U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2008–1269

Prepared in cooperation with the National Park Service

Protocol for Monitoring Metals in Ozark National Scenic 

Riverways, Missouri: Version 1.0
Cover Photographs. Shells of Asian clams (Corbicula fluminea) on the bottom of a stream (background). Photograph by B. Poulton, 

U.S. Geological Survey. Asian clams, Corbicula fluminea (top left inset). Photograph by B. Poulton, U.S. Geological Survey. The Current 

River at Cave Spring (right inset). Photograph by D. Hardesty, U.S. Geological Survey. Golden crayfish, Orconectes luteus (bottom left 

inset). Photograph courtesy of R. DiStefano, Missouri Department of Conservation.
Protocol for Monitoring Metals in Ozark 

National Scenic Riverways, Missouri: 

Version 1.0

By Christopher J. Schmitt, William G. Brumbaugh, John M. Besser, Jo Ellen 

Hinck, David E. Bowles, Lloyd W. Morrison, and Michael H. Williams

Prepared in cooperation with the National Park Service

Open-File Report 2008–1269

U.S. Department of the Interior

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

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2008

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Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Schmitt, C.J., Brumbaugh, W.G., Besser, J.M., Hinck, J.E., Bowles, D.E., Morrison, L.W., and Williams, M.H., 2008, 

Protocol for monitoring metals in Ozark National Scenic Riverways, Missouri: Version 1.0: U.S. Geological Survey 

Open-File Report 2008–1269, 42 p.
iii

Contents

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

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

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

Sampling Design ............................................................................................................................................2

Spatial Design........................................................................................................................................2

Selecting the Stretches to be Sampled ...................................................................................3

Temporal Design ...................................................................................................................................3

Monitoring Organism Selection .........................................................................................................3

Crayfish..........................................................................................................................................6

Asian Clam ....................................................................................................................................6

Methods...........................................................................................................................................................7

Field Season and Sampling Preparations.........................................................................................7

Field Methods........................................................................................................................................7

Crayfish..........................................................................................................................................7

Asian Clams ..................................................................................................................................8

Alternate Species ........................................................................................................................8

Chemical Analysis and Laboratory Quality Control.........................................................................8

Overview........................................................................................................................................8

General Analytical Quality Control............................................................................................9

Instrument Calibration and Calibration Verification......................................................9

Blanks and Blank Verification...........................................................................................9

Detection Limits ..................................................................................................................9

Post-Digestion Spikes........................................................................................................9

Standard Additions.............................................................................................................9

Method Precision .............................................................................................................10

Standard Reference Materials.......................................................................................10

Pre-Digestion Spikes........................................................................................................10

Instrumental Precision.....................................................................................................10

Background and Spectral Interference Check............................................................10

Procedural Errors and Deviations..................................................................................10

Analytical Data Review and Completeness .................................................................10

Data Management..............................................................................................................................11

Overview of Database Design .................................................................................................11

Data Entry....................................................................................................................................11

Data Verification and Editing ...................................................................................................11

Metadata Procedures...............................................................................................................12

Database Versions.....................................................................................................................12

Database Security .....................................................................................................................12

Data Analysis and Interpretation .....................................................................................................12

Control Charts.............................................................................................................................12

Toxicity Threshold Development.............................................................................................13
iv

Assumptions and Uncertainties ..............................................................................................13

Reporting.....................................................................................................................................15

Personnel Requirements and Training............................................................................................15

Roles and Responsibilities .......................................................................................................15

Qualifications and Training.......................................................................................................15

Operational Requirements..........................................................................................................................15

Annual Workload and Field Schedule .............................................................................................15

Facility, Equipment, and Personnel Needs .....................................................................................16

Procedure for Revising the Protocol and Archiving Previous Versions of the Protocol .................16

Standard Operating Procedure 1: Preparation for Sampling, Version 1.0 (July 28, 2008)...............18

Revision History Log...........................................................................................................................18

General Preparation and Review.....................................................................................................18

Scheduling Field Work .......................................................................................................................19

Organizing Supplies and Equipment................................................................................................19

Pre-Sampling Procedures.................................................................................................................19

Standard Operating Procedure 2: Collecting, Processing, and Preparing Samples of Crayfish 

and Asian Clams, Version 1.0 (July 28, 2008) .............................................................................21

Revision History Log...........................................................................................................................21

Safety Note ..........................................................................................................................................21

Additional Notes..................................................................................................................................21

Procedures...........................................................................................................................................21

Crayfish........................................................................................................................................21

Asian Clams ................................................................................................................................22

Standard Operating Procedure 3: After the Field Collections, Version 1.0 (July 28, 2008) .............24

Revision History Log...........................................................................................................................24

Procedures...........................................................................................................................................24

Standard Operating Procedure 4: Data Management, Version 1.0 (July 28, 2008)..........................25

Revision History Log...........................................................................................................................25

Data Model...........................................................................................................................................25

Data Preparation.................................................................................................................................25

Data Entry.............................................................................................................................................26

Standard Operating Procedure 5: Data Analysis, Version 1.0 (July 28, 2008)...................................27

Revision History Log...........................................................................................................................27

Parameter Estimation.........................................................................................................................27

Control Charts......................................................................................................................................27

Example Based on 2005 Data............................................................................................................28

Constructing the Control Charts.......................................................................................................28

Standard Operating Procedure 6: Reporting, Version 1.0 (July 28, 2008)..........................................30

Revision History Log...........................................................................................................................30

Report Format......................................................................................................................................30

Template ......................................................................................................................................30

Style. .........................................................................................................................................30

Types of Reports and the Review Process .....................................................................................30

Report Distribution..............................................................................................................................30

Standard Operating Procedure 7: After the Field Season, Version 1.0 (July 28, 2008)....................31

Revision History Log...........................................................................................................................31
v

Procedures...........................................................................................................................................31

Standard Operating Procedure 8: Revising the Protocol, Version 1.0 (July 28, 2008) .....................32

Revision History Log...........................................................................................................................32

Procedures...........................................................................................................................................32

Acknowledgments.......................................................................................................................................32

References Cited..........................................................................................................................................33

Appendix 1. Invertebrate field data form, sample labels, and chain-of-custody record.................39

Figures

1. Map of Ozark National Scenic Riverways (ONSR)..................................................................4

2. Example control chart for tracking concentrations of metals in crayfish and Asian 

clam...............................................................................................................................................27

Tables

1. Reaches and stretches selected for monitoring.....................................................................5

2. Estimated personnel, travel, and analytical resources, per collection.............................16

3. Field equipment and supplies for collecting crayfish and Asian clams ............................20

4. Species codes for invertebrate samples................................................................................23

5. No observed adverse effect level toxicity reference values for metals in mammals and 

birds, and maximum 2005 concentrations in crayfish (Orconectes luteus) and Asian 

clam (Corbicula fluminea) from Ozark National Scenic Riverways ...................................28

6. No-effect hazard concentrations and safety factors of metals in crayfish (Orconectes 

spp.) and Asian clam (Corbicula fluminea) for receptor wildlife species .........................29
vi

Conversion Factors

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

kilometer (km) 0.6214 mile (mi)

meter (m) 1.094 yard (yd) 

Area

square meter (m2) 0.0002471 acre 

hectare (ha) 2.471 acre

hectare (ha) 0.003861 square mile (mi2) 

square kilometer (km2) 0.3861 square mile (mi2)

Flow rate

cubic meter per second (m3/s) 22.83 million gallons per day (Mgal/d) 

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

milligram (mg) 0.000035 ounce (oz)

kilogram (kg) 2.205 pound avoirdupois (lb)

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

°F=(1.8×°C)+32

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

°C=(°F-32)/1.8

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

Concentrations of chemical constituents are given in micrograms per gram (μg/g; parts-permillion).
Abstract

The National Park Service is developing a monitoring 

plan for the Ozark National Scenic Riverways in southeastern Missouri. Because of concerns about the release of lead, 

zinc, and other metals from lead-zinc mining to streams, the 

monitoring plan will include mining-related metals. After 

considering a variety of alternatives, the plan will consist of 

measuring the concentrations of cadmium, cobalt, lead, nickel, 

and zinc in composite samples of crayfish (Orconectes luteus

or alternate species) and Asian clam (Corbicula fluminea) collected periodically from selected sites. This document, which 

comprises a protocol narrative and supporting standard operating procedures, describes the methods to be employed prior 

to, during, and after collection of the organisms, along with 

procedures for their chemical analysis and quality assurance; 

statistical analysis, interpretation, and reporting of the data; 

and for modifying the protocol narrative and supporting standard operating procedures. A list of supplies and equipment, 

data forms, and sample labels are also included. An example 

based on data from a pilot study is presented. 

Introduction

Ozark National Scenic Riverways (ONSR) is situated 

within the Ozark Plateau in southeastern Missouri. Managed by the National Park Service (NPS), ONSR comprises 

approximately 33,265 hectares and includes 216 km of 

the Current River and the Jacks Fork. Famous for its large 

freshwater springs, caves, spring-fed rivers, and recreational 

opportunities, ONSR lies in a landscape of oak-hickory forest 

and pastoral river valleys. Canoeing, camping, and sport fishing are especially popular recreational activities. The Current River and Jacks Fork support populations of the Ozark 

hellbender (Cryptobranchus alleganiensis bishopi), a large, 

predatory salamander considered endangered in Missouri that 

has been proposed for Federal listing (Missouri Department of 

Conservation, 2007). 

The highly mineralized Ozark Plateau contains economically significant lead-zinc deposits of the Mississippi Valley 

Type (Goldhaber and others, 1995). Surface lead-zinc deposits 

were discovered by early French explorers; these and subsequently discovered deposits have been exploited at varying 

levels of intensity since the early 1700s. Advances in mining 

technology facilitated deep mining, which was and remains 

focused in two southeast Missouri districts: the “Old Lead 

Belt,” primarily located in Washington and St. Francois counties, was active from about 1700 until the early 1970s (Schmitt 

and others, 1984; Gale and others, 2004); and the “New Lead 

Belt,” primarily located in Crawford, Iron, and Reynolds 

counties, which became active in the 1960s and where lead 

and zinc are still mined (Wixson and Jennett, 1975; Wixson, 

1978; Proctor, 1984; Imes, 2002). These metals, along with 

other potentially toxic byproduct metals including cadmium, 

cobalt, and nickel, are released to the environment from mining and ore processing. 

Most mining and ore processing in the New Lead Belt 

occurs on lands that formerly were part of the Mark Twain 

National Forest. The southernmost extent of the New Lead 

Belt is located only about 32 km northeast of ONSR. Additional potentially exploitable deposits have been discovered 

within the Mark Twain National Forest in Shannon County 

(Imes, 2002). Although the exploration area lies in the surfacewater drainage of the Eleven Point River, it is relevant to 

ONSR because it is in the ground-water recharge area of Big 

Spring, an important feature within ONSR that contributes 

substantially to flows in the Current River (Imes, 2002; Imes 

and others, 2007). 

Ambient concentrations of lead and other metals from 

natural sources in the Ozarks have been augmented by releases 

from mining and ore processing activities. Karst features such 

as sinkholes, conduits, and springs in the area may facilitate the transport of mining-associated contaminants from 

runoff and discharges (Imes, 2002; Imes and others, 2007). 

Although lead does not accumulate to high concentrations in 

aquatic organisms, elevated concentrations and effects such 

as biochemical responses in fish and altered benthic fish 

and macro-invertebrate community composition have been 

Protocol for Monitoring Metals in Ozark National Scenic 

Riverways, Missouri: Version 1.0

By Christopher J. Schmitt1, William G. Brumbaugh1, John M. Besser1, Jo Ellen Hinck1, David E. Bowles2, 

Lloyd W. Morrison2, and Michael H. Williams2

1

 U.S. Geological Survey

2

 National Park Service
2 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

associated with the release of metals from the Old Lead Belt 

and New Lead Belt (Schmitt and Finger, 1982, 1987; Whelan, 

1983; Schmitt and others, 1984, 1987, 1992, 1993, 2005, 

2006, 2007a, 2007b, 2007c; Dwyer and others, 1988; Gale and 

others, 2004; Brumbaugh and others, 2005; Besser and others, 

2007a; Allert and others, 2008). A human consumption advisory also has been issued for fish from some mining-contaminated streams because of elevated lead concentrations (Missouri Department of Health and Senior Services, 2008). ONSR 

managers have expressed a need to define and track concentrations of lead and other potentially toxic metals. These needs 

may become more acute if additional economically significant 

lead-zinc deposits are located and exploited. Out of concern 

for human and ecological health, monitoring would enable 

ONSR to take all necessary actions to meet applicable federal, 

state, and local laws and regulations and better support the 

management goals of ONSR in accordance with the park’s 

enabling legislation and NPS management policies.

Purpose and Scope

The National Parks Omnibus Management Act of 1998 

and NPS policy require that park managers know the condition of natural resources under their stewardship, and monitor 

long-term trends in those resources to fulfill the agency mission of conserving parks unimpaired. Accordingly, a comprehensive monitoring plan developed by the Heartland Inventory and Monitoring Network (HTLN) of the NPS calls for 

periodic assessments of stream condition at ONSR (DeBacker 

and others, 2005). The U.S. Geological Survey (USGS), in 

cooperation with HTLN, developed this protocol for monitoring metals in waters of ONSR. The specific goals of monitoring conducted under this protocol are to: determine the current 

environmental concentrations of lead and other metals in the 

rivers of ONSR to provide reference points for future comparisons; determine the natural variability of metals concentrations 

in the biota inhabiting these streams; and document changes in 

the environmental concentrations of lead and other metals to 

better understand the dynamic nature and condition of ONSR 

streams and risks to biota.

Monitoring will consist of measuring concentrations of 

metals in samples of invertebrates collected periodically from 

ONSR. A pilot study using a draft version of this protocol was 

conducted to document baseline (2005) concentrations of lead 

and other metals (Schmitt and others, 2007b). Version 1.0 of 

the protocol reflects the results of and experience gained from 

the pilot study. Future monitoring will determine if concentrations are increasing and if riparian wildlife, aquatic organisms, or both are exposed to potentially toxic concentrations 

of lead or other metals. Monitoring conducted under related 

protocols will determine the status of fish and benthic macroinvertebrate communities (Bowles and others, 2007; Petersen 

and others, 2007).

The protocol is comprised a protocol narrative, eight 

standard operating procedures (SOPs), and three appendices 

(at the back of the report). This organization will facilitate 

modification of individual SOPs by the NPS.

Sampling Design

An integrated aquatic monitoring plan is being developed for ONSR that will include the co-location and potential co-visitation of sites to document “vital signs” (Fancy, 

2001). Vital signs are measurement variables selected to 

reflect the condition of National Parks. The framework for the 

ONSR monitoring plan was conceived during a workshop of 

biologists, statisticians, and administrators held in July 2004 

(McDonald, 2004). The protocol described here focuses on 

contamination from lead-zinc mining, which is one of the vital 

signs selected for ONSR.

Spatial Design

A common sample unit has been defined to accommodate 

the field protocols for monitoring all vital signs. The common sample unit is defined as a “stretch” of contiguous river 

of some minimum and maximum length. The geomorphology 

of the waterways within ONSR and the resulting biological 

processes are scale-dependent; that is, the distances associated with pool-riffle sequences increase as river size and 

flow increase. A key characteristic of the overall monitoring 

design is that all aquatic studies should be capable of producing unbiased estimates that are applicable to the entire stretch. 

Although stretches must be long enough to accommodate 

sampling to produce unbiased estimates for all studies, they 

do not have to be the same length. Once defined, sample unit 

boundaries remain fixed, and are used by all studies conducted 

under the unified monitoring design. 

Two categories of stretch sizes have been established. In 

tributaries and upper main stems, stretch lengths are 1 to 2 km; 

in lower main stems, stretch lengths are 3 to 5 km. Within 

categories, stretch length is not fixed, but varies depending on 

several factors. Stretches were broken at natural features, such 

as confluences and springs. They also were delimited based on 

Valley Segment Type (VST) information. The initial sample 

frame of stretches was developed through a collaborative 

agreement with the Missouri Resource Assessment Partnership (MORAP). Missouri Aquatic Gap datasets were used; 

these are the same datasets used by the Missouri Department 

of Natural Resources (DNR) and the Missouri Department of 

Conservation (MDC). MORAP used a subset of data from the 

1:100,000 National Hydrography Dataset (NHD) developed 

by USGS and the U.S. Environmental Protection Agency 

(USEPA). The dataset includes arcs representing the centerlines of wide streams and the segments of single-line streams. 

Selected attributes of the segments were extracted from various NHD tables using an Arc/Info® [Environmental Systems 
Sampling Design 3

Research Institute (ESRI), Redlands, Calif.] macro; the attributes were attached directly to the arc component on the arc 

segments. These stream segments were classified according to 

variables such as temperature, stream size, flow, geology, soil 

texture, relative gradient, valley wall interaction (a surrogate 

for potential bluff pool habitat), stream size discrepancy, and 

channel type. Thus, each stretch has associated with it characteristics based on geographic information system (GIS) 

data that could be used in statistical analyses as covariates or 

domains (that is, subpopulations of interest for which parameter estimates are desired). The dataset was restricted to those 

stream segments that touched the ONSR jurisdictional boundary or other adjacent public lands. The final sample frame was 

established by removing all stretches that were not entirely 

or partially within ONSR boundaries (the MORAP dataset 

included adjacent public lands). All secondary channels, which 

occur where a waterway splits and flows around an island, 

were then removed because they transport the smaller volume 

of water.

Selecting the Stretches to be Sampled

Spatial balance is important because all responses are 

known to be spatially autocorrelated (that is, units close to 

one another tend to respond similarly). When responses are 

correlated in space, spatial balance can greatly improve the 

precision of the resulting estimates. The Generalized Random 

Tessellation Stratified (GRTS) method of sample selection 

(Stevens and Olsen, 1999, 2004) was employed to achieve 

spatial balance. The GRTS technique, which generates a random sample that is spatially balanced, allows multiple studies 

to maximize overlap of selected streams by utilizing a common sample. It also allows units to be added without bias after 

an initial sample has been drawn. Additionally, because GRTS 

samples are not evenly spaced, sample locations are not in 

phase with a longitudinally cyclic response. Perhaps the most 

desirable characteristic of GRTS is that for any sample size, 

any subset of stretches in the ordered GRTS sample constitutes 

a spatially balanced sample. This characteristic is desirable 

because it allows multiple studies to maximize overlap and 

add stretches in a way that guarantees spatial balance. 

The GRTS samples were drawn with the S-Draw program 

(Western Ecosystem Technologies, Inc., 2008), with the main 

stem sites weighted by stretch length. The S-Draw program 

allows for several options in drawing the sample. The hierarchical structure was randomized (Stevens and Olsen, 1999). 

The reverse hierarchical ordering option, which assures that 

any contiguous set of stretches will be spatially balanced, was 

used (Stevens and Olsen, 2004) with a random number seed 

generated from the system clock (the default option).

All GRTS draws were “oversampled” (that is, more sites 

were selected and ordered than will be immediately sampled). 

This allows for an increase in the number of sites in the future 

(if budget allows) without decreasing the overall degree of 

spatial balance. Oversampling also provides the flexibility 

not to sample certain sites. In such a situation, sampling can 

progress to the next site in the ordered GRTS list while sacrificing only a small degree of spatial balance.

A greater degree of control was desired for the main stem 

than was possible by selecting all stretches from the same 

pool with GRTS (which has a strong random element). The 

Jacks Fork, Upper Current River, and Lower Current River 

(upstream and downstream, respectively, of the confluence 

with the Jacks Fork) differ profoundly, primarily due to the 

influence of large springs. A total of 130 stretches comprised 

the sample frame for these main stems. Stretches on the Jacks 

Fork (n=39) and Upper Current River (n=53) are approximately 1 to 2 km long. Stretches on the Lower Current River 

upstream from Van Buren (where a break in the ONSR boundary occurs) were approximately 1 to 2 km long, and stretches 

below Van Buren were approximately 3 to 5 km long. The 

Current River below Van Buren has greater flows, in large 

part because of the input of Big Spring [12.2 cubic meters 

per second (m3/s)]. A total of 38 stretches were identified on 

the Lower Current River. An equal number of sample sites 

on each of these three main stem sections was desired. Thus, 

the main stems within ONSR were divided into three groups 

[Jacks Fork, Upper Current River (above confluence with the 

Jacks Fork), and Lower Current River (below the Jacks Fork)] 

before the GRTS sample was selected. Following these criteria, GRTS was used to order 64 main stem stretches within 

ONSR. Although immediate sampling is planned only for the 

first nine stretches (fig. 1, table 1), this procedure will allow 

an increase in the number of stretches sampled in the future or 

integration with other studies with a larger sample size without 

sacrificing spatial balance.

The total number of stretches to be sampled annually in 

the unified monitoring design is limited primarily by budget 

and personnel. Taking into account the other HTLN protocols 

that will be implemented at ONSR, it was determined that 12 

total stretches could be sampled (nine main stem stretches and 

three tributary stretches). However, metals monitoring will 

focus on the nine main stem stretches, with the addition of 

the downstream-most non-floodplain stretch of Blair Creek, 

a tributary of the Current River (fig. 1, table 1). There is 

substantial interest in Blair Creek because of its proximity to 

active mining in the New Lead Belt.

Temporal Design

All nine main stem stretches and Blair Creek will be 

sampled at least once every 5 years. There are presently 

(2008) no mining activities in any ONSR surface watersheds; 

however, in the event that mining activity expands, the sampling frequency can be increased.

Monitoring Organism Selection

Metal concentrations can be monitored in samples 

representing virtually all living and non-living components 

of aquatic ecosystems (water, sediment, plants, and animals). 
4 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Mountain

View

Summersville

TEXAS

COUNTY

Birch Tree

HOWELL

COUNTY

CURRT06

JACKM03

JACKM02

JACKM01

CURRM06

CURRM06

CURRM04 CURRM05

CURRM03

CURRM02

CURRM01

SHANNON

COUNTY

CARTER

COUNTY

REYNOLDS

COUNTY

DENT

COUNTY

OREGON

COUNTY

RIPLEY COUNTY

Winona Van Buren

Eminence

Ellington

Bunker

Centerville

Jack’s Fork

Current River

Current River

Blair Creek

0 2 4 6 8 MILES

0 2 4 6 8 KILOMETERS

EXPLANATION

Current River watershed

Ozark National Scenic Riverways

Sample site and map identifier

91°40' 30' 20' 10' 91°

37°20'

10'

37°

36°50'

Index map

MISSOURI

Study area

Current River

watershed

Base from U.S. Geological Survey digital data, 1:100,000, 2000

Universal Transverse Mercator projection,

Zone 15

Horizontal coordinate information is referenced to the North American

 Datum of 1983 (NAD 83)

Figure 1. Ozark National Scenic Riverways (ONSR).
Sampling Design 5

Each sample type has advantages and disadvantages that differ 

among the parameters (in this example, metals) to be monitored. General criteria for selecting a sample type or types 

include spatial and temporal variability, analytical detection 

thresholds, costs, and the degree to which concentrations in a 

given matrix represent “environmental conditions,” including 

site fidelity—the extent to which an organism represents the 

location from which it was collected. Effects of contaminant 

exposure at many levels of biological organization (biochemical, organ, organism, population, and community) also can be 

monitored; however, except for certain specific biochemical 

responses (Schmitt and others, 1984, 2007c), most effects are 

not contaminant-specific and can be affected by many factors 

in addition to chemical exposure. 

Crawford and Luoma (1993) completed an extensive literature review pertaining to organic and inorganic contaminant 

monitoring in biota as part of the USGS National Water-Quality Assessment Program (NAWQA) development (Hirsch and 

others, 1988). The following criteria were identified for choosing species for chemical analysis: Chemical concentrations in 

the organisms should be responsive to environmental exposure; uptake of contaminants by organisms should be rapid; 

concentrations in the organisms should be greater than those 

in water; the organisms must not be killed by exposure to low 

levels of the contaminants to be monitored; concentrations 

in organisms should vary little within a site; the organisms 

should be relatively sedentary so as to reflect concentrations 

in the collection locale; the organisms should be abundant 

and widespread in the study area to facilitate comparisons; 

the organisms should be sufficiently large, abundant, or both 

to provide adequate tissue for analysis; the organisms should 

be sufficiently long-lived to integrate exposure concentrations 

over at least several months; and the organisms should be easy 

to sample (Crawford and Luoma, 1993). An additional consideration of the NPS is to minimize the removal of aquatic 

organisms, especially those considered to be rare, recreationally significant, or ecologically significant. 

A problem shared by all programs and protocols relying 

on the collection of indigenous organisms is that there are no 

truly ubiquitous large, long-lived, abundant, and sedentary 

species that accumulate all contaminants equally well. Of the 

organisms available for consideration in ONSR, certain invertebrates meet most requirements for trace-metal monitoring. 

Most aquatic macroinvertebrates are comparatively sedentary, 

and generally they do not regulate metals as well as fish. 

Many invertebrates also ingest varying amounts of particulate material (and its contaminants) from the water column, 

bed sediment, or both depending on feeding guild (Goodyear 

and McNeill, 1999). Consequently, metals concentrations in 

invertebrates tend to reflect environmental concentrations 

comparatively well (Crawford and Luoma, 1993; Goodyear 

and McNeill, 1999). The NAWQA program considered sampling fish, invertebrates (mollusks, crayfish, aquatic insect 

larvae), and plants (attached algae, macroalgae, macrophytes), 

and ultimately adopted a step-down approach for trace metals 

depending on taxa available at a site. Organisms are sampled 

by NAWQA in the following order of preference: Asian clams 

(Corbicula fluminea), aquatic insects, target fish species (as 

identified by Crawford and Luoma, 1993), and aquatic plants. 

In the Ozark Plateaus Study Unit (Petersen and others, 1998), 

Table 1. Reaches and stretches selected for monitoring.

[Easting and northing; Universal Transverse Mercator Projection, NAD83, Zone 15N]

Reach ID 

(fig. 1)

River 

basin Site type Site number County Stretch ID

Lower 

stretch 

easting

Lower 

stretch 

northing

Lower 

reach 

easting

Lower reach 

northing

CURRM01 Current Main stem 01 Shannon 14 623336.28 4141730.82 623330.810 4141819.358

CURRM02 Current Main stem 02 Shannon 35 637468.41 4131822.17 637113.180 4131465.688

CURRM03 Current Main stem 03 Shannon 42 643252.57 4126300.08 642631.838 4126422.535

CURRM04 Current Main stem 04 Shannon 67 661785.40 4111783.86 662104.165 4112724.645

CURRM05 Current Main stem 05 Shannon 71 666195.85 4111128.94 666087.431 4111411.528

CURRM06 Current Main stem 06 Carter 97 684220.63 4078321.12 685209.621 4079792.188

JACKM01 Jacks Fork Main stem 01 Shannon 105 619895.88 4101117.92 619901.956 4101344.874

JACKM02 Jacks Fork Main stem 02 Shannon 114 627768.31 4102604.03 627579.024 4102191.921

JACKM03 Jacks Fork Main stem 03 Shannon 123 633244.58 4108431.69 633452.088 4108220.025

CURRT06 Current Tributary (Blair Creek) 06 Shannon 917 659132.19 4116340.75 659132.190 4116340.750
6 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

which included waters of the ONSR, Asian clams were collected and analyzed by NAWQA where the species could 

be found. Liver tissue from several fish species (Crawford 

and Luoma, 1993) were sampled elsewhere; samples were 

obtained preferentially from longear sunfish (Lepomis megalotis) and, secondarily, from smallmouth bass (Micropterus 

dolomieu). Crayfish met all the monitoring criteria identified 

by NAWQA, but they were not selected because information was not available on the uptake and retention of organic 

chemicals by these organisms (Crawford and Luoma, 1993). 

Although organic chemicals remain a priority for NAWQA, 

they are not presently (2008) an issue in ONSR. Based on a 

discussion of these factors with NPS personnel, crayfish and 

Asian clams were selected for monitoring at ONSR. Attributes 

of these organisms for metals monitoring are summarized in 

the following sections.

Crayfish

Crayfish constitute a large percentage of the invertebrate biomass in Ozark streams (Hobbs, 1993; Momot, 

1995; Rabeni and others, 1995; Whitledge and Rabeni, 1997; 

DiStefano, 2005). They are considered “ecological dominants” 

(Simberloff, 1998) that shape the entire aquatic community. 

Crayfish are opportunistic omnivores that feed on varying proportions of fish, aquatic invertebrates, periphyton, and detritus 

during their life cycle (Hobbs, 1993; Momot, 1995; Whitledge 

and Rabeni, 1997; Parkyn and others, 2001). Crayfish process 

large quantities of organic material (including macrophytes, 

attached algae, and detritus) and represent a significant food 

source for smallmouth bass, other fishes, and riparian wildlife (Probst and others, 1984; Whitledge and Rabeni, 1997; 

DiStefano, 2005). Crayfish tend to accumulate non-essential 

metals such as lead and cadmium in proportion to exposure, 

but may be able to regulate copper and zinc (Gillespie and 

others, 1977; Dickson and others, 1979; Knowlton and others, 1983; Crawford and Luoma, 1993; Schmitt and others, 

2007b). Lead concentrations in crayfish from mining-affected 

streams in southeast Missouri were well correlated with those 

in sediments, but were lower by a factor of about five (Schmitt 

and Finger, 1982). Most metals in crayfish concentrate in 

the hepatopancreas, antennal (green) gland, exoskeleton, 

and digestive tract (Dickson and others, 1979; Roldan and 

Shivers, 1987; Crawford and Luoma, 1993); however, mercury 

also accumulates in muscle (Allard and Stokes, 1989). The 

NAWQA program considered dissecting the hepatopancreas 

from crayfish for analysis (Crawford and Luoma, 1993); however, metals in whole crayfish (including the contents of the 

digestive tract) represent the concentrations to which higherlevel organisms are exposed (Schmitt and others, 2006). In 

addition, there is a large body of extant data describing metals 

concentrations in whole crayfish from streams representing the 

range of metals concentrations present in Missouri to which 

concentrations in ONSR crayfish can be compared (Wixson, 

1978; Schmitt and Finger, 1982; Whelan, 1983; Allen and 

Wilson, 1992; Wildhaber and others, 1997; Besser and others, 

2007a; Allert and others, 2008). There also are uptake data 

from locally relevant controlled studies (Knowlton and others, 

1983; Besser and Rabeni, 1987), and the analysis of metals in 

crayfish represents an important component of ongoing USGS 

studies related to lead-zinc mining in southern Missouri (Imes, 

2002; Besser and others, 2007a; Allert and others, 2008). 

Riffle-dwelling crayfish of the genus Orconectes, which 

are distributed across the Ozark Plateau, were targeted for 

monitoring. The golden crayfish (O. luteus) is widespread 

in the Ozarks (Pflieger, 1996) and is the prevalent species 

at ONSR (Rabeni and others, 1995). Consequently, and 

even though it does not grow as large as some other species, 

O. luteus is the preferred species. The spothanded crayfish 

(O. punctimanus) is the primary alternate species in the 

event that golden crayfish are not available at a site, followed 

by the Ozark crayfish (O. Ozarkae) and Hubbs’ crayfish 

(Cambarus hubbsi). 

Crayfish molt several times during the growing season, 

which may affect metals concentrations; however, reported 

effects of size on metals concentrations vary. For example, 

Knowlton and others (1983) indicated that lead concentrations 

decreased with size, but Dickson and others (1979) reported 

no size-related concentration effects for metals. Samples 

should nevertheless be collected as consistently as possible 

with respect to sampling period and crayfish size. 

Asian Clam

The Asian clam is an exotic species that has become 

established throughout much of North America. It is extremely 

invasive and is regarded as a threat to indigenous mussels 

and other native aquatic organisms (McMahon, 1983; Oesch, 

1995). In 1992, when NAWQA sampled the Current River 

and Jacks Fork for contaminants in biota, Asian clams were 

obtained only at the downstream-most site on the Current 

River at Van Buren (Petersen and others, 1998). This species 

has since been found throughout most of the Current River and 

Jacks Fork within ONSR (Schmitt and others, 2007b). 

The use of the Asian clam for contaminants monitoring 

has grown in popularity as the species has spread. As a result, 

and as noted by Crawford and Luoma (1993), protocols for 

monitoring with Asian clams are well documented (Graney 

and others, 1983; Foe and Knight, 1987; Leland and Scudder, 

1990). Asian clams accumulate most metals in proportion to 

exposure, with the possible exception of zinc (Crawford and 

Luoma, 1993). Laboratory exposure studies indicate a concentration-dependent linear uptake of mercury and cadmium, but 

with a plateau effect for cadmium, and that concentrations persist for at least 30 days post-exposure (Inza and others, 1998). 

Studies with caged, transplanted Asian clams indicate that 

tissue concentrations may not reflect zinc gradients in water or 

sediment as well as those of cadmium (Baudrimont and others, 

1999). Schmitt and others (2007b) reported that cobalt concentrations in Asian clams at sites within and outside ONSR 

were relatively uniform, indicating that internal concentrations 

may be regulated; however, zinc concentrations in Asian clams 
Methods 7

differed. Data from mining-affected parts of the Spring River 

Basin (Missouri, Kansas, and Oklahoma) indicate that concentrations of lead, cadmium, and zinc in wild Asian clams are 

correlated with concentrations in sediment (Angelo and others, 

2007). Concentrations of cadmium, but not of lead or zinc, 

also increase with an increase of shell diameter (Angelo and 

others, 2007). Consequently, and as described for crayfish, the 

collection of similar sized Asian clams should be the objective.

Protocols for metals monitoring with Asian clams vary 

with respect to the incorporation of a depuration period. The 

NAWQA program incorporated a 24-hour depuration period; 

however, concentrations of lead, but not cadmium or zinc, 

may decline after depuration for 24 hours because of the 

preferential binding of lead to sediment particles (Angelo and 

others, 2007). Nevertheless, a depuration period is included 

in the protocol for ONSR for consistency with previous studies, with the recognition that it probably reduces the original 

lead concentration.

Methods

Field Season and Sampling Preparations

A list of field equipment and supplies is provided in 

SOP 1; data forms (appendix 1) and sample labels (appendix 2) can be found at the back of this report. Extreme care 

must be exercised to avoid external contamination, including 

cross-contamination between samples, when sampling for 

trace metals (Schmitt and Finger, 1987). Sampling equipment 

and containers must be thoroughly cleaned between samples 

and sites and stored in plastic bags, and personnel handling 

samples will wear disposable gloves. Work surfaces must 

be covered with disposable material (paper or polyethylene) 

that is cleaned or replaced between samples, and should be 

used only at one site if samples from multiple sites will be 

processed. Because pre-cleaning containers requires acid 

solutions and ultra-pure water that are generally not available 

to NPS personnel, the protocol maximizes the use of commercially available pre-cleaned containers and disposable items 

that do not have to be acid-cleaned.

Preventing the introduction of invasive species and 

mitigating against the further spread of those already present 

at ONSR is a tacit objective of this and other NPS monitoring protocols. The Asian clam is present at ONSR, but other 

aquatic nuisance species such as the zebra mussel (Dreissena 

polymorpha) and didymo algae (Didymosphenia geminate) are 

not; however, both species are present in northern Arkansas, 

and zebra mussels are present elsewhere in Missouri. To prevent their introduction, all equipment, containers, boots, and 

personal flotation devices (PFDs) used for sampling at ONSR 

should be labeled as such and not used elsewhere, and should 

be thoroughly cleaned and air-dried for at least 48 hours 

between uses.

The preferred collection period is September, but sampling during August or October is acceptable (see SOP 2). 

Weekday sampling is preferable to weekends, to minimize 

contact with and interference from ONSR visitors. For safety 

reasons, a two-person team is recommended for aquatic field 

collections. Correctly sized PFDs are required for all personnel working in or near water regardless of season. Personnel 

that will collect crayfish and Asian clams should be able to 

identify them to species in the field and be familiar with the 

procedures described here and in the related SOPs. A resume 

of all personnel involved in the study should be on file at the 

HTLN office along with documentation that these individuals 

have been trained in all pertinent procedures. The movement 

of samples also should be documented; an example chain-ofcustody record, which accompanies samples as they change 

hands, is given in appendix 3 (at the back of this report).

Depending on the personnel involved and collection 

locations, scientific collection permits from MDC, the NPS, or 

both may also be required. Collections in MDC Natural Areas 

adjacent to ONSR (such as Blue Spring) may also require 

special permission. Applications for these permits must be 

submitted early in the sampling year to ensure approval, and 

both permits require an annual report. The address for obtaining MDC permits is given in SOP 1. Under an MDC permit, 

the Conservation Agent in the county of collection must also 

be notified in advance, and the permittee must be present during the collections. 

Field Methods

For each selected stretch, three consecutive riffles will 

be chosen to represent a sample reach at the time of collection. Riffle selection is determined before sampling; the three 

riffles should be those located in consecutive order upstream 

from the first riffle above the lower geographic boundary of 

the selected stretch. Sampling procedures are described in 

more detail in SOP 2. Sample storage and shipment and other 

procedures to be followed upon completion of sampling are 

described in SOP 3.

Crayfish

Crayfish are captured by kick-seining or kick-netting 

in riffles containing coarse substrate or along the margins of 

emergent vegetation beds. They are removed from the nets and 

held alive in lined plastic buckets containing ambient water. 

Crayfish of 18 to 25 millimeters (mm) carapace length, which 

are typically young-of-the-year, are preferred. Crayfish of this 

size were present at all ONSR sites sampled in 2005 (Schmitt 

and others, 2007b). When at least 10 crayfish of the appropriate size have been collected from each riffle, they are transported to shore where the identification (species) of each animal is confirmed and it is measured (carapace length, in mm). 

Following identification and measurement, each individual crayfish is then carefully washed with ambient water and 
8 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

the combined sample of 10 specimens is either double-bagged 

in zip-seal plastic freezer bags or placed in pre-cleaned, prelabeled polyethylene jars. Crayfish that are not the preferred 

species or size are released and more are collected until the 

three samples are completed. Samples may be either chilled 

(on ice) or frozen (in dry ice) in the field, then transferred to 

a freezer [–20° C (degrees C)] upon return to the duty station. 

They are kept frozen until sent to the analytical laboratory, and 

then shipped frozen (in dry ice). The crayfish samples will be 

freeze-dried, homogenized, and analyzed for metals; moisture 

content (percent) may or may not be determined and reported. 

Concentrations are reported as micrograms per gram (μg/g) 

dry-weight (see section on Chemical Analysis and Quality 

Control later in this report). 

Asian clams

Asian clams usually are harvested by gloved hand or 

with dip nets. They may be difficult to locate; during pilot 

studies, Asian clams were found along sandy margins or in 

sandy interstices among cobble. Three composite samples of 

nominally 10 animals each are collected for analysis, with 

10 specimens coming from each of the three riffles described 

previously. The preferred size range based on the extant 

literature is 15 to 25 mm shell width; Asian clams obtained 

from ONSR sites in 2005 by Schmitt and others (2007b) were 

15.5 to 21.4 mm. As described for crayfish, Asian clams are 

held in lined plastic buckets containing ambient water until at 

least 10 specimens of the preferred size have been obtained 

from each riffle. They are transferred to shore where as much 

external material (sediment, algae) as possible is removed 

by hand from the external surfaces of the shell, the identity 

is confirmed, and the specimen is measured. Maximum shell 

diameter is recorded for each clam in each sample, and the 

specimens are transferred to a new, pre-labeled polyethylene 

bag about one third full of ambient water. The bag is then 

filled with oxygen, sealed, and placed in another bag containing a sample label; a sheet of sample labels is appended to the 

back of this report (appendix 2). Remaining specimens are 

released. The labeled, double-bagged sample is transferred to 

a cooler containing ambient water, and the process is repeated 

until three samples of 10 specimens have been obtained. The 

Asian clam samples remain in the bags of ambient water in the 

coolers for 24 hours, which allows them to depurate (that is, 

purge themselves of particulate material). 

Following depuration, the samples either are doublebagged in zip-seal freezer bags or transferred to pre-labeled, 

pre-cleaned polyethylene jars and frozen and shipped as 

described for crayfish. In the laboratory, the soft tissues are 

separated from the shells, freeze-dried, and analyzed for trace 

metals. Metals concentrations are reported as μg/g dry-weight. 

Moisture content (percent) may or may not be determined 

and reported (see section on Chemical Analysis and Quality 

Control later in this report).

Alternate Species

The collection of the target species is desirable, but not 

always possible, and alternate species can be substituted. 

Presently there is no alternate for Asian clam; lacking this 

species, no sample will be collected. If the full complement 

of golden crayfish or spothanded crayfish are not available at 

a site, other crayfish (Ozark crayfish, Hubbs crayfish) may be 

substituted; however, under no circumstance should a composite sample contain more than one species.

Chemical Analysis and Laboratory Quality 

Control

Overview

The Asian clam and crayfish samples from ONSR will 

be analyzed for lead, cadmium, cobalt, zinc, and nickel by 

inductively-coupled plasma mass spectrometry (ICP-MS), the 

most sensitive multi-elemental method currently available to 

most laboratories. High sensitivity is necessary because concentrations are expected to be low based on samples obtained 

in 2005 (Schmitt and others, 2007b). Statistical analysis and 

trend detection based on censored values [that is, less than 

the method detection limits (MDLs)] are inherently difficult 

(Bauch and others, 2005), a problem that is exacerbated when 

detection limits change with time. Censored values greater 

than toxicity thresholds also are inherently problematic 

Accordingly, target MDLs are 0.05 μg/g dry weight (approximately 0.01 μg/g wet weight) for cadmium, cobalt, nickel, and 

lead; and 1.0 μg/g dry weight (0.2 μg/g wet weight) for zinc. 

These can be achieved by ICP-MS. 

Chemical analyses generally should follow Crawford and 

Luoma (1993), May and others (1997), Brumbaugh and others 

(2005), Besser and others (2007a), and Schmitt and others 

(2007b). Composite samples of whole crayfish (with intact 

exoskeletons and digestive tracts) are prepared for analysis by 

lyophilization followed by homogenization to a powder with 

a cryogenic mill. Asian clams are lyophilized whole and the 

soft tissues subsequently separated from the shells. The soft 

tissues of all the clams or crayfish in the composite sample are 

homogenized to a coarse powder by pulverizing them with a 

glass rod. Alternately, if enough mass is available, clam tissue 

is processed with a cryo mill in the same manner as whole 

crayfish. Dried, homogenized samples are digested in nitric 

acid and hydrogen peroxide in a laboratory microwave oven, 

and the digestates are analyzed for lead, cadmium, cobalt, 

zinc, and nickel by ICP-MS (Brumbaugh and others, 2005; 

Besser and others, 2007a).

The chemical analyses can be performed by government, 

university, or private (contract) laboratories. Regardless of 

the laboratory performing the analyses, the analytical plan 

should incorporate field and laboratory replication, procedural blanks, fortified samples (spikes), and standard reference materials. The data should be examined for repeatability 
Methods 9

(mean, minimum, and maximum percent deviation of replicates), blank contamination, recovery efficiency (from spikes), 

and conformance with certified concentrations in reference 

materials. Samples analyzed with associated quality control 

(QC) checks that fail to meet acceptance criteria should be reanalyzed or, in some instances “flagged”—that is, identified as 

having failed one or more QC parameters. Flagged values may 

be used, but may be eliminated from consideration when the 

data are reviewed, interpreted, and reported. Analytical laboratories routinely analyze blanks, standards, and duplicates to 

meet their needs, but not necessarily in the number necessary 

for the monitoring program; however, the cost of analyzing 

field replicates and any additional QC beyond that normally 

performed will have to be budgeted. 

The field and laboratory procedures described in this 

document and its associated SOPs conform to USEPA Good 

Laboratory Practices (USEPA, 1989) and are in general accordance with USEPA Contract Laboratory Program guidelines 

(USEPA, 2004). If implemented as described, these procedures will yield defensible data suitable for monitoring and 

information purposes; however, these data may not be entirely 

adequate to support regulatory procedures or litigation, 

which generally require the development of and documented 

conformance with a Quality Assurance (QA) Plan. General 

guidelines for analytical QC are summarized in the following 

section. 

General Analytical Quality Control

Quality-control measures incorporated at the digestion 

stage of the analyses should include digestion blanks, certified reference materials, replicates, and pre-digestion fortified samples (spikes). A calibration blank and an independent 

calibration verification standard should be analyzed regularly 

to confirm the calibration status of the ICP-MS throughout 

the instrumental analyses. As a check for potential interferences, post-digestion spikes should be performed, and dilution 

percent differences based on five-fold dilutions of the sample 

digestates should be determined. Instrument detection limits 

(IDLs), MDLs, and method quantitation limits (MQLs) should 

be estimated for each element in each type of sample based on 

the standard deviations of the measured concentrations in the 

blanks, which should be computed according to standard published methods (USEPA, 2004). Instrumental precision should 

be determined as the relative percent difference between duplicate analyses of digestates. 

Instrument Calibration and Calibration Verification

At least three standard concentrations and a calibration 

blank should be used for instrument calibration; the coefficient 

of determination (r2) of the regression for each group of measurements should be at least 0.995. A calibration verification 

solution should be prepared to a mid-range concentration from 

an independent stock source and be measured after every 10 

sample analyses. The measured value for this solution should 

be within 10 percent of the expected value. If these criteria 

are not met, the check solution should be re-analyzed. If it 

still fails, the instrument calibration should be repeated, and 

any samples analyzed after the previous acceptable calibration verification should be re-analyzed. If recalibration fails to 

correct the discrepancy, new standards should be prepared, and 

the samples should be re-analyzed. 

Blanks and Blank Verification

Procedural blanks (preferably three per preparation set) 

should be analyzed to provide measurements needed for determining the MDL. Blank equivalent concentrations (BECs) 

should be no more than 10 times the average of the three most 

recently determined IDLs. Re-preparation of all samples in 

non-conforming preparation sets is preferable to flagging; 

however, flagging may be acceptable under some circumstances depending on the concentration of the analyte the relative percent error estimated for blank equivalent concentrations (BECs). If the BECs exceed the previously stated criteria 

but all sample concentrations are at least 10 times greater 

than the greatest BEC, then flagging may be acceptable. If all 

sample concentrations are at least 20 times greater than the 

greatest BEC, no corrective action usually is required. 

Detection Limits

The IDLs and MDLs should be determined periodically 

according to USEPA or other standard, published methods. 

The IDLs should be determined every 6 months. The MDLs 

should be determined for each sample preparation set. In the 

event that a measured MDL fails to meet a target, the analyses 

required for the MDL determination should be repeated. If the 

MDL still fails to meet the criteria, the reason should be determined (for example, procedural contamination or poor instrument performance) and corrected. If necessary, the samples 

should be re-prepared, re-analyzed, or both.

Post-Digestion Spikes

At least one post-digestion spike should be analyzed for 

every 20 samples of each sample type (Asian clam, crayfish) 

to check for sample matrix effects. For each metal in each 

sample type, a suggested target for average recovery of postdigestion spikes is 100 percent ± (plus-or-minus) 10 percent, 

with no individual recovery exceeding ±20 percent. If upon 

re-analysis an individual spike fails to meet the latter criterion, 

or if the average recovery criteria are not met, all samples of 

a similar type should be analyzed by the method of standard 

additions. Alternatively, those results should be flagged.

Standard Additions

Samples for which conventional calibration procedures 

yield unsatisfactory QC results may be re-analyzed by the 

method of standard additions. A blank (“0-X”) and spikes of 

“0.5-X” and “1-X,” where “X” is approximately the low-end 

instrument calibration concentration, will be analyzed. The 
10 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

r2 for each regression should be at least 0.995, otherwise the 

sample should be re-analyzed. If the calculated sample concentration is more than twice X, it should be diluted as necessary and re-analyzed. A calibration verification solution also 

should be analyzed by this method; the result should be within 

+10 percent of the true value. 

Method Precision

An estimate of method precision should be determined 

either from at least one triplicate (preferred) or duplicate preparation (digestion and analysis) per 20 samples of each sample 

type. Computation of the percent relative standard deviation 

(percent RSD) or the relative percent difference (RPD) should 

then be evaluated. Typical targets for biological tissue for the 

mean percent RSD or RPD are ±20 percent; however, these 

targets are valid only for the estimation of methods precision 

if the concentration in the replicated sample exceeds the MDL 

by at least 10-fold. To evaluate instrument precision, at least 

one sample digestate solution should be prepared for duplicate 

analysis for every 20 samples of each type. Reproducibility 

of duplicate readings should be within ±10 percent except at 

concentrations that are ≤ (less than or equal to) 20-fold greater 

than the IDL, which can be greater. Groups of samples for 

which these criteria are exceeded should be re-analyzed; if 

they fail a second time, they should be flagged.

Standard Reference Materials

At least one representative reference tissue should be 

prepared for each type of sample. Appropriate reference materials for crayfish and Asian clams include National Institute of 

Standards and Technology (NIST) oyster (NIST 1566a, 1566b) 

and National Research Council Canada (NRCC) lobster 

hepatopancreas (NRCC TORT-1). For each measured result, 

the suggested target criterion is the certified range (usually 

represented by the 95% confidence interval about the mean) 

extended by ±10 percent of the certified mean. This approach 

provides an allowance for measurements of certified reference 

materials for which the certified 95 percent confidence limits 

are unusually narrow. In addition, these criteria should only 

be applied if the concentration of the analyte in the reference 

material is at least 10-fold greater than the target MDL. 

Pre-Digestion Spikes 

At least two pre-digestion spikes for each analyte should 

be prepared for every 20 samples of each type. The target for 

mean recovery of pre-digestion spikes is 100 ± 20 percent 

with not more than 20 percent of individual spike recoveries exceeding ±50 percent error; however, the second criterion does not apply if effective concentration in the spike is 

not at least 50 percent of the concentration measured in the 

un-spiked sample. Spiked samples meeting this condition but 

failing the acceptance criteria should be flagged. If more than 

one analyte spike recovery result per preparation set fails the 

second criterion, then all samples in the preparation set should 

be re-digested and re-analyzed. Alternatively, all results in the 

digestion set should be flagged for the suspect analyte(s).

Instrumental Precision

At least one sample digestate solution should be prepared 

for duplicate analysis for every 20 samples of each type. 

Reproducibility of duplicate readings should be within ±10 

percent except at concentrations that are ≤20-fold greater than 

the IDL. For concentrations between 10- and 20-fold greater 

than the IDL, agreement should be within ±20 percent. No 

criteria are suggested for values less than 10 times the IDL. 

A duplicate analysis producing variation outside these criteria 

should be re-run. If the result is still outside the criteria, all 

samples in the group should be flagged.

Background and Spectral Interference Check

Spectral interferences from representative samples of 

each type should be checked by one or more of three following methods: (a) analysis at a secondary mass; (b) re-analysis 

after dilution by at least five-fold; and (c) for determinations 

having known interferences, a check solution containing the 

analyte and interfering element(s) can be analyzed. Results 

for (a), (b), or (c) with the original analysis should be within 

10 percent of the alternate, undiluted, or true value. If not, 

troubleshooting and elimination of the interference must be 

documented or an alternate method of analysis performed; 

otherwise, all samples in the group should be flagged.

Procedural Errors and Deviations 

Procedural errors occasionally may occur during sample 

preparation or analysis. In these instances, any corrective actions should be documented. The corrective action 

employed depends upon the stage at which the suspicion 

arose. Procedural errors arising at the chemical preparation 

stage, such as accidental sample spilling or dropping, digestion losses, incorrect spiking or dilution, or possible mislabeling should be documented. Corrective action might include 

continuation of process with sample flagging or complete repreparation. 

Procedural errors at the instrumental analysis stage can 

be indicated by a variety of reasons including unusually large 

variations between duplicate analyses, off-scale concentrations, and unusual concentration compared to other samples. 

The corrective action for re-analysis at this stage should 

include written documentation of the reason or reasons for 

rejecting the original analysis. All re-analyses should be identified as such.

Analytical Data Review and Completeness

Data related to sample processing and analysis should 

be reviewed, approved, and retained at the laboratory. Any 

errors that are discovered should be rectified and documented 

during the review process. Corrections should become part of 

the permanent records associated with the samples. At least 
Methods 11

95 percent completeness (percent of analytical results reported 

without flags) should be obtainable.

Data Management

Overview of Database Design 

All data management activities related to this protocol 

are described in SOP 5. Microsoft Access 2003 is the primary 

software for managing ONSR elemental contaminants data. 

ESRI ArcInfo 9 serves as a tool for validation of spatial data 

residing in Access 2003. Data products are posted at the NPS 

Inventory and Monitoring (I&M) website (http://science.

nature.nps.gov/nrdata). Metadata for ONSR elemental 

contaminants monitoring are available on the NPS I&M 

application server (http://science.nature.nps.gov/im/monitor/

protocoldb.cfm). The data management activities described in 

this document are based on the recommendations of Rowell 

and others (2005) and the NPS I&M Program (http://science.

nature.nps.gov/im/monitor).

The general data model for ONSR elemental contaminants monitoring consists of two core sets of tables. One set 

manages species data (Asian clam shell diameters and crayfish 

carapace lengths) and the other associated dry-weight concentrations of specific metals. These data are linked in time and 

space by way of standardized event and location tables that 

provide detailed information associated with each sampling 

point and time. Supporting tables include species attributes, 

observers, and administrative information; look-up tables are 

linked to relevant tables to provide the values for pick-lists on 

data-entry forms, thereby reducing possible error during data 

entry (see Data Verification and Editing, later in this report).

Data Entry

Several features have been designed into the database 

to minimize errors that occur when field data are transcribed 

to the database for storage and analysis. Forms are used as 

portals for data entry into the database. Standardized identifiers (such as sample location and event) are selected from a 

list of easily interpreted codes. Species measurement data are 

entered into fields linked to appropriate tables. Look-up tables 

contain project-specific data and prohibit entry of data into a 

field if a corresponding value is not included in the look-up 

table. Consequently, only valid names or measures may be 

entered and spelling mistakes are eliminated. Species measures are selected using a pick list or by typing the beginning 

of the name. 

Data Verification and Editing

Data verification immediately follows data entry, 

and involves checking the accuracy of computerized 

records against the original source, usually paper field 

records. Although the goal of data entry is to achieve 

100 percent correct entries, this rarely is accomplished. To 

minimize transcription errors, NPS policy is to verify 100 percent of records to their original source by staff familiar with 

project design and field implementation. Further, 10 percent 

of records are reviewed a second time by the Project Manager 

and the results of that comparison reported with the data. If 

errors are found in the Project Manager’s review, then the 

entire data set is verified again. Once the computerized data 

are verified as accurately reflecting the original field data, the 

paper forms are archived and the electronic version is used for 

all subsequent data activities.

Although data may be transcribed correctly from the 

original field forms, they may not be accurate or logical. For 

example, an Asian clam shell measurement of 333 mm instead 

of 33 mm may be illogical and almost certainly incorrect, 

whether or not it was properly transcribed from field forms. 

The process of reviewing computerized data for range and 

logic errors is the validation stage. Certain components of data 

validation are built into data entry forms (for example, range 

limits). Data validation also can be extended into the design 

and structure of the database. As much as possible, values for 

data-entry forms have been limited to valid entries stored in 

the look-up tables. 

Additional data validation can be accomplished during verification if the operator is sufficiently knowledgeable 

about the data. The project manager will validate the data after 

verification is complete. Validation procedures seek to identify 

generic errors (such as missing, mismatched, or duplicate 

records) as well as errors specific to particular projects. For 

example, one database query detects ambiguous records with 

a location ID and an event ID; another counts the number of 

observations sampled per site to be sure all data were collected 

and entered. 

During the entry, verification, and validation phases, 

the project manager is responsible for the data. The project 

manager must assure consistency between field forms and the 

database by noting how and why any changes were made to 

the data on the original field forms. In general, changes made 

to the field forms should not be made via erasure, but rather 

through marginal notes or attached explanations. Once validation is complete, the data set is turned over to the data manager for archiving and storage.

Spatial validation of database sample coordinates can 

be accomplished using ArcGIS (ESRI, Inc.). Because this is 

an Access-maintained database, it can be integrated directly 

with ArcCatalog (ArcGIS, ESRI, Inc.) as an object linking 

and embedding database (OLE DB) object that allows part of 

an object (such as a database or spreadsheet) to be exported 

and then re-imported into a different object. Coordinate data 

[Universal Transverse Mercator (UTM) northing and easting] 

of the locations table can then be used to validate the UTM 

coordinate values for sample locations stored in Access against 

the original Global Positioning System (GPS) coordinates. 
12 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Metadata Procedures

Biological and spatial metadata are developed in accordance with guidelines established in Rowell and others (2005) 

using the NPS Database Metadata Extractor (a Microsoft 

Access add-in), ESRI ArcCatalog, and the NPS Metadata 

Editor and Tools extension. Metadata follow the Federal 

Geographic Data Committee (FGDC) standards and include 

Integrated Taxonomic Information System (ITIS) biological 

profile elements that allow integration of the metadata record 

with the NPS I&M NPSpecies and NatureBib online databases. Metadata are then parsed using a USGS metadata parser 

to check for errors.

Database Versions

Changes in database structure and functionality require 

a versioning system. This allows for the tracking of changes 

with time. With proper controls and communication, versioning ensures that only the most current version is used in any 

analysis. Versioning of archived data sets is handled by adding 

a two digit number separated by a period to the file name, 

with the first version being numbered XXXXXX1.0. Minor 

changes such as revisions in forms and report content should 

be noted by an increase of the number to the right of the 

period. Major changes such as migration between Access versions or database normalization across multiple tables should 

be indicated by an increase in the number to the left of the 

period. Frequent users of the data are notified of the updates, 

and provided with a copy of the most recent archived version.

Database Security

Secure data archiving is essential for protecting data files 

from corruption. No versions of the database should be deleted 

under any circumstance. Monitoring databases are small and 

do not require significant computer drive space or resources. 

On the other hand, they represent primary data and are expensive to create and impossible to replace. Multiple backup 

copies of all program data are maintained at the HTLN offices, 

at the Wilson’s Creek National Battlefield in Republic, Missouri, and at the Missouri State University campus offices in 

Springfield. Tape backups of the databases are made weekly. 

Each weekly full backup copy is maintained at the Wilson’s 

Creek National Battlefield. Once a month, one tape copy is 

stored offsite. 

Data currently (2008) are available on request for 

research and management applications for database versions 

where all QA/QC has been completed and the data have been 

archived. Most data requests currently are met using file transfer protocol (FTP) services. Parts of the monitoring data collected under this protocol will be made available for download 

directly from the NPS I&M Monitoring webpage. Information 

related to location and persistence of species determined to 

be threatened or endangered will not be made available for 

download by the general public. In addition, metadata will be 

available directly from the NPS I&M NR-GIS Metadata and 

Data metadata server (http://science.nature.nps.gov/nrdata). 

Data requests should be directed to:

Data Manager

Wilson’s Creek National Battlefield

6424 W. Farm Road 182

Republic, MO 65738-9514

(417) 732–6438

Data Analysis and Interpretation

One of the primary recommendations from a workshop 

on environmental monitoring organized by the Ecological Society of America was that trend studies should focus 

on description of trends and their uncertainty rather than 

on hypothesis testing (Olsen and others, 1997). Core data 

analyses and presentation methods are, therefore, focused on 

parameter estimates; they will provide a standard format for 

evaluation of all metals concentrations, are straightforward 

to interpret, and can quickly be updated whenever additional 

data become available. They will primarily consist of point 

estimates of metals concentrations and the associated uncertainties. In the 2005 ONSR pilot project, a sample size of three 

replicates per stretch resulted in relatively small amounts of 

variability among replicates (Schmitt and others, 2007b).

Control Charts

A control chart approach will be employed for long-term 

analyses. Control charts represent a basic summary of a data 

set that indicates which variables are in the greatest need of 

more in-depth analyses or management action. Developed for 

industrial applications, control charts indicate when a system is going “out of control” by plotting through time some 

measure of a stochastic process with reference to its expected 

value (Beauregard and others, 1992; Gyrna, 2001; Montgomery, 2001). Control charts have been applied to ecological data 

(McBean and Rovers, 1998; Manly, 2001), including fish communities (Petterson, 1998; Anderson and Thompson, 2004) 

and other natural resources (Atkinson and others, 2003). In 

this application, control charts will contain only upper control 

limits, which will specify toxicity thresholds for invertebrate 

metals concentrations that represent dietary risks of metals 

to riparian wildlife. Construction of the control chart and an 

example are presented in SOP 5.

The toxicity thresholds have been determined through 

food chain analysis using procedures developed for conducting ecological risk assessments (USEPA, 1992, 1997, 1999, 

2007b). Cadmium, cobalt, nickel, lead, and zinc are natural 

constituents of the earth’s crust. Although cobalt, nickel, and 

zinc are essential elements, these metals all are potentially 

toxic to wildlife at concentrations that can occur in the environment. Because of the extent of the river system at ONSR 

(the main stems of the Current River and Jacks Fork extend 
Methods 13

for more than 200 km within the ONSR boundaries), averaging over all stretches to obtain a single ONSR-wide estimate 

will not be attempted. No individual point estimate would be 

representative of most of the river(s). Such an approach would 

be problematic, especially if concentrations were low in most 

stretches but were high in a few. Thus, the primary focus will 

be on evaluating each stretch individually with time. This 

approach will allow evaluation of the potential effect of tributaries with confluences between sampled main stem stretches, 

and signal tributaries that require additional monitoring.

Toxicity Threshold Development

Risk analysis for warm-blooded vertebrates (homeotherms) is based on food chain analysis, the foundation 

of which is the toxicity reference value (TRV). The TRV 

is the daily contaminant intake rate in milligrams (mg) of 

contaminant per kilogram (kg) body weight per day associated with a toxicity threshold. The threshold can be either 

the no-observed-adverse-effect-level (NOAEL) or the 

lowest-observed-adverse-effect-level (LOAEL) for a specific 

contaminant in a relevant wildlife species as reported in the 

scientific literature. Toxicity reference values have been tabulated for a variety of birds and mammals (Sample and others, 

1996; USEPA, 1993, 2005a, 2005b, 2005c, 2007a, 2007c). By 

definition, NOAEL-based TRVs are lower than LOAEL-based 

TRVs and are used for screening-level assessments, which 

are preliminary evaluations of ecological risk. NOAEL-based 

TRVs for wildlife recently (2007) have been approved by the 

USEPA as part of a process for establishing ecological soil 

screening levels (Eco-SSLs) for metals and other contaminants. The Eco-SSL TRVs for lead, zinc, cadmium, cobalt, 

and nickel are incorporated into the assessment described in 

SOP 5, which also contains a control chart example. 

For analyses of homeotherms, receptor wildlife species 

are selected by the investigator to be representative of the 

ecosystem being modeled. Daily contaminant intake in each 

selected species is estimated and evaluated relative to the TRV. 

To estimate daily contaminant intake, the measured concentrations of the metal in the food source or sources being evaluated (crayfish and Asian clam, in units of mg/kg wet-weight) 

is multiplied by the weight-normalized food ingestion rate 

(kg food/kg body weight/day) of the species. Weight-normalized food intake rates in homeotherms decrease with body 

weight; that is, small animals consume a greater proportion of 

their body weight per day than large animals (USEPA, 1993). 

With all other factors being equal, risk associated with dietary 

exposure to metals tends to be greatest in small mammals and 

birds. Weight-normalized food intake rates and body weights 

for wildlife as reported in the scientific literature also have 

been tabulated (Sample and others, 1996; USEPA, 1993). 

Species bracketing the size range of birds and mammals 

likely to consume stream-dwelling aquatic invertebrates in 

southern Missouri were selected as receptors in ONSR. Great 

blue heron (Ardea herodias) and American mink (Mustela 

vison) were chosen as representative large, adult riparian birds 

and mammals, respectively. The American robin (Turdus 

migratorius) and short-tailed shrew (Blarina brevicauda) 

were selected to represent smaller birds and mammals. These 

small species are not riparian. They were selected based on 

body weight to represent organisms such as the least sandpiper 

(Calidris minutilla), spotted sandpiper (Actitis macularia), and 

southeastern shrew (Sorex longirostris) that might be more 

ecologically relevant, but for which no or only limited life history data are available. Organisms of this size also are representative of the young of larger birds and mammals that might 

regularly consume locally procured aquatic invertebrates. A 

scenario in which the receptor organism is a shrew-sized mammal or robin-sized bird consuming a diet composed entirely 

of the most contaminated organisms from the stretch or reach 

being considered typically yields a conservative wildlife risk 

estimate (that is, greatest indicated hazard) that is suitable for 

screening-level assessments. Such screening-level assessments 

based on conservative assumptions typically are conducted to 

determine whether or not more detailed investigations are warranted (USEPA, 1998).

Daily contaminant intake rates estimated as described 

can be compared to the TRVs either directly or as a ratio. 

The ratio, or hazard quotient (HQ), is obtained by dividing 

the daily contaminant intake rate by the TRV. HQ values that 

exceed 1.0 indicate risk. The no effect hazard concentration 

(NEHC) also can be computed. The NEHC is the dietary concentration at which the HQ = 1.0 in the species being evaluated; it is computed as the product of the weight-normalized 

food intake rate and the TRV (Hinck and others, 2006). 

Food chain analysis has not yet been extended to coldblooded vertebrates (poikilotherms). Instead, measured 

concentrations in prey items often are compared to toxicity benchmarks as published in the scientific literature. The 

limited literature available (for example Woodward and others, 

1994; Linder and others, 1998; Nagel and others, 2001; Hopkins and others, 2002) indicates that fish, reptiles, and amphibians generally are less sensitive to naturally incorporated 

dietary metals than small mammals and birds. Consequently, 

the NEHCs developed for robins and shrews (see SOP 5) are 

lower than published toxicity benchmarks for poikilotherms. 

Cadmium is an exception; James and others (2004) reported 

reduced survival of American toads (Bufo americanus) fed a 

diet of earthworms containing as little as 4.7 μg/g dry-weight. 

This value is virtually identical to the lowest NEHC computed 

for homeotherms (4.8 μg/g dry-weight in crayfish, in the robin 

model), indicating that a lower NEHC ultimately may need to 

be developed for cadmium.

Assumptions and Uncertainties

Diet is the only exposure pathway considered in the 

screening-level assessments described here; however, the 

incidental ingestion of contaminated sediments by warm- and 

cold-blooded animals feeding on aquatic organisms from 

contaminated areas, along with the inhalation of contaminated dust and any water consumed by wildlife either through 
14 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

drinking or while feeding, may represent additional and 

important routes of exposure. For example, as much as 30 percent of the diet of sandpipers (Calidris spp.) may be composed 

of sediment (Beyer and others, 1994). When information on 

these other exposure pathways is available, daily contaminant 

intake rates computed from maximum measured concentrations typically are evaluated relative to LOAEL-based TRVs; 

daily intake rates based on mean concentrations are evaluated 

against NOAEL-based values (USEPA, 1993). However, the 

invertebrate metals data from ONSR represent composite 

samples of 10 animals of the same species, which are in effect 

means. Consequently, and lacking contemporaneous data 

describing the other exposure pathways, comparisons of daily 

contaminant intake rates computed from concentration maxima in composite samples to NOAEL-based TRVs represent 

conservative (that is, maximum) estimates of risk. Similarly, 

and as noted, exposure of fish and amphibians to waterborne 

metals also occurs; however, and with the possible exception 

of nickel and cobalt, significant waterborne exposure to metals 

would not be expected under the hard water conditions typical 

of ONSR.

There are numerous additional assumptions and sources 

of uncertainty inherent in wildlife food chain exposure 

analysis (Schmitt and others, 2006). First, it also is important 

to recognize that although there are consensus values for all 

five metals, alternative TRVs have been proposed and used 

elsewhere. Some TRVs are lower than the values used in this 

report; consequently, an evaluation that results in an HQ near 

or exceeding 1.0 (or a safety factor near or less than 1.0) based 

on these TRVs should be considered reason for further monitoring, investigation, or both. For example, and although the 

maximum 2005 ONSR concentrations in crayfish and Asian 

clams were low relative to mining areas in southern Missouri 

(Besser and others, 2007a; Schmitt and others, 2007b), some 

safety factors were small (see example in SOP 5). Further 

evaluation relative to other TRVs and exposure scenarios 

would, therefore, be warranted.

Crayfish were selected for monitoring because they 

represent a food source for many species. In contrast, Asian 

clams are not a significant component of the riparian wildlife 

food chain. The Asian clam was selected because this species 

accumulates most metals and because there are extant data for 

comparison. Nevertheless, the Asian clam is a filter-feeder, as 

are many other aquatic invertebrates. Metals concentrations 

in benthic insect larvae from streams draining the New Lead 

Belt of southeastern Missouri typically exceed those in crayfish; and concentrations of lead and cadmium, but not zinc, 

in small fish (largescale stoneroller, Campostoma oligolepis; 

and juvenile longear sunfish) typically exceed concentrations 

in invertebrates (Besser and others, 2007a). Consequently, 

metals concentrations in Asian clams may accurately represent dietary exposure concentrations for wildlife species that 

consume aquatic organisms containing greater concentrations 

than crayfish. Although the Asian clams are depurated prior to 

analysis, which can lower the concentration of lead relative to 

freshly collected clams, concentrations of other metals are not 

likely to be affected (Gunther and others, 1999; Angelo and 

others, 2007). 

A wide range of body weights characterize the various 

life stages of birds and mammals. Moisture content of potential prey items also can vary. Values believed to be representative of southern Missouri were chosen from available data (see 

footnotes of table 6 in SOP 5). As noted, the exposure of and 

corresponding risk to smaller, younger animals would exceed 

that of adult animals, which could be especially problematic 

for nestling birds fed contaminated invertebrates. 

As also noted, the assessment described here is based 

on one exposure pathway (diet). The incidental ingestion of 

contaminated sediments, inhalation of contaminated dust, and 

drinking water may represent additional and important routes 

of exposure that are not accounted for. Absent data for these 

pathways, the conservative alternative of comparing daily 

ingestion rates assuming a diet comprising 100 percent Asian 

clam or crayfish containing the maximum composite concentrations is employed. Although reasonable as a screening tool, 

this approach indicates only if harmful effects are possible, not 

if they are probable (Sample and Suter, 1999). Conversely, it 

is unlikely that the diet of adult animals of the receptor species 

modeled (or those they might represent as surrogates) would 

be entirely composed of invertebrates from one site, which is 

tacitly assumed in this approach. Nevertheless, the assumption 

may be more valid for nestling birds, which may be fed a large 

percentage of locally procured food items.

Much of the toxicological literature on metal toxicity to 

fish and amphibians is based on waterborne exposures. Collectively, these studies indicate that amphibians are at least 

as sensitive to dissolved metals as fish. The uptake of toxic, 

dissolved ionic metals by fish and amphibians across the gills 

or skin represents a significant route of exposure in situations where geochemical conditions favor these metal forms; 

however, the dissolved ionic forms of most metals associated with lead-zinc mining are generally not present in hard, 

carbonate-dominated, alkaline streams such as those in ONSR 

(Barks, 1978). Under these conditions, food chain transfer and 

dietary exposure are more important than waterborne exposure (Woodward and others, 1994; Farag and others, 1999; 

Besser and others, 2007a). Cobalt and nickel are exceptions; 

although present in relatively small amounts, these metals are 

more labile than lead, cadmium, and zinc, and can occur in 

dissolved form as free metal ions. Elevated concentrations of 

cobalt and nickel have been reported in sediment pore waters 

and biota in streams draining the Viburnum Trend (Brumbaugh and others, 2007; Schmitt and others, 2007a). In addition, much of what is known about dietary metals exposure 

in fish is based on studies conducted with Western salmonids 

(Woodward and others, 1994; Farag and others, 1999). More 

recent studies indicate that sculpins (Cottus spp.), which typically are abundant in Ozark streams, may be more sensitive to 

metals than salmonids (Woodling and others, 2002; Besser and 

others, 2007b). Sculpin density is low in streams draining the 

Viburnum Trend (Allert and others, 2005) and other mining 

areas (Woodling and others, 2002).
Methods 15

As a final consideration, this assessment assumes that the 

five metals act independently. Lead, cadmium, zinc, and other 

metals co-occur throughout the mineralized areas of southern 

Missouri, and their effects are not necessarily independent 

or strictly additive either among themselves or together with 

other environmental factors such as temperature, nutrition, 

and disease (Joselow, 1980; Schmitt and others, 1984, 1993). 

Nevertheless, and in spite of the numerous assumptions and 

uncertainties noted, the values in table 6 represent conservative screening-level criteria for the assessment of potential 

adverse effects in wildlife from metals in aquatic invertebrates. 

As indicated by the example in SOP 5, application of the values to the 2005 ONSR pilot data (Schmitt and others, 2007b) 

indicates that the margin of safety for wildlife consumers of 

aquatic invertebrates is small for some metals associated with 

lead-zinc mining. Continued monitoring of metals in ONSR 

is, therefore, warranted. In addition, the field of ecological 

risk assessment is evolving rapidly, especially for reptiles and 

amphibians. Considering the apparent sensitivity of amphibians to cadmium (James and others, 2004), the present status 

of the Ozark hellbender in ONSR (MDC, 2007) and the fact 

that Ozark hellbenders feed predominantly on crayfish (Nickerson and Mays, 1973), pertinent sections of SOP 5 (including 

tables 5 and 6) should be reviewed and revised as necessary in 

association with each invertebrate collection cycle.

Reporting

Reports will summarize the most recent monitoring 

results and include a summary of data collected previously. 

The primary audience for these reports will be NPS resource 

managers and external scientists. An Executive Summary, 

highlighting key points for non-technical audiences, will 

be produced with each report. The primary audience for the 

Executive Summary will be Park Superintendents, interpreters, 

and the general public. Peer review for both will be conducted 

internally by HTLN staff. Reports will be produced each time 

monitoring is conducted (every 5 years). To disseminate findings in a timely manner, reports will be completed by September 1 of the year following data collection. Report distribution 

is described in SOP 6. 

Personnel Requirements and Training

Roles and Responsibilities

The aquatic program leader serves as the project manager 

for implementing this monitoring protocol, and is supervised 

by the HTLN network coordinator. Because of the need for 

consistency in implementing the protocol, the project manager 

usually will oversee sample collection. The data management 

aspects of the monitoring effort is the shared responsibility 

of the project manager and the data manager. Typically, the 

project manager is responsible for data collection, entry, verification, validation, summarization, analysis, and reporting. 

The data manager is responsible for data archiving, security, 

dissemination, and database design. The data manager, in collaboration with the project manager, also develops data entry 

forms and other database features as part of quality assurance 

and automates report generation. The data manager is ultimately responsible for ensuring that QA and QC procedures 

are built into the database management system and that appropriate data handling procedures are followed.

Qualifications and Training

The collection of credible, high-quality trace-metal data 

requires attention to detail with respect to where and when 

samples are collected, protection of the samples from external 

contamination, and data recording and transcription. Therefore, it is essential that the samples be collected from the 

assigned sites at the designated time of year, that the collection 

locations are well documented, and that personnel follow the 

clean procedures identified in this protocol and its associated 

SOPs to prevent external contamination. Information with 

which to determine whether or not trace metal concentrations 

vary among the crayfish and freshwater bivalve species of 

ONSR is limited; consequently, it may or may not be important to be able to correctly identify crayfish and Asian clams in 

the field. It is nevertheless best to err conservatively by assuming that species-level differences exist; specimens should be 

correctly identified in the field, and samples should not comprise mixed species. Differences in trace metal concentrations 

between genders of crayfish or Asian clams have not been 

reported; however, differences between genders have been 

reported in fish (Schmitt, 2004), so it would not be unreasonable to expect similar differences in freshwater invertebrates. 

Regardless, differentiating between invertebrate genders is 

often not possible, especially during the recommended late 

summer-early fall collection period. Also important is attention to detail with respect to recording and data entry. Time 

will be budgeted for field personnel to learn to identify crayfish and mollusks, and to verify data entry.

Operational Requirements

Annual Workload and Field Schedule

Crayfish and Asian clam collection should begin no 

sooner than the last full week of August and be completed by 

the end of the first full week of October to minimize exposure 

variation among sites and to ensure that crayfish of the correct 

size are obtained. Inclement weather, high water levels, and 

personnel workloads will preclude the scheduling of sampling to specific dates. Sampling dates should be scheduled 

and logistics organized before the start of each field season 

(SOP 1). 
16 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Facility, Equipment, and Personnel Needs

Invertebrate collections do not require special facilities 

beyond normal office space and equipment storage needs. 

A chest-type freezer with enough capacity to store samples 

before shipment to the analytical laboratory is required. SOP 1 

contains a list of field equipment and supplies needed for one 

crew. If two or more crews work simultaneously, equipment 

requirements will increase accordingly. 

Personnel estimates for field work are based on a crew 

of two people: an aquatic ecologist to collect and identify the 

organisms, and a bio-technician to record data and process 

samples (table 2). 

Time and budget are contingent upon the amount of time 

required to travel to the sites and to locate and collect the 

animals. Sample processing requires about 1 hour per site in 

the field. Another hour (for each person) is required at the end 

of each trip to log in the samples; verify, copy, and file data 

sheets; enter data; and clean and store equipment. Startup costs 

include the purchase of the equipment and supplies listed in 

SOP 1, as well as maintenance and replacement of equipment 

shared among multiple projects (such as GPS units, cameras, 

waders, and raingear). As noted, equipment should be labeled 

for use only at ONSR to minimize the potential spread of 

invasive species.

Procedure for Revising the Protocol 

and Archiving Previous Versions of the 

Protocol

Revisions to the Protocol Narrative and specific SOPs 

are to be expected. Careful documentation of changes, and a 

library of previous protocol versions are essential for maintaining consistency in data collection, and for appropriate 

treatment of the data during data summary and analysis. The 

Microsoft (MS) Access database for each monitoring component contains a field that identifies which version of the 

protocol was being used when the data were collected.

The rationale for dividing a sampling protocol into a

Protocol Narrative with supporting SOPs is based on the 

following:

1. The Protocol Narrative is a general overview of the 

protocol that gives the history and justification for doing 

the work and an overview of the sampling methods, but 

that does not provide all of the methodological details. 

The Protocol Narrative will be revised only if substantial 

changes to the protocol are required.

2. The SOPs, in contrast, are specific step-by-step instructions for performing a given task. They are expected to be 

revised more frequently than the protocol narrative. 

3. When a SOP is revised, in most instances it is not necessary to revise the Protocol Narrative to reflect the specific 

changes made to the SOP.

Table 2. Estimated personnel, travel, and analytical resources, per collection.

[Collection period varies; based on 30 composite samples from five locations with three samples of each taxon from each location]

Budget item Quantity Estimated cost (2008)

Personnel

Field crew

Aquatic program leader 80 hours $2,504

Aquatic ecologist, STF 72 hours 1,270

Seasonal biological technician 72 hours 1,075

Administrative support

Coordinator 20 hours 744

Quantitative ecologist 8 hours 250

GIS specialist 8 hours 173

Data manager 8 hours 250

Administrative assistant 16 hours 262

Travel and miscellaneous

Per diem 5 days/person $475

Vehicle use 300 miles 124

Expendables and overnight shipping 30 samples 100

Analytical

Chemical analysis (includes quality assurance and sample containers) 30 samples $16,000

Total $23,227
Procedure for Revising the Protocol and Archiving Previous Versions of the Protocol 17

4. All versions of the Protocol Narrative and SOPs will be 

archived by the HTLN.

The steps for changing the protocol (either the Protocol Narrative or the SOPs) are outlined in SOP 8. Each SOP 

contains a Revision History Log that should be filled out 

each time a SOP is revised to explain why the change was 

made, and to assign a new version number to the revised 

SOP. The new version of the SOP or Protocol Narrative will 

then be placed on the NPS Inventory & Monitoring website 

(http://science.nature.nps.gov/im/monitor/protocoldb.cfm). 

Revised versions of the protocol and SOPs will not be subjected to USGS peer review and, therefore, will not constitute 

USGS publications. 
18 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

General Preparation and Review

1. Before the field season each year, all study participants 

should review the current version of the entire protocol, 

including the SOPs. 

2. Review of crayfish and mussel identification is important; 

misidentification of a species may affect trace metal concentrations. In addition, a rare animal may inadvertently 

be killed. Misidentification is more serious than slight 

deviations from specified organism size or failing to collect the 30 specimens of each type. Be sure to make a positive identification, and do not combine different species 

within a composite sample. Fourteen species of crayfish 

are known to inhabit the Current River/Jacks Fork system, 

including several rare species (see Pflieger, 1996, or 

http://mdc.mo.gov/nathis/arthopo/crayfish/varcraw.htm). 

Many are similar in appearance. In contrast, Asian clams 

are readily distinguished from the other 13 (indigenous) 

mussel species present in the Current River/Jacks Fork 

system by their thick shells of rounded triangular shape 

and the series of heavy concentric ridges originating at 

the hinge area (Oesch, 1995; see photo at http://www.mdc.

mo.gov/fish/watershed/current/biotic/). These sources 

should be consulted before starting each collection. 

3. Review the instructions for using the GPS units, located 

online at:

http://science.nature.nps.gov/im/units/htln/

datamanagement.cfm

4. Obtain the necessary scientific collection permits. Permit requirements vary depending on the organization 

or agency doing the collection. Applications for these 

permits must be submitted early in the sampling year to 

ensure approval. Missouri Department of Conservation 

(MDC) permit requests should be submitted to: 

Missouri Department of Conservation

P.O. Box 180

Jefferson City, MO 65102-0180

(573) 751-4115

Permit applications can also be obtained online at: 

http://mdc4.mdc.mo.gov/Documents/8908.doc

5. Preseason planning facilitates the successful and timely 

completion of collections; scouting sites in advance to 

locate crayfish and Asian clams usually is advantageous.

6. Notebooks from previous collections should be reviewed 

if available to identify any unique events that may be 

encountered. A field notebook for the survey year should 

be prepared with pages for entry of sampling schedules, 

observer names, field hours, and unique happenings that 

may have affected how the data are reported. Trip reports 

are based on information recorded in field notebooks so 

it is imperative that they are clearly organized for ease of 

field note entry. 

7. Prior knowledge of species most likely to be encountered will aid collectors in preparing for the field season; 

therefore, species lists from previous collection efforts in 

the park or local area should be compiled and compared 

to reference manuals to identify species not recorded that 

have a probability of being recorded. Copies of these 

combined species lists should be carried into the field as 

quick references in helping to identify unknown species. 

8. A list of waypoints representing the downstream-most 

boundaries of stretches to be sampled should be carried 

into the field to ensure sampling begins at the appropriate locations. A list of waypoints representing crayfish or 

Asian clam concentration areas within each stretch also 

may be helpful for locating target organisms in subsequent years.

Standard Operating Procedure 1: Preparation for Sampling, Version 1.0 

(July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
Standard Operating Procedure 1: Preparation for Sampling, Version 1.0 19

Scheduling Field Work

1. Crayfish and Asian clam collections should begin no 

sooner than the last full week of August and be completed 

by the end of the first full week of October. Inclement 

weather, high water levels, and personnel workloads will 

preclude the scheduling of sampling events to specific 

annual dates. Sampling dates should be scheduled and 

logistics organized before the start of each field season. 

2. Sample collection requires a two person crew (both members to sample; then one to identify and measure organisms, the other to record data). Time per site depends on 

availability of crayfish and Asian clams; processing time 

is 1 hour or less. 

3. If access across private lands is necessary to reach sites, 

the prior consent of landowners should be obtained before 

entry; record the time and place of such entries along with 

the name, address, and telephone number of the person 

providing the consent on the data sheet.

Organizing Supplies and Equipment

1. All of the equipment and supplies listed in this SOP 

should be organized and made ready for the field season several weeks in advance. This allows time to make 

needed repairs and order equipment and supplies. A list 

of field equipment and supplies needed for one crew (two 

people) to collect crayfish and Asian clams at one site is 

given in table 3. If two or more crews work simultaneously, the equipment needs will change accordingly. 

2. Copies of the field data forms and sample labels (appendixes 1 and 2) should be made on Rite-in-the-Rain® paper 

in advance of need. A supply of invertebrate sampling 

data forms (appendix 1) and sample labels (appendix 2), 

the protocol narrative, and the SOPs copied to or printed 

on Rite-in-the-Rain paper also should be available for 

field use.

Pre-Sampling Procedures

1. Review the list of equipment and supplies (table 3) to be 

certain that everything on the list is available. Note that 

numbers in the tables are minima; spares of important 

items (such as calipers) are advisable. Check the status 

and condition of the oxygen bottle and regulator; if in 

doubt, get the bottle refilled. Safety note: Secure the oxygen bottle in the vehicle during transport.

2. Prior to the day of sampling, determine which sites will 

be sampled, and make a list of the easting and northing 

(UTMX and UTMY) coordinates for those sites; bring 

the list into the field. Reminder: The Asian clams need 

to be allowed to depurate (purge particulate material) for 

24 hours following collection; plan accordingly (that is, it 

is best to sample not later in the week than Thursday).

3. If sampling is to be conducted under an MDC Wildlife 

Collectors Permit, notify the Conservation Agent in 

the county where sampling will occur in advance of sampling. Reminder: The permittee must be present during 

the collections.

4. Crayfish samples need to be either stored on ice or frozen 

in dry ice while in the field. Ascertain that you have sufficient ice or dry ice for the number of sites to be visited. 

Check the ice or dry ice regularly, and know where to 

get more should the need arise. Safety note: Dry ice is 

dangerously cold; avoid skin and eye contact. Personnel 

handling samples and containers in dry ice should wear 

insulated gloves. Dry ice also needs to vent CO2 to the 

atmosphere; do not store it in tightly sealed containers or 

in confined spaces.

5. It generally is advantageous to prepare labels on Rite-inthe-Rain paper (appended to the protocol) in advance for 

each site. Each label should bear the site number, taxon 

(crayfish or Asian clam), date, and the composite sample 

identification (letter A, B, or C). Write with a ball-point 

pen or permanent felt-tipped marking pen (Sharpie® or 

equivalent). Labels should be attached to jars using 2-inch 

clear plastic tape; wrap completely around the jar to protect the label from moisture and abrasion. If such tape is 

unavailable, write on both the jar and lid with a felt-tipped 

pen. To prevent contamination, labels must never be 

placed inside of bags or jars containing samples.
20 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Table 3. Field equipment and supplies for collecting crayfish and Asian clams.

[Minimum quantities; *, items for which spares or extra are advised]

Number 

required Description

Equipment and supplies (for multiple sites)

1 (each) Mussel and crayfish references (Oesch, 1995; Pflieger, 1996)

1 (each) Copy of current protocol narrative and SOPs (on Rite-in-the-Rain® paper)

1 Field notebook

1 Clip board for recording data

1* Ball-point pen (black) or felt-tipped permanent marker, fine (Sharpie® or equivalent ) for recording data

1* Felt-tipped permanent marker, medium (Sharpie® or equivalent) for marking bags and other containers

1* Felt-tipped permanent marker, coarse, for labeling bags

1* Roll of 2-inch clear plastic tape (NSN/Part No: MMM3750260CR or equivalent), to protect jar labels

1* Scissors (to cut labels)

2 D-frame net (Wildco 425-D11) or bait net (Memphis Net and Twine BN10-BN13 or equivalent) with a mesh size of 1/8”-3/8” 

(square) 

1* Seine, 10-foot X 4-foot (max) X 1⁄4-inch (square) mesh (minnow seine; Memphis Net and Twine CSS1 or CSS2); or Zo seine 

(Wildco 4278-B35 or equivalent)

1* Cultivating rake (Ames A42 or equivalent)

3–5 Polyethylene bucket, 5-gal

1* Dial calipers (mm), measuring board (Wildco 118-E40 or equivalent), or ruler (mm), for measuring crayfish and Corbicula

1* Hand lens

1 Oxygen bottle, full, with regulator and hose1

2–3 Polyethylene coolers

1* GPS unit (with spare batteries)

1 Digital camera

1 Personal floatation device (pfd), correctly sized for each person (mandatory)

1 Boots, raingear, and waders as necessary, correctly sized for each person (optional)

1* Insect repellent

1* Sunscreen

1 First aid kit

1* Tube or bottle of disinfectant hand cream

1 Radio (for communication, if available)

1* Roll of polyethylene sheeting or paper to cover work surface(s)

1* Roll of plastic flagging tape

1 Freezer in which to store frozen samples pending shipment to the laboratory

1 Watch or clock (for determining depuration start time)

Supplies (per site)

As needed Maps of ONSR and collection sites

As needed Species lists and locations/waypoints from previous collections

1* Sheet of Rite-in-the-Rain® paper or pre-printed sample labels

2* Invertebrate collection data sheets, on Write-in-the-Rain paper

1 Roll or package of paper towels

1* Bag or block of ice or dry ice, 5 lb

6 Pre-cleaned polyethylene jars, 250-mL (VWR 15900-196 or equivalent)

2* Polyethylene zip-seal freezer bags, 1-gallon

3* Large plastic (trash or leaf) bags for transporting nets

3–5 Plastic trash can liner (for buckets and solid waste disposal)

1 Box of disposable gloves (powder-free latex, nitrile, or pvc; MicroFlex Diamond Grip DGP-350 or equivalent), sized to fit 

sample processing personnel

6* Heavy duty plastic bags for Asian clams (Associated No. 64-6-50 or equivalent)

1* De-ionized or distilled water, 1 gal, in a polyethylene bottle with spigot

1* Light- or medium duty clear plastic bags to cover measuring board (Associated Nol 26-6-775 or equivalent)

6* Heavy duty rubber bands 1

Oxygen bottle to be secured in vehicle during transit.
Standard Operating Procedure 2: Collecting, Processing, and Preparing Samples of Crayfish and Asian Clams, Version 1.0 21

Safety Note

Because working in or near water is potentially hazardous, a minimum of two people is required to conduct these 

procedures. Personal flotation devices (PFDs) are required 

for all personnel regardless of season. Note also that kicknetting or seining may require the manipulation of rocks and 

other debris with the feet; boots or waders are, therefore, also 

strongly recommended regardless of season. 

Additional Notes

The following should be kept in mind while collecting 

and processing specimens:

1. Transport the collection equipment (nets) in plastic 

trash bags between sites to keep them clean (and other 

items dry). 

2. Cover your work surface (table, cooler top, vehicle tailgate) with a clean piece of plastic sheeting or paper. Wear 

disposable gloves when processing samples, and replace 

them between sites or when they become torn.

3. Place the measuring board inside a clear plastic bag. To 

prevent contamination, perform all measurements on the 

bag-covered board on the covered work surface.

4. Line buckets to be used for transporting organisms 

with a plastic bag (polyethylene tall-kitchen trash bag 

or equivalent). 

Procedures

5. Find the stretch of river to be sampled. Use GPS to locate 

the lower geographic boundary of the stretch, which is 

given in table 1 of the protocol narrative. (Note: beginning and ending points of stretches often correspond to 

tributary confluences, which may help to locate the lower 

boundary in the field). For instructions on using the GPS, 

refer to the GPS SOP available online at http://science.

nature.nps.gov/im/units/htln/datamanagement.cfm

6. Locate the first riffle above the lower geographic boundary. The area that will be sampled includes the next three 

riffles located in consecutive order upstream from the first 

riffle above the lower boundary of the stretch.

7. Site names are designated by the first four characters 

of the river name (CURR, JACK), Mainstem (M) or 

Tributary (T), and the site number. Mainstem sites will be 

labeled in successive order from upstream to downstream. 

Example: JACKM03 is the 3rd mainstem site on the Jacks 

Fork. The locations of sampling sites, sample site codes, 

and GPS coordinates can be found in fig. 1 and table 1 of 

the protocol narrative. 

Crayfish

1. Crayfish are obtained by kick-netting or kick-seining in 

coarse-substrate riffles or along the edges of emergent 

vegetation beds. Note: avoid walking through areas to 

be sampled. To kick-seine, unwind the net to a manageable length (usually 1 to 2 m). Orient the net vertically 

(weighted line down) and perpendicular to the current; 

stretch it as wide as one person can manage, and leave 

the excess net wrapped around the poles. Ensure that the 

weighted line remains on the bottom by placing rocks 

along the inside of the net. While the net is held open, the 

other team member walks downstream toward the net, 

overturning rocks or disturbing vegetation with his or her 

feet along the way to dislodge the crayfish and allow them 

to be swept into the net by the current. If more personnel 

are available, the net can be expanded to sample a larger 

area. A cultivating rake also can be used in lieu of the feet 

to manipulate the rocks. When the kicker(s) reach(es) the 

net, all team members lift the net in unison from the water 

Standard Operating Procedure 2: Collecting, Processing, and Preparing 

Samples of Crayfish and Asian Clams, Version 1.0 (July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
22 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

horizontally and take it to shore, where the crayfish are 

removed by gloved hand, rinsed, and placed into a plasticlined bucket containing stream water. Fish and other 

organisms are immediately released back into the stream. 

The kick-netting process is similar, but the D-frame net 

can be operated by one person. The lower edge of the net 

is placed on the stream bottom, downstream from rocks 

or vegetation, which are then dislodged with the foot. 

When at least 10 specimens of suitable size (18 to 25 mm 

carapace length) have been obtained, return to shore with 

the bucket to begin processing the crayfish. 

2. Remove crayfish from the bucket one at a time with 

a gloved hand and examine each specimen (with a 

hand lens if necessary) to confirm species identities 

according to the characteristics presented by Pflieger 

(1996). This reference also contains color photographs 

and range maps of Missouri crayfish species. The photographs and range maps can also be viewed online at 

http://www.mdc.mo.gov/nathis/arthopo/crayfish/

varcraw.htm It is important to note that many of the structural features that definitively distinguish crayfish species 

are not evident during late summer and early fall. Nevertheless, and as noted by Pflieger (1996), knowledge of 

the collection location (that is, river basin) and information on coloration can distinguish between most species 

at a given site. Fourteen species of crayfish are known to 

inhabit the Current River and Jacks Fork. Crayfish of the 

recommended size obtained from riffles within ONSR 

will most likely be golden crayfish. This species is similar 

in size and general appearance to the Ozark crayfish 

(O. ozarkae), which is usually less abundant. Both species 

bear two dark, saddle-like marks that differentiate them 

from other species likely to be encountered within ONSR. 

However, the claws (chelae) of the golden crayfish lack 

spots or blotches; those of Ozark crayfish are usually 

speckled (covered with black spots), which may also be 

present on the abdomen. Spothanded crayfish (O. punctimanus), which also are common, is readily distinguished 

from golden and Ozark crayfish by the presence of a 

single dark spot at the joint of the claws. The only other 

species likely to be encountered is Hubbs’ crayfish (Cambarus hubbsi), which can be distinguished by its stout 

body form and claws relative to the Orconectes spp. In 

the event that golden crayfish (the preferred species) are 

not available, the order of preference for alternated species is spothanded, crayfish, Ozark crayfish, and Hubbs’ 

crayfish. Note: if a specimen cannot be identified in the 

field, release it and collect more if necessary to fill out the 

sample. However, do not mix species within a sample.

3. After confirming the identity of the specimen, rinse it 

carefully in stream water, measure its carapace length 

(mm) with the calipers (on the bag-covered measuring 

board) and record the species and carapace length on the 

data sheet. Carapace length is the distance from the tip 

of the rostrum to the posterior edge of the carapace; a 

diagram illustrating how it is measured appears on p. 34 

of Pflieger (1996). Use the following codes, which correspond to the genus and species names (table 4), to identify 

the species: golden crayfish, OL; spothanded crayfish, 

OP; Ozark crayfish, OO; Hubbs’ crayfish, CH. 

4. Place the 10 crayfish collected per riffle into a precleaned, pre-labeled jar. Repeat this procedure until all 

three samples (one per riffle) of 10 specimens of the 

appropriate size (18 to 25 mm) have been obtained.

5. Crayfish are separated into lots of 10 specimens per each 

of three riffles sampled. Again, do not mix species within 

a sample; if 10 specimens of golden crayfish cannot be 

obtained in a given riffle, retain all that are collected. The 

alternate species may be substituted in the order of preference identified previously, but different species should 

be placed in separate containers (that is, they are separate 

samples).

6. Thoroughly clean the net(s) and bucket(s) in stream 

water to remove all vegetation and sediment. Rinse the 

bag-covered measuring board in stream water, then with 

de-ionized or distilled water; replace the bag if it is damaged or dirty.

7. Rinse your gloved hands in stream water, then with deionized or distilled water. Inspect and replace the gloves if 

they are torn or damaged.

8. If the samples will be frozen in dry ice, place them 

directly in the cooler. If wet ice is used, put the three jars 

in a plastic bag to protect them from water, then place 

them in the cooler.

Asian Clams

1. Asian clams (Corbicula fluminea) are located visually, 

then collected from riffles, their associated backwaters, 

or vegetation beds by hand and held in lined buckets of 

ambient water for processing. Asian clams are readily 

distinguished from the indigenous mussel species present 

in the Current River Basin by their thick shells of rounded 

triangular shape and the series of heavy concentric ridges 

originating at the hinge area. Photographs are available in 

Oesch (1995) and at http://cars.er.usgs.gov/corbicula4.

pdf Asian clams often occur in dense colonies. The clams 

attain a maximum size of about 38 mm in diameter. 

Specimens of 15 to 25 mm (greatest diameter) are suitable 

for monitoring. If Asian clams cannot be obtained, there 

will be no sample; there is no alternate species, but Asian 

clams outside the preferred size range are preferable to no 

sample.

2. Asian clams are processed in a manner similar to that 

described for crayfish, except that they must be depurated 

(allowed to purge themselves of particulate material) 
Standard Operating Procedure 2: Collecting, Processing, and Preparing Samples of Crayfish and Asian Clams, Version 1.0 23

for 24 hours before processing. Each Asian clam sample 

comprises 10 specimens from a riffle (one sample from 

each of the three riffles sampled). After 10 clams have 

been collected from a riffle, they should be transferred to 

shore in the plastic-lined bucket. While wearing gloves, 

begin processing the specimens by manually removing 

as much debris from the shells as possible. Rinse them 

in stream water, then confirm the identity of each specimen. Measure its greatest shell diameter (width, in mm) 

either with the calipers or with the bag-covered measuring 

board. Record the species (use CF as the code for Asian 

clam; table 4) and diameter on the data sheet. Place the 

specimens in an 8 x 6 x 18 inch, 4-mil gusseted polyethylene bag (Associated Bag Co., Milwaukee, Wisconsin 

no. 64-6-50, or equivalent) about one-third full of ambient 

water (one for each composite sample). When 10 specimens have been placed in the bag, fill the bag with 

oxygen from the bottle, double-twist the end, and seal the 

bag tightly with a heavy-duty rubber band. Place this bag 

in another bag, add the sample label, and seal the outer 

bag with another rubber band. Place the labeled, doublebagged samples in a cooler containing ambient water. 

Keep the cooler in a shaded location. 

3. Rinse the bucket in stream water. Rinse the bag-covered 

measuring board in stream water, then with de-ionized or 

distilled water; replace the bag(s) if damaged or dirty.

4. Rinse your gloved hands in stream water, then with deionized or distilled water. Inspect and replace the gloves if 

they are torn or damaged. Repeat steps 1–4 until all three 

samples have been obtained.

5. When sampling has been completed, the team leader 

should review and sign the data sheet after ascertaining 

that all required information has been recorded; be certain 

that the collection times have been recorded, especially 

for the Asian clam samples. Remove the cover from the 

work surface and place it and all other disposable items 

(gloves, paper towels, etc.) in one of the bucket liners 

and retain all for later disposal. If time permits, prepare a 

sketch of the site in the notebook or on the back of one of 

the data sheets; indicate in the sketch the general locations 

from which the samples were obtained. 

6. Thoroughly clean and inspect all sampling equipment to 

ensure that no organisms remain attached.

Table 4. Species codes for invertebrate samples.

Species code Common name Scientific name

OL Golden crayfish Orconectes luteus

OP Spothanded crayfish O. punctimanus

OO Ozark crayfish O. ozarkae

CH Hubbs’ crayfish Cambarus hubbsi

CF Asian clam Corbicula fluminea
24 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Procedures

1. Upon return to the duty station, all crayfish samples and 

the Asian clams that have completed depuration (see 

item 2, following) should immediately be transferred to 

a freezer (–20° C). If a freezer is not immediately available, the samples should be frozen in dry ice until one is 

available. Samples should be inventoried by checking the 

appropriate box on the data sheet as each is transferred 

from the cooler(s) to the freezer. Samples should be 

maintained frozen (–20° C) until they are shipped to the 

analytical laboratory. Review and sign the field data sheet 

again, verifying that the samples have been logged in and 

frozen. Photocopy the data sheets and store them in properly designated areas, preferably in different buildings to 

protect against loss, fire, flood, theft, or other unforeseen 

events. Dispose of all used expendables as trash.

2. After 24 hours of depuration, transfer the Asian clams to 

jars. Transfer each sample label from the outer holding 

bag to a jar and fasten the label to the jar with clear polyethylene tape. While wearing disposable gloves, transfer 

the Asian clams from the bag to the labeled jar. Repeat for 

all samples. If the samples will be stored on wet ice, place 

the three jars from the site in a plastic bag to protect them 

from water; otherwise, freeze them immediately. Record 

the time of the transfer on the data sheet. 

3. Complete any logbook or notebook entries. Photocopy 

appropriate logbook pages and file the copies with the 

duplicate data sheets. At the end of each field trip, file a 

trip report with the data manager outlining hours worked, 

field-crew members and their responsibilities on the 

project, and any unique situations encountered. File a 

copy of the report with the copies of the data sheets. This 

information is incorporated in the database and used during data analysis; it is critical for identifying discrepancies 

and inconsistencies in the data. The project manager is 

responsible for filing all field reports.

4. Samples are shipped to the analytical laboratory only 

upon receipt of instructions. (Note: the analytical laboratory may not be the same for all collections.) Ship 

samples frozen, in properly labeled coolers containing 

enough dry ice to maintain them frozen in transit (generally a quantity equal to the weight of the samples) to the 

analytical laboratory via Federal Express (FEDEX). Initiate a chain-of-custody form (appendix 3) and ship it with 

the sample. Request that it be signed and returned upon 

receipt by the laboratory, then copy and file it with the 

data sheets. Note: Dry ice is considered a hazardous material for which special shipment restrictions and regulations apply. The regulations are subject to change; consult 

FEDEX (http://www.fedex.com) for current instructions 

before shipment. 

5. Thoroughly rinse (in tap water) and re-inspect all watercontact equipment (including boots, nets, buckets, coolers, and PFDs) for the presence of attached organisms. 

Spread the equipment and allow it to air-dry completely.

Standard Operating Procedure 3: After the Field Collections, Version 1.0 

(July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
Standard Operating Procedure 4: Data Management, Version 1.0 25

This SOP explains procedures for the management of 

ONSR metals data. It includes a general description of the data 

model and procedures for data entry, data verification and validation, and data integrity for the primary metals data.

Data management can be divided into the initial design 

phase that involves defining the data model, its entities and 

their relations; and the procedures necessary to implement the 

database. Microsoft (MS) Access 2003 is the primary software used for maintaining metals data. The metals data will 

be stored in the National Park Service’s NPStoret database. 

Spatial data associated with field sampling locations are managed with ESRI ArcInfo 9.x. Data products derived from this 

project will be available at the NPS I&M Data Store and the 

USEPA STORET National Data Warehouse (http://www.epa.

gov/storet/dw_home.html). Quality-assurance (QA) and quality-control (QC) guidelines for data management described in 

this document are based on recommendations of Rowell and 

others (2005) and references cited therein.

Data Model

The NPS I&M program has designed the Natural 

Resource Database Template (NRDT) to be used as a database 

model for storing vital signs monitoring data in MS Access 

(NPS, 2006). The template has a core database structure that 

standardizes location and observation data to facilitate the 

integration of datasets. Developed in MS Access, the database 

allows users to enter, edit, display, summarize, and generate 

reports, as well as integrate with other Natural Resource data 

systems such as NPStoret. The NRDT data dictionary follows 

standards identified in the NRDT phase 2 and is modified 

where required. Naming conventions follow I&M recommendations. 

The aquatics program relies on distributed databases 

to allow remote users to enter data and is accomplished via 

database replication consisting of a design master and replicas. 

The design master is stored on the server at Missouri State 

University in Springfield, Missouri, and is used for local data 

entry, revisions, and design changes. A replica is created for 

users without access to the server and is distributed for data 

entry activities. When a user has finished data activities with 

the replica, it is returned and synchronized with the design 

master. The NPS Water Resource Division (WRD) also has 

designed the NPStoret database to facilitate archiving NPS 

data in the EPA Storet database. NPStoret is a series of Accessbased templates patterned after the NRDT that includes data 

entry templates and an import module. It supports the core 

data management objectives of data entry and verification/

validation in a referentially constrained environment (that is, 

related locations, events, and primary data elements) (NPS, 

2007). The sampling events (tbl_SamplingEvents) and locations (tbl_Locations) tables are the two core tables and contain 

general information pertaining to the field sample occasion; 

they include information such as date and time, river stretch 

ID and UTM coordinates, and park/project codes. Detailed 

information pertaining to the Asian clam (Corbicula fluminea) 

or crayfish sample is maintained in tbl_CorbiculaData, tbl_

CorbiculaMetals, tbl_CrayfishData, and tbl_CrayfishMetals. 

Other tables include administrative and personnel data and a 

species lookup table.

Data Preparation

Procedures for QA and QC related to data recording are 

important components of any project. Sampling data (such 

as sample methods, water-quality conditions, and speciesmeasurement data) are recorded and checked for completeness 

either before leaving a site or within 24 hours of data recording. This will aid in verification and validation of the data after 

entry into the database. To prevent the complete loss of field 

form data because of unforeseen circumstances such as fire or 

flood in the workplace, all field sheets are photocopied, and 

a hard copy located in a location separate from the originals. 

Field sheets are scanned, and electronic copies of the data 

sheets are stored on the HTLN server located at Missouri State 

University. This will ensure that at least one copy is available 

for data entry and verification. 

Standard Operating Procedure 4: Data Management, Version 1.0 

(July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
26 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Data Entry

Data entry is accomplished in either the design master or 

replica databases using a tiered set of forms. Upon opening 

the replica, a “switchboard” directs the user to a preliminary 

set of forms that define the sampling occasion and requires the 

input of sampling periods and events before entry of additional data. The other forms address the details of elemental 

contaminant occurrence, organism measurements, observers, 

and so forth, and have data entry instructions. Once all fields 

for the preliminary set of forms have been completed, data can 

be entered for the remaining forms. When using replicas for 

data entry, the replica is synchronized with the master database so that new data are added. Note: The “Prevent Deletes” 

option is enforced in replica databases to ensure that data are 

not inadvertently lost.

Several features are built-in to form properties that enable 

the user to maximize data entry efforts while minimizing 

error. Data input masks for ease of viewing multi-part data 

(park/project codes and date in PeriodID or EventID), “fillin-as-you-type” to automatically complete a field, limiting 

input values to known ranges (or restricting null values) or 

providing “drop-down boxes,” highlighting data entry boxes 

being edited, and tab indexes to control the order of data entry. 

Forms also contain fields that require data input and/or are 

constrained to properties and integrity of related tables.
Standard Operating Procedure 5: Data Analysis, Version 1.0 27

Parameter Estimation

The most basic data analyses will consist of plotting 

means and ranges of metal concentrations in Asian clams 

and crayfish from each stretch with time. If desired, standard 

errors and confidence intervals also can be computed around 

the means; however, and as noted in the narrative, an overall 

mean for ONSR will not be computed.

Control Charts

The construction and interpretation of control charts is 

described in many texts focusing on industrial quality control 

(Beauregard and others, 1992; Gyrna, 2001; Montgomery, 

2001). The application of control charts for environmental 

monitoring is discussed in texts by McBean and Rovers (1998) 

and Manly (2001), and in a review by Morrison (2008). These 

sources, or an analyst familiar with control charts, should be 

consulted before actual control chart construction.

Although control limits often are based on values from a 

statistical sampling process, the control limits will be set based 

on toxicity thresholds developed as described in the protocol 

narrative. It is important to recognize that this approach is not 

based on a statistical distribution, and therefore probabilities 

cannot readily be associated with the observations. A generic 

control chart is illustrated in figure 2. If variations are desirable or necessary, a statistical analyst familiar with control 

charts should be consulted.

The control chart represents a basic data set summary 

indicating which variables are in the greatest need of more indepth analyses or management action. Control charts contain 

only upper control limits representing toxicity thresholds for 

invertebrate metals concentrations associated with dietary risk 

to riparian wildlife. The toxicity thresholds have been determined through food chain analysis using procedures developed for conducting ecological risk assessments (USEPA, 

1992, 1997, 1999, 2007b). 

The rationale and assumptions associated with food chain 

analysis for warm-blooded vertebrates are described in the 

“Toxicity Threshold Development” section of the protocol 

narrative. Food chain analysis is based on TRVs for the metals of interest. These represent daily contaminant intake rates 

associated with toxicity in representative wildlife species. The 

TRVs used here are consensus NOAEL values developed by 

the USEPA in support of Ecological Soil Screening Levels 

(Eco-SSLs) for metals. Current (May 2008) values are listed 

in table 5.

The other component(s) of food chain analysis are the 

daily food intake (DI) rates of the wildlife species being 

modeled. Values for the receptor species used here (American 

robin, Turdus migratorius; great blue heron, Ardea herodias; 

short-tailed shrew, Blarina brevicauda; and American mink, 

Mustela vison) were obtained from the “Wildlife Exposure 

Factors Handbook” (USEPA, 1993) and are identified in the 

footnotes of table 6. 

The measured concentrations in Asian clams and crayfish are compared to the toxicity thresholds, which represent 

NEHCs. The NEHCs in table 6 have been computed from 

Standard Operating Procedure 5: Data Analysis, Version 1.0 (July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number

TIME

METAL CONCENTRATION

Warning limit for small mammals

Warninglimitfor small birds

Crayfish and Asian clam samples

Figure 2. Example control chart for tracking concentrations 

of metals in crayfish and Asian clam.
28 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

the TRVs and DIs as wet-weight and dry-weight values as 

described in the “Toxicity Threshold Development” section 

of the protocol narrative. The comparisons can be made either 

directly (between measured concentrations and NEHCs) or by 

computing “safety factors” (maximum measured concentration 

÷ NEHC). 

Example Based on 2005 Data

Maximum 2005 concentrations of cadmium, cobalt, 

lead, nickel, and zinc in crayfish and Asian clams from ONSR 

(Schmitt and others, 2007b) are presented in table 5. The 

NEHCs computed for the four receptor species are presented 

in table 6. The NEHCs are presented in wet weight and dryweight units to facilitate comparisons with invertebrate metals 

data from ONSR, which are measured in dried samples and 

reported in dry-weight units; for budgetary reasons, moisture 

(water) content is not determined. Dry-weight NEHCs for 

Asian clams and crayfish were computed using approximate moisture values obtained from the scientific literature 

(Belanger and others, 1986; Graney and Giesy, 1988; Schmitt 

and others, 2006; table 6). The generally large safety factors 

(>10) indicate a relatively low risk level for most metals in 

both taxa (table 6). The exceptions are for cadmium and zinc 

risk to small mammals and birds and nickel risk to small mammals (table 6). These comparatively small safety factors (<10) 

reflect both the large uncertainty in the process, but also that 

further monitoring of cadmium and zinc is warranted.

As noted previously, the NEHCs for the metals and 

wildlife species identified in table 6 were based on TRVs and 

exposure factors that were current at the time this document 

was prepared (May, 2008); however, the Eco-SSLs and the 

TRVs upon which they are based, along with other data used 

in the analysis, are subject to revision. In addition, the process 

of risk assessment as it applies to cold-blooded vertebrates 

(fishes, amphibians, and reptiles) is evolving rapidly. Consequently, the relevant information source(s) should be consulted to ascertain the current values each time an analysis is 

performed (see urls in the references identified in the headnotes and footnotes of tables 5 and 6). If necessary, the tables 

and control charts should be updated to reflect the most recent 

values in a revised version of this SOP. 

Constructing the Control Charts

1. Determine the contaminant, stretch, and species of interest. Separate control charts should be constructed for each 

contaminant, stretch, and monitored species. Use figure 2 

as a guide.

2. Plot the mean and range (or individual sample values) of 

the contaminant of interest (on the y-axis) against time 

(on the x-axis). Standard errors can be indicated by error 

bars surrounding each mean.

3. Determine a “center-line” value for the contaminant, 

which in general represents a long-term mean, a target value, or some other value. Here the most obvious 

“center-line” values are the 2005 concentrations for each 

stretch reported by Schmitt and others (2007b). Generally, determining an appropriate center-line value contains 

inherent pitfalls, and an analyst who is familiar with 

control charts should be consulted. In the context of this 

protocol, the center-line value is not as critical because the 

upper control limit is not based on any statistical property 

of the center-line.

4. Establish the upper control limit. (Lower control limits will not be necessary; low concentrations are not a 

concern.) The upper control limit will be the current 

dry-weight NEHC for the metal of interest in each taxon 

(table 6). It should be noted that control limits constructed 

in this way should be considered as “warning” limits 

rather than “action” limits because the thresholds represent NOAELs; the inherent assumptions (such as the 

receptor species consuming only the monitored species) 

will, therefore, likely result in conservative limits. For 

most metals, two such warning limits ideally would be 

indicated on each control chart: one representing the 

threshold for the small bird (robin) category, and a second 

representing the threshold for the small mammal (shrew) 

category. For cadmium, a third warning level representing 

the NEHC for amphibians also may be indicated.

5. Continue to plot values of the contaminant with time 

as new data become available. An observation that 

approaches or exceeds the warning limit(s) should trigger 

management concern. Any general increase with time in 

the contaminant concentrations beyond the 2005 baseline data (Schmitt and others, 2007b), even if below the 

warning limits, may be cause for concern. Update tables 5 

and 6 and the control charts (and revise this SOP) as 

necessary.

Table 5. No observed adverse effect level toxicity reference 

values for metals in mammals and birds, and maximum 2005 

concentrations in crayfish (Orconectes luteus) and Asian clam 

(Corbicula fluminea) from Ozark National Scenic Riverways. 

[TRV, toxicity reference value; from the U.S. Environmental Protection 

Agency (USEPA, 2005a, 2005b, 2005c, 2007a, 2007c). Concentrations in 

crayfish and Asian clam from Schmitt and others (2007c)]

Metal

TRV (mg/kg/d) Maximum concentration 

(mg/kg dry weight)

Mammals Birds Crayfish Asian clam

Cadmium 0.77 1.47 0.77 3.34

Cobalt 7.33 7.61 1.27 2.00

Lead 4.70 1.63 .37 .51

Nickel 1.70 6.71 1.25 2.00

Zinc 75.4 66.1 84.9 226
Standard Operating Procedure 5: Data Analysis, Version 1.0 29

Table 6. No-effect hazard concentrations and safety factors of metals in crayfish (Orconectes spp.) and Asian clam (Corbicula 

fluminea) for receptor wildlife species.

[NEHC, no-effect hazard concentration; SF, safety factor; ww, wet weight; dw, dry-weight]

Species NEHC

(μg/g ww)1

Crayfish NEHC

(μg/g dw)1, 2 Crayfish SF1 Asian clam NEHC(μg/g dw)1, 2 Asian clam SF1

Cadmium

Robin3 1.0 4.8 6.3 6.4 1.9

Heron4 8.2 40.8 53.0 54.4 16.3

Shrew5 1.2 6.2 8.1 8.3 2.5

Mink6 5.5 27.5 35.7 36.7 11.0

Cobalt

Robin3 5.0 25.0 19.7 33.4 16.7

Heron4 42.3 211 166 282 141

Shrew5 11.8 59.1 46.5 78.8 39.4

Mink6 52.4 262 206 349 175

Lead

Robin3 1.1 5.4 14.5 7.1 14.0

Heron4 9.1 45.3 122 60.4 118

Shrew5 7.6 37.9 102 50.5 99.1

Mink6 33.6 168 454 224 439

Nickel

Robin3 4.4 22.1 17.7 29.4 14.7

Heron4 37.3 186 149 249 124

Shrew5 2.7 13.7 11.0 18.3 9.1

Mink6 12.1 60.7 48.6 81.0 40.5

Zinc

Robin3 43.5 217 2.6 290 1.3

Heron4 367 1,836 21.6 2,448 10.8

Shrew5 122 608 7.2 811 3.6

Mink6 539 2,693 31.7 3,591 15.9

1

 NEHC=Toxicity reference value (TRV)/daily food ingestion (DI); SF=NEHC/maximum 2005 concentration in crayfish and Asian clams from Ozark 

National Scenic Riverways (Schmitt and others, 2007c); all assuming a diet of 100 percent crayfish or Asian clam.

2

 dw values computed assuming a moisture content of 80 percent for crayfish (Schmitt and others, 2006) and 85 percent for Asian clam (Belanger and others, 1986; Graney and Giesy, 1988; Jacomini and others, 2006).

3

 American robin, Turdus migratorius; DI=1.52 kg/kg/d (USEPA, 1993).

4

 Great blue heron, Ardea herodias; DI=0.18 kg/kg/d (USEPA, 1993).

5

 Short-tailed shrew, Blarina brevicauda; DI=0.62 kg/kg/d (USEPA, 1993).

6

 American mink, Mustela vison; DI=0.14 (USEPA, 1993).
30 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Report Format

Template

The report template for regional natural resource technical reports should be followed (http://www.nature.nps.gov/

publications/NRPM/index.cfm). Natural resource reports are 

the designated medium for disseminating high priority, current 

natural resource management information with managerial 

application. The NPS Natural Resource Technical Reports

series is used to disseminate the peer-reviewed results of scientific studies in the physical, biological, and social sciences 

for the advancement of science and the achievement of the 

NPS mission. 

Style

Standards for scientific writing as recommended in the 

Council of Science Editors (CSE) Style Manual (CSE, 2006) 

should be followed. Reports should be direct and concise. 

Authors of reports also should refer to Mack (1986), Goldwasser (1999), Strunk and White (2000), and Day and Gatsel 

(2006) for grammar and writing style.

Types of Reports and the Review Process

The HTLN monitoring plan (DeBacker and others, 

2005) specifies that, for vital signs monitored annually, 

two main types of reports will be produced: annual status 

reports, and comprehensive trends and analysis and synthesis 

reports (every 5 to 7 years). Because metals monitoring will 

be conducted every 5 years, a modified reporting strategy is 

necessary; a report will be produced each time monitoring 

is conducted (every 5 years). This report will summarize the 

most recent monitoring results, provide an update on the status 

of selected natural resources, and highlight concerns that may 

require management action. It also should include a summary 

of data collected previously. After several monitoring events, 

it may be possible to begin to describe and interpret trends in 

metal concentrations. The primary audience for these reports 

will be park resource managers and external scientists. An 

Executive Summary, which highlights key points for nontechnical audiences, will be produced with each report. The 

primary audience for this Executive Summary will be Park 

Superintendents, interpreters, and the general public. Peer 

review for both will be conducted internally by HTLN staff.

Report Distribution

After review, annual reports will be distributed by September 1 of the year following data collection. Reports will 

be provided to ONSR, and a copy will be kept on file with 

the HTLN office of the National Park Service in Republic, 

Missouri, and can be made available to interested parties upon 

request. All data are public property and subject to requests 

under the Freedom of Information Act (FOIA); however, 

sensitive data, such as the location of rare species, must be 

withheld in some situations. Reports containing non-sensitive 

data will be disseminated through the HTLN network website 

(http://science.nature.nps.gov/im/units/htln/).

Standard Operating Procedure 6: Reporting, Version 1.0 (July 28, 2008) 

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
Standard Operating Procedure 7: After the Field Season, Version 1.0 31

Procedures

1. Thoroughly clean, inspect, and repair or replace as 

necessary all equipment (such as nets, buckets, personal 

flotation devices, and boots) before storing it for future 

use. All reference manuals should be re-shelved on their 

appropriate bookshelf. Other reference materials and extra 

data sheets need to be filed in their appropriate filing 

cabinet. Clean the insides and outsides of all vehicles used 

in the field.

2. Organize field data sheets and check that they have been 

filled out completely. As a rule, all data sheets need to 

be reviewed for completeness before the crew leaves the 

field, then again as samples are logged in and frozen. 

However, because of the number of field days and crew 

members, some deficiencies in data recording may not be 

identified until all data sheets have been organized and 

reviewed as a group.

3. It also is best to enter data into computer files and verify 

the data as they are collected rather than letting it accumulate until after all the field work is done. This also is 

a good check on completeness, and will allow questions 

to be resolved while memories are fresh and personnel 

involved with the project are available. Ascertain that all 

data have been entered and verified by signing a “Supervisor Check” block on the Invertebrate Field Data Form.

4. When the laboratory data become available, check them 

over carefully and resolve any discrepancies before merging them with the field data. 

Standard Operating Procedure 7: After the Field Season, Version 1.0 

(July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
32 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Personnel requested to edit the Protocol Narrative or any 

of the SOPs need to follow this procedure to eliminate confusion about how data are collected and analyzed. All personnel 

should be familiar with this SOP to identify and use the most 

current methodologies.

Procedures

1. This protocol and accompanying SOPs have attempted to 

incorporate the most sound methodologies for collecting 

and analyzing trace metal data. Nevertheless, all protocols 

require updating and editing as new and different information becomes available. Required edits should be made in 

a timely manner and appropriate reviews undertaken.

2. All edits require review for clarity and technical soundness. Small changes or additions to existing methods 

will be reviewed in-house by HTLN staff. However, if a 

complete change in methods is sought, an outside review 

is required. Regional and National NPS staff with familiarity in toxicology and data analysis will be utilized as 

reviewers. Experts outside of the NPS also will be utilized 

in the review process.

3. Document edits and protocol versions in the Revision 

History Log within the Protocol Narrative and each SOP. 

Log the changes in the Protocol Narrative or SOP being 

edited only. Version numbers increase incrementally (for 

example version 1.1, version 1.2, ...) for minor changes. 

Major revisions should be designated with the next whole 

number (version 2.0, version 3.0, version 4.0...). Record 

the previous version number, date of the revision, and 

author of the revision; identify paragraphs and pages 

where changes are made and the reason for making the 

changes; record the new version number.

4. Inform the Data Manager about changes to the Protocol Narrative or SOP so the new version number can be 

incorporated in the Metadata of the project database. The 

database may have to be edited by the Data Manager to 

accompany changes in the Protocol Narrative and SOPs.

5. Post new versions on the internet (http://science.nature.

nps.gov/im/monitor/protocoldb.cfm) and forward electronic copies to all known individuals in possession of 

a previous version of the affected Protocol Narrative or 

SOP.

Acknowledgments

This study was supported by the Contaminants Biology 

and Status and Trends of Biological Resources Programs of 

the U.S. Geological Survey and through an interagency agreement with the National Park Service. G. Linder and A.L. Allert 

provided useful comments on earlier versions of this report.

Standard Operating Procedure 8: Revising the Protocol, Version 1.0 

(July 28, 2008)

Revision History Log

Previous version 

number Revision date Author Changes made Reason for change New version number
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Appendix 1. Invertebrate Field Data Form, 

Sample Labels, and Chain-of-Custody Record
40 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0 Invertebrate Field Data Form: Collecting, Processing, and Preparing Crayfish and Asian clams (Corbicula fluminea) for Metals Analysis Park ______ Nominal site location ________________________________________________________________________ Date (mm/dd/yyyy): _____________ Start Time (hhmm): __________ Personnel: __________________________________________ Protocol Version: _______________ End Time (hhmm):__________ Sample Type (Crayfish or Corbicula): _____________________ Sample A Species Carapace length of shell dia. (mm) Sample B Species Carapace length of shell dia. (mm) Sample C Species Carapace length of shell dia. (mm) Comments

1 1 12 2 23 3 34 4 45 5 56 6 67 7 78 8 89 9 9

10 10 10

UTM-X UTM-X UTM-X GPS Datum:UTM-Y UTM-Y UTM-Y GPS Accuracy:Corbicula

start/end date, time

Freezer date 

and time

Supervisor Check: _________________________________________________ Supervisor Check: _________________________________________________Supervisor Check: _________________________________________________ Supervisor Check: _________________________________________________Supervisor Check: _________________________________________________ Supervisor Check: _________________________________________________Supervisor Check: _________________________________________________ Supervisor Check: _________________________________________________
Appendix 1. Invertebrate Field Data Form, Sample Labels, and Chain-of-Custody Record 41

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42 Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0

Chain-of-Custody Record

Study No. Study Name Control No.

page___ of ____

Samplers: (Signatures)

Sample Identification Date Time Type * Remarks and Observations

Relinquished by:

(Signature) Date/Time Received by:(Signature)

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(Signature)

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(Signature)

Relinquished by:

(Signature) Date/Time Received for Laboratoryby:(Signature) Date/Time Remarks

* CR=crayfish; CO=Asian clam (Corbicula); O=other, define in remarks
Publishing support provided by:

Rolla Publishing Service Center

For more information concerning this publication, contact:

Director, USGS Columbia Environmental Research Center

4200 New Haven Road

Columbia, MO 65201

(573) 875–5399

Or visit the Columbia Environmental Research Center Web site at:

http://www.cerc.usgs.gov
Schmitt and others—Protocol for Monitoring Metals in Ozark National Scenic Riverways, Missouri: Version 1.0—Open-File Report 2008–1269