Managing Aquatic Mercury Pollution:Strategies to Quantify Mercury Biomethylation Potential

in Sediments

1

Helen Hsu-Kim, DUKE UNIVERSITYhsukim@duke.edu

Udonna Ndu, Natalia Neal-Walthall, Marc Deshusses, DUKE UNIVERSITY

Dwayne Elias, Geoff Christensen, Caitlin Gionfriddo, OAK RIDGE NATIONAL LABORATORY

R01ES024344

Engstrom, 2007, PNAS

• Mercury biomagnifies in aquatic food webs as monomethylmercury (MeHg)

• MeHg is produced by anaerobic microorganisms

2

Methylmercury: the driver of risk at Hg-contaminated sites

East Fork Poplar Creek (Tennessee)cleanup estimate : ~$3 billion

Onondaga Lake (NY)cleanup estimate: ~$500 million

Management of Mercury-Contaminated sites

Penobscot River estuary (Maine)cleanup estimate : >$130 million

3

Management of Mercury-Contaminated sitesBenchmarks for Site Assessment

Challenges:• Total Hg content is a poor predictor

of risk• Current water quality standard:

MeHg in fish

Needs: • More functional shorter-term

target for watershed management & remediation(e.g., Biomethylation potential of Hg)

0.001

0.01

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000 10000

Sedi

men

t MeH

g (n

g g-1

)Sediment Total Hg (µg g-1)

UrbanIndustrial AreasRice/AgricultureMiningReservoirs

103

10-3

10-2

10-1

100

101

102

100 101 102 103 10410-110-210-3

4Hsu-Kim et al. (2018) Challenges and Opportunities in Managing Aquatic Mercury Pollution. Ambio. 47: 141-169

Why do we need a model to predict Hg methylation potential?

Hg Bioavailability

Prod

uctiv

ity o

f Hg-

met

hyla

ting

mic

robe

s

low high

low

high

mesohaline marsh

HgS contaminated tidal marsh

Hg(0) discharge in low order stream

Cinnabar mine drainage

Total Hg Methylation Risk Profile

high %MeHg (d[MeHg]/dt)

low %MeHg(d[MeHg]/dt)

moderate %MeHg(d[MeHg]/dt)

Why do we need a model to predict Hg methylation potential?

Hg Bioavailability

Prod

uctiv

ity o

f Hg-

met

hyla

ting

mic

robe

s

low high

low

high

mesohaline marsh

HgS contaminated tidal marsh

Hg(0) discharge in low order stream

Cinnabar mine drainage

Total Hg Methylation Risk Profile

Site A (original status)e.g. upland, unsat’d soil

wetland creation, flooding, sea level rise, sulfate deposition

Water column aeration

?

Impacts of Remediation Activities and Other Perturbations

activated carbon amendment

high %MeHg (d[MeHg]/dt)

low %MeHg(d[MeHg]/dt)

moderate %MeHg(d[MeHg]/dt)

Hg Bioavailabilitylow high

low

high

Prod

uctiv

ity o

f Hg-

met

hyla

ting

mic

robe

s

Methods for Quantifying Mercury Biomethylation Potential

The conventional approach: Equilibrium speciation

weakly complexed

Hg(OH)2, HgCl2, HgCl3-

strongly complexed

Hg(HS)x, Hg-DOM

Mineral phaseHgS(s) Ksp or Kd

Hg2+ + xHS- ↔ Hg(HS)x2-x KHg(HS)x

Hg2+ + DOM ↔ Hg-DOM KHgDOM

DissolvedParticulate

Bioavailable

SorbentOrganic matter

FeS(s)

7

•All have the HgcA and HgcB proteins•Ubiquitous in anaerobic niches(sulfate reducers, Fe reducers, methanogens)

Parks et al. 2013 ScienceGilmour et al. 2013 ES&T

Hg-methylating microbes:

MeHgphoto credit: Poulain and Barkay, 2013, Science

Sediment porewater of a freshwater lake

0

1000

2000

3000

3000 g 20 min

6,700 g 5 min

370,000 g 2 hr

<0.2μm filter

[Hg]

in s

uper

nata

nt (

ng/L

)

Site 2 (lake center)

Most of the mercury in porewater is bound to particles

HgT = 700 ng/g

Hg speciation in benthic settings

8

dissolved Hg(II)

complexesligand-promoted or inhibited dissolution

Stable colloid and nanoparticle suspension

bioavailable

Aggregated colloids and nanoparticles

not bioavailable

chelation by DOM

clus

ter f

orm

atio

n in

pr

esen

ce o

f DO

M

ripening or aging

dissolution

dissociation

“free” ion, weak or

labile complexes

aggregation

Heterogeneous amorphous HgSnanoparticles

precipitation

with DOM

sorption of DOM

DOM-capped polynuclearHg-sulfide clusters

Aiken et al., ES&T 2011

Bioavailability of Mercury for Methylation: An Alternative Approach

Rates of these reactions at microbial interfaces determine bioavailability 9

Inorganic Hg is primarily associated with particles

Slurry sample

Add GSH (1 mM)

end-over-end mix anaerobic for 30 min

Quantify Hg in <0.2 µm fraction

Thiol-based selective extraction

Glutathione (GSH) Extraction

Methods to Quantify Hg Bioavailability

dissolved Hg(II)

bioavailable HgSR

Ticknor et al., Env Engr Sci (2015)10

Methods to Quantify Hg Bioavailability

polypropylene cover

0.45-µm membrane filteragarose diffusion layerthiolated silica resin layer

polypropylene support base

Conventional approach: derive a ‘truly dissolved’ concentration

Area ADg

Dissolved Hg in bulk solution, Cb

Diffusive Gradient in Thin-film (DGT) samplers

11

Dg

Hg concentration

dept

h

Soluble Hg concentration in DGT agarose diffusion layer

Sediment or surface water Mass of Hg

uptake mreactive Hg fraction

Thiolated resin interface

Cagarose

Hg uptake into DGT:

Our approach:

polypropylene cover

0.45-µm membrane filteragarose diffusion layerthiolated silica resin layer

polypropylene support base

Area ADg

Method testing: sediment microcosms

Added Hg: (100-200 ng g-1 dw per species)

dissolved 204Hg-nitratedissolved 196Hg-humic199Hg adsorbed to FeShumic-coated nano-200HgS

sediment slurry with DGT (sample origin: tidal marsh, freshwater lake)

Testing Methods of Quantifying Hg Methylation PotentialDiffusive Gradient in Thin-Films (DGT) passive samplers

Slurry sample

Add GSH (1 mM)

end-over-end mix anaerobic for 30 min

Glutathione (GSH) Selective Extraction

Quantify Hg in <0.2 µm fraction

Quantify over time:• MeHg from each isotopic

endmember• Hg on DGTs• GSH-extractable Hg fraction• hgcA gene copy number and

microbial community composition

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0

0.2

0.4

0.6

0.8

1

0 2 4 6

MeH

g (%

of t

otal

Hg)

Time (days)

0

0.2

0.4

0.6

0.8

1

0 2 4 6

MeH

g (%

of t

otal

Hg) A

²⁰⁴Hg²⁺ ¹⁹⁹Hg-FeS ¹⁹⁸Hg-native¹⁹⁶Hg-humic nano-²⁰⁰HgS

Mesohaline

Type of Hg added:

Methylation of Hg added to slurries

Ndu et al., ES&T, 2018.13

Tidal marsh (mesohaline) sediment slurry

0

0.1

0.2

0.3

0 2 4 6H

g on

DG

T(%

of T

ot H

g)Time (days)

Uptake of total Hg in DGTs

Testing Methods of Quantifying Hg Methylation Potential

Hg uptake in DGTs correlates with MeHg production

Net MeHg production: • correlated with uptake on the DGT sampler • did not correlate with the <0.45 µm or the

GSH-extractable fraction

DGT Sampler Filter-passing fraction

GSH-extractable fraction

14Ndu et al., ES&T, 2018.

0

5

10

15

20

0 2 4 6

MeH

g (%

of t

otal

Hg)

Time (days)

Freshwater Lake Sediment Slurry with 1 mM pyruvate

DGT Sampler

Filter-passing fraction

GSH-extractable fraction

Hg uptake in DGTs correlates with MeHg production

0

0.2

0.4

0.6

0.8

1

0 2 4 6

MeH

g (%

of t

otal

Hg) A

²⁰⁴Hg²⁺ ¹⁹⁹Hg-FeS ¹⁹⁸Hg-native¹⁹⁶Hg-humic nano-²⁰⁰HgS

Mesohaline

Type of Hg added:

Mesohaline Slurry

Freshwater Slurry

Comparing the Hg-Methylating Microbial Communities

Difference because of abundance of hgcAB+ microbes?

Ndu et al., ES&T, 2018.

dissolved Hg(II)

Hg-DOM, Hg(HS)2

Bioavailable Hg

Inorganic Hg Speciationin anaerobic settings

Anaerobic Microbiome

amorphous, hydratednanostructuredweakly sorbed

well-crystallinemacrostructuredstrongly sorbed

MeHgPolymerization & Sorption: Sulfide, NOM

ripening

or aging

dissolution and

desorption

aggregation

Biogenic sulfide, organic carbon, redox gradients

hgcAB+d-Proteobact.

hgcAB

+

Firmicute

hgcAB+methanogenhgcAB+

microbial community composition

hgcAB+

Geochemical vs. Microbiome Controls on Mercury Methylation

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Mesohaline Slurry

Freshwater Slurry

Comparing the Hg-Methylating Microbial Communities

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0

20,000

40,000

60,000

0 2 4 6

hgcA

cop

y nu

mbe

r pe

r ng

DN

A(D

elta

prot

eoba

cter

ia)

Day

MesohalineFreshwater

hgcA+ Deltaproteobacteria

0

500

1000

1500

2000

0 2 4 6

hgcA

cop

y nu

mbe

r pe

r ng

DN

A(A

rcha

ea)

Day

limit of quantification

hgcA+ methanogenic Archaea

qPCR hgcA genes

Diversity and abundance of methylators from DNA-based approaches

Ndu et al., ES&T, 2018.

Next Steps

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Can DGTs work in the real world?

Outdoor freshwater wetland mesocosms.

Added Hg:

dissolved 202Hg2+

dissolved 201Hg-humic199Hg adsorbed to FeSnano-200HgS

Next StepsCan DGTs work in the real world?

0

1

2

3

0 5 10 15 20 25

MeH

g in

sedi

men

t (ng

g-1

)Inorganic Hg flux into DGT

(ng Hg m-2 h-1)

mesocosm 1

mesocosm 2

mesocosm 3

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Semi-Mechanistic Model

Model for Hg Methylation Potential

A possible simplification…..

Next Steps

Summary

• Quantifying MeHg potential in ecosystems:1.Hg bioavailability (Hg uptake rate in DGTs)2.Productivity of the methylating microbiome

(hgcA gene expression?)

• Hg bioavailability for methylation:Controlled by reactivity of Hg-S-NOM phases at microbial interfaces

Mercury: Strategies to Quantify Methylation Potential in the Environment

• Needs for site management & remediation: functional measures of MeHgproduction potential

22R01ES024344

Additional questions are welcome!Helen Hsu-Kim

hsukim@duke.edu

References

Aiken, G.R.; Hsu-Kim, H.; Ryan, J.N. (2011). Influence of dissolved organic matter for the environmental fate of metals, nanoparticles, andcolloids. Environ. Sci. & Technol. 45, 3196–3201.DOI: 10.1021/es103992s.

Engstrom, D.R., 2007. Fish respond when the mercury rises. Proceedings of the National Academy of Sciences, 104(42), pp.16394-16395.

Gilmour, C.C., Podar, M., Bullock, A.L., Graham, A.M., Brown, S.D., Somenahally, A.C., Johs, A., Hurt Jr, R.A., Bailey, K.L. and Elias, D.A., 2013.Mercury methylation by novel microorganisms from new environments. Environmental science & technology, 47(20), pp.11810-11820.

Hsu-Kim, H.; Eckley, C.S.; Achá, D.; Feng, X; Gilmour, C.C.; Jonsson, S.; Mitchell, C.P.J. (2018). Challenges and Opportunities for ManagingAquatic Mercury Pollution in Altered Landscapes. Ambio. 47(2). 141-169. DOI: 10.1007/s13280-017-1006-7

Hsu-Kim, H.; Kucharzyk, K.H.; Zhang, T.; Deshusses, M.A. (2013). Mechanisms regulating mercury bioavailability for methylatingmicroorganisms in the aquatic environment: A critical review. Environ. Sci. & Technol. 47(6), 2441-2456. DOI: 10.1021/es304370g.

Ndu, U.; Christensen, G.A.; Rivera, N.A.; Gionfriddo, C.M.; Deshusses, M.A.; Elias, D.A.; Hsu-Kim, H. (2018). Quantification of MercuryBioavailability for Methylation Using Diffusive Gradient in Thin-Film Samplers. Environ. Sci. & Technol. 52, 8521-8529. DOI:10.1021/acs.est.8b00647

Parks, J.M., Johs, A., Podar, M., Bridou, R., Hurt, R.A., Smith, S.D., Tomanicek, S.J., Qian, Y., Brown, S.D., Brandt, C.C. and Palumbo, A.V.,2013. The genetic basis for bacterial mercury methylation. Science, 339(6125), pp.1332-1335.

Poulain, A.J. and Barkay, T., 2013. Cracking the mercury methylation code. Science, 339(6125), pp.1280-1281.

Ticknor, J.L; Kucharzyk, K.H.; Porter, K.A.; Deshusses, M.A.; Hsu-Kim, H. (2015). Thiol-based selective extraction assay to comparativelyassess bioavailable mercury in sediments. Environ. Engr. Sci. 32(7), 564-573. DOI: 10.1089/ees.2014.0526

Zhang, T.; Kim, B.; Levard, C.; Reinsch, B.C.; Lowry, G.V.; Deshusses, M.A.; Hsu-Kim, H. (2012). Methylation of mercury by bacteria exposedto dissolved, nanoparticulate, and microparticulate mercuric sulfides. Environ. Sci. & Technol. 46(13), 6950-6958. DOI: 10.1021/es203181m

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