U.S. Department of the Interior

U.S. Geological Survey

Data Series 705

National Water-Quality Assessment Program

Mercury Bioaccumulation Studies in the 

National Water-Quality Assessment Program—

Biological Data From New York and 

South Carolina, 2005–2009
Cover. Fishing Brook (County Line Flow outlet) near Newcomb, New York. (Photograph by Dennis A. Wentz, U.S. Geological Survey)
Mercury Bioaccumulation Studies in 

the National Water-Quality Assessment 

Program—Biological Data From New York 

and South Carolina, 2005–2009

By Karen M. Beaulieu, Daniel T. Button, Barbara C. Scudder Eikenberry, 

Karen Riva-Murray, Lia C. Chasar, Paul M. Bradley, and Douglas A. Burns

National Water-Quality Assessment Program

Data Series 705

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

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Suggested citation:

Beaulieu, K.M., Button, D.T., Scudder Eikenberry, B.C., Riva-Murray, Karen, Chasar, L.C., Bradley, P.M., and Burns, D.A., 

2012, Mercury bioaccumulation studies in the National Water-Quality Assessment Program—Biological data from 

New York and South Carolina, 2005–2009: U.S. Geological Survey Data Series 705, 13 p., at http://pubs.usgs.gov/

ds/705/.
iii

Foreword

The U.S. Geological Survey (USGS) is committed to providing the Nation with reliable scientific 

information that helps to enhance and protect the overall quality of life and that facilitates effective 

management of water, biological, energy, and mineral resources (http://www.usgs.gov/). Information 

on the Nation’s water resources is critical to ensuring long-term availability of water that is safe for 

drinking and recreation and is suitable for industry, irrigation, and fish and wildlife. Population growth 

and increasing demands for water make the availability of that water, measured in terms of quantity 

and quality, even more essential to the long-term sustainability of our communities and ecosystems.

The USGS implemented the National Water-Quality Assessment (NAWQA) Program in 1991 to 

support national, regional, State, and local information needs and decisions related to water-quality 

management and policy (http://water.usgs.gov/nawqa). The NAWQA Program is designed to answer: 

What is the quality of our Nation’s streams and groundwater? How are conditions changing over 

time? How do natural features and human activities affect the quality of streams and groundwater, 

and where are those effects most pronounced? By combining information on water chemistry, physical 

characteristics, stream habitat, and aquatic life, the NAWQA Program aims to provide sciencebased insights for current and emerging water issues and priorities. From 1991 to 2001, the NAWQA 

Program completed interdisciplinary assessments and established a baseline understanding of 

water-quality conditions in 51 of the Nation’s river basins and aquifers, referred to as Study Units 

(http://water.usgs.gov/nawqa/studies/study_units.html).

In the second decade of the Program (2001–2012), a major focus is on regional assessments of 

water-quality conditions and trends. These regional assessments are based on major river basins 

and principal aquifers, which encompass larger regions of the country than the Study Units. Regional 

assessments extend the findings in the Study Units by filling critical gaps in characterizing the quality 

of surface water and groundwater, and by determining water-quality status and trends at sites that 

have been consistently monitored for more than a decade. In addition, the regional assessments 

continue to build an understanding of how natural features and human activities affect water quality. 

Many of the regional assessments employ modeling and other scientific tools, developed on the basis 

of data collected at individual sites, to help extend knowledge of water quality to unmonitored, yet 

comparable areas within the regions. The models thereby enhance the value of our existing data and 

our understanding of the hydrologic system. In addition, the models are useful in evaluating various 

resource-management scenarios and in predicting how our actions, such as reducing or managing 

nonpoint and point sources of contamination, land conversion, and altering flow and (or) pumping 

regimes, are likely to affect water conditions within a region.

Other activities planned during the second decade include continuing national syntheses of 

information on pesticides, volatile organic compounds (VOCs), nutrients, trace elements, and aquatic 

ecology; and continuing national topical studies on the fate of agricultural chemicals, effects of 

urbanization on stream ecosystems, bioaccumulation of mercury in stream ecosystems, effects of 

nutrient enrichment on stream ecosystems, and transport of contaminants to public-supply wells.

The USGS aims to disseminate credible, timely, and relevant science information to address practical 

and effective water-resource management and strategies that protect and restore water quality. We 

hope this NAWQA publication will provide you with insights and information to meet your needs, 

and will foster increased citizen awareness and involvement in the protection and restoration of our 

Nation’s waters. 

The USGS recognizes that a national assessment by a single program cannot address all waterresource issues of interest. External coordination at all levels is critical for cost-effective 

management, regulation, and conservation of our Nation’s water resources. The NAWQA Program, 

therefore, depends on advice and information from other agencies—Federal, State, regional, 

interstate, Tribal, and local—as well as nongovernmental organizations, industry, academia, and 

other stakeholder groups. Your assistance and suggestions are greatly appreciated.

 William H. Werkheiser

USGS Associate Director for Water
THIS PAGE INTENTIONALLY LEFT BLANK
v

Contents

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

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

Site Selection..................................................................................................................................................1

Data Collection...............................................................................................................................................4

Fish and Macroinvertebrate Collection and Processing ...............................................................4

Age Determination of Predatory Game Fish.....................................................................................5

Periphyton Collection and Processing..............................................................................................5

Detritus and Leaf Litter Collection and Processing ........................................................................5

Analytical Methods........................................................................................................................................6

Mercury in Fish......................................................................................................................................6

Mercury in Macroinvertebrates, Periphyton, and Leaf Litter .......................................................6

Chlorophyll a and Ash-Free Biomass ................................................................................................6

Stable Isotopes in Biological Tissues and Plant Material .............................................................6

Quality Assurance and Quality Control ......................................................................................................6

Standard Reference Material and Certified Reference Material.................................................6

Fish Tissue.....................................................................................................................................6

Macroinvertebrates ....................................................................................................................7

Stable Isotopes......................................................................................................................................7

Summary..........................................................................................................................................................7

References Cited............................................................................................................................................8

Glossary.........................................................................................................................................................11

Appendixes 1–13..........................................................................................................................................13

Figures

1. Map showing overview of locations of New York and South Carolina study basins........2

2. Map showing the Fishing Brook subbasin in New York and the McTier Creek subbasin 

in South Carolina...........................................................................................................................3

Table

1. Sites for streams sampled in New York and South Carolina during the U.S. Geological 

Survey mercury studies from 2005 through 2009.....................................................................4
vi

Conversion Factors, Datum, and Abbreviations

Multiply By To obtain

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft)

kilometer (km) 0.6214 mile (mi)

square kilometer (km2) 247.1 acre

square centimeter (cm2) 0.001076 square foot (ft2)

square meter (m2) 10.76 square foot (ft2)

gram (g) 0.03527 ounce, avoirdupois (oz)

nanogram (ng) 0.03527 × 10-9 ounce, avoirdupois (oz)

microgram (μg) 0.03527 × 10-6 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound, avoirdupois (lb)

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

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

or micrograms per liter (μg/L).

Abbreviations

CL confidence limit

CRM certified reference material

δ13C stable isotope ratio of carbon (13C/12C) expressed in per mil

δ15C stable isotope ratio of nitrogen (15N/14N) expressed per mil

DOC dissolved organic carbon

DTH depositional-targeted habitat (periphyton or invertebrates)

IAEA International Atomic Energy Agency

MeHg methylmercury

NAWQA National Water-Quality Assessment

NIST National Institute of Standards and Technology

NRCC National Research Council Canada

NWIS National Water Information System

PVC polyvinyl chloride

QA/QC quality assurance and quality control

RM reference material

RTH richest targeted habitat (periphyton or invertebrates)

SAV submerged aquatic vegetation

SRM standard reference material

THg total mercury

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey
Mercury Bioaccumulation Studies in the National WaterQuality Assessment Program—Biological Data From 

New York and South Carolina, 2005–2009

By Karen M. Beaulieu, Daniel T. Button, Barbara C. Scudder Eikenberry, Karen Riva-Murray, Lia C. Chasar, 

Paul M. Bradley, and Douglas A. Burns

Abstract

The U.S. Geological Survey National Water-Quality 

Assessment Program conducted a multidisciplinary study 

from 2005–09 to investigate the bioaccumulation of mercury 

in streams from two contrasting environmental settings. Study 

areas were located in the central Adirondack Mountains region 

of New York and the Inner Coastal Plain of South Carolina. 

Fish, macroinvertebrates, periphyton (attached algae and 

associated material), detritus, and terrestrial leaf litter were 

collected. Fish were analyzed for total mercury; macroinvertebrates, periphyton, and terrestrial leaf litter were analyzed for 

total mercury and methylmercury; and select samples of fish, 

macroinvertebrates, periphyton, detritus, and terrestrial leaf 

litter were analyzed for stable isotopes of carbon (δ13C) and 

nitrogen (δ15N). This report presents methodology and data 

on total mercury, methylmercury, stable isotopes, and other 

ecologically relevant measurements in biological tissues.

Introduction

Bioaccumulation of mercury in fish and other aquatic 

organisms has generated increasing public concern during 

the past decade. Recent advances have been made in 

describing some of the factors that influence the transport and 

geochemical cycling of mercury in freshwater ecosystems. 

Even so, specific environmental controls and the relative 

importance of abiotic and biotic processes responsible for 

the uptake and biomagnification of mercury by aquatic 

organisms remain poorly characterized, especially in stream 

ecosystems. From 2005 through 2009, the U.S. Geological 

Survey (USGS) National Water-Quality Assessment 

(NAWQA) Program conducted studies of stream ecosystems 

across two distinct environmental settings in New York 

(Adirondack Mountains) and South Carolina (Inner Coastal 

Plain) to address this information gap. Aquatic organisms, 

surface water, groundwater, and streambed sediment were 

collected seasonally from multiple sites in each watershed. 

Aquatic consumers representing different trophic (feeding) 

levels and habits were collected, as were potential dietary 

sources of mercury. Consumers were fish (top predators and 

midlevel forage fish) and macroinvertebrates (that is, insects 

and other aquatic invertebrates longer than 2 millimeters 

(mm)). Potential dietary sources were periphyton (algae and 

associated inorganic and organic material attached to various 

substrates) and organic detritus. In addition, terrestrial leaf 

litter was sampled from selected locations to characterize the 

potential input of particulate organic matter to streams. Many 

physical and chemical characteristics were also characterized 

as part of this study. In surface water, these characteristics 

include dissolved and particulate forms of total mercury 

(THg) and methylmercury (MeHg), streamflow, pH, water 

temperature, dissolved organic carbon (DOC), sulfide and 

sulfate, major ions, and nutrients. This report presents the 

biological data. Geochemical data for streambed sediment, 

surface water, and groundwater are presented in Feaster and 

others (2010), Bradley and others (2011), and Nystrom and 

Burns (2011). Further detail regarding the environmental 

setting is presented in Scudder Eikenberry and others (2012). 

These and other reports are available from the U.S. Geological 

Survey (2012).

Site Selection

Sites were sampled in the upper Hudson River Basin, 

located in the Adirondack region of New York, and in the 

Edisto River Basin, located in the Coastal Plain of South 

Carolina (figs. 1 and 2; table 1). While the environmental characteristics differ greatly in these regions, both are “biological 

mercury hotspots,” having resident fish and other biota with 

mercury concentrations that are elevated above both human 

and wildlife fish consumption advisory levels and landscapes 

that favor the conversion of atmospherically deposited inorganic mercury to organic methylmercury and its transport to 

aquatic systems (Driscoll and others, 2007; Evers and others, 

2007; Glover and others, 2010; New York State Department 

of Health, 2010; South Carolina Department of Health and 

Environmental Control, 2010; Bradley and others, 2011).
2 Mercury Bioaccumulation Studies in the National Water-Quality Assessment Program—N.Y. and S.C., 2005–2009

Figure 1. Overview of locations of New York and South Carolina study basins.

NEW YORK

SOUTH

CAROLINA

Greenville

Columbia

New York

Albany

Buffalo

Syracuse

ATLANTIC

OCEAN

Upper Hudson River Basin

Fishing Brook subbasin (fig. 2)

McTier Creek

at New Holland, SC

Edisto River Basin

McTier Creek

subbasin (fig. 2)

Base from U.S. Geological Survey Watershed Boundaries Dataset.

Albers Conical Equal Area Projection, North American Datum of 1983

0 8 KILOMETERS

0 4

4

8 MILES

0 20 40 MILES

0 20 40 KILOMETERS

0 100 200 KILOMETERS

0 100 200 MILES

11 1

17

In-channel sampling location and identifier—

Number refers to site listed in table 1

EXPLANATION

85° 80° 75°

45°

40°

35°
Site Selection 

3

Figure 2. The Fishing Brook subbasin in New York and the McTier Creek subbasin in South Carolina.

Fishing Brook Sixmile Brook

PickwacketPond

County LineFlow

McTier Creek

NEW YORK

SOUTH CAROLINA

Fishing Brook

subbasin outlet

McTier Creeksubbasin outlet

00

2

2

EXPLANATION

75° 80°

45°40°35°30°

2 3

4

5

7

8

9 10

6

In-channel sampling location—

Number refers to site listed intable 1

Subbasin boundary

16

14

12 15

15

13

4 MILES

4 KILOMETERS

00 200 KILOMETERS

200 MILES 100

100

Base from U.S. Geological Survey Watershed Boundaries Dataset.Albers Conical Equal Area Projection, North American Datum of 1983

ATLANTIC OCEAN

ll u

G

y

C

er ek
4 Mercury Bioaccumulation Studies in the National Water-Quality Assessment Program—N.Y. and S.C., 2005–2009

Nine sites were sampled across the Fishing Brook subbasin, which drains 65.6 square kilometers (km2

) in the western 

portion of the upper Hudson River Basin, and 5 sites were 

sampled in the McTier Creek subbasin, which drains 79.4 km2

in the northwestern portion of the Edisto River Basin. A site 

was located at the outlet of each of these larger basins; these 

were USGS station identification number (ID) 01312000 in a 

493-km2 drainage area on the Hudson River near Newcomb, 

N.Y., and USGS station ID 02174175 in a 5,341-km2 drainage area on the Edisto River near Cottageville, S.C. Data are 

also provided for an additional site, the Hudson River near 

Winebrook Hills, N.Y., which was sampled only once for 

top predator fish in 2005 as part of a national occurrence and 

distribution study (Scudder and others, 2009).

Data Collection

Trace-metal clean techniques were used for all 

samples collected and processed for mercury analysis (U.S. 

Environmental Protection Agency, 1996; Cleckner and others, 

1999; Olson and DeWild, 1999; Lewis and Brigham, 2004; 

Scudder and others, 2008). A list of the taxa collected in 

both study areas is provided in appendix 1. The list includes 

available descriptions of associated habitat, life history, and 

feeding strategies.

Fish and Macroinvertebrate Collection and 

Processing

Fish taxa were targeted to represent predatory game fish 

species and forage fish (appendix 2). Forage fish are generally smaller, midtrophic level species that can be consumed 

by predatory game fish species. Macroinvertebrate taxa were 

targeted to represent a range of consumer functional feeding 

groups and trophic positions, including shredders, scrapers, 

collector-gatherers, filterer-gatherers, omnivores, and predators (appendix 3).

Fish were collected by electrofishing (backpack, barge, or 

boat), trapping, seining, and angling. Specimens were sorted in 

the field and held in site water with aeration or in plastic bags 

of site water placed on wet ice until processing. Samples were 

typically processed within 4 hours of collection. Each forage 

fish specimen was rinsed three times in deionized water, 

weighed, and measured for total length. Specimens were 

Table 1. Sites for streams sampled in New York and South Carolina during the U.S. Geological Survey mercury studies from 2005 

through 2009.

[Map code refers to USGS station ID shown in figures 1 and 2. USGS, U.S. Geological Survey; ID, identification number; km2, square kilometer]

USGS station ID USGS station name Map 

code Latitude1 Longitude1

Drainage 

basin area 

(km2)

01311951 Hudson River near Winebrook Hills, N.Y. 1 43.95841667 -74.09375000 223

01311990 Fishing Brook at 28N near Long Lake, N.Y. 2 43.97805556 -74.33666667 27.1

0131199010 Sixmile Brook at 28N near Long Lake, N.Y. 3 43.97566667 -74.36130556 4.56

0131199020 Sixmile Brook Tributary near Long Lake, N.Y. 4 43.99127778 -74.35119444 6.89

0131199021 Sixmile Brook below Sixmile Brook Tributary near Long Lake, N.Y. 5 43.98777778 -74.34555556 17.0

0131199022 Sixmile Brook near Long Lake, N.Y. 6 43.98819444 -74.34100000 17.7

0131199035 Pickwacket Pond Outlet at mouth near Long Lake, N.Y. 7 43.99284085 -74.30904714 8.42

0131199040 Fishing Brook above County Line Flow near Long Lake, N.Y. 8 43.98758333 -74.29133333 60.6

0131199045 Unnamed tributary to County Line Flow near Long Lake, N.Y. 9 43.97900000 -74.28588889 0.96

0131199050 Fishing Brook (County Line Flow outlet) near Newcomb, N.Y. 10 43.97738889 -74.27041667 65.6

01312000 Hudson River near Newcomb, N.Y. 11 43.96617378 -74.13070448 493

02172300 McTier Creek (State Highway 209) near Monetta, S.C. 12 33.75347736 -81.60177142 40.5

02172304 McTier Creek above Hunt Shed near New Holland, S.C. 13 33.74597760 -81.59704916 79.1

02172305 McTier Creek near New Holland, S.C. 14 33.71750000 -81.60750000 79.4

3345100813509 Gully Creek at Bridge on Shoals Road near Monetta, S.C. 15 33.75277778 -81.58583333 25.9

3344250813538 Gully Creek at McTier Creek near New Holland, S.C. 16 33.74027778 -81.59388889 29.9

02174175 Edisto River near Cottageville, S.C. 17 33.05461187 -80.44926614 5,341

1

Latitude and longitude are referenced to the North American Datum 1927 (NAD27).
Data Collection 5

either processed as individual samples or as taxon-specific 

composites of 2 to 15 individuals of similar size. Samples 

were double-bagged in fresh zip-seal bags and immediately 

frozen on dry ice. Select game fish, typically top predators, 

were processed as individual samples of skin-off fillet. 

Fish specimens were rinsed three times in deionized water, 

weighed, and measured for total length. A skinless fillet was 

removed from one side, as described in Scudder and others 

(2008). The fillet was weighed, rinsed three times in deionized 

water, double-bagged, and immediately frozen. Samples were 

kept frozen until analysis. In the case of predatory game fish, 

the sagittal otoliths were removed from the head, cleaned, 

dried, and stored in plastic vials for age analysis.

Macroinvertebrates were collected by handpicking and 

by kick-net capture (for example, jabbing at banks, kicking 

substrate), using trace-metal clean techniques and equipment. 

Specimens were held in site water and processed generally 

within 4 hours of collection. Field processing involved sorting 

by taxon (and grouping within taxon by size, in some cases), 

picking clean of visible debris, and rinsing with deionized 

water a minimum of three times. Specimens within each taxon 

(and size grouping) were then divided into composites (target 

of three composites per taxon and size grouping), with targeted 

per-composite mass of at least 1 gram wet weight and targeted 

count of at least 15 individuals. Composite samples were 

stored in plastic scintillation vials, immediately frozen on dry 

ice, and kept frozen until analysis. Additional details regarding 

the collection and field processing of macroinvertebrate and 

fish samples can be found in Scudder and others (2008).

Age Determination of Predatory Game Fish

Age determination for fish samples collected during 

2007–09 was conducted at the Florida Fish and Wildlife 

Conservation Commission Laboratory in Eustis, Fla., using 

methods described in Taubert and Tranquilli (1982), Porak 

and others (1986), and Hall (1991). For fish samples collected 

during 2005, age determination was conducted at the USGS 

Cooperative Wildlife Research Unit Laboratory at Clemson 

University in Clemson, S.C., using a standard technique 

described in Jearld (1983).

Periphyton Collection and Processing

Periphyton (attached algae and associated material) 

were collected from rocks, woody snags, unconsolidated 

substrates in depositional zones, and macrophytes (appendix 

4). At least two of these habitats were targeted at most sites 

where periphyton samples were collected. These samples were 

referred to as (1) depositional-targeted habitat samples (DTH) 

of episammon or epipelon, collected from fine, unconsolidated 

substrates in depositional areas and (2) richest targeted habitat 

samples (RTH), comprising epilithon from cobbles, epidendron from woody snags, or (in a few cases) epiphyton from 

submerged aquatic vegetation (SAV).

Each DTH sample was a composite of algal material collected from at least five locations throughout the stream reach. 

DTH samples were collected by gently inserting a Teflon petri 

dish lid into the desired substrate, sliding a spatula under the 

dish, lifting the trapped material out of the water, and rinsing 

the material into a sample bottle. Samples were elutriated in 

the field to separate periphyton from sand and debris by shaking and decanting twice, adding 50 milliliters (mL) of reagent 

water, and shaking and decanting two more times.

RTH samples collected from cobbles or woody snags 

were composited from 10 locations throughout the stream 

reach; RTH samples collected from SAV were collected from 

7 to 10 locations. RTH samples were collected by removing 

the desired substrate from the stream reach, then the substrate 

was gently scraped or brushed to dislodge periphyton into a 

plastic bin and lastly, the substrate was rinsed with site water. 

For samples collected from cobble substrate, the periphyton 

was scraped or brushed from a known area, delineated by a 

polyvinyl chloride (PVC) cylinder. For samples collected from 

woody snags and SAV, the area was determined by measuring 

the length and width of each snag and leaf. Once the 

periphyton was removed, subsamples were vacuum filtered on 

precombusted 47-mm quartz fiber filters (mercury and stable 

isotope analysis) or on 47-mm glass fiber filters (chlorophyll a

and ash-free dry mass analysis). The filters were stored in petri 

dishes (Teflon for mercury, polystyrene for other analyses) 

and frozen until analysis. Additional detail regarding field 

protocols for collecting periphyton can be found in Bell and 

Scudder (2005).

Detritus and Leaf Litter Collection and 

Processing

Detritus samples, consisting of conditioned leaf packs, 

were collected along channel margins and other depositional 

zones of selected stream reaches (appendix 5). Areas were 

targeted to include macroinvertebrate collection zones. 

Samples were gently and repeatedly rinsed in a clean bucket 

of site water to dislodge invertebrates. After dislodging 

invertebrates, conditioned leaf packs were rinsed with site 

water through a nested series of precleaned plastic sieves. 

The four sieves, stacked largest to smallest, allowed for four 

size increments of particulate organic material (very coarse 

(>3.4 mm), coarse (between 2 and 3.4 mm), fine (between 

1 and 2 mm), and very fine (between 0.5 and 1 mm)). The 

contents of each sieve were placed in a bin and rinsed three 

times, again removing any unwanted material (invertebrates 

or debris). The contents of each sieve were placed in a zip-seal 

bag and frozen until analysis.

In the New York study area, leaf litter was collected 

for mercury analysis by picking up leaves immediately as 

they fell to the ground on a day near the time of peak fall 

rate (appendix 6). Deciduous leaves were separated into the 

two dominant species sugar maple (Acer saccharum) and 

American beech (Fagus grandifolia) before processing. 
6 Mercury Bioaccumulation Studies in the National Water-Quality Assessment Program—N.Y. and S.C., 2005–2009

Samples of important shrub and ground cover were 

collected for mercury analysis by picking the leaves from 

live vegetation of speckled alder (Alnus incana), wood fern 

(Dryopteris sp.), and hobblebush (Viburnum lantanoides). 

Coniferous litter from balsam fir (Abies balsamea) was 

collected by stretching a piece of plastic sheet (either 

25 meters (m) × 25 m or 50 m × 50 m) beneath the collection 

stand for a few hours, then retrieving the fallen needles. In 

the South Carolina study area, peach baskets were placed in 

a mixed coniferous tree stand and a mixed deciduous tree 

stand to collect leaf litter samples. Baskets were retrieved after 

30 days. All litter was handled with clean nitrile gloves and 

placed in plastic bags. Samples were freeze-dried overnight, 

then pulverized to a powder with a mortar and pestle. Each 

sample was then placed in a glass tube for shipment for 

mercury analysis.

Analytical Methods

Mercury in Fish

Before analysis, fish samples were freeze dried to 

constant weight and ground in their entirety to a fine 

powder, using a stainless-steel ball mill (Retsch MM200) 

or an ultracentrifugal mill (Retsch ZM200). Samples were 

analyzed for THg by the Texas A&M University Trace 

Element Research Laboratory in College Station, Tex., by 

combustion and atomic absorption, using a direct mercury 

analyzer (Milestone DMA–80) following U.S. Environmental Protection Agency (USEPA) method 7473 (U.S. 

Environmental Protection Agency, 1998). Fish were analyzed 

for THg only because MeHg composes more than 90 percent 

of the mercury in fish tissue (Grieb and others, 1990; Bloom, 

1992; Southworth and others, 1995; Hammerschmidt and 

others, 1999; Marrugo-Nagrete and others, 2008).

Mercury in Macroinvertebrates, Periphyton, and 

Leaf Litter

Before analysis, macroinvertebrates were freeze dried 

to constant weight and ground in their entirety to a fine 

powder, using a stainless-steel ball mill (Retsch MM200). 

Macroinvertebrates and leaf litter samples were analyzed for 

THg and MeHg by the USGS Wisconsin Mercury Research 

Laboratory in Middleton, Wisc., using a dilute nitric acid 

extraction and cold-vapor atomic fluorescence spectroscopy 

(Hammerschmidt and Fitzgerald, 2006). The same laboratory 

also analyzed periphyton samples for THg and MeHg using 

methods outlined in Olund and others (2004) and DeWild and 

others (2002), respectively.

Chlorophyll a and Ash-Free Biomass

Subsamples of the periphyton samples were analyzed 

for chlorophyll a and ash-free biomass at the National Water 

Quality Laboratory (NWQL) in Denver, Colorado, using a 

spectrofluorometric method described in USEPA method 445 

(Arar and Collins, 1997).

Stable Isotopes in Biological Tissues and Plant 

Material

Select subsamples of fish, macroinvertebrates, 

periphyton, detritus, and terrestrial leaf litter were analyzed 

for stable isotopes of carbon (δ13C) and nitrogen (δ15N) at 

the Stable Isotope Geochemistry Laboratory at the Florida 

State University National High Magnetic Field Laboratory in 

Tallahassee, Fla. The particulates of a sample were scraped 

with a stainless steel spatula from a filter and loaded into 

a silver cup. All the sample cups were placed in wells of a 

plexiglas plate. An appropriate amount of deionized water 

was added to moisten samples. Then the plate was placed 

in a desiccator overnight (approximately 20 hours), with 

approximately 50 mL of hydrochloric acid. After fumigation, 

the samples were wrapped into a tin cup and dried in an 

oven at 70°C. Sample capsules were stored in a desiccator 

if they were not analyzed the same day (Harris and others, 

2001). Samples were analyzed with a Thermo Quest NC2500 

elemental analyzer interfaced with a Thermo Fisher Scientific, 

Inc. Finnigan DELTAplus XP isotope ratio mass spectrometer.

Quality Assurance and Quality Control

Standard Reference Material and Certified 

Reference Material

Laboratory quality assurance and quality control 

(QA/QC) results for mercury and stable isotopes included 

the analysis of field-submitted single-blind certified reference 

material (CRM) and standard reference material (SRM). CRM 

and SRM results, along with the results for laboratory internal 

CRM and SRM analysis are provided in the appendix tables.

Fish Tissue

Samples submitted for fish QA/QC were sent to the Texas 

A&M University Trace Element Research Lab. Reference 

material included various forms of biological tissue inoculated 

with known quantities of THg (National Research Council 

Canada, 1993, 1994, 2003; National Institute of Standards 

and Technology, 2008). Tissue types included mussel tissue in 

the form of marine bivalve mollusk tissue (National Institute 
Summary 7

of Standards and Technology standard (NIST 2976), dogfish 

liver (National Research Council Canada standard (NRCC) 

DOLT–3), lobster hepatopancreas (NRCC TORT–2), and 

dogfish muscle (DORM–2). The target values for CRM and 

SRM analysis, individual results, percentage recovery, and 

comparison to target confidence limits (CLs) for THg are 

found in appendix 7.

Summary statistics for THg in fish are provided in 

appendix 8. The median recovery for THg in CRM and SRM 

submitted samples was 94.6, 99.4, and 107.4 percent for NIST 

2976, NRCC DOLT–3, and NRCC TORT–2, respectively. 

The mean measured value for NIST 2976 was less than 

the lower 95 percent of the CL of the target value, while 

all others were within the range of the confidence limits. 

For the combined CRM and SRM samples, 47.4 percent of 

the samples were within 95 percent of the CL of the target 

value, and 89.5 percent were within 25 percent of the target 

value. For internal laboratory reference samples, 84.7 percent 

were within 95 percent of the CL of the target value, and 

all samples were within 25 percent of the target value. The 

median recovery for laboratory internal reference samples was 

100.4 percent for NRCC DOLT–3 and 96.8 percent for NRCC 

DORM–2.

Macroinvertebrates

Samples submitted for macroinvertebrate QA/QC 

included reference material with known quantities of THg 

and MeHg (National Research Council Canada, 1994, 2003; 

National Institute of Standards and Technology, 2008) and 

were sent to the USGS Wisconsin Mercury Research Laboratory for analysis. The target values for CRM/SRM analysis, 

individual results, percentage recovery, and comparison to 

target CL are found in appendix 9 for THg and appendix 10 

for MeHg.

Summary statistics for THg and MeHg in 

macroinvertebrates are provided in appendix 11. The median 

recovery for THg in the CRM and SRM samples was 94.9, 

92.0, and 100 percent for NIST 2976, NRCC DOLT–3, 

and NRCC TORT–2, respectively, and 48.8 percent of the 

samples were within 95 percent of the CL of the target value, 

and 92.7 percent were within 25 percent of the target value. 

Internal laboratory analysis of macroinvertebrate tissue for 

THg used an SRM (IAEA–407) from the International Atomic 

Energy Agency (2003); 12.2 percent of samples were within 

95 percent of the CL of the target value, and 95.1 percent were 

within 25 percent of the target value. The median recovery 

for MeHg analysis was 93.6, 81.1, and 97.7 percent for NIST 

2976, NRCC DOLT–3, and NRCC TORT–2, respectively. 

For the combined CRM and SRM samples, 26.0 percent of 

the measured MeHg values were within 95 percent of the CL 

of the target value, and 78.0 percent were within 25 percent 

of the target value. Internal laboratory analysis of MeHg for 

macroinvertebrate tissue also used IAEA–407; 46.2 percent 

of the samples were within 95 percent of the CL of the target 

value, and 94.2 percent were within 25 percent of the target 

value. The median recovery for laboratory internal reference 

samples was 89.0 percent for THg and 98.2 percent for MeHg.

Stable Isotopes

Stable isotope ratios were measured relative to reference 

gases and calibrated to known carbon and nitrogen standards 

[δ13C and δ15N, ranging from -12.7 to -32.1 per mil (per thousand) and -5.3 and 2.5 per mil, respectively]. Twenty reference 

material (RM) single-blind samples of USGS40 L-glutamic 

acid (National Institute of Standards and Technology, 2007) 

were submitted to the Florida State University Stable Isotope 

Laboratory. RM samples accounted for approximately 1 of 

every 10 regular samples submitted (appendix 12). The range 

between the target value and measured value differences was 

0.47 and 0.046 per mil for δ13C and 0.69 and 0.028 per mil for 

δ15N. Recoveries ranged from 98.22 to 101.15 percent for δ13C 

and 90.72 and 115.25 percent for δ15N.

Summary results are presented in appendix 13. Of the 

samples submitted, the results for three δ13C samples and 

five δ15N samples were within 95 percent of the CL of the 

standards. Results for all δ13C samples and 11 δ15N samples 

were within 10 percent of the target value. The mean measured 

value for δ13C was -26.33 per mil (target value: -26.39), 

and the mean measured value for δ15N was -4.64 per mil 

(target value: -4.52). Appendix 13 also provides a summary 

of internal laboratory results for RM. The laboratory uses a 

variety of internally created RM and those received from other 

stable isotope labs (Biasatti, 2009; Y. Wang, Stable Isotope 

Geochemistry Laboratory at the Florida State University 

National High Magnetic Field Laboratory in Tallahassee, 

Fla., oral commun., January 27, 2012). The laboratory does 

not use USGS40 L-glutamic acid. Therefore, samples are not 

directly comparable with submitted samples. Over the study 

period, 81 internal RM samples were analyzed for δ13C and 

69 were analyzed for δ15N. Overall, for the five RM types, 

median percent recovery ranged from 99.4 to 101.1 for δ13C 

and 80.1 to 100.8 for δ15N. For the combined lab internal δ13C 

results, 65.4 percent of samples were within 95 percent of the 

CL of the target values, and all were within 10 percent of the 

target value. For δ15N, 60.9 percent of samples were within 

95 percent of the CL of the target values, and 66.7 percent 

were within 10 percent of the target value.

Summary

Bioaccumulation of mercury in fish and other aquatic 

organisms has generated increasing public concern during the 

past decade. Recent advances have been made in describing 

some of the factors that influence the transport and geochemical cycling of mercury in freshwater ecosystems. The 

U.S. Geological Survey National Water-Quality Assessment 

Program conducted a multidisciplinary study in 2005–09 to 

investigate the bioaccumulation of mercury in streams from 
8 Mercury Bioaccumulation Studies in the National Water-Quality Assessment Program—N.Y. and S.C., 2005–2009

two contrasting environmental settings. Study areas were 

located in the central Adirondack Mountains region of New 

York and the Inner Coastal Plain of South Carolina. Fish, 

macroinvertebrates, periphyton (attached algae and associated 

material), detritus, and terrestrial leaf litter were collected. 

Fish were analyzed for total mercury; macroinvertebrates, 

periphyton, detritus, and terrestrial leaf litter were analyzed for 

total mercury and methylmercury; and select samples of fish, 

macroinvertebrates, periphyton, detritus, and terrestrial leaf 

litter were analyzed for stable isotopes of carbon (δ13C) and 

nitrogen (δ15N).

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sec. 5.6.4.B of Wilde, F.D., Radtke, D.B., Gibs, Jacob, and 

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Benitez, L.N., 2008, Total mercury and methylmercury 

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Hambrook, J.A., 2002, Revised protocols for sampling 

algal, invertebrate, and fish communities as part of the 

National Water-Quality Assessment Program: U.S. 

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Glossary 11

Glossary

benthic Associated with (living on or near) 

the bottom of an aquatic habitat (Thorp and 

Covich, 2001; Wehr and Sheath, 2003).

bioaccumulation Gradual increase in the 

amount of a substance in the tissue(s) of 

an organism that occurs when the rate of 

intake (through respiration, ingestion, dermal 

contact, and other mechanisms) exceeds 

removal (International Union of Pure and 

Applied Chemistry, 1993).

confidence limits (95 percent) Upper and 

lower limits of the 95-percent confidence 

level. This is an estimate of the interval 

that contains the true mean value with a 

95-percent degree of certainty and is defined 

as follows:

 X

t N s

N

±

− 



 



 

2

, 1

(1)

where

X is the sample mean,

t is the critical value of the 

t-distribution,

α is the significance level 

(for example, for 

95-percent confidence 

level, α = 0.05),

N is the number of samples, and

s is the standard deviation.

depositional-targeted habitat A habitat 

where fine sediment, such as sand and silt, is 

deposited (Moulton and others, 2002).

ecosystem The collective term describing 

biota (all biological organisms) and their 

associated abiotic environment.

epidendron Benthic habitat that consists 

of woody substrates on which organisms are 

attached or loosely associated.

epilithon Benthic habitat that consists 

of natural, coarse-grained substrates (for 

example, gravels, cobbles, or boulders) or 

bedrocks or artificial, hard substrates (such as 

submerged concrete) on which organisms are 

attached or loosely associated.

epipelon Benthic habitat that consists of 

silt-sized (less than 0.064-millimeter (mm) 

diameter) streambed sediments on which 

organisms are loosely associated. This habitat 

is commonly found in areas of low flow 

velocities, such as pools and side-channel 

areas, where silt can deposit.

epiphyton Benthic habitat hat consists of 

plants on which organisms are attached or 

loosely associated.

epipsammon Benthic habitat that consists 

of sand-sized (between 0.064- and 2-mm 

diameter) particles on which organisms are 

attached or loosely associated.

forage fish Primary (herbivores) and secondary consumers (omnivores or carnivores) 

that are generally smaller fecund species that 

are forage (prey) for larger predaceous fish.

macroinvertebrate Invertebrates (organisms 

without a spinal column) larger than 

microinvertebrates; that is, organisms larger 

than 2-mm diameter retained on a sieve 

(Thorp and Covich, 2001).

periphyton Commonly used to indicate algal 

cells; however, periphyton collectively refers 

to fungi, bacteria, algae, and detritus attached 

to any substrate in an aquatic system (Wehr 

and Sheath, 2003).

predatory game fish The predaceous fish 

species that occupy the highest trophic level 

in a given community or ecosystem.

richest targeted habitat Usually riffles or 

woody snags; this is the habitat type where 

the taxonomically richest (greatest number 

of species) algal or invertebrate community 

is located in a given stream (Moulton and 

others, 2002).
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Appendixes 1–13 13

Appendixes 1–13

The following data files are included as part of the U.S. Geological Survey (USGS) Data Series 705 and are available for download 

from http://pubs.usgs.gov/ds/705/.

Appendix 1. Ecological information for fish and macroinvertebrate taxa collected in New York and South Carolina.

Appendix 2. Fish results and field measurements for samples collected in New York and South Carolina.

Appendix 3. Macroinvertebrate results and field measurements for samples collected in New York and South Carolina.

Appendix 4. Periphyton results for samples collected in New York and South Carolina.

Appendix 5. Detritus results for samples collected in New York and South Carolina.

Appendix 6. Leaf litter results for samples collected in New York and South Carolina.

Appendix 7. Quality assurance and quality control results of reference material for the assessment of mercury in fish.

Appendix 8. Summary of results for reference material submitted in assessment of mercury in fish.

Appendix 9. Quality assurance and quality control results of reference material for the assessment of mercury in 

macroinvertebrates.

Appendix 10. Quality assurance and quality control results of reference material for the assessment of methylmercury in 

macroinvertebrates.

Appendix 11. Summary of results for reference material submitted in assessment of mercury and methylmercury in 

macroinvertebrates.

Appendix 12. Quality assurance and quality control results of reference material for the assessment of stable isotopes 

in tissue.

Appendix 13. Summary of results for reference material submitted in assessment of stable isotopes in tissue.
Prepared by the Pembroke Publishing Service Center.

For more information concerning this report, contact:

Director

U.S. Geological Survey

Connecticut Water Science Center

101 Pitkin Street

East Hartford, CT 06108

dc_ct@usgs.gov

or visit our Web site at:

http://ct.water.usgs.gov
Beaulieu and others—Mercury Bioaccumulation Studies in the National Water-Quality Assessment Program—Biological Data From N.Y. and S.C., 2005–2009—Data Series 705