U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2018–5063

Prepared in cooperation with Naval Facilities Engineering Command

Mercury Methylation and Bioaccumulation in Sinclair Inlet, 

Kitsap County, Washington
Cover:

Top (from left to right): 

Box-corer being prepared for sediment sampling in Operable Unit B, Puget Sound Naval Shipyard, 

Sinclair Inlet, Washington. Photograph by James Foreman, U.S. Geological Survey, August 4, 2009.

Intact incubation tube removed from box-core sample used in mercury flux experiments, Sinclair Inlet, 

Washington. Photograph by James Foreman, U.S. Geological Survey, August 4, 2009. 

Vegetation restoration along north shoreline of Inner Sinclair Inlet, south facing. Photograph by 

Anthony Paulson, U.S. Geological Survey, April 23, 2008. 

Sub-samples being collected from intact box core sample from Sinclair Inlet sediments. Photograph 

by James Foreman, U.S. Geological Survey, August 4, 2009. 

Bottom: 

North shoreline of Sinclair Inlet at low tide, near Inner Sinclair Inlet station, facing East. Photograph 

by Anthony Paulson, U.S. Geological Survey, October 27, 2008. 
Mercury Methylation and Bioaccumulation in 

Sinclair Inlet, Kitsap County, Washington

By A.J. Paulson, M.C. Marvin-DiPasquale, P.W. Moran, J.F. Stewart, J.F. DeWild, 

J. Toft, J.L. Agee, E. Kakouros, L.H. Kieu, B. Carter, R.W. Sheibley, J. Cordell, and 

D.P. Krabbenhoft

Prepared in cooperation with Naval Facilities Engineering Command

Scientific Investigations Report 2018–5063

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

RYAN K. ZINKE, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2018

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Suggested citation:

Paulson, A.J., Marvin-DiPasquale, M.C., Moran, P.W., Stewart, J.F., DeWild, J.F., Toft, J., Agee, J.L., Kakouros, E., 

Kieu, L.H., Carter, B., Sheibley, R.W., Cordell, J., and Krabbenhoft, D.P., 2018, Mercury methylation and 

bioaccumulation in Sinclair Inlet, Kitsap County, Washington: U.S. Geological Survey Scientific Investigations Report 

2018–5063, 66 p., https://doi.org/10.3133/sir20185063.

ISSN 2328-0328 (online)
iii

Contents

Abstract...........................................................................................................................................................1

I. Introduction and Methods.....................................................................................................................2

Purpose and Scope ..............................................................................................................................2

Site Description ....................................................................................................................................3

Sinclair Inlet..................................................................................................................................3

Bremerton Naval Complex .........................................................................................................3

Representative Bays ...................................................................................................................3

History of Remediation and Environmental Investigations Related to Mercury ...............3

Field Sampling .......................................................................................................................................7

Sediment Sampling .....................................................................................................................7

Marine Water Sampling..............................................................................................................9

Statistical Methods.............................................................................................................................11

II. Methylation Potential of Mercury in Sediments............................................................................12

Sediment Laboratory Methods ........................................................................................................12

Mercury Species and Mercury Methylation.........................................................................12

Physical Characteristics of Sediment and Speciation of Iron and Sulfur........................13

Regional Analysis of Sinclair Inlet Compared to the Representative Bays .............................14

Spatial Analysis of Sediment from Bremerton Naval Complex Compared to Greater

Sinclair Inlet ...........................................................................................................................15

Seasonal Analysis...............................................................................................................................16

Controls on Gross Methylmercury Production ..............................................................................18

III. Release of Mercury Species from Sediment to Water Column ................................................22

Porewater and Water Laboratory Methods ...................................................................................23

Porewater Sampling and Analysis..........................................................................................23

Incubation Experiments............................................................................................................23

Tumbling Core Experiments......................................................................................................24

Laboratory Analyses..................................................................................................................25

Mercury Concentrations in Porewater...........................................................................................25

Redox State of Porewater.................................................................................................................28

Fluxes of Total Mercury and Methylmercury from Sediment......................................................29

Release of Total and Methylmercury from Sediment During Tumbling .....................................30

Mercury Concentrations in Water Column.....................................................................................30

Water Column Mercury in Sinclair Inlet Compared to Representative Bays..................31

Importance of Sedimentary Sources of Mercury Species................................................31

Correlations Between Porewater, Fluxes, and Water Column Constituents ............................36

Comparison of Porewater Concentrations with Fluxes......................................................40

Factors Controlling Porewater Concentrations and Fluxes...............................................41
iv

Contents

IV. Methylmercury Accumulation 

in the Base of an Estuarine Food Web........................................................................................43

Methods for Food Web Study ...........................................................................................................43

Water-Column Sample Processing and Laboratory Analysis ............................................43

Zooplankton Sample Collection...............................................................................................43

Results of Food Web Study ...............................................................................................................44

Spatial Sampling in August 2008 .............................................................................................44

Monthly Sampling in Sinclair Inlet...................................................................................................47

Estimating Zooplankton Mercury.....................................................................................................52

V. Synthesis ...............................................................................................................................................54

Correlations between Methylation, 

Release, and Bioaccumulation ...........................................................................................54

Summary...............................................................................................................................................56

Acknowledgments.......................................................................................................................................57

References Cited..........................................................................................................................................58

Appendix 1. Supplementary Figures and Tables.....................................................................................63

Figures

1. Map showing Sinclair Inlet and locations of representative bays in Holmes Harbor,

Budd Inlet, Liberty Bay, Puget Sound, Washington, August 2008 ........................................4

2. Map showing marine sediment stations and frequency of sample collection in

greater Sinclair Inlet, Kitsap County, Washington, 2008, 2009, 2010 ....................................5

3. Map showing marine sediment stations in the OU B Marine, Bremerton naval

complex, Kitsap County, Washington, 2008 and 2009 .............................................................6

4. Schematic diagram showing sediment methylation potential, porewater analyses,

and tumbling-core and incubation experiments .....................................................................8

5. Map showing locations of marine water-column stations sampled in Sinclair Inlet,

Kitsap County, Washington, 2008–10.......................................................................................10

6. Boxplots showing sediment methylmercury concentration and sediment

methylmercury production potential rates at Sinclair Inlet stations, Washington,

2009 ..............................................................................................................................................16

7. Graph showing simulated and measured sediment methylation rate constant from

the sediment reactive mercury pool developed for representative bays, Bremerton

naval complex and greater Sinclair Inlet, Kitsap County, Washington, 2008–09 ............18

8. Scatterplot showing sediment reactive inorganic mercury as a percentage of

sediment total mercury concentration compared to acid-volatile sulfur

concentration for all stations sampled in Sinclair Inlet, Kitsap County, Washington,

during August 2008 ....................................................................................................................19

9. Scatterplot plot showing simulated and measured sediment total mercury

concentration, Sinclair Inlet, Kitsap County, Washington ..................................................20

10. Graph showing simulated and measured sediment methylmercury concentration,

Sinclair Inlet, Kitsap County, Washington .............................................................................20
v

11. Graph showing simulated and measured sediment methylmercury production

potential rates, Sinclair Inlet, Kitsap County, Washington ..................................................21

12. Graphs showing filtered total mercury and filtered methylmercury concentrations

in porewater for four seasonal sampling periods, Sinclair Inlet, Kitsap County,

Washington, between August 2008 and August 2009 ..........................................................27

13. Graph showing daily fluxes of filtered methylmercury from triplicate experiments

during 3-day core-incubation experiments from Bremerton naval complex 

(station BNC-60), Sinclair Inlet, Kitsap County, Washington, June 2009...........................30

14. Boxplot showing filtered total mercury in near-surface and near-bottom water in

representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair 

Inlet, Kitsap County, Washington, August 2008–August 2009 ............................................32

15. Boxplot showing filtered methylmercury in near-surface and near-bottom water 

in representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair 

Inlet, Kitsap County, Washington, August 2008–August 2009 .............................................33

16. Boxplot showing total mercury of suspended solids in near-surface and

near-bottom water in representative bays (Holmes Harbor, Liberty Bay, and 

Budd Inlet) and Sinclair Inlet, Kitsap County, Washington, August 2008–

August 2009 .................................................................................................................................34

17. Boxplot showing methylmercury of suspended solids in near-surface and

near-bottom water in representative bays (Holmes Harbor, Liberty Bay, and 

Budd Inlet) and Sinclair Inlet, Kitsap County, Washington, August 2008–

August 2009..................................................................................................................................35

18. Graph showing filtered total mercury concentrations in porewaters compared to

dissolved organic carbon concentrations grouped by two redox conditions,

Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009 ..............................37

19. Graph showing filtered methylmercury concentrations in porewaters compared to

dissolved organic carbon concentrations grouped by two redox conditions,

Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009...............................39

20. Graph showing fluxes of filtered methylmercury compared to gradients of filtered

methylmercury between porewater and the water column, Sinclair Inlet, Kitsap

County, Washington, August 2008–August 2009 ...................................................................41

21. Graph showing concentrations of selected surface-water constituents 

associated with carbon, Puget Sound, Washington, August 2008 ....................................45

22. Graph showing concentrations of methylmercury in bulk zooplankton tissue,

Sinclair Inlet, Kitsap County, Washington, August 2008 ......................................................46

23. Graph showing comparison of stable nitrogen isotopeand stable carbon isotope 

of ratios in zooplankton tissue for stations in Sinclair Inlet and representative 

bays, Puget Sound, Washington, August 2008 ......................................................................47

24. Graph showing average chlorophyll a concentrations for selected stations in 

and adjacent to, Sinclair Inlet, Kitsap County, Washington, August 2008–

August 2009 .................................................................................................................................48

25. Graph showing particulate methylmercury (mass/volume) concentrations for

selected stations in and adjacent to Sinclair Inlet, Kitsap County, Washington ............49

Figures
vi

26. Graph showing filtered methylmercury concentrations in seawater for selected

stations in and adjacent to Sinclair Inlet, Kitsap County, Washington, 

August 2008–August 2009 .........................................................................................................50

27. Graphs showing average ratios of stable isotopes of nitrogen and carbon in

suspended solids for selected stations in and adjacent to Sinclair Inlet, Kitsap

County, Washington, August 2008–August 2009....................................................................51

28. Graph showing average methylmercury concentrations in zooplankton tissue for

selected stations in and adjacent to Sinclair Inlet, Kitsap County, Washington,

August 2008–August 2009 .........................................................................................................52

29. Graph showing averaged concentrations of filtered methylmercury in seawater,

particulate material, and zooplankton for selected stations in and adjacent to

Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009 ..............................53

30. Boxplot showing methylmercury production potential and median fluxes of

sediment at OU B Marine, and greater Sinclair Inlet stations, Kitsap County, Puget

Sound, Washington during seasonal sampling events, August 2008–August 2009 .......55

31. Boxplot showing filtered methylmercury concentration in porewater and in

sediment at OU B Marine, and greater Sinclair Inlet stations Kitsap County,

Washington, August 2008–August 2009 .................................................................................55

32. Graph showing methylmercury concentrations in porewater compared to

methylmercury concentrations in sediment, Sinclair Inlet, Kitsap County,

Washington, August 2008–August 2009..................................................................................56

Figures

Tables

1. Methods summary and abbreviations used for sediment parameters .............................13

2. Wilcoxon Rank-Sum test results for all Sinclair Inlet stations and representative

bays sampled during August 2008, Puget Sound, Washington...........................................14

3. Wilcoxon Rank-Sum test results comparing sediment mercury and non-mercury

parameters from Operable Unit B Marine and Greater Sinclair Inlet stations, Kitsap

County, Washington ...................................................................................................................15

4. Kruskal-Wallis Rank Sum test results comparing combined data by month for all

Sinclair Inlet stations, Kitsap County, Washington, 2009 .....................................................17

5. Predominant redox conditions, dissolved organic carbon and mercury

concentrations in porewater, and releases during core incubation and tumbling

experiments from sediment collected from Sinclair Inlet, Kitsap County,

Washington, 2008 and 2009.......................................................................................................26

6. Non-parametric regression and parametric correlation statistics of filtered total

mercury, filtered methylmercury, and percentage of methylmercury in porewaters

compared to dissolved organic carbon categorized by porewater sulfide

concentration, Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009 ...38

7. Statistics for filtered total mercury, methylmercury, percentage of methylmercury,

and dissolved organic carbon categorized by redox state, Sinclair Inlet, Kitsap

County, Washington, August 2008–August 2009....................................................................40

8. Analysis of variance of porewater concentrations and releases of mercury from

sediment in Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.........42
vii

Conversion Factors

U.S. customary units to International System of Units

Multiply By To obtain

Length

foot (ft) 0.3048 meter (m)

Volume

gallon (gal) 3.785 liter (L)

International System of Units to U.S. customary units

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in)

micrometer (μm) 0.003937 inch (in)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

kilometer (km) 0.6214 mile (mi)

Area

square kilometer (km2) 0.3861 square mile (mi2)

square centimeter (cm2) 0.001076 square foot (ft2)

Volume

kilogram per liter (kg/L) 8.3454 pound per gallon (lb/gal)

milliliter (mL) 0.0338 ounce, fluid (fl. oz)

liter (L) 0.2642 gallon (gal)

Mass

milligram (mg) 3.527 ounce (oz)

Flow rate

meter per second (m/s) 3.2808 foot per second (f/s)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as: 

°F = (1.8 × °C) + 32.

Datums

Vertical coordinate information is referenced to National Geodetic Vertical Datum of 1929 

(NGVD 29).

Horizontal coordinate information is referenced to North American Datum of 1983 (NAD 83).

Altitude, as used in this report, refers to distance above the vertical datum.
viii

Supplemental Information

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L), 

micrograms per liter (μg/L), or nanograms per liter (ng/L).

Concentrations of chemical constituents of solids are given in either percentage of dry weight, 

milligrams per kilogram (mg/kg) or nanograms per milligram (ng/mg), which are equivalent. 

Nanogram per gram (ng/g) approximately equals parts per billion. Nanogram per square meter 

per day ([ng/m2]/d) is concentration produced per area per day.

Abbreviations

AIC Akaike Information Criterion

ANOVA analyses of variance

ASSR ArcSine square root (data transformation) 

AVS acid-volatile sulfur (sediment)

BD bulk density

BNC Bremerton naval complex 

BI Budd Inlet

CERCLA Comprehensive Environmental Response, Compensation, and Liability Act

CTD conductivity, temperature, and depth

CVAFS cold-vapor atomic fluorescence spectrometry

CZ convergence zone

DOC dissolved organic carbon

Eh reduction-oxidation potential

ENVVEST ENVironmental inVESTment

Fe(II) ferrous iron

Fe(II)AE acid-extractable ferrous iron (sediment)

Fe(III)a amorphous (poorly crystalline) ferric iron (sediment)

Fe(III)c crystalline ferric iron (sediment)

FeT total iron (sediment)

FMHg filtered methylmercury

FP fluorocarbon polymer

FTHg filtered total mercury

GSI greater Sinclair Inlet

H2S hydrogen sulfide

HCl hydrochloric acid

HDPE high-density polyethylene

Hg0 elemental mercury
ix

Abbreviations

hgcAB mercury (II)-methylation gene cluster

Hg(II) mercury(II), an oxidative state commonly found in inorganic salts of 

mercury

HH Holmes Harbor

kmeth methylmercury production rate constant

KOH potassium hydroxide

KWRS Kruskal-Wallis Rank Sum

LB Liberty Bay

ln Natural log

LOI loss of ignition

MHg methylmercury

Mn manganese

MPP methylmercury production potential

NBK Naval Base Kitsap Bremerton

N2 nitrogen gas

NRP National Research Program (USGS)

OU B Operable Unit B (includes “OU B Marine” and “OU B Terrestrial”)

PCB polychlorinated biphenyl

PETG polyethylene terephthalate glycol

PFA perfluoroalkoxy copolymer

PMHg particulate methylmercury

PSNS Puget Sound Naval Shipyard

PTFE polytetrafluoroethylene

PTHg particulate total mercury

QFF quartz fiber filter

r Pearson correlation coefficient

R2 coefficient of determination

redox reduction-oxidation

rpm revolution per minute

RPD relative percent difference

SI Sinclair Inlet

SI-IN Sinclair Inlet-Inner

SI-OUT Sinclair Inlet-Outer

SI-PO Sinclair Inlet-Port Orchard

SMHg sediment methylmercury

SRHg sediment reactive inorganic mercury

STHg sediment total mercury
x

Abbreviations

δ13C stable isotope of carbon

δ15N stable isotope of nitrogen

THg total mercury

TRS total reduced sulfur (sediment)

USGS U.S. Geological Survey

WAWSC Washington Water Science Center (USGS)

WMRL Wisconsin Mercury Research laboratory (USGS)

WRS Wilcoxon Rank Sum

YSI Yellow Springs Instruments Company, Incorporated
Mercury Methylation and Bioaccumulation in 

Sinclair Inlet, Kitsap County, Washington

By A.J. Paulson1, M.C. Marvin-DiPasquale2, P.W. Moran2, J.F. Stewart2, J.F. DeWild2, J. Toft3, J.L. Agee2, 

E. Kakouros2, L.H. Kieu2, B. Carter4, R.W. Sheibley2, J. Cordell4, and D.P. Krabbenhoft2

1U.S. Geological Survey, retired.

2U.S. Geological Survey.

3University of Washington, School of Fisheries and Aquatic Sciences, Wetland Ecosystem Team.

4Washington State Department of Health, Office of Drinking Water, Northwest Regional Office.

Abstract

The U.S. Geological Survey evaluated the transformation 

of mercury to bioavailable methylmercury in Sinclair 

Inlet, Kitsap County, Washington, and assessed the effect 

of the transformation processes on the mercury burden in 

marine organisms and sediment. In August 2008, samples 

of sediment, water, and biota from six sites in Sinclair Inlet 

and three bays representative of Puget Sound embayments 

were collected. The extensive sediment sampling included 

analysis of methylmercury in sediment and porewater, 

estimates of methylation production potential, and analyses 

of ancillary constituents associated with organic carbon 

and reduction-oxidation (redox) conditions to assist in 

interpreting the mercury results. Analyses of methylmercury 

in water overlying incubated cores provided an estimate of 

the release of methylmercury to the water column. Collection 

of samples for mercury species in the aqueous, particulate 

(suspended solids), and biological phases, and for ancillary 

carbon and nitrogen constituents in surface water, continued, 

on about a monthly schedule, at four stations through 

August 2009. In February, June, and August 2009, seasonal 

sediment samples were collected at 20 stations distributed 

between greater Sinclair Inlet and Operable Unit B Marine 

of the Bremerton naval complex, Bremerton, Washington, 

to examine geographical and seasonal patterns of mercury 

biogeochemistry of sediment in Sinclair Inlet. At six of these 

seasonal sediment stations, porewater was collected and 

triplicate core incubation experiments were done.

Median sediment-methylmercury concentrations were 

not statistically different between the representative bays and 

Sinclair Inlet. The percentage of sediment methylmercury 

(relative to total mercury) was actually lower in the Sinclair 

Inlet sites compared with the representative bays, reflecting 

the higher sediment total mercury concentration for the 

Sinclair Inlet stations compared with the representative bays. 

Likewise, median sediment methylmercury concentrations 

were not statistically different between the greater Sinclair 

Inlet stations and the Bremerton naval complex stations; 

whereas the percentage of sediment methylmercury to total 

mercury was lower in the Bremerton naval complex due to 

higher sediment total mercury concentrations than the greater 

Sinclair Inlet stations. The biogeochemical characteristics 

of each station, measured by redox, organic carbon, and the 

seasonal availability of nutrients controlled methylmercury 

biogeochemistry. Mercury methylation production potential 

was a function of temperature, concentration of total mercury 

in sediment, and the percentage of ferrous iron (relative to 

total measured iron) across all sites. Methylmercury porewater 

concentrations were best described by using concentrations of 

dissolved organic carbon and reduction-oxidation conditions. 

Likewise, the variable fluxes of methylmercury from 

incubated cores were best described using dissolved organic 

carbon and reduction-oxidation conditions.

Sinclair Inlet exhibited the classic Puget Sound biological 

cycle, with spring and autumn phytoplankton blooms 

resulting in depletion of nitrate, orthophosphate, and silicate 

in the surface water. Although variable in timing between 

2008 and 2009, a strong corresponding seasonal trend of 

increased availability, incorporation, and bioaccumulation of 

methylmercury into the food web of Sinclair Inlet occurred 

during the early spring and summer growing season.
2 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

I. Introduction and Methods

By A.J. Paulson, M.C. Marvin-DiPasquale, and 

P.W. Moran

As early as the 1980s, the sediment in Sinclair Inlet was 

identified as having increased concentrations of a number 

of elements and organic compounds (Malins and others, 

1982). A remedial investigation of the marine waters off the 

Bremerton naval complex (BNC), Bremerton, Washington, 

was completed in 1996 (U.S. Navy, 2002), and the Record 

of Decision (U.S. Environmental Protection Agency, 2000) 

was issued as final in 2002. The remediation option included 

isolating a considerable volume of contaminated sediment 

from interactions with the benthic food web by capping and 

disposing of dredge spoils in a covered, confined aquatic 

disposal pit in 2001. The primary objective of the marine 

sediment cleanup was to address the potential risk to 

humans, particularly those engaged in a subsistence lifestyle, 

from consumption of bottom-dwelling fish known to have 

polychlorinated biphenyls (PCBs) in their tissues (U.S. Navy, 

2002). Three pathways were identified as having the capability 

to transport chemicals from the terrestrial landscape of the 

BNC to the marine environment, and thus as having the 

potential to re-contaminate the recently remediated marine 

sediment. The pathways included discharge directly from dry 

docks, discharge of groundwater directly to marine waters, and 

discharge of stormwater from facilities handling surface-water 

runoff.

As lead agency for environmental cleanup of the BNC, 

the U.S. Navy completed the second 5-year review of the 

remedial actions of the marine sediment in the boundary of 

the BNC (U.S. Navy, 2007); pursuant to Section 121(c) of 

the Comprehensive Environmental Response, Compensation, 

and Liability Act (CERCLA; Public Law 107-377) and the 

National Oil and Hazardous Substances Pollution Contingency 

Plan (40 Code of Federal Regulations Part 300). One of 

the issues in the second 5-year review highlighted by the 

cooperator, Naval Facilities Engineering Command was that, 

“There is insufficient information to determine whether the 

remedial action taken at OU [Operable Unit] B Marine with 

respect to mercury in sediment is protective of ingestion of 

rockfish by subsistence finfishers” (U.S Navy, 2007, p. 5). 

Recommendations and follow-up actions in the 5-year review 

were: 

• Revisit Remedial Investigation/Feasibility Study 

(RI/FS) ground-water-to-surface-water transport 

evaluations in light of total mercury concentrations in 

two long-term monitoring wells, 

• Perform trend analyses and assess functionality and 

protectiveness of remedy for marine sediment, and

• Collect additional information necessary to perform 

a risk evaluation and reach conclusions regarding the 

protectiveness of the remedy (U.S. Navy, 2002) with 

respect to total mercury concentrations in Sinclair Inlet 

sediment and fish tissue.

Purpose and Scope

 Since 2007, the U.S. Geological Survey (USGS) and 

the U.S. Navy have started several multi-year studies. The 

objectives were to (1) estimate the magnitudes of the different 

predominant sources of total mercury to Sinclair Inlet, 

including those from the BNC, (2) evaluate the transformation 

of mercury to a bioavailable form in Sinclair Inlet, and 

(3) assess the effect of the sources and transformation 

processes on the mercury burden in marine organisms and 

sediment. The initial Watershed Sources Project, which 

focused on the first objective, synthesized existing data of total 

mercury (THg) in sediment, water, and biota of Sinclair Inlet 

(Paulson and others, 2010) and assessed sources of filtered 

and particulate (suspended solids) mercury to Sinclair Inlet5

(Paulson and others, 2012, 2013).

This report documents the Methylation and 

Bioaccumulation Project, which focused on the second and 

third objectives. The specific tasks completed to achieve these 

objectives were:

• Task 1—Assess the seasonal probability that 

sedimentary Hg throughout Sinclair Inlet may be 

methylated.

• Task 2—Confirm Task 1 by intensively examining the 

porewaters of Sinclair Inlet sediments and the releases 

of total mercury and methylmercury from Sinclair Inlet 

sediments using incubated sediment-core experiments.

• Task 3—Determine the spatial and temporal variability 

of methylmercury concentrations in zooplankton and, 

as feasible, phytoplankton in Sinclair Inlet relative 

to the spatial and temporal variability of dissolved 

and particulate concentrations of total mercury and 

methylmercury in the water.

5 Several types of mercury measurements were collected during this study. Various forms of mercury herein are abbreviated as total mercury (THg), 

methylmercury (MHg), particulate (typically collected onto a filter) total mercury (PTHg), particulate methylmercury (PMHg), filtered total mercury (FTHg), 

and filtered methylmercury (FMHg).
I. Introduction and Methods 3

Site Description 

Sinclair Inlet

Sinclair Inlet (SI), a shallow embayment (maximum 

depth of 20 meters [m]) is on the west side of the Puget 

Sound lowlands, (fig. 1). The Puget Sound lowland is a 

long, northward-trending structural depression between the 

Cascade Mountains on the east and the Olympic Mountains 

on the west. Most of the Puget Sound lowland physiographic 

province is mantled with thick glacial and postglacial deposits.

The Sinclair Inlet-Dyes Inlet system is hydrologically 

complex not only because of the geometry of the Sinclair 

Inlet-Dyes Inlet connection, but Bainbridge Island blocks 

the connection between the Dyes Inlet-Sinclair Inlet system 

and central Puget Sound (fig. 1). The Dyes Inlet-Sinclair 

Inlet system is connected to central Puget Sound through 

Port Orchard Passage on the north side of Bainbridge Island 

and through Rich Passage on the south side of Bainbridge 

Island (fig. 1). The maximum depth of Rich Passage is 20 m 

and the maximum depth of Port Orchard Passage is 6 m. The 

shallowness of these passages results in extensive vertical 

mixing of the incoming tidal water. Tides in Puget Sound are 

mixed diurnally and have a maximum tidal range of about 5 m 

relative to a maximum depth of about 20 m for Sinclair Inlet. 

The relative proportion of tidal volumes through Port Orchard 

Passage and Rich Passage is unknown. Because the tidal prism 

volume of Dyes Inlet is about three times that of Sinclair Inlet, 

tidal currents in Port Washington Narrows (fig. 2), which 

connects Dyes Inlet to Sinclair Inlet, often lag those of Sinclair 

Inlet (Wang and Richter, 1999). Further, the convergence of 

strong tidal currents south of Port Washington Narrows, which 

drains Dyes Inlet, and east of Sinclair Inlet proper where 

strong tides and extensive mixing has been shown (Wang and 

Richter, 1999), is defined here as the convergence zone (CZ). 

Bremerton Naval Complex

The Bremerton naval complex (approximately about 

2 square kilometers [km2]) is located on the north shore of 

Sinclair Inlet in Bremerton, Washington (fig. 3) and contains 

Puget Sound Naval Shipyard (PSNS) and the Naval Base 

Kitsap Bremerton (NBK Bremerton).

The primary role of PSNS (1.5 km2) is to provide 

overhaul, maintenance, conversion, refueling, defueling, and 

repair services to the naval fleet. The PSNS, which can dry 

dock and maintain all classes of Navy vessels, is the Nation’s 

sole nuclear submarine and ship recycling facility. The PSNS 

occupies the eastern part of the complex and has six dry docks, 

eight piers and moorings, and numerous shops to support its 

industrial operations. This fenced high-security area hosts 

many tenant commands. 

The primary role of NBK Bremerton, which occupies 

the western part of the naval complex, is to serve as a deepdraft homeport for aircraft carriers and supply ships. The 

facility is a fenced and secure area that extends into Sinclair 

Inlet. Facilities on the NBK Bremerton property (0.4 km2) 

include six piers and moorings, a steam plant, parking lots, 

housing, stores, recreation areas, and eateries. NBK Bremerton 

is responsible for providing long-term care of inactive naval 

vessels. For the purposes of environmental remediation, the 

BNC was divided into Operable Units (OU) OU A, OU B, 

OU C, OU D, and OU NSC. Subsequently, OU B was further 

divided into OU B Terrestrial and OU B Marine. Of the OUs, 

only data previously collected from OU B Marine (fig. 3) are 

addressed in this report. For the purposes of this report, the 

greater Sinclair Inlet (GSI) is defined as the area outside of 

OU B Marine of the BNC and includes the station in the CZ. 

Representative Bays

The three representative bays (fig. 1) selected for this 

study are similar to Sinclair Inlet in size, depth, and geometry. 

Holmes Harbor is an embayment adjacent to rural Whidbey 

Island, whereas Budd Inlet (BI) is adjacent to Olympia, the 

capital city of Washington. Similar to Sinclair Inlet, Liberty 

Bay is connected to Port Orchard Passage and is adjacent to 

the suburban town of Poulsbo.

History of Remediation and Environmental 

Investigations Related to Mercury

A synthesis of data related to THg concentrations 

in sediment throughout Puget Sound indicated that THg 

concentrations in sediment in OU B Marine were higher 

than other urban areas of Puget Sound (Evans-Hamilton, 

Inc., and D.R. Systems, Inc., 1987). In 1989, the State of 

Washington Puget Sound Ambient Monitoring Program began 

monitoring the marine waters and sediment of Puget Sound. 
4 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig01

Study area

123° 122°

48°

47°

WASHINGTON

Figure location

0 10 20 30 KILOMETERS

0 10 20 30 MILES

EXPLANATION

U.S. Geological Survey

 representative bays, August 2008

HH

Nisqually

Seattle

Tacoma

Kent

Manchester

Everett

Bellevue

Lacey

Bellingham

Mt Vernon

Renton

Bremerton

Poulsbo

Port

Orchard

Olympia

Lakewood

Sammamish

Eagle

Harbor

Dyes

Inlet

Elliot

Bay

Duwamish

Waterway

Sinclair

Inlet

Rich

Passage

Commencement

Bay

Bellingham

Bay

Port

Gardner

Port

Orchard

Passage

Port

Gamble

Holmes

Harbor

Vendovi

Island

Vancouver Island

Budd

Inlet

Hood Canal

Strait of Georgia

Strait of Juan de Fuca

BRITISH COLUMBIA

CANADA

UNITED STATES

WAHINGTON

Raft

Island

Bainbridge

Island

Blake Island

OLYMPIC

MOUNTAINS

CASCADE MOUNTAINS

HH

LB

BI

Whidbey Island

Figure 1. Sinclair Inlet and locations of representative bays in Holmes Harbor (HH), Budd Inlet (BI), Liberty Bay (LB), 

Puget Sound, Washington, August 2008.
I. Introduction and Methods 5

tac17-1117_fig02

 

Wright

 Creek

oR ss ree C k

 l B ca kcaj k r C ee k

 enl O y keer C

s oli pannA

C er ke

Ander os n er C ke

pS

keer C gnir

Gorst

Creek

Sinclair 

 

 

 

 Inlet

Sinclair 

 

 

 

 Inlet

Cable area seaward boundary

CENTRALPUGETSOUND

PortWashingtonNarrows

4

5

9

8

1

3

2

7

6

11

25

17

18

29

31

24

13

30

14

32

12

28

10

21

19

26

23

20

15

22

27

16

122°36' 122°38' 122°40' 122°42'

47°

34'

47°

32'

0 .5

.5

1 2 MILES

2 KILOMETERS 0 1

EXPLANATION

OU AOU B MarineOU B TerrestrialOU COU DOther

Operable units

1,500-footgrid cell

OU NSC

1

U.S. Navy confined aquatic disposal pit

U.S. Geological Survey marine sediment site and sampling period

500 1,000 1,500 METERS 0

0 1,000 2,000 3,000 4,000 5,000 FEET

SI-6

SI-4

SI-PO

CZ

SI-4B

SI-28

SI-25

SI-18

SI-11

SI-OUT

SI-IN

August

2008

June

2009

February

2009

(crossmarks

represent

February

2010)

August

2009

Figure 2.

Marine sediment stations and frequency of sample collection in greater Sinclair Inlet, Kitsap County, Washington, 2008, 2009, 2010.
6 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig03

Sinclair 

 

 

 Inlet

500 1,000 1,500 METERS 0

0 1,000 2,000 3,000 4,000 5,000 FEET

122°40' 122°39' 122°38'

47°

33'

4

3 8

2

6

7

5 1

9

11

67

14

43

58

68

31

57

16

10

36

32

55

28 35

21

30

26

64

53

61

52

47

25

34

19

60

46

56

50

27

42

69

37

20

51

54

29

48

65

15

23

71

41

22

18

62

44

63

66

17

70

49

40

33

12

38

59

24

45 39

13

4

3 8

2

6

7

5 1

9

11

67

14

43

58

68

31

57

16

10

36

32

55

28 35

21

30

26

64

53

61

52

47

25

34

19

60

46

56

50

27

42

69

37

20

51

54

29

48

65

15

15

23

71

41

22

18

62

44

63

66

17

70

49

40

33

12

38

59

24

45 39

13

Dry dock

Dry dock

Dry dock

Dry dock

Dry dock

Dry dock

Western

PSNS & IMF

Naval Base Kitsap

Bremerton

Operable Unit A

Central

PSNS & IMF

Eastern

PSNS & IMF

BNC-28 BNC-39

BNC-6

BNC-4 BNC-3

BNC-71

BNC-61

BNC-60

BNC-52

BNC-42

BNC-38

BNC-26

BNC-25

BNC-19

BNC-18

BNC-18

BNC-11

August

2008

June

2009

February

2009

August

2009

Bremerton naval complex (BNC) boundaryPuget Sound Naval Shipyard PSNS —

IMF —

500-foot grid

OU AOU B MarineOU B TerrestrialOU COU DOther

EXPLANATION

Operable units

OU NSC

ShorelineIntermediate Maintenance Facility

U.S. Geological Surveymarine sediment station

Figure 3.

Marine sediment stations in the OU B Marine, Bremerton naval complex, Kitsap County, Washington, 2008 and 2009.
I. Introduction and Methods 7

The sediment of Sinclair Inlet had the highest concentrations 

of THg and PCBs of all the long-term sediment-monitoring 

stations in the first Puget Sound-wide sampling effort (Tetra 

Tech, Inc., 1990). Mercury concentrations in sediment 

samples collected from Sinclair Inlet and BNC during the 

Remedial Investigation/Feasibility Study during the 1990s, 

are summarized in Paulson and others (2010). During 

screening of marine sediment proposed to be dredged for 

navigational purposes, a considerable volume of sediment 

was determined unsuitable for open-water disposal. A Navy 

confined aquatic disposal pit (fig. 2) was developed in 2000 

for disposal of dredge spoils, and dredging of contaminated 

sediment for CERCLA purposes and was used to fill the 

excess capacity in the confined aquatic disposal pit. Even 

after the navigational and CERCLA dredging was completed, 

the level of THg contamination was of the same magnitude 

as reported in sediment from Bellingham Bay associated 

with the Georgia-Pacific chlor-alkali plant and in sediment 

from Commencement Bay (fig. 1; Paulson and others, 2010). 

The State of Washington continues long-term monitoring of 

sediment at one station in Sinclair Inlet and one station in 

Dyes Inlet, and the U.S. Navy determines THg concentrations 

at 32 sites in greater Sinclair Inlet and 71 sites with OU B 

Marine included as part of the monitoring plan outlined in the 

record of decision (U.S. Environmental Protection Agency, 

2000). The second 5-year review for the BNC (U.S. Navy, 

2007) identified mercury contamination in marine sediments 

and groundwater as an ongoing concern.

The ENVironmental inVESTment (ENVVEST) project 

was developed between Federal, State, and local partners 

to specifically address the development of Total Maximum 

Daily Loads for the Sinclair/Dyes Inlet watershed adjacent to 

PSNS. The final Project Agreement was signed in September 

2000 (Washington State Department of Ecology, 2009). The 

ENVVEST project documented fecal coliform contamination 

(Cullinan and others, 2007) and measured contaminants of 

concern, including THg, discharged to Sinclair and Dyes Inlets 

(Paulson and others, 2010). Since the completion of the USGS 

marine sampling described in this report and in Paulson and 

others (2010), the ENVVEST project continues monitoring 

Sinclair Inlet through an ambient monitoring program 

(Johnston and others, 2009).

Field Sampling

Sediment, water, and biota were sampled in August 2008 

in the three bays discussed in section, “Representative Bays” 

(fig. 1), three greater Sinclair Inlet stations (fig. 2), and three 

OU B Marine stations (fig. 3). The Puget Sound embayments, 

spanning a north-south distance of approximately 70 km, were 

selected to represent various conditions and tidal exchange 

regimes that are present across the Puget Sound region. This 

sample collection was intended to give a regional perspective 

on the sampling effort in Sinclair Inlet. Starting in September 

2008, sampling focused exclusively on stations in Sinclair 

Inlet. Near-surface water and biota sampling occurred at about 

monthly intervals. Near-bottom water and sediment sampling 

occurred on a seasonal basis and were coordinated with the 

near-surface and biota monthly sampling.

Sediment Sampling 

In August 2008, sediment was sampled at three bays 

discussed in section, “Representative Bays” (fig. 1), three 

greater Sinclair Inlet stations (Sinclair Inlet-Inner [SI-IN], 

Sinclair Inlet-Outer [SI-OUT], and Sinclair Inlet-Port Orchard 

[SI-PO]) (fig. 2), and three OU B Marine stations (BNC‐39, 

BNC-52, and BNC-71) (fig. 3). Sediment was sampled 

in Sinclair Inlet during four subsequent surface sediment 

sampling periods (figs. 2 and 3): February 2009 (4 greater 

Sinclair Inlet stations sites and 16 OU B Marine station); June 

2009 (9 greater Sinclair Inlet stations and 11 OU B Marine 

stations); August 2009 (10 greater Sinclair Inlet stations 10 

OU B Marine stations); and February 2010 (3 greater Sinclair 

Inlet stations). The sampling details and quality-assurance data 

are reported in Huffman and others (2012). Bottom sediment 

and overlying water were sampled using a 13.5 × 13.5 × 

23-centimeter (cm) deep Eckman-style box corer (Wildlife 

Supply Company, Buffalo, New York). For each station with 

incubation experiments, multiple intact sediment cores with 

at least 10 cm of overlaying water were isolated by sealing an 

interior sediment core in a 6.35-cm-diameter acrylic core liner 

with rubber end caps over Parafilm®. Shorter intact cores with 

less overlying water for reduction-oxidation (redox) sensitive 

species were isolated in a similar manner. Cores were stored 

upright in a caddy over ice and transported to the USGS 

Washington Water Science Center laboratory. The sediment 

sampling schematic for physical characteristics, mercury, 

sulfur, and iron species, sediment methylation potential, 

porewater analyses, tumbling core experiments and incubation 

experiments are shown in figure 4.
8 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig04

0

10 cm 20 cm

0

10 cm 20 cm

10 cm 

0 

20 cm

0

10 cm 

20 cm

Intact core with overlying water for 

incubation core experiments

Core equipped with stirrer for 

incubation core experiment

FTHg and FMHg in 

overlying water

Trip blank for 

porewater Composite porewater 

sample for FTHg, 

FMHg, DOC, TN

Intact box core isolated from corer

Extruder plate

Core liner removed from 

corer and positioned 

on extruder plate

Thin plastic slicing plate to isolate top 2 cm

Top 2-cm of core isolated on slicing plate

Fifteen 50-mL 

Oak-Ridge TeflonTM

tubes filled with 

sediment and 

then centrifuged 

One Oak-Ridge 

TeflonTM centrifuge 

tube of pre-tested 

water filled 

in the field

Methylation 

rate constant,

Hg, S, Fe species 

in sediment 

physical properties

Mason jar 

(250-mL) 

filled to top

Hg species, 

physical 

properties

TeflonTM jar (500-mL)

 for tumbling 

core experiment

2/3 sediment

 and 

1/3 site water

FTHg, FMHg 

and 

redox species 

(August 2009)

Redox cores being isolated 

with intact overlying water

Zip-lock®

plastic bag

Redox core in glove box being loaded 

into 100-mL plastic centrifuge 

tubes in a nitrogen atmosphere

Centrifuged sediment (66-mL of sediment)

Porewater analyses of sulfide, ferrous Fe, filtered total Fe and Mn, and filtered nutrients

EXPLANATION

FTHg filtered total mercuryFMHg filtered methylmercuryDOC dissolved organic carbonTN total nitrogenHg mercuryS sulfurFe ironMn manganesemL millilitercm centimeter

Figure 4. Sediment methylation potential, porewater analyses, and tumbling-core and incubation experiments.
I. Introduction and Methods 9

Sediment in the top 2 cm of the square box corer was 

collected for all other constituents. Water overlying the 

sediment from the entire area (13.5 × 13.5 cm) of multiple 

box cores was removed and saved only for tumbling core 

experiments. The top 2 cm of sediment from each core 

was isolated onto an acid-clean sheet of plastic open to the 

atmosphere. Sediment was collected in (1) two glass jars for 

analyses of physical characteristics including mercury, sulfur, 

and iron species, and methylation rates were determined by the 

USGS National Research Program (NRP) laboratory, Menlo 

Park, California; and (2) two subsamples for the analyses 

of mercury species were analyzed by the USGS Wisconsin 

Mercury Research Laboratory (WMRL). For the subset of 

stations with incubation experiments, sediment was collected 

in perfluoroalkoxy copolymer (PFA) beakers for sediment 

tumbling core experiments and in fifteen 50-milliliter (mL) 

PFA Oak-Ridge-type centrifuge tubes chilled in the field for 

extraction of porewater for the analysis of (1) mercury species 

by the WMRL and (2) dissolved organic carbon (DOC) by 

NRP laboratory, Boulder, Colorado. In 2009, paired sets of 

sediment samples were collected randomly from each of two 

sheets containing the top 2-cm of sediment for duplicate box 

cores as replicates. All containers were chilled on ice in the 

field until further processing (except for the subsamples in 

2009, which were frozen over dried ice in the field).

Marine Water Sampling

A data sonde (Yellow Springs Instruments Company, 

Inc.) was used at the three Puget Sound representative bays 

in August 2008 (fig. 1) to collect water-column profiles of 

depth, salinity, temperature, dissolved oxygen, turbidity, and 

fluorescence samples before water chemistry and zooplankton 

samples were collected. Similarly, vertical profiles were 

measured monthly at discrete depths in Sinclair Inlet between 

August 2008 and January 2009. From February 2009 to 

August 2009, an SBE 19plus (Seabird Electronics., Inc., 

Bellevue, Washington) conductivity, temperature, and depth 

(CTD) sensor package was used to collect the same types of 

samples (for a complete list of dates and locations of sample 

collection, see Huffman and others [2012], appendix A). Water 

samples were collected at a minimum of four, occasionally as 

many as seven, stations in Sinclair Inlet (fig. 5). Near-bottom 

and near-surface water was collected in August 2008 and 

February, June, and August 2009 before sediment sampling 

began. 

After collecting profile data, marine water was 

peristaltically pumped through C-Flex® tubing connected to 

PFA tubing, which was attached to a polytetrafluoroethylene 

(PTFE) port that was lowered to the appropriate depth in the 

following sequence: 

1. Raw water was pumped into polyethylene terephthalate 

glycol (PETG) bottles for mercury species (FTHg, 

FMHg, PTHg, and PMHg). 

2. Water was filtered through Pall Aqua-Prep 

0.45 micrometer (μm) pore size, 79-millimeter (mm) 

diameter, polyester polysulfone disk filter into separate 

high-density polyethylene (HDPE) bottles for nutrients 

and total manganese (Mn; acidified in the field). 

3. Raw water was pumped in baked amber glass bottles for 

DOC and total particulate carbon and nitrogen. 

4. Raw water was pumped in baked amber glass bottles 

from the near-surface sites for chlorophyll a and isotopes 

of particulate carbon and nitrogen. 

5. Raw water for total suspended solids measurements 

was pumped in separate HDPE bottles in August and 

September 2008, after which total suspended solids were 

measured in every bottle in which PTHg and PMHg 

samples were collected. 

Seawater was processed for analyses of various constituents 

in a mobile laboratory in August 2008 and at the USGS 

Washington Water Science Center (WAWSC) laboratory 

between September 2008 and August 2009. Water-column 

sampling methods included analysis of nitrate, ammonia, 

total nitrogen, orthophosphate, total particulate carbon and 

nitrogen, DOC, and suspended solids.

Zooplankton was collected monthly between August 

2008 and November 2009 (except for December 2008) at the 

BNC-52, SI-IN, SI-PO, and CZ stations. On each sampling 

date, vertical plankton tows were collected for quantitative 

analysis at each station using a 0.5 m diameter, 0.1 mm mesh 

plankton net with an attached TSK flowmeter (Tsurumi Seiki 

Co., Ltd., North Bend, Washington). The net was lowered to 

the bottom, depth, data were recorded, and then the net was 

pulled to the surface at a speed of approximately 0.5 meter 

per second (m/s). Samples were fixed in 10 percent volume/

volume buffered formalin solution. Between three and six 

additional vertical tows were made with the 0.1 mm mesh net 

and several vertical tows were made with a 0.75 m diameter, 

0.253 mm mesh plankton net to collect live material. The 

number of net tows depended on the density of organisms 

observed in the nets. Live specimens were retained and placed 

on ice in 1-gallon glass jars for less than 24 hours until sample 

processing began. 
10 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig05

 Wright

oR ss ree C k

 l B ca kcaj k r C ee k

 enl O y keer C

pannA il o s C keer

Ander os n er C ke

pS

C gnir keer

Gorst

Creek

Sinclair 

 

 

 

 Inlet

Cable Area Seaward Boundary

CETRALPUGETSOUND

Creek

PortWashingtonNarrows

122°36' 122°38' 122°40' 122°42'

47°

34'

47°

32'

0 .5

.5

1 2 MILES

2 KILOMETERS 0 1

EXPLANATION

Bremerton Naval Complex U.S. Geological Survey marine

water column station (near surface)

BNC-52

SI-IN

BNC-60

U.S. Geological Survey marine station

(near surface and near bottom)

U.S. Geological Survey marine

water column station (near bottom)

BNC-52

BNC-60

CZ

SI-IN

SI-PO

SI-OUT

BNC-39

BNC-71

Location figure 2

Figure 5. Locations of marine water-column stations sampled in Sinclair Inlet, Kitsap County, Washington, 2008–10.
I. Introduction and Methods 11

Samples of suspended solids and zooplankton from the 

monthly sampling were also analyzed for stable isotopes to 

explain the food quality of the suspended solids (particulates) 

material and its trophic relation to the zooplankton. Stable 

nitrogen isotope ratios (δ15N) provide a spatially and 

temporally integrated measure of trophic relations in a 

food web (that is, primary producers to invertebrates to 

fish) because δ15N becomes enriched by 2.5–5 parts per 

thousand between prey and predator (Peterson and Fry, 1987). 

Stable carbon isotope ratios (δ13C) tend to show little or no 

enrichment (<1 part per thousand [‰]) with each trophic 

level, but can identify contributions of different foods (that 

is, carbon sources), if foods have distinct isotopic signatures 

(France, 1995).

Statistical Methods

Type II error probability was set at p<0.05 for all 

statistical tests, unless otherwise noted. All data evaluated 

were reviewed for normality, and appropriate parametric or 

non-parametric tests were selected based on data distribution 

characteristics. Data transformations were used, as needed, to 

meet assumptions of parametric statistics when non-parametric 

approaches were not, or were less, suitable.

The non-parametric Wilcoxon Rank Sum (WRS) test was 

used to compare two medians: for sediment data grouped by 

region (representative bays compared to SI [GSI and OU B 

Marine combined]) and water column data grouped by depth 

(surface compared to bottom) in a specific region. The nonparametric Kruskal-Wallis Rank Sum (KWRS) test was used 

to compare multiple medians for sediment data grouped by 

sampling period only for the 2009 periods (February, June, 

and August). In instances where left-censored (less than 

reporting limit) data existed for a given parameter, summary 

statistics (medians and interquartile ranges) were calculated 

using “maximum likelihood estimation” (Helsel, 2005) subroutines developed by the USGS for the S-Plus statistical 

platform (ver. 8.1, 2008).

Predictive correlations for surface-sediment mercury 

metrics were systematically developed. First, potential 

explanatory X-variables were assessed for normality. In 

instances where parameters were not normally distributed, 

various transformations were assessed (that is, ln(X), X-1, 

X2, and X1/2), and the most appropriate transformation was 

selected for each parameter either to achieve normality or 

most closely approach it. In the case of percentage data, the 

ArcSine square root (ASSR) transformation was used (that 

is, ArcSine(X%/100)1/2). Second, the Akaike Information 

Criterion (AIC) approach (Akaike, 1974) was used to 

complete all potential models containing as many as four 

explanatory variables. The most parsimonious model (that 

with the minimum number of explanatory variables necessary) 

was selected after comparing AIC rankings (those with 

lower AIC scores outrank those with a higher scores), after 

excluding any candidate models where there was correlation 

between any two predictor variables exceeding a Pearson 

correlation coefficient (r) of r greater than +0.7 or r less than 

-0.7. Third, the model residuals (unexplained error) were 

examined for normality. Any candidate models with residuals 

that did not conform to the normal distribution were rejected. 

For the sediment porewater parameters, FTHg and 

FMHg, non-parametric Spearman and Kendal tau correlation 

analyses were done to determine if correlations were 

significant. The slope of the parametric Pearson regression is 

given for datasets in which one or both of the non-parametric 

tests were significant. A variety of non-parametric analyses 

of variance was done using geographic and biogeochemical 

categorical variables. Tukey’s multi-comparison method is an 

ANOVA method to create confidence intervals for all pairwise 

differences between factor level means resulting in Tukey 

categories that are significantly different from one another. 

Water column concentrations greater than the 75th percentile 

value by more than one interquartile range were considered 

exceptionally high concentrations and were not included in 

the ANOVA test. Parameters indicative of accumulation of 

mercury in aquatic food webs were evaluated using ordinary 

least squares and stepwise least squares regression techniques. 

Data were analyzed using TICBO Spotfire S+ (version 8.1, 

Palo Alto, CA), SYSTAT software (ver. 13, San Jose, CA), or 

JMP (version 11.2.1; SAS Institute Inc., Cary, NC) statistical 

software.
12 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

II. Methylation Potential of Mercury

in Sediments

By M.C. Marvin-DiPasquale, J.L. Agee, E. Kakouros, 

L.H. Kieu, D.P. Krabbenhoft, J.F. DeWild, and 

A.J. Paulson 

As part of Task 1 of the Methylation and 

Bioaccumulation Project (to describe and quantify the 

biogeochemical cycling of mercury throughout Sinclair Inlet), 

the study focused on four primary objectives associated 

with surface sediment. The primary tasks involved (1) 

quantifying mercury species concentrations; (2) quantifying 

methylmercury production potential (MPP) rates; (3) 

examining the extent to which mercury species concentrations 

and MPP rates vary spatially and seasonally; and (4) 

examining these spatial and seasonal trends in terms of the 

sediment carbon, sulfur, and iron biogeochemistry. Sediment 

sampling sites and sampling periods are shown in figures 2

and 3.

Although net MHg production reflects the balance of 

gross MHg production and degradation (Marvin-DiPasquale 

and Agee, 2003; Marvin-DiPasquale and others, 2003), the 

gross production of MHg is ultimately a function of both the 

activity of the inorganic-mercury (II) (within the mercury 

methylating microbial community) and the availability of 

Hg(II) to those microbes (Marvin-DiPasquale, Lutz, and 

others, 2009). In terms of controls on the activity of the 

Hg(II)-methylating microbial community, the factors most 

commonly cited are the availability of electron acceptors 

(Gilmour and others, 1992; Kerin and others, 2006), electron 

donors (that is, labile organic matter; Lambertsson and 

Nilsson, 2006), and temperature (Heyes and others, 2006). 

Less is known about the controls on Hg(II) availability to the 

resident community of Hg(II)-methylating bacteria; however, 

the specific chemical forms (species) of mercury compounds 

(Benoit and others, 1999) and dissolved organic matter (Dong 

and others, 2010; Slowey, 2010) have been cited as playing 

important roles in this process. In the current study, an attempt 

was made to determine which environmental variables exert 

the strongest control on the activity of the Hg(II)-methylating 

community in surface sediment (as assessed by stableisotope incubation-derived measurements of methylmercury 

production rate [kmeth]) and on the availability of Hg(II) to 

those microbes (as assessed by the sediment reactive inorganic 

mercury [SRHg] metric). Two primary drivers of MHg 

production, as methylation rate (kmeth) and methylmercury 

production potential (MPP), both vary spatially (between 

OU B Marine, GSI, and at a subset of representative bays 

outside of the Sinclair Inlet) and seasonally (February, June, 

and August 2009).

Sediment Laboratory Methods 

Field samples were subsampled for specific analytes 

under anoxic conditions in a nitrogen gas (N2) flushed glove 

bag. Except for August 2008, when most sub-sampling was 

done at a local off-site staging area within hours of sample 

collection, sediment was shipped to the USGS Menlo Park, 

California, laboratory and subsampled within 1–6 days 

(median = 2 days, n = 75) from the time of field collection. 

Unless otherwise noted in Huffman and others (2012), samples 

typically were homogenized in a large glass bowl with a 

PTFE spatula. Quality-assurance data presented in Huffman 

and others (2012) indicate adequate, but occasionally high, 

variability between homogenized replicates, with THg having 

0.4, 4.2, and 59 for minimum relative percent difference 

(RPD), median RPD, and maximum RPD, respectively, 

and MHg having 1, 17, and 70 RPDs, respectively. Details 

about sediment initial subsampling, preservation, and 

analysis are available in Huffman and others (2012). All 

sediment parameters analyzed as part of this study, along 

with analyte names and abbreviation used for each in this 

text, and a citation for the full method details are listed in 

table 1. All surface sediment laboratory analyses described 

in “Methylation Potential of Mercury in Sediments” were 

completed at the USGS NRP laboratory in Menlo Park, 

California, unless otherwise indicated.

Mercury Species and Mercury Methylation

For sediment total mercury (STHg), sediment was first 

digested in concentrated hydrochloric acid (HCl) and nitric 

acid. The digestate was subsequently subsampled, chemically 

reduced with tin-chloride, and THg quantified by cold-vapor 

atomic fluorescence using a Tekran® 2600 automated total 

mercury analyzer according to U.S. Environmental Protection 

Agency method 1631 (U.S. Environmental Protection Agency, 

2002). For SRHg, thawed sediment was transferred to an 
II. Methylation Potential of Mercury in Sediments 13

Table 1. Methods summary and abbreviations used for sediment parameters.

Parameter 

abbreviation 

Parameter

name

Method

citation 

Mercury parameters 

STHg Sediment total mercury (Marvin-DiPasquale and others, 2011) 

SMHg Sediment methylmercury (Marvin-DiPasquale and others, 2011) 

Hg(II)R Inorganic reactive mercury (sediment) (Marvin-DiPasquale and others, 2011) 

kmeth Methylmercury (MHg) production potential rate constant (Marvin-DiPasquale and others, 2011) 

MPP Methylmercury production potential rate (calculated) (Marvin-DiPasquale and others, 2011) 

Non-mercury parameters 

AVS Acid-volatile sulfur (Marvin-DiPasquale and others, 2008) 

TRS Total reduced sulfur (Marvin-DiPasquale and others, 2008) 

Fe(II)AE Acid-extractable ferrous iron [Fe(II)] (Marvin-DiPasquale and others, 2008) 

Fe(III)a Amorphous (poorly crystalline) ferric iron [Fe(III)] (Marvin-DiPasquale and others, 2008) 

Fe(III)c Crystalline ferric Iron [Fe(III)] (Marvin-DiPasquale and others, 2008) 

FeT Total iron (calculated; sum of measured three iron fractions) (Marvin-DiPasquale and others, 2008) 

%LOI Percentage of weight loss on ignition (Marvin-DiPasquale and others, 2008) 

BD Bulk density (Marvin-DiPasquale and others, 2008) 

%FINES Percent fines, grain size (percent, less than 63 micrometers) (Matthes and others, 1992) 

Eh Oxidation-reduction potential (Marvin-DiPasquale and others, 2008) 

pH pH units (Marvin-DiPasquale and others, 2008)

N2-flushed bubbler containing 0.5 percent HCl, reduced with 

tin-chloride, and purged with N2 for 15 minutes, and the 

resulting elemental mercury was collected on an in-line gold 

trap and subsequently quantified by using cold-vapor atomic 

fluorescence spectrometry (CVAFS) (Marvin-DiPasquale 

and Cox, 2007). Sediment methylmercury (SMHg) was first 

extracted with a solution of 25 percent potassium hydroxide 

(KOH) in methanol (Xianchao and others, 2005); SMHg is 

used here to denote MHg extracted from sediments, whereas 

PMHg is used to denote MHg measured on particles from 

collected from the water column. The extraction solution 

subsequently was subsampled, ethylated with sodium 

tetraethyl borate in a sealed vessel, and quantified for SMHg 

using a MERX (Brooks Rand, Seattle, Washington) automated 

MHg analyzer.

Surface sediment Hg(II)-methylation rate constants 

(kmeth) were assessed with 4–5 hour bottle incubation 

core experiments composed of native sediment and 

site water (fig. 4), amended with short-term (4–5 hour) 

stable isotope amendments (Huffman and others, 2012). 

Incubation temperature was set at ±1 degree Celsius (°C) 

of the average sediment temperature measured in the field 

at the time of collection for all sites sampled. The labeled 

200Hg-methylmercury, an isotopically enriched form of 

mercury, was extracted with KOH/methanol and quantified 

using isotope-dilution inductively coupled plasma with mass 

spectrometry. The MPP rates were subsequently calculated 

using independently measured kmeth and SRHg data for each 

site (referred to as Hg(II)R in Huffman and others [2012]).

Physical Characteristics of Sediment and 

Speciation of Iron and Sulfur

Acid-volatile sulfur (AVS) concentration, a measure of 

substances that generate hydrogen sulfide (H2S) gas upon 

HCl addition, was measured on wet sediment collected during 

only August 2008, using hot acid distillation, H2S–trapping, 

and colorimetric quantification of sulfide (Marvin-DiPasquale 

and others, 2008). Total reduced sulfur (TRS) concentration 

was measured on all sediment samples using a single-step hot 

acid, chromium-reduction distillation with H2S-trapping and 

colorimetric quantification of sulfide (Marvin-DiPasquale and 

others, 2008). Three iron species were measured by chemical 

extraction and colorimetric quantification (Marvin-DiPasquale 

and others, 2008), which included acid-extractable ferrous 

iron (Fe(II)AE), amorphous (poorly crystalline) ferric iron 

(Fe(III) a), and crystalline ferric iron (Fe(III)c). The sum of 

these three iron fractions is calculated as a measure of total 

iron (FeT = Fe(II)AE + Fe(III)a + Fe(III)c). The percentage 

of Fe(II) AE relative to FeT (%Fe(II)AE = Fe(II)AE/FeT × 

100) also was calculated. Sediment bulk density (BD), dry 

weight, porosity, and organic content (as percentage of weight 

loss on ignition; LOI) were measured in sequence from a 

single sediment subsample (Marvin-DiPasquale and others, 

2008). Sediment grain size, assessed as the sand/silt split and 

expressed as percentage of fines (<63 μm), was done by wet 

sieving and subsequent drying (Matthes and others, 1992). 

Sediment redox potential and pH were measured by electrode 

(Marvin‐DiPasquale and others, 2008).
14 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Regional Analysis of Sinclair Inlet 

Compared to the Representative Bays

Statistical results of the 2008 (reconnaissance sampling) 

sediment parameters from the four representative bays (fig. 1) 

located outside of Sinclair Inlet (CZ, HH, LB, BI) for the 

comparisons with the stations located inside of Sinclair Inlet 

(see fig. 2) and the sites within the Operable Unit shown in 

figure 3. Concentrations of STHg were significantly greater 

in the Sinclair Inlet stations than those measured at the 

representative stations, with the median value for the greater 

Sinclair Inlet stations (305 ng/g) being approximately six-fold 

greater than the median value for the representative stations 

(55 ng/g). Similarly, median SRHg concentrations were eightfold greater in the Sinclair Inlet stations (0.57 ng/g) compared 

to the representative bays (0.07 ng/g), with an eight-fold 

higher median concentration in the Sinclair Inlet (table 2). The 

only other significant difference detected was for SMHg as a 

percentage of STHg, for which the median value was more 

than five-fold greater for the representative bays (3 percent 

of STHg as SMHg) compared to the Sinclair Inlet stations 

with 0.6 percent of STHg as SMHg. That is, the percentage of 

sediment total mercury in the methyl form in representative 

bays was much higher than in Sinclair Inlet. In contrast, a 

number of key mercury parameters were not significantly 

different inside than outside of Sinclair Inlet during the 2008 

reconnaissance sampling, including SMHg concentrations, 

SRHg as a percentage of STHg, the experimentally derived 

rate constants for Hg(II)-methylation (kmeth), and MPP rates. 

Furthermore, none of the non-mercury sediment parameters 

assayed for August 2008 were significantly different inside 

than outside of Sinclair Inlet (see table 2, not all parameters 

shown).

Although more THg (and SRHg) is in the sediment 

collected from Sinclair Inlet compared to representative bays 

outside of Sinclair Inlet, this limited dataset provides little 

evidence that SMHg concentrations or production rates vary 

significantly inside than outside of Sinclair Inlet. The one 

exception is MHg as a percentage of STHg, which has been 

considered a measure of the Hg(II)-methylation efficiency in 

some instances (Gilmour and others, 1998; Krabbenhoft and 

others, 1999; Domagalski, 2001). These results imply that 

as a group, the sediment associated with the representative 

bays had a higher efficiency for MHg production than did 

stations in Sinclair Inlet. However, that interpretation was 

not consistent with the MHg concentrations or measured 

MPP rates. It is more likely that both groupings had a similar 

propensity for MHg production, based on the activity of 

the resident Hg(II)-methylating microbes (as implied by 

similar kmeth values) and similar MHg concentrations. 

However, because the THg concentrations inside Sinclair 

Inlet were so much greater than in the representative bays, the 

calculated percentage of MHg (that is, percentage of MHg 

= [MHg]/ [THg] × 100 percent) values were much lower for 

stations inside Sinclair Inlet.

Table 2. Wilcoxon Rank-Sum test results for all Sinclair Inlet stations and representative bays sampled during August 2008, Puget 

Sound, Washington.

[Parameter definitions are given in table 1. Bold values indicate statistically significant difference. The non-parametric Wilcoxon Rank Sum comparison of 

medians included all eleven sites sampled during the August 2008 reconnaissance sampling event, for data grouped by greater Sinclair Inlet and Bremerton 

naval complex sites (combined; n=7) and by representative sites located outside of the Sinclair Inlet (n=4). The first quartile (25th percentile), median (50th 

percentile), third quartile (75th percentile) are shown. Wilcoxon Rank Sum: ***, significant differences between sediment mercury parameter groupings at the 

probability levels of p < 0.05; NS, non-significant differences. All non-mercury sediment parameter comparisons were NS. Abbreviations: ng/g, nanogram per 

gram; (pg/g)/d, picogram per gram per day]

Parameter (units)

Representative bays Greater Sinclair Inlet and 

Bremerton naval complex sites Wilcoxon 

25th Rank Sum

percentile Median 75thpercentile 

25th

percentile Median 75thpercentile

STHg1 (ng/g) dry weight 15 55 94 191 305 659 ***

SRHg (ng/g) dry weight 0.04 0.07 0.12 0.38 0.57 0.60 ***

%Hg(II)R (percentage of STHg) 0.16 0.18 0.22 0.08 0.15 0.29 NS

SMHg (ng/g) dry weight 0.88 1.90 2.93 1.47 1.79 3.81 NS

%SMHg (percentage of STHg) 2.71 3.29 5.22 0.51 0.61 1.12 ***

kmeth

1 (day) 0.007 0.016 0.024 <0.0014 0.011 0.014 NS

MPP rate1 ([pg/g]/d) dry weight 0.3 1.7 3.1 <0.84 2.7 6.9 NS

1 Parameter contained left-censored (less than) data. Interquartile range and medians calculated using maximum likelihood estimate statistics (Helsel, 2005).
II. Methylation Potential of Mercury in Sediments 15

Spatial Analysis of Sediment from 

Bremerton Naval Complex Compared to 

Greater Sinclair Inlet

Comparing BNC stations to GSI stations across all 

sampling dates, STHg and SRHg concentrations were 

significantly greater in the BNC than in the GSI sediment 

stations. Non-mercury sediment parameters, TRS, Fe(II) AE, 

FeT, the percentage of Fe(II)/FeT, the percentage of fines 

(<63 μm), Eh, and pH, also were greater in the BNC than 

in the GSI sediment (table 3), based on Wilcoxon ranksum test. The only sediment mercury parameter that was 

significantly lower for BNC than the GSI was the percentage 

of SMHg/ STHg, as were non-mercury parameters Fe(II)a

and field temperature. No other sediment mercury parameters 

(SMHg, the percentage of SRHg/STHg, kmeth, and MPP rate) 

or non-mercury parameters showed significant difference 

between BNC stations and GSI stations. These results suggest 

that although the absolute concentration of STHg and SRHg is 

higher in the BNC, than in the GSI, the MPP rate and SMHg 

concentration is not. The higher STHg and SRHg content in 

the BNC may be partially due to smaller sediment particles 

(higher percentage of fines <63 μm) in this area; an increase 

in STHg concentration typically is observed as particle 

size decreases (Bengtsson and Picado, 2008; Fleck and 

others, 2011) due to the increase in the ratio of particle surface 

area to volume.

Table 3. Wilcoxon Rank-Sum test results comparing sediment mercury and non-mercury parameters from Operable Unit B Marine and 

Greater Sinclair Inlet stations, Kitsap County, Washington.

[Parameter definitions are given in table 1. Bold values indicate statistically significant difference. The non-parametric Wilcoxon Rank Sum comparison of 

medians included all eleven sites sampled during the August 2008 reconnaissance sampling event, for data grouped by greater Sinclair Inlet and Bremerton 

naval complex sites (BNC; n=38) and greater Sinclair Inlet (n=32). The first quartile (25th percentile), median (50th percentile), third quartile (75th percentile) 

are shown along with all results from all mercury metric comparisons. Wilcoxon Rank Sum: ***, significant differences between sediment mercury parameter 

groupings at the probability levels of p < 0.05; NS, non-significant differences. Abbreviations: °C, degrees Celsius; μm, micrometer; μmol/g, micromole 

per gram; mg/g, milligrams per gram; ng/g, nanogram per gram; n.c., not calculated due to the number of left-censored (less than); pg/g, picogram per gram; 

(pg/g)/d, picogram per gram per day; <, less than] 

Parameter (units)

Bremerton naval complex Greater Sinclair Inlet

Wilcoxon 

Rank Sum 25th

percentile Median 75thpercentile 

25th

percentile Median 75thpercentile

Sediment mercury parameters

STHg (ng/g) dry weight 510 674 756 152 457 649 ***

Hg(II)R

1 (ng/g) dry weight 0.32 0.42 0.49 0.19 0.28 0.39 ***

%Hg(II)R

1 (percentage of STHg) 0.050 0.063 0.093 0.045 0.058 0.126 NS

SMHg (ng/g) dry weight 1.95 2.69 4.09 0.95 2.86 5.29 NS

%SMHg (percentage of STHg) 0.28 0.42 0.66 0.56 0.90 1.12 ***

kmeth

1 (day) n.c. <0.00079 0.012 <0.00077 0.0048 0.016 NS

MPP rate1 ([pg/g]/d) dry weight n.c. <0.21 3.60 <0.01 0.84 5.23 NS

Sediment non-mercury parameters

TRS (μmol/g) dry weight 218 249 290 80 163 277 ***

Fe(II)AE (mg/g) dry weight 3.3 4.0 4.5 1.5 3.2 3.8 ***

Fe(III)a

1 (mg/g) dry weight 0.010 0.012 0.030 0.01 0.02 0.11 ***

FeT (mg/g) dry weight 3.6 4.6 5.1 2.7 3.5 4.8 ***

%Fe(II)/FeT (percent) 80 91 99 63 81 99 ***

%FINES (percent, <63 μm) 72 84 87 36 74 86 ***

Eh laboratory (millivolt) -17 1 24 -69 -16 4 ***

pH units (pH units) 7.13 7.20 7.25 7.01 7.09 7.17 ***

Field temperature (°C) 7.6 11.3 13.8 11.3 13.5 7.17 ***

1Parameter contained left-censored (less than) data. Interquartile range and medians calculated using maximum likelihood estimate statistics (Helsel, 2005).
16 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Seasonal Analysis

A non-parametric 

statistical analysis (KWRS 

test) of mercury and nonmercury sediment parameters 

was done on data grouped 

by month (February, June, 

and August 2009) for all 

dates and all stations in the 

BNC and GSI (combined; 

table 4). Significant temporal 

trends were indicated in all 

mercury (Hg) parameters 

except in STHg and SRHg 

concentrations. Overall, 

SMHg concentration, 

percentage of SMHg/STHg, 

kmeth, and MPP rates were 

lowest in February and 

higher in June and August. 

Temperature also was low 

during these months, which 

suggests the ever-present 

influence of temperature on 

microbial Hg(II)-methylation. 

Additionally, surface 

sediment organic content 

(as percentage of LOI) was 

highest in June (median = 

10.5 percent) and similar 

in February and August 

(medians = 8.6 percent and 

8.9 percent, respectively). 

This temporal trend may 

partially reflect the deposition 

of phytodetritus associated 

with a spring bloom in 

phytoplankton (see section, 

“Release of Mercury Species 

from Sediment to the Water 

Column”), which was evident 

by a spike in chlorophyll a

in the water column with 

a concentration of 16 μg/L 

during April. However, 

much larger chlorophyll a

spikes were seen in the 

late summer and autumn 

(August and September) in 

Sinclair Inlet (see section, 

“Release of Mercury Species 

from Sediment to the Water 

Column,” and Huffman and 

others, 2012). Thus, the 

combination of increasing 

temperature and the episodic 

loading of labile organic 

material to the sediment 

surface during spring 

and summer likely drive 

the higher overall SMHg 

concentrations and MPP 

rates in this system. A more 

frequent temporal sediment 

sampling program (for 

example, monthly) would be 

required to further resolve 

the relative importance of 

temperature compared with 

organic loading on SMHg 

production dynamics in the 

Sinclair Inlet, see figure 6.

100

10

1

0.1

0.01

February June August

Sediment methylmercury production potential, in picograms per gram of dry weight Sediment methylmercury, in nanograms per gram of dry weight

11

10

9

8

7

6

5

4

3

2

1

0

B

A

2009

EXPLANATION

75th percentile

90th percentile

10th percentile

Data point

50th percentile

(median)

25th percentile

Interquartile

range 

Figure 6. (A) Sediment 

methylmercury concentration 

and (B) sediment 

methylmercury production 

potential rates at Sinclair Inlet 

stations, Washington, 2009.
II. Methylation Potential of Mercury in Sediments 17

Table 4. Kruskal-Wallis Rank Sum test results comparing combined data by month for all Sinclair Inlet stations, Kitsap County, Washington, 2009.[Parameter definitions are given in table 1. Bold values indicate statistically significant difference. The non-parametric Kruskal-Wallis Rank Sum test comparison of medians grouped by sampling season (month) [February (n=23), June (n=20) and August (n=27)] for all dates and all sites within both Bremerton Naval Complex and greater Sinclair Inlet (combined). The first quartile (25th percentile), median (50th percentile), third quartile (75th percentile) are shown along with all results from all mercury metric comparisons. Kruskal-Wallis Rank Sum: ***, significant differences between groupings at a probability level of p < 0.05; NS, non-significant differences. Only significant results for non-mercury metrics are shown. Abbreviations: d, day; °C, degrees Celsius; g/cm3, gram per cubic centimeter; μmol/g, micromole per gram; ng/g, nanogram per gram; n.c., not calculated due to the number of left-censored (less than) data; nanogram per gram; pg/g, picogram per gram; (pg/g)d, picogram per gram per day; <, less than]

Parameter (units)

February June August KruskalWallis 

Rank Sum

25th

percentile Median 75th percentile 25th percentile Median 75th percentile 25th percentile Median 75th percentile

Sediment mercury parameters

STHg (ng/g) dry weight 442 658 768 461 530 712 240 535 649 NSHg(II)R1 (ng/g) dry weight 0.23 0.33 0.40 0.23 0.35 0.46 0.28 0.43 0.58 NS%Hg(II)R1 (percentage of STHg) 0.04 0.05 0.06 0.05 0.06 0.08 0.06 0.09 0.13 ***SMHg (ng/g) dry weight 1.32 2.11 2.33 1.55 4.53 6.07 1.86 3.63 4.41 ***%SMHg (percentage of STHg) 0.24 0.30 0.44 0.41 0.79 1.12 0.57 0.83 1.13 ***kmeth1 (day) n.c. <0.00077 0.0057 <0.00082 0.0069 1.12 <0.0010 0.0049 0.017 ***MPP rate1 ([pg/g]/d) dry weight n.c. <0.014 1.83 <0.22 1.00 4.30 4.30 1.66 7.21 ***

Sediment non-mercury parameters

BD (g/cm3) wet weight 1.16 1.20 1.28 1.11 1.12 1.12 1.12 1.15 1.21 ***TRS (μmol/g) dry weight 182 271 300 196 262 262 109 197 243 ***% LOI (percentage of dry weight 

loss on ignition)

5.9 8.6 9.4 8.5 10.5 10.5 7.7 8.9 9.9 ***

Eh laboratory (millivolt) -6 29 84 -57 -6 0 -54 -17 3 ***Field temperature (°C) 7.5 7.6 8.0 11.3 11.5 11.5 13.9 14.1 14.6 ***

1Parameter contained left-censored (less than) data. Interquartile range and medians calculated using maximum likelihood estimate statistics (Helsel, 2005).
18 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Controls on Gross Methylmercury Production

Gross SMHg production in sediment is essentially a 

function of the activity of the Hg(II)-methylating microbial 

community and the availability of Hg(II) to that community, 

with many factors mediating both terms. To better 

understand which environmental factors play a significant 

role in the temporal and spatial variations for surface SMHg 

concentrations in the current study, the factors most influential 

in controlling the activity of the Hg(II)-methylating microbial 

community (as approximated by kmeth) and the availability 

of Hg(II) to that community (as approximated by SRHg 

concentration) were examined. Then how the calculated MPP 

rate, which is a function of both kmeth and SRHg, compares to 

SMHg concentration was examined to draw some inferences 

on the potential role of SMHg degradation, a pathway that was 

not directly examined in this study.

The best linear regression model describing kmeth, based 

on AIC analysis (comparing all possible models with as 

many as four explanatory variables), included the sediment 

variables, temperature (used for incubations), reductionoxidation (redox as Eh), and percentage of Fe(II)AE (ASSRtransformed). The resulting final regression equation, using 

data for all stations and sampling periods, accounted for 

37 percent of the measured variability in ln[kmeth] (fig. 7). 

The signs on the respective coefficients indicate that ln[kmeth] 

is negatively related to sediment Eh and positively related to 

incubation temperature and the percentage of Fe(II)AE/FeT, the 

latter reflecting the extent to which Fe(III) has been reduced 

to Fe(II), presumably largely by microbial iron reduction 

(Marvin-DiPasquale and others, 2014). Because concentration 

data (for example, Fe(II)AE and FeT data used to calculate 

the percentage of Fe(II)AE/FeT) are not microbial rate data, 

the positive correlation between kmeth and the percentage 

of Fe(II) AE/FeT cannot be inferred. As revealed by the best 

regression model, but rather indicates that kmeth is a direct 

function of microbial iron-reduction in the sites sampled for 

this study. However, we note that various other candidate 

models that included sediment TRS (a metric often associated 

with microbial sulfate reduction [Marvin-DiPasquale and 

others, 2014]) were ranked lower than the best model 

presented here. A recent study of freshwater wetland sediment 

(Marvin-DiPasquale and others, 2014) determined that kmeth

was positively correlated with sediment TRS concentration, 

as well as with net iron-reduction as assessed by the seasonal 

change in Fe(II)AE concentration between sampling dates. 

A similar AIC competitive model approach was used to 

develop a predictive function for ln-transformed sediment 

reactive inorganic mercury (ln[SRHg]). The resulting top 

model, using data for all stations and sampling events, 

calculated 45 percent of the measured variability in ln[SRHg] 

with sediment Eh and STHg (ln-transformed) as positive 

variables and sediment wet bulk density (ln-transformed) 

(ln[BD]) as a negative variable (fig. 7). The negative 

correlation between sediment SRHg and bulk density is 

because sediment bulk density is a function of both sediment 

organic content and sediment grain size. This is strongly 

negatively correlated with both r = -0.94 and r = -0.88, 

respectively (not shown) and because sediment SRHg 

exhibited weak positive correlations with sediment organic 

content (r = 0.46) and grain size (r = 0.50) for the complete 

dataset. Thus, sediment with a low bulk density tends to have 

high organic content and (or) a high percentage of fine grained 

particles (as opposed to sand).

Because SRHg concentrations were significantly higher 

in the BNC and Sinclair Inlet stations (combined), compared 

to the representative bays during August 2008 (table 2), 

another model was run that excluded the representative bays 

to determine if the same parameters caused the variation in 

SRHg concentration in the BNC and GSI stations, but not the 

representative bays. The resulting best-fit equation was less 

strong (model R2 = 0.37, not shown), with ln[SRHg] being 

a positive function of both sediment Eh and ln[BD]. Thus, 

sediment redox potential plays a consistent role in estimating 

both the activity of the Hg(II)-methylating community and the 

availability of Hg(II) to those microbes. However, as indicated 

–5 –4 –3 –2 –1 0 1

–4

–5

–3

–2

–1

0

1

Simulated sediment reactive mercury (SRHg)

concentrations*

ln[SRHg] simulated = 0.0039 * [Eh] + 0.354 *

ln[STHg] – 1.73 * In[BD] – 2.94 

Measured sediment reactive mercury concentrations*

R2 = 0.4501

Representative bays

Greater Sinclair Inlet

Bremerton naval complex

EXPLANATION

Figure 7. Simulated and measured sediment methylation rate 

constant from the sediment reactive mercury pool developed 

for representative bays, Bremerton naval complex and greater 

Sinclair Inlet, Kitsap County, Washington, 2008–09. (Modelsimulated values for ln-transformed sediment reactive mercury 

(ln[SRHg]) are based on the least squares multiple linear 

regression equation, with X variables: sediment redox (Eh), 

ln-transformed sediment total mercury concentration (ln[STHg] 

and ln-transformed sediment bulk density (ln[BD]).) Data 

include all samples collected from study area. The coefficient of 

determination (R2) for the regression is given.
II. Methylation Potential of Mercury in Sediments 19

by the multiple regression equations, 

as sediment Eh increases (sediment 

becomes more oxidized), SRHg 

increases (positive Eh coefficient in 

fig. 7). This partially explains why 

estimating SMHg concentration or 

MPP rates, based on sediment redox 

conditions alone, is difficult if not 

impossible.

The observation that the 

availability of Hg(II) for Hg(II)-

methylation can be affected by 

sediment redox conditions or redoxsensitive sediment constituents has 

been previously reported. In a study 

of eight diverse stream systems, 

sediment SRHg concentration was 

a positive function of sediment 

Fe(III)a concentration, whereas 

the percentage of SRHg was a 

negative function of sediment TRS 

concentration (Marvin‐DiPasquale, 

Lutz, and others, 2009). In studies 

of freshwater wetlands in the 

Central Valley of California, the 

percentage of sediment SRHg/STHg 

increased with increasing sediment 

Eh values (Marvin-DiPasquale, 

Alpers, and Fleck, 2009), whereas 

the concentration of sediment 

SRHg decreased with increasing 

sediment AVS (Marvin-DiPasquale, 

Alpers, and Fleck, 2009) and TRS 

(Marvin-DiPasquale and others, 

2014) concentrations. These 

previous reports are consistent with 

the current study, where sediment 

Eh was a positive term in the 

multivariable model describing 

SRHg concentration and where 

the percentage of SRHg/STHg 

decreased with increasing sediment 

AVS concentration during August 

2008 (R2 = 0.84, n=11), which was 

the only sampling event for which 

AVS was assayed (fig. 8). 

Because Hg(II) availability is 

an important factor that mediates 

SMHg production, and because 

SRHg concentration is partially a 

tac17-1117_fig08

R2 = 0.84

Sediment reactive inorganic mercury,

as a percentage of total mercury concentration 

Acid-volatile sulfur concentration,

in micromoles per gram of dry weight

0.3

0.4

0.1

0

0.2

0 10 20 30 40 50 60

Representative bays

Greater Sinclair Inlet

Bremerton naval complex

EXPLANATION

Figure 8. Sediment reactive inorganic mercury [SRHg] as a percentage 

of sediment total mercury concentration compared to acid-volatile sulfur 

concentration for all stations sampled in Sinclair Inlet, Kitsap County, Washington, 

during August 2008.

function of STHg concentration across all stations and dates, then the next question 

is “What controls STHg concentration across the study stations?” Using the AIC 

competitive model approach, the top ranking model included sediment bulk density 

only and explained 69 percent of the STHg concentration for all BNC and GSI sites 

(fig. 9). Sediment bulk density reflects (and is negatively correlated with) both sediment 

organic content and grain size. Lower ranking models did include these terms, and both 

terms have been cited as primary controls of STHg concentration (Horowitz, 1985; 

Scudder and others, 2009). When the representative bay sites were included, the top 

model used both sediment TRS and grain size (positive coefficients for both); however, 

the model residuals (unexplained error) were not normally distributed and the model 

was rejected. 

The top ranked model for SMHg concentration (ln-transformed) included 

sediment Eh and bulk density, both as negative terms, and explained 83 percent of 

the variability in SMHg across all BNC and GSI sites (fig. 10). When representative 

bay sites were included, the top model contained Eh and bulk density as negative 

terms, but also included STHg as a positive term (model R2 = 0.81, not shown). The 

negative correlation between SMHg and sediment Eh is consistent with high SMHg 

concentration at locations with high rates of microbial Fe(III)-reduction and sulfate 

reduction, which typically are more chemically reducing. The negative correlation 

between SMHg and sediment bulk density also is consistent with high MHg 

concentrations at locations with high organic content and (or) a high proportion of 

fine‐grained particulates. 
20 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

TAC_17-1117_fig09

4.5

3.5

5.5

6.5

7.5

Simulated sediment total mercury (STHg) concentration*

Measured sediment total mercury concentration*

ln[STHg] simulated = –4.54 * In[BD] + 6.99

R2 = 0.69

3.5 4.5 5.5 6.5 7.5

Greater Sinclair Inlet

Bremerton naval complex

EXPLANATION

Figure 9. Simulated and measured sediment total 

mercury concentration, Sinclair Inlet, Kitsap County, 

Washington. (Simulated values of sediment total 

mercury (ln-transformed) (ln[STHg]) are based on 

the least squares linear regression equation, with 

sediment wet bulk density (ln-transformed) (ln[BD]) 

as the X variable.) Data include all sampling periods 

and from all greater Sinclair Inlet and Bremerton naval 

complex stations.

0

–1

–2

1

2

3

Simulated sediment methylmercury (SMHg) concentration*

Measured sediment methylmercury concentration*

ln[SMHg] simulated –0.0045 * [Eh] – 4.58 * In[BD] + 1.80

–2 –1 0 1 2 3

R2 = 0.8336

Greater Sinclair Inlet

Bremerton naval complex

EXPLANATION

Figure 10. Simulated and measured sediment 

methylmercury concentration, Sinclair Inlet, Kitsap 

County, Washington. (Simulated sediment methylmercury 

concentrations (ln-transformed) (ln[SMHg]) are based 

on the least squares multiple linear regression equation, 

with X variables: sediment redox (Eh) and wet bulk 

density (ln-transformed) (ln[BD]).) Data are from all 

sampling periods and from all greater Sinclair Inlet and 

Bremerton naval complex stations.
II. Methylation Potential of Mercury in Sediments 21

TAC_17-1117_fig11

–6 –4 –2 0 2 4 6

Measured sediment methylmercury production potential rate*

0

–1

–2

–3

1

2

3

Simulated sediment methylmercury production potential (MPP) rate*

ln[MPP] simulated = 0.23 * [temp] + 0.70 * In[STHg] + 1.68 *

ASSR[%Fe(II)AE] – 8.65

R2 = 0.3525

Representative bays

Greater Sinclair Inlet

Bremerton naval complex

EXPLANATION

Figure 11. Simulated and measured sediment methylmercury production potential (MPP)

rates, Sinclair Inlet, Kitsap County, Washington. (Simulated values of sediment methylmercury 

production potential rates (ln-transformed) (ln[MPP]) are based on the least-squares multiple 

linear regression equation, with X variables: incubation temperature (temp), sediment total mercury 

(ln-transformed) (ln[STHg]), and percentage of Fe(II)AE/FeT (arcsine square root [ASSR] transformed) 

(ASSR[%Fe(II)AE]).) Data are from all sampling periods and all sites, including representative bays, 

greater Sinclair Inlet and the Bremerton naval complex stations.

The top ranked model for MPP rates (ln-transformed) 

included incubation temperature, STHg (ln-transformed), 

and the percentage of Fe(II)AE/FeT (ASSR transformed), 

with all three positive terms (model R2 = 0.35; fig. 11). When 

representative bays were excluded from the analysis, the 

top ranked model for MPP rates (ln-transformed) was only 

marginally improved (model R2 = 0.36), with X variables 

of incubation temperature and grain size (as percentage 

of FINES; ASSR transformed), both as positive terms. 

FeT (square root transformed) as a negative term also was 

used for all BNC and GSI sites combined (not shown). As 

for both MPP and SRHg top ranked models, STHg was a 

dominant explanatory variable only when representative 

bays were included in the analyses. This reflects the fact that 

median STHg concentrations were significantly lower for 

representative bays than all Sinclair Inlet stations (GSI and 

BNC combined, table 2), although, within the primary study 

area, median STHg concentrations also were significantly 

lower for GSI stations than BNC stations (table 3).

The best ranked model for kmeth, which included the 

percentage of Fe(III)AE/FeT as an explanatory variable, 

does not necessarily indicate the top model for MPP (all 

sites; fig. 11), as MPP is primarily driven by microbial ironreduction in the sites sampled for this study. The ambiguous 

or co-occurring roles of iron reduction or sulfate-reduction as 

the dominant microbial process that leads to methylation rates 

in marine estuaries has been noted in recent literature (Merritt 

and Amirbahman, 2008, 2009; Faganeli and others, 2012). 

Recent advances in the identification of the Hg(II)-methylation 

gene cluster (hgcAB) have demonstrated that hgcAB is 

abundant in marine sediment overall, and that contaminated 

marine sediment contains representatives of both iron- and 

sulfate-reducing bacteria that possess the hgcAB gene cluster 

(Podar and others, 2015).
22 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

III. Release of Mercury Species from 

Sediment to Water Column 

By A.J. Paulson, B. Carter, J.F. DeWild, and 

R.W. Sheibley

Methylation of inorganic mercury in sediment (see 

section, “Methylation Potential of Mercury in Sediments”) is 

the first step in the geochemical transformation and transport 

of MHg to the base of the pelagic food web in the water 

column. Then MHg must be released into sediment porewater 

and into the water column.

The maximum porewater FTHg and FMHg 

concentrations have been detected near the sediment-water 

interface of most open-water estuarine sediment (Gill and 

others, 1999; Choe and others, 2004; Muresan and others, 

2007; Schäfer and others, 2010). The environmental and 

geochemical factors controlling porewater FTHg and FMHg 

in estuarine sediments are unclear. Lower porewater FTHg 

and FMHg concentrations were measured in winter relative 

to concentrations measured during warmer periods in San 

Francisco Bay, California (Choe and others, 2004); Grado 

Lagoon, Adriatic Sea, Italy (Covelli and others, 2008); New 

York/New Jersey Harbor (Hammerschmidt and others, 

2008); and Thau Lagoon, France (Muresan and others, 2007). 

However, season does not appear to influence FTHg and 

FMHg concentrations from sediment in Long Island Sound 

(Hammerschmidt and Fitzgerald, 2008). In this study, FTHg 

and FMHg concentrations in porewaters from six stations 

in Sinclair Inlet (fig. 2) were measured in August 2008, and 

February, June, and August 2009. Additionally, porewater 

concentrations of FTHg and FMHg were measured in 

sediment from three Puget Sound representative bays (fig. 1) 

in August 2008.

Many estuarine chemists interpret porewater 

concentrations as in equilibrium with the solid phase of 

sediment. In this interpretation, porewater concentrations 

are controlled by solid-phase mercury concentrations and 

the organic matter of the sediment (Bloom and others, 1999; 

Hammerschmidt and others, 2008; Hollweg and others, 

2009). However, other investigators have reported that 

porewater FTHg and FMHg concentrations are controlled 

by other factors associated with the redox condition of the 

porewaters. Rothenberg and others (2008) concluded FMHg 

concentrations were related to dissolved porewater ferrous 

iron (Fe[II]) rather than organic matter or AVS in sediment. 

In contrast, Hollweg and others (2009) concluded that 

methylation and accumulation of FMHg in porewaters was 

favored when dissolved H2S concentrations ranged between 

3 and 320 μg/L and that FMHg in porewaters is partially 

controlled by the quality of organic matter in sediment. In this 

study, DOC was collected from the same composite sample 

as FTHg and FMHg samples. Additionally, the redox state 

of the sediment was categorized by the presence of reduced 

species (ammonia, Fe(II), and H2S), increased concentrations 

of filtered total iron and manganese, and the absence of nitrate 

(an oxidizer).

In order for FTHg and FMHg to be released to the water 

column, the rate of exchange of porewaters with the overlying 

water by diffusion or advection must be more rapid than 

geochemical processes that are removing these species from 

the aqueous phase at the sediment water interface. Similar 

to porewater concentrations, seasonal patterns in the flux 

of FMHg from porewater into the water column have been 

observed. Gill and others (1999) measured the maximum 

fluxes in spring and diurnal fluxes of FMHg from sediment in 

a shallow Texas bay. Hammerschmidt and Fitzgerald (2008) 

measured higher fluxes in August 2003 than in February 2004. 

Merritt and Amirbahman (2008) and Benoit and others (2006) 

suggest that the depth of oxygen penetration into the sediment 

is a major factor in controlling mercury fluxes from the 

sediment. In incubation core experiments, Mason and others 

(2006) detected maximum FMHg concentrations in overlying 

waters after 20 hours and maximum FTHg concentrations 

after 60 hours. In this study, fluxes of FMHg and FTHg were 

calculated from changes in concentrations in water overlying 

cores incubated in the dark, typically for 1 day.

Of particular note in the literature was the reference to 

demethylation at the sediment water interface. In one instance, 

high concentrations of FTHg and FMHg in porewater 

were measured in White Slough, San Francisco Bay-Delta, 

California, but the FMHg flux measured by flux chambers 

was low and the FTHg flux was high (Choe and others, 2004). 

The authors attributed this discrepancy to either oxygen 

penetration, lack of bioirrigation, or demethylation at the 

sediment-water interface.

The reproducibility of replicate flux measurements 

varied greatly among studies. The relative standard deviation 

of flux measurements from shipboard incubation studies 

with triplicate cores collected from New York/New Jersey 

Harbor generally were less than 10 percent. In contrast, 

highly variable fluxes, relative standard deviation exceeding 

100 percent in some cases, were measured by benthic flux 

chambers in a Texas bay (Gill and others, 1999), by incubation 

of cores from Baltimore Harbor, Maryland, and by diffusive 
III. Release of Mercury Species from Sediment to Water Column 23

flux calculations from porewater concentrations collected 

from Thau Lagoon, France. Because previous studies showed 

large variations of sediment properties in Sinclair Inlet over 

short distances (Paulson and others, 2012), triplicate core 

incubation studies were done at each of the six stations 

for the three seasons in 2009 after the initial pilot study in 

August 2008, in which only one core incubation experiment 

could be completed for each site. The STHg of sediment 

samples collected in greater Sinclair Inlet and OU B Marine 

in 2009 (fig. 1-1) were within the 95 percent confidence 

level of the correlation of STHg and total organic carbon of 

greater Sinclair Inlet sediment collected in 2007 (Paulson and 

others, 2010).

Porewater and Water Laboratory Methods

Porewater Sampling and Analysis

The collection methods for porewater, core incubation 

experiments, and tumbling core experiments are shown in 

figure 4. Details of sample collection, sample processing, and 

analytical results is available in Huffman and others (2012) 

along with extensive quality-assurance data and assessments. 

Intact and sealed duplicate cores from each station for 

determination of predominant redox conditions were placed 

in a nitrogen atmosphere, where the top 2-cm of sediment 

was packed into polypropylene centrifuge tubes and then 

centrifuged at 2,000 revolutions per minute (rpm) for 20 

minutes. Immediately after opening the centrifuge tube and 

isolating the unfiltered porewater in a syringe, sulfide and 

ferrous iron concentrations were measured at various dilutions 

with laboratory water purged with N2. After the spectrometric 

measurements (Huffman and others, 2012) were completed, 

porewater was filtered through a 0.45 μm, 25-mm, Millex®

disk filter into HDPE bottles for analysis of nutrients and FeT

and Mn.

After centrifugation, the supernatant in the 15 PFA tubes 

was filtered through multiple quartz fiber filters (QFF) held in 

a fluorocarbon polymer (FP) filtering tower into a 500-mL FP 

bottle in the vacuum desiccator in a laminar flow hood. About 

60-mL of filtered porewater was transferred to an amber prepacked bottle for analysis of DOC and total nitrogen by the 

NRP laboratory in Boulder, Colorado, and the remainder of 

the porewater in the FP bottle was acidified and analyzed for 

FTHg and FMHg at the WMRL.

Incubation Experiments

 Single cores at all nine stations sampled in August 

2008 were incubated at ambient temperatures for Sinclair 

Inlet in a manner similar to Hammerschmidt and Fitzgerald 

(2008). Incubation experiments generally were done on three 

cores (A, B, and C) at each of the six Sinclair Inlet stations 

during February, June, and August 2009. An initial sample of 

filtered overlying water (one-half to two-thirds of the volume 

of overlying water) was collected for analysis of FTHg and 

FMHg. Filtered, near-bottom water from their respective 

stations (replacement water) was added in a manner that 

minimized disturbance of the sediment-water interface. After 

stirring the overlying water for typically 1 day (2 days in some 

instances in August 2008), the process of filtering the water 

overlying the core, adding replacement water, and stirring in 

the incubator continued daily until the fourth day. All water 

added and removed, and the sediment held in the apparatus 

with no overlying water at the end of the experiment were 

weighed to account for the increase in mass of FTHg and 

FMHg in the overlying water needed to calculate release rates. 

Dissolved-oxygen concentrations of overlying water from a 

subset of cores were always greater than 1 mg/L measured 

using 0–1 mg/L R-7501 CHEMets (Chemetrics, Calverton, 

Virginia) and 0–12 mg/L R-7512 CHEMets immediately after 

the sample was collected on the last day.

Fluxes of FTHg and FMHg were calculated based on 

the change in concentrations in the test, overlying water 

during each period between observations over the time of the 

experiment (dc/dt in nanograms per liter per day):

 / * dc Release flux V A = dt tot , (1)

where

dc/dt is the rate of change in concentration in the 

test overlying water, in nanograms per liter 

per day;

Vtot is the total volume, in liters, of water 

overlying the core; and

A is the cross sectional area of the inside of the 

core liner (33.3 square centimeters).
24 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

The flux rate was calculated between each measurement by 

substituting:

∆ ∆= c t d dt / – / (CC t – ) ∆ , (2)

where

Cd is the concentrations of FTHg or FMHg, in 

nanograms per liter, in the overlying water 

on the day of the measurement at the end 

of the elapsed time (Δt); and 

Cd-t is the calculated concentration of the test 

overlying water immediately after taking 

the prior measurement and filling the 

core liner with replacement water and is 

calculated by mass balance as 

C C V C Vr V d t p p r − * * / ( ) ( ) tot   =  + , (3)

where

Cp is the concentrations of FTHg or FMHg, 

in the water overlying the core liner 

after withdrawing water for the prior 

measurement;

Cr is generally the average of the FTHg and 

FMHg concentrations of near-bottom water 

collected in the field and the replacement 

water in PETG bottles stored in the 

refrigerator for 3 days; and

Vp and Vr are the volume, in liters, of overlying water 

remaining after the prior measurement, and 

the volume of replacement water added, 

respectively, where Vtot is equal to the sum 

of Vp and Vr.

Substituting equation 3 into equation 2, and then substituting 

Δc/Δt in equation 2 for dc/dt in equation 1 results in: 

Release flux ( / ) /

( * ) [( * ) ( * )

ng m d

C V d tot C V p p C V r r

2 

 

     

 / *   area t . (4)

If Cd was less than 0.04 ng/L or (Cd – Cd–t) was less than 

0.07 ng/L (1.66 times the reporting level), the release flux was 

reported as not detected. Negative release fluxes indicate that 

FTHg and FMHg were removed from the overlying water by 

the sediment or the apparatus.

One control-core incubation experiment, with Purelab®

Ultra water in a clean core liner sealed at the bottom with a 

machined polycarbonate plug, was done in August 2008 and 

four control-core incubation experiments were done in 2009. 

For the August 2008 control core-incubation experiments, no 

initial measurements of Cp and Cr were made. After 2 days, 

FTHg and FMHg concentrations were 0.71 and 0.10 ng/L, 

respectively. Setting Cd and Cp to zero results in maximum 

control fluxes of 5 and 32 (ng/m2)/d for FMHg and FTHg, 

respectively, for the 2008 control incubation experiments.

For the four control incubation experiments completed 

in 2009, water in the core liner was measured 15 minutes 

after filling the apparatus with the Purelab® Ultra water. After 

3 days of stirring, FTHg concentrations ranged from 0.12 to 

0.71 ng/L. Using the FTHg concentration at 15 minutes for 

Cp, the median FTHg release flux for the control experiment 

was 2.5 (ng/cm2)/d and ranged from 25 to +19 (ng/m2)/d. 

The FTHg release flux of the 2009 control experiments for 

qualification of data was set at the maximum release flux of 

19 (ng/m2)/d. After 3 days, FMHg concentrations ranged 

from less than 0.04 to 0.15 ng/L. The median FMHg release 

rate was 2.5 (ng/m2)/d and ranged from not detectable to 

6 (ng/ m2)/d. For consistency with 2008 data, 5 (ng/m2)/d was 

used for qualifying FMHg release fluxes. Only in six instances 

when the value of (Cd – Cd–t) was 0.07 ng/L or greater, did the 

calculated FMHg release flux of the 2009 control cores exceed 

5 (ng/m2)/d.

Tumbling Core Experiments

On the evening of sample collection, station water 

was added to fill the 500-mL FP beakers containing the 

sediment for the tumbling core experiment. After 15 minutes 

tumbling end-over-end on a 1-m diameter wheel rotating at 7 

revolutions per minute (rpm) for 15 minutes to allow mixing 

of porewater and added water, the sediment slurry was allowed 

to settle and the supernatant was filtered through multiple 

QFF filters. The beaker was filled to the rim with replacement 

water from the station, sealed, bagged, and secured to the 

rotating wheel at room temperature. After 2 days, the slurry 

was allowed to settle and was filtered through multiple QFF 

filters. Similar to the incubation experiment, the mass of the 

slurry was recorded at every step, and solids in the beaker 

during the experiment were dried at 60 °C to calculate the 

ratio of solids to liquid. The ratio of solids to liquid during the 

tumbling experiment of Sinclair Inlet samples ranged from 

0.09 to 0.2 kg/L, and the ratio was near 1 kg/L for the sandy 
III. Release of Mercury Species from Sediment to Water Column 25

CZ sediment. In February, dissolved oxygen concentrations 

in overlying water from three tumbling cores were greater 

than 1 mg/L using R-7512 CHEMets immediately after 

withdrawing a sample for FTHg and FMHg. In August 2009, 

the overlying water was processed for redox-sensitive species 

described for the porewater sampling.

The release rate was calculated as follows:

( 3 ) ( 15 ) ( ) ( )

Release rate, in nanograms per gram 

* * – – * / solids CV C V CV d tot m p r r

  =  , (5)

where C15m, C3d, and Cr are the concentrations of FTHg or 

FMHg, in nanograms per liter after 15 minutes of mixing, 

on day three after 2 days of tumbling, and the replacement 

water, respectively. Volumes (Vtot, Vp, and Vr in L), and solids 

(in grams dry weight) are as indicated for the incubation 

experiment. During 2 days of tumbling Purelab® Ultra water 

in a clean beaker without sediment, FTHg increased from 0.22 

to 0.26 ng/L.

Laboratory Analyses

The UW Chemical Oceanography Laboratory analyzed 

nutrients for all porewaters and marine water column samples 

following the methods of Armstrong and others (1967), and 

Slawyk and MacIsaac (1972), Bernhardt and Wilhelms (1967). 

Samples for particulate total carbon and total nitrogen were 

collected on 0.45-mm pore size, 25-mm diameter, baked glassfiber filters in a PFA filter holder and measured using U.S. 

Environmental Protection Agency (EPA) method 440.0 (U.S 

Environmental Protection Agency, 1997). The filtrate (125 

mL) was collected into a baked, glass bottle and acidified with 

HCl for analysis of DOC (see “Methylmercury Accumulation 

in the Base of an Estuarine Food Web”). Concentrations of 

total suspended soldis were measured gravimetrically (6-place 

balance (precision of 0.001 mg) before and after filtering water 

through a NucleporeTM 0.4-μm pore size, 47-mm diameter, 

polycarbonate filter. After August 2008, total suspended 

solids measurements were taken from every bottle in which 

particulate Hg measurements were made. 

All marine water samples were filtered through a QFF 

held in a filtering tower into a 500-mL FP bottles in the 

vacuum desiccator in a laminar flow hood. The filtrates were 

collected in FP bottles and acidified, and some QFF filter 

were placed in FP petri dishes and frozen. Total mercury in 

filtered water (FTHg) in all other samples was measured by 

the WMRL using the EPA method 1631, revision E (U.S. 

Environmental Protection Agency, 2002) that includes 

oxidation, purge and trap, desorption, and cold vapor atomic 

fluorescence spectrometry (CVAFS). Methylmercury in 

filtered water (FMHg) was first distilled to reduce matrix 

affects, then ethylated and trapped onto reaction traps (DeWild 

and others, 2002). The mercury species were thermally 

desorbed, separated by gas chromatography, and analyzed 

by CVAFS. The reporting levels for FTHg and FMHg are 

0.04 ng/L, 

Analysis of total mercury in suspended solids (PTHg) are 

prepared by room-temperature acid digestion and oxidation 

with aqua regia followed by overnight heating (50 °C) in a 

5-percent (w/v) bromine monochloride solution to ensure 

complete oxidation (Olund and others, 2004). The solution 

was analyzed by CVAFS. The method detection limit of PTHg 

is 0.059 ng of mercury per filter. Analysis of methylmercury 

in suspended solids (PMHg) was prepared by extraction with 

potassium bromide, copper sulfate, and methylene chloride 

(DeWild and others, 2004). The methylmercury was solvent 

extracted with methylene chloride and back extracted in water 

by evaporation of the methylene chloride. The methylmercury 

was then analyzed in a manner similar to the ethylation, 

chromatographic separation, and analysis by CVAFS. The 

reporting level for PTHg is 0.01 ng of mercury per filter. More 

detailed methods and quality assurance measures are reported 

in Huffman and others (2012).

Mercury Concentrations in Porewater

The biochemical accumulation of FTHg and FMHg 

in the porewater of sediment is necessary to release FTHg 

and FMHg to the water column of Sinclair Inlet. FTHg and 

FMHg are released to the water column when the porewater 

is transported out of the sediment by physical processes. In 

August 2008 (table 5, fig. 12), high FTHg concentrations 

(35.9 –59.8 ng/L) and high FMHg concentrations 

(20– 28 ng/L) were measured in porewater from the two 

stations with highly reducing sediment (SI-PO and LB) and 

from one station with moderately reducing sediment (SIIN). For the other two representative bays (HH and BI) and 

the other greater Sinclair Inlet station (SI-OUT) that were 

categorized as moderately reducing, FTHg concentrations 

ranged from 3.19 to 10.8 ng/L and FMHg concentrations 

ranged from 0.08 to 1.30 ng/L. For the three OU B 

Marine stations that were characterized as reducing, FTHg 

concentrations in porewater ranged from 2.23 to 6.21 ng/L and 

FMHg concentrations ranged from 0.09 to 0.36 ng/L.
26 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Table 5. Predominant redox conditions, dissolved organic carbon and mercury concentrations in porewater, and releases during core 

incubation and tumbling experiments from sediment collected from Sinclair Inlet, Kitsap County, Washington, 2008 and 2009.

[Station: BI, Budd Inlet; BNC, Bremerton naval complex; CZ, convergence zone; HH, Holmes Harbor; LB, Liberty Bay; OU B Marine, Operable Unit B 

Marine; SI-IN, Sinclair Inlet-inner; SI-OUT, Sinclair Inlet-outer; SI-PO, Sinclair Inlet-Port Orchard; p value, probability of difference between representative 

bays and Sinclair Inlet including OU B Marine. Area: SI, Sinclair Inlet; RB, representative bay. Redox: HR, highly reducing; MR, moderately reducing; R, 

reducing; WR, weakly reducing. N: Number of core incubation experiments. Abbreviations: mg/L, milligram per liter; ng/L, nanogram per liter; mg/kg, 

milligram per kilogram; [ng/m2]/d, nanogram per square meter per day; NA, not analyzed; nd, not detected]

Station Area Redox

Porewater concentrations

N

Median flux from 

sediment ([ng/m2]/d)

Release during

tumbling (mg/kg)

Dissolved 

organic 

carbon

(mg/L)

Filtered 

total 

mercury 

(ng/L)

Filtered 

methylmercury 

(ng/L)

Filtered 

total 

mercury

Filtered 

methylmercury

Filtered 

total 

mercury

Filtered 

methylmercury

August 2008

SI-OUT SI MR 7.6 9.97 0.08 1 nd nd NA NA

SI-PO SI HR 4.0 35.9 20 1 491 353 NA NA

SI-IN SI MR 8.7 59.8 28 1 338 5 NA NA

BNC-52 OUB Marine R 3.8 6.21 0.09 1 nd nd NA NA

BNC-39 OUB Marine R 4.3 2.86 0.2 1 nd nd NA NA

BNC-71 OUB Marine R 3.8 2.23 0.36 1 E 32 nd NA NA

HH RB MR 4.5 10.8 1.30 1 48 nd NA NA

LB RB HR 8.9 58.1 26 1 NA 329 NA NA

BI RB MR 4.9 3.19 0.52 1 108 nd NA NA

p value 0.3 0.41 0.45

February 2009

SI-OUT SI R 2.5 8.62 0.04 3 67 nd 0.007 0.000

SI-PO SI WR 3.3 6.18 0.07 3 22 nd 0.011 0.000

SI-IN SI R 2.5 3.1 0.1 3 nd nd 0.004 0.001

BNC-39 OU B Marine MR 4.1 16.9 0.14 3 41 nd NA 0.001

BNC-60 OU B Marine R 2.8 2.72 0.24 3 73 nd NA 0.000

BNC-71 OU B Marine MR 3.1 6.99 0.09 3 nd nd 0.008 0.005

CZ SI NA NA NA NA NA NA NA 0.070 0.001

June 2009

SI-OUT SI R 5.5 7.87 0.58 3 nd nd -0.0053 0.0026

SI-PO SI R 7.5 28.9 18 2 75 128 NA NA

SI-IN SI R 6.6 47.3 20 3 1,462 1,079 1.635 0.929

BNC-39 OUB Marine MR 4.9 4.47 0.63 3 25 6 0.143 0.098

BNC-60 OUB Marine R 7.8 41.7 15 3 333 372 0.563 0.269

BNC-71 OUB Marine MR 4.6 22.7 1.5 3 100 8 0.454 0.349

CZ SI NA NA NA NA NA NA 0.093 0.007

August 2009

SI-OUT SI WR 3.6 10.9 0.1 94 nd -0.001 0.002

SI-PO SI WR 5.4 4.56 2.8 247 264 0.60 0.44

SI-IN SI WR 2.7 2.95 0.36 200 212 0.07 0.06

BNC-39 OUB Marine MR 4.5 5.89 0.46 nd nd 0.02 0.01

BNC-60 OUB Marine R 5.0 5.07 1.1 26 nd 0.21 0.12

BNC-71 OUB Marine MR 5.6 16.2 1.4 nd nd 0.43 0.15

CZ SI NA NA NA NA NA NA 0.02 0.00
III. Release of Mercury Species from Sediment to Water Column 27

tac17-1117_fig12

 ▪

 ▪

▪ ▪

 ▪

 ▪

BNC-52

60

40

20

0

60

40

20

0

20

15

10

5

0

20

15

10

5

0

Mercury concentration, in nanograms per liter

August 2008

February 2009

June 2009

August 2009

BNC-60 BNC-39 BNC-71 Outer

OU B Marine Port Orchard

Greater Sinclair Inlet

Inner

Budd Inlet

Liberty Bay

Representative bays

Holmes Harbor

ND

Reduction-oxidation category

Different reduction-oxidation 

state for duplicate core

Filtered total mercury

Filtered methylmercury

EXPLANATION

Highly reducing

Reducing

Moderately reducing

Weakly reducing

Figure 12. Filtered total mercury and filtered methylmercury concentrations in porewater for four 

seasonal sampling periods, Sinclair Inlet, Kitsap County, Washington, between August 2008 and 

August 2009. Representative bays Holmes Harbor (HH), Liberty Bay (LB) and Budd Inlet (BI) stations 

were sampled only in August 2009. Only one methylmercury value was not detectable. Note 

changes in the range of mercury concentrations (y-axis) among seasons.
28 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

In February 2009, FMHg concentrations in porewater 

were low in OU B Marine (0.09–0.14 ng/L) and greater 

Sinclair Inlet (<0.04–0.1 ng/L) stations. No clear trend 

between FTHg concentrations and redox state was 

observed during February 2009. Considerable overlap of 

FTHg concentrations was noted across redox states for all 

stations (reducing [2.72–8.62 ng/L], moderately reducing 

[6.99–16.9 ng/L], and weakly reducing [6.18 ng/L]). The 

highest FTHg concentrations (28.9–47.3 ng/L) and FMHg 

concentrations (15–20 ng/L) in porewater measured in June 

2009 were collected from stations with reducing sediment 

(the most reducing redox condition for this sampling 

period). Similar to August 2008, a high FTHg concentration 

(22.7 ng/L) was measured in porewater in June 2009 from a 

station with moderately reducing sediment (BNC-71). Unlike 

porewater from SI-IN collected in August 2008, low FMHg 

(1.5 ng/L) was measured in moderately reducing sediment 

from BNC-71 in June 2009. Reducing porewater from 

SI-OUT contained lower concentrations of FTHg (7.87 ng/L) 

and FMHg (0.58 ng/L).

Sediment collected in August 2009 was less reducing 

than that collected at the same stations in August 2008. 

The FMHg concentrations (average 1.0 ng/L; range 0.1 to 

2.8 ng/L) in porewater collected in August 2009 was lower 

than corresponding concentrations (average 9.7 ng/L; range 

0.08 to 28 ng/L) collected in August 2008 from five common 

stations. Unlike the results from August 2008, high FTHg 

concentrations were measured in porewater at stations with 

moderately reducing sediment (5.89–16.2 ng/L) and weakly 

reducing sediment (10.9 ng/L).

Redox State of Porewater

The method for assessing the predominant redox 

conditions of the sediments was based on the presence or 

absence of electron acceptors (nitrate) or the presence of 

reduced byproducts (H2S; and dissolved ammonia, Fe, and 

Mn). The predominant redox state of a station is not an exact 

measurement because of vertical and horizontal heterogeneity. 

The top 2 cm of sediment likely contains more than one of 

the redox states, so species (for example, nitrate and sulfide) 

that are not chemically stable in the presence of each other 

may nevertheless co-exist at low concentrations during the 

short time that the 2-cm interval of porewater is extracted. 

Therefore, the identified predominant redox state at a station 

at a particular time may not be entirely consistent with the 

presence or absence of all chemical species. If duplicate 

measurements were available for a sampling station, they were 

included in all statistics that involved categorical summaries 

such as comparison between seasons or between location 

groups to fully represent the site-specific variability.

Because concentrations of nitrate and sulfide were critical 

to categorizing the predominant redox state of porewater, 

threshold values of nitrate and sulfide were established for 

each redox category. During August 2008, and February and 

June 2009, nitrate concentrations were relatively low and 

less than three times the interquartile range greater than the 

means for the sampling event (0.018, 0.007, and 0.009 mg/L 

as nitrogen, respectively). During August 2009, one of 

two replicate cores from each of the three greater Sinclair 

Inlet stations contained substantially more nitrate (0.107 to 

0.365 μg/L as N). The threshold nitrate concentration for the 

other nine samples was 0.105 mg/L as nitrogen. When the 

spectrometric reading for sulfide concentrations was adjusted 

for the dilution factor of the sample, a natural break in the data 

was measured at 0.26 mg/L (8 micromolar) and was used as 

threshold for the presence of sulfide. This sulfide concentration 

is similar to sulfide concentrations critical to methylation 

processes (Hollweg and others, 2009). 

Detectable concentrations of dissolved Fe and Mn 

also were measured in most porewaters, but provided little 

information on identifying predominant redox state. Dissolved 

Mn was detected in all porewater samples at concentrations 

ranging from the reporting limit (20 μg/L) to 2,840 μg/L. 

After adjusting for dilution, reporting levels for dissolved 

Fe generally were 400 or 800 μg/L, which were too high to 

determine the onset of ferric iron reduction. 

Four sediment redox states were established. Sediment 

cores containing nitrate, dissolved Mn but no sulfide greater 

than the threshold were classified as “weakly reducing.” For 

the “Moderately reducing” category, cores did not contain 

nitrate, did contain dissolved Mn, and did not contain sulfide 

greater than threshold values. “Reducing” cores did not 

contain nitrate, did contain dissolved Mn and sulfide greater 

than threshold values. When sulfide concentrations generated 

by sulfate reduction become sufficiently high, Fe and Mn 

sulfides begin to precipitate, leading to decreased Fe and 

Mn concentrations. “Highly reducing” cores contain (1) 

high levels of sulfide (greater than 2.4 mg/L overage range 

of the measurement at a dilution of 1:4), (2) nitrate and Fe 

concentrations less than the reporting level, and (3) Mn 

concentrations less than or equal to 100 μg/L. For 19 of 24 

duplicate cores, the redox state of the duplicate cores was the 

same (Huffman and others, 2012). If the redox state of the 

duplicate cores differed, the redox state of the station was set 

at the more reducing state. 
III. Release of Mercury Species from Sediment to Water Column 29

In August 2008, highly reducing sediments were detected 

only at stations SI-PO and LB (table 5). Two representative 

bays (HH and BI) and two greater Sinclair Inlet stations 

(-IN and -OUT) were classified as moderately reducing. The 

three BNC stations were classified as reducing due to sulfide 

concentrations greater than the threshold of 0.26 mg/L. 

During 2009, the redox state of the greater Sinclair 

Inlet stations changed throughout the year. In February, the 

predominant redox state of the SI-PO station was “weakly 

reducing,” whereas it was “reducing” at stations SI-IN and 

SI-OUT. In June, the predominant redox state at the three 

greater Sinclair stations was reducing, and weakly reducing 

in August 2009. The predominant redox state at the OU B 

Marine stations remained unchanged during 2009; which was 

reducing at BNC-60 and moderately reducing at BNC-39 and 

BNC-71.

Fluxes of Total Mercury and Methylmercury 

from Sediment

Fluxes of FTHg and FMHg occur through the physical 

and biological processes that transport porewaters across the 

sediment-water interface into the water column. The stirring 

conditions of the core incubation experiments for this study 

reflect the low tidal-energy conditions often observed in 

Sinclair Inlet (Gartner and others, 1998). For each season, 

there were similarities in the trends of the release fluxes of 

FTHg and FMHg with the trends in porewater concentrations; 

however, there also were notable exception to the trends.

Similar to the high FTHg and FMHg porewater 

concentrations in August 2008, high fluxes of FTHg in August 

2008 (table 5) were measured from cores collected from the 

two stations with highly reducing sediment, SI-PO and LB. 

Flux of FMHg in August. Fluxes of FMHg in August 2008 

could not be determined due to analytical difficulty. Similarly, 

a relatively high flux of FTHg was measured from moderately 

reducing sediment from SI-IN that also contained high 

porewater concentration of FTHg. However, the flux of FMHg 

from the SI-IN core was at the detection limit of the flux 

measurement (5 [ng/m2]/d), even though FMHg porewater 

concentrations (28 ng/L) from SI-IN were the highest 

measured during the study. Fluxes of FMHg were less than 

the detection limit for the other six cores. The FTHg fluxes 

of 48 and 108 (ng/m2)/d were measured from the two other 

representative bays (HH and BI, respectively).

For the six SI stations sampled in February, June, and 

August 2009, the fluxes of FTHg and FMHg from triplicate 

cores were measured over 3 days. The variability of FMHg 

fluxes among the triplicate cores from BNC-60 collected in 

June 2009 were typical of the daily fluxes results detected 

among other triplicate cores. The FMHg fluxes were 

significantly different between the three cores during the first 

day of the experiment (day 0–1; fig. 13). Fluxes near the end 

of the experiment for cores A and C were less than fluxes on 

the first day, but the FMHg flux of core B was negative on the 

second day. The FTHg and FMHg fluxes generally decreased 

as experiments progressed in the laboratory. Because the 

flux measured between sample collection and 1 day later 

probably best reflects natural conditions, the first acceptable 

measurement from each replicate core is described and 

presented in table 1-1.

Similar to the low porewater concentration of FMHg, 

median FMHg fluxes for all six stations in February 2009 

(median concentrations of the triplicate cores are presented 

table 5) were less than the detection limit. Only 1 of 16 FMHg 

flux measurements was detectable (6.4 [ng/m2]/d) at rates 

only slightly greater than the fluxes from the control cores 

(table 1-1). Median fluxes of FTHg from all stations were low 

(<75 [ng/m2]/d) and dependent on redox conditions. 

In June 2009, high fluxes of FTHg (table 1-1) were 

released from reducing sediment from two of three cores from 

SI-IN (median 1,462 [ng/m2]/d; table 5) and from two of three 

cores from BNC-60 (median 333 [ng/m2]/d). Lower fluxes 

of FTHg were measured from reducing sediment from SI-PO 

(median 75 [ng/m2]/d) and moderately reducing sediment 

from BNC-71 (median 100 [ng/m2]/d). Fluxes of FMHg 

greater than 100 (ng/m2)/d were measured from five of the 

eight cores from Sinclair Inlet stations with reducing sediment 

(SI-PO, SI-IN, BNC-60 in table 1-1). 

The median flux of FTHg from weakly reducing sediment 

collected in August 2009 from greater Sinclair Inlet ranged 

from 94 to 247 (ng/m2)/d (table 5). The fluxes of FMHg from 

SI-PO and SI-IN were similar to FTHg, but no FMHg was 

released from SI-OUT sediment. The median flux of FTHg 

from sediment from OU B Marine stations was less than 

30 (ng/m2)/d and a detectable flux of FMHg was measured 

from only one of nine cores from OU B Marine (table 1-1). 

Overall, the measured fluxes were highly variable.
30 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

TAC_17-1117_fig13

1,400

1,200

1,000

800

600

400

200

0

0–1

1–2

Days

2–3

A

B

Core

C

Methylmercury, in nanograms per square meter per day

*

Figure 13. Daily fluxes of filtered methylmercury from triplicate experiments during 3-day coreincubation experiments from Bremerton naval complex (station BNC-60), Sinclair Inlet, Kitsap 

County, Washington, June 2009. (*Indicates a negative value, -200 [ng/m2]/d, for this core and time 

point.) 

Release of Total and Methylmercury from 

Sediment During Tumbling

High-energy windstorms occasionally occur in the 

Sinclair Inlet area. To assess release of mercury when 

sediment is suspended into the water column during such high 

energy storms, water from each station was added to the top 

2-cm of sediment and the slurry was mixed by slow endover-end tumbling for 2 days. The supernatant of the slurry 

at the beginning of the experiment represents the weighted 

average of sediment porewater and site water; FTHg and 

FMHg concentrations generally were less than 9 and 1 ng/L, 

respectively. Decomposition of organic matter in the slurry in 

the sealed beaker could change the redox conditions over the 

2-day experiment. In August 2009, concentrations of redoxsensitive species were measured in the slurry at the end of the 

experiment. Except for the slurry containing SI-PO sediment 

where significant concentrations of sulfide were measured, the 

slurries were categorized as moderately reducing.

Little THg or MHg (less than 0.07 ng/g) was released 

from sediment collected during February 2009 (table 5). 

Sediment collected in June 2009 released the most FTHg and 

FMHg after tumbling, with four of the six sediment slurries 

releasing 0.1 ng/g or more (table 5). The slurry of sediment 

from SI-IN tumbled for 2 days release of 1.635 ng/g of FTHg. 

In August 2009, greater than 0.1 ng/g of THg was released 

from three of the seven samples. Little THg or MHg was 

released from sediment from the open water stations CZ and 

SI-OUT. When more than 0.1 ng/g THg was released, the ratio 

of MHg release to THg release was generally greater than 

50 percent.

Mercury Concentrations in Water Column

The concentrations of mercury in the water column reflect 

a baseline marine concentration with inputs from sediment 

and near-surface terrestrial and atmospheric sources that are 

moderated by biological processes and turbulent mixing by 
III. Release of Mercury Species from Sediment to Water Column 31

tidal forces (Paulson and others, 2012). The rates of inputs 

from sedimentary and terrestrial sources and biotransformation 

processes in the water column were manifested if their relative 

rates were much faster than physical processes that tend to 

mask these sources and processes. Examining the differences 

between near-surface and near-bottom concentrations 

provides insight into the relative rates of physical mixing and 

other factors. When sufficient ancillary data are available, 

the probable causes of exceptionally high concentrations 

(outliers) of any of the four mercury species (FTHg, FMHg, 

PTHg, and PMHg) were examined. Although some seasonal 

differences in surface-water concentrations are apparent in 

graphs in this section, seasonal biological processes that affect 

Hg species are described in detail in section, “Methylmercury 

Accumulation in the Base of an Estuarine Food Web.” 

Water Column Mercury in Sinclair Inlet 

Compared to Representative Bays

In August 2008, there was no significant difference (p

values in table 1-2) in FTHg concentration in both nearsurface or near-bottom waters between Sinclair Inlet and 

the representative bays (fig. 14). The range of the FTHg 

concentrations in representative bays (0.13–0.42 ng/L) 

encompassed the range for Sinclair Inlet (about 0.25–

0.40 ng/L) for the August 2008 sampling period. As expected 

for the representative bays, Holmes Harbor had the lowest 

FTHg concentrations (0.13–0.16 ng/L), whereas FTHg 

concentrations in Budd Inlet were slightly higher than 

Holmes Harbor (0.20–0.24 ng/L) and FTHg concentrations 

in Liberty Bay were about 0.42 ng/L. The ranges of FMHg 

concentrations in the representative bays were also similar 

to those in Sinclair Inlet for near-surface and near-bottom 

samples (fig. 15).

Concentrations of THg of suspended solids (table 1-3) 

from the representative bays ranged from 0.017 to 0.270 μg/g 

(fig. 16). Concentrations of THg of suspended solids in 

Sinclair Inlet in August 2008 (0.066–0.392 μg/g for nearsurface solids and from 0.122 to 1.025 μg/g for near-bottom 

solids from Paulson and others, 2012) were not significantly 

different from those of representative bays (p= 0.25 and 

0.14, respectively, table 1-2). No significant differences (p = 

0.29 and 0.55, respectively; table 1-2) were noted between 

concentrations of MHg of suspended solids (table 4) between 

the representative bays and Sinclair Inlet for near-surface and 

near-bottom waters (fig. 17), respectively.

Importance of Sedimentary Sources of 

Mercury Species

Statistical examination of differences between 

near-surface and near-bottom waters provides an overall 

perspective on the general importance of sedimentary sources 

relative to near-surface terrestrial and atmospheric sources. 

For each of the four seasonal sampling periods, differences 

between near-surface and near-bottom concentrations in 

Sinclair Inlet were examined for each parameter. This seasonal 

grouping by water column layers emphasizes the overall 

stratification of the water column in Sinclair Inlet rather than 

differences between layers at a specific site. This grouping 

is appropriate because of the large tidal excursion in Sinclair 

Inlet (kilometers in scale) and the difference in time of 

collection between the near-surface and near-bottom samples 

(several hours in some instances).

Despite significant differences (table 1-2) in some 

biogeochemical constituents (that is, filtered ammonia 

[fig. 1-2A], filtered nitrate plus nitrite, [fig. 1-2C], filtered 

orthophosphate [fig. 1-2B], and filtered silicate [fig. 1-2D]), 

few significant differences in mercury concentrations between 

near-surface and near-bottom concentrations were noted in 

Sinclair Inlet (figs. 14–17, table 1-2). Only 3 of 12 seasonal 

comparisons of FTHg, PTHg and THg of suspended solids 

(mass of particulate total mercury per mass of suspended 

solids) between near-surface and near-bottom concentrations 

were significantly different. The FTHg concentrations in 

the near-surface layer of Sinclair Inlet in August 2009 were 

significantly higher than concentrations in the near-bottom 

layer. In contrast, both concentrations of PTHg and THg of 

solids (particulate total mercury of a mass per volume of 

water and per mass of suspended solids, respectively) in the 

near-bottom layer during June 2009 were significantly higher 

than concentrations in the near-surface layer. Both these 

instances of significant vertical differences were associated 

with water column layers containing exceptionally high 

concentrations. There were no significant differences (table 2) 

in concentrations of FMHg (fig. 15) and of MHg of suspended 

solids (fig. 17) between near-surface and near-bottom 

layers. Only the suspended solid concentration of MHg (in 

nanograms per liter) for the August 2009 sampling period was 

significantly different (p = 0.04 in table 2).
32 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig14

0.0

0.2

0.4

0.6

0.8

1.0

Representative

bays

Sinclair Inlet

August

2008

February

2009

June

2009

August

2009

SI-OUT

BNC-39

SI-IN

SI-IN SI-OUT

SI-PO

Filtered total mercury, in nanograms per liter

Near-surface

Near-bottom

EXPLANATION

Value outside the 90th percentile

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

Figure 14. Filtered total mercury in near-surface and near-bottom water in 

representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair Inlet, 

Kitsap County, Washington, August 2008–August 2009.
III. Release of Mercury Species from Sediment to Water Column 33

tac17-1117_fig15

SI-PO

BNC-71

SI-IN

0.0

0.2

0.4

0.6

0.8

1.0

Representative

bays

Sinclair Inlet

August

2008

February

2009

June

2009

August

2009

Filtered methylmercury, in nanograms per liter

Near-surface

Near-bottom

Single valve

EXPLANATION

Value outside the 90th percentile

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

SI-IN

Figure 15. Filtered methylmercury in near-surface and near-bottom water in 

representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair Inlet, 

Kitsap County, Washington, August 2008–August 2009. (In February and June 2009, one 

near-surface filtered methylmercury concentration slightly above the detection limit were 

measured and are represented as unlabeled squares.)
34 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig16

BNC-71

0.0

0.2

0.4

0.6

1.0

0.8

1.2

Representative

bays

Sinclair Inlet

August

2008

February

2009

June

2009

August

2009

Particulate total mercury of suspended solids, in mircograms per gram

BNC-39

EXPLANATION

Value outside the 10th or 90th 

percentile

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

BNC-39

Near-surface

Near-bottom

Figure 16. Total mercury of suspended solids in near-surface and near-bottom water in 

representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair Inlet, Kitsap 

County, Washington, August 2008–August 2009.
III. Release of Mercury Species from Sediment to Water Column 35

tac17-1117_fig17

BNC-39

BNC-39

SI-IN

BNC-60

0.0

0.05

0.10

0.15

0.20

Representative

bays

Sinclair Inlet

August

2008

February

2009

June

2009

August

2009

Particulate methylmercury of suspended solids, in mircograms per gram

EXPLANATION

Value outside the 90th percentile

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

SI-IN

Near-surface

Near-bottom

Figure 17. Methylmercury of suspended solids in near-surface and near-bottom water in 

representative bays (Holmes Harbor, Liberty Bay, and Budd Inlet) and Sinclair Inlet, Kitsap 

County, Washington, August 2008–August 2009.
36 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Isolated instances of exceptionally high or low 

concentrations of a mercury species at a specific site 

(table 1-5) provide insights into local biogeochemical 

processes affecting a mercury source. Increased concentrations 

of FTHg and FMHg in surface water were measured at 

station SI-IN in August 2009 (figs. 14–15). Although the 

highest orthophosphate concentration of this seasonal dataset 

(0.17 mg/L as phosphate) was associated with near-surface 

layer sample in August 2009 SI-IN (fig. 1-2B), the silicate 

concentration was not elevated (fig. 1-2D) and nitrate plus 

nitrite essentially was depleted (0.002 mg/L; fig. 1-2C). 

These observations indicate that the FTHg and FMHg did 

not originate from a sedimentary source. Exceptionally 

high concentrations of MHg of suspended solids (0.083 and 

0.119 μg/g in table 1-4), total suspended solids (average of 

13.1 in table 1-4), an atomic ratio of carbon to nitrogen of 

the suspended solids (9.33 in table 1-4) and chlorophyll a 

(150 μg/L in Huffman and others, 2012) were measured at 

SI-IN during August 2009. Thus, the phytoplankton bloom 

conditions in August 2009 accumulated carbon, nitrogen, 

and phosphorus, THg, and MHg in the particulate phase. 

As the phytoplankton died, they released these species into 

aqueous or colloidal phases. Nitrate is limiting and is reused 

to produce new phytoplankton, which leaves an accumulation 

of filterable orthophosphate. Colloidal THg and MHg from 

the phytoplankton breakdown debris that can pass through 

the pores of the quartz-fiber filter probably are responsible for 

the elevated FTHg and FMHg concentrations during bloom 

conditions at SI-IN during August 2009.

The second highest FTHg concentration of the seasonal 

Sinclair Inlet set (0.84 ng/L) was measured in near-bottom 

waters of SI-PO in June 2009 and was associated with 

an exceptionally high FMHg concentration (0.58 ng/L). 

Ammonia, orthophosphate, silicate, and manganese are species 

that are released to the water column of Puget Sound as a 

result of diagenetic processes occurring in sediment (Paulson 

and others, 1988; Brandes and Devol, 1997; Kuwabara and 

others, 2009). Near-bottom waters collected from station 

SI-PO in June 2009 contained high concentrations of filtered 

ammonia, orthophosphate, and silicate (table 1-2 and fig. 1-2), 

and the highest concentrations of manganese detected during 

the study (fig. 1-3). These elevated near-bottom concentrations 

of diagenetic species and FMHg suggest enhanced release of 

porewaters to the water column. The most likely conditions 

are (1) submarine discharge of groundwater that advects 

porewater with high concentrations of nutrients, manganese, 

FTHg, and FMHg deeper in the sediment column to the 

sediment-water interface and into the water column, or (2) 

enhanced exchange of porewater and near-bottom water by 

biological or physical processes.

Data from a CTD cast at SI-PO collected in early May as 

part of a special study on THg concentrations of solids show 

evidence of submarine groundwater discharge (fig. 1-4). In 

the bottom 0.3 m of the water column, salinity decreases from 

29.3 to 28.7 PSU. The large decrease in dissolved-oxygen 

concentrations near the sediment-water interface indicates the 

upward movement of oxygen-depleted porewaters into the 

water column. The consistency of the turbidity values (not 

shown) in the bottom 0.3 m indicates that the CTD sensors 

had not entered the nepheloid layer above the sedimentwater interface. Activity of invertebrates in the sediment can 

irrigate sediment, and thus enhance water exchange across 

the sediment-water interface. The low fluxes of FTHg from 

the incubation experiments with SI-PO cores collected during 

June 2009 argues against enhanced biological mixing.

Correlations Between Porewater, Fluxes, and 

Water Column Constituents

Examination of correlations between FTHg, FMHg 

and DOC concentrations is procedurally consistent because 

these analyses were done on aliquots from the same bottle of 

porewater composited from the supernatant from the 15 tubes 

of centrifuge sediment from multiple box cores collected at 

each station. The statistical correlation between FTHg and 

DOC in 27 porewater samples collected between August 

2008 and August 2009 is highly influenced by samples with 

reducing and highly reducing sediment (fig. 18). The nonparametric correlation coefficient (r) (table 6) between FTHg 

and DOC in reducing and highly reducing sediment (sulfide 

concentrations greater than 0.26 mg/L, 11 samples) is greater 

than the r for all 27 samples. The non-parametric correlation of 

between FTHg and DOC in weakly and moderately reducing 

sediment (sulfide concentrations less than 0.26 mg/L, 14 

samples) was not significant for both non-parametric tests.

The FMHg concentrations were correlated with DOC 

(fig. 19) regardless of redox state of the sediments. However, 

the correlation coefficients generally were higher for reducing 

and highly reducing sediment than for weakly and moderately 

reducing sediment. Similar to FTHg, the percentage of 

FTHg in the FMHg form was not significant for weakly and 

moderately reducing sediment. 

Non-parametric multi-comparison Tukey tests were done 

to examine the correlations between FTHg, FMHg and DOC 

and respective redox states (table 7) because the redox state 

is a categorical variable; samples for redox constituents were 

collected from different sediment cores than samples collected 

for mercury and DOC. The only significant difference in 

FTHg concentrations among redox state was the difference 
III. Release of Mercury Species from Sediment to Water Column 37

tac17-1117_fig18

70

60

50

40

30

20

10

0

Filtered total mercury, in nanograms per liter

0 2 4

Dissolved organic carbon, in milligrams per liter

6 8 10

SI-IN (August 2008)

SI-PO (August 2008)

SI-OUT (August 2008)

Parametric regression

FTHg = –18.5 + 7.0 DOC

R = 0.75, p < 0.001 BNC-60 (June 2009)

LB (August 2008)

SI-PO (June 2009)

SI-Inner (June 2009)

Reducing and highly reducing

Weakly and moderately reducing

Different results from duplicate cores

EXPLANATION

Figure 18. Filtered total mercury concentrations in porewaters compared to 

dissolved organic carbon concentrations grouped by two redox conditions, 

Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009. Sample 

with filtered total mercury concentrations greater than 25 ng/L and samples 

separated from the regression line are labeled.

between moderately and weakly reducing sediment (Tukey 

multi-comparison category B) and highly reducing sediment 

(Tukey multi-comparison category A). The absence of other 

significant differences was a result of the large range in FTHg 

concentrations for the 11 samples of reducing sediment (2.23–

47.3 ng/g) and 10 samples of moderately reducing sediment 

(3.19–59.8 ng/L). The FMHg concentrations in the two highly 

reducing sediment samples were significantly greater than 

samples of other redox states. Similar to FTHg, the variation 

in FMHg concentrations in reducing sediment (<0.04–20 

ng/g) and in moderately reducing sediment (0.08–28 ng/L) 

resulted in no significant differences between these two redox 

categories. There were no differences in percentage of mercury 

in the MHg form or DOC among redox states.
38 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Table 6. Non-parametric regression and parametric correlation statistics of filtered total mercury, filtered methylmercury, 

and percentage of methylmercury in porewaters compared to dissolved organic carbon categorized by porewater sulfide 

concentration, Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.

[Bold values indicate a statistically significant difference. Abbreviations: BNC, Bremerton naval complex; mg/L, milligram per liter; ng/mg, nanogram 

per milligram; p, probability; r; correlation coefficient; >, greater than; < , less than; –, parametric regression data are not given for data sets in which both 

non-parametric tests indicate the correlation is not significant]

Sulfide

grouping

Number of 

samples

Non-parametric Parametric

Spearman Rho Kendall Tau b Pearson

r p r p r p Intercept Slope

(ng/mg)

Filtered total mercury

All samples 127 0.58 0.002 0.42 0.003 0.75 < 0.0001 -18.5 7.0

Sulfide > 0.26 mg/L2 11 0.69 0.019 0.55 0.019 0.79 0.003 -16.2 7.3

Sulfide < 0.26 mg/L3 14 0.28 0.329 0.21 0.297 0.67 0.008 – –

Filtered methylmercury

All samples 27 0.71 < 0.0001 0.58 < 0.0001 0.77 < 0.0001 -12.0 3.5

Sulfide > 0.26 mg/L 11 0.76 0.006 0.61 0.010 0.76 0.007 -9.2 3.5

Sulfide < 0.26 mg/L 14 0.59 0.027 0.50 0.014 0.69 0.006 12.1 3.1

Methylmercury (percent)

All samples 27 0.58 0.003 0.42 0.003 0.60 0.008 -14.0 6.6

Sulfide > 0.26 mg/L 11 0.69 0.022 0.55 0.019 0.69 0.02 -9.9 6.9

Sulfide < 0.26 mg/L 14 0.36 0.200 0.26 0.100 0.48 0.08 – –

1Stations BNC-39 and -71 in August 2008 are not included in either category because sulfide concentrations from duplicate cores produced different 

sulfide categories.

2Reducing and highly reducing conditions.

3Moderately and weakly reducing conditions.
III. Release of Mercury Species from Sediment to Water Column 39

tac17-1117_fig19

e

SI-IN (August 2008)

SI-PO (August 2008)

Parametric regression

FMHg = –12.0 + 3.5 DOC

R = 0.77, p < 0.001

BNC-60 (June 2009)

LB (August 2008)

SI-PO (June 2009)

SI-IN (June 2009)

30

20

10

0

Filtered methylmercury, in nanograms per liter

0 2 4

Dissolved organic carbon, in milligrams per liter

6 8 10

SI-OUT (August 2008)

Reducing and highly reducing

Weakly and moderately reducing

Different results from duplicate cores

EXPLANATION

Figure 19. Filtered methylmercury concentrations in porewaters compared 

to dissolved organic carbon concentrations grouped by two redox conditions, 

Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009. Sample 

with filtered methylmercury concentrations greater than 10 ng/L and samples 

separated from the regression line are labeled. 
40 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Table 7. Statistics for filtered total mercury, methylmercury, percentage of methylmercury, and dissolved organic carbon categorized 

by redox state, Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.

[Abbreviations: mg/L, milligram per liter; ng/L, nanogram per liter; <, less than]

Redox

condition

Number of 

samples Mean Standarddeviation Median Range

Tukey multicomparison 

category

Filtered total mercury (ng/L)

Highly reducing 2 47 15.7 47 35.9–58.1 A

Reducing 11 14.23 16.77 6.21 2.23–47.3 AB

Moderately reducing 10 15.69 16.69 10.39 3.19–59.8 B

Weakly reducing 4 6.15 3.43 5.37 2.95–10.9 B

Filtered methylmercury (ng/L)

Highly reducing 2 23 4.24 23 20–26 A

Reducing 11 5.06 8.18 0.36 <0.04–20 B

Moderately reducing 10 3.41 8.66 0.58 0.08–28 B

Weakly reducing 4 0.83 1.32 0.23 0.07–2.8 B

Methylmercury (percent)

Highly reducing 2 50.2 7.7 50.2 44.8–55.7 A

Reducing 11 18.8 20 8.8 0.5–62.3 A

Moderately reducing 10 11.5 13.6 8.2 0.8–46.8 A

Weakly reducing 4 18.9 28.8 6.7 0.9–61.4 A

Dissolved organic carbon (mg/L)

Highly reducing 2 6.5 3.5 6.5 4–8.9 A

Reducing 11 4.7 1.9 4.3 2.5–7.8 A

Moderately reducing 10 5.3 1.7 4.8 3.1–8.7 A

Weakly reducing 4 3.8 1.2 3.5 2.7–5.4 A

Comparison of Porewater Concentrations 

with Fluxes

Sediment geochemical principles indicate that the 

diffusive flux of any constituent out of the sediment is 

proportional to its vertical concentration gradient below 

the sediment-water interface (Li and Gregory, 1974). The 

proportionality constant in an abiotic system is based on 

sediment texture and the molecular diffusivity of the element 

of interest. Organisms living in and on the sediment can 

inhibit or enhance the abiotic flux by a number of physical 

and biological mechanisms. The data from this project were 

examined for consistency with these principles by plotting 

the fluxes of FTHg and FMHg from the core incubation 

experiments versus the differences in concentrations between 

porewater and bottom waters at each station. This estimate of 

gradient is the only measure available from the data collected 

and may be imprecise estimate of the actual gradient at the 

sediment-water interface.

Generally, reducing and highly reducing sediments with 

gradients in FMHg concentration greater than 10 ng/L (SI-PO, 

August 2008 and June 2009; LB, August 2008; SI-IN, June 

2009; BNC-60, June 2009) resulted in fluxes of FMHg greater 

than 200 (ng/m2)/d (fig. 20). The anomalous low release 

from moderately reducing sediment from SI-IN collected 

in August 2008 was near the detection limit in this single 

core incubation experiment, even though sediment from this 

station had the highest porewater FMHg concentrations of 

the study. As indicated by the variation in the first-day release 

on core incubation experiment triplicates and the variation 

over the course of the experiment (fig. 13) among replicate 

cores, the variation in flux from multiple cores for a site can 

be high. If triplicate core incubation experiments were done 

in August 2008, similar high, but variable, fluxes may have 

been observed, which suggests that porewater FMHg may 

be demethylated at the sediment-water interface by benthic 

infauna (Choe and others, 2004).

In contrast, FMHg fluxes greater than 200 (ng/m2)/d 

were measured in August 2009 in weakly reducing sediment 

collected from SI-PO and SI-IN in which porewater FMHg 

concentrations were less than 3 ng/L. Benthic infauna may 

enhance or inhibit the transfer of FTHg and FMHg from the 

sediment to the water column (Benoit and others, 2009). The 

lack of correlations between fluxes measured during core 

incubation experiments and the porewater concentrations 

in the top 2 cm may be a result of significant horizontal 

heterogeneity in sediments. Additionally, 2-cm section may 

not provide the vertical resolution to measure the actual 

concentration gradient at the sediment-water interface.
III. Release of Mercury Species from Sediment to Water Column 41

0 10 20 30

LB

2,000

1,500

1,000

500

0

–500

Filtered methylmercury flux, in nanograms per square meters per day

Filtered methylmercury gradient, in nanograms per liter

Highly reducing

Reducing

Moderately reducing

Weakly reducing

Minimum and maximum of triplicate

 core incubation experiments

EXPLANATION

SI-PO (August 2009)

BNC-60

(June 2009) SI-PO

(June 2009)

SI-IN

(June 2009)

SI-PO

(August 2008)

SI-IN (August 2009)

SI-IN (August 2008)

TAC_17-1117_fig20

Figure 20. Fluxes of filtered methylmercury compared to gradients of 

filtered methylmercury between porewater and the water column, Sinclair 

Inlet, Kitsap County, Washington, August 2008–August 2009.

Factors Controlling Porewater 

Concentrations and Fluxes

The most conspicuous feature of the seasonal data is the 

near total absence of MHg in porewater, bottom water and 

surface water in Sinclair Inlet during winter (February 2009, 

figs. 12 and 15). In August 2008, June 2009, and August 2009, 

methylation in sediment of some stations led to increased 

concentrations of FMHg in porewaters (fig. 12). Based on 

these observations, analyses of variance (ANOVA) were done 

using models based on categorical variables of time (four 

seasonal events) and space (table 8). In the first model in 

which general area (greater Sinclair Inlet and OU B Marine) 

was the spatial categorical variable, the only significant 

relation for season was DOC. Season or area was not 

correlated with any mercury concentration or release value.

In the second spatial model, the six individual stations 

were used as values for the categorical spatial variable. In 

this model, season was a significant parameter for porewater 

DOC, FMHg concentrations and percentage of FMHg, 

whereas site was only a significant variable for percentage of 

FMHg. Releases during the core incubation and tumbling core 

experiments were not correlated with either season or site. 

Because DOC was always correlated with season in 

these temporal-spatial models, season may be a surrogate for 

the geochemical variable DOC. Because the correlation of 

porewater FTHg and FMHg with DOC differed by porewater 

sulfide concentrations category (table 6), a model based on 

the geochemical parameters DOC and the redox condition of 

each of the 27 sample collections (n = 27) was developed. The 

FTHg and FMHg in porewater were correlated with both DOC 

and redox; and the geochemical-based event model produced 

the best simulations. Although the percentage of mercury 

in the MHg form was correlated with DOC and redox, the 

geochemical based event model did not produce the best 

prediction. The fluxes measured during the core incubation 

experiments were not correlated with either DOC or redox 

porewater conditions, and the standard error of the predictions 

were only slightly lower than those of the site model. 

The geochemical model was not applied to the tumbling 

experiment because DOC and redox conditions during 

tumbling were considerably different from isolated cores.
42 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Table 8. Analysis of variance of porewater concentrations and releases of mercury from sediment in Sinclair Inlet, Kitsap 

County, Washington, August 2008–August 2009.

[Bold values indicate statistically significant difference. Parameter: DOC, dissolved organic carbon; FTHg, filtered total mercury; FMHg, filtered 

methylmercury; %FMHg, percentage of filtered methylmercury. f: model a function (what is “a”)?. p: probability of being correlation to DOC. 

Abbreviations: redox, reduction-oxidation; >, greater than; –, not measured]

Parameter

f (season, area) f (season, site) f (DOC, redox)

p 

(season)

p

(area)

Standard

error

p

(season)

p

(site)

Standard

error

Dissolved 

organic 

carbon

Redox Standard

error

DOC 0.01 0.44 1.45 0.021 0.95 1.59 – – –

FTHg 0.11 0.22 14.9 0.11 0.43 15 0.0004 0.034 10.1

FMHg 0.09 0.07 7.3 0.04 0.069 6.5 > 0.0001 0.0005 4.2

%FMHg 0.15 0.09 18.8 0.019 0.0033 12.9 0.0005 0.008 13.5

Flux FTHg 0.23 0.18 190 0.19 0.15 230 0.3 0.07 220

Flux FMHg 0.37 0.34 240 0.38 0.32 180 0.18 0.11 160

Tum FTHg 0.6 0.76 0.48 0.74 0.64 0.50 – – –

Tum FMHg 0.11 0.82 0.21 0.11 0.45 0.21 – – –
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 43

IV. Methylmercury Accumulation 

in the Base of an Estuarine Food Web

By P.W. Moran, A.R. Stewart, J. Toft, J. Cordell, and 

A.J. Paulson

Concern over mercury concentrations in finfish from 

Sinclair Inlet has prompted a more detailed investigation into 

the patterns of methylmercury accumulation in the base of the 

marine food web in and around Sinclair Inlet. As recognized 

by the research community, and more recently by U.S. 

Environmental Protection Agency (2010) with regard to new 

methylmercury water-quality criteria, bioaccumulation from 

water into increasingly higher trophic levels exerts a primary 

control over the concentration of methylmercury in fish and 

shellfish tissue (see section, “Introduction and Methods”). The 

largest of these bioaccumulation steps occurs in the lowest 

level of the food web (Pickhardt and others, 2005; Mason 

and others, 2006; Stewart and others, 2008). In response to 

Task 3 in section, “Purpose and Scope,” a monthly sampling 

regime was implemented to evaluate the temporal and spatial 

variability in methylmercury accumulation in suspended solids 

and zooplankton of Sinclair Inlet.

The food webs of the neritic zone of Puget Sound 

embayments are characterized as a classic phytoplanktonto-zooplankton-to-planktivore system with strong seasonal 

swings in abundance, and, relative to adjacent benthic 

or littoral food webs, tightly coupled temporal relations 

(Simenstad and others, 1979; Strickland, 1983; Olson and 

others, 2001). Calanoid copepods have been repeatedly 

shown to be a dominant consumer of phytoplankton in Puget 

Sound (Simenstad and others, 1979; Dagg and others, 1998; 

Olson and others, 2001); however, they also consume ciliates 

in the heterotrophic cycle of bacteria or ultraphytoplankton 

to ciliates (Cowlishaw, 2004), and some larger types of 

calanoid copepods are known to be predatory on smaller 

copepods. The life histories of copepods common to Puget 

Sound also show (1) extensive use of diapause periods 

followed by growth and reproduction periods coincident 

with phytoplankton bloom seasons, and (2) more limited, 

low-density persistence at depth across years (Osgood and 

Frost, 1994). The dual characteristics of both responsive to 

changes in primary production and yet more persistent, and 

their key role as the primary food for forage and juvenile fish 

(Simenstad and others, 1977; Simenstad and others, 1979; 

Penttila, 2007), suggests that copepods make a key sentinel 

species for monitoring bioaccumulative compounds such as 

mercury. A one-time spatial sampling in Sinclair Inlet and 

three representative bays (fig. 1) was completed in August 

2008. From August 2008 through August 2009, four stations 

in Sinclair Inlet were sampled monthly (fig. 5 and, specifically, 

sites Sinclair Inlet, SI-IN, SI-PO, BNC-52, and the CZ). 

Methods for Food Web Study

Water-Column Sample Processing and 

Laboratory Analysis

Dissolved organic carbon in filtrate from the TPCN 

methods (see section, “Release of Mercury Species from 

Sediment to Water Column”) was analyzed at the NWQL 

using ultraviolet (UV)-promoted persulfate oxidation and 

infrared spectrometry methods (U.S. Environmental Protection 

Agency, 1997). The 25-mm diameter filters holding suspended 

solids were subsampled with a 6.3-mm diameter hole-punch 

(two per 25-mm filter). To prepare suspended solids samples 

for isotopic analysis, sub-sampled filters were moistened 

with Milli-Q® water (approximately 100 microliters [μL]) 

and fumed overnight in a desiccator with concentrated HCl 

to remove carbonates. The following morning, subsamples 

were dried in an oven at 60 °C for 1 hour and then cooled in a 

desiccator. The subsamples from each 25-mm filter were then 

loaded into tin capsules and held in desiccators until analyzed 

for carbon and nitrogen isotopes.

Duplicate chlorophyll samples were filtered within 

2 hours of collection onto precombusted, 25-mm-diameter, 

glass fiber filters (nominal pore size of 0.7 μm), frozen on 

dry ice, and then either stored briefly at -20 °C, or placed in 

longer term storage at -80 °C. Chlorophyll a concentrations 

were determined fluorometrically from the 25-mm filters 

(Holm‐Hansen and Riemann, 1978) within 6 months of 

collection by the USGS NRP laboratory in Menlo Park, 

California. The reporting level was 0.1 μg/L; values less than 

1.0 μg/L were qualified as estimated. 

Zooplankton Sample Collection

Live zooplankton samples returned to the laboratory 

and were immediately sorted into whole community and 

species-specific zooplankton samples for mercury analyses. 

Zooplankton sorting and taxonomy was done by the Wetland 

Ecosystem Team in the School of Aquatic and Fishery Science 

at the University of Washington. Species and bulk zooplankton 

identification and sorting was completed by trained technicians 

under low power magnification.

Material from the 0.1 mm mesh net was used for 

obtaining “whole community” samples. For these community 

samples, zooplankton were first separated from phytoplankton 
44 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

as much as possible by swirling aliquots of the sample in a 

petri dish, causing heavier diatoms to separate from the lighter 

zooplankton. Representative samples of the zooplankton 

then were moved using plastic forceps onto 0.073-mm mesh 

pieces of net and concentrated for freezing and later analysis. 

When approximately 10–12 milligrams (mg) wet weight of 

organisms were obtained, they were placed on a small sheet 

of filter paper (about 5 × 8 mm), put into labeled 1.5-mL 

centrifuge tubes, and frozen for later analysis.

The 0.253-mm mesh samples were used to obtain 

individuals of larger species for analysis. The most abundant 

organism was used (for example, calanoid copepods [Acartia

sp.]). If possible, the same species was used across all 

sampling stations for a given date, but this was not often 

possible because of low numbers of the selected organism 

at one or two stations. Individual species were collected by 

pipet from the sample jar, placed on 0.073-mm mesh pieces 

of net, and individually sorted until approximately 10–12 mg 

wet weight of a given species was obtained; species were then 

frozen using the same method as for the whole community 

samples. Vertical hauls that had been fixed in the field were 

quantitatively subsampled if necessary in the laboratory, using 

a Hensen’s Stempel pipette to obtain approximately 200 of the 

most abundant taxon. Plankton taxa were enumerated and all 

adult copepods were identified to genus or species.

Composite samples of zooplankton representing either 

the whole community or individual species were freeze-dried 

(using a Virtis Genesis 35EL) prior to processing for stable 

isotope and MHg analysis. Zooplankton samples for stable 

isotopes were weighed using a microbalance (1–2 mg) and 

packed into tin capsules.

Stable isotopic analysis of the suspended solids and 

zooplankton was completed on frozen samples by the 

University of California at Davis Stable Isotope Facility 

(University of California, 2011) following established 

methods. Suspended solids samples were evaluated in 

triplicate; zooplankton replication depended on organism 

availability, but ranged from two to five replicates per 

sampling station and time. Isotope ratios of carbon (δ13C) 

and nitrogen (and δ15N) of the suspended solids and 

zooplankton were determined using a Europa Scientific Hydra 

20/20 continuous flow isotope ratio mass spectrometer in 

conjunction with a Europa ANCA-SL elemental analyzer 

to convert organic carbon and nitrogen into carbon dioxide

and N2 gas. Nitrogen isotope samples were standardized 

against N2 in air as follows: 

δ

15N ‰ = [(Rsample/Rstandard) – 1] × 1,000, (6)

where

R (ratio)equals 15N/14N, and

‰ is parts per thousand.

Likewise, δ13C (R = 13C/12C) or (carbon with atomic weight 

of 13/carbon with an atomic weight of 12) with a similar 

formula as above is reported here as a standardized carbon 

isotope ratio against the international standard, Vienna Pee 

Dee Belemnite.

Methylmercury concentrations in whole community and 

species-specific zooplankton samples were determined by 

the USGS Mercury Laboratory in Menlo Park, California. 

Subsamples of freeze-dried zooplankton (whole community 

and species specific) were weighed by using a microbalance 

(1–2 mg) into 1.5 mL acid-washed centrifuge tubes. 

Methylmercury was extracted from the samples by adding 

200 μL of 25-percent (weight/volume) KOH/methanol 

to the centrifuge tubes. The sample and extractant were 

homogenized by vortexing and then samples were heated in 

an oven at 60 °C for 4 hours, with samples being vortexed 

once after 2 hours. Digested samples were cooled and 

stored frozen (-80 °C) until analysis. On the day of analysis, 

samples were thawed, and 8 μL of low-DOC laboratory water 

was added to each centrifuge tube. The samples were then 

homogenized by vortexing and then centrifuged at 4,000 rpm 

for 15 minutes to compress the remaining solid material into 

a pellet. A 0.15-mL aliquot of the extractant was subsampled 

into a trace-metal-clean glass I-ChemTM vial along with a drop 

(about 25 μL) of a silica-based antifoaming agent. The vial 

was nearly filled with laboratory water, the pH was adjusted 

to 4.9 by using acetate buffer, and an ethylated agent (sodium 

tetraethyl borate) was added. The vial then was topped off 

with laboratory water, capped with a septa screw-top cap, and 

shaken. MHg was converted in the vial to volatile methylethyl-mercury, which was subsequently analyzed by MERX 

Automated Methyl Mercury Analytical System (Brooks Rand 

Laboratories, Seattle, Washington) by using CVAFS detection. 

Each batch of analytical samples was accompanied with 

analysis of the minimum following quality assurance samples: 

(1) two certified reference materials—National Research 

Council Canada DORM-2 Dogfish muscle and NIST-Research 

Material 2890 Mussel Tissue, (2) one matrix spike sample, 

(3) one analytical duplicate, and (4) one method blank. 

Mercury analysis in water and filtered particulates (solids) 

is discussed in section “Release of Mercury Species from 

Sediment to Water Column.” 

Results of Food Web Study

Spatial Sampling in August 2008

With the exception of chlorophyll, the synoptic sampling 

during August 2008 showed water column constituents 

associated with carbon to be relatively consistent at all 

stations (fig. 21). Chlorophyll, in contrast, was variable across 

stations, and had significantly higher concentrations (one-way 
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 45

TAC_17-1117_fig21

Dissolved organic carbon, filtered particulate organic carbon, and

total suspended solids concentration, in milligrams per liter

BI BNC-52 CZ HH LB SI-IN SI-OUT SI-PO

Characteristics of Puget Sound embayments

16

12

8

4

0

Chlorophyll a, in micrograms per liter

A

C

B

B B

B

B

10

8

6

4

2

0

Chlorophyll a

Dissolved organic carbon

Particulate organic carbon

Total suspended solids

EXPLANATION

Note: Letters indicate significance

(p-value < 0.05) in ANOVA for Chlorophyll,

with Tukey’s adjustment for multiple

comparison, F-statistic 102, p < 0.00)

Figure 21. Concentrations of selected surface-water constituents associated with carbon, Puget 

Sound, Washington, August 2008. Stations include three representative bays (Budd Inlet [BI]; Holmes 

Harbor [HH]; and Liberty Bay [LB]); and five greater Sinclair Inlet (SI) stations: (OU B- Marine station 

(Bremerton naval complex [BNC-52]), CZ (convergence zone), Inner [SI-IN], Outer [SI-OUT], and Port 

Orchard [SI-PO]. No samples were collected at SI-PO in August 2008.

ANOVA with Tukey’s adjustment for multiple comparisons, 

F-statistic of 102, p-value <0.01) at BI, followed by the CZ, 

as compared to other study sites in the large-scale spatial 

comparison of August 2008. Intense spring and summer algal 

blooms observed during this study period also were described 

in Winters and others (1975), with phytoplankton production 

rates of 460–470 grams of carbon per square centimeter per 

year reported in central Puget Sound. The largest chlorophyll 

values in August 2008 were from BI and were consistent with 

the warmest temperature (22.3 °C) and highest orthophosphate 

concentration (0.135 mg/L) (see Huffman and others, 2012, 

appendix G), relative to all stations sampled in August 2008 

(n=7). Similarly, total particulate carbon (table 1-4) and 

particulate organic carbon concentrations (fig. 21) were 

highest in Budd Inlet during that month. However, nitrate, 

as nitrate plus nitrite, did not differ substantially between the 

seven sites in August 2008 (see Huffman and others, 2012, 

appendix G). Dissolved organic carbon was unremarkable at 

Budd Inlet and fairly consistent across all stations in August 

2008, ranging from less than 0.4 to 0.9 mg/L. Zooplankton 

tissue concentrations of MHg were not significantly different 

across the stations in August 2008 (Scheffe post hoc test, 

ANOVA, F-ratio 24.2, p-value < 0.01; fig. 22).
46 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

BI BNC-52 CZ HH LB SI-IN SI-OUT

Puget Sound embayments

80

60

40

20

0

Methylmercury concentrations in bulk zooplankton tissue,

in nanograms per gram dry weight

TAC_17-1117_fig22

Figure 22. Concentrations of methylmercury in bulk zooplankton tissue, Sinclair 

Inlet, Kitsap County, Washington, August 2008. Stations include three representative 

bays (Budd Inlet [BI], Holmes Harbor [HH], and Liberty Bay [LB]); greater Sinclair 

Inlet (SI) stations (OU B- Marine station (Bremerton naval complex [BNC-52], CZ, 

convergence zone Inner [SI-IN], and Outer [SI-OUT]).

Despite a lack of significant difference in MHg 

concentrations in zooplankton tissue collected from the seven 

stations in August 2008, stable isotope signatures of δ15N 

and δ13C indicate significant spatial differences in carbon and 

nitrogen sources (fig. 23). In particular, the greatest relative 

degree of separation is for Budd Inlet and Holmes Harbor, 

which are spatially the most distant stations. Intermediate 

between them, primarily in 13C values, are the SI stations 

and the adjacent LB station. Although the concentrations 

from the CZ and LB stations plot near the SI stations, they 

are somewhat distinctive in this group of five. This grouping 

is consistent with spatial differences and the understanding 

of tidal exchange dynamics in the area. That is, the location 

of the CZ just outside the mouth of Sinclair Inlet, at the 

intersection of Port Washington Narrows and Rich Passage 

(figs. 1 and 5), has considerably greater tidal exchange than 

the three more western SI stations (Gartner and others, 1998; 

Wang and Richter, 1999).

As demonstrated in the studies of Kidd and others 

(1995) and Stewart and others (2008), there often is a relation 

between the 15N value of an organism and its methylmercury 

concentration. This relation is somewhat evident, although not 

statistically significant, in samples collected in August 2008 

(fig. 23); (spearman rank correlation of -0.61, p-value =0.34). 
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 47

–22 –21 –20 –19 –18 –17 –16 –15

Zooplankton, in δ13C (per mil) average and standard deviation

Zooplankton, in δ15N (per mil) average and standard deviation

12

11

10

9

8

Holmes Harbor (1.7)

Budd Inlet (9.1)

SI-Inner (30.9) Convergence zone (16.3)

Liberty Bay (37.2)

SI-Outer

(19.6)

BNC-52

(18.0)

(1.7)

tac17-1117_fig23

EXPLANATION

Average methylmercury concentration,

in nanograms per gram dry weight

Figure 23. Comparison of stable nitrogen isotope (δ15N) and stable carbon isotope of (δ13C)

ratios in zooplankton tissue for stations in Sinclair Inlet and representative bays, Puget 

Sound, Washington, August 2008.

Monthly Sampling in Sinclair Inlet

Approximately monthly (December 2008 was not 

sampled) samples were collected between August 2008 

and August 2009 at the four Sinclair Inlet stations: SI-IN, 

SI-PO, BNC-52, and the CZ (figs. 5). This sampling 

included numerous water column constituents, four mercury 

constituents in water and the collection, identification, sorting, 

and sub-sampling of zooplankton for tissue concentrations. 

These monthly samples also corresponded to the quarterly 

benthic sediment sampling. Strong seasonal trends in 

chlorophyll were measured and visually observed in summer, 

at the four Sinclair Inlet stations, with concentrations ranging 

from less than 1 μg/L in middle of winter to 150 μg/L in late 

summer (fig. 24).

Strong and spatially similar phytoplankton growth in 

Sinclair Inlet during spring and late summer is indicated 

in figure 24 (see “Seasonal Analysis”). This growth phase 

is then interrupted with abrupt “crashes” in chlorophyll 

concentrations in mid- to late summer followed by rebounds 

in concentrations. This classic phytoplankton bloom and 

chlorophyll crash pattern previously has been described in 

Puget Sound (Winters and others, 1975; Strickland, 1983). 

Although adequate data were not available preceding the 

August 2008 sampling to confirm the phenomena, the 

autumn “bloom” or apparent rebound in the September 2008 

chlorophyll samples (fig. 24) is reflected in the methylmercury 

concentrations of suspended solids from August to October 

2008 (fig. 25).
48 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig24

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

2008–09

Average chlorophyll a concentration, in micrograms per liter

0.1

1

10

100

1,000

BNC-52

Convergence zone

SI-Inner

SI-Port Orchard

Minimum and maximum of triplicate core 

 incubation experiments

Bud Inlet

Sinclair outer

Liberty Bay

Holmes Harbor

EXPLANATION

Figure 24. Average chlorophyll a concentrations for selected stations in and adjacent to, Sinclair 

Inlet, Kitsap County, Washington, August 2008–August 2009.
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 49

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

2008–09

Particulate methylmercury in seawater, in nanograms per liter

BNC-52

Convergence zone

SI-Inner

SI-Port Orchard

Standard deviations of duplicate analyses

EXPLANATION

TAC_17-1117_fig25

0.001

0.01

0.1

10

1

Range of detection limits

(non-detections shown at 0.5

detection limit for graph)

Figure 25. Particulate methylmercury (mass/volume) concentrations for selected stations in and 

adjacent to Sinclair Inlet, Kitsap County, Washington. Data are logarithmic transformed.
50 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

The coupling of the overall pattern of increasing mercury 

concentrations as phytoplankton concentrations increase 

has been described in several freshwater systems (Miles and 

others, 2001; Stewart and others, 2008), but the phenomena 

in coastal marine waters is less well described. This seasonal 

trend in increases of methylmercury during the growing 

season is also visible, although less clear, when evaluating the 

concentration of filtered methylmercury in seawater over the 

sampling period (fig. 26).

Similar to the strong seasonal trends in chlorophyll 

(fig. 24) and dissolved nutrients (fig. 1-2), the ratio of 15N to 

14N of suspended solids increased during the growing season, 

suggesting an increased coupling of the nutrient cycle in the 

water column. These seasonal trends are seen at four Sinclair 

Inlet stations. An anomaly to this trend is SI-IN in February 

2009 with a significant decrease in the 15N ratio (fig. 27).

In February 2009, SI-IN (fig. 1-2A) also had dramatically 

higher filtered ammonia values (0.22 mg/L compared to an 

average of 0.03 mg/L for the other 11 months) and a filtered 

orthophosphate value of 0.11 mg/L (fig. 1-2B) that was the 

highest measured for any of the stations during that month, 

and the third highest in this study. Orthophosphate values 

at the other three stations ranged from 0.08 to 0.09 mg/L in 

February 2009. These observations suggest an additional 

nutrient source was present during the February 2009 

sampling. A review of the tidal and weather data for February 

2009 indicates nothing remarkable.

Long term detection limit,

non-detections plotted at 0.05 ng/L

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

2008–09

Filtered methylmercury in seawater, in nanograms per liter

BNC-52

Convergence zone

SI-Inner

SI-Port Orchard

EXPLANATION

0.01

0.1

1

Figure 26. Filtered methylmercury concentrations in seawater for selected stations in and adjacent 

to Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 51

tac17-1117_fig27

-25

-29

-21

-17

-13

-3

-5

-1

1

3

5

7

9

11

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

2008–09

A

B

Nitrogen isotopic ratio (N15) of

particulate matter, in per mil

Carbon isotopic ratio (C13) of

particulate matter, in per mil

BNC-52

Convergence zone

SI-Inner

SI-Port Orchard

Standard deviations of triplicate analyses

Low particulate matter where isotope could not be measured

EXPLANATION

Figure 27. Average ratios of stable isotopes of (A) nitrogen (δ15N) and (B) carbon (δ13C) in suspended solids for selected 

stations in and adjacent to Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.
52 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

Estimating Zooplankton Mercury

In addition to the monthly sampling of nutrients and 

chlorophyll, other parameters were co-sampled at the time of 

zooplankton collection that were expected to aid in estimating 

zooplankton mercury concentrations. The distribution of these 

explanatory parameters were reviewed and transformed as 

needed to meet assumptions of normality. In order to explain 

zooplankton tissue concentrations of MHg, several predictor 

variables were evaluated. Their significance in ordinary least 

squares regression were as follows- natural log (ln) of average 

chlorophyll a (multiple R2 0.125, p-value 0.012), ln FMHg 

in nanograms per liter (R2 0.30, p-value 0.019), ln of average 

PMHg in nanograms per liter (R2 0.345, p-value 0.001), 

and average δ15N (R2 0.159, p-value 0.004), respectively. 

Stepwise regression also was done with a combination of 

two or three of these parameters, excluding FMHg or PMHg, 

which were correlated to zooplankton MHg (Spearman’s 

rho of 0.54–0.52). No stepwise combination improved upon 

the single, average PMHg model in estimating zooplankton 

mercury concentrations. The importance of suspended solids 

to estimate the accumulation to the next trophic level has been 

demonstrated by Stewart and others (2008). Despite the recent 

examples of “bloom dilution” of methylmercury in freshwater 

primary consumers (Pickhardt and others, 2002; Karimi and 

others, 2007), bulk zooplankton tissue concentrations of 

methylmercury in this study (fig. 28) generally track, albeit 

with a 30–45 day delay, concentrations observed in the 

particulate phase (fig. 25) and chlorophyll (fig. 24).

A review of the August 2008 synoptic sampling and the 

year-long monthly sampling results suggests that although 

there are some spatial differences in mercury concentrations 

across Puget Sound, the differences appear to be smaller, or 

similar in magnitude, than differences at a given site over 

the course of the year. The strong seasonal phytoplankton 

growth, as indicated by chlorophyll concentration, provides 

an overriding control on mercury uptake and accumulation 

into the lower estuarine trophic levels. These seasonal peaks 

in MHg concentrations are weak, but evident, in the FMHg 

concentrations, and stronger in the PMHg and zooplankton 

concentrations (fig. 29). These trends correspond to the 

quarterly sediment sample results that indicated production 

and release of methylmercury from the sediments and that 

increased in strength from February to June and then leveled 

off or decreased slightly by August. Temperature alone has 

strong controls on MHg production (Fagerstrom and Jernelov, 

1972; Wright and Hamilton, 1982), and likely is somewhat 

responsible for the increase in MHg availability in sediments. 

Near-bottom temperatures varied from 7 to about 14 °C during 

the year; however, the pelagic production, senescence, and 

2008–09

0

10

20

30

40

50

60

Methylmercury concentration in zooplankton tissue, in nanograms per gram in dry weight

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

BNC-52

Convergence zone

SI-Inner

SI-Port Orchard

Standard deviations of 

triplicate anlyses

EXPLANATION

Figure 28. Average methylmercury concentrations in zooplankton tissue for selected stations in and adjacent to Sinclair 

Inlet, Kitsap County, Washington, August 2008–August 2009.
IV. Methylmercury Accumulation in the Base of an Estuarine Food Web 53

deposition of phytoplankton to the rather shallow sediments 

appears to have a stimulatory effect on sulfate-reducing 

bacteria and, therefore, methylmercury production.

A comparison of methylmercury concentrations in 

particles and in zooplankton tissue for the same sampling 

date (fig. 29) indicates a several order of magnitude increase 

in concentration on a dry weight basis. This phenomenon 

of bioaccumulation in the marine food web has been well 

described; however, given the high frequency of nondetectable concentrations in the filtered water sample 

collected, a bioaccumulation factor from water to zooplankton 

could not be calculated.

The FMHg concentrations greater than 0.25 ng/L were 

measured in one-half of the surface water samples collected 

in the greater Sinclair Inlet in August 2009. The highest 

concentration of FMHg during this study (0.99 ng/L) was 

measured at SI-IN. The ancillary data indicate that the 

extremely high concentrations of FTHg and FMHg in surface 

water measured at station SI-IN in August 2009 (figs. 14–15) 

was associated with biological processes and high chlorophyll 

concentration. Thus, the phytoplankton bloom conditions in 

August 2009 accumulated carbon, nitrogen and phosphorus, 

THg, and MHg in the particulate phase. As the phytoplankton 

died, there is an apparent release of these elements into 

aqueous or colloidal phases. Nitrate is limiting and is reused 

to produce new phytoplankton, which leaves an accumulation 

of filterable orthophosphate. Colloidal THg and MHg from the 

phytoplankton breakdown that pass through the pores of the 

QFF filters used likely are responsible for the increased FTHg 

and FMHg concentrations during bloom conditions at SI-IN 

during August 2009.

2008–09

Filtered methylmercury in seawater, in nanograms per liter

0.0

0.2

0.4

0.6

0.8

1.0

-0.1

0.1

0.3

0.5

0.7

0.9

0.01

0.001

0.1

1.0

10.0

100.0

Particluate and zooplankton methylmercury, in nanograms per gram dry weight

Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

Filtered methylmercury in seawater

Particulate methylmercury of solids

Zooplankton methylmercury in tissues

Methylmercury = Standard deviation of duplicate samples

Zooplankton= Standard deviation of triplicate samples

EXPLANATION

Figure 29. Averaged concentrations of filtered methylmercury in seawater, particulate material, and zooplankton for 

selected stations in and adjacent to Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009.
54 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

V. Synthesis

By A.J. Paulson, P.W. Moran, and 

M.C. Marvin-DiPasquale 

Water column processes acting on the aqueous and 

particulate phases of mercury control the bioaccumulation of 

methylmercury into the base of the pelagic food web in Puget 

Sound. The concurrent annual cycling of biological indicators 

(chlorophyll a, orthophosphate, nitrate, and silicate) at each 

of three stations in Sinclair Inlet suggests that the near-surface 

water of Sinclair Inlet is well mixed by tidal dispersion. Thus, 

seasonal biological productivity, rather than geographical 

location, generally controls mercury bioaccumulation in 

the surface layer of Sinclair Inlet. The CZ station outside of 

Sinclair Inlet seems to be responding in a slightly different 

manner than Sinclair Inlet stations (fig. 5).

Correlations between Methylation, 

Release, and Bioaccumulation

The MHg concentrations of suspended solid in the 

near-surface water of Sinclair Inlet increased between August 

and September 2008 (fig. 25, and summarized in fig. 29). 

Likewise, the average MHg concentrations of zooplankton 

increased slightly (from 21 to 26.5 ng/g). Throughout winter 

(November 2008–March 2009), MHg concentrations of 

solids were low and near the detection limits. The MHg 

concentrations of zooplankton also decreased during winter, 

reaching their lowest median concentrations in April 2009 

(fig. 28). Beginning in April 2009, MHg concentrations 

of solids increased through August 2009, the end of the 

sampling effort. Beginning in April, zooplankton MHg also 

increased and reached a plateau of about 35 ng/g zooplankton 

tissue in July 2009. Measurable concentrations of FMHg 

throughout Sinclair Inlet were detected only in October 

2008 and May 2009, near or after the time of the chlorophyll 

maximum. Thus, MHg concentrations of suspended solids 

are better indicators of bioaccumulation into the food web 

than MHg concentrations in water, as FMHg is quickly taken 

adsorbed by particles. Methylmercury has particularly strong 

affinity for sulfhydryl groups common to biological molecules 

(Ravichandran, 2004). The use of MHg of suspended solids 

as an indicator of bioaccumulation into the food web could be 

further expanded by lowering the reporting limit of MHg of 

suspended solids simply by filtering more water.

The seasonal trends in fluxes of FMHg in sediment 

suggest that sedimentary processes are producing the 

MHg that is being accumulated in suspended solids and 

zooplankton. High, but also variable, fluxes of FMHg from 

sediments (fig. 30) were detected when MHg of suspended 

solids were high. The fluxes of FMHg (fig. 30) were 

controlled by accumulation of FMHg in porewater (fig. 31). 

In reducing and highly reducing sediment, porewater 

FMHg concentrations were highly correlated with SMHg 

concentrations (fig. 32). Likewise, the seasonal trends 

in SMHg (fig. 31) were similar to the seasonal trends in 

methylmercury production potential (fig. 30).

Sedimentary diagenetic processes likely are controlled 

by biological processes in the water column. Analysis of 

covariance suggests that MMP, SMHg, FMHg in porewater, 

and fluxes out of the sediment are primarily controlled by 

seasonal factors, redox-sensitive constituents, and organic 

carbon (figs. 10–11; tables 6–8). In turn, sedimentary redox 

conditions are controlled by the flux of labile carbon to the 

sediment, especially after a phytoplankton die-off. Baker 

and others (1985) demonstrated that the flux of labile carbon 

(concentration of pigments in sediment trap material) to 

the sediment-water interface of Puget Sound sediment 

was controlled by bloom-induced increases in pigment 

concentrations in the euphotic zone during short periods in 

June, August, and September. The increased incorporation of 

reduced Mn into shell of bivalves in the Bay of Seine, France, 

followed maximums in the water column chlorophyll a (Barats 

and others, 2008). The time history of Mn in bivalve shells, 

which was co-incident with high ammonia concentrations 

in the sediments, following phytoplankton blooms clearly 

demonstrates the linkage between biological process in the 

water column and redox conditions in the sediment.

Specifically, the high porewater FMHg concentrations 

and FMHg fluxes from the sediment samples collected in June 

2009 may have been a result of the supply of labile carbon 

from the phytoplankton die-off as a remarkable decrease 

was observed in chlorophyll a concentrations in May 2009 

(fig. 24). The data from four seasonal sampling periods in 

2008 and 2009 are limited and somewhat variable. However, 

the opinion that plankton die-off stimulates microbial activity 

in shallow estuary systems has been described in Chesapeake 

Bay, Maryland (Kemp and Boynton, 1981). Therefore, the 

production and cessation of phytoplankton may stimulate the 

subsequent production of MHg in the sediment and thus its 

transfer back to the pelagic food web.
V. Synthesis 55

tac17-1117_fig30

August

2008

February

2009

June

2009

August

2009

0

20

40

60

80

100

0

400

800

1,200

Methylmercury product potential, in picograms per dry gram per day, dry weight

Methylmercury flux, in nanograms per square meter per day

Potential production

Flux

EXPLANATION

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

Figure 30. Methylmercury production potential and median fluxes of sediment at OU B Marine, and greater Sinclair Inlet 

stations, Kitsap County, Puget Sound, Washington during seasonal sampling events, August 2008–August 2009.

0

10

20

30

0

6

4

2

8

10

Filtered methylmercury in porewater, in nanograms per liter

Methylmercury in sediment, in nanograms per dry gram

August

2008

February

2009

June

2009

August

2009

Sediment methylmercury concentration

Porewater methylmercury concentration

EXPLANATION

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

Figure 31. Filtered methylmercury concentration in porewater and in sediment at OU B Marine, and greater Sinclair Inlet 

stations Kitsap County, Washington, August 2008–August 2009.
56 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_fig32

0 5 10 15

0

10

20

30

40

Methylmercury in sediment, in nanograms per gram

Methylmercury in porewater, in nanograms per liter

For reducing and highly reducing sediment

FMHg = -2.75 + 2.58 * SMHg

Weakly and moderately reducing sediment

(r = 0.26, p = 0.43) 

Reducing and highly reducing sediment

(r = 0.77, p = 0.002) 

EXPLANATION

Figure 32. Methylmercury concentrations in porewater compared to methylmercury 

concentrations in sediment, Sinclair Inlet, Kitsap County, Washington, August 2008–August 2009. 

The correlation coefficients (r) and the probabilities of the slope being about zero (p) for two 

groupings of redox conditions are given. The regression line for the more reducing grouping is 

shown with the significant regression.

Summary

In August 2008, samples of sediment, water, and biota 

from three bays representative of Puget Sound, Washington, 

embayments and from six stations in Sinclair Inlet were 

collected. The representative bays ranged from locations in 

Holmes Harbor on remote Whidbey Island to Liberty Bay, 

which was adjacent to a suburban town and contained highly 

reducing sediments. Substantially higher concentrations of 

total mercury and reactive mercury in sediment were measured 

in Sinclair Inlet relative to the representative bays. In 

contrast, there was no difference in sediment methylmercury 

concentrations, in methylmercury concentrations in porewater, 

or in the water-column methylmercury concentrations of 

Sinclair Inlet relative to the representative bays in August 

2009. Although the inorganic reactive mercury (SRHg) metric 

provides some measure of the pool of inorganic mercury 

(Hg(II)) that is potentially available for Hg(II)-methylation, 

there is no evidence that methylmercury (MHg) production 

rates are significantly higher in Sinclair Inlet than in the 

representative Puget Sound embayments in this study. 

Likewise, broad-scale sampling of sediments over 1 year 

detected significantly higher concentrations of total and 

reactive mercury in Bremerton naval complex (BNC) sediment 

compared to greater Sinclair Inlet sediment. Previous analysis 

of the total mercury (THg) concentrations of solids also 

indicated that suspended matter collected from near-bottom 

waters of BNC stations were higher than those of greater 

Sinclair Inlet (GSI). Yet, there was no difference in sediment 

methylmercury (SMHg) concentrations or methylmercury 

production potential (MPP) rates between BNC and GSI. 

A model that examined differences among the individual 

stations, rather than differences between GSI and BNC, 

better described the factors controlling porewater filtered 

methylmercury (FMHg) concentrations and methylmercury 

(MHg) release from sediments. 

Rather than detecting differences based on geographical 

distinctions, these observations are consistent with the concept 

that sediment total mercury (STHg) has only a minor effect 

on net MPP rates and SMHg concentrations. The Akaike 

Information Criterion competitive model approach used to 

assess key MHg-metrics in sediments from this study support 

this concept. The proxy measure for the activity of the Hg(II)-

methylating microbial community (methylmercury production 

rate constant [kmeth]), was best described by a function that 

included temperature, sediment redox, and the percentage 

of acid-extractable ferrous iron/total iron (sediment) 

(Fe(II) AE/ FeT) as significant variables, consistent with the 

role of iron-reducing bacteria in the Hg(II)-methylation 

process. However, the importance of sulfate-reducing bacteria 

cannot be ruled out in this analysis. The proxy measure 

for the availability of Hg(II) to those Hg(II)-methylating 

bacteria across all sites, namely SRHg, was best described 

by a function that included sediment redox (Eh), STHg, 

and sediment bulk density, the latter parameter reflecting 

the combined influence of sediment organic content and 
Acknowledgments 57

grain size. When representative bays were excluded from 

the analysis, STHg was no longer a significant variable for 

describing sediment SRHg concentrations. The analysis of 

both kmeth and SRHg data point to the dominant role sediment 

redox has on the SMHg production process. The MPP rates 

were a function of temperature, STHg concentration, and the 

percentage of Fe(II)AE/FeT across all sites, but at the local 

scale (GSI and BNC stations only, excluding representative 

bays) the importance of STHg was again eliminated from 

the best fitting model. Although only 35–36 percent of the 

variability in sediment MPP rates could be explained by the 

final, best fit models, 83 percent of the variability in SMHg 

concentration for Sinclair Inlet (GSI and BNC stations 

combined) was explained by sediment redox and bulk density 

alone.

Methylmercury concentrations in sediment porewaters 

also were correlated with dissolved organic carbon, and 

the correlation was especially strong for reducing and 

highly reducing sediments. The correlation between MHg 

concentrations in porewaters and the fluxes of methylmercury 

out of incubated cores was not straightforward. Little 

methylmercury was released from an incubated core collected 

from the station with moderately reducing sediment and with 

the highest porewater methylmercury concentration of the 

study. In contrast, two incubated cores collected from stations 

with weakly reducing sediment and low methylmercury 

porewater concentrations released as much methylmercury 

as most of the stations with reducing and highly reducing 

sediment. The role of benthic fauna in inhibiting 

methylmercury release through demethylation or enhancing 

methylmercury through biological irrigation of the sediments 

needs further study.

Water column samples collected at four stations 12 

times at about monthly intervals between August 2008 and 

August 2009 helped to develop a better understanding of the 

seasonal processes affecting mercury in Sinclair Inlet. The 

variation in water-quality parameters, including mercury, 

between the Sinclair Inlet stations, or between BNC stations 

and GSI stations, was similar to variations across Puget 

Sound. Most notable was the strong seasonal trends in 

nutrient concentrations from the monthly sampling. Seasonal 

biological activity also resulted in significant differences 

between near-bottom and near-surface samples, especially 

in August 2008 and June 2009. Although several parameters 

displayed a winter minima, the order of the appearance 

of that minima is led by the chlorophyll and particulate 

MHg concentrations and followed by zooplankton mercury 

concentrations. Concentrations then begin to increase for 

chlorophyll in January or February, for 15N in March, 

particulate methylmercury in April, and zooplankton in May. 

Filtered methylmercury trends were difficult to interpret 

due to a predominance of samples with results less than the 

detection limit. This order of the seasonal inflection point in 

these parameters is consistent with the hypothesis that the 

water column production of phytoplankton has a 1 to 2 month 

delayed stimulatory effect on benthic microbial activity.

Unlike many reactive biogeochemicals, few significant 

differences of FMHg or particulate methylmercury (PMHg) 

exist for any of the four seasonal sampling periods. Two 

instances of extremely high concentrations of FTHg and 

FMHg provide insight into processes affecting methylmercury 

in Sinclair Inlet. Vertical water column data suggested that 

the extremely high concentrations of FTHg and FMHg in 

near-bottom water from Sinclair Inlet-Port Orchard station 

in June 2009 was consistent with indications that submarine 

groundwater discharge pushed reduced porewater into the 

water column. In contrast, the high concentrations of FTHg 

and FMHg in near-surface water from the Sinclair Inlet-Inner 

station in August 2009 were consistent with accumulation of 

both dissolved and particulate constituents in the surface layer 

because of a rapid phytoplankton growth (chlorophyll a of 

150 μg/L) prior to that date. These single outlier observations 

suggest insight into the system, but provide insufficient 

evidence for definitive conclusions about those set of 

conditions. 

Acknowledgments

The authors would like to thank the numerous individuals 

and organizations who assisted with the sampling and sample 

processing during the course of this study. In particular, 

from the USGS, Washington Water Science Center, Reagan 

Huffman, James Foreman, Kathy Conn, Karen Payne, Greg 

Justin, Sarah Henneford, Kelly Sholtting and Morgan Keys 

from the assisted with sample collection, sample processing 

and overall project support. Gary Rust, Peter Laird, and 

Greg Justin of the USGS operated the boats. Theresa Olsen 

provided GIS support and generated maps. Reagan Huffman 

and Rick Wagner provided data management. Lonna Frans 

assisted with statistical analysis. Jeffery Corbet, William Gibbs 

and Luis Menoyo provided graphical support. Dissolved 

organic carbon analyses of porewaters was performed by 

Kenna Butler and George Aiken with the USGS National 

Research Program and Center for Water, Earth Science and 

Technology (CWEST) laboratory in Boulder, Colorado. The 

following USGS NWQL personnel provided analytical support 

for ancillary environmental constituents: Carl Harris, Mark 

Cree, Armin Burdick, Mark Hill, Jim Kammer, Mary Alice 

Carey and Angie White. Amy Kleckner with the USGS Menlo 

Park National Research Program and several staff members 

of the University of Washington’s Wetland Ecosystem Team 

assisted with sample collection, processing and taxonomic 

identification. 
58 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

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Appendix 1. Supplementary Figures and Tables 63

Appendix 1. Supplementary Figures and Tables

Appendix 1 tables (Microsoft® Excel file) are available for download at https://doi.org/10.3133/sir20185063.

0 2 4 6

0

1

2

3

4

Total organic carbon, in percentage by weight

Total mercury, in milligrams per kilogram

Greater Sinclair Inlet

OU B Marine

EXPLANATION

95 percent confidence interval

Figure 1-1. Total mercury concentrations and total organic 

carbon in sediment in and adjacent to Sinclair Inlet, Kitsap 

County, Washington, 2009. Regression and upper confidence 

interval is from the 2007 U.S. Navy Long-term Monitoring 

Program (Paulson and others, 2010).
64 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_appendix_fig 1-2

SI-IN

SI-PO

BNC-39

BNC-71

*

*

*

*

Verticle lines—Lines from

rectangle extend to the limit of

the data

75th percentile

25th percentile

Semiquartile

range

*

*

*

*

*

0

0.05

0.10

0.15

0.20

0.25

Aug.

2008

Feb.

2009

June

2009

Aug.

2009

Aug.

2008

Feb.

2009

June

2009

Aug.

2009

Filtered ammonia, in milligrams per liter as nitrogen

EXPLANATION

Aug.

2008

Feb.

2009

June

2009

Aug.

2009

Filtered silicate, in milligrams per liter

0

1

2

3

4

5

SI-PO

SI-PO

SI-PO

Aug. 

2008

Feb. 

2009

June 

2009

Aug. 

2009

Monthly: Near-surface stations—Asterisk (*) 

indicates an extremely high concentration 

for the depth and season

Convergence zone (CZ)

Bremerton Naval Complex 39 (BNC-39)

Sinclair Inlet-Port Orchard (SI-PO)

Sinclair Inlet-Inner (SI-IN)

Seasonal (six stations)

Near-surface

Near-bottom

SI-IN

SI-PO

SI-PO

BNC71

SI-IN

0

0.04

0.08

0.12

0.16

0.20

Filtered orthophosphate, in milligrams per liter as phosphorus

Filtered nitrate plus nitrite, in milligrams per liter as nitrogen

0.5

0.4

0.3

0.2

0.1

0

SI-PO

A

B

C

*

* *

*

D

Figure 1-2. Filtered (A) ammonia, (B) orthophosphate, (C) nitrate plus nitrite, and (D) silicate for four monthly near-surface 

stations and boxplots for six seasonal near-surface and near-bottom stations Sinclair Inlet, Kitsap County, Washington.
Appendix 1. Supplementary Figures and Tables 65

Filtered manganese, in micrograms per liter

0

10

20

30

40

SI-PO

SI-PO

BNC-39

Verticle lines—Lines from

rectangle extend to the limit of

the data

75th percentile

25th percentile

Semiquartile

range

EXPLANATION

Monthly: Near-surface stations—Asterisk (*) 

indicates an extremely high concentration 

for the depth and season

Convergence zone (CZ)

Bremerton Naval Complex 39 (BNC-39)

Sinclair Inlet-Port Orchard (SI-PO)

Sinclair Inlet-Inner (SI-IN)

Seasonal (six stations)

Near-surface

Near-bottom

Aug. 

2008

Feb. 

2009

June 

2009

Aug. 

2009

*

*

Figure 1-3. Filtered manganese for four monthly near-surface stations and boxplots for six seasonal near-surface and nearbottom stations, Sinclair Inlet, Kitsap County, Washington.
66 Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington

tac17-1117_appendix_fig 1-4 Depth, in meters

12

4

8

0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Sediment

Salinity, in Practical Salinity Units

28.6 28.8 29 29.2 29.4

EXPLANATION

Salinity

Dissolved oxygen

Dissolved oxygen, in milligrams per liter

Figure 1-4. Vertical profile of salinity and dissolved 

oxygen at Sinclair Inlet station SI-PO, May 9, 2009 (1230), 

showing evidence of submarine discharge of freshwater 

near the sediment water interface. Data from Huffman 

and others (2012, appendix I).
Publishing support provided by the U.S. Geological Survey 

Science Publishing Network, Tacoma Publishing Service Center

For more information concerning the research in this report, contact the 

Director, Idaho Water Science Center

U.S. Geological Survey 

230 Collins Road

Boise, Idaho 83702

http://id.water.usgs.gov
Paulson and others--Mercury Methylation and Bioaccumulation in Sinclair Inlet, Kitsap County, Washington--Scientific Investigations Report 2018-5063

ISSN 2328-0328 (online)

https://doi.org/10.3133/sir20185063