Method 1668A Interlaboratory 

Validation Study Report

March 2010
Method 1668A Interlaboratory Validation Study

March 2010 i

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
Method 1668A Interlaboratory Validation Study

March 2010 ii

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
Method 1668A Interlaboratory Validation Study

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Table of Contents

Executive Summary......................................................................................................................................ii

Acknowledgments.........................................................................................................................................ii

Disclaimer ..........................................................................................................................................ii

Contacts ..........................................................................................................................................ii

Section 1 Introduction And Background.................................................................................................. 1

1.1 Introduction ..................................................................................................................... 1

1.2 Background...................................................................................................................... 2

1.3 2003 Revision to Method 1668A..................................................................................... 2

Section 2 Study Management, Objectives, Design, and Implementation ................................................ 3

2.1 Study Management.......................................................................................................... 3

2.2 Study Objectives and Design........................................................................................... 3

2.3 Laboratory Selection........................................................................................................ 3

2.4 Sample Selection ............................................................................................................. 4

2.5 Preparation of Study Samples.......................................................................................... 5

2.5.1 Biosolids and Tissues.............................................................................................5

2.5.2 Wastewater ............................................................................................................6

2.5.3 Labeling and Shipping...........................................................................................7

2.6 Sample Analysis and Data Reporting.............................................................................. 7

2.7 Deviations from the Method or Study Design................................................................. 8

2.7.1 Instrument Calibration ...........................................................................................8

2.7.3 Tissue...................................................................................................................10

2.7.4 Wastewater ..........................................................................................................10

Section 3 Data Review and Validation .................................................................................................. 11

Section 4 Results and Discussion........................................................................................................... 13

4.1 Background and Homogeneity Testing ......................................................................... 13

4.1.1 Wastewater Sample Homogeneity.......................................................................13

4.1.2 Tissue and Biosolids Sample Homogeneity ........................................................13

4.2 Congener Concentrations in Samples............................................................................ 13

4.3 Congener Concentrations in Blanks.............................................................................. 15

4.4 Wastewater Sample Recovery and Precision ................................................................ 15

4.5 Variability as a Function of Concentration.................................................................... 17

4.5.1 Variability vs. Concentration for Wastewater .....................................................17

4.5.2 Variability vs. Concentration for Tissue..............................................................19

4.5.3 Variability vs. Concentration for Biosolids.........................................................20

4.6 Labeled Compound Recovery and Precision................................................................. 21

Section 5 Revision of Quality Control Acceptance Criteria .................................................................. 23

5.1 Calibration ..................................................................................................................... 23

5.2 Calibration Verification................................................................................................. 23

5.3 Initial Precision and Recovery....................................................................................... 23

5.4 Ongoing Precision and Recovery .................................................................................. 24

5.5 Labeled Compound Recovery from Samples, Blanks, and IPR and OPR Standards.... 24

Section 6 Conclusions............................................................................................................................ 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
Method 1668A Interlaboratory Validation Study

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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
Method 1668A Interlaboratory Validation Study

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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.
Method 1668A Interlaboratory Validation Study

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 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. 
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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).1

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).2,3 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 SPBOctyl 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 

 

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

2 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.

3 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

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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

Phone: 706-355-8807

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.”
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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 
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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. 
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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 
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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 samplespecific 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 studyspecific 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 lesssensitive 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.
Method 1668A Interlaboratory Validation Study

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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 
Method 1668A Interlaboratory Validation Study

March 2010 10

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 dryweight 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.
Method 1668A Interlaboratory Validation Study

March 2010 11

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 
Method 1668A Interlaboratory Validation Study

March 2010 12

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.
Method 1668A Interlaboratory Validation Study

March 2010 13

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 
Method 1668A Interlaboratory Validation Study

March 2010 14

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.
Method 1668A Interlaboratory Validation Study

March 2010 15

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

10

3

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.
Method 1668A Interlaboratory Validation Study

March 2010 16

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 
Method 1668A Interlaboratory Validation Study

March 2010 17

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. 
Method 1668A Interlaboratory Validation Study

March 2010 18

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 
Method 1668A Interlaboratory Validation Study

March 2010 19

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.)
Method 1668A Interlaboratory Validation Study

March 2010 20

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 
Method 1668A Interlaboratory Validation Study

March 2010 21

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
Method 1668A Interlaboratory Validation Study

March 2010 22

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.

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
Method 1668A Interlaboratory Validation Study

March 2010 23

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).
Method 1668A Interlaboratory Validation Study

March 2010 24

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.
Method 1668A Interlaboratory Validation Study

March 2010 25

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-HxCB4156 50 16 87 – 108 78 – 117

2,3,3',4,4',5'-HxCB4157 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
Method 1668A Interlaboratory Validation Study

March 2010 26

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

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
Method 1668A Interlaboratory Validation Study

March 2010 27

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. 
Method 1668A Interlaboratory Validation Study

March 2010 A-1

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:

where: X 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:

where: X 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.

n

1

4

1

X  t

(0.975,n1)

*s * 

n

1

*s * 1

(0.975,n 1)

X t 





* F *100%

X

s

(0.95,n1)

n

1

X  t

(0.975,n1)

*s * 1
Method 1668A Interlaboratory Validation Study

March 2010 B-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

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
Method 1668A Interlaboratory Validation Study

March 2010 B-2

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.
Method 1668A Interlaboratory Validation Study

March 2010 B-3

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.4

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 Adioxin-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.

 

4Although some congeners have only a single chlorine atom, the entire suite of 209 chlorinated biphenyl congeners 

will be referred to as APCBs@ in the remainder of this study plan for consistency with common usage.
Method 1668A Interlaboratory Validation Study

March 2010 B-4

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

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.5 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 

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.6,7,8 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 

 

5The 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.

6Guidelines 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.

7

AReport of the Committee on Collaborative Interlaboratory Studies,@ J. Assoc. Office. Anal. Chem., 67, (2), 1984
Method 1668A Interlaboratory Validation Study

March 2010 B-5

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 

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.

 

8ASTM Standard D2777-98, AStandard 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

March 2010 B-6

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 

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 Anot 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 
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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

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 Awall 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

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 
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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

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

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

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;
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 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 Azero,@ Atrace,@ or AND (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

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

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 
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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.