Quality Assurance Project Plan for

Fish Sample Preparation and Analysis of Mercury,
Perfluorinated Compounds (PFCs), Polybrominated Diphenyl
Ethers (PBDEs), Polychlorinated Biphenyls (PCBs), and Fatty
Acids in Fish Tissue from the Great Lakes Human Health

Fish Tissue Study

Revision 2

April 18, 2012

Prepared for:

United States Environmental Protection Agency
Office of Water
Office of Science and Technology
Standards and Health Protection Division

Prepared jointly by:

Tetra Tech, Inc.
under:

Office of Science and Technology
Contract No. EP-C-09-019

and

CSC

under:

Technical, Analytical, and Regulatory Mission Support for the Water Security Division

Contract No. EP-C-10-060

EPA-822-B-24-001

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GLHHFTS Sample Preparation and Analysis QAPP

Revision 2
Date: April 18, 2012
Page i of 47

Revision History

June 21, 2011 - Original QAPP signed

October 20, 2011 - Revision 1 prepared

•	This revision added summary level information on PBDE analysis to Sections A4, A6, A7,
Bl, and B3 and details on the analysis of PBDEs in tissue samples by a commercial
laboratory to Sections B4, including the decision that 20-gram aliquots will be analyzed for
PBDEs.

•	It added the associated QC discussion and acceptance limits for PBDEs as a new Section
B5.4, including an update to the 2-tiered PBDE method blank acceptance criteria that
lowered the threshold for the second tier from 5 times the minimum level (ML) to 2 times the
ML.

•	It deleted all references to future PBDE analyses by NERL-Cincinnati.

•	It corrected the list of omega-3 fatty acid target analytes in Table 2 to reflect the final list for
which the laboratory can obtain authentic standards.

•	It added the list of PBDE target analytes as Table 3, which resulted in renumbering all
subsequent tables in the document.

•	It added new references associated with the PBDE analyses.

•	It revised Table 1 in Appendix B to reflect changes to the target mass to be collected for each
sample aliquot and added instructions to archive all remaining tissue mass available after
production of the second archive jar.

•	The revision number and the date in the header and on the title page were changed to reflect
the fact that the QAPP has been revised.

•	The title was updated to include addition of PBDE analysis to the GLHHFTS.

•	NERL-Cincinnati staff were removed from the Distribution List.

•	Appendix C was added to provide method detection limits (MDLs) and minimum level (ML)
summaries for all GLHHFTS target analytes.

•	References were added to Sections A4 and A6 to indicate the need for a second revision to
the QAPP to add PCB analysis to the GLHHFTS.

April 18, 2012 - Revision 2 prepared

•	This revision added summary level information on PCB analysis to Sections A4, A6, A7, Bl,
and B3 and details on the analysis of PCBs in tissue samples by a commercial laboratory to
Section B4.

•	It added the associated QC discussion and acceptance limits for PCBs as a new Section B5.5.

•	It added the list of PCB target analytes to Appendix C (avoiding inserting a very long table in
the middle of the document).

•	It added new references associated with the PCB analyses.

•	The revision number and the date in the header and on the title page were changed to reflect
the fact that the QAPP has been revised.

•	The title was updated to include addition of PCB analysis to the GLHHFTS.

•	Appendix C was revised to include PCB method detection limits (MDLs) and minimum
levels (MLs).

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GLHHFTS Sample Preparation and Analysis QAPP

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Fish Sample Preparation and Analysis of Mercury,

Perfluorinated Compounds (PFCs), Polybrominated Diphenyl Ethers (PBDEs),
Polychlorinated Biphenyls (PCBs), and Fatty Acids in Fish Tissue
from the Great Lakes Human Health Fish Tissue Study

A. PROJECT MANAGEMENT

This Quality Assurance Project Plan (QAPP) presents performance criteria, acceptance criteria,
and objectives for the analysis of mercury, perfluorinated compounds (PFCs), polybrominated
diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and omega-3 fatty acids in fish
composites collected for the Great Lakes Human Health Fish Tissue Study under the National
Coastal Condition Assessment (NCCA). This QAPP also describes the methods and procedures
that will be followed during the Great Lakes Human Health Fish Tissue Study (GLHHFTS) to
ensure that the criteria and objectives are met. This document addresses mercury, PFCs, PBDEs,
PCBs, and omega-3 fatty acid analytical activities only.

This QAPP was prepared in accordance with the most recent version of EPA QA/R-5, EPA
Requirements for Quality Assurance Project Plans (USEPA 2001), that was reissued in 2006. In
accordance with EPA QA/R-5, this QAPP is a dynamic document that is subject to change as
analytical activities progress. Changes to procedures in this QAPP must be reviewed by the EPA
Project Manager and the EPA Standards and Health Protection Division (SHPD) Quality
Assurance Coordinator for the GLHHFTS to determine whether the changes will impact the
technical and quality objectives of the project. If so, the QAPP will be revised accordingly,
circulated for approval, and forwarded to all project participants listed in the QAPP distribution
list (Section A3). Key project personnel and their roles and responsibilities are discussed in the
QAPP section to follow (Section A4), and project background perspective and description is
provided in Sections A5 and A6, respectively.

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Al. Approvals

Wanni. mh!

Leanne Stah], OST Project Manager, EPA

. i" -J-k J.

Denise Hawkins, Chief, FSBOB, EPA

/? / /

Robert Shippen, SHPD QA-Coordinator, EPA

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""	. * '	1	I ^ -f ' • '.

Marion Kelly, OST QA Officer. EPA

—	

Blaine Snyder. Tetra/fech Project Leader

0	U i (j

Susan Lanberg, Tetra Tech QA^Officer

Harr>'(MeCarty, C3C Project Uader

.1	„ /-

Marguerite Jones, CSC QA Officer

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GLHHFTS Sample Preparation and Analysis QAPP	Revision 2

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A2. Table of Contents

A.	PROJECT MANAGEMENT	1

Al. Approvals	2

A2. Table of Contents	3

A3. Di stributi on Li st	7

A4. Project/Task Organization	9

A5. Problem Definition/Background	15

A6. Project/Task Description	15

A7. Quality Objectives and Criteria	17

A8. Special Training/Certification	17

A9. Documents and Records	18

B.	DATA GENERATION AND ACQUISITION	18

B1. Sampling Process Design (Experimental Design)	18

B2. Sampling Methods	20

B3. Sample Handling and Custody	20

B4. Analytical Methods	21

B5. Quality Control	28

B5.1 Mercury	29

B5.2 PFCs	30

B5.3 Fatty Acids	31

B5.4 PBDEs	33

B5.5 PCBs	35

B6. Instrument/Equipment Testing, Inspection, and Maintenance	37

B7. Instrument/Equipment Calibration and Frequency	37

B8. Inspection/Acceptance of Supplies and Consumables	37

B9. Non-direct Measurements	38

BIO. Data Management	38

C.	AS SES SMENT AND OVERSIGHT	40

CI. Assessments and Response Actions	40

Cl.l Surveillance	40

C1.2 Product Revi ew	41

C1.3 Quality Systems Audit	42

C1.4 Readiness Review	42

CI.5 Technical Systems Audit	42

C1.6 Data Quality Assessment	43

C2. Reports to Management	43

D.	DATA VALIDATION AND USABILITY	43

Dl. Data Review, Verification, and Validation	43

Dl.l Data Review	43

D1.2 Data Verification	44

D1.3 Data Validation	44

D2. Verification and Validation Methods	44

D2.1 Verification Methods	44

D2.2 Validation Methods	45

D3. Reconciliation with User Requirements	45

References	46

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List of Tables

Table 1. PFC Target Analytes and Identifiers	23

Table 2. Target Omega-3 Fatty Acids and Identifiers	24

Table 3. PBDE Target Analyte List	25

Table 4. QC Samples and Acceptance Criteria for Mercury Analysis of Fish Tissue	29

Table 5. QC Samples and Acceptance Criteria for Mercury Analysis of Rinsates	29

Table 6. QC Samples and Acceptance Criteria for PFC Analysis of Fish Tissue	30

Table 7. QC Samples and Acceptance Criteria for PFC Analysis of Rinsates	31

Table 8. QC Samples and Acceptance Criteria for Fatty Acid Analysis of Fish Tissue	32

Table 9. QC Samples and Acceptance Criteria for Fatty Acid Analysis of Rinsates	32

Table 10. QC Samples and Acceptance Criteria for PBDE Analysis of Fish Tissue	33

Table 11. Laboratory Control Sample Limits (%) for PBDE Analysis	33

Table 12. Labeled Compound Recovery Limits (%) for PBDEs in Samples	35

Table 13. QC Samples and Acceptance Criteria for PCB Analysis of Fish Tissue	35

Table 14.	Calibration Verification Limits (%), Laboratory Control Sample Recovery

Limits (%), and Labeled Compound Recovery Limits (%) for PCB Analyses	36

List of Figures

Figure 1. GLHHFTS project team organization	11

Figure 2. Location of the 157 randomly selected nearshore Great Lakes sampling locations,

a statistical subset of NCCA Great Lake sites	16

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List of Acronyms and Abbreviations

AO AC	Association of Official Analytical Chemists

CAS	Chemical Abstract Service

CEC	Contaminant of emerging concern

DDT	Di chl orodiphenyltri chl oroethane

ECO	Ecological

EPA	Environmental Protection Agency

FSBOB	Fish, Shellfish, Beach, and Outreach Branch

GC/FID	Gas chromatography/flame ionization detector

GLHHFTS	Great Lakes Human Health Fish Tissue Study

GLNPO	Great Lakes National Program Office

HDPE	High density polyethylene

HPLC-MS/MS	High performance liquid chromatography-tandem mass spectrometry

ID	Identification

LCS	Laboratory control sample

MDL	Method detection limit

MS/MSD	Matrix spike/matrix spike duplicate

NCCA	National Coastal Condition Assessment

NERL	National Exposure Research Laboratory

NHEERL	National Health and Environmental Effects Research Laboratory

ORD	Office of Research and Development

OST	Office of Science and Technology

OW	Offi ce of Water

OWOW	Office of Wetlands, Oceans, and Watersheds

PBDE	Polybrominated diphenyl ether

PCB	Polychlorinated biphenyl

PFC	Perfluorinated compound

PFOA	Perfluorooctanoic acid

PFOS	Perfluorooctanesulfonic acid

PTFE	Polytetrafluoroethylene

QA	Quality assurance

QAPP	Quality Assurance Proj ect Plan

QC	Quality control

QSA	Quality system audit

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RF

Response factor

RPD

Relative percent difference

SHPD

Standards and Health Protection Division

SOP

Standard operating procedure

SOW

Statement of work

SPE

Solid-phase extraction

SWRI

Southwest Research Institute

USEPA

U.S. Environmental Protection Agency

VCSB

Voluntary consensus standard body

WED

Western Ecology Division

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A3. Distribution List

Michael Arbaugh
Microbac Laboratories
2101 VanDeman Street
Baltimore, MD 21224
(Contact Harry McCarty at CSC)

Louis Blume
USEPA/GLNPO (G-17J)
77 West Jackson Boulevard
Chicago, IL 60604
312/353-2317
blume.louis@epa.gov

Gregory Colianni
USEPA/OWOW (4503T)
1200 Pennsylvania Ave., N.W.
Washington, DC 20460
202/566-1249
colianni.gregory@epa.gov

Jacqueline Fisher
USEPA/GLNPO (G-17J)
77 West Jackson Boulevard
Chicago, IL 60604
312/353-1481
fi sher .j acqueline@epa. gov

Michael Flournoy
TestAmerica Laboratories
880 Riverside Parkway
West Sacramento, CA 95605
(Contact Harry McCarty at CSC)

Denise Hawkins
USEPA/OST (4305T)
1200 Pennsylvania Ave., N.W.
Washington, DC 20460
202/566-1384
hawkins.denise@epa.gov

Marguerite Jones
CSC

6101 Stevenson Avenue
Alexandria, VA 22304
703/461-2247
mjones214@csc.com

Marion Kelly

USEPA/OST (4303T)

1200 Pennsylvania Avenue, N.W.

Washington, DC 20460

202/566-1045

kelly.marion@epa.gov

Susan Lanberg

Tetra Tech, Inc.

10306 Easton Place, Suite 340

Fairfax, VA 22030

703/385-6000

susan. 1 anb erg@tetratech. com

Sarah Lehmann
USEPA/OWOW (4503T)
1200 Pennsylvania Avenue, N. W.
Washington, DC 20460
202/566-1379
lehmann.sarah@epa.gov

Harry McCarty
CSC

6101 Stevenson Avenue
Alexandria, VA 22304
703/461-2392
hmccarty@csc.com

Rod McLeod
ALS Canada Ltd.

5420 Mainway Drive
Burlington ON, Canada L7L 6A4
(Contact Harry McCarty at CSC)

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Elizabeth Murphy

Blaine Snyder

USEPA/GLNPO (G-17J)

Tetra Tech, Inc.

77 West Jackson Boulevard

400 Red Brook Blvd., Suite 200

Chicago, IL 60604

Owings Mills, MD 21117

312/353-4227

410/356-8993

murphy.elizabeth@epa.gov

blaine.snyder@tetratech.com

Mari Nord

Leanne Stahl

USEPA Region 5 (WQ-16J)

USEPA/OST (4305T)

77 West Jackson Boulevard

1200 Pennsylvania Ave., N.W.

Chicago, IL 60604

Washington, DC 20460

312/886-3017

202/566-0404

nord.mari@epa.gov

stahl.leanne@epa.gov

John O'Donnell

Cynthia Tomey

Tetra Tech, Inc.

AXYS Analytical Services, Ltd.

10306 Easton Place, Suite 340

2045 Mills Road W.

Fairfax, VA 22030

Sidney, BC, Canada V8L 5X2

703/385-6000

(Contact Harry McCarty at CSC)

j ohn. odonnell @tetratech. com



Tony 01 sen

Lynn Walters

USEPA/ORD/NHEERL/WED

CSC

200 S.W. 35th Street

6101 Stevenson Avenue

Corvallis, OR 97333

Alexandria, VA 22304

541/754-4790

703/461-2060

ol sen.tony@epa. gov

1 walters3 @csc. com

Lorraine Scheller

John Wathen

Southwest Research Institute

USEPA/OST (4305T)

6220 Culebra Road

1200 Pennsylvania Ave., N.W.

San Antonio, TX 78238

Washington, DC 20460

(Contact Harry McCarty at CSC)

202/566-0367



wathen.j ohn@epa.gov

Robert Shippen

Tom Yoder

USEPA/OST (4305T)

TestAmerica Laboratories

1200 Pennsylvania Ave., N.W.

5815 Middlebrook Pike

Washington, DC 20460

Knoxville, TN 37921

202/566-0391

(Contact Harry McCarty at CSC)

shippen.robert@epa.gov



Edwin Smith



USEP A/GLNPO (G-17J)



77 West Jackson Boulevard



Chicago, IL 60604

312/353-6571

smith.edwin@epa.gov

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A4. Project/Task Organization

EPA's National Coastal Condition Assessment (NCCA) is a probability-based survey designed
to assess the condition of coastal waters of the United States. It includes collection and analysis
of physical, chemical, and biological indicator data that will allow a statistically-valid
characterization of the condition of the Nation's coastal waters. EPA used an unequal
probability design to select 682 marine sites along the coasts of the contiguous United States and
225 freshwater sites from nearshore areas throughout the Great Lakes. The Office of Wetlands,
Oceans, and Watersheds (OWOW) within the Office of Water (OW) is responsible for the
overall planning and implementation of the NCCA.

One national and three regional fish contamination studies are being conducted under the NCCA.
The national assessment is using fish collected from all sampling sites in outer coastal waters as
indicators of ecological (ECO) contamination, based on whole body contaminant concentrations.
The ECO fish samples will be analyzed for 12 metals (including mercury), 21 polychlorinated
biphenyl (PCB) congeners, and 14 pesticides (including DDT and its metabolites). Results from
these analyses of whole body tissue will be used in conjunction with data from other indicators
(e.g., water chemistry) to determine the ecological integrity of all U.S. coastal resources. The
three other fish tissue surveys are regional studies of the Great Lakes, which involve two
ecological assessments and one assessment of fish contamination relevant to human health. The
first ecological assessment is the Great Lakes Embayment Enhancement study. It includes 150
randomly selected sites in embayments across all five Great Lakes to improve the ability to
assess the lake-wide condition of bays and harbors, and it focuses on whole-body analyses of fish
for evaluation of ecological condition.

The second ecological study and the human health study involve collection of fish from Great
Lakes nearshore sites (depths up to 30 m or distances up to 5 km from shore). Fish for the
second ECO fish study were collected from the full complement of 225 randomly selected
nearshore sites (45 sites per lake) and will be analyzed for the same group of chemicals identified
above for the national assessment. The Great Lakes Human Health Fish Tissue Study
(GLHHFTS) sample collection effort targeted game fish from a statistically representative subset
of about 150 nearshore sites (about 30 sites per lake). Field crews collected fish composite
samples for the GLHHFTS during a June through November 2010 sampling season. Routine
composite samples for this study consist of five similarly-sized adult fish of a single species
commonly consumed by humans. All of the samples collected for the GLHHFTS were shipped
as whole fish to a central storage facility at Microbac Laboratories in Baltimore, Maryland, and
staff at this laboratory will be preparing the fish samples for analysis (i.e., filleting the fish
samples and homogenizing the fillet tissue). The fillet tissue from these fish samples will be
analyzed for mercury, perfluorinated compounds (PFCs), omega-3 fatty acids (hereafter simply
referred to as fatty acids), polybrominated diphenyl ethers (PBDEs), and polychlorinated
biphenyls (PCBs). Other contaminants are under consideration for future analysis (e.g.,
pharmaceutical compounds).

EPA's Office of Science and Technology (OST) within OW is collaborating with the Great
Lakes National Program Office (GLNPO) in Chicago, Illinois and with the Office of Research
and Development (ORD) Western Ecology Division in Corvallis, Oregon to conduct the
GLHHFTS under the NCCA. OST is responsible for management of the GLHHFTS under the
NCCA with financial and technical support from GLNPO. ORD's Western Ecology Division in
Corvallis, Oregon developed the study design and selected all the sampling locations for the

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NCCA, including the 157 GLHHFTS sites. Statisticians in the Western Ecology Division will
also be analyzing the fish tissue concentration data.

In 2010, OWOW developed the NCCA Quality Assurance Project Plan (QAPP) (USEPA 2010a)
that describes the procedures and associated quality assurance/quality control (QA/QC) activities
for collecting and shipping NCCA fish tissue samples. It includes the human health fish
collection and shipping procedures that OST developed for the GLHHFTS based on the
protocols used for the National Lake Fish Tissue Study. In June 2011, OST developed the first
version of this QAPP that covers laboratory activities associated with GLHHFTS fish sample
preparation and analysis of fillet tissue for mercury, PFCs, and fatty acids. The first revision of
the OST QAPP added PBDE analyses and addresses other minor changes to the original QAPP
(see the revision history at the front). The current document represents the second revision to the
QAPP that adds PCB analyses to the GLHHFTS.

The GLHHFTS project team currently consists of managers, scientists, statisticians, and QA
personnel in OST, the ORD Western Ecology Division, and GLNPO, along with contractors
providing scientific and technical support to OST from CSC and Tetra Tech, Inc. (Figure 1).
Project team members from GLNPO are providing support for developing and reviewing
technical and program information related to all aspects of the study, including training
materials, standard operating procedures, QAPPs, analytical QA reports, briefings and reports on
study results, and outreach materials. Responsibilities for other key members of the project team
are described below.

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Figure 1. GLHHFTS project team organization

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Leanne Stahl of OST is the GLHHFTS Project Manager who is providing overall direction for
planning and implementation of this regional Great Lakes study being conducted under the
NCCA. This role involves the following responsibilities related to the GLHHFTS:

•	developing technical information for GLHHFTS fish sample collection that includes
preparation of the sampling SOP and coordination with the NCCA Project Leaders in
OWOW to integrate field sampling technical information for the GLHHFTS into NCCA
documents and training materials

•	providing technical support to conduct training on the GLHHFTS field sampling
requirements in coordination with the NCCA Project Leaders in OWOW

•	developing the fish preparation SOP, implementing training for laboratory processing of
NCCA fish samples, and providing technical direction for and oversight of fish
preparation activities, including technical support for review of fish preparation QA data

•	managing analysis of fish samples for target chemicals, including obtaining technical
support for chemical analysis of fish tissue, directing development of this QAPP,
providing for QA review of the analytical results, developing the data files for statistical
analysis of the data, reviewing and approving the final analytical QA report, and
providing oversight for development of the database to store GLHHFTS fish tissue
results

•	facilitating communication among GLHHFTS project team members and coordinating
with all of these individuals to ensure technical quality and adherence to QA/QC
requirements

•	developing and managing work assignments under OST contracts to provide technical
support for the GLHHFTS, providing oversight of all OST contractor activities, and
reviewing and approving study deliverables for each work assignment

•	scheduling and leading meetings and conference calls with project team members for
planning study activities, reporting progress on study tasks, and discussing and resolving
technical issues related to the study

•	working with QA staff to identify corrective actions necessary to ensure that study
quality objectives are met

•	managing the development of and/or reviewing and approving all major work products
associated with the GLHHFTS

•	collaborating with the GLHHFTS project team for reporting the study results in technical
journal articles and federal technical reports

Marion Kelly is the OST Quality Assurance Officer who is responsible for reviewing and
approving all Quality Assurance Project Plans (QAPPs) that involve scientific work being
conducted by OST. Robert Shippen is the Standards and Health Protection Division QA
Coordinator who is responsible for reviewing and recommending approval of all QAPPs that
include scientific work being conducted by the Standards and Health Protection Division
(SHPD) within OST. The OST QA Officer and SHPD QA Coordinator are also responsible for
the following QA/QC activities:

•	reviewing and approving this QAPP

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•	reviewing and evaluating the QA/QC requirements and data for all the GLHHFTS
activities and procedures

•	conducting external performance and system audits of the procedures applied for all
GLHHFTS activities

•	participating in Agency QA reviews of the study

Blaine Snyder is the Tetra Tech Project Leader who is responsible for managing all aspects of
the technical support being provided by Tetra Tech staff for the GLHHFTS. His specific
responsibilities include the following:

•	providing direct technical support for the following GLHHFTS activities or providing
leadership and oversight for Tetra Tech staff supporting these activities:

developing standard operating procedures for field sampling and fish preparation

preparing GLHHFTS documents (including this QAPP) or project information to
incorporate into NCCA documents

providing field sampling and fish preparation training

planning and implementing GLHHFTS logistics

conducting field sampling at Great Lake sites designated by the OST Project Manager

obtaining and performing QA reviews of Great Lakes human health field sampling
data

preparing fish preparation instructions for human health fish samples collected from
Great Lakes nearshore sites

evaluating weekly fish processing reports for adherence to the technical and quality
requirements in the fish preparation SOP

preparing summary project information and graphics for development of project fact
sheets, presentations, and other EPA meeting and outreach materials

developing technical journal articles and final project reports

•	monitoring the performance of Tetra Tech staff participating in this study to ensure that
they are following all QA procedures described in this QAPP that are related to Tetra
Tech tasks being performed to support this study (see list above)

•	ensuring completion of high-quality deliverables within established budgets and time
schedules

•	participating in meetings and conference calls with project team members for planning
study activities, reporting progress on study tasks, and discussing and resolving technical
issues related to the study

Susan Lanberg is the Tetra Tech QA Officer whose primary responsibilities include the
following:

•	assisting Tetra Tech's Project Leader with the development and review of this QAPP

•	approving this QAPP

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•	providing oversight for the implementation of QA procedures related to Tetra Tech tasks
that are described in this QAPP

•	reporting deviations from this QAPP to the Tetra Tech Project Leader and assisting in
implementing corrective actions to resolve these deviations

Harry McCarty is the CSC Project Leader who is responsible for managing all aspects of the
technical support being provided by CSC staff for the GLHHFTS. His specific responsibilities
include the following:

•	providing direct technical support for the following GLHHFTS activities or providing
leadership and oversight for CSC staff supporting these activities:

preparing information related to technical and quality assurance requirements for
preparation and chemical analysis of fish tissue samples for target chemicals,
validation of analytical data, and database development to support project planning
and development of GLHHFTS documents (including this QAPP) or characterization
of the GLHHFTS in NCCA documents

conducting reviews of fish preparation QA/QC data associated with each batch of up
to 20 fish samples and preparing a report about the results of each batch review for
distribution to the OST Project Manager and the fish preparation laboratory

obtaining subcontractor laboratory services to analyze urban river water and fish
tissue samples for mercury, PFCs, fatty acids, PBDEs, and PCBs, and providing
technical and QA oversight of laboratory operations

completing analytical data review for all target chemicals and developing the
analytical data QA report

formatting the analytical data files for statistical analysis and preparing raw
(unweighted) data files for public release

developing and maintaining a project database for storing GLHHFTS field and
analytical data and initiating queries of the database to respond to data requests from
Agency and external data users

obtaining freezer space that meets the requirements for long-term storage of archived
fish tissue samples, organizing the archived fish tissue samples by project to facilitate
retrieval of the samples, and developing and maintaining an inventory of the archived
samples

preparing summary project information and graphics for development of project fact
sheets, presentations, and other EPA meeting and outreach materials

supporting development of technical journal articles and final project reports

•	monitoring the performance of CSC staff participating in this study to ensure that they are
following all QA procedures described in this QAPP that are related to CSC tasks being
performed to support this study (see list above)

•	ensuring completion of high-quality deliverables within established budgets and time
schedules

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•	participating in meetings and conference calls with project team members for planning
study activities, reporting progress on study tasks, and discussing and resolving technical
issues related to the study

Marguerite Jones is the CSC QA Officer whose primary responsibilities include the following:

•	assisting CSC's Project Leader with the development and review of this QAPP

•	approving this QAPP

•	providing oversight for the implementation of QA procedures related to CSC tasks that
are described in this QAPP

•	reporting deviations from this QAPP to the CSC Project Leader and recommending
corrective actions to resolve these deviations

Tony Olsen is the Senior Statistician at the ORD Western Ecology Division in Corvallis,
Oregon who is supporting the GLHHFTS by providing technical expertise for study planning
and implementation and by assuming responsibility for the following activities:

•	study design development for the NCCA, including statistically representative national
and regional studies being conducted under the NCCA, such as the GLHHFTS

•	site selection and tracking for final statistical classification of sites

•	statistical analysis of analytical data for GLHHFTS fish tissue samples

•	development of cumulative density functions for analytical data sets with sufficient data
points

•	participation in development of technical journal articles and final reports for publication
A5. Problem Definition/Background

Obtaining statistically representative environmental data on mercury and chemicals of emerging
concern (CECs) is a priority area of interest for EPA. Since 1998, OW has collaborated with
ORD to conduct the first national-scale assessments of mercury in fish tissue through
statistically-based studies of U.S. lakes and rivers. These studies are referred to as the National
Lake Fish Tissue Study and the National Rivers and Streams Assessment, respectively. The
Great Lakes were excluded from the National Lake Fish Tissue Study because assessment of a
freshwater system of that magnitude required a separate sampling design. The probability-based
Great Lakes sampling design developed for the NCCA offered the opportunity to conduct the
GLHHFTS, which is the first representative study of chemical residues in fish relevant to human
health for this region. The GLHHFTS will also provide the first lake-wide data on the
occurrence and distribution of CECs (e.g., PFCs) in the Great Lakes. In addition, the GLHHFTS
will generate species-specific data on fatty acids to address an existing data gap and to identify
fish with higher omega-3 levels and potentially greater health benefits.

A6. Project/Task Description

OST is collaborating with the Great Lakes National Program Office and with ORD's Western
Ecology Division in Corvallis, Oregon, to conduct the GLHHFTS within the framework of the

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NCCA. Fish composite samples were collected from June through November 2010 at a
statistical subset of NCCA Great Lakes sites, which consisted of over 150 randomly selected
nearshore sites distributed throughout the five Great Lakes (Figure 2).

NCCA Great Lakes Human Health Fish Tissue Study

Sampling Locations

Figure 2. Location of the 157 randomly selected nearshore Great Lakes sampling locations, a statistical subset of
NCCA Great Lake sites

Following are the key design components for the GLHHFTS:

•	sampling over 150 randomly selected sites (about 30 sites per lake) in the nearshore
regions (depths up to 30 m or distances up to 5 km from shore) during 2010 (Appendix
A).

•	collecting one fish composite sample for human health applications (i.e., five similarly
sized adult fish of the same species that are commonly consumed by humans) from each
site.

•	shipping whole fish samples to a commercial laboratory for storage and fish sample
preparation, which includes filleting the fish, homogenizing the fillet tissue composites,
and preparing fillet tissue aliquots for analysis of specific contaminants, along with a
series of archive samples that may be used for future analyses of other contaminants.

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•	analyzing the fillet tissue samples for mercury (total), 13 perfluorinated compounds
(including PFOA and PFOS), 5 fatty acids, 52 PBDE congeners (and 2 other brominated
compounds), and 209 PCB congeners.

Microbac Laboratories in Baltimore, Maryland, is storing the GLHHFTS fish samples and
preparing the fish tissue samples for analysis as outlined in the third bullet above. As shown in
Figure 1, commercial laboratories under subcontract to CSC will be analyzing the GLHHFTS
fish tissue samples for mercury, PFCs, fatty acids, PBDEs, and PCBs. For each sample,
Microbac staff are also preparing and holding multiple aliquots of archived fillet tissue in a
freezer at their facility to allow for further analyses of GLHHFTS samples if resources can be
identified in the future to support these analyses.

Note: The sample aliquots that will be analyzed for PCBs are those originally prepared for the
analysis of pharmaceuticals and personal care products (PPCPs).

A7. Quality Objectives and Criteria

The overall quality objective for the analysis of the GLHHFTS fish tissue samples for mercury,
PFCs, fatty acids, PBDEs, and PCBs is to obtain a complete set of data for each chemical or
chemical group and to produce data of known and documented quality. Completeness is defined
as the percentage of samples collected in the study for which usable analytical results were
produced. The goal for completeness is 95% and it is calculated at the sample-analyte level,
such that an issue with the quality of one analyte out of many does not invalidate the entire
sample.

Commercial laboratories proposed analytical methods and quality control acceptance criteria for
analyses of GLHHFTS fish tissue samples for mercury, PFCs, fatty acids, and PBDEs. The
information describing the proposed methods has been added to Section B4 of this QAPP. Data
usability for each analysis will be assessed using QC criteria established by the respective
laboratories and summarized in Section B.5. For the PCB analyses, EPA specified the use of
Method 1668C and the QC acceptance criteria from that method are summarized in Section
B.5.5.

A8. Special Training/Certification

Fish Tissue Sample Preparation

All laboratory staff involved in the preparation of fish tissue samples must be proficient in the
associated tasks, as required by the NCCA GLHHFTS Tissue Preparation, Homogenization, and
Distribution Procedures (Appendix B).

Specialized training was provided for laboratory technicians who will be preparing fish tissue
fillets and homogenates for this project. This training was conducted at Microbac in Baltimore,
Maryland, for all laboratory staff involved with GLHHFTS fish tissue sample preparation to
accomplish the following objectives:

•	present GLHHFTS fish tissue preparation, homogenization and distribution procedures
described in Appendix B,

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•	demonstrate filleting and homogenizing techniques with fish from invalid GLHHFTS
samples, and

•	provide hands-on opportunities for fish preparation laboratory staff to develop
proficiency with filleting and homogenizing fish samples.

Analysis of Fish Tissue Samples

All laboratory staff involved in the analysis of fish tissue samples must be proficient in the
associated tasks, as required by each analytical laboratory's existing quality system. All
contractor staff involved in analytical data review and assessment will be proficient in data
review, and no specialized training is required for data reviewers for this project.

A9. Documents and Records

The Statements of Work (SOWs) for the analytical subcontracts provide the specific
requirements for laboratory deliverables. The major points are summarized below:

•	The laboratory must provide reports of all results required from analyses of
environmental and QC samples.

•	Summary level data must be submitted in electronic format and must include the
following information: EPA sample number, analyte name and CAS number, laboratory
sample ID, measured amount, reporting units, sample preparation date, and analytical
batch ID (if applicable).

•	The laboratory shall provide raw data in the form of direct instrument readouts with each
data package. Raw data include:

Copy of traffic report, chain-of-custody records, or other shipping information

Instrument readouts and quantitation reports for analysis of each sample, blank,
standard and QC sample, and all manual worksheets pertaining to sample or QC data
or the calculations thereof

Copies of bench notes, including preparation of standards and instrumental analyses

The laboratories will maintain records and documentation associated with these analyses for a
minimum of five years after completion of the study. Additional copies will be maintained by
CSC for at least five years and will be transferred to EPA on request.

B. DATA GENERATION AND ACQUISITION

Bl. Sampling Process Design (Experimental Design)

The objective of the GLHHFTS is to investigate the occurrence of mercury, PFCs, fatty acids,
PBDEs, and PCBs in the edible tissue (fillets) of harvestable-sized adult freshwater fish that are
typically consumed by humans. The study will provide:

•	statistically representative data on the levels of mercury, PFCs, PBDEs, and PCBs in
Great Lakes fish commonly consumed by humans,

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•	information on the potential for the target PFCs to bioaccumulate in fish tissue,

•	data to answer questions concerning the occurrence of PFCs in fish and the potential for
human exposure through fish consumption, and

•	species-specific information on fatty acid content of Great Lakes fish that are commonly
targeted by fishermen and consumed by humans.

The details of the sampling process design, sampling methods, and sample handling and custody
procedures are described in EPA's National Coastal Condition Assessment Quality Assurance
Project Plan prepared by OWOW (USEPA 2010a). However, to provide some context for the
readers of this QAPP, those aspects of the NCCA are summarized below.

The NCCA target population included nearshore areas of U.S. waters in the Great Lakes. The
Great Lakes survey design was stratified by lake and country (U.S. and Canada) with unequal
probability of selection based on state (or province) shoreline length within each stratum. The
nearshore zone was defined as the region from the shoreline to a depth of 30 m or to a distance of
5 km from the shoreline in shallower waters of the Great Lakes (e.g., Lake Erie). NCCA sites
were randomly selected in the five Great Lakes (Lakes Superior, Michigan, Huron, Erie, and
Ontario) bordered by eight Great Lakes states (Illinois, Indiana, Michigan, Minnesota, New
York, Ohio, Pennsylvania, and Wisconsin). The sample frame for the NCCA was derived by
ORD's Western Ecology Division in Corvallis, Oregon. The target population for the
GLHHFTS consists of a statistically representative subset of 157 NCCA sites distributed
throughout the U.S. nearshore zone of the five Great Lake (about 30 sites per lake). Sampling at
the GLHHFTS locations included collection of fish for analysis of mercury, PFCs, fatty acids,
PBDEs, and PCBs in the fillets.

To meet the study objective, one fish sample was collected from each site. A routine fish
composite sample consists of five fish of adequate size to provide a minimum of 300 grams of
edible tissue for analysis. Fish are selected for each composite applying the following criteria:

•	all are of the same species

•	all satisfy legal requirements of harvestable size (or weight) for the sampled lake, or at
least be of consumable size if no legal harvest requirements are in effect,

•	all are of similar size, so that the smallest individual in a composite is no less than 75% of
the total length of the largest individual, and

•	all are collected at the same time, i.e., collected as close to the same time as possible, but
no more than one week apart. (Note: Individual fish may have to be frozen until all fish
to be included in the composite are available for delivery to the designated laboratory).

Accurate taxonomic identification is essential in preventing the mixing of closely related target
species. Under no circumstances are individuals from different species used in a composite
sample.

Initially, OWOW designated June through September 2010 as the sampling period for the
NCCA, including the Great Lakes region. Field crews in the Great Lakes scheduled fish
collection at the majority of the nearshore sites during June and July, which turned out to be a
period when many of the target species for the GLHHFTS are difficult to find in shallow waters.

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Consequently, OST arranged re-sampling at over 50 nearshore sites after July and extended the
sampling period through mid-November to complete the goal of collecting fish for the
GLHHFTS from at least 150 nearshore sites.

B2. Sampling Methods

Sampling method procedures and requirements for collection of human health fish samples are
detailed in EPA's National Coastal Condition Assessment Quality Assurance Project Plan
(USEPA 2010a) and National Coastal Condition Assessment Field Operations Manual (USEPA
2010b). These sampling procedures and requirements are summarized below.

The field objective was for sampling teams to obtain one representative fish composite sample
from each sampling site. Collecting fish composite samples is a cost-effective means of
estimating average chemical concentrations in the tissue of target species, and compositing fish
ensures adequate sample mass for analysis of multiple chemicals. The sampling procedures
specified that each composite should consist of five similarly sized adult fish of the same species.
OST developed a recommended fish species list with GLNPO concurrence that contained 26
priority target fish species and 18 alternative fish species. Fish teams used this list as the basis
for selecting appropriate fish species for the GLHHFTS samples. The method applied for fish
collection was at the discretion of the field team, but it typically involved angling or gillnetting
and occasionally trawling.

In preparing fish samples for shipping, field teams recorded sample number, species name,
specimen length, sampling location and sampling data and time on a fish collection form. Each
fish was wrapped in solvent-rinsed, oven-baked aluminum foil, with the dull side in using foil
sheets provided by EPA. Individual foil-wrapped specimens were placed into a length of food-
grade polyethylene tubing, each end of the tubing was sealed with a plastic cable tie, and a fish
specimen label was affixed to the outside of the food-grade tubing with clear tape. All of the
wrapped fish in the sample from each site were placed in a large plastic bag and sealed with
another cable tie, then placed immediately on dry ice for shipment to Microbac in Baltimore,
Maryland. Field crews were directed to pack fish samples on dry ice in sufficient quantities to
keep samples frozen for up to 48 hours (50 pounds were recommended), and to ship them via
priority overnight delivery service (e.g., Federal Express), so that they could arrive at Microbac
in less than 24 hours from the time of sample collection. CSC was responsible for receiving and
examining the fish samples at Microbac before they were stored in a walk-in freezer at the
laboratory.

B3. Sample Handling and Custody

This section describes the sample handling and custody procedures that apply once the
homogenized fish tissue samples are shipped from Microbac to each of the analytical
laboratories selected for analysis of GLHHFTS fish tissue samples for mercury, PFCs, fatty
acids, and PBDEs. All sample handling and custody procedures prior to those described here are
discussed in the QAPP prepared by OWOW (USEPA 2010a) for other portions of the study and
are not repeated here.

CSC will ship the fish tissue homogenates from Microbac to the GLHHFTS analytical
laboratories. Samples will be packaged in sturdy coolers for shipping and wrapped with bubble
wrap or other suitable packaging to protect the samples in transit. Samples will be shipped

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frozen with sufficient dry ice in the coolers to ensure that the samples remain frozen for at least
48 hours. Step 29 of the fish preparation procedures (Appendix B) provides specific information
about dry ice requirements for shipping fish tissue samples. CSC will prepare sample tracking
paperwork and include it in each shipment.

When received at the respective analytical laboratories, the samples are inspected for damage,
logged into the laboratory, and immediately placed into freezers. Because the samples are
shipped frozen, typical temperature blanks consisting of a bottle of water are not practical (they
may break due to expansion), nor required. The laboratories measure and record the temperature
of the coolers containing the samples on receipt using an infrared temperature sensor or other
suitable device. CSC is notified of the receipt of samples by email. CSC will advise EPA of
tissue sample receipt. Any questions from the laboratories regarding sample paperwork or
condition will be sent to CSC, routed to OST or Tetra Tech as appropriate, and CSC will send
the answers back to the appropriate laboratory.

Fish tissue samples will be stored frozen at < -20ฐC until analyzed. There are no formal holding
time studies or requirements that apply to these analytes, except mercury and PCBs. EPA's
Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories: Volume 1
(USEPA 2000) recommended a 28-day holding time for fish tissue mercury analysis, i.e., from
receipt of the fish at the sample processing laboratory to analysis. Recently, Peterson et al.
(2007) conducted a holding time study focused specifically on mercury. They reported that
results for frozen tissue homogenates retained at -20ฐC between their original analysis in 2002
and a subsequent analysis in 2006 revealed no statistical differences in mercury concentrations
over time. They concluded that wet fish tissue homogenates can be held frozen for at least four
years without affecting analytical results for mercury. Considering those findings, a 1-year
administrative holding time (from homogenization to analysis) will be applied for GLHHFTS
mercury analyses in order to adhere to the study schedule and ensure sufficient time for data
compilation, review, and statistical analysis. For PCBs, EPA Method 1668C specifies a 1-year
holding time for solid samples, including tissues. For this study, that holding time will begin at
the completion of the homogenization of the composite sample.

EPA will note any results for either mercury or PCBs generated outside of these 1-year holding
times, but will not preclude use of such results for the purposes of this project.

B4. Analytical Methods

Fish Sample Preparation

Microbac was selected as the fish sample preparation laboratory (prep lab) for the GLHHFTS.
In this role, Microbac is responsible for filleting each valid fish sample, homogenizing the fillet
tissue, preparing the required number of fish tissue aliquots for analysis and archive, shipping the
fish tissue aliquots for each analysis to the designated analytical laboratory, and storing archived
fish tissue samples in a freezer at their facility. The specific procedures for all GLHHFTS fish
sample preparation activities are described in Appendix B.

Fish are filleted by qualified prep lab technicians using thoroughly clean utensils and cutting
boards (cleaning procedures are detailed in Appendix B). Each fish is weighed to the nearest
gram wet weight, rinsed with deionized water, and filleted on a glass cutting board. For the

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GLHHFTS, fillets from both sides of each fish are prepared with scales removed, skin on, and
belly flap (ventral muscle and skin) attached. Fillets are composited using the "batch" method,
in which all of the individual specimens that comprise the sample are homogenized together,
regardless of each individual specimen's proportion to one another (as opposed to the
"individual" method, in which equal weights of each specimen are added together).

An electric meat grinder is used to prepare homogenate samples. Entire fillets (with skin and
belly flap) from both sides of each fish are homogenized, and the entire homogenized volume of
all fillets from the fish sample is used to prepare the tissue sample. Tissues are mixed thoroughly
until they are completely homogenized as evidenced by a fillet homogenate that consists of a fine
paste of uniform color and texture. The collective weight of the homogenized tissue from each
sample is recorded to the nearest gram (wet weight) after processing. Microbac prepares fillet
tissue aliquots according to the specifications listed in Step 15 of the fish sample preparation
procedures in Appendix B.

Fish Tissue Analysis

Mercury

Fish tissue samples will be analyzed by TestAmerica - Knoxville using a microwave-assisted
strong acid digestion, followed by cold-vapor atomic absorption detection of mercury (CAS
Number 7439-97-6). The digestion and analysis procedures are based on SW-846 Method
3051A and Method 7470A, respectively. Approximately 0.5 g of tissue is used for the analysis.
Because the microwave-assisted digestion procedure dissolves all of the tissue, the digestate can
be analyzed as a liquid sample.

The rinsate samples for mercury are being analyzed by Microbac Laboratories, during the course
of the homogenization of the fish tissue samples. Microbac is analyzing these aqueous samples
using EPA Method 245.1, a cold-vapor atomic absorption procedure applicable to water samples.

Tissue sample results are reported based on the wet weight of the tissue sample, in micrograms
per kilogram (|ig/kg). Mercury method detection limits (MDLs) and minimum levels (MLs) are
listed in Appendix C.

Rinsate results are reported based on the volume of the rinsate sample, in micrograms per liter
(^g/L).

PFCs

There are no formal analytical methods from EPA or any voluntary consensus standard bodies
(VCSBs) for the PFC analyses. Therefore, fish tissue samples will be analyzed by the
TestAmerica - West Sacramento using procedures developed, tested, and documented in that
laboratory. The SOPs for those procedures are considered proprietary by the laboratory and
therefore are not attached to this QAPP. However, the SOPs have been reviewed by CSC and
the analytical procedures are briefly described below.

The 13 target PFC analytes are shown in Table 1 below.

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Table 1. PFC Target Analytes and Identifiers

Name

Abbreviation

Formula

CAS Number

Perfluorobutyric acid

PFBA

c3f7cooh

375-22-4

Perfluoropentanoic acid

PFPeA

c4f9cooh

2706-90-3

Perfluorohexanoic acid

PFHxA

c5f„cooh

307-24-4

Perfluoroheptanoic acid

PFHpA

CsF^COOH

375-85-9

Perfluorooctanoic acid

PFOA

C7F15COOH

335-67-1

Perfluorononanoic acid

PFNA

C8F17COOH

375-95-1

Perfluorodecanoic acid

PFDA

C9F19COOH

375-76-2

Perfluoroundecanoic acid

PFUnA

c10f21cooh

2058-94-8

Perfluorododecanoic acid

PFDoA

c„f23cooh

307-55-1

Perfluorobutanesulfonic acid

PFBS

c4f9so3h

375-73-5

Perfluorohexanesulfonic acid

PFHxS

CsF^SOsH

355-46-4

Perfluorooctanesulfonic acid

PFOS

c8f17so3h

1763-23-1

Perfluorooctanesulfonamide

PFOSA

c8f17so2nh2

754-91-6

13

The concentration of each PFC is determined using the responses from the Ci2-labeled
standards added prior to sample extraction, applying the technique known as isotope dilution. As
a result, all of the target analyte concentrations are corrected for the recovery of the labeled
standards, thus accounting for extraction efficiencies and losses during cleanup.

Approximately 1 to 5 g of fish tissue are required for analysis. The sample is spiked with twelve
isotopically labeled standards and extracted by shaking the tissue in a caustic solution of
methanol, water, and sodium hydroxide. The hydroxide solution breaks down the tissue and
allows the PFCs to be extracted into the methanol/water.

After extraction, the solution is centrifuged to remove the solids and the supernatant liquid is
diluted with dilute hydrochloric acid (HC1) to a pH < 2. That diluted extract is processed by
solid-phase extraction (SPE). The PFCs are eluted from the SPE cartridge and the eluant is
spiked with additional labeled recovery standards and analyzed by HPLC-MS/MS.

A 250-mL aliquot of an aqueous rinsate sample is spiked with the labeled standards and acidified
with dilute hydrochloric acid (HC1) to a pH < 2. That diluted extract is processed by solid-phase
extraction (SPE), in a similar manner as the tissue samples. The PFCs are eluted from the SPE
cartridge and the eluant is spiked with additional labeled recovery standards and analyzed by
HPLC-MS/MS.

Tissue sample results are reported based on the wet weight of the tissue sample, in nanograms
per kilogram (ng/kg). MDLs and MLs for PFCs are listed in Appendix C. Aqueous rinsate
results are reported based on the volume of the rinsate sample, in nanograms per liter (ng/L).

Fatty Acids

As with the PFCs, there are no formal analytical methods from EPA for the fatty acids, largely
because they are natural products and not environmental contaminants. However, there are
procedures for analysis of fats and oils available from some VCSBs, including the Association of
Official Analytical Chemists (AOAC). The fatty acid samples will be analyzed by Southwest
Research Institute (SwRI) using a combination of an extraction procedure from the literature and
an AO AC analytical method, as described below. The SOPs for those procedures are considered

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proprietary by the laboratory and therefore are not attached to this QAPP. However, the
analytical procedures are briefly described below.

The 5 target fatty acid analytes are shown in Table 2 below.

Table 2. Target Omega-3 Fatty Acids and Identifiers

Common name

Abbreviation

Structural Shorthand

CAS Number

alpha-Linolenic acid

ALA

18:3 (n-3)

463-40-1

Eicosatrienoic acid

ETE

20:3 (n-3)

2091-27-2

Eicosapentaenoic acid

EPA

20:5 (n-3)

10417-94-4

Docosapentaenoic acid

DPA

22:5 (n-3)

24880-45-3

Docosahexaenoic acid

DHA

22:6 (n-3)

6217-54-5

EPA initially planned to include four other fatty acids as target analytes, but neither the
laboratory nor CSC could locate authentic standards for those four fatty acids, so they have been
dropped from the list in Table 2.

The method used to extract the fatty acids from the fish tissue samples is based on the procedure
described by Sathivel et al. (2002). Briefly, a 1-g aliquot of homogenized fish tissue is placed in
a centrifuge tube and spiked with a surrogate solution containing triheneicosanoin
(a C21-triglyceride). The sample is extracted with 25 mL of a 1:4:4 solution of distilled water,
chloroform, and methanol and vortexed for 1 minute. The sample is placed on a mechanical
shaker for 15 minutes. After shaking, the mixture is filtered through Whatman No. 1 filter paper
to remove the solids, and the filtrate is collected in a separatory funnel, where it separates into
two layers. If needed, additional water is added to the separatory funnel to ensure phase
separation. The chloroform layer is drawn off from the bottom of the separatory funnel and
passed through anhydrous sodium sulfate to remove any remaining water. The extract is reduced
to dryness using nitrogen evaporation.

An internal standard is added to the extract and the fatty acids are derivatized to their methyl
esters by adding 1.5 mL of 0.5 N methanolic sodium hydroxide solution. The sample is
blanketed with either nitrogen or argon to prevent oxidation and heated to 100 ฐC for 30 min.
The sample is cooled to about 40 ฐC and 2 mL of isooctane are added. The sample is vortexed
for 30 sec. and 5 mL of saturated NaCl solution is added to the isooctane, followed by another 1
min of vortexing, after which the layers are allowed to separate. The isooctane layer is
transferred to a clean vial and the process is repeated once. The isooctane aliquots are combined
and the volume is adjusted to 1 mL and analyzed by GC/FID, using a DB-23 GC column.

For the rinsate samples, an aliquot of the rinsate will be evaporated to dryness with nitrogen and
the fatty acids will be derivatized following AO AC Method 991.39. That method uses boron
trifluoride (BF3) to derivatize the fatty acids to their methyl esters. As with the tissue samples,
the methyl esters are extracted with isooctane, concentrated, and analyzed by GC/FID.

Tissue results will be reported as the fractional percentage of each fatty acid methyl ester, based
on the wet weight of the sample. MDLs and MLs for fatty acids are listed in Appendix C.
Rinsate results will be reported as the fractional percentage of each fatty acid methyl ester, based
on the volume of the hexane rinsate sample.

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PBDEs

The PBDE samples will be prepared and analyzed by ALS - Canada in general accordance with
EPA Method 1614A (USEPA 2010c) and as detailed in the laboratory's proprietary SOP. The
ALS SOP deviates from the published EPA method in several aspects, including:

•	The use of more 13C-labeled extraction standards than called for in the method

•	Approximately 20 g of fish tissue is used for the analysis

•	GC performance criteria are monitored for every 12-hour run sequence instead of
requiring that the absolute retention time for decabromodiphenyl be at least 48 minutes

•	The concentrations of labeled and native spiking solutions differs from those listed in
Method 1614A

•	The labeled clean-up standard hexabromo-BDE-139L has been replaced with
hexabromo-BDE-13 8L

13

•	The list of injection standards has been enhanced to include four C-labeled BDEs
(BDE-79L, -139L, -180L, and -206L), rather than two labeled PCBs

•	The initial calibration range has been narrowed from 1 to 2500 ng/mL to 1 to 500 ng/mL,
with the CS4 standard at 150 and CS5 at 500 ng/mL

These changes fall within the method's established allowance for flexibility, and EPA has
accepted these deviations from Method 1614A for the purposes of this study.

The target analytes are listed in Table 3 and include 52 PBDE congeners and two other
brominated analytes. Of the 47 PBDE congeners, 41 are determined as individual congeners and
6 are determined as coeluting pairs that cannot be separated chromatographically. MDLs and
MLs for target PBDEs are listed in Appendix C.

Table 3. PBDE Target Analyte List

Full name

Abbreviation

CAS Number*

2,4-Dibromodiphenyl ether

BDE-7

171977-44-9

2,4'-Dibromodiphenyl ether coeluting with 3,3'-Dibromodiphenyl ether

BDE-8/11

147217-71-8/
6903-63-5

2,6-Dibromodiphenyl ether

BDE-10

51930-04-2

3,4-Dibromodiphenyl ether coeluting with 3,4'-Dibromodiphenyl ether

BDE-12/13

189084-59-1/
83694-71-7

4,4'-Dibromodiphenyl ether

BDE-15

2050-47-7

2,2',4-Tribromodiphenyl ether coeluting with
2,3',4-Tribromodiphenyl ether

BDE-17/25

147217-75-2/
147217-77-4

2,4,4'-Tribromodiphenyl ether coeluting with
2',3,4-Tribromodiphenyl ether

BDE-28/33

41318-75-6/
147217-78-5

2,4,6-Tribromodiphenyl ether

BDE-30

155999-95-4

2,4',6-Tribromodiphenyl ether

BDE-32

189084-60-4

3,3',4-Tribromodiphenyl ether

BDE-35

147217-80-9

3,4,4'-Tribromodiphenyl ether

BDE-37

147217-81-0

2,2',4,4'-Tetrabromodiphenyl ether

BDE-47

5436-43-1

2,2',4,5'-Tetrabromodiphenyl ether

BDE-49

243982-82-3

2,2',4,6'-Tetrabromodiphenyl ether

BDE-51

189084-57-9

2,3',4,4'-Tetrabromodiphenyl ether

BDE-66

189084-61-5

2,3',4',6-Tetrabromodiphenyl ether

BDE-71

189084-62-6

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Full name

Abbreviation

CAS Number*

2,4,4',6-Tetrabromodiphenyl ether

BDE-75

189084-63-7

3,3 ',4,4'-Tetrabromodiphenyl ether

BDE-77

93703-48-1

3,3',4,5'-Tetrabromodiphenyl ether

BDE-79

446254-48-4

2,2',3,4,4'-Pentabromodiphenyl ether

BDE-85

182346-21-0

2,2',4,4',5-Pentabromodiphenyl ether

BDE-99

60348-60-9

2,2',4,4',6-Pentabromodiphenyl ether

BDE-100

189084-64-8

2,3,3 ',4,4'-Pentabromodiphenyl ether

BDE-105

373594-78-6

2,3,4,5,6-Pentabromodiphenyl ether

BDE-116

189084-65-9

2,3',4,4',5-Pentabromodiphenyl ether

BDE-118

446254-80-4

2,3',4,4',6-Pentabromodiphenyl ether coeluting with
2,3',4,5,5'-Pentabromodiphenyl ether

BDE-119/120

189084-66-0/
417727-71-0

3,3',4,4',5-Pentabromodiphenyl ether

BDE-126

366791-32-4

2,2',3,3',4,4'-Hexabromodiphenyl ether

BDE-128

NA

2,2',3,4,4',5'-Hexabromodiphenyl ether coeluting with
2,3,4,4',5,6-Hexabromodiphenyl ether

BDE-138/166

182677-30-1/
189084-58-0

2,2',3,4,4',6'-Hexabromodiphenyl ether

BDE-140

243982-83-4

2,2',4,4',5,5'-Hexabromodiphenyl ether

BDE-153

68631-49-2

2,2',4,4',5',6-Hexabromodiphenyl ether

BDE-154

207122-15-4

2,2',4,4',6,6'-Hexabromodiphenyl ether

BDE-155

35854-94-5

2,3,3 ',4,4',5-Hexabromodiphenyl ether

BDE-156

NA

2,2',3,4,4',5,6-Heptabromodiphenyl ether

BDE-181

189084-67-1

2,2',3,4,4',5',6-Heptabromodiphenyl ether

BDE-183

207122-16-5

2,2',3,4,4',6,6'-Heptabromodiphenyl ether

BDE-184

117948-63-7

2,3,3',4,4',5,6-Heptabromodiphenyl ether

BDE-190

189084-68-2

2,3,3',4,4',5',6-Heptabromodiphenyl ether

BDE-191

NA

2,2',3,3',4,4',5',6-Octabromodiphenyl ether

BDE-196

NA

2,2',3,3',4,4',6,6'-Octabromodiphenyl ether

BDE-197

NA

2,2',3,4,4',5,5',6-Octabromodiphenyl ether

BDE-203

337513-72-1

2,2',3,3',4,4',5,5',6-Nonabromodiphenyl ether

BDE-206

63387-28-0

2,2',3,3',4,4',5,6,6'-Nonabromodiphenyl ether

BDE-207

437701-79-6

2,2',3,3',4,5,5',6,6'-Nonabromodiphenyl ether

BDE-208

NA

2,2',3,3',4,4',5,5',6,6'-Decabromodiphenyl ether

BDE-209

1163-19-5

Pentabromoethylbenzene

PBEB

85-22-3

Hexabromobenzene

HBB

87-82-1

* CAS numbers for coeluting congeners are shown for information only and will not be used in the project database
NA = Not available - no CAS number found for this congener

PCBs

The PCB samples will be prepared and analyzed by AXYS Analytical Services, in general
accordance with EPA Method 1668C (USEPA 2010d). The samples will be analyzed for all 209
PCB congeners, and reported as either individual congeners or coeluting groups of congeners.
The following method modifications have been reviewed, found to be within the allowance for
flexibility in Section 9.1.2 of Method 1668C, supported by performance data that are maintained
on file at the laboratory, and have been approved for use in this study:

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Section

Modification

Original Method Approach

Table 2

The use of 34 13C-labeled extraction standards

32 13C-labeled extraction standards

4.2.1,
4.2.2

The protocol for washing reusable glassware
includes a detergent wash, water rinse and baking at
a minimum of 300 ฐC for 8 h. Immediately prior to
use, glassware is solvent rinsed with toluene and
hexane.

Glassware should be rinsed with solvent and washed with a
detergent solution. After detergent washing, glassware should be
rinsed immediately, first with methanol, then with hot tap water.
The tap water rinse is followed by another methanol rinse, then
acetone, and then methylene chloride.

4.7

The first cleanup column for tissue extracts is a
gravity gel permeation column (SX-3 Biobeads). An
anthropogenic isolation column 7.5.3 is not used.

Lipids must be removed by the anthropogenic isolation column
procedure in Section 13.6, followed by the gel permeation
chromatography procedure.

6.5.1

Glass wool is cleaned by rinsing twice with toluene
and twice with hexane.

Glass wool is solvent-extracted using a Soxhlet or SDS extractor for
3 h minimum.

7.2.1

Sodium sulfate is baked at a minimum of 300 ฐC for
8 h.

Sodium sulfate, reagent grade, granular, anhydrous, rinsed with
methylene chloride (20 mL/g), baked at 400 ฐC for 1 h minimum.

7.5.1

Silica is activated by baking at 450 ฐC in a muffle
oven for at least 8 h.

Activated silica gel, rinsed with methylene chloride, baked at
180 ฐC for a minimum of 1 h.

7.5.4.1.1

Florisil is baked at 450 ฐC in a muffle oven for at
least 8 h, then deactivated with water to 2.1 %
deactivation.

Place in an oven at 130-150 ฐC for a minimum of three days to
activate the Florisil.

7.12,

7.13,
9.0,11.0

The concentration of the labeled toxics/level of
chlorination and the cleanup standard spiking
solutions is 100 ng/mL and the sample spiking
volume is 20 |xL. The resulting final concentrations
in the extracts are as specified in the method.

The concentration of the labeled toxics/level of chlorination and the
cleanup standard spiking solutions is 2 ng/mL and the sample
spiking volume is 1 mL.

7.14

Concentration of the labeled injection internal
standard spiking solution is modified so that a
volume of 5 |iL is added. The resulting amount of
standard added to the final extract is the same as
specified in the method. The solution is spiked into
a 15 |iL extract volume for a final extract volume of
20 |xL.

Concentration of the injection internal standards is 1000 ng/mL.
When 2 |xL of this solution is spiked into a 20 |xL extract, the
concentration of each injection internal standard will be nominally
100 ng/mL.

10.3.3,
15.3.3

S:N ratio of 3:1 for di-PCBs and nona-PCBs in
CS0.2 calibration solution is acceptable. (Note, this
standard is 5 times lower than the standard in the
method, hence the lesser S:N requirement)

The peaks representing the CBs and labeled compounds in the CS-1
calibration standard must have signal-to-noise ratios (S/N) >10.

11.5,
11.5.2,
11.5.5,
12.3

Solid samples are dried by mixing with anhydrous
sodium sulfate. The dried solid is extracted using a
Soxhlet extraction apparatus. The surrogate spike is
incorporated after the drying step. Equilibration
time for the surrogate is 30 minutes. The extracting
solvent for solids is dichloromethane. (Note, the
method warns that the use of toluene and SDS may
result in loss of some of the mono- through
trichlorinated analytes)

Weigh a well-mixed aliquot of each sample sufficient to provide
10 g of dry solids into a clean beaker or glass jar. Spike with
labeled compounds and stir or tumble and equilibrate the aliquots
for 1 to 2 h. Extract the sample using the SDS procedure using
toluene.

11.8,
12.4

The surrogate spike is incorporated into the sample
after the drying step to eliminate the possibility of
disproportional loss of volatile labeled and target
compounds.

Spike the labeled compounds, then mix in the sodium sulfate drying
agent.

12.4.2

The precleaning of the Soxhlet apparatus is carried
out using toluene instead of dichloromethane,
for 2 h.

Pre-extract the Soxhlet apparatus using methylene chloride
(dichloromethane).

12.6.1.1

Rotary evaporation is done at 30 ฐC. Daily cleaning
of the rotary evaporators include dismantling and
rinsing/soaking with solvent. Proofs are run
periodically but are not archived daily.

Rotary evaporation - Concentrate the extracts in a water bath at
45 ฐC. Pre-clean the rotary evaporator daily by concentrating
100 mL of clean extraction solvent through the system. Archive
both the concentrated solvent and the solvent in the catch flask for a
contamination check if necessary.

12.7.4

Before Florisil or alumina cleanup procedures, a
solvent exchange is done by reducing under
nitrogen to 300 |xL and bulking up to 1 mL in
hexane. If toluene is present, the extract is reduced
to 50 |xL under nitrogen and bulked up to 1 mL.

When the volume of the liquid is approximately 100 |iL, add 2 to
3 mL of the hexane and continue concentration to approximately
100 |iL. Repeat the addition of solvent and concentrate once more.
Bring the final volume to 1.0 mL with hexane.

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Section

Modification

Original Method Approach

12.7.7

Toluene (1 mL) is added to the eluate from the final
column prior to rotary evaporation and nitrogen
concentration steps. Transfer the extract to the
autosampler vial, rinsing with hexane and adding
the rinse to the vial. Add 15 |xL of nonane to the
vial, and evaporate the solvent to the level of the
nonane. Add 5 |xL of labeled injection internal
standard spiking solution, as described in 7.14
above.

Quantitatively transfer the extract to a 0.3-mL conical vial for final
concentration, rinsing the larger vial with hexane and adding the
rinse to the conical vial. Add 20 |xL of nonane to the vial, and
evaporate the solvent to the level of the nonane.

13.3.1

Routine layered silica column is as follows: 0.5 g
neutral silica, 2 g 28% basic silica, 0.5 g neutral
silica, 4 g 44% acidic silica, 4 g 22% acidic silica,
1 g neutral silica. (Note, these masses are half of
those in the method, but in the same relative
proportions)

Pack the column with 1 g silica gel, 4 g basic silica gel, 1 g silica
gel, 8 g acid silica gel, 2 g silica gel.

13.3.4

The sample is loaded onto the column, followed by
2-3 rinses of a least 1 mL, and eluted with 100 mL
of hexane.

Rinse the receiver twice with 1-mL portions of hexane, and apply
separately to the column. Elute the PCBs with 25 mL of hexane
and collect the eluate.

14.2

The volume of labeled injection internal standard
added to the extract is 5 |xL, for a final extract
volume of 20 |xL. Hexane, rather than nonane, is
used as the solvent to bring extract back to volume
for re-analysis or to dilute extracts.

Add 2 |xL of the labeled injection internal standard spiking solution
to the 20 |iL sample extract immediately prior to injection to
minimize the possibility of loss by evaporation, adsorption, or
reaction. If an extract is to be reanalyzed and evaporation has
occurred, do not add more labeled injection internal standard
spiking solution. Rather, bring the extract back to its previous
volume with pure nonane.

15.3

The calibration solution containing all 209 PCB
congeners is used as the CAL/VER solution.

Table 4 of the method includes only 27 PCB congeners.

17.0

The concentrations of target analytes, and the
labeled compound concentrations and recoveries,
are calculated using slight variations of the
equations described in the method. The modified
procedures are equivalent to those described in the
method, but are more direct.

Note, neither set of equations is reproduced in this QAPP, given
their length, but they are on file at CSC.

17.5

Extracts are diluted with hexane. The concentration
of the labeled injection internal standard is not
re-adjusted to 100 pg/|iL when dilutions are
performed.

Dilute the sample extract by the factor necessary to bring the
concentration within the calibration range, adjust the concentration
of the labeled injection internal standard to 100 pg/|xL in the extract.

Note: Given the large number of target analytes involved, the final list of PCB congeners and
coelutions is provided in Appendix C of this QAPP.

B5. Quality Control

The analytical procedures being applied by the laboratories designated for analysis of GLHHFTS
fish tissue samples include many of the traditional EPA analytical quality control activities. For
example, all samples are analyzed in batches and each batch includes:

•	up to 20 samples, including both field samples and QC samples

•	blanks - 5% of the samples within a batch are method blanks

Other common quality control activities vary by the analysis type, as described in the subsections
below.

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B5.1 Mercury

Quality control samples associated with each batch of tissue samples analyzed for mercury are
summarized in Table 4 below.

The cold-vapor atomic absorption instrument is calibrated daily, as described in SW-846 Method
7470A and the laboratory's SOP. At least five calibration standards and a blank are used for
calibration, and the resulting calibration curve must have a correlation coefficient of at least
0.995. The calibration is verified after every 10 samples by the analysis of a mid-range standard.
The results for the verification standard must fall within 20% of the true value.

The rinsate samples are prepared and analyzed individually, not in batches of up to 20, in order
to provide timely feedback of the cleanliness of the homogenization equipment. Therefore, the
quality control samples associated with the rinsate samples analyzed for mercury are usually
analyzed with each rinsate sample, and are summarized in Table 5 below.

Table 4. QC Samples and Acceptance Criteria for Mercury Analysis of Fish Tissue

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

One per sample batch

As noted elsewhere, all results, including blanks, are
reported down to the MDL.

-	If the method blank result is above the MDL, but below
the laboratory's nominal quantitation limit, the laboratory
will flag all associated field sample results as having a
detectable method blank for mercury. (Subsequent
validation of the results by EPA or its contractors will
evaluate the potential contribution of the blank to such
field sample results.)

-	If the method blank result is above the quantitation limit,
the laboratory will reanalyze the method blank.

•	If the reanalysis result is below the quantitation limit,
then the laboratory will reanalyze all of the associated
field and QC samples.

•	If the reanalysis result is still above the quantitation
limit, then the laboratory will redigest and reanalyze
all field samples with original results above the MDL.

Laboratory control
sample

One per sample batch

80 - 120% recovery of mercury. Otherwise, correct
instrumental problems, and redigest and reanalyze the batch
of field samples and QC samples.

Matrix spike sample

One per sample batch

75 - 125% recovery of mercury. Otherwise, compare spiking
level to background concentration in unspiked sample. If
spiked too low, adjust spiking level for future batches. If
spiked at least 5x background, and spike recovery criteria is
not met, flag all associated sample results and contact CSC
to discuss options for future batches.

Duplicate sample

One per sample batch

RPD < 20%. Flag results outside the limit.

Table 5. QC Samples and Acceptance Criteria for Mercury Analysis of Rinsates

Quality Control Sample

Frequency

Acceptance Criteria

Instrument blank

With each rinsate
sample

Result must be less than the MDL. Otherwise redigest and
reanalyze the rinsate sample.

Laboratory control
sample

With each rinsate
sample

80 - 120% recovery of mercury. Otherwise, correct
instrumental problems, and redigest and reanalyze the
rinsate sample.

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Because the rinsates are prepared in reagent water, there is little chance of a "matrix effect" and
the laboratory control sample, which is also prepared in reagent water, provides sufficient
information on the performance of the method and the laboratory in reagent water.

The rinsate samples for mercury are being analyzed by Microbac Laboratories, during the course
of the homogenization of the fish tissue samples. Microbac is analyzing these aqueous samples
using EPA Method 245.1, a cold-vapor atomic absorption procedure applicable to water samples.
As is the case for the tissue sample analyses, the instrument is calibrated daily as described in
Method 245.1

B5.2 PFCs

Quality control samples associated with each batch of tissue samples analyzed for PFCs are
summarized in Table 6 below.

Table 6. QC Samples and Acceptance Criteria for PFC Analysis of Fish Tissue

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

One per sample batch

As noted elsewhere, all results, including blanks, are
reported down to the MDL.

-	If the method blank result for any PFC is above the MDL,
but below the laboratory's nominal quantitation limit, the
laboratory will flag all associated field sample results as
having a detectable method blank for that analyte.
(Subsequent validation of the results by EPA or its
contractors will evaluate the potential contribution of the
blank to such field sample results.)

-	If the method blank result is above the quantitation limit,
the laboratory will reanalyze the method blank.

•	If the method blank reanalysis result is below the
quantitation limit, then the laboratory will reanalyze
all of the associated field and QC samples.

•	If the method blank reanalysis result is still above the
quantitation limit, then the laboratory will re-extract
and reanalyze all field samples with original results
above the MDL.

Laboratory control
sample

One per sample batch

60 - 140% recovery of each analyte quantified by isotope
dilution, and 50 -150% for each analyte quantified by
internal standard. Otherwise, correct instrumental
problems, and re-extract and reanalyze the batch of field
samples and QC samples.

Matrix spike and matrix
spike duplicate samples

One pair per sample
batch

60 - 140% recovery of each analyte quantified by isotope
dilution, and 50 - 150% for each analyte quantified by
internal standard. Otherwise, compare spiking level to
background concentration in unspiked sample. If spiked
too low, adjust spiking level for future batches. If spiked at
least 5x background, then correct instrumental problems,
and re-extract and reanalyze the batch of field samples and
QC samples. If spike recovery criteria still cannot be met,
flag all associated sample results.

RPD of MS/MSD pair < 30%. Otherwise, flag results
outside the limit.

Labeled compound
recovery

Every field and QC
sample

25 - 150% recovery of each labeled compound. For
recoveries <25%, examine results for native analyte and
assess impact. Contact CSC for direction, as needed.

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The rinsate samples are prepared and analyzed individually, not in batches of up to 20, in order
to provide timely feedback of the cleanliness of the homogenization equipment. Therefore, the
quality control samples associated with the rinsate samples analyzed for PFCs are summarized in
Table 7 below.

Table 7. QC Samples and Acceptance Criteria for PFC Analysis of Rinsates

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

With each rinsate
sample

As noted elsewhere, all results, including blanks, are
reported down to the MDL.

-	If the method blank result for any PFC is above the MDL,
but below the laboratory's nominal quantitation limit, the
laboratory will flag all associated field sample results as
having a detectable method blank for that analyte.
(Subsequent validation of the results by EPA or its
contractors will evaluate the potential contribution of the
blank to such field sample results.)

-	If the method blank result is above the quantitation limit,
the laboratory will reanalyze the method blank.

•	If the method blank reanalysis result is below the
quantitation limit, then the laboratory will reanalyze
all of the associated field and QC samples.

•	If the method blank reanalysis result is still above the
quantitation limit, then the laboratory will re-extract
and reanalyze the rinsate sample if the original
rinsate sample results were above the MDL.

Laboratory control
sample

With each rinsate
sample

60 - 140% recovery of each analyte quantified by isotope
dilution, and 50 - 150% for each analyte quantified by
internal standard. Otherwise, correct instrumental
problems, and re-extract and reanalyze the rinsate sample.

Labeled compound
recovery

Every rinsate sample

25 - 150% recovery of each labeled compound. For
recoveries <25%, examine results for native analyte and
assess impact. Contact CSC for direction, as needed.

Because the rinsates are prepared in reagent water, there is little chance of a "matrix effect" and
the laboratory control sample, which is also prepared in reagent water, provides sufficient
information on the performance of the laboratory in reagent water.

The HPLC-MS/MS instrument is calibrated using nine calibration standards and modeling the
instrument response with either a linear regression through the origin that is based on calculating
the response factor (RF) for each standard, or a linear regression not through the origin. For a
calibration employing response factors, the relative standard deviation of the nine response
factors must be < 35%. If a linear regression that does not pass through the origin is employed,
the correlation coefficient must be > 0.995.

The calibration is verified at the beginning of each analytical shift and after every 10 samples,
using a single standard that must meet ฑ 40% for each analyte quantified by isotope dilution, and
ฑ 50% for each analyte quantified by internal standard.

B5.3 Fatty Acids

Quality control samples associated with each batch of tissue samples analyzed for fatty acids are
summarized in Table 8 below.

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Table 8. QC Samples and Acceptance Criteria for Fatty Acid Analysis of Fish Tissue

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

One per sample
batch

As noted elsewhere, all results, including blanks, are reported
down to the MDL.

-	If the method blank result for any fatty acid is above the MDL,
but below the laboratory's nominal quantitation limit, the
laboratory will flag all associated field sample results as having
a detectable method blank for that analyte. (Subsequent
validation of the results by EPA or its contractors will evaluate
the potential contribution of the blank to such field sample
results.)

-	If the method blank result is above the quantitation limit, the
laboratory will reanalyze the method blank.

•	If the reanalysis result is below the quantitation limit, then
the laboratory will reanalyze all of the associated field and
QC samples.

•	If the reanalysis result is still above the quantitation limit,
then the laboratory will re-extract and reanalyze all field
samples with original results above the MDL.

Surrogate

Every field and
QC sample

70 - 130% recovery in each sample. Otherwise re-extract and
reanalyze the sample.

Laboratory control
sample

One per sample
batch

70 - 130% recovery of each target fatty acid. Otherwise, correct
instrumental problems, and reanalyze the batch of samples.

Reference material (NIST
SRM 1946 Lake Superior
fish tissue)

One per sample
batch

Within ฑ 30% of the certified value for each analyte. Otherwise,
correct instrumental problems, and re-extract and reanalyze the
batch of samples.

Duplicate sample

One per sample
batch

RPD < 20%. Flag results outside the limit.

The rinsate samples are prepared and analyzed individually, not in batches of up to 20, in order
to provide timely feedback of the cleanliness of the homogenization equipment. Therefore, the
quality control samples associated with the rinsate samples analyzed for fatty acids are
summarized in Table 9 below.

Table 9. QC Samples and Acceptance Criteria for Fatty Acid Analysis of Rinsates

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

With each rinsate
sample

Result must be less than the MDL. Otherwise reanalyze the
associated rinsate sample.

Surrogate

Every field and QC
sample

70 - 130% recovery in each sample. Otherwise reanalyze
the sample.

Laboratory control
sample

With each rinsate
sample

70 - 130% recovery of each analyte Otherwise, correct
instrumental problems, and reanalyze the rinsate sample.

Because the rinsates and blanks are prepared from hexane and no sample extraction is required,
"matrix effects" are highly unlikely. Therefore matrix spike and duplicate samples are not
required for these hexane samples. Analysis of a LCS prepared in hexane with each set of
rinsate samples will provide sufficient evidence of the performance of the method in hexane.

The GC/FID is calibrated using a series of six standards of the target analytes that are carried
through the esterification process along with the samples. The calibration must attain a
correlation coefficient of at least 0.995. The calibration is verified at the beginning and end of
each analytical sequence and after every 15 injections using a mid-level calibration standard that
must meet ฑ 20% for each analyte.

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B5.4 PBDEs

Quality control samples associated with each batch of tissue samples analyzed for PBDEs are
summarized in Table 10 below.

Table 10. QC Samples and Acceptance Criteria for PBDE Analysis of Fish Tissue

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

One per sample
batch

As noted elsewhere, all results, including blanks, are reported
down to the MDL. Given the ubiquitous nature of PBDEs, it can
be difficult to produce method blanks that are completely free of
these analytes. The following scheme will be used by the
laboratory to evaluate method blank results:

•	If any PBDE congener other than 28, 47, 99, 100, and 209 is
found in the method blank above the concentration equivalent
to the low point of the initial calibration (e.g., EPA's
Minimum Level in Method 1614A), analysis will be halted
until the source of the contamination can be identified and
corrected. Samples associated with a contaminated method
blank will be re-extracted and reanalyzed.

•	For the frequently occurring congeners 28, 47, 99, 100, and
209, similar corrective action will be taken if the results are
greater than 2 times the low point of the initial calibration.

Note: CSC will evaluate the sample results based on the
relation between the reported detection limit for each congener in
the method blank and the results in the field sample, and qualify
sample results accordingly.

Laboratory duplicate
sample

One per sample
batch

The acceptance criterion for the relative percent difference (RPD)
is 50% for results that are above the low point of the calibration
range. Results below that level will be compared, but may not
meet the acceptance criterion in every case.

Laboratory control
sample

One per sample
batch

See acceptance criteria in Table 11

Labeled compounds

Spiked into
every field
sample

See acceptance criteria in Table 12

Table 11. Laboratory Control Sample Limits (%) for PBDE Analysis

Compound

Lower Limit

Upper Limit

Source

Native Analytes

BDE-7

20

150

In-house

BDE-8/11

20

150

In-house

BDE-10

20

150

In-house

BDE-12/13

20

150

In-house

BDE-15

50

150

In-house

BDE-17/25

50

150

In-house

BDE-28/33

50

150

1614A

BDE-30

20

150

In-house

BDE-32

50

150

In-house

BDE-35

50

150

In-house

BDE-37

50

150

In-house

BDE-47

50

150

1614A

BDE-49

50

150

In-house

BDE-51

50

150

In-house

BDE-66

50

150

In-house

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Compound

Lower Limit

Upper Limit

Source

BDE-71

50

150

In-house

BDE-75

50

150

In-house

BDE-77

50

150

In-house

BDE-79

50

150

In-house

BDE-85

50

150

In-house

BDE-99

50

150

1614A

BDE-100

50

150

1614A

BDE-105

50

150

In-house

BDE-116

50

150

In-house

BDE-118

50

150

In-house

BDE-119/120

50

150

In-house

BDE-126

50

150

In-house

BDE-128

50

150

In-house

BDE-138/166

50

150

In-house

BDE-140

50

150

In-house

BDE-153

50

150

1614A

BDE-154

50

150

1614A

BDE-155

50

150

In-house

BDE-156

50

150

In-house

BDE-181

50

150

In-house

BDE-183

50

150

1614A

BDE-184

50

150

In-house

BDE-190

50

150

In-house

BDE-191

50

150

In-house

BDE-196

50

150

In-house

BDE-197

50

150

In-house

BDE-203

50

150

In-house

BDE-206

50

200

In-house

BDE-207

50

200

In-house

BDE-208

50

200

In-house

BDE-209

50

200

1614A

PBEB

50

150

In-house

HBB

50

150

In-house

Extraction Standards

13C12-BDE-3

10

140

In-house

13C12-BDE-15

20

140

In-house

13C12-BDE-28

30

140

1614A

13c6-hbb

30

140

In-house

13C12-BDE-47

30

140

1614A

13C12-BDE-77

30

140

In-house

13C12-BDE-99

30

140

1614A

13c12-bde-ioo

30

140

1614A

13C12-BDE-126

30

140

In-house

13C12-BDE-153

30

140

1614A

13C12-BDE-154

30

140

1614A

13C12-BDE-183

30

140

1614A

13C12-BDE-197

30

140

In-house

13C12-BDE-207

25

200

In-house

13C12-BDE-209

25

200

1614A

Cleanup Standard

13C12-BDE-138

40

125

1614A

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Table 12. Labeled Compound Recovery Limits (%) for PBDEs in Samples

Compound

Lower Limit

Upper Limit

Source

Extraction Standards

13C12-BDE-3

10

150

In-house

13C12-BDE-15

20

150

In-house

13C12-BDE-28

25

150

1614A

13c6-hbb

25

150

In-house

13C12-BDE-47

25

150

1614A

13C12-BDE-77

25

150

In-house

13C12-BDE-99

25

150

1614A

13c12-bde-ioo

25

150

1614A

13C12-BDE-126

25

150

In-house

13C12-BDE-153

25

150

1614A

13C12-BDE-154

25

150

1614A

13C12-BDE-183

25

150

1614A

13C12-BDE-197

25

150

In-house

13C12-BDE-207

20

200

In-house

13C12-BDE-209

20

200

1614A

Cleanup Standard

13C12-BDE-138

40

125

1614A

B5.5 PCBs

Quality control samples associated with each batch of tissue samples analyzed for PCBs are
summarized in Table 13, below, and are based on the QC requirements of Method 1668C, with
the project-specific addition of one laboratory duplicate sample per batch.

Table 13. QC Samples and Acceptance Criteria for PCB Analysis of Fish Tissue

Quality Control Sample

Frequency

Acceptance Criteria

Method blank

One per
sample batch

5 times the MDL for each congener (As noted elsewhere, all
results, including blanks, are reported down to the MDL.) If the
method blank result is above 5 times the MDL, the laboratory will
reanalyze the method blank extract.

•	If the reanalysis result is below 5 times the MDL, then the
laboratory will reanalyze all of the associated field and QC
samples.

•	If the reanalysis result is still above 5 times the MDL, then the
laboratory will re-extract and reanalyze all field samples with
original results above the MDL.

Laboratory duplicate
sample

One per
sample batch

The RPD of the duplicate measurements must be:

•	< 50% for sample concentrations greater than or equal to
5 times the MDL, and

•	<100% for sample concentrations less than 5 times the MDL.

(When comparing the sample concentration to the MDL, use the

lower of the two concentrations in the paired samples.)

If the RPD exceeds the acceptance limit, the laboratory will

reanalyze the laboratory duplicate extract.

•	If the reanalysis result does not exceed the RPD limit, then the
laboratory will reanalyze all of the associated field and QC
samples.

•	If the reanalysis result still exceeds the RPD limit, then the
laboratory will re-extract and reanalyze all field samples with
original results above the MDL.

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Quality Control Sample

Frequency

Acceptance Criteria

Laboratory control

One per

Per Table 14 below

sample

sample batch



Labeled compounds

Spiked into
every field
sample

Per Table 14 below

Calibration verification

At the

Per Table 14 below

(VER)

beginning of
every 12-h
analytical shift



Table 14. Calibration Verification Limits (%), Laboratory Control Sample Recovery

Limits (%), and Labeled Compound Recovery Limits (%) for PCB Analyses1

Compound

CAL VER

LCS Recovery

Labeled Compound Recovery in Samples

PCB-1

75 - 125

60 - 135



PCB-3

75 - 125

60 - 135



PCB-4

75 - 125

60 - 135



PCB-15

75 - 125

60 - 135



PCB-19

75 - 125

60 - 135



PCB-37

75 - 125

60 - 135



PCB-54

75 - 125

60 - 135



PCB-77

75 - 125

60 - 135



PCB-81

75 - 125

60 - 135



PCB-104

75 - 125

60 - 135



PCB-105

75 - 125

60 - 135



PCB-114

75 - 125

60 - 135



PCB-118

75 - 125

60 - 135

Limits for each labeled compound
are shown below

PCB-123

75 - 125

60 - 135

PCB-126

75 - 125

60 - 135

PCB-155

75 - 125

60 - 135



PCB-156

75 - 125

60 - 135



PCB-157

75 - 125

60 - 135



PCB-167

75 - 125

60 - 135



PCB-169

75 - 125

60 - 135



PCB-188

75 - 125

60 - 135



PCB-189

75 - 125

60 - 135



PCB-202

75 - 125

60 - 135



PCB-205

75 - 125

60 - 135



PCB-206

75 - 125

60 - 135



PCB-208

75 - 125

60 - 135



PCB-209

75 - 125

60 - 135



uC-Labeled Compounds

13c12-pcb-i

50 - 145

15 - 145

5 -145

13C12-PCB-3

50 - 145

15 - 145

5 -145

13C12-PCB-4

50 - 145

15 - 145

5 -145

13C12-PCB-15

50 - 145

15 - 145

5 - 145

13C12-PCB-19

50 - 145

15 - 145

5 - 145

13C12-PCB-37

50 - 145

15 - 145

5 - 145

13C12-PCB-54

50 - 145

15 - 145

5 - 145

13C12-PCB-77

50 - 145

40 - 145

10 - 145

13C12-PCB-81

50 - 145

40 - 145

10 - 145

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Compound

CAL VER

LCS Recovery

Labeled Compound Recovery in Samples

13C12-PCB-104

50 - 145

40 - 145

10 - 145

13C12-PCB-105

50 - 145

40 - 145

10 - 145

13C12-PCB-114

50 - 145

40 - 145

10 - 145

13C12-PCB-118

50 - 145

40 - 145

10 - 145

13C12-PCB-123

50 - 145

40 - 145

10 - 145

13C12-PCB-126

50 - 145

40 - 145

10 - 145

13C12-PCB-155

50 - 145

40 - 145

10 - 145

13C12-PCB-156

50 - 145

40 - 145

10 - 145

13C12-PCB-157

50 - 145

40 - 145

10 - 145

13C12-PCB-167

50 - 145

40 - 145

10 - 145

13C12-PCB-169

50 - 145

40 - 145

10 - 145

13C12-PCB-170

50 - 145

40 - 145

10 - 145

13C12-PCB-180

50 - 145

40 - 145

10 - 145

13C12-PCB-188

50 - 145

40 - 145

10 - 145

13C12-PCB-189

50 - 145

40 - 145

10 - 145

13C12-PCB-202

50 - 145

40 - 145

10 - 145

13C12-PCB-205

50 - 145

40 - 145

10 - 145

13C12-PCB-206

50 - 145

40 - 145

10 - 145

13C12-PCB-208

50 - 145

40 - 145

10 - 145

13C12-PCB-209

50 - 145

40 - 145

10 - 145

Cleanup Standards

13C12-PCB-28

65 - 135

15 - 145

5 - 145

13C12-PCB-111

75 - 125

40 - 145

10 - 145

13C12-PCB-178

75 - 125

40 - 145

10 - 145

Adapted from Table 6 of Method 1668C

B6. Instrument/Equipment Testing, Inspection, and Maintenance

The inspection, testing, and maintenance of all laboratory equipment and instrumentation is
addressed in the individual laboratory operating procedures to be used, or in each laboratory's
existing overall quality system documentation (TestAmerica 2010, TestAmerica 2011,

Southwest Research Institute 2010, ALS 2009, and AXYS 2012). There are no additional
requirements specific to this project, and therefore, none are described here.

B7. Instrument/Equipment Calibration and Frequency

Each laboratory's instrument calibration procedures and frequency are included in Section B5 of
this QAPP and in the laboratory's existing overall quality system documentation (TestAmerica
2010, TestAmerica 2011, Southwest Research Institute 2010, ALS 2009, and AXYS 2012). No
additional discussion is required.

B8. Inspection/Acceptance of Supplies and Consumables

The inspection and acceptance of any laboratory supplies and consumables are addressed in the
individual laboratory operating procedures to be used, and/or in the laboratory's existing overall
quality system documentation (TestAmerica 2010, TestAmerica 2011, Southwest Research
Institute 2010, ALS 2009, and AXYS 2012). There are no additional requirements specific to
this project, and therefore, none are described here.

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B9. Non-direct Measurements

Non-direct measurements are not required for this project.

BIO. Data Management

Data management practices employed in this study will be based on standard data management
practices used for EPA's National Lake Fish Tissue Study. The data management (i.e., sample
tracking, data tracking, data inspection, data quality assessment, database development)
procedures have been regularly applied to other technical studies. These procedures are being
employed because they are effective, efficient, and have successfully withstood repeated internal
and external audits, including internal review by EPA Quality Staff, public review and comment,
judicial challenge, and the Government Accountability Office audit. These procedures, as
implemented for the GLHHFTS, are summarized below.

Laboratory Data Management

Laboratory data management procedures include the following:

•	Each laboratory is required to maintain all records and documentation associated with the
preparation and analysis of study samples for a minimum period of five years after
completion of the study.

•	To facilitate data tracking, each laboratory is required to use EPA-assigned sample
numbers when reporting results.

•	All results of field sample analyses and QC sample analyses must be reported on
electronic media.

•	All required reports and documentation, including raw data, must be sequentially
paginated and clearly labeled with the laboratory name, contract number, episode
number, and associated EPA sample numbers. Any electronic media submitted must be
similarly labeled.

•	Each laboratory will adhere to a comprehensive data management plan that is consistent
with the principles set forth in Good Automated Laboratory Practices, EPA Office of
Administration and Resources Management, October 10, 1995 (USEPA 1995). Those
data management plans are incorporated in their overall quality system documentation
(e.g., their quality management plan, TestAmerica 2010, TestAmerica 2011, Southwest
Research Institute 2010, ALS 2009, and AXYS 2012).

CSC Data Management

Data management procedures employed by CSC include the use of 1) standardized data review
guidelines to promote consistency in data quality audits (data reviews) across reviewers and over
time, 2) a multi-stage data review process designed to maximize the amount of useable data
generated in each study, and 3) a standardized database development process that facilitates rapid
development of a database with at least 99.9% accuracy.

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Standardized data review guidelines will be used in this study to facilitate rapid, consistent,
accurate, and thorough data quality audits. The data review guidelines are those that were
employed for the National Lake Fish Tissue Study and are in use for a variety of analyses
performed for EPA programs. These guidelines detail method-specific data review procedures
for commonly used methods and more general procedures that can be applied to less frequently
used methods. Where appropriate, CSC will modify existing data review guidelines as necessary
to reflect the methods, method modifications, and data quality objectives for the GLHHFTS.
Descriptions of any modifications will be retained in CSC's project records.

Although each guideline is written for a specific method, technique, or group of analytes, all
guidelines specify a general review process that ensure data are in proper format, are complete,
are contractually compliant, and are usable. CSC data review chemists use this multi-stage
process to verify the quality of each laboratory submission under the GLHHFTS. If an error is
detected in any stage of the review, the CSC data review chemist and the CSC Project Leader
will initiate corrective action procedures to obtain the maximum amount of usable data from the
study. These actions may serve to obtain missing data, correct typographical or transcription
errors on data reporting forms, or initiate reanalysis of field or QC samples that do not meet the
performance criteria for this study. Any such actions will be documented in CSC's project
records.

Concurrent with the performance of data quality audits, CSC will begin developing a MS Access
database of combined field and analytical results for tissue samples. At a minimum, this
database will be formatted in a manner that is consistent with the National Lake Fish Tissue
Study. At a minimum, each record should include fields containing the following information
for each tissue sample:

•	the site identification number assigned by EPA;

•	the EPA sample number;

•	sample matrix (tissue);

•	sample type (indicates the type of sample, whether it was a primary composite, duplicate
composite, matrix spike, etc);

•	fish species (scientific and common names);

•	fish specimen number;

•	length of fish specimen;

•	weight of fish specimen;

•	retention of fish specimen for homogenization;

•	method of collection;

•	sample collection date;

•	Great Lake from which samples were collected;

•	state where site is located;

•	latitude/longitude where site is located;

•	year samples were collected;

•	ecological group for fish samples (predator or bottom dweller);

•	sample analysis date;

•	measured value for each target analyte; and

•	fish tissue lipid content measurements.

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The MS Access database will contain the field and analytical results from all study samples until
the complete data set is transmitted to EPA. The database also will contain data for the QC
samples described in Section B, associated with the field sample results, as well as applicable
surrogates and labeled compounds. The structure of the database will allow CSC to segregate
these QC results from those in the field samples.

Results for the rinsate samples will not be included in the same database, but will be used to
assess the homogenization and equipment cleaning procedures employed during sample
preparation activities before the analyses take place.

As with the data quality audits, a multi-stage process of inspections and corrective actions are
used to facilitate timely, efficient construction of databases that are least 99.9% accurate. The
database development process will begin with a completeness check to verify the laboratory has
submitted data on an electronic medium that contains all data in an appropriate format. If
deficiencies are found, appropriate corrective action measures will be initiated.

The CSC data review chemist responsible for performing the data quality audit will verify that
the electronic data accurately reflect the hard copy submission. Accuracy will be confirmed by
spot checking at least 10% of all results that were downloaded directly from an analytical
instrument in the laboratory and by performing a 100% QC check of data that were manually
entered by the laboratory or CSC. Corrective actions will be taken as needed to resolve
deficiencies. Following completion of the data quality audit, the CSC data review chemist and
the CSC database administrator will modify the database to reflect data usability determinations.
A report, generated to reflect the modified database, will then be reviewed by the CSC data
review chemist to verify database accuracy before submission to EPA. These reports are
maintained in CSC's project files.

C. ASSESSMENT AND OVERSIGHT

CI. Assessments and Response Actions

The laboratory contracts prepared to support this study stipulate that each laboratory has a
comprehensive QA program in place and operating at all times during the performance of their
contract, and that in performing laboratory work for this study, each laboratory shall adhere to
the requirements of that QA program (TestAmerica 2010, TestAmerica 2011, Southwest
Research Institute 2010, ALS 2009, and AXYS 2012).

Sections Cl.l through CI.6 describe other types of assessment activities and corresponding
response actions identified to ensure that data gathering activities in the GLHHFTS are
conducted as prescribed and that the performance criteria defined for the study are met.

Cl.l Surveillance

The CSC Project Leader will schedule and track all analytical work performed by laboratories
for mercury, PFC, fatty acid, and PBDE analyses. The Project Leader will coordinate with staff
at Microbac regarding fish tissue sample shipments.

When samples are shipped to an analytical laboratory, the Project Leader will contact designated
laboratory staff by email to notify them of the forthcoming shipment(s) and request that they

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contact CSC if the shipments do not arrive intact as scheduled. Within 24 hours of scheduled
sample receipt, CSC will contact the laboratory to verify that the samples arrived in good
condition, and if problems are noted, will work with the laboratory and EPA to resolve the
problem as quickly as possible to minimize data integrity problems.

CSC also will communicate periodically with laboratory staff by telephone or email to monitor
the progress of analytical sample preparation, sample analysis, and data reporting. If technical
problems are encountered during sample preparation and analysis, CSC will identify a technical
expert within CSC to assist in resolving the problem, and work with EPA to identify and
implement a solution to the problem. If the laboratory fails to deliver data on time, or if the
laboratory notifies CSC of anticipated reporting delays, CSC will notify the EPA Project
Manager of the situation. To the extent possible, CSC will adjust schedules and shift resources
within CSC as necessary to minimize the impact of laboratory delays on EPA schedules. CSC
also will immediately notify the Project Manager of any laboratory delays that are anticipated to
impact EPA schedules.

Finally, the CSC Project Leader will monitor the progress of the data quality audits (data
reviews) and database development to ensure that each laboratory data submission is reviewed in
a timely manner. In the event that dedicated staff are not able to meet EPA schedules, CSC will
identify additional staff who are qualified and capable of reviewing the data in a timely manner.
If such resources cannot be identified, and if training new employees is not feasible, CSC will
meet with the EPA Project Manager to discuss an appropriate solution.

CI.2 Product Review

Product reviews for validated analytical data packages will be performed within CSC to verify
that the CSC data reviews are being performed consistently over time and across data reviewers,
that the review findings are technically correct, and that the reviews are being performed in
accordance with this QAPP. Product reviewers will be charged with evaluating the completeness
of the original CSC data review, the technical accuracy of the reviewer's findings, and the
technical accuracy of the analytical database developed to store results associated with the data
package. The CSC data reviewers will be responsible for identifying and assigning qualified
product reviewers and for selecting packages to be product reviewed. Qualified product
reviewers will include any staff members that have been trained in CSC data review procedures,
that are experienced in reviewing data similar to those being reviewed, and are familiar with the
requirements of this QAPP. To ensure the findings of each data review are documented in a
consistent and technically accurate manner, CSC staff will review 100% of the data qualifier
flags entered into the project database.

The EPA Project Manager and SHPD QA Coordinator will review the analytical QA report
developed by CSC, and the EPA Project Manager will approve the final analytical QA report.
The GLHHFTS data files prepared by CSC for statistical analysis of the data will be reviewed
internally by CSC staff and independently by the EPA Project Manager with support from Tetra
Tech before being forwarded to Tony Olsen at NHEERL-Corvallis, who will complete statistical
analysis of the GLHHFTS data and deliver the results to the EPA Project Manager.

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C1.3 Quality Systems Audit

A quality system audit (QSA) is used to verify, by examination and evaluations of objective
evidence, that applicable elements of the quality system are appropriate and have been
developed, documented, and effectively implemented in accordance and in conjunction with
specified requirements. The focus of these assessments is on the quality system processes - not
on evaluating the quality of specific products or judging the quality of environmental data or the
performance of personnel or programs.

The SHPD QA Coordinator may perform a QSA of the GLHHFTS mercury, PFC, fatty acid, and
PBDE analyses portion of the NCCA.

CI.4 Readiness Review

A readiness review of each laboratory's capability to produce precise and accurate results with
the methods specified in this study will be performed before the laboratory is allowed to analyze
field samples collected during the study. Because there are no formal EPA methods for most of
these analytes in tissue samples, there are no independent acceptance criteria by which to judge
laboratory performance. As part of the laboratory contracting process, CSC has requested
information from each laboratory regarding their demonstrated detection and quantitation limits
in tissue samples, as well as their in-house QC acceptance criteria for all QC operations
associated with the methods that each laboratory proposes to use for this project. CSC has also
requested information regarding each laboratory's capacity for the analyses (e.g., how many
samples per month), any relevant proficiency testing results in tissue samples, and any
accreditations relevant to tissue analyses. CSC will examine all information provided by each
laboratory to determine if the laboratory is capable of supporting the project.

Readiness reviews will be performed by CSC data reviewers. If problems are identified during
these reviews, CSC will work with the laboratory, to the extent possible, to resolve the problem.
If the problem cannot be resolved within the time frame required by EPA or within the scope of
the laboratory's existing contract, CSC will notify the EPA Project Manager immediately.
Records of these reviews and any corrective actions are maintained by CSC separate from the
analytical results for the field samples. CSC staff will document their findings and
recommendations concerning the readiness review as part of a written analytical QA report to
EPA.

CI.5 Technical Systems Audit

Each laboratory contract will require that the laboratory be prepared for and willing to undergo
an on-site, or technical systems, audit of its facilities, equipment, staff, and sample analysis,
training, record keeping, data validation, data management, and data reporting procedures. An
audit will be conducted only if the results of the readiness reviews, data quality audits, and
surveillance suggest serious or chronic laboratory problems that warrant on-site examinations
and discussion with laboratory personnel.

If such an audit is determined to be necessary, a standardized audit checklist may be used to
facilitate an audit walkthrough and document audit findings. Audit participants may include the
EPA Project Manager or the SHPD QA Coordinator (or a qualified EPA staff member
designated by the OST QA Officer) and a CSC staff member experienced in conducting

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laboratory audits. One audit team member will be responsible for leading the audit and
conducting a post-audit debriefing to convey significant findings to laboratory staff at the
conclusion of the audit. The other audit team member will be responsible for gathering pre-audit
documentation of problems that necessitated the audit, customizing the audit checklist as
necessary to ensure that those problems are addressed during the audit, documenting audit
findings on the audit checklist during the audit, and drafting a formal report of audit findings for
review by EPA.

CI.6 Data Quality Assessment

Upon completion of data verification and validation procedures (see Section Dl), CSC will
create an analytical database that contains all field sample results from the GLHHFTS (see
Section BIO).

At selected intervals and upon completion of the study, CSC's database development staff will
perform statistical analyses to verify the accuracy of the database. The statistical procedures will
be directed at evaluating the overall quality of the database against data quality objectives
established for the study, and in identifying trends in field and QC results obtained during the
study. CSC staff will document their findings and recommendations concerning this data quality
assessment as part of a written analytical QA report to EPA.

C2. Reports to Management

Following data verification and validation of all project data, CSC will apply standardized data
qualifier flags to the results in the project database that describe data quality limitations and
recommendations concerning data use. The data qualifier flags are based on those developed for
the National Lake Fish Tissue Survey and the complete list of qualifier flags and their
implications for data use will be summarized in a report to EPA at or near the end of the data
assessment process.

On request, CSC also will provide a report that describes the status of all current analysis and
data review activities, and periodic database status reports that provide up-to-date information
concerning database revisions that occurred since distribution of previous reports.

D. DATA VALIDATION AND USABILITY

This QAPP addresses the generation of data for mercury, PFCs, fatty acids, PBDEs, and PCBs in
fish tissue samples. Sections Dl, D2, and D3 of this QAPP apply to all of the analytical data
generation for the GLHHFTS.

Dl. Data Review, Verification, and Validation

Dl.l Data Review

All laboratory results and calculations will be reviewed by the Laboratory Manager prior to data
submission. Any errors identified during this peer review will be returned to the analyst for
correction prior to submission of the data package. Following correction of the errors, the
Laboratory Manager will verify that the final package is complete and compliant with the

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contract, and will sign each data submission to certify that the package was reviewed and
determined to be in compliance with the terms and conditions of the contract.

D1.2 Data Verification

The basic goal of data verification is to ensure that project participants know what data were
produced, if they are complete, if they are contractually compliant, and the extent to which they
meet the objectives of the study.

Every laboratory data package submitted under this study will be subjected to data verification
by qualified CSC staff who have been trained in procedures for verifying data and who are
familiar with the laboratory methods used to analyze the samples. This includes all of the
mercury, PFC, fatty acid, PBDE, and PCB analysis results generated under this QAPP. The
verification process is designed to identify and correct data deficiencies as early as possible in
order to maximize the amount of usable data generated during this study.

CSC staff will also conduct reviews of the QC sample results for homogenized fish tissue
samples prepared by Microbac. This will involve review of data for percent lipid measurements
that serve as a surrogate for homogeneity testing and review of the results from reagent water
rinsates, methanol rinsates, and hexane rinsates of the sample processing equipment, which are
analyzed by Microbac and the other contract laboratories. The CSC Project Leader will verify
the summary level results for these QC samples, determine if they meet the project objectives in
this QAPP, and report the verification findings to OST.

D1.3 Data Validation

Data validation is the process of evaluating the quality of the results relative to their intended
use. Data need not be "perfect" to be usable for a particular project, and the validation process is
designed to identify data quality issues uncovered during the verification process that may affect
the intended use. One goal of validation is to answer the "So what?" question with regard to any
data quality issues.

CSC data review chemists will validate all of the mercury, PFC, fatty acid, PBDE, and PCB
analysis results to be generated under this QAPP.

D2. Verification and Validation Methods

D2.1 Verification Methods

In the first stage of the data verification process, CSC data review chemists will perform a "Data
Completeness Check" in which all elements in each laboratory submission will be evaluated to
verify that results for all specified samples are provided, that data are reported in the correct
format, and that all relevant information, such as preparation and analysis logs, are included in
the data package. Corrective action procedures will be initiated if deficiencies are noted.

The second stage of the verification process will focus on an "Instrument Performance Check" in
which the CSC data review chemists will verify that calibrations, calibration verifications,
standards, and calibration blanks were analyzed at the appropriate frequency and met method or

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study performance specifications. If errors are noted at this stage, corrective action procedures
will be initiated immediately.

Stage three of the verification process will focus on a "Laboratory Performance Check" in which
CSC data review chemists will verify that the laboratory correctly performed the required
analytical procedures and was able to demonstrate a high level of precision and accuracy. This
stage includes evaluation of QC elements such as the laboratory control samples, method blanks,
matrix spike samples and/or reference samples, where applicable. Corrective action procedures
will be initiated with the laboratories to resolve any deficiencies identified.

In stage four of the verification process, the CSC data review chemist will perform a
"Method/Matrix Performance Check" to discern whether any QC failures are a result of
laboratory performance or difficulties with the method or sample matrix. Data evaluated in this
stage may include matrix spike, matrix spike duplicate, duplicate sample, reference sample,
labeled compound, and surrogate spike results. The CSC data review chemist also will verify
that proper sample dilutions were performed and that necessary sample cleanup steps were taken.
If problems are encountered, the CSC data review chemist will immediately implement
corrective actions.

D2.2 Validation Methods

CSC data review chemists will perform a data quality and usability assessment in which the
overall quality of data is evaluated against the performance criteria (see Section B5 for a
description of performance criteria). This assessment will strive to maximize use of data
gathered in this study based on performance criteria established for this study. This will be
accomplished by evaluating the overall quality of a particular data set rather than focusing on
individual QC failures. Results of this assessment will be documented in a project QA report
developed after all of the results have been evaluated, and before they are used in any final
decision making.

D3. Reconciliation with User Requirements

As data qualifier flags are applied to the project results to identify any results that did not meet
the method- or project-specific requirements, CSC data review chemists still may also apply
additional qualifiers that indicate an assessment of the impact of the problem. For example,
individual sample results are often qualified based on the presence of the analyte in a method
blank associated with samples prepared together (e.g., extracted or digested in the same batch).
While it is important to identify any result associated with the presence of the analyte in the
blank, the relative significance of the potential for sample contamination will be assessed using
commonly accepted "rules." In instances where the amount of the analyte found in the method
blank has very limited potential to affect the field sample result, an additional data qualifier will
be applied to that field sample result to indicate that the result was not affected by the observed
blank contamination. Similar assessments made for other data quality concerns may result in the
application of additional flags that reconcile the observed data quality concerns with the user
requirements and warn the end user of any limitations to the results (i.e., potential low or high
bias, qualitative uncertainty, etc.). All of the data qualifiers will be included in the database
along with summary level comments that explain the implication in relatively plain English.

-------
GLHHFTS Sample Preparation and Analysis QAPP

Revision 2
Date: April 18, 2012
Page 46 of 47

Where data quality concerns suggest that no valid result was produced for a given analyte, the
result for that analyte will be excluded from the database, and the comments will provide the
rationale for the exclusion. As noted earlier, the overall verification and validation process is
designed to maximize the amount of usable data for the project, and excluding results from the
final database is intended as a last resort.

References

AO AC. 1995. Official Method 991.39, Fatty Acids in Encapsulated Fish Oils and Fish Oil
Methyl and Ethyl Esters.

ALS. 2009. ALS Quality Manual, NAQM1,Version 03, June 1, 2009.

AXYS. 2012. Quality Assurance/Quality Control Policies and Procedures Manual: QDO-001,
Revision No. 20, February 22, 2012.

Peterson, S.A., D.V. Peck, J. Van Sickle, and R.M. Hughes. 2007. Mercury contamination in
frozen whole-fish homogenates is insensitive to holding time. Environmental contamination and
Toxicology 53(3):411-417.

Sathivel, S., W. Prinyawiwatkuk, C. Grimm, J. King, and S. Lloyd. 2002. FA Composition of
Crude Oil Recovered from Catfish Viscera. JAOCS 2002:79, 989-992.

Southwest Research Institute. 2010. Quality Assurance Plan for Analytical and Environmental
Services, Revision 9, August 2010.

TestAmerica. 2010. West Sacramento, Quality Assurance Manual, Revision 4. July 2010.

TestAmerica. 2011. Knoxville, Quality Assurance Manual, Revision 2.2. February 2011.

USEPA. 1974. Method 245.1, Mercury (Manual Cold Vapor Technique).

USEPA. 1994. Method 7470A, Mercury in Liquid Waste, Test Methods for Evaluating Solid
Waste, Physical/Chemical Methods (SW-946), September.

USEPA. 1995. Good Automated Laboratory Practices. U.S. Environmental Protection Agency,
Office of Administration and Resources Management, Washington, DC, October.

USEPA. 1998. Method 3051 A, Microwave Assisted Acid Digestion of Sediments, Sludges,
Soils, and Oils, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods (SW-
946), January.

USEPA. 2000. Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories,
Volume 1: Fish Sampling and Analysis, Third Edition. U.S. Environmental Protection Agency,
Office of Water, Office of Science and Technology, Washington, DC. EPA/823-B-00-007.

USEPA. 2001. EPA Requirements for Quality Assurance Project Plans. EPAQA/R-5. U.S.
Environmental Protection Agency, Office of Environmental Information, Washington, DC.
EPA/240/B-01/003.

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GLHHFTS Sample Preparation and Analysis QAPP

Revision 2
Date: April 18, 2012
Page 47 of 47

USEPA. 2010a. National Coastal Condition Assessment Quality Assurance Project Plan, U.S.
Environmental Protection Agency, Office of Water, Office of Wetlands, Oceans, and
Watersheds. Washington, DC. EPA/84l-R-09-004.

USEPA. 2010b. National Coastal Condition Assessment: Field Operations Manual, U.S.
Environmental Protection Agency, Office of Water and Office of Research and Development.
Washington, DC. EPA/84l-R-09-003.

USEPA. 2010c. Method 1614A, Brominated Diphenyl Ethers in Water, Soil, Sediment and
Tissue by HRGC/HRMS, March 2010.

USEPA. 2010d. Method 1668C, Chlorinated Biphenyl Congeners in Water, Soil, Sediment,
Biosolids, and Tissue by HRGC/HRMS, April 2010.

-------
Appendix A

List of Great Lakes Human Health Fish Tissue Study

Sampling Locations

-------
List of Great Lakes Human Health Fish Tissue Study Sampling Locations

Site ID Number

Lake

State

Latitude

Longitude

NCCAGL10-1001

Lake Super

or

Minnesota

47.14114

-91.45036

NCCAGL10-1002

Lake Super

or

Minnesota

47.55628

-90.86774

NCCAGL10-1003

Lake Super

or

Michigan

47.38864

-87.92476

NCCAGL10-1005

Lake Super

or

Wisconsin

46.77051

-91.62224

NCCAGL10-1006

Lake Super

or

Michigan

46.88719

-88.32472

NCCAGL10-1007

Lake Super

or

Michigan

47.28380

-88.51741

NCCAGL10-1008

Lake Super

or

Michigan

46.68530

-86.16970

NCCAGL10-1009

Lake Super

or

Wisconsin

46.95817

-90.84602

NCCAGL10-1010

Lake Super

or

Michigan

46.92451

-87.84378

NCCAGL10-1011

Lake Super

or

Minnesota

46.79049

-92.04478

NCCAGL10-1012

Lake Super

or

Michigan

46.79342

-85.23359

NCCAGL10-1013

Lake Super

or

Wisconsin

46.67280

-90.81696

NCCAGL10-1014

Lake Super

or

Michigan

47.04289

-88.98127

NCCAGL10-1015

Lake Super

or

Michigan

46.51201

-87.14860

NCCAGL10-1018

Lake Super

or

Michigan

46.48751

-86.74091

NCCAGL10-1019

Lake Super

or

Wisconsin

46.72925

-91.78798

NCCAGL10-1021

Lake Super

or

Michigan

46.72029

-85.76284

NCCAGL10-1022

Lake Super

or

Michigan

46.73077

-89.96820

NCCAGL10-1023

Lake Super

or

Michigan

46.84623

-89.57309

NCCAGL10-1024

Lake Super

or

Michigan

46.68694

-85.50666

NCCAGL10-1025

Lake Super

or

Michigan

46.58207

-90.40632

NCCAGL10-1026

Lake Super

or

Michigan

47.11605

-88.91043

NCCAGL10-1028

Lake Super

or

Wisconsin

46.81943

-91.33555

NCCAGL10-1029

Lake Super

or

Minnesota

47.71590

-90.47964

NCCAGL10-1030

Lake Super

or

Michigan

46.71267

-86.00895

NCCAGL10-1031

Lake Super

or

Minnesota

47.24485

-91.31001

NCCAGL10-1032

Lake Super

or

Michigan

46.87086

-88.23574

NCCAGL10-1033

Lake Super

or

Wisconsin

46.67815

-90.78103

NCCAGL10-1034

Lake Super

or

Michigan

46.90615

-89.34169

NCCAGL10-1035

Lake Super

or

Michigan

46.54149

-86.96612

NCCAGL10-1036

Lake Super

or

Michigan

46.91310

-89.26228

NCCAGL10-1037

Lake Super

or

Michigan

46.65890

-87.46141

NCCAGL10-1039

Lake Super

or

Minnesota

47.78138

-90.16989

NCCAGL10-1040

Lake Super

or

Michigan

47.23721

-88.63144

NCCAGL10-1041

Lake Super

or

Wisconsin

46.71701

-91.99021

NCCAGL10-1043

Lake Super

or

Michigan

46.79607

-84.98365

NCCAGL10-1044

Lake Super

or

Wisconsin

46.61594

-90.55170

NCCAGL10-1046

Lake Michigan

Michigan

45.93795

-84.99405

NCCAGL10-1048

Lake Michigan

Wisconsin

42.61470

-87.81058

NCCAGL10-1051

Lake Michigan

Michigan

45.76909

-86.74218

NCCAGL10-1054

Lake Michigan

Michigan

45.00071

-85.47705

NCCAGL10-1055

Lake Michigan

Michigan

44.94403

-85.84072

NCCAGL10-1056

Lake Michigan

Michigan

44.39603

-86.30882

NCCAGL10-QLM-10-01

Lake Michigan

Illinois

42.46726

-87.77979

NCCAGL10-1058

Lake Michigan

Michigan

45.88809

-86.25744

NCCAGL10-1059

Lake Michigan

Wisconsin

43.32892

-87.86407

NCCAGL10-QLM-10-05

Lake Michigan

Illinois

42.34262

-87.82165

NCCAGL10-1062

Lake Michigan

Michigan

42.94420

-86.24677

NCCAGL10-1064

Lake Michigan

Michigan

45.79750

-84.79227

NCCAGL10-1065

Lake Michigan

Michigan

43.10240

-86.27177

NCCAGL10-1067

Lake Michigan

Michigan

45.93451

-85.71997

NCCAGL10-1068

Lake Michigan

Michigan

45.09776

-85.69919

NCCAGL10-1069

Lake Michigan

Indiana

41.66361

-87.26672

List of GLHHFTS Sampling Locations

A-l

-------
List of Great Lakes Human Health Fish Tissue Study Sampling Locations

Site ID Number

Lake

State

Latitude

Longitude

NCCAGL10-1071

Lake Michigan

Wisconsin

43.71907

-87.65705

NCCAGL10-1072

Lake Michigan

Michigan

44.31255

-86.29955

NCCAGL10-1073

Lake Michigan

Michigan

46.05134

-85.24134

NCCAGL10-1074

Lake Michigan

Michigan

45.73460

-86.77751

NCCAGL10-1075

Lake Michigan

Wisconsin

44.69307

-87.76815

NCCAGL10-1077

Lake Michigan

Michigan

45.69042

-86.90180

NCCAGL10-1078

Lake Michigan

Michigan

45.93913

-85.91562

NCCAGL10-1081

Lake Michigan

Wisconsin

43.04134

-87.86151

NCCAGL10-1082

Lake Michigan

Wisconsin

44.01482

-87.62677

NCCAGL10-1083

Lake Michigan

Michigan

42.20083

-86.41114

NCCAGL10-1084

Lake Michigan

Michigan

45.73852

-86.46434

NCCAGL10-1085

Lake Michigan

Michigan

42.73211

-86.26326

NCCAGL10-QLM-10-20

Lake Michigan

Illinois

41.91638

-87.60207

NCCAGL10-1088

Lake Michigan

Indiana

41.70618

-87.50341

NCCAGL10-1090

Lake Michigan

Michigan

42.11036

-86.50359

NCCAGL10-1091

Lake Huron

Michigan

43.66117

-83.81375

NCCAGL10-1093

Lake Huron

Michigan

45.75036

-84.56395

NCCAGL10-1094

Lake Huron

Michigan

44.83935

-83.24250

NCCAGL10-1096

Lake Huron

Michigan

45.96307

-84.71430

NCCAGL10-1097

Lake Huron

Michigan

45.37810

-83.64797

NCCAGL10-1099

Lake Huron

Michigan

44.00505

-83.22758

NCCAGL10-1100

Lake Huron

Michigan

45.93965

-84.66939

NCCAGL10-1101

Lake Huron

Michigan

45.00660

-83.35906

NCCAGL10-1102

Lake Huron

Michigan

43.87991

-83.43664

NCCAGL10-1103

Lake Huron

Michigan

44.01350

-82.76739

NCCAGL10-1104

Lake Huron

Michigan

45.96071

-84.41915

NCCAGL10-1105

Lake Huron

Michigan

45.18651

-83.33389

NCCAGL10-1106

Lake Huron

Michigan

44.26273

-83.47572

NCCAGL10-1107

Lake Huron

Michigan

45.36534

-83.55898

NCCAGL10-1108

Lake Huron

Michigan

43.93339

-83.37510

NCCAGL10-1110

Lake Huron

Michigan

44.97528

-83.44224

NCCAGL10-1113

Lake Huron

Michigan

45.70090

-84.35747

NCCAGL10-1115

Lake Huron

Michigan

44.04340

-82.72642

NCCAGL10-1116

Lake Huron

Michigan

45.97315

-84.56772

NCCAGL10-1120

Lake Huron

Michigan

43.77893

-83.88343

NCCAGL10-1121

Lake Huron

Michigan

44.00634

-83.16876

NCCAGL10-1122

Lake Huron

Michigan

45.76363

-84.64443

NCCAGL10-1124

Lake Huron

Michigan

45.68168

-84.48027

NCCAGL10-1125

Lake Huron

Michigan

45.13464

-83.32074

NCCAGL10-1126

Lake Huron

Michigan

45.95788

-84.16044

NCCAGL10-1127

Lake Huron

Michigan

44.84472

-83.30373

NCCAGL10-1130

Lake Huron

Michigan

45.50311

-83.92721

NCCAGL10-1131

Lake Huron

Michigan

44.85847

-83.31801

NCCAGL10-1136

Lake Er

e

Ohio

41.74625

-83.37917

NCCAGL10-1137

Lake Er

e

Ohio

41.51048

-82.13912

NCCAGL10-1138

Lake Er

e

New York

42.73212

-78.97097

NCCAGL10-1139

Lake Er

e

New York

42.53829

-79.27534

NCCAGL10-1140

Lake Er

e

Michigan

41.85549

-83.37181

NCCAGL10-1141

Lake Er

e

Ohio

41.50063

-82.21454

NCCAGL10-1142

Lake Er

e

New York

42.68146

-79.08613

NCCAGL10-1144

Lake Er

e

Ohio

41.63394

-83.16825

NCCAGL10-1146

Lake Er

e

Pennsylvania

42.21606

-79.90829

NCCAGL10-1148

Lake Er

e

Michigan

41.97839

-83.22607

List of GLHHFTS Sampling Locations

A-2

-------
List of Great Lakes Human Health Fish Tissue Study Sampling Locations

Site ID Number

Lake

State

Latitude

Longitude

NCCAGL10-1149

Lake Er

e

Ohio

41.56669

-82.76520

NCCAGL10-1152

Lake Er

e

New York

42.75304

-78.92968

NCCAGL10-1155

Lake Er

e

New York

42.64527

-79.13889

NCCAGL10-1157

Lake Er

e

Ohio

41.71204

-83.24907

NCCAGL10-1161

Lake Er

e

Ohio

41.43843

-82.92615

NCCAGL10-1163

Lake Er

e

Ohio

41.73072

-83.44522

NCCAGL10-1165

Lake Er

e

Ohio

41.86898

-81.10047

NCCAGL10-1168

Lake Er

e

Michigan

41.92854

-83.23335

NCCAGL10-1169

Lake Er

e

Ohio

41.46351

-82.92912

NCCAGL10-1170

Lake Er

e

New York

42.35038

-79.60465

NCCAGL10-1173

Lake Er

e

Pennsylvania

42.06438

-80.38134

NCCAGL10-1174

Lake Er

e

Ohio

41.50192

-82.13009

NCCAGL10-1175

Lake Er

e

New York

42.83341

-78.89074

NCCAGL10-1176

Lake Er

e

Pennsylvania

42.21322

-80.05048

NCCAGL10-1178

Lake Er

e

Ohio

41.74465

-81.39602

NCCAGL10-1179

Lake Er

e

Ohio

41.51697

-82.17261

NCCAGL10-1181

Lake Ontario

New York

43.96827

-76.1154

NCCAGL10-1182

Lake Ontario

New York

43.91360

-76.18341

NCCAGL10-1183

Lake Ontario

New York

43.35820

-78.70273

NCCAGL10-1185

Lake Ontario

New York

43.50622

-76.48772

NCCAGL10-1188

Lake Ontario

New York

43.25480

-77.48873

NCCAGL10-1189

Lake Ontario

New York

43.58759

-76.25065

NCCAGL10-1190

Lake Ontario

New York

44.07588

-76.37700

NCCAGL10-1191

Lake Ontario

New York

43.38128

-78.08532

NCCAGL10-1192

Lake Ontario

New York

43.43145

-76.62718

NCCAGL10-1193

Lake Ontario

New York

43.80338

-76.25182

NCCAGL10-1195

Lake Ontario

New York

43.36138

-77.93097

NCCAGL10-1196

Lake Ontario

New York

43.31913

-76.87901

NCCAGL10-1200

Lake Ontario

New York

43.38012

-78.59547

NCCAGL10-1201

Lake Ontario

New York

43.29485

-77.35129

NCCAGL10-1202

Lake Ontario

New York

43.68424

-76.23963

NCCAGL10-1204

Lake Ontario

New York

43.36850

-76.69966

NCCAGL10-1205

Lake Ontario

New York

43.75382

-76.25459

NCCAGL10-1206

Lake Ontario

New York

44.00692

-76.28367

NCCAGL10-1207

Lake Ontario

New York

43.28966

-77.21855

NCCAGL10-1209

Lake Ontario

New York

43.95098

-76.24824

NCCAGL10-1210

Lake Ontario

New York

43.28736

-77.60575

NCCAGL10-1211

Lake Ontario

New York

43.51761

-76.29959

NCCAGL10-1213

Lake Ontario

New York

43.82965

-76.32422

NCCAGL10-1214

Lake Ontario

New York

44.06539

-76.41119

NCCAGL10-1216

Lake Ontario

New York

43.29208

-79.04195

NCCAGL10-1217

Lake Ontario

New York

43.34313

-77.80294

NCCAGL10-1218

Lake Ontario

New York

43.30384

-77.07563

NCCAGL10-1221

Lake Ontario

New York

43.38191

-78.36226

NCCAGL10-1222

Lake Ontario

New York

43.26149

-77.47764

NCCAGL10-1223

Lake Ontario

New York

43.34353

-76.76442

NCCAGL10-1224

Lake Ontario

New York

43.96971

-76.20600

NCCAGL10-1225

Lake Ontario

New York

43.29486

-76.90896

NCCAGL10-2005

Lake Superior

Minnesota

46.82415

-92.01783

NCCAGL10-2093

Lake Huron

Michigan

45.68080

-84.33828

NCCAGL10-2140

Lake Erie

Ohio

41.50642

-82.15552

List of GLHHFTS Sampling Locations

-------
Appendix B

GLHHFTS Fish Tissue Preparation, Homogenization,
and Distribution Procedures

Note: The discussion of the tissue preparation, homogenization, and

distribution procedures in this appendix represents the approach that was
implemented at the time that the samples were prepared. EPA
subsequently decided to use the sample aliquots prepared for the
pharmaceuticals and personal care products (PPCPs) for the analysis of
the PCBs. Because the procedures described in this appendix were
already complete when that decision was made, the text of this appendix
has not been modified other than to add a brief note below the table of
sample aliquots.

-------
National Coastal Condition Assessment
Great Lakes Human Health Fish Tissue Study
Tissue Preparation, Homogenization, and Distribution Procedures

I.	PURPOSE

This document describes the procedures that Microbac Laboratories will follow when preparing
fish tissue samples for EPA's National Coastal Condition Assessment (NCCA) Great Lakes
Human Health Fish Tissue Study (GLHHFTS) under contract to CSC. Adherence to these
procedures will ensure that fish tissue preparation activities are performed consistently across all
study samples and in a manner consistent with previous EPA fish tissue studies. The effort is
divided into four components:

•	A kickoff meeting and workshop involving all study participants, including Microbac
staff, EPA, CSC, and Tetra Tech

•	An initial demonstration of capabilities, also referred to as the QA study

•	Normal fish tissue processing and distribution procedures, including quality control steps

•	Analyses of rinsate samples and blanks for mercury and polybrominated diphenyl ethers
(PBDEs)

Each of these components is described in detail below.

II.	KICKOFF MEETING AND WORKSHOP

EPA held a kickoff meeting and workshop at Microbac on February 23, 2011. Staff from all
study participants, including Microbac, EPA, CSC, and Tetra Tech, met at Microbac to review
the overall GLHHFTS project goals, the roles of each participant, the fish sample preparation
procedures, and the communication strategies necessary to ensure successful completion of the
project. In conjunction with that meeting, CSC provided whole fish samples that will be used
during a hands-on workshop on the specific procedures for fish sample preparation. All
Microbac staff involved in the preparation of fish samples attended the kickoff meeting and
workshop.

III.	INITIAL DEMONSTRATION OF CAPABILITIES

A routine aspect of any procedure for sample preparation or analysis is an initial demonstration
of capabilities, or QA study. For the GLHHFTS project, Microbac will receive three whole large
fish provided by Tetra Tech. Each of these fish will be treated as a separate project sample and
will be prepared using the procedures detailed in Section IV (i.e., Steps 1 to 20). In between
each fish, Microbac will prepare the entire series of equipment rinsate samples and blanks
described in Section IV, Steps 22 to 23, but analyze only the rinsates and blanks for mercury and
PBDEs (Steps 24, 25, and Section V). Microbac will perform triplicate determinations of lipids
on each test sample, as described in Step 21. The results of the QA study will be reported to
CSC.

Note: Microbac will not be authorized to process actual project samples until CSC determines
that the QA study results meet the project objectives, including the adequacy of
Microbac's equipment cleaning and homogenization procedures.

GLHHFTS Fish Tissue Sample Preparation, Homogenization, and Distribution Procedures

B-l

-------
The sample aliquots prepared from these samples will be stored frozen at Microbac for possible
future use by EPA. Each of the samples prepared for the QA study will be billable under the
CSC subcontract at the cost for a normal project sample.

IV. NORMAL FISH TISSUE PROCESSING AND DISTRIBUTION PROCEDURES

The procedures for processing and distributing GLHHFTS composite fish tissue samples are
described below. The process description is organized into the following eight components,
including the quality control (QC) procedures:

A.	Sample Handling

B.	Filleting and Homogenization Procedures

C.	Aliquoting and Distribution Procedures

D.	Equipment Cleaning between Composite Samples

E.	Lipid Determination on Every Homogenized Composite Sample

F.	Quality Control (QC) Procedures

G.	Reporting Requirements

H.	Shipping Samples

The individual steps in the overall process are presented as a series of numbered steps across the
eight components listed above.

Note: Microbac may not process any fish tissue samples until directed by CSC to proceed. No
normal samples may be processed until after the kickoff meeting and workshop and until
CSC reviews the results of the initial demonstration of capabilities (QA study) described
in Section III above.

Composite Sample Classifications

For the purposes of the GLHHTS, EPA has classified each valid sample as a "routine" composite
sample, or a "non-routine" composite sample, based on the following definitions:

Routine sample - A routine composite sample consists of five individual adult fish of a
single species that meet EPA's length requirements (i.e., length of the smallest specimen
in the composite is at least 75% of the length of the largest individual). Fillets from both
sides of all five fish will be removed (total of 10 fillets) and homogenized to prepare one
composite fillet sample.

Non-routine sample - A non-routine sample is any sample that does not meet the
definition of a routine sample, including those that do not meet the 75% rule and those
with fewer or greater than five fish. When non-routine samples are sent to the prep lab,
EPA and CSC will provide instructions for processing the non-routine samples. These
instructions may include discarding some of the fish in the composite sample based on
size before proceeding with filleting and homogenizing. In cases when fewer or more
than five fish were collected, instructions may include processing some or all of those
fish in the composite sample.

Each of the five fish in the routine samples must be filleted before homogenization. For non-
routine composites, only the designated specimens (identified by specimen number) will be
filleted and homogenized. For both types of samples, the specimens to be included in each

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composite must be scaled (i.e., scales removed) and both fillets from each specimen prepared as
skin-on fillets (belly-flap included) to form the fillet composites.

Note: The classifications described above do not include samples that were collected from an
incorrect sampling location, were an unnecessary duplicate sample, or contained an
inappropriate fish species. EPA does not plan on using these "invalid" samples for the
GLHHFTS, so it is imperative that Microbac not process any sample without specific
instructions from CSC. Therefore, samples will be retained in frozen storage and
processed only upon receipt of CSC-issued instructions. If the status of any composite
sample in the instructions is not clear, contact CSC and wait for clarification.

IV.A. Sample Handling

The whole fish collected for the GLHHFTS are being stored frozen (e.g., -20 ฐC) at CSC's
sample repository in stackable trays. Samples to be processed must be retrieved from the
freezer, with their associated paperwork, and allowed to partially thaw before they can be
filleted.

1.	CSC will send sample processing instructions to the laboratory The instructions consist of an
Excel spreadsheet file that details the site and sample identifiers for fish that EPA has
determined are routine valid five-fish composites, or non-routine composites to be prepared.
At a minimum, the Excel file will list the following fields for each individual fish specimen
in a given composite sample:

Site ID (NCCAGL10XXXX = National Coastal Condition Assessment Great Lakes,
2010, and a 4-digit site number, except for three samples from Illinois that use a different
format)

Lake

Date (of collection)

Sample ID (526###.X, where X usually ranges from 1 to 5 specimens in the composite,

but can range up to 10)

Species (scientific and common names)

Total Length (mm) of each specimen

Composite Type (predator or bottom dweller)

Composite Classification (Routine, Non-Routine, or Invalid)

Deviation (e.g., why it is not routine or not valid)

Instructions (sample-specific details about which fish to process)

CSC will provide the storage tray number for each sample, as part of the instructions, or
separately.

2.	When retrieving samples from the freezer, the sample custodian must:

-	Verify that all associated hardcopy paperwork stored with the samples is complete,
legible, and accurate.

-	Compare the information on the label on each individual fish specimen to the processing
instructions and notify CSC of any discrepancies between the sample labels and the Excel
file of instructions. Problems involving sample paperwork, sample integrity, or custody
inconsistencies for all fish tissue samples should be reported to CSC in writing (e.g., by

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email) as soon as possible following sample retrieval and inspection. Do not proceed
with sample processing until discrepancies are resolved.

Note: The hardcopy paperwork generated by the field samplers and stored with the
samples does not contain all of the information in the Excel instruction files.
Therefore, lack of information on hardcopy field paperwork regarding the
composite type, composite classification, or deviation is not a discrepancy that
must be reported.

IV.B. Filleting and Homogenization Procedures

3.	Prior to preparing each composite sample, thoroughly clean utensils and cutting boards using
the following series of procedures:

-	Wash with a detergent solution (phosphate- and scent-free) and warm tap water

-	Rinse three times with warm tap water

-	Rinse three times with DI water

-	Rinse with acetone

-	Rinse three times with DI water

-	Rinse with (not soak in) 5% nitric acid

-	Rinse three times with DI water

To control contamination, separate sets of utensils and cutting boards must be used for
scaling fish and for filleting fish.

4.	Put on powder-free nitrile gloves before unpacking individual fish specimens for filleting and
tissue homogenization. As samples are unpacked and unwrapped, inspect each fish carefully
to verify that it has not been damaged during collection or shipment. If damage (e.g., tearing
the skin or puncturing the gut) is observed, document it in the laboratory project logsheet and
notify CSC.

5.	Weigh each fish to the nearest gram (wet weight) prior to any sample processing. Enter
weight information for each individual fish into a laboratory project logsheet. Individual
specimen weights eventually will be transferred to spreadsheets for submission to CSC.

6.	Rinse each fish with deionized water as a precautionary measure to treat for possible
contamination from sample handling in the field. Use HDPE wash bottles for rinsing fish
and for cleaning homogenization equipment and utensils. Do NOT use Teflonฎ wash bottles
for these procedures, because PFCs are among the target analytes for this study.

7.	Put on new powder-free nitrile gloves during the scaling and filleting processes. Fish with
scales must be scaled (and any adhering slime should be removed) prior to filleting. Scale
each fish by laying it flat on a clean glass cutting board and scraping from the tail to the head
using a stainless steel scaler or the blade-edge of a clean stainless steel knife. Rinse the
cutting board and scaler or knife with deionized water between fish to minimize the risk of
cross-contamination. Filleting can proceed after all scales have been removed from the skin
and a separate clean cutting board and scaler or fillet knife are prepared or available.

8.	Place each fish on a clean glass cutting board in preparation for the filleting process. Note
that filleting should be conducted under the supervision of an experienced fisheries biologist,

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if possible. Ideally, fish should be filleted while ice crystals are still present in the muscle
tissue. Fish should be thawed only to the point where it becomes possible to make an
incision into the flesh. Remove both fillets (lateral muscle tissue with skin attached) from
each fish specimen using clean, high-quality stainless steel knives. Include the belly flap
(ventral muscle and skin) with each fillet. Care must be taken to avoid contaminating fillet
tissues with material released from inadvertent puncture of internal organs. In the event that
an internal organ is punctured, rinse the fillet with deionized water immediately after filleting
and make a note on the laboratory project logsheet that a puncture has occurred. Bones still
present in the tissue after filleting should be carefully removed.

9.	Samples should be homogenized partially frozen for ease of grinding. Composite fillets
using the "batch" method, in which all of the fillets from the individual specimens that
comprise the sample are homogenized together, regardless of each individual specimen's
proportion to one another (as opposed to the "individual" method, in which equal weights of
tissue from each specimen are added together).

10.	Process each sample using a size-appropriate homogenization apparatus (e.g., automatic
grinder or high-speed blender). Entire fillets (with skin and belly flap) from both sides of
each fish must be homogenized, and the entire homogenized volume of all fish fillets from
the composite will be used to prepare the composite. Mix the tissues thoroughly until they
are completely homogenized as evidenced by a final composite sample that consists of a fine
paste of uniform color and texture. Chunks of skin or tissue will hinder extraction and
digestion and, therefore, are NOT acceptable. Grinding of tissue may be easier when tissues
are partially frozen. Chilling the grinder briefly with a few chips of dry ice may also keep
the tissue from sticking to the equipment.

11.	Grind the sample a second time, using the same grinding equipment. This second grinding
should proceed more quickly. The grinding equipment does not need to be cleaned between
the first and second grinding of the sample. The final sample must consist of a fine paste of
uniform color and texture. If there are obvious differences in color or texture, grind the
entire sample a third time.

12.	Measure the collective weight of the homogenized tissue from each composite to the nearest
gram (wet weight) after processing and record the total homogenized tissue weight of each
composite on a laboratory project log sheet. The collective weight of the homogenized tissue
from each sample will be transferred to spreadsheets for submission to CSC. At least 300 g
of homogenized tissue will be needed to fill all of the containers in Table 1 below. If a
sample does not yield at least 300 g of homogenized tissue, contact CSC via email
immediately and await instructions. As appropriate, place any less-than-300-g
homogenized samples in the freezer while waiting for instructions, which are likely to
involve preparing smaller archive aliquots.

13.	After the final (second or third) grinding, clean the grinding equipment and all other
sample preparation equipment using the procedures described in Step 19.

14.	Once in every batch of 20 samples, verify the continued absence of equipment contamination
and uniformity of homogenization using the procedures described in Steps 22 to 27.

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IV.C. Aliquoting and Distribution Procedures

15. The prep lab will prepare one bulk homogenate tissue aliquot per fish composite sample and
use it to fill the pre-cleaned sample containers specified for each type of sample listed in
Table 1, following the procedures described in Step 16. All containers will be provided by
the prep lab. Documentation of their cleanliness provided by the vendor (i.e., certificates of
analysis) must be retained by the prep lab and provided to CSC on request.

Table 1. NCCA Initial Tissue Sample Aliquot Requirements

Analysis

Target Mass

Container Type

Destination

Mercury

5-10g

50-mL HDPE straight-sided jar with foil-lined lid, or
conical HDPE tube with snap top

TestAmerica-
Knoxville

Fatty acids

25 - 30 g

125-mL straight-sided amber or clear glass jar with
PTFE-lined lid

Southwest
Research
Institute

PFCs

20 - 25 g

50-mL HDPE straight-sided jar with foil-lined lid, or
conical HDPE tube with snap top. PTFE lid liners not
allowed.

Test America-

West
Sacramento

PBDEs

20 - 25 g

125-mL straight-sided amber or clear glass jar with
PTFE-lined lid

ALS - Canada

PPCPs

20 - 25 g

125-mL straight-sided amber or clear glass jar with
PTFE-lined lid

To be
determined

Lipids

10 - 15 g

Lab's choice, as this aliquot will be used in-house to
determine the lipid content of the sample

In-house

Archive 1

100 g

250-mL straight-sided amber or clear glass jar with foil-
lined lid

CSC Sample
Repository

Archive 2

100 g

250-mL straight-sided amber or clear glass jar with foil-
lined lid

CSC Sample
Repository

Total*

300-330g



Additional
Archives

All available
tissue is used to
fill jars up to
80% full

500-mL straight-sided amber or clear glass jars with foil-
lined lids

CSC Sample
Repository

* In the event that insufficient fish tissue mass exists to prepare the required number of aliquots, contact CSC
for instructions, per Step 12.

Note: After the fish preparation procedures were completed, EPA decided to utilize the samples
prepared for the PPCPs for the analysis of PCBs. Therefore, this document has not been
modified beyond the addition of this note. The procedures were implemented as
described, but the PPCP aliquot was sent to the laboratory performing the PCB analyses.

16. Prepare the sample aliquots for mercury, fatty acids, PFCs, PBDEs, and PPCPs. Weigh
an appropriate clean sample container (Table 1) to the nearest 0.5 g and record the weight.
Transfer sufficient aliquots of ground sample to the container to achieve the minimum mass
for that container in Table 1, weigh the container again, record the weight, and determine the
weight of the aliquot to the nearest 0.5 g by difference. The prep lab must use foil-lined
lids for jars containing the tissue aliquots for PFC analysis and the archived tissue
samples, as specified in Table 1.

Note: The archive sample jars are not filled until after sufficient volume for lipids

determination has been collected, as described in Step 18. For the sample used for

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homogeneity testing, the archive jars are not filled until triple the lipid mass is
collected (see Step 26).

When filling jars, leave sufficient space at the top of each jar to allow for expansion of the
tissue as it freezes. In no case shouldjars be filled beyond 80% capacity, as this may result
in breakage on freezing. Wipe off the outside of the jars to remove any tissue residue or
moisture. Fill out a label for each container using a waterproof marker. Include the
following information (at a minimum) on each label:

-	site identification number (e.g., NCCAGL10-1023),

-	sample identification number (e.g., 560208),

-	analysis type (e.g., mercury, PFCs, PBDEs, etc.),

-	aliquot weight (to the nearest 0.5 gram),

-	preparation batch ID, and

-	and preparation date (e.g., mm/dd/yyyy)

Affix the label to the container with clear wide tape. Place each container inside one heavy-
weight food-grade self-sealing plastic freezer bag to avoid sample loss due to breakage.
Freeze the tissue aliquots at -20 ฐC, and maintain samples in the freezer until directed by CSC
to ship them to the analytical laboratories. (CSC will not issue such instructions until
equipment rinsate and homogeneity tests described in Steps 22 to 27 have been completed,
reported, evaluated, and determined to be acceptable.)

17.	After filling all of the containers for the aliquots for mercury, fatty acids, PFCs, PBDEs, and
PPCPs, remove 5 to 10 g of homogenized tissue to be used by the prep lab to determine the
lipid content of each sample. Place this aliquot in a clean glass or plastic container of
suitable size and label it with the site ID and sample number. Transfer the lipid aliquot to the
appropriate staff performing the lipid determinations described in Steps 21, 26, and 27.

18.	The archive sample jars are not filled until after sufficient volume for lipids determination
has been collected. Once the aliquots for mercury, fatty acids, PFCs, PBDEs, PPCPs, and
lipids have been collected, the remaining tissue mass is used to create at least two archive
samples. Transfer 100 g of tissue to each of the first two archive containers, and seal and
label the containers as described in Step 16 for the other aliquots.

Note: Step 12 requires that the laboratory contact CSC whenever a sample does not yield at
least 300 g of tissue. CSC will provide direction to the laboratory regarding samples
yielding less than 300 g of tissue that must be followed at this point in the procedure.

If additional tissue mass remains after filling the first two archive sample containers, use it to
fill successive 500-mL glass jars with foil-lined lids until all of the excess tissue has been
archived. Label the containers as described in Step 16 for the other aliquots. No tissue will
be discarded without written EPA approval.

IV.D. Equipment Cleaning between Composite Samples

19.	All of the homogenization equipment must be thoroughly cleaned between each composite
sample. Once all of the fillets from the individual specimens in a given composite sample
have been homogenized, disassemble the homogenization equipment (i.e., blender, grinder,

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or other device) and thoroughly clean all surfaces and parts that contact the sample.
Similarly, clean all knives, cutting boards, and other utensils used. At a minimum:

-	Wash with a detergent solution (phosphate- and scent-free) and warm tap water

-	Rinse three times with warm tap water

-	Rinse three times with deionized (DI) water

-	Rinse with acetone

-	Rinse three times with DI water

-	Rinse with (not soak in) 5% nitric acid

-	Rinse three times with DI water

-	Allow the components to air dry

20.	Reassemble the homogenization equipment and proceed with homogenization of the next
sample in the batch (e.g., begin with Step 4 above).

IV.E. Lipid Determination on Every Homogenized Composite Sample

The procedures for determining the lipid content of every fillet composite are described in Step
21 below. (Additional lipid determinations are required for one sample in every preparation
batch, as described in Steps 26 and 27.)

21.	Use the 5 to 10 g of homogenized tissue collected in Step 17 to determine the lipid content of
the sample. Extract the aliquot using the method of the laboratory's choice. (This method
was previously pre-approved by CSC and EPA.) Determine the lipid content of that aliquot
and record it in units of percent (i.e., grams of lipid per gram of tissue x 100), and provide the
results to CSC by email, as described in Section IV.G. These results may be used by the
laboratories conducting the other analyses to lipid-normalize their results.

IV.F. Quality Control (QC) Procedures

The project-specific QC procedures include preparation and testing of equipment rinsate samples
and homogeneity testing, using lipids as a surrogate. The QC procedures are performed in two
distinct phases: (1) as part of an initial demonstration of capabilities after the kickoff meeting
and workshop with EPA, and (2) during normal operations.

Initial demonstration of capabilities: After the kickoff meeting and workshop, Microbac
staff will prepare three test fish samples provided by Tetra Tech. Each test sample will
consist of a single large fish which will be processed separately. Each of these test samples
will be carried through the entire sample preparation and aliquoting procedures separately.
The resulting sample aliquots will not be distributed to other laboratories at this time, but
stored frozen. In between processing each individual fish sample, Microbac staff will clean
all of the sample preparation equipment as described in Step 19 above. After each cleaning,
Microbac staff will prepare the entire series of equipment rinsates and solvent blanks
described in Step 22 below.

Microbac also will collect three lipid aliquots from each sample prepared during the initial
demonstration and use them for triplicate determinations of lipids, as described in Step 26
below.

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The results of the analyses of the rinsates and the homogeneity testing (three sets each) will
be submitted to CSC for review. Microbac may not begin Great Lakes Human Health Fish
Tissue Study sample preparation until CSC and EPA determine that Microbac has
successfully demonstrated proficiency in meeting QC requirements for equipment cleaning
and tissue homogenization.

Normal Operations: During normal sample preparation efforts, Microbac will prepare one
set of rinsate samples and will conduct one set of triplicate lipid determinations per batch of
20 composite fish samples, as described in Steps 22 to 27, below. The batch-specific rinsate
and homogeneity results will be reviewed by CSC and EPA. Microbac may continue to
process up to one additional batch of 20 samples (based on sample preparation instructions
provided by CSC) during that review process. However, Microbac may not continue beyond
that next batch of samples until receiving notification from CSC that review of the prior
batch rinsate and homogeneity test results is complete and the results were deemed
satisfactory.

Thus, continued sample processing is dependent on both the quality of Microbac's efforts
and on the timeliness of their delivery of QC results.

Rinsate Sample Production

22 Prior to reassembling the homogenization equipment (Step 20) between each of the samples
processed during the initial demonstration of capabilities, and once per batch during normal
operations, prepare five rinsate samples, as follows:

- Prepare two hexane rinsate samples by pouring two 100-mL portions of pesticide-grade
hexane over all parts of homogenization equipment, including the cutting boards and
knives, and collect each 100-mL portion in a separate clean glass container. Place two
additional 100-mL aliquots of clean hexane in similar glass containers for use as solvent
blanks. Allow the solvent to evaporate from the equipment. One set of these rinsates and
blanks will be analyzed by Microbac for PBDEs and the other set will be archived for
analysis of fatty acids by a laboratory to be determined later. CSC will provide Microbac
with the name and shipping information for the fatty acids laboratory as soon as it is
available. Label and store the fatty acids rinsate and blank as described in Step 23.

Label, store, and analyze the PBDE rinsate and blank as described in Step 24.

Once the hexane has evaporated off the equipment, prepare the methanol rinsate in a
similar fashion, using 100-mL of pesticide-grade methanol. Collect that rinsate in a clean
glass container and place a second aliquot of methanol in a separate similar clean glass
container for use as a solvent blank. This rinsate and blank will be analyzed for PPCPs
by a laboratory to be determined later. CSC will provide Microbac with the PPCP
laboratory name and shipping information as soon as it is available. Label and store these
PPCP rinsate and blanks as described in Step 23.

Once the methanol has evaporated, prepare the first DI water rinsate using 250 mL of
DI water. Collect the DI water rinsate in a clean glass or HDPE container. Place a
second aliquot of DI water in a separate similar clean container for use as a blank.

Acidify these two samples to pH < 2 with nitric acid. Analyze these rinsate and blank
samples for mercury as described in Step 25.

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- Prepare the second DI water rinsate using an additional 250 mL of DI water. Collect
this rinsate in a clean glass container with a non-PTFE lid liner. Place a second aliquot
of DI water in a separate similar clean glass container for use as a blank. This rinsate and
blank will be analyzed for PFCs by a laboratory to be determined later, thus the non-
PTFE lid liners are essential. CSC will provide Microbac with the PFC laboratory name
and shipping information as soon as it is available. Label and store these PFC rinsates
and blanks as described in Step 23.

Note: In order to minimize the number of project samples that might be affected by
cross contamination, collect the normal rinsate samples on the first day that
samples in a batch of 20 are processed. Ideally, the laboratory will vary the point
at which the rinsates are collected on that first day over the course of the project
(e.g., between the 1st and 2nd samples for one batch, the 2nd and 3rd samples for
another batch, etc.).

23.	Label each container as either "rinsate - [insert name of solvent]" or "blank - [insert name of
solvent]," and include the date it was prepared (mm/dd/yyyy), the analysis type (Hg, PBDE,
PPCP, PFC, or fatty acids), and the preparation batch identifier. Store the rinsates and blanks
cold (<6 ฐC).

Rinsate Analyses

24.	As part of the initial demonstration of capabilities, Microbac will analyze three sets of
hexane rinsate and blank samples for PBDEs using a GC/ECD procedure (e.g., one set
prepared after each tissue sample prepared during the initial demonstration process). During
normal operations, Microbac will analyze one set of the hexane rinsate and blank samples per
batch for PBDEs using a GC/ECD procedure. That procedure will require concentration of
the hexane to a final volume of 1 mL, and analysis on two dissimilar GC columns, in order to
identify the PBDE congeners of interest by retention time. Requirements for the PBDE
analyses are provided in Section V.

25.	As part of the initial demonstration of capabilities, Microbac will analyze three sets of DI
water rinsate and blank samples for mercury using a cold-vapor atomic absorption procedure
(e.g., one set prepared after each tissue sample prepared during the initial demonstration
process). During normal operations, Microbac will analyze one set of the DI water rinsate
and blank samples for mercury using a cold-vapor atomic absorption procedure.

Requirements for the mercury analyses are provided in Section V.

Corrective Actions for Rinsates

CSC will evaluate the rinsate results based on the mass of each analyte detected, and
assuming that all of the apparent contamination could be transferred to a nominal 300-g mass
of homogenized tissue. Results for mercury or any PBDEs above the anticipated reporting
limits for these analytes in tissue samples may be cause for corrective actions by Microbac.
Such corrective actions may include revisions to Microbac's equipment cleaning procedures,
followed by a successful demonstration of the revised cleaning procedures through
preparation and analysis of additional rinsate samples.

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Lipid Determination to Confirm Homogeneity

26.	For each of the samples processed during the initial demonstration of capabilities, and for
one sample in every batch of 20 composite samples prepared during normal operations,
Microbac will conduct triplicate analyses of the lipid content of samples to confirm that the
samples are homogeneous.

As with the collection of rinsate samples, the homogeneity testing must be performed on the
first day on which samples in a batch of 20 are processed. However, the sample chosen for
homogeneity testing must be one that yields enough tissue mass to support the added mass
needed for triplicate lipid aliquots (15 to 30 g). Therefore, unless otherwise directed by CSC
for a particular batch of samples, Microbac will select one sample processed on the first day
of every batch that will provide well over 300 g of total tissue mass.

From that sample, remove three 5- to 10-g aliquots of tissue before filling the archive sample
containers. Place these three aliquots in clean glass or plastic containers of suitable size and
label each with the site ID, sample number, and an aliquot identifier of the lab's choice.
Transfer the lipid aliquot to the appropriate staff performing the lipid determination.

27.	From the lipid results, calculate the mean lipid content (in percent), the standard deviation
(SD), and the relative standard deviation (RSD) using the formulae below, or the
corresponding functions in Excel.

y,(% lipids),
mean % lipids = —	

SD =

Z(% lipids -meanlipids)2

RSD = ^-

mean

If the RSD of the triplicate results is less than or equal to 15%, then the homogenization
effort is judged to be sufficient for all samples in that preparation batch. For this sample
analyzed in triplicate, the mean lipid content will be the value reported for that sample,
following the requirements described in Step 28.

Corrective Actions for Homogeneity

If the RSD is greater than 15%, then corrective action is required for all samples in that
preparation batch. Corrective actions will be determined by CSC in direct consultation with
the laboratory and EPA, but the default corrective action consists of regrinding all of the
aliquots from each composite sample in the affected batch until the RSD criterion is met.

This may entail retrieving all sample aliquots (see Table 1) from the freezer, allowing them
to partially thaw, and homogenizing them again, beginning at Step 10. In these instances, all
of the equipment cleaning procedures will be repeated between each composite sample, new
lipids results will be determined for each composite, and a new homogenization QC

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determination (triplicate lipids on one sample per batch) will be performed. New sample
containers will be required for any rehomogenized samples.

IV.G. Reporting Requirements

28.	Microbac will prepare a weekly progress report to document the status of fish preparation
activities and forward the report electronically to CSC. The format of the weekly progress
report will be as an Excel spreadsheet. For each composite processed during that period,
include at least the following information in the report:

-	site identification number (e.g., NCCAGL10-1023),

-	sample identification number (e.g., 560208),

-	specimen numbers of the fish homogenized for the composite,

-	species name (both scientific and common names)

-	lengths and weights of individual specimens that were filleted and homogenized

-	total composite sample (i.e., homogenate) weight (to the nearest gram),

-	analysis type (e.g., mercury, PFCs, archive sample, etc.),

-	aliquot weight (to the nearest 0.5 gram),

-	preparation batch ID,

-	preparation date (e.g., mm/dd/yyyy),

-	QC sample identifiers associated with the batch of composite samples,

-	lipid results for each composite sample, and

-	airbill numbers for all sample shipments that week (these may include samples prepared
during previous weeks).

(Much of the sample-specific information above will be provided to Microbac electronically
in the sample processing instructions from CSC.)

The weekly report will be due by COB Monday, or as agreed to in writing by CSC after
consultation with the laboratory, and will document sample preparation progress for the
previous week.

In addition, the laboratory must report the results of the rinsate analyses for mercury and
PBDEs and the triplicate lipid results associated with the sample batch. Those results must
be reported to CSC as soon after the analyses as practical to facilitate CSC's timely review
and to minimize delays in receiving instructions to process future batches.

Note: As specified in the QC section of this document, Microbac may not continue beyond
the next batch of samples until receiving notification from CSC that review of the
prior batch rinsate and homogeneity test results is complete and the results were
deemed satisfactory.

IV.H. Shipping Samples

29.	No samples may be shipped until CSC and EPA have reviewed the sample homogeneity
testing and rinsate results. CSC will notify Microbac by email when specific samples may
be shipped, and to whom.

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When shipping batches of pre-frozen tissue aliquots, keep the individual containers bagged in
the food-grade plastic freezer bags. Place these bags in a cooler with adequate space for the
tissue containers, packing materials, and dry ice. (CSC will provide suitable coolers from
existing stocks.) Secure each of the tissue containers with packing materials (e.g., bubble
wrap or foam) before adding the dry ice. Place a modest layer of newspaper on top of the
containers before adding the dry ice, as this can prevent cracking the lids. A single "section"
of the local newspaper will usually suffice.

The amount of dry ice required for shipping will depend on the number of tissue samples in
the cooler and the time of year. It should be an adequate supply to keep the tissue samples
frozen for 48 hours (i.e., a minimum of 25 pounds of dry ice per cooler for up to 10 pounds
of tissue samples).

Record the samples contained in the cooler on a chain-of-custody form provided by CSC and
place the form in a plastic bag taped to the inside lid of the cooler. Secure the outside of the
cooler with sealing tape, address it to the sample recipient identified by CSC, and attach a dry
ice (dangerous goods) label. Ship the cooler via an overnight express carrier on a date that
will allow delivery of the cooler to the analytical lab on a normal business day (e.g., no
Saturday deliveries and no deliveries on U.S. Federal holidays without express
permission from CSC). Provide the air bill number for each shipment to CSC via email on
the day that the shipment occurs. CSC will provide the prep lab with a third-party FedEx
account to which each shipment will be billed.

V. ANALYSES OF RINSATES AND BLANKS FOR MERCURY AND PBDEs

This section describes the analyses of rinsate samples and blanks generated during the composite
fish sample preparation process. The results of those analyses are important in demonstrating
that the sample preparation laboratory's equipment cleaning procedures are effective at
preventing cross-contamination between fish tissue samples.

V.A. EQUIPMENT AND MATERIALS:

-	Mercury analyzer suitable for aqueous samples. Cold-vapor atomic absorption (CVAA)
instruments compatible with EPA Method 254 are acceptable. Must be capable of achieving
an MDL of approximately 1 |ig/L.

-	Gas chromatograph with an electron-capture detector (GC/ECD) and two dissimilar GC
columns suitable for analysis of organohalide compounds such as PBDEs.

-	Solvent concentration equipment suitable for reducing hexane rinsates to final volumes of 1
to 10 mL.

-	A PBDE standard solution containing at least the following PBDE congeners: 47, 66, 99,
100, 138, 153, 154, and 183, to be used to establish retention times and perform at least a 3-
point calibration of the GC/ECD.

-	Assorted glassware, syringes, etc.

V.B. RINSATE AND BLANK ANALYSES

During the initial demonstration of capabilities, the laboratory will prepare three sets of rinsate
samples, i.e., one set after each fish prepared as part of that demonstration. Each set of rinsate
samples will include:

GLHHFTS Fish Tissue Sample Preparation, Homogenization, and Distribution Procedures

B-13

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-	Two de-ionized water (DI) rinsate samples and two DI water blanks per sample for
analysis of mercury and for analysis of PFCs.

-	Two hexane rinsate samples and two hexane blanks per sample for analysis of PBDEs
and fatty acids.

-	One methanol rinsate sample and a methanol blank per sample for analysis of PPCPs.

During normal sample preparation efforts, the laboratory will prepare rinsates at a frequency of
one set for each batch of 20 fish tissue samples prepared. Up to 8 sets of rinsates are anticipated.

The laboratory will digest and analyze the mercury rinsates and blanks by CVAA. The
laboratory will concentrate the PBDE rinsates and blanks to a final volume of 1 mL and analyze
the concentrated samples by GC/ECD. For each analysis, the laboratory will determine the mass
of each analyte (mercury or PBDE congener) in the total volume of each rinsate or blank sample,
rather than the concentration of each analyte.

The laboratory must be able to achieve an MDL of approximately 1 |ig/L. Mercury results will
be reported down to the mass equivalent to the mass at the method detection limit (MDL) for
aqueous samples.

Because the PBDE rinsates are not aqueous samples that are extracted, a traditional MDL study
for aqueous samples does not apply. Therefore, the laboratory must perform an instrument
detection limit (IDL) study before beginning any rinsate analyses. The IDL study will consist of
analyzing 7 low-level standards containing the PBDEs of interest, determining the standard
deviation of results for each PBDE across all 7 analyses, and multiplying the standard deviation
times 3.143, the Student's t-value for 7 replicates. The laboratory must achieve an IDL on the
order of 0.5 ng/mL, for a 1-mL final volume.

PBDE congeners will be identified based on retention time windows on both GC columns (see
EPA Methods 608 or 8000C for examples of procedures for determining retention time
windows).

PBDE results in the rinsates and blanks will be reported down to the mass equivalent to the IDL.
Any PBDEs detected on one GC column must be confirmed by the analysis of the sample on a
second GC column with a different stationary phase. Alternatively, GC/ECD analyses may be
conducted on an instrument set up for simultaneous dual-column analyses.

The rinsates for PPCPs, PFCs, and fatty acids will not be analyzed by the laboratory, but will be
held by Microbac.

V.C. QUALITY CONTROL

The quality control (QC) procedures required for the rinsate analyses include:

-	MDL or IDL studies, as described above

-	Instrument calibration (see Method 245.1 and Method 608 for procedures and acceptance
criteria)

-	Instrument blanks for both mercury and PBDE analyses

-	Calibration verification (once per analysis batch) for both mercury and PBDE analyses

-	Laboratory control sample (LCS) once per analysis batch, for mercury only.

GLHHFTS Fish Tissue Sample Preparation, Homogenization, and Distribution Procedures

B-14

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The MDL and DDL results will be reviewed by CSC as soon as they become available, and the
laboratory will not be authorized to prepare additional fish tissue samples until that review is
complete and the results are acceptable.

The matrix for the mercury rinsates is reagent water, which should not adversely affect method
performance. Therefore, matrix spike samples are not required for mercury.

Because the PBDE rinsates do not involve extraction of an environmental matrix, matrix spike
samples are not applicable. Likewise, laboratory control samples are not applicable to PBDEs.

The instrument blanks for mercury and PBDEs take the place of a traditional method blank that
would be extracted along with environmental samples.

V.D. DELIVERABLES

Summary data from the rinsate analyses are to be delivered to CSC in an Excel file. That file
must contain the following information, at a minimum:

•	Batch ID - to be established by the laboratory, but a simple approach would be to number or
letter each sample batch (e.g., A to H, or 1 to 8). The batch ID for the rinsates prepared
during the initial demonstration results may be reported as "QA study"

•	Sample ID - as described in the instructions for preparing the rinsates

•	Lab sample ID - unique internal identifier used by the laboratory, in any

•	Prep date - Date (MM/DD/YYYY) on which the rinsate or solvent blank was prepared

•	Analysis type - either "Mercury" or "PBDE"

•	Analysis date - Date (MM/DD/YYYY) on which the rinsate or solvent blank was analyzed

•	Analyte name - PBDE congeners may be abbreviated as PBDE-047, etc.

•	Mass of analyte found - in micrograms for mercury, and either micrograms or nanograms for
the PBDEs, provided that the reporting units for PBDEs are consistent throughout the effort

•	Retention time on GC Column 1 - PBDEs only

•	Retention time on GC Column 2 - PBDEs only

•	Lab qualifiers - as needed to describe any analytical concerns. A complete list of the
qualifiers and their meanings must be included with each data submission (e.g., in a separate
tab on the Excel file)

•	Reporting limit (i.e., MDL) for each analyte - in the same mass units used for the results.

•	Instrument calibration data - Submit as a separate tab in the Excel file. Must include results
for the initial calibrations for mercury and PBDEs, as well as any relevant calibration
verifications associated with the analyses. Include calibration equations (e.g., regressions)
and metrics (e.g., correlation coefficient or calibration factor), and for PBDEs, the retention
times of the analytes in each calibration standard on both GC columns.

Raw data supporting each analysis (e.g., chromatograms or instrument printouts) must be
retained by the laboratory and made available to CSC when requested. If requested, raw data
may be submitted in hard copy, or as a PDF file.

GLHHFTS Fish Tissue Sample Preparation, Homogenization, and Distribution Procedures

B-15

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Appendix C

Method Detection Limits and Minimum Levels for
GLHHFTS Target Analytes

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Method Detection Limits (MDLs) and Minimum Levels (MLs)
for GLHHFTS Target Analytes



Metals





(based on a 0.5-g sample)

Analyte

MDLa (|jg/kg)

ML(|jg/kg)

Mercury

Underdevelopment

Under development

PFCs

(based on a 5-g sample)

Analyte

MDLa (|jg/kg)

ML" (|jg/kg)

PFBS

0.10

1

PFBA

0.07

1

PFDA

0.06

1

PFDoA

0.12

1

PFHpA

0.09

1

PFHxS

0.12

1

PFHxA

0.07

1

PFNA

0.08

1

PFOS

0.08

1

PFOS

0.13

1

PFOA

0.10

1

PFPeA

0.13

1

PFUnA

0.11

1

Fatty Acids

(based on a 1-g sample)

Analyte

MDLa (weight/weight %)

MLb (weight/weight %)

ALA

0.0027

0.0053

ETE

0.0027

0.0053

DHA

0.0027

0.0053

EPA

0.0026

0.0053

DPA

0.0026

0.0053

a All MDLs are based on the EPA procedure described at 40 CFR 136, Appendix B.
b ML values are based on the concentration of the lowest calibration standard.

MDLs and MLs for GLHHFTS Target Analytes

C-l

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PBDEs

(based on a 20-g sample)

Analyte

MDLa (ng/kg)

ML" (ng/kg)

BDE-7

0.46

5

BDE-10

0.33

5

BDE-8/BDE-11

0.47

5

BDE-12/BDE-13

0.58

5

BDE-15

0.12

5

BDE-17/BDE-25

0.48

10

BDE-28/BDE-33

0.50

2.5

BDE-30

0.57

5

BDE-32

0.25

5

BDE-35

0.15

5

BDE-37

0.21

5

BDE-47

0.96

5

BDE-49

0.29

5

BDE-51

0.28

5

BDE-66

0.32

2.5

BDE-71

0.24

5

BDE-75

0.30

5

BDE-77

0.27

5

BDE-79

0.20

5

BDE-85

0.55

5

BDE-99

2.99

5

BDE-100

0.66

2.5

BDE-105

0.42

5

BDE-116

0.75

5

BDE-118

0.48

5

BDE-119/BDE-120

0.61

5

BDE-126

0.26

5

BDE-128

0.87

5

BDE-138/BDE-166

1.33

5

BDE-140

0.73

5

BDE-153

0.39

2.5

BDE-154

0.69

2.5

BDE-155

0.47

5

BDE-156

0.86

5

BDE-181

0.57

5

BDE-183

0.49

5

BDE-184

0.89

5

BDE-190

0.98

2.5

BDE-191

0.84

5

BDE-196

0.9

10

BDE-197

0.38

10

BDE-203

0.32

10

BDE-206

1.36

25

BDE-207

1.56

25

BDE-208

1.76

25

BDE-209

6.06

100

HBB

0.81

5

PBEB

0.26

5

a All MDLs are based on the EPA procedure described at 40 CFR 136, Appendix B.
b ML values are based on the concentration of the lowest calibration standard.

MDLs and MLs for GLHHFTS Target Analytes

C-2

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PCBs

(based on a 10-g sample



Analyte

MDLa (ng/kg)

ML" (ng/kg

PCB-1

0.13

0.5

PCB-2

0.14

0.5

PCB-3

0.20

0.5

PCB-4

0.27

1

PCB-5

0.24

1

PCB-6

0.22

0.5

PCB-7

0.35

1

PCB-8

0.29

1

PCB-9

0.19

0.5

PCB-10

0.29

1

PCB-11

0.24

1

PCB-12/PCB-13

0.36

1

PCB-14

0.31

1

PCB-15

0.14

0.5

PCB-16

0.45

2

PCB-17

0.29

1

PCB-18/PCB-30

0.66

2

PCB-19

0.27

1

PCB-20/PCB-28

0.45

1

PCB-21/33

0.57

2

PCB-22

0.30

1

PCB-23

0.31

1

PCB-24

0.34

1

PCB-25

0.27

1

PCB-26/PCB-29

0.52

2

PCB-27

0.32

1

PCB-31

0.20

0.5

PCB-32

0.30

1

PCB-34

0.27

1

PCB-35

0.31

1

PCB-36

0.40

1

PCB-37

0.33

1

PCB-38

0.30

1

PCB-39

0.32

1

PCB-40/PCB-41 /PCB-71

1.33

5

PCB-42

0.44

1

PCB-43

0.52

2

PCB-44/PCB-47/PCB-65

1.23

5

PCB-45/PCB-51

0.87

2

PCB-46

0.33

1

PCB-48

0.43

1

PCB-49/PCB-69

0.85

2

PCB-50/PCB-53

0.72

2

PCB-52

0.50

2

PCB-54

0.15

0.5

PCB-55

0.42

1

PCB-56

0.54

2

PCB-57

0.37

1

PCB-58

0.26

1

PCB-59/PCB-62/PCB-75

1.23

5

MDLs and MLs for GLHHFTS Target Analytes

C-3

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PCBs

(based on a 10-g sample



Analyte

MDLa (ng/kg)

ML" (ng/kg

PCB-60

0.51

2

PCB-61/PCB-70/PCB-74/PCB-76

1.81

5

PCB-63

0.43

1

PCB-64

0.36

1

PCB-66

0.43

1

PCB-67

0.26

1

PCB-68

0.32

1

PCB-72

0.36

1

PCB-73

0.32

1

PCB-77

0.17

0.5

PCB-78

0.39

1

PCB-79

0.33

1

PCB-80

0.44

1

PCB-81

0.20

0.5

PCB-82

0.20

0.5

PCB-83/PCB-99

0.66

2

PCB-84

0.50

2

PCB-85/PCB-116/PCB-117

0.68

2

PCB-86/PCB-87/PCB-97/PCB-109/PCB-119/PCB-125

1.41

5

PCB-88/PCB-91

0.91

2

PCB-89

0.50

2

PCB-90/PCB-101/PCB-113

0.43

1

PCB-92

0.51

2

PCB-93/PCB-95/PCB-98/PCB-100/PCB-102

2.19

5

PCB-94

0.51

2

PCB-96

0.32

1

PCB-103

0.37

1

PCB-104

0.10

0.2

PCB-105

0.17

0.5

PCB-106

0.21

0.5

PCB-107

0.77

2

PCB-108/PCB-124

0.57

2

PCB-110/PCB-115

0.52

2

PCB-111

0.21

0.5

PCB-112

0.32

1

PCB-114

0.21

0.5

PCB-118

0.28

1

PCB-120

0.32

1

PCB-121

0.53

2

PCB-122

0.42

1

PCB-123

0.34

1

PCB-126

0.17

0.5

PCB-127

0.28

1

PCB-128/PCB-166

0.50

2

PCB-129/PCB-138/PCB-160/PCB-163

1.54

5

PCB-130

0.28

1

PCB-131

0.41

1

PCB-132

0.29

1

PCB-133

0.32

1

PCB-134/PCB-143

0.59

2

MDLs and MLs for GLHHFTS Target Analytes

C-4

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PCBs

(based on a 10-g sample



Analyte

MDLa (ng/kg)

ML" (ng/kg

PCB-135/PCB-151/PCB-154

1.59

5

PCB-136

0.32

1

PCB-137

0.26

1

PCB-139/PCB-140

1.28

5

PCB-141

0.35

1

PCB-142

0.26

1

PCB-144

0.42

1

PCB-145

0.42

1

PCB-146

0.35

1

PCB-147/PCB-149

0.75

2

PCB-148

0.34

1

PCB-150

0.26

1

PCB-152

0.37

1

PCB-153/PCB-168

0.92

2

PCB-155

0.12

0.5

PCB-156/PCB-157

0.32

1

PCB-158

0.27

1

PCB-159

0.36

1

PCB-161

0.25

1

PCB-162

0.32

1

PCB-164

0.30

1

PCB-165

0.26

1

PCB-167

0.22

0.5

PCB-169

0.15

0.5

PCB-170

0.73

2

PCB-171/PCB-173

0.32

1

PCB-172

0.26

1

PCB-174

0.58

2

PCB-175

0.11

0.5

PCB-176

0.27

1

PCB-177

0.41

1

PCB-178

0.25

1

PCB-179

0.28

1

PCB-180/PCB-193

1.53

5

PCB-181

0.34

1

PCB-182

0.26

1

PCB-183/PCB-185

0.43

1

PCB-184

0.15

0.5

PCB-186

0.25

1

PCB-187

0.43

1

PCB-188

0.12

0.5

PCB-189

0.28

1

PCB-190

0.18

0.5

PCB-191

0.26

1

PCB-192

0.19

0.5

PCB-194

0.38

1

PCB-195

0.26

1

PCB-196

0.35

1

PCB-197/PCB-200

1.34

5

PCB-198/PCB-199

0.45

2

MDLs and MLs for GLHHFTS Target Analytes

C-5

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PCBs

(based on a 10-g sample



Analyte

MDLa (ng/kg)

ML" (ng/kg

PCB-201

0.39

1

PCB-202

0.41

1

PCB-203

0.22

0.5

PCB-204

0.17

0.5

PCB-205

0.17

0.5

PCB-206

0.31

1

PCB-207

0.21

0.5

PCB-208

0.38

1

PCB-209

0.31

1

a All MDLs are based on the EPA procedure described at 40 CFR 136, Appendix B.

b The minimum level (ML) for the NLFTS was calculated by EPA based on a tissue MDL study. The ML
values shown above are equivalent to 10 times the standard deviation from that MDL study, rounded to the
nearest multiple of 1, 2, or 5, consistent with the approach used in both the NLFTS and during the
development of EPA Method 1668C.

MDLs and MLs for GLHHFTS Target Analytes

C-6

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