

Cu+/2+ 1.2 × 10−8–2.8 × 10−8 2.2 × 10−8

Ni−2+ 4.6 × 10−9–1.6 × 10−8 9.5 × 10−9

Cd2+ 5.2 × 10−8–1.3 × 10−9 8.7 × 10−10

Pb2+ 6.1 × 10−10–1.3 × 10−9 5.2 × 10−10

UO2+2 1.7 × 10−8–2.2 × 10−8 2.0 × 10−8

Fe3+ 2.6 × 10−8–2.8 × 10−8 A 2.7 × 10−8

Dissolved Hg – present work 6 × 10−11–4 × 10−10

– measured in 1998[3,5] 1 × 10−9–4 × 10−9

Dissolved CH3Hg+ 1 × 10−12–5 × 10−12

pH 7.5–8.2 7.8DOC (mg L−1) 1.3–1.8 1.5 (∼3 mg L−1 DOM)Estimated reactive thiols 4 × 10−9–6 × 10−8 B

Estimated ionic strength (I) 0.01

AEstimated from the solubility of amorphous iron oxides (Fe(OH)3) under similar water conditions in EFPC.BCalculated based on a DOC content of 1.5 mg L−1 (see text for details).

mercury in fresh-water aquatic ecosystems. In the EFPC water,the total DOM concentration ranges from 2.5 to 3.5 mg L−1,but little is known about how this DOM, among other dissolvedionic species, influences the speciation of Hg2+ or CH3Hg+ inthe system. This study was therefore undertaken to determinethe aqueous speciation of Hg2+ and CH3Hg+ as part of a majorstudy of biogeochemical and molecular mechanisms of Hg trans-formation in the most contaminated upper EFPC ecosystem.Our specific objectives are to evaluate: (i) how DOM affectsthe speciation of Hg2+ and CH3Hg+ through the complexa-tion with thiol functional groups; and (ii) the competitive effectsof coexisting metal ions on the Hg-DOM complexation and itsenvironmental implications in the EFPC ecosystem.

Experimental methodsWater sampling and analysisSurface water samples were collected from the EFPC site in OakRidge, Tennessee, between November 2007 and October 2008.Samples were filtered using either 0.45-µm polycarbonate filtersor 0.2-µm cellulose nitrate membrane filters.[25] Both filteredand unfiltered samples were collected in precleaned Teflon bot-tles, placed on ice and transported to the laboratory immediately,and then kept at 4◦C until analysis.

Dissolved Hg in filtrates and total Hg in unfiltered sampleswere measured by cold vapour atomic fluorescence spectrometry(CVAFS) following the US EPA Method 1631.[26] Briefly, sam-ples were oxidised overnight by bromine monochloride (BrCl),

and hydroxyammonium hydrochloride (NH2OH·HCl) was thenadded to remove free halogens. Stannous chloride (SnCl2) wasused to reduce Hg2+ species to gaseous Hg0, which was trappedonto gold and subsequently thermally desorbed into an N2 gasstream and analysed by CVAFS. Dissolved methylmercury con-centrations were determined following the US EPA Method1630.[27] This particular set of samples was preserved in HCl(∼0.05 M) before analysis.

Filtered samples were also analysed for dissolved organiccarbon (DOC), alkalinity, major anions and cations,[28,29] whichare given in Table 1. DOC was analysed using a ShimadzuTOC-5000 Total Organic Carbon Analyzer (Shimadzu ScientificInstruments, Columbia, MD, USA), and the alkalinity as HCO−

3was determined with the alkalinity test kit (HACH, Loveland,CO, USA) and performed within 24 h after samples were col-lected. Major dissolved anions (Cl−, SO2−

4 , PO3−4 , and F−) and

total elemental composition (Ca, Mg, Zn, Cu, Ni, Cd, Pb, Fe, andU) were analysed by ion chromatography (IC) (Dionex DX-120,Sunnyville, CA, USA) and inductively coupled plasma-massspectrometer (ICP-MS, Perkin-Elmer, Waltham, MA, USA)respectively.

Mercury speciation analysis and modellingThe aqueous speciation distribution of Hg2+ and CH3Hg+ inthe EFPC water was calculated using the equilibrium geochem-ical speciation computer code PHREEQC.[30] Water chemistrydata (Table 1) and selected thermodynamic reaction constantsof Hg2+ were used in the aqueous speciation modelling.

95

W. Dong et al.

Table 2. Selected thermodynamic reaction constants (log β) of Hg2+and MeHg+ with major inorganic ligands at temperature (T) = 25◦C,

ionic strength (I) = 0, and pressure = 105 PaRecalculated to zero ionic strength using Davies equation from A, I = 0.5 M;

B, I = 0.1 M; and C, I = 0.7 M

Reaction Log β Ref.

Selected Hg2+ reactionsHg2+ + H2O = HgOH+ + H+ −3.40 [31]Hg2+ + 2 H2O = Hg(OH)2 + 2 H+ −5.98 [31]Hg2+ + 3 H2O = Hg(OH)−3 + 3 H+ −21.1 [31]Hg2+ + Cl− = HgCl+ 7.31 [31]Hg2+ + 2 Cl− = HgCl2 14.00 [31]Hg2+ + 3 Cl− = HgCl−3 14.93 [31]Hg2+ + 4 Cl− = HgCl2−

4 15.54 [31]Hg2+ + Cl− + H2O = HgOHCl + H+ 4.27A [31]Hg2+ + H2O + CO2 = HgCO3 + 2 H+ −6.68 [31]Hg2+ + H2O + HCO−

3 = Hg(OH)CO−3 + 2 H+ −5.00 [31]

Hg2+ + H2O + CO2 = HgHCO+3 + H+ −2.35 [31]

Hg2+ + HPO2−4 = HgHPO4 8.8 [31]

Hg2+ + HPO2−4 = HgPO−

4 + H+ 3.25 [31]Hg2+ + SO2−

4 = HgSO4 1.4 [31]Hg2+ + 2 SO2−

4 = Hg(SO4)2−2 2.4 [31]

Hg2+ + F− = HgF+ 1.57A [32]Selected CH3Hg+ reactions

CH3Hg+ + H2O = CH3HgOH + H+ −4.53B [32]2 CH3Hg+ + H2O = (CH3Hg)2OH+ + H+ −2.11B [32]CH3Hg+ + Cl− = CH3HgCl 5.39B [32]CH3Hg+ + F− = CH3HgF 1.71B [32]CH3Hg+ + SO2−

4 = CH3HgSO−4 1.37C [32]

CH3Hg+ + HPO2−4 = CH3HgHPO4 5.4C [32]

Selected solubilty reactionsHgO(s) + 2 H+ = Hg2+(aq) + H2O 2.37 [31]HgO(s) + H2O = Hg(OH)2(aq) −3.62 [31]HgCl2(s) = HgCl2(aq) −0.58 [31]HgCO3·2HgO(s) + 3 H2O = 3 Hg(OH)2(aq) + CO2(g) −11.27 [31]Hg3(PO4)2(s) + 2 H+ = 3 Hg2+ + 2 HPO2−

4 −24.6 [31](HgOH)3PO4(s) + 4 H+ = 3 Hg2+ + HPO2−

4 + 3 H2O −9.4 [31]HgHPO4(s) = Hg2+ + HPO2−

4 −13.1 [31]

As concentrations of DOC, major anions and cations in EFPCvaried in a narrow range, the average values were used in modelcalculations. Dissolved sulfide was not considered because ofrelatively high levels of dissolved oxygen (>8 mg L−1) andredox potential (∼96 to 226 mV) in the creek, in which dis-solved sulfide species would not be stable.[25] Provided in Tables2 and 3 are comprehensive lists of reaction constants of Hg2+and CH3Hg+ with various inorganic ligands and natural organicmatter (NOM) of both aquatic and soil origin.

Data for the Hg2+ complexation with inorganic ligands suchas Cl−, OH−, CO2−

3 , SO2−4 , and PO3−

4 and the solubility con-stants of solid phases of Hg2+ were obtained from the recentlyrecommended and critically reviewed IUPAC database.[31] Thestability constants of CH3Hg+ and Hg2+ complexes with F−are from the National Institute of Standards and Technology(NIST) standard reference database[32] and corrected to zeroionic strength using the Davies equation.[33] As NOM is a com-plex mixture of polyelectrolytes with heterogeneous functionalgroups and chemical structures,[34,35] their thermodynamicstability constants with Hg2+ cannot be obtained using simpleactivity correction methods but are generally given in condi-tionally determined forms.[9] In the present work, emphasis wasgiven to the complexation of Hg2+ and CH3Hg+ with reducedsulfur groups (RS−) or thiol-like groups in DOM, where the

conditional stability constants were given based on calculatedconcentrations of RS− in DOM and defined by the followingreactions:

Hg2+ + RS− = Hg(RS)+, KHg(RS)+ = [Hg(RS)+][Hg2+][RS−] (1)

Hg2+ + 2RS− = Hg(RS)2, KHg(RS)2 = [Hg(RS)2][Hg2+][RS−]2

(2)

HgCH2+3 +RS− = HgCH3(RS), KHgCH3(RS) = [CH3Hg(RS)]

[Hg2+][RS−](3)

RSH = RS− + H+, Ka = [RS−][H+][RSH] (4)

Results and discussionComplexation models and reactive thiols in DOMThe complexation between Hg2+ and DOM and the speciationanalysis depend on the use of specific complexation models andknowledge of the binding sites in DOM.[9,10] Because literature-reported values of conditional stability constants (log K) varyover many orders of magnitude for the complexation betweenNOM and Hg2+ or CH3Hg+ (Table 3a, b), it is challenging toselect an appropriate binding constant (log K) for the calcula-tion of the Hg2+ speciation in EFPC. Most of the log K valueswere determined based on reactions of Hg2+ with reduced sul-fur (RS−) functional groups (Table 3a). Reported log K valuesvary from 28.7 to 47 for the 1 : 2 complexes of Hg2+ bindingwith either two RS− or one RS− and one other RX− group(RX−, X = O, N, S) in DOM. For the 1 : 1 or the monodentatecomplexes, the log K values vary from 21 to 33.5. In compari-son with Hg2+, CH3Hg+ only forms 1 : 1 complexes with RS−and exhibits much lower conditional binding constants, rangingfrom 10.7 to 17.1 (Table 3b). Direct comparison and validation ofthese binding constants from different investigators are difficultbecause of the complexity of DOM and different methodolo-gies and assumptions used in the experiments as well as models(1 : 1 or 1 : 2) used in calculations. Experimental conditions suchas the Hg2+ concentration, pH, ionic strength, and compositionare also expected to affect the determination of stability con-stants. Recent studies using EXAFS spectroscopy demonstratethat the Hg-DOM complexation depends on the ratios of Hg2+to reduced sulfur (Sred) in DOM.[8,16,18] EXAFS results pro-vided direct evidence that Hg2+ forms 1 : 2 complexes with twoRS− at molar ratios of Hg2+/Sred below 0.1–0.15.[18] Abovethis ratio, the Hg2+ is thought to form complexes with oneRS− and an additional functional group of RX− (X = O orN),[8,16,18] although the formation of such mixed O–Hg–S orN–Hg–S complexes has never been experimentally validated.The RO− or RN− functional groups (i.e. carboxylic, phenolic,and amino) become the dominant binding sites once the RS−is saturated, presumably at a Hg2+/Sred ratio >0.4, resulting inlower log K values.

Accordingly, the determination of the abundance of Sredin DOM is critical for the Hg2+ speciation analysis in theEFPC water. The measured DOC concentration in EFPC rangesfrom 1.3 to 1.8 mg C L−1, with a mean value of 1.5 mg C L−1

(Table 1), which is equivalent to a DOM concentration of∼3 mg L−1 by assuming that DOM consists of 50% organic car-bon (or DOC) by weight.[36] The following equation (Eqn 5) isestablished to estimate the concentration of Sred in DOM:

[Sred] = [DOM] × F1 × F2/W (5)

96

Mercury and methylmercury speciation

Tab

le3.

Mea

sure

dco

ndit

iona

lst

abili

tyco

nsta

nts

(log

K)

for

the

1:2

and

1:1

com

plex

esbe

twee

nH

g2+an

dre

acti

vefu

ncti

onal

grou

ps(i

.e.

redu

ced

thio

lgr

oup

(RS−

),ox

ygen

-or

nitr

ogen

-con

tain

ing

func

tion

algr

oup

(RO

−an

dR

N− )

)in

natu

ralo

rgan

icm

atte

r(N

OM

)an

dN

OM

isol

ates

(a).

Mea

sure

dco

ndit

iona

lsta

bilit

yco

nsta

nts

(log

K)

for

the

1:1

com

plex

esbe

twee

nC

H3H

g+an

dre

duce

dth

iolg

roup

(RS−

)in

NO

M(b

)S

OM

,soi

lorg

anic

mat

ter;

DO

M,d

isso

lved

orga

nic

mat

ter;

HA

,hum

icac

id;C

LE

,com

peti

tive

liga

ndex

chan

ge;S

PE

,sol

id-p

hase

extr

acti

on;E

DL

E,e

quil

ibri

umdi

alys

isli

gand

exch

ange

;SS

E,s

olve

nt–s

olve

ntex

trac

tion

;S

EM

,sor

ptio

neq

uili

briu

mm

odel

ling

;na,

nota

vail

able

;SE

C,s

orpt

ion

equi

libr

ium

calc

ulat

ion;

IHS

S,I

nter

nati

onal

Hum

icS

ubst

ance

sS

ocie

ty;F

A,f

ulvi

cac

id

Type

ofN

OM

Rea

ctio

nlo

gK

pHI

(M)

Met

hodA

Ref

.

(a)

HA

isol

ates

and

Min

neso

tafo

rest

peat

Hg2+

+R

S2− 2

=H

gS2R

38.2

–40.

43–

5∼0

.5C

LE

-SE

M[1

3]R

SH

=R

S−

+H

+−8

.4D

OM

isol

ates

from

Flo

rida

Eve

rgla

des

Hg2+

+R

S−

+R

X−

=H

g(R

S)(

RX

)28

.7±

0.1

4–7

0.1

ED

LE

[12]

RX

H=

RX

−+

H+

(X=

O,N

,S)

−6.3

RS

H=

RS

−+

H+

−10.

3S

OM

inM

inne

sota

Mar

cell

Exp

erim

enta

lFor

est

Hg2+

+R

S−

+R

O−

=H

g(R

S)(

RO

)31

.6–3

2.2

3–3.

4∼0

.6C

LE

-SE

M[5

8]R

OH

=R

O−

+H

+−3

.44

RS

H=

RS

−+

H+

−9.9

6D

OM

inC

alif

orni

ana

tura

lwat

ers

Hg2+

+R

S−

=H

g(R

S)+

29.9

–33.

57.

4–7.

8na

CL

E-S

PE

[41]

RS

H=

RS

−+

H+

−9.9

6D

OM

inTe

xas

estu

arin

ean

dco

asta

lwat

ers

Hg2+

+R

S−

=H

g(R

S)+

26.1

–26.

97.

0–7.

50.

6C

LE

-SS

E[1

4]26

.8–2

9.2

9.5–

9.8

naR

SH

=R

S−

+H

+−1

0D

OM

infr

esh

and

sea

wat

ers

Hg2+

+R

S−

=H

g(R

S)+

21–2

47.

5na

Red

ucib

leH

g[1

5]D

OM

isol

ate

inF

lori

daE

verg

lade

s(1

)H

g2++

RS

−=

Hg(

RS

)+28

.57.

04.

9–5.

60.

10.

1E

DL

E[1

1]R

SH

=R

S−

+H

+−1

0(2

)H

g2++

RO

−=

Hg(

RO

)+10

RO

H=

RO

−+

H+

−4.5

Peat

san

dD

OM

inF

lori

daE

verg

lade

s(1

)H

g2++

RS

−=

Hg(

RS

)+25

.8–2

7.2

6.0

0.01

SE

M[2

2]R

SH

=R

S−

+H

+−1

0(2

)H

g2++

RO

−=

Hg(

RO

)+7.

3–11

.8R

OH

=R

O−

+H

+−4

.25

DO

Mis

olat

esin

Flo

rida

Eve

rgla

des

Hg2+

+R

S−

=H

g(R

S)+

20.6

–22.

44–

60.

06C

LE

-SS

M[3

7]21

.4–2

3.8

0R

SH

=R

S−

+H

+−1

0

(b)

SO

Man

dD

OM

inS

wed

enpe

atso

ils

CH

3H

g++

RS

−=

CH

3H

g(R

S)

15.6

–17.

12–

5.1

0.25

CL

E[3

9]R

SH

=R

S−

+H

+−8

.5,−

9.95

SO

Min

Sw

eden

peat

soil

sC

H3H

g++

RS

−=

CH

3H

g(R

S)

16.3

–16.

73–

4.3

0.01

SE

C[1

7]R

SH

=R

S−

+H

+−9

.96

HA

isol

ates

from

IHS

SC

H3H

g++

RS

−=

CH

3H

g(R

S)

10.3

9–14

.96

5–9

0.00

1E

DL

E[6

]R

SH

=R

S−

+H

+−4

,−7,

−10

HA

and

FAis

olat

esfr

omO

ntar

iola

kes,

Can

ada

CH

3H

g++

RS

−=

CH

3H

g(R

S)

13.0

2–14

.56

(str

ong

site

)6.

50.

0001

ED

LE

[44]

RS

−he

rede

note

sbo

thst

rong

and

wea

kbi

ndin

gsi

tes

inD

OM

12.1

5–13

.07

(wea

ksi

te)

HA

and

FAis

olat

esfr

omFa

wn

Lak

e,C

anad

aC

H3H

g++

RS

−=

CH

3H

g(R

S)

12.1

1(s

tron

gsi

te)

6.5

0.00

1E

DL

E[4

3]R

S−

here

deno

tes

both

stro

ngan

dw

eak

bind

ing

site

sin

DO

M10

.7(w

eak

site

)A

vera

ge∼1

4

97

W. Dong et al.

1 2 3 4 5 6 7 8 9 1010�30

10�25

10�20

10�15

10�10

10�5

54

Hg-DOM(carboxyl)Hg(OH)2

Hg2�

spe

cies

(m

ol L

�1 )

Hg-DOM(thiol)

HgCl2

1

3

67

89 10

2

11

pH

Fig. 1. Calculated aqueous species of Hg2+ as a function of pHbased on geochemical parameters and reactions listed in Tables 1–3,the total Hg2+ concentration of 0.4 nM and an ionic strength of0.01 mol L−1 in the EFPC (East Fork Poplar Creek) water. Model para-meters reported by Haitzer et al.[11,12] in Table 3a were used in cal-culations, including log K = 28.7 and pKa = 10 for the 1 : 2 Hg2+ : thiolcomplexes, and log K = 10 and pKa = 4.5 for the 1 : 1 Hg : carboxyl com-plexes with DOM (dissolved organic matter). Numbers in the figuredenote: 1 = HgCl−3 ; 2 = HgCl+; 3 = Hg(OH)Cl; 4 = HgCl2−

4 ; 5 = Hg2+;6 = Hg(OH)+; 7 = HgHPO4; 8 = HgPO−

4 ; 9 = HgCO3; 10 = Hg(OH)CO−3 ;

and 11 = Hg(OH)−3 . The estimated total reactive thiol concentration in DOMin EFPC is 4 nM and the carboxyl content is 16 µM.[11]

where F1 is the percentage fraction of total sulfur content inDOM by weight, F2 is the percentage fraction of reduced sul-fur content (relative to total sulfur by weight), and W = atomicweight of sulfur. DOM usually contains less than 2% sulfur,with a mean value of F1 = ∼0.86% estimated from literaturedata (n = 32, range = 0.3–2%, with a standard deviation of0.1).[6,12,17,19,22,37–39] About 50% of the total sulfur in DOMis present in the reduced form (F2 = 0.5).[12,13,18,40] Based onthe above calculations, the Sred content in DOM in the EFPCwater is ∼200 nM, and the estimated molar ratio of Hg2+/Sredis <<0.1 at the Hg2+ concentration of 0.06–0.4 nM (Table 1).

However, studies of the complexation between Hg2+ andNOM also indicate that only a small fraction (∼2%) of Sredin DOM, presumably that with the highest binding affinitiesfor Hg2+, actively takes part in the reactions with Hg2+ orCH3Hg+, especially at low Hg concentrations typically found inthe environment.[6,11,12] Direct EXAFS spectroscopic measure-ments suggest that ∼20–30% of Sred is involved in the formationof Hg-thiol complexes.[18] In other words, the reactive reducedsulfur groups (RSrx) may vary between 2 and 30% of Sred, andit gives 4–60 nM RSrx in the EFPC water. To be conservative,we assume that only 2% of Sred is available as reactive thiols(i.e. RSrx = 4 nM) for binding with Hg2+. This value still givesa Hg2+/RSrx ratio of ≤0.1 (Table 1), indicating the presenceof abundant DOM capable of forming either bidentate (1 : 2) ormonodentate (1 : 1) Hg-DOM complexes in the EFPC water.

Speciation analysis of Hg2+ and CH3Hg+ in EFPCUsing the 1 : 2 complexation model and thermodynamic data ofHg2+ complexes with inorganic ligands listed in Tables 1 and2, we show that Hg-DOM(thiol) complexes are the dominant

20 21 22 23 33 340

20

40

60

80

100

log K

Hg-DOM(thiol)

Hg(OH)Cl

Hg2�

spe

cies

(%

)

Hg(OH)2

Fig. 2. Dominant Hg2+ species in the EFPC (East Fork Poplar Creek)water as a function of log K (based on the 1 : 1 complexation between Hg2+and thiols in DOM (dissolved organic matter) (Table 2a)). The total Hg2+concentration is 0.4 nM, and pH is 7.8. The estimated total reactive thiolconcentration in DOM is 4 nM, and the ionic strength is 0.01 M (Table 1).

aqueous species of Hg2+ in EFPC (Fig. 1). Nearly 100% Hg2+ iscomplexed with DOM in the pH range from 2 to 10, even thougha relatively low log K value of 28.7[11,12] was used in calcula-tions as a conservative estimate. This log K value was selectedpartly because experimental conditions and the DOM used inthese previous studies are more representative of the reactionsoccurring in the EFPC water.The estimated concentration of Hg-DOM complexes is at least six orders of magnitude higher thanother inorganic Hg2+ complexes such as Hg(OH)2, Hg(OH)Cl,and HgCl2. As can be expected, the abundance of Hg2+ com-plexes with carboxyl groups is very low owing to their weakinteractions (log K ≈ 10) compared with Hg2+ binding with thiolfunctional groups in DOM. Similarly, complexes of Hg2+ withCl−, OH−, CO2−

3 , PO3−4 , SO2−

4 , and F− are at very low concen-trations or negligible. The free ionic Hg2+ concentrations are atthe level of only ∼10−27–10−28 M and decrease with increasingpH in EFPC. Obviously, if a higher log K value is selected fromTable 3a for the model calculation, an even higher fraction ofHg-DOM is expected, resulting in even lower or negligible dis-tributions of ionic Hg2+ and its complexes with other inorganicligands. For example, the conditional stability constants of com-plexes of Hg2+ with humics from the peat and organic soilswere reported to be as high as 38.2–40.4 (Table 3a).[13] Thehigh affinity of Hg2+ with these peat humic materials is likelyattributed to their different structural and compositional prop-erties (such as molecular weight and aromaticity) and thereforetheir strong binding characteristics. Skyllberg[10] suggested thata log K value of 43–47 is reasonable based on spectroscopicmeasurements and comparisons of Hg-thiol complexes in DOMand low molecular-weight thiol compounds.

Even by assuming a 1 : 1 complexation[11,14,22,37,41] andby using the lowest log K value of 21 (Table 3a),[15,37] themodel predicts that the Hg-DOM complexes dominate in theEFPC water, with only ∼15% of Hg2+ occurring as neutralHg(OH)2 and Hg(OH)Cl species (Fig. 2). Conditional stabil-ity constants for the 1 : 1 complexation were reported to rangefrom 20.6 to 33.5 (Table 3a). At log K = 22, nearly 100% ofHg2+ is in the form of Hg-DOM(thiol) complexes. The neutralspecies of Hg(OH)2 and Hg(OH)Cl are negligible in the EFPC

98

Mercury and methylmercury speciation

2 4 6 8 100

20

40

60

80

100

10 12 14 16

CH3HgCl

CH3Hg-DOM

CH3HgOH

CH3Hg�CH3HgOHC

H3H

g� s

peci

es (

%)

pH

CH3HgCl

CH3Hg-DOM

(a) (b)

log K

Fig. 3. Dominant species of methylmercury (CH3Hg+) (5 pM) in theEFPC (East Fork Poplar Creek) water as a function of pH with an aver-age log K = 14 (a); and as a function of log K at pH 7.8 (b) using the 1 : 1complexation model (Table 3b). Calculations were based on geochemicalparameters and reactions listed in Tables 1 and 2. The estimated total reac-tive thiol concentration is 4 nM, and the ionic strength is 0.01 M (Table 1).DOM, dissolved organic matter.

water (Fig. 2). We can thus conclude that Hg-DOM(thiol) com-plexes are the dominant Hg2+ species in the EFPC surface waterregardless of the use of the 1 : 1 or 1 : 2 complexation model.

As for the complexation between CH3Hg+ and DOM, recentstudies using EXAFS showed that CH3Hg+ only forms a1 : 1 complex with one reactive RS− functional group,[17,19,42]and the formation stability constants are from 10.7 to 17.1(Table 3b).[6,17,24,39,43] An average log K value of 14[44] wasfirst used for the calculation of the complexation betweenCH3Hg+ and DOM in EFPC (Fig. 3a). Results indicate thatthe CH3Hg-DOM(thiol) and neutral CH3HgOH complexes arethe major aqueous species of CH3Hg+ at pH ∼7.8 in the EFPCwater (Table 1). Approximately 60% of CH3Hg+ is complexedwith DOM, and the remaining 40% is in the form of neutralCH3HgOH. However, at low pH conditions (<6), the neutralCH3HgCl complex becomes the dominant species.

Given the wide variation of log K values (10.7–17.1) reportedin the literature (Table 3b), we further examined the aqueousspeciation of CH3Hg+ as a function of log K values in the EFPCwater at pH 7.8 (Fig. 3b). Results show that the concentration ofCH3Hg-DOM(thiol) increased as expected with increasing log Kvalues and becomes dominant when log K is >14.At log K > 16,almost 100% of CH3Hg+ is bound to thiols in DOM in the EFPCwater. At log K ≤ 12, the neutral CH3HgOH is the dominantspecies, followed by the CH3HgCl complex. These observationsindicate that the use of different log K values could result insignificantly different distributions of CH3Hg+ speciation in theEFPC water.

Effect of coexisting cations on Hg2+ and CH3Hg+speciationThe effect of coexisting metal ions (i.e. Zn2+, Cd2+, Ni2+,Pb2+, Cu2+, Fe3+, UO2+

2 , Ca2+, and Mg2+) on the speciationof Hg2+ and CH3Hg+ was further evaluated because concentra-tions of these ions in EFPC are orders of magnitude higher thanthose of Hg2+ and CH3Hg+ (Table 1). Multivalent metal ions,particularly Zn2+ owing to its relatively high concentrations,

are expected to compete with Hg2+ and CH3Hg+ for bindingwith DOM.[45] However, little information is currently availablefor the complexation between these ions and thiol-like func-tional groups in DOM.[46] Previous studies of the complexationbetween these metal ions and DOM have been largely focussedon carboxyl and hydroxyl functional groups because of theirhigh abundance.[47,48] In order to evaluate their potential com-petitive effects on the complexation between DOM thiols andHg2+ or CH3Hg+, complexes of these ions with cysteine (CYS)and glutathione (GS) were used as analogues, and the best avail-able formation constants (log K) were selected and are tabulatedin Table 4.[32,49] Only 1 : 1 and 1 : 2 complexes of metal ions(M) with these thiol ligands (L) were used in the calculations tomimic the competitive complexation of metal ions with thiols inDOM.The average log K values for the 1 : 1 (21.3) and 1 : 2 (34.0)complexes in Table 4 are comparable with those of Hg-DOMcomplexes listed in Table 3a. Complexes between CH3Hg+ andCYS or GS show a log K value of ∼16.8.

Fig. 4 shows the distribution of thiol ligands (L) associatedwith dominant metal ions and the Hg2+ species in the pres-ence or absence of coexisting metal ions as a function of totaldissolved Hg concentration (0.1–4 nM) at pH = 7.8 observedin the EFPC water (Table 1). The total concentration of L was4 nM, which is equivalent to the total RSrx content in DOM inEFPC, as described earlier. Results (Fig. 4a) indicate that the 1 : 2complexes of HgL2 are the dominant Hg2+ species at relativelylow Hg2+ concentrations (<3 nM) but decrease with increasingconcentrations. The 1 : 1 HgL complex was a minor fraction,which increased slightly to ∼12% with an increasing Hg2+ con-centration. Similarly, Hg(OH)2 increased with the dissolved Hgconcentration. The presence of competing metal ions had a rela-tively small impact on Hg2+ speciation and caused only ∼5–10%decrease in the percentage of HgL2 distribution (Fig. 4a) whenthe average concentration of coexisting metal ions (Table 1) wasused in calculations.This decrease was primarily attributed to thepresence of Zn2+, which exists in EFPC at relatively high con-centrations (210 nM,Table 1).At a low level of Hg2+ (≤0.1 nM),nearly 50% of thiol ligands were complexed with Zn2+ as ZnL,but this decreased to ∼10% as the dissolved Hg2+ concentrationincreased to 4 nM (Fig. 4b). Our results are supported by previ-ous studies,[46] in which Zn2+ was found to be coordinated by amixture of S and O or N ligands in the first coordination shell atZn2+ concentrations between 500 and 10 000 µg g−1 DOM. Theeffect of other coexisting metal ions (Cd2+, Ni2+, Pb2+, Cu2+,Fe3+, UO2+

2 , Ca2+, and Mg2+) on the complexation of Hg2+and thiol ligands is insignificant, and the estimated thiol ligandsthat are associated with these ions amount to <1%.

The effect of coexisting metal ions on the complexationbetween CH3Hg+ and thiols in DOM was estimated simi-larly and found to be negligible (data not shown) becausethe available thiols (Fig. 4b) are sufficiently abundant forbinding the very low concentration of CH3Hg+ (1–5 pM) inEFPC (Table 1). These results demonstrate that the coexistingmetal ions can affect the complexation of DOM with Hg2+ orCH3Hg+, but the extent depends on their relative concentra-tions and binding affinities. In EFPC, Zn2+ is the only metalion that is likely to compete with Hg2+ for binding with DOM(Fig. 4b).

Conclusions and implications

Despite relatively low DOM concentrations (∼3 mg L−1)(Table 1), complexes of Hg-DOM and CH3Hg-DOM are

99

W. Dong et al.

Table 4. Thermodynamic formation constants (log β) for the 1 : 1 and 1 : 2 complexes between selected metal ionsand Cysteine (CYS) or Glutathione (GS) at temperature, T = 25◦C and ionic strength, I = 0 unless noted otherwise

L, thiol groups in CYS and GS

Cation CYS2− GS3− Average log β Ref.

Complex log β Complex log β

H+ HL− 10.74 HL2− 10.15A 10.44 [32]Hg2+ HgL 15.26A HgL− 27.28A 21.27 [32]

HgL2−2 HgL4−

2 34.04A 34.04 [32]CH3Hg+ CH3HgL− 16.89A CH3HgL2− 16.64A 16.76 [32]Ca2+ CaL 3.36A,E CaL− 5.23B,F 4.30 [32]Mg2+ MgL 3.61A,E MgL− 3.61 [32]Zn2+ ZnL 9.97A ZnL− 9.41B,F 9.69 [32]

ZnL2−2 18.98A ZnL4−

2 13.21B,F 16.1 [32]Cu2+ CuL CuL− 12

CuL2−2 16.95 CuL4−

2 16.95 [49]Ni2+ NiL 10.65A NiL− 8.80B,F 9.72 [32]

NiL2−2 20.76A NiL4−

2 11.16B,F 15.96 [32]Cd2+ CdL 11.05B,F CdL− 10.18D 10.62 [32]

CdL2−2 19.6D CdL4−

2 15.35D 17.48 [32]Pb2+ PbL 13.06A PbL− 10.6D 11.83 [32]

PbL2−2 16.72C PbL4−

2 15.0D 15.86 [32]UO2+

2 UO2L2−2 6.70A UO2L4−

2 6.70 [49]UO2L2−

2 12.71A UO2L4−2 10.19A 11.45 [32]

Fe3+ FeL+ 12.28B,E FeL 12.28 [49]FeL−

2 16.40B,E FeL3−2 16.40 [49]

AI = 0.1 mol L−1, recalculated to I = 0 using Davies equation.BI = 0.15 mol L−1, recalculated to I = 0 using Davies equation.CI = 1.0 mol L−1, uncorrected.DI = 3.0 mol L−1, uncorrected.E20◦C.F37◦C.

0 1 2 3 40

20

40

60

80

100

0

20

40

60

80

100

Com

plex

ed L

(%

)

Dissolved [Hg2�] (nmol L�1)

Hg(OH)2

HgL

HgL2

Hg2�

spe

cies

(%

)

Line only: no metal ions

Symbol � line: with metal ions

HgL2

HL

ZnL HgL

(a)

(b)

Fig. 4. The effects of coexisting metal ions (Table 1) on (a) the distributionof major Hg2+ species; and (b) the distributions of thiol ligands (L) associ-ated with major metal ions as a function of dissolved Hg2+ concentrations.Calculations were based on the 1 : 1 and 1 : 2 complexation models for allmetals ions with L (Table 4) and geochemical parameters and reaction con-stants listed in Tables 1 and 2. The estimated total reactive thiol ligands [Lor RSrx] = 4 nM, pH = 7.8, and ionic strength, I = 0.01 M.

predicted to dominate Hg speciation in EFPC, using currentlyavailable thermodynamic data (Tables 2–3). Neutral species suchas Hg(OH)2, Hg(OH)Cl, HgCl2, CH3HgOH, and CH3HgClwere predicted to be negligible or orders of magnitude lower

than the Hg-DOM complexes when a log K value of 28.7 wasused in calculations.[12] The coexisting metal ions, particularlyZn2+ at concentrations of 1.6–2.6 × 10−7 M, may compete withHg2+ for binding with DOM and cause a decreased HgII-DOMcomplexation. This study concludes that, in the absence ofsulfide (due to oxidising conditions in EFPC), the complexa-tion between Hg2+ and DOM could significantly control thereactivity and transformation of mercury, such as the methy-lation process. Whether DOM in EFPC makes Hg2+ speciesmore or less bioavailable for microbial methylation requires fur-ther investigation. However, strong complexation between Hg2+and thiols in DOM is recognised to suppress the uptake andbioavailability of Hg2+ species for methylation[23,24,50] whereasthe formation of CH3Hg-DOM complexes has been shown toinhibit the bioaccumulation of CH3Hg+.[51,52] Results of thepresent study may thus offer an explanation for relatively lowCH3Hg+ concentrations in fish in the EFPC.[3] However, givenour limited understanding of the methylation process and thecomplexity or heterogeneous nature of DOM, the presence oflow DOM has also been shown to enhance the bioaccumula-tion of mercury.[53,54] For example, competitive adsorption oflipophilic Hg2+ species (including neutral species of Hg(OH)2,HgCl2, Hg(OH)Cl, CH3HgOH, CH3HgCl) by biotic ligandsmay occur, resulting in promoted bioaccumulation of Hg2+ andCH3Hg+.[52,53,55] DOM itself could be adsorbed onto cell sur-faces and may alter the permeability of cell membranes, therebyfacilitating the uptake of lipophilic Hg species to cells.[56,57]These factors have been suggested to exert a greater role overthe inhibitory influence of Hg-DOM complexes, particularly atrelative low DOM concentrations (e.g. DOC < 5 mg L−1).[53,54]

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Mercury and methylmercury speciation

AcknowledgementsThis research is part of the Science Focus Area (SFA) at Oak Ridge NationalLaboratory (ORNL) supported by the Office of the Biological and Environ-mental Research, US Department of Energy (DOE). ORNL is managed byUT-Battelle LLC for US DOE under contract DE-AC05–00OR22725.

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Manuscript received 14 July 2009, accepted 13 January 2010

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