Method 1668A Interlaboratory Validation Study Report

		

March 2010



  SEQ CHAPTER \h \r 1 U.S. Environmental Protection Agency

Office of Water

Office of Science and Technology

Engineering and Analysis Division (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC  20460

EPA-820-R-10-004  SEQ CHAPTER \h \r 1 



Executive Summary

	This report presents the results of EPA’s interlaboratory validation
of EPA Method 1668A   Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, and Tissue by HRGC/HRMS.  This study was conducted in
2003-2004 to validate the performance of EPA Method 1668A in municipal
wastewater, fish tissue, and biosolids matrices.  

EPA used the results of the study to evaluate and revise Method 1668A
quality control (QC) acceptance criteria for initial precision and
recovery, ongoing precision and recovery, and labeled compound recovery
from real world samples.  These interlaboratory criteria (Table 5-1)
replace the single-laboratory criteria, and are published in Table 6 of
the revised version of this PCB-congener method, EPA Method 1668B.

Acknowledgments

This report was written under contract for EPA by CSC Systems &
Solutions, LLC, and Interface, Inc.  EPA acknowledges the volunteer
laboratories that participated in the study and, in particular, those
laboratories that took the extra effort to comment on EPA Method 1668A
and to provide suggestions for improvements.

Disclaimer

Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

Contacts

Richard Reding, Ph.D., Chief

Engineering & Analytical Support Branch

Engineering and Analysis Division (4303T) 

Office of Science and Technology, Office of Water

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue NW

Washington, DC 20460

http://www.epa.gov/waterscience

ostcwamethods@epa.gov



Table of Contents

  TOC \o "1-3" \u  Executive Summary	ii

Acknowledgments	ii

Disclaimer			ii

Contacts			ii

Section 1	Introduction And Background	  PAGEREF _Toc260302750 \h  1 

1.1	Introduction	  PAGEREF _Toc260302751 \h  1 

1.2	Background	  PAGEREF _Toc260302752 \h  2 

1.3	2003 Revision to Method 1668A	  PAGEREF _Toc260302753 \h  2 

Section 2	Study Management, Objectives, Design, and Implementation	 
PAGEREF _Toc260302754 \h  3 

2.1	Study Management	  PAGEREF _Toc260302755 \h  3 

2.2	Study Objectives and Design	  PAGEREF _Toc260302756 \h  3 

2.3	Laboratory Selection	  PAGEREF _Toc260302757 \h  3 

2.4	Sample Selection	  PAGEREF _Toc260302758 \h  4 

2.5	Preparation of Study Samples	  PAGEREF _Toc260302759 \h  5 

2.5.1	Biosolids and Tissues	  PAGEREF _Toc260302760 \h  5 

2.5.2	Wastewater	  PAGEREF _Toc260302761 \h  6 

2.5.3	Labeling and Shipping	  PAGEREF _Toc260302762 \h  7 

2.6	Sample Analysis and Data Reporting	  PAGEREF _Toc260302763 \h  7 

2.7	Deviations from the Method or Study Design	  PAGEREF _Toc260302764
\h  8 

2.7.1	Instrument Calibration	  PAGEREF _Toc260302765 \h  8 

2.7.3	Tissue	  PAGEREF _Toc260302766 \h  10 

2.7.4	Wastewater	  PAGEREF _Toc260302767 \h  10 

Section 3	Data Review and Validation	  PAGEREF _Toc260302768 \h  11 

Section 4	Results and Discussion	  PAGEREF _Toc260302769 \h  13 

4.1	Background and Homogeneity Testing	  PAGEREF _Toc260302770 \h  13 

4.1.1	Wastewater Sample Homogeneity	  PAGEREF _Toc260302771 \h  13 

4.1.2	Tissue and Biosolids Sample Homogeneity	  PAGEREF _Toc260302772 \h
 13 

4.2	Congener Concentrations in Samples	  PAGEREF _Toc260302773 \h  13 

4.3	Congener Concentrations in Blanks	  PAGEREF _Toc260302774 \h  15 

4.4	Wastewater Sample Recovery and Precision	  PAGEREF _Toc260302775 \h 
15 

4.5	Variability as a Function of Concentration	  PAGEREF _Toc260302776
\h  17 

4.5.1	Variability vs. Concentration for Wastewater	  PAGEREF
_Toc260302777 \h  17 

4.5.2	Variability vs. Concentration for Tissue	  PAGEREF _Toc260302778
\h  19 

4.5.3	Variability vs. Concentration for Biosolids	  PAGEREF
_Toc260302779 \h  20 

4.6	Labeled Compound Recovery and Precision	  PAGEREF _Toc260302780 \h 
21 

Section 5	Revision of Quality Control Acceptance Criteria	  PAGEREF
_Toc260302781 \h  23 

5.1	Calibration	  PAGEREF _Toc260302782 \h  23 

5.2	Calibration Verification	  PAGEREF _Toc260302783 \h  23 

5.3	Initial Precision and Recovery	  PAGEREF _Toc260302784 \h  23 

5.4	Ongoing Precision and Recovery	  PAGEREF _Toc260302785 \h  24 

5.5	Labeled Compound Recovery from Samples, Blanks, and IPR and OPR
Standards	  PAGEREF _Toc260302786 \h  24 

Section 6	Conclusions	  PAGEREF _Toc260302787 \h  27 

 

Appendix A	Statistical Procedures Used to Develop QC Acceptance Criteria
A-1

Appendix B	Study Plan for Interlaboratory Validation of EPA Method 1668A
for Determination of Chlorinated Biphenyl Congeners in Water, Biosolids,
and Tissue by HRGC/HRMS	B-1

List of Tables

Table 2-1.	Laboratories Participating in the Method 1668A Validation
Study

Table 2-2.	Congener Concentrations in Wastewater Samples by Level of
Chlorination

Table 2-3.	Sample Pairs for Distribution to 14 Participant Laboratories

Table 3-1.	Summary of Data Received from Participant Laboratories

Table 4-1.	Congener Concentrations in Study Samples by Level of
Chlorination

Table 4-2.	Congener Concentrations in Blanks by Level of Chlorination

Table 4-3.	Wastewater Sample Recovery and Precision by Level of
Chlorination

Table 4-4.	Labeled Compound Recovery and Precision by Level of
Chlorination

Table 5-1.	Revised QC Acceptance Criteria for IPR, OPR, and Labeled
Compounds in Samples

List of Figures

Figure 4-1.	Mean Recovery vs. Spike Concentration, PCB Congeners in
Wastewater

Figure 4-2.	Concentration Standard Deviation vs. Spike Concentration,
PCB Congeners in Wastewater

Figure 4-3.	Relative Standard Deviation vs. Spike Concentration, PCB
Congeners in Wastewater

Figure 4-4.	Mean vs. Standard Deviation of Measured Tissue Results

Figure 4-5.	Mean vs. Relative Standard Deviation of Measured Tissue
Results

Figure 4-6.	Mean vs. Standard Deviation of Measured Biosolids Results

Figure 4-7.	Mean vs. Relative Standard Deviation of Measured Biosolids
Results

Section 1

Introduction and Background

1.1	Introduction

	This report describes the interlaboratory validation study of EPA
Method 1668A that EPA conducted in 2003 - 2004 on municipal wastewater,
biosolids and fish tissue matrices.  The study was conducted according
to the Study Plan for Interlaboratory Validation of EPA Method 1668A for
Determination of Chlorinated Biphenyl Congeners in Water, Biosolids, and
Tissue by HRGC/HRMS, November 2003, which is an appendix to this report.
 A draft of this report was peer reviewed, and the following changes
were made as a result of this review:

Rounded all numbers to 3 significant figures maximum.

Expanded discussion of how QC acceptance criteria were generated

Moved the definition of “Youden pair” from Section 2.5.1 to its
first use in this paragraph. 

Table 4-4:  Truncated numbers at the decimal point in the “# pairs”
column.

Section 2.4 was expanded to give greater detail about the nature of the
fish and biosolids samples.

Section 2.5.2 was expanded to give greater detail about how the
wastewater sample was prepared.

A paragraph was inserted into Section 2.7.2 stating that the
participating labs were required to determine the solids content of the
biosolids sample and report the result in units of dry weight.

A result in Table 4-1 was corrected.  

Footnote 1 to Table 4-1 was revised to make clear that results for
biosolids are in units of dry weight and results for fish are in units
of wet weight.

Footnote 2 to Table 4-1 was revised to state that the mean, median, and
maximum concentrations at each LOC are based on any detected congeners
in that LOC and when coelution of two or more congeners occurred, the
combined value of those co-eluted congeners was used.

The same Footnote 2 was applied to Table 4-2 because it is called out in
the header row.  The existing “2” applied to the cell with the
number of sand/oil blanks was changed to a “3” in that cell and the
existing Footnote 2 was renumbered as Footnote 3.

Section 4.3 was expanded to clarify that blanks were to be analyzed in
the same way as samples.

The header to column 3 in Table 4-2 was changed from “# labs” to
“# blanks” to indicate the number of blanks analyzed in the study.

Footnote 1 to Table 4-2 was expanded to indicate that results for the
sand/oil blank were to be reported as wet weight.

Section 4.4 was expanded to explain that, even though the native
congeners weren't recovered within the range expected, the labeled
congeners were, thus indicating that the native congeners most likely
were lost in transit.

Table 4-3 was expanded to include recovery and precision for the labeled
compounds by level of chlorination

Section 4.6 and Table 4-4 were expanded to present results for the 27
individual labeled rather than by level of chlorination. 

A fifth footnote was called out in the original Table 5-1, for labeled
congeners 156L and 157L, but did not appear below the table.  The
missing Footnote 5 was added in this version.

 

EPA used the results of the study to revise Method 1668A and publish, in
2008, Method 1668B.  Quality control (QC) acceptance criteria for
initial precision and recovery, ongoing precision and recovery, and
labeled compound recovery from real world samples are in Table 5-1 of
this report.  These interlaboratory criteria replace the
single-laboratory criteria, and are published in Table 6 of EPA Method
1668B. 

1.2	Background

	Method 1668A is for determination of chlorinated biphenyl congeners
(PCBs) in water, soil, sediment, biosolids, and tissue by high
resolution (capillary column) gas chromatography combined with high
resolution mass spectrometry (HRGC/HRMS).  These 209 PCB-congeners are
the individual chemicals that comprise a class of pollutants known as
Aroclors.  Since publication in 1999, Method 1668A has been used to
measure PCBs in biosolids in EPA’s 2001 National Sewage Sludge Survey,
and fish tissue in EPA’s four-year National Study of Chemical Residues
in Lake Fish Tissue.  Additional background on the nature and
determination of PCBs and on the history of development, validation, and
peer-review of EPA Method 1668A is in the study plan.

1.3	2003 Revision to Method 1668A

	Minor revisions to Method 1668A were made in August 2003 for use in
this interlab study.  The changes corrected technical and typographical
errors and reflected practice of the method by laboratories based on
comments received.  

Section 2

Study Management, Objectives, Design, and Implementation

2.1	Study Management

	This study was designed and managed by the Engineering and Analytical
Support Branch (EASB, formerly the Statistics and Analytical Support
Branch) of the Engineering and Analysis Division in the Office of
Science and Technology within EPA's Office of Water.  Day-to-day
coordination of study activities was performed by the
contractor-operated Sample Control Center (SCC).

	Preliminary results of this study were presented at the 2004 National
Environmental Monitoring Conference in Washington, DC, July 20, 2004. 
Since that presentation, the results have been further evaluated and
presented in this report.  Therefore, this report supersedes any
material previously presented or published.

2.2	Study Objectives and Design

	Objectives of this study were to 1) characterize the performance of
Method 1668A in multiple laboratories and matrices, and 2) evaluate and,
if appropriate, revise the QC acceptance criteria in the method.

	EASB designed the study in accordance with guidelines published by EPA
and ASTM International (ASTM).,  These guidelines recommend a minimum of
six complete data sets for evaluation of a method.  To allow for some
loss of data due to error, lost samples, outlier removal, or other
unforeseen causes, EPA included 14 participant laboratories in the
study.  The study design is detailed in an appendix to this report.  

2.3	Laboratory Selection

	EPA used volunteer laboratories for participation in the study.  Each
interested laboratory was asked to demonstrate that it had recent
experience in using HRGC/HRMS to determine chlorinated pollutants in
environmental samples and confirm that it would determine all 209
congeners using an SPB-Octyl column, as described in Method 1668A.  The
intent was to ensure that study participants already possessed the
facilities, equipment, and trained staff necessary to implement the
method.  

	Fourteen (14) volunteer laboratories were selected to participate in
this study. The laboratories were notified of their selection at least
two weeks before the study began, so that they would have time to review
the method and study-specific instructions.  Of the 14 laboratories
selected, 11 were commercial laboratories and 3 were EPA Regional
laboratories.  Laboratories were not required to validate the method in
all three matrices; as a result, the number of participant laboratories
varied, depending on the matrix tested.  As discussed in section 3 of
this report, because of scheduling problems 3 of these 14 volunteer labs
did not submit data.

	To offset costs to the laboratories, EPA provided each laboratory with
a set of analytical standards necessary to identify and measure the 209
PCB congeners targeted by Method 1668A.  EPA also provided the
laboratories sets of standard solutions containing native and carbon-13
labeled compounds necessary to calibrate their instruments and to
conduct all analyses.  The packaged sets of standards were purchased
from Cambridge Isotope Laboratories (CIL) and AccuStandard, Inc. 
Laboratories were provided with detailed instructions for combining and
diluting standards to preclude injudicious use of standards.  The
instructions were based on procedures given in Method 1668A.

	In addition to the 14 volunteer participant laboratories, a sample
processing laboratory was contracted to perform all activities necessary
to ensure that the participant laboratories received homogenized,
spiked, and aliquoted samples.  Homogenization of bulk sample volume was
necessary to prepare replicate samples for analysis by participant
laboratories.  The participant and sample processing laboratories are
listed in Table 2-1.

Table 2-1.	Laboratories Participating in the Method 1668A Validation
Study

Alta Analytical Laboratory Inc.

1104 Windfield Way

El Dorado Hills, CA 95762

Phone:  916-933-1640	Battelle-Columbus Laboratories

505 King Avenue

Columbus, OH 43201

Phone:  614-424-7884	Philip Analytical Services Corporation

5555 North Service Road

Burlington, ON L7L 5H5 CANADA

Phone:  800-668-0639

EPA Region 7

300 Minnesota Ave.

Kansas City, KS 66101

Phone:  913-551-5120	Columbia Analytical Services

10655 Richmond Avenue, Suite 130A

Houston, TX 77042

Phone:  713-266-1599	Enviro-Test Laboratories

9936-67th Avenue

Edmonton, AB T6E OP5 CANADA

Phone:  780-413-6481

AXYS Analytical Services, Ltd.

2045 Mills Road West

Sidney, BC V8L 3S8 Canada

Phone:  250-655-5800	Severn Trent Laboratories – Knoxville

5815 Middlebrook Pike

Knoxville, TN 37921

Phone:  865-291-3000	EPA Region 3

701 Mapes Road

Fort Meade, MD 20755-5350

Phone:  410-305-2606

Pace Analytical Services 

1700 West Albany

Broken Arrow, OK 74012

Phone:  918-251-2858	Paradigm Analytical Laboratories, Inc.

5500 Business Drive

Wilmington, NC 28405

Phone:  910-350-1903	Severn Trent Laboratories – Sacramento

880 Riverside Parkway

West Sacramento, CA 95605

Phone:  916-374-4433

Pacific Analytical, Inc.

6056 Corte del Cedro

Carlsbad, CA 92009

Phone:  760-496-2200	Data Analysis Technologies, Inc.

7715 Corporate Blvd.

Plain City, OH 43064

Phone:  800-733-8644	EPA Region 4

980 College Station Rd.

Athens, GA 30605-2720

 

Note:	The primary purpose of this study was to evaluate the performance
of Method 1668A.  While results obtained by individual laboratories were
used relative to this purpose, no attempt was made to assess performance
of individual laboratories.  No endorsement of these laboratories is
implied, nor should any be inferred.  To preserve confidentiality,
laboratories that volunteered for this study, including three that did
not submit lab data, were assigned numbers randomly from 1 to 14.  The
lab identities and that of the sample processing laboratory are not
revealed in the data or lists in this report.

2.4	Sample Selection

	To minimize burden on volunteer laboratories, the study was designed so
that no more than two samples of each matrix type would be analyzed,
with each sample containing varying concentrations of the target PCB
congeners.  EPA provided existing (archived) fish tissue and biosolids
samples to the sample processing laboratory to prepare study samples
representing these matrices.  No archived sample volume was available
for wastewater, therefore, the sample processing laboratory prepared the
wastewater samples.  In preparing study samples, EPA’s objective was
to ensure that the congeners present in each sample matrix would span
the anticipated measurement range of Method 1668A, from the upper end of
the calibration range down to “not detected.”

	Tissue and biosolids samples were generated from excess samples
collected during EPA’s 1999-2000 National Lake Fish Tissue Study
(NLFTS) and EPA’s 2001 National Sewage Sludge Survey, respectively. 
These samples had been stored in freezers at an EPA sample repository. 
All of the tissue samples used in the validation study were from
bottom-dwelling fish species and were originally prepared as whole-fish
composite samples.  The samples for the NLFTS were prepared as finely
ground tissue by a single laboratory.  Excess sample beyond that shipped
to the laboratories during the NLFTS was archived in 500-mL jars and
stored frozen.  The NLFTS also collected samples of predator species,
from which fillets were taken and composited for analysis.  The
bottom-dweller samples provided more tissue than the predator samples,
and thus a greater excess that was available for other purposes such as
a Method 1668A validation study.  The tissue samples used for the
validation study were prepared from the 500-mL archive jars. 

	The original biosolids samples used to prepare the study samples were
collected as solid sewage sludges, as opposed to the pourable liquid
sewage sludges that may be produced at some wastewater treatment
facilities.  Each laboratory that analyzed the biosolids samples
determined the percent solid contents.  The reported values were in the
range of 30 to 40% for the two samples, amounts that are typical for
many biosolids produced in the U.S.

	So that a sufficient amount of each sample was available to support the
study, EPA identified several samples of each matrix type that could be
combined to produce large volumes of Youden pairs with the desired
congener distribution.  (Youden pairs are defined as two samples of the
same matrix containing similar, but not exact, concentrations of the
analytes of interest.)  Once these stored samples were identified, they
were forwarded on ice to the sample processing laboratory.  Although
PCBs are stable and do not require preservation, ice was used to prevent
decomposition of fish tissue and to retard gas production in the
biosolids.  For wastewater, amounts of effluent grab samples were
collected from a publicly owned treatment works (POTW) that were
sufficient to provide enough samples for all of the participant
laboratories, and excess sample in case of breakage, spillage, or other
problems.  Bulk wastewater was collected in polyethylene carboys and
shipped overnight to the sample processing laboratory for spiking and
distribution.

2.5	Preparation of Study Samples

	The sample processing laboratory was provided with a detailed set of
instructions for:

(	Combining and homogenizing the biosolids samples

(	Combining and homogenizing fish tissues

(	The number of aliquots to be prepared from each combined/homogenized
matrix

(	Aliquoting and spiking the wastewater samples

(	Labeling and shipping the prepared sample aliquots. 

2.5.1	Biosolids and Tissues

	Because the biosolids and tissue samples used in this study were
already known to contain PCBs at levels sufficient to cover the
analytical range of Method 1668A, the sample processing laboratory did
not have to spike these matrices with PCBs.  This eliminated concerns
about how well spiked constituents would be incorporated into these
matrices and whether spiked samples were representative of real-world
samples.  The goal of the mixing and aliquoting scheme for biosolids and
tissues was to obtain Youden pairs for each matrix of interest (i.e.,
composites A and B).  As described in ASTM Practice D2777, the
concentrations of Youden pairs should differ by no more than 20%. 
Because the available “excess” volumes of the biosolids and fish
tissues were limited and the number of laboratory participants was
relatively large, the Youden pairs were prepared in a multi-step
process.  For the biosolids, the first step was to combine and
homogenize five biosolids samples to form a composite.  This composite
was then divided approximately in half.  One half of the composite was
designated as biosolids sample “A” while the other half was used to
prepare biosolids sample “B.”  Biosolids sample “B” was prepared
by adding material from a sixth existing biosolids sample, plus some
clean sand, to produce a composite with PCB congener concentrations that
were approximately 20 % different from those in sample “A.”  For the
tissue samples, two existing tissue samples were homogenized.  The
composite was then divided approximately in half, with one half being
designated as tissue sample “A.”  Tissue sample “B” was prepared
by adding tissue from a third existing sample to the remaining half of
the initial composite.

	The sample processing laboratory was required to perform background and
homogeneity analyses of both the biosolids and tissue matrices.  The
laboratory was instructed to analyze one 10-g dry weight aliquot of
sample “A” as the background analysis, and two 10-g dry weight
aliquots of sample “B” as the homogeneity aliquots for both the
biosolids and tissue matrices.  Because of the mixing scheme for both of
these matrices, it was assumed that if the homogeneity for sample
“B” is found to be acceptable, the homogeneity of sample “A”
would also be acceptable.  This approach was used to preserve sample
mass.  Results of tissue and biosolids background and homogeneity
analyses are discussed in Section 4.1.

2.5.2	Wastewater

	Based on previous experience, municipal wastewater discharges would be
unlikely to contain PCB congeners at concentrations sufficient to
adequately test the capabilities of the method.  Thus, the sample
processing laboratory was instructed to first analyze an aliquot of
wastewater from a publicly owned treatment works (POTW) to determine
background PCB congener levels.  Following a review of the background
results by SCC, EPA defined the spiking levels, and provided the sample
processing laboratory with detailed instructions to divide the unspiked
POTW matrix into the required number of aliquots and spike each aliquot
separately (rather than spiking a bulk volume of wastewater and then
subdividing the spiked sample into replicate aliquots) to the
appropriate concentrations.  Spiking each aliquot separately avoids
problems with “wall effects,” whereby organic pollutants spiked into
a bulk sample tend to adhere to the walls of the container, making it
difficult to divide a bulk sample into multiple aliquots containing the
same analyte concentrations.

	In addition, the study-specific instructions provided to each
participating laboratory required that the laboratory filter the
wastewater sample prior to extraction and treat both the filtrate and
any solids on the filter in the manner described in Method 1688A.  This
instruction was included to prevent problems in which some laboratories
followed the method as written, and others deciding to skip the
filtration step if the wastewater did not appear turbid.

	The unspiked wastewater sample was analyzed by the sample preparation
laboratory using Method 1668A.  Out of the 209 PCB congeners, 39
congeners were detected in the sample.  All of those congeners were
between PCB 001 and PCB 168, and all of the concentrations were between
17 and 247 pg/L well below the concentrations of the spikes of the
congeners into the wastewater (see Table 2-2 of the Report for the
spiking levels).  Results for the blanks were all below the calibration
range of Method 1668A as practiced by the sample preparation laboratory.
 The sample preparation lab thus flagged results for the blanks as
estimates.  In preparing the actual study samples, EPA decided that
these background levels were low enough that adjustments need not be
made to the amount of each analyte spiked into the samples.  The solvent
used for spiking the congeners into wastewater was acetone.  The volume
of acetone used to spike the Youden pair samples was either 0.5 or 0.6
mL per 1-L volume of wastewater.  

	Because of the difficulty that would be encountered in preparing custom
spiking solutions, wastewater samples were spiked with varying amounts
of “individual native CB congener solutions” A2 through E2 listed in
Table 4 of EPA Method 1668A.  Concentrations of the congeners in the
wastewater samples, by level of chlorination, are given in Table 2-2. 

Table 2-2.	Spiked Congener Concentrations in Wastewater Samples (by
Level of Chlorination)

Congeners	Concentration (pg/L)

	Youden Pair #1	Youden Pair #2

24	Mono- through Trichlorinated biphenyl congeners	900	750

6	Mono- through Dichlorinated biphenyl congeners	1,200	1,000

9	Mono- through Trichlorinated biphenyl congeners	1,500	1,250

74	Tetra- through Heptachlorinated biphenyl congeners	1,800	1,500

38	Tetra- through Heptachlorinated biphenyl congeners	2,400	2,000

42	Tetra- through Heptachlorinated biphenyl congeners	3,000	2,500

13	Octa- through Decachlorinated biphenyl congeners	2,700	2,250

3	Octachlorinated biphenyl congeners	4,500	3,750



	The sample processing laboratory analyzed two random aliquots of one
concentration level for homogeneity determination.  Results of the
homogeneity analyses are discussed in Section 4.1.

2.5.3	Labeling and Shipping

	SCC provided the sample processing laboratory with a unique 5-digit
sample number for each sample.  After the aliquots were prepared, the
sample processing laboratory labeled each sample container and cap with
the corresponding unique sample number.  The sample processing
laboratory then shipped the prepared, numbered samples to the
participant laboratories via air courier.  Although PCBs are persistent,
and thus do not require preservation, biosolids and tissue samples were
shipped on ice to hinder decomposition of the tissues and gas formation
in the biosolids.  The sample processing laboratory notified SCC of the
shipping date, and SCC notified participant laboratories of the shipping
and scheduled arrival dates.  Table 2-3 lists the numbers of wastewater,
biosolids, and tissue samples that were prepared for distribution to the
14 participant laboratories.

Table 2-3.	Sample Pairs for Distribution to 14 Participant Laboratories

Matrix	Samples per Laboratory	Number of Aliquots Distributed

Wastewater	2 (1 Youden Pair)	28

Biosolids	2 (1 Youden Pair)	28

Tissue	2 (1 Youden Pair)	28

All Three Matrices	6	84



2.6	Sample Analysis and Data Reporting

	Participant laboratories did not know the concentration of PCBs in the
samples received, and were instructed to prepare and analyze the samples
according to Method 1668A procedures, except where stated otherwise in
the participant’s scope of work.  In addition to the analysis of study
samples, laboratories also were required to prepare and analyze two
ongoing precision and recovery (OPR) samples in reagent water, one
reagent water blank, and one solids/tissue blank (playground sand mixed
with corn oil).

	Because study results were to be used to evaluate or further develop QC
acceptance criteria, laboratories were prohibited from performing
multiple analyses to improve results.  Laboratories were, however,
allowed to implement corrective action and reanalyses for QC failures
attributable to analyst error, instrument failure, or identified
contamination. The laboratories also were instructed that any deviations
from the method and Statement of Work (SOW) must be pre-approved by EPA.

	Laboratories were required to submit electronic and hard copies of
summary sample results, and hard copies of all supporting raw data, run
chronologies, chromatograms, example equations, and case narratives to
SCC for review and data validation.  Additionally, laboratories were
asked to provide a detailed narrative describing any problems or
recommendations and a description of any modifications to procedures
specified in the method.  All submitted data were reviewed against the
study and method requirements prior to use for evaluation of method
performance.  Laboratories were asked to adhere to the following rules
in reporting results:

Report results to the lowest level possible, using a signal-to-noise
ratio of 3 as the sample-specific detection limit.

For congeners that are not detected, report as “<nn”, where nn is an
estimate of the detection limit at S/N=3.  Do not use the terms
“zero,” “trace,” or “ND” (not detection).

Report PCB congener concentrations in pg/L for aqueous samples or in
ng/kg for biosolids and tissue samples.

Report individual values, including results for congeners found in
blanks.

Do not average or perform other data manipulations unless required by
the method or study-specific instructions.  Report data to three
significant figures, rounding or truncating the data only after all
calculations have been completed.

Report data in the electronic format provided to the laboratory by SCC.

If data are reported in hardcopy form, paginate all data packages.

2.7	Deviations from the Method or Study Design

	Although Method 1668A explicitly allows use of a five-point calibration
for less-sensitive instruments (e.g., VG70) and a six point calibration
for more-sensitive instruments (e.g., Micromass Autospec Ultima),
laboratories interpreted this option differently.  This, and other
deviations from the study design are described below.  Most of these
deviations involved use of smaller sample volumes and/or diluted
extracts.

2.7.1	Instrument Calibration

	Section 10.4 of Method 1668A states that the relative response (RR)
(labeled to native) vs. concentration in the calibration solutions
should be determined using a five-point calibration for less-sensitive
HRMS instruments and a five- or six-point calibration for more-sensitive
instruments.  Laboratories used the following calibration approaches in
this study:

Laboratories 7 and 8 used a six-point calibration (CS-0.2 through CS-5).

Laboratories 2, 10, and 13 used a five-point calibration (CS-1 through
CS-5).

Laboratory 4 used a six-point calibration with a CS-5 standard at 1/4
the concentration given in the method to prevent saturation of their
HRMS instrument. 

Laboratory 12 performed two sets of calibrations, a high and a low. 
This laboratory applied a high calibration range (CS-1 through CS-5),
except in cases where a signal was observed below the CS-1 point, in
which case it applied a low calibration range (CS-0.2 through CS-4).

Laboratory 6 did not provide calibration data.

Provided the instruments were calibrated using a consistent injection
volume, these differences in the calibrations used by the laboratories
had little or no effect on the results of study samples.  

2.7.2	Biosolids

	The study-specific instructions stipulated that each laboratory
determine the percent solids of the samples, using no more than 2.5 g
for that purpose, to ensure that sufficient material was available for
several analyses by Method 1668A.  The reporting instructions also
stipulated that the biosolids results be reported on a dry-weight basis.

Some laboratories submitted results for the analysis of biosolids
samples that: used a smaller sample size than suggested in the method,
analyzing more dilute extracts than suggested in the method, or both.

Laboratory 2 used a 15-g (wet weight) sample as opposed to the 30-g
sample suggested by the method, resulting in a two-fold dilution.

Laboratory 12 used a 6-g (wet weight) sample as opposed to the 30-g
sample, resulting in a five-fold dilution.

Laboratory 7 used a 10-g (wet weight) sample as opposed to the 30-g
sample and concentrated the extract to a final volume of 100 µL, as
opposed to 20 µL, resulting in a 15-fold dilution.

Laboratory 8 used the full sample size, but concentrated the extract to
a final volume of 200 µL as opposed to 20 µL, resulting in a ten-fold
dilution.

Laboratory 13 used a 1-g (wet weight) sample as opposed to the 30-g
sample, and concentrated the extract to a final volume of 100 µL as
opposed to 20 µL, resulting in a 150-fold dilution.  Discussions with
this laboratory revealed no attempt to analyze a 30-g sample.  Instead,
based on past experience with GC/HRMS analyses, the laboratory used a
1-g sample, and concentrated the extract to 100 µL.  Their general
experience has been that using a 30-g sample results in difficulties
during instrumental analysis (lock-mass problems).   Based on their
GC/HRMS experience, Laboratory 13 also did not use the prescribed sample
amounts for the fish tissue and wastewater samples.

	Two laboratories (4 and 10) did not submit biosolids data because of
difficulties encountered with clean-up and analysis.  Both of these
laboratories attempted analyses on 30-g samples, as suggested in the
method.

Laboratory 4 reported difficulties with the cleanup of both the
biosolids samples.  In one of the biosolids samples, upon the first acid
wash, the sample appeared black in color and the phases could not be
distinguished.  The laboratory proceeded with the addition of sodium
chloride in an attempt to mitigate the problem.  During the subsequent
acid wash steps (second, third and fourth) no color appeared in the
aqueous layer.  The extract layer contained suspended particles and had
a tar-like appearance and viscosity.  The sample was then put through an
acid/base silica column before the gel permeation chromatography (GPC)
step in hopes that the extract would then not plug the filter used in
the GPC.  In the case of the second biosolids sample, an emulsion
resulted during back-extraction with base (Section 12.5 in the method). 
The laboratory unsuccessfully attempted to break the emulsion by adding
sodium chloride and cooling, and tried diluting the extract with sodium
chloride solution and hexane, followed by hexane rinses, and addition of
sulfuric acid.  The extract was drained into a round bottom flask and
concentrated by heating mantle.  The sample was then washed with the
maximum number (4) of acid washes suggested in the method.

Laboratory 10 reported difficulties with the cleanup and extraction of
both biosolids samples and reported that, despite having made two
separate attempts to cleanup and extract the biosolids samples, they
were not able to obtain reportable results.  The samples were initially
extracted using approximately 22 grams of each sample (dry weight
basis).  A total of six cleanup steps were applied to each sample. 
According to the laboratory narrative, even after these measures, the
final extracts contained significant amounts of white crystals.  The
remaining liquid portions of the extracts were separated from the
crystals and injected.  These extracts did not yield reportable results.
 The laboratory attempted to extract the samples a second time, this
time using 2 grams each.  Two cleanup steps were applied to these
samples.  No crystals were present in the final extracts; however the
laboratory was still unable to obtain reportable results. 

Laboratories 7 and 12 reported biosolids results on a wet-weight basis,
whereas laboratories 8 and 13 reported biosolids results on a dry-weight
basis.  The dry-weight data for laboratory 8 were corrected to wet
weight based on percent solids data provided by laboratory 8 (33.3%
solids for Youden 1 and 39.3% for Youden 2).  Because laboratory 13 did
not provide percent solids data, the laboratory 13 dry-weight data were
corrected to wet weight, based on the mean of the percent solids data
provided by the:  sample preparation laboratory, laboratory 2, and
laboratory 8.  These three laboratories were the only labs that provided
percent solids data (33.3% solids for Youden 1 and 35.9% for Youden 2).

	The laboratory narratives suggest that many laboratories lacked
experience extracting and cleaning up a biosolids matrix.  The resulting
deviations from the method and study-specific instructions for analysis
of biosolids samples by different laboratories resulted in some unusable
and inconsistent data.  Thus, EPA excluded some biosolids results, as
described in Section 3 of this report.  

2.7.3	Tissue

	Two of the seven laboratories that submitted usable tissue data used a
smaller sample size than that suggested in the method, or analyzed a
more dilute extract than suggested in the method.

Laboratory 7 used a 5-g (wet weight) sample as opposed to the 10-g
sample suggested in the method, resulting in a two-fold dilution.

For reasons similar to their deviation in biosolid sample volume (i.e.,
previous experience with GC/HRMS analyses), Laboratory 13 concentrated
the extract to a final volume of 100 µL as opposed to the 20-µL volume
suggested in the method, resulting in a 5-fold dilution.  

Laboratory 6 did not submit tissue data, and reported difficulties with
the analysis of this matrix due to interferences from lipids.  The
laboratory reported unsuccessful use of an acid-base wash extraction,
and two rounds of silica gel cleanup. 

2.7.4	Wastewater

	One of the eight laboratories that submitted usable wastewater data
analyzed a more dilute extract than suggested in the method. 
Specifically Laboratory 13, for reasons explained previously (GC/HRMS
experience), concentrated the extract to a final volume of 100 µL as
opposed to the 20-µL volume suggested in the method, resulting in a
5-fold dilution.

Section 3

Data Review and Validation

	Three of the 14 volunteer laboratories that were selected to
participate in this study failed to submit data despite repeated
requests and offers to extend the submission deadlines.  In all three
cases, the laboratories cited scheduling conflicts as the reason for
their inability to complete the study.

	Data from the 11 laboratories that submitted results were reviewed and
validated by SCC as soon as possible after receipt.  Data packages
included sample tracking logs, summary results, QC summaries, raw data,
sample calculations, laboratory narratives (including descriptions of
any problems encountered, corrective actions taken, and comments on
method procedures), and electronic data reporting spreadsheets.  Data
were reviewed against requirements in the study plan and the method to
ensure that results from each laboratory were complete (i.e., that all
required data were present, including results of all required tests,
sample lists, run chronologies, summaries of analytical results, raw
data, example questions).  This included verification that: all samples
were analyzed properly; appropriate spike levels were used; the
analytical systems were properly calibrated; results calculation
procedures were followed correctly; and that raw data supported the
results.  A fundamental objective of this review was to maximize data
use, and every attempt was made to resolve data discrepancies with
laboratories.  This review disclosed the following facts:

Data from Laboratories 3 and 11 failed to meet one or more of the
chromatographic resolution requirements in Section 6.9.1.1.2 of Method
1668A.  This section specifies that the SPB-Octyl GC column must resolve
congener pairs 34/23 and 187/182, and that congener pair 156/157 must
coelute.

Laboratory 3 data showed coelutions across several chlorination levels,
inability to detect many of the congeners in the low (CS-1) calibration
standard, and high baseline noise that made integration difficult. 
Laboratory 3 also reported loss of sensitivity, column deterioration,
and expressed general dissatisfaction with the method.  Laboratory 3
reported results for only 1 wastewater sample, 1 blank sample, and no
other QC or sample results.

Laboratory 11 data indicated an inability to recover 25 of the 34
labeled compounds spiked into the biosolids samples without an
acknowledgment or explanation of the difficulties, and incomplete raw
supporting data.  For example, selected ion current profiles for samples
in which the laboratory reported very high recoveries of some labeled
compounds (e.g., 572%) were not provided.  SCC contacted the laboratory
repeatedly, but did not receive an explanation.

Laboratory 2 submitted only summary level sample results, and provided
little or no calibration data.  During attempts to obtain details and
raw supporting data, SCC learned that the laboratory manager was no
longer with the company and that the laboratory was closing.  Without
sufficient information to support the summary level results submitted,
it was not possible to investigate potential causes of the observed low
recoveries.  Sample results for this laboratory were consistently below
those for all other laboratories, and the labeled compound recoveries
were generally low in both study and QC samples.

Laboratory 6 did not submit tissue sample results and did not provide
OPR results associated with the wastewater samples.  In e-mail
correspondence, the laboratory indicated that the lighter PCB congeners
were lost during final transfer, and therefore, results were not
submitted.  This laboratory provided some raw data (e.g., selected ion
current profiles) for some QC samples, but only summary level data for
the results of calibration, calibration verification, and field samples.
 SCC was unable to obtain additional information or supporting data. 
The laboratory reported results for 167 peaks containing one or more
congeners.  This is more than most other laboratories, and more than the
159 peaks described in the method, making a side-by-side comparison with
data from other laboratories difficult.

Mean relative response (RR) and response factor (RF) values reported by
Laboratory 14 were reported inconsistently across the laboratory’s
report forms.  For example, page 318 of the laboratory’s data package
lists the RR for 13C-labeled PCB congener 81 as 2.7956 and page 319 has
RR values ranging from 2.01 to 2.28 (with a mean of 2.09).  Many of the
congeners have only 5 RR values, while many others appear to have 6 RR
values.  Conversely, for congener 77L, SCC could reproduce the mean RR
value of 2.16 reported on page 319, but this value does not match the
value of 2.8026 on report Form 3B.  Results differ most for the
early-eluting labeled congeners.  SCC examined the calibration data for
these congeners and compared them to calibration data from other
laboratories in the study.  Although some differences in the responses
are expected between different GC/MS instruments, results from
Laboratory 14 were inconsistent with results from the other
laboratories.

	Of the remaining six laboratories:

Four laboratories (7, 8, 11, and 13) provided usable data for all three
of the matrices used in this study, and

Two laboratories (4 and 10) provided usable data for wastewater and
tissue matrices only.

	Thus, this validation study using volunteer labs yielded six usable
data sets for wastewater and tissue matrices, and four for biosolids. 
Data obtained from these laboratories followed the requirements of the
study plan and the method and included results for the required
accompanying QC analyses; i.e., calibration, calibration verification
(where submitted), OPR, reagent water blank, and solids/tissue blank
(playground sand mixed with corn oil).  Table 3-1 summarizes the status
of results from the laboratories.

Table 3-1.	Summary of Data Received from Participant Laboratories

Laboratory	Submitted data?

	Wastewater	Tissue	Biosolids

1	No	No	No

2	Yes, but unusable	Yes, but unusable	Yes, but unusable

3	Yes, but unusable	No	No

4	Yes	Yes	No

5	No	No	No

6	Yes, but unusable	No	Yes, but unusable

7	Yes	Yes	Yes

8	Yes	Yes	Yes

9	No	No	No

10	Yes	Yes	No

11	Yes, but unusable	Yes, but unusable	Yes, but unusable

12	Yes	Yes	Yes

13	Yes	Yes	Yes

14	Yes, but unusable	Yes, but unusable	Yes, but unusable

Total usable data packages 	6	6	4



	Study samples were assessed for outlying results using Grubbs’
outlier test, performed in accordance with Standard Practice for
Determination of Precision and Bias of Applicable Test Methods of
Committee D-19 on Water (ASTM D2777-98).  Details on the outlier
analyses are presented in Appendix A.

Section 4

Results and Discussion

4.1	Background and Homogeneity testing

	As described in Section 2.3 of this report, the sample processing
laboratory was required to perform background analyses of the wastewater
matrix, and homogeneity analyses of the tissue and biosolids matrices. 

4.1.1	Wastewater Sample Homogeneity

	Wastewater samples were prepared: by determining the background
concentration to select   appropriate spike levels, by spiking and
aliquoting the samples as described in Section 2.3, and analyzing two
random aliquots for homogeneity verification.  Relative percent
differences (RPDs) for all congener concentrations between wastewater
homogeneity test aliquots B1 and B2 were less than 16%, and all but five
were less than 10%, confirming the adequacy of the homogenization and
aliquoting process.

4.1.2	Tissue and Biosolids Sample Homogeneity

	For tissue and biosolids, the sample processing laboratory was
instructed to analyze one 10-g dry weight aliquot of sample “A” as
the background analysis, and two 10-g dry weight aliquots of sample
“B” as the homogeneity aliquots for both the biosolids and tissue
matrices.  Because of the mixing scheme for the tissue matrix it was
assumed, if the homogeneity for sample “B” was found acceptable,
that the homogeneity of sample “A” would be acceptable.  This
approach was used to preserve mass of sample for the study itself by
taking the two homogeneity aliquots from the larger aliquot (sample
“B”).  Relative percent differences (RPDs) between tissue
homogeneity test aliquots B1 and B2 were calculated to verify the
homogenization and aliquoting scheme.  All but five RPD values were 20%;
the remaining five were associated with sample concentrations below the
sample-specific minimum level of quantitation (ML; see Table 2 of Method
1668 for MLs), where greater uncertainty is expected.

4.2	Congener Concentrations in Samples

	The frequency of detection and the mean, median, and maximum
concentrations of the congeners found in tissue, wastewater, and
biosolids samples by level of chlorination (LOC) are in Table 4-1.  The
total number of congeners analyzed reflects the total number of
congeners or coeluted congener groups analyzed by all labs in both
samples for the given chlorination level.  For example, 12 congeners
were analyzed in water for LOC 10.  This equates to one congener
reported by six labs in two samples (12 = 1 x 6 x 2).  Although the same
six labs provided usable data for tissue and water, there are
differences between these matrices in the number of congeners analyzed
for a given LOC.  For example for LOC 4, a total of 356 tetrachlorinated
congeners (or co-eluting congeners) were analyzed in the wastewater
Youden pairs, but only 352 tetrachlorinated congeners were analyzed in
the in tissue Youden pair.  The difference is attributable to the
removal of outliers.  The next two columns in the table provide
information on the number of detected congeners in each LOC, and the
percentage of analyzed congeners that were detected.  Finally, the mean,
median, and maximum concentrations in each LOC represent all congeners
within that level; when coelutions of two or more congeners occurred,
the combined value of those co-eluted congeners was used.

	In wastewater samples, all congeners at LOCs 4 and higher were detected
by all laboratories. Only LOC 1 had a rate of detection below 90%.  The
rate of congener detection across laboratories was generally consistent
for the different LOCs in biosolids and tissue samples, ranging between
69% and 100% for tissue and between 70% and 100% for biosolids.  With
the exception of LOCs 9 and 10 (which include only congeners 3 and 1,
respectively), at least one congener was not detected in the solids
matrices by at least one laboratory for each LOC.  The reason that all
laboratories do not detect the same congeners in each sample is likely
due to differences in coelutions and because some laboratories
concentrated extracts to 100 or 50 µL instead of 20 µL as required by
EPA Method 1668A.  Those laboratories that did not concentrate extracts
to 20 µL would not measure to as low a level as laboratories that did,
and low concentration congeners would, therefore, not be detected by
these laboratories.

Table 4-1.	Congener Detection Rates and Concentrations in Study Samples
(by Matrix and Level of Chlorination)

Matrix	LOC	# Labs	# Congeners Analyzed	# Congeners Detected	% Congeners
Detected	Concentration (Detects Only)1,2







Mean	Median	Maximum

Biosolids	1	4	24	23	96	142	159	281

	2

88	64	73	494	265	2780

	3

160	134	84	972	482	7130

	4

240	195	81	1270	372	12400

	5

237	166	70	2070	742	13400

	6

254	196	77	1120	407	12300

	7

169	129	76	665	344	4810

	8

81	72	89	377	259	1750

	9

24	23	96	280	230	821

	10

8	8	100	313	299	493

Tissue	1	6	36	26	72	4	3	12

	2

131	90	69	47	27	188

	3

232	181	78	267	150	1610

	4

352	288	82	402	130	3330

	5

347	258	74	418	128	15700

	6

362	270	75	429	108	10700

	7

240	182	76	276	120	3560

	8

114	105	92	157	108	709

	9

35	35	100	162	137	390

	10

12	12	100	200	201	236

Water	1	6	36	25	69	27	20	106

	2

128	118	92	533	505	1460

	3

233	223	96	1100	946	3430

	4

356	356	100	2850	2170	15300

	5

344	344	100	2660	1750	21800

	6

362	362	100	2190	1660	11800

	7

235	235	100	1750	1420	7370

	8

116	116	100	2410	1740	9560

	9

35	35	100	1760	1520	3350

	10

12	12	100	1740	1510	3170



1	Biosolids (dry weight) and tissue (wet weight) concentrations in ng/kg
(pg/g); water concentration in pg/L

2	Mean, median, and maximum concentrations at each LOC are based on any
detected congeners in that LOC. When coelution of two or more congeners
occurred, the combined value of those co-eluted congeners was used.

4.3	Congener Concentrations in Blanks

	Table 4-2 gives mean, medium, and maximum congener concentrations found
in the water and sand/corn oil blanks, by level of chlorination.  PCBs
can be ubiquitous in the laboratory environment.  Congener detection
rates in blank samples ranged from 8-33%, with most of the detected
congeners being reported at very low concentrations relative to the
concentrations reported in samples.  The relatively low frequency of
detection of congeners in blanks by all laboratories is thought to be
attributable to the failure by some laboratories to concentrate extracts
to 20 µL and to lesser PCB backgrounds in some laboratories.

	Method 1668A requires that blanks be analyzed in the same way as
environmental and IPR/OPR samples, including use of a 1-L aliquot for
water or 10 g of an appropriate reference matrix for solids (see Section
7.6 of Method 1668A), and including concentration of extracts of blanks
to 20 µL.

Table 4-2.	Congener Detection Rates and Concentrations in Blanks (by
Matrix and Level of Chlorination)

Matrix	LOC	# Blank samples	# Congeners Analyzed	# Congeners Detected	%
Congeners Detected	Concentration (Detects Only)1,2







Mean	Median	Maximum

Sand/oil	1	103	50	14	28	4.0	3.5	9.6

	2

130	20	15	5.3	5.1	12.9

	3

227	61	27	2.5	1.4	12.3

	4

328	77	23	4.4	2.0	29.0

	5

370	70	19	6.7	3.3	37.7

	6

355	100	28	5.7	0.4	60.8

	7

245	65	27	3.3	0.6	20.0

	8

122	30	25	1.3	0.2	5.6

	9

50	4	8	2.0	2.1	3.0

	10

20	3	15	0.7	0.1	1.9

Water	1	6	30	10	33	25.8	15.1	82.1

	2

79	9	11	34.8	21.3	113

	3

135	33	24	17.3	11.0	57.7

	4

197	43	22	79.5	10.0	2280

	5

220	29	13	23.9	20.2	74.2

	6

213	52	24	12.2	2.2	85.0

	7

146	35	24	6.7	2.5	39.2

	8

74	15	20	9.4	9.9	28.4

	9

30	3	10	25.4	32.0	33.2

	10

12	2	17	13.2	13.2	26.1



1	Sand/oil concentration in ng/kg (pg/g) (wet weight); water
concentration in pg/L

2	Mean, median, and maximum concentrations at each LOC are based on any
detected congeners in that LOC. When coelution of two or more congeners
occurred, the combined value of those co-eluted congeners was used..

3	Six labs provided usable data for sand/oil blanks.  Four of the six
labs (Labs 7, 8, 10, and 13) analyzed two sand/oil blanks, yielding a
total of ten sand/oil blanks.

4.4	Wastewater Sample Recovery and Precision

	Table 4-3 summarizes the laboratories’ ability to recover congeners
from the wastewater samples, presenting the recovery and precision of
congener determination by level of chlorination.

	The mean and median recoveries of nearly all native congeners were in
the 60 - 110 percent range, typical for recovery of organic compounds
extracted from wastewater.  Excluding data at LOCs 1 and 2, the median
recovery across all native congeners and all labs is approximately 75%,
and the median RSD is approximately 10%.  Low recoveries of the native
congeners at LOCs 1 and 2 (Table 4-3) may be due to loss during
transport from the sample preparation laboratory to the participant
laboratories.  (Data reported by the sample prep laboratory indicate
that the congeners were present immediately after shipment.)  Even
though the native congeners were not recovered within the range
expected, the labeled congeners were recovered from spikes into the
wastewater samples by the laboratories.  Recoveries of the labeled
compounds are shown by LOC in Table 4-3 for comparison.  Recovery of the
labeled congeners indicates that the loss of the native congeners could
not be by evaporation during the solvent evaporation step because the
labeled congeners would have been lost also.

The exact reason for loss of the native congeners at LOCs 1 and 2 is not
known.  Possible causes may be loss by evaporation into the headspace of
the sample container during shipment, with subsequent release to the
atmosphere when the container is opened, or to biological or other
degradation during transit, although selective degradation of congeners
at LOCs 1 and 2 only would be unusual.  In Figure 4-1, low recoveries
for congeners in the 750 - 900 pg/L range are, almost exclusively,
attributable to these partial losses of the mono- and dichloro-
congeners.

Table 4-3.	Wastewater Sample Recovery and Precision by Level of
Chlorination

LOC	Percent Recovery (%)	Within-pair Relative Standard Deviations (%)

	# Results	Mean	Median	Min.	Max.	# Pairs	Pooled*	Median	Min.	Max.

 Native congeners spiked by sample prep lab

1	25	3.15	2.71	0.49	11.8	11	29.7	17.5	5.8	80.8

2	118	54.2	44.6	2.63	162	57	12.2	4.42	0.17	62.4

3	223	89.5	82.8	34.2	164	111	7.62	5.06	0.02	24

4	356	95.6	91.4	38.1	201	178	7.36	6.16	0	20.6

5	344	81.4	72.2	30.6	182	170	10.8	7.99	0.06	40.5

6	362	75.3	68.8	8.14	196	178	12.1	8.64	0.16	46.8

7	235	72.3	64.4	10.4	155	114	9.63	5.24	0.14	39.3

8	116	68	59.3	18.1	135	56	11.3	7.26	0.12	32.3

9	35	70.8	57.8	44.4	126	17	8.91	6.66	0.57	18.5

10	12	70	59.1	49.3	118	6	5.67	4.23	0.37	9.05

 Labeled congeners spiked by participant labs

1	24	51.4	48.9	21.0	84	12	19.7	13.8	3.45	42.9

2	24	58.5	55.1	25.0	90	12	15.7	11.9	0	29.2

3	36	67.4	62.6	26.0	108	18	12.5	5.32	0.516	33.3

4	33	60.5	57.5	35.0	101	15	8.24	3.34	0.873	17.7

5	83	77.2	81.0	41.0	110	41	11.0	2.61	0	28.7

6	50	75.6	74.3	38.6	106	25	9.83	7.13	1.32	21.8

7	36	76.9	77.0	5.00	123	18	30.4	3.72	0	126

8	23	76.6	79.5	38.4	94	11	6.20	5.12	2.29	12.4

9	24	71.2	70.0	49.1	98	12	6.33	4.24	0	9.92

10	12	73.1	74.5	52.8	98.2	6	6.81	5.26	4.07	12.0



* Pooled RSD calculated as the square root of the mean of the squared
within-pair RSDs

	Recovery, as a function of concentration, is plotted in Figure 4-1. 
(Plots of absolute and relative precision as a function of concentration
are addressed with precision for biosolids and tissue samples in Section
4.5.)  The spike concentrations displayed in Figure 4-1 do not match
exactly the concentrations that were spiked (see Table 2-2) because
coelutions result in combined concentrations.

Figure 4-1.  Mean Recovery vs. Spike Concentration, PCB Congeners in
Wastewater

4.5	Variability as a Function of Concentration

	Because true congener concentrations in the tissue and biosolids
samples were not known, it was not possible to calculate recoveries of
congeners from tissue and biosolids.  Variability (precision) vs.
concentration was determined for wastewater, soil, and, tissue matrices.
 The following subsections present plots of absolute precision (as
standard deviation of the determined concentrations) and relative
precision (as relative standard deviation) as functions of congener
concentration for each of these matrices.  For the three matrices,
standard deviation increased approximately linearly with increasing
concentration.  It was expected that, at the very low concentrations in
the tissue and biosolids samples, standard deviation would become
constant and the plots would resemble a “hockey stick.”  (The
wastewater sample was not spiked at low enough concentrations to
demonstrate this effect.)  The lack of a hockey stick appearance for the
tissue and biosolids; i.e., the lack of constant standard deviation at
low concentrations, is good because this indicates that measurements are
being made in the quantitative range for the congeners.  This is not
surprising because the rigorous congener identification criteria in
Method 1668A are that the signal-to-noise ratio must be greater than 3
and ratio of the peak heights or areas for the 2 exact m/z’s must be
within 15% of theoretical, in addition to the requirement that the
relative retention time of the congener must be within a specified
window based on a calibration or calibration verification standard. 
Thus, the identification criteria raise the lowest level of congeners
that are determined to levels above the region of constant standard
deviation.

4.5.1	Variability vs. Concentration for Wastewater

4.5.1.1	Absolute variability vs. concentration for wastewater

	Figure 4-2 is a plot of the standard deviation as a function of
concentration for the congeners spiked into wastewater.  The congener
concentrations are defined by the spiking solutions, as described in
Section 2.5.2.  Results appear slightly skewed to lower standard
deviation at low concentration. The skewed appearance is likely due to
the higher concentrations of the coeluted congeners. 

Figure 4-2.	Concentration Standard Deviation vs. Spike Concentration,
PCB Congeners in Wastewater

4.5.1.2	Relative variability vs. concentration for wastewater

	Figure 4-3 is a plot of RSDs as a function of concentration for the
congeners spiked into wastewater.  The congener concentrations are
defined by the spiking solutions, as described in Section 2.5.2.  The
variability is somewhat higher than expected at the higher
concentrations, with RSDs of approximately 40%.  The reason for these
higher than expected RSDs is not known.

Figure 4-3.	Relative Standard Deviation vs. Spike Concentration, PCB
Congeners in Wastewater   

 

4.5.2	Variability vs. Concentration for Tissue

4.5.2.1	Absolute Variability vs. Concentration for Tissue

	Figure 4-4 is a plot of standard deviation as a function of
concentration for congeners detected in tissue.  Congeners were detected
in tissue from as low as a few parts-per-trillion (ppt; pg/g) to well
into the part-per-billion (ppb; ng/g) range.

Figure 4-4.	Mean vs. Standard Deviation of Measured Tissue Results

4.5.2.2	Relative Variability vs. Concentration for Tissue

	Figure 4-5 is a plot of RSD as a function of concentration for the
congeners detected in tissue.  RSDs are mostly between 10 and 30
percent, as expected, with a few outlying high values. The unusually
high RSDs occurred in congeners that are only rarely detected (2-3
laboratories.)

Figure 4-5.	Mean vs. Relative Standard Deviation of Measured Tissue
Results

4.5.3	Variability vs. Concentration for Biosolids

4.5.3.1	Absolute Variability vs. Concentration for Biosolids

	Figure 4-6 is a plot of standard deviation as a function of
concentration for congeners detected in biosolids.  Congeners were
detected in biosolids from as low as a few ppt to well into the ppb
range.

Figure 4-6.	Mean vs. Standard Deviation of Measured Biosolids Results

4.5.3.2	Relative Variability vs. Concentration for Biosolids

	Figure 4-7 is a plot of RSDs as a function of concentration for the
congeners detected in biosolids.  Unlike the plots of relative
variability for tissue and wastewater samples, this plot does not
suggest a strong relationship between variability and concentration.
RSDs are mostly between the expected ranges of 10 to 30 percent, with a
few outlying high values.

Figure 4-7.	Mean vs. Relative Standard Deviation of Measured Biosolids
Results

4.6	Labeled Compound Recovery and Precision

	Table 4-4 lists labeled compound recovery and precision for the 27
labeled congeners spiked into wastewater, biosolids, and tissue samples.
 Except for congener 1L, median recoveries ranged from 56 to 94 percent.
 Except for congener 178, pooled  RSDs ranged between 5 and 22 percent. 
The low recovery of congener 1L and, to some extent other congeners at
low chlorination levels, is thought to be caused by loss in the solvent
evaporation step.  The reason for the high RSD for congener 178 is not
known.

Table 4-4.	Recovery and Precision of Labeled Compounds Spiked into
Samples1

LOC2	Labeled Congener3	Recovery (%)	Within-pair RSD (%)



# Results	Mean	Median	Min.	Max.	# Pairs	Pooled4	Median	Min.	Max.

1	1L	32	52.9	48.5	13.0	95.0	16	22.0	17.4	0.81	43.4

1	3L	32	60.4	61.1	15.9	107	16	18.9	10.9	2.70	47.6

2	4L	32	60.8	56.1	32.0	120	16	16.7	11.9	1.30	31.6

2	15L	32	64.8	69.0	25.0	96.3	16	13.7	2.8	0	31.4

3	19L	32	59.0	59.0	4.5	112	16	12.0	5.4	0.69	26.5

3	28L	32	78.8	87.8	25.0	118	16	11.7	7.8	1.40	33.3

3	37L	32	77.2	77.6	35.0	110	16	10.0	2.4	0.52	23.6

4	54L	31	61.6	59.1	25.5	109	15	10.3	5.4	0.87	22.0

4	77L	31	72.7	67.3	43.0	106	15	9.1	5.7	1.10	19.8

4	81L	31	74.9	71.0	26.0	129	15	9.0	6.6	1.30	18.8

5	104L	32	76.5	80.0	41.0	102	16	10.1	4.8	0	24.4

5	105L	31	81.9	86.3	52.7	103	15	10.9	5.2	0.50	25.2

5	111L	29	85.2	86.3	63.0	110	13	5.4	3.7	0.29	12.7

5	114L	32	83.0	86.3	48.0	119	16	10.8	5.2	1.00	28.3

5	118L	32	83.9	87.5	53.8	120	16	10.8	6.4	0.36	26.6

5	123L	32	84.7	88.8	53.8	116	16	11.4	6.5	0.48	23.4

5	126L	32	81.0	80.8	59.0	123	16	11.8	4.5	0	28.7

6	155L	32	77.3	81.3	33.0	106	16	7.8	3.6	0	21.8

6	156L+157L	32	87.8	76.5	44.0	216	16	12.9	8.0	1.10	30.3

6	167L	31	84.3	82.0	57.3	110	15	9.9	8.6	1.30	22.6

6	169L	32	78.3	75.3	30.5	112	16	14.8	8.0	2.40	37.5

7	178L	30	89.1	93.8	5.0	126	14	34.6	4.7	0.69	126

7	188L	32	78.0	85.2	32.0	136	16	8.5	4.6	0.22	17.4

7	189L	32	82.8	82.0	54.0	118	16	8.1	4.8	0	18.2

8	202L	31	84.9	87.8	38.4	145	15	9.2	5.4	1.30	19.9

8	205L	32	82.3	81.5	51.0	118	16	7.9	4.8	0.25	17.3

9	206L	32	80.8	81.3	50.0	115	16	9.2	7.7	0	19.8

9	208L	32	80.4	84.7	49.0	129	16	7.6	4.9	0	17.2

10	209L	32	78.7	79.8	47.7	110	16	9.2	6.3	2.20	18.9



1	Wastewater, biosolids, and tissue 

2	Level of chlorination

3	Labeled analog of World Health Organization dioxin-like (Toxic)
congener shown in bold

4	Pooled RSD calculated as the square root of the mean of the squared
within-pair RSDs

Section 5

Revision of Quality Control Acceptance Criteria

	Interlaboratory quality control (QC) acceptance criteria were developed
for initial precision and recovery (IPR), on-going recovery (OPR;
laboratory control sample, LCS), and for recovery of labeled compounds
from samples.  These revised criteria are in Table 5-1 of this report,
and Table 6 of the successor method, 1668B.  The statistical details for
development of these criteria are in an appendix to this report.  The
tests to which these criteria are applied are discussed in this Section
of this report.

5.1	Calibration

	The study plan and study-specific instructions suggested a 5- or 6-
point calibration.  Laboratories did not provide enough calibration data
to permit revision of the QC acceptance criterion for calibration
linearity.  Therefore, the criterion for which an average relative
response may be used for a given congener remains at 20%, as stated in
Section 10.4.4 of EPA Method 1668A; otherwise, a calibration curve must
be used for that congener.  This calibration linearity criterion applies
to congeners determined by isotope dilution only (i.e., the
“toxics,” “level of chlorination,” and “GC window-defining”
congeners) because all other congeners are calibrated at a single point.

5.2	Calibration Verification

	The study plan and study-specific instructions suggest single
calibration verification after calibration.  Because only two
laboratories submitted calibration verification data, EPA did not revise
the calibration verification QC acceptance criteria.  The calibration
verification QC acceptance criteria in Table 5-1 remain unchanged from
previous revisions of EPA Method 1668A.  If EPA receives calibration
verification data from enough laboratories, EPA may revise these
criteria in future versions of 1668.

5.3	Initial Precision and Recovery

	To minimize resource burden on volunteer participants, laboratories
were not required to prepare and analyze IPR samples.  Instead, EPA used
the OPR data gathered in the study to develop revised IPR and OPR QC
acceptance criteria.  In addition, results from the aqueous and solids
(sand/corn oil) OPRs were combined to yield a single set of OPR QC
acceptance criteria that would be applicable to aqueous, solids, and
tissue samples.  Two laboratories resolved labeled and native congeners
156 and 157, while the other laboratories reported these congeners as
coeluting pairs.  Similarly, one laboratory reported coelution of
congeners 4 and 10, one laboratory reported coelution of congeners 114
and 122, and two laboratories reported congener 106 coeluting with
either congener 107 or 109.  Because calculations of IPR/OPR QC
acceptance criteria were based on recoveries, coelution was ignored when
generating the revised criteria.

	Data from Laboratories 2, 3, 6, 11, and 14 were excluded for the
reasons described in Section 3 of this report.  The remaining dataset
yielded a total of 15 usable reagent water and solid matrix OPR samples.
 After performing Grubbs' outlier tests on these OPRs, a total of 13
individual data points were identified as outliers and removed from the
dataset prior to development of revised IPR/OPR QC acceptance criteria. 
Table 5-1 presents revised IPR QC acceptance criteria.  When compared to
QC acceptance criteria in Method 1668A, recoveries windows are generally
narrower than those in the method.  Recoveries for low molecular weight
congeners are centered lower than for the other congeners and for
recoveries of low molecular weight congeners in Method 1668A.  These
lower recovery windows reflect that these congeners are partially lost
in the solvent evaporation step(s).

	QC acceptance criteria for IPR precision, as relative standard
deviation (RSD) of recoveries, are also presented in Table 5-1.  The
RSDs generally are higher than those in Method 1668A for some of the low
molecular weight congeners, and lower for some of the other congeners. 
The higher RSDs for the low molecular weight congeners reflect partial
loss of these congeners in the solvent evaporation step(s) in Method
1668A, resulting in greater variability in results for these congeners.

5.4	Ongoing Precision and Recovery

	Each participating laboratory was required to spike and analyze two
reagent water OPR samples.  These samples were used to evaluate
laboratory and method performance and to update IPR and OPR QC
acceptance criteria.  Although not required by the study design, four
laboratories analyzed at least one solids matrix OPR sample.  In some
cases, laboratories provided one solids matrix OPR and one reagent water
OPR instead of two reagent water OPRs.  In other cases, laboratories
supplemented the two reagent water OPRs with one or more solids matrix
OPRs.  

	Revised OPR QC acceptance criteria are in Table 5-1.  As with the IPR
QC acceptance criteria, OPR recovery windows are, generally, narrower
than those in Method 1668A, and centered lower for some of the low
molecular weight congeners.

5.5	Labeled Compound Recovery from Samples, Blanks, and IPR and OPR
standards

	Labeled compound recovery data from samples were used to construct
revised QC acceptance criteria for labeled compound recoveries.  Results
from a total of 24 analyses were used to develop the labeled compound
recovery QC acceptance criteria (Table 5-1.)  The IPR and OPR recovery
windows are centered lower, for the low molecular weight congeners.



Table 5-1.	Revised QC Acceptance Criteria for IPR, OPR, and Labeled
Compounds in Samples

Congener	Congener number1	Test conc. (ng/mL)2	IPR	OPR

Recovery (%)3	Labeled Compound Recovery in Samples and Blanks (%)3



	RSD (%)	Recovery (%)3



2-MoCB	1	50	25	84 – 119	71 – 132	NA

4-MoCB	3	50	22	83 – 112	72 – 123

	2,2'-DiCB	4	50	18	82 – 105	73 – 114

	4,4'-DiCB	15	50	17	85 – 107	76 – 116

	2,2',6-TrCB	19	50	13	86 – 103	79 – 109

	3,4,4'-TrCB	37	50	26	77 – 109	64 – 122

	2,2',6,6'TeCB	54	50	17	84 – 106	76 – 114

	3,3',4,4'-TeCB	77	50	20	81 – 106	71 – 116

	3,4,4',5-TeCB	81	50	20	81 – 106	70 – 116

	2,2',4,6,6'-PeCB	104	50	19	83 – 107	74 – 117

	2,3,3',4,4'-PeCB	105	50	19	83 – 107	73 – 117

	2,3,4,4',5-PeCB	114	50	18	83 – 105	74 – 113

	2,3',4,4',5-PeCB	118	50	13	88 – 105	81 – 112

	2',3,4,4',5-PeCB	123	50	16	82 – 102	74 – 109

	3,3',4,4',5-PeCB	126	50	17	82 – 104	74 – 113

	2,2',4,4',6,6'-HxCB	155	50	15	86 – 105	79 – 112

	2,3,3',4,4',5-HxCB4	156	50	16	87 – 108	78 – 117

	 2,3,3',4,4',5'-HxCB4	157	50	16	87 – 108	78 – 117

	2,3',4,4',5,5'-HxCB	167	50	13	85 – 101	79 – 107

	3,3',4,4',5,5'-HxCB	169	50	16	80 – 100	73 – 108

	2,2',3,4',5,6,6'-HpCB	188	50	14	88 – 106	81 – 113

	2,3,3',4,4',5,5'-HpCB	189	50	16	85 – 106	77 – 114

	2,2',3,3',5,5',6,6'-OcCB	202	50	17	82 – 104	74 – 112

	2,3,3',4,4',5,5',6-OcCB	205	50	15	87 – 107	79 – 115

	2,2',3,3',4,4',5,5',6-NoCB	206	50	17	85 – 106	76 – 115

	2,2',3,3,'4,5,5',6,6'-NoCB	208	50	17	86 – 108	77 – 116

	DeCB	209	50	20	81 – 106	71 – 116

	Labeled Compounds

13C12-2-MoCB	1L	100	78	21 – 100	2 – 100	4 – 100

13C12-4-MoCB	3L	100	63	31 – 100	13 – 100	11 – 106

13C12-2,2'-DiCB	4L	100	56	35 – 100	18 - 100	14 – 107

13C12-4,4'-DiCB	15L	100	70	34 – 100	10 – 118	19 – 107

13C12-2,2',6-TrCB	19L	100	68	32 – 100	10 – 106	1 – 108

13C12-3,4,4'-TrCB	37L	100	57	47 – 104	24 – 128	25 – 123

13C12-2,2',6,6'-TeCB	54L	100	62	37 – 100	16 – 111	13 – 105

13C12-3,3',4,4'-TeCB	77L	100	35	57 – 100	43 – 105	31 – 109

13C12-3,4,4',5-TeCB	81L	100	33	57 – 100	44 – 102	14 – 127

13C12-2,2',4,6,6'-PeCB	104L	100	48	49 – 100	30 – 115	36 – 115

13C12-2,3,3',4,4'-PeCB	105L	100	31	66 – 101	52 – 116	50 – 111

13C12-2,3,4,4',5-PeCB	114L	100	41	57 – 100	39 – 117	41 – 121

13C12-2,3',4,4',5-PeCB	118L	100	33	65 – 102	51 – 117	49 – 111

13C12-2',3,4,4',5-PeCB	123L	100	32	66 – 103	52 – 118	49 – 116

13C12-3,3',4,4',5-PeCB	126L	100	29	67 – 100	54 – 113	50 – 106

13C12-2,2',4,4',6,6'-HxCB	155L	100	42	58 – 103	40 – 121	25 – 124

13C12-2,3,3',4,4',5 –HxCB5	156L	100	35	61 – 100	46 – 115	40 –
120

13C12-2,3,3',4,4',5'-HxCB5	157L	100	35	61 – 100	46 – 115	40 – 120

13C12-2,3',4,4',5,5'-HxCB	167L	100	24	74 – 103	63 – 115	45 – 118

13C12-3,3',4,4',5,5'-HxCB	169L	100	33	66 – 103	51 – 117	37 – 117

13C12-2,2',3,4',5,6,6'-HpCB	188L	100	47	53 – 102	33 – 121	23 – 125

13C12-2',3,3',4,4',5,5'-HpCB	189L	100	28	68 – 100	55 – 112	47 –
116

13C12-2,2',3,3',5,5',6,6'-OcCB	202L	100	50	56 – 113	33 – 136	31 –
134

13C12-2,3,3',4,4',5,5',6-OcCB	205L	100	21	70 – 100	61 – 103	46 –
115

13C12-2,2',3,3',4,4',5,5',6-NoCB	206L	100	29	64 – 100	51 – 107	38
– 122

13C12-2,2',3,3',4,5,5',6,6'-NoCB	208L	100	32	62 – 100	48 – 111	31
– 126

13C12-2,2',3,3',4,4',5,5',6,6'-DeCB	209L	100	30	65 – 100	52 – 111	43
– 115

Cleanup standards







13C12-2,4,4'-TrCB	28L	100	63	43 – 106	18 – 131	14 – 131

13C12-2,3,3',5,5'-PeCB	111L	100	23	75 - 102	64 – 113	57 – 112

13C12-2,2',3,3',5,5',6-HpCB	178L	100	30	78 - 117	62 – 133	57 – 125



1	Suffix “L” indicates labeled compound.

2	See Table 5 in EPA Method 1668A.

3	Where necessary, the limit was increased to include 100% recovery.

4	PCBs 156 and 157 are tested as the sum of the two concentrations.

5	Labeled PCBs 156L and 157L are tested as the sum of the two
concentrations.

	NA = Not applicable

Section 6

Conclusions

	This study demonstrated that PCB congeners can be measured in water,
biosolids, and tissue in multiple laboratories using EPA Method 1668A. 
Results show that recovery is nearly constant as a function of
concentration, and that precision is proportional to concentration.  Of
significance with this method is the benefit that measured
concentrations are corrected by the isotope dilution technique, even
when the recovery of the labeled compounds is low.  

The results of this interlaboratory study met our objectives to
characterize the performance of Method 1668A in several laboratories and
matrices, and use the results to replace the single-laboratory QC
acceptance criteria in 1668A with interlaboratory criteria.  These new
interlaboratory QC criteria are in Table 6 of the successor EPA Method,
1668B.  Appendix A

Statistical Procedures Used to Develop QC Acceptance Criteria

1.0	Initial Precision and Recovery (IPR) and Ongoing Precision and
Recovery (OPR)

	

IPR and OPR QC acceptance criteria were calculated using OPR results for
all matrix types for each given congener. The acceptance criteria were
calculated as prediction limits for mean and individual recoveries, set
at the 95% confidence level.  

	Prior to calculation of QC acceptance criteria, Grubbs’ outlier test,
as described in ASTM E178-02, was first run on the individual OPR sample
recoveries.  Based on Grubbs’ test, a single outlying recovery was
removed for 13 of the native or labeled congeners. These results were
not included in the subsequent IPR and OPR QC acceptance criteria
calculations.

Upper and lower limits for IPR samples were calculated as:

 is the overall mean of all OPR recoveries for the given congener, 

s is the standard deviation of all OPR recoveries for the given
congener, and

	n is the number of OPR recoveries for the given congener.			

Upper and lower limits for OPR samples were calculated as:

The maximum RSD for IPR samples was calculated as:

2.0	Labeled Compound Recovery from Samples and Blanks

	

QC acceptance criteria for the recovery of labeled compound from samples
and blanks were calculated using all labeled sample results for all
matrix types for the given congener.  The acceptance criteria were
calculated as prediction limits for mean and individual recoveries, set
at the 95% confidence level.  

	Prior to calculation of QC acceptance criteria, the Grubbs’ outlier
test, described in ASTM E178-02, was first run on the individual labeled
sample recoveries. Based on Grubbs’ test, two outlying recoveries were
each removed for two of the native or labeled congeners. These results
were not included in the subsequent labeled sample recovery QC
acceptance criteria calculations.

Upper and lower limits for IPR samples were calculated as:

 is the overall mean of all labeled sample recoveries for the given
congener, 

s is the standard deviation of all labeled sample recoveries for the
given congener, and

	n is the number of labeled sample recoveries for the given congener. 

Appendix B

Study Plan

for

Interlaboratory Validation of EPA Method 1668A

for Determination of Chlorinated Biphenyl Congeners in Water,

Biosolids, and Tissue by HRGC/HRMS

Prepared for:

William A. Telliard, Director of Analytical Methods

Engineering and Analysis Division (4303T)

Office of Science and Technology

Office of Water

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue NW

Washington, DC  20460

Prepared by:

DynCorp Systems & Solutions LLC

6101 Stevenson Avenue

Alexandria, VA 22304

May 2003



Acknowledgments

This study plan was prepared under the direction of William A. Telliard
of the Engineering and Analysis Division within the U.S. Environmental
Protection Agency (EPA) Office of Water.

Disclaimer

This study plan has been reviewed and approved by EPA’s Engineering
and Analysis Division. Mention of company names, trade names, or
commercial products does not constitute endorsement or recommendation
for use.

1.0	Introduction  tc \l1 "1.0	INTRODUCTION 

This study plan is for interlaboratory validation of EPA Method 1668,
Revision A:  Chlorinated Biphenyl Congeners in Water, Soil, Sediment,
and Tissue by HRGC/HRMS (“Method 1668A”).  Method 1668A is for
determination of the 12 polychlorinated biphenyl (PCB) congeners
designated as “toxic” by the World Health Organization (WHO), and
the remaining 197 chlorinated biphenyl (CB) congeners, either as
individual congeners or as congener groups.

2.0	Background tc \l1 "2.0	BACKGROUND 

From the 1940s into the early 1980s, PCBs were manufactured under
several trade names, most predominantly “Aroclor” in the U.S.  The
Aroclor name was accompanied by a four-digit number indicating the
degree of chlorination of the commercial mixture (e.g., Aroclor 1016,
Aroclor 1260, etc.).  In general, the higher the number, the higher the
degree of chlorination.

From the late 1950s through the 1970s, PCBs were determined as Aroclors
by low resolution (packed column) GC with an electron capture detector
(ECD).  In the late 1970s and early 1980s, heightened interest in PCBs
and ambiguities in PCB identification led several researchers to
separate and identify all 209 PCB congeners using high resolution (open
tubular capillary) GC columns coupled with low resolution mass
spectrometry (LRMS).  In the early to mid-1990s, researchers began to
investigate use of high resolution mass spectrometry (HRMS) more
intensely as a means to reduce or eliminate interferences that
compromise measurement of PCBs by ECD or LRMS.

In 1995, EPA developed Method 1668, which uses high resolution GC (HRGC)
combined with HRMS for determination of 13 dioxin-like PCBs that the
World Health Organization (WHO) designated as “toxic” in 1994. 
Method 1668 was based on data from studies conducted at Pacific
Analytical, Inc., Carlsbad, CA.  In 1997, interest in additional
congeners led EPA to investigate determination of as many congeners as
possible in a single HRGC/HRMS run.  This led to draft Revision A of EPA
Method 1668.  At about the same time that Method 1668A was drafted, WHO
modified the list of dioxin-like congeners by adding congener 81 and
deleting congeners 170 and 180, resulting in the current list of 12 PCBs
that exhibit (dioxin-like( toxicity.

Method 1668A was validated in a single-laboratory study at AXYS
Analytical Services Ltd., Sidney, BC, Canada.  AXYS Analytical produced
a report that was subsequently published in March, 2000, in two parts
titled:  Development of a Full-Congener Version of EPA Method 1668 and
Application to Determination of 209 CB Congeners in Aroclors (Part I)
and Development of Method 1668A (Part II).

Draft Method 1668A was subjected to formal peer review in
September-October of 1999.  The peer review was conducted in accordance
with EPA's Science Policy Council Peer Review Handbook (EPA
100-B-98-001, January 1998).  Based on the peer review, EPA revised and
published Method 1668A without the word “Draft” in December of 1999
(EPA 821-R-00-002).  EPA also published a report titled Peer Review of
Draft EPA Method 1668, Revision A:  Chlorinated Biphenyl Congeners in
Water, Soil, Sediment, and Tissue by HRGC/HRMS in February 2000.

3.0	Study Objectives  tc \l1 "3.0	STUDY OBJECTIVES 

The objectives of this study are to 1) characterize the performance of
EPA Method 1668A in multiple laboratories and matrices and 2) evaluate
and, if appropriate, revise the quality control (QC) acceptance criteria
in the method.  The ultimate objective is to propose and promulgate
Method 1668 at 40 CFR part 136 for use in EPA's Clean Water Act
programs.

	To ensure that these study objectives are met, EPA will require that:

Each laboratory follow all analytical and quality control procedures in
EPA Method 1668A and study-specific instructions,

Any laboratory that wishes to deviate from the procedures in Method
1668A or the study-specific instructions obtain prior approval of the
changes and document those approved changes in detail

All data produced be capable of being verified by an independent person
reviewing the analytical data package

Each laboratory has a comprehensive quality assurance (QA) program in
place and operating throughout the study.  This QA program will ensure
that the data produced are of appropriate and documented quality.

4.0	Study Management  tc \l1 "4.0	STUDY MANAGEMENT 

The study will be managed by the Statistics and Analytical Support
Branch (SASB) in the Engineering and Analysis Division within EPA's
Office of Science and Technology.  Day-to-day management and
coordination of study activities will be performed by the
contractor-operated Sample Control Center (SCC) under SASB guidance. 
SCC will coordinate the purchase of standards, sample collection, sample
and data tracking, and monitor day-to-day study activities.  SCC also
will establish schedules for activities given in this study plan and
will keep SASB informed as to the status of the study.   SASB will draw
conclusions from the study and produce a report presenting study
results.  If appropriate, SASB will revise Method 1668A as necessary to
reflect study findings.

5.0	Study Design  tc \l1 "5.0	STUDY DESIGN 

The design of this study is intended to provide EPA with a sufficient
amount of data to evaluate method performance in accordance with the
guidelines published by EPA, AOAC-International, and ASTM
International.,,  These guidelines recommend a minimum of six data sets
for evaluation of a method.  In order to allow for some loss of data due
to error, lost samples, outlier removal, or other unforeseen causes, EPA
plans to identify at least nine laboratories willing and able to
participate in the study.  However, given the relatively limited number
of laboratories with the equipment and experience necessary to analyze
for PCBs using HRGC/HRMS, it may not be possible to identify nine
laboratories willing to participate as volunteers or to obtain at least
six usable sets of data.  If it is not possible to obtain at least six
usable data sets, EPA may utilize any Method 1668A data available to
assess method performance, develop revised QC acceptance criteria, and
for other purposes.

Due to budget limitations, EPA intends to seek as much volunteer
participation as possible in this study.  To help offset study costs,
EPA will provide volunteer laboratories with a set of analytical
standards necessary to implement Method 1668A.  Even so, it is not
reasonable to expect laboratories to donate tens of thousands of dollars
worth of analyses.  Therefore, the number of analyses will be balanced
against the need to obtain a sufficient number of participant
laboratories.

An interlaboratory study designed in accordance with ASTM standard
D-2777 would involve spikes of all 209 congeners at multiple and
replicate concentrations in multiple matrices, plus initial and batch
QC.  The total number of analyses per laboratory could be upwards of 75
if calibration, QC, and a method detection limit (MDL) study are
included.  Given that a single HRGC/HRMS analysis costs $750 - 1200, the
cost for such a study in a minimum of nine laboratories would exceed
available EPA resources and be impractical for volunteers.

To address these cost concerns, EPA intends to include no more than two
samples of each matrix type, with each sample containing varying
concentrations of the target PCB congeners.  EPA anticipates validating
the method in wastewater, biosolids, and fish tissue.  To further reduce
study costs, EPA plans to use excess sample volume collected from
previous studies of biosolids and fish.  Biosolid samples or sample
locations will be selected based on results of EPA(s 2001 National
Sewage Sludge Survey; tissue samples or sample locations will be
selected based on results of EPA(s ongoing National Study of Chemical
Residues in Fish Tissue. EPA does not have a similar supply of stored
wastewater sample volume.  Therefore, EPA plans to collect and spike
wastewater samples with PCBs.

Given the above considerations, EPA believes that the study can be
conducted with a total of 10 analyses per laboratory (in addition to 5
runs necessary to determine the absolute and relative retention time for
each congener, and an initial 6-point instrument calibration) as
follows:

2 reagent water samples,

2 biosolid samples,

2 tissue samples,

2 wastewater samples,

1 reagent water blank, and

1 solids/tissue blank (playground sand spiked with corn oil).

EPA believes that increasing the number of samples beyond the numbers
described above would significantly limit the number of laboratories
willing to participate in the study, even with enticements offered by
the recognition gained through participation in the study and
EPA-provided standards.

6.0	Study Implementation  tc \l1 "6.0	STUDY IMPLEMENTATION 

The study will be conducted in four phases: (1) identifying and
selecting the participant laboratories; (2) collecting, preparing, and
shipping standards and samples; (3) sample analysis and data reporting;
and (4) data review and assessment.  Details of each phase are
summarized below.

6.1	Phase 1 - Laboratory Identification and Selection tc \l2 "6.1	Phase
1 - Laboratory Identification and Selection 

The study will involve one sample processing laboratory and a group of
participant laboratories.   The total number of participant laboratories
will depend upon laboratory capability, availability, cost, and
scheduling constraints.  Participant laboratories may include commercial
laboratories, academic laboratories, State laboratories, EPA
laboratories, and/or municipal laboratories.  EPA will also request
participation by international laboratories so that study results
reflect worldwide application of EPA Method 1668A.  EPA recognizes
international environmental concerns and abilities to implement
laboratory analytical techniques targeting PCBs, and successfully
included international participation in validating EPA Method 1613
(chlorinated dibenzo-p-dioxins and dibenzofurans), EPA Method 1622
(Cryptosporidium), EPA Method 1623 (Cryptosporidium and Giardia), and
EPA Method 1631 (mercury).  As noted in Section 5 above, EPA 1) plans to
identify at least nine laboratories willing and able to participate in
the study and 2) intends to seek as much volunteer participation as
possible.

All laboratories that participate in the study will be required to
demonstrate that they have recent experience in analyzing for
chlorinated pollutants in environmental samples by HRGC/HRMS with
selected ion monitoring (SIM).  This is intended to ensure that study
participants already have the facilities, equipment, and trained staff
necessary to implement Method 1668A.  Once qualified participant
laboratories have been identified, they will be provided with at least
two weeks notice of their selection to participate in the study before
the study begins.  This is intended to provide study participants with a
reasonable amount of time to review any study-specific instructions.

Note:	Given the relatively limited number of laboratories with HRGC/HRMS
instrumentation, and EPA(s desire to obtain volunteer support, it may
not be possible to achieve a sufficient number of laboratories to meet
the study design.  If a sufficient number of volunteer laboratories are
not identified, EPA may consider issuing contracts with one or more
qualified laboratories through a competitive bidding process.

6.2	Phase 2 - Collection, Preparation, and Shipment of Samples and
Standard Solutions  tc \l2 "6.2	Phase 2 - Collection, Preparation, and
Shipment of Samples and Standard Solutions  

6.2.1	Sample Identification and Collection tc \l3 "6.2.1	Sample
Identification and Collection 

Biosolid samples will be generated from excess sample volume collected
during EPA(s 2001 National Sewage Sludge Survey.  Tissue samples will be
generated from excess sample volume collected during EPA(s National
Study of Chemical Residues in Fish Tissues.  Excess sample volume from
both studies is currently stored in freezers at an EPA sample
repository.  As described in Section 5, EPA will examine biosolids and
fish tissue data from these studies to identify samples that contain PCB
congeners at concentrations of interest.  In selecting the samples,
EPA(s objective will be to maximize the number of congeners represented
and ensure that the congeners span the anticipated measurement range of
the method, ranging from the upper end of the calibration range down to
(not detected.(  In order to ensure that a sufficient volume of each
sample is available to support the needs of this study, EPA will
identify several samples of each matrix type that can be combined to
produce large volumes of Youden pairs with the desired congener
distribution.  Once these frozen, stored samples are identified, they
will be forwarded on ice to the Sample Processing laboratory.  (Although
PCBs are stable and do not require preservation, ice will be used to
prevent decomposition of the fish and retard gas production in the
biosolids.)

Because EPA does not have a stored supply of excess wastewater sample
volume, wastewater will be collected for this study from a publicly
owned-treatment works (POTW) located near the sampling organization. 
Based on previous experience, EPA believes that municipal wastewater
discharges are unlikely to contain a sufficient number of PCB congeners
at concentrations to adequately test the capabilities of the method. 
Depending on available resources and the selected site location, these
wastewater samples may be collected by SCC, the Sample Processing
Laboratory, States, or EPA Regional staff.  Samples will be collected by
individuals trained in appropriate sample collection and handling
procedures.  The sampling team will collect a sufficient volume to allow
for testing in all laboratories and to provide extra volume in case of
sample breakage, lost shipment, or other unforeseen problems.  Samples
will be collected into pre-cleaned bottles (e.g., from a
bottle-manufacturing process that includes high-temperature annealing or
that have been cleaned by a laboratory experienced in the determination
of PCBs by HRGC/HRMS).  Because PCBs are known to be persistent in the
environment, wastewater samples will not be stored on ice.

Immediately after sample shipment (i.e., as soon as samples are in the
custody of the carrier), the sample repository or sampling team will
call SCC and provide information on the shipment, including sample
numbers, numbers of coolers, and courier and air bill number.  SCC will
notify the processing laboratory of the scheduled shipment and confirm
that samples have arrived in good condition and as scheduled.  If
necessary, SCC will implement tracking activities to locate any lost
shipment(s).

6.2.2	Sample Processing at the Processing Laboratory tc \l3 "6.2.2
Sample Processing at the Processing Laboratory 

Each set of tissue and biosolid samples sent to the sample processing
laboratory will be accompanied by a detailed set of instructions
concerning combination and homogenization of sample volumes, the number
of aliquots to be prepared from each combined/homogenized sample, and
instructions for labeling the prepared sample aliquots.  These
instructions will reflect the considerations described in Section 6.2.1
(i.e., creating sufficient volume of samples that contain a large number
of PCB congeners at a wide range of concentrations).  The sample
processing laboratory will combine and homogenize the tissue and
biosolid samples according to these instructions.

EPA also will direct the sample processing laboratory to divide the
unspiked wastewater into the required number of aliquots and spike each
aliquot separately (rather than spiking a bulk volume wastewater and
then subdividing the spiked sample into replicate aliquots).  Spiking
each aliquot separately avoids the problems with (wall effects,( whereby
organic pollutants spiked into a bulk sample in a solvent tend to adhere
to the walls of the container, making it difficult, if not impossible,
to divide the bulk sample into multiple aliquots containing the same
concentration.  EPA will provide detailed instructions to the sample
processing laboratory regarding the number of aliquots, the PCBs to be
used for spiking, and spiking concentrations.  In developing those
instructions, EPA will assume that any background concentration of PCB
congeners in the wastewater samples is minimal.

Because PCBs are ubiquitous in the environment, including laboratories,
the sample processing laboratory must judiciously guard against sample
contamination.  To minimize contamination, the processing facility will
homogenize the samples and divide the homogenized samples into replicate
aliquots under controlled conditions.

Note:	It is not necessary that the exact congener concentrations of each
sample be known because 1) each sample will have been designed to ensure
that a wide variety of congeners and concentrations are present, 2) the
purpose of the study is to compare interlaboratory measurements rather
than to definitively characterize specific samples, and 3) spikes of
labeled compounds into these matrices will be used to measure recovery.

6.2.3	Sample Shipment tc \l3 "6.2.3	Sample Shipment 

Once the study samples have been prepared, aliquoted, and labeled, the
sample processing laboratory will ship the samples to the participant
laboratories via air courier.  Because of the stability of PCBs, the
samples will not require preservation.  Biosolids and tissue samples
will be shipped on ice, however, to hinder decomposition of tissues and
gas formation in the biosolids.  The processing laboratory will notify
SCC of the shipping date so that SCC can notify all participant
laboratories of the shipping and scheduled arrival dates, and if
necessary, implement tracking procedures for any lost shipments.

Note:	If overseas laboratories are included in the study, biosolids and
tissues may be freeze dried so that they can be shipped without concern
that ice may melt during extended transit times.

6.2.4	Standards Acquisition, Packaging, and Shipment tc \l3 "6.2.4
Standards Acquisition, Packaging, and Shipment 

To reduce the cost to volunteer laboratories, EPA will provide each
volunteer laboratory with a single set of standards sufficient to
calibrate their instrumentation and conduct all analyses.  Sets of
standards solutions will be acquired from suppliers of native and
carbon-13 labeled compounds.  If possible, a single supplier will
aggregate all standards solutions into a set, and package a set of
standards for shipment to each laboratory.  To preclude injudicious use
of standards, EPA will remind laboratories of the instructions given in
Method 1668A for combining and diluting standards.

6.3	Phase 3 - Sample Analysis and Data Reporting tc \l2 "6.3	Phase 3 -
Sample Analysis and Data Reporting 

6.3.1	Sample Analysis tc \l3 "6.3.1	Sample Analysis 

Participant laboratories will be required to analyze samples in a timely
fashion in accordance with the study schedule, and will follow
procedures for preparation, handling, and analysis of standards
solutions and samples provided in EPA Method 1668A.

If analytical results appear unreasonable, laboratories will be
instructed to investigate possible causes, first by checking for
transcription and calculation mistakes, and then by reanalysis. 
Although laboratories will be prohibited from performing multiple
analyses to improve results, they will be allowed to implement
corrective action and reanalysis for QC failures that are attributable
to instrument failure or to analyst error (e.g., incorrect spiking
levels).

6.3.2	Data Reporting tc \l3 "6.3.2	Data Reporting 

Specific reporting requirements will be provided in detailed
instructions to the laboratories.   Gathering data from analyses of 209
congeners in IPR, OPR, blank, and the study sample(s) could represent a
formidable challenge because of the multiplicity of possible data
reporting formats.  To simplify data evaluation, EPA will provide an
electronic spreadsheet template and request that laboratories submit
data in this suggested format.

Each laboratory will be asked to report the following:

Summary level data in spreadsheet format;

Summary level and raw data in hardcopy format;

Individual results, including results for all congeners found in all
blanks.

	(Note:  Laboratories will not be allowed to average results or perform
other data manipulations beyond those described in Method 1668A.  When
results are below the minimum level of quantitation but are detected,
laboratories will be required to report the actual calculated result,
regardless of its value);

A list of the composition and concentrations of PCB congeners in the
calibration, IPR, blank(s), OPR, samples analyzed, and a run chronology;

Copies of all raw data, including chromatograms, quantitation reports,
spectra, bench sheets, and laboratory notebooks showing weights,
volumes, and other data that will allow verification of the calculations
performed and will allow the final results reported to be traced to the
raw data.  Each data element must be clearly identified in the
laboratory's data package;

A written report that details any problems associated with analysis of
samples or standard solutions.   The written report also must provide
comments on the performance of any part of Method 1668A; and

A detailed description of any modifications to the procedures specified
in Method 1668A.  Details and raw data from all runs will be reviewed
for determination as to whether further testing is required.

Laboratories also will be instructed to use the following rules in
reporting results:

Quantitative results above or at the MDL - report value;

Quantitative results below the MDL - report value but flag with footnote
giving the MDL;

Nonquantitative results - report as less than the MDL value and state
the MDL value; and

The terms (zero,( (trace,( or (ND (not detected)( are not to be used.

EPA will request that laboratories submit analytical results within 45
days of receipt of samples.

6.4	Phase 4 - Data Review and Assessment tc \l2 "6.4	Phase 4 - Data
Review and Assessment 

Upon receipt of laboratory data packages, SCC will review the data to
ensure all results were generated in accordance with the method and with
the requirements of this study plan and any associated laboratory
instructions.  An objective of this review will be to maximize data use.
 If a discrepancy occurs, it will be resolved with the laboratory, where
possible.  Data and laboratory comments and recommendations will be
assessed in the context of the objectives of the study and the ultimate
uses of EPA Method 1668A under the Clean Water Act.

The objective of this assessment will be to evaluate the precision,
recovery, and comparability of results obtained by multiple laboratories
employing the method, and to determine if the QC acceptance criteria in
Method 1668A should be revised based on study results.  Results of this
assessment will be published in a study report.

EPA plans to perform a statistical analysis of the data to determine
acceptability and suitability for use.  This statistical analysis will
be performed in accordance with Standard Practice for Determination of
Precision and Bias of Applicable Test Methods of Committee D-19 on Water
(ASTM D2777) or other accepted statistical practice.

7.0	LIMITATIONS tc \l1 "7.0	LIMITATIONS 

The study design does not include a requirement that each laboratory
perform an MDL or IPR study in each reference matrix.  In order to
ensure that the MDL and IPR specifications published in the final method
can be achieved in these matrices by multiple laboratories, EPA intends
to supplement this study with MDL and IPR data gathered from at least
three sources.  One of these sources will include existing MDL data
generated in reference tissue, solids, and aqueous matrices.  EPA
already has such data from AXYS Analytical Services Ltd., and will
contact other laboratories to determine if such existing data are
available.

Given the cost of Method 1668A analyses, EPA believes it is neither
feasible nor necessary to validate the method in each and every possible
matrix or to validate each and every congener at low, medium and high
concentrations.  This study design focuses on representative matrices
and concentrations.  EPA believes that application of a method to one or
more matrices in multiple laboratories usually can reflect the
performance of the method across multiple matrices.  EPA also believes
that PCBs are extremely stable and are not subject to adsorption and
other processes that cause percent recovery to vary as a function of
concentration for some analytes (e.g., nitrophenols).  If EPA is able to
gather data from other matrices and concentrations not tested in this
study, EPA will make such data available to interested parties, either
upon request or as an addendum to the final study report.  EPA also is
willing to consider expanding the study if the additional analyses can
be justified in terms of the additional information that they will
provide, and if external funding can be found to support the additional
analyses.

 The Sample Control Center (SCC) is operated by CSC Systems & Solutions,
LLC under contract to EPA.

 Guidelines for Selection and Validation of US EPA’s Measurement
Methods, U.S. EPA Office of Acid Deposition, Environmental Monitoring
and Quality Assurance (OADEMQA), Office of Research and Development,
U.S. Environmental Protection Agency, August 1987 Draft.

 ASTM Standard D2777-98, “Standard Practice for Determination of
Precision and Bias of Methods of Committee D-19 on Water,” Annual Book
of ASTM Standards, Vol. 11.01, ASTM International, West Conshohocken, PA
19428.

Although some congeners have only a single chlorine atom, the entire
suite of 209 chlorinated biphenyl congeners will be referred to as
(PCBs( in the remainder of this study plan for consistency with common
usage.

The Sample Control Center (SCC) is operated by DynCorp Systems &
Solutions LLC under EPA Contract No. 68-C-01-091.  All SCC activities
are performed under the direction and guidance of EPA SASB.

Guidelines for Selection and Validation of US EPA(s Measurement Methods,
U.S. EPA Office of Acid Deposition, Environmental Monitoring and Quality
Assurance (OADEMQA), Office of Research and Development, U.S.
Environmental Protection Agency, August 1987 DRAFT.

(Report of the Committee on Collaborative Interlaboratory Studies,( J.
Assoc. Office. Anal. Chem., 67, (2), 1984

ASTM Standard D2777-98, (Standard Practice for Determination of
Precision and Bias of Methods of Committee D-19 on Water,( Annual Book
of ASTM Standards, Vol. 11.01, ASTM International, West Conshohocken, PA
19428.

Method 1668A Interlaboratory Validation Study Results	

Method 1668A Interlaboratory Validation Study

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