U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 5 A–7

Methods for the Preparation and Analysis of Solids 

and Suspended Solids for Methylmercury

Chapter 7 of

Book 5, Laboratory Analysis

Section A, Water Analysis
Methods for the Preparation and 

Analysis of Solids and Suspended 

Solids for Methylmercury

By John F. DeWild, Shane D. Olund, Mark L. Olson, and Michael T. Tate

Chapter 7 of

Book 5, Laboratory Analysis 

Section A, Water Analysis

Techniques and Methods 5 A–7

U.S. Department of the Interior

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

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia: 2004

For more information about the USGS and its products:

Telephone: 1-888-ASK-USGS

World Wide Web: http://www.usgs.gov/

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.
iii

Contents

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

Introduction ................................................................................................................................................... 1

Part 1. Method for the Extraction and Analysis of Methylmercury from Solids ................................. 2

Scope and Application ........................................................................................................................ 2

Method Summary ................................................................................................................................ 2

Sample Collection and Preservation ................................................................................................ 2

Method Detection Limit ...................................................................................................................... 2

Sample Preparation ............................................................................................................................. 3

Reagents ...................................................................................................................................... 3

Extraction Equipment ................................................................................................................. 3

Extraction Procedure ................................................................................................................. 4

Sample Analysis ................................................................................................................................... 4

Data Analysis ........................................................................................................................................ 5

Acceptance Criteria ............................................................................................................................ 6

Method Performance .......................................................................................................................... 6

Part 2. Method for the Extraction and Analysis of Methylmercury from Suspended Solids ........... 8

Scope and Application ........................................................................................................................ 8

Method Summary ................................................................................................................................ 8

Sample Collection and Preservation ................................................................................................ 8

Method Detection Limit ...................................................................................................................... 8

Sample Preparation ............................................................................................................................. 9

Reagents ...................................................................................................................................... 9

Distillation Equipment ................................................................................................................ 9

Distillation Procedure .............................................................................................................. 10

Sample Analysis ................................................................................................................................. 10

Data Analysis ...................................................................................................................................... 10

Acceptance Criteria .......................................................................................................................... 11

Method Performance ........................................................................................................................ 11

Summary ...................................................................................................................................................... 12

References ................................................................................................................................................... 12
iv

Tables

 1. Methylmercury concentrations from multiple analyses of bottom sediment 

for the determination of the method detection limit .............................................................. 3

 2. Methylmercury concentrations for multiple analyses of IAEA-405 

(certified reference material) and Sleepers River sediment. ............................................... 6

 3. Methylmercury concentration and percent recovery data for IAEA-405, 

Sleepers River sediment and upland soil samples ................................................................ 7

 4. Mass of methylmercury per filter from artificial water sample, after conversion 

to 100.0 mL equivalency, for detection limit assay ................................................................. 9

 5A. Methylmercury concentrations and percent recoveries from multiple analyses 

of range A artificial water sample .......................................................................................... 12

 5B. Methylmercury concentrations and percent recoveries from multiple analyses 

of range B artificial water sample .......................................................................................... 12
v

Conversion Factors

Multiply By To obtain

Length

centimeter (cm) 3.94 x 10-1 inch (in)

micrometer (μm) 3.94 x 10-5 inch (in)

millimeter (mm) 3.94 x 10-2 inch (in)

Mass

gram (g) 3.53 x 10-2 ounce, avoirdupois (oz)

nanogram (ng) 3.53 x 10-11 ounce, avoirdupois (oz)

milligram (mg) 3.53 x 10-5 ounce, avoirdupois (oz)

Volume

liter (L) 2.64 x 10-1 gallon (gal)

liter (L) 3.38 x 10-1 ounce, fl uid (oz)

microliter (μL) 2.64 x 10-7 gallon (gal)

milliliter (mL) 2.64 x 10-4 gallon (gal)

Flow

milliliters per minute (mL/min) 6.10 x 10-2 cubic inch per minute (in3

/min)

Resistivity

megohm centimeter (MΩ-cm) 3.94 x 10-1 megohm inch (MΩ-in)

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

 °F=(1.8×°C)+32

Concentrations for suspended solids samples are in nanograms per liter (ng/L).

Concentrations for solids samples are in nanograms per grams (ng/g).
vi

Abbreviated Water-Quality Units

g gram

mg milligram (10-3 grams)

ng nanograms (10-9 grams)

pq picograms (10-12 grams)

L liters

mL milliliters (10-3 liters)

μL microliters (10-6 liters)

ng/L nanograms per liter (parts per trillion)

ng/g nanograms per gram (parts per billion)

cm centimeters (10-2 meters)

mm millimeters (10-3 meters)

μm micron (10-6 meters)

MΩ-cm megohm centimeter

mL/min milliliters per minute

M molar (mole per liter)

Abbreviations and Acronyms

Ar argon

CH2

Cl2 methylene chloride

CRM certified reference material

CuSO4

 copper sulfate

CVAFS cold vapor atomic fluorescence spectroscopy

DDL daily detection limit

DQO data quality objective

GC gas chromatography

HCl hydrochloric acid

Hg mercury

HPLC high pressure luquid chromatography

IAEA International Atomic Energy Agency

I.D. inside diameter

KCl potassium chloride

KOH potassium hydroxide

MDL method detection limit

MeHg methylmercury

N2

 nitrogen

N2

SO4 sulfuric acid

NaBEt4

 sodium tetraethyl borate

NH2

OH*HCl hydroxylamine hydrochloride

NIST National Institute of Standards and Technology

O.D. outside diameter 

QA quality assurance

QFF quartz fiber filter

RPM revolutions per minute

RSD relative standard deviation

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

WDML Wisconsin District Mercury Lab
Abstract

This report presents the methods and method performance data for the determination of methylmercury 

concentrations in solids and suspended solids. Using 

the methods outlined here, the U.S. Geological Survey’s 

Wisconsin District Mercury Laboratory can consistently 

detect methylmercury in solids and suspended solids at 

environmentally relevant concentrations. Solids can be 

analyzed wet or freeze dried with a minimum detection 

limit of 0.08 ng/g (as-processed). Suspended solids must 

first be isolated from aqueous matrices by filtration. The 

minimum detection limit for suspended solids is 0.01 ng 

per filter resulting in a minimum reporting limit ranging 

from 0.2 ng/L for a 0.05 L filtered volume to 0.01 ng/L for 

a 1.0 L filtered volume. Maximum concentrations for both 

matrices can be extended to cover nearly any amount of 

methylmercury by limiting sample size. 

Introduction

Since the industrial revolution, pronounced increases 

(approximately three-fold) in atmospheric mercury emissions, transport, and subsequent deposition have yielded 

what is now considered a global contamination issue. 

Although most surface waters and sediments are now 

(2004) enriched with mercury (Hg) relative to historic 

times, environmental Hg concentrations remain a substantial analytical challenge to quantify accurately. These difficulties are primarily due to sample contamination, artifact 

formation of methylmercury (MeHg) during distillation, 

matrix interferences, and natural heterogeneity of samples. 

Naturally occurring microbial processes increase Hg 

toxicity by the conversion of inorganic mercury to MeHg. 

Methylmercury, the most toxic and bioaccumulative 

form of mercury in food webs (Wiener and Spry, 1996; 

Brumbaugh and others 2001; Wiener and others 2003), is 

generally about 0.1 to 5 percent of the total mercury pool 

in most waters and sediments (Krabbenhoft and others, 

1999; Wiener and others, 2003). Although MeHg represents greater than 95 percent of the mercury in consumable 

game fish tissues and commonly reaches concentrations 

at the low part per million level (Brumbaugh and others, 

2001; Wiener and others, 2003), MeHg concentrations in 

water from the aquatic ecosystems these fish inhabit range 

from about 0.04 to 0.8 nanograms per liter (ng/L) (St. 

Louis and others, 1994; Hurley and others, 1995; Gilmour and others 1998; Babiarz and others, 1998; Bodaly 

and others, 1998; Krabbenhoft and others, 1999; Waldron 

and others, 2000). Concentrations of MeHg in sediment 

range from about 0.1 to 10 nanograms per gram (ng/g) dry 

weight (Gilmour and others 1998; Krabbenhoft and others, 

1999). Methylmercury concentrations in anoxic waters 

or waters affected by industrial pollution (for example, 

chloralkali plants) can reach levels near 10 ng/L (Bloom 

and Effler, 1990; Krabbenhoft and others 1998; Brigham 

and others, 2002) and sediment MeHg concentrations can 

reach approximately 100 ng/g dry weight (Benoit and others, 2003).

Researchers from the U.S. Geological Survey (USGS) 

Wisconsin District Mercury Laboratory (WDML) published a method for the determination of MeHg concentrations in natural waters (DeWild and others, 2002). The 

current report describes the methods used by the WDML 

to analyze solids (bed sediments from aquatic ecosystems 

and soil from terrestrial ecosystems) and suspended solids 

(solids isolated from natural waters by filtration) for MeHg 

concentration, and documents the method detection limit 

(MDL) for these sample media using the described techniques. Because the WDML employs different procedures 

for the preparation of solids and suspended solids, the 

procedures are presented separately in this report. 

Methylmercury can be a difficult parameter to 

measure in solids because of matrix interferences and the 

possibility of unintentionally producing MeHg during 

Methods for the Preparation and Analysis of Solids and 

Suspended Solids for Methylmercury

By John F. DeWild, Shane D. Olund, Mark L. Olson, and Michael T. Tate
2 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

distillation (Bloom and others, 1997; Hintelmann and 

others, 1997; Hammerschmidt and Fitzgerald, 2001). 

Researchers at the WDML have adopted a previously 

published technique for extracting MeHg from solids that 

eliminates formation of MeHg in samples with high inorganic mercury levels (Hintelmann, 1999).

The WDML gratefully acknowledges support for 

this study from the USGS Toxic Substances Hydrology 

Program.

Part 1. Method for the Extraction 

and Analysis of Methylmercury from 

Solids

Scope and Application

The method presented here is suitable for the detection of MeHg in solid samples collected from terrestrial 

and aquatic ecosystems. Material consists of bed sediment 

samples and soil samples. Samples are processed and analyzed as they are received, which may be as wet sediment 

or freeze-dried. Method performance therefore is evaluated 

on an as-processed basis to eliminate factors not related 

to sample processing and analysis. Minimum detectable 

concentration is 0.08 ng/g as-processed, with the maximum concentration dependent on mass of sample extracted 

and volume of extractant analyzed. Results are reported on 

a dry weight basis by dividing the concentration as-processed by the percent dry weight.

Method Summary

Solids (0.5 to 1.0 g) are placed into a centrifuge tube. 

Potassium bromide (KBr), copper sulfate (CuSO4), and 

methylene chloride (CH2Cl2) are sequentially added. The 

mixture is allowed to react for an hour and then is shaken 

for an hour to ensure complete extraction of the MeHg. 

Following the shaking, the samples are centrifuged to 

break any emulsion that has formed. An aliquot of the 

CH2

Cl2 is cleanly transferred to a vial containing reagent 

water. These vials are placed in a heating block until all 

CH2

Cl2 has been evaporated and the MeHg has been backextracted into the reagent water. The pH of the extractant is adjusted to 4.9 (to maximize ethylation potential) 

using acetate buffer. The extractant then is ethylated using 

sodium tetraethyl borate (NaBEt4

) and allowed to react for 

15 minutes. After reaction with NaBEt4, the extractant is 

purged with nitrogen gas (N2) for 20 minutes and the ethylated Hg species are collected on a sample trap containing Carbotrap. These ethylated Hg species are desorbed 

thermally from the sample trap, separated using a gas 

chromatographic (GC) column, reduced using a pyrolytic 

column, and detected using cold vapor atomic fluorescence 

spectrometry (CVAFS). This extraction procedure (Bloom 

and others, 1997) eliminates all known interfereneces from 

organic matter, particulates, and sulfides in addition to 

greatly reducing potential positive artifact generated from 

interaction of ambient inorganic Hg with organic compounds.

Sample Collection and Preservation

Methylmercury analysis is extremely sensitive to 

contamination; therefore, care must be taken to avoid contamination in sample collection and analysis. Sample collection should be conducted using clean hands/dirty hands 

protocol (Olson and DeWild, 1999). Solids samples are 

collected and placed into precleaned vials. Collection and 

analysis equipment is cleaned according to the procedures 

outlined in DeWild and others (2002). Vials can consist of 

Teflon, cleaned according to DeWild and others (2002); 

baked glass vials (prepared by heating to 550°C for four 

hours); or acid rinsed polycarbonate vials. Samples are 

frozen as soon as possible after collection, shipped to the 

lab on dry ice by overnight mail, and held at a temperature 

of -15°C or less until processing. The WDML has not performed a holding time study; however, a frozen reference 

material (CRM) certified for Hg is available through the 

National Institute of Standards and Technology (NIST), 

and is stable for nine years.

Method Detection Limit

MDL of 0.08 ng/g (as-processed) was determined 

according to U.S. Environmental Protection Agency 

(USEPA) protocol (U.S. Environmental Protection Agency, 

1990) from multiple analyses of a bottom sediment sample 

(table 1) collected from Sleepers River in New Hampshire. 

Because samples are received with varying water content 

(including freeze-dried), MeHg per gram of sediment 

as-processed is the most useful way to evaluate the MDL. 

The sample was homogenized with a Teflon policeman 

and seven aliquots, ranging from 0.696 to 1.22 g, were 

transferred from the sample container to individual centrifuge tubes that were then refrozen until processed. The 

subsamples were processed and analyzed over a period of 

several days. 
Sample Preparation

As practiced at the WDML, an extraction batch 

consists of 22 environmental samples, four method blanks, 

two triplicate environmental sample sets, and two CRMs. 

All reagent additions, sample transfers, and the backextraction are carried out inside a certified fume hood. 

Reagents: 

A. Reagent water. Ultra-pure reagent water containing less than 0.1 ng/L Hg with a resistance greater 

than 18 MΩ-cm starting from a prepurified source 

(distilled, reverse osmosis, and others). The water 

is delivered through a 0.2 μm filter, as obtained 

from a Millipore Academic water-purification 

system or equivalent.

B. Potassium bromide extraction solution. Dissolve 

180 g of reagent grade KBr in reagent water, add 

50 mL of concentrated sulfuric acid (H2

SO4) and 

dilute to 1 liter. Adding 0.2 g of hydroxylamine 

hydrochloride (NH2

OH*HCl) stabilizes this solution. This solution should prepared in either glass 

or Teflon, be stored refrigerated and in the dark, 

and made fresh monthly.

C. 1M Copper sulfate. Dissolve 125 g of reagent 

grade CuSO4

 in 500 mL of reagent water. This 

solution is stored in a mercury clean Teflon bottle 

for up to six months.

D. Methylene chloride. HPLC grade CH2

Cl2 that has 

been blank tested and found to be low in methylmercury.

E. Nitrogen. Prepurified or reagent grade N2

 passed 

through a gold bead trap attached to the outlet of 

the tank to remove any Hg. 

F. Acetate buffer. 11.8 mL of glacial acetic acid and 

27.2 g reagent grade sodium acetate trihydrate 

diluted to 100 mL with reagent water.

G. Ethylating Reagent. 1 g of Sodium Tetraethyl 

Borate (NaBEt4

; Strem 11-0575) dissolved in 

100 mL of 2 percent Potassium Hydroxide (KOH), 

weight to weight (w/w), solution that has been 

chilled to form slush. The NaBEt4

 solution is 

divided equally among 10 clean 15 mL Teflon 

vials that then are capped and frozen. This solution 

should be kept frozen and made fresh every two 

weeks. Never use NaBEt4

 solid or solutions that 

are yellow in color. Note: NaBEt4

 is toxic, gives off 

toxic gases (triethylboron) and is spontaneously 

combustible. Any NaBEt4

 use should take place 

in a high-volume fume hood. To discard unused 

portions of ethylating reagent, empty bottles into a 

large beaker of 6N hydrochloric acid (HCl) inside 

a high-volume fume hood. Place beaker on a hotplate and boil down to half-volume, then discard 

the remaining solution as an acid waste. Triethylboron will boil off into the air where it is oxidized 

to harmless boric acid.

H. Argon (Ar). Ultra high purity grade 5.0 Ar passed 

through a gold bead trap attached to the outlet of 

the tank to remove any Hg. 

I. Nitrogen. Ultra high purity grade 5.0 N2

 passed 

through a gold bead trap attached to the outlet of 

the tank to remove any Hg.

Extraction Equipment:

A. An analytical balance capable of weighing sediment samples to the nearest mg.

B. Pneumatic fixed-volume and variable pipettes ranging from 5 μL to 5 mL. Pipettes need to be calibrated monthly to ensure accuracy and precision of 

+/- 3 percent.

Table 1. Methylmercury concentrations from multiple analyses of bottom sediment for the determination of the method 

detection limit

[All concentrations in nanograms per gram (ng/g)]

Sleepers River s-1 0.132

Sleepers River s-1 0.144

Sleepers River s-1 0.171

Sleepers River s-1 0.108

Sleepers River s-1 0.146

Sleepers River s-1 0.097

Sleepers River s-1 0.123

Average 0.132

Standard deviation 0.025

Detection limit (standard deviation x 3.143*) 0.078

*students T-value at the 99 percent confidence interval for n=7

Part 1. Method for the Extraction and Analysis of Methylmercury from Solids 3
4 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

C. Repipet capable of consistent (+/- 3 percent) delivery of 10 mL of organic solvent. 

D. Teflon centrifuge tubes (35 mL). Oak-Ridge type 

or equivalent.

E. Laboratory shaker capable of sustaining vigorous 

agitation for one hour.

F. Centrifuge capable of maintaining 3000 RPM for 

20 minutes.

G. Teflon distillation tubes (60 mL). Savillex, Inc. part 

# 0202 or equivalent.

H. Midget impingers for distillation tubes. Savillex, 

Inc. part # 338 or equivalent.

I. Flow meters capable of maintaining N2

 flow at 100 

+/- 20 mL per minute.

J. A custom-fabricated aluminum block heated with 

a Thermolyne type 2200 (or equivalent) hot plate 

is used during the back-extraction step. A probe 

placed in the center of the block monitors block 

temperature.

Extraction Procedure:

A. Homogenize the sample with a mercury-clean 

Teflon policeman in the original sample collection 

container. Weigh approximately 0.5 to 1.0 g of 

material into each centrifuge tube. 

B. Pipette 5 mL of the KBr extraction solution, 1 mL 

of CuSO4

, and 10 mL of CH2Cl2 into each tube. 

C. Allow the tubes to sit at room temperature for one 

hour.

D. Place the tubes on a lab shaker and shake vigorously for 1 hour.

E. Place the tubes in a centrifuge and spin at 3000 

RPM for 20 minutes to break any emulsion that 

may have formed. 

F. Prepare back-extraction vials by adding 40.0 mL 

of reagent water to each 60 mL Teflon vial with a 

repipettor.

G. Carefully pipette 2 mL of the CH2

Cl2 (lower layer) 

into a back-extraction vial. Care must be taken to 

pipette only clean, clear CH2

Cl2 from the centrifuge 

tube and a new pipette tip should be used for each 

sample. Pasteur-type pipette tips are recommended 

for pipetting the CH2

Cl2 due to its low surface tension. 

H. Place the back-extraction vials into the heating 

block and attach N2 lines to inlets of impingers. 

Ensure block temperature is at 45°C and that flow 

is at 100 mL per minute.

I. After all of the CH2

Cl2 has been purged (approximately one hour) the samples can be removed from 

the block and stored in the dark at 4°C for up to 48 

hours prior to analysis.

Sample Analysis

After the samples have been back-extracted, they are 

ready for analysis and should be analyzed within 48 hours. 

The analysis is a two-step process consisting of purging 

the mercury species from the sample and detecting the 

mercury species with a cold vapor atomic fluorescence 

detector (DeWild and others 2002). All chemical additions 

to the reaction vessels are carried out in a fume hood and 

then the vessels are transferred to a clean bench below a 

laminar-flow hood equipped with a HEPA filter which is 

99.99 percent efficient on particles less than 0.3 microns in 

diameter.

A. Create a standard curve by adding varying amounts 

of working standard (typically 100, 50, 25, and 

10 pg, but the range needs to cover the expected 

concentrations in the analytical batch) to approximately 100 mL of reagent water in each of the 

reaction vessels. Pipette 200 μL of acetate buffer 

and 100 μL of NaBEt4

 into each of the reaction 

vessels. The NaBEt4

 reagent serves to derivatize the 

two remaining ionic Hg species after the extraction step (inorganic Hg(II) and MeHg) to their 

ethylated forms (diethyl Hg and methylethyl Hg, 

respectively). Elemental Hg does not react with the 

NaBEt4

. Note: The NaBEt4 needs to remain near 

0°C. It should be removed from the freezer approximately 3 minutes before being added to the reaction vessels and placed in a dark place to partially 

thaw. A new vial of NaBEt4

 should be used each 

day. 

B. Tighten the sparging stoppers, ensure the four-way 

valve is in the closed position, gently swirl the 

reaction vessels, and allow the reaction to proceed 
for 15 minutes. After the reaction time has elapsed, 

remove the plugs from the ends of the sample 

traps. Place the sample traps onto the outlet of the 

reaction vessels, with the identification number 

downstream, turn the four-way valve to the open 

position, and allow grade 5 N2

 to purge the vessel 

at a rate of 250 mL/min for 20 minutes.

C. After the samples have been purged, turn the 

four-way valve to the closed position and remove 

the sample trap from the reaction vessel outlet. 

Remove the N2

 line from the inlet of the four-way 

valve and place the sample trap on the end of the 

N2

 line. Allow the N2 to flow through the sample 

traps at 250 mL/min for 7 minutes to remove any 

water vapor that has collected on the sample trap.

D. Four ethylation blanks are prepared by adding 

approximately 100 mL of reagent grade water, 

200 μL of acetate buffer, and 100 μL of NaBEt4

to separate reaction vessels. Then proceed as in 

step B.

E. While one set of reaction vessels and sample traps 

are being used to collect the purged sample, the 

other set can be desorbed and analyzed. Remove 

the sample traps from the N2

 lines, attach the N2

lines to the inlets of the four-way valves, and cap 

both ends of the sample traps.

F. To desorb and analyze the traps, remove the plugs 

from the ends of the first trap and place it into the 

analytical train by threading it, with the identification number upstream, through the center of 

the nichrome wire coil. Center the nichrome wire 

over the Carbotrap, allow the flow to stabilize for 

approximately 30 seconds, and start the system. 

The nichrome wire will heat to 250°C with a ramp 

time of 30 seconds to desorb the Hg species from 

the sample trap. As the Hg species are desorbed 

from the sample trap they are carried by the Ar carrier gas at a flow of 25 mL/min into the GC column 

where the elemental Hg, methylethyl Hg and the 

diethyl Hg are separated. Following separation, the 

individual Hg species are carried into the pyrolytic 

column where the methylethyl and diethyl Hg species are reduced thermally to elemental Hg. The 

CVAFS detector can only detect elemental mercury. The detector then outputs a millivolt signal to 

the peak integration software resulting in three distinct peaks (the center peak represents the MeHg).

G. After the standard curve and the ethylation blanks 

have been analyzed, and found to meet the dataquality objectives (DQO), the extractants from the 

batch can be analyzed. The procedure for analyzing 

the method blanks, environmental samples, and 

CRM samples are identical to the procedure used 

for the standards and ethylation blanks. Simply dispense an appropriate amount of extractant into the 

reaction vessel, add reagent water to bring volume 

in reaction vessel to approximately 100 mL, add 

the acetate buffer and the NaBEt4

, and proceed as 

in step B. An appropriate amount of sample would 

be an amount that produces a MeHg peak with 

an area that falls within the calibration range (for 

most samples this amount is the entire extractant 

volume).

Data Analysis

Peak areas obtained from the CVAFS detector are 

used in the following series of calculations to determine 

the concentration of MeHg in the original sediment 

sample.

MASS OF MERCURY IN ALIQUOT ANALYZED

MA = PA/S, (1)

where

MA = mass per aliquot (ng)

PA = peak area

S = slope of calibration line

MASS OF MERCURY IN BACK-EXTRACTION VIAL

MV = (MA * (40 /(WB – WA))) – MBAVE , (2)

where

MV = mass in back-extraction vial (ng)

MA = mass per aliquot (ng)

The factor of 40 represents the total volume of water 

 in the back-extraction vial

WB = weight of receiving vessel before pouring off 

 aliquot to be ethylated (g)

WA = weight of receiving vessel after pouring off 

 aliquot to be ethylated (g) Note: Because water 

 has a specific gravity of 1, 1 gram of water is 

 assumed to equal to 1 mL of water.

MBAVE = average mass in method blanks (ng)

Part 1. Method for the Extraction and Analysis of Methylmercury from Solids 5
6 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

CONCENTRATION OF METHYLMERCURY IN ORIGINAL 

SAMPLE 

C = (MV * 5)/ WS , (3)

where

C = concentration (ng/g)

MV = mass of MeHg in back-extraction vial (ng)

WS = weight of sample added to centrifuge tube (g)

The factor of 5 represents the correction for taking 

 2 mL from the total of 10 mL of CH2Cl2 for the 

 back-extraction

Acceptance Criteria

Included with each batch of environmental samples 

are method blanks, replicate analyses, and CRM samples. 

Each of these samples provides quality-control information 

used to evaluate the acceptability of the analytical run.

A. Method blanks. Four method blanks are included 

in each sample batch and are used to evaluate 

potential contamination during the extraction and 

analytical steps. The daily detection limit (DDL) is 

defined as three standard deviations of all method 

blanks. The WDML DQO for DDL is 0.08 ng.

B. Replicate analyses. Two samples from each batch 

are set up in triplicate to evaluate the precision of 

the method. DQOs for replicate analyses are a relative standard deviation of no more than 25 percent.

C. Certified reference material. There are two CRM 

samples included in each batch that are used to 

evaluate the accuracy of the analytical run. DQOs 

for CRMs are 55 to 95 percent recovery because 

the certified value was established using a distillation technique known to produce positively biased 

results (Bloom, 1997; Hintelmann, 1997, 1999; 

Hammerschmidt, 2001).

Method Performance

Precision and accuracy for this method were determined by multiple analyses of a CRM, a bed material 

sample from Sleepers River, Vermont, and an upland sandy 

soil. The CRM used to evaluate this method was IAEA405 (a polluted marine sediment obtained from the International Atomic Energy Agency) that has a certified value 

of 5.49 ng/g and a range of 4.96–6.02 ng/g. The certified 

value was determined using a distillation procedure that 

has been shown to cause significant amounts of artifact 

MeHg formation when the MeHg fraction is less than 1 

percent of the inorganic pool (Hintelmann, 1999), which 

is the case with IAEA-405. Using the method described in 

this report, multiple analyses (19) by the WDML resulted 

in a 76 percent recovery of the CRM as compared to the 

literature value. Because the potential exists for the certified value to be biased high, and multiple analyses from 

this lab produced a consistently lower value, the accepted 

range of recovery for the WDML has been established 

at 55 to 95 percent. Method precision was evaluated by 

determining the percent relative standard deviation of the 

concentrations obtained from all analyses of the CRM 

and the Sleepers River sample (table 2). The upland sandy 

soil sample was not used to evaluate precision because the 

MeHg concentration was below detection limit. Accuracy 

was evaluated by calculating the percent recovery from 

analyses of the CRM and spiked environmental samples 

(table 3). The environmental samples were spiked with 

standard by weighing out the aliquots and adding 2.547 ng 

Table 2. Methylmercury concentrations for multiple 

analyses of IAEA-405 (certified reference material) and 

Sleepers River sediment.

[all concentrations in nanograms per gram (ng/g)]

Concentration 

in IAEA-405

Concentration in 

Sleepers River sediment 

 4.50

 4.84

 4.21

 4.01

 4.46

 4.30

 4.09

 3.95

 4.00

 3.84

 3.78

 4.70

 4.89 0.132

 3.68 0.144

 3.82 0.171

 3.98 0.108

 3.95 0.146

 4.18 0.097

 3.95 0.123

Average 4.16 0.132

Standard deviation 0.359 0.025

Percent relative

standard deviation 8.63 19.0
of MeHg to each aliquot before adding reagents. Percent 

recovery was calculated using equation 4 for the CRM and 

equation 5 for the spiked environmental samples. 

PERCENT RECOVERY FOR CRM ANALYSIS

%R = (Ca /Cc ) * 100, (4)

where

%R = percent recovery

Ca = analytically determined concentration (ng/g)

Cc = certified concentration of CRM (ng/g)

PERCENT RECOVERY FOR SPIKED SAMPLES

%R = ((MMs – (Ca * Ms)) / Sm) * 100, (5)

where

%R = percent recovery

MMs = analytically determined methylmercury mass 

 in spiked aliquot (ng)

Ca = average concentration of unspiked sample (ng/g)

Ms = mass of sample aliquot (g)

Sm = mass of spike added (ng)

Table 3. Methylmercury concentration and percent recovery data for IAEA-405, Sleepers River sediment and 

upland soil samples

[all concentrations in nanograms per gram (ng/g)]

Concentration 

in IAEA-405 

Percent 

recovery

Concentration in 

spiked Sleepers 

River sediment 

Percent 

recovery

Concentration 

in spiked 

upland soil

Percent 

recovery

 4.50 82.0

 4.84 88.3

 4.21 76.7

 4.01 73.0

 4.46 81.3

 4.30 78.3

 4.09 74.5

 3.95 72.0

 4.00 72.8

 3.84 69.9

 3.78 68.9

 4.70 85.7

 4.89 89.1 2.05 80.4

 3.68 67.0 2.32 100.6 2.08 81.6

 3.82 69.6 2.16 96.9 1.69 66.2

 3.98 72.5 3.97 98.1 2.26 88.8

 3.95 72.0 3.04 89.0 2.20 86.2

 4.18 76.1 2.28 84.9 2.26 88.8

 3.95 72.0 3.22 89.4 2.09 82.2

Average 4.16 75.9 93.2 82.0

Standard deviation 0.359 6.6 6.2 7.8

Part 1. Method for the Extraction and Analysis of Methylmercury from Solids 7
8 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

Part 2. Method for the Extraction 

and Analysis of Methylmercury from 

Suspended Solids

Scope and Application

The method presented in this report is suitable for 

suspended solids samples isolated from aqueous samples 

by filtration. Samples are collected onto baked (prepared 

by firing to 550°C) quartz fiber filters (QFF) and submitted 

to the laboratory frozen. Performance tests on this method 

show it can be used to determine MeHg concentrations for 

filter-collected suspended solids with a MDL of 0.01 ng of 

MeHg on a filter. Because suspended particulate loads vary 

considerably within hydrologic settings, varying amounts 

of sample water need to be filtered; therefore, mass of 

MeHg per filter is the most useful way to evaluate the 

MDL. A direct comparison of mass detected on the filter 

to the MDL eliminates the need for a volume-predicated 

MDL. Results are reported on a ng/L basis by dividing the 

mass of MeHg on a filter by the volume filtered; therefore, 

the minimum reporting limit ranges from 0.2 ng/L for a 

0.05 L filtered volume to 0.01 ng/L for a 1.0 L filtered 

volume.

Method Summary

Filters containing the suspended solids are placed in 

distillation bottles, reagents are added, and the samples are 

distilled. The distillation procedure extracts MeHg from 

the solid matter into the dissolved phase, converts MeHg 

into methyl mercury chloride, and removes potential 

interferences. Analysis of the distillate follows the method 

described in DeWild and others (2002) and summarized 

above, with minor modifications to the data calculations. 

Sample Collection and Preservation

To provide reliable concentrations for MeHg in 

suspended solids, the WDML has developed a method 

to concentrate suspended solids from unfiltered water by 

either in-line filtration or by vacuum filtration. In either 

case, samples should be collected using clean hands/dirty 

hands sampling protocols (Olson and DeWild, 1999) 

to ensure sample integrity. Sample size is dependent on 

suspended solids load and MeHg concentration and can 

range from 0.05 to 1 L. The suspended solids are retained 

on baked QFFs. To provide MeHg concentrations for 

suspended solid samples in mass per unit volume (for 

example, ng/L), field crews must measure the volume of 

water filtered. Individual filters are placed into stackable 

Teflon petri dishes, double bagged in sealable plastic bags, 

and frozen. Samples are shipped to the lab on dry ice by 

overnight mail, and held frozen at a temperature of -15°C 

or less until processing. The WDML has not performed a 

holding time study; however, a frozen CRM for Hg, available through NIST is stable for 9 years.

Method Detection Limit

To document the WDML’s ability to provide quality 

data at commonly observed natural levels of MeHg in suspended solids (about 0.04 to 25 ng MeHg/L), an artificial 

whole-water sample was created by adding 0.20814 g of a 

CRM to 2.0 L of reagent water. A polluted marine sediment (IAEA-405) was used as the surrogate for suspended 

solids. The artificial water sample was mixed thoroughly, 

and filtered using vacuum filtration. The suspended solids 

retained on the filters were analyzed using the method 

presented in this report, and unfiltered and filtered water 

samples were analyzed using the methods described by 

DeWild and others (2002). The suspended solids samples 

were processed in five separate distillation batches and 

analyzed over five days. Unfiltered and filtered water 

samples were collected to determine the concentrations of 

the slurry and the filtrate. The water samples were distilled 

during a single distillation.

Because suspended particulate loads vary considerably within hydrologic settings, varying amounts of 

sample water need to be filtered; therefore, mass of MeHg 

per filter is the most useful way to evaluate the MDL. A 

direct comparison of mass detected on the filter to the 

MDL eliminates the need for a volume-predicated MDL. 

For this study, the target volume per filter was 100.0 mL. 

This volume was not accurately achieved for each filter; 

therefore, the analytically determined mass per filter was 

converted to a 100.0 mL equivalency to accurately determine a mass-based MDL. The MDL was determined to be 

0.01 ng per filter from multiple analyses of the particulate 

filters (table 4).
Sample Preparation

Samples must be distilled prior to analysis (see details 

in DeWild and others, 2002) to extract the MeHg from 

the suspended solids into the dissolved phase, to convert 

MeHg to methyl mercury chloride, and to remove potential 

interferences.

Reagents:

A. Reagent water. Ultra-pure reagent water containing less than 0.1 ng/L Hg with a resistance greater 

than 18 MΩ-cm starting from a prepurified source 

(distilled, reverse osmosis, and others). The water 

is delivered through a 0.2 μm filter, as obtained 

from a Millipore Academic water-purification 

system or equivalent.

B. 8M Sulfuric acid. Equal volumes of reagent water 

and trace pure H2

SO4. This reagent should be 

prepared in a glass or Teflon container as need to 

prepare the combined reagent. 

C. 20 percent Potassium chloride. 20 g reagentgrade KCl diluted to 100 mL total volume with 

reagent water. This reagent should be prepared in 

a glass or Teflon container as need to prepare the 

combined reagent.

D. 1M Copper sulfate. Dissolve 125 g of reagent 

grade CuSO4 in 500 mL of reagent water. This 

solution is stored in a mercury clean Teflon bottle 

for up to six months.

E. Combined reagent. Combine 200 mL of 8M 

H2

SO4, 100 mL of 20 percent KCl, and 200 mL of 

CuSO4

. This solution is stored in a mercury clean 

Teflon bottle for up to six months.

Distillation Equipment

The distillation system (DeWild and others, 2002) 

consists of a solid aluminum heating block, a hot plate, a 

small refrigerator, Teflon distillation and receiving vessels, 

and Teflon transfer lines.

A. A custom-fabricated aluminum block with 40 positions is heated with a hotplate capable of maintaining 125 +/- 5°C. Two probes are used to monitor 

block temperature.

B. A small commercially available refrigerator is used 

to hold the receiving vials, aid in condensation, 

maintain distillate at 4°C, and protect the distillate from exposure to light. Small holes are drilled 

in the side of the refrigerator to accommodate the 

transfer lines.

C. The distillation and receiving vessels are 125 mL 

Teflon bottles (Nalgene catalog number 1630-0004 

or equivalent). Distillation vessel caps are custommade Teflon caps with two ports integrated into the 

cap itself. One port is a 1⁄4-in compression fitting, 

into which a 1⁄4-in outside diameter (O.D.) Teflon 

tube is inserted so that it will extend to within 

2 mm of the bottom of the distillation vessel to 

insure complete sample purging. The other port 

is a 1⁄8-in compression fitting, which the transfer 

line connects to. The receiving vessel caps consist 

of a Teflon insert (Savillex part number 0738-4-2 

or equivalent) molded integrally with two transfer ports equipped with compression fittings for 

1⁄4-in O.D. tubing. A length of 1⁄4-in O.D. tubing is 

inserted into one of the ports so that it will extend 

to within 2 mm of the bottom of the receiving 

vessel to insure complete sample recondensation. 

Teflon transfer lines of 1⁄8-in O.D. are connected by 

friction fit from the outlet tubing of the distillation 

vessels to the inlet tubing of the receiving vessels.

Part 2. Method for the Extraction and Analysis of Methylmercury from Suspended Solids 9

Table 4. Mass of methylmercury per filter from artificial 

water sample, after conversion to 100.0 mL equivalency, for 

detection limit assay

[all concentrations in nanograms (ng)]

Particulate CRM

 0.054

 0.056

 0.046

 0.047

 0.050

 0.045

 0.048

 0.049

 0.050

Average 0.049

Standard deviation 0.004

Percent relative

standard deviation 7.300

Detection limit

(standard deviation x 2.896*) 0.01

*students T-value at the 99 percent confidence interval for n=9
10 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

D. Flowmeters capable of maintaining a flow of 60 

mL/min of N2 are placed immediately upstream 

of the distillation vials to maintain constant and 

equal flow to all distillation vials. Gas is supplied 

through 1⁄8-in O.D. Teflon line inserted into the 

inlet tubing of the distillation vessel.

Distillation Procedure

A WDML suspended solids distillation batch consists 

of 34 environmental samples, 4 filter blanks, and 2 CRMs. 

A filter blank consists of a baked QFF and the CRM consists of a pre-weighed mass into the distillation vessel.

A. Using clean Teflon tweezers, transfer the QFF sample from the petri dish into a distillation vessel. Use 

reagent water to rinse the petri dishes three times 

back into the distillation vessel. A total volume of 

50.0 mL of reagent water is added to each vessel. 

Add 2.0 mL combined reagent to each bottle and 

cap tightly with the distillation cap corresponding 

to the block position to be occupied by that vial.

B. Dispense 50 mL of reagent water to each receiving 

vessel. Record the bottle identifier of each. Cap 

each vessel with the receiving cap corresponding to 

the block position occupied by the matching distillation vessel.

C. Place the distillation vials in their respective positions in the distillation block and thread the transfer 

lines through the numbered holes in the refrigerator.

D. Turn on the N2

 flow to the flowmeters and connect 

the gas lines to the inlet ports of the distillation 

caps.

E. Place the receiving vial tray in the refrigerator and 

begin placing the receiving vials into the tray. As 

the receiving vials are placed into the tray, connect 

the transfer lines to the inlet ports of the receiving 

caps. Check for bubbling in the reagent water to 

verify unrestricted flow.

F. Adjust the flow on the flowmeters to 60 mL/min. 

Adjust the hot plate temperature to maintain a 

block temperature of 125 +/- 5°C. This temperature 

should result in a distillation rate of 6–8 mL per 

hour but adjustments may be needed for individual 

systems.

G. Periodically throughout the distillation, check the 

receiving vials to ensure unrestricted flow, the distillation vials to ensure no leakage, and the block 

temperature for stability.

H. Remove the transfer lines from the receiving vessels and the distillation vessels from the block 

when approximately 25 percent of the volume in 

the distillation vessel remains. The distillation caps 

and the inside of the transfer lines should be rinsed 

thoroughly with reagent water.

I. Weigh the receiving vessels and record the weight 

for later determination of the percent of the original 

sample that was distilled. Cap the bottles and place 

in a refrigerator at 4°C until analysis (distillates 

should be analyzed within 48 hours).

Sample Analysis

Sample analysis is performed following the method 

described in DeWild and others (2002) and outlined previously.

Data Analysis

Peak areas obtained from the CVAFS detector are 

used in the following series of calculations to determine 

the concentration of MeHg in the original sediment 

sample.

FRACTION DISTILLED:

D = VS /(WF2 – WW), (6)

where

D = fraction distilled

VS

 = sample volume (mL). Equal to 50 mL as 

 dispensed from repipettor.

WF2 = weight of receiving vessel after distillation (g)

WW = weight of receiving vessel with reagent water 

 before distillation (g). Note: Because water has a 

 specific gravity of 1, 1 gram of water is assumed 

 to equal to 1 mL of water.

MASS OF METHYLMERCURY IN ALIQUOT ANALYZED

MA = (PA /S), (7)

where

MA = mass per aliquot (ng)

PA = peak area

S = slope of calibration line
MASS OF METHYLMERCURY IN FILTER BLANK

MB = (MA/D) * ((WF2 – WT2)/(WF2 – WA)), (8)

where

MB = mass in filter blank (ng)

MA = mass per aliquot (ng)

D = fraction distilled

WF2 = weight of receiving vessel after distillation (g)

WT2 = weight of receiving vessel (g)

WA = weight of receiving vessel after pouring off 

 aliquot to be ethylated (g)

MASS OF METHYLMERCURY IN ORIGINAL SAMPLE 

MS = (MA/D) * ((WF2 – WT2)/(WF2 – WA)) – MBAVE, (9)

where

MS

 = mass in original sample (ng)

MA = mass per aliquot (ng)

D = fraction distilled

WF2 = weight of receiving vessel after distillation (g)

WT2 = weight of receiving vessel (g)

WA = weight of receiving vessel after pouring off 

 aliquot to be ethylated (g)

MBAVE = average mass found in filter blanks (ng)

FINAL METHYLMERCURY CONCENTRATION

C = MS / VF, (10)

where

C = concentration (ng/L)

MS = mass in original sample (ng)

VF = volume filtered during filtration process (L)

Acceptance Criteria

Included with each batch of environmental samples 

are filter blanks and CRM samples. Each of these samples 

provides quality-control information used to evaluate the 

acceptability of the analytical run.

A. Filter blanks. Four filter blanks are included 

in each sample batch and are used to evaluate 

potential contamination during the distillation and 

analytical steps by providing a DDL. The DDL is 

defined as three standard deviations of all method 

blanks. A DDL less than or equal to 0.01 ng/filter 

meets laboratory DQO.

B. Certified reference material. There are two CRM 

samples included in each batch that are used to 

evaluate the accuracy of the analytical run. DQOs 

Part 2. Method for the Extraction and Analysis of Methylmercury from Suspended Solids 11

for CRMs are 75 to 125 percent recovery. Values 

are calculated using equation 4.

Method Performance

To evaluate precision and accuracy for this method, 

two artificial water samples (range A and range B) were 

created by adding different amounts of CRM (IAEA-405) 

to approximately 2 L of reagent water. These artificial 

water samples were then filtered by vacumm filtration to 

create 10 suspended sediment samples from each water 

sample. Subsamples of the unfiltered and filtered artificial 

water samples were collected and analyzed according to 

DeWild and others (2002) to determine the concentration 

of MeHg in the artificial water samples. Methylmercury 

concentration of the suspended solids was calculated by 

subtracting the filtered water concentration from the unfiltered water concentration. The unfiltered concentration 

for range A was 0.614 ng/L, the filtered concentration was 

0.101 ng/L, and the calculated suspended solids concentration was 0.513 ng/L. For range B the unfiltered concentration was 2.62 ng/L, the filtered concentration was 0.274, 

and the calculated suspended solids concentration was 2.35 

ng/L. 

Precision was evaluated by examining the percent 

relative standard deviation (RSD) of the concentrations 

obtained from all analyses of the particulate filters for each 

concentration range (tables 5A and 5B). The percent RSDs 

were 6.84 and 18.3 for range A and range B, respectively. 

Accuracy was evaluated by calculating the percent 

recovery of the CRM from each filter (tables 5A and 5B). 

Percent recoveries were calculated using the formula:

PERCENT RECOVERY

R = (CP /(CU – CF )) * 100, (11)

where

R = percent recovery

CP = concentration of suspended solids sample 

 (ng/L)

CU = concentration of unfiltered artificial water 

 sample (ng/L)

CF = concentration of filtered artificial water sample 

 (ng/L)

Applying the equation above, percent recoveries 

ranged from 88.7 to 108.9, with an average of 95.4 percent 

and a standard deviation of 7.9 for range A. For range B, 

percent recoveries ranged from 65.2 to 117.5, with an average of 92.5 percent and a standard deviation of 17.1.
12 Methods for the Preparation and Analysis of Solids and Suspended Solids for Methylmercury

Summary

This document describes the methods used by the 

USGS Wisconsin District Mercury Laboratory to analyze 

solids and suspended solids for Methylmercury (MeHg) 

concentration. Because the procedures used to process and 

analyze solids and suspended solids differ, two distinct 

analytical performance studies were conducted and the 

results are presented. 

The method detection limit (MDL) established for 

the solids procedure as outlined in this report is 0.08 ng 

MeHg/g (as-processed), which was considered acceptable because it is substantially below the levels commonly 

encountered in natural samples from a wide range of 

environments. The method precision, calculated as the 

percent relative standard deviation (RSD), ranged from 

8.63 to 19.0 percent. The accuracy of the procedure, which 

was determined from recovery tests for spiked samples and 

certified reference material (CRM) replicates, ranged from 

75.0 to 93.2 percent and was considered acceptable. 

The second method performance test documented in 

this report is for the sample preparation and analysis of 

suspended solids on baked quartz fiber filters. An artificial raw water sample was created by suspending CRM in 

reagent water, which was then filtered to create suspended 

solid samples. The MDL was established by analyses 

of multiple filters, and a limit of 0.01 ng per filter was 

achieved. Precision was evaluated by calculating percent 

relative standard deviation from analyses of replicate filters. The percent RSD ranged from 6.84 to 18.3. Accuracy 

was evaluated from percent recovery of MeHg on the filters from their target value. Target values were determined 

by subtracting the analytically determined concentration 

of the filtrate from the raw water sample concentration. 

Recoveries ranged from 65.2 to 117.5 percent, with an 

overall mean and standard deviation of 94.5 percent and 

12.7, respectively.

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References 13
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