Fish Sampling and Analysis:
A Guidance Document
for Issuing Fish Advisories
Prepared by
Research Triangle Institute
Research Triangle Park, NC 27709'
* ' "'
Prepared for
,*' : - t
U.S. Environmental Protection-Agency
Office of Science and Technology
Washington,. DC
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82OD70001
TABLE OF CONTENTS
Section Page
1 INTRODUCTION 1-1*
1.1 Background 1-2
1.2 Objectives 1-4
1.3 Applicability of this Manual 1-6
1.4 Relationship of Manual to Other EPA Documents 1-6
1.5 Organization of the Manual 1-7
2 MONITORING STRATEGY 2-1
2.1 Initial Screening 2-4
2.1.1 Objective 2-4
2.1.2 Target Species 2-8
2.1.3 Target Contaminants 2-9
2.1.4 Contaminant Trigger Values 2-10
2.1.5 Sampling Locations -2-11
2.1.6 Sampling Times 2-12
2.1.7 Sample Type 2-14
2.1.8 Sample Replication 2-17
2.1.9 Sample Analysis 2-18
2.1.10 Data Reporting and Evaluation 2-19
2.1.11 Summary of Assumptions and Limitations 2-20
2.2 Intensive Monitoring 2-21
2.2.1 Objective 2-21
2.2.2 Target Species 2-22
2.2.3 Target Contaminants 2-23
2.2.4 Contaminant Trigger Values 2-23
2.2.5 Sampling Locations 2-24
2.2.6 Sampling Times 2-25
2.2.7 Sample Type 2-26
2.2.8 Sample Replication 2-31
2.2.9 Sample Analysis 2-33
2.2.10 Data Reporting and Evaluation 2-34
3 RECOMMENDED TARGET SPECIES 3-1
3.1 Utility of Using Target Species 3-1
3.2 Criteria for Selection of Target Species 3-2
3.2.1 Initial Screening Study 3-3
3.2.2 Intensive Monitoring Study 3-5
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TABLE OF CONTENTS (continued)
Section Page
3.3 Freshwater Target Species 3-6
3.3.1 Initial Screening Study 3-6
3.3.2 Intensive Study 3-15
3.4 Estuarine/Marine Target Species 3-15
3.4.1 Initial Screening Study 3-15
3.4.2 Intensive Study 3-21
4 TARGET CONTAMINANTS 4-1
4.1 Development of Target Contaminant List 4-1
4.1.1 Metals 4-15
4.1.2 Pesticides 4-23
4.1.3 Base/Neutral Organic Compounds 4-33
4.1.4 Dioxins and Furans 4-40
4.1.5 Chlorinated Phenolic Compounds 4-41
4.1.6 Volatile Organic Compounds 4-43
4.2 Development of Trigger Values for Target Contaminants .... 4-43
4.2.1 General Equations for Calculating Trigger Values . . . 4-43
4.2.1.1 Noncarcinogens 4-46
4.2.1.2 Recommended Values for Variables in
TV Equations 4-46
4.2.2 Estimating Trigger Values for Initial Screening 4-47
4.2.3 Estimating Trigger Values for Intensive Monitoring ... 4-52
4.2.4 Comparison of EPA Trigger Values with Other
Health Protection Criteria 4-53
5 FIELD PROCEDURES 5-1
5.1 Sampling Design 5-1
5.1.1 Initial Screening Study 5-2
5.1.2 Intensive Monitoring 5-11
5.2 Sample Collection 5-17
5.2.1 Sampling Equipment and Use 5-18
5.2.2 Preservation of Sample Integrity 5-26
5,2.3 Field Recordkeeping 5-27
5.3 Simple Processing, Preservation, and Shipping 5-38
5.3.1 Sample Processing 5-38
5.3.2 Sample Preservation 5-44
5.3.3 Sample Shipping 5-47
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TABLE OF CONTENTS (continued)
Section Page
6 LABORATORY PROCEDURES , 6-1
6.1 Sample Receipt and Chain-of-Custody 6-1
6.2 Sample Processing 6-3
6.2.1 General Considerations 6-3
6.2.2 Fish Samples 6-6
6.2.3 Shellfish Samples 6-12
6.3 Sample Distribution 6-17
6.3.1 Sample Aliquotting 6-17
6.3.2 Sample Transfer 6-20
6.4 Sample Analyses 6-20
6.4.1 Target Analytes 6-20
6.4.2 Analytical Methods 6-20
6.4.3 General QA/QC Considerations for Sample Analysis . 6-31
7 DATA REPORTING, ANALYSIS AND EVALUATION 7-1
7.1 Data Reporting 7-1
7.1.1 Initial Screening 7-1
7.1.2 Intensive Monitoring 7-3
7.2 Data Analyses and Evaluation 7-5
7.2.1 Initial Screening 7-5
7.2.2 Intensive Study-Phase I 7-7
7.2.3 Intensive Study-Phase II 7-11
7.2.4 Issuance of Fish/Shellfish Consumption Advisories ..7-16
8 LITERATURE CITED 8-1
Appendixes Page
A Freshwater Species for Which State Consumption Advisories
Have Been Issued A-1
B West Coast Estuaries Br1
C Gulf Coast Estuaries C-1
in
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TABLE OF CONTENTS (continued)
Appendix Page
D Summary of Chemical Contamination Results from National and
Regional Fish/Shellfish Contaminant Monitoring Programs D-1
E IRIS Printouts E-1
F General Quality Assurance/Quality Control Considerations F-1
G Forms G-1
GG Recommended Procedures for Preparing Whole Fish Composite
Samples GG-1
H Example Procedure for Analysis of Percent Lipid in Tissue
Samples H-1
I Example Procedure for Analysis of Cadmium by Graphite
Furnace Atomic Absorption (GFAA) Spectrometry 1-1
J Example QA/QC Procedures and Requirements for Analysis of
Organic Compounds J-1
K Example QA/QC Procedures and Requirements for Analysis
of Metals K-1
L Sources of EPA-Certified Reference Materials and Standards L-1
M Definition and Procedure for the Determination of the Method
Detection Limit M-1
N Example Data Forms for Analysis of Metals and Organic Target
Contaminants N-1
O Statistical Tables O-1
IV
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LIST OF FIGURES
Number Page
5-1 Example of a sample request form 5-3
5-2 Spawning period of primary freshwater target species 5-9
5-3 Sampling station layouts for probability sampling
in two dimensions 5-14
5-4 Field record for fish contaminant monitoring program--
screening study 5-29
5-5 Field record for shellfish contaminant monitoring program--
screening study 5-30
5-6 Field record for fish contaminant monitoring program--
intensive study 5-31
5-7 Field record for shellfish contaminant monitoring program--
intensive study 5-33
5-8 Sample identification label 5-35
5-9 Example of a chain-of-custody tag or label 5-36
5-10 Example of a chain-of-custody form 5-37
5-11 Recommended measurements of body length and size for fish
and shellfish 5-40
6-1 Laboratory sample preparation and handling for fish fillet
composite samples 6-7
6-2 Sample processing record for fish contaminant monitoring program--
fish fillet composites 6-9
6-3 Laboratory sample preparation and handling for shellfish
edible tissue composite samples 6-14
6-4 Sample processing record for shellfish contaminant monitoring
program-edible tissue composites 6-15
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LIST OF FIGURES
Number Page
6-5 Fish/shellfish monitoring program sample aliquotting record 6-19
6-6 Fish/shellfish monitoring program sample transfer record 6-21
7-1 Sample output from the database-Current State Fish and
Shellfish Consumption Advisories and Bans 7-18
VI
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LIST OF TABLES
Number Page
5-1 Primary Target Species Recommended for Initial Screening
Studies 5-7
5-2 Summary of Fish Sampling Equipment 5-19
5-3 Summary of Shellfish Sampling Equipment 5-21
5-4 Checklist of Field Sampling Equipment and Supplies for
Fish/Shellfish Contaminant Monitoring Programs 5-23
5-5 Safety Considerations for Field Sampling Using a Boat 5-24
5-6 Recommendations for Preservation of Fish/Shellfish Samples
from Time of Collection to Delivery at Central
Processing Laboratory 5-46
6-1 Recommendations for Container Materials, Preservation,
and Holding Times for Fish/Shellfish Tissues from
Delivery at Central Processing Laboratory to Analysis 6-3
6-2 Individual Weights (g) of Homogenate Required for a
Composite Sample 6-13
6-3 Summary of Basic Sample Preparation and Analytical
Techniques for Organic Target Contamination 6-23
6-4 Contract Laboratories Conducting Dioxin/Furan Analysis in
Fish/Shellfish Tissues 6-25
6-5 Recommended Methods for Analysis of Target Contaminants 6-26
6-6 Comparison of Target Contaminant Trigger Values (TVs) with Typical
Detection Limits for Organic Compounds in Tissue Samples 6-27
6-7 Comparison of Target Contaminant Trigger Values (TVs) with
Typical Detection Limits for Trace Metals in Tissue Samples 6-28
6-8 Recommended Quality Assurance/Quality Control (QA/QC)
Samples 6-33
VII
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6-9 Marine/Estuarine Tissue Reference Materials t 6-40
7-1 ANOVA Table for Single-Factor Study 7-13
7-2 ANOVA Table for the Phase II Toxaphene Study 7-14
7-3 Recommended Guidelines for Issuing Various Types of
Fish/Shellfish Advisories 7-17
VIII
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SECTION 5
FIELD PROCEDURES
The major objective of this section is to provide guidance to States on (1) sampling
design for initial screening and intensive monitoring phases of fish contaminant monitoring
programs and (2) field procedures for collecting, processing, preserving, and shipping samples
to a central processing laboratory for pollutant analysis. This guidance emphasizes planning
and documentation of all field procedures to ensure that collection activities are cost-effective
in meeting sampling objectives and that sample integrity is preserved during all phases of the
sampling process, from collection to delivery of samples to the central processing laboratory.
The format of the Work/QA Project Plan outlined in Appendix F is recommended for
documenting the specific procedures used in State fish/shellfish contaminant monitoring
programs. In addition, protocols for sample collection procedures should be prepared to
document the methods used by each State and to allow assessment of final data quality and
comparability.
5.1 SAMPLING DESIGN
Prior to making a field collection trip, the program manager and field sampling staff
should meet to develop a detailed plan for sampling at the proposed sample collection sites.
In preparation for these planning meetings, staff should review all pertinent information on the
sites that have been selected for inclusion in the contaminant monitoring studies. Historic
information on water and sediment quality and any previously conducted tissue contaminant
monitoring data should be reviewed. Existing data on pollutant inputs to the waterbody from
point and nonpoint sources should also be reviewed. In addition, personnel roles and
responsibilities with respect to all phases of the fish/shellfish sampling effort should be clearly
defined. All aspects of the final sampling design for a State's fish/shellfish contaminant
monitoring program should be documented clearly by the program manager in the Work/QA
Project Plan (see Appendix F).
In the recommended two-tiered monitoring strategy described in Section 2, there are
six major parameters directly associated with sample collection that must be specified for each
sampling site during the planning stage and prior to the initiation of any field collection
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activities. The following parameters must be selected for each site:
Site location
Target species
Target contaminants
Sampling times
Sample type
Sample replication.
After reviewing the objectives of initial screening studies or intensive monitoring studies
(Section 2) and all relevant information on the sites to be monitored, States should plan the
specific aspects of field collection activities for each site by considering all of those
parameters that influence sample collection procedures. The program manager should
document specific aspects of each parameter in a sample request form (Figure 5-1) for each
sampling site. (A copy of this form is available in Appendix G.) The sample request form
should provide the field collection team with readily available information on the project
objective, sample type to be collected, target contaminants to be evaluated, site
name/number, site location, target species and alternate species to be collected, sampling
date, sampling method to be used, number of replicates to be collected, and number of
samples to be collected for each composite. The original sample request form should be
maintained on file with the program manager and a copy taken into the field by the field
sampling team and maintained with the field logbook. Each of the major parameters that
influence sample collection procedures is discussed for initial screening studies in Section
5.1.1 and for intensive monitoring studies in Section 5.1.2.
5.1.1 Initial Screening Study
The primary objective of initial screening studies is to monitor probable worst-case
exposure situations and some reference sites for a wide range of target contaminants to
identify hot spots for more intensive followup monitoring. Analyses of fish fillets (skin-on
including belly flap tissue and edible portions of shellfish) are recommended for
screening studies to estimate worst-case exposures of the general U.S. population.
Note: To provide an indication of potential exposures of the subpopulations of sport or
subsistence fishermen (e.g., certain ethnic groups) who do consume whole fish or parts other
than fillets, States may deem it necessary to collect whole fish and/or shellfish for analyses
during the initial screening study and/or intensive study.
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Sample Request Form
Project
Objective
Sample
Type
Target
Contaminants
D Screening Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than
whole _ )
D Whole fish or portions other
than fillet (Specify tissues used
if other than whole
D All target contaminants
D Additional contaminants
(Specify
D Intensive Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than whole
D Whole fish or portions other than fillet (Specify
tissues used if other than whole
D Contaminants exceeding screening study TVs
(Specify
J
INSTRUCTIONS TO SAMPLE COLLECTION TEAM
Project Number:.
County/Parish:
Target Species:
D Freshwater
D Estuarine
Site (Name/Number):
Lat./Long.:
Alternate Species: (in order of preference)
Proposed Sampling Dates:.
Proposed Sampling Method:
Electrofishing
D Seining
D Trawling
D Other (Specify,
D Mechanical grab or tongs
D Biological dredge
D Hand collection
Number of Sample Replicates: D No field replicates (1 composite sample only)
D field replicates
(Specify number for each target species)
Number of Individuals
per Composite:
.Fish per composite (10 fish optimum)
.Shellfish per composite (specify number to obtain 500 grams of tissue)
Figure 5-1. Example of a sample request form.
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[Reviewers, please comment on fillet type to use In both initial screening and in
intensive studies. We are recommending skin on-but should bellyflap be Included for
worst case scenario?]
5.1.1.1 Site Selection--
The field collection staff should review historical data on each screening site using a
recent hydrologic map of the site, of the appropriate scale, to ensure that the sampling site is
Located downstream of target point source discharges such as
-- Industrial or municipal dischargers
-- Combined sewer overflows (CSOs)
- Urban storm drains
Located downstream of target nonpoint source inputs such as
-- Landfill, RCRA, or CERCLA sites
-- Areas of intensive agricultural activities, mining activities, or urban land
development
-- Areas receiving inputs through multimedia mechanisms such as atmospheric
deposition or hydrogeologic connections
Located in an area acting as a potential pollutant sink where contaminated
sediments accumulate and bioaccumulation potential might be enhanced
Located in an unpolluted area that can serve as a reference site for subsequent
intensive studies.
Although the procedures required to identify candidate hot spot sites in proximity to
significant point source discharges are usually straightforward, it is often more difficult to
identify clearly defined hot spot areas associated with nonpoint sources. In these instances,
assessment information summarized in State Section 305(b) reports or Section 319 nonpoint
source assessment reports should be reviewed before site locations are selected.
The ultimate selection of any sampling site location must be a site-specific
decision based on the best professional judgment of the field sampling staff. Several
site-specific considerations have been identified that should be evaluated (Versar, 1982):
Proximity of sites for sampling water and sediments
Availability of data on fish or shellfish community structure
Bottom condition
Type of sampling equipment available
Accessibility of the site.
5-4
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The most important benefit of locating fish or shellfish sampling sites near sites
selected for water and sediment sampling is the possibility of correlating contaminant
concentrations in different environmental compartments (water, sediment, and fish). Selecting
sampling sites in proximity to one another is also more cost-effective in that it provides
opportunities to combine sampling trips for different matrices.
Availability of data on the indigenous fish and shellfish communities should be
considered in final site selection. Information on preferred feeding areas, spawning areas, and
migration patterns of target species is a valuable asset in locating populations of the target
species (Versar, 1982). Knowledge of habitat preference provided by fishery biologists or
commercial fishermen may significantly reduce the time required to locate a suitable
population of the target species at a given site.
Bottom condition is another site-specific factor that is closely related to the ecology of
a target fish or shellfish population (Versar, 1982). For example, if only soft-bottom areas are
available at an estuarine site, neither oysters (Crassostrea virginica) nor mussels (Mytilus
edulis and M. califomianus) would likely be present at the site because these species prefer
hard substrates. Bottom condition also must be considered in the selection and deployment of
sampling equipment. Navigation charts provide depth contours and the locations of large
underwater obstacles in coastal areas and larger navigable rivers. Sampling staff might also
consult commercial fishermen familiar with the candidate site to identify localized areas where
the target species congregates and the appropriate sampling equipment to use.
Another factor closely linked to equipment selection is the accessibility of the sampling
site. For some small streams or land-locked lakes (particularly in mountainous areas), it is
often impractical to use a boat (Versar, 1982). In such cases the sampling site should be
located where there is good land access. When a site must be reached by land,
consideration should be given to the type of vegetation and local topography that could make
transport of collection equipment difficult. If access to the sampling site is by water,
consideration should be given to the location of boat ramps and marinas and the depth of
water (during the proposed sampling time) required to deploy the selected sampling gear
efficiently and to operate the boat safely.
All of the factors described above should be given consideration when selecting
sampling sites. Once the site has been selected, it should be plotted and numbered on the
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most accurate, up-to-date map of appropriate scale available. Recent 7.6-minute (1:24,000
scale) maps from the U.S. Geologic Survey (USGS) or National Ocean Survey or blue line
maps produced by the U.S. Army Corps of Engineers are of sufficient detail and accuracy for
sample site positioning. The type of sampling to be conducted, water depth, and estimated
time to the station from an access point should be noted. The availability of known targets for
visual or range fixes should be determined for each sampling site. Biological trawl paths (or
other sampling gear transects) and navigational hazards should also be indicated. Additional
information on site-positioning methods are described in Battelle (1986), Tetra Tech (1986),
and Puget Sound Estuary Program (1990a).
An accurate description of each sampling site is important since State fish/shellfish
contaminant monitoring data will be stored in the Ocean Data Evaluation System (ODES)
database available to a broad spectrum of users nationwide. Each sampler should provide a
detailed description of each site and should refer to a 7.5-minute USGS map to determine the
exact latitude and longitude coordinates for the site. This information should be documented
in the sample request form and on the field record sheets (see Section 5.2.3).
5.1.1.2 Target Species Selection--
After reviewing information on each sampling site, the field collection staff should
identify the target species that can be expected to be collected at the site. The national
target species recommended for initial screening studies In freshwater systems are
shown In Table 5-1. For bottom feeders, the order of preference is carp, channel catfish, and
white sucker. No preferred order is given for predator species. In estuarlne/marine
ecosystems, one of the three bivalve species or a finfish species listed In Table 5-1
should be collected. If a recommended national target species is not available for collection,
a contingency plan for species selection should be decided upon at the planning meeting.
Field collection staff should select a second and third choice for the target species if none of
the national target species are available at the site. The alternate species for collection
should be selected from the regional target species lists presented in Table 3-6 for freshwater
and Tables 3-7 through 3-13 for estuarine/marine systems.
[Reviewers, please recommend estuarine finfish species as national target
species. These recommended species should be widely distributed geographically,
5-6
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TABLE 5-1. NATIONAL TARGET SPECIES RECOMMENDED FOR
INITIAL SCREENING STUDIES
Freshwater systems0 Estuarine/marine systems"
Bottom feeders:
Carp (Cyprinus carpio) Blue mussel (Mytilus edulis)
Channel catfish (Ictalurus punctatus) California mussel (Mytilus califomianus)
White suckers (Catostomus commersoni) American oyster (Crassostrea virginica)
Predators:
Largemouth bass (Micropterus salmoides)
Smallmouth bass (Micropterus dolomieui)
White crappie (Pomoxis annularis)
Northern pike (Esox lucius)
Flathead catfish (Pylodictus olivarus)
Brown trout (Salmo trutta)
Walleye (Stizostedion vitreum)
White bass (Morone chrysops)
fl For freshwater systems, one bottom-feeder and one predator species should be collected
at each site.
" For estuarine/marine systems, one bivalve species and one finfish species should be
collected at each site.
preferably demersal, nonmlgratory species with a known ability to bioconcentrate
pollutants. The National Bioaccumulation Study recommended the following estuarlne
species: hardhead catfish (Arlus fells), blue catfish (Ictalurus furcatus), freshwater
drum (Aplodlnotus grunnlens), spot (Lelostomus xanthurus), southern flounder
(Parallctys lethostlgma), and black drum (Pogonias cromls). The NOAA Status and
Trends Program recommended the following estuarine species: winter flounder
(Pseudopleuronectes amerlcanus), Atlantic croaker (Mlcropogonias undulatus), spot
(Lelostomus xanthurus), in Atlantic and Gulf waters and starry flounder (Platlchthys
stellatus), English sole (Parophrys vetulus), white croaker (Genyonemus llneatus),
barred sandbass (Paralabrax nebullfer), black croaker (Chellotrema saturnum),
hornyhead turbot (Pleuronlchthys vertlcalls) in Pacific waters. Recommendations are
needed for all estuarine waters.
5.1.1.3 Target Contaminant Selection-
For initial screening studies, all of the recommended target contaminants In
Table 4-3 should be analyzed. During the planning meeting, State staff should consider
whether additional contaminants should be analyzed. Historic data on water, sediment, and
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tissue contamination should be reviewed. In addition, priority pollutant scans from known
point source discharges should be examined to determine whether additional contaminant
analysis is warranted.
5.1.1.4 Sampling Times-
If program resources are sufficient, biennial screening of waterbodies where
commercial, recreational, or subsistence harvesting is practiced (as identified by the State) is
recommended. This recommended screening frequency will allow screening data to be used
in the biennial State 305(b) reports to document the extent of support of Clean Water Act
goals. At a minimum, these waterbodies should be screened once every 3 to 5 years.
Selection of the most appropriate sampling period is very important, particularly when
screening sampling will be conducted no more often than biennially. For initial screening
studies, the recommended sampling period Is from late summer to fall (i.e., September
to October). This sampling period avoids the spawning periods of most of the target species
except the brown trout (Figure 5-2). Water levels in many waterbodies are typically lower
during this time, which may simplify sampling procedures. In addition, this sampling period is
recommended to simulate a worst-case exposure scenario for organic pollutants (see Section
2.1.6).
Exceptions to this recommended sampling period for national target species should be
made only when important regional or site-specific factors favor alternative sampling periods.
For many States, budgetary constraints may require that a major portion of their sampling
efforts be carried out during July and August when they are able to employ temporary help or
student interns. When sampling is not conducted during the recommended late summer to fall
sampling period, the actual sampling period and the rationale for its selection should be
documented fully and the final data evaluation should include an assessment of how the
results may have been affected by sampling at a less than optimal time.
5.1.1.5 Sample Type-
Composite samples of homogenates of fish fillets (skin-on and including
bellyflap tissue) or the edible portions of shellfish are recommended for the analysis of
target contaminants in initial screening studies. Fish or shellfish collected for tissue
analysis should satisfy any legal requirements for harvestable sizes or weights and at least be
5-8
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01
CO
BOTTOM FEEDERS
Carp
Channel catfish
White sucker
PREDATORS
Largemouth bass
Smallmouth bass
White bass
White crappie
Walleye
Northern pike
Flathead catfish
Brown trout*
JAN
FEB
MAR
APR
-
MAY
~
-
JUNE
JULY
AUG
SEP ] OCT
1
I
Reconrti
Sampling
tnitteFSt
Stu
i
i
1
i
i
i
letlded
J«rlod for
reeflirtg
Ite*
NOV
DEC
Source: U.S. EPA, 1991. National Bioaccumulation Study (Draft Report). Office of Water Regulations and Standards, Washington, DC.
'Great Lakes only? Reviewers please comment on the use of brown trout as national target species and when they should be sampled. Please specify whether you are
referring to Great Lake populations or other riverine populations of brown trout.
Figure 5-2. Spawning period of national freshwater target species.
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of consumable size where no legal harvestable requirements are in effect. Given the aim of
screening studies to identify worst-case exposure conditions, it is recommended that the
largest available individuals of the target species be selected because larger (older) organisms
generally show the highest bioaccumulation levels (Phillips, 1980).
It is extremely important that the individual organisms used in composite samples be of
similar length or size (Wisconsin Department of Natural Resources, 1988). For fish or
shellfish, it is recommended that the total length (size) of the smallest individual in a
composite sample be no less than 75 percent of the total length (size) of the largest
individual in the composite sample (U.S. EPA, 1990b).
A minimum of 500 grams of tissue homogenate is recommended for each
composite sample so that sufficient material will be available for the number of
analyses required for the recommended target contaminants (Versar, 1982; 1984). If, in
addition to the recommended target contaminants, a State has included other pollutants for
analysis to address regional or site-specific concerns, a larger composite sample mass may
be required, and the estimated numbers of individuals required for each composite sample
noted in the following paragraphs may also need to be increased.
The number of individual organisms from a given species required to prepare a 500-g
composite whole-body sample will depend primarily on the target species and the age of the
individuals in the sample. For this reason, only approximate ranges can be suggested for the
numberofindividual organisms to collect (Versar, 1982; U.S. EPA, 1989d). For fish, 6 to 10
Individuals (10 is the preferred number for each composite) of legal harvestable size or
at least of consumable size should be collected for a given target species, with
preference given to the largest available individuals.
For shellfish, composite samples should be prepared from 10 to 50 individuals,
although for smaller shellfish (e.g., mussels, shrimp, crayfish) more than 50 individuals
may be needed to obtain the required 500-g composite sample.
Whenever possible, the same number of individuals should be used to prepare each
composite sample for a given target species for all sites. The number of individuals actually
used to prepare each composite sample should be clearly documented. If this number is
outside the recommended range, the reasons for this deviation should be recorded.
Recommended sample preparation procedures are discussed in Section 6.2.
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5.1.1.6 Sample Replication--
Sample replication requires the collection of sufficient numbers of individual organisms
from a target species at a target site to allow for the independent preparation of more than
one composite sample. Sample replication is optional in initial screening studies. If
resources are available, however, single replicate (i.e., duplicate) composite samples should
be collected for QA/QC purposes at a minimum of 10 percent of the screening sites (U.S.
EPA, 1990b). These sites should be identified during the planning phase and sample
replication specifications noted on the sample request form. If replicate field samples are to
be collected, the relative difference (in percent) between the overall mean length (size) of the
replicate samples and mean length (size) of any individual replicate sample should be no
greater than 10 percent (U.S. EPA, I990b). Note: Additional replicates must be collected at
each site for each target species if statistical comparisons to the target contaminant TVs are
required in the State monitoring programs. The statistical advantages of replicate sampling
are discussed in detail in Section 2.2.8 and Section 7.2.
5.1.2 Intensive Monitoring
The primary objective of intensive followup monitoring is to characterize the magnitude
and geographic extent of contamination in a range of legal size classes of harvestable
fish/shellfish species at those initial screening sites where concentrations of specific target
contaminants in tissues were found to be above recommended TVs. Intensive monitoring
focuses on the edible tissues of shellfish and fish (fillets) In order to assess whether
the contamination poses an unacceptable health risk to local fish/shellfish consumers
and whether a consumption advisory should be Issued. Rather than discouraging all fish
consumption, intensive monitoring studies should be designed to identify those specific fish
and shellfish species or age classes for which advisories should be issued. In addition,
intensive monitoring studies should be designed to tailor advisories to the consumption habits
or sensitivities of specific local human subpopulations.
Fillets (skin-on and including belly flap tissue) are recommended for analyses
because they are most representative of what the general U.S. population consumes.
However, if local subpopulations are known to consume whole fish or other specific parts
(e.g., heads, livers) of certain target species, the screening and intensive monitoring programs
should be expanded to include composites of those portions consumed in addition to fillet
5-11
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composites. The specific tissue type(s) to be collected should be noted on the sample
request form.
After reviewing the objectives of intensive monitoring studies (Section 2.2) and
reviewing the fish contaminant data obtained in the initial screening studies, State staff should
plan the specific aspects of field collection activities for each intensive monitoring site by
considering all the parameters that influence sample collection activities. Specific aspects of
each parameter should be documented clearly by the program manager for each site on a
sample request form.
5.1.2.1 Site Selection--
In planning the intensive followup monitoring that is required at all sites where TVs for
one or more target contaminants are exceeded, the field collection staff should review a
7.5-minute (1:24,000 scale) USGS hydrologic map of the potential hot spot and all relevant
water, sediment, and tissue contaminant data related to the site. Many of the same
considerations of site selection evaluated in the initial screening must be reevaluated before
sampling is initiated in the intensive study, including
Bottom conditions
Type of sampling equipment available
Accessibility of the screening site used in the initial screening study and Phase I
intensive study as well as additional sites where sampling efforts may be conducted
to bracket the geographic extent of the contamination as part of the Phase II
intensive study.
To the extent that program resources allow, intensive monitoring studies should be
conducted in two phases. Phase I of the intensive monitoring study should be designed to
identify the magnitude of tissue contamination (in edible tissues) in key species and size
classes of fish/shellfish of commercial, recreational, or subsistence fishing value at the site
sampled in the initial screening study. Phase II of the intensive monitoring study should be
designed to define clearly the geographic extent of the suspected contamination at the
targeted site and should include the Phase I intensive study site and additional sites located in
the waterbody under study. This may be quite straightforward where the sources of pollutant
introduction are highly localized or if site-specific hydrologic features create a significant
pollutant sink where contaminated sediments accumulate and the bioaccumulation potential
5-12
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might be enhanced (U.S. EPA, 1986b). For example, upstream and downstream monitoring to
bracket point source discharges, outfalls, and regulated disposal sites showing contaminants
from surface runoff or leachate can often be used to characterize the geographic extent of the
contaminated area. Within coves or small embayments where streams enter large lakes,
estuaries, or harbors, the geographic extent of contamination may also be characterized via
multilocational sampling to bracket the areas of concern. Such sampling designs are clearly
most effective where the target species are sedentary or of limited mobility.
Although bracketing approaches work best where the ultimate sources of
contamination can be associated with spatially well-defined hot spots, alternative sampling
designs are usually required where hot spots are not suspected. In the absence of historic
data, other appropriate sampling designs may be used to determine the geographic extent of
contamination in monitoring larger reservoirs, estuaries, or near-coastal areas. Several of the
more common recommended sampling designs are discussed in Gilbert (1987). Guidelines
for selecting appropriate sampling designs are summarized briefly here.
Although Gilbert (1987) discusses several sampling designs, systematic and two-stage
sampling appear to be the approaches most applicable for fish and shellfish contaminant
monitoring programs and the easiest to implement in the field. Where the target species are
widely and homogeneously distributed throughout the study area, a systematic sampling
design is often appropriate. This approach, which consists of sampling target species at
locations using a spatial pattern, is appropriate only for species with limited mobility (i.e.,
shellfish and fish with limited home ranges) so that the contaminant concentration in their
tissues is characteristic of the sampling site. For example, a State may select locations at
equidistant intervals in a river downstream from a suspected point source of contamination as
shown in Figure 5-3A. Or, the State may overlay a grid on a map of a large reservoir and
then systematically sample locations for collecting the target species (Figure 5-3B).
If habitat requirements or other life history features indicate that target species are
restricted to specific identifiable habitats within the study areas (e.g., the target species are
found in shallow water areas only), a two-stage sampling design may be considered. Using
this design, the State would first identify the shallow areas of a waterbody (e.g., estuary) on a
hydrologic map (see Figure 5-3C). Then, a probability sample of these shallow water areas
(first-stage units) would be selected prior to the initiation of field collection activities. In the
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A) Systematic Sampling
B) Grid Sampling
C) Two-stage Sampling
6m Depth Contour
Figure 5-3. Sampling station layouts for probability sampling in two dimensions.
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second stage, the target species would be sampled within the selected shallow water habitats
(second-stage units) (see Figure 5-3C).
Data collected from such complex sampling designs must be analyzed with statistical
estimation procedures that incorporate the complex design. The reader is referred to Gilbert
(1987) for details on proper estimation procedures. In all cases where intensive monitoring
studies are conducted, the program manager should enlist the assistance of a qualified
statistician in the initial sampling design phase through to the final data analysis and
interpretation phase.
5.1.2.2 Target Species Selection-
The main goal of intensive monitoring is to expand the range of fish and shellfish
species examined in initial screening studies to include as target species those species most
frequently consumed by the local population or specific subpopulations. The regional target
species recommended for sampling in intensive monitoring studies In freshwater
systems are listed in Table 3-6. The recommended regional target species that should
be considered for sampling in estuarine/marine waters are listed in Tables 3-7 through
3-9 for Atlantic Coast estuaries, in Table 3-10 for Gulf Coast estuaries, and in Tables
3-11 through 3-13 for Pacific Coast estuaries.
Final selection of regional target species must be the responsibility of State fisheries
personnel who have the expertise to identify local fish/shellfish species of commercial,
recreational, or subsistence value as a human food source in the study area and who are also
most familiar with local consumption patterns. In the event that the selected target species
are not available for collection, a contingency plan for collecting alternative species should be
decided upon at the planning meeting and the selection of species documented by the
program manager on the sample request form.
5.1.2.3 Target Contaminants--
Intensive monitoring at a given site should focus on those target contaminants
found in the initial screening study to be present In fish/shellfish tissue at
concentrations exceeding EPA-recommended TVs (Sections 2.1.4 and 4.2). Thus, in
general, the number of target contaminants evaluated in intensive followup monitoring studies
will be significantly smaller than the number evaluated in initial screening studies.
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5.1.2.4 Sampling Times-
To the extent that program resources allow, sampling times in intensive monitoring
studies should cover the principal period or periods when the target species is most
frequently harvested for human consumption and should ensure the collection of
appropriate samples of size and/or age classes over the legal harvestable size.
5.1.2.5 Sample Type-
The type of sample required for analysis of target contaminants in intensive
monitoring studies should be prepared from edible fish and/or shellfish tissue. For
finfish, edible tissue is defined as the fillet portion (skin-on and bellyflap tissue included).
[Reviewers: Please indicate whether belly flap should be Included in the intensive
study design.] It is extremely important that the individual specimens used in the composite
sample be of the same species and that the total length (size) of the smallest individual in the
composite be >75 percent of the total length (size) of the largest individual (U.S. EPA, 1990b).
Composite samples should be prepared for each target fish species from equal weights of
individual homogenates of fillets from 6 to 10 fish.
For shellfish, the tissues considered to be edible will vary depending on the target
species used and regional or local dietary preferences. For each target shellfish species, a
clear description of the edible tissue selected for analysis and the rationale for selection
should be provided in the Work/QA Project Plan. Because of the small size of shellfish, it is
not practical to prepare homogenates of individual organisms. Composite samples should be
prepared for each regional target shellfish species from the homogenization of the combined
edible tissue from enough organisms to produce a 500-g-minimum composite sample.
Separate composite samples are required for all subgroups (e.g., size or age class)
within a target species population that have been selected for evaluation in the intensive
monitoring study. For example, if three size classes of a specific target species are of
interest, then the sampling design should allow for the collection of a sufficient number of
individuals in each size class to allow for the preparation of composite samples for each class.
The same number of Individual organisms should be used to prepare all
replicate composite samples for a given target species at a given site. If this number is
outside the recommended range, the reasons for this deviation should be documented clearly.
Recommended sample preparation procedures are discussed in Section 6.2.
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5.1.2.6 Sample Replication-
A minimum of five replicate composite samples (each composed of equal
numbers of 6 to 10 Individual fish) for each selected size or age class of each target
species is recommended for both Phase I and Phase II of intensive monitoring studies.
For shellfish, five replicate composite samples (each composed of the same number of
individuals) should be used. Each composite may contain from 10 to 50 individuals
depending on the species and size class being sampled. The relative difference (in
percent) between the overall mean length (size) of the replicate samples and the mean length
(size) of any individual replicate should be no greater than 10 percent (U.S. EPA, 1990b).
Previous EPA guidance (U.S. EPA, 1987c; 1987g; 1989d) has emphasized the
importance of using replicate samples to permit the analysis of the data by statistical methods
(e.g., ANOVA, power analysis, and trend analysis techniques) to detect differences in mean
concentrations among sites. These types of statistical analyses are essential in characterizing
the geographic extent of fish consumption advisories and in assessing the effectiveness of
management efforts to protect fishery resources from contaminants or to mitigate existing
pollution problems.
Selection of the appropriate number of replicate composites for the intensive
monitoring study depends on site-specific levels of sample variability in target contaminant
tissue concentration and is discussed in detail in Sections 2.2.8 and 7.2.3. Replicate
composite sampling is most appropriate for intensive monitoring studies that have as a
primary objective the determination of differences in contaminant tissue concentrations among
sampling locations (e.g., using multilocational sites to determine the geographic extent of
contamination).
5.2 SAMPLE COLLECTION
After all sampling parameters have been reviewed and specified, sample collection
activities can be initiated in the field. This section discusses recommended sampling
equipment and its use, considerations for ensuring preservation of sample integrity, and field
recordkeeping and chain-of-custody procedures associated with sample processing,
preservation, and shipping.
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5.2.1 Sampling Equipment and Use
In response to the variations in environmental conditions and target species of interest,
fisheries biologists have had to devise methods that are intrinsically selective for certain
species and sizes of fish and shellfish (Versar, 1982). Although this selectivity can be a
hindrance in an investigation of community structure, it is not a problem where tissue
contaminant analysis is of concern because tissue contaminant data can be compared only if
factors such as differences in taxa and size are minimized.
Collection methods can be divided into two major categories, active and passive.
Each collection method has advantages and disadvantages. Various types of sampling
equipment, their use, and their advantages and disadvantages are summarized in Table 5-2
for fish and in Table 5-3 for shellfish.
A basic checklist of field sampling equipment and supplies appropriate to field
collection activities is shown in Table 5-4. Safety considerations associated with the use of a
boat in sample collection activities are summarized in Table 5-5.
5.2.1.1 Active Collection--
Active collection methods encompass a wide variety of fish sampling devices, including
Electroshocking units
Seines
Trawls
Angling equipment (hook and line),
and shellfish (e.g., bivalves and crustaceans) sampling devices, including
Seines
Trawls
Mechanical grabs (e.g., pole-operated grab buckets and tongs and line- or
cable-operated grab buckets)
Biological dredges
Scoops and shovels
Rakes
Dip nets
Manual collection by SCUBA divers.
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TABLE 5-2. SUMMARY OF FISH SAMPLING EQUIPMENT
Device
Use
Advantages
Disadvantages
ACTIVE METHODSI
Electrofishing
Seines
Trawls
Angling
Purchasing specimens
from commercial
fishermen
PASSIVE METHODS r.Z".
Gill nets
Trammel nets
Shallow rivers, lakes, and streams.
Shallow rivers, lakes, and streams.
Shoreline areas of estuaries.
Various sizes can be used from boats
in moderate to deep open bodies
of water (1 0 to >70 m depths).
Generally species selective involving
use of hook and line.
Can be used where sampling sites
are located in areas where target
species are commercially harvested
,
Lakes, rivers, and estuaries. Where
fish movement can be expected or
anticipated.
Lakes, rivers, and estuaries. Where
fish movement can be expected or
anticipated. Frequently used
where fish may be scared into the net.
Most efficient nonselective method. Minimal
damage to fish. Adaptable to a number of
sampling conditions (e.g., boat, wading, shore-
lines). Particularly useful at sites where other
active methods cannot be used (e.g., around
snags and irregular bottom contours).
Relatively inexpensive and easily operated.
Mesh size selection available for target species.
Effective in deep waters not accessible by
other methods. Allows collection of a large
number of samples.
Most selective method. Does not require use
of large number of personnel or expensive
equipment.
Most cost-effective and efficient means of
obtaining commercially valuable species
from harvested waters
Effective for collecting pelagic fish species.
Not particularly difficult to operate. Requires
less fishing effort than active methods. Selec-
tivity can be controlled by varying mesh size.
Slightly more efficient than a straight gill net.
Nonselective stuns or kills most fish. Cannot
be used in brackish, salt, or extremely soft
water. Requires extensive operator training.
DANGEROUS when not used properly.
Cannot be used in deep water or over substrates
with an irregular contour. Not completely efficient
as fish can get over, around, and under the net
during seining operation.
Requires boat and personnel with operator
training.
Inefficient and not dependable.
Commercially harvested areas may not include
sampling sites chosen for fish contaminant
monitoring. The field collection staff must
accompany the commercial fishermen and should
remove the required samples from the collection
device. This will ensure the proper handling of
the specimens and accurate recording of the
collection time and sampling location.
Not effective for bottom-dwelling fish or popula-
tions that do not exhibit movement patterns. Nets
prone to tangling or damage by large and Sharp
spined fish. Gill nets will kill captured specimens,
which, when left for extended periods, may
undergo physiological changes.
(Same as for gill nets.) Tangling problems may
be more severe. Method of scaring fish into net
requires more personnel or possibly boats in
deep water areas.
V
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TABLE 5-2. (continued)
Device
Use
Advantages
Disadvantages
PASSIVE METHODS EZ
Hoop, Fyke and
Pound Nets
D-Traps
%
Shallow rivers, lakes, and estuaries
where currents are present or when
movements of fish are predictable.
Frequently used in commercial
operations.
Used for long-term capture of slow
moving fish, particularly bottom
species. Can be used in all environ-
ments.
Unattended operation. Very efficient in regard
to long-term return and expended effort.
Particularly useful in areas where active
methods are impractical.
Easy to operate and set. Unattended operation.
Particularly useful for capturing bottom dwelling
organisms in deep waters or other types of
inaccessible areas. Relatively inexpensive
often can be hand made.
Inefficient for short term. Difficult to set up and
maintain.
Efficiency is highly variable. Not effective for
pelagic fish or fish that are visually oriented.
Less efficient for all species when water is clear
rather than turbid. Not a good choice for a
primary sampling technique, but valuable as
backup for other methods.
Source: Versar, Inc. 1982. Sampling Protocols for Collecting Surface Water, Bed Sediment, Bivalves, and Fish for Priority Pollutant AnalysisFinal Draft Report. EPA Contract
68-01-6195. Prepared for U.S. Environmental Protection Agency, Office of Water Regulations and Standards. Versar. Inc. Springfield, VA.
en
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TABLE 5-3. SUMMARY OF SHELLFISH SAMPLING EQUIPMENT
Device
ACTIVE METHODSL-
Seines
Trawls
Mechanical grabs
Double-pole-
operated grab
buckets
Tongs or double-
handled grab
sampler
Line or Cable-Operated
Grab Buckets:
Ekman grab
Peterson grab
Ponar grab
Orange peel grab
Use
Shallow shoreline areas of
estuaries.
Various sizes can be used from boats
in moderate to deep open bodies
of water (10 to >70 m depths).
Used from boat or pier. Most useful
in shallow water areas less than
6 m deep including lakes, rivers,
and estuaries.
Most useful in shallow water, lakes,
rivers, and estuaries. Generally used
from a boat.
Used from boat or pier to sample soft
to semisoft substrates.
Deep lakes, rivers, and estuaries for
sampling most substrates.
Deep lakes, rivers, and estuaries for
sampling sand, silt or clay substrates.
Deep lakes, rivers, and estuaries for
sampling most substrates.
Advantages
Relatively inexpensive and easily operated.
Mesh size selection available for target crusta-
cean species (e.g., shrimp and crabs).
Effective in deeper waters not accessible by
other methods. Allows collection of a large
number of samples.
Very efficient means of sampling bivalves
(e.g., clams and oysters) that are located on
or buried in bottom sediments.
Very efficient means of sampling oysters, clams,
and scallops. Collection of surrounding or
overlying sediments is not required and the
jaws are generally open baskets. This reduces
the weight of the device and allows the washing
of collected specimens to remove sediments.
Can be used in water of varying depths in
lakes, rivers, and estuaries.
Large sample is obtained; grab can penetrate
most substrates.
Most universal grab sampler. Adequate on
most substrates. Large sample is obtained
intact.
Designed for sampling hard substrates.
Disadvantages
Cannot be used in deep water or over substrates
with an irregular contour. Not completely efficient
as crustaceans can get over, around, and under
the net during seining operation.
Requires boat and personnel with operator
training.
At depths greater than 6 m, the pole-operated
devices become difficult to operate manually.
At depths greater than 6 m, the pole-operated
devices become difficult to operate manually.
Possible incomplete closure of jaws can result in
sample loss. Must be repeatedly retrieved and
deployed. Grab is small and is not particularly
effective in collecting large bivalves (clams and
oysters).
Grab is heavy, may require winch for deploy-
ment. Possible incomplete closure of jaws can
result in sample loss. Must be repeatedly retrieved
and deployed.
Possible incomplete closure of jaws can result in
sample loss. Must be repeatedly retrieved and
deployed.
Grab is heavy, may require winch for deployment.
Possible incomplete closure of jaws can result in
sample loss. Must be repeatedly retrieved and
deployed. Grab is small and not particularly
effective in collecting large bivalves (clams
and oysters).
01
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TABLE 5-3. (continued)
Device
Use
Advantages
Disadvantages
Biological dredge
Dragged along the bottom of deep
waterbodies to collect large stationary
invertebrates.
Qualitative sampling of large area of bottom
substrate and benthic community. Length of
tows can be relatively short if high density
of shellfish exists in sampling area.
If the length of the tow is long, it is difficult to
pinpoint the exact location of the sample collec-
tion area Because of the scouring operation of
the dredge, bivalve shells may be damaged. All
bivalve specimens should be inspected and
individuals with cracked or damaged shells
should be discarded.
Scoops, shovels
Used in shallow waters accessible by
wading or SCUBA equipment for
collection of hard clams (Mercenaria
mercenaria) or soft-shell clam (Mya
arenaria)
Does not require a boat; sampling can be
done from shore.
Care must be taken not to damage the shells of
bivalves while digging in substrate.
Scrapers
Used in shallow waters accessible by
wading or SCUBA equipment for
collection of oysters. (Crassostrea
virginica) or mussels (Mytilus sp)
Does not require a boat; sampling can be
done from shore.
Care must be taken not to damage shells of
bivalves while removing them from hard
substrate.
Rakes
01
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Used in shallow waters accessible by
wading or can be used from a boat.
Does not require a boat; sampling can be done
close to shore. Can be used in soft sediments
to collect clams or scallops, and can also be
used to dislodge oysters or mussels that are
attached to submerged objects such as rocks
and pier pilings.
Care must be taken not to damage the shells of
the bivalves while raking or dislodging them from
the substrate.
Purchasing specimens
from commercial
fishermen
Can be used where sampling sites
are located in areas where target
species are commercially harvested.
Most cost-effective and efficient means of
obtaining bivalves for pollutant analysis from
commercially harvested waters.
Commercially harvested areas may not include
sampling sites chosen for shellfish contaminant
monitoring. The field collection staff must
accompany the commercial fishermen and shoulc
remove the required samples from the collec-
tion device. This will ensure the proper handling
of the specimens and accurate recording of the
exact collection time and sampling location.
PASSIVE METHODS L
D-traps
Used for capture of slow-moving
crustaceans (crabs and lobsters)
that move about on or just above
the substrate.
Can be used in a variety of environments.
Particularly useful for capturing bottom
dwelling organisms in deep water or other
inaccessible areas. Relatively inexpensive,
can be hand made.
Catch efficiency is highly variable. Not a good
choice for a primary sampling technique, but
valuable as a backup for other methods.
Source: Versar, Inc. 1982. Sampling Protocols for Collecting Surface Water, Bed Sediment, Bivalves, and Fish for Priority Pollutant AnalysisFinal Draft Report. EPA
Contract 68-01-6195. Prepared for U.S. Environmental Protection Agency, Office of Water Regulations and Standards. Versar, Inc. Springfield, VA.
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TABLE 5-4. CHECKLIST OF FIELD SAMPLING EQUIPMENT A*ND SUPPLIES
FOR FISH/SHELLFISH CONTAMINANT MONITORING PROGRAMS
Boat supplies
- Fuel supply (primary and auxiliary supply)
- Spare parts repair kit
- Life preservers
- First aid kit (including emergency phone numbers of local hospitals, family
contacts for each member of the sampling team)
- Spare oars
- Nautical charts of sampling site locations
Collection equipment (e.g., nets, traps, electroshocking device)
Recordkeeping/documentation supplies
- Field logbook
- Sample request forms
- Specimen identification labels
- Chain-of-Custody (COC) Forms and COC tags or labels
- Indelible pens
Sample processing equipment and supplies
- Holding trays
- Fish measuring board (metric units)
- Calipers (metric units)
- Balance to weigh representative specimens for estimating tissue weight (metric
units)
- Aluminum foil (extra heavy duty)
- Freezer tape
- String
- Large plastic bags for holding composite samples
- Resealable watertight plastic bags for storage of Field Records, COC Forms, and
Sample Request Forms
Sample preservation and shipping supplies
- Ice (wet ice, blue ice packets, or dry ice)
- Ice chests
- Filament-reinforced tape to seal ice chests for transport to the central processing
laboratory
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TABLE 5-5. SAFETY CONSIDERATIONS FOR FIELD SAMPLING* USING A BOAT
Field collection personnel should not be assigned to duty alone in boats.
Life preservers should be worn at all times by field collection personnel near the
water or onboard boats.
If electrofishing is the sampling method used, there must be two shut-off
switches-one at the generator and a second on the bow of the boat.
All deep water sampling should be performed with the aid of an experienced,
licensed boat captain.
Minimize or eliminate all sampling during nondaylight hours, during severe weather
conditions, or during periods of high water when the safety of field collection
personnel might be jeopardized.
All field collection personnel should be trained in first aid procedures to allow proper
response in the event of an accident. Personnel should have local emergency
numbers readily available for each sampling trip and know the location of the
hospitals or other medical facilities nearest each sampling site.
For fish/shellfish contaminant monitoring programs, EPA recommends that
active collection methods be used whenever possible. Although active collection requires
greater fishing effort, it is usually more efficient than passive collection for covering a large
number of sites and catching the relatively small number of individuals needed from each site
for tissue analysis (Versar, 1982). Active collection methods are particularly useful in shallow
waters (e.g., streams, along lake shorelines, and shallow coastal areas of estuaries).
When sampling must be conducted in deep water, however, active collection methods
have distinct disadvantages because they are more resource-intensive, requiring larger
numbers of field personnel and expensive equipment. This problem may be overcome by
coordinating sampling efforts with commercial collection efforts. Purchasing fish/shellfish
from commercial fishermen using active collection devices Is acceptable only when
field sampling staff accompany the commercial fishermen during the collection
operation to ensure that proper collection and handling techniques are observed. Thus,
trained personnel can remove the target species directly from the nets and ensure that sample
collection, processing, and preservation are conducted as prescribed in sample collection
protocols, with minimal chance of contamination. This is an excellent method of obtaining
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specimens of commercially important target species, particularly from the Great Lakes and
coastal estuarine areas (Versar, 1982).
One active collection method that is not recommended by EPA involves the use of
chemical poisons to stun or kill fish. EPA strongly advises against the use of chemical
poisons as a technique for collecting fish and shellfish for contaminant monitoring
programs because these toxicants may induce physiological changes that could alter
contaminant concentrations in the tissues.
A more detailed description of active sampling devices and their use is provided in
Bennett (1970); Weber (1973); Battelle (1975); Mearns and Allen (1978); Pitt, Wells and
McKrone (1981); Versar (1982); Hayes (1983); Gunderson and Ellis (1986); and Puget Sound
Estuary Program (1990b).
5.2.1.2 Passive Collection--
Passive collection methods encompass a wide array of sampling gears for fish and
shellfish including
Gill nets
Fyke nets
Trammel nets
Hoop nets
Pound nets
D-traps.
Passive methods of fish and shellfish collection generally require less fishing effort than active
methods but are usually less desirable for shallow water sample collection because of the
ability of many species to evade these entanglement and entrapment devices. These
methods normally yield a much greater catch than would be required for a contaminant
monitoring program and are time consuming to deploy. In deep water, however, passive
collection techniques are generally more efficient than active methods. A more detailed
description of passive sampling devices and their use is provided in Versar (1982 and 1984)
and Hubert (1983).
The following procedures should be observed when passive collection devices
must be deployed:
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Target fish and shellfish must be removed from the passive collection device (i.e.,
nets) at frequent intervals (<1 hour) during daylight sample collection hours to avoid
physiological stress associated with capture (U.S. EPA, 1991c).
Target fish and shellfish captured during the night in nets must be discarded before
daylight sample collection activities are initiated because there is no way to
determine the length of time the specimen was in the collection device (U.S. EPA,
1991c).
Target shellfish species (lobster, crabs, crayfish) captured using D-traps must be
removed at an interval not to exceed 48 hours.
All target species captured using passive collection devices must be alive at the
time of retrieval of the sampling equipment. If they are not alive, they must be
discarded.
Purchasing fish/shellfish from commercial fishermen using passive collection
methods is acceptable only when field sampling staff accompany the fishermen during
both the deployment and collection operations. Thus, the field sampling staff can verify
that proper collection processing and preservation techniques were used and that specimens
were alive at the time of collection.
Although passive methods for sample collection may be needed in some
environmental situations for some target species, EPA recommends that passive methods
be used only as a last resort.
5.2.2 Preservation of Sample Integrity
The primary QA/QC consideration when defining sample collection, processing,
preservation, and shipping procedures is the preservation of sample integrity to ensure the
accuracy of target contaminant analyses. Sample integrity is preserved by
Prevention of extraneous tissue contamination
Prevention of loss of contaminants already present in the tissues (Smith, 1985).
In the field, sources of contamination include sampling gear, boats and motors, grease from
ship winches or cables, spilled engine fuel (gasoline or diesel), engine exhaust, dust, ice
chests, and ice used for cooling. Care must be taken during handling to avoid these and any
other sources of contamination. For example, during sampling, the boat should be positioned
so that engine exhausts do not fall on the deck. Ice chests should be scrubbed clean with
detergent and rinsed with distilled water after each use to prevent contamination. To avoid
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contamination from melting ice, samples should be placed in watertight plastic bags (Stober,
1991). Sampling equipment that has been obviously contaminated by oils, grease, diesel fuel,
or gasoline should not be used. All utensils or equipment that will be used directly in handling
fish or shellfish (e.g., fish measuring board or calipers) should be cleaned in the laboratory
prior to each sampling trip, rinsed in acetone and pesticide-grade hexane, and stored in
aluminum foil until use (Versar, 1982). Between sampling stations, the field collection team
should clean each measurement device by rinsing it with ambient water and rewrapping it in
aluminum foil to prevent contamination. All potential sources of contamination in the field
should be identified and steps taken to minimize or eliminate them.
In addition to controlling sources of contamination during the sample collection
process, many sources of contamination can be avoided by resecting (i.e., surgically
removing) tissues in a controlled laboratory environment. EPA recommends that all
resecting of fish fillets or of shellfish edible portions be conducted in a clean area of
the central processing laboratory to reduce contamination of specimens (Stober, 1991).
Procedures for laboratory processing and resection are described in Section 6.2. Procedures
for assessing sources of sample contamination through the analyses of field and processing
blanks are described in Section 6.4.3.5.
5.2.3 Field Recordkeeping
Thorough documentation of the sample collection and processing work done in the
field is necessary for interpretation of the results of a field survey. For fish and shellfish
contaminant monitoring studies, it is advisable to have preprinted, waterproof data forms and
writing implements that produce indelible markings and can function when wet (Puget Sound
Estuary Program, 1990b). When multicopy forms are required, no-carbon-required (NCR)
paper is recommended because it allows information to be forwarded on the desired schedule
while allowing retention of the data for the project file.
Four distinct preprinted sample tracking forms should be used for each sampling site to
document field activities from the time the sample is collected through processing and
preservation until the sample is delivered to the central processing laboratory. These are
Field record form
Sample identification label
5-27
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Chain-of-custody (COC) label or tag
Chain-of-custody (COC) form.
Full-sized copies of each of these forms for use in both the initial screening and intensive
studies are included in Appendix G for use by the States.
5.2.3.1 Field Record Form-
The following information is recommended for inclusion on the field record for each
sampling site in both the initial screening (Figures 5-4 and 5-5) and intensive followup studies
(Figures 5-6 and 5-7):
Project number
Sampling date and time
Sampling site location (including site name and number, county/parish,
latitude/longitude, State waterbody segment number, waterbody type, and site
description)
Collection method
Collectors' names and signatures
Agency (including telephone number and address)
Species collected (including species scientific name, composite sample number [5
digits] and individual specimen suffix number [3 digits], number of individuals per
composite, number of replicate samples, total length/size [cm], sex [male, female,
indeterminate])
Compute percent difference in size between the smallest and largest specimens to
be composited (smallest individual length [or size] divided by the largest individual
length [or size] x 100 £ 75 percent) and compute mean composite length or size.
Notes (including visible morphological abnormalities, e.g., fin erosion, skin ulcers,
cataracts, skeletal and exoskeletal anomalies, neoplasms, or parasites).
5.2.3.2 Sample Identification Label-
The following information should be included on the sample identification label:
Species scientific name or code number
Total length/size of specimen (cm)
Composite number (5 digits) and individual specimen suffix number (3 digits)
5-28
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Field Record for Fish Contaminant Monitoring Program Screening Study
Project Number:
Samolina Date and Time:
SITE LOCATION
Site Name/Number:
County/Parish:
State Waterbody Segment Number:
Waterbody Type: D RIVER D
Site Description:
Lat./Lona:
LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Aaency:
Address:
Phone: ( )
FISH COLLECTED 1 i
Bottom Feeder Species Name:
Composite Sample #:
Fish # Length (cm) Sex
001
002
003
004
005
Minimum size
x 100 = %
Maximum size
Notes (e.g., morphological anomalies):
Predator Soecles Name:
Composite Sample #:
Fish # Length (cm) Sex
001
002
003
004
005
Minimum size ,_,. ^ __0/
x 1 00 = > 75%
Maximum size
Notes (e.g., morphological anomalies):
Number of Individuals:
Fish # Length (cm) Sex
006
007
008
009
010
Composite mean lenoth cm
Number of Individuals:
Fish* Length (cm) Sex
006
007
008
009
010
Composite mean lenoth cm
Figure 5-4.
5-29
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Field Record for Shellfish Contaminant Monitoring Program Screening Study
Project Number:
Sampling Date and Time:
SITE LOCATION
Site Name/Number:
County/Parish:
State Waterbody Segment Numb
WaterbodyType: D RIVER
Site Description:
er:
D
Lat./Lona:
LAKE p ESTUARY
Collection Method:
Collector Name:
(print and sign)
Aoencv:
Address:
Phone: ( )
SHELLFISH COLLECTED 1 1
Bivalve Species Name:
Composite Sample #:
Bivalve # Size (cm)
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Minimum size w ftn
Maximum size
Notes (e.g., morphological anoma
Bivalve #
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
2 75%
lies):
Number of Individuals:
Size (cm) Bivalve # Size (cm)
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
Composite mean size cm
Figure 5-5.
5-30
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Field Record for Fish Contaminant Monitoring Program Intensive Study
Project Number
Sampling Date and Time
SITE LOCATION
Site Name/Number
County/Parish:
State Waterbody Segment Number
Waterbody Type: D RIVER
Site Description:
LatAong.:
D LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Agency:
Address:
Phone: ( )
FISH COLLECTED ! 1
Species Name:
Composite Sample #:
Fish # Length (cm) Sex (M,
001
002
003
004
005
Minimum length
x 1 00 =
Maximum length
Notes (e.g., morphological anomalies):
Species Name:
Composite Sample #:
Fish « Length (cm) Sex (M,
001
002
003
004
005
Minimum length
x 1 00 = 2.
Maximum length
Notes (e.g., morphological anomalies):
Number of Individuals:
F, or I) Fish # Length (cm)
006
007
008
009
010
% Composite mean length
Number of Individuals:
F, or 0 Fish * Length (cm)
006
007
008
009
010
75% Composite mean length
Replicate Number:
Sex (M, F, or 0
cm
Replicate Number:
Sex (M, F, or I)
cm
page Iof2
Figure 5-6.
5-31
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Field Record for Fish Contaminant Monitoring Program Intensive Study (con.)
Project Number
SITE LOCATION:
Site Name/Number
County/Parish:
Sampling Date and Time
LatAong.:
4
FISH COLLECTED !
Species Name:
Composite Sample #:
Fish * Length (cm) Sex (M, F, or 1)
001
002
003
004
005
Minimum length K100_ /
Maximum length
Notes (e.g., morphological anomalies):
Species Name:
Composite Sample #:
Fish * Length (cm) Sex (M, F, or 0
001
002
003
004
005
Minimum length
Maximum length
Notes (e.g.. morphological anomalies):
Species Name:
Composite Sample #:
Fish * Length (cm) Sex (M, F, or 0
001
002
003
004
005
Minimum length .
; x 1 oo -
-------
Field Record for Shellfish Contaminant Monitoring Program Intensive Study
Project Number
SITE LOCATION
Site Name/Number
County/Parish:
State Waterbody Segment Number _
Waterbody Type: D RIVER
Site Description:
D
«
Sampling Date and Time:
LatAong.:
LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Agency:
Address:
Phone: ( )
SHELLFISH COLLECTED 1 1
Species Name:
Composite Sample #:
Shellfish # Size (cm) Sex Shellfish
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Minimum size
x 1 00 = 2
Maximum size
Notes (e.g., morphological anomalies]
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
75%
Replicate Number:
Number of Individuals:
# Size (cm) Sex Shellfish # Size (cm) Sex
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
Composite mean size cm
Figure 5-7.
5-33
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Sample type: F (fish fillet analysis only)
S (shellfish edible portion analysis only)
W (whole fish analysis)
O (other fish tissue analysis)
Sampling site-name and/or identification number
Sampling date/time (24-h clock).
Information on this label should be completed in indelible ink after each individual fish or
shellfish specimen is processed to identify each sample uniquely (Figure 5-8). The sample
identification label should then be taped to each aluminum-foil-wrapped specimen.
5.2.3.3 Chain-of-Custody Label or Tag-
The information to be completed for each composite fish or shellfish sample on the
chain-of-custody (COG) label or tag is shown in Figure 5-9. After all information on a specific
composite sample has been completed, the COC label or tag should be taped or attached
with string to the outside of the water-proof plastic bag containing the composite sample.
Information on the COC tag/label also should be recorded on the COC form.
5.2.3.4 Chain-of-Custody Form--
Information recommended for documentation on the chain-of-custody form (Figure
5-10) is necessary to track all composite samples from field collection to receipt at the central
processing laboratory. In addition, this form can be used for tracking samples through initial
laboratorynDrocessing (e.g., resection) as described in Section 6.2.
One copy of the COC form and a copy of the field record sheet should be sealed in a
resealable watertight plastic bag and placed in the ice chest with the samples being tracked
prior to sealing the ice chests. Ice chests should be sealed with reinforced tape for shipment.
In addition to the four sample tracking forms discussed above, the field collection team
should document in a field logbook any additional information on sample collection activities,
hydrologic conditions (e.g., tidal stage), weather conditions, boat or equipment operations, or
any other unusual problems encountered that would be useful to the program manager in
evaluating the quality of the fish/shellfish contaminant monitoring data.
5-34
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Species Name or Code
lucius
Sample Type
Total Length or Size (cm)
Sampling Site (name/number)
Specimen Number
3340001
Sampling Date/Time
Figure 5-8. Sample identification label.
5-35
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Project Number
Sampling Site (name and/or ID number)
Collecting Agency (name, address, phone)
Sampler (name and signature)
Composite Number
Sampling Date/Time
Species Name or Code
Chemical Analyses
O All target contaminants
|~] Others (specify)
Processing
Whole Body
Comments
Resection
Study Type
Screening
Intensive
Type of Ice
Wet
Dry
Figure 5-9. Example of a chain-of-custody tag or label.
5-36
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Chain-of-Custody Record
Project Number
Collecting Agency (name, address, phone)
Samplers (print and sign)
Composite
Number
Sample
Nos.
Sampling
Time
Study Type
Scr
Int
Sampling Date
Container
of
Sampling Site (name/number)
J/ /
/sS /
f / O /
//
f/
. - / Comment*
Delivery Shipment Record
Delivery Method D Hand ""V
D Shipped
Deliver/Ship to: (name, address and phone)
Oate'/Time Shipped:
Relinquished by: (signature) Date/Time Received by: (signature)
Relinquished by:
(signature)
Date / Time
Received by: (signature)
Relinquished by: (signature)
Date / Time
Received for Central Processing
Laboratory by: (signature)
Date / Time
Remarks:
Laboratory Custody:
Released
Name/Date
Received
Name/Date
Purpose
Location
Figure 5-10. Example of a chain-of-custody form.
5-37
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5.3 SAMPLE PROCESSING, PRESERVATION, AND SHIPPING
5.3.1 Sample Processing
As soon as individual fish specimens are removed from the collection device or water,
they should be stunned by a sharp blow to the base of the skull with a wooden stick or metal
rod. This rod should be used solely for the purpose of stunning fish, and care should be
taken to keep it reasonably clean to prevent contamination of the samples (Versar, 1982).
Each fish should then be rinsed in ambient water to remove any foreign material. Individual
specimens of the target species should be grouped by species and general size class and
placed in clean holding trays to prevent contamination.
As soon as shellfish are removed from the collection device, they should be rinsed in
ambient water to remove any sediment deposits. Bivalves (oysters and mussels) should be
separated when found to be adhering to one another and scrubbed with a nylon or natural
fiber brush to remove any adhering detritus or fouling organisms from the exterior shell
surface (NOAA, 1987). All bivalves should be inspected carefully to ensure that the shells
have not been cracked or damaged by the sampling equipment; damaged specimens should
be discarded (Versar, 1982). Bivalves should never be removed from their shells in the field.
A few specimens may be shucked to determine the wet weight of the edible portion (meats).
This will provide an estimate of the number of individuals required to ensure that the minimum
sample weight (500 g) can be attained.
Crustaceans, including shrimp, crabs, crayfish, and lobsters, should be rinsed in
ambient water to remove any foreign material from their external surface. All crustaceans
should be inspected to ensure that their exoskeletons have not been cracked or damaged
during the sampling process; damaged specimens should be discarded.
After they have been rinsed, individual shellfish specimens should be grouped by
species and general size class and placed in a clean holding tray to prevent contamination. A
few specimens may be resectioned (edible portions removed) to determine wet weight of the
edible portions. This will provide an estimate of the number of individuals required to ensure
that the minimum sample weight (500 g) can be attained. For blue crabs (Callinectes
sapidus), the edible meat (claw and back fin meat) constitute approximately 10 percent of the
overall body weight including the carapace (Sean McKenna, North Carolina Division of Marine
5-38
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Fisheries, personal communication). Thus, a 100-g adult crab will yield approximately 10 g of
edible tissue and 50 crabs would be required to obtain the minimum sample weight (500 g).
5.3.1.1 Species Identification--
Species identification should be conducted only by experienced personnel
knowledgeable of the taxonomy of species in the waterbodies included in the fish/shellfish
contaminant monitoring program. Taxonomic keys, appropriate for the waters being sampled,
should be consulted for species identification. Because the objective of both the screening
and intensive monitoring studies is to determine the magnitude of contamination in specific
fish and shellfish species, it is necessary that all individuals used in a composite sample be of
a single species. Correct species identification is important and different species should
never be combined in a single composite sample for any reason.
When sufficient numbers of the target species have been identified to make up a
composite sample, a member of the field collection team should record the species name and
all other appropriate information on the field record sheet (Figures 5-4 through 5-7).
5.3.1.2 Length or Size Measurements--
Each individual fish within the target species selected for analysis should be measured
to determine total body length (cm). To be consistent with the convention used by most
fisheries biologists in the United States, maximum total length should be measured as shown
in Figure 5-11. The maximum body length is defined as the length from the anterior-most part
of the fish to the tip of the longest caudal fin ray (when the lobes of the caudal fin are
compressed dorsoventrally) (Anderson and Gutreuter, 1983).
For shellfish, after initial processing, each individual specimen selected for analysis
should be measured to determine total body size (cm). As shown in Figure 5-11, the
recommended body measurements differ depending on the type of shellfish being collected.
Height is a standard measurement of size for oysters, mussels, clams, scallops, and other
bivalve molluscs (Galtsoff, 1964; Abbott, 1974). The height is the distance from the umbo to
the anterior shell margin. For crabs, the lateral width of the carapace is a standard size
measurement (U.S. EPA, 1990c), and for shrimp, lobster and crayfish, a standard
measurement of body size is the length from the rostrum to the tip of the telson (Texas Water
Commission, 1990).
5-39
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Maximum body length2
Fish
Carapace widthb
Crab
Height0
Bivalves
Rostrum
,\
QZI
Lengthd
Telson
Shrimp, Lobster, Crayfish
aMaximum body length is the length from the anterior-most part of the fish to the tip of the longest
caudal fin ray (when the lobes of the caudal fin are compressed dorso ventrally) (Anderson and
Gutreuter, 1983).
"Carapace width is the lateral distance across the carapace (from tip of spine to tip of spine) (U.S.
EPA, 1990c).
cHeight is the distance from the umbo to the anterior shell margin (Galtsoff, 1964).
dLength is the distance from the tip of the rostrum to the tip of the telson (Texas Water Commis-
sion, 1990).
Figure 5-11. Recommended measurements of body length and
size for fish and shellfish.
5-40
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[Reviewers, please comment on acceptability of shellfish size measurements. If
different from above, please provide complete literature citation.]
5.3.1.3 Sex Determination (Optional)--
An experienced fisheries biologist can often make a preliminary sex determination for
fish by visual inspection. Under no circumstances, however, should the body of the fish
be dissected in the field to determine sex; sex can be determined through internal
examination of the gonads during laboratory processing (Section 6.2).
For shellfish, a preliminary sex determination can be made by visual inspection only for
crustaceans. Sex determination cannot be made in bivalve molluscs without shucking the
bivalves and microscopically examining gonadal material. Under no circumstances should
bivalves be shucked In the field to determine sex; sex determination through exami-
nation of the gonads can be performed during laboratory processing (Section 6.2).
5.3.1.4 Morphological Abnormalities (Optional)--
If resources allow, States may wish to consider documenting external gross
morphological conditions in fish from contaminated waters. Severely polluted aquatic habitats
have been shown to produce a higher frequency of gross pathological disorders than similar,
less polluted habitats (Sinderman et al., 1980; Sinderman, 1983; Malins et al., 1984 and 1985;
Mix, 1986; Krahn et al., 1986).
Sinderman et al. (1980) reviewed the literature on the relationship of fish pathology to
pollution in marine and estuarine environments, and identified four gross morphological
conditions acceptable for use in monitoring programs:
Fin erosion
Skin ulcers
Skeletal anomalies
Neoplasms (i.e., tumors).
Fin erosion is the most frequently observed gross morphological abnormality in
polluted areas and is found in a variety of fishes (Sinderman, 1983). In demersal fishes, the
dorsal and anal fins are the fins most frequently affected; in pelagic fishes, the caudal fin is
primarily affected.
5-41
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Skin ulcers have been found in a variety of fishes from polluted waters and are the
second most frequently reported gross abnormality. Prevalence of ulcers generally varies with
season and is often associated with organic enrichment (Sinderman, 1983).
Skeletal anomalies involve the spinal column and include fusions, flexures, and
vertebral compressions. Skeletal anomalies also include abnormalities of the head, fins, and
gills.
Neoplasms or tumors have been found at a higher frequency in a variety of polluted
areas throughout the world. The most frequently reported visible tumors are liver tumors, skin
tumors (i.e., epidermal papillomas and/or carcinomas), and neurilemmomas.
The occurrence of fish parasites and other gross morphological abnormalities that are
suspected at a specific site location should be noted on the field record sheet. States
interested in documenting morphological abnormalities in fish should review the recommended
protocols for fish pathology studies used in the Puget Sound Estuary Program (1990c).
Although gross morphological observations generally are not definitive evaluations of
fish health, they may be very useful in uncovering previously unknown pathological conditions
in fishes from polluted areas (Puget Sound Estuary Program, 1990c). These relatively quick
examinations are very cost-effective because they do not require specialized equipment or
preparation techniques and can be made as the specimens are sorted from the catch. In
addition, gross external observations generally do not require that a trained pathologist be
aboard the sampling boat. However, it is extremely important that at least one member of the
collecting team be trained by a qualified pathologist to identify the various kinds of
pathological conditions that may be encountered, because at least two pathological conditions
(fin erosion and skin ulcers) can easily be confused with the external damage that fishes may
suffer as they are dragged along the seafloor in an otter trawl (Puget Sound Estuary Program,
1990c).
Given the potential usefulness of gross observations and the need for accurate and
verifiable determinations, it is recommended that representative fishes having each kind of
pathological condition be archived for each major sampling survey, and that the conditions be
confirmed by a qualified pathologist. This verification step is especially important if different
personnel make the gross observations during different surveys. For all suspected
pathological conditions that cannot be identified in the field, representative specimens should
5-42
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be archived for later evaluation by a qualified pathologist (Puget Sound Estuary Program,
1990C).
5.3.1.5 Composite Samples-
For each target fish species (or age class of target fish species) sampled, 10
individual fish of the same species and similar size should be composited. However,
samples containing 6 fish are minimally acceptable. The smallest individual fish used in a
target species composite sample should be no less than 75 percent of the total length of the
largest individual. For example, if the largest fish is 40 cm, then the smallest individual
included in the composite sample should be no smaller than 30 cm (U.S. EPA, 1990b).
For each shellfish species (or age class of shellfish species) sampled, 10 to 50
individual specimens of the same species and similar size should be composited. The
number of specimens to be composited cannot be specified for shellfish because the number
will depend on the size of the specimens collected and the weight of the edible portion. For
small shellfish, larger numbers of specimens (>50) may have to be composited to achieve the
minimum tissue mass of 500 g (excluding bivalve shell weight). For shellfish, the smallest
individual specimen used in the composite should be no less than 75 percent of the total size
of the largest individual. In some State sampling programs such as the California Mussel
Watch Program, a predetermined size range (55 to 65 mm) for the target bivalves (Mytilus
californianus and M. edulis) is used as a sample selection criterion at all sampling sites to
reduce size-related variability (Phillips, 1986).
5.3.1.6 Replicate Samples-
If replicate field samples for target fish or shellfish species are to be collected, the
relative difference between the overall mean length of the replicate samples and the mean
length of any individual replicate sample should be no greater than 10 percent. In the
following example, the overall mean length (±10 percent) of five replicate composite samples
is calculated to be 31 (±3.1) cm.
Mean Length of
Replicates Composite Fish Sample (cm)
1 30
2 32
3 33
4 28
5 32
Overall mean length (± 10%) = 31 (± 3.1) cm.
5-43
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Therefore, the acceptable range for the mean length of individual composite samples is 27.9
to 34.1 cm and the five replicate composite samples listed above all fall within this acceptable
size range.
5.3.2 Sample Preservation
After initial processing, each, fish should be individually wrapped in extra heavy duty
aluminum foil. Spines on fish should be sheared to minimize punctures in the aluminum foil
packaging (Stober, 1991). The sample identification label shown in Figure 5-8 should be
taped to the outside of each aluminum foil package.
After wrapping and labeling each individual fish in the composite sample, all of the
wrapped specimens in the composite sample should be secured with string or tape. If tape is
used, care should be taken not to tape over any of the individual sample identification labels.
The COC tag or label (Figure 5-9) should be completed for the composite sample and the
appropriate information should be recorded on both the field record sheet and COC form
(Figure 5-10). The composite fish sample should be placed into a watertight plastic bag and
sealed, and the COC tag should be attached to the outside of the plastic bag with string or
tape. Once packaged, the composite sample should be cooled on ice immediately.
After processing, each shellfish specimen should be wrapped individually in extra
heavy duty aluminum foil. A completed sample identification label should be taped to the
outside of each aluminum foil package. NOTE: Some crustacean species (e.g., blue crabs
and spiny lobsters) have sharp spines on their carapaces that might puncture the aluminum
foil wrapping. For such species, samplers may use one of the following procedures to reduce
punctures to the outer foil wrapping:
Double-wrap the entire specimen in extra heavy duty aluminum foil.
Place clean cork stoppers over the protruding spines prior to wrapping the
specimen in aluminum foil.
Selectively wrap the spines with multiple layers of foil prior to wrapping the entire
specimen in aluminum foil.
Carapace spines should never be sheared off as this would destroy the integrity of the
carapace. A COC tag or label should be completed for the composite sample and
appropriate information should be recorded on the field record sheet and COC form. After
wrapping and labeling each shellfish specimen in the composite sample, all of the wrapped
5-44
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specimens in the composite sample should be placed in a plastic watertight bag. The COC
label or tag should be completed for the composite sample and appropriate information should
be recorded on both the field record sheet and COC form. The COC label or tag should be
attached to the outside of the plastic bag with string or tape. Once packaged, the composite
sample should be cooled on ice immediately.
5.3.2.1 Preservation of Fish or Shellfish for Resection--
The type of ice to be used for shipping should be determined by the length of time the
samples will be in transit to the central processing laboratory and the sample type to be
analyzed (Table 5-6). Fish and shellfish specimens should not be frozen prior to resection if
analyses will include internal tissue (e.g., fillets or edible tissues) because freezing may cause
some internal organs to rupture and contaminate fillets or other edible tissues (Tetra Tech,
1989). If fish fillet samples or edible portions of shellfish are to be analyzed, wet ice or blue
ice (sealed prefrozen ice packets) should be used and samples should be delivered to the
processing laboratory within 24 hours. Wet Ice or blue Ice is recommended as the
preservative of choice when the fish fillet or shellfish edible portions are the primary
tissues to be analyzed.
5.3.2.2 Fish or Shellfish for Whole-Body Analysis-
At some sites, States may deem it necessary to collect fish for whole-body analysis, if
a specific human subpopulation typically consumes whole fish or shellfish. If whole fish or
shellfish samples are to be analyzed, either wet ice, blue Ice, or dry ice is
recommended. If shipping time to the laboratory will take more than 24 hours, dry ice
must be used.
Dry ice requires special packaging precautions before shipping to comply with U.S.
Department of Transportation (DOT) regulations. The Code of Federal Regulations classifies
dry ice as ORM-A (Other Regulated Material). These regulations specify the amount of dry
ice that may be shipped by air transport and the type of packaging required. For any amount
of dry ice to be shipped by air, advance arrangements must be made with the carrier and not
more than 440 pounds of dry ice may be shipped by air freight unless the shipper has made
special arrangements with the aircraft operator. Quantities of dry ice for tissue preservation
are usually considerably less than 440 pounds (Versar, 1982).
5-45
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TABLE 5-6
RECOMMENDATIONS FOR PRESERVATION OF FISH/SHELLFISH SAMPLES FROM TIME OF COLLECTION TO
DELIVERY AT CENTRAL PROCESSING LABORATORY
01
-K
O)
Sample
Type
FISH* I
Whole fish
(to be filleted)
Whole fish
Number per
Composite
6-10
6-10
Container
Same as above
Extra heavy duty aluminur
of each fish. All fish in a a
taped together and placec
watertight plastic bag
Preservation Maximum Holding Time
Cool on wet ice or 24 hours
blue ice packets
n foil wrap Cool on wat ice, or 1. ~« hnnrc
)mposrte blue ice packets, '
to the processing laboratory
SHELLFISH* 1
Whole shellfish
(to be resected
for edible
portion)
Whole shellfish
10-50
(species and
size dependent)
10-50
(species and
size dependent)
Same as above
Extra heavy duty aluminur
of each specimen. All she
composite placed in a wat
plastic bag
Cool on wet ice 24 hours
or blue ice packets
n foil wrap Cool on wet ice or \ _, . . ^, 21 hours
(fish in a blue ice packets, J
ertight or on dry ice for transport >> 48 hours
to the processing laboratory
'Use only individuals that have attained at least legal or consumable size.
-------
The regulations further specify that the packaging must be constructed in a manner to
permit the release of carbon dioxide gas which, if restricted, could cause rupture of the
package. If samples are being transported in a cooler, several vent holes should be drilled to
allow carbon dioxide gas to escape. The vents should be near the top of the vertical sides of
the cooler, rather than in the cover, to prevent debris from falling into the cooler. Furthermore,
wire screen or cheesecloth should be installed to help keep foreign materials from entering the
vents. When the samples are packaged, care should be taken to keep these vents open to
prevent the buildup of pressure.
Dry ice is exempted from shipping paper and certification requirements if the amount is
less than 440 pounds and the package meets design requirements. The package must be
marked "Carbon Dioxide, Solid" or "Dry Ice" with a statement indicating that the material being
refrigerated is to be used for diagnostic or treatment purposes (e.g., frozen tissue).
5.3.3 Sample Shipping
The fish/shellfish samples should be hand-delivered or shipped to the central
processing laboratory as soon as possible after collection. The time of collection and
time of arrival at the processing laboratory should be recorded on the COC form (Figure
5-10).
If the sample Is to be shipped rather than hand-delivered to the processing
laboratory, field collection staff must ensure the samples are packed properly with
adequate ice layered between samples so that sample degradation does not occur. In
addition, a member of the field collection staff should call ahead to the central processing
laboratory to alert them to the anticipated delivery time of the samples and the name and
address of the carrier to be used. Field collection staff should avoid shipping samples for
weekend delivery to the central processing laboratory unless prior plans for such a delivery
have been agreed upon with the central processing laboratory staff.
5-47
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SECTION 6
LABORATORY PROCEDURES
This section provides guidance to States on laboratory procedures followed from the
time a field sample is received at the central processing facility, through sample analysis for
target analytes, to final archiving. It includes recommended procedures for chain-of-custody,
sample processing, sample distribution, and sample analyses. Planning, documentation, and
quality assurance/quality control (QA/QC) of all laboratory activities are emphasized to ensure
that the integrity of samples is preserved during all phases of sample preparation and
chemical analyses, that chemical analyses are performed cost-effectively and meet program
data quality objectives, and that the data produced by different States and Regions are
comparable.
Laboratory procedures used in State fish/shellfish contaminant monitoring programs
should be documented in a Work/QA Project Plan as described in Appendix F and all routine
sample processing and analysis procedures should be prepared as standard operating
procedures (SOPs) (U.S. EPA, 1984b).
6.1 SAMPLE RECEIPT AND CHAIN-OF-CUSTODY
Collected samples are shipped or hand-carried from the field according to one or more
of the following pathways:
From the field to a State laboratory for sample processing and analysis
From the field to a State laboratory for sample processing and shipment of
composite sample aliquots to a contract laboratory for analysis
From the field to a contract laboratory for sample processing and analysis.
In each case, sample processing and distribution for analysis, if necessary, must be
performed by one central processing laboratory. Because EPA recommends that dioxin
analyses be performed by a contract laboratory (see Section 6.4.2), aliquots of each
composite sample designated for dioxin analyses must be shipped from the sample
processing laboratory to a contract laboratory.
Transportation of the samples from the field must be coordinated by the sampling team
supervisor and the laboratory responsible for sample processing (see Section 5.3.3). An
6-1
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accurate written record must be maintained so that possession and treatment of each sample
can be traced from the time of collection through analysis and final archiving, if applicable.
The fish and shellfish samples should be brought to or shipped to the sample
processing laboratory in sealed containers accompanied by a copy of the sample request form
(Figure 5-1), a chain-of-custody (COC) form (Figure 5-10), and the field records (Figures 5-4
through 5-7). Each time a sample or group of samples changes hands, the Personnel
Custody Record of the COC form must be completed and signed by both parties. Corrections
to the COC form should be made by drawing a line through and initialing and dating the error
and then entering the correct information.
When custody is transferred from the field to the sample processing laboratory, the
following procedure should be used:
Check that each shipping container has arrived undamaged and that the seal is
intact.
Open each shipping container and remove the copy of the sample request form,
the COC form, and the field records.
Note the general condition of the shipping container (samples iced properly with no
leaks, etc.) and the accompanying documentation (dry, legible, etc.).
Locate each composite sample listed on the COC form and note the condition of
its container. Composite sample containers should be properly sealed and
labeled. Note any problems (container punctured, illegible labels, etc.) on the COC
jrm.
Check the contents of each composite sample container against the field record for
that sample to ensure that the individual specimens are properly wrapped and
labeled. Note any discrepancies or missing information.
Initial the COC form and record the date and time of sample receipt.
Enter the following information for each composite sample into a permanent
laboratory record book and, if applicable, a computer database:
-- Sample identification number (5-digit composite sample number and 3-digit
sample suffix)
- Collection date
- Collection site (name and number)
-- Fish species (scientific name or code number)
6-2
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-- Total length of each fish (cm) or size of each shellfish (cm)
Store samples according to the procedures described in Section 6.2 and in Table
6-1. If the fish are on wet ice or blue ice and fillets are to be resected, distribute
the samples immediately to the biologist responsible for resection. Note: Samples
must remain iced until they are placed in a freezer for longer term storage.
TABLE 6-1. RECOMMENDATIONS FOR CONTAINER MATERIALS, PRESERVATION,
AND HOLDING TIMES FOR FISH/SHELLFISH TISSUES FROM DELIVERY
AT CENTRAL PROCESSING LABORATORY TO ANALYSIS
Analyte
Matrix
Sample
container
Storage
Preservation Holding time
Trace metals
(except Hg)
Hg
Organics
Tissue (whole
specimens, edible
portions, homogenates)
Tissue (whole
specimens,
edible portions,
homogenates)
Tissue (whole
specimens,
edible portions,
homogenates)
Plastic, glass Freeze at £-20 °C 1 year
Plastic, glass Freeze at £-20 °C 28 days
Glass, teflon Freeze at £-20 °C 1 year
6.2 SAMPLE PROCESSING
This section describes recommended procedures for preparing composite samples of
fish fillets (skin on and belly flap included) and edible portions of shellfish as required in initial
screening studies and intensive followup monitoring studies (Phases I and II, see Section 7).
Recommended procedures for preparing whole fish/shellfish composite samples are included
in Appendix GG for use when States determine that it is necessary to assess the potential risk
to local subpopulations that are known to consume whole fish or shellfish.
6.2.1 General Considerations
Avoiding contamination is one of the most important considerations in sample
processing. All instruments, work surfaces, and containers used in processing a sample must
be composed of materials that can be cleaned easily and that are not themselves potential
sources of contamination. Sources of contamination by organics are different from sources of
6-3
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contamination by trace metals. Therefore, if time and funding permit, it is recommended that
duplicate samples be collected for the initial screening; one sample to be processed and
analyzed for organics and the other to be processed independently and analyzed for trace
metals. Alternatively, for fish of adequate size, separate composites of right and left fillets
may be prepared and analyzed independently for trace metals and organics. If only one
composite sample is prepared for screening analysis, the processing equipment must be
chosen and cleaned carefully to avoid contamination by both organics and trace metals.
Intensive monitoring focuses on target contaminants identified in the initial screening
study. If intensive monitoring samples are to be analyzed only for organics or trace metals,
processing equipment and procedures should be chosen accordingly. If intensive monitoring
samples are to be analyzed for both organics and trace metals, fish may be filleted and the
left fillet processed and analyzed for organics and the right fillet processed and analyzed for
trace metals.
Suggested sample processing equipment and cleaning procedures by analysis type
are discussed in more detail in Sections 6.2.1.1, 6.2.1.2, and 6.2.1.3. Variations of these
procedures may be used if it can be demonstrated, through the analysis of sample blanks,
that no contamination is introduced (see Section 6.4.3.5). To avoid cross-contamination, all
equipment used in sample handling should be thoroughly cleaned between samples.
6.2.1.1 Samples for Organic Analysis--
Equipment used in processing samples for organic analysis should be constructed of
stainless steel, anodized aluminum, borosilicate glass, and/or quartz. Polypropylene and
polyethylene (plastic) surfaces and implements are a potential source of contamination by
organics and should not be used.
A suggested cleaning procedure is to wash with detergent solution, rinse with tap
water, soak in isopropanol (distilled in glass or pesticide grade), and rinse with organic-free,
distilled, deionized water. Work surfaces should be cleaned with isopropanol, washed with
distilled water, and allowed to dry completely (Stober, 1991). Alternative washing procedures
may be used if it can be demonstrated, through the analysis of appropriate processing blanks,
that all surfaces and equipment are free of organic contaminants (see Section 6.4.3.5).
Filleting should be done on cutting boards covered with heavy duty aluminum foil,
which is changed between each composite sample. Tissue removal should be done with
clean stainless steel or quartz instruments. Knives, fish sealers, measurement boards, etc.,
6-4
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should be cleaned with pesticide-grade isopropanol followed by a rinse with distilled water
between each composite sample (Stober, 1991).
Samples may be stored in glass or Teflon containers with Teflon-lined lids.
6.2.1.2 Samples for Trace Metals Analysis--
Equipment used in processing samples for trace metal analyses should be made of
quartz, TFE (tetrafluoroethylene), polypropylene, or polyethylene. Stainless steel that is
resistant to corrosion may be used if necessary. Stainless steel scalpels have been found not
to contaminate mussel samples (Stephenson et al., 1979). However, other biological tissues
(e.g., fish muscle) containing low concentrations of heavy metals may be contaminated
significantly by any exposure to stainless steel. The predominant metal contaminants from
stainless steel are chromium and nickel. If these metals are not of concern, the use of
stainless steel for sample processing is acceptable. Quartz utensils are ideal but expensive.
To control contamination when resecting tissue, separate sets of utensils should be used for
removing outer tissue and for removing tissue for analysis. For bench liners and bottles,
borosilicate glass is preferred over plastic (Stober, 1991).
Prior to use, utensils and bottles should be cleaned thoroughly with a detergent
solution, rinsed with tap water, soaked in acid, and then rinsed with metal-free water. For
quartz, TFE, or glass containers, 50% HN03, 50% HC1, or aqua regia (3 parts cone HC1 + 1
part cone HN03) should be used for soaking. For plastic material, 50% HN03 or 50% HC1 is
appropriate. Reliable soaking conditions are 24 h at 70 °C (Greenburg et al., 1985). Chromic
acid should not be used for cleaning any materials. Acids used should be at least reagent
grade. Metal parts may be cleaned as stated for glass or plastic, omitting the acid soaking
step (Stober, 1991).
6.2.1.3 Samples for Organics and Trace Metals Analyses--
Several established monitoring programs, including the Puget Sound Estuary Program
(1990c,d), the NOAA Mussel Watch Program (Battelle, 1989), and the California Mussel
Watch Program (California, 1990) recommend that different procedures be used to process
samples for organics analysis and for trace metals analysis. However, this may not always be
feasible, especially in a screening program where only one shellfish composite is collected
and processed or where fish are not of adequate size to allow the preparation of separate
composites from right and left fillets. In these cases, precautions must be taken to use
6-5
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materials and cleaning procedures that are noncontaminating for both organics and trace
metals. (Corrosion-resistant stainless steel, quartz, and Teflon are recommended materials.)
A suggested procedure for cleaning sample processing instruments is to wash them
with a detergent solution, rinse with tap water, and rinse with organics- and metal-free water.
Work surfaces may be cleaned with isopropanol, washed with distilled water, and allowed to
dry. Borosilicate glass bench liners are recommended.
Homogenates and composites should be stored in clean glass, quartz, or Teflon
containers with Teflon-lined lids. All containers should be thoroughly cleaned with a detergent
solution, rinsed with tap water, soaked in acid (50% HN03, 50% HCI, or aqua regia), and then
rinsed with organics- and metal-free water. Reliable soaking conditions are 24 h at 70 °C
(Greenburg et al., 1985).
Composite sample aliquots taken for metals analysis may be stored in plastic
containers that have been cleaned according to the procedure given above for glass, with the
exception that aqua regia must not be used for the acid soaking step.
6.2.2 Fish Samples
Processing in the laboratory to prepare fish fillet composite samples (diagrammed in
Figure 6-1) involves
Weighing individual fish
._Removing scales and/or otoliths for age determination
Determining the sex of each fish (optional)
Removing skin of catfish, bullheads, and sturgeons and scaling all other fish
(leaving belly flap on)
Filleting the fish
Weighing individual fillets
Homogenizing individual fillets
Preparing a composite homogenate
Aliquotting the composite homogenate for analysis
Shipping frozen aliquots to one or more contract laboratories for analysis as
necessary.
6-6
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Log in fish samples using COC procedures
Unwrap individual fish and record weight (g)
Remove and archive scales and/or otoliths for age determination
Determine sex (optional)
Remove scales for all fish except
catfish, bullheads, and sturgeons
Remove skin of catfish,
bullheads, and sturgeons
Fillet fish
Weigh individual fillets (g)
Homogenize individual fillets
Divide ground sample into quarters, mix opposite
quarters and then mix halves (3 times)
Composite equal weights (g) of
homogenized fillet tissues from 6-10
fish of the same species and of
similar size (500-g minimum)
Optional
Save remainder of fillet
homogenate from each
individual fish
COC - Chain of Custody
Seal and label (500-g minimum)
homogenate in appropriate
container(s) and store at -20 °C until
analysis (See Table 6-1 for
recommended container materials
and holding times)
Seal and label individual fillet
homogenate in appropriate
container(s) and archive at
-20 °C (See Table 6-1 for
recommended container
materials and holding times)
Figure 6-1. Laboratory sample preparation and handling for
fish fillet composite samples.
6-7
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Whole fish samples should be shipped or brought to the processing laboratory on wet
or blue ice and fillets resected within 48 hours of sample collection. Fish should not be frozen
prior to resection because freezing may cause internal organs to rupture and contaminate
edible tissue (Stober, 1991). Fish arriving in the laboratory should be weighed, scales and/or
otoliths removed, the sex of each fish determined, and fillets (with belly flap) taken within 48
hours of sample collection. Individual fillets then should be frozen at S-20 °C in the laboratory
prior to being homogenized. The grinding/homogenization procedure can be carried out more
easily if the sample is frozen (Stober, 1991). If resection cannot be performed within 48
hours, the samples should be frozen at the sample site and shipped to the central sample
processing laboratory on dry ice. The fish should then be partially thawed prior to resection.
If rupture of organs is noted for an individual fish, the specimen should be eliminated from the
composite sample.
The thawed or partially thawed fillets should be homogenized individually, and portions
of each homogenate should be combined and mixed to form the composite sample. Individual
homogenates and/or composite homogenates may be refrozen; however, frozen individual
homogenates must be rehomogenized before compositing, and frozen composite
homogenates must be rehomogenized before aliquotting, extraction, and analysis. The
maximum holding time from sample collection to analysis for mercury is 28 days at ^-20 °C;
for all other analytes, the holding time is 6 months to 1 year at ^-20 °C (Stober, 1991).
Sample processing procedures are discussed in more detail in the following sections. Data
from each procedure should be recorded directly in a bound laboratory notebook or on forms
that can be taped or pasted into the laboratory notebook. A sample processing record for fish
fillet composites is shown in Figure 6-2.
6.2.2.1 Sample Weighing-
A wet weight should be determined for each fish collected. If the fish has been
shipped on wet or blue ice, it should be unwrapped and placed on a foil-lined balance tray
and the weight recorded to the nearest gram on the sample processing record and/or in the
laboratory notebook. To avoid contamination, the foil lining should be replaced between each
weighing. Frozen fish should be weighed in clean, tared containers if thawing is expected
before the weighing can be completed. Liquid associated with the sample when thawed must
be maintained in the container as part of the sample because it will contain lipid material that
has separated from the tissue (Stober, 1991).
6-8
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Sample Processing Record for Fish Contaminant Monitoring Program Fish Fillet Composites
Project Number: Sampling Date and Time:
STUDY PHASE: Initial Screening ; Intensive Monitoring: Phase IPhase II
SITE LOCATION
Site Name/Number:
County/Parish: LatAong.:
State Waterbody Segment Number: Waterbody Type:.
Sample Type (bottom feeder, predator, etc.) Species Name:
Composite Sample #: Replicate Number: Number of Individuals:
Left Fillet Right Fillet
Weight Scatos/Otoltths Sex Resection Weight Homogenate Wt.ofHomog. Weight Homogenate Wt.ofHomog.
Rsh f (g) Removed (/) (M,F) Performed (/) (g) Prepared (/) for Composite (g) Prepared (/) for Composite
o>
001
002
003
004
005
006
007
008
009
010
Analyst
Date
Total Composite Weight (g) (left) (right).
Notes:
Figure 6-2.
-------
6.2.2.2 Removal of Scales and/or Otoliths for Aging-
A few scales or otoliths should be removed from each fish for the purpose of age
determination by a fisheries biologist. Aging provides a good indication of the length of
exposure to pollutants (Versar, 1982). For most warm water inland gamefish, 5 to 10 scales
should be removed from below the lateral line and behind the pectoral fin. On softrayed fish
such as trout and salmon, the scale sample should be taken just above the lateral line
(Wisconsin, 1988). For catfish and other scaleless fish, the pectoral fin spines should be
clipped and saved (Versar, 1982). Otoliths are another indicator of age that may be collected
(Jearld, 1983). The scales, spines, or otoliths may be stored by sealing in small envelopes
(such as coin envelopes) or plastic bags labeled with, and cross-referenced by, the
identification number assigned to the tissue specimen (Versar, 1982). Removal of scales,
spines, or otoliths from each fish should be noted (by a check mark) on the sample
processing record.
6.2.2.3 Sex Determination--
Fish sex may be determined during or after filleting. To determine the sex of each
individual fish, an incision should be made on the ventral surface of the body from a point
immediately anterior to the anus toward the head to a point immediately posterior to the pelvic
fins. If necessary, a second incision should be made on the left side of
the fish from the initial point of the first incision toward the dorsal fin. The resulting flap should
be folded back to observe the gonads. Ovaries appear whitish to greenish to golden brown
and have a granular texture. Testes appear creamy white and have a smooth texture (Texas
Water Commission, 1990). The sex of each fish should be recorded on the sample
processing form.
6.2.2.4 Sample Resection (Filleting)--
Resection should be carried out by or under the supervision of an experienced
fisheries biologist. Tissue should be removed with carefully cleaned instruments (see Section
6.2.1), and the specimens should come into contact with noncontaminating surfaces only. To
control contamination when resecting tissue, technicians should use separate sets of utensils
for removing outer tissue and for resecting tissue for analysis.
Special care must be taken to avoid contaminating targeted tissues with material
adhering to the fish exterior. The proper handling of fish tissue to prevent contamination
during laboratory processing cannot be overemphasized. Filleting should be conducted on
6-10
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cutting boards covered with heavy duty aluminum foil that is changed between samples
(Puget Sound Estuary Program, 1990d,e). For catfish, bullheads, and sturgeon, the skin
should be removed before filleting. Belly flaps should be included with all fillets.
The FDA method (1990) for filleting fish is as follows:
Remove and discard heads, scales, tails, fins, guts, and inedible bones;
do not remove skin; fillet.and obtain all flesh and skin from head to tail
and from top of back to belly on both sides.
A comparable fillet can be obtained from the other side of the fish and can be composited with
the first fillet, kept separate for duplicate quality assurance analysis, analyzed for different
analytes, or archived.
Large fish should be sectioned according to the following FDA (1990) method:
Clean, scale, and eviscerate fish. Take 1-inch thick slices, one from
behind the pectoral fins, one from halfway between the first slice and
the vent, and one from behind the vent. Remove bones from each
slice before combining.
Care must be exercised not to puncture any of the internal organs. If the body cavity
is inadvertently penetrated, the fillet should be rinsed with distilled water. This skin-on fillet
deviates from the skin-off fillets analyzed in the National Bioaccumulation Study (U.S. EPA,
1991C); however, skin-on is recommended because that is the way most sport anglers
prepare their fillets.
Each fillet should be weighed and the weight recorded to the nearest gram on the
sample processing record. If the fillets are to be homogenized later, they should be wrapped
individually in aluminum foil and labeled with the sample identification number, the weight (g),
and the date of resection. The designation "L" (for left fillet) or "R" (for right fillet) should be
added to the composite sample identification number at this time. The right and left fillets from
each fish should be kept together, all fillets from a composite should be placed in a labeled
plastic bag, and the bag stored at <-20 °C until homogenization.
6.2.2.5 Preparation of Individual Homogenates-
Small fish fillets (<300 g) should be ground in a hand crank meat grinder and fillets
(300 to 1,000 g) should be ground in a food processor. Larger fillets may be cut into 2.5-cm
cubes with a food service band saw (e.g., Hobart Model 5212) and then ground in either a
small (e.g., Hobart, 1/4 hp, Model 4616) or large (e.g., Hobart, 1 hp, Model 4822) meat
grinder. Homogenizers used to grind tissue should have tantalum or titanium parts if possible.
6-11
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The ground sample should be divided into quarters, opposite quarters mixed together by
hand, and the two halves mixed back together. The grinding, quartering, and hand-mixing
steps should be repeated two more times. If chunks of tissue are present at this point,
grinding/homogenizing should be repeated. No chunks should be discarded because they will
not be extracted efficiently. If the sample is to be analyzed for trace metals only, the ground
tissue may be mixed by hand in a polyethylene bag. As each individual fish is homogenized,
it should be noted (marked with a check) on the sample processing record.
Individual fish fillet homogenates may be either composited or frozen individually and
stored at <-20 °C.
6.2.2.6 Preparation of Composite Homogenates-
If individual fish fillet homogenates are frozen they should be thawed partially and
rehomogenized prior to compositing. Any associated liquid should be maintained as a part of
the sample. Equal weights from each individual homogenate should be removed and blended
to provide a composite sample of sufficient size (500 g minimum) to perform all necessary
analyses. Weights of individual homogenates required for a composite sample, based on the
total number of fish per composite and the quantity of composite prepared, are given in Table
6-2. The actual weight of each individual homogenate that is used in the composite sample
should be recorded, to the nearest gram, on the sample processing record. The remaining
individual homogenates should be archived at <-20 °C with the designation "Archive" and the
expiration date added to each sample label. Location of the archived samples should be
indicated on the sample processing record under "Notes." Each composite sample should be
divided into quarters, opposite quarters mixed together by hand, and the two halves mixed
together. The quartering and mixing should be repeated two more times. If the sample is to
be analyzed only for trace metals, the composite sample may be mixed by hand in a
polyethylene bag. At this point, the composite sample may be frozen and stored at <-20 °C or
processed for analysis.
6.2.3 Shellfish Samples
Laboratory processing of shellfish to prepare edible tissue composites (diagrammed in
Figure 6-3) involves
Removing the edible parts from each shellfish in the composite sample (10 to 50
individuals, depending upon the species)
Combining the edible parts in an appropriate noncontaminating container
6-12
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TABLE 6-2. INDIVIDUAL WEIGHTS (g) OF HOMOGENATE
REQUIRED FOR A COMPOSITE SAMPLE6
Total
number of
fish per
sample
6
7
8
9
10
Total homogenate weight
500 g
(minimum)
84
72
63
56
50
1,000 g
(average)
167
143
125
112
100
2,000 g
334
286
250
223
200
Based on total number of fish per composite and the total homogenate
weight required for analysis.
Homogenizing the composite sample
Aliquotting the composite homogenate for analysis
Shipping frozen aliquots to one or more contract laboratories for analysis as
necessary.
Sample aliquotting and shipping are discussed in Section 6.3; all other processing
steps are discussed in this section. A sample processing record for shellfish edible tissue
composite samples is shown in Figure 6-4.
Shellfish samples collected for intensive monitoring studies should be shipped to the
sample processing laboratory either on wet ice or blue ice (if next-day delivery is assured) or
on dry ice (see Section 5.3.2). Shellfish samples arriving on wet ice or blue ice should have
edible tissue removed and should be frozen to <-20 °C within 48 hours after collection.
Shellfish samples that arrive frozen at the central processing laboratory should be placed in a
freezer for storage until edible tissue is removed. Thawing of frozen shellfish samples should
be kept at a minimum during tissue removal procedures to avoid loss of liquids. Shellfish
should be rinsed well with organic- and metal-free water to remove any loose external debris.
6-13
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Log in shellfish samples using COC procedures
Remove edible parts from each shellfish specimen
Combine edible parts from all 10-50 shellfish
in a tared container (g)
Weigh the filled container (g)
Homogenize the composite sample
Divide ground sample into quarters, mix opposite
quarters and then mix halves (3 times)
Seal and archive (500-g minimum)
homogenate in appropriate
container(s) and store at -20 °C until
analysis (See Table 6-1 for
recommended container materials
and holding times)
Seal and archive remaining
homogenate in appropriate
container(s) and store at -20 °C
(See Table 6-1 for recommended
container materials and holding
times)
COC - Chain of Custody
Figure 6-3. Laboratory sample preparation and handling for
shellfish edible tissue composite samples.
6-14
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Sample Processing Record for Shellfish Contaminant Monitoring Program Edible Tissue Composites
Project Number:
STUDY PHASE: Initial Screening
SITE LOCATION
Site Name/Number:
County/Parish:
State Waterbody Segment Number
SHELLFISH COLLECTED
Species Name:
Composite Sample #:
Shellfish Included In
* Composite (/)
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Preparation of Composite:
Weight of container + shellfish
Weight of container
Total weight of composite
Analyst
D:
Shellfish #
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
Sampling Date and Time:
Intensive Monitoring Phase I I I Phase II I I
Lat./Lona:
Waterbodv Tvoe:
Number of Individuals:
Included In Included In
Composite (/) Shellfish* Composite (/)
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
g
g
a +
f of specimens Average weight
of specimen
Date
Rgure 6-4.
6-15
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Edible parts from all shellfish constituting a composite (10-50 individuals) should be
placed in an appropriate preweighed and labeled noncontaminating container. The weight
ofthe empty container should be recorded on the sample processing record. All fluids
accumulated during removal of edible tissue are considered part of the sample. As the edible
portion of each shellfish is placed in the container, it should be noted on the sample
processing record. When the edible tissue has been removed from all shellfish in the
composite, the container should be reweighed and the weight recorded on the sample
processing record. At this point, the composite sample may be frozen and stored at <-20 °C
or processed for analysis.
Each composite sample should be homogenized to a paste-like consistency in a
Polytron or blender before aliquots are taken for analysis. Composite homogenates may be
refrozen; however, they must be rehomogenized before aliquotting. The maximum holding
time from sample collection to analysis for mercury is 28 days at <-20 °C. For all other
analytes, the holding time is 6 months to 1 year at ^-20 °C (Stober, 1991). Bivalve sample
processing procedures are discussed in more detail in the section below. Performance of
each procedure should be documented in the laboratory notebook or on an appropriate form
that can be taped or pasted in the laboratory notebook (see Figure 6-4).
6.2.3.1 Removal of Edible Parts-
For the intensive study, analysis of shellfish is restricted to tissues that consumers
might reasonably be expected to eat. Edible portions should be clearly defined in sample
processing protocols by each State because the definition of edible parts may be site- or
region-specific. Bivalve molluscs (oysters, clams, mussels, and scallops) typically are
prepared by severing the adductor muscle, prying open the shell, and removing the soft
tissue. The soft tissue includes viscera, meat, and body fluids (U.S. EPA, 1985c). Byssal
threads from mussels should be removed with a knife before shucking and should not be
included in the composite sample. Edible tissue for crabs typically includes all leg and claw
meat, back shell meat, and body cavity meat. Internal organs generally are removed. A
decision on inclusion of the hepatopancreas should be based upon the eating habits of the
local population or subpopulations of concern. If the crab is soft-shelled, the entire crab
should be used in the sample. Hard- and soft-shelled crabs must not be combined in the
same composite (U.S. EPA, 1985c). Typically, shrimp and crayfish are prepared by removing
the cephalothorax and removing the tail meat from the shell. Only the tail meat with the
6-16
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section of intestine passing through the tail muscle is retained for analysis (U.S. EPA, 1985c).
Edible tissue for lobsters may include tail meat, claw meat, tomalley (hepatopancreas), and
gonad or ovaries (Duston, 1990).
6.2.3.2 Preparation of Composite Homogenate--
Grinding of tissue is easier when the tissue is partially frozen (Stober, 1991). Chilling
the grinder briefly with a few chips of dry ice will reduce the tendency of the tissue to stick to
the grinder. However, do not freeze the grinder because it will make it difficult to force frozen
tissue through the chopper plate.
Tissue for trace metals analysis may be homogenized in 4-oz polyethylene jars
(California, 1990) using a Polytron (e.g., Brinkman Model PT10-35) equipped with a titanium
generator (e.g., Brinkman Model PTA 20). If the tissue is to be analyzed for organics only, or
if chromium and nickel contamination are not of concern, a commercial food chopper with
stainless steel blades and glass container may be used. The edible parts of all samples in
the composite should be ground together to a paste-like consistency. Larger samples may be
cut into 2.5-cm cubes before grinding. If samples were frozen after dissection, they can be
cut without thawing with either a knife-and-mallet or a clean bandsaw. Samples should be
homogenized in a grinder, blender, or chopper that has been cooled briefly with dry ice (U.S.
EPA, I985c). The ground sample should be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed back together. The quartering and mixing should
be repeated two more times. At this point, the composite sample may be frozen and stored at
£-20 °C (see Table 6-2) or processed for analysis.
6.3 SAMPLE DISTRIBUTION
The central processing laboratory should prepare aliquots of the composite
homogenates for analysis, transfer the aliquots to the appropriate laboratory (or laboratories),
and archive the remainder of each composite sample.
6.3.1 Sample Aliquotting
If composite homogenate tissue samples have been frozen, they must be thawed and
rehomogenized before aliquots are prepared. Samples may be thawed overnight in an
insulated cooler or refrigerator and then homogenized. Suggested aliquot weights and
appropriate containers are as follows:
6-17
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Analysis Aliquot Weight Shipping/Storage Container
Trace metals 1-5 g Polystyrene jar
Organics 20-50 g Glass or Teflon jar with Teflon-
lined lid
Dioxins 20-50 g Glass or Teflon jar with Teflon-
lined lid
*
It has been recommended (Stober, 1991) that the exact quantity of tissue required for
extraction and analysis be weighed and placed in an appropriate container that has been
labeled with the sample ID and the exact tissue weight. The analytical laboratory can then
recover the entire sample, including any liquid from thawing, by rinsing the container directly
into the digestion or extraction vessel with the appropriate solvent. If this procedure is used, it
is the responsibility of the central processing laboratory to provide a sufficient number of
duplicate aliquots and aliquots for matrix spikes so that the QA/QC requirements of the
program can be met. It is extremely important that accurate records be maintained when
samples are aliquotted for analysis (see Section 6.4.3). It is recommended that a carefully
designed form be used to ensure that all the necessary information is recorded. Several
programs have designed sample aliquotting forms to fit particular needs. An example of a
sample aliquotting record for a fish/shellfish monitoring program is presented in Figure 6-5.
The composite sample identification number is assigned to the composite sample at
the time of collection and carried through sample processing (plus "L" or "R," if the composite
represents a left fillet or right fillet, respectively). The aliquot identification number should
indicate analyte class (e.g., TM for trace metals, OR for organics, DX for dioxin, etc.) and the
sample type (e.g., R for routine sample; RS for a routine sample that is split for analysis by a
second laboratory; MS1 and MS2 for sample pairs, one of which will be prepared as a matrix
spike). The composite sample identification number may be of the form WWWWWX-YY-ZZZ,
where WWWWW is the sample composite identification number, X indicates the left or right
fillet, if applicable, YY is the analyte code, and Z is the sample type.
"Blind" duplicates may be introduced by preparing two separate aliquots of the same
composite homogenate andlabeling one aliquot with a "dummy" composite sample
identification. However, the analyst who prepares the sample aliquots must be careful to
assign a "dummy" identification number that has not been used for an actual sample and to
indicate clearly on the processing records that the samples are blind duplicates. The
analytical laboratory should not receive this information.
6-18
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Fish/Shellfi^^/lonitoring Program
Sample Aliquotting Record
Aliquotted by
Date
Time
(name)
Comments
Samples from:
Project No.
Site#
D Screening study
D Intensive study
Composite Sample ID
Archive Location:
Analyte Code
Aliquot ID
Aliquot Weight
Analyze for:
Ship to:
Analyte Code
Aliquot ID
Aliquot Weight
Analyze for:
Ship to:
Analyte Code
Aliquot ID
Aliquot Weight
>
*
Analyze for:
Ship to:
1
CO
Page.
of
Figure 6-5.
-------
When the appropriate number of aliquots of a composite sample have been prepared
for all analyses to be performed on that sample, the remainder of the composite sample
should be labeled "ARCHIVE" and placed in a secure location in the sample processing
laboratory. The expiration date also should be added to the sample label. The location of the
archived samples should be indicated on the sample aliquotting record. Aliquots for sample
analysis should be frozen at ^-20 °C before they are transferred or shipped to the appropriate
analytical laboratory.
6.3.2 Sample Transfer
When all composite homogenates have been aliquotted for analysis, the frozen
aliquots should be transferred on dry ice to the analytical laboratory (or laboratories)
accompanied by a sample transfer record such as the one shown in Figure 6-6. Further
details on Federal regulations for shipping biological specimens in dry ice are given in Section
5.3.2.1. The sample transfer record may include a section to serve as the analytical
laboratory COC record. The COC record must be signed each time the samples change
hands for preparation and analysis.
6.4 SAMPLE ANALYSES
6.4.1 Target Analvtes
In initial screening studies, composite samples of fish fillets or edible portions of
shellfish should be analyzed for all target contaminants listed in Table 4-3 and for any
additional site-specific target contaminants that have been identified by States or Regions. In
intensive monitoring studies, composite samples of edible portions of fish or shellfish should
be analyzed only for those target contaminants that were found to exceed recommended
trigger values (TVs) in initial screening studies (see Section 4.2).
All samples analyzed for organic target contaminants in initial screening studies and
intensive monitoring studies should also be analyzed for percent lipid to allow data users to
normalize organic target contaminant data if desired (e.g., for trend analysis or model
validation) (see Sections 2.1.9 and 2.2.9).
6.4.2 Analytical Methods
A recommended procedure for lipid analysis is given in Appendix H.
6-20
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Fish/Shellfish Monitoring Program
Sample Transfer Record
Date
DO MM
Released by:
At:
Shipment Method.
Shipment Destination
YY
Date
DD MM
Received by:
YY
At:
Comments
Time
(24-h clock)
HH MM
(name)
(location)
Time
(24-h clock)
HH MM
(name)
(location)
Study Type: D ScreeningAnalyze for: D Trace metals
D IntensiveAnalyze for (specify)
Organics
Sample IDs:
Laboratory Chain of Custody
Relinquished by
Received by
Purpose
Location
Figure 6-6.
6-21
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{Reviewers' comments are requested regarding this procedure. If modifications
or alternative methods are recommended, please be specific and Include full literature
citations.]
At present, no procedures have been approved officially by the EPA or other regulatory
agencies for the analysis of low parts-per-billion concentrations of organic contaminants in fish
and shellfish tissues (Puget Sound Estuary Program, 1990d), and only interim procedures
have been proposed for the analysis of metals in tissue samples (U.S. EPA, 1981). However,
based on a review of EPA guidance for bioaccumulation monitoring programs (U.S. EPA,
1986a) and of analytical methods currently used or recommended in a variety of these
programs (Puget Sound Estuary Program 1990d,e; California, 1990; U.S. EPA, 1989a-c; U.S.
FDA, 1990; Krahn et al., 1988; MacLeod et al., 1985), It is recommended that organic
target contaminants be analyzed by gas chromatography/mass spectrometry (GC/MS)
or gas chromatography/electron capture detection (GC/ECD) methods using the sample
preparation techniques shown In Table 6-3.
Because of the relatively poor sensitivity of GC/MS for analysis of chlorinated
compounds, PCBs and chlorinated pesticides should be quantified by GC/ECD. However,
analysis by GC/ECD does not provide definitive compound identification, and false positives
due to interferences have been commonly reported. Therefore, confirmation by GC/MS using
selected ion monitoring or by using an alternative GC column phase (with ECD) is required for
positive identification of chlorinated pesticides and PCBs. The large number of congeners of
PCBs anoTheir chemical nature present serious analytical difficulties. Quantitation of
individual congeners, or even individual aroclors, is tedious and expensive. It is therefore
recommended that total PCB analysis be performed routinely, especially in initial screening
studies. If initial screening study results indicate significant PCB contamination, more detailed
analyses of PCB isomer distributions may be performed during intensive followup monitoring
studies.
[Reviewers are asked to provide recommendations as to which chemical analysis
procedures to use for the analysis of PCB congeners and which PCB congeners are
most important to monitor.]
All other organic compounds should be analyzed by GC/MS (U.S. EPA, 1985b). The
determination of individual PAHs is not recommended in initial screening studies. However, if
initial screening study results indicate a high level of PAH contamination in the target species,
identification and quantitation of individual PAH compounds should be performed with
6-22
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TABLE 6-3. SUMMARY OF BASIC SAMPLE PREPARATION AND ANALYTICAL
TECHNIQUES FOR ORGANIC TARGET CONTAMINATION
Procedural step Recommended technique
Sample drying Centrifugation or sodium sulfate
Extraction Shaker/roller; Soxhlet, sonication
Extract drying Separatory funnel partitioning as needed to remove water
(pH must be controlled); sodium sulfate for all other extract
drying. Kuderna-Danish apparatus (to ca. 1 mL), rotary
evaporation (to 2 mL) or comparable technique; purified
nitrogen gas for concentration to smaller volumes
Extract cleanup Removal of organic interferents with GPC, size exclusion
chromatography (e.g., phenogel, Sephadex), bonded
octadecyl columns, HPLC, silica gel, or alumina
Extract analysis GC/MS for volatiles and semivolatiles, GC/ECD for
chlorinated pesticides, PCBs, and aroclor mixtures
GPC - Gel permeation chromatography.
HPLC = High performance liquid chromatography.
GC/MS = Gas chromatography/mass spectrometry.
GC/ECD = Gas chromatography/electron capture detection.
PCB = Polychlorinated biphenyls.
Source: Puget Sound Estuary Program (1990a).
6-23
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particular attention given to benzo[a]pyrene and related compounds (e.g.i 1,2-benzanthracene;
3,4-benzpyrene; 3-methylcholanthrene; 5,6-dimethylphenanthrene).
[Reviewers are asked to provide recommendations as to which chemical analysis
procedures to use for the analysis of individual PAHs and which PAH compounds (in
addition to benzo[a]pyrene) are most important to monitor.]
Because of the toxiclty of dioxins and the difficulty and cost of analysis for
dioxins and furans (U.S. EPA, 1989b), it is recommended that tetra- through
octa-chlorinated dibenzo-p-dioxins and dibenzofurans be analyzed by a contract
laboratory with demonstrated expertise in these analyses. If resources are limited, the
2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,7,8-tetrachlorodibenzofuran
(2,3,7,8-TCDF) congeners should be analyzed for at a minimum. Contract laboratories
currently performing dioxin/furan analyses are listed in Table 6-4. This list is included
for information purposes only and should not be construed as an endorsement of
laboratories.
It is recommended that all metal target contaminants except mercury be
analyzed by graphite furnace atomic absorption (GFAA) spectrophotometric methods.
Mercury analysis should be performed by cold vapor atomic absorption (CVAA)
spectrophotometric methods (U.S. EPA, 1989a). GFAA requires a separate determination
for each analyte, which increases the time and cost relative to broad-scan methods such as
inductively coupled plasma emission spectrometry (ICP). However, because detection limits
typically achieved with GFAA are significantly lower than those achieved with ICP, GFAA is
recommended for the analysis of target metal contaminants (U.S. EPA, 1985b).
Recommended methods for the analysis of target contaminants are summarized in
Table 6-5. As shown in Tables 6-6 and 6-7, these methods have demonstrated detection
limits in the low parts-per-billion range, which is well below the screening study target
contaminant TVs (see Section 4.2). Alternative methods of analysis may be used if
comparable detection limits and acceptable accuracy and precision can be demonstrated (see
Sections 6.4.3.3 and 6.4.3.4). If lower TVs are used (e.g., for susceptible populations in
intensive monitoring studies), it is the responsibility of the program manager to ensure that the
detection and quantitation limits of the analytical methods are sufficiently low to allow reliable
quantitation of target analytes at or below these TVs (see Section 6.4.3.3).
Because of the lack of official EPA-approved methods and to allow States and Regions
flexibility in developing their analytical programs, specific step-by-step procedures for the
6-24
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TABLE 6-4. CONTRACT LABORATORIES CONDUCTING DIOXIN/FURAN ANALYSES
IN FISH/SHELLFISH TISSUES8
Alta Analytical Laboratory"
5070 Robert J. Matthews Parkway, Suite 2
Eldorado Hills, CA 95630
916/933-1640
FAX: 916/933-0940
Bill Luksemburg
Battelle-Columbus Laboratories''
505 King Avenue
Columbus, OH 43201
614/424-7379
Karen Riggs/Gerry Pitts
Enseco-California Analytical Labs"
2544 Industrial Blvd.
West Sacramento, CA 95691
916/372-1393
916/372-1059
Kathy Gill/Michael Filigenzi/Mike Millie
IT Corporation
Technology Development Laboratory*3
304 Directors Drive
Knoxville.TN 37923
615/690-3211
Duane Root/Nancy Conrad/Bruce Wagner
Midwest Research Institute15
425 Volker Boulevard
Kansas City, MO 64110
816/753-7600 ext. 190/ext. 160
Paul Kramer/John Stanley
New York State Department of Health*3
Wadsworth Laboratories
Empire State Plaza
P.O. Box 509
Albany, NY 12201-0509
518/474-4151
Arthur Richards/Kenneth Aldous
Pacific Analytical lnc.b
1989-B Palomar Oaks Way
Carlsbad, CA 92009
619/931-1766
Phil Ryan/Bruce Colby
Seakem Analytical Services'3
P.O. Box2219
2045 Mills Road
Sidney, BC V8L 351
Canada
604/656-0881
Valerie Scott/Allison Peacock/Coreen Hamilton
TMS Analytical Services'3
7726 Moller Road
Indianapolis, IN 46268
317/875-5894
FAX: 317/872-6189
Dan Denlinger/Don Eickhoff/
Kelly Mills/Janet Sachs
Triangle Laboratories'3
Alston Technical Park
801 Capitola Drive, Suite 10
Research Triangle Park, NC 27713
919/544-5729
Steve Guyan/Diane Williford/
Bill Hurst/Mary Collins
Twin City Testing Corporation13
662 Cromwell Avenue
St. Paul, MN 55114
612/649-5502
Chuck Sueper/Fred DeRoos
University of Nebraska
Mid-West Center for Mass Spectrometry
12th and T Street
Lincoln, NE 68588
402/472-3507
Michael Gross
Wellington Environmental Consultants'3
395 Laird Road
Guelph, Ontario N1G 3X7
Canada
519/822-2436
Judy Sparling/Brock Chittin
Wright State University*3
175 Brehm Laboratory
3640 Colonel Glen Road
Dayton, OH 45435
513/873-2202
Thomas Tiernan/Garrett Van Ness
aThls list should not be construed as an endorsement of these laboratories, but Is provided for Information
purposes only.
^Laboratory participating in Method 1613 interiaboratory (round- robin) dioxin study (May 1991).
6-2E
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TABLE 6-5. RECOMMENDED METHODS FOR ANALYSIS OF
TARGET CONTAMINANTS
Analyte type
Recommended analytical method
Metals (except mercury)
Mercury
Semivolatile organics
(PAHs, chlorinated aromatics, phenols)
PCBs
Pesticides
Dioxins/furans
GFAA
CVAA
GC/MS
GC/ECD
GC/ECD
GC/MSa'b
GFAA = Graphite furnace atomic absorption spectrophotometry.
CVAA = Cold vapor atomic absorption spectrophotometry.
GC/MS = Gas chromatography/mass spectrometry.
GC/ECD = Gas chromatography/electron capture detection.
PAH = Polycyclic aromatic hydrocarbons.
PCB = Polychlorinated biphenyls.
a For the analysis of tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and
dibenzofurans (PCDFs) using isotope dilution. Note: If resources are limited,
2,3,7,8-TCDD and 2,3,7,8-TCDF should be analyzed for at a minimum.
b Because of the difficulty and cost of the analysis, and human health considerations, it
is recommended that dioxins and furans be analyzed by a contract laboratory expert
in conducting dioxin/furan analyses (see Table 6-4; this list is provided for
information purposes only and is not to be construed as an endorsement of
laboratories).
6-26
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TABLE 6-6. COMPARISON OF TARGET CONTAMINANT TRIGGER VALUES (TVs)' WITH
TYPICAL DETECTION LIMITS" FOR ORGANIC COMPOUNDS IN TISSUE SAMPLES
Detection limits
(ppm; ng/g wet weight)1''0
Compound type TV ~~ ,
(target contaminant) (ppm; ng/9 wet weight) GC/MS GC/ECD
Phenols 0.02*
Pentachlorophenol 320 0.08
i
Aromatic hydrocarbons (low and high 0.01
molecular weight)
PAHs 0.095
1,2-Dichlorobenzene 970
1,4-Dichlorobenzene 143
Hexachlorobenzene 8.6
Pentachlorobenzene 8.6
1,2,4,5-Tetrachlorobenzene 3.2
1,2,4-Trichlorobenzene 215
PCBs
Pesticides
Aldrin
Chlordane
DDT
Dieldrin
Endosulfan
Endrin
Heptachlor
Heptachlor epoxide
Lindane
Mirex
Toxaphene
0.14
0.063
0.65
3.2
0.067
0.54
3.2
0.23
0.12
0.82
0.02
0.98
0.02
0.05 0.0001 -0.005s
GC/MS - Gas chromatography/mass spectrometry.
GC/ECD - Gas chromatography/electron capture detection.
PAH - Polycyclic aromatic hydrocarbons.
PCB Polychlorinated biphenyls.
' From Table 4-6.
b From U.S. EPA (1985b). Values in boldlace type are typically achievable detection limits for methods
recommended in this guidance document for the analysis of organic compounds in tissue samples.
° Detection limits are based on a 25-g (wet weight) tissue sample extracted, concentrated to 0.5 mL after gel
permeation chromatography cleanup, and 1 uL injected. Bonded, fused silica capillary GC columns, which
provide better resolution than packed columns, are assumed for analyses of semivolatile compounds.
* Extract cleanup (e.g., removal of polar interferences by alumina column chromatography) is assumed.
* Substantially increased detection limits (ppm) are observed for 4-nitrophenol (0.1), 2,4-nitrophenol (0.1), and
pentachlorophenol (0.08).
1 No detection limits provided because methodology does not allow adequate recovery and/or detection.
8 The higher range of detection limits are appropriate for pesticides such as mirex, methoxychlor, the DDTs,
and endosulfans, and for chlorinated butadienes. Compounds such as lindane, aldrin, heptachlor, and
hexachlorobenzene can be detected at the lower limit. Toxaphene (a mixture) may require a higher
detection limit than the other organochlorine pesticides.
6-27
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TABLE 6-7. COMPARISON OF TARGET CONTAMINANT TRIGGER VALUES
(TVs)8 WITH TYPICAL DETECTION LIMITS FOR TRACE METALS
IN TISSUE SAMPLES"
Element
Arsenic
Cadmium
Lead
Mercury
Selenium
TV
(ppm; M9/g wet weight)
0.61
11
d
3.2
43
Recommended detection limit0
(ppm; iug/g wet weight)
0.02
0.01
0.03
0.01
0.02
8 From Table 4-6.
b From U.S. EPA (1985b). Based on detection levels normally achieved in methods commonly used for
tissue analyses in environmental laboratories: Graphite furnace atomic absorption (GFAA) analysis
for arsenic, cadmium, lead, selenium; cold vapor atomic absorption (CVAA) analysis for mercury.
Lower detection limits may be achieved by experienced analysts with state-of-the-art equipment.
0 Detection limits are based on 5 g (wet weight) of muscle tissue, digested and diluted to 50 mL.
d No reference dose (RfD) available at this time for calculating the TV (see Section 4.2).
analysis of target contaminants in fish/shellfish monitoring programs are not included in this
guidance document. Instead, a performance-driven analytical program is recommended. This
recommendation is based on the assumption that the analytical results produced by different
laboratories and/or different methods will be comparable if appropriate minimum QA/QC
procedures are implemented within each laboratory and if comparable analytical performance
on round-robin comparative analyses of standard reference materials or split sample analyses
of field samples can be demonstrated. Performance-based analytical programs currently are
used in several fish/shellfish monitoring programs (e.g., NOAA Status and Trends Program
[NOAA, 1987; Battelle, 1989; Cantillo, 1991], E-MAP Program [REF], Puget Sound Estuary
Program [1990a-e]).
Analytical methods and QA/QC procedures described in the following documents are
recommended as guidelines for methods used by State or Regional laboratories or by
selected contract laboratories for the analyses of target contaminants in fish or shellfish
tissues:
Bioaccumulation Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority
Pollutants and 301 (h) Pesticides in Tissues from Marine and Estuarine Organisms
(U.S. EPA, 1986a)
6-28
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Quality Assurance/Quality Control (QA/QC) for 301 (h) Monitoring Programs: Guidance
on Field and Laboratory Methods (U.S. EPA, 1987e)
U.S. EPA Method 1624: Volatile Organic Compounds by Isotope Dilution GC/MS.
Method 1625: Semivolatile Organic Compounds by Isotope Dilution GC/MS (U.S. EPA,
1989c)
U.S. EPA Interim Methods for the Sampling and Analysis of Priority Pollutants in
Sediments and Fish Tissue (U.S. EPA, 1981)
Puget Sound Estuary Program Plan (1990d,e)
U.S. EPA Contract Laboratory Program Statement of Work for Inorganic Analysis (U.S.
EPA, 1991 a)
U.S. EPA Contract Laboratory Program Statement of Work for Organic Analysis (U.S.
EPA, 1991b)
U.S. Food and Drug Administration Pesticide Analytical Manual (PAM Vols. I and II)
(U.S. FDA, 1990)
Standard Analytical Procedures of the NOAA National Analytical Facility (Krahn et al.,
1988; MacLeod et al., 1985)
Official Methods of Analysis of the Association of Official Analytical Chemists (Williams,
1984)
Analytical Procedures and Quality Assurance Plan for the Determination of Mercury in
Fish (U.S. EPA, 1989a).
Analytical Procedures and Quality Assurance Plan for the Determination of
PCDD/PCDF in Fish (U.S. EPA, 1989b)
Analytical Procedures and Quality Assurance Plan for the Determination of Xenobiotic
Chemical Contaminants in Fish (U.S. EPA, 1989c)
U.S. EPA Test Methods for the Evaluation of Solid Waste, Physical/Chemical Methods
(U.S. EPA, 1986b)
Standard Methods for the Examination of Water and Wastewater (Greenburg et al.,
1985)
U.S. EPA Test Methods for the Chemical Analysis of Municipal and Industrial
Wastewater (U.S. EPA, 1982b)
U.S. EPA Methods for the Chemical Analysis of Water and Wastes (U.S. EPA, 1979b)
State of California, Department of Fish and Game, Laboratory Quality Assurance
Program Plan (California, 1990)
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A recent evaluation of current methods for the analyses of organic and trace metal target
contaminants in fish tissue (Capuzzo et al., 1990) provides useful guidance on method
selection, validation, and data reporting procedures. Laboratories should select or develop
analytical procedures for routine analyses of target contaminants that are most appropriate for
their programs based on available resources, experience, program objectives, and data quality
requirements.
All methods used by a laboratory for the analyses of target contaminants and
llpld content must be validated by the laboratory prior to routine sample analysis. That
is, the detection and quantitation limits and accuracy and precision of each method must be
assessed and documented to be sufficient for reliable quantitation of all target contaminants at
or below their estimated TVs (see Sections 6.4.3.3 and 6.4.3.4).
All analytical methods used routinely for the analyses of fish and shellfish
tissues should be documented thoroughly, preferably as formal standard operating
procedures (SOPs) (U.S. EPA, 1984b). Analytical SOPs should include the following
information:
Scope and application
Method performance characteristics (accuracy, precision, and method detection and
quantitation limits) for each analyte
Interferences
Equipment, supplies, and materials
Sample preservation and handling
Instrument calibration procedures
Sample preparation procedures
Sample analysis procedures
Quality control procedures
Data reduction and analysis procedures (with example calculations)
Recordkeeping procedures (with standard data forms, if applicable)
Safety procedures and/or cautionary notes
References.
6-30
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A published method may serve as an analytical SOP only if the analysis ie performed exactly
as described.
Analytical SOPs must be followed exactly as written. Any deviations should be
documented in the laboratory records (signed and dated by the responsible person)
and noted in the final data report. Adequate evidence must be provided to demonstrate
that SOP deviations did not adversely affect method performance (i.e., detection or
quantitation limits, accuracy, precision), or the effect on data quality must be assessed and
documented and all suspect data identified.
Examples of SOPs for the analysis of cadmium by GFAA (California, 1990) are
included in Appendix I as a guide to laboratories for developing their own analytical SOPs.
They are intended to illustrate the kind of information and level of detail that is required in an
SOP to permit a suitably trained person to conduct the analysis accurately and reproducibly.
6.4.3 General QA/QC Considerations for Sample Analysis
Definitions of QA/QC terminology (including QA/QC samples) used in this section are
included in the Glossary. [Note to reviewers: The Glossary will be Included in the next
iteration of this document.]
Each laboratory performing target contaminant analyses for fish consumption
advisory programs should have a formal QA/QC program as described in Appendix F
(U.S. EPA, 1984b). It is the responsibility of each program manager, in consultation
with the analytical laboratory staff, to ensure that appropriate detection and quantitation
limits and QA/QC requirements have been established for each analytical method prior
to beginning routine sampling and analysis. In particular, the QA/QC guidelines in the
EPA Contract Laboratory Program (CLP) (U.S. EPA, 1991a,b), the Puget Sound Estuary
Program (1990d,e), the NOAA Status and Trends Program (NOAA, 1987; Battelle, 1989;
Cantillo, 1991), and the EPA 301 (h) Monitoring Programs (U.S. EPA, 1987e) are
recommended as a basis for developing program-specific QA/QC programs. The Puget
Sound Estuary Program QA/QC requirements for organic and metal analyses are included in
Appendixes J and K, respectively, as specific examples of the application of EPA CLP QA/QC
requirements to a bioaccumulation monitoring program.
The QA/QC program for each analytical laboratory should be documented fully in
a QA/QC plan or in a combined Work/QA Project Plan (U.S. EPA, 1980c). (See
Appendix F.) Each QA/QC requirement or procedure should be described clearly and
6-31
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the rationale for each provided. Documentation should clearly demonstrate that the
QA/QC program meets overall program objectives and data quality requirements.
For sample analyses, minimum QA/QC requirements consist of initial demonstration of
laboratory capability and routine analyses of appropriate QA/QC samples to document data
quality and to demonstrate continued acceptable performance. The QA/QC requirements for
the analyses of target contaminants in tissues should be based on specific performance
criteria, or control limits, for data quality indicators such as accuracy and precision.
Typically, control limits for accuracy are based on the historical mean recovery plus or
minus three standard deviation units, and control limits for precision are based on the
historical standard deviation or coefficient of variation (or mean relative percent difference for
duplicate samples) plus three standard deviation units. Procedures should be in place for
monitoring historical performance and should include control charts (Taylor, 1985; ASTM,
1976) and/or tabular presentations of the data. When established control limits are not met,
appropriate corrective action should be taken and, if possible, all suspect samples reanalyzed.
If reanalyses cannot be performed, all suspect data should be identified clearly.
Recommended QA/QC samples, suggested frequencies of analyses, example control
limits (performance criteria), and appropriate corrective actions are summarized in Table 6-8.
It is the responsibility of program managers to ensure that appropriate QA/QC
programs are developed for all participating analytical laboratories to ensure the quality
and comparability of reported data.
The following QA/QC procedures are necessary to ensure the quality and intra- and
interlaboratory comparability of the data obtained by various analytical methods used for
analyzing target contaminants in fish by consumption advisory programs (Battelle, 1989):
Instrument calibration and calibration checks
Assessment of method detection and quantitation limits
Assessment of method accuracy and precision
Routine monitoring of interferences and contamination
Regular external QA assessment of analytical performance-interlaboratory
comparison programs
Appropriate documentation and reporting of data (including QA/QC data).
6-32
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TABLE 6-8. RECOMMENDED QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) SAMPLES
Sample type"
Objective
Suggested frequency
of analysis'*
Example
control limits"
Corrective action
Calibration Standards
(3-5 standards over the
expected range of
sample concentrations,
with the lowest
concentration standard at
or near the MDL).
Calibration Check
Standards
(minimum of one mid-
range standard prepared
independently from initial
calibration standards, or
a mid-range laboratory
control sample [see
below])
Matrix Spikes
(one spike for each
analyte at 3-5 times the
estimated MDL)
(0.5 to 5 times the
concentration of the
analyte of interest or 5
times the PQL)
Full calibration: Establish
relationship between
instrument response and
analyte concentration (i.e.,
r2, slope, or relative
response factor [RRF]).
Verify initial calibration.
Establish or confirm MDL
for analyte of interest.
Assess matrix effects and
accuracy (percent
recovery).
Instrument/method dependent; follows
manufacturers recommendations or
procedures in specific analytical
protocols. At a minimum, perform a 3-
point calibration at beginning of project,
after each major equipment change or
disruption, and when routine calibration
check exceeds specific action limits.
Organics (GC/MS): At beginning and
end of each work shift, and once
every 12 hours (or every 10-12
analyses, whichever is more
frequent).
Organics (GC/ECD): At beginning and
end of each work shift, and once
every 6 hours (or every 6 samples,
whichever is less frequent).
Metals: Every 10 samples or every 2
hours, whichever is more frequent.
Seven replicate analyses prior to use
of method for routine analyses.
One per 20 samples or one per batch,
whichever is more frequent.
Organics: RSD of RRFs
>30%.
Metals: %R of all standards
95-105.
Recalibrate; prepare new
calibration standards if
necessary. Reanalyze all
samples from last acceptable
calibration or calibration check,
or flag all suspect data.
Organics: Percent difference
between the average RRF
from initial calibration and
the RRF from the calibra-
tion check >25%.
Mercury. %R - 80-120
Other Metals: %R - 90-110
Recalibrate; prepare new
calibration standards if
necessary. Reanalyze all
samples from last acceptable
calibration or calibration check,
or flag all suspect data.
Determined by program manager. Redetermine MDL.
Organics: Determined by
program manager. % R >
50 with good precision is
acceptable.
Metals: %R - 75-125
Determine cause of problem
(e.g., incomplete extraction or
digestion, contamination), take
appropriate corrective action,
and reanalyze all suspect
samples or flag all suspect data.
Zero percent recovery requires
rejection of all suspect data.
(continued)
-------
TABLE 6-8. (continued)
Sample type"
Objective
Suggested frequency
of analysis'*
Example
control limits0
Corrective action
Matrix Spike Duplicates
(0.5 to 5 times the
concentration of the
analyte of interest or 5
times the PQL)
Assess method precision.
Blanks (Method, Field,
Processing, Bottle)
Assess contamination from
equipment, reagents, etc.
One per 20 samples or one per batch,
whichever is more frequent.
One method blank and one field blank
per 20 samples or one per batch,
whichever is more frequent. At least
one processing blank per study. At
least one bottle blank per lot or per
study, whichever is more frequent.
Organics: A difference of no
more than a factor of 2
among replicates (i.e.,
approximately 50% coefficient
of variation). NOTE: pooling
of variances in duplicate
analyses from different sample
batches is recommended for
estimating the standard
deviation or coefficient of
variation of replicate analyses.
Metals: +20 RPD for duplicates.
Concentration of any analyte
or PQL, or £10-30 % of sample
concentration as determined by
program manager.d
Determine cause of problem
(e.g., incomplete extraction or
digestion, contamination,
instrument instability or
malfunction), take appropriate
corrective action, and reanalyze
all suspect samples or flag all
suspect data.
Determine cause of problem
(e.g., contaminated reagents,
equipment), take appropriate
corrective action, and reanalyze
all suspect samples or flag all
suspect data.
Reagent Blanks
Surrogate Spikes
Laboratory Control
Samples
(Spiked method blanks
or QC check smples)
Check purity of reagents.
Assess method
performance and estimate
the recovery of target
analytes.
Assess method
performance (initial method
validation and ongoing
assessment); check
calibration.
Prior to use of a new batch of reagent
and whenever method blank exceeds
action limits.
In every sample analyzed for organics,
unless isotope dilution technique is
used:
Semivolatiles: 3 for neutral fraction
+2 for acid fraction
Volatile* 3
Pesticides/PCBs: 1
Method validation: as many as
required to establish confidence in
method before routine analysis of
samples (i.e., when using a method for
the first time or after any method
modification).
Concentration of any target analyte
k MDL or PQL.
Determined by program manager
according to EPA CLP guidelines".
Determined by program manager.
Discard and use new batch of
reagent, or purify.
Determine cause of problem
(e.g., incomplete extraction or
digestion, contamination, inaccu-
rate preparation of surrogates),
take appropriate corrective
action, and reanalyze all suspect
samples or flag all suspect data.
(continued)
-------
TABLE 6-8. (continued)
Sample type*
Objective
Suggested frequency
of analysis"
Example
control limits0
Corrective action
Laboratory Control
Samples (continued)
Reference Materials'
Assess method performance
(initial method validation and
ongoing assessment).
O5
CO
01
Laboratory Replicates9 Assess method precision.
Routine assessment and calibration
check one per 20 samples or one per
batch, whichever is more frequent.
Method validation, as many as required
to assess accuracy (and precision) of
method before routine analysis of
samples (i.e., when using a method for
the first time or after any method
modification)
Routine assessment one (preferably
blind) per 20 samples or one per batch,
whichever is more frequent.
One blind duplicate sample per 20
samples or one per batch, whichever is
more frequent.
Organics: determined by
program manager.
Metals: 80% to 120% recovery.
Organics: <95% confidence
intervals, if certified, or
determined by program
manager.
Metals: 80% to 120% accuracy.
Organics: <95% confidence
interval, if certified, or
determined by the program
manager.
Metals: 80% to 120% accuracy.
Organics: A difference of no
more than a factor of 2 among
replicates (i.e., approximately
50% coefficient of variation).
NOTE: pooling of variances in
duplicate analyses from different
sample batches is recommend-
ed for estimating the standard
deviation or coefficient of
variation of replicate analyses.
Metals: +20 RPD for duplicates.
Determine cause of problems
(e.g., inaccurate calibration,
inaccurate preparation of control
samples), take appropriate
corrective action, and reanalyze all
suspect samples or flag all
suspect data. Zero percent
recovery requires rejection of al
suspect data.
Determine cause of problem (e.g.,
inaccurate calibration,
contamination), take appropriate
corrective action, and reanalyze all
suspect samples or flag all
suspect data.
Determine cause of problem (e.g.,
composite sample not
homogeneous, instalment
instability or malfunction), take
appropriate corrective action, and
reanalyze all suspect samples or
flag all suspect data
Analytical Replicates
Assess analytical precision. Duplicate injections for all metal
analyses."
Determined by program manager.d
Determine cause of problem (e.g.,
instrument instability or
malfunction), take appropriate
corrective action, and reanalyze
sample.
(continued)
-------
TABLE 6-8. (continued)
Sample type"
Objective
Suggested frequency
of analysis6
Example
control limits0
Corrective action
Field Replicates
Assess total sample
variability (i.e., population
variability, field or sampling
variability, and analytical
method variability).
Reid Blanks
Split Samples
Assess contamination in the
field.
Assess interlaboratory
comparability.
Initial screening: OPTIONAL; if
program resources allow, a minimum
of one replicate (i.e., duplicate) for
each primary target species at 10
percent of screening sites.
Intensive monitoring: five blind
replicate samples for each target
species (and size, age or sex class, if
appropriate) at each sampling location.
One field blank per sampling location.
5-10 percent of field samples split
between States and/or Regions that
routinely share monitoring results, or
as determined by program managers.h
Determined by program manager.
Determined by program manager.
Concentration of any target analyte
2 MDL or POL
Determined by program managers.
Determined by program
manager.
Determined by program
manager.
Identify and remove sources of
field contamination. Flag all
suspect data.
Review sampling and analytical
methods. Identify sources of
noncomparability. Standardize
and validate methods to
document comparability.
RSD - Relative standard deviation (see Section 6.4.3.4.2 and Glossary).
%R - Percent recovery (see Section 6.4.3.4.1 and Glossary).
RRF - Relative response factor (see Section 6.4.3.2.2 and Glossary).
MDL - Method detection limit (see Section 6.4.3.3.1 and Glossary).
RPD - Relative percent difference (see Section 6.4.3.4.2 and Glossary).
POL - Practical quantitation limit (see Section 6.4.3.3.2 and Glossary).
* Definitions of QA/QC samples are given in the Glossary. [Note: A Glossary of Terms will be included in the next iteration of this document].
b Suggested frequencies are based primarily on recommendations in U.S. EPA, 1986b, 1987e, 1991a,b, 1989c; Puget Sound Estuary Program, 1990d,e; and Batelle, 1989.
It Is the responsibility of each program manager to determine the appropriate level of QA/QC needed to meet program objectives.
" From Puget Sound Estuary Program (1990 d,e) control (action) limits, except where otherwise noted. Individual programs may require different control limits. It is the
responsibility of each program manager to set control limits that will ensure that the measurement data meet program data quality objectives.
"From U.S. EPA, 1987e.
' From U.S. EPA, 1991 a,b.
1 As available (see Table 6-9). If available, SRMs or CRMs should be used.
9 Sometimes referred to as Analytical Replicates (e.g., in Puget Sound Estuary Program [1990d]).
h Recommended in this guidance document.
-------
These procedures should be documented thoroughly (e.g., as part of the analytical
SOPs or as separate SOPs) and approved by appropriate supervisory personnel prior to
initiation of sample analyses. A more detailed discussion of recommended QA/QC
procedures and the use of appropriate QA/QC samples is provided in Sections 6.4.3.2 through
6.4.3.6. Recommended procedures for documenting and reporting analytical and QA/QC data
are given in Section 6.4.3.7. Because of their importance in assessing data quality and
interlaboratory comparability, reference materials are discussed separately in Section 6.4.3.1.
6.4.3.1 Reference Materials-
The appropriate use of reference materials is an important part of good QA/QC
practices for analytical chemistry. The following definitions of reference materials, taken from
the Puget Sound Estuary Program (1990d), are used in this guidance document:
A reference material is any material or substance of which one or more properties
have been sufficiently well established to allow its use for instrument calibration,
method evaluation, or characterization of other materials.
A certified reference material (CRM) is a reference material of which the value(s)
of one or more properties have been certified by a technically valid procedure,
accompanied by or traceable to a certificate or other documentation that is issued
by the certifying organization (e.g., U.S. EPA; National Institute of Standards and
Technology [MIST]; National Research Council of Canada [NRCC]).
A standard reference material (SRM) is a CRM issued by the NIST.
Reference materials may be used to (1) provide information on method accuracy and,
when analyzed in replicate, on precision, and (2) obtain estimates of intermethod and/or
interlaboratory comparability. An excellent discussion of the use of reference materials in
QA/QC procedures is given in Taylor (1985). The following general guidelines should be
followed to ensure proper use of reference materials (UNESCO, 1990):
When used to assess the accuracy of an analytical method, the matrix of the
reference material should be as similar as possible to that of the samples of
interest. If reference materials in matrices other than fish or shellfish tissue are
used, possible matrix effects should be addressed in the final data analysis or
interpretation.
Concentrations of reference materials should cover the range of possible
concentrations in the samples of interest. However, because there is a lack of low-
and high-concentration reference materials for most analytes in tissue matrices,
potential problems at low or high concentrations often cannot be documented.
6-37
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Reference materials should be analyzed regularly to detect and document any
changes in the analytical procedure over time. Appropriate corrective action should
be taken whenever changes are observed outside specified performance limits
(e.g., accuracy, precision, detection limit). Note: Because of the limited number of
certified marine/estuarine tissue reference materials available, the results of
analyses of these materials may be biased by an analyst's increasing ability to
recognize these materials with increased use. If possible, reference material
samples should be introduced into the sample stream as double blinds, that is, with
identity and concentration unknown to the analyst.
Results of reference material analyses are essential to assess the comparability of
data from different laboratories and/or from different methods. However, the results
of sample analyses should not be corrected based on percent recoveries of
reference materials. Final reported results should include both uncorrected sample
results and percent recoveries of reference materials.
Sources of EPA-certified analytical reference materials for priority pollutants and
selected related compounds are given in Appendix L. In addition, the following
comprehensive publications on certified standards and reference materials are recommended:
Standard and Reference Materials for Marine Science (UNESCO, 1990).
Available from
Dr. Adrianna Cantillo
National Ocean Service
National Oceanic and Atmospheric Administration
U.S. Department of Commerce
6001 Executive Blvd., Room 323
Rockville, MD 20852
This catalog lists over 900 reference materials and includes information on their
producers, sources, matrix type, analyte concentrations, proper use, availability,
and costs. Reference materials are categorized as follows: ashes, gases,
instrumental performance, oils, physical properties, rocks, sediments, sludges,
tissues, and waters.
Biological and Environmental Reference Materials for Trace Elements,
Nuclides and Organic Microcontaminants (Toro et at., 1990). Available from
Dr. R.M. Pan-
Section of Nutritional and Health-Related Environmental Studies
International Atomic Energy Agency
P.O. Box 100
A-1400 Vienna, Austria
This report contains approximately 2,700 analyte values for 117 analytes in 116
biological and 77 nonbiological environmental reference materials from more
6-38
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than 20 sources. Additional information on cost, sample size available, and
minimum amount of material recommended for analysis is also provided.
Currently available marine or estuarine tissue reference materials that may be
appropriate for use by analytical laboratories in fish and shellfish consumption advisory
programs are listed in Table 6-9.
6.4.3.2 Instrument Calibration and Calibration Checks--
Specific calibration procedures and requirements for recommended analytical methods
(i.e., GFAA, CVAA, GC/MS, GC/ECD) are included in the methods referenced in Section
6.4.2. It Is the responsibility of each program manager to ensure that proper
calibration procedures are developed and followed for each analytical procedure to
ensure the accuracy of the measurement data.
6.4.3.2.1 General Guidelines-The following general guidelines should be followed in
developing calibration procedures and requirements.
All analytical instruments and equipment should be maintained properly and calibrated
to ensure optimum operating conditions throughout a measurement program. Calibration and
maintenance procedures should be performed according to SOPs based on the
manufacturers' specifications and the requirements of specific analytical procedures.
Calibration procedures must include provisions for documenting calibration frequencies,
conditions, standards, and results to describe adequately the calibration history of each
measurement system.
An established schedule for the routine calibration and maintenance of analytical
instruments should be followed, based on manufacturers' specifications, historical data, and
specific procedural requirements. At a minimum, calibration should be performed each time
an instrument is set up for analysis, after any major disruption or failure, and after any
unacceptable calibration check.
Calibration standards of known and documented accuracy must be used to ensure the
accuracy of the analytical data. Each laboratory should have a program for verifying the
accuracy and traceability of calibration standards against the highest quality standards
available. If possible, EPA-certified standards should be used for calibration standards (see
Appendix L). A log of all calibration materials and standard solutions should be maintained.
Appropriate storage conditions (i.e., container specifications, shelf-life, temperature, humidity,
light condition) should be documented and maintained.
6-39
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TABLE 6-9. MARINE/ESTUARINE TISSUE REFERENCE MATERIALS
Identification
code
DOLT-1
DORM-1
LUTS-1
TORT-1
MA-A-1/OC
MA-A-3/OC
MA-B-3/OC
MA-M-2/OC
MA-A-1fTM
MA-A-2/TM
MA-B-3/TM
MA-B-3/RN
CRM-278
EPA-RSH
RM-50
SRM-1566
NIESNo_6L
Analyte type
Elements
Elements
Elements
Elements
Organic compounds
Organic compounds
Organic compounds
Organic compounds
Elements
Elements
Elements
Isotopes
Elements
Pesticides
Elements
Elements
Elements
Source
NRCC
NRCC
NRCC
NRCC
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
BCR
EPA
NIST
NIST
NIES
Matrix
Dogfish liver (freeze-dried)
Dogfish muscle (freeze-dried)
Non-defatted lobster hepatopancreas
Lobster hepatopancreas
Copepod homogenate
Shrimp homogenate
Fish tissue
Mussel tissue
Copepod homogenate
Fish flesh homogenate
Fish tissue
Fish tissue
Mussel tissue
Fish tissue
Albacore tuna (freeze-dried)
Oyster tissue (freeze-dried)
Mussel tissue
Sources:
NRCC - National Research Council of Canada, Institute for Environmental Chemistry, Marine
Analytical Chemistry Standards Program, Division of Chemistry, Montreal Road, Ottawa,
Ontario K1A OR9, Canada.
IAEA - International Atomic Energy Agency, Analytical Quality Control Service, Laboratory
Seibersdorf, P. O. Box 100, A-1400 Vienna, Austria.
BCR - Community Bureau of Reference, Commission of the European Communities, Directorate
General for Science, Research and Development, 200 rue de la Loi, B-1049 Brussels,
Belgium.
EPA - U.S. Environmental Protection Agency, Quality Assurance Branch, EMSL-Cincinnati,
Cincinnati, Ohio, 45268, USA. (Material now available from Supelco, Inc., Supelco Park,
Bellefonte, Pennsylvania, 16823-0048, USA.)
NIST - National Institute of Standards and Technology, Office of Standard Reference Materlais,
Gaithersburg, Maryland, 20899, USA.
NIES - National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki, 305, Japan.
6-40
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A minimum of three (and preferably five) calibration standards should be used to
construct a calibration curve covering the normal working range of the instrument or
bracketing the concentration range of the samples to be analyzed. The lowest-concentration
calibration standard should be at or near the estimated detection limit (see Section 6.4.3.3.1).
Calibration standards should be prepared in the same matrix as the prepared sample extract
or digestate. Criteria for acceptable calibration (e.g., acceptable limits for r2, slope, intercept,
response factors) should be established for each analytical procedure. If these criteria
(control limits) are exceeded, the source of the problem should be identified (e.g., inaccurate
standards, instrument instability or malfunction) and appropriate corrective action taken. No
analyses should be performed until acceptable calibration has been achieved and
documented.
After initial calibration has been achieved and prior to the routine analyses of samples,
the accuracy of the calibration should be verified by the analysis of a mid-range calibration
standard that has been prepared independently (i.e., using a different stock) from the initial
calibration standards, or by the analysis of a mid-calibration-range laboratory control sample
(i.e., a sample consisting of a known matrix spiked with compounds representative of the
target analytes). Thereafter, routine calibration checks should be performed using a mid-
range calibration check standard or laboratory control sample at a frequency that has been
documented to provide adequate assurance of maintaining instrument calibration (e.g., once
every 10 samples or every 2 hours during an analysis run, whichever is more frequent [U.S.
EPA, 1991a,b]; or once every 20 samples or once every sample batch, whichever is more
frequent [California, 1990]). A calibration check should always be performed after analyzing
the last sample in a batch.
If a calibration check does not fall within the calibration control limits specified in the
method, the source of the problem should be determined and appropriate corrective action
taken. After acceptable calibration has been achieved, all suspect analyses should be
reperformed. If reanalysis is not possible, all suspect data should be identified clearly.
All reported data should be within the calibration range. That is, data above or
below the range of calibration standards should not be reported. If a sample
concentration occurs outside the calibration range, the sample volume must be adjusted
appropriately and the sample reanalyzed, or the calibration range must be extended.
Extremely high concentrations of organic compounds may indicate that the extraction
capabilities of the method have been saturated and extraction of a smaller sample size or
6-41
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modification of the extraction procedure may be required (U.S. EPA, 1982b). If, for any
«
reason, data outside the calibration range are reported, they must be clearly qualified
(e.g., as greater than the concentration of the highest calibration standard).
All calibration and maintenance procedures and results should be documented clearly
in the laboratory records. Calibration and maintenance records should be inspected regularly
to ensure that these procedures are being performed at the required frequency and according
to established SOPs. Any deficiencies in the records or deviations from established
procedures should be documented and appropriate corrective action taken.
6.4.3.2.2 Calibration and Performance Evaluation of GC/MS Systems-Trie general
guidelines presented above pertain to external calibration procedures, which involve the
analysis of standard solutions, independent of the samples, to determine the relationship
between instrument response and the concentration of the analyte being measured. Internal
standard calibration involves the determination of relative response factors (RRFs), that is, of
instrument responses from target analytes relative to the responses from one or more internal
standards added to every sample prior to sample preparation.
Both external-standard and internal-standard calibration procedures are used for the
analysis of organic compounds by GC/MS. Ideally, the chemical and physical properties of an
internal standard should be as similar as possible to those of the target analyte. A stable
isotope-labeled analog of the target analyte is an ideal internal standard, and, if resources
permit, an isotope dilution technique is recommended for the analysis of organic compounds
for which isotope-labeled analogs are available (Puget Sound Estuary Program, 1990d; U.S.
EPA, 1987e,f; U.S. EPA, 1991a,b; U.S. EPA, 1989c,e). Acceptance criteria for the RRF of
each target analyte should be established consistent with program data quality
requirements.
When an isotope dilution technique is used for the analysis of organic target
contaminants, an instrument internal standard (e.g., 2,2'-difluorobiphenyl) must be added to
the final sample extract prior to actual analysis to determine the physical percent recoveries of
isotopically labeled internal standards added prior to extraction. Instrument internal standards
are used only for QA/QC purposes (i.e., to assess the quality of data) and not to quantify
analytes. Acceptance limits for percent recovery and recommended corrective actions are
given in EPA Method 1625c (U.S. EPA, 1987f, 1989e).
If the isotope dilution technique cannot be used (e.g., for chlorinated pesticides and
PCBs analyzed by GC/ECD), surrogate spikes must be added as internal standards to each
6-42
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sample prior to extraction. As noted above, surrogate compounds should have chemical and
physical properties similar to the target analytes. In addition, surrogates should be
compounds not expected to be present in the original samples. The percent recovery (% RJ
of each surrogate spike should be determined for all samples as follows:
% R8 = 100 (Cm/Ca)
where
% Rs = surrogate percent recovery
Cm = measured concentration of surrogate
Ca = actual concentration of surrogate added to the sample.
Acceptance criteria for the percent recovery of each surrogate compound should be
established consistent with program data quality requirements.
The following additional procedures are required to evaluate the performance of GC/MS
systems. In the discussion below, procedural details and performance criteria are those
recommended for Phase II of the National Dioxin Study (U.S. EPA, 1989c), unless otherwise
noted, it is the responsibility of each program manager to determine specific GC/MS
evaluation procedures and criteria appropriate for their data quality requirements.
Evaluation of the GC System
The GC performance should be evaluated by determination of the number of theoretical
plates of resolution and by the relative retention times of the internal standards.
Column Resolution: The number of theoretical plates of resolution, N, should be
determined at the time the calibration curve is generated (using chrysene-d10) and
monitored with each sample set. The value of N should not decrease by more than
20percent during an analysis session. The equation for N is given as follows:
N - 16 (RT/W)2
where
RT = retention time of chrysene-d10 (s)
W = peak width of chrysene-d10 (s).
Relative Retention Time: Relative retention times of the internal standards should not
deviate by more than ±3 percent from the values calculated at the time the calibration
curve was generated.
6-43
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If the column resolution or relative retention times are not within the specified performance
criteria, appropriate corrective action (e.g., adjust GC parameters, flush GC column,
replace GC column) should be taken.
Evaluation of the MS System
The performance of the mass spectrometer should be evaluated for sensitivity and
spectral quality.
Sensitivity: The signal-to-noise value must be at least 3.0 or greater for m/z 198 from an
injection of 10 ng decafluorotriphenylphosphine (DFTPP).
Spectral Quality: The intensity of ions in the spectrum of DFTPP must meet the criteria
listed below (U.S. EPA, 1987f):
m/z Criteria
51 30-60% mass 198
68 <2% mass 69
70 <2% mass 69
127 40-60% mass 198
197 <1% mass 198
199 5-9% mass 198
275 10-30% mass 198
365 >1% mass 198
441 present and 40% mass 198
443 17-23% mass 442
If the^performance criteria for MS sensitivity or spectral quality are not met, appropriate
corrective action (e.g., clean MS, retune MS) should be taken.
Evaluation of Cleanup Columns
Because the fatty content of many tissue samples may overload the cleanup columns,
these columns should be calibrated and monitored regularly to ensure that target
contaminants are consistently collected in the proper fraction. The gel permeation
columns should be monitored by visual inspection (for column discoloration, leaks, cracks,
etc.) and by measurement of flow rate, column resolution, collection cycle, and method
blanks (see Section 6.4.3.5). Silica gel columns should be evaluated by their ability to
resolve cholesterol from a selected target analyte.
6-44
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6.4.3.3 Assessment of Detection and Quantitation Limits-
EPA has previously issued guidance on recommended detection limits for trace metal
and organic compound analytical methods used in bioaccumulation monitoring programs (U.S.
EPA, 1985b). These recommended detection limits are summarized in Tables 6-6 and 6-7.
Several factors influence achievable detection and quantitation limits regardless of the specific
analytical procedure. These include amount of sample available, matrix interferences, and
stability of the instrumentation (measurement precision). The limits of detection given in
Tables 6-6 and 6-7 are representative of typically attainable values.
It Is the responsibility of each laboratory to determine appropriate detection and
quantitation limits for each analytical method for each target analyte In a fish or
shellfish tissue matrix and to ensure that these limits are sufficiently low to allow
reliable quantitation of the analyte at or below the recommended TVs (see Section 4.2).
Detection and quantitation limits must be determined prior to use of a new method for
routine analyses and after any significant changes are made to an existing method.
At present there is no clear consensus among analytical chemists on a standard
procedure for determining and reporting the limits of detection and quantitation of analytical
procedures. Furthermore, the bases for detection and quantitation limits reported in the
literature are seldom given. Reported detection limits may be based on instrument sensitivity
or determined from the analyses of method blanks or low-level matrix spikes; quantitation
limits may be determined from the analyses of method blanks or low-level matrix spikes
(Puget Sound Estuary Program, 1990d).
6.4.3.3.1 Detection Limits-Three types of detection limits have been defined by the
American Chemical Society Committee on Environmental Improvement (Keith et al., 1983):
Instrument Detection Limit (IDL): The smallest signal above background noise
that an instrument can detect reliably.
Limit of Detection (LOD): The lowest concentration that can be determined to be
statistically different from a method blank. The recommended value for the LOD is
3 times the standard deviation of the blank in replicate analyses, corresponding to a
99 percent confidence level.
Method Detection Limit (MDL): The minimum concentration of an analyte in a
given matrix that can be measured and reported with 99 percent confidence that the
concentration is greater than zero. The MDL is determined by multiplying the
appropriate (i.e., n-1 degrees of freedom) one-sided 99 percent student's t-statistic
(Vw) by tne standard deviation (S) obtained from a minimum of seven replicate
analyses of a spiked matrix sample containing the analyte of interest at a
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concentration 3 to 5 times the estimated MDL (Glaser et al., 1981; 40 CFR, Part
136, App. B, 1987):
MDL - (t0.J (S).
It is important to emphasize that all sample processing steps of the analytical
method (e.g., digestion, extraction, cleanup) must be included in the determination
of the MDL.
Each of these estimates has its practical limitations. The IDL does not account for
possible blank contaminants or matrix interferences. The LOD accounts for blank
contaminants but not for matrix effects or interferences. In some instances, the relatively high
value of the MDL may be too stringent and result in the rejection of valid data; however, it is
the only detection limit estimate that accounts for matrix effects and interferences and
provides a high level of statistical confidence in sample results. Therefore, it Is
recommended that the MDL be used to define the limits of detection for the analytical
methods used for routine analyses of all target contaminants. An EPA-recommended
procedure for determining and reporting the MDL (U.S. EPA, 1982a) is given in
Appendix M.
The MDL, expressed as the concentration of target contaminant fish tissue, should be
calculated from the measured MDL of the target analyte in the sample extract or digestate
according to the following equation:
MDL»88ue (PPm or PPb) [MDL,xtr«c. (PPm or PPb) * V]/W
where
V = final extract or digestate volume, after dilution or concentration (mL)
W = weight of sample digested or extracted (g).
This equation clearly indicates that the MDL in tissue may be improved (lowered) by
increasing the sample weight (W) and/or decreasing the final extract or digestate volume (V).
Experienced analysts may use their best professional judgment to adjust the
measured MDL to a lower "typically achievable" detection limit (U.S. EPA, 1985b; Puget
Sound Estuary Program, 1990e) or to derive other estimates of detection limits. For
example, EPA recommends the use of lower limits of detection (LLDs) for methods used to
analyze organic pollutants in bioaccumulation monitoring programs (U.S. EPA, 1986a).
Estimation of the LLD for a given analyte involves determining the noise level in the retention
window for the quantitation mass of the analyte for at least three field samples in the sample
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set being analyzed. The LLD is then estimated as the concentration corresponding to the
signal required to exceed the average noise level observed by at least a factor of 2. Based
on the best professional judgment of the analyst, this LLD is applied to samples in the set with
comparable or lower interference; samples with significantly higher interferences (i.e., by at
least a factor of 2) are assigned correspondingly higher LLDs. LLDs are greater than IDLs,
but usually less than the more rigorously defined MDLs. Thus, data quantified between the
LLD and the MDL have a lower statistical confidence associated with them than data
quantified above the MDL. However, these data are considered valid and useful in assessing
low-level environmental contamination.
Similarly, in EPA 1600 series methods (e.g., U.S. EPA 1987f, 1989e), EPA
recommends the use of a minimum level of detection, which is defined as the minimum
concentration of the analyte of interest at which the entire GC/MS system must give a
recognizable (background corrected) mass spectrum and acceptable calibration points. Thus,
a minimum level of detection is the concentration of a target contaminant in a sample that is
equivalent to the concentration of the lowest acceptable calibration standard.
If estimates of detection limits other than the MDL are developed and used to
qualify reported data, they should be clearly defined In the analytical SOPs and In all
data reports, and their relationship to the MDL should be clearly described.
6.4.3.3.2 Quantitation Limits-ln addition to the method detection limits (e.g., MDL or
LLD), a method limit of quantitation (MLQ), or minimum concentration allowed to be reported
at a specified level of confidence without qualifications, should be derived for each analyte.
Ideally, MLQs should account for matrix effects and interferences. The MLQ can be greater
than or equal to the MDL (or LLD). No consistent guidance for determining MLQs has been
found in the recent literature; therefore, it is not possible to provide specific recommendations
for determining these limits at this time.
[Reviewers' comments or recommendations are requested regarding definition of
MLQs and procedures for calculating them.]
The American Chemical Society Committee on Environmental Improvement (Keith et
al., 1983) has defined one type of quantitation limit:
Limit of Quantitation (LOQ): The concentration above which quantitative results
may be obtained with a specified degree of confidence. The recommended value
for the LOQ is 10 times the standard deviation of a method blank in replicate
analyses, corresponding to an uncertainty of +30 percent in the measured value
(10a ± 3a) at the 99 percent confidence level.
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However, the LOQ does not account for matrix effects or interferences.
The U.S. EPA (1986b) has defined another type of quantitation limit:
Practical Quantitation Limit (PQL): The lowest concentration that can be reliably
reported within specified limits of precision and accuracy under routine laboratory
operating conditions.
The Puget Sound Estuary Program (1990d) and the National Dioxin Study (U.S. EPA,
1989c) use a PQL based on the lowest concentration of the initial calibration curve, the
amount of sample typically analyzed, and the final extract volume of that method. However,
the PQL is also applicable only to samples without substantial matrix effects or interferences.
Analysts must use their expertise and professional judgment to determine the
best estimate of the MLQ for each target analyte. MLQs, including the estimated degree
of confidence in analyte concentrations above the quantitation limit, should be clearly
defined in the analytical SOPs and in all data reports.
6.4.3.3.3 Use of Detection and Quantitation Limits-Method detection and quantitation
limits should be used to qualify reported data as follows:
No detected concentrations should be reported below the MLD.
Concentrations between the MLD and the MLQ should be reported with the
qualification that they are below the quantitation limit.
Concentrations above the MLQ may be reported and used without qualification.
6.4.3.4 Assessment of Analytical Accuracy and Precision-
The accuracy and precision of each analytical method should be assessed and
documented for each target analyte of Interest prior to the performance of routine
analyses and on a regular basis during routine analyses.
6.4.3.4.1 Accuracy-Analytical accuracy may be assessed through analyses of
appropriate reference materials (e.g., SRMs or CRMs) (see Section 6.4.3.1), laboratory control
samples, matrix spikes, and/or surrogate spikes.
Accuracy is calculated from the results of the analyses of reference materials or
laboratory control samples as follows:
% Accuracy - [(M - T)fT\ x 100
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where
M = measured value of the concentration of analyte i
T = "true" value of the concentration of analyte i.
Accuracy is calculated as percent recovery from the analyses of spiked samples as
follows:
% Recovery = [(M8 - MU)/TJ x 100
where
Ms = measured concentration of analyte i in the spiked sample
Mu = measured concentration of analyte i in the unspiked sample
T8 = "true" concentration of analyte i in the spiked sample.
When sample concentrations are less than the MDL, the value of zero should be used
as the concentration of the unspiked sample (Mu) in calculating spike recoveries (California,
1990).
The concentrations of target analytes in reference materials should fall within the range
of concentrations found in the field samples; however, this is often not possible because of the
limited number of certified marine/estuarine tissue reference samples available (see Table
6-9).
Matrix spike samples should be prepared using spike concentrations approximately
equal to the concentration found in the unspiked sample. An acceptable range of spike
concentrations is 0.5 to 5 times the sample concentrations (U.S. EPA, 1987e).
Method accuracy should be assessed initially by analyses of appropriate reference
materials, preferably SRMs or CRMs, in a tissue matrix. The actual number of reference
samples required to be analyzed for the initial assessment of method accuracy should be
determined by each laboratory for each analytical procedure.
Laboratory control samples and matrix spikes or surrogate spikes should be used for
ongoing assessment of accuracy during the routine analyses. It is recommended that, at a
minimum, one laboratory control sample and one matrix spike sample be analyzed with
every 20 samples or with each sample batch, whichever Is more frequent (Puget Sound
Estuary Program, 1990d,e). Ideally, CRMs or SRMs should also be analyzed at this
recommended frequency; however, limited availability and cost of these materials often
make this impractical. For organic compounds not analyzed by isotope dilution
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techniques (i.e., PCBs and pesticides), surrogate spikes should be added to each
«
sample to assess accuracy.
Spikes should be added to the sample homogenates prior to digestion or extraction
and dilution steps to provide an assessment of total method (i.e., sample preparation and
analysis) accuracy. Percent recovery values for spiked samples must fall within control limits
specified in the Work/QA Project Plan and in individual analytical SOPs. If the percent
recovery falls outside the acceptable recovery range, the analyses should be discontinued,
appropriate corrective action taken, and, if possible, the samples associated with the spike
reanalyzed. If reanalysis is not possible, all suspect data should be clearly identified.
Poor performance on the analysis of reference materials or poor spike recovery may be
caused by inadequate mixing of the sample before aliquotting, inconsistent contamination,
inconsistent digestion or extraction procedures, matrix interferences, or instrument problems.
If replicate analyses are acceptable (see Section 6.4.3.3.5), matrix interferences or loss of
target analytes during sample preparation are indicated.
To check for loss of target analytes during sample preparation, a step-by-step
examination of the procedure using spiked blanks should be conducted. For example, to
check for loss of metal target analytes during digestion, a postdigestion spike should be
prepared and analyzed and the results compared with those from a predigestion spike. If the
results are different, the digestion technique should be modified to obtain acceptable
recoveries. If there is no difference in the results of pre- and postdigestion spikes, the sample
should be diluted by at least a factor of 5 and reanalyzed. If spike recovery is still poor, then
the method of standard addition or use of a matrix modifier is indicated (U.S. EPA, 1987e).
6.4.3.4.2 Precision-Precision is defined as the agreement among a set of replicate
measurements without assumption of knowledge of the true value. Method precision (i.e.,
variability due to sample preparation and analysis) is estimated by means of the analyses of
duplicate or replicate aliquots of samples containing concentrations of analyte above the MDL
The most commonly used estimates of precision are the relative standard deviation (RSD) or
the coefficient of variation (CV),
RSD - CV = 100 S/x,
where
x, = arithmetic mean of the x, measurements
S = standard deviation of the x, measurements
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and the relative percent difference (RPD) when only two samples are available,
«
RPD = 100[(x1-x2)/{(x1 + x2)/2}].
Method precision may be assessed prior to routine sample analyses by the analysis of
replicate samples of reference materials, preferably in tissue matrices, and/or laboratory
control samples. Ongoing assessment of method precision during routine analysis should be
performed by the analysis of duplicate (or replicate) aliquots of samples (laboratory duplicates)
and matrix spike duplicates (or replicates).
For ongoing assessment of method precision, It is recommended that, at a
minimum, one laboratory duplicate and one matrix spike duplicate be analyzed with
every 20 samples or with each sample batch, whichever Is more frequent. In addition, it
is recommended that a laboratory control sample be analyzed at the above frequency to
allow an ongoing assessment of method performance, including an estimate of method
precision over time. Specific procedures for estimating method precision by laboratory
and/or matrix spike duplicates and laboratory control samples are given in ASTM (1983). This
reference also includes procedures for estimating method precision from spike recoveries and
for testing for significant change in method precision.
Precision estimates obtained from the analyses of laboratory duplicates, matrix spike
duplicates, and repeated laboratory control sample analyses must fall within control limits
specified in the Work/QA Project Plan and in individual analytical SOPs. If these values fall
outside the control limits, the analyses must be discontinued, appropriate corrective action
taken, and, if possible, the samples associated with the duplicates reanalyzed. If reanalysis is
not possible, all suspect data should be clearly identified.
Unacceptable precision estimates derived from the analysis of duplicate or replicate
samples may be caused by inadequate mixing of the sample before aliquotting; inconsistent
contamination; inconsistent digestion, extraction, or cleanup procedures; or instrumentation
problems (U.S. EPA, 1987e).
An alternative approach to assessing laboratory performance using laboratory
duplicates, based on testing the null hypothesis that the mean difference in the concentrations
of a target contaminant in a number of laboratory duplicates is zero, is given in Section
7.2.1.1.
The analysis of replicate aliquots of final sample extract or digestate solutions
(analytical replicates) provides only an estimate of analytical precision; it does not provide an
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estimate of total method precision. For organic target analytes, such analyses may be
<
conducted at the discretion of the program manager or laboratory supervisor. For the
analysis of target metal analytes by GFAA and CVAA, It is recommended that duplicate
injections of each sample be analyzed and the mean concentration be reported. The
RPD should be within established control limits or the sample should be reanalyzed
(U.S. EPA, 1987e).
Estimates of the variability of pollutant concentrations in the sample population and of
the sampling and analysis procedures can be obtained by the collection and analysis of
replicate field samples. Replicate field samples are optional in initial screening studies;
however, if resources permit, it is recommended that duplicate samples be collected at 10
percent of the screening sites (see Section 2.1.8). In intensive monitoring studies, five
replicate samples should be collected at each sampling location for target contaminant
analyses (see Section 2.2.8).
6.4.3.5 Routine Monitoring of Interferences and Contamination-
Because contamination can be a limiting factor in the reliable quantitation of target
contaminants in tissue samples, the recommendations for proper materials and handling and
cleaning procedures given in Sections 5.2.2. and 6.2 should be followed carefully to avoid
serious contamination of samples in the field and laboratory. In addition, the following blank
samples should be analyzed prior to beginning the sample collection and analyses program
and on a routine basis during each monitoring study (U.S. EPA, 1987e):
Field blanks - Rinsates of empty field sample containers (i.e., aluminum foil
packets and plastic bags) that are prepared, shipped, and stored as actual field
samples should be analyzed to evaluate field sample packaging materials as
sources of contamination. Each rinsate should be collected and the volume
recorded. The rinsate should be analyzed for target contaminants of interest and
the total amount of target contaminant in the rinsate recorded. It is recommended
that one field blank be analyzed with every 20 samples or with each batch of
samples, whichever is more frequent.
Processing blanks - Rinsates of utensils and equipment used for dissecting and
homogenizing fish and shellfish should be analyzed, using the procedure described
above for field blanks, to evaluate the efficacy of the cleaning procedures used
between samples. It is recommended that processing blanks be analyzed at
least once at the beginning of a monitoring study and preferably once with
each batch of 20 or fewer samples.
Bottle blanks - Rinsates of empty bottles used to store and ship sample
homogenates should be analyzed, using the procedure described above for field
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blanks, to evaluate these sample containers as sources of contamination. It Is
recommended, at a minimum, that one bottle blank be analyzed for each lot
of sample bottles used and preferably once with each batch of 20 or fewer
samples.
Method blanks - Blank samples, consisting of an analyte-free matrix to which all
reagents are added in the same proportion as used in sample preparation, should
be analyzed to evaluate contaminants resulting from the total analytical method
(e.g., contaminated glassware, reagents, solvents, column packing materials,
processing equipment). Note that the method blank is carried through the
complete analytical method. It is recommended that one method blank be
analyzed with every 20 samples or with each batch of samples, whichever is
more frequent.
In addition to the routine analysis of the blank samples described above, it is also
recommended that each lot of analytical reagents be analyzed for target contaminants of
interest prior to use to prevent a potentially serious source of contamination. For organic
analyses, each lot of alumina, silica gel, sodium sulfate, or Florasil used in extract drying and
cleanup should also be analyzed for target analyte contamination and cleaned as necessary.
Surrogate mixtures used in the analysis of organic target analytes have also been found to
contain contaminants and interfering impurities and should be verified prior to use (U.S. EPA,
1987e).
In the analysis of organic contaminants by GC/MS or GC/ECD, cross-contamination
should be avoided during all steps of analysis. Injection micro-syringes must be cleaned
thoroughly between uses. If separate syringes are used for the injection of solutions, possible
differences in syringe volumes should be assessed and, if present, corrected for. Particular
care should be taken to avoid carryover when high- and low-level samples are analyzed
sequentially. Analysis of an appropriate method blank following the analysis of a high-level
sample may be required to assess carryover (U.S. EPA, 1987e).
Ideally, there should be no detectable concentration of any target analyte in any blank
(i.e., the concentration of target analytes in all blanks should be less than the MDL).
However, program managers may set higher acceptance limits (e.g., £10-30 percent of
sample concentration [U.S. EPA, 1987e]), depending on overall data quality requirements of
the monitoring program. If the concentration of any blank is greater than the established
acceptance limit, appropriate corrective action should be taken and, if there is sufficient
sample material, all samples associated with the blank should be reanalyzed. If reanalysis is
not possible, all suspect data should be identified clearly. Data should not be corrected for
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blank contamination by the reporting laboratory. The blank concentrations should
always be reported with each associated sample value.
If the concentration of a target analyte in a blank is greater than the MDL, all steps in
the relevant sample handling, processing, and analysis procedures should be reviewed. Many.
trace metal contamination problems are due to airborne dust. High zinc blanks may result
from airborne dust or galvanized iron, while high chromium and nickel blanks often indicate
contamination from stainless steel. In the field, the use of mercury thermometers should be
avoided, because broken thermometers can be a source of serious mercury contamination. In
the laboratory, samples to be analyzed for mercury should be isolated from materials and
equipment (e.g., polarograms) that are potential sources of mercury contamination. In organic
analyses, phthalates, methylene chloride, and toluene are common laboratory contaminants
that are often detected in blanks at concentrations above the MDL. Chromatographic
interference by natural substances in the tissue (e.g., fatty acids) may require additional
cleanup procedures (U.S. EPA, 1987e).
6.4.3.6 Regular External QA Assessment of Analytical Performance--
Participation in an external QA program by all analytical laboratories in State fish and
shellfish consumption advisory programs is recommended for several reasons:
To enhance the comparability of data between States and Regions.
To identify potential analytical problems prior to conducting routine analyses and to
provide technical assistance to correct these problems.
To provide an independent ongoing assessment of each laboratory's capability to
perform the required analyses.
Two types of external QA programs are recommended to establish most reliably the
comparability of data reported from different State and Regional Laboratories: round-robin
interlaboratory comparisons and spilt sample interlaboratory comparisons.
6.4.3.6.1 Round-Robin Analysis Interlaboratory Comparison Program-At present, the
only external round-robin QA program available for analytical laboratories conducting
fish/shellfish tissue analyses for environmental pollutants is the QA program administered by
NOAA in conjunction with its National Status and Trends Program. This QA program has
been designed to ensure proper documentation of sampling and analysis procedures and to
reduce intra- and interlaboratory variations among participating laboratories (Cantillo, 1991).
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Each laboratory participating in the National Status and Trends QA program is required
«
to conduct yearly analyses of one set each of three organic and three inorganic (i.e., trace
metals) environmental and standard reference samples. The organic analytical
intercomparison program is coordinated by NIST, and the inorganic analytical intercomparison
program is coordinated by the NRCC. The sample types and matrices vary yearly. Sample
types include freeze-dried sediments, extracted freeze-dried tissues, and frozen tissues.
Sample matrices include mussel, oyster, and fish tissue, and sediments from pristine and
contaminated areas. Individual laboratory performance is evaluated against the consensus
values (i.e., grand means) of the results reported by all participating laboratories. A second
set of samples is provided to a laboratory only after the first set is analyzed successfully. NIST
and NRCC also provide technical assistance to participating laboratories that may have
problems with the intercomparison analyses. Results of the QA analyses are reviewed by
NIST, NRCC, and participants at an annual National Status and Trends QA meeting.
Analytical methods are not specified by NOAA; participants in the QA program may use
any analytical method, provided its QA results are within established limits of the consensus
values. However, all analytical and sampling protocols used must be documented thoroughly
for future reference. Participants in the National Status and Trends QA program are also
required to analyze reference materials such as the NIST SRMs and NRCC CRMs (see Table
6-9) as part of routine sample analysis. Results of the routine analysis of reference or control
materials must be reported to NOAA. These results and the results of the QA samples are
stored electronically in the National Status and Trends database.
Participation in the National Status and Trends QA program is strongly
recommended to enhance the credibility and comparability of analytical data among
different fish/shellfish monitoring programs. However, because of NOAA's budget
constraints, there are only a limited number of openings in the National Status and Trends QA
program at present. To address this problem, NOAA is considering expanding the program
and charging each participating laboratory a fee to cover administrative costs; however, no
estimates of the cost per laboratory are available at this time.
To apply for participation in the National Status and Trends QA program or for
additional information, contact Dr. Adriana Cantillo, QA Manager, NOAA/National Status and
Trends Program, N/OMA3, Rockville, MD 20852, Tel: 301-443-8655.
6.4.3.6.2 Split Sample Analysis Interlaboratory Comparison Programs-Another useful
external QA procedure for assessing interlaboratory comparability of analytical data is a split
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sample analysis program in which a percentage (usually 5 to 10 percent) of all field samples
analyzed by each State or Region are divided and distributed for analyses among laboratories
from other States or Regions. Because actual field samples are used in a split-sample
analysis program, the results of the split-sample analyses provide a more direct assessment
of the comparability of the reported monitoring results from different States or Regions.
The NOAA National Status and Trends QA program described above does not include
an interlaboratory split-sample analysis program. At a minimum, it is recommended that
split-sample analyses be conducted regularly by States and/or Regions that routinely
share monitoring results.
6.4.3.7 Documentation and Reporting of Data-
The results of all chemical analyses (i.e., percent lipid and all target contaminant
analyses) must be documented adequately and reported properly to ensure the proper
evaluation and interpretation of the data.
Because all analytical data from State fish consumption advisory programs will be
stored eventually in the Ocean Data Evaluation System (ODES) database for nationwide use,
it will be essential that laboratory documentation procedures be consistent with ODES data
reporting requirements.
The documentation of analytical data for each method should include, at a minimum,
the following information (U.S. EPA, 1984a):
Description of the procedure used, including documentation and justification of any
deviations from the standard procedure
Method accuracy and precision for each target analyte
Method detection limit and limit of quantitation for each target analyte
Discussion of any analytical problems and corrective action taken
Sample identification numbers
Sample weights
Final dilution volumes
Date(s) of analysis
Identification of analyst
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Identification of instrument used (manufacturer, model number, serial number,
location)
Summary calibration data, including identification of calibration materials, dates of
calibration and calibration checks, and calibration range(s); for GC/MS analyses,
include DFTPP and bromofluorobenzene (BFB) spectra and quantitation report
Reconstructed ion chromatograms for each sample analyzed by GC/MS
Mass spectra of detected target compounds for each sample analyzed by GC/MS
Chromatograms for each sample analyzed by GC/ECD and/or gas
chromatography/flame ionization detection (GC/FID)
Raw data quantitation reports for each sample
Description of all QA/QC samples associated with each sample (e.g., field blanks,
rinsate blanks, method blanks, duplicate or replicate samples, spiked samples,
laboratory control samples) and results of all QA/QC analyses. QA/QC reports
should include quantitation of all target analytes in each blank, recovery
assessments for all spiked samples, and replicate sample summaries. Laboratories
should report all surrogate spike recovery data for each sample; the range of
recoveries should be included in any reports using these data.
Analyte concentrations with reporting units identified (as jag/g wet weight to two
significant figures unless otherwise justified). NOTE: Reported data should not
be blank-corrected.
Percent lipid associated with each sample. NOTE: Reported data should not be
normalized for lipid concentration.
Specification of all tentatively identified compounds (if requested) and any
quantitation data.
Data qualifications (including qualification codes and their definitions, if applicable,
and a summary of data limitations).
To ensure completeness and consistency, standard forms should be developed and
used by each laboratory for recording and reporting data from each analytical method.
Standard data forms used in the EPA Contract Laboratory Program (U.S. EPA, 1991a,b) are
included in Appendix N as examples of the types of forms that analytical laboratories should
use.
All analytical data should be reviewed thoroughly by the analytical laboratory supervisor
and, ideally, by a qualified chemist who is independent of the laboratory. In some cases, the
analytical laboratory supervisor may conduct the full data review, with a more limited QA
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review provided by an independent chemist. The purpose of the data review is to evaluate
the data relative to the data quality specifications (e.g., detection and quantitation limits,
precision, and accuracy) and other performance criteria established in the Work/QA Project
Plan. In many instances, qualifiers may be necessary for reported data values; these
qualifiers should always be defined clearly and included in the database.
Summaries of analytical data should be prepared for each target species at a specific
sampling location and should include sample size (i.e., number of individuals in each
composite sample), measured concentration of each target analyte (for intensive monitoring
studies and initial screening studies where replicate QA samples are collected, the arithmetic
mean and range of measured concentrations of each target analyte), and a measure of
variance (standard error or 95 percent confidence limits). Specific data reporting requirements
for the initial screening and followup intensive monitoring phases of these programs are given
in Sections 2.1.9, 2.2.9, and 7.
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SECTION 7
DATA REPORTING, ANALYSIS AND EVALUATION
This section provides guidance on data reporting, data analysis procedures for both
initial screening studies and intensive followup studies (Phase I and II) of fish/shellfish
contaminant programs, and procedures for evaluating residue data for the issuance of fish or
shellfish consumption advisories. A discussion of four types of consumption advisories and
bans currently issued by States is provided in Section 7.2.4.
All data reporting, analysis, and evaluation procedures should be documented fully as
part of the Work/QA Project Plan for each study, prior to initiating the study. All routine data
reporting, analysis, and evaluation procedures should be described in Standard Operating
Procedures (SOPs). In particular, the procedures to be used to determine if the concentration
of a target contaminant differs significantly from the recommended TV, and the specific
decision rules to be used by the State to determine if a consumption advisory should be
issued must be clearly documented. EPA has provided guidelines for evaluating fish/shellfish
contaminant monitoring data (U.S. EPA, 1989d). Additional recommended data evaluation
procedures are included in Sections 4.2 and 7.2 of this guidance document.
7.1 DATA REPORTING
7.1.1 Initial Screening
Data reports should be prepared for each target species sampled at each screening
site. These reports should include, at a minimum, the following information:
Site location (e.g., waterbody name, river mile, latitude/longitude, reach number or
State waterbody identification number)
Scientific name and common name of national target species
Sampling dates (including rationale for sampling outside of the recommended
sampling period [late summer to fall])
Number of QA/QC replicates (optional; a minimum of one field replicate at 10
percent of the sites is recommended if resources permit)
Number of individual organisms used in the composite sample (and in the QA/QC
replicate, if applicable)
7-1
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Characteristics of each individual used in the composite sample (and in the QA/QC
replicate, if applicable) (e.g., age, sex, total body length or size, total weight,
percent lipid)
For each target contaminant:
- Measured concentration (ppm) in the composite sample
- Measured concentration (ppm) in the QA/QC replicate, if applicable
- Evaluation of laboratory performance (i.e., description of all QA/QC samples
associated with the sample(s) and results of all QA/QC analyses)
- Comparison of measured concentration with EPA-recommended TV and clear
indication of whether TV was exceeded.
In the initial screening study, if a reported contaminant concentration exceeds the TV,
a State should initiate an intensive followup study (Phase I, see Section 5.1.2.1) to verify the
contamination in species of economic, sport fishing, or subsistence value. If a reported
contaminant concentration from the initial screening study is close to the TV but does not
exceed the TV (e.g., a reported value of 1.98 as compared to a TV of 2.00), the criteria used
to determine if additional Phase I intensive monitoring is warranted should be documented
clearly by the State. In this case, a State should reexamine historic data on water, sediment,
and fish tissue contamination at the site as well as evaluate data on laboratory performance.
If these data indicate that further examination of the site is warranted, the State should initiate
a Phase I intensive study to verify the magnitude of the contamination. In Phase I studies, the
State may wish to assess the tissue residue concentrations in additional target species or
additional age classes of the target species for the contaminant of concern.
States are reminded that several aspects of the EPA-recommended screening study
design presented in this guidance document are conservative in nature and are intended to
protect the public health because they are based on worst-case exposure assumptions.
These include
Use of target species known to bioaccumulate environmental contaminants
Use of fish fillets with skin-on and belly flap included
Use of the oldest individuals in the target species to represent longest exposure
times
Late summer/fall sampling to maximize concentration of bioaccumulants in target
species
7-2
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Targeting suspected hot spots for sampling
Use of the 70-yr exposure rate to calculate TVs for carcinogens.
There are, however, several aspects of the screening study design that are of concern
either because they are not based on worst-case exposure assumptions or because they
preclude valid statistical analyses of the data. These include the
Use of fillet tissue samples rather than whole fish which may underestimate
contaminant exposures in subpopulations that consume the whole fish.
Use of composite samples which results in loss of information on the range and
variance of the underlying population of individual samples. Such information is
critical in bioaccumulation monitoring programs as an early warning sign of
potentially harmful levels of contamination (U.S . EPA, 1989d).
Use of a single sample per site for each target species which precludes estimating
the variability of the contamination level at that site and, consequently, of
conducting valid statistical comparisons to the target contaminant TVs.
Use of trigger values calculated for the general U.S. population and not for local
populations or subpopulations of recreational or subsistence fishermen.
Use of a risk level of 10"4 for calculating TVs for carcinogens (i.e., as cancer
incidence of 1 in 10,000 individuals). Some States are currently using more
conservative risk levels of 10~5 (9 States) and 10~6 (8 States) for calculating TVs
for carcinogens (Cunningham et al., 1990).
States should consider the potential effects of these design features on screening
study results and should make modifications as appropriate to achieve the specific objectives
of their contaminant monitoring programs.
7.1.2 Intensive Monitoring
For each intensive monitoring study (Phase I and Phase II, see Section 5.1.2.1), data
reports should be prepared for each target species (by size or age class, if appropriate) at
each sampling site within the waterbody under investigation. Note that in Phase II intensive
studies, each sampling location is considered to be a separate site. These reports should
include, at a minimum, the following information:
Site location (e.g., waterbody name, river mile, latitude/longitude, reach number or
State waterbody identification number)
Scientific name and common name of regional target species
Sampling dates (including rationale for sampling periods chosen for target species)
7-3
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Sampling design (e.g., two-stage sampling, systematic grid sampling)
Number of replicates (five minimum)
Number of individual organisms used in each composite sample (6 to 10 fish; 10 to
50 shellfish)
Characteristics of individuals used in each composite sample (e.g., age, sex, total
length or body size, total weight, percent lipid) and description of fish fillet or edible
parts of shellfish used
For each target contaminant in Phase I or Phase II of the intensive study:
- Measured contaminant concentrations (ppm) in individual replicate composite
samples
- Mean (arithmetic) contaminant concentration for each set of replicate composite
samples
- Range of the contaminant concentrations for each set of replicate composite
samples
- Standard deviation of the contaminant concentrations
- Comparison of the mean contaminant concentration with the appropriate TV,
and clear indication of whether the TV was exceeded.
If the reported mean contaminant concentration is near the TV, the criteria used to
determine if the TV was in fact exceeded should be documented clearly. If the study design
includes a Phase II intensive study with the specific objectives of making multilocational
comparisons to determine the geographic extent of the contamination or performing trend
analyses, these results should be presented along with any appropriate statistical results (e.g.,
analysis of variance [ANOVA], nonparametric multiple comparisons, or trend tests).
NOTE: EPA is currently in the process of modifying the Ocean Data Evaluation
System (ODES) database so that it can be used as a national repository for fish and shellfish
contaminant monitoring data for both inland and coastal waters. Additional information on data
reporting requirements for the ODES database will be available in subsequent drafts of this
guidance document.
7-4
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7.2 DATA ANALYSES AND EVALUATION
7.2.1 Initial Screening
The primary objective of the initial screening study is to assist States in identifying
potential hot spots where further investigation of fish/shellfish contamination may be
warranted. If the State deems that the measured concentration of a target contaminant in fish
or shellfish obtained during the initial screening study warrants further investigation, then the
State should initiate a Phase I intensive study at that site. The purpose of the Phase I
intensive study is to confirm the findings of the initial screening and to assess the magnitude
of the contamination in selected species and age classes of fish and shellfish of commercial,
sportfishing, or subsistence importance.
Because duplicate or replicate field composite samples are not required as part of the
initial screening study, estimating the variability of the composite contaminant concentration at
any site is precluded. States may use duplicate laboratory samples to evaluate a laboratory's
performance (see Section 6.4.3).
7.2.1.1 Laboratory Replicates--
States are required to process laboratory duplicate samples as part of the QA/QC
protocol for the initial screening study. Consequently, States can conduct quality assurance
investigations of their own laboratory or a contract laboratory by pooling duplicate results from
multiple sites. Consider, for example, the following concentrations of toxaphene found in
duplicate laboratory analyses of field composite samples from n=8 sites:
Concentration of Toxaphene (ppm)
Site i
1
2
3
4
5
6
7
8
Duplicate #1
1.00
0.91
0.79
1.17
0.85
0.75
0.63
1.02
Duplicate #2
0.91
1.12
0.93
1.19
0.67
0.82
0.53
0.73
Difference (d,)
0.09
-0.21
-0.14
-0.02
0.18
-0.07
0.10
0.29
7-5
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Note that the last column contains the difference (dj) between the duplicate laboratory
concentrations of toxaphene from screening site i. The average difference (d) in the
concentrations of toxaphene over the eight sites is
6
c
M
= 0.03 ppm
and the standard deviation (s) is
,.-c02/(8-1) =0.17 ppm
If the participating laboratory (either the State laboratory or a contract laboratory) is
performing adequately, the mean difference in toxaphene concentrations is expected to be
zero. Data collected at the eight sampled sites are used to test the null hypothesis that the
mean difference in the concentration of toxaphene in laboratory duplicates is zero
versus the two-sided alternative hypothesis that the mean difference is not zero. First, the
State should calculate the statistic t* as
r =
= (0.03)/(0.17/
= 0.50 .
Under the assumption of the null hypothesis, t* has a Student's t-distribution with
7 (=8-1) degrees of freedom. Appendix 0 contains a table of the percentage points for
various Student's t-distributions. If the State sets the Type I error rate (i.e., the probability of
rejecting the null hypothesis when, in fact, it is true) at 0.05 (a=0.05) and considers a
two-sided alternative hypothesis, then the null hypothesis is rejected if
r f(7,0.975) = 2.45 .
In this example, the State would not reject the null hypothesis that the mean difference in
toxaphene concentrations found in duplicate laboratory samples is zero. The State could
interpret this result as suggesting, with some caution, that the laboratory's analytical
performance is acceptable. The cautionary note is deemed necessary because of the small
7-6
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sample size (i.e., n=8)--there may not be adequate power to detect a difference when a true
difference does exist.
Under a different scenario, the State may reject the null hypothesis in favor of the
alternative that the mean difference in toxaphene concentrations found in duplicate laboratory
samples is not zero. This conclusion makes the laboratory results for toxaphene
concentration suspect. In such cases, EPA advises the States to review all field and
laboratory procedures for a potential explanation of this finding.
[Reviewers, please provide additional methods for using laboratory replicates
data to assess laboratory performance.]
7.2.1.2 Field Replicates-
Field sample replication is optional for the initial screening study. If resources permit
and States collect a minimum of five replicate composite samples at a suspected hot spot,
then States may pursue the statistical analysts described in the subsequent section for
Phase I intensive monitoring studies.
[Reviewers, please provide additional methods for using limited field replicate
data to assess total error.]
7.2.2 Intensive Study-Phase I
In the intensive study (Phase I), EPA recommends that the States analyze at least five
replicate composite samples for each target species and/or size class of target species at
each sampling site. Replicate samples must be as similar to each other as possible. EPA
recommends that replicate samples of fish/shellfish be defined as follows:
All replicate composite samples contain specimens of only a single species.
Each fish composite sample contains a minimum of 6 fish, with 10 individuals
being the optimal number and with each replicate composite containing equal
numbers of individuals; each shellfish composite sample contains 10 to 50
individuals, with each replicate composite containing an equal number of
individuals
The smallest individual in any replicate is no less than 75 percent of the total size
of the largest individual in the composite sample.
The relative difference between the overall mean length or size of the replicate
samples and the mean length or size of any individual replicate sample is no
greater than 10 percent.
The specimens in all replicates are collected at the same sampling site and within
24 hours of each other (U.S. EPA, 1990b).
7-7
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States should analyze at least five replicate samples of each species of commercial,
sportfishing, or subsistence value in the study area to determine whether fish fillets or edible
parts of shellfish (as well as whole fish or shellfish in specific cases) contain tissue residues
above the TV for any target contaminant identified in the initial screening study.
The following case studies include appropriate equations and resulting calculations of
contaminant tissue residues.
Case Study I
EPA recommends that States collect a minimum of five replicate composite field
samples for each secondary target species at each sampling location during the Phase I
intensive study. Suppose a State finds the following toxaphene concentrations in 10 replicate
composite samples collected at one suspect site during a Phase I intensive study:
Composite Concentration of
sample (i) toxaphene (ppm)(x))
1 0.89
2 1.03
3 1.08
4 0.89
5 0.84
6 0.92
7 0.85
8 0.89
9 0.84
10 1.08
Let j index the composite sample and Xj describe the concentration of toxaphene found in the
composite sample. Then the mean toxaphene concentration over the 10 samples (x) is
10
x = ]Tx//10 = 0.93 ppm
and the standard deviation (s) is
7-8
-------
10
1) = 0.10
The State is interested in comparing the average toxaphene concentration at the
suspect site to the TV for toxaphene. The TV for toxaphene is 0.98 ppm (see Table 4-6).
This TV was calculated using the following equation for carcinogens (see Section 4.2.1.1).
7VC = [(RLIqV) xBW\ICR
where
TVC = trigger value for a carcinogen (m/kg; ppm)
RL = maximum acceptable risk level (10'4)
q1* = carcinogenic potency factor for toxaphene (1.1 mg/kg/d)'1 from IRIS
BW = mean body weight, estimated for the general population (70 kg)
CR = mean daily fish/shellfish consumption rate averaged over a 70-year lifetime for
the general population (0.0065 kg/d).
Specifically, the State will test the null hypothesis that the mean toxaphene
concentration at the suspect site is greater than the TV for 70-kg adults in the general
population versus the one-sided alternative hypothesis that the mean site-specific toxaphene
concentration is less than the TV for these individuals. To accomplish this, the State first
calculates the statistic t* as
r = (x -
= (0.93 - 0.98)/(0.10//10)
= - 1.58 .
Under the assumption of the null hypothesis, t* has a Student's t-distribution with
9 (=10-1) degrees of freedom. Appendix O contains a table of the percentage points for
various Student's t-distributions. If the State selects 0.05 (ot=0.05) as the Type I error rate,
then the null hypothesis is rejected if
r
-------
TV for the general population (since t* = -1.58 is not less than t(9,0.05) - -1,83). The TV for
the general population is within sampling variability of the site-specific mean tissue
concentration. Thus, the State might consider issuing a no-consumption advisory for the
general population (see Section 7.2.4) because the toxaphene TV based on 0.0065 kg/d
consumption by a 70-kg adult has been exceeded. Furthermore, the State should proceed to
a Phase II intensive study to determine the geographic extent of the contamination.
Case Study II
States may be confronted with another scenario. Suppose the results of an initial
screening study suggest that a State should initiate a Phase I intensive study of toxaphene at
a suspect location. During the Phase I study, the State collects nine composite samples at the
site:
Composite Concentration of
sample (i) toxaphene (ppmUx,)
1 0.58
2 0.83
3 0.85
4 0.71
5 0.90
6 0.57
7 0.79
8 0.96
9 1.01
where the mean toxaphene concentration over the nine samples (x) is
9
x = XI9 = °-80
and the standard deviation (s) is
^T ~
1) = 0.16 ppm .
As in Case Study I, the State will test the null hypothesis that the mean toxaphene
concentration at the suspect site is greater than the TV for adults in the general
population versus the one-sided alternative that the mean site-specific toxaphene
7-10
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concentration is less than the TV for adults in the general population. The statistic t* is
calculated from the data collected by the State:
r = (x - 0.98)/(s/v/7?)
= (0.80 - 0.98)/(0.16/\/9)
= -3.38 .
Under the assumption of the null hypothesis, t* has a Student's t-distribution with 8 (=9-1)
degrees of freedom and a Type I error rate of 0.05 (oc=0.05). Then the null hypothesis is
rejected if
r =
-------
The State's objective for Phase II monitoring is to determine the extent of the
geographical area over which the consumption advisory should extend. The location of each
sampling station must be determined by State personnel familiar with the specific hydrologic
aspects of the waterbody and with the location of additional anthropogenic sources of
contamination to the waterbody under investigation. State staff should consult a qualified
statistician both in designing Phase II intensive monitoring studies and in interpreting tissue
residue data collected at multiple sites throughout a given waterbody.
For some small lakes, States may opt to issue fish consumption advisories after
analyzing results of Phase I monitoring rather than conducting further resource-intensive
multilocational Phase II monitoring. For large lakes or reservoirs and for riverine or estuarine
areas, however, where the economic impact of a commercial or recreational closure may be
devastating to the local economy, the State may want to conduct extensive multilocational and
multispecies sampling of the targeted hot spot to determine the geographic extent of the
advisory and the specific species and age classes affected. The complexity of the monitoring
design depends on the complexity of the hydrologic processes operating in the affected
waterbody (e.g., estuaries with strong tidal influence or coastal sites influenced by long shore
currents).
The following case study illustrates the methodology proposed by EPA for analyzing
Phase II intensive study data.
Case Study III
Suppose a State determines in a Phase I study that the mean tissue concentration of
toxaphene in a target species exceeds the TV for toxaphene (0.98 ppm) at a riverine site. In
this case, EPA recommends that the State proceed to a Phase II intensive study to determine
the geographic extent of the contamination at the site.
The State selects four additional riverine sites downstream from the Phase I study site
and then collects five replicate composite samples at each of the five sites. For the following
fish tissue residue data, Site 1 is the Phase I (and initial screening) site and Sites 2 through 5
are located progressively farther downstream.
7-12
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Concentration of Toxaphene (ppm) in Replicate Samples
Sitef
1.12
1.40
1.33
1.35
1.29
Site 2
1.27
1.32
1.35
1.17
1.21
Site 3
1.30
1.22
1.13
1.29
1.44
Site 4
1.13
1.28
1.45
1.31
1.29
Site5
0.60
0.50
0.55
0.57
0.62
To analyze these data, EPA recommends that States use a single-factor ANOVA
(Neter and Wasserman, 1974). The appropriate ANOVA table for investigating the null
hypothesis that the mean toxaphene concentration is the same across all sites versus
the alternative hypothesis that at least one site-specific mean toxaphene concentration is
different is shown in Table 7-1.
In Table 7-1, j indexes the sites, i indexes the composite samples, r is the number of
sites, n is the total number of composite samples, PJ is the number of composite samples at
site j, Xjj is the concentration of toxaphene in the rth sample at the jth site, xt is the mean
toxaphene concentration over all sites and samples, and x; is the mean toxaphene
concentration at site j.
In Case Study III, r=5; n=25; nj=5 for j=1,2,3,4 and 5; x§ =1.14 ppm; x .,=1.30 ppm;
x>2=1.29 ppm; x>3=1.28 ppm; x4=1.26 ppm; and x_5=0.57 ppm. The ANOVA table for the
Case Study III example is shown in Table 7-2.
TABLE 7-1. ANOVA TABLE FOR SINGLE-FACTOR STUDY
Source of Sum of squares Degrees of freedom
variation (SS) (df)
Mean square
(MS)
Between sites SSTR - Ln/xj - x J
r- 1
MSTR=
SSTR
r-\
Error (within
sites)
n -r
MSE=
SSE
n-r
Total
SSTO-LL(x,j-xJ
n-1
7-13
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TABLE 7-2. ANOVA TABLE FOR THE PHASE II TOXAPHENE STUDY
Source of
variation
Between sites
Error
Total
SS
2.045
0.181
2.226
df
4
20
24
MS
0.511
0.009
To test the null hypothesis described above, the State will first calculate the statistic F*
as the ratio of the mean square for treatments (MSTR) to the mean square error (MSE).
F' = MSTRIMSE = 0.511/0.009 = 56.6 .
Under the assumption of the null hypothesis, F* has an F-distribution with (r-1 ,n-r) degrees of
freedom. Appendix O contains a table of the percentage points for various F-distributions. If
the Type I error rate is selected to be 0.05 (oc=0.05), then the null hypothesis is rejected if
F' >F(4,20,0.95) = 2.87 .
In this case study, the State would reject the null hypothesis that the mean toxaphene
concentrations across the five sites are equal and conclude that the mean toxaphene
concentration at least one site is different from the other sites. If the State were unable to
reject the null hypothesis, the State would conclude that either the null hypothesis was true or
that there were not enough data (e.g., replicates) to detect the differences to be tested. At
this point, the State might consider revising their Phase II sampling protocol to include
additional replicates at each site. In the future, the EPA would like all State fish and shellfish
contaminant monitoring data to be entered into the national database, ODES, which contains
a statistical power analysis tool for designing contaminant monitoring programs (U.S. EPA,
1987a). Specifically, States may use ODES to determine how best to use their limited
monitoring resources (i.e., when to increase the sample size collected in order to achieve
adequate power for a statistical test.) States should review the discussion of power analysis
provided in U.S. EPA (1989) for the ODES database.
Rejection of the null hypothesis allows the State to pursue pairwise comparisons to
determine where the difference in the mean concentration of toxaphene exists (i.e., the
geographic extent of the contamination). EPA recommends that States employ Scheffe's
7-14
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method of multiple comparisons to examine mean concentration differences between sites
(Kleinbaum and Kupper, 1978). Scheffe's method, which involves constructing and comparing
confidence intervals for all the comparisons, accommodates unequal numbers of samples at
each site.
In the illustration, the State may be concerned with the following comparisons: Site 1
vs. Site 5, Site 1 vs. Site 4, Site 1 vs. Site 3, Site 1 vs. Site 2, Site 2 vs. Site 5, Site 2 vs. Site
4, Site 2 vs. Site 3, Site 3 vs. Site 5, Site 3 vs. Site 4, and Site 4 vs. Site 5. For investigating
the difference in the mean toxaphene concentrations between Sites 1 and 5, the form of the
Scheffe-type confidence interval for this pairwise comparison is
(x5 -x,) ± Sx[MSEx(Vns
where
x 5 = 0.57
x-, = 1.30
S = [(M)xF(r-1,n-r,1-a)]^
= (4 x 2.87)^
= 3.39
MSE = 0.009 (from Table 7-2)
n5 «= n-, = 5
Thus, the confidence interval for comparing Sites 1 and 5 is
(0.57 - 1.30) ± 3.39 x [0.009 x (^ + -1)]*
w O
= -0.73 ± 3.39 x 0.06
= -0.73 ± 0.20
= (-0.93, -0.53) .
This interval does not contain the value zero, which supports rejection of the null hypothesis
that the mean toxaphene concentrations at Sites 1 and 5 are equal.
Repeating the above calculation for the comparison between Sites 1 and 4 yields the
confidence interval
7-15
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(-0.19,0.21)
which does contain the value zero. Thus, the State cannot reject the null hypothesis that the
mean toxaphene concentrations at Sites 1 and 4 are equal. After investigating for all other
site differences similarly, the State would conclude (1) that the mean toxaphene concentration
at Sites 1, 2, 3, and 4 are similar and (2) that the mean toxaphene concentration at Site 5 is
different (lower) than the mean toxaphene concentration at the other sites. After confirming
that the mean toxaphene concentration at Site 5 is less than the toxaphene TV (using the
methodology discussed in Section 7.2.2), the State should consider issuing a no-consumption
advisory for the general population for Site 1 extending downstream to Site 4 but excluding
Site 5 (see Section 7.2.4).
7.2.4 Issuance of Fish/Shellfish Consumption Advisories
After analyzing Phase I and/or Phase II contaminant residue results, a State may find
that there is justification for issuing a fish/shellfish consumption advisory. States should
review the discussion of estimating TVs for intensive monitoring studies in Section 4.2.3.
There are four specific types of advisories that States have issued:
No consumption advisory that advises against consumption of fish or shellfish
species by the general population (NCGP)
No consumption advisory that advises against consumption of fish or shellfish
species by a subpopulation that could be at greater risk (e.g., pregnant women,
nursing mothers or children) (NCsp)
Restricted consumption advisory that advises restricted consumption (e.g., limited
number of meals and/or size of meals per unit time) of fish or shellfish species by
the general population (RGP)
Restricted consumption advisory for a subpopulation that advises restricted
consumption (e.g., a limited number of meals or size of meals per unit time) of fish
or shellfish species by a subpopulation that could be at greater risk (e.g., pregnant
women, nursing mothers, and children) (Rsp)
Guidance on issuing each type of advisory is given in Table 7-3.
The four advisory categories were identified from a review of consumption advisories
and bans listed by the 50 States, the District of Columbia, and the Virgin Islands and Puerto
Rico in their 1990 305(b) reports and were used to develop a database-Current State Fish
and Shellfish Consumption Advisories and Bans (RTI, 1991). EPA's Office of Science and
7-16
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TABLE 7-3. RECOMMENDED GUIDELINES FOR ISSUING VARIOUS TYPES
OF FISH/SHELLFISH ADVISORIES"
Type of advisory
Conditions under which to Issue advisory
NCGP The contaminant TV is calculated using a 0.0065 kg/d consumption
rate for a 70-kg adult and the null hypothesis cannot be rejected--
States would conclude that the mean contaminant concentration is
greater than the contaminant TV.
NCspb The contaminant TV is calculated using a consumption rate >0.0065
kg/d for ethnic or subsistence subpopulations or for individuals <70-
kg body weight (e.g., women and children) and the null hypothesis
cannot be rejected-States would conclude that the mean
contaminant concentration is greater than the contaminant TV
calculated for the potentially more sensitive subpopulation.
RGP The contaminant TV is calculated using a 0.0065 kg/d consumption
rate for a 70-kg adult and the null hypothesis is rejected-States
would conclude that the mean contaminant concentration is less
than the TV; however, it is approaching a level of concern based on
the best professional judgment of the project team.
Rsp" The contaminant TV is calculated using a consumption rate >0.0065
kg/d for ethnic or subsistence subpopulations or for individuals <70-
kg body weight (e.g., women and children) and the null hypothesis is
rejected-States would conclude that the mean contaminant
concentration is less than the TV; however, it is approaching a level
of concern based on the best professional judgment of the project
team.
a Based on EPA recommended risk assessment procedures discussed in Section 4.2 of this
guidance document, assuming a 10"4 risk factor for carcinogens. States may employ
lower (i.e., 10"6 or 10~6) but not higher risk factors.
b Subpopulations may include ethnic or subsistence populations that consume more than
0.0065 kg/d of fish or shellfish or individuals with <70 kg body weight such as pregnant
women, nursing mothers, and children who may be at potentially greater risk particularly
for contaminants that are developmental (fetal) toxicants.
Technology is in the process of developing an electronic bulletin board that will contain this
advisory database and will be available to the States in October 1991. For each State
advisory listing, the pollutant that triggered the advisory, the type of advisory, the species of
fish or shellfish (and for some States, size class of fish affected) for which the advisory was
issued, and the waterbody name and extent of the contamination are presented. A sample of
the information contained in the consumption advisory database is shown in Figure 7-1. The
name, address, and telephone number of a contact person in each State who can discuss the
basis of the consumption advisories will also be added to the database.
7-17
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PACE NO. 21
06/29/91
STATE POLLUTANT
VA
VA
VA
VA
W
VI
A
W
ay
wV
w
v
wV
ay
«
NI
WI
NI
NI
NI
NI
NI
M
NI
NI
NI
NI
NI
NI
M
PCBs
Oioxtna
Dioxlna a
Dioxlna a
Dloxlne
Dioxlna
Dioxlna
Dloxlna
Oioxlna
Dloxlna
PCBs, chlordana
KBs
KBs, pasticld**
PCBs, pasticidas
KBs, pasticidas
KBs, pasticldas
KBs, pasticidas
KBs, pasticldas
KBs, pasticldas
KBa, pasticldas
KBs, pasticldas
KBs. pasticldas
KBs, pasticldsa
KBs, pasticldas
KBs, pasticidas
KBs, pasticldas
KBs, pasticidas
KBs, pasticldas
N*URE OF
ADVISORY
NCCP
NCCP
NCCP
NCCP
NCCP
Nona
Nona
NOGP
NCCP
NCCP
NCCP
NCCP
NCCP
NOGP
NCsp, RCP
NOGP
NCsp, RCP
NOGP
NCsp, RCP
NCCP
NCsp, RCP
NOGP
NCsp, RCP
NOGP
NCsp, RGP
NOGP
NCsp, RCP
NOGP
NCsp, RCP
NCCP
OflRBVT STATE FISH AK> St&LFtSH CONSUmON ADVISORIES AND
BANS
FISH (commn nan*) WATOTCDY HUE
All flah apaciaa
Bottom fasdlng apsclas
Alt flshspaclos
Bottom fasdlng spaclas
All flah apaciaa
Bottom faadara
All flah apselaa
All flah apaciaa
Bottom fisilars
Bottom faadara
Charmal catfish, carp
Carp, suckars. chamal cat* Ian
Lako trout 20 to 23*. cohe salmon > 20*,
chinook salmon 21 to 32*. broan trout to 23*
Lako trout > 23*, chinook aalmon >32*,
broan trout > 23*. carp, catfish
Splaka up to 10*
Rainbow trout > 22*, chinook salmon > 26*
brown trout > 12*. brook trout > IS*, carp.
splaka > 10*, northarn piha > 2ft*,
aallaya > Vf , whlta bsas
Northarn ptka, whit* suckar, aallayo 16-18*
Wilt* baa>, walloya > 18*, carp, drum.
ehannal catfish
Nalloya > 16*, bullhsad
Carp > 17*
Laka trout 2O to 23*. coho aalaen > 28*
Chinook salmon 21 to 32*, broan trout to 23*
Carp, catfish, laka trout > 23*, chinook
alnon > 32*. brown trout > 23*
Laka trout 20 to 23*. coho aalmon > 28*.
chinook salimn > 21*, brown trout to 23*
Catfiah, laka trout > 23*.
chinook salmon > 32*, brown trout > 23*.
Rslnbow trout, brook trout.
coho aalaon > 28*. chinook salaon 21-32*
Bluagill, crappla, reck baas, carp.
amallmouth bsaa, walloya, northarn plka,
brown trout, catfish, chinook salami > 32*
Lak* trout 20 to 23*. coho aslant > 24*,
chinook salmon 21-32*. brown trout to 23*
Laka trout > 23*, chinook a* law > 32*
brown trout > 23*. carp, cmtf lah, crsppla.
northarn pika, radhoraa, smallsoth bsaa.
hit* auckar
N. F. Shanandoah Rivar
Blackwatar Rivar
Jackson Rivar
Nottcmay Rivar
Jamas Rivar
Kanawha Rivar
Pocatalico Rivar
Armour Craak
North Br. Potomac
Potomac Rivar
Ohio Rivar
Shanandoah Rivar
Laka Michigan
Laka Michigan
Groan Bsy
Graan Bay
Lowar Fox Rivar
Lowar Fox Rivar
Luwsr Fox Rivar
Lu.ai Fox Rivar
East and Wast Twin Rivara
East and Watt Twin Rivara
Manltowoc Rivar
Msnltowoc Rivar
Shaboygan Rivar
Shaboygan Rivar
Milwaukaa Rivar
Mllwauko* Rivar
CHXRArWIC BOBff
Passaga Cr to confl. with Shsnandoah R
Union Camp plant to Nottoway R (6 mi)
From dam abova Dunlsp Cr to Jamas Rivar
Can. Vaughan Bridga (U.S. 268) to NC faordar
Conf lu*no* with J*ckson Rivar downstroam
to Snowdan Dam
(48 mi)
(2.0 ml)
(2.O ml)
(60.6 mi)
(39 mi)
(277 mi)
(19.46 mi)
Uanominaa, Ooonto, A Paahtigo Rivars
Manomin**, Ooonto, m Poshtigo Rivars
From mouth at Gr**n Bsy up to DaPara Dam
From mouth at Graan Bsy up to Da?ara Dam
From mouth* upstraam to first dam
From mouth* upstraam to first dam
Mouth upstraam to first dam
Mouth upstraam to first dam
Fr Shaboygan Fal Is/Gnaandal* * Naadans Cr
Fr Shaboygan Fal Is/Graandala Naadans Cr
From mouth to North Avanua Dan
From mouth to North Avanua Dam
Figure 7-1. Sample output from the database-Current State Fish and Shellfish
Consumption Advisories and Bans (RTI, 1991).
-------
States will be able to access information from this electronic bulletin board on
advisories issued in adjacent States with which they may share waters and may use this
information to direct their own contaminant monitoring program. For example, the issuance of
an advisory by one State on the upper reaches of a particular river can provide information to
an adjacent State on contaminants that might be anticipated in the same fish species at
monitoring sites downstream. This is especially important in cases where the geographic
extent of the fish consumption advisory for a State ends at the State border because the
State's monitoring activities and jurisdiction end at the State line. However, the actual
geographic extent of the contamination may continue into the adjacent State's waters for
many miles downstream. In addition, information on the bulletin board will allow States to
review differences in fish consumption advisories in shared (interstate) waters, where, for
example, one State may issue a consumption advisory for one side of a river, while an
adjacent State may have no consumption advisory for its jurisdictional waters on the other
side of the river. Such inconsistencies in consumption advisories in interstate waters
undermine public confidence in State regulations designed to protect public health.
7-19
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SECTION 8
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Availability. Federal Register, Vol. 45, No. 231, Part V, pp. 79318-79379. U.S.
Environmental Protection Agency, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1980b. TSCA Chemical Assessment
Series. Assessment of Testing Needs: Chlorinated Benzene Support Document for
Proposed Health Effects. Test Rule Toxic Substances Control Act. EPA-
560/11-80-014, Section 4. Office of Pesticides and Toxic Substances. Washington,
DC.
U.S. EPA (U.S. Environmental Protection Agency). 1980c. Interim Guidelines and
Specifications for Preparing Quality Assurance Project Plans. QAMS-005/80. Quality
Assurance Management Staff, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1981. Interim Methods for the Sampling
and Analysis of Priority Pollutants in Sediments and Fish Tissue. EPA- 600/4-81-005.
Washington, DC.
8-10
-------
U.S. EPA (U.S. Environmental Protection Agency). 1982a. Methods for*Organic Chemical
Analysis of Municipal and Industrial Wastewater. James E. Longbottom and James J.
Lichtenberg (eds.). EPA-600/4-82-057. Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
U.S. EPA (U.S. Environmental Protection Agency). 1982b. Test Methods for the Chemical
Analysis of Municipal and Industrial Wastewater. EPA-600/4-82-057.
U.S. EPA (U.S. Environmental Protection Agency). 1982c. Toxaphene: Decision Document.
Office of Pesticide Programs, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1984a (revised January 1985). Contract
Laboratory Program Statement of Work for Organics Analysis, Multi-Media, Multi-
Concentration. IFB WA 85-T176, T177, T178. Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1984b. Policy and Program Requirements
to Implement the Quality Assurance Program, EPA Order 5360.1. Quality Assurance
Management Staff, Washington, DC. April 3.
U.S. EPA (U.S. Environmental Protection Agency). 1985a. Bioaccumulation Monitoring
Guidance: 1. Estimating the Potential for Bioaccumulation of Priority Pollutants and
301 (h) Pesticides Discharged to Marine and Estuarine Waters. EPA-503/3-90-001.
U.S. EPA (U.S. Environmental Protection Agency). 1985b. Bioaccumulation Monitoring
Guidance: 3. Recommended Analytical Detection Limits. EPA-503/6-90-001.
U.S. EPA (U.S. Environmental Protection Agency). 1986a. Bioaccumulation Monitoring
Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants and 301 (h)
Pesticides in Tissues from Marine and Estuarine Organisms. EPA-503/6-90-002.
U.S. EPA (U.S. Environmental Protection Agency). 1986b. Test Methods for the Evaluation
of Solid Waste, Physical/Chemical Methods. SW-846; 3rd Edition.
U.S. EPA (U.S. Environmental Protection Agency). 1986c. Work/Quality Assurance Project
Plan for the Bioaccumulation Study. Office of Water Regulations and Standards,
Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1987a. Bioaccumulation Monitoring
Guidance: 2. Selection of Target Species and Review of Available Data.
EPA-430/9-86-005. Office of Marine and Estuarine Protection, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1987b. Bioaccumulation Monitoring
Guidance: 2. Selection of Target Species and Review of Available Data-Appendix.
EPA-430/9-86-006.
U.S. EPA (U.S. Environmental Protection Agency). 1987c. Bioaccumulation Monitoring
Guidance: 5. Strategies for Sample Replication and Compositing. EPA-430/9-87-003.
Office of Marine and Estuarine Protection, Washington, DC.
8-11
-------
U.S. EPA (U.S. Environmental Protection Agency). 1987d. National Dioxin Study.
EPA-440/4-87-003.
U.S. EPA (U.S. Environmental Protection Agency). 1987e. Quality Assurance/Quality Control
(QA/QC) for 301 (h) Monitoring Programs: Guidance on Field and Laboratory Methods.
EPA-430/9-86-004.
U.S. EPA (U.S. Environmental Protection Agency). 1987f. Methods 1624 and 1625 Revision
C. Volatile and semivolatile organic compounds by isotope dilution GC/MS. Federal
Register, Part 136. Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1987g. Technical Support Document for
ODES Statistical Power Analysis. EPA-430/09-87-005. Office of Water, Washington,
DC.
U.S. EPA (U.S. Environmental Protection Agency). 1989a. Analytical Procedures and Quality
Assurance Plan for the Determination of Mercury in Fish. EPA-600/3-90- .
U.S. EPA (U.S. Environmental Protection Agency). 1989b. Analytical Procedures and Quality
Assurance Plan for the Determination of PCDD/PCDF in Fish. EPA-600/3-90-002.
U.S. EPA (U.S. Environmental Protection Agency). 1989c. Analytical Procedures and Quality
Assurance Plan for the Determination of Xenobiotic Chemical Contaminants in Fish.
EPA-600/3-90-023.
U.S. EPA (U.S. Environmental Protection Agency). 1989d. Assessing Human Health Risks
from Chemically Contaminated Fish and Shellfish: A Guidance Manual.
EPA-503/8-89-002. Office of Water Regulations and Standards, Office of Marine and
Estuarine Protection, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1989e. Method 1624: Volatile organic
compounds by isotope dilution GC/MS. Method 1625: Semivolatile organic compounds
by isotope dilution GC/MS. Office of Water Regulations and Standards, Industrial
Technology Division, Washington, DC. 75 pp.
U.S. EPA. 1990a. Exposure Factors Handbook. EPA-600/8-89/043. Office of Health and
Environmental Assessment, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1990b. Work Plan for FY 91 Regional
Ambient Fish Tissue Monitoring Program Activity No. ELR 80. Region VII,
Environmental Monitoring and Compliance Branch, Kansas City, KS.
U.S. EPA (U.S. Environmental Protection Agency). 1990c. Tetrachlorodibenzo-p-dioxins and
-dibenzofurans in edible fish tissue at selected sites in Arkansas and Texas. Region 6.
Water Quality Management Branch and Surveillance Branch, Dallas, TX.
8-12
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U.S. EPA (U.S. Environmental Protection Agency). 1991 a. Contract Laboratory Program
Statement of Work for Inorganic Analysis, Multi-Media, Multi-Concentration. SOW 788,
July. Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1991b. Contract Laboratory Program
Statement of Work for Organic Analysis, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1991c. National Bioaccumulation Study.
Draft Report. Office of Water Regulations and Standards, Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). 1991d. Origin of Human Health Criteria,
April 1991. Office of Criteria and Standards, Washington, DC.
U.S. FDA (U.S. Food and Drug Administration). 1984. Shellfish Sanitation Interpretation:
Action Levels for Chemical and Poisonous Substances, June 21, 1984. Shellfish
Sanitation Branch, Washington, DC.
U.S. FDA (U.S. Food and Drug Administration). 1990. Pesticide Analytical Manual, Volumes I
and II. Report No. FDA/OMO-90/15A. U.S. Department of Health and Human
Services, Washington, DC.
Versar, Inc. 1982. Sampling Protocols for Collecting Surface Water, Bed Sediment, Bivalves
and Fish for Priority Pollutant Analysis-Final Draft Report. EPA Contract 68-01-6195.
Prepared for U.S. EPA Office of Water Regulations and Standards. Versar, Inc.,
Springfield, VA.
Versar, Inc. 1984. Sampling Guidance Manual for the National Dioxin Study-Final Draft
Report. EPA Contract 68-01-6160. Prepared for U.S. EPA Office of Water Regulations
and Standards. Versar, Inc., Springfield, VA.
Ware, G. W. 1978. The Pesticide Book. W. H. Freeman and Company, San Francisco, CA.
Weber, C. I. (ed.) 1973. Biological Field and Laboratory Methods for Measuring the Quality
of Surface Waters and Effluents. Office of Research and Development, U.S.
Environmental Protection Agency, Cincinnati, OH. 670/4-73-001.
Williams, C. D., D. M. Nelson, M. E. Monaco, S. L Stone, C. lancu, L. Coston-Clements, L. R.
Settle, and E. A. Irlandi. 1990. Distribution and Abundance of Fishes and
Invertebrates in Eastern Gulf of Mexico Estuaries. ELMR Report No. 6. Strategic
Assessment Branch, National Oceanic and Atmospheric Administration, Rockville, MD.
Williams, S. (ed.). 1984. Official Methods of Analysis of the Association of Official Analytical
Chemists. Fourteenth edition. The AOAC, Inc., Arlington, VA.
Wisconsin Department of Natural Resources. 1988. Fish Contaminant Monitoring
Program-Field and Laboratory Guidelines (1005.1). Madison, Wl.
8-13
-------
Wolf, R. J., and R. J. Walker. 1987. Economies in Alaska: Productivity, geography, and
development impacts. Arctic Anthropology 24:56-81.
Wood, J. M. 1974. Biological cycles for toxic elements in the environment. Science
183:1049-1052.
Wood, J. M., F. Kennedy, and C. G. Rosen. 1968. Synthesis of methyl mercury compounds
by extracts of a methanogenic bacterium. Nature 220:173-174.
Worthing, C. R. (ed.). 1991. The Pesticide Manual. The British Crop Protection Council,
Surrey, England.
8-14
-------
APPENDIX G
FORMS
-------
Sample Request Form
Project
Objective
Sample
Type
D Screening Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than
whole
Target
Contaminants
Whole fish or portions other
than fillet (Specify tissues used
if other than whole
D All target contaminants
D Additional contaminants
(Specify
D Intensive Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than whole
__ _ _ _ __ __ __ ^
D Whole fish or portions other than fillet (Specify
tissues used if other than whole
D Contaminants exceeding screening study TVs
(Specify
J
INSTRUCTIONS TO SAMPLE COLLECTION TEAM
Project Number:.
County/Parish:
Target Species:
D Freshwater
D Estuarine
Site (Name/Number):
LatAong.:
Alternate Species: (in order of preference)
Proposed Sampling Dates
Proposed Sampling Method:
D Electrofishing
D Seining
D Trawling
D Other (Specify.
D Mechanical grab or tongs
D Biological dredge
D Hand collection
Number of Sample Replicates: D No field replicates (1 composite sample only)
Number of Individuals
per Composite:
field replicates
(Specify number for each target species)
.Fish per composite (10 fish optimum)
.Shellfish per composite (specify number to obtain 500 grams of tissue)
-------
Field Record for Fish Contaminant Monitoring Program Screening Study
Project Number.
Sampling Date and Time:.
SITE LOCATION
Site Name/Number
County/Parish:
LatAong.:.
State Waterbody Segment Number.
WaterbodyType: D RIVER
Site Description:
D LAKE
ESTUARY
Collection Method:
Collector Name: _
(print and sign)
Agency:
Address:
Phone: (.
FISH COLLECTED
Bottom FeederSpecies Name: _
Composite Sample #:
Fish # Length (cm) Sex
Number of Individuals:
Fish # Length (cm)
Sex
001
002
003
004
005
006
007
008
009
010
Minimum size
x100 =
Maximum size
Notes (e.g., morphological anomalies):
Composite mean length.
cm
PredatorSpecies Name:
Composite Sample #:
Fish # Length (cm) Sex
Number of Individuals:
Fish # Length (cm)
Sex
001
002
003
004
005
006
007
008
009
010
Minimum size
x100 =
> 75%
Maximum size
Notes (e.g., morphological anomalies):
Composite mean length.
.cm
-------
Field Record for Shellfish Contaminant Monitoring Program Screening Study
Project Number:
Sampling Date and Time:
SITE LOCATION
Site Name/Number
County/Parish:
State Waterbody Segment Numb
WaterbodyType: D RIVER
Site Description:
en
D
LatAong.:
LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Aaency:
Address:
Phone: ( )
SHELLFISH COLLECTED 1 1
Bivalve Species Name:
Composite Sample #:
Bivalve # Size (cm)
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Minimum size
x 1 00 =
Maximum size
Notes (e.g., morphological anoma
Bivalve #
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
£ 75%
lies):
Number of Individuals:
Size (cm) Bivalve # Size (cm)
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
Composite mean size cm
-------
Field Record for Fish Contaminant Monitoring Program Intensive Study
Project Number,
Sampling Date and Time:
SITE LOCATION
Site Name/Number
County/Parish:
LatAong.:.
State Waterbody Segment Number
Waterbody Type: D RIVER
Site Description:
D LAKE D ESTUARY
Collection Method:
Collector Name: _
(print and sign)
Agency:
Address:
Phone: (.
FISH COLLECTED
Species Name:
Replicate Number:
Composite Sample #:
Fish# Length (cm)
Number of Individuals:
Sex(M, F.orl) Fish* Length (cm) Sex(M, F, orl)
001
002
003
004
005
006
007
008
009
010
Minimum length
x100 =
Maximum length
Notes (e.g., morphological anomalies):
Composite mean length.
cm
Species Name:
Replicate Number:
Composite Sample #:
Fish# Length (cm)
Number of Individuals:
Sex (M, F, or I) Fish # Length (cm) Sex (M, F, or I)
001
002
003
004
005
006
007
008
009
010
Minimum length
x100 =
Maximum length
Notes (e.g., morphological anomalies):
page 1of2
.£75%
Composite mean length.
cm
-------
Field Record for Fish Contaminant
Project Number:
SITE LOCATION:
Monitoring Program Intensive Study (con.)
Sampling Date and Time:
Site Name/Number
County/Parish:
LatAong.:
FISH COLLECTED ! !
Species Name:
Composite Sample #:
Fish # Length (cm) Sex (M, F, or 1)
001
002
003
004
005
Minimum length K10Q_ 0/
Maximum length
Notes (e.g., morphological anomalies):
Species Name:
Composite Sample #:
Fish # Length (cm) Sex (M, F, or I)
001
002
003
004
005
Minimum length
...,_.. x 100= %
Maximum length
Notes (e.g.. morphological anomalies):
Species Name:
Composite Sample #:
Fish # Length (cm) Sex (M, F, or 0
001
002
003
004
005
Minimum length
x 1 00 = * 75%
Maximum length
Notes (e.g.. morphological anomalies):
Replicate Number:
Number of Individuals:
Fish # Length (cm) Sex (M, F, or 0
006
007
008
009
010
Composite mean length cm
Replicate Number:
Number of Individuals:
Fish # Length (cm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length cm
Replicate Number:
Number of Individuals:
Fish # Length (cm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length cm
page 2 of 2
-------
Field Record for Shellfish Contaminant Monitoring Program Intensive Study
Project Number
Samplina Date and Time:
SITE LOCATION
Site Name/Number
County/Parish:
State Waterbody Segment Number
WaterbodyType: D RIVER
Site Description:
D
LatAong.:
LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Agency:
Address:
Phone: ( )
SHELLFISH COLLECTED mmmmmmm
Species Name:
Composite Sample #:
Shellfish # Size (cm) Sex Shellfish
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Minimum size
x100= £
Maximum size
Notes (e.g., morphological anomalies)
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
75%
Replicate Number:
Number of Individuals:
# Size (cm) Sex Shellfish # Size (cm) Sex
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
Composite mean size cm
-------
Species Name or Code
Sample Type
Total Length or Size (cm) Sampling Site (name/number)
Specimen Number
Sampling Date/Time
-------
Project Number
Sampling Site (name and/or ID number)
Collecting Agency (name, address,
shone)
Sampler (name and signature)
Composite Number
Sampling Date/Time
Species Name or Code
Chemical Analyses
Q All target contaminants
[~| Others (specify)
Processing
Whole Body
Comments
Resection
Study Type
Screening
Intensive
Type of Ice
Wet
Dry
-------
Chain-of-Custody Record
Project Number
Collecting Agency (name, address, phone)
Samplers (print and sign)
Composite
Number
Sample
Nos.
Sampling
Time
Study Type
Scr Int
Sampling Date
Container
of
Sampling Site (name/number)
Chemical
Analyses
Comments
Delivery Shipment Record
Delivery Method D Hand carry
D Shipped
Deliver/Ship to: (name, address and phone)
Date/Time Shipped:
Relinquished by: (signature) Date/Time Received by: (signature)
Relinquished by:
(signature)
Date/Time
Received by: (signature)
Relinquished by: (signature)
Data/Time
Received for Central Processing
Laboratory by: (signature)
Date/Time
Remarks:
Laboratory Custody:
Released
Name/Date
Received
Name/Date
Purpose
Location
-------
Sample Processing Record for Fish Contaminant Monitoring Program Fish Fillet Composites
Project Number: Sampling Date and Time:
STUDY PHASE: Initial Screening ; Intensive Monitoring: Phase I Phase II
SITE LOCATION
Site Name/Number: _
County/Parish: _ LatAong.:
State Waterbody Segment Number: _ Waterbody Type:
Sample Type (bottom feeder, predator, etc.) _ Species Name:
Composite Sample #: Replicate Number: Number of Individuals:
Analyst
Date
Lett Fillet Right Fillet
Weight Scales/Otoltths Sex Resection Weight Homogenate Wt.ofHomog. Weight Homogenate Wt. ofHomog.
Fish* (g) Removed (S) (M,F) Performed (
-------
Sample Processing Record for Shellfish Contaminant Monitoring Program Edible Tissue Composites
Project Number:
STUDY PHASE: Initial Screening
SITE LOCATION
Site Name/Number:
Countv/Parish:
State Waterbody Segment Number:
SHELLFISH COLLECTED
Species Name:
Composite Sample #:
D;
Shellfish Included In
# Composite (/) Shellfish*
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Preparation of Composite:
Weight of container + shellfish
Weight of container
Total weight of composite
Analvst
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
«
Sampling Date and Time:
Intensive Monitoring Phase I I I Phase II I I
Lat./Lona:
Waterbodv Type:
Number of Individuals:
Included in Included In
Composite (/) Shellfish # Composite (/)
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
g
g
a +
# of specimens Average weight
of specimen
Date
-------
Sample Processing Record for Fish Contaminant Monitoring ProgramWhole Fish Composites
Project No..
Sampling Date and Time:.
STUDY PHASE: Initial Screening
SITE LOCATION
Site Name/Number:
County/Parish:
Intensive Monitoring Phase I
Phase II
State Waterbody Segment Number:.
LatAong.:
Waterbody Type:.
Bottom Feeder- Species Name:
Composite Sample #:
Number of Individuals:
Fish*
001
002
003
004
005
006
007
008
009
010
Analyst
Initials/Date
Weight, g
Scales/Otollths
Removed (/)
Sex
Homogenate
Prepared (/)
Weight of homogenate
taken for composite
Total Composite Homogenate Weight
Predator - Species Name:
Composite Sample #:
Number of Individuals:
Fish*
001
002
003
004
005
006
007
008
009
010
Analyst
Initials/Date
Weight, g
Scales/Otollths
Removed (/)
Homogenate
Sex Prepared (/)
Weight of homogenate
taken for composite
Total Composite Homogenate Weight
-------
Fish/Shellfish Monitoring Program
Sample Allquotting Record
Aliquotted by
Dale
Time
(name)
Comments
Samples from:
Project No.
Site*
D Screening study
D Intensive study
Composite Sample ID
Archive Location:
AnatyteCode
Aliquot ID
Aliquot Weight
*
Analyze for
Ship to:
AnalyteCode
Aliquot ID
Aliquot Weight
Analyze for
Ship to:
Analyte Code
Aliquot ID
Aliquot Weight
*
Analyze for
Ship to:
Page.
of
-------
Fish/Shellfish Monitoring Program
Sample Transfer Record
Date
DD MM
Released by:
YY
Time : (24-h clock)
HH MM
(name)
At:
(location)
Shipment Method.
Shipment Destination
Date
DO MM
Released by:
Time : (24-h clock)
YY HH MM
(name)
At:
(location)
Shipment Method.
Shipment Destination
Comments
Study Type: Q ScreeningAnalyze for: D Trace metals D Organics D Dioxins
D IntensiveAnalyze for (specify)
Sample IDs:
Laboratory Chain of Custody
Relinquished by
Received by
Purpose
Location
-------
APPENDIX GG
RECOMMENDED PROCEDURES FOR PREPARING
WHOLE FISH COMPOSITE SAMPLES
GG-1
-------
Laboratory processing to prepare whole fish composite samples (diagrammed in Figure
GG-1) involves
Inspecting individual fish for foreign material on the surface and rinsing if
necessary
Weighing individual fish
Removing scales or otoliths for age determination
Determining the sex of each fish (optional)
Preparing individual whole fish homogenates
Preparing a composite whole fish homogenate.
Whole fish samples should be shipped on wet ice or blue ice packets from the field to
the sample processing laboratory if next-day delivery is assured (see Section 5.3.2). Fish
samples arriving in this manner (chilled but not frozen) should be weighed, scales and/or
otoliths removed, and the sex of each fish determined within 24 hours after receipt by the
central processing laboratory. The samples should then be frozen (-20 °C) in the laboratory
prior to being homogenized. (The grinding/homogenization procedure may be carried out
more easily and efficiently if the sample has been frozen previously [Stober, 1991].)
If the fish samples arrive frozen at the sample processing laboratory, precautions
should be taken during weighing, removal of scales and/or otoliths, and sex determination to
ensure that any liquid formed in thawing remains with the sample. The liquid will contain lipid
material that should be included in the analysis scheme.
The thawed or partially thawed whole fish should then be homogenized individually,
and equal weight portions of each homogenate should be combined and mixed to form the
composite sample. Individual homogenates and/or composite homogenates may be refrozen;
however, frozen individual homogenates must be rehomogenized before compositing, and
frozen composite homogenates must be rehomogenized before aliquotting for analysis. The
maximum holding time from sample collection to analysis for mercury is 28 days at <-20 °C;
for all other analytes, the holding time is 6 months to 1 year at £-20 °C (Stober, 1991).
Recommended container materials, preservation methods, and holding times are given in
Table GG-1. Fish sample processing procedures are discussed in more detail in the sections
below. Each time the samples are transferred from one person to another during processing,
the COC form that originated in the field must be signed so that possession and location of
the samples can be traced at all times. As each procedure is performed, it should be
GG-2
-------
Log in fish samples using COC procedures
Unwrap individual fish, weigh, and record weight (g)
Remove scales and/or otoliths for age determination
Determine sex (optional)
Fish < 1,000 g
Fish >1,000 g
Partially thaw
*
Grind whole fish in a.hand crank
meat grinder (<300 g) or a food
processor (300-1000 g)
Divide ground sample into
quarters, mix opposite quarters
and then mix halves
Repeat from * two more times
Partially thaw
Chop sample into ~2.5-cm
cubes
Pass entire chopped sample
through a meat grinder
Composite equal weights (g) of
homogenized tissues from 6-10
fish of the same species and of
similar size (500-g minimum)
Optional
Save remainder of
homogenate from each
individual fish
Seal and label (500-g minimum)
homogenate in appropriate
container(s) and store at -20 °C
until analysis (See Table 6-1 for
recommended container materials
and holding times)
Seal and label individual fillet
homogenate in appropriate
container(s) and archive at
-20 °C until analysis (See
Table 6-1 for recommended
container materials and
holding times)
COC «= Chain of Custody
Figure GG-1. Laboratory sample preparation and handling for
whole fish composite samples.
GG-3
-------
TABLE GG-1. RECOMMENDATIONS FOR CONTAINER MATERIALS,
PRESERVATION, AND HOLDING TIMES FOR FISH/SHELLFISH TISSUES
FROM DELIVERY AT CENTRAL PROCESSING LABORATORY TO ANALYSIS
Analyte
Matrix
Sample
container
Storage
Preservation Holding time
Trace metals Tissue (whole
(except Hg) specimens, edible
portions, homogenates)
Hg
Organics
Tissue (whole
specimens,
edible portions,
homogenates)
Tissue (whole
specimens,
edible portions,
homogenates)
Plastic, glass Freeze at £-20 °C 1 year
Plastic, glass Freeze at £-20 °C 28 days
Glass, teflon Freeze at £-20 °C 1 year
documented directly in a bound laboratory notebook or on forms that can be taped or pasted
into the notebook. Several existing programs have developed forms similar to the sample
processing record for whole fish composite samples shown in Figure GG-2. The use of a
form is recommended to ensure consistency and completeness of the record.
Sample Weighing-A wet weight should be determined for each fish collected. If the
fish has been shipped on wet ice, it should be unwrapped, placed on a foil-lined balance tray,
and the weight recorded to the nearest gram on the sample processing record and/or in the
laboratory notebook. To avoid contamination, the foil lining should be replaced between each
weighing. Frozen fish should be weighed in tared containers if thawing is expected before the
weighing can be completed. Liquid associated with the sample when thawed must be
maintained in the container as part of the sample because it will contain lipid material that has
separated from the tissue (Stober, 1991).
Removal of Scales and/or Otoliths for Aging-It is recommended that a few scales or
otoliths be removed from each fish for age determination by a fisheries biologist. Aging
provides a good indication of the length of exposure to pollutants (Versar, 1982). For most
warmwater inland gamefish, 5 to 10 scales should be removed from below the lateral line and
behind the pectoral fin. On softrayed fish such as trout and salmon, the scale sample should
be taken just above the lateral line (Wisconsin, 1988). For catfish and other scaleless fish,
GG-4
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Sample Processing Record for Fish Contaminant Monitoring ProgramWhole Fish Composites
Project No..
Sampling Date and Time:.
STUDY PHASE: Initial Screening
SITE LOCATION
Site Name/Number:
County/Parish:
Intensive Monitoring Phase I
Phase 2
LatAong.:
State Waterbody Segment Number:.
Waterbody Type:.
Bottom Feeder - Species Name:
Composite Sample #:
Number of Individuals:
Fish*
001
002
003
004
005
006
007
008
009
010
Analyst
Initials/Date
Weight, g
Scales/Otollths
Removed (/)
Sex
Homogenate
Prepared (/)
Weight of homogenate
taken for composite
Total Composite Homogenate Weight
Predator - Species Name:
Composite Sample #:
Number of Individuals:
Fish*
001
002
003
004
005
006
007
008
009
010
Analyst
Initials/Date
Weight, g
Scales/OtolHhs
Removed (/)
Sex
Homogenate
Prepared (/)
Weight of homogenate
taken for composite
Total Composite Homogenate Weight
Figure GG-2.
GG-5
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the pectoral fin spines should be clipped and saved (Versar, 1982). Otoliths are another
indicator of age that may be collected (Jearld, 1983). The scales, spines, or otoliths may be
stored by sealing in small envelopes (such as coin envelopes) or plastic bags labeled with,
and cross-referenced by, the identification number assigned to the tissue specimen (Versar,
1982). Removal of scales, spines, or otoliths from each fish should be noted on the sample
processing record.
Sex Determination-To determine the sex of each individual fish, an incision should be
made on the ventral surface of the body from a point immediately anterior to the anus toward
the head to a point immediately posterior to the pelvic fins. If necessary, a second incision
should be made on the left side of the fish from the initial point of the first incision toward the
dorsal fin. The resulting flap should then be folded back to observe the gonads. Ovaries
appear whitish to greenish to golden brown and have a granular texture. Testes appear
creamy white and have a smooth texture (Texas Water Commission, 1990). The sex of each
fish should be recorded on the sample processing record.
Preparation of Individual Homogenates-Grinding of biological tissue, especially skin
from whole fish samples, is easier when the tissue is partially frozen (Stober, 1991). Chilling
the grinder briefly with a few chips of dry ice will reduce the tendency of the tissue to stick to
the grinder. However, do not freeze the grinder because it will make it difficult to force frozen
tissue through the chopper plate.
Smaller whole fish may be ground in a hand crank meat grinder (fish < 300 g) or a
food processor (fish 300-1,000 g). Larger fish may be cut into 2.5-cm cubes with a food
service band saw (e.g., Hobart Model 5212) and then ground in either a small (e.g., Hobart,
1/4 hp, Model 4616) or large (e.g., Hobart, 1 hp, Model 4822) meat grinder. To avoid
contamination by metals, homogenizers used to grind tissue should have tantalum or titanium
parts. The ground sample should be divided into quarters, opposite quarters mixed together
by hand, and the two halves mixed back together. The grinding, quartering, and hand mixing
should be repeated two more times. If chunks of tissue are present at this point, the
grinding/homogenizing should be repeated. No chunks should be discarded. If the sample is
to be analyzed for trace metals only, the ground tissue may be mixed by hand in a
polyethylene bag (Stober, 1991). Homogenization of each individual fish should be noted on
the sample processing record.
Individual whole fish homogenates may be either composited or frozen and stored at
<-20 °C in cleaned containers that are noncontaminating for the analyses to be performed.
GG-6
-------
Preparation of Composite Homogenates-lf individual whole fish homogenates are
frozen, they should be thawed partially and rehombgenized prior to compositing. Any
associated liquid should be maintained as a part of the sample. Equal weights should be
taken from each individual homogenate and blended to provide a composite sample of
sufficient size (500 g minimum) to perform all necessary analyses. Weights of individual
homogenates required for a composite sample, based on the total number of fish per
composite and the quantity of composite needed, are given in Table GG-2. The actual weight
of each individual homogenate that is taken for the composite sample should be recorded on
the sample processing record. The remaining individual homogenates should be archived in a
freezer at <-20 °C, with the designation "Archive" and the expiration date added to each
sample label. Location of the archived samples should be indicated on the sample processing
record under "Notes." Each composite sample should be divided into quarters, opposite
quarters mixed together by hand, and the two halves mixed together. The quartering and
mixing should be repeated two more times. If the sample will be analyzed for trace metals
only, the composite sample may be mixed by hand in a polyethylene bag. At this point, the
composite sample may be frozen and stored at S-20 °C or processed for organics and trace
metals analyses.
TABLE GG-2. INDIVIDUAL WEIGHTS (g) OF HOMOGENATE
REQUIRED FOR A COMPOSITE SAMPLE"
Total
number of
fish per
sample
6
7
8
9
10
Total homogenate weight
500 g
(minimum)
84
72
63
56
50
1,000g
(average)
167
143
125
112
100
2,000 g
334
286
250
223
200
* Based on total number of fish per composite and the total homogenate
weight required for analysis.
GG-7
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APPENDIX H
EXAMPLE PROCEDURE FOR ANALYSIS OF
PERCENT LIPID IN TISSUE SAMPLES
[From: State of California. 1990. Laboratory Quality
Assurance Program Plan. Department of Fish and Game,
Environmental Services Division. March.]
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Method f 2-LIPID
Determination of Percent Lipid in Tissue Samples.
1.0 Scope and Application
1.1 This method determine percent lipid in fish and
wildlife tissue samples.
2.0 Summary of Method
2.1 The tissue sample is dried using anhydrous grannular
sodium sulfate and the lipid extracted with petroleum
ether (PE). The petroleum ether is evaporated and the
residue is weighed.
3.0 Interferences
3.1 Each lot of petroleum ether must be tested by
evaporating 250 mL of PE to dryness, the residue must
be less than 10 mg.
4.0 Apparatus and Materials
4.1 Balance, capable of weighing to the nearest mg.
4.2 Beaker, 250 mL borosilicate glass.
4.3 Buchner Funnel, 8 cm.
4.4 Filter Flask, 500 mL
4.5 Filter Paper, Whatman #42, 8 cm.
4.6 Aluminum Dish, 50 mL.
4.7 Water Bath, heated, with concentric ring cover, capable
of temperature control (±2 °C), installed in fume hood.
4.8 Desiccator.
4.9 Waring Blender - Glass or stainless steel blender with
stainless steel blades and carbon bearings. The motor
of the blender must be explosion proof.
5.0 Reagents
5.1 Sodium sulfate, anhydrous, granular meets ACS
specifications.
5.2 Petroleum ether, distilled in glass.
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6.0 Sample collection, Presevation, and Handling
«
6.1 All samples must have been collected using a
sampling plan that addresses the considerations
discussed in this manual.
6.2 All sample containers must be prewashed with
detergents, acids, and Type II water. Plastic and
glass containers are both suitable.
7.0 Procedure for Sample Preparation
7.1 Weigh 5.0 g of tissue into a 250 mL beaker,
and record weight in notebook.
7.2 Add approximately 50 g of anhydrous sodium
sulfate and macerate sample with a glass rod 'co remove
moisture. Continue to add sodium sulfate as necessary
until sample is free flowing.
7.3 Transfer sample and sodium sulfate to the blender.
7.4 Add 150 mL of petroleum ether (PE) to blender and
blend for two minutes at high speed.
7.5 Decant the PE to a Buchner Funnel fitted with the
#42 Whateman filter paper. Use vacuum to expedite the
filtration process.
7.6 Repeat steps 7.4 and 7.5 using 100 mL PE.
7.7 Preconcentrate the filtrate to 25 mL on a steam bath.
Quantitively transfer the 25 mL into a preweighed
aluminum dish.
7.8 Evaporate the PE extract on a steam bath, dry in a
oven at 103 °C, store in a desiccator, and reweigh
the aluminum dish with the lipid material.
8.0 Analytical Procedure
8.1 Calculation of Per Cent Lipid.
i Lipid = Wt of. Al dish with lipid (a) - wt of Al dish (a) & 100
Weight of sample (g)
9.0 Quality Control
9.1 All quality control data should be maintained and
available for easy reference.
9.2 Analyze at least one blank per batch of samples. See
Section 3.0.
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9.3 Analyze one duplicate sample for every twenty samples.
9.4 The analytical balance shall be serviced by a qualified
service engineer every twelve to twenty-four months.
10.0 Method Performance
10.1 Laboratory duplicates.
i. of pairs Relative Standard Deviation
68.3% Confidence limit- 6.4%
10 95.5% Confidence limit- 9.0%
99.9% Confidence limit-11.6%
11.0 References
11.1 U.S Food and Drug Administration 1970b Method of
Analysis. AOAC-Eleventh Edition PAM. Vol. 1,
Section 160.
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APPENDIX I
EXAMPLE PROCEDURE FOR ANALYSIS OF CADMIUM
BY GRAPHITE FURNACE ATOMIC
ABSORPTION (GFAA) SPECTROMETRY
[From: State of California. 1990. Laboratory Quality
Assurance Program Plan. Department of Fish and Game,
Environmental Services Division. March.]
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trelesop.dbc
METHOD TRELEDIG
DIGESTION AND ANALYSIS OF TRACE ELEMENTS IN 'TISSUES BY
FLAME AAS AND GRAPHITE FURNACE AAS
1.0 SCOPE AND APPLICATION
1.1 This procedure utilizes an open tube nitric acid
digestion for the determination of: aluminum (Al);
cadmium (Cd); chromium (Cr); copper (Cu); lead (Pb);
manganese (Mn); nickel (Ni); silver (Ag); and zinc
(Zn); in whole fish, fish liver, and mussel tissues by
flame (FAAS) and graphite furnace (GFAAS) atomic
absorption spectrophotometry.
2.0 SUMMARY OF METHOD
2.1 Samples are prepared for analysis by digesting the
tissue with concentrated nitric acid in a glass tube
inserted in a heating block at elevated temperature.
Samples are refluxed for 2-3 hours or until no more
nitrogen oxides (reddish brown vapors) are observed In
the tubes. The liquid digestate is then evaporated to
about 0.5 ml, to remove most of the acid, and then is
diluted with 1.0% nitric acid to a final volume of
40.0 ml.
2.2 Whole Fish and Fish Liver
Digestates of whole fish and fish liver are analyzed
first by graphite furnace atomic absorption spectro-
photometry (GFAAS) on a Perkin-Elmer Model 3030 with
Zeeman background correction for Cd, Ag, Pb, Cr, Ni,
and Cu. The samples are then analyzed by flame atomic
absorption spectrophotometry on a Varian Spectra 300
with deuterium arc background correction for Cu, Zn,
and any of the trace elements analyzed by GFAAS
present in the samples at a high enough concentration
to be detected by flame AAS.
2.3 Mussels
Mussel tissue digestates are analyzed by GFAAS on a
Perkin-Elmer Model 3030 Zeeman for Pb, Cr, Ag, and
Ni. The samples are then analyzed by flame AAS on a
Perkin-Elmer Model 2280 for Cd, Cu, Mn, Zn, and Al.
-------
2.4 The detection limits for this method are as follows?
Whole Fish and Fish Liver ucr/q (ppm) wet*
«
Cadmium 0.01
Silver 0.01
Lead 0.1
Chromium 0.02
Nickel 0.1
Copper 0.02
Zinc 0.05
* based on 1.0 g sample weight and final volume of
40 ml.
Mussels uq/q (ppm) dry''
Aluminum 1.0
Cadmium 0.01
Chromium 0.02
Copper 0.02
Lead 0.1
Manganese 0.1
Nickel 0.1
Silver 0.01
Zinc 0.05
* based on 3.0 g sample weight and final volume of
20 mL.
3.0 INTERFERENCES
3.1 Sample Digestion
3.1.1 Tissue samples can cause various problems
especially with GFAAS due to the complex matrices
involved. A fairly rigorous digestion is needed to
remove as much of the sample matrix as possible. The
matrix problems can also be addressed by using stand-
ard reference materials of similar matrix to the
sample and by using the method of standard additions.
3.1.2 Special care must be used in selecting the
acid used for digestion. Only redistilled HNO-,
should be used because other reagent grade acids are
frequently contaminated with trace levels of metals,
especially chromium. Prior to use all acids used in
the digestion should be checked for contamination.
3.2 Direct aspiration flame AAS
3.2.1 The most troublesome type of interference in
atomic absorption spectrophotometry is usually termed
"chemical" and is caused by lack of absorption of
atoms bound in molecular combination in the flame.
-------
This phenomenon can occur when the flame is not suffi-
ciently hot to dissociate the molecule, as in the case
of phosphate interference with magnesium, or when the
dissociated atom is immediately oxidized to a compound
that will not dissociate further at the temperature of
the flame. The addition of lanthanum will overcome
phosphate interference in magnesium, calcium, and
barium determinations. Similarly, silica interference
in the determination of manganese can be eliminated
by the addition of calcium.
3.2.2 Chemical interferences may also be eliminated
by separating the metal from the interfering material.
Although complexing agents are employed primarily to
increase the sensitivity of the analysis, they may
also be used to eliminate or reduce interferences.
3.2.3 The presence of high dissolved solids in the
sample may result in an interference from nonatomic
absorbance such as light scattering. If background
correction is not available, a nonabsorbing wavelength
should be used. Preferably, samples containing high
solids should be extracted.
3.2.4 lonization interferences occur when the flame
temperature is sufficiently high to generate the re-
moval of an electron from a neutral atom, giving a
positively charged ion. This type of interference
can generally be controlled by the addition, to both
standard and sample solutions, of a large excess
(1,000 mg/L) of an easily ionized element such as K,
Na, Li, or Cs.
3.2.5 Spectral interference can occur when an ab-
sorbing wavelength of an element present in the sample
but not being determined falls within the width of the
absorption line of the element of interest. The
results of the determination will then be erroneously
high, due to the contribution of the interfering
element to the atomic absorption signal. Interference
can also occur when resonant energy from another
element in a multielement lamp, or from a metal impu-
rity in the lamp cathode, falls within the bandpass of
the slit setting when that other metal is present in
the sample. This type of interference may sometimes
be reduced by narrowing the slit width.
3.2.6 Samples and standards should be monitored for
viscosity differences that may alter the aspiration
rate.
3.2.7 Some sample solutions may have solids suspend-
ed in them from incomplete digestion. These solids
can plug the nebulizer tubing and slow or stop the
aspiration of sample.
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3.2.8 All metals are not equally stable in the
digestate, especially if it contains only HN03, not
HN03 and HC1. The digestate should be analyzed as
soon as possible, with preference given to Ag, Cd,
and Pb.
3.3 Furnace procedure
3.3.1 Although the problem of oxide formation is
greatly reduced with furnace procedures because atoro-
ization occurs in an inert atmosphere, the technique
is still subject to chemical interferences. The
composition of the sample matrix can have a major
effect on the analysis. It is those effects which
must be determined and taken into consideration in the
analysis of each different matrix encountered. To
help verify the absence of matrix or chemical inter-
ference, the serial dilution technique (see Paragraph
9.7) may be used. Those samples which indicate the
presence of interference should be treated in one or
more of the following ways:
1. Successively dilute and reanalyze the samples to
eliminate interferences.
2. Modify the sample matrix either to remove inter-
ferences or to stabilize the analyte. Examples
are the addition of ammonium nitrate to remove
alkali chlorides and the addition of ammonium
phosphate to retain cadmium. The mixing of
hydrogen with the inert purge gas has also been
used to suppress chemical interference. The
hydrogen acts as a reducing agent and aids in
molecular dissociation.
3. Analyze the sample by method of standard additions
while noticing the precautions and limitations of
its use (see Paragraph 9.8).
3.3.2 Gases generated in the furnace during atom-
ization ion may have molecular absorption bands encom-
passing the analytical wavelength. When this occurs,
use either background correction or choose an alter-
nate wavelength. Background correction may also
compensate for nonspecific broad-band absorption
interference.
3.3.3 Continuum background correction cannot correct
for all types of background interference. When the
background interference cannot be compenstated for,
chemically remove the analyte or use an alternate form
of background correction, e.g., Zeeman background cor-
rection.
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3.3.4 Interference from a smoke-producing sample
matrix can sometimes be reduced by extending the char-
ring time at a higher temperature or utilizing an ash-
ing cycle in the presence of air. Care must be taken,
however, to prevent loss of the analyte.
3.3.5 Samples containing large amounts of organic
materials should be oxidized by conventional acid di-
gestion before being placed in the furnace. In this
way, broad-band absorption will be minimized.
3.3.6 Anioh interference studies in the graphite
furnace indicate that, under conditions other than
isothermal, the nitrate anion is preferred. There-
fore, nitric acid is preferable for any digestion or
solubilization step. If another acid in addition to
HN03 is required, a minimum amount should be used.
This applies particularly to hydrochloric and, to a
lesser extent, to sulfuric and phosphoric acids.
3.3.7 Carbide formation resulting from the chemical
environment of the furnace has been observed. Molyb-
denum may be cited as an example. When carbides form,
the metal is released very slowly from the resulting
metal carbide as atomization continues. Molybdenum
may require 30 sec or more atomization time before the
signal returns to baseline levels. Carbide formation
is greatly reduced and the sensitivity increased with
the use of pyrolytically coated graphite. Elements
that readily form carbides are: Ba, Mo, Ni, and V.
3.3.8 For comments on spectral interference, see
Paragraph 3.1.4.
3.3.9 Cross-contamination and contamination of the
sample can be major sources of error because of the
extreme sensitivities achieved with the furnace. The
sample preparation work area should be kept scrupu-
lously clean. All glassware should be cleaned as dir-
ected in Paragraphs 4.11 and 7.1. Pipet tips are a
frequent source of contamination. If suspected, they
should be acid soaked with 1:5 HMO-, and rinsed
thoroughly with tap and deionized (Type II) water.
The use of a better grade of pipet tip can greatly
reduce this problem. Special attention should be
given to reagent blanks in both analysis and in the
correction of analytical results. Lastly, pyrolytic
graphite, because of the production process and
handling, can become contaminated. As many as five to
ten high-temperature burns may be required to clean
the tube before use.
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4.0 APPARATUS AND MATERIALS
4.1 Atomic absorption spectrophotometer
«
4.1.1 FAAS
Varian Spectra 300 with data system and Mark VI
burners for air- and nitrous oxide-acetylene flames -
or a Perkin-Elmer Model 2280 spectrophotometer with
deuterium arc background corrector and digital display.
4.1.2 GFAAS
Perkin-Elmer Model 3030 spectrophotometer with
Zeeman effect background correction, HGA-60 furnace
controller, AS-60 autosampler, EDL power supply, and
PR-100 printer.
4.2 Hollow cathode lamps: Single-element lamps are used
and are preferred over multi-element lamps which may
be used occasionally. Electrodless discharge lamps
may also be used for certain elements.
4.3 Graphite furnace parts:
Perkin-Elmer P/N
Pyrolytic coated graphite tubes 091504
Pyrolytic coated graphite tubes(grooved) 109322
L'vov platforms 109324
4.4 Pressure-reducing valves: The supplies of fuel and
oxidant should be maintained at pressures somewhat
higher than the controlled operating pressure of the
instrument by suitable valves. (See manufacterer's
specifications.)
4.5 Block Thermostat: Liebisch model 2102 with model
2279 programmable controller.
4.6 Digestion Tubes: 25x200 mm glass test tubes with bead-
ed rim.
4.7 Polyethylene caps: Wheaton part No. 227720 caps for
BOD bottles, with bottom ring removed.
4.8 Polyethylene (HOPE) bottles: Nalgene part No.2002-002,
2 oz., 60 mL polyethylene (HOPE) bottles.
4.9 Polyethylene cups for AS-60 autosampler: Evergreen
part No. 127-0018-020 (case of 1000).
4.10 Pipetors: Preferably all plastic/teflon of various
sizes from 100-1000 uL with polyethylene tips. Do
not use yellow pipet tips, they are commonly contami-
nated with cadmium.
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4.11 Glassware: All glassware, polypropylene, polyethylene,
and Teflon containers, excluding HOPE sample bottles
and polyethylene cups for AS-60 autosaropler, should be
washed in the following sequence: detergent, tap
water, 1:1 nitric acid, tap water, 1:1 hydrochloric
acid, tap water, and Type II water. (Chronic acid
should not be used as a cleaning agent for glassware
if chromium is to be included in the analytical
scheme.) If it can be documented through an active
analytical quality control program using spiked
samples and reagent blanks that certain steps in the
cleaning procedure are not required for routine
samples, those steps may be eliminated from the
procedure.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests.
Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the
Committee on Analytical Reagents of the American Chem-
ical Society, where such specifications are available.
Other grades may be used, provided it is first ascer-
tained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of
the determination.
5.2 Type II water (ASTM D1193): Use Type II water for the
preparation of all reagents and calibration standards
and as dilution water.
5.3 Concentrated nitric acid (HN03): Use a spectrograde
acid certified for AA use. For graphite furnace work
all acids should be checked using reagent blanks for
all of the analytes to be reported. Prepare a 1:1
dilution with Type II water by adding the concentrated
acid to an equal volume of water.
5.4 Hydrochloric acid (HC1, 1:1): Use a spectrograde acid
certified for AA use. Prepare a 1:1 dilution with
Type II water by adding the concentrated acid to an
equal volume of water.
5.5 Fuel and oxidant: Commercial grade acetylene is
generally acceptable. Air may be supplied from a com-
pressed air line, a laboratory compressor, or a
cylinder of compressed air. Reagent grade nitrous
oxide is also required for certain determinations.
Standard commercially available argon and nitrogen are
required for furnace work.
5.6 Stock standard metal solutions: Stock standard solu-
tions are prepared from high purity metals, oxides, or
nonhygroscopic reagent-grade salts using Type II water
and redistilled nitric or hydrochloric acids. (See
-------
individual methods for specific instructions.)
Sulfuric or phosphoric acids should be avoided as they
produce an adverse effect on many elements. The
stock solutions are prepared at concentrations of
1,000 mg of the metal per liter. Commercially avail-
able standard solutions may also be used if standards
from two different vendors are checked against one
another and are in agreement. Standards available
from the U.S. National Institute of Standards and
Technology (NIST) are also acceptable and do not have
to be verified. Where the sample viscosity, surface
tension, and components cannot be accurately matched
with standards, the method of standard additions may
be used (see Paragraph 9.8).
5.7 Calibration standards: For those instruments which do
not read out directly in concentration, a calibration
curve is prepared to cover the appropriate concentra-
tion range. Usually, this means the preparation of
standards which produce an absorbance of 0.0 to 0.7.
Calibration standards are prepared by diluting the
stock metal solutions at the time of analysis. For
best results, calibration standards should be prepared
fresh each time a batch of samples is analyzed or dem-
onstrate that the standards are still good by compar-
ing the standard absorbances with those of SRM 1643b
"Trace Elements in Water". ' The expiration date on the
SRM 1643b should be used to validate its use for this
purpose. If the standards cannot be validated using
the SRM 1643b then the following can be used as a
guideline:
less than 0.1 ppm - prepare daily
0.1 to 1 ppm - prepare weekly
1.0 to 10 ppm - prepare monthly
10 to 100 ppm - prepare quarterly
100+ ppm - prepare yearly (at a minimum)
Prepare a blank and at least three calibration stan-
dards in graduated amounts in the appropriate range of
the linear part of the curve. The calibration stan-
dards should be prepared using the same type of acid
or combination of acids and at the same concentration
as will result in the samples following processing,
1% HNO-j (14 mL concentrated HN03/L) for tissues.
Beginning with the blank and working toward the
highest standard, aspirate the solutions and record
the readings. Repeat the operation with both the
calibration standards and the samples a sufficient
number (minimum of two) of times to secure a reliable
average reading for each solution. Calibration
standards for furnace procedures should be prepared
as described on the individual sheets for that metal.
-------
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling
plan that addresses the considerations'discussed in
this procedure.
6.2 All sample containers must be prewashed with deter-
gents, acids, and Type II water. Plastic, glass,
HOPE, and Teflon containers are suitable. Only new
HOPE bottles (Nalgene part No. 2002-002) will be used
for sample digestates and should not be washed with
soap and water. These HDPE bottles should be cleaned
and checked according to 7.1.2 and 7.2.
6.3 Samples shall be double-wrapped in aluminum foil or
placed in polyethylene bags (do not use polyethylene
bags if samples are to be analyzed for organics) and
frozen as soon as possible after collection and remain
frozen until dissection. After dissection and
homogenization the samples should be refrozen until
analysis.
7.0 PROCEDURE FOR SAMPLE PREPARATION
7.1 Preparation of glassware
7.1.1 Digestion tubes:
1. Tubes should first be cleaned using the procedure
described in section 4.11.
2. Prior to starting digestion add 10 mL 6N nitric
acid to tube and fill to the top with with Type II
water. Cover with polyethylene cap and leave
overnight.
3. The next day discard the acid solution and rinse
the tube and the cap three times with Type II
water. The tubes are now ready to be checked.
7.1.2 Polyethylene (HDPE) bottles:
1. Fill polyethylene (HDPE) bottles with 6 N nitric
acid, cap, and allow to soak for at least 48 hours.
2. Prior to using for digestion tube blanks, empty
the bottles, rinse with 0.1 N nitric acid and with
Type II water.
7.1.3 Polyethylene digestion tube caps: Store caps
in a large polyethylene bottle filled with 1 N nitric
acid. Remove from acid bath and rinse with Type II
water just prior to use.
-------
7.1.4 Teflon policemen:
1. Policemen should have previously been washed with
soap and tap water after the last use.
2. Add fresh solution of 1 N HN03 to a milk dilution
bottle.
3. Soak Teflon policeman in the acid solution.
4. Rinse the policeman with Type II water.
7.2 Preparation of digestion tube and polyethylene (HOPE)
bottle blanks:
1. Each digestion tube should have a corresponding
polyethylene (HDPE) bottle with the same number or
letter. Each tube/bottle pair should have a unique
number or letter to distinguish it from all other
tube/bottle pairs.
2. Add 1 mL of redistilled nitric acid to each tube
and place in aluminum block with temperature set
at 160°C. Heat tubes for about 2 hours. Remove
tubes from block and allow to cool. Fill tubes to
20 mL with Type II water and use vortex mixer on
slow speed to mix. Transfer solution to pre-
cleaned 60 mL Nalgene polyethylene (HDPE) bottle,
re-fill tube to 20 mL with Type II water, mix on a
vortex mixer on slow speed, and combine with
solution in polyethylene (HDPE) bottle.
3. Analyze solution by GFAAS for elements to be
analyzed in samples by GFAAS using the procedure
described in 8.5.
4. Rinse digestion tubes with 1% redistilled nitric
acid and store with caps covering tubes.
5. After analyzing the tube/bottle blanks remove any
tube/bottle pairs that are contaminated with any of
the elements to be analyzed in the samples. These
tube/bottle sets should be taken through the
cleaning procedure and rechecked at a later date.
It is good policy to clean and check approximately
20% more tube/bottle pairs than will be needed for
the current set of samples.
7.3 Weighing Procedures
7.3.1 Prior to weighing samples, prepare lab note-
book (i.e. list sample numbers and any special in-
structions) .
-------
7.3.2 Record tube/bottle pair identification number
next to the blank or sample identification number as
the samples are weighed.
«
7.3.3 Preparation of method blanks: Prepare two
blanks for each set of samples.
1. Add 5 mL of concentrated redistilled HNO3 to
digestion tube, cover with polyethylene cap, and
place in heating block.
7.3.4 Standard reference material (SRM): Use refer-
ence materials with matrix as close as possible to
that of the samples to be analyzed. Weigh at least
two SRM's for each set of samples.
1. Weigh 0.25+0.05 g of SRM into tared digestion
tube. Be careful to place the sample on the bottom
of the tube and not on the sides.
2. Record weight of sample in notebook to at least
two places to the right of the decimal point.
3. Add 5 mL of concentrated redistilled HNCK to the
tube, cover with polyethylene cap, and place in
the heater block.
7.3.5 Fish liver samples:
1. Using a clean Teflon policeman, mix liver sample
thoroughly.
2. Weigh 1.00+0.10 g of fish liver into a tared
digestion tube. Be careful to place the sample on
the bottom of the tube and not on the sides.
3. Record weight of sample in notebook to at least
two places to the right of the decimal point.
4. Add 5 mL of concentrated redistilled HNOo to the
tube, cover with polyethylene cap, and place in the
heater block.
7.3.6 Whole fish samples:
(Whole fish samples are homogenized using fish:water
[1:1]).
1. Using a clean Teflon policeman, mix whole fish
thoroughly. Some of the whole fish samples are
very watery and should be shaken to thoroughly mix.
2. Weigh 2.00+0.10 g of whole fish homogenate into a
tared digestion tube. Be careful to place the
sample in the bottom of the tube and not on the
sides.
-------
3. Record weight of sample in notebook to at least
two places to the right of the decimal point.
4. Add 5 roL of concentrated redistilled HN03, cover
with polyethylene cap, and place in heater block.
7.3.7 Mussel tissue:
1. Using a clean Teflon policeman, mix mussel sample
thoroughly.
2. Weigh 3.00±0.10 g of mussel tissue into a tared
digestion tube. Be careful to place the sample
into the bottom of the tube and not on the sides.
3. Record weight of sample in notebook to at least
two places to the right of the decimal point.
4. Add 5 mL of concentrated redistilled HN03, cover
with polyethylene cap, and place in heater block.
7.4 Sample digestion procedure
7.4.1 Program temperature of heating block to 70°C
at a rate of 600°C/hour. Leave heating block at 70°C
for at least one hour.
7.4.2 Program temperature of heating block to 160°C
at a rate of 300°C/hour. Leave block at 160°C until
no more NOX (reddish brown fumes) are observed in the
tube (at least 2 hours).
7.4.3 After the digestion is completed remove the
polyethylene caps from the tubes to allow the acid
digestate to evaporate. Evaporate the solution until
about 0.5 roL remains in the tube. It may be necessary
to elevate the temperature of the block to 170°C.
7.4.4 After the evaporation is completed remove the
tubes from the block and allow them to cool. Dilute
the remaining digestate with 1% redistilled HN03 to
the 20 mL mark on the digestion tube. Mix the sample
on the vortex mixer and transfer the solution to the
60 mL polyethylene (LPE) bottle. Immediatly add
another 20 mL of 1% HN03 to the tube, mix on the
vortex mixer, and add to the LPE bottle and mix thor-
oughly. The sample is now ready for analysis.
8.0 ANALYTICAL PROCEDURE
8.1 Sample digestion and dilution steps should result in
an extract that is clear and free of undissolved solid
materials. If the sample solution is cloudy or has
solid materials suspended in solution at the time of
analysis, it should be noted in the laboratory note-
-------
book under a "comments" column.
8.2 Samples should be analyzed for silver, cadmium, and
lead within 48 hours of digestion. Ttie remaining
elements should be analyzed as soon as possible.
8.3 All graphite furnace analyses should be done prior to
the flame analyses to prevent cross-contamination
between bottles from the aspirator tubing.
8.4 Direct aspiration (flame) procedure
8.4.1 Differences between the various makes and
models of atomic absorption spectrophotometers prevent
the formulation of detailed instructions applicable to
every instrument from being included in this document.
Good laboratory practice is to have detailed instruc-
tions for the operation of each instrument kept with
the instrument for the analyst to use during
operation. These instructions should follow the
manufacturer's operating instructions for a particular
instrument. In general, after choosing the proper
lamp for the analysis, allow the lamp to warm up for a
minimum of 15 minutes, unless operated in a double-
beam mode. During this period, align the instrument,
position the monochronometer at the correct
wavelength, select the proper monochronometer slit
width, and adjust the current according to the
manufacturer's recommendation. Some or all of these
parameters may be done by the instrument automat-
ically. Subsequently, light the flame and regulate
the flow of fuel and oxidant. Adjust the burner and
nebulizer flow rate for maximum percent absorption and
stability. Balance the photometer. Run a series of
standards of the element under analysis. Construct a
calibration curve by plotting the concentrations of
the standards against absorbances or have the data
system construct it. Aspirate the samples and
determine the concentrations either directly or from
the calibration curve. Standards must be run each
time a sample or series of samples is run.
8.5 Furnace procedure
8.5.1 Furnace devices (flameless atomization) are the
most useful means of extending detection limits. Be-
cause of differences between various makes and models
instruments, no detailed operating instructions can be
given for each instrument in this document. Detailed
operating instructions following the instructions
provided by the manufacturer of each instrument are
kept with each instrument for the analyst to use
during the analysis.
-------
8.5.2 Background correction is important when using
flameless atomization, especially below 350 nm. Cer-
tain samples, when atomized, may absorb or scatter
light from the lamp. This can be caused by the pres-
ence of gaseous molecular species, salt particles, or
smoke in the sample beam. If no correction is made,
sample absorbance will be erroneously high. Zeeman
background correction is effective in overcoming com-
position or structured background interferences. It
is particularly useful when analyzing for As in the
presence of Al and when analyzing for Se in the pres-
ence of Fe.
8.5.3 Memory effects occur when the analyte is not
totally volatilized during atomization. This condi-
tion depends on several factors: volatility of the
element and its chemical form, whether pyrolytic
graphite is used, the rate of atomization, and furnace
design. This situation is detected through blank
burns. The tube should be cleaned by operating the
furnace at full power for the required time period,
as needed, at regular intervals during the series of
determinations.
8.5.4 Inject a measured microliter aliquot of sample
into the furnace and atomize. If the concentration
found is greater than the highest standard, the sample
should be diluted in the same acid matrix and reanal-
yzed. The use of multiple injections can improve ac-
curacy and help detect furnace pipetting errors.
8.5.5 To verify the absence of interference, follow
the serial dilution procedure given in Section 9.7.
8.5.6 A check standard should be run after approx-
imately every 10 sample injections. Standards are run
in part to monitor the life and performance of the
graphite tube. Lack of reproducibility or significant
change in the signal for the standard indicates that
the tube should be replaced. Tube life depends on
sample matrix and atomization temperature. A conser-
vative estimate would be that a tube will last at
least 50 firings. A pyrolytic coating will extend
that estimated life by a factor of three.
8.6 Calculation
8.6.1 For determination of metal concentration by
direct aspiration and furnace: Read the metal value
in mg/L from the calibration curve or directly from
the read-out system of the instrument.
8.6.2 Different injection volumes must not be used
for samples and standards. Instead, the sample shou
be diluted and the same size injection volume be usea
-------
for both samples and standards. If dilution of sample
was required:
mg/L metal in sample = A (C+B)
C
where:
A = mg/L of metal in diluted aliquot from calibration
curve.
B = Acid blank matrix used for dilution, mL.
C = Sample aliquot, mL.
8.6.3 For solid samples, report all concentrations
as ug/g based on wet. Hence:
ug metal g sample = AxV
W
where:
A = mg/L of metal in processed sample from calibra-
tion curve.
V = Final volume of the processed sample, mL.
W = Weight of sample, grams.
9.0 QUALITY CONTROL
9.1 All quality control data should be maintained and
available for easy reference or inspection.
9.2 A calibration curve must be prepared at least twice
each day (one at the beginning and one at the end of
each set of samples) for each element analyzed with a
minimum of a reagent blank and three standards. The
calibration curve should be verified by the use of at
least a reagent blank and one quality control check
standard at or near the mid-range every 15 samples.
Checks throughout the day must be within 20% of the
original curve.
9.3 If 20 or more samples per day are analyzed, the work-
ing standard curve must be verified by running an ad-
ditional standard at or near the midrange every 10
samples. Checks must be within + 20% of the true
value.
9.4 Employ a minimum of one reagent blank per sample batch
to determine if contamination or any memory effects
are occuring.
9.5 At least one spiked matrix and one replicate sample
should be run every 10 samples or per analytical
batch, whichever is greater. At least one spiked
replicate sample should also be run with each matrix
type to verify precision of the method.
-------
9.6 Where the sample matrix is so complex that viscosity,
surface tension, and components cannot be accurately
matched with standards, the method of standard addi-
tion may be used (see Step 9.8 below).'
9.7 Serial dilution - Withdraw from the sample two equal
aliquots.To one of the aliquots add a known amount
of analyte and dilute both aliquots to the same pre-
determined volume. (The dilution volume should be
based on the analysis of the undiluted sample. Pre-
ferably, the dilution should be 1:4, while keeping in
mind that the diluted value should be at least 5 times
the instrument detection limit. Under no circum-
stances should the dilution be less than 1:1.) The
diluted aliquots should then be analyzed, and the un-
spiked results, multiplied by the dilution factor,
should be compared to the original determination.
Agreement of the results (within 10%) indicates the
absence of interference. Comparison of the actual
signal from the spike with the expected response from
the analyte in an aqueous standard should help confirm
the finding from the dilution analysis.
9.8 Method of standard additions - The standard addition
technique involves adding known amounts of standard to
one or more aliquots of the processed sample solution.
This technique compensates for a sample constituent
that enhances or depresses the analyte signal, thus
producing a different slope from that of the calibra-
tion standards. It will not correct for additive
interferences which cause a baseline shift.
9.8.1 In the simplest version of this technique is
the single addition method, in which two identical
aliquots of the sample solution, each of volume Vx,
are taken. To the first (labeled A) is added a known
volume Vs of a standard analyte solution of concentra-
tion Cs. To the second aliquot (labeled B) is added
the same volume Vs of the solvent. The analytical
signals of A and B are measured and corrected for non-
analyte signals. The unknown sample concentration Cx
is calculated:
Cx = SBVSCS/(SA-SB)VX
where SA and SB are the analytical signals (corrected
for the blank) of solutions A and B, respectively. Vs
and Cs should be chosen so that SA is roughly twice SB
on the average, avoiding excess dilution of the sam-
ple. If a separation or concentration step is used,
the additions are best made first and carried through
the entire procedure.
9.8.2 Improved results can be obtained by employing
a series of standard additions. Equal volumes of the
-------
sample are added to a series of standard solutions
containing different known quantities of the test ana-
lyte, all diluted to the same volume. For example,
addition 1 should be prepared so that the resulting
concentration is approximately 50 percent of the ex-
pected sample absorbance. Additions 2 and 3 should be
prepared so that the concentrations are approximately
100 and 150 percent of the expected sample absorb-
ances, respecively. The absorbance of each solution
is determined and then plotted on the vertical axis
(ordinate) of a graph, with the concentrations of the
known standards plotted on the horizontal axis (ab-
scissa) . When the resulting line is extrapolated back
to zero absorbance, the point of interception of the
abscissa is the concentration of the unknown. The
abscissa on the left of the ordinate is scaled the
same as on the right side, but in the opposite
direction from the ordinate. An example of a plot so
obtained is shown in Figure 1. Some of the newer
instruments (Perkin-Elmer 3030) have standard addition
software built into the data system. The AS-60 auto-
sampler on the Perkin-Elmer 3030 will automatically
make the standard additions in the graphite tube. All
of the calculations for the standard additions tech-
nique are done for the operator by the instrument.
9.8.3 For the results of this technique to be valid,
the following limitations must be taken into consider-
ation:
1. The absorbance plot of sample and standards must
be linear over the concentration range of concern.
For best results, the slope of the plot should be
nearly the same as the slope of the standard curve.
If the slope of the standard addition plot is
significantly different (greater than 20%) caution
should be exercised.
2. The effect of the interference should not vary as
the ratio of analyte concentration to sample matrix
changes, and the standard addition should respond
in a similar manner as the analyte.
3. The determination must be free of spectral inter-
ference and corrected for nonspecific background
interference.
9.9 Dilute samples if they are more concentrated than the
highest standard or if they fall on the plateau of a
calibration curve.
9.10 Duplicates, spiked samples, standard reference mate-
rials, and check standards should be routinely ana-
lyzed.
-------
9.11 Atomic absorption spectrophotometers (AAS) should be
serviced on a regular basis by qualified technicians
as part of a regularly scheduled preventive mainte-
nance program.
9.12 A log book should be kept for each AAS that includes:
1 Standard absorbances, photomultiplier voltages,
detection limits, maintenance information, and any
problems that might occur each time the instrument is
used.
10.0 METHOD PERFORMANCE
10.0 See individual methods.
11.0 REFERENCES
1. U.S. Environmental Protection Agency, Test Methods
for Evaluating Solid Waste, SW-486 Third Ed., Revision
1, December 1987.
-------
cdgfsop
METHOD CDQFAA
4
CADMIUM (ATOMIC ABSORPTION, FURNACE TECHNIQUE)
1.0 SCOPE AND APPLICATION
1.1 See Section 1.0 of Method TRELEDIG.
2.0 SUMMARY OF METHOD
2.1 See Section 2.0 of Method TRELEDIG.
3.0 INTERFERENCES
3.1 See section 3.0 of Method TRELEDIG if interferences are
suspected.
3.2 In addition to the normal interferences experienced
during graphite furnace analysis, cadmium analysis can
suffer from severe nonspecific absorption and light
scattering caused by matrix components during atora-
ization. Simultaneous background correction is re-
quired to avoid erroneously high results.
3.3 Cadmium is the most volatile element commonly deter-
mined by GFAA. Simple aqueous solutions of Cd prccu ..a
ashing losses starting at 300°C or 400°C. With
the addition of monobasic or dibasic ammonium phos-
phate, Cd is not lost until about 600°C. If in
addition, Mg(N03)2 is added to the phosphate, Cd is >ot
lost until 900°C.
3.4 Contamination;
3.4.1 Many plastic tips (yellow) contain cadmium. Use
"cadmium free" tips.
3.4.2 The pouring surfaces of glass and plastic ware
may be contaminated with cadmium. When pouring solu-
tions to be analyzed by graphite furnace pour a small
amount and discard to rinse the pouring surface prior
collecting the liquid.
4.0 APPARATUS AND MATERIALS
4.1 For basic apparatus, see Section 4.0 of Method
TRELEDIG.
-------
4.2 Instrument parameters (general):
4.2.1 Drying tin* and traps 60 sec at 120°C.
4.2.2 Ashing tine and temps 45 sec at 900°C.
4.2.3 Atomizing time and temps 5 sec at 2500°C.
4.2.4 Purge gass Argon.
4.2.5 Wavelengths 228.8 run.
4.2.6 Background corrections Required.
4.2.7 Other operating parameters should be set as
specified by the particular instrument manufacturer.
NOTE: The above concentration values and instrument
conditions are for a Perkin-Elmer HGA-600, based on
a 10-uL injection, stop internal gas flow during atom-
ization, pyrolytic coated graphite tube with L'vov
platform.
5.0 REAGENTS
5.1 See section 5.0 of Method TRELEDIG.
5.2 Preparation of standards;
5.2.1 Stock solutions Dissolve 1.000 g cadmium
metal (analytical reagent grade) in 20 mL of 1:1 HN03
and dilute to 1 liter with Type II water. Alterna-
tive, procure a standard from a commercial supplier.
Analytical standards prepared in the laboratory or
purchased from a commercial vendor should be verified
by comparison with a second standard. Standards
purchased from the U.S. Institute of Standards and
Technology (NIST) are certified and do not need to be
verified using standards from a second source.
1.0 Standard suppliers and part numbers:
National Institute of Standards and Technology (NIST)
Part No. SRM 3108 (Cd-10 mg/mL in 10% HNO3).
5.2.2 Prepare dilutions of the solution to be used as
calibration standards at the time of analysis. The
calibration standards should be prepared using the same
type of acid and at the same concentration as will
result in the sample to be analyzed after processing
(1.0% HN03).
5.2.3 Ammonium phosphate-magnesium nitrate solutions
Dissolve 2.42 g of NH4H2PO4 and 0.173 g of Mg(NO3)2
H,O in 100 mL of Type II water. A 10-uL injection of
this solution contains 200 ug PO4 and 10 ug Mg(NO3)2«
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See Chapter Three, Section 3.1.3, Sample Handling and
Preservation.
-------
7.0 PROCEDURE
7.1 Sample preparation; The procedures for preparation
of the sample are given in Method TRELEDIG.
4
7.2 See Method TRELEDIG Paragraph 8.5, Furnace procedure.
8.0 QUALITY CONTROL
8.1 See section 9.0 of Method TRELEDIG.
9.0 METHOD PERFORMANCE
9.1 The performance characteristics for an aqueous sample
free of interferences are:
Optimum concentration range: 0.5-10 ug/L (ppb).
Detection limit: 0.1 ug/L (ppb)
9.2 The performance characteristics for a tissue sample
free of interferences are:
Optimum concentration range: 0.02 mg/kg (ppm)
Detection limit: 0.01 mg/kg (ppm).
9.3 Precision and accuracy data:
9.3.1 Duplicate data (fish liver-mg/kg):
Duplicates x s %RSD
0.04 0.03 0.035 0.007 20.3%
0.06 0.07 0.065 0.007 10.9%
0.04 0.04 0.04 0.000 0.0%
0.99 1.00 0.995 0.007 0.7%
0.009 0.011 0.010 0.0008 8.0%
0.008 0.001 0.0045 0.0049 109. %
0.052 0.053 0.0525 0.0007 1.3%
9.3.2 Procedural blanks:
n = 11 x - 0.0094 s = 0.0223
9.3.3 Standard Reference Materials:
SRM cert val(mq/kq) matrix n x s
DOLT-1 (4.18±0.28) liver 4 5.81 0.31
DORM-1 (0.086+0.012) muscle 15 0.098 0.017
NIES#6 (0.82+0.03) mussel 5 0.96 0.14
9.3.4 Recovery data from spiked samples (fish liver):
\
Level (ppm) %Recoverv
0.005 100%
0.025 96.0%
-------
10.0 REFERENCES
1. U.S. Environmental Protection Agency, Test Methods for
Evaluating Solid Waste, SW-486 Third Ed., Revision 1,
December 1987.
2. Slavin, W., G.R. Carnick, O.C. Manning, and E.
Pruszkowska, Perkin-Elmer Corp., Recent Experiences with the
Stabilized Temperature Platform Furnace and Zeeman Back-
ground Correction, Atomic Spectroscopy, Vol. 4, No. 3, 69,
1983.
-------
APPENDIX J
EXAMPLE QA/QC PROCEDURES AND
REQUIREMENTS FOR ANALYSIS
OF ORGANIC COMPOUNDS
[From: Puget Sound Estuary Program. 1990 (revised). Recommended
Guidelines for Measuring Organic Compounds in Puget Sound
Sediments and Tissue Samples. Prepared by PTI Environmental
Services, Bellevue, WA. In: Recommended Protocols and
Guidelines for Measuring Selected Environmental Variables
in Puget Sound, U.S. EPA, Region 10, Seattle, WA. (Looseleaf)]
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
QA/QC PROCEDURES AND REQUIREMENTS
QA/QC requirements are the foundation of this guidance document because they provide
information necessary to assess the comparability of data generated by different laboratories or
different analytical procedures. The following QA/QC variables are discussed in the order noted:
Initial and ongoing calibrations (used to establish and verify the quantification
technique)
Surrogate spike compounds (used to evaluate the analytical recovery of each sample)
Method blanks and field blanks (used to evaluate possible sources of laboratory and
field contamination)
Reference materials (used to evaluate laboratory accuracy)
Matrix spikes (used to evaluate the effect of sample matrix on the compound of
interest)
Spiked method blanks (used as a procedural check to evaluate method performance
prior to and during routine analysis of samples) (also called check standards)
Analytical replicates (used to evaluate precision of the analytical method and
instrumentation)
Field replicates (used to evaluate field variability).
Data for all QA/QC variables should be submitted by the laboratory as part of the data package.
Program managers and project coordinators should verify that requested QA/QC data are included
in the data package as supporting information for the summary data, and may wish to review key
QA/QC data (e.g., analytical replicate data or surrogate spike recoveries). Acceptable limits for
these variables are discussed in the following sections and summarized in Tables 6 and 8. A
detailed QA/QC review of the entire data package, especially original quantification reports and
standard calibration data, should be conducted by a technical expert. Guidelines on laboratory
data validation are available in U.S. EPA (1988).
Screening level analyses (see Table 4) should be conducted according to the QA/QC require-
ments of the most recent EPA CLP program document. The guidance provided in this section is
applicable to low parts-per-billion analyses of both sediment and tissue unless specifically noted.
Warning limits are numerical criteria that serve to alert data reviewers and users to possible
problems within the analytical system. When a warning limit is exceeded, the laboratory is not
obligated to halt analyses, but the reported data may be qualified during subsequent QA/QC
review. Action limits are numerical criteria that, when exceeded, require specific action by the
laboratory before data may be reported. Action limits are intended to serve as contractual controls
on laboratory performance. The warning and action limits are summarized in Table 8.
31
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
TABLE 8. SUMMARY OF WARNING AND ACTION LIMITS
FOR QUALITY CONTROL SAMPLES
Analysis Type*
Recommended Warning Limit Recommended Action Limitb
Ongoing calibration
Surrogate spikes
Method blanks
Reference materials
Matrix spikes
Spiked method blanks
(check standards)
Analytical replicates
Field replicates
Project manager decision
50 percent recovery1
Exceeds the limit of
detection*1
95 percent confidence
interval, if certified
50-150 percent
50-150 relative percent
difference
35 percent coefficient
of variation
Project manager decision
25 percent of initial
calibration
Follow EPA CLP guidelines0
Exceeds the practical
quantification limitd
Project manager decision
Project manager decision6
Project manager decision
50 percent coefficient
of variation (or a factor of
2 for duplicates)
Project manager decision
" The definition of each quality control sample is given in the QA/QC Procedures and
Requirements section of this report.
b Recommendations for corrective action when action limits are exceeded are given in text.
c Except when using the isotope dilution technique; see Appendix C for a summary of
acceptance limits and recommended corrective action for EPA Method 1625C.
"See Table 5.
e Zero percent spike recovery requires rejection of data.
32
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Organic Compounds
QA/QC Procedures and Requirements
4 Revised December 1989
CALIBRATION
The procedure used for calibration of analytical instruments can affect the accuracy of
analytical results and therefore can be considered an element of QA/QC. Both external standard
calibration and internal standard calibration procedures are used for organic analyses. External
standard calibration involves the preparation of standard solutions, independent of the samples, that
are used to determine the relationship between instrument response and concentration for the
substance being measured. Internal standard calibration is a procedure in which the instrument
responses from analytes are determined relative to the responses from one or more internal
standards added to every sample prior to extraction and sample processing. An ideal internal
standard has chemical and physical properties similar to those of the analyte. This latter calibration
technique is discussed in the section entitled Method Calibration Using the Isotope Dilution
Techniques.
Specific criteria for initial and continuing calibrations using the external standard calibration
technique are not supplied in this document because of the diversity of methods that might be
used. However, it is critical to adhere to the calibration criteria specified in the analytical method
being used.
Initial Calibration Using the External Standard Technique
Initial calibration is performed to determine the response of the instrument across a range of
concentrations of each analyte of interest. The relationship between response and concentration is
often called linearity. Response factors (RF) for analytes relative to standards at various concen-
trations are established by calibration.
The procedures and requirements in this section are generally for GC/MS determinations and
are consistent with the CLP requirements for external standard calibration of analytical instruments.
FrequencyEquipment should be subject to initial calibration at the beginning of the project
before any samples are analyzed, after each major equipment disruption, and when ongoing
calibration does not meet criteria.
Number of Calibration PointsRF values must be determined for at least three concentration
levels (five concentration levels or a five-point calibration, is preferable). The standard concentra-
tions tested should encompass the range of expected sample concentrations. The lowest standard
in this curve is analyzed at an on-coiumn concentration equivalent to the PQL for the sample set.
Reporting of results for an additional standard analyzed near the LOD (e.g., a sample
concentration equivalent to approximately 1-5 ng on-column for many compounds on GC/MS) is
recommended to provide evidence of the ability to report estimated quantities in the low concen-
tration range between the LOD and PQL. The use of this standard in the calibration curve is not
33
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
recommended because random error becomes relatively more significant at concentrations
approaching the ultimate detection limit of an instrument, and any random error in the determina-
tion of the calibration factor becomes a systematic error when used to calculate concentrations of
samples.
Warning LimitWarning limits are determined at the discretion of the project manager.
Action LimitFor most compounds, action limits are based on the variation among the RRF
calculated during the initial calibration. The percent relative standard deviation (percent RSD)
obtained from the RRF in the initial calibration should not exceed 30 percent.
Corrective ActionIf the percent RSD for the RRF exceeds 30 percent, the initial calibration
should be repeated. Failure to meet this calibration before analysis of samples may be cause for
omitting the data from regional databases.
ReportInitial calibration results within acceptable limits must be verified prior to the analysis
of samples. Summary data documenting initial calibration and any episodes requiring recalibration
and the corresponding recalibration data should be included with analytical results.
Ongoing Calibration Using the External Standard Technique
The ongoing calibration (single point) is used to check that the original three-point calibration
curve continues to be valid.
FrequencyFor GC/MS analyses, compare all area counts of the internal standard to those in
the standard for the day.
For GC/MS or GC/FID analyses, calibration should be checked at the beginning of each work
shift, at least once every 12 hours (or every 10-12 analyses, whichever is more frequent), and after
the last sample of each work shift.
For GC/ECD analyses, calibration should be checked at the beginning of each shift, every
6 hours (or every six samples, whichever is less frequent), and after the last sample of each shift.
Warning LimitWarning limits are determined at the discretion of the project manager.
Action LimitThe RRF determined for specific compounds should meet the following action
limits. The RRF determined for PCB and pesticides analyzed with GC/ECD should be within 25
percent of the initial calibration RRF, as specified in EPA CLP protocols. Those semivolatile and
34
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
volatile compounds that must meet the ongoing calibration 25 percent control limits per EPA CLP
are shown in Table 9.
Corrective ActionIf the action limit is not met, the initial three-point calibration will have
to be repeated. The last sample analyzed before the standard analysis that failed criteria should
then be reanalyzed. The results from the reanalysis should be within 15 percent of the results from
the original analysis. (The expected agreement between replicate injections of a complex extract
is 15 percent). If the results exceed a 25 percent difference, the instrument is assumed to have
been out of control during the original analysis. Reanalysis of samples should progress in reverse
order until it is determined that there is <25 percent difference between initial and reanalysis
results. In some cases results from reanalysis may exceed a 25 percent difference because of matrix
effects. If the next sample reanalyzed meets the 25 percent requirement, evidence exists for
assuming a matrix effect. Requirements for additional reanalysis should be at the discretion of the
program manager or project coordinator. For GC/MS, monitor the integrated area for the response
of all internal standards. Repeat the initial calibration or reanalyze the sample, if the observed
area/amount for any internal standard response varies by more than a factor of 2 when compared
to the observed area/amount for the response of the same internal standard of the standard mix
analyzed at the beginning of the shift.
ReportSamples requiring reanalysis should be identified. Reanalysis results should be
provided with the sample results. A discussion of the values causing exceedance of limits and
corrective actions taken should also be provided.
Method Calibration Using the Isotope Dilution Technique
The following introduction to calibration using the isotope dilution technique is excerpted
from Kirchmer et al. (1986). Isotope dilution mass spectrometry is a type of internal standard
calibration and analysis, in which the internal standard is an isotopically labeled analog of the
analyte. When added initially to the sample, the internal standard serves to correct for losses
during the processing of samples, and to compensate for errors owing to differences in injected
volume and unnoticed variations in instrument sensitivity. A stable isotope-labeled analog of the
analyte is an ideal internal standard, because its chemical and physical properties can be expected
to be almost identical to the analyte, thus assuring negligible differences in extraction, cleanup, and
chromatographic properties during sample processing (Watson 1976).
Internal standard calibration requires both a calibration solution for instrument calibration and
a spiking solution. The calibration standard is used to determine the relative responses of an
analyte and an internal standard, while the spiking solution is used to add a known amount of
internal standard to each sample prior to extraction, processing, and analysis. EPA Methods 1624C
and 1625C contain isotope dilution calibration and analysis procedures. In these procedures, the
isotopically labeled internal standards are added to the sample prior to extraction, and the results
are corrected for losses that occur during sample processing but do not occur in the instrument
calibration standards because they are not processed before analysis.
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QA/QC Procedures and Requirements
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TABLE 9. MINIMUM COMPOUNDS REQUIRED TO MEET
ONGOING CALIBRATION CONTROL LIMITS
Semivolatiles Volatile;
phenol vinyl chloride
1,4-dichlorobenzene 1,1 -dichloroethane
2-nitrophenol chloroform
2,4-dichlorophenol 1,2-dichloropropane
hexachlorobutadiene toluene
4-chloro-3-methylphenol ethylbenzene
2,4,6-trichlorophenol
acenaphthene
N-nitrosodiphenylamine
pentachlorophenol
fluoranthene
di-n-octyl phthalate
benzo(a)pyrene
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Organic Compounds
QA/QC Procedures and Requirements
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It is important to note that the isotope dilution technique (or any other spiking technique)
can not be used to correct for the efficiency of extraction because some analytes may be more
tightly bound to particles in the sample than are the isotopically labeled internal standards spiked
into the sample. Hence, tested or proven extraction procedures are considered essential to ensure
complete extraction of all analytes from the sample matrix.
In all methods used for the analysis of volatile organic compounds, including the isotope
dilution technique, the procedures used for calibration are identical to those used for analysis.
Because calibration bias can only occur when the procedures used for instrument calibration
standards differ from those used for complete analysis of samples, an isotope dilution technique
such as EPA Method 1624C offers no substantial reduction in calibration bias when compared to
a non-isotope dilution technique such as EPA Method 624. However, because random errors in
calibration can be converted to a bias for quantification of sample responses, it is important that
a sufficient number of calibrations standards be run to reduce bias.
In EPA Method 1625C, an instrument internal standard (2,2'-difluorobiphenyl) is added to the
final extract prior to instrument analysis to determine the physical percent recoveries of the
isotopically labeled internal standards that were added to the sample prior to extraction. The
physical percent recoveries of the isotopically labeled internal standards should meet QA/QC
criteria for the isotope dilution technique to be valid. Acceptance limits and recommendations for
corrective action are given in EPA Method 1625C and are reproduced in Appendix B.
Use of an instrument internal standard is only to obtain QA/QC data and not to measure the
analytes in the sample. The instrument internal standard is used to quantify selected analytes
under the following conditions:
An isotopically labeled analog of an analyte is not available, and there is no closely
eluting and structurally similar surrogate that can be substituted for an isotopically
labeled analog of the anaiyte (e.g., dg-naphthalene could be used to quantify
2-methylnaphthalene)
Certain QA/QC criteria specified in the method are not met for an analyte.
SURROGATE SPIKE COMPOUNDS
A surrogate is a type of check standard that is added to each sample in a known amount prior
to extraction or purging. The surrogate is not one of the target compounds for the analyses, but
should have analytical properties similar to those compounds. Because surrogate spikes are the
only means of checking method performance on a sample-by-sample basis, they are required for
all methods except isotope dilution methods.
Frequency
Surrogate spikes should be added to each sample unless the isotope dilution technique is used.
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Organic Compounds
QA/QC Procedures and Requirements
'Revised December 1989
Compound Type
A minimum of five surrogate spikes should be added to each sample (three neutral and two
acid compounds) when analyzing for semivolatile organic compounds. These surrogate spikes
should cover a wide elution range and include one of the more volatile compounds (e.g., ds-
phenol) as well as a degradable PAH [e.g., du-perylene or d12-benzo(a)pyrene]. Three surrogate
spikes are required for the analysis of volatile compounds.
Surrogates need not be isotopically labeled. They need only be compounds that are physically
and chemically similar to the analytes. Surrogates should be compounds that are not expected to
be present in the samples.
At least one surrogate spike is required as a check on recovery of pesticides and PCB
mixtures. This compound must be well-resolved, must not co-elute with any PCB or pesticide
analyte, and should behave similarly to the analytes. This surrogate will likely not be a perfect
PCB/pesticide analog. Possible standards are dibutylchlorendate (used in the EPA CLP), hexa-
bromobenzene (used at EPA/Ecology Manchester laboratory), dibromooctofluorobiphenyl (used by
Northwest NMFS and by EPA/Ecology Manchester laboratory), and isodrin (the endo-endo isomer
of aldrin).
Warning and Action Limits
The warning and action limits in the most recent EPA CLP methods are recommended for use
in evaluating surrogate recoveries. These limits are only valid if surrogates are added at the
concentrations specified in the CLP methods.
Corrective Action
The corrective actions specified in the most recent EPA CLP protocols should be followed
when action limits for surrogate recoveries are exceeded.
Report
Percent recovery values in sample and method blanks for all surrogate compounds analyzed
should accompany the data. Data are not to be recovery corrected.
METHOD BLANKS AND FIELD BLANKS
Method blanks are analyzed to assess possible laboratory contamination of samples
associated with all stages of preparation and analysis of sample extracts. Contamination is of
concern because it can result in a false positive result (i.e., erroneous reports of the compound as
present in the sample) or overestimates of sample concentrations. Alternatively, it is possible that
38
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
method blanks could incorrectly indicate contamination to be present in a sample. If analyte data
are incorrectly rejected on the basis of method blank results, then a false negative result would
occur. Protection against false positive results is given greatest weight in programs that generate
data for possible use in litigation. Guidelines consistent with EPA CLP functional guidelines for
QA review (EPA 1988) are recommended in this section for qualifying data associated with
significant blank contamination.
Frequency
At a minimum, one method blank should be run for every extraction batch (or for volatile
compound analyses, every 12-hour shift, whichever is more frequent).
Warning Limit
The warning limit is reached for a contaminant in a blank when its concentration exceeds the
LOD.
Action Limit
The action limit for a contaminant is reached when its concentration in a blank exceeds the
PQL.
Corrective Action
If any warning limit is exceeded, likely sources of contamination should be discussed in the
cover letter of the data report. If action limits are exceeded, analyses should be halted until the
contaminant source is eliminated or greatly reduced, or the data recipient has been notified and an
acceptable plan of action has been determined.
The following compounds are some of the common laboratory contaminants that often appear
in method blanks: methylene chloride, acetone, toluene, 2-butanone (all volatile compounds), and
selected phthalate esters (semivolatile compounds including bis-ethylhexyl phthalate, butyl benzyl
phthalate, and di-n-octyl phthalate). Sample data should be qualified as undetected at either the
higher of the sample results or at the PQL when the sample concentration is less than 10 times the
blank concentration for these compounds (i.e., the blank response is >10 percent of the sample
response). The appropriate qualifiers for such data are ZU, indicating a detection limit established
because of significant blank contamination.
Sample data for other contaminants should be qualified as undetected (ZU qualifiers) at the
higher of the sample result or the PQL when the sample concentration is less than 5 times the
blank concentration (i.e., the blank response is >20 percent of the sample response). If gross
contamination exists (i.e., saturated peaks by GC/MS in the method blank), concentration data for
39
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Organic Compounds
QA/QC Procedures and Requirements
' Revised December 1989
all compounds affected should be rejected (R qualifier) and not incorporated into regional
databases.
Report
Laboratories should report original sample data without blank correction and should report
data for all method blanks such that the contribution to associated samples can be determined. If
contamination exists but does not exceed the guidelines in this section, then corrections may be
applied to the data during independent QA review at the discretion of the project manager to
minimize the effects of laboratory contamination on what may otherwise be unqualified analyte
concentrations. For such corrections, the blank analyses are assumed to be representative of the
potential contamination in sample extracts. However, blank correction is not acceptable under the
EPA CLP (U.S. EPA 1988).
Any reported concentrations that have been blank-corrected must be qualified with a Z
qualifier. Data sets that have not been blank-corrected must be explicitly identified as such. In
all cases, results for method blanks and a cross-reference to identify associated samples for each
method blank analysis must be summarized in data reports.
Blank analyses may not involve the same weight, volume, or dilution factors as the associated
samples. These factors must be taken into consideration when blank-correcting data or applying
the following guidelines for data qualification, such that a comparison of the total amount of
contamination is actually made.
REFERENCE MATERIALS
The following definitions of reference materials will be adhered to throughout these guide-
lines:
Reference MaterialA material or substance, one or more properties of which are
sufficiently well established to be used for the calibration of an apparatus, the
assessment of a measurement method, or for assigning values to materials. In Puget
Sound, a regional reference material (RRM) has been developed for marine
sediments by NOAA/NMFS for EPA, NOAA, and other agencies and laboratories.
The RRM is a fresh-frozen sediment homogenate from Sequim Bay, spiked with
selected organic acid and neutral compounds at low concentrations. Available
samples of the RRM can be requested from the EPA Region 10 Office of Puget
Sound. This RRM has been analyzed in interlaboratory studies using NOAA
methods, the results of which have ben compared with analyses by various investi-
gators using different methods. Although not certified, this RRM is useful for
intercomparing Puget Sound studies and is strongly recommended in every project.
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
Certified Reference Material (CRM)A reference material, one or more of whose
property values are certified by a technically valid procedure, accompanied by or
traceable to a certificate or other documentation that is issued by a certifying body
(e.g., National Research Council of Canada, National Institute of Standards and
Technology). A standard reference material is a CRM issued by the National
Institute for Standards and Technology. There is no marine sediment CRM available
for organic compounds of concern in Puget Sound, except for a marine sediment
certified by the National Research Council (Canada) for organotin compounds (i.e.,
PACS-1). Tissue homogenates are sometimes available as reference materials (e.g.,
mega mussel sample, EPA, Environmental Research Laboratory, Narragansett, Rhode
Island). An oyster CRM may be available by special request for selected organic
contaminants.
RM and CRM provide information on the accuracy (i.e., how near the measurement is to its true
value) as opposed to precision (i.e., how near replicate measurements are to each other). When
analyzed in replicate, RM and CRM provide information on both accuracy and precision for a
particular matrix type. Routine analysis of the RRM for Puget Sound sediment is recommended
to provide data for interlaboratory comparisons.
Frequency
If five or fewer samples are submitted for analysis, one RM (or CRM, if available) is recom-
mended, at the discretion of the project coordinator. If analysis of an available reference material
is not included, the data may be qualified before entry in regional databases. If 6-50 samples are
submitted, at least one RM should be analyzed. For submittals of more than SO samples, one RM
should be analyzed for each 50 samples.
Warning Limits
For analyses of CRM, the reported values should be within the 95 percent confidence interval
certified by the agency dispensing the CRM. If more than two anaiytes fall outside of the 95
percent confidence interval, corrective action should be taken. If CRM are unavailable, control
limits may not be appropriate, but analyses of RM can still be used to assess overall accuracy or
method bias (in conjunction with matrix spikes and surrogate compounds).
Action Limits
Action limits are only appropriate for analysis of CRMs (i.e., action limits are not recom-
mended for RM analyses). Action limits may be determined at the discretion of the project
manager.
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Organic Compounds
QA/QC Procedures and Requirements
' Revised December 1989
Corrective Action
It is recommended that the RM, if available, be analyzed prior to analysis of any samples.
If values are outside the action limits, the RM should be reanalyzed to confirm the results. If the
values are still outside action limits in the repeat analysis, the samples may be analyzed and
reported with statements that describe the possible bias of the results in the cover letter accom-
panying the data. Alternatively, the laboratory may be required to repeat the analyses until action
limits are met before continuing with sample analyses. Determination of the appropriate corrective
action is the responsibility of the program manager or project coordinator and should be specified
in the statement of work for the laboratory.
Report
The laboratory should keep a running record of results obtained for each analysis of a RM.
Observed results should be compared to the mean provided by the originator of the RM, the
observed mean obtained from repeated analyses by the laboratory, and acceptable range limits.
Minimum reporting of RM results with laboratory data should include observed and expected
values and the acceptable range limits. The steps for corrective action and observed bias relative
to existing RM values should be reported and discussed in the cover letter.
MATRIX SPIKES
Matrix spike results are a common form of recovery data provided by laboratories, and are
required by the EPA CLP protocol for screening level analyses. Matrix spike results are of less
value than RM results, because the efficiency of the extraction of the compounds of interest from
the sample matrix is not accounted for in matrix spike results. Matrix spikes are preferred as QC
samples only in the absence of a suitable RM. Matrix spikes should include a wide range of
representative analyte types (preferably all analytes). Compounds should be spiked at ca. 5 times
the concentration of compounds in the sample or 5 times the PQL.
It was agreed in a 1989 work group that matrix spike samples will be recommended to provide
data for cross-comparing the isotope dilution technique and matrix spike results, which are usually
obtained at different concentration levels and serve different purposes. Matrix spike results are
used to provide an indication of interferences during sample processing and analysis using native
compounds typically at moderate concentrations. The isotope dilution technique is used to correct
for losses during the processing of samples and to compensate for sample-specific instrument
analysis errors using isotopically-labeled analogs at moderate to low concentrations.
Spiking concentrations that are low relative to sample concentrations increase random error in
the matrix spike analysis. Spiking concentrations that are too high reduce the value of matrix spike
analyses for interpreting sample interferences at representative concentrations of pollutants.
For comparison, EPA CLP spiking levels for sediments (U.S. EPA 1988) result in approxi-
mately 100 ng on-column for organic base/neutral compounds and 200 ng on-column for organic
acids assuming a 1-mL final dilution volume, 100 percent recovery, and undetected concentrations
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Organic Compounds
QA/QC Procedures and Requirements
'Revised December 1989
in the unspiked sample. These levels represent approximately 6,700-13,000 A«g/kg dry weight
assuming a 30-gram sediment sample with 50 percent moisture, or approximately 10-20 times the
lowest contract required limit of 330 /ig/kg wet weight (660 /*g/kg dry weight in this example).
The same spiked amount in a 100-gram sample with 50 percent moisture would result in
approximately 2,000-4,000 /Jg/kg dry weight concentrations under the same assumptions for other
variables. This spiking level would be approximately 40-80 times an LOD of 50 pg/kg dry weight
for mortified CLP procedures (i.e., assuming lowest calibration at 10 ng on-column and 0.5-mL
final dilution volume). Matrix spikes for marine samples should be similar to the levels that are
expected in the environment, assuming that the analytical technique is sufficient to produce
reproducible results at these concentrations. In many areas of Puget Sound, environmental
concentrations of organic contaminants are closer to the PQL than to the spiking levels used for
hazardous waste samples in the CLP. The EPA CLP spiked amount is most appropriate for highly
contaminated samples that occur in small areas of Puget Sound.
The range of LOD in Table 5 will bracket or exceed the concentrations of many organic
compounds in Puget Sound reference area sediments. Concentrations of compounds in contam-
inated urban bay samples may exceed 10-100 times reference area concentrations, and will often
exceed the PQL in Table 5. Ideally, matrix spike results would be obtained for a range of sample
types. Given limited resources, it is probably of greater value to assess possible interferences in
moderately contaminated samples than in reference area samples.
Frequency
If fewer than 20 samples are submitted, at least one matrix spike and one matrix spike
duplicate should be run. If 20 or more samples are submitted, one matrix spike and one matrix
spike duplicate should be run for each 20 samples.
Warning and Action Limits
Recovery of >50 percent of matrix spike compounds accompanied by good precision is
considered to be acceptable. Low matrix spike recoveries may result from matrix interferences in
the sample. Therefore, poor results alone should not be cause for data qualification. Rigorous
control limits for qualifying data are not recommended because of the potential difficulty in
determining when matrix spike results indicate bias due to sample interferences rather than the
expected random error of the difference between sample results before and after spiking.
However, sample data should be rejected whenever zero percent recovery of an associated matrix
spike compound has occurred.
Corrective Action
In the event of poor matrix spike performance, alternative QA measures should be considered
before any associated sample data are qualified as estimates (£) or underestimates (C), or in very
extreme cases, rejected (R). These measures include results of reference material analyses.
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
surrogate recoveries, and the physical percent recoveries of isotopically labeled internal standards,
if using the isotope dilution technique. Professional judgment must be used to determine which
samples should be associated with each matrix spike analysis.
Report
An explanation of low percent recovery values for matrix spike results should be discussed in
the cover letter accompanying the data package.
SPIKED METHOD BLANKS
Spiked method blanks, sometimes called check standards, are method blanks spiked with
surrogate compounds and analytes. Such samples are useful in verifying acceptable method
performance prior to and during routine analysis of samples. Spiked method blanks do not take
into account sample matrix effects, but can be used to identify basic problems in procedural steps.
Spiked method blanks can also provide minimum recovery data when no suitable RM is available
or when insufficient sample size exists for matrix spikes. Target analyte compounds and surrogate
compounds should be added to a method blank prior to extraction.
Frequency
A spiked method blank should be analyzed before analysis of samples when a method is used
for the first time in a project and after each method modification.
Warning and Action Limits
The.warning and action limits in the most recent EPA CLP methods are recommended for use
in evaluating spiked method blank recoveries. These limits are only valid if spiked compounds are
added at the concentrations specified in the CLP methods.
Corrective Action
Analysis of actual samples should not begin until results are within action limits.
Report
Detailed notes should be kept in a laboratory notebook. The notes should discuss method
spike results exceeding recommended limits, corrective action, and verification of instrument
response within acceptable criteria. This information need not be included with data package
results because analysis cannot continue until all results are within action limits.
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
ANALYTICAL REPLICATES
Analytical replicates (usually duplicates are sufficient when using a protocol that is well
proven in the laboratory) provide precision information on the actual samples. Replicate analyses
are useful in assessing potential sample heterogeneity and matrix effects.
Frequency
If five or fewer samples are submitted for analysis, a minimum of one replicate is recom-
mended, at the discretion of the program manager or project coordinator. If 6-19 samples are
submitted, at least one analytical replicate should be analyzed. If at least 20 samples are submitted,
one blind replicate (i.e.. unknown to the laboratory) analysis should be required, for a minimum
replication of 5 percent overall.
Pooling of variances in duplicate analyses from different sample batches is recommended for
estimating the standard deviation of replicate analyses. This technique is preferred to the analysis
of a blind triplicate sample. Blind replicates also provide information on potential laboratory bias
in analyzing known QA samples. Because there are limited numbers of blind replicates analyzed
in a sample case, there is some value in analyzing a triplicate measurement (i.e., there may be no
other blind replicates that can be pooled). However, the use of a triplicate analysis is at the
discretion of the project manager.
Warning and Action Limits
Based on data of Horwitz et al. (1980), who charted interlaboratory precision as a function of
concentration, a 30 percent coefficient of variation (a statistical measure of precision) is expected
for concentrations ranging between 1 and 50 jig/kg dry weight. Compound-specific advisory limits
are provided in the EPA CLP protocols.
These advisory limits are recommended as warning limits. Extensive discussion of precision
requirements occurred at a Puget Sound organics workshop in 1985 and in subsequent work
sessions. Based on professional judgment of analysts and regional program managers in attendance,
it was decided that a difference of no more than a factor of 2 among replicates would be the basis
for the laboratory action limit (i.e., approximately 50 percent coefficient of variation). Exceedance
of the action limit would require automatic reanalysis to confirm the results. Many compound
analyses are more precise. There was discussion about easing the action limit if the results were
well beyond some regulatory guideline for acceptable contamination, and tightening the action limit
if the results were close to some regulatory guideline. However, most data will have multiple uses
and adjustable limits will be difficult to apply as a laboratory control.
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Organic Compounds
QA/QC Procedures and Requirements
Revised December 1989
Corrective Action
If results fall outside the action limit for more than two compounds, a repeat analysis is
required to determine the origin of the problem before any data can be reported. If results
continue to exceed action limits, subsequent corrective action is at the discretion of the program
manager or project coordinator.
Report
A discussion of the results of duplicate sample analysis should include probable sources of
laboratory error and an assessment of natural sample variability. If data are to be qualified on the
basis of duplicate results, justification for assigning the data qualifier should be provided.
FIELD REPLICATES
Field replicates are separate samples collected at the identical station in the field and
submitted for analysis. These QA samples are useful in determining total sample variability (i.e.,
analytical variability plus field variability).
Frequency
The program manager or project coordinator determines the frequency with which field
replicates are collected. Laboratory replicates must be coordinated with field replicates so that
sampling and analytical variability will be measured for the same station.
Warning and Action Limits
Warning and action limits are not appropriate when measuring field sampling variability since
the analytical laboratory does not have control over the variability due to field sampling.
Corrective Action
No corrective action is recommended for field replicate analyses.
Report
If it is determined that variability observed in field duplicate results can be partially explained
by analytical or sampling variability, it should be noted and discussed in a QA/QC evaluation of
the data.
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Organic Compounds
Data Reporting Requirements
Revised December 1989
DATA REPORTING REQUIREMENTS
The following items are recommended to be provided by the analytical laboratory. The items
listed below include most, but not all, of the documentation required by the EPA CLP. This
documentation is necessary for independent QA/QC review of the data, and its delivery (or
availability for inspection at the laboratory) should be required in the original statement of work
if an independent QA/QC review is to be conducted:
A cover letter discussing analytical problems (if any) and referencing or describing
the procedure used
Reconstructed ion chromatograms for GC/MS analyses for each sample
Mass spectra of detected target compounds (GC/MS) for each sample
GC/ECD or GC/FID chromatograms for each sample
Raw data quantification reports for each sample
A calibration data summary reporting the calibration range used [and for GC/MS,
spectra and quantification reports for decafluorotriphenylphosphine (for semivolatile
analyses), bromofluorobenzene (for volatile analyses), or an appropriate substitute
standard]
Final dilution volumes, sample size, wet-to-dry ratios, and instrument detection
limit
Analyte concentrations with reporting units identified to two significant figures
unless otherwise justified
Quantification of all analytes in method blanks (ng/sample rather than using a
hypothetical sediment weight to calculate ng/g)
Method blanks associated with each sample
Tentatively identified compounds (if requested) and methods of quantification
(include spectra)
Recovery assessments and a replicate sample summary (laboratories should report all
surrogate spike recovery data for each sample; a statement of the range of recoveries
should be included in reports using these data)
Data qualification codes and their definitions (qualifier codes used by PSEP are
shown in Table 10).
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Organic Compounds
Data Reporting Requirements
Revised December 1989
TABLE 10. QUALIFIER CODES USED BY PSEP
Qualifier
Code Description
C Combined with unresolved substances
E Estimate
G Estimate is greater than value shown
K Detected at less than detection limit shown
L Value is less than the maximum shown
M Value is a mean
Q Questionable value
T Detected below quantification limit shown
U Undetected at the detection limit shown
X Recovery less than 10 percent
Z Blank-corrected
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Organic Compounds
Data Reporting Requirements
Revised December 1989
RECOVERY AND BLANK CORRECTIONS
Recovery corrections based on a limited number of internal standards should not be applied.
However, correction for bias due to compound losses in sample processing is inherent to isotope
dilution techniques such as EPA Method 1625C, and is acceptable.
Blank corrections should not be applied by the laboratory. Concentrations of analytes in
method blanks should be reported by the laboratory as pan of the data report; corrections may then
be made by program or project data managers. All such corrections must be indicated by assigning
the Z data qualifier to the data value (or to the detection limit if the contamination is significant
as described in the QA/QC Procedures and Requirements section). Whether data are corrected or
not, the concentration of analytes in method blanks should always be given in reports. Results for
several analytes are often suspect because they are commonly reported in method blanks. For
example, reported concentrations of phthalates, methylene chloride, acetone, chloroform, benzene,
2-butanone, and toluene in samples should be carefully compared to those in the method blank
before the compounds are assessed as environmental contaminants of concern.
DETECTION AND QUANTIFICATION LIMITS
Concentrations, LOD, and PQL are reported in terms of fig/kg dry weight sediment and
/ig/kg wet weight tissue. No detected concentrations should be reported below the LOD.
Concentrations reported between the LOD and PQL are usable after qualification as estimates using
the T qualifier (Table 10). Concentrations reported above the PQL are usable without qualification
unless qualification is deemed appropriate during QA review. Laboratory statements of work that
reference PSEP protocols for low-level analyses must, at a minimum, specify the PQL as the
maximum acceptable limit to be reported for samples without significant interferences.
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Organic Compounds
Cost Implications
Revised December 1989
COST IMPLICATIONS
Higher analytical costs may be required to achieve lower LOD and to increase the precision
of results (Table 11). Lowering LOD to achieve project goals can increase costs, particularly if
additional sample cleanup is required. Additional sample cleanup may also improve precision
because interferences are removed. However, the range of precision expected at a given detection
limit in Table 11 reflects primarily differences in the analytical variability of a set of diverse
compound types. For example, hydrocarbons can typically be recovered at the lower end of each
range of precision estimates shown, while phthalates and some acid compounds are often analyzed
much less precisely (i.e., higher coefficient of variation). Hence, a wide range of precision may
be found at constant cost when analyses cover a wide range of compounds.
The major determinants of the range of analytical costs at a given detection limit are
individual laboratory efficiencies and the specific analytical technique used (i.e., methods having
large differences in cost can yield similar detection limits and precision of results). Nevertheless,
lowering the required detection limits tends to raise the minimum cost expected for the analysis;
a range in costs can still be expected above this minimum for different laboratories.
The major goal of QA/QC activities is to improve and control the accuracy of results. A
successful QA/QC program will minimize the quantity of data that are rejected (a waste of
sampling and analysis resources), improve the legal defensibility of the data set, and enable an
assessment of comparability among data sets. Additional analytical costs are incurred to achieve
these^oals because QC samples must be analyzed with each sample set. The percent of the total
analytical cost attributed to QC samples as a function of the number of samples submitted for
analysis is shown in Figure 1. The number of QC samples for each sample set is based on the
minimum frequency of analysis recommended in the QA Procedures and Requirements section.
The percent of total costs attributed to QC samples rapidly declines as the number of samples
submitted for analysis increases from 1 to 20. The percent QC cost is constant at 10 to 15 percent
of total costs (depending on whether matrix spike analyses are conducted) in sets of greater than
50 samples.
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Organic Compounds
Cost Implications
.Revised December 1989
TABLE 11. APPROXIMATE COST RANGE OF ANALYSES
AS A FUNCTION OF MATRIX, DETECTION LIMITS,
AND PRECISION'
Matrix
Sediments
Extractable acid/base/neutrals
PCB/pesticides
Volatiles
Practical
Quantitation
Limit
(Mg/kg
dry weight)
>200
<200
0.01 - 15
2-10
Typical
Precision
(Mg/kg
dry weight)
±20% - >±100%
±20% - >±100%
<±5% - >±50%
<±5% - >±50%
Approximate
Cost Rangeb
(Per Analysis)
$404 - >$600
$404 - >$700
$153 - $555
$213 - $300
Tissues
Extractable acid/base/neutrals
PCB/pesticides
Volatiles
>330
20 - 100
0.1 - 20
5 - 20
<±5% - >±100%
<±5% - >±100%
<±5% - >±100%
<±10% - >±100%
$454 - $900
$454 - $700
$203 - >$555
$263 - >$400
1 Cost range is based on multiple quotes compiled in 1989 for specific applications and
greater than five samples. The actual costs may vary from the range shown. The table
provides a general perspective of the relative difference in costs.
b NOTE: Each cost range is mainly the result of laboratory differences in technique pricing
and number of analytes, not the range in precision or detection limits shown.
51
-------
Organic Compounds
Cost Implications
Revised December 1989
UJ
en
in
en
u.
o
o
o
u.
O
m
U
cc
UJ
OL
80
70
60
50
40
30
20
10
0
1 RM
1 REP
1 MS
1 RM
1 REP
1 MS
1 RM
2 REP
1 MS
1 RM
3 REP
3 MS
2RM
5 REP
SMS
4RM
10 REP
10 MS
20
50
100
200
NUMBER OF FIELD SAMPLES IN SET
Assumes calibration runs and method blanks included in
per sample cost, and method has been checked with
spiked blanks.
RM - Certified Reference Material
REP - Replicate Analysis
MS - Matrix Spike Sample
Figure 1. Percent of the total analytical cost attributed to QC samples for analysis of organic
compounds in Puget Sound as a function of the number of samples submitted for analysis
52
-------
APPENDIX K
EXAMPLE QA/QC PROCEDURES AND REQUIREMENTS
FOR ANALYSIS OF METALS
[From: Puget Sound Estuary Program. 1990 (revised). Recommended
Protocols for Measuring Metals in Puget Sound Water,
Sediments, and Tissue Samples. Prepared by PTI Environmental
Services, Bellevue, WA. In: Recommended Protocols and
Guidelines for Measuring Selected Environmental Variables
in Puget Sound, U.S. EPA, Region 10, Seattle, WA. (Looseleaf)]
-------
Metals
Quality Assurance/Quality Control
Revised December 1989
QUALITY ASSURANCE/QUALITY CONTROL
QUALITY ASSURANCE/QUALITY CONTROL MEASURES INITIATED IN THE ANALYTICAL
LABORATORY
Standard laboratory practices for cleanliness of laboratory ware, reagents, solvents, gases, and
instruments must be followed. For additional guidelines not covered in this report, see Sections 4
and 5 of Handbook for Analytical Quality Control in Water and Wastewater Laboratories (U.S. EPA
1979b) and U.S. EPA (1988).
Instrument Quality Assurance/Quality Control Checks
Instrument QA/QC checks necessary for all the EPA-approved methods discussed in the
previous section include:
Calibration blank
Initial calibration and initial calibration verification
Continuing calibration verification
CRMs
ICP interference check sample analysis (for ICP only).
Guidelines for instrument calibration are given in Methods for Chemical Analysis of Water
and Wastes (U.S. EPA 1979a). In general, calibrations must be conducted each time the instrument
is set up, and or on a daily basis when analyses are in progress. Calibration procedures should
follow the procedures specified for each analysis in the EPA protocols. In addition, as specified
for the CLP (U.S. EPA 1987), after an instrument system has been calibrated, the accuracy of the
initial calibration should be verified and documented for every analyte by the analysis of EPA
quality control solutions. Where a certified solution of an analyte is not available from EPA or any
source (e.g., tin), analyses should be conducted on an independent standard at a concentration other
than that used for calibration, but within the calibration range. When measurements for the
certified components exceed the action limits, the analysis must be terminated, the problem
corrected, the instrument recalibrated, and the recalibration verified.
For ICP and AA analyses, all work should be performed using continuing calibration as
outlined in the EPA CLP Statement of Work. Frequency of continuing calibration analysis is
10 percent of the samples or every 2 hours during an analysis run, whichever is more frequent.
23
-------
Metals
Quality Assurance/Quality Control
Revised December 1989
Method Quality Assurance/Quality Control Checks
Laboratories should perform the quality control checks listed below:
Procedural or method blank
Spiked sample analysis
Replicate or duplicate sample analysis
GFAA method of standard addition (if necessary)
Laboratory control sample or CRM analysis.
Details on the use and application of these checks, along with action limits, reporting
requirements, and corrective actions, are discussed in U.S. EPA (1987). The frequencies of
application of these checks are 5 percent or one per batch, whichever is more frequent. The action
limits are ±20 relative percent difference for duplicates, 75-125 percent recovery for spikes, and
80-120 percent recovery for the analysis of CRMs. Other recovery limits may be accepted if they
are specified for a particular CRM. For the purpose of QA/QC, the required LODs are listed in
Table A-6 of Appendix A. For batches of five or fewer samples, the minimum QC checks should
be two blanks and the analysis of a CRM. Since spiked metals do not necessarily equilibrate with
metals in the original matrix, a CRM from an ambient sample is preferred over a CRM with spiked
metals. For the analysis of total or dissolved metals in ambient estuarine or coastal seawater, the
National Research Council of Canada's CASS-1 nearshore seawater reference material has become
the accepted standard of trace metal chemists (NRCC Marine Chemistry Standards, Ottawa, Canada
K1A OR6). If an analyte is not in the CRM, a matrix spike must be analyzed for that particular
analyte.
In general, for small batches of fewer than five samples, the priority of QC checks should be:
CRMs > check standards > analytical duplicates > matrix spikes. If several small batches of the
same matrix are analyzed sequentially (e.g., for several small projects), a CRM can be analyzed at
a frequency of 5 percent overall, with at least one sample duplicate analyzed per individual batch.
If any QA/QC check does not meet the established criteria, the laboratory QA officer should notify
the project QA coordinator and the methods should be adjusted or, if necessary, data qualifications
and reasons for noncompliance with QA/QC criteria should be submitted with the analytical data
report.
QUALITY ASSURANCE/QUALITY CONTROL MEASURES INITIATED IN THE FIELD
In addition to the QA/QC checks listed above, the following five checks may be initiated at
the time of sample collection (Plumb 1981):
Transfer (preservation) blanks
Cross-contamination blanks
Blind replicate samples
24
-------
Metals
Quality Assurance/Quality Control
Revised December 1989
Field replicate samples
Blind CRMs.
These checks may not replace any of the QA/QC measures outlined previously, but may be
included as part of the overall QA/QC program.
Transfer (Preservation) Blanks
Reagents used for sample preservation can become contaminated after a period of use in the
field. Analysis of the transfer blank will enable detection of contaminants in reagents and
contaminants introduced during shipping.
To obtain a transfer blank, a sample container is filled in the field with deionized or distilled
water to the same volume as that of samples. The transfer blank is preserved as if it were a normal
water sample and sent to the laboratory for analysis.
Cross-Contamination Blanks
Carry-over from one sample to the next can occur if field equipment is not thoroughly
cleaned between samples. The cross-contamination blank is designed to verify the absence of
carry-over.
To obtain a cross-contamination blank, decontaminated sample-handling equipment (e.g.,
spatulas, augers, core barrels) is rinsed with deionized or distilled water, and the rinse water is
collected. This sample is preserved as if it were a normal water sample and sent to the laboratory
for analysis.
Blind Replicate Samples
To obtain blind replicates (i.e., replicates that are not known to be replicates by the
laboratory) a collected sample is homogenized and split in the field into at least two identical
aliquots, and each aliquot is treated and identified as a separate sample. Caution must be exercised
to prevent field contamination. The replicates are sent blind to the laboratory. Homogenization
and splitting may also be done in the laboratory. However, the identity of such samples must be
unknown to the analyst. The mean, standard deviation, and relative percent standard deviation are
calculated by the project QA coordinator.
In addition, a collected sample may be split in the field into two aliquots, and one aliquot sent
to a different laboratory for analysis. The relative percent difference is calculated by the project
QA coordinator. If project constraints require the use of more than one laboratory, comparability
of the laboratories must be established using CRMs.
25
-------
Metals
Quality Assurance/Quality Control
Revised December 1989
Field Replicate Samples
Field replicates are separate samples collected at the identical station and submitted for
analysis. These samples are useful in determining total sample variability (i.e., analytical variability
plus field variability).
Blind Certified Reference Materials
Blind analysis of CRMs can be conducted to determine the accuracy of laboratory analyses.
To conduct analysis of a blind CRM sample, a subsample of a CRM is placed in a sample container
and sent blind to the laboratory. The percent recovery is calculated by the project QA coordinator.
It is recommended that the same action limits be established for blind analysis of CRMs as for
the laboratory QA/QC checks. The project QA coordinator must inform the laboratory if the
action limits are exceeded and corrective actions must be taken (see below).
CORRECTIVE ACTIONS
If the concentration of the field or laboratory blank is greater than the detection limit required
in the contract, all steps in the sample handling should be reviewed. For blank contamination, see
U.S. EPA (1988) for appropriate corrective action. Many trace metal contamination problems are
due to airborne dust. Contamination from airborne dust can be minimized by keeping containers
closed and by rinsing all handling equipment immediately before use. See the Contaminant Sources
section of this report (above) for further information.
Poor replication may be caused by inadequate mixing of the sample before taking aliquots,
inconsistent contamination, inconsistent digestion procedures, or instrumentation problems.
Instrumentation problems may be corrected by recalibration and calibration blank analysis.
Inconsistent digestion may be caused by analyte loss during digestion; see below for corrective
action. Also, hotplates may not hold a constant temperature across their surfaces. This problem
can be alleviated by changing the position of digestion vessels at regular intervals during heating.
Poor performance on the analysis of CRMs or poor spike recovery may be caused by the same
factors that were discussed above for poor replication. However, if replicate results are acceptable,
poor CRM performance or spike recovery may be caused by loss of analyte during digestion. To
check for analyte loss during digestion and for low recovery due to interferences during analysis,
spike the sample after digestion and compare results to those of the predigestion spike. If the
results are different, the digestion technique should be adjusted. If the results are not significantly
different, dilute the sample by at least a factor of 5 and reanalyze. If spike recovery is still poor,
standard additions, matrix modifiers, or the use of another method is required.
26
-------
Metals
Data Reporting
Revised December 1989
DATA REPORTING
Concentrations of elements in sediment, tissue, and water samples should be corrected for
method (or procedural) blanks. However, some EPA programs do not allow for blank correction
of results (e.g., CLP). Therefore, to distinguish these data, any result that has been blank corrected
should be qualified with the Z qualifier (blank correction). Results for sediments should be
reported on a dry-weight basis. Results for tissues should be reported on a wet-weight basis along
with the percent moisture content (wet/dry ratio) of the tissue. If the tissue sample is too small
to do both a metals analysis and a moisture determination, omit the latter analysis.
DATA REPORT PACKAGE
The data report package for analyses of each sample should include the following items:
A summary of the digestion procedure.
Tabulated results in units of mg/kg (dry weight) for sediment, mg/kg (wet weight)
for tissue, and pg/L for water (validated and signed in original by the laboratory
manager or designee).
Method blanks for each batch of samples.
Results from analysis of CRMs and matrix spikes.
All data qualifications and explanations for all departures from the analytical
protocols.
Results for all the QA/QC checks initiated by the laboratory.
Tabulation of instrument detection limits and LODs achieved for the samples. The
LOD value reported by the laboratory for the analyses should be calculated as three
times the standard deviation of the method blanks. A minimum of three method
blanks need to be analyzed to calculate the LOD. When the concentration of the
metal in a sample is less than the LOD after the method blank is subtracted, the ZV
qualifiers (blank corrected to the detection limit) should be entered together with
the LOD in the data report. Data reviewers may qualify data for which the method
blank concentration exceeds 20 percent of the original metal concentration.
27
-------
Metals
Data Reporting
Revised December 1989
BACKUP DOCUMENTATION
All laboratories are required to submit results that are supported by sufficient backup data and
quality assurance results to enable independent QA reviewers to conclusively determine the quality
of the data.
Legible photocopies of original data sheets should be available from the laboratory with
sufficient information to identify unequivocally the following items:
Calibration results
Calibration and method blanks
Samples and dilutions
Duplicates and spikes
Control samples or CRMs
Any anomalies in instrument performance or unusual instrument adjustments.
28
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APPENDIX L
SOURCES OF ERA-CERTIFIED
REFERENCE MATERIALS AND STANDARDS
-------
EPA-certified analytical reference materials for priority pollutants and related compounds are
currently produced under Cooperative Research and Development Agreements (CRADAs) by the
following organizations:
EPA-certified organic quality control samples, including standards for pesticides in fish tissue,
are produced by:
Supelco, Inc.
Supelco Park
Bellefonte, PA 16823-0048
TEL: 1 -800-247-6628 or 1 -814-359-3441
FAX: 1-814-359-3044
Contact: Linda Alexander
EPA-certified organic solution standards for toxic and hazardous materials (formerly the EPA
Toxic and Hazardous Materials Repository) are produced by:
NSI Environmental Solutions, Inc.
P.O. Box12313
2 Triangle Drive
Research Triangle Park, NC 27709
TEL: 1 -800-234-7837 or 1 -919-549-8980
FAX: 1-919-544-0334
EPA-certified neat organic standards, including neat pesticide standards (formerly the EPA
Pesticide Repository), are produced by:
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
TEL: 1-401-294-9400
FAX: 1-401-295-2330
Contact: Dr. Bill Russo
EPA-certified inorganic quality control samples, including trace metals, minerals, and nutrients,
are produced by:
SPEX Industries, Inc.
3880 Park Avenue
Edison, NJ 08820
TEL: 1 -201 -549-7144 or 1 -800-GET-SPEX
FAX: 1-201-549-5125
The most recent information on EPA-certified materials is available on the EPA Electronic
Bulletin Board (Modum No. 513-569-7610). Names and addresses of retailers of EPA-certified CRADA
QA/QC samples or standards as of February 20, 1991, are given below. When ordering these
materials, specify "EPA Certified Materials."
-------
Retailers of EPA-Certlfied Organic Quality Control Samples
Accurate Chemical and Scientific
300 Shamee Drive
Westbury, NY 11590
TEL: 516-443-4900
FAX: 516-997-4938
Contact: Rudy Rosenberg
Accustandard
25 Science Park Road
New Haven, CT 06511
TEL: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Aidrich Chemical Company, Inc.
940 West Saint Paul Avenue
Milwaukee, Wl 53233
TEL: 414-273-3850
FAX: 800-962-9591
Contact: Roy Pickering
Adtech Associates/Applied
Science/Wescan Instruments
2051 Waukegan Road
Deerfield, IL 60015
TEL: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Analytical Products Group
2730 Washington Boulevard
Belpre, OH 45714
TEL: 614-423-4200
FAX: 614-423-5588
Contact: Tom Coyner
Bodman Chemicals
P. 0. Box 2221
Aston, PA 19014
TEL: 215-459-5600
FAX: 215-459-8036
Contact: Kirk Lind
Chemical Research Supply
P. O. Box 888
Addison, IL 60101
TEL: 708-543-0290
FAX: 708-543-0294
Contact: Nelson Armstrong
Crescent Chemical Corporation
1324 Motor Parkway
Hauppauge, NY 11788
TEL: 516-348-0333
FAX: 516-348-0913
Contact: Eric Rudnick
Curtis Matheson Scientific
P. O. Box 1546
9999 Veterans Memorial Drive
Houston, TX 77251-1546
TEL: 713-820-9898
FAX: 713-878-2221
Contact: Mitchel Martin
Environmental Research Associates
5540 Marshall Street
Arvada.CO 80002
TEL: 303-431-8454
FAX: 303-421-0159
Contact: Mark Carter
Restek Corporation
110 Benner Circle
Bellefonte, PA 16823
TEL: 814-353-1300
FAX: 814-353-1309
Contact: Eric Steindle
Supelco
Supelco Park
Bellefonte, PA 16823-0048
TEL: 800-247-6628 or 814-359-3441
FAX: 814-359-3044
Contact: Linda Alexander
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
TEL: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
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Retailers of EPA-Certified Organic Solution Standards
(Formerly the EPA Toxic and Hazardous Materials Repository)
Absolute Standards
498 Russel Street
New Haven, CT 06513
TEL: 800-368-1131
FAX: 203-468-7407
Contact: Jack Ciscio
Accustandard
25 Science Park Road
New Haven, CT 06511
TEL: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Alltech Associates
2051 Waukegan Road
Deerfield, IL 60015
TEL: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Alameda Chemical and Scientific
922 East Southern Pacific Drive
Phoenix, AZ 85034
TEL: 602-256-7044
FAX: 602-256-6566
Cambridge Isotope Laboratories
20 Commerce Way
Woburn, MA 01801-9894
TEL: 800-322-1174 or 617-938-0067
FAX: 617-932-9721
NSI Environmental Solutions, Inc.
P.O. Box 12313
2 Triangle Drive
Research Triangle Park, NC 27709
TEL: 800-234-7837 or 919-549-8980
FAX: 919-544-0334
Contact: Zora Bunn
Promochem
Postfach 1246
D 4230 Wesel
West Germany
TEL: 0281/530081
FAX: 0281/89991-93
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
TEL: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
Bodman Chemicals
P.O. Box 2221
Aston, PA 19014
TEL: 215-459-5600
FAX: 215-459-8036
Contact: Kirk Lind
Retailers of EPA-Certifled Neat Organic Standards
(Including the Former EPA Pesticide Repository Standards)
Absolute Standards
498 Russel Street
New Haven, CT 06513
TEL: 800-368-1131
FAX: 203-468-7407
Contact: Jack Ciscio
Accustandard
25 Science Park Road
New Haven, CT 06511
TEL: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Alltech Associates
2051 Waukegan Road
Deerfield, IL 60015
TEL: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
TEL: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
-------
Retailers of EPA-Certlfied Inorganic Quality Control Samples
SPEX Industries, Inc.
3880 Park Avenue
Edison, NJ 08820
TEL: 1-201-549-7144 or 1-800-GET-SPEX
FAX: 1-201-549-5125
-------
APPENDIX M
DEFINITION AND PROCEDURE FOR THE
DETERMINATION OF THE METHOD DETECTION LIMIT
[From: U.S. Environmental Protection Agency. 1982. Methods
for Organic Chemical Analysis of Municipal and Industrial
Wastewater. James E. Longbottom and James J. Lichtenberg (eds.).
EPA-600/4-82-057. Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.]
-------
Definition and Procedure for the Determination
of the Method Detection Limit
The method detection limit (MOD is defined as the minimum concentration of a
substance that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero and determined from analysis of a
sample m a given matrix containing analyte.
Scope and Application
This procedure is designed for applicability to a wide variety of sample types
ranging from reagent (blank) water containing analyte to wastewater containing
analyte. The MDL for an analytical procedure may vary as a function of sample
type. The procedure requires a complete, specific and well defined analytical
method It is essential that all sample processing steps of the analytical method be
included in the determination of the method detection limit.
The MDL obtained by this procedure is used to judge the significance of a single
measurement of a future sample.
The MDL procedure was designed for applicability to a broad variety of physical
and chemical methods. To accomplish this, the procedure was made device- or
instrument-independent.
Procedure
1. Make an estimate of the detection limit using one of the following:
(a) The concentration value that corresponds to an instrument signal/noise
ratio in the range of 2.5 to 5. If the criteria for qualitative identification of
the analyte is based upon pattern recognition techniques, the least
abundant signal necessary to achieve identification must be considered in
making the estimate.
-------
(b) If the MDL is to be determined in another sample matrix, analyze the
sample H the measured level of the analyte is in the recommended range
of one to five times the estimated MOL. proceed to Step 4 ,
If the measured concentration of analyte is less than the estimated MDL.
add a known amount of analyte to bring the concentration of analyte to
between one and five times the MDL. In the case where an interference is
coanalyzed with the analyte
If the measured level of analyte is greater than five times the estimated
MDL. there are two options:'
(1) Obtain another sample of lower level of analyte in same matrix if
possible.
(2) The sample may be used as is for determining the MDL if the analyte
level does not exceed 10 times the MOL of the analyte in reagent
water. The variance of the analytical method changes as the analyte
concentration increases from the MDL. hence the MDL determined
under these circumstances may not truly reflect method variance at
lower analyte concentrations.
(a) Take a minimum of seven atiquots of the sample to be used to calculate
the MDL and process each through the entire analytical method. Make all
computations according to the defined method with final results in the
method reporting units. If blank measurements are required to calculate
the measured level of analyte. obtain separate blank measurements for
each sample aliquot analyzed. The average blank measurement is
subtracted from the respective sample measurements.
(b) It may be economically and technically deirable to evaluate the estimated
MDL before proceeding with 4a. This will: (1 ) prevent repeating this entire
procedure when the costs of analyses are high and (2) insure that the
procedure is being conducted at the correct concentration. It is quite
possible that an incorrect MDL can be calculated from data obtained at
many times the real MDL even though the background concentration of
anaiyte is less than five times the calculated MDL. To insure that the
estimate of the MDL is a good estimate, it is necessary to determine that a
lower concentration of analyte will not result in a significantly lower MOL
Take two aliquots of the sample to be used to calculate the MDL and
process each through the entire method, including blank measurements
as described above in 4a. Evaluate these data:
(1 ) If these measurements indicate the sample is in the desirable range for
determining the MDL. take five additional aliquots and proceed. Use
all seven measurements to calculate the MDL
(2) If these measurements indicate the sample is not in the correct range.
reestimate the MDL obtain new sample as in 3 and repeat either 4a or
4b.
Calculate the variance (S2) and standard deviation (S) of the replicate
measurements, as follows:
where: the x., i = 1 to n are the analytical results in the final method reporting
units obtained from the n sample aliquots and j X* refers to the sum of
the X values from i = 1 to n. ' = 1
6. (a) Compute the MDL as follows:
MDL = tin-1. 1-0 . 98i (S)
A.2 July 1982
-------
where
MDL = the .method detection
tii.-» )-o . M> = the students' t value appropriate for a 99% confidence
level and a standard deviation estimate with n-1 degrees
of freedom See Table
S = standard deviation of the replicate analyses.
(b) The 95% confidence limits for the MDL derived in 6a are computed
according to the following equations derived from percentiles of the chi
square over degrees of freedom distribution (XVdl) and calculated as
follows:
MDLLCL = 0.69 MDL
MDLoci = 1.92 MDL
where MDLLci and MDLuci are the lower and upper 95% confidence limits
respectively based on seven aliquots.
Optional iterative procedure to verify the reasonableness of the estimated
MDL and calculated MDL of subsequent MDL determinations.
(a) If this is the initial attempt to compute MDL based on the estimated MDL
in Step 1. take the MDL as calculated in Step 6. spike in the matrix at the
calculated MDL and proceed through the procedure starting with Step 4.
(b) If the current MDL determination is an iteration of the MDL procedure for
which the spiking level does not permit qualitative identification, report the
MDL as that concentration between the current spike level and the
previous spike level which allows qualitative identification.
(c) If the current MDL determination is an iteration of the MDL procedure and
the spiking level allows qualitative identification, use S1 from the current
MDL calculation and SJ from the previous MDL calculation to compute the
F ratio.
if fr < 3.05
Se
then compute the pooled standard deviation by the following equation:
S.
S*
if -rj > 3.05. respike at the last calculated MDL and process the samples
Si
through the procedure starting with Step 4.
(c) Use the SPOMM as calculated in 7b to compute the final MDL according to
the following equation:
MDL = 2.681
where 2.681 is equal to tnZ. i-» . .MI.
(d) The 95% confidence limits for MDL derived in 7c are computed according
to the following equations derived from percentiles of the chi squared over
degrees of freedom distribution.
= 0.72 MDL
MDLucL = 1 .65 MDL
where LCL and UCL are the lower and upper 95% confidence limits
respectively based on 14 aliquots.
Reporting
The analytical method used must be specifically identified by number or title and
the MDL for each analyte expressed in the appropriate method reporting units. If
the analytical method permits options which affect the method detection limit.
these conditions must be specified with the MDL value. The sample matrix used to
A-3 July 1982
-------
determine the MDL must also be identified with the MDL value Report the mean
analyte level with the MDL If a laboratory standard or a sample that contained a
known amount analyte was used for this determination, report the, mean recovery
and indicate if the MDL determination was iterated
If the level of the ana I vie in the sample matrix exceeds 10 times the MDL of the
analyte in reagent water, do not report a value for the MDL.
Reference
Glaser. J. A.. Foerst. D. L.. McKee. G 0.. Quave, S. A., and Budde. W L. "Trace
Analysis for Wastewaters " Environmental Science and Technology. 15. 1426
(1981).
Table of Students' t Values at the 99 Percent Confidence Level
Number of Degrees of freedom
Replicates t.n-1) {<., ,.0 . 99I
7 6 3.143
8 7 2.998
9 8 2.896
10 9 2.821
11 10 2.764
16 15 2.602
21 20 2.528
26 25 2.485
31 30 2.457
61 60 2.390
* « 2.325
A-4 July 1982
>.lit.042/044)
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APPENDIX N
EXAMPLE DATA FORMS FOR ANALYSIS OF
METALS AND ORGANIC TARGET CONTAMINANTS
[From: U.S. Environmental Protection Agency. 1991. Contract
Laboratory Program Statement of Work for Inorganic Analysis,
Multi-Media, Multi-Concentration, SOW #788, July, and Contract
Laboratory Program Statement of Work for Organic Analysis,
February. Washington, DC.]
-------
EXAMPLE DATA FORMS FOR
METALS ANALYSIS
-------
U.S. EPA - CLP
EPA SAMPLE NO.
INORGANIC ANALYSIS DATA SHEET
Name:
Lab Code:
Contract:
Case No.
SAS No.:
SDG No
Matrix (soil/water):
Level (low/med):
% Solids:
Lab Sample ID:
Date Received:
Concentration Units (ug/L or mg/kg dry weight):
1 1
|CAS No. | Analyte
1 1
J7429-90-5 (Aluminum
(7440-36-0 | Antimony
(7440-38-2 JArsenic
17440-39-3 | Barium
17440-41-7 | Beryllium
17440-43-9 ICadmium
17440-70-2 | Calcium
17440-47-3 | Chromium
17440-48-4 | Cobalt
17440-50-8 | Copper
17439-89-6 | Iron
J7439-92-1 |Lead
17439-95-4 (Magnesium
17439-96-5 | Manganese
J7439-97-6 | Mercury
17440-02-0 (Nickel
(7440-09-7 (Potassium
(7782-49-2 (Selenium
(7440-22-4 (Silver
(7440-23-5 (Sodium
(7440-28-0 (Thallium
(7440-62-2 (Vanadium
(7440-66-6 (Zinc
I (Cyanide
1 1
Concentration
C
Q
M
Color Before:
Color After:
Comments:
Clarity Before:
Clarity After:
Texture:
Artifacts:
FORM I - IN
7/88
-------
U. S. EPA - CLP
COVER PAGE - INORGANIC ANALYSES DATA PACKAGE
Lab Name: Contract:
Lab Code: Case No.: SAS No.: SDG No.:
SOW No.
EPA Sample No. Lab Sample ID
Were ICP interelement corrections applied? Yes/No
Were ICP background corrections applied? Yes/No
If yes, were raw data generated before
application of background corrections? Yes/No
Comments:
I certify that this data package is in compliance with the terms and
conditions of the contract, both technically and for completeness, for
other than the conditions detailed above. Release of the data contained
in this hardcopy data package and in the computer-readable data submitted
on floppy diskette has been authorized by the Laboratory Manager or the
Manager's designee, as verified by the following signature.
Signature: Name:
Date: Title:
COVER PAGE - IN
-------
U.S. EPA - CLP
2A
INITIAL AND CONTINUING CALIBRATION VERIFICATION
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Initial Calibration Source:
Continuing Calibration Source:
Concentration Units: ug/L
I
Initial Calibration |
Continuing Calibration
JAnalyte
1
|Aluminum_
| Antimony
I Arsenic
| Barium
| Beryllium
Cadmium
fcalcium
"hromium
I Cobalt
I Copper
llron
ILead
{Magnesium
(Manganese
I Mercury
| Nickel
I Potassium
(Selenium
I Silver
I Sodium
(Thallium
j Vanadium
IZinc
j Cyanide
1
True
Found
%R(D
True
Found
%R(1)
Found
II I
M
(1) Control Limits: Mercury 80-120; Other Metals 90-110; Cyanide 85-115
FORM II (PART 1) - IN
7/88
-------
U.S. EPA - CLP
2B
CRDL STANDARD FOR AA AND ICP
Lab Name:
Lab Code:
Case No.:
AA CRDL Standard Source:
ICP CRDL Standard Source:
Contract:
SAS No.:
SDG No.:
Concentration Units: ug/L
1
1
1
|Analyte
1
| Aluminum
(Antimony
| Arsenic
j Barium
| Beryllium
.dmium
| Calcium
| Chromium
| Cobalt
| Copper
|Iron
(Lead
(Magnesium
(Manganese
| Mercury
(Nickel
(Potassium
(Selenium
(Silver
| Sodium
(Thallium
| Vanadium
(Zinc
1
CRDL S
True
tandard fc
Found
>r AA
%R
True
CRDL Standard for ICP
Initial Final
Found %R Found %R
FORM II (PART 2) - IN
7/88
-------
U.S. EPA - CLP
3
BLANKS
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.
Preparation Blank Matrix (soil/water):
Preparation Blank Concentration Units (ug/L or mg/kg)
Analyte
Aluminum
j Antimony
(Arsenic
] Barium
^Jerylliura
^Kadmium
^Jalcium
j Chromium
| Cobalt
| Copper
llron
ILead
j Magnesium
1 Manganese
| Mercury
(Nickel^
j Potassium
(Selenium
(Silver ""
(Sodium
(Thallium
(Vanadium
(Zinc
j Cyanide
1
Initial
Calib.
Blank
(ug/L) C
-
-
-
Continuing Calibration
Blank (ug/L)
1 C 2 C 3 C
-
"
"
"
"
~
Prepa-
ration
Blank C
-
M
.
FORM III - IN
7/88
-------
U.S. EPA - CLP
ICP INTERFERENCE CHECK SAMPLE
Lab Name:
Lab Code:
Case No:
Contract:
SAS No.:
SDG No.
ICP ID Number:
ICS Source:
Concentration Units: ug/L
1
1
1
| Analyte
1
| Aluminum_
| Antimony
(Arsenic
| Barium
| Beryllium
| Cadmium
j Calcium
j Chromium
| Cobalt
| Copper
jlron
ILead
(Magnesium
| Manganese
(Mercury
(Nickel
j Potassium
(Selenium
(Silver ~
( Sodium
(Thallium^
(Vanadium^
(Zinc
1
True
Sol. Sol.
A AB
Initial Found
Sol. Sol.
A AB %R
Final Found
Sol. Sol.
A AB %R
FORM IV - IN
7/88
-------
U. S. EPA - CLP
5A
SPIKE SAMPLE RECOVERY
EPA SAMPLE NO.
Lab Name:
Lab Code:
Matrix:
Case No.:
Contract:
SAS No.:
SDG No.:
Level (low/med):
% Solids for Sample:
Concentration Units (ug/L or ng/kg dry weight):
Analyte
Aluminum
| Antimony
(Arsenic
Barium
Beryllium
Cadmium
Ifalcium
Bfnromium
Cobalt
Copper
Iron
Lead
| Magnesium
| Manganese
1 Mercury
1 Nickel
| Potassium
Selenium
Silver
I Sodium
Thallium
[Vanadium
IZinc
1 Cyanide
1
Control
Limit
%R
Spiked Sample
Result (SSR)
c
Sample
Result (SR)
C
Spike
Added (SA)
%R
Q
M
_s
Comments:
FORM V (Part 1) - IN
-------
U. S. EPA - CLP
SB
POST DIGEST SPIKE SAMPLE RECOVERY
EPA SAMPLE NO.
Lab Name:
Lab Code:
Matrix:
Case No.:
Contract:
SAS No.:
SDG No.:
Level (low/Bed):
Concentration Units: ug/L
Analyte
Aluminum
| Antimony
I Arsenic
I Barium
Beryllium
"admium
, calcium
Chromium
Cobalt
| Copper
Iron
Lead
j Magnesium
| Manganese
I Mercury
Nickel
Potassium
| Selenium
1 Silver
1 Sodium
[Thallium
| Vanadium
IZinc
1
Control
Limit
%R
Spiked Sample
Result (SSR)
C
Sample
Result (SR)
C
Added (SA)
%R
Q
M
_!
\
Comments:
FORM V (Part 2) - IN
-------
U.S. EPA - CLP
DUPLICATES
EPA SAMPLE NO.
r
Name:
Lab Code:
Contract:
Case No.:
SAS No.:
SDG No.:
Matrix (soil/water):
% Solids for Sample:
Level (low/Bed):
% Solids for Duplicate:
Concentration Units (ug/L or ng/kg dry weight):
1
1
(Analyte
1
(Aluminum
j Antimony
(Arsenic
1 Barium
(Beryllium
(Cadmium
(Calcium
| Chromium
I Cobalt
1 Copper
(Iron
(Lead
j Hagnesiumj
(Manganese)
(Mercury
1 Nickel
j Potassium
(Selenium
(Silver
j sodium
(Thallium
(Vanadium
(Zinc
(Cyanide
I
Control
Limit
Sample (S)
c
Duplicate (D)
C
RPD
'
.
Q
M
FORM VI - IN
7/88
-------
U.S. EPA - CLP
LABORATORY CONTROL SAKPLE
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.
Solid LCS Source:
Aqueous LCS Source:
1
1
| Analyte
1
|Aluroinum_
j Antimony"
(Arsenic
| Barium
j Beryllium
| Cadmium
(Calcium
j Chromium
(CUfelt
|cW>er
(Iron
ILead
(Magnesium
(Manganese
(Mercury
| Nickel^
j Potassium
j Selenium
(Silver
j Sodium
(Thallium_
j Vanadium"
(Zinc ""
j Cyanide
1
Aqueous (ug/L)
True Found *R
Solid
True Found C
"
^
(mg/kg)
Limits %R
FORM VII - IN
7/88
-------
U.S. EPA - CLP
8
STANDARD ADDITION RESULTS
Lab Name:
Lab Code:
Case No. :
Contract :
SAS No.:
SDG No.:
Concentration Units: ug/L
EPA
Sample
No.
|An
|0 ADD
ABS
1 ADD
CON ABS
2 ADD
CON ABS
3 ADD
CON ABS
Final
Cone.
r
Q
M»
-
"
~
-
FORM VIII - IN
7/88
-------
U.S. EPA - CLP
ICP SERIAL DILUTION'S
. EPA SAMPLE NO.
LdflkName:
Lab Code:
Contract:
Case No.:
SAS No.:
SDG Nc.:
Matrix (soil/water):
Level (low/ined):
Concentration Units: ug/L
1
1
(Analyte
1
| Aluminum
j | Antimony
(Arsenic
1 1 Barium
(Beryllium
| Cadmium
I Calcium
j Chromium
1 Cobalt
I Copper
(Iron
(Lead
(Magnesium
| Manganese
1 Mercury
(Nickel
j Potassium
(Selenium
(Silver
(Sodium
(Thallium
(Vanadium
(Zinc
I
(Initial Sample
Result (I)
c
Serial
Dilution
Result (S)
c
%
Differ-
ence
Q
M
FORM IX - IN
7/88
-------
D. S. EPA - CLP
Lab Name:
Lab Code:
10
Instrument Detection Limits (Quarterly)
Case No.:
ICP ID Number:
Flame AA ID Number:
Furnace AA ID Number:
Contract:
SAS No.:
Date:
SDG No,
Analyte
(Aluminum
(Antimony
(Arsenic
(Barium
Beryllium
(Cadmium
(Calcium
j Chromium
1 Cobalt
I Copper
Iron
Lead
| Magnesium
| Manganese
I Mercury
I Nickel
j Potassium
(Selenium
{Silver
Sodium
Thallium^
Vanadium
Zinc
Wave-
length
(nm)
Back-
ground
CRDL
(ug/L)
200
0
10
200
£
5
5000
10
5J>
25
100
5QOO
15
0.2
40
5000
5
1C
5000
10
25
IDL
(ug/L)
M
:omments:
FORM X - IN
-------
U, S. EPA - CLP
11A
ICP Interelement Correction Factors (Anriually)
Mane:
Lab Code:
Case No.:
ICP ID Number:
Contract:
SAS No.:
Date:
SDG No.:
Analyte
(Aluminum
(Antimony
(Arsenic
Barium
Beryllium
Cadmium
1 Calcium
Chromium
Cobalt
opper
iiron
Lead
Mgnesium
ramganeee
1 Mercury
1 Nickel
Potassium
[Selenium
Silver
I Sodium
[Thallium
[Vanadium
IZinc
Wave-
length
(nm)
In1
Al
:erelement (
Ca
Jorrection 1
Fe
^actors for:
Mg
-
Comments:
FORM XI (Part 1) - IN
-------
U. S. EPA - CLP
11B
ICP Interelement Correction Factors (Annually)
Lab Name:
Lab Cods:
Case No.:
ICP ID Number:
Contract:
SAS No.:
Date:
SDG No.:
Analyte
Aluminum
j Antimony
I Arsenic
Barium
Beryllium
Cadmium
Calcium
j Chromium
"obalt
, Jopper
Iron
Lead
j Magnesium
I Manganese
[Mercury
[Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
Wave-
length
(nm)
In1
Lerelement (
:orrection 1
'actors for:
»
t
Comments:
FORM XI (Part 2) - IN
-------
EXAMPLE DATA FORMS FOR
ORGANICS ANALYSIS
-------
EPA SAMPLE HO.
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
b Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol: (g/"»L)__
Level: (low/med)
% Moisture: not dec. dec._
Extraction: (SepF/Cont/Sonc)
GPC Cleanup: (Y/N) pH:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
CAS NO.
COMPOUND
Dilution Factor:
CONCENTRATION UNITS:
(ug/L or ug/Kg)
108-95-i' Ph.enol
111-44-4 bis (2-Chloroethyl) ether
95-57-8 2-Chlorophenol
541-73-1 1,3-Dichlorobenzene
106-46-7 1,4-Dichlorobenzene
100-51-6 Benzyl alcohol
95-50-1 1,2-Dichlorobenzene
95-48-7 2-Methylphencl
108-60-1 bis(2-Chlorcisopropyl)ether_
106-44-5 4-Methylphenol
621-64-7 N-Nitrcso-di-n-propylamine
67-72-1 Hexachloroe thane
98-95-3 Nitrobenzene .
78-59-1 Isophorone
88-75-5 2-Nitrophenol
105-67-9 2,4-Dimethylphenol
65-85-0 Benzoic acid
111-91-1 bis (2 -Chloroethoxy) methane
120-83-2 2,4-Dichlorophenol
120-82-1 1,2,4-Trichlorobenzene
91-20-3 Naphthalene
106-47-8 4-Chloroaniline__
87-68-3 Hexachlorobutadiene
59-50-7 4-Chloro-3-methylphenol
91-57-6 2-Methylnaphthalene
77-47-4 Hexachlorocyclopentadiene
88-06-2 2, 4, 6-Trichlorophenol
95-95-4 2 , 4 , 5-Trichlorophenol
91-58-7 2-Chloronaphthalene
88-74-4 2-Nitroaniline
131-11-3 Dimethylphthalate
208-56-8 Acsnaphthylene
606-20-2 2, 6-Dinitrotoluene
ropy, l SV-1
1/S7 Re-.
-------
EPA SAMPLE NO.
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol:
Level: (low/med)
% Moisture: not dec.
.(g/mL).
dec.
Extraction: (SepF/Cont/Sonc)
GFC Cleanup: (Y/N) pH:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
CAS NO.
COMPOUND
Dilution Factor:
CONCENTRATION UNITS:
(ug/L or ug/Kg)
99-09-2 3-Nitroaniline
83-32-9 Acenaphthene
51-28-5 2,4-Dinitrophenol
100-02-7 4-Nitrophenol
132-64-9 Dibenzofuran
121-14-2 2,4-Dinitrotoluene
84-66-2 Diethylphthalate
7005-72-3 4-Chlorophenyl-phenylether
86-73-7 Fluorene
100-01-6 4-Nitroaniline
534-52-1 4 , 6-Dinitro-2-methylphenol
86-30-6- N-Nitrosodiphenylamine (1)'
101-55-3 4-Bromophenyl-phenylether
118-74-1 Hexachlorobenzene ^
87-86-5 Pentachlorophenol
85-01-8 Phenanthrene
120-12-7 Anthracene
84-74-2 Di-n-butylphthalate
206-44-0 Fluoranthene
129-00-0 Pyrene
85-68-7 Butylbenzylphthalate
91-94-1 3,3'-Dichlorobenzidine
56-55-3 Benzo (a) anthracene
218-01-9 Chrysene
117-81-7 bis(2-Ethylhexyl)phthalate
117-84-0 Di-n-octylphthalate
205-99-2 Benzo (b) f luoranthene
207-08-9 Benzo(k) f luoranthene
50-32-8 Benzo(a)pyrene
193-39-5 Indeno(l,2.3-cd)pyrene
53-70-3 Dibenz (a,h) anthracene
191-24-2 Benzo(g,h, i) perylene
(1) - Cannot be separated from Diphenylamine
FORM I SV-2
1/37 Re-,
-------
EPA SAMPLE NO.
PESTICIDE ORGANICS ANALYSIS DATA SHEET
ab Name:
Lab Code:
Case No.:
Contract:_
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol: (g/mL)
Level: (low/med)
% Moisture: not dec. dec.
Extraction: (SepF/Cont/Sonc)
GPC Cleanup: (Y/N) pH:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
CAS NO.
COMPOUND
Dilution Factor:
CONCENTRATION UNITS:
(ug/L or ug/Kg)
319-84-6 alpha-EHC
319-85-7 beta-BHC
319-86-8 delta-BHC
58-89-9 -gamma-BHC (Lindane)
76-44-8 Heptachlor '
309-00-2 Aldrin
1024-57-3 Heptachlor epcxide_
959-98-8 Endosulfan I
60-57-1 Dieldrin
72-55-9 4 , 4 ' -DDE
72-20-8 Endrin
33213-65-9 Endosulfan II
72-54-8 4 , 4 ' -ODD
1031-07-8 Endosulfan sulfate_
50-29-3 4 , 4 ' -DDT
72-43-5 Methoxychlor
53494-70-5 Endrin ketone
5103-71-9 alpha-Chlordane
5103-74-2 gamma-Chlordane
8001-35-2 Toxaphene
12674-11-2 Aroclor-1016
11104-28-2 Aroclor-1221
11141-16-5 Aroclor-1232
53469-21-9 Aroclor-1242
12672-29-6 Aroclor-1248
11097-69-1 Aroclor-1254
11096-82-5 Aroclor-1260
FORM I PEST
1/87 Rev
-------
EPA SAMPLE NO.
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
TENTATIVELY IDENTIFIED COMPOUNDS
Name:
l*.-> Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol:
Level: (low/med)
* Moisture: not dec.
dec.
Kxtraction: (SepF/Cont/Sonc)
-Ji-'C Cleanup: (Y/N) pH:
Number TICs found:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:.
Date Analyzed:
Dilution Factor:
CONCENTRATION UNITS:
(ug/L or ug/Kg)
CAS NUMBER
1.
2.
»3.
A.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
«'
'.
"0.
COMPOUND NAME
RT
=SS=ST = S = =
EST. CONC.
Q
1
i
1
FORM I SV-TIC
1/67 Rev
-------
SEMIVOLATILE SURROGATE RECOVERY
> Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Level:(low/med)
| EPA
| SAMPLE NO.
on
02|
03|
04|
05|
06|
07|
08|
09|
10|
HI
12|
13|
14|
15|
16|
17|
18|
191
20|
211
22|
23|
24|
25|
26|
27 |
28|
29|
30|
SI
(NBZ)#
S2
(FBP)#
S3
(TPH)#
~" "
S4
(PHL)#
S5
(2FP)f
S6
(TBP)#
OTHER
TOT|
OUT
SI (NBZ) = Nitrobenzene-d5
S2 (FBP) «= 2-Fluorobiphenyl
S3 (TPH) = Terphenyl-dl4
S4 (PHL) = Phenol-d6
S5 (2FP) = 2-Fluorophenol
S6 (TBP) = 2 , 4 , 6-Tribromophenol
QC LIMITS
(23-120)
(30-115)
(18-137)
(24-113)
(25-121)
(19-122)
41 Column to be used to flag recovery values
* Values outside of contract required QC limits
D Surrogates diluted out
page
of
FORM II £V-2
1/87 Re\
-------
Name:
Lab Code:
PESTICIDE SURROGATE RECOVERY
Case No.:
Contract:
SAS No.:
SDG No.:
| EPA
j SAMPLE NO.
| ============
oil
021
031
041
051
06|
07|
08|
091
10|
111
12|
13|
14|
151
16|
17|
18|
191
20!
211
221
23|
241
251
261
27|
281
29|
301
SI
(DBC)f
| OTHER
=====
ADVISORY
QC LIMITS
Si (DEC) = Dibutylchlorendate (24-154)
I Column to be used to flag recovery values
* Values outside of QC limits
D Surrogates diluted out
page of
FORM II PEST-1
1/87 Rev.
-------
SEMIVOLATILE MATRIX SPIKE/MATRIX SPIKE DUPLICATE RECOVERY
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix Spike - EPA Sample No.:
COMPOUND
====«===================
Phenol
2-Chlorophenol
1,4-Dichlorobehzene
N-Nitrpso-di-n-prop. (1)
1 , 2 , 4-Trichlorobenzene
4-Chloro-3-methylphenol
Acenaphthene
4-Nitrophenol
2 , 4-Dinitrotoluene
Pentachlorophenol
Pyrene
SPIKE
ADDED
(ug/L)
=5 sr =?===>===
SAMPLE
CONCENTRATION
(ug/L)
=============
MS
CONCENTRATION
(ug/L)
=============
MS
%
REC *
======
QC
LIMITS
REC.
======
12- 89
27-123
36- 97
41-116
39- 98
23- 97
46-118
10- 80
24- 96
9-103
26-127
COMPOUND
Phenol
2-Chlorophenol
1 , 4-Dichlorobenzene
N-Nitroso-di-n-prop. (l)
1 , 2 , 4-Trichlorobenzene
4-Chloro-3-methylphenol
Acenaphthene
4-Nitrophenol
2 , 4-Dinitrotoluene
Pentachlorophenol
Pyrene
SPIKE
ADDED
(ug/L)
MSD
CONCENTRATION
(ug/L)
MSD
%
REC |
%
RPD *
QC LI
RPD
======
42
40
28
38
28
42
31
50
38
50
31
EMITS
REC.
12- 89
27-123
36- 97
41-116
39- 98
23- 97
46-118
10- 80
24- 96
9-103
26-127
(1) N-Nitroso-di-n-propylamine
f Column to be used to flag recovery and RPD values with an asterisk
* Values outside of QC limits
RPD: out of outside limits
Spike Recovery: out of outside limits
COMMENTS:
FORM III SV-1
1/37 Re--.-.
-------
PESTICIDE MATRIX SPIKE/MATRIX SPIKE DUPLICATE RECOVERY
Lab Name:,
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix Spike - EPA Sample No.:
COMPOUND
gamma -BHC (Lindane)
Heptachlor
Aldrin
Dieldrin
Endrin
4,4' -DDT
SPIKE
ADDED
(ug/L)
SAMPLE
CONCENTRATION
(ug/L)
MS
CONCENTRATION
(ug/L)
MS .| QC. |
% | LIMITS |
REC l| REC. 1
|56-123|
140-1311
|40-120|
152-1261
156-1211
|38-127|
1 1
COMPOUND
gamma-BHC (Lindane)
Heptachlor
Aldrin
Dieldrin
Endrin
4,4' -DDT
SPIKE
ADDED
(ug/L)
MSD
CONCENTRATION
(ug/L)
MSD
%
REC I
'
%
RPD 1
QC LIMITS
RPD | REC.
15 |56-123
20 (40-131
22 |40-120
18 |52-126
21 156-121
27 |38-127
1
I Column to be used to flag recovery and RPD values with an asterisk
* Values outside of QC limits
RPD: out of outside limits
Spike Recovery: out of outside limits
COMMENTS:
FORM III PEST-1
8/87 Rev.
-------
SEMIVOLATILE METHOD BLANK SUMMARY
,b Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No. :
Lab File ID:
Date Extracted:
Date Analyzed:
Matrix: (soil/water)
Instrument ID:
Lab Sample ID:
Extraction:(SepF/Cont/Sonc)
Time Analyzed:
Level:(low/med)
THIS METHOD BLANK APPLIES TO THE FOLLOWING SAMPLES, MS AND MSD:
SMMENTS:
| EPA
I SAMPLE NO.
| ============
oil
02|
03|
04|
05|
06|
07|
08 |
09|
10|
HI
12|
131
14|
15|
161
17|
18|
19|
20|
211
22|
23|
24|
25|
26|
27|
28|
29|
30|
LAB
SAMPLE ID
= 35 S= = = = = == ====
LAB
FILE ID
s===s=s===:==s==:= =
DATE
ANALYZED
page
of
FORM IV SV
1/87 Rev
-------
Lab Name:,
Lab Code:
PESTICIDE METHOD BLANK SUMMARY
Contract:_
SAS No.:
Case No.:
SDG Nc.:
Lab Sample ID:
Matrix:(soil/water)
Date Extracted:
Date Analyzed (1):
Time Analyzed (1):
Instrument ID (2):
GC Column ID (1):
Lab File ID:
Level:(low/med)
Extraction: (SepF/Cont/Sonc)
Date Analyzed (2):
Time Analyzed (2):
Instrument ID (2):
GC Column ID (1):
THIS METHOD BLANK APPLIES TO THE FOLLOWING SAMPLES, MS AND MSD:
| EPA
J SAMPLE NO.
on
021
03|
04|
05|
06|
07|
08 |
09f
101
HI
121
131
141
151
16|
17|
18|
191
20|
211
221
231
24|
251
261
LAB
SAMPLE ID
= ======r=S === = =;
DATE
ANALYZED 1
ssssssssas
DATE
ANALYZED 2
COMMENTS:
age
of
FORM IV PEST
1/87 Rev.
-------
U. S. EPA - CLP
12
ZCP Linear Ranges (Quarterly)
Lab Name:
Lab Code:
Case No.:
ZCP ID Number:
Contract:
SAS No.:
Date:
SDG No.:
Analyte
Aluminum
| Antimony
(Arsenic
Barium
Beryllium
| Cadmium
Calcium
Chromium
Cobalt
| Copper
Iron
Lead
| Magnesium
[Manganese
1 Mercury
1 Nickel
Potassium
I Selenium
1 Silver
I Sodium
I Thallium
j Vanadium
IZinc
Integ.
Time
(sec.)
Concentration
(ug/L)
M
:omment«:
FORM XII - IN
-------
U.S. EPA - CLP
13
PREPARATION LOG
Lab Name:
Lab Code:
Method:
Case No
EPA
Sample
No.
*
Preparation
Date
Contra<
SAS No
Weight
(gram)
:t:
Volume
(mL)
SDG No.:
FORM XIII - IN
7/88
-------
U.S. EPA - CLP
14
ANALYSIS RUN LOG
«1
>
Jaae:
ib Code:
Case No.:
istrunent ID Number:
:art Date:
Contract:
SAS No.:
Method: _
End Date:
SDG No.
EPA
Sample
No.
D/F
Time
% R
A
L
S
B
A
S
B
A
B
E
C
D
C
A
C
R
C
0
Ar
C
u
ia]
F
E
yt
p
B
:es
K
G
>
M
N
H
G
N
I
K
S
E
A
G
N
A
T
L
V
2
N
C
N
FORM XIV - IN
7/88
-------
EXAMPLE DATA FORMS FOR
METALS ANALYSIS
-------
U.S. EPA - CLP
EPA SAMPLE NO',
INORGANIC ANALYSIS DATA SHEET
Lab Name:
Lab Code:
Contract:
Case No.:
SAS No.:
SDG No.:
Matrix (soil/water):
Level (low/med):
% Solids:
Lab Sample ID:
Date Received:
Concentration Units (ug/L or mg/kg dry weight):
1 1
|CAS No. | -Analyte
1 1
(7429-90-5 (Aluminum
(7440-36-0 (Antimony
(7440-38-2 (Arsenic
(7440-39-3 (Barium
(7440-41-7 | Beryllium
(7440-43-9 (Cadmium
j 7440-70-2 (Calcium
(7440-47-3 (Chromium
(7440-48-4 (Cobalt
(7440-50-8 (Copper
17439-89-6 I Iron
17439-92-1 (Lead
J 7439-95-4 (Magnesium
(7439-96-5 (Manganese
(7439-97-6 (Mercury
17440-02-0 (Nickel
(7440-09-7 (Potassium
[7782-49-2 (Selenium
(7440-22-4 (Silver
(7440-23-5 (Sodium
(7440-28-0 (Thallium '
(7440-62-2 (Vanadium
(7440-66-6 (Zinc
| | Cyanide
1 1
Concentration
C
Q
M
:olor Before:
:olor After:
:omments:
Clarity Before:
Clarity After:
Texture:
Artifacts:
FORM I - IN
ILM02.0
-------
U.S. EPA - CLP
2A
INITIAL AND CONTINUING CALIBRATION VERIFICATION
Lab Name:
Lab Code:
Case No.:
Initial Calibration Source:
Continuing Calibration Source:
Contract:
SAS No.:
SDG No.:
Concentration Units: ug/L
1
1
I Analyte
1
| Aluminum_
| Antimony
| Arsenic
| Barium
j Beryllium
j Cadmium
j Calcium
| Chromium
I Cobalt
| Copper
| Iron
Lead
| Magnesium
j Manganese
| Mercury
' Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
Cyanide
Initial Calibration
True Found %R ( 1 )
Continuing Calibration
True Found %R(1) Found %R(1)
'
M
(1) Control Limits: Mercury 80-120; Other Metals 90-110; Cyanide 85-115
FORM II (PART 1) - IN
ILM02.0
-------
U.S. EPA - CLP
2B
CRDL STANDARD FOR AA AND ICP
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
AA CRDL Standard Source:
ICP CRDL Standard Source:
SDG No.:
Concentration Units: ug/L
1
1
1
I Analyte
1
| Aluminum
| Antimony
| Arsenic
| Barium
| Beryllium
«dmium
Icium
romium
| Cobalt
| Copper
| Iron
ILead
(Magnesium
| Manganese
I Mercury
I Nickel
j Potassium
| Selenium
| Silver
| Sodium
(Thallium
| Vanadium
(Zinc
1
CRDL S
True
tandard fo
Found
r AA
%R
True
CRDL Standard for ICP
Initial Final
Found %R Found %R
CRDL= Contract required detection limit.
FORM II (PART 2) - IN
ILM02.0
-------
U.S. EPA - CLP
BLANKS
Lab Name:
Lab Code:
Case No*:
Contract:
SAS No.:
Preparation Blank Matrix (soil/water):
Preparation Blank Concentration Units (ug/L or mg/kg):
SDG No.:
1
1
1
1
| Analyte
1
| Aluminum
| Antimony
I Arsenic
| Barium
| Beryllium
j Cadmium
I Calcium
I Chromium
| Cobalt
| Copper
| Iron
ILead
(Magnesium
j Manganese
I Mercury
| Nickel
j Potassium
j Selenium
i Silver
I Sodium
(Thallium
| Vanadium
| Zinc
I Cyanide
1
Initial
Calib.
Blank
(ug/L) C
__
Continuing Calibration
Blank (ug/L)
1 C 2 C 3
C
1
Prepa-
ration
Blank C
M
FORM III - IN
ILM02.0
-------
'.S. EPA - CLP
Lab Name:
Lab Code:
5A
SPIKE SAMPLE RECOVERY
Contract:
EPA SAMPLE NO.
Case No.:
SAS No.:
SDG No.:
Matrix (soil/water):
% Solids for Sample:
Level (low/med):
Concentration Units (ug/L or mg/kg dry weight):
1
1
1
I Analyte
1
| Aluminum
| Antimony
| Arsenic
| Barium
| Beryllium
| Cadmium
| Calcium
| Chromium
I Cobalt
I Copper
I Iron
|Lead
(Magnesium
| Manganese
| Mercury
1 Nickel
| Potassium
| Selenium
I Silver
I Sodium
(Thallium
| Vanadium
I Zinc
| Cyanide
1
Control
Limit
%R
Spiked Sample
Result (SSR)
C
Sample
Result (SR)
C
Spike
Added (SA)
%R
Q
M
1
Comments:
FORM V (PART 1) - IN
ILM02.0
-------
U.S. EPA - CLP
5B
POST DIGEST SPIKE SAMPLE RECOVERY
EPA SAMPLE NO.
Name:
Lab Code:
Matrix (soil/water):
Contract:
Case No.:
SAS No.:
SDG No.:
Level (low/med):
Concentration Units: ug/L
1
1
j Analyte
1
| Aluminum
| Antimony
| Arsenic
| Barium
| Beryllium
| Cadmium
| Calcium
| Chromium
I Cobalt
| Copper
| Iron
IL^d
(HVfnesium
| Manganese
| Mercury
1 Nickel
| Potassium
j Selenium
(Silver
j Sodium
(Thallium
j Vanadium
| Zinc
I Cyanide
1
Control
Limit
%R
Spiked Sample
Result (SSR)
c
Sample
Result (SR)
C
Spike
Added (SA)
%R
Q
M
Comments:
FORM V (PART 2) -IN
ILM02.0
-------
U.S. EPA - CLP
DUPLICATES
EPA SAMPLE NO.
Lab Name:
Lab Code:
Contract:
Case No.:
SAS No.:
SDG No.:
Matrix (soil/water):
% Solids for Sample:
Level (low/med):
% Solids for Duplicate:
Concentration Units (ug/L or mg/kg dry weight):
1
1
| Analyte
1
| Aluminum
| Antimony
| Arsenic
| Barium
| Beryllium
| Cadmium
I Calcium
| Chromium
I Cobalt
I Copper
llron
ILead
(Magnesium
| Manganese
| Mercury
1 Nickel
j Potassium
| Selenium
I Silver
j Sodium
| Thallium
(Vanadium
(Zinc
I Cyanide
1
Control
Limit
.-Sample (S)
C
Duplicate (D)
C
RPD
Q
M
FORM VI - IN
ILM02.0
-------
Lab Name:
Lab Code:
U.S. EPA - CLP
7
LABORATORY CONTROL SAMPLE
Contract:
Case No.: SAS No.:
Solid LCS Source:
Aqueous LCS Source:
SDG No.:
1
1
I Analyte
1
| Aluminum
| Antimony
j Arsenic
| Barium
j Beryllium
I Cadmium
I Calcium
| Chromium
| Cobalt
I Copper
llron
(Lead
(Magnesium
j Manganese
j Mercury
1 Nickel
j Potassium
j Selenium
| Silver
j Sodium
j I Thallium ..
| Vanadium
IZinc
I Cyanide
1
Aqueous (ug/L)
True Found %R
Sol]
True Found
C Limits
%R
FORM VII - IN
ILM02.0
-------
U.S. EPA - CLP
8
STANDARD ADDITION RESULTS
Lab Name
Lab Code
Case No
Contract
SAS No . :
SDG
No. :
Concentration Units: ug/L
EPA
Sample
No.
An
0 ADD
ABS
1 ADD
CON ' ABS
"
2 ADD
CON ABS
3 ADD
CON ABS
Final
Cone.
-
.
r
Q
~
~
FORM VIII - IN
ILM02.0
-------
U.S. EPA - CLP
10
INSTRUMENT DETECTION LIMITS (QUARTERLY)
Lab Name:
Lab Code:
Case No.
ICP ID Number:
Flame AA ID Number:
Furnace AA ID Number:
Contract:
SAS No.:
Date:
SDG No.
Comments:
1 1
1
1
| Analyte
1
| Aluminum
| Antimony
| Arsenic
| Barium
(Beryllium
| Cadmium
| Calcium
| Chromium
I Cobalt
I Copper
llron
(Lead
(Magnesium
| Manganese
I Mercury
1 Nickel
| Potassium
| Selenium
I Silver
| Sodium
(Thallium
| Vanadium
IZinc
Wave-
length
(nm)
Back-
ground
CRDL
(ug/L)
200
60
10
200
5
5
5000
10
50
25
100
3
5000
15
0.2
40
5000
5
10
5000
10
50
20
IDL
(ug/L)
M
FORM X - IN
ILM02.0
-------
13
PREPARATION LOG'
Lab Name:
Lab Code:
Method:
Case No.:
Contract:
SAS No.:
SDG No,
EPA
Sample
No.
Preparation
Date
Weight
(gram)
Volume
(mL)
FORM XIII - IN
ILM02.0
-------
U.S. EPA - CLP
14
ANALYSIS RUN LOG
Lab Name:
Lab Code: Case No.:
Instrument ID Number:
Start Date:
Contract :
SAS No. :
Method :
End Date:
SDG NO.
EPA
Sample
No.
D/F
Time
% R
A
L
S
IB
A
S
IB
A
B
E
C
D
C
A
C
R
C
0
Al
C
u
na.
F
E
Lyi
P
B
be:
M
G
3
M
N
H
G
N
I
K
S
E
A
G
N
A
T
L
|V
Z
N
C
N
FORM XIV - IN
.ILM02.0
-------
EXAMPLE DATA FORMS FOR
ORGANICS ANALYSIS
-------
IB
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol: (g/mL)
Level:' (low/med)
% Moisture: decanted: (Y/N)
Concentrated Extract Volume: (uL)
Injection Volume: (uL)
GPC Cleanup: (Y/N) pH:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
Dilution Factor:
CAS NO.
COMPOUND
CONCENTRATION UNITS:
(ug/L or ug/Kg)
108-95-2 Phenol
111-44-4 bis (2-Chloroethyl) ether
95-57-8 2-Chlorophenol '
541-73-1 1,3-Dichlorobenzene
106-46-7 1,4-Dichlorobenzene
95-50-1 1,2-Dichlorobenzene
95-48-7 2-Methylphenol
108-60-1 2,2 ' -oxybis (1-Chloropropane)
106-44-5 4-Methylphenol
621-64-7 N-Nitroso-di-n-propylamine
67-72-1 Hexachloroethane
98-95-3 Nitrobenzene
78-59-1 Isophorone
88-75-5 2-Nitrophenol
105-67-9 2,4-Dimethylphenol
111-91-1 bis (2-Chloroethoxy) methane
120-83-2 2,4-Dichlorophenol
120-82-1 1,2,4-Trichlorobenzene
91-20-3 Naphthalene
106-47-8 4-Chloroaniline
87-68-3 Hexachlorobutadiene
59-50-7 -4-Chloro-3-methylphenol
91-57-6 2-Methylnaphthalene
77-47-4 Hexachlorocyclopentadiene
.88-06-2 2,4,6-Trichlorophenol
95-95-4 2,4,5-Trichlorophenol
91-58-7 2-Cnloronaphthalene
88-74-4 2-Nitroaniline
131-11-3 Dimethylphthalate
208-96-8 Acenaphthylene
606-20-2 2,
-------
EPA SAMPLE NO.
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix: (soil/water)
Sample wt/vol: (g/mL)
Level: ' (low/med)
% Moisture: decanted: (Y/N)
Concentrated Extract Volume:
Injection Volume: (uL)
GPC Cleanup: (Y/N) pH:
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
Dilution Factor:
CAS NO.
COMPOUND
CONCENTRATION UNITS:
(ug/L or ug/Kg)
51-28-5 2,4-Dinitrophenol
100-02-7 4-Nitrophenol
132-64-9 Dibenzofuran
121-14-2 2,4-Dinitrotoluene
84-66-2 Diethylphthalate
7005-72-3 4-Chlorophenyl-phenylether
86-73-7 Fluorene
100-01-6 4-Nitroaniline
534-52-1 4,6-Dinitro-2-methylphenol
86-30-6 N-Nitrosodiphenylamine (1)
101-55-3 4-Bromophenyl-phenylether
118-74-1 Hexachlorobenzene .
87-86-5 Pentachlorophenol
85-01-8 Phenanthrene
120-12-7 Anthracene
86-74-8 Carbazole
84-74-2 Di-n-butylphthalate
206-44-0 Fluoranthene "-
129-00-0 Pyrene
85-68-7 Butylbenzylphthalate
91-94-1 3,3 '-Dichlorobenzidine
56-55-3 Benzo (a) anthracene
218-01-9 Chrysene
117-81-7 bis(2-Ethylhexyl)phthalate
117-84-0 Di-n-octylphthalate .
205-99-2 Benzo (b) f luoranthene
207-08-9 Benzo (k) f luoranthene
50-32-8 Benzo(a)pyrene
193-39-5 Indeno(l,2,3-cd)pyrene
53-70-3 Dibenz(a,h) anthracene
191-24-2 Benzo (g,h, ijperylene
(1) - Cannot be separated from Diphenylamine
FORM T SV-?
/ar»
-------
ID
PESTICIDE ORGANICS ANALYSIS DATA SHEET
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
Matrix: (soil/water)
Sample wt/vol: (g/roL)
% Moisture: decanted: (Y/N)
Extraction: (SepF/Cont/Sonc)
Concentrated Extract Volume:
Injection Volume: (uL)
GPC Cleanup: (Y/N)
PH:
(uL)
SDG No. :
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
Dilution Factor:
Sulfur Cleanup: (Y/N)
CAS NO.
COMPOUND
CONCENTRATION UNITS;
(ug/L or ug/Kg)
319-84-6 alpha-BHC
319-85-7 beta-BHC
319-86-8 delta-BHC
58-89-9 gamma-BBC (Lindane)
76-44-8 Heptachlor '
309-00-2 Aldrin
1024-57-3 Heptachlor epoxide__
959-98-8 Endosulfan I
60-57-1 Dieldrin
72-55-9 4, 4 ' -DDE
72-20-8 Endrin
33213-65-9 Endosulfan II
72-54-8 4 ,4 ' -ODD
1031-07-8 Endosulfan sulfate_
50-29-3 4 , 4 ' -DDT
72-43-5 Methoxychlor
53494-70-5 Endrin ketone
7421-36-3 Endrin aldehyde
5103-71-9 alpha-Chlordane
5103-74-2 gamma-Chlordane
8001-35-2 Toxaphene
12674-11-2 Aroclor-1016
11104-28-2 Aroclor-1221
11141-16-5 Aroclor-1232
53469-21-9 Aroclor-1242
12672-29-6 Aroclor-1248
11097-69-1 Aroclor-1254
11096-82-5 Aroclor-1260
FORM I PEST
3/90
-------
IF
SEMIVOLATILE ORGANICS ANALYSIS DATA SHEET
TENTATIVELY IDENTIFIED COMPOUNDS
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.
Matrix: (soil/water)
Sample wt/vol:
Level:. (low/med)
% Moisture: decanted: (Y/N).
Concentrated Extract Volume:
Injection Volume: (uL)
GPC Cleanup: (Y/N) pH:
Number TICs found:
(uL)
Lab Sample ID:
Lab File ID:
Date Received:
Date Extracted:
Date Analyzed:
Dilution Factor:
CONCENTRATION UNITS:
(ug/L or ug/Kg)
CAS NUMBER
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
COMPOUND NAME
.
RT
EST. CONC.
Q
FORM I SV-TIC
3/90
-------
2C
SEMIVOLATILE SURROGATE RECOVERY
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No,
| EPA
1 SAMPLE NO.
on
02|
03|
04|
05|
06|
07|
08]
09|
10|
HI
12|
131
14|
15|
16|
17|
181
191
20|
211
22|
23|
24|
25|
26|
27|
28|
29|
30|
SI
(NBZ)ff
S2 -| S3
(FBP) | | (TPH) «
.
S4
(PHL) f
S5
(2FP)|
S6
(TBP) |
S7
1 (2CP) |
| S8
I (DCB)|
ITOTI
OUT
QC LIMITS
SI (NBZ) « Nitrobenzene-dS (35-114)
S2 (FBP) = 2-Fluorobiphenyl (43-116)
S3 (TPH) = Terphenyl-dl4 (33-141)
S4 (PHL) = Phenol-d5 (10-110)
S5 (2FP) = 2-Fluorophenol (21-110)
S6 (TBP) = 2,4,6-Tribroraophenol (10-123)
S7 (2CP) = 2-Chlorophenol-d4 (33-110)
S8 (DCB) = l,2-Dichlorobenzene-d4 (16-110)
(advisory)
(advisory)
n Column to be used to flag recovery values
* Values outside of contract required QC limits
D Surrogate diluted out
page
of
FORM II SV-1
3/90
-------
2E
PESTICIDE SURROGATE RECOVERY
Lab Name:
Lab Code:
GC Column(1):
Case No.:
ID:
Contract:
SAS No.:
SDG No.:
(mm) GC Column(2):
ID:
(nun)
| EPA
j SAMPLE NO.
on
02|
03|
04|
05|
06|
07|
08 |
09|
10|
HI
121
13|
141
15|
16|
17|
18|
191
20|
211
22|
23|
24|
25|
26|
27|
28|
291
30|
TCX 1
%REC 1
|TCX 2
%REC *
DCB 1
%REC |
-
DCB 2
%REC |
»-
OTHER
(1)
OTHER
(2)
..
ITOTI
OUT
TCX = Tetrachloro-m-xylene
DCB = Decachlorobiphenyl
ADVISORY
QC LIMITS
(60-150)
(60-150)
I Column to be used to flag recovery values
* Values outside of QC limits
D Surrogate diluted out
page of
FORM II PEST-1
3/90
-------
3C
SEMIVOLATILE MATRIX SPIKE/MATRIX SPIKE DUPLICATE RECOVERY
Lab Name:
Code:
Case No.:
Matrix Spike - EPA Sample No.:
Contract:
SAS No.:
SOG No.:
COMPOUND
Phenol
2-Chlorophenol
1 , 4-Dichlorobenzene
N-Nitroso-di-n-prop. (1)
1,2, 4-Trichlorobenzene
4-Chloro-3-methylphenol
Acenaphthene
4 -Nitrophenol
2 , 4-Dinitrotoluene
Pentachlorophenol
Pyrene
SPIKE
ADDED
(ug/L)
| SAMPLE
| CONCENTRATION
(ug/L)
p
MS
CONCENTRATION
(ug/L)
MS | QC.
% | LIMITS
REC S| REC.
112-110
127-123
136- 97
141-116
|39- 98
123- 97
146-118
|10- 80
|24- 96
I 9-103
126-127
1
1
| COMPOUND
Phenol
2-Chlorophenol
1, 4-Dichlorobenzene
N-Nitroso-di-n-prop. (i'j
1,2,4 -Trichlorobenzene
4-Chloro-3-methylphenol
Acenaphthene
4 -Nitrophenol
2 , 4-Dinitrotoluene
Pentachlorophenol
Pyrene
SPIKE
ADDED
(ug/L)
| MSD
| CONCENTRATION
(ug/L)
MSD
1 %
REC *
%
RPD #
1
QC LIMITS j
RPD | REC. |
42 |12-110|
40 |27-123|
28 |36- 97 |
38 |41-116|
28 |39- 98 |
42 |23- 97|
31 |46-118|
50 |10- 80|
38 |24- 96|
50 | 9-103|
31 |26-127|
1 1
(1) N-Nitroso-di-n-propylamine
# Column to be used to flag recovery and RPD values with an asterisk
* Values outside of QC limits
RPD: out of outside limits
Spike Recovery: out of outside limits
COMMENTS:
FORM III SV-1
3/90
-------
3E
PESTICIDE MATRIX SPIKE/MATRIX SPIKE DUPLICATE RECOVERY
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Matrix Spike - EPA Sample No.:
COMPOUND
ganuna-BHC (Lindane)
Heptachlor
Aldrin
Dieldrin
Endrin
4,4' -DDT
SPIKE
ADDED
(ug/L)
SAMPLE
CONCENTRATION
(ug/L)
__ ,- F - , ...._.,.
MS
1 CONCENTRATION
(ug/L)
MS | QC. |
| % | LIMITS |
REC #| REC. |
156-1231
140-1311
140-1201
152-1261
|56-121|
138-1271
1 1
COMPOUND
ganuna-BHC (Lindane)
Heptachlor
Aldrin
Dieldrin
Endrin
4,4' -DDT
SPIKE
ADDED
(ug/L)
| MSD
| CONCENTRATION
(ug/L)
MSD
1 *
REC #
%
RPD «
1
QC LIMITS |
RPD | REC. |
, 1 1
15 |56-123|
20 |40-131|
22 |40-120|
18 |52-126|
21 |56-121|
27 | 38-127|
1 1
# Column to be used to flag recovery and RPD values with an asterisk
* Values outside of QC limits
RPD: out of outside limits
Spike Recovery: out of outside limits
COMMENTS:
FORM III PEST-1
3/90
-------
4B
SEMIVOLATILE METHOD BLANK SUMMARY
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No. :
Contract:
SAS No. : S
SDG No.:
Lab File ID:
Instrument ID:
Matrix: (soil/water)
Level:(low/med)
Lab Sample ID:
Date Extracted:
Date Analyzed:
Time Analyzed:
THIS METHOD BLANK APPLIES TO THE FOLLOWING SAMPLES, MS AND MSD:
| EPA
| SAMPLE NO.
on
02|
03|
04|
05|
06|
07|
08!
091
10|
111
121
131
141
15|
16|
17|
181
19|
20|
211
221
23|
241
25|
26|
27|
281
291
30|
LAB
SAMPLE ID
LAB
FILE ID
DATE
ANALYZED
COMMENTS:
page of
FORM IV SV
3/90
-------
4C
PESTICIDE METHOD BLANK SUMMARY
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No.:
Contract:
SAS No.:
SDG No.:
Lab Sample ID:
Lab File ID:
Ma-trix: (soil/water)
Sulfur' Cleanup: (Y/N)
Date Analyzed (1):
Time Analyzed (1) :
Instrument ID (1):
GC Column (1):
ID:
Extraction:(SepF/Cont/Sonc)
Date Extracted:
Date Analyzed (2):
Time Analyzed (2):
Instrument ID (2):
(mm) GC Column (2):
ID:
(mm)
THIS METHOD BLANK APPLIES TO THE FOLLOWING SAMPLES, MS AND MSD:
| EPA
| SAMPLE NO.
on
02|
03|
04|
05|
06|
07|
08|
09|
101
HI
121
131
141
15|
161
17|
1S|
191
20|
211
22|
23|
24|
25|
261
LAB
SAMPLE ID
DATE
ANALYZED 1
DATE
[ANALYZED 2
COMMENTS:
page of
FORM IV PEST
3/90
-------
5B
SEMIVOLATILE ORGANIC INSTRUMENT PERFORMANCE CHECK
DECAFLUOROTRIPHENYLPHOSPHINE (DFTPP)
Lab Name: Contract:
Lab Code: Case No.: SAS No.: SDG No.:
Lab File ID: DFTPP Injection Date:
Instrument ID: DFTPP Injection Time:
m/e
=====
51
68
69
70
127
197
198
199
275
365
441
442
443
. .
ION ABUNDANCE CRITERIA
30.0 - 80.0% of mass 198
Less than 2.0% of mass 69
Mass 69 relative abundance
Less than 2.0% of mass 69
25.0 - 75.0% of mass 198
Less than 1.0% of mass 198
Base Peak, 100% relative abundance
5.0 to 9.0% of mass 198
10.0 - 30.0% of mass 198
Greater than 0.75% of mass 198
Present, but less than mass 443
40.0 - 110.0% of mass 198
15.0 - 24.0% of mass 442
% RELATIVE
ABUNDANCE
( ) 1
( ) 1
( )2
1 -Value is % mass 69
2-Value is % mass 442
CHECK APPLIES TO THE FOLLOWING SAMPLES, MS, MSD, BLANKS, AND STANDARDS:
| EPA
j SAMPLE NO.
1 -
oil
02|
031
04|
05|
06 |
07|
08 |
09 |
10|
HI
12|
131
141
15|
16|
17|
18|
191
20|
211
22|
LAB
SAMPLE ID
LAB
FILE ID
DATE
ANALYZED
TIME
ANALYZED
page of
FORM V SV
3/90
-------
6D
SEMIVOLATILE ORGANICS INITIAL CALIBRATION DATA
Lab Name:
ib Code:
Case No.:
Contract:
SAS No.:
SDG No.
Instrument ID:
Calibration Date(s):
Calibration Times:
ILAB FILE ID: RRF20 = RRF50 =
|RRF80 - RRF120= RRF160=
1
1 1
| COMPOUND IRRF20
I Phenol *
1 bis (2-Chloroethyl) ether *
I 2-Chlorophenol *
|l,3-Dichlorobenzene *
I 1,4-Dichlorobenzene *
| 1 , 2-Dichlorobenzene *
I 2-Methylphenol *
1 2 , 2 ' -oxybis (1-Chloropropane) |
|4-Methylphenol *
|N-Nitroso-di-n-propylamine *
| Hexachloroethane *
j Nitrobenzene *
| Isophorone *
I^Nitroohenol *
IW4-Dimethylphenol *
j bis (2-Chloroethoxy ) methane *
1 2 , 4 -Dichlorophenol *
1 1 , 2 , 4-Trichlorobenzene *
| Naphthalene *
J4-Chloroaniline |
j Hexachlorobutadiene j
|4-Chloro-3-methylphenol *
|2-Methylnaphthalene *
JHexachlorocyclopentadiene |
|2,4,6-Trichlorophenol *
1 2 , 4 , 5-Trichlorophenol *
j 2-Chloronaphthalene *
|2-Nitroaniline I
| Dimethylphthalate I
j Acenaphthylene *
|2,6-Dinitrotoluene *
| 3-Nitroaniline I
j Acenaphthene *
1 2 , 4-Dinitrophenol I
|4-Nitrophenol |
| Dibenzof uran *
|2,4-Dinitrotoluene *
1 1
RRF50
RRF30
|RRF120
-~
1
[RRF160
RRF
%
RSD
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
^
*
*
Compounds with required minimum RRF and maximum %RSD values.
Ail other compounds' must meet a minimum RRF of 0.010.
FORM VI SV-1
3/90
-------
6C
SEMIVOLATILE ORGANICS INITIAL CALIBRATION DATA
Lab Name:_
Code:
Case No.:
Contract:
SAS No.:
SDG No.
Instrument ID:
Calibration Date(s):
Calibration Times:
LAB FILE ID: RRF20 '
RRF80 = RRF120=
= RRF50 =
RRF160=
COMPOUND
Diethylphthalate
RRF20
4-Chlorophenyl-phenylether *
Fluorene *
4-Nitroaniline
4 , 6-Dinitro-2-methylphenol
N-Nitrosodiphenylamine (1)
4 -Broroophenyl-pheny lether '
Hexachlorobenzene '
k
k
Pentachlorophenol *
Phenanthrene *
Anthracene *
Carbazole
p^-n-butylphthalate
^borantnene *
Pyrene *
Butylbenzylphthalate
3,3' -Dichlorobenzidine
Benzo (a) anthracene *
Chrysene *
bis (2-Ethylhexyl) phthalate
Di-n-octylphthalate
Benzo (b) f luoranthene *
Benzo (k) f luoranthene *
Benzo(a)pyrene *
Indeno(l,2,3-cd)pyrene *
Dibenz (a , h) anthracene *
Benzo (g,h,i)perylene *
Nitrobenzene-d5 |
2-Fluorobiphenyl *
Terphenyl-dl4 *
Phenol-<35 *
2-Fluorophenol *
2,4, 6-Tribromophenol |
2-Chlorophenol-d4 *
l,2-Dichlorobenzene-d4 *
1
RRF50
RRF80
PJIF120
RRF160
RRF
%
RSD
i
4
*
*
It
*
*
*
*
*
*
4
*
*
*
it
*
*
*
*
*
*
*
*
(1) Cannot be separated from Diphenylamine
*
« pounds with required minimum RRF and maximum %RSD values.
other compounds must meet a minimum RRF of 0.010.
FORM VI SV-2
3/90
-------
PESTICIDE INITIAL CALIBRATION OF SINGLE COMPONENT ANALYTES
ab Name:
ab Code:
nstrument ID:
C Column:
Contract:
Case No.: SAS No.:
Level (x low): low
ID: (mm)
Date(s) Analyzed:
COMPOUND
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC (Lindane)
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin
4,4' -DDE
Endrin
Endosulfan II
4,4' -ODD
Endosulfan sulfate
4,4' -DDT
Methoxychlor
Endrin ketone
Endrin aldehyde
alpha-Chlordane
gamma-Chlordane
Tetrachloro-m-xylene
Decachl orobipheny 1
RT O
LOW
F STAND-
.MID
&RDS
HIGH
MEAN
RT
RT W
FROM
IN DOW
TO
* Surrogate retention times are measured from Standard Mix A analyses.
Retention time windows are +0.05 minutes for all compounds that elute
before Heptachlor epoxide, ±0.07 minutes for all other compounds,
except ±0.10 minutes for Decachlorobiphenyl.
FORM VI PEST-1
3/90
-------
6E
PESTICIDE INITIAL CALIBRATION OF SINGLE COMPONENT ANALYTES
Name:
Lab Code:
Contract:
SAS No.:
Instrument ID:
GC Column:
Case No.:
Level (x low): low
ID: (mm) Date(s) Analyzed:
SDG No.: _
mid high
COMPOUND
alpha-BHC
beta-BHC
delta-BHC
gamma-BHC (Lindane)
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan I
Dieldrin
4,4' -DDE-
Endrin
Endosulfan II
4,4' -ODD
Endosulfan sulfate
4,4' -DDT
Methoxychlor
Endrin ketone
Endrin aldehyde
alpha-Chlordane
gamma-Chlordane
Tetrachloro-m-xylene
Decachlorobiphenyl
LOW
CALIBRATK
MID
DN FACTORS
HIGH
MEAN
%RSD
* Surrogate calibration factors are measured from Standard Mix A analyses
%RSD must be less than or equal 20.0 % for all compounds except the
surrogates, where %RSD must be less than or equal to 30.0%. Up to
two target compounds, but not surrogates, may have %RSD greater than
20.0% but less than or equal to 30.0%.
FORM VI PEST-2
3/90
-------
6F
PESTICIDE INITIAL CALIBRATION OF MULTICOMPONENT ANALYTES
Lab Name:
Lab Code:
Instrument ID:
GC Column:
Contract:
Case No.: SAS No.: SDG No.:
Date(s) Analyzed:
ID: (mm)
COMPOUND
Toxaphene
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
AMOUNT
(ng)
PEAK | RT
*1
*2
*3
4
5
*1
*2
*3
4
5
*1
*2
*3
4
5
*1
*2
*3
4
5
*1
*2
*3
4
5
*1
*2
*3
4.
5
*1
*2
*3
4
5
*1
*2
*3
4
5
*
RT W
FROM
INDOW
TO
.«
CALIBRATION
FACTOR
* Denotes required peaks
FORM VI PEST-3
3/90
-------
6G
PESTICIDE ANALYTE RESOLUTION SUMMARY
Lab Name:
Lab Code:
Case No. :
Contract:
SAS No. : S
SDG No.:
GC Column (1):
ID:
EPA Sample No. (Standard 1) :
Date Analyzed (1):
(mm) Instrument ID (1):
Lab Sample ID (1):
Time Analyzed (1):
1
| ANALYTE
01!
021
031
04 |
05|
06|
07|
08 |
091
RT
RESOLUTION
(%)
GC Column (2):
ID:
EPA Sample No. (Standard 2) :
(mm) Instrument ID (2):
Lab Sample ID (2):
fzed (2) :
1
| ANALYTE
on
02|
03|
04|
05|
06|
07|
08 |
09|
rime An.
RT
alyzed (2) :
RESOLUTION
(%)
Resolution of two adjacent peaks must be calculated as a percentage of the
height of the smaller peak, and roust be greater th?.r or equal to 60.0%.
FORM VI PEST-4
3/90
-------
7D
SEMIVOLATILE CONTINUING CALIBRATION CHECK
,ab Name:
Code:
Case No.:
Contract:
SAS No.:
Instrument ID:
File ID:
Calibration Date:
SDG No.
Time:
Init. Calib. Date(s):
Init. Calib. Times:
1
| COMPOUND
I Phenol
1 bis (2-Chloroethyl) ether
| 2-Chlorophenol
1 1 , 3-Dichlorobenzene
1 1 , 4 -Dichlorobenzene
| 1,2-Dichlorobenzene
|2-Methylphenol
| 2 , 2 ' -oxybis ( 1-Chloropropane)
| 4-Methylphenol
| N-Nitroso-di-n-propylamine
| Hexachloroe thane
| Nitrobenzene
| Isophorone
| 2-Nitrophenol
| 2 , 4 -Diraethylphenol
| bis ( 2-Chloroethoxy) methane
1 2 , 4-Dichlorophenol
| 1 , 2 , 4 -Trichlorobenzene
| Naphthalene
| 4-Chloroaniline
| Hexachlorobutadiene
| 4-Chloro-3-methylphenol
J 2-Methylnaphthalene
| Hexachlorocyclopentadiene
| 2 , 4 , 6-Trichlorophenol
1 2 , 4 , 5-Trichlorophenol
| 2-Chloronaphthalene
I 2-Nitroaniline
1 Dimethylphthalate
| Acenaphthylene
| 2 , 6-Dinitrotoluene
| 3-Nitroaniline
| Acenaphthene
| 2 , 4 -Dinitrophenol
I 4-Nitrophenol
| Dibenzofuran
j 2,4-Dinitrotoluene
1
RJRF
| MIN |
RRF50 | RRF j
====== | ===== |
| 0.800|
|0.700|
|0.800|
|0.600|
|0.500|
|0.400|
|0.700|
| MAX|
%D | %D |
====== |====|
|25.0|
I25.0J
|25.0|
|25.0|
|25.0|
125. OJ
|25.0|
II II
|0.600|
|0.500|
|0.300|
I0.200J
|0.400|
|0.100|
|0.200|
10.3001
(0.2001
|0.200|
|0.700|
|25.0|
|25.0|
|25.0|
|25.0|
|25.0|
|25.0|
|25.0|
125.01
|25.0|
|25.0|
|25.0|
II II
II II
10.2001
|0.400|
|25.0|
125.01
II II
10.2001
10.200)
10.800]
125.01
|25.0|
|25.0|
II II
II II
11.3001
|0.200|
125.01
125.01
II II
10.800)
1 1
1 1
10.8001
10.2001
125.01
1 1
1 1
125.01
125.01
II II
All other compounds must meet a minimum RRF of 0
.010.
FORM VII SV-1
3/90
-------
7C
SEMI VOLATILE CONTINUING CALIBRATION CHECK
.b Name:
ib Code:
Case No.:
Contract:
SAS No.:
istrument ID:
ib File ID:
Calibration Date:
SDG No.
Time:
Init. Calib. Date(s):
Init. Calib. Times:
1
| COMPOUND
1 Diethvlphthalate
1 4-Chlorophenyl-phenylether
J Fluorene
1 4-Nitroaniline
| 4 , 6-Dinitro-2-methylphenol
IN-Nitrosodiphenylaraine (1)
I 4-Bromophenyl-phenylether
| Hexachlorobenzene
. | Pentachlorophenol
I Phenanthrene
| Anthracene
| Carbazole
j Di-n-butylphthalate
| Fluoranthene
| Pyrene
| Butylbenzylphthalate
| 3 , 3 ' -Dichlorobenzidine
| Benzo (a) anthracene
| Chrysene
| bis (2-Ethylhexyl) phthalate
1 Di-n-octylphthalate
J Benzo (b) fluoranthene
| Benzo (k) fluoranthene
| Benzo (a) pyrene
| Indeno (1,2,3 -cd) pyrene
1 Dibenz (a , h) anthracene
| Benzo (g,h, ijperylene
j Nitrobenzene-d5
| 2-Fluorobiphenyl
|Terphenyl-dl4
|Phenol-d5
| 2-Fluorophenol
| 2 , 4 , 6-Tribromophenol
| 2-Chlorophenol-d4
I l,2-Dichlorobenzene-d4
1
RRF
| MIN |
RRF50 | RRF
1 1
|0.400|
|0.900|
1 1
1 1
1 1
10.1001
(0.1001
|0.050|
|0.700|
|0.700|
1 1
1 1
|0.600|
|0.600|
1 1
1 1
|0.800|
10.7001
1 1
1 1
10.7001
|0.700|
10.7001
10.5001
(0.4001
10.5001
10.2001
10.7001
10.5001
|0.800|
|0.600|
1 1
10.800)
|0.400|
1 1
| MAX|
%D | %D |
1 1
1 1
|25.0|
|25.0|
1 1
1 1
1 1
|25.0|
125.01
125.01
125.01
|25.0|
1 1
1 1
125.01
125.01
1 1
1 1
125.01
|25.0|
1 1
1 1
125.01
125.01
125.01
125.01
125.01
125.01
= = 1 1
|25.0|
125.01
125.01
125.01
125.01
1 1
125.01
125.01
1 I'
(1) Cannot be separated from Diphenylamine
All other compounds must meet a minimum RRF of 0.010
FORM VII SV-2
3/90
-------
7D
PESTICIDE CALIBRATION VERIFICATION SUMMARY
Lab Name:_
Lab Code:
GC Column:
Contract:
SAS No.:
Case No.: SAS No.: SDG No.
ID: (mm) Init. Calib. Date(s):
EPA Sample No.(PIBLK):
Lab Sample ID (PIBLK):
EPA Sample No.(PEM): _
Lab Sample ID (PEM):
Date Analyzed
Time Analyzed
Date Analyzed
Time Analyzed
PEM
COMPOUND
alpha-BHC
beta-BHC
gamma-BHC (Lindane)
Endrin
4,4' -DDT
| Methoxychlor
RT
RT W
FROM
ENDOW
TO
CALC
AMOUNT
(ng)
NOM
AMOUNT
(ng)
RPD
4,4'-DDT % breakdown (1) :
Combined % breakdown (1):
Endrin % breakdown (1)
QC LIMITS:
RPD of amounts in PEM must be less than or equal to 25.0%
4,4'-DDT breakdown must be less than or equal to 20.0%
Endrin breakdown must be less than or equal to 20.0%
Combined breakdown must be less than or equal to 30.0%
FORM VII PEST-1
3/90
-------
7E
PESTICIDE CALIBRATION VERIFICATION SUMMARY
Lab Name:_
Lab Code:
GC Column:
Contract:
SAS No.:
Case No.: SAS No.: SDG No.:
_ ID: (mm) Init. Calib. Date(s):
EPA Sample No.(PIBLK):
Lab Sample ID (PIBLK):
EPA Sample No.(INDA):
Lab Sample ID (INDA):
Date Analyzed :
Time Analyzed :_
Date Analyzed :_
Time Analyzed :_
INDIVIDUAL MIX A
COMPOUND
alpha-BHC
qamma-BHC (Lindane)
Heptachlor
Endosulfan I
Dieldrin
Endrin
4.4' -ODD
4,4' -DDT
Methoxychlor
Tetrachloro-m-xylene
Decachlorobiphenyl
RT
RT W]
FROM
.
ENDOW
TO
-
-
CALC
AMOUNT
(ng)
NOM
AMOUNT
(ng)
RPD
EPA Sample No.(INDB):
Lab Sample ID (INDB):
Date Analyzed :
Time Analyzed :
INDIVIDUAL MIX B
COMPOUND
beta-BHC
delta-BHC
Aldrin
Heptachlor epoxide
4, 4 '-DDE
Endosulfan II
Endosulfan sulfate
Endrin ketone
Endrin aldehyde
alpha-Chlordane
gamma-Chlordane
Tetrachloro-m-xylene
Decachlorobiphenyl
RT
RT W]
FROM
ENDOW
TO
CALC
AMOUNT
(ng)
NOM
AMOUNT
(ng)
RPD
QC LIMITS:
RPD of amounts in the Individual Mixes must be less than
or egual to 25.0%.
FORM VII PEST-2
3/90
-------
8B
SEMIVOLATILE INTERNAL STANDARD AREA AND RT SUMMARY
Lab Name:_
ib Code:
Case No.:
Contract:
SAS No.:
SDG No. :
Lab File ID (Standard):
Instrument ID:
Date Analyzed:
Time Analyzed:.
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
12 HOUR STD
UPPER LIMIT
LOWER LIMIT
EPA SAMPLE
NO.
ISl(DCB)
AREA *
RT |
IS2 (NPT)
AREA |
-
'
RT £
IS3 (ANT)
AREA #
-
RT «
IS1 (DCB) = l,4-Dichlorobenzene-d4
IS2 (NPT) - Naphthalene-d8
IS3 (ANT) - Acenaphthene-dlO
AREA UPPER LIMIT = +100% of internal standard area
AREA LOWER LIMIT = - 50% of internal standard area
RT UPPER LIMIT = +0.50 minutes of internal standard RT
RT LOWER LIMIT = -0.50 minutes of internal standard RT
I Column used to flag internal standard area values with an-asterisk.
* Values outside of QC limits.
page of
FORM VIII SV-1
3/90
-------
8C
SEMIVOLATILE INTERNAL STANDARD AREA AND RT SUMMARY
Lab Name:
^feab Code:
w
Lab File ID
.
Case No. :
Contract:
SAS No. :
(Standard) :
SDG No.
Date Analyzed:
*
Instrument ID:
Time Analyzed:
01
02
03
04
05
06
07
08
09 |
10|
HI
12|
13|
14|
15|
16|
17|
18|
19|
20|
21|
22|
12 HOUR STD
UPPER LIMIT
LOWER LIMIT
============
EPA SAMPLE
-NO.
IS4 (PHN)
AREA £
==========
RT g
ISS(CRY)
AREA %
....
RT S
IS6(PRY)
AREA $
RT f
=======
-
IS4 (PHN) = Phenanthrene-dlO
IS5 (CRY) = Chrysene-dl2
IS6 (PRY) = Perylene-dl2
AREA UPPER LIMIT = +100% of internal standard area
AREA LOWER LIMIT = - 50% of internal standard area
RT UPPER LIMIT = +0.50 minutes of internal standard RT
RT LOWER LIMIT = -0.50 minutes of internal standard RT
U Column used to flag internal standard area values with an asterisk.
* Values outside of QC limits.
of
FORM VIII SV-2
3/90
-------
,ab Name:_
,ab Code:
;c Column:
8D
PESTICIDE ANALYTICAL SEQUENCE
Contract:
SAS No.:
Case No.: SAS No.: SDG No.:
ID: (mm) Init. Calib. Date(s):
nstrument ID:
THE ANALYTICAL SEQUENCE OF PERFORMANCE EVALUATION MIXTURES, BLANKS,
SAMPLES, AND STANDARDS IS GIVEN BELOW:
| MEAN SURROGATE RT FROM INITIAL CALIBRATION
I TCX: DCB:
1
| EPA
| SAMPLE NO.
oil
02|
03|
04]
05|
06|
07|
08|
09|
X0|
HI
121
13|
141
151
16|
17|
18|
19|
20|
211
22|
23|
24|
25|
26|
27|
28|
29|
30|
311
32|
LAB
SAMPLE ID
.
DATE
ANALYZED
TIME
ANALYZED
TCX
RT g
DCB
RT S
TCX = Tetrachloro-m-xylene
DCB = Decachlorobiphenyl
QC LIMITS
(+ 0.05 MINUTES)
( + 0.10 MINUTES)
c Column used to flag retention time values with an asterisk.
* Values outside of QC limits.
page of
FORM VIII PEST
3/90
-------
Lab Name:
b Code:
9A
PESTICIDE FLORISIL CARTRIDGE CHECK
Contract:
SAS No.:
Case No.:
SDG No.:
Florisil Cartridge Lot Number:
GC Column(l): ID:
Date of Analysis:
(mm) GC Column(2):
ID:
(mm)
' COMPOUND
alpha-BHC
garoma-BHC (Lindane)
Heptachlor
Endosulfan I
Dieldrin
Endrin
4 , 4 « -ODD
4,4' -DDT
Methoxychlor
Tetrachloro-m-xylene
Decachlorobiphenyl
SPIKE
ADDED
(ng)
.
SPIKE
RECOVERED
(ng)
1 *
REC $
1
-QC
LIMITS
80-120
80-120
80-120
80-120
80-120
80-120
80-120
80-120
80-120
80-120
80-120
£ Column to be used to flag recovery with an asterisk.
* Values outside of QC limits.
THIS CARTRIDGE LOT APPLIES TO THE FOLLOWING SAMPLES, BLANKS, MS, AND MSD:
| EPA
| SAMPLE NO.
I
on
02|
03|
04|
05|
06|
07|
08|
09|
10|
HI
12|
13|
14|
15|
16|
17|
18|
19|
20|
211
22|
23|
LAB
SAMPLE ID
DATE
ANALYZED 1
DATE
ANALYZED 2
page of
FORM IX PEST-1
3/90
-------
9B
PESTICIDE GPC CALIBRATION
Lab Name:
Lab Code:
Case No. :
Contract:
SAS No. : S
GPC Column:
GC Column(1):
ID:
SDG No. :
Calibration Date:
(mm) GC Column(2): _
ID:
(mm)
COMPOUND
gamroa-BHC (Lindane)
Heptachlor
Aldrin
Dieldrin
Endrin
4,4' -DDT
SPIKE
ADDED
(ng)
| SPIKE
| RECOVERED
(ng)
1 QC. |
1 % | LIMITS |
REC $| REC. |
180-1101
180-1101
180-1101
180-1101
180-1101
(80-1101
1 1
# Column to be used to flag recovery values with an asterisk
* Values outside of QC limits
THIS GPC CALIBRATION APPLIES TO THE FOLLOWING SAMPLES, BLANKS, MS AND MSD:
| EPA
| SAMPLE NO.
oil
02|
03|
04 |
05|
06 |
07|
08|
09|
10|
111
12|
13|
14|
15|
16|
17|
18|
19|
20|
21|
22|
23|
24|
25|
26|
LAB
SAMPLE ID
DATE
ANALYZED 1
DATE
ANALYZED 2
-
page of
FORK IX PEST-2
3/90
-------
Lab Name:
Lab Code:
10A
PESTICIDE IDENTIFICATION SUMMARY
FOR SINGLE COMPONENT ANALYTES
Contract:
SAS No.:
Case No. :
SDG No. :
Lab Sample ID :
Instrument ID (1):
GC Column(1):
ID:
Date(s) Analyzed:
Instrument ID (2):
(mm) GC Column(2):
ID:
(nun
ANALYTE
1 1
|COL| RT
1
2
1
1
2
1
2
1
2
1
2
1
2
1
2
RT W
FROM
-
'
INDOW
TO
i
I
CONCENTRATION
%D
page of
FORM X PEST-1
3/90
-------
10B
PESTICIDE IDENTIFICATION SUMMARY
FOR MULTICOMPONENT AHALYTES
EPA SAMPLE NO.
Lab Name:
Lab Code:
Case No. :
Contract :
SAS No. : S
SDG No.:
Lab Sample ID :
Instrument ID (1) :
GC Column(1):
ID:
Date(s) Analyzed:
Instrument ID (2):
(mm) GC Column(2):
ID:
(mm)
ANALYTE
COLUMN 1
COLUMN 2
COLUMN 1
COLUMN 2
COLUMN 1
COLUMN 2
1
IPEAKI RT
i
2
3
4
5
1
2
3
4
5
1
2
3
4
5
I
2
3
4
5
1
2
3
4
5
1
2
3
4
5
======
======
RT W
FROM
======
======
IN DOW
TO
======
CONCENTRATION
=============
=============
MEAN
CONCENTRATION
%D
_
At least 3 peaks are required for identification of multicomponent analytes
page of
FORM X PEST-2 3/90
-------
SAMPLE LOG-IT- Slie.e.1
l^hNmmc- P«Ee of
Received By ( Print Nine): _ . I ng-Jn Dale:
Case Number.
Sample Delivery
Group No_-
SAS Number
REMARKS:
I. Cuaody SeaJ(x) Present/Absent"
Inlact/Brokeo
2. Cnaody Seal Not*
3. Chairn-oC-Cueody Present/Absent*
Records
4. Traffic Reporu or Present/ Absent*
Packing Lia
5. Airbill Airbill/Slicker
Present/Absent*
6. Airbill No.:
7. Sampk Tags Preieal/Abxeol*
SuopleTic Listed/Not Listed
{ Numben oo Chaia-of-
Cooody
'. 8. Sample Condition: Inuet/Brokea*/
I 9. Does informatioa oo
teporti,aad cample
up tfftc.1 Tei/rio*
11. Time Received:
Sample Transfer
Friction*
Am t- . ,.
Bv ..,..._.. . .
On-
EPA
SAMPLE
.. -
CORRESPONDING
SAMPLE
TAG
«
ASSIGNED
LAB
"
REMARKS:
CONDITION
OF SAMPLE
SHIPMENT, ETC.
Cooua SMO wid «O»
-------
APPENDIX O
STATISTICAL TABLES
-------
TABLE A-2 Percentiles of the t distribution
T.
(a) Student's t distribution
X
1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
35
40
45
50
60
70
80
90
100
120
140
160
180
200
OB
55
0.158
0.142
0137
0.134
0.132
0.131
0.130
0.130
0.129
0.129
0.129
0.128
0.128
0.128
0.128
0.128
0.128
0.127
0.127
0.127
0.127
0.127
0.127
0.127
0.127
0127
0.127
0.127
0127
0.127
0127
0.126
0.126
0.126
0.126
0.126
0126
0.126
0126
0.126
0.126
0.126
0.126
0.126
0.126
66
0.510
0.445
0.424
0.414
0.408
0.404
0.402
0.399
0398
0.397
0.396
0.39S
0.394
0.393
0.393
0.397
0.392
0.392
0.391
0.391
0.391
0390
0.390
0.390
0.390
0.390
0.389
0.389
0389
0.389
0.388
0.388
0.388
0.388
0.387
0.387
0387
0387
0.386
0.386
0.386
0.386
0.386
0.386
0.385
75
1.000
0.816
0.765
0.741
0.727
0.718
0.711
0.706
0.703
0.700
0.697
0.695
0.694
0.692
0.691
0.690
0.689
0.688
0.688
0.687
0686
0.686
0.685
0.685
0.684
0.684
0.684
0.683
0.683
0.683
0.682
0.681
0.680
0.679
0.679
0.678
0678
0677
0.677
0.677
0.676
0.676
0.676
0.676
0.674
85
1.963
1.38C
1.250
1.190
1.156
1.134
1.119
1.108
1.100
1.093
1.088
1.083
1.079
1.076
1.074
1.071
1.069
1.067
1.066
1.064
1.063
1.061
1.060
1.059
1.058
1.058
1.057
1.056
1.055
1.055
1.052
1 050
1.049
1.047
1.045
1.044
1.043
1.042
1.042
1.041
1.040
1.040
1.039
1.039
1.036
90
3078
1386
1.638
1.533
1.476
1.440
1.415
1497
1.383
1.372
1.363
1.356
1.350
1.345
1.341
1437
1.333
1.330
1.328
1.325
1.323
1321
1.319
1.318
1.316
1315
1.314
1.313
1311
1310
1.306
1303
1.301
1299
1296
1.294
1.292
1.291
1.290
1.289
1.288
1287
1286
1.286
1.282
95
6.314
2.920
2353
2.132
2.015
1.943
1.895
1.860
1.833
1.812
1.796
1.782
1.771
1.761
1.753
1.746
1.740
1.734
1.729
1.726
1.721
1.717
1.714
1.711
1.708
1.706
1.703
1.701
1.699
1.697
1.690
1.684
1.679
1.676
1.671
1.667
1.664
1.662
1.660
1.658
1.656
1.654
1.653
1.653
1.645
97.S
12.706
4303
3.182
2.776
2.571
2.447
2.365
2306
2.262
2228
2201
2.179
2.160
2.145
2.131
2.120
2.110
2.101
2.093
2.086
2.060
2.074
2.069
2.064
2.060
2.056
2.052
2.048
2.045
2.042
2030
2.021
2.014
2.009
2.000
1594
1.990
1387
1.984
1.980
1.977
1.975
1.973
1.972
1.960
99
31321
6.965
4.541
3.747
3.365
3.143
2.998
2396
2.821
2.764
2.718
2.681
2.650
2.624
2.602
2583
2.567
2.552
2.539
2.528
2.518
2.508
2.500
2.492
2.485
2.479
2.473
2.467
2.462
2.457
2.438
2.423
2.412
2.403
2.390
2.381
2374
2.368
2.364
2.358
2.353
2.350
2.547
2345
2.326
99.5
63.657
9925
5341
4.604
4.032
3.707
3.499
3.355
3.250
3.169
3.106
3.055
3.012
2.977
2.947
2.921
2 £98
2.878
2.861
2.845
2331
2319
2.807
2.797
2.787
2.779
2.771
2.763
2.756
2.750
2.724
2.704
2.690
2.678
2.660
2.648
2639
2632
2.626
2.617
2.611
2.607
2.603
2.601
2.576
99.96
636.619
31.599
12.924
8.610
6.869
5.959
5.408
5.041
4.781
4.587
4.437
4.318
4.221
4.140
4073
4.015
3.965
3.922
3.883
3350
3.819
3.792
3.768
3.745
3.725
3.707
3.690
3.674
3.659
3.646
3.591
3.561
3.520
3.496
3.460
3.435
3.416
3.402
3.390
3.373
3.361
3.352
3.345
3.340
3.291
495
-------
TABLE A-4 Percentiles of the F distribution
"t.fif
Upptr 25% point of the F dittribulion
(c) F distribution
too
175
150
TOO
300
500
IOOO
DEGREES OF FREEDOM FOft NUMERATOR
II II
13
75
30
40
5O
10O ISO 70O
147
146
I «5
1 47
14?
750
300
7.78
700
186
I 76
1.70
166
16?
160
158
156
155
153
152
151
1 51
ISO
149
149
148
148
147
I 47
I 47
146
146
I 46
I 4ft
145
145
1 38
1 J7
1 37
I 37
I 16
1 43
143
143
143
143
14?
I 41
I 34 141
I 34 141
114 141
1 34 1 40
I 33 1 40
133 140
I 33 I 39
I 33 1 39
I 35
135
820
3 15
236
205
1 88
1 78
I 72
167
163
160
158
156
155
IS?
1.52
1.51
1.50
149
1 49
1.48
148
I 47
1 47
146
1 46
145
I 45
145
I 45
I 44
1 44
143
143
I 43
142
I 42
142
1 42
1 41
I 41
858
321
239
208
189
I 79
1 72
166
163
I 59
1.57
155
153
1 S?
1 51
I5O
149
148
1 47
147
146
145
1 45
I 44
I 43
1 43
1 43
1 42
142
142
I 41
I 41
I 40
1 40
I 40
I 40
I 39
1.39
682
328
241
207
I 89
I 79
1 71
166
162
1.59
156
1 54
I 52
151
149
148
1 47
1 46
I 46
145
144
141 1 38
1 40 1 38
140 138
139 I 37
I 39 137
I 39 I 36
I 38 I 36
I 38 1 36
I 38 I 35
137 135
898
331
247
208
189
1 78
1 71
165
161
1 58
1.55
1 53
1 SI
1.50
1.48
147
146
145
9.10 9 19
3 34 3 35
243 2 44
208 208
I 89 I 89
I 78 1.78
1 7O I 70
164 164
1 60 1 60
157 156
1 54 1.53
152 151
I SO 1.49
1.49 148
1.47 146
146 1.45
1 45 1 44
I 44 1 43
I 43 1 42
143 1.42
I 43
143
I 42
142
I 42
1 41
1 41
I 41
1 40
1.40
I 19
I 39
I 39
I 38
1 38
I 38
1 38
137
1 37
I 36
I 36
1 IS
1 35
1 34
I 34
I 34
I 33
I 33
142
1 4?
I 41
I 41
1 41
140
1 40
1 40
I 39
1 39
1 18
I 38
I 37
I 37
137
1 36
I 36
1 16
I 16
1 IS
I 14
I 34
1 11
1 31
1 33
1 12
1 12
I 12
1 It
141 1 40
141 1 40
I 40 1 39
1 40 I 39
1 19
1 39
1 39
I 38
I 38
I 17
1 17
I 16
1 36
I 36
1 35
I 35
I 35
I 35
1.34
I 38
1 38
1 38
1 37
I 37
1 36
1 36
1 35
I 35
I 35
I 34
I 34
I 14
1 33
1 33
1 33 1 32
I 33 1 32
13? Ill
1 12 131
1 32 1.30
131 110
111 I 30
1 30 1 79
I 30 1 79
I 1O 1 78
9 26 9 12
337 318
2 44 7 44
706 208
189 189
1.77 177
1 69 1 69
163 163
1.59 1.59
1 56 1.55
153 1.52
1.51 ISO
1.49 148
1.47 1.46
937
339
245
708
189
1 77
169
163
1.58
1.55
I 52
1.49
I 47
1 46
1 46 145 1 44
1 44 1 44
I 43 I 43
147 14?
141 141
141 I 40
I 40 1 39
139 139
39 1 38
38 1 38
38 1 37
37 I 37
37 I 36
37 I 36
36 I 35
36 I 35
35 I 34
35 I 34
34 133
34 I 33
34 1 13
33 13?
33 132
31 1 32
32 131
3? I 31
143
142
1 41
1 40
1 39
1 39
I 38
1 31
I 37
1 36
1 36
1 35
1 35
I 35
1.34
I 34
I 11
I 33
1 3?
I 32
I 32
1.31
1 31
1 31
1 30
941
3 39
245
208
1 89
1 77
168
16?
1 58
1 54
1 51
1 49
1 47
1 45
1 44
1 43
141
I 40
140
1 39
1 38
I 37
I 37
1.36
1 36
1 35
I 35
t 34
1 34
I 34
1 31
1 13
1 3?
1.3?
1 31
1 31
I 31
1 30
1 30
1 30
31 130 179
31 I 30 1 79
30 I 79 I 78
10 1 79 1 78
79 1.78 1.77
79 I 78 I 77
78 1 77 1 77
78 1 77 1 76
77 I 76 I 76
77 I 76 1 75
944
340
745
2O8
1 89
1 77
I 68
162
1 58
I 54
151
I 49
1 47
1 45
1.43
142
1 41
1 40
I 39
I 38
I 37
I 37
I 16
1 16
I 35
I 35
I 34
I 34
I 11
1 33
1 32
I 32
I 31
1 31
I 31
1 30
1 30
1 10
I 79
1 79
947 949
141 341
745 746
708 208
189 189
1 76 I 76
1 68 1 68
1.67 I 6?
I.S7 157
1 54 153
1.51 ISO
I 48 1 48
1 46 1 46
95?
141
746
70S
188
1 76
1 68
16?
I 57
1 S3
I 50
1 48
1 46
.4? 141
41 140
40 I 39
39 1 38
138 137
I 37
I 36
I 37
I 36
136 135
I 35 1 35
1 34
I 34
1 33
I 13
I 33
1 3?
1 II
I 11
I 30
I 10
I 10
I 79
1 79
I 79
1 28
1 14
1 33
1 33
1 3?
1 3?
I 31
1 II
I 30
1 30
1 30
1 79
I 79
I 78
1 78
1 7B
953
347
746
708
1 88
1 76
1 67
1 61
1 57
1 53
1 SO
1 47
1 45
43 143 14? 14?
I 41
1 40
I 39
I 18
I 37
1 36
1.36
1 35
1 34
I 34
I 33
1 33
I 37
13?
1 3?
1 31
I 30
1 30
I 79
I 79
I 79
1 78
1 78
1 78
I 71
141
1 19
1 38
1 37
1 17
.16
.35
35
34
33
33
33
37
37
31
31
30
30
79
79
78
78
.78
27
77
179 178 1.77 177 176
I 78 I 77 1 77 I 76 1 76
177 177 1.76 176 175
1 77 1 76 1.76 I 75 I 75
I 77 1 76 1 T5 l.TS 1.74
1.76 1 75 I 75 1 74 1 74
I 76 1 75 1 74 I 24 I 21
1.25 I 24 I 74 1.71 1 71
1.75 I 74 I 73 I 73 17?
I 74 I 74 I 73 17? 17?
955
34?
746
708
I 88
I 76
167
161
I 56
I 53
ISO
1 47
1 45
143
14?
140
1 19
I 38
I 37
1.36
I 36
1.35
1 34
1 34
1 31
1 11
1 37
I 37
I 31
I 31
1.30
1 30
I 79
1 79
1 28
I 28
I 78
1.77
I 77
1 77
957
34?
746
208
1 88
1 76
167
161
1 56
1 S3
1 49
1 47
I 45
143
1 41
1 40
1 39
1 18
1 17
1 36
1 35
1 35
1 34
1 33
1 11
1 32
1 32
1 31
1 31
I 31
1 30
I 29
1 79
I 78
1 78
I 78
I 77
1 77
1 77
1 76
958
343
746
JOB
168
1 76
1 67
1 61
1 56
1 5?
I 49
1.47
1 45
1 43
1 41
I 40
1 39
1 38
I 37
1 36
I 35
I 34
1 34
1 33
1 11
1 3?
1 37
I 31
I 31
I 30
1.30
I 79
1 79
1 28
1 78
1 27
I 77
I 76
1 76
I 76
963
344
746
7 OB
188
1 75
I 67
160
I 55
1 S?
I 49
I 46
1 44
1 4?
1 40
1 39
1 38
1 37
1 36
1 35
1 34
I 33
1 33
I 3?
1 31
1 31
I 30
1 3O
I 30
I 79
I 78
1 78
1 27
1 77
1 76
1 76
I 75
1 75
I 75
1 75
967 9 71
3 44 3 45
747 747
708 708
188 1 88
1 75 I 75
1 66 I 66
I 6O I 59
1 55 I 54
151 1.51
1 48
1 45
1 43
1 41
1 40
38
37
36
35
34
33
3?
3?
31
31
30
10
79
79
78
78
77
76
76
75
76 1 76 1 75 1 75 I 71
.75 1 75 I 74 1 74 1 73
75 1 74 1 74 I 73 1 7?
74 I 24 I 73 I 73 1.71
74 1 73 1 23 1 73 I 71
73 I 73 I 7? 1.7? I 70
73 1 77 1 7? 1.21 1.70
.7? 12? I 71 I 71 1 19
7? I 71 I 71 1 70 1 19
71 I 71 I 70 1 70 1 18
1 47
1 45
1 4?
141
1 39
1 37
1.36
I 35
1 34
1 13
13?
131
1 31
1 30
I 79
I 29
I 28
1 78
I 77
I 77
1 76
I 76
I 75
I 74
1 74
75 1 74
74 I 73
24 123
24 1.22
23 I 22
9 74
346
247
208
I 88
1 75
I 66
I 59
1 54
1 50
147
1 44
1 42
I 40
1 38
1 37
1 36
1 34
1 33
1 32
1 32
1 31
1 30
I 79
I 79
1 78
I 78
1 77
1 77
1.76
I 75
I 25
1 24
I 74
I 73
I 23
I 2?
1.7?
I 71
1 71
980
347
747
708
I 6'
I 74
I 65
158
1 S3
I 49
I 46
1 43
1 41
1 39
1 37
1 36
1 34
I 33
13?
1 31
1 30
I ?9
1 79
1 78
1.77
I 77
1 76
1 75
1 75
I 75
1 74
I 73
1 7?
1 7?
1 71
1 71
I 70
1 70
I 19
I 19
981 98?
147 147
747 747
708 708
187 I 87
1 74 1 74
1 65 1 65
1 58 1 58
1 51 I 53
1 49 1 49
1 46
1 43
1 41
1 39
137
1 35
1 34
1 33
1 31
1 30
1 30
1 79
1 78
I 77
1 77
1 76
1 75
1 75
1 74
1 74
1 73
1 7?
I ??
1 71
I 70
1 70
1 19
1 19
1 19
I 18
I 46
1 43
1 40
1 38
1 37
1.35
1 34
1 3?
I 31
I 30
1 29
1 28
1 28
I 27
1 76
1 26
1 25
1 75
I 74
1 24
I 73
I 77
1 71
I 71
I 70
1 70
1 19
I 19
1 18
I 18
7? I 71 1 70 1 18 117 I 16
71 I TO 1 19 1 16 1 16 ,1 15
71 I 19 I 18 I 16 1 15 1 14
70 1 19 1 18 I 15 1 14 1 13
70 1 18 1 17 1.14 1 13 1 13
19 I 17 1.16 1 14 11? 11?
19 1 17 1 16 1 13 11? Ill
18 I 16 1 15 11? Ill 1 10
17 I 15 I 14 III I 10 I 09
17 115 1 14 110 1 09 1 08
13? 139 137 135 113 131 179 178 177 176 ITS 1 74 173 123 122 121 121 120 120 119 118 116 114 113 110 108 107
-------
TABLE A-4 Percentiles of the F distribution (continued)
Upp»r 10% point of tt* f dittritution
DEGREES OF FREEDOM FOR NUMERATOR
10
It
13
14
IS
IT
19
2O
30
4O SO IOO ISO 2OO
40
/90
ISO
700
300
500
399
8 S3
5.54
'4.54
406
3 78
3.59
346
336
3.29
3?3
3 18
3 14
3.10
3.07
305
303
301
299
297
296
295
294
293
292
291
290
289
289
288
287
286
284
284
283
281
281
2 79
2 78
277
2 76
2 76
1 72
272
495
900
548
432
3.78
346
3.26
3 11
301
292
288
281
2.76
2.73
2.70
167
264
262
2.61
259
257
2.56
2.55
254
253
252
2.51
250
2.50
249
248
247
246
245
244
243
243
242
24?
241
239
238
2.37
2.36
236
235
2 34
233
232
231
536
916
539
4 19
3.62
329
307
292
281
273
266
261
2.56
2.52
249
246
2.44
242
240
2.38
2.36
235
2.34
2.33
232
231
230
229
228
228
276
225
7 74
273
223
772
721
771
7 70
770
7 IB
7 16
7 15
7 1*
7 14
2 13
2 17
7 11
7 10
7.09
558
9.24
5.34
411
352
3.18
2.96
2.81
269
261
2.54
2.48
743
2.39
2.36
233
2.31
229
2.27
2.25
2.23
2.22
221
2 19
7.18
2 17
2 17
2 16
2 15
7 14
7 13
2 17
7 II
7 10
709
70S
708
707
707
706
704
7.03
707
701
700
199
I 98
197
1 96
1.96
572
9.29
53»
405
345
3.11
788
2.73
761
752
245
239
735
231
227
224
222
7.20
7 18
7 16
2 14
2 13
2 II
7 10
209
708
707
706
706
205
204
7 O2
701
701
7OO
199
I 98
I 98
1 97
197
195
193
1 97
1 91
I 91
189
1 89
I 88
I 87
1 86
587
933
578
401
3.40
306
2.83
267
255
246
239
233
278
724
771
2 18
2 15
2.13
7.11
209
2.08
706
705
204
2.02
201
200
200
1 99
196
1 91
196
1 94
194
I 93
1 97
I 91
I 91
I 9O
1 90
1 87
1 86
185
184
183
182
I 81
1 80
1 79
I 79
589
935
527
398
337
301
2 78
262
251
241
2.34
278
273
7.19
7 16
7.13
7.10
208
206
204
202
201
1 99
198
1 97
196
195
194
1 93
1 93
191
I 90
I R9
1 88
1 H7
I 86
I 86
1 85
I 85
1 84
I 82
180
79
78
78
77
76
75
74
73
594
937
5.75
395
3.34
7.98
2.75
259
2.47
238
2.30
224
2.20
2.15
2 12
709
206
704
702
200
198
1.97
195
1 94
1 93
192
1 91
190
189
I 88
187
186
I 85
184
1 8)
I 87
I 81
I 81
1 80
180
I 77
1.76
1 75
1 74
I 73
1 77
I 71
1 '0
169
168
599
938
574
3.94
3.37
296
2 72
2.56
744
235
227
771
7 16
7 12
209
206
2.03
200
1 98
196
1.95
1 93
192
1 91
189
188
187
I 87
I 86
185
183
187
1 81
180
I 79
i 78
I 78
I 77
I 77
l 76
1.74
1 77
I 71
I 70
1 69
168
1 67
166
I 65
164
602
939
523
392
330
294
270
254
747
737
225
2 19
2.14
2 10
206
203
700
198
196
194
1 92
1 90
1.89
188
1 87
1 86
1 85
1 84
183
187
1 81
1 79
I 78
1.77
1 76
1 74
1 7i
1 74
1 73
1 73
1 71
169
168
167
166
165
1 64
163
167
1 61
605
940
5.22
3.91
3 78
797
768
757
740
730
723
7 17
2 12
207
204
201
198
1 95
193
1.91
190
1 88
1 87
1 85
184
1 83
I 82
1 81
1 80
I 79
I 78
1 77
I 76
I 75
I 74
I 73
1 11
I 71
1 71
1 70
168
1 66
165
164
1 64
1 67
161
1 60
1.59
I 58
607
9.41
5.22
390
3.27
290
267
750
7 38
778
771
7 15
7 10
205
207
1.99
1 96
I 93
1.91
1 89
1 87
1 86
1 84
183
1.87
1 81
1 80
1 79
I 78
I 77
I 76
1 75
I 73
1 77
I 71
I 71
1 >0
169
1 69
1 68
I 66
I 64
1 63
167
161
1 60
1.59
1 58
I 57
I 56
60.9.
941
5.21
389
3.26
289
265
249
236
2 27
2 19
7 13
708
704
7OO
I 97
I 94
1 92
1 89
1.87
1 86
1 84
1.83
I 81
180
1 79
1 78
1 77
I 76
1 75
I 74
1 73
I 71
I 70
1 70
1 69
1 68
I 67
1 67
1 66
164
I 62
1 61
I 60
1 59
1 58
1 57
I 56
1 55
1 54
61.1
947
5 70
388
3.75
788
7.64
748
2 35
276
7 18
7 17
707
207
1 99
95
93
90
88
86
84
83
81
80
.79
77
76
75
75
74
77
M
JO
I 69
168
i e;
1 66
I 65
1 65
164
167
1 60
1 59
1 58
I 57
1 56
I 55
1 54
1.53
I 57
612
9.42
5 TO
387
3.24
287
263
246
234
224
2 17
7 10
705
701
1 97
1 94
1 91
1 89
1 86
1 84
1.83
1 81
1 80
I 78
I 77
I 76
1 75
1 74
I 73
1 7?
1 M
1 69
I 68
I 67
I 66
1 65
1 lib
I 64
1 63
1 63
160
1 59
1 57
I 56
I 56
1 54
1 53
I 57
I 51
1 SO
61.3
943
570
386
373
286
262
745
7.33
7 73
7 16
709
7O4
700
1 96
I 93
190
I 87
1 85
1 83
1 81
1 80
1 78
1 77
1 76
1 75
1 74
1 73
1 72
1 71
I b9
1 68
I 67
I 66
I 65
I b4
I bJ
I 63
1 62
1 61
1 59
1 57
I 56
I 55
I 54
153
I 57
1 51
1 49
I 49
771 2.31 2O9 195 185 178 1.77 168 164 161 158 155 153 151 149 148
615
943
519
386
377
7.85
761
745
732
277
7 15
708
703
199
1 95
197
1 89
1 86
I 84
1 87
1 80
1 79
1 77
1 76
1 75
1 73
I 77
I 71
I 71
1 70
1 68
1 67
I 66
1 65
1 64
I 6J
1 67
I 61
1 61
160
1 58
I 56
1 55
54
53
51
50
49
48
1 47
1 46
616
944
519
385
3.72
285
261
244
231
722
7 14
708
707
1 98
I 94
191
188
1 85
I 83
I 81
1 79
1 78
I 76
1 75
1 74
1 77
1 71
1 70
1 69
1 69
I 67
I 66
1 65
I 63
I 67
I 67
1 61
I 60
I S9
I 59
1 56
1 55
I 53
I 52
1 57
1 5O
1 49
1 48
I 47
1 46
61.7
944
5 19
385
321
284
2.60
243
230
2 71
7 13
707
7 O1
1 97
193
1 9O
1 87
1 84
1 87
1 80
I 78
1 77
1 75
1 74
1 73
I 71
1 70
1 69
1 68
1 68
I 66
1 65
I 64
I b?
I bl
I 61
I 6O
I S9
I 58
I 5R
I 55
I 54
1 57
1 51
1 5O
1 49
1 48
I 47
1 46
1 45
61.7
944
5.18
384
371
784
759
247
230
770
7 17
706
701
1 96
I 92
I 89
1 86
1 84
1 81
1 79
1 78
1 76
1 74
1 73
1 77
1 71
1 70
1 69
1 68
1 61
I 65
I 64
I bJ
1 til
I 61
I tin
I S9
I 58
1 57
I 57
1 54
I S3
1 51
1 50
1 49
1 48
I 47
I 46
I 45
1 44
62 1
945
5 17
383
3.19
281
257
240
277
7 17
7 1O
703
1 98
1 93
1 89
86
83
80
78
76
74
73
71
70
1 68
167
1 66
1 65
1 64
1 63
1 6?
I 60
I 5'J
I 58
I 5/
I 56
I 5S
I 54
I 54
1 53
1 5O
1 49
I 47
I 46
I 45
I 44
I 43
I 41
1 40
I 39
673
946
5 17
387
317
780
756
218
775
716
708
701
I 96
I 91
I 87
1 84
1 81
1 78
1 76
1 74
1 72
1 70
1 69
1 67
1 66
165
I 64
1 63
1 67
I 61
1 59
1 58
I 5b
I 55
1 54
I 53
I 57
1 VI
I 51
1 50
I 48
1 46
1 44
1 43
I 47
I 41
I 40
1 38
I 37
1 36
67.5
9.47
5 16
380
3 16
7.78
754
736
7 73
7 13
705
1 99
1 93
I 89
1 85
I 81
I 78
I 75
I 73
I 71
I 69
1 67
I 66
I 64
163
1 61
I 60
I 59
1 58
1 57
I 56
1 54
I bl
I 57
I 51
I 50
I 49
1 48
I it
1 46
1 44
I 47
I 40
I 39
I 38
1 36
I 35
1 34
1 37
I 31
67 7
947
5.15
380
3 15
2 77
252
2 35
277
7 17
7O4
I 97
I 97
1 87
1 83
1 79
I 76
1 74
I 71
1 69
1 67
1 65
1 64
1 67
1 61
59
58
57
56
55
53
57
51
49
48
4>
4b
46
45
44
41
39
38
36
35
34
33
31
V9
78
630
948
5 14
3 78
3.13
7 75
75O
7 37
7 19
7O9
701
94
88
83
79
76
73
I 70
I 67
165
I 63
I 61
I 59
I 58
1 56
1 55
1 54
I 53
I 5?
1 51
I 49
I 4;
1 46
1 45
I 43
I *J
1 4 I
1 40
I 40
1 J9
1 J6
I J4
1 37
I 30
1 79
1 11
1 76
I ?4
i 77
I 71
63 1
948
5 14
3 77
3 17
7 74
7 49
731
7 18
208
I 99
1 93
I 87
1 82
1 78
I 74
1 71
1 68
1 66
1 64
I 67
1 60
1 58
1 56
I 55
1 54
1 57
1 51
1 50
I 49
1 4/
I 46
1 44
1 43
1 47
1 4U
I iy
I .19
I IH
I J>
I 34
1 M
I 30
1 78
I 77
I 75
I 73
I ?l
I 19
I 18
637
949
5 14
3 77
3 17
7 73
748
731
7 17
207
1 99
I 97
1 86
I 87
1 77
I 74
I 71
1 68
1 65
1 63
1 61
1 59
1 57
1 56
1 54
1 53
I 52
1 5O
1 49
I 48
I 4b
I 45
1 4J
1 47
I 41
I 4O
I J9
I 38
1 II
I J6
I 33
I JO
1 78
I 7/
I 76
1 73
I 77
I ?O
I 18
I 16
I 45 I 44 143 1 38 I 35 I 30 177 I ?O I 16 i 15
-------
TABLE A-4 Percentiles of the F distribution (continued)
Upptr 5* point of thf F dittribution
DEGREES OF f BttOOM F OR NUMC BATOR
10
11
16
17
4O
1OO 1M >OO
161
185
101
7.71
661
599
559
532
512
496
4S4
4 75
4.67
460
4.54
449
4.45
441
438
435
4.37
430
478
476
4.74
473
471
470
18
17
IS
13
11
10
408
407
406
46
48
50
60
70
80
90
100
175
ISO
^200
300
500
1000
404
700
190
955
6.94
5 79
5 14
4 74
4.46
476
4 10
398
389
3.81
3 74
3.68
363
359
355
3.57
3.49
347
344
347
340
339
337
335
334
333
3.37
3.79
378
376
3.74
323
377
3 71
3 70
3 19
403 318
400 3 15
3 93x 3 13
396 311
3 95 310
394 309
392 307
390 306
389 304
38' 303
386 301
716 775
19.7 19.7
978 917
6.59 6.39
5.41 519
4.76 4.53
4.35 4.17
407 384
386 363
371 348
359 336
349 376
341 3 18
334 3 11
379 305
374 301
370 796
3.16 793
3.13 790
310 787
307 784
305 787
3 03 7 80
301 2.78
799 775
798 7 74
796 7 73
795 7.71
7 93 7 70
7.97 7 69
790 767
7 88 7 65
787 763
7 85 7 67
784 761
7 83 7 59
287 -758
781 757
7 80 757
7 79 7 56
7 76 7 53
7 74 75O
277 7 49
771 747
2.70 745
7 68 7 44
766 743
765 747
763 740
767 739
230
19.3
901
6.76
5.05
4.39
397
3.69
348
3.33
370
3.11
303
796
7.90
7.85
7.81
2.77
2.74
7 71
768
766
764
767
760
759
757
756
755
253
7.51
749
748
746
745
744
743
747
741
740
737
735
773
737
731
779
777
776
774
773
734
193
894
6.16
4.95
4.28
387
3.58
3.37
327
309
300
797
7.85
7.79
2 74
2.70
266
263
760
757
7.55
753
751
749
747
746
7.45
743
7.47
740
738
736
735
734
737
731
730
7 79
729
225
773
7.71
770
7 19
7 17
7 16
7 14
7 13
7 17
737
194
889
6.09
488
4.71
3 79
3.50
379
3.14
3.01
791
7.83
2 76
7 71
766
761
758
754
751
7.49
746
2.44
242
740
739
737
7.36
735
7.33
731
779
728
776
775
774
7 73
777
771
2 70
7 17
7 14
7 13
7 II
7.10
708
707
706
704
203
239
194
885
6.04
487
4.15
3 73
344
373
3.07
295
285
7.77
7.70
764
259
755
751
248
245
747
740
7.37
7.36
734
737
731
779
778
7.77
7 74
773
771
7 19
7 18
7 17
7 16
7 15
7 14
7 13
7 10
707
706
704
703
701
7OO
198
I 97
I 96
241
194
881
600
4.77
4 10
368
3.39
3 18
307
7.90
780
7.71
765
759
754
749
746
747
739
7.37
734
232
230
278
777
7.75
774
277
771
7 19
7 17
7 15
7 14
2 17
7 11
7.1O
709
708
707
704
707
700
1 99
1.97
196
I 94
I 93
I 91
I 9O
747
194
8.79
6.96
4.74
408
364
335
3 14
2.98
285
2 75
7.67
7.60
754
749
2.45
2.41
238
235
737
730
727
2.25
224
277
770
7 19
7.18
2 16
2 14
7 17
7 II
709
708
706
7O5
7O4
703
703
I 99
1 97
1 95
1 94
I 93
1 91
I 89
I 88
1 86
1 85
743
19.4
876
594
4 70
403
360
331
3 10
294
787
7 77
763
757
7.51
746
7.41
737
734
731
778
7.76
774
7.77
2.70
7 18
7 17
7 15
7 14
7 13
7 10
708
707
705
7O4
703
701
700
I 99
I 99
I 95
I 93
1 91
1 90
1 89
1 87
I 85
1 84
1 87
1 81
744
194
8.74
591
468
400
3.57
3.78
307
791
779
769
760
753
748
747
7.38
734
731
278
775
773
7.70
7 18
7 16
7 IS
7.13
7 17
7 10
7.09
707
705
703
707
700
99
98
97
96
95
1 97
1 89
I 88
1 86
1 85
83
82
80
78
77
745
194
8 73
589
466
398
355
3.26
305
2.89
7.76
766
7.58
751
745
740
735
731
778
775
777
770
7 18
7 15
7 14
7 17
2 10
209
208
706
704
707
700
1 99
1 97
1 96
95
94
93
92
89
86
(J4
83
87
I 80
I 79
1 77
1 75
1 74
745
194
8.71
587
4.64
396
353
374
3.03
286
7.74
7.64
7.55
748
7.47
737
233
7.79
776
777
770
7 17
7 15
7 13
7.11
709
708
706
705
704
701
99
98
96
95
94
97
91
90
89
86
84
87
80
79
77
76
74
72
1 71
746
194
870
586
467
3.94
3.51
3.77
301
785
7.77
767
753
746
7.40
7.35
731
727
223
720
7.18
7 15
7 13
7 11
709
707
706
704
703
701
1 99
197
1 95
1 94
1 97
I 91
1 90
I H9
I 88
1 8/
1 84
1 81
1 79
1 78
I 77
1 75
1 n
1 77
1 70
169
746
194
869
584
460
397
349
3.70
799
283
7 70
760
751
744
7.38
733
279
775
771
7 18
7 16
7 13
7 II
709
207
705
704
707
701
1 99
1 97
1 95
1 93
I 97
1 90
I 89
1 88
I 87
1 86
I 85
I 87
1 79
1 77
1 76
1 75
1 73
1 71
I 69
168
1 66
747
194
868
5.83
4.59
391
348
3 19
797
781
7.69
758
7.50
743
737
737
777
773
770
7 17
7 14
2 II
209
207
705
703
70?
700
1.99
1 98
1 95
1 93
I 97
I 90
I 89
1 87
I 86
I Kb
1 b4
1 83
I 80
I 77
I 75
I 74
I 73
1 71
1 69
I 67
1 66
I 64
747
194
867
687
4.58
3.90
347
3.17
796
780
767
757
748
741
735
730
226
272
2 18
2 15
7 17
7 10
708
70S
704
202
700
I 99
97
96
94
97
90
86
84
83
H7
81
78
75
73
72
71
69
67
66
164
162
248 748
194 194
867 866
581 580
4.57 456
388 3.87
346 344
3 16 315
7.95 794
7 79 7 77
7.66 765
756 754
7.47 246
740 739
7.34 2 33
7 79 2 28
2 24 2 23
77O 219
7 17 2 16
7 14 712
211 2 10
2O8 2U7
206 205
2O4 203
702 201
2OO I 99
99 I 97
97 1 96
96 I 94
95 1 93
92 1 91
90 1 89
88 I 87
87 I 85
85 184
84
83
82
81
80
76
74
72
70
69
67
66
64
6?
61
I 83
I 81
I HO
1 79
I '8
I 75
1 72
1 70
I 69
168
1 66
I 64
I 67
I 61
I 59
249
195
863
5.77
4.52
3.83
3.40
3.11
789
7.73
760
750
741
7.34
778
773
7 18
7 14
7 II
707
705
707
700
I 97
I 96
1 94
I 9?
I 91
I 89
1 88
I 85
1 83
I 81
I 80
1 78
I 77
I 76
I 75
I 74
t 73
1 69
I 66
I 64
1 63
I 67
I 59
1 58
1 SO
I b4
I 53
750
195
867
5.75
4.50
381
338
308
7.86
7.70
757
7.47
738
731
7 75
7 19
7.15
7 11
707
704
701
1 98
1 96
1 94
1 97
1 90
I 88
I 87
I 85
I 84
I 82
1 80
1 78
I 76
1 74
I 73
1 72
1 71
1 7O
I 69
1 65
I 62
1 60
I 59
1 57
I 55
I 54
I b?
1 50
I 48
251
19.5
8.59
5.72
4.46
3.77
3.34
3.04
283
266
253
243
2.34
7.77
77O
2 15
7 10
706
703
1.99
1 96
1 94
1 91
I 89
1 87
1 85
1 84
1 87
1 81
I 79
1 77
1 75
I 73
I 71
I 69
I 68
I 6/
I 65
I 64
I 63
I 59
I 57
1 54
I S3
1 57
1.49
1 48
I 46
I 43
1 47
757
195
8.58
5.70
4.44
3.75
332
3.07
7.80
7.64
7.51
740
7 31
7.74
7 18
7 17
708
704
700
1 97
1 94
1 91
I 88
1 86
I 84
1 87
1 81
1 79
1 77
1 76
1 74
1 71
I 69
1 68
1 66
1 65
1 63
1 67
I 61
I 6O
I 56
I 53
1 51
I 49
1 48
1 45
I 44
I 41
I 39
1 38
753
195
8.55
5.66
4.41
3 71
3.27
2.97
7.76
2.59
2.46
7.35
776
7 19
7 I?
707
70?
1 98
1 94
I 91
I 88
1 85
I 8?
I 80
1 78
I 76.
I 74
1 73
1 71
1 7O
1 67
1 65
1 62
1 61
I 59
I 57
I 56
I 55
I 54
I 52
1 48
1 45
1 43
I 41
I 39
1 36
1 34
1 3?
I JO
1 78
753
195
854
5.65
4.39
3 70
3 76
796
7.74
7.57
744
733
7.74
7 17
7 10
705
700
1 96
19?
1 89
I 86
I 83
1 80
I 78
1 76
1 74
1 72
I 70
I 69
I 67
1 64
1 6?
I 60
I 58
I b6
I 55
I 53
I 52
I 51
1 50
I 45
I 42
1 39
I J8
1 36
1 33
I 31
I 28
I 26
I 2J
254
195
854
5.65
439
369
325
795
2.73
256
243
232
223
2 16
2 10
2O4
I 99
1 95
1 91
1 88
1 84
I 82
1 79
1 77
1 75
1 73
1 71
1 69
1 67
1 66
1 63
1 61
1 59
I 57
I 55
I 53
I 57
I 51
1 49
I 48
I 44
I 40
I 38
I 36
I 34
I 31
I 29
1 26
I ?3
I 21
3 85 3 00 261 2 38 2 22 711 70? I 95 1 89 I 84 I 80 I 76 I 73 I 7O 1 68 I 65 I 63 161 1 60 1 58 I 57 147 141 I 36 I ?6 I 72 I 19
-------
TABLE A-4 Percentiles of the F distribution (continued)
Upper 2.5% point of the F dittribution
o
4
*
I
O
z
ut
O
(C
o
It.
3
8
tu
(X
u.
O
i/t
Ul
o
o
1
7
3
4
S
6
;
a
9
10
11
1?
13
14
IS
16
17
18
19
70
71
7?
23
24
25
26
27
28
29
30
32
34
36
38
40
42
44
46
48
SO
60
10
80
/90
/
1
100
125
ISO
700
3OO
SOO
000
DEGREES OF FREEDOM FOR NUMERATOR
548 800 864 900 97? 937 948 957 983 969 973 977 98O 983 985 987 989 99O 99? 993 998 1001 10O6 1OO8 1013 1015 1016
385 390 39.7 39? 39.3 393 39.4 394 394 394 39.4 394 39.4 39.4 39.4 394 394 394 394 394 395 395 395 395 395 395 395
174 160 154 151 149 14.7 146 14.5 145 14.4 144 143 143 14.3 143 142 14.7 14 7 142 142 141 141 14 O 140 140 139 139
172 106 998 960 936 9 TO 907 898 8.90 884 8.79 875 8.71 868 866 8.63 861 859 858 856 850 846 841 838 8.3? 830 879
100 843 7.76 739 7.15 898 6.85 6.76 6.68 66? 6.57 6.57 6.49 6.46 6.43 6.40 638 6.36 634 6.33 677 673 6
.18 614 6O8 606 605
881 776 6.60 6.73 S.99 58? S 70 560 S.5? 5.46 5.41 537 533 530 577 574 5.7? 570 5.18 517 5.11 507 5 O1 498 49? 489 488
807 654 589 55? 579 512 4.99 4.9O 48? 476 4.71 4.67 4.63 4 6O 457 454 45? 450 448 447 440 4
36 431 4 78 4 71 4 19 4
18
757 606 54? SOS 48? 465 453 443 436 430 474 470 416 413 410 408 4 OS 403 40? 4 OO 394 389 384 381 374 37? 370
771 571 508 47? 4.48 437 470 410 403 396 3.91 387 383 380 3/7 3.74 37? 3/0 368 367 360 356 351 347 3 4O 338 337
6.94 546 4.83 447 474 407 395 385 3/8 3.7? 366 362 358 355 352 350 347 345 344 342 3.35 331 3
6.72 626 463 428 404 388 376 366 3.59 353 3.47 3.43 339 336 333 3.30 378 376 3.74 373 3 16 3
76 3.7? 315 3 13 31?
1? 3 O6 303 296 793 79?
655 510 447 41? 3.89 3.73 361 361 344 337 33? 378 3.74 3.71 318 315 313 311 3 O9 307 301 796 791 ?8/ 7 8O 7/8 7/6
641 497 435 400 377 3 6O 348 3.39 331 325 370 3.15 3.1? 308 3 OS 3 O3 300 798 296 795 788 784 778 774 767 765 763
630 *B6 474 389 366 3 SO 338 379 371 315 3.09 305 301 798 795 79? 790 788 786 784 7.78 7/3 767 764 756 754 753
6.70 477 415 380 358 341 379 370 3.1? 306 301 796 292 789 786 784 781 779 7.77 776 769 764 759 255 747 745 744
6.1? 469 408 373 350 334 37? 3.12 305 799 793 789 285 78? ?./9 776 774 77? 770 768 761 757 751 747 740 7.37 736
604 46? 401 366 344 3.78 316 306 798 79? 7.87 282 779 775 272 770 767 765 763 76? 755 750 744 741 733 ? 3O 779
598 4.56 395 361 338 37? 3.10 301 7.93 787 781 777 773 770 767 7.64 76? 7 6O 758 7.56 749 744 738 735 777 774 ? 73
59? 451 390 356 333 317 3 OS 796 788 7.8? 776 77? 768 265 2.6? 7.59 757 755 753 751 744 ? 39 733 730 ?.?? 719 718
587 446 386 351 3.79 313 301 7 91 2 84 777 7.7? 768 764 760 757 7.55 ? 5? 750 748 746 740 735 779 775 717 714 713
583 44? 38? 348 375 3 O9 797 787 780 773 768 764 ? 6O 756 753 751 748 746 744 74? 736 731 775 771 713 710 ? O9
579 438 3/8 344 37? 305 793 784 776 770 765 760 756 753 2 SO 247 245 243 241 239 73? 777 771 717 709 706 2 OS
575 435 375 341 318 30? 7.90 781 773 767 76? 757 7.53 7 5O 747 744 74? 739 737 736 ? 79 774 2.18 214 2 O6 203 201
572 432 372 338 315 799 787 773 770 764 759 754 750 747 744 741 739 736 735 733 776 ? 71 715 711 70? 7 OO
569 479 369 335 313 797 785 775 768 761 756 751 748 744 741 738 736 734 73? 730 773 718 71? 7 O8 7 OO 197
566 4?7 367 333 310 794 78? 773 765 759 754 ? 49 745 74? 739 736 734 731 229 228 221 216 2 O9 205
563 24 365 331 308 79? 780 7/1 763 757 751 747 743 739 736 734 731 779 727 275 718 213 707 703
561 7? 363 379 3 O6 7 9O 778 769 761 755 749 745 741 737 734 73? 779 777 775 773 716 211 70S 701
559 70 361 377 304 788 776 7.67 759 753 748 743 739 736 73? 730 727 7 75 773 771 714 ? O9 703
557 18 359 375 3 O3 787 775 765 757 751 746 741 737 734 731 778 776 773 221 2 2O 21? 207 201
553 15 356 322 3 OO 284 271 262 754 748 743 738 734 731 778 775 7 ?? 770 718 716 7 O9 7 O4
98
550 1? 353 319 797 781 769 759 75? 745 7 4O 735 731 778 775 222 770 717 715 213 2 O6 201 195
547 4O9 350 317 294 278 266 2 S/ 249 243 237 233 279 775 ? 77 770 717 715 713 711 7 O4
545 407 348 315 79? 776 764 755 747 7.41 735 731 777 773 ? 70 717 715 713 711 7 O9 701
54? 405 346 313 ?9<> 774 76? 753 745 739 733 779 775 ? 71 718 715 713 711 209 207 199
54O 403 345 311 289 773 ?6I 251 243 237 232 277 773 ? 70 716 714 711 7 O9 7 O/ 705 198
539 40? 3 43 309 707 771 759 750 74? 736 230 276 77? 718 715 212 210 20/ 205 203 196
53/ 4OO 342 308 286 2/0 258 248 241 234 229 224 220 217 213 711 7 O8 206 2 O4 202 194
535 J99 340 30/ 284 269 256 247 239 233 227 223 219 215 212 209 207 2 OS 202 201 193
534 397 339 JOS 283 ?6f 255 246 738 73? ? 76 7 77 718 714 711 7 O8 706 703 701 199 19?
S 79 393 334 301 779 763 751 741 733 777 77? 717 713 7 O9 ? O6 703 2 Ol 198 96 94 187
5 7S 389 3 31 7 9f ? 75 ? 59 7 47 7 38 7 30 7 74 2 18 2 14 2 10 2 O6 203 2 OO 97 1 95 93 91 183
522 3 B6 378 795 2 /3 257 245 7 35 2 28 2 21 ? 16 2 11 207 2O3 7 00 9/ 95 1 92 9O 88 1 81
S 70 384 3 76 7 9J 7 /I 755 743 7 34 7 26 2 19 2 14 209 2 OS 2 O2 US 95 93 91 88 86 1 /9
5 18 3 83 3 25 292 2 /O 254 74? 2 32 2 74 2 18 2 12 2 O8 2 O4 2 OO 9/ 94 91 89 8/ 85 1 77
SIS 3 HO 322 7H9 267 251 239 230 222 215 7 IO 2 OS 201 97 94 91 B9 86 84 82 174
S 13 3 IH 3 20 787 265 249 2 3/ 2 28 2 2O 2 13 2 OS 203 1 99 95 97 89 8' 84 82 80 1/2
S 10 3 16 3 18 28S 263 247 2 35 2 26 2 18 2 II 706 201 1 9/ 93 90 8/ 84 82 80 /8 1 JO
SO/ J /J 316 283 261 245 233 273 216 2 O9 2 O4 199 195 91 88 85 82 1 80 // IfS 1 6/
SOS 3/7 314 281 7 SU 243 731 77? 714 ?0/ 70? 197 193 89 86 83 BO 1/8 76 1/4 165
504 370 3 13 J BO 758 74? 730 770 713 706 201 196 19? 188 185 18? 179 1/7 1/4 If? 164
99
96
94
9?
91
89
88
B/
8?
/8
/S
/3
71
68
b .'
64
67
6O
9?
90
88
86
84
8?
81
8O
74
71
68
66
64
61
59
56
54
52
99
97
93
90
88
85
83
81
80
78
/7
75
70
66
63
61
59
56
54
51
48
46
.97
94
9?
9O
88
85
8?
79
76
74
7?
70
69
6/
66
60
56
53
SO
48
45
4?
39
36
J4
94
91
89
8/
85
8?
78
76
/3
/I
69
67
98
95
9?
90
88
86
84
80
77
/4
/I
69
6f
65
65 1 63
64 1 6?
6?' 1 60
56
S2
49
46
44
4O
38
35
31
78
58 1 SO 1 45 13? 1 76
54
SO
4f
44
4?
38
JS
J?
?B
2S
2J
-------
TABLE A-4 Percentiles of the F distribution (continued)
Upper 1% point of thf f dittribution
DEGREES OF FREEDOM FOR NUMERATOR
8
9 10 II I? 13 14 IS 16 17 18 19 70 K 30 «0 SO IOO ISO TOO
90
05?
98 5
21 1
163
13 1
177
II 3
106
100
965
933
907
8 OH
868
8 S3
840
879
18
8 IO
807
795
788
787
7 77
7 77
768
764
760
7S6
750
744
740
735
731
778
7 75
7 7?
7 19
7 I?
7 OH
;oi
696
693
690
684
6 76
6 I'l
669
SOOO S403
990 997
308 795
180 167
13.3 17.1
109 9.78
9 55 8 45
8 65 1 59
807 699
756 655
771 677
6 93 5 95
6 70 5 74
651 556
6 36 547
6 73 5 79
611 5 19
601 5O9
593 501
5 85 * 94
578 487
577 487
566 4 76
561 4 77
557 46fl
5 53 4 64
5 49 4 60
54S 457
547 4 S4
539 4 SI
5 34 4 46
5 79 4 47
5 75 4 38
571 4 34
5 18 4 31
515 4 79
517 4 76
5 10 4 74
5 08 4 77
506 4 70
4 9H 4 IJ
497 4 O 7
4 88 4 04
485 4OI
487 3 98
4 78 3 94
4/5 391
471 3 88
4 68 J85
465 3 B?
5675 5764
99 7 99.3
78 7 78 7
160 155
114 110
915 875
785 746
701 663
647 6O6
599 564
567 537
541 506
571 486
504 469
4 89 4 56
4 77 444
467 434
458 4 75
4 SO 417
443 4 10
4 37 4O4
4 31 3 99
476 394
477 390
4 18 3 85
4 14 3 87
411 3 78
407 3 75
404 3 73
4 07 3 70
39' 365
393 361
389 357
386 354
3 83 3 51
3 BO 3 49
3 78 347
3 76 3 44
374 343
3 77 341
3 65 1 34
3 GO 3 79
356 376
3 53 J 73
351 371
347 317
345 3 1«
341 311
3 .18 3 OS
3 36 3 05
5859
993
779
157
10.7
647
1 19
637
580
539
507
487
467
446
437
4 70
4 IO
401
394
387
381
3 76
3 M
367
363
359
356
353
3 50
347
343
339
335
3 37
3 79
3 77
3 74
3 77
J 70
3 19
3 17
30/
304
301
799
795
797
789
786
784
5978
994
77 7
150
105
876
699
6 IB
561
570
489
464
4 44
4.78
4 14
403
393
384
3 77
3 10
364
359
354
350
346
34?
339
336
333
330
376
37?
3 18
3 15
3 17
3 10
308
306
304
307
?9S
7!il
787
7B4
787
7 79
7 >6
7 13
? 10
768
5981
994
775
148
10.3
8.10
684
603
547
5O6
4 74
450
4 30
4 14
4OO
389
3 79
3 71
363
356
351
3*5
341
336
33?
379
376
3 73
3 TO
3 17
3 13
309
305
30?
799
797
795
793
791
789
787
? IK
? 74
7 7?
769
766
76.1
76O
7 57
? 55
607?
994
773
.14 7
10?
798
6 77
591
535
494
463
439
4.19
4O3
389
3 78
368
360
357
346
340
335
330
3 76
37?
3 18
3 15
3 I?
309
307
30?
798
795
79?
789
786
784
78?
780
? >8
7 7?
?6<
764
761
759
755
753
750
747
744
6056 6083
994 994
77 7 771
145 145
10 I 996
787 7 79
66? 654
581 573
576 5.18
485 477
4 54 446
4 30 4.??
4 10 40?
394 386
3.80 3 73
369 36?
3 59 35?
351 343
3 43 3 36
337 3 79
331 374
3 76 3 18
3 71 3 14
317 3O9
3 13 3O6
3O9 3O?
3O6 799
303 796
3 00 ? 93
7 98 791
793 786
7 89 78?
786 ? 79
7 83 7 75
780 773
7 78 7 70
775 768
773 7 66
7 71 764
? 10 763
763 ? 56
759 751
? 55 7 48
75? 745
7 50 743
747 739
744 ?37
741 734
7 38 731
736 778
6106
994
77 1
144
989
7.7?
647
567
5 II
4 71
440
4 16
396
380
367
355
346
3 37
330
373
3 17
3 I?
307
303
799
796
793
790
787
784
780
7 76
7 7?
769
766
764
767
760
758
756
75O
745
7 4?
739
7.17
733
731
777
774
77?
6176
994
770
14 3
98?
766
641
561
SOS
465
434
4 IO
391
3 75
361
350
340
3 3?
374
3 18
3.17
307
30?
798
794
790
787
784
781
7 79
7 74
7 70
767
764
761
759
756
7 54
753
751
744
740
736
? 33
731
778
? 75
77?
7 19
? 17
6143
.994
769
14 7
9 77
760
636
556
501
460
479
405
386
3 70
356
345
335
3 77
3 19
3 13
3.07
30?
797
793
789
786
78?
7 79
? 11
1 74
7 70
766
76?
759
756
754
75?
750
748
746
739
7 JS
731
779
777
773
770
7 17
7 14
7 1?
6157
994
769
14?
97?
756
631
55?
496
456
475
401
38?
366
35?
341
331
3 73
3 IS
309
303
798
793
789
785
781
7 78
7 75
? 73
7 70
765
761
758
755
75?
750
747
745
744
74?
735
7 31
7 ?/
774
7 ??
7 19
7 16
7 13
7 10
6170 6181
994 994
76.8 768
14 7 14 I
968 964
7 5? 7 48
6 78 6 74
5 48 S 44
49? 4 89
45? 4 49
4.71 4 18
397 394
3 78 3 75
36? 3 59
3 49 3 45
337 334
377 374
3 19 3 16
3.1? 3O8
305 30?
799 796
794 791
789 786
7 85 78?
781 778
7 78 7 75
7 75 771
7 77 768
7 69 7 66
? 66 ? 63
76? 758
758 754
754 ?bl
751 748
7 48 7 45
746 743
7 44 7 40
74? ? 38
?40 737
7 38 7 35
7 !l I 7 78
? ? 7 7 73
7 73 7 7O
771 717
7 19 715
? 15 711
? I? 709
?O9 706
7O6 703
7O4 700
619? 6701
994 994
768 76.7
141 140
961 958
7 45 74?
671 618
541 538
486 4 83
4 46 4 43
4 IS 4 1?
391 388
37? 3 69
356 3 53
34? 340
331 378
3 71 3 19
313 3 10
305 3 03
799 796
793 790
? 88 7 85
783 780
7 79 7 76
7 75 7.7?
77? 769
768 766
7 65 7 63
7 63 7 60
760 757
? 55 7.53
751 749
748 745
745 74?
74? 739
740 737
737 7 35
? 35 7 33
733 731
73? 7 79
7 75 77?
? 70 7 18
7 If 7 14
7 14 711
71? 709
708 70S
7O6 703
703 70O
1 99 197
197 1 94
6709 6740
994 995
76 7
140
955
740
6 16
536
481
4 41
766
139
945
7 30
606
576
4 71
431
4 10 401
3 86 3 76
366
351
3 37
357
341
3 78
3 76
3 16
308
300
794
788
783
7 78
7 74
? 7O
766
763
760
757
755
750
746
743
740
737
734
73?
730
7 70
? 15
? 1?
7O9
707
703
700
1 97
1 94
I 9?
3 16
307
798
791
784
7 79
? 73
769
764
760
757
754
751
748
745
741
737
7 33
730
777
775
7 77
770
7 18
7 17
? 10
7 US
701
1 99
1 97
193
1 90
1 87
1 84
1 81
6761
995
765
13.8
938
7.73
599
570
465
4 75
394
3 10
3 51
335
371
3 10
30O
79?
784
7.78
7 7?
767
76?
758
754
7 SO
747
744
741
739
734
730
776
773
7.70
7 18
7 IS
7 13
7 I?
7 IO
703
1 98
1 94
I 9?
1 89
85
83
79
76
74
6787
995
764
13 7
979
7 14
591
5 1?
457
4 17
386
36?
343
3 77
3 13
30?
79?
784
7 76
769
76-1
758
754
749
745
74?
738
735
733
730
775
771
7 18
7 14
7 II
709
707
704
70?
701
1 94
89
HS
8?
80
76
73
69
66
63
6303
995
764
13 7
974
709
586
so;
45?
4 I?
381
3 57
338
3 ??
308
797
787
? 78
? 71
764
758
753
748
744
740
736
733
73O
??7
775
770
? 16
7 I?
?O9
?O6
703
701
1 99
97
95
88
83
79
76
74
69
66
63
59
57
6334
995
76 ?
136
9 13
699
5 75
496
4 41
401
3.71
347
377
3 II
798
786
? 76
768
760
754
748
74?
737
733
779
775
?.??
7 19
? 16
? 13
708
704
7OO
1 97
1 94
1 91
I 89
1 86
I 84
1 8?
1 75
70
65
67
60
55
5?
48
44
41
6345 6350
995 995
76?
135
9O8
76?
135
909
695
5 77
493
4 38
398
367
343
3 ?4
308
794
783
7 n
764
757
7 SO
744
738
734
779
775
7 71
7 18
7 IS
7 I?
709
704
700
1 96
1 93
1 90
I 87
I 84
1 8?
1 80
1 >8
I 7O
1 65
1 61
1 57
I 55
1 50
1 46
I 47
1 38
1 34
693
5 70
491
4 36
396
366
341
3 7?
306
79?
781
7 71
76?
755
748
74?
736
73?
777
773
7 19
? 16
7 13
7 10
707
70?
I 98
1 94
1 90
I 87
1 85
1 8?
1 80
I '8
I 16
1 68
I 6?
1 58
I 55
I 57
1 47
I 43
1 39
I 35
I 31
IOOO
666 4.63-380 334. J O4 ? 8? 766 753 743 734 77; 770 715 710 7 O6 70? 198 195 197 190 179 17? 161 154 138 13? 178
-------
TABLE A-4 P,ercentiles of the f distribution (continued)
S Upper 0.5* point of the F dittribution
DECREES OF FREEDOM FOR NUMERATOR
5O 1OO ISO IOO
60
80
90
100
175
150
700
300
500
1000
199
556
31.3
778
186
16?
14.7
136
178
17?
108
106
104
10.7
101
994
983
9 73
963
955
9.48
941
9.34
978
973
9 18
909
888
883
8 78
8 74
8 70
866
863
8.49
840
8.33
878
874
8 17
8.1?
806
800
7.95
199 199
498 ..475
76 3 74.3
183 165
14S 179
17.4 109
110 960
101 877
943 a 08
891 760
851 773
819 693
797 668
7 70 6 48
751 6.30
735 616
771 603
709 597
699 5.87
689 573
681 565
6 73 558
666 557
66O 5.46
654 541
6 49 5 36
644 537
6 40 5 78
6 35 5 74
6 78 517
6 ?7 511
6 16 506
611 507
607 498
603 494
599 491
596 4 88
593 485
590 483
5 79 4 J3
5 77 466
567 461
567 457
5.59 4 54
55.' 449
549 4.45
5.44 441
S.39 4 36
5.35 4.33
199
467
73 7
156
170
10 1
881
796
734
688
657
673
600
580
564
550
537
577
517
509
507
495
489
484
4'79
4 74
4.70
466
462
456
450
446
4.41
437
434
4 31
478
475
4 73
4 14
408
4O3
399
396
3.91
388
384
380
3 76
199 199
45 4 44 a
775 720
149 14.5
115 M 1
9.52 9 16
830 7.95
747 713
687 654
6.47 6 10
607 5 76
5.79 548
556 576
537 5.07
521 491
507 4 78
4.96 4.66
4.85 4.56
4.76' 447
4.68 4.39
461 432
4 54 4 76
4.49 470
4 43 4 15
4 38 4 10
4 34 4 06
3O 407
76 398
73 395
17 389
M 384
4 06 3 79
4 07 3 75
399 371
3 95 3 68
3 97 3 65
3.90 367
387 360
385 358
376 349
370 343
3 65 3 39
367 335
3 59 3 33
3 54 3 78
351 375
3.47 371
343 317
340 3 14
199 199
44 4 44.1
716 714
142 140
108 106
8.89 868
769 750
688 669
630 617
586 568
557 535
575 508
5 03 4.86
485 467
469 457
456 439
4 44 4 78
434 4 18
476 4.09
418 401
4.11 3.94-
405 3.88
399 383
394 378
3 89 3 73
3 85 3 69
3.81 365
3 77 3.61
374 358
368 357
363 3.47
358 3.47
354 339
J 51 335
3 48 3.37
345 379
347 3 76
3.40 3.74
3.38 377
329 313
373 308
3 19 303
3 15 300
313 297
3 08 7.93
3 05 2 89
301 786
797 782
2.94 279
199
439
21 I
13.8
104
8.51
734
654
597
554
5.20
494
4 77
4.54
4 38
475
4.14
4O4
396
388
381
3 75
369
364
360
3 56
357
348
345
339
3.34
3.30
3.76
327
3 19
3.16
3 14
3 11
309
301
795
791
787
785
780
7 77
7 73
769
766
199
43 7
710
136
10.3
8.38
7.71
647
585
542
509
487
4.60
4.47
4 77
4 14
403
393
385
3 77
3 70
364
359
354
349
345
3.41
3.38
334
3.79
374
3 19
3 15
3 17
309
306
303
301
799
790
785
780
7 77
774
770
767
763
759
756
199
435
708
135
101
8.77
7.10
631
5.75
537
4.99
4 72
451
433
4.18
405
394
384
3 76
368
361
3 55
350
3.45
340
336
3.32
379
375
3.70
3.15
3 10
306
303
300
297
794
797
790
787
2 76
7 77
768
766
761
758
754
751
748
199
43.4
70.7
134
100
8 18
701
6.73
566
5.74
491
4.64
443
4.75
4 10
397
386
3 76
3.68
3 BO
354
347
3.47
337
3.33 '
3 78
375
3.71
3 18
3 12
307
303
299
795
797
789
787
785
787
7 74
768
764
761
758
754
751
747
743
7.40
199
43.3
706
133
995
8 10
694
6 15
559
5.16
484
4.57
436
4.18
403
39O
3 79
3 70
361
354
347
341
335
330
'376
327
3 18
3.15
3.11
306
3.01
296
292
789
786
783
2 BO
2 78
7.76
768
762
758
754
752
747
744
740
737
734
199
43.7
705
137
988
8.03
687
609
5.53
5.10
4 77
451
430
4.17
397
394
3 73
364
355
3.48
3.41
3.35
330
375
370
3.16
3 17
309
306
300
7.95
790
787
783
780
7 77
2 75
2.72
2 70
767
756
757
749
746
747
738
7.35
731
778
199
43.1
704
13 I
981
797
681
603
547
5O5
4 77
446
4.75
4.07
397
3 79
3.68
3.59
350
?,f»
336
330
375
3.20
3.15
311
3.07
304
3.01
295
2.90
785
782
7 78
7 75
7 77
7 70
767
765
257
2.51
247
744
741
737
733
230
2 76
773
199
43.0
704
13.1
976
791
6 76
598
547
500
467
4 41
470
407
387
3 75
364
354
346
338
331
375
3-70
3.15
3 11
307
3.03
799
296
290
2.85
2.81
277
7.74
7 71
768
765
763
2.61
253
247
243
739
737
7.37
779
7.75
771
7 19
199
479
703
13.0
9 71
787
6.77
594
5.38
496
4.63
4 37
4 16
398
383
3 71
360
3 SO
347
3.34
3.77
3 71
3 16
3.11
307
303
299
795
7.92
786
781
2 77
2 73
7 70
767
764
761
759
7.57
749
243
739
735
733
778
725
771
2 17
2 14
199
42.9
20.3
130
966
783
668
590
534
4.97
4.59
433
4 17
395
380
367
356
3.46
3.38
3 31
3.74
3.18
3.17
308
3.03
799 .
795
7.92
289
2.83
7 78
7 73
7 70
766
763
760
758
755
753
2.45
739
235
232
229
224
771
7.18
7 14
7.11
199
478
707
179
967
779
664
586
531
489
4 56
4 30
4O9
391
3 76
364
353
343
335
3.77
3 71
3 15.
3.09
3O4
300
796
297
288
785
780
2.75
2 70
266
263
260
257
254
252
250
242
736
732
278
776
771
7.18
7 14
7 10
707
199
47.8
7O.7
17.9
959
7 75
661
583
577
486
453
4 27
4 O6
368
3 73
361
3 50
340
3 37
3 74
3 18
3 17'
3 06
301
797
793
789
7.86
787
7 77
7 77
767
763
760
757
754
751
749
747
239
233
279
275
7 73
7 18
7 15
211
707
7O4
199
476
TOO
178
945
76?
648
5 71
5/5
4 74
441
4 15
394
3 77
36?
349
3 38
379
3 ?O
3 13
306
300
7.95
290
785
781
? 77
7 74
2.71
199 199
42.5 42.3
199 198
127 17.5
9.36 9 74
7 53 7 47
6 40 6.79
567 557
5.07 4 97
465 455
433 473
< 07 3.97
386 3 76
369 358
354 344
341 331
3 30 3 70
3 71 311
317 302
3 05 7 95
798 788
797 787
7,87 777
787 7 72
7 77 767
7 73 :s 7.63
769 759
7 66 7 56
763 757
,199 199
477 470
197 195
175 173
9 17 .903,
7 35 J 7?
677 6O9
545 537
49O 4 77
4 49 4 36
417 4O4
3 91 3 78
3.70 357
3 57 3 39
337 375
3 75 317
3 14 3OI
3O4 791
796 783
7 88 7 75
2 82 2 69
276 262
2 70 2 57
2 65 2 52
761 247
757 743
2 53 2 39
2 49 2.36
746 737
199 199
470 419
194 194
17? 17?
898 895
717 7 15
6O4 607
578 576
4 73 4 71
431 4 79
399 397
3 74 3 71
353 350
3 35 3 33
3 7O 3 18
307 3OS
7.96 794
787 785
7 78 7 76
7 71 768
764 76?
758 756
757 750
747 745
7 43 ? 40
7 38 2 36
2 35 73?
? 31 229
2 28 2 75
765 257
260 252
256 2.48
7 5? 7 44
7 48 7 40
245 737
24? 734
240 23?
737 779
735 777
777 719
771 713
717 708
713 7O5
711 7 O?
706
7.O3
1 99
I 95
1 9?
247
242
737
233
230
226
274
271
7 19
7 16
70S
?O?
I 97
I 94
1 91
1 86
1 83
1 79
1 75
I 7?
240
2.35
230
277
773
770
7.17
7 14
7 1?
7 IO
701
95
9O
87
84
79
76
71
67
64
? 76 7 7?
??1 716
717 712
71? 7O8
709 704
706 7OO
703
700
1 97
I 95
I 86
1 80
75
71
68 6?
63
59
54
50
I 46
97
9S
9?
90
81
74
69
65
43
39
? 19
? 14
709
705
701
98
95
9?
90
87
78
71
66
6?
59
53
49
44
39
35
7.91 533 430 3.74 337 3.11 292 777 264 254 245 2.38 23? 776 771 716 7.1? 709 70S 70? 190 181 169 161 143 136 131
-------
TABLE A-4 Percentites of the F distribution (continued)
Uppfr 0.1% point of «f>« F dittribution
DEGREES OF FREEDOM FOR NUMERATOR
1OO 160 ZOO
16?
74 1
47.7
35.5
79?
754
779
' 90
iop
175
150
700
300
500
IOOO
197
186
178
17 1
16.6
16 I
15.7
154
15 1
148
146
144
14.7
140
139
13.7
136
135
134
133
13 I
130
178
I? 7
176
175
174 ;
174
173
17?
170
II 8
II 7
116
II 5
11 4
II 3
II 7
11 0
11 0
999
148
61 7
37 1
770
71 7
185
164
149
138
130
17.3
118
11.3
999
141
56.7
33.7
73.7
188
158
139
176
116
10.8
107
9.73
934
110 901
107 873
10.4 849
10 7 8 78
995 RIO
9 77 7.94
961 780
947 767
934 7.55
9.77 745
917 736
907 7 77
893 7.19
885 717
8.77 7.O5
864 694
8 57 6 83
8 47 6 74
8 33 6 66
8 75 6 59
818
812
8O6
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7 77
764
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7 15
707
700
653
648
647
638
6 34
617
606
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591
586
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577
5 71
563
556
551
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137 135
534 SI 7
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71 9 70.8
177 16.7
144 135
176 11 7
113 105
103 958
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907 8.35
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7 94 7.77
768 707
7.46 681
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584 576
5 76 5 19
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546 490
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5 17 458
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325
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740
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