United States Office of Water DRAFT Reviews & Responses 

Environmental Protection Agency (4303) April 2010

Peer Review of the Method 1668A 

Interlaboratory Validation Study

April 2010
United States Office of Water

Environmental Protection Agency (4303) April 2010

ii

U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA 820-R-10-007
Method 1668A Interlab Study Peer Review

April 2010 iii

Table of Contents

DISCLAIMER............................................................................................................................ IV

INTRODUCTION ....................................................................................................................... 1

EPA’S CHARGE TO THE FIRST TWO PEER REVIEWERS..................................................... 2

EPA’S CHARGE TO THE THIRD PEER REVIEWER................................................................ 3

PEER REVIEW.......................................................................................................................... 5

ISSUES RAISED BY BOTH REVIEWERS AND EPA’S RESPONSES ..................................... 5

PEER REVIEW COMMENTS FROM M ICHAEL C. NEWMAN ................................................. 7

PEER REVIEW COMMENTS FROM CHARLES S, WONG .....................................................17

PEER REVIEW COMMENTS FROM KURUNTHACHALAM KANNAN....................................21

ORIGINAL PEER REVIEW COMMENTS .................................................................................35
Method 1668A Interlab Study Peer Review

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Disclaimer 

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

use.

Contacts

The first two peer reviews in this document were conducted under EPA Contract # 68-C-02-091 by 

Versar, Inc., 6850 Versar Center, Springfield, Virginia 22151. The third and final peer review in this 

document was conducted under Contract No. EP-C-07-059 by Eastern Research Group, Inc. 110 Hartwell 

Avenue. Lexington, MA 02421. Please direct your questions concerning this document to:

Clean Water Act methods Group, Brian Englert or Richard Reding

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

OSTCWAMethods@epa.gov, englert.brian@epa.gov or reding.richard@epa.gov
Method 1668A Interlab Study Peer Review

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Introduction

This document contains the comments and EPA’s responses to comments of a peer review of the Method 

1668A Interlaboratory Validation Study Report. In 2007, a contractor obtained three independent peer 

reviewers and conducted this review for EPA using EPA’s Science Policy Council Peer Review 

Handbook (EPA/100/B-06/002, 3rd Edition, the “Peer Review Handbook”).

For the review, the contractor sent the Study Report to each reviewer with a “charge.” Two reviewers 

provided written comments and suggestions for improvement of the Study Report on the Method 1668A 

Interlaboratory Validation Study. After a third reviewer dropped out for personal reasons, EPA solicited a 

third reviewer in April of 2010.

In our responses to peer reviewer comments and suggestions, we either accept a suggestion or provide an 

explanation why the suggestion was not accepted. Based on the suggestions and on further data gathered, 

EPA has revised Method 1668A and the “Method 1668A Interlaboratory Validation Study Report” 

(March 2010, EPA-820-R-10-004.) This Study Report and Method 1668C are posted on the CWA 

methods website at http://www.epa.gov/waterscience/methods/method/other.html.
Method 1668A Interlab Study Peer Review

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EPA’s Charge to the first two Peer Reviewers

EPA developed the following charge for the first two peer reviewers:

You are requested to peer review “Report of the Method 1668A Interlaboratory Validation Study”. Please 

prepare a summary of comments based on the EPA specified Charge. The Comments Summary should 

include a cover page with the title of the document, your name and affiliation and comments organized by 

the format described in the cover letter. 

Note: You may use this draft to become familiar with Method 1668A Interlaboratory Validation Study, 

but please do not distribute it. We shall provide you with a revised copy after changes resulting from 

comments have been incorporated. 

EPA Method 1668A was developed in the late 1990s and a draft version was peer reviewed in 1999. 

Results of that peer review were published in February, 2000, in Peer Review of Draft EPA Method 1668, 

Revision A: Chlorinated Biphenyl Congeners in Water, Soil, Sediment, and Tissue by HRGC/HRMS. 

Draft EPA Method 1668A was revised as a result of the peer review and the revised version, dated 

December 1999 (EPA-821-R-00-002) was published in January of 2000.

After publication of the December 1999 draft of EPA Method 1668A, EPA distributed the method 

extensively and planned inter-laboratory validation. In preparation for the inter-laboratory validation, 

EPA updated, revised, and released Method 1668A on August 20, 2003. A copy of that revision is 

included with this letter. The interlaboratory validation study was conducted in 2003 - 2004. Results of 

the study were analyzed and the enclosed Report of the Method 1668A Interlaboratory Validation Study 

was drafted in 2006. The major result of the interlaboratory study is a revision to the quality control 

acceptance criteria (QCAC) in EPA Method 1668A based on interlaboratory study results. These QCAC 

are performance specifications within which Method 1668A must be operated and performance yardsticks 

against which a method modification must measured.

EPA received comments as a result of the interlaboratory study and other comments between release of 

the August 20, 2003, revision and the present. EPA has included with this letter a list of changes that 

EPA plans to make to EPA Method 1668A based on the revised QCAC and comments received. These 

changes are listed in the document titled: Changes to be made to EPA Method 1668A from the 8/20/03 

revision based on the Method 1668A interlaboratory validation study and comments and corrections 

received from laboratories, included with this letter. Because of changes to Method 1668A, primarily as a 

result of the revised QCAC, and based on your peer review, EPA plans to revise EPA Method 1668 to 

Revision B (EPA Method 1668B). In your review, please respond to the following questions:

1. Please review EPA's Report of the Method 1668A Interlaboratory Validation Study for the 

objectives of the study, the soundness of the technical approach, and the results produced.

2. Please render an opinion as to suitability for use of the data from the study for revision of the 

QCAC in Method 1668 to make it Method 1668B. 

3. If you have a suggestion for an improvement of the interlaboratory study report, please provide 

suggested text by section number.

4. If you believe that a given item should be clarified, please provide the clarification by section 

number or define the clarification needed to a sufficient level of detail that we can make the 

necessary changes. 

5. Please point out errors or omissions.
Method 1668A Interlab Study Peer Review

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EPA’s Charge to the third Peer Reviewer

EPA developed the following charge for the third and final peer reviewer:

BACKGROUND

EPA Method 1668A was developed in the late 1990s and a draft version was peer reviewed in 1999. 

Results of that peer review were published in February, 2000, in “Peer Review of Draft EPA Method 

1668, Revision A: Chlorinated Biphenyl Congeners in Water, Soil, Sediment, and Tissue by 

HRGC/HRMS.” A copy of the peer-review report is included. Draft EPA Method 1668A was revised as 

a result of the peer review and the revised version, dated December 1999 (EPA-821-R-00-002) and was 

published in January of 2000.

After publication of the December 1999 draft of EPA Method 1668A, EPA distributed the method 

extensively and planned an inter-laboratory validation. In preparation for the inter-laboratory validation, 

EPA updated, revised, and released Method 1668A on August 20, 2003. The interlaboratory validation 

study was conducted in 2003-2004 to validate the performance of EPA Method 1668A in municipal 

wastewater, fish tissue, and biosolids matrices. EPA received comments as a result of the interlaboratory 

study and other comments after the release of the August 20, 2003 revision of 1668A. Results of the 

study were analyzed and the “Report of the Method 1668A Interlaboratory Validation Study was drafted 

in 2006 and peer reviewed in 2008.” The major result of the interlaboratory study is a revision to the 

quality control acceptance criteria (QAQC) in EPA Method 1668A based on interlaboratory study results. 

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. EPA subsequently revised 1668A to 1668B based on the 

Interlaboratory study, and comments received on 1668A. EPA published 1668B with a list of changes in 

November 2008. These interlab criteria (Table 5-1 of the study report) replace the single-lab criteria, and 

are published in Table 6 of the revised working draft version of PCB-congener method, EPA Method 

1668B (Method VIII). 

“Method 1668A Interlaboratory Validation Study 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. 

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. 

EPA designed the Method 1668A Interlaboratory Validation Study in accordance with guidelines 

published by EPA and ASTM International (ASTM). These guidelines recommend a minimum of six 

complete data sets for evaluation of a method. To allow for some loss of data due to error, lost samples, 

outlier removal, or other unforeseen causes, EPA included 14 participant laboratories in the study. The 

study design and report are detailed in the document, “Method 1668A Interlaboratory Validation Study 

Report.”

The QC acceptance criteria developed in the interlaboratory method validation study did not allow the 

upper recovery limit for some congeners to be above 100 percent. The QC acceptance criteria were 

recently revised based on data from the interlaboratory study and data from two laboratories with 

extensive experience in use of Method 1668A. Data were provided by TestAmerica, Knoxville, 

Tennessee and AXYS Analytical Services, Ltd., Sidney, BC, Canada and are included in the attached 

excel file. Method 1668B was subsequently revised. A draft version of that as yet unpublished revision to 
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1668B is attached (See Method VIII). The revisions to that method are summarized in the introduction of 

the method. The addendum describing the revised QC acceptance criteria, “Addendum to the Method 

1668A Interlaboratory Validation Study Report Draft, August 2009” is included in this review. Data 

supporting the addendum can be found in the file, “OPRresults_Labs1&2.”

EPA peer reviewed the “Method 1668A Interlaboratory Validation Study Report” in late 2007 but was 

unable to obtain three peer reviews and is attempting to obtain a third peer review now.

CHARGE QUESTIONS

Please refer to the Review Guidelines and Formatting Instructions found on pages 4 and 5 of this 

Charge.

Please review the attached documents “Method 1668A Interlaboratory Validation Study Report” and 

“Addendum to the Method 1668A Interlaboratory Validation Study Report Draft, August 2009.” In your 

review, please respond to the following questions:

1. Please review EPA's Report of the Method 1668A Interlaboratory Validation Study and its 

addendum. Comment on the objectives of the study, the soundness of the technical approach, and 

the results produced.

2. Please render an opinion as to suitability for use of the data from the study and the addendum for 

revision of the QCAC in Method 1668 to make it Method 1668B. 

3. If you have a suggestion for an improvement of the interlaboratory study report or addendum, 

please provide suggested text by section number.

4. If you believe that a given item should be clarified, please provide the clarification by section 

number or define the clarification needed to a sufficient level of detail that we can make the 

necessary changes. 

5. Please point out errors or omissions.

6. Please provide any additional comments you would like to make regarding the documents under 

review.
Method 1668A Interlab Study Peer Review

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Peer Review

Dr. Michael Newman, Dr. Charles Wong and Dr. Kurunthachalam Kannan peer-reviewed the Study 

Report. This document has three parts. The first section is EPA’s responses to some common issues 

form more than one reviewer. The second section is EPA’s responses to specific comments from each 

reviewer. The last section contains the complete review of each reviewer. 

Section One: Selected Issues raised by more than one reviewer and EPA’s responses

Comment: It would have been useful if the laboratories were asked to provide measurement data on 

QC Check samples (Method 1668A, Section 7.15) from standardized reference materials 

(e.g., NIST SRMs or Canadian NRC SRMs).

Response: EPA asked laboratories to analyze spiked and typical Clean Water Act matrices, i.e., 

wastewater, tissue, and biosolids (sewage sludge.) These samples better represented the 

intended application of this method for environmental monitoring.

In addition, laboratories used to using the available QC check samples prepared in 

reagent waters or solvents might be overly familiar with their content. Method 1668A 

has been available for use by laboratories since 2000. Because it contains the suggestion 

for a QC Check sample in Section 7.15, many labs had been using a NIST SRM or NRC 

CRM, and might recognize the sample from having analyzed it previously. 

Comment: Laboratory participation was disappointing and this was the source of numerous issues. 

For example, six of 14 laboratories declined to participate in the biosolids analysis.

Response: The high failure-to-report rate is attributable to (1) the extensive effort needed to set up 

the extraction, concentration, cleanup, and HRGC/HRMS systems for determination of 

209 PCB congeners, (2) a lack of familiarity with the biosolids matrix, and (3) the 

voluntary nature of participation.

Comment: Outlier deletion, as discussed in Grubbs' paper and many others, should be done very 

thoughtfully according to clear rules. For example, the "outliers" detected based on the 

assumption of a normal distribution might come from a population more appropriately 

described as lognormal.

Response: Outlier tests were applied to both blind sample data (as presented in Table 4-1 of the 

report) and QC data. In both cases, Grubbs' test was applied conservatively, following 

guidelines for maximum outlier removal as specified by ASTM D2777-98.

Comment: Round all numbers to 3 significant figures.

Response: We rounded all results to three significant figures maximum in the final report.

Comment: On page 4, paragraph 2, please consider replacing "properly homogenized" with 

"homogenized."

Response: We have removed "properly."

Comment: It is extremely important in judging the generalizability of the report conclusions that the 

laboratory participation in generating the final QC criteria is very clearly discussed. At 

present, it is not easy to follow.

Response: Where possible, we revised the report to clarify this discussion. EPA discusses exclusion 

of a laboratory's data is in Section 3 of the report. EPA generally rejected a laboratory 

because the laboratory submitted incomplete data sets, insufficient raw data to verify a 

laboratory's results, or did not follow the method correctly.
Method 1668A Interlab Study Peer Review

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Comments: Move the definition of "Youden pair" from Section 2.5.1 to its first use in Section 2.4.

Response: We moved the definition to its first use.
Method 1668A Interlab Study Peer Review

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Section Two: Comments by each reviewer and EPA’s responses

PEER REVIEW COMMENTS FROM MICHAEL C. NEWMAN

NAME: Michael C. Newman, Ph.D.

AFFILIATION: 121 York Point Dr., Seaford, VA, also Professor at the College of William and Mary, 

Virginia Institute of Marine Science

DATE: September 18, 2007

COMMENTS ON: Method 1668A Interlaboratory Validation Study

I. GENERAL IMPRESSIONS

Comment 1: Overall, this Report of the Method 1668A Interlaboratory Validation Study is a 

sufficiently accurate, clear, and thorough analysis discussing the results of a very difficult 

task. The study objectives were to (1) characterize the performance of Method 1668A for 

multiple laboratories and matrices (wastewater, biosolid, fish tissue), and (2) evaluate and 

possibly revise the QC acceptance criteria. These objectives were met with some 

qualification. It is important to emphasize prior to discussing the specific qualifications 

that this effort does appear to be generally well-intended, -planned and -interpreted.

Response: No response needed.

Comment 2: Some challenges to applying the results do exist. The laboratory participation was 

disappointing and this was the source of numerous issues. For example, six of 14 

laboratories declined to participate in the biosolids analysis. An additional four produced 

unusable numbers, resulting in only four of 14 potential sets of results being applicable to 

the task at hand. For wastewater and fish tissue, only six laboratories produced the data 

used in this exercise. Four laboratories successfully analyzed all three sample types and, 

therefore, have a very strong influence on the results. The candidate laboratories 

included government and private organizations which might be expected to have 

differences in response; however, it is unclear from the report if the final subset was 

biased toward one group or another. The very high failure-to-report rate for this process 

suggests that failure to achieve useful analyses with 1668A warrants more discussion. 

Was the high failure rate normal for such voluntary validation exercises or a feature to be 

anticipated for Method 1668A as currently written?

Response: The high failure-to-report rate is attributable to (1) the extensive effort needed to set up 

the extraction, concentration, cleanup, and HRGC/HRMS systems for determination of 

209 PCB congeners, (2) a lack of familiarity with the biosolids matrix, and (3) the 

voluntary nature of participation. When participating voluntarily, a laboratory has no 

financial incentive to complete the work and submit a complete set of results. Thus, when 

the laboratory encounters difficulty, as with the biosolids matrix, the laboratory may 

simply stop the analysis rather than time-consuming corrective actions.

Comment 3: Some laboratories deviated from 1668A for some analyses. Also, outlier tests that require 

extremely thoughtful application were applied to cull away some data. Together, these 

features do not permit as high confidence in the study’s generalizations as might be 

desired.

Response: Within limits, laboratories were allowed to deviate from prescribed procedures under the 

performance-based provisions of Method 1668A. EPA applied the same outlier removal 

procedures that we have applied to EPA wastewater methods for the past approximately 
Method 1668A Interlab Study Peer Review

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20 years. These procedures are based on ASTM D2777-981, which ensures that outlier 

removal be limited to a small percentage of the data, and therefore the removal would 

have a small effect on the study results. These outlier procedures remove true outliers yet 

retain valid data. Additionally, some of the QC sample results removed as outliers were 

“better” (i.e., closer to 100% recovery) than the remaining results, which means that their 

removal would not portray method performance as unrealistically good. 

Comment 4: The studies conclusions are not unequivocal at this point. The resulting, central concern 

can be articulated with a quote from Stuart’s Basic Ideas of Scientific Sampling (1968): 

“the credentials of a sample are not only important for the interpretation of results, but are 

in principle the only information of value we can have in this respect.”

Response: Since EPA completed this study and peer review, EPA has obtained additional data that 

demonstrate that laboratories can overcome blank contamination, and otherwise perform 

Method 1668 under tighter specifications than we observed in this study.

Comment 5: My general conclusion is that the results are adequate for meeting the study objectives but 

should be augmented in the near future in order to enhance confidence in the general 

validity of the conclusions. Detailed below are specific examples from within the report 

and associated recommendations.

Response: EPA has since obtained additional data that demonstrate that laboratories can perform 

Method 1668 under tighter specifications than we observed in this study. Subsequent to 

this study, EPA has also obtained MDL data from several contract laboratories. In a 

study of data comparability between two laboratories on samples collected from the 

Passaic River in New Jersey, in which 151 PCB congeners were identified and measured, 

accuracy as measured by analysis of a NIST SRM was 15% or better. Recoveries of the 

PCB congeners ranged from 90% to 124% and averaged 105%; precision ranged from 4.2 

to 23%.

II. RESPONSE TO CHARGE QUESTIONS

1. Please review the EPA's Report of the Method 1668A Interlaboratory Validation Study for the 

objectives of the study, the soundness of the technical approach, and the results produced.

Comment 6: The Interlaboratory Validation Study attempts to characterize the performance of Method 

1668A in multiple laboratories and matrices (wastewater, biosolid, fish tissue), and to 

evaluate and possibly revise the QC acceptance criteria. As stated above, my overarching 

opinion is that the study met these goals but further work is required to enhance 

confidence in the generality of the conclusions. The goals for this complicated method 

were extremely challenging so the implied incremental generation of sufficient 

characterization/validation information is, in my opinion, reasonable. My suggestion that 

more work is required is not an oblique inference about the work quality of the study 

leaders. This was a very difficult task.

Response: We appreciate your understanding of the difficulty of conducting the study and the 

analyses.

2. Please render an opinion as to suitability for use of the data from the study for revision of the QCAC 

in Method 1668 to make it Method 1668B. 

 

1 American Society for Testing and Materials. 1998. Annual Book of ASTM Standards, Vol. 11.01. Standard 

Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D-19 on Water, ASTM 

D2777-98, October 1998.
Method 1668A Interlab Study Peer Review

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Comment 7: Yes. The study data are adequate, although lacking in some ways, for revision of the QC 

in Method 1668.

Response: EPA has since obtained additional data and revised the QC data in the 2009 version of 

Method 1668.

3. If you have a suggestion for an improvement, please provide suggested text by section number.

Comment 8: A wide range of suggestions and insights will be addressed below regarding the 

improvement of the study or existing study presentation.

Response: Our responses to this group of comments follow.

Comment 9: Page 6, Top paragraph and first full paragraph - This is the first place in the report that 

the issue of sample homogeneity emerged. It is also discussed in Section 4.1. Described 

here is the process in which a “sample processing laboratory was required to perform 

background and homogeneity analyses of both the biosolids and 

tissue matrices.” The text does not convince me that the samples 

were “homogeneous” in a strict sense relative to the analytes. 

For example, some fatty tissues might contain more analyte than 

others and similar-sized pieces of fatty tissues might not be 

homogeneously distributed in a well-mixed homogenate. The 

biosolid to which sand was added might experience densitydriven sample component segregation (i.e., sinking sand) so that 

the sample aliquot analyzed by the volunteer laboratory might 

not have been homogeneous (well-mixed?) relative to the 

analyte. There are very specific methods of determining this 

sample quality (well- or poorly-mixed) yet they are not applied 

by the sample processing laboratory. It is not clear how this 

might or might not have contributed to differences in results 

among laboratories. This concern is also inherently linked to 

sample aliquot size which 1668 prescribes versus and the actual 

sample sizes used by the volunteer laboratories. (I assume that the recommendations of 

sample sizes were based only on anticipated analyte concentrations in typical samples and 

the mechanics of the sample processing.)

The point being made above can be illustrated easily using the conventional explanation 

as depicted in the figure to the left. The analyte (dot) is homogeneously distributed in 

this figure=s top panel; therefore, sample aliquot size illustrated in the figure as different 

size boxes will not influence the measured analyte concentration in an analyzed aliquot. 

(Homogeneity is a material quality in which the analyte is distributed in a completely 

homogeneous manner throughout the material.) In the bottom panel, a heterogeneous 

material is depicted in which sample aliquot size becomes critical depending on whether 

the heterogeneous material is well- or poorly-mixed. If sample aliquot sizes are too small 

for a poorly-mixed, heterogeneous material (small squares in figure bottom panel), 

variation among reported analyte concentrations will be higher than that derived with 

aliquot sizes of sufficient size (large square in the bottom panel) (Newman 1995). To 

conclude that a heterogeneous sample is sufficiently well-mixed to permit use of a 

specified aliquot size (e.g., 20 g of fish tissue) requires some conventional calculations. 

The homogeneity analyses described in this report do not include the associated statistics 

permitting such decisions. This is especially relevant to the biosolid and tissue samples, 

and with sufficiently high solids content, also to wastewater samples. The pertinent 

approach to this problem and associated statistical computations are described in classic 
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papers by Visman (1969), Ingamells (1973) Ingamells and Switzer (1974), and Wallace 

and Kratochil (1987). 

Response: We are confident that the samples were homogeneous. although using the standard that 

you have developed, those that you reference, and existing data, it is impossible to prove. 

The sample-processing laboratory has extensive experience in sample homogenization, 

and homogenization procedures are provided in Section 11.7 of Method 1668A.

Comment 10: Page 8, Top Bullets - The laboratories were told to report all “>nm” values as quantified 

concentrations and the statistical analysis of these measured values was treated such that

all “>nm” values possessed the same quality of measurement. It is incorrect to assume 

that all values above “nm” had the same measurement quality (e.g., Keith et al. (1983)). 

The relative uncertainty of a measured value can be estimated relative to the standard 

deviation of the analytical process baseline noise (See also figure on page 6),

N

Z

lative Uncerta y

2

Re int (%)  100

where Z= Z-statistic value for a confidence level of  (conventionally 95%), and N is the 

measured signal expressed in units of multiples of the standard deviation of the baseline 

noise, e.g., N=3 and 10 are associated with the signal at the detection limit and limit of 

quantification, respectively. Interpretation of the study results is very difficult if this fact 

is not addressed in the discussions and shown in graphs of results for the various samples 

and sample types. An example of this will be discussed below relative to the figures in 

the report.

Response: An analysis of this type would require knowledge of the error function for the analytical 

system in each laboratory in the interlab study. Differences in sample amounts and 

volumes, and final extract volumes, used by the laboratories, as these amounts and 

volumes would shift the results upward or downward on the concentration-error function 

would be further complicate the suggested analysis. Therefore, this type of analysis is 

impractical and beyond the scope of the study.

Comment 11: Page 12, First paragraph after bullet list and also last sentence - Here and elsewhere, my 

assumption is that the study results are intended to provide measurement quality 

characteristics anticipated for “in-control” analyses of the specified sample types by a 

laboratory represented by the initial 14 laboratories. It is critical that the population for 

which these final QC criteria are generated be clearly defined given the variations in 

reporting, method application, and data selected for final QC calculations. 

Response: EPA intends that any single laboratory using Method 1668, not a hypothetical population 

derived from the laboratories in the study, will use the final QC acceptance criteria. Our 

experience is that QC acceptance criteria generated from inter-laboratory data are 

approximately a factor of 2 broader than the same criteria generated from data from a 

single laboratory. Note that EPA derived the QC acceptance criteria for the recovery of 

labeled compounds from results from all three matrices in the study. And EPA derived 

the QC acceptance criteria for IPR and OPR sample recovery from results of spiked water 

and solid matrices. Therefore, although the reduced data set may narrow the criteria 

somewhat, the QC acceptance criteria are sufficiently generous that future laboratory will 

not have difficulty meeting them.

Comment 12: Here and elsewhere, Grubbs’ single outlier test (Grubbs 1969) is applied to cull away 

apparent outliers. Outlier deletion, as discussed in his paper and many others, should be 
Method 1668A Interlab Study Peer Review

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done very thoughtfully according to clear rules. For example, the “outliers” detected 

based on the assumption of a normal distribution might come from a population more 

appropriately described as lognormal. The “outlier” would be falsely removed based on 

the assumption of the wrong distribution. The rules for handling outliers in Grubbs 

(1969) and other papers should be considered prior to applying any outlier tests. The 

results of such application should then be described in the report. Classic papers that 

might be useful are Anscombe (1960) and Rosner (1983). As multiple outliers might be 

masking each other in these data sets, Rosner’s method (1983) for multiple outliers might 

be useful. Personally, I think that Woodworth’s (2004) classification of outliers and the 

associated approach based on inter-quartile ranges is more relevant as it is more open to 

intelligent application than the above hypothesis-based, parametric tests. This point will 

be repeated later in discussing the absence of quartile (Q1 and Q3) values in tables of 

summary statistics.

Response: Outlier tests were applied to both blind sample data (as presented in Table 4-1 of the 

report) and QC data. In both cases, EPA applied the Grubbs test conservatively, 

following guidelines for maximum outlier removal as specified by ASTM D2777-98. In 

the case of sample data, these results were only used to calculate summary statistics of the 

sample results by chlorination level, and therefore outlier removal would not strongly 

affect conclusions about method performance. For QC data, outlier removal was limited 

to one result per congener. For labeled compound samples, there were a greater number 

of results, and a maximum of two results were removed per congener. As stated 

elsewhere, EPA had no strong evidence that these data did not follow a normal 

distribution, and therefore EPA’s use of the Grubbs test is appropriate. We are familiar 

with outlier tests based on the interquartile range, but these tests tend to classify an 

unrealistically large percentage of data as outliers.

Comment 13: Page 13, Section 4.2.1, line 4 - The “homogeneity” was found acceptable. Respectfully, 

my guess would be that this statement is correct; however, I disagree that enough 

information was extracted to make a definitive statement. I base this disagreement on the 

material already presented above.

Response: We agree that the samples could have been homogeneous but we lack sufficient data to 

prove it. At this point, we must assume homogeneity because further testing is not 

possible.

Comment 14: Page 14, Table 4-1- Here and elsewhere, it would be very helpful to have the first and 

third quartile values (Q1 and Q3) or, minimally, the inter-quartile range (|Q3-Q1|) in the 

table. One immediate use would be to apply inter-quartile range-based methods to 

explore potential outliers or outlying laboratories. The inclusion of these statistics is 

highly recommended.

Response: Application of these ranges would alter the results or conclusions significantly or 

appreciably alter the QC acceptance criteria to justify the effort to do so.

Comment 15: As an aside, the differences between means and medians for congeners with 3 to 8 

chlorination levels (biosolids and tissues) suggest a skewed sample set. Are these data 

skewed for any reason or is this simply a spurious trend in the tabulated data?

Response: We agree that the distribution of results presented in Table 4-1 were often skewed. This 

was most likely due to the difference in prevalence and coelution of the various 

congeners within each chlorination level. This skewness was not observed with a single 

congener in the QC sample data, and therefore this skewness would not reflect a 

departure from the normality assumption used in the developing QC acceptance criteria.
Method 1668A Interlab Study Peer Review

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Comment 16: Page 29, Last line of text - I generally agree but feel that a less definitive, and more 

conditional, conclusion can be reached.

Response: Because you did not provide an alternative conclusion, we continue to conclude that QC 

acceptance criteria developed from multi-laboratory data more accurately reflect the 

performance of an analytical method than QC acceptance criteria developed from singlelaboratory data. The QC acceptance criteria in Method 1668A were estimates resulting 

from testing the method in a single laboratory. The acceptance criteria in revise Method 

1668 are based on data from several laboratories. 

Comment 17: Appendix A. Section 1.0., 95% Confidence Intervals - The section begins by describing 

conventional, parametric calculations for all congeners in all matrices at all 

concentrations. Was the assumption of normality tested and met, or found to be 

unimportant? Some discussion seems warranted.

Response: While a statistical test of normality is not extremely powerful for a small set of data, the 

spiked QC sample results for nearly all congeners passed the Shapiro-Wilk test at alpha = 

0.05. Those that did not pass generally had one or two extreme results, or systematic 

differences in recovery between matrices. A normal distribution of QC spike recoveries is 

also consistent with experience with past environmental studies. While results of 

environmental samples tend to follow a skewed distribution, percent recoveries of QC 

samples spiked at approximately the same concentration level tend to follow a more 

symmetric distribution.

Comment 18: Appendix A Section 1.0, Application of Grubbs’ outlier test - Again, outlier tests should 

be used very carefully and interpretation of any consequent data sets based on truncated 

distribution statistics (see Grubbs (1969) and other sources for discussion).

Response: Outlier tests were applied to both blind sample data (as presented in Table 4-1 of the 

report) and QC data. In both cases, EPA applied the Grubbs’ test conservatively, 

following guidelines for maximum outlier removal as specified by ASTM D2777-98. In 

the case of sample data, these results were only used to calculate summary statistics of the 

sample results by chlorination level, and therefore outlier removal would not strongly 

affect conclusions about method performance. For IPR and OPR data, outlier removal 

was limited to one result per congener. For labeled compound data, there were more 

results, and a maximum of two results were removed per congener. As stated elsewhere, 

EPA had no strong evidence that these data did not follow a normal distribution, and 

therefore EPA’s use of the Grubbs’ test is appropriate. 

Comment 19: As a simple example, what is the n in the equation given in Section 2.0? This is not as 

easy a question to answer as might be suspected since some observations were thrown 

out.

Response: For most analytes, the n in the equation given in Section 2.0 was 24, as stated in Section 

5.5 on page 27 of the report. Two outliers each were removed for labeled congeners 111 

and 178, so n would be 22 for these two congeners. 

Comment 20: Also, was the assumption of a normal distribution (upon which Grubbs’ test depends) 

appropriate?

Response: The assumption of normality was appropriate because the testing was performed in the 

center of the analytical range. See also the response to the comment on Appendix A. 

Section 1.0., 95% Confidence Intervals (Comment 17), above.

Comment 21: Finally, what Type I error rate was selected? Likely, the typical 0.05 was used yet one 

could argue for a more conservative error rate.
Method 1668A Interlab Study Peer Review

April 2010 13

Response: Yes, EPA selected a Type I error rate of 0.05. A more conservative Type I error rate 

would yield QC inappropriately wide acceptance criteria, and would not be useful in 

detecting laboratories that are not performing the method appropriately or are out of 

control.

4. If you believe that a given item should be clarified, please provide the clarification by section number 

or define the clarification needed to a sufficient level of detail that we can make the necessary 

changes. 

Comment 22: A wide range of issues will be addressed below as suggestions for clarifying presentation.

Response: Our responses to this group of comments follow.

Comment 23: Section 1.2, Line 3 - Change “seen” to “seven”?

Response: We have corrected this.

Comment 24: Page 4, Paragraph 2 - Please consider replacing “properly homogenized” with 

“homogenized.” The reasons are given above for removing the vague word from this 

sentence. One could easily argue that the samples were not shown to be “properly 

homogenized.”

Response: We have removed "properly."

Comment 25: Page 5, Paragraph 2 of Section 2.4 - The term, “Youden pairs” is first used here but not 

defined until Section 2.5.1. Please move the definition up to the first use of the term.

Response: We have moved the definition to its first use.

Comment 26: Table 2-1 (page 4) and Table 3-1 (page 12) - It is extremely important in judging the 

generalizability of the report conclusions that the laboratory participation in generating 

the final QC criteria is very clearly discussed. At present, it is not easy to follow. 

Generally, one starts with 14 laboratories (government and private) and ends up with data 

from 4 to 6 labs. It isn’t easy to judge how (1) withdrawal of the various laboratories 

from providing results, (2) the study author’s judgment about reported data usefulness, 

and (3) the final data inclusion influences the statistical population of laboratories for 

which inferences about QC are being made. Several issues to be addressed include the 

following. Are the QC conclusions relevant to only the best or best equipped 

laboratories? Are they representative of primarily government laboratories, private 

laboratories, or both? A very clear and concise summary addressing this linchpin issue is 

needed. In developing this summary, discussion of the influence of outlier rejection is 

also warranted.

Response: Where possible, we revised the report to clarify this discussion. EPA discusses exclusion 

of a laboratory's data is in Section 3 of the report. EPA generally rejected a laboratory 

because the laboratory submitted incomplete data sets, insufficient raw data to verify a 

laboratory's results, or did not follow the method correctly. Exclusion of these 

laboratories does not mean that the conclusions are relevant only to the best laboratories, 

but to laboratories that perform the method correctly. 

Comment 27: Page 8, Section 2.7, first paragraph - The requirements were given for calibration curves 

but the laboratories interpreted the requirements differently. I saw no obvious reason for 

the misinterpretation; however, some thought should be put into rewriting the associated 

text so it is even clearer. Also, relative to Section 2.7.1. and 2.7.2, were the consequences 

of any of the various deviations quantifiable? For example, the differences due to 
Method 1668A Interlab Study Peer Review

April 2010 14

different aliquot weights used by the various laboratories might be quantified with the 

methods cited above for Page 6, Top paragraph and first full paragraph.

Response: We were unable to establish any clear relationship between the various deviations and the 

results. The primary reason is that neither we nor the laboratories linked a specific 

deviation to specific data. If a laboratory chose to calibrate its analytical system different 

from the calibration suggested in Method 1668A, or chose not to follow a procedure, we 

had no way to force the laboratory to re-calibrate or to repeat the analysis.

Comment 28: Page 9, Section 2.7.2 - Different problems were encountered by different laboratories. 

Different weight normalizations were done by the different laboratories. It isn’t clear to 

me how this directly influenced statistical estimates and associated inferences. Also, the 

numerous deviations suggest that even more clarity needs to be placed into 1668A. 

Perhaps more discussion is needed about what deviations should not be considered.

Response: In principle, we agree. Although Method 1668A is clear about the amount of material to 

be extracted and the final extract volume, in revising the method we have provided more 

clarity about where flexibility is allowed and not allowed in conducting the method. We 

control laboratories under contract to us, so these deviations would not occur with our 

routine data gathering nor should they with others who contract for lab work. In this 

interlab study, although we asked the participants to follow Method 1668A, we had no 

control over the deviations that these volunteers eventually chose. Therefore, we used the 

data as provided except where we could justify finding it lacking, inconsistent with other 

data, or outlying.

Comment 29: Page 11, Section 3, first paragraph - This section begins by describing three laboratories 

that did not submit data. It is important to know if non-reporters are considered a biased 

or random subset of the population of possible responders. Was there a risk of biased 

sampling of possible laboratories using this method as a result of biased non-response?

Response: No. The laboratories that did not submit data were government laboratories, but we have 

no evidence that their skill level differs from other private or commercial laboratories.

Comment 30: Page 11, Section 3, fourth bullet - How was this issue handled during the analysis of 

results? Would omission of an outlier laboratory create a biased sample of laboratories?

Response: Results from the laboratory were not considered in the analysis for the reasons given in 

this bullet in the report. The omission of this laboratory would not bias the results. We 

could not question the laboratory about poor recoveries or other problems. In addition, 

because the laboratory is no longer in business, the laboratory is no longer part of the 

population of laboratories performing this method. This laboratory is not an "outlier 

laboratory." We did not have any such laboratories because there were not enough 

laboratories in the study with which to perform an outlying laboratory test.

Comment 31: Page 15, Table 4-2 and all other tables - Univariate statistics are applied throughout these 

tables. Some graphs suggest skewed distributions for some data. Was the assumption of 

normality or the robustness of conclusions to violations of this assumption assessed?

Response: Many of the univariate statistics and graphs combine congeners, and as such the 

distribution of results would be affected by differences in the prevalence of congeners in 

the environment and in coelution. While a statistical test of normality is not extremely 

powerful for a small set of data, the spiked QC sample results of most congeners passed 

the Shapiro-Wilk test at alpha = 0.05. Those that did not pass generally had one or two 

extreme results, or systematic differences in recovery between matrices. A normal 

distribution of QC spike recoveries is also consistent with experience with past 

environmental studies. While results of environmental samples tend to follow a skewed 
Method 1668A Interlab Study Peer Review

April 2010 15

distribution, percent recoveries of QC samples spiked at approximately the same level 

tend to follow a more symmetric distribution.

Comment 32: Page 16, first lines - This discussion is too speculative in my opinion.

Response: The sample-preparation lab prepared a solution containing all of the congeners. The 

laboratory then lined up all of the sample bottles, filled each with POTW effluent, and 

spiked an aliquot of the solution into each bottle. The lab then analyzed two of the 

samples and found all congeners to be present in the concentrations spiked (within 

experimental error). The lab shipped the bottles to the participant labs. The mean of the 

concentrations found by the participant labs for the mono- and di- CB congeners was 3 

and 54 %, respectively, whereas the mean for the tri- through deca- CB congeners ranged 

between 68 and 96 %. The most logical explanation is that the congeners were lost during 

transit or when the bottles were opened at the participant laboratories. Remember, EPA 

recovery corrected all results using the labeled compounds as reference, and labeled 

compound recoveries were within the normal range, so the losses were real and occurred 

prior to the time the samples were spiked with labeled compounds at the participant labs.

Comment 33: Page 17, Figure 4-1 (and also 4-3, 4-5, and 4-7) - As dictated by the generally-accepted 

equation given above, the relative uncertainty of a measurement will rapidly increase as 

concentration decreases. The following figure 

gives the general shape of such a relationship. 

Depending on the positions of the detection 

limit (LOD in this figure) and limit of 

quantitation (LOQ in this figure) (from 

Newman (1995)), the variation in the y 

direction as a function of measured 

concentration would be totally expected based 

on this relationship. Would it be possible to 

insert vertical lines at the LOD and LOQ in 

these figures so a reader could judge this issue? 

As a good example of how this would help, it is 

difficult to decide if the explanation for the 

Figure 4-5 outliers (Page 20, first few lines) is 

reasonable or whether the variations at low concentrations (in lower part of the Region of 

Less-Certain Quantitation? are what you would expect for any measurement process.

Response: It would not be possible to insert lines at the LOD and/or LOQ in Figure 4-1 or the other 

figures, because different participant labs used different sample volumes and different 

final extract volumes. Further, and most unfortunate, the congeners at low spike 

concentration were also those at low chlorine level. Therefore, the apparent resemblance 

to the Newman model is misleading.

Comment 34: Page 18, Figure 4-2 (and also 4.4, 4.6) - These figures do not convey any useful 

information. Relative Standard Deviations (e.g., 4-3) are much more useful for 

conveying the associated information (that variation can change with concentration).

Response: These figures can be useful in determining where in the concentration scale absolute 

variability begins to increase linearly with concentration.

Comment 35: Page 21, Table 4-4 - A simple mistake was made here such that the “# pairs” column has 

two digits after the decimal point, e.g., 32.00 pairs.

Response: We have corrected the number of pairs to a whole number.
Method 1668A Interlab Study Peer Review

April 2010 16

Comment 36: Page 29 - The conventional “This page intentionally blank” statement is made but, in fact, 

there is text on the page.

Response: We have corrected the offset between this page and the previous page.

5. Please point out errors or omissions.

Comment 37: Please see comments above.

Response: We responded to these in the preceding section.

III. SPECIFIC OBSERVATIONS

[none submitted]

IV. ADDITIONAL REFERENCES

1. Anscombe, F.J. 1960. Rejection of outliers. Technometrics 2: 123-147.

2. Grubbs, F.E. 1969. Procedures for detecting outlying observations in samples. Technometrics 11: 

1-21.

3. Ingamells, C.O. 1974. New approaches to geochemical analysis and sampling. Talanta 21: 141-155.

4. Ingamells, C.O. and P. Switzer. 1973. A proposed sampling constant for use in geochemical 

analysis. Talanta 20: 547-568.

5. Keith, L.H., W. Crummett, J. Deegan, Jr., R.A. Libby, J.K. Taylor, and G. Wentler. 1983. 

Principles of environmental analysis. Anal. Chem. 55: 2210-2218.

6. Newman, M.C. 1995. Quantitative Methods in Aquatic Ecotoxicology. Lewis/CRC Publishers, 

Boca Raton, FL, pp. 426. 

7. Rosner, B. 1983. Percentage points for a generalized ESD many-outlier procedure. Technometrics 

25: 165-172.

8. Stuart, A. 1968. Basic Ideas of Scientific Sampling, Hafner Publishing Co., New York, pp. 99.

9. Visman, J. 1969. A general sampling theory. Mat. Res. Stand. 9:9-64.

10. Wallace, D. and B. Kratochvil. 1987. Visman equations in the design of sampling plans for 

chemical analysis of segregated bulk materials. Anal. Chem. 59: 226-232.

11. Woodworth, G.G. 2004. Biostatistics. A Bayesian Introduction. John Wiley & Sons/
Method 1668A Peer Review

October 2007 17

PEER REVIEW COMMENTS FROM

CHARLES S, WONG

NAME: Charles S. Wong, Ph.D.

AFFILIATION: University of Alberta, Dept. of Chemistry

DATE: September 18, 2007

COMMENTS ON: Method 1668A Interlaboratory Validation Study

I. GENERAL IMPRESSIONS

Comment 1: This document is a synopsis of the interlaboratory validation study intended to improve 

EPA method 1668A, a protocol for congener-specific measurement of polychlorinated 

biphenyls (PCBs) in water, soil, sediment, biosolids, and tissue by high resolution gas 

chromatography/high resolution mass spectrometry. Fourteen laboratories were invited 

to participate, and samples as well as instructions and required reporting information was 

provided to them. Reported data was compiled and summarized.

Comment 2: The document provides a useful but succinct summary of the interlaboratory validations 

very large dataset. Sufficient detail is provided on the background on Method 1668A and 

the methodology used to invite participating laboratories to ascertain the effectiveness of 

the validation study in characterizing measurement of PCBs in the samples chosen. 

Methods for selection of samples for the laboratories are clear and reasonable, including 

explicit discussion of limitations for logistical and technical purposes. A clear and 

detailed discussion of deviations from the required protocol is evident, so that the reader 

can clearly understand limitations in the reported data. For the most part, the discussion 

of the usable data is clear and pertinent to the intended goal of determining possible 

changes to the protocol and to acceptance criteria.

Response: No response needed.

II. RESPONSE TO CHARGE QUESTIONS

1. Please review the EPA’s Report of the Method 1668 Interlaboratory Validation Study for the 

objectives of the study, the soundness of the technical approach, and the results produced.

Comment 3: The objectives of the study are sound. Should Method 1668A be revised, and if so, what 

protocol changes and/or acceptance criteria should be modified? This can only be done 

with an interlaboratory validation of a standard set of samples.

Comment 4: Sample selection is reasonable in my opinion. Biosolids and tissues were prepared by 

EPA at two distinct concentrations, properly homogenized, and shipped to each 

laboratory with appropriate safeguards against decompositions to ensure that that the 

integrity of the samples upon receipt by the participating laboratory was maintained. 

While it would have been ideal to have wastewater samples with PCBs shipped to the 

laboratories, it is reasonable to assume that testing of Method 1668 capabilities would not 

be feasible. Thus, providing instructions to spike the wastewater effluent shipped to the 

laboratory was reasonable.

Response: The wastewater samples were spiked with PCBs at a sample-preparation laboratory. The 

participant laboratories spiked the 13C-labeled PCBs in their laboratory.
Method 1668A Interlab Study Peer Review

April 2010 18

Comment 5: As previously stated, deviations from the method were characterized in a succinct but 

meaningful manner. This allowed the flagging of suspect data as unusable for further 

purposes. Many of the deviations noted had details on the underlying rationale, e.g., 

problems with cleanup of samples encountered by some of the laboratories. However, 

not all such deviations were detailed in this manner, which I would have found useful. It 

is troubling that there were sufficient problems in sample processing, measurement, and 

even data reporting (e.g., no calibration reporting from one laboratory???) that less than 

half the data from the participating laboratories was usable (Table 3-1), as part of the 

point of having a large number of independent participants was to assess the robustness of 

both the overall Method as well as the validation for improving the Method. That having 

been said, I feel that the most problematic deviations were flagged unusable, and that the 

usable data is sufficient for the intended purpose.

Response: You have verified our conclusions concerning problems encountered with the samples 

and data reporting.

Comment 6: It would have been useful if the laboratories were asked to provide measurement data on 

QC Check samples (Method 1668A, Section 7.15) from standardized reference materials 

(e.g., NIST SRMs or Canadian NRC SRMs) in addition to the other QC data, e.g., OPR 

samples in reagent water, and blanks. This is important because as noted in the report, 

PCB concentrations in the shipped samples were unknown, and having this check would 

be useful. If this was actually done, this was not clear from the report.

Response: EPA asked laboratories to analyze spiked and typical Clean Water Act matrices, i.e., 

wastewater, tissue, and biosolids (sewage sludge.) These samples better represented the 

intended application of this method for environmental monitoring. 

In addition, laboratories used to using the available QC check samples prepared in 

reagent waters or solvents might be overly familiar with their content. Method 1668A 

has been available for use by laboratories since 2000. Because it contains the suggestion 

for a QC Check sample in Section 7.15, many labs had been using a NIST SRM or NRC 

CRM, and might recognize the sample from having analyzed it previously. 

Comment 7: The reported results provided a good summary of the variability observed with PCB 

measurement in the various matrices supplied to the participating laboratories. This 

information is most useful for resetting IPR/OPR guidelines. The summary and 

recommendations appear reasonable, e.g., using average relative responses under specific 

circumstances, noting poorer and more variable recoveries for lower chlorinated 

congeners, and somewhat tighter recovery windows particularly for heavier congeners.

Response: No response needed.

2. Please render an opinion as to suitability for use of the data from the study for revision of the QCAC 

in Method 1668 to make it Method 1668B.

Comment 8: I feel that this data, despite limitations noted above and in the report, are of sufficient 

quality to revise the method for the next version of this Method.

Response: We agree that EPA should move forward with the revised QC acceptance criteria and 

other changes to Method 1668A.

3. If you have a suggestion for an improvement, please provide suggested text by section number.

Comment 9: In Method 1668, Section 23.0, page 61: It is useful to provide the PCB numbering 

system used, to avoid confusion. I realize that CAS registry numbers are provided, which 
Method 1668A Interlab Study Peer Review

April 2010 19

eliminate ambiguity; however, most discussion of PCBs is by the numbering system, so 

having that clarified explicitly in the document is very useful. See comments below on

references.

Response: It is important to link the PCB numbers to the widely-used and internationally-recognized 

CAS Registry numbers to minimize ambiguity about the identity of specific congeners.

4. If you believe that a given item should be clarified, please provide the clarification by section number 

of define the clarification needed to a sufficient level of detail that we can make the necessary 

changes.

Comment 10: It would be useful to provide more details on the instructions to the participating 

laboratories as how to spike the shipped wastewaters with PCBs.

Response: The wastewater samples were spiked at the sample-processing laboratory. See Section 

2.5.2 on page 6 of the report.

Comment 11: Also, as stated previously, additional QC data from the laboratories should be given to 

help ensure that the usable data was of high quality.

Response: None of the participant laboratories submitted data for a QC Check sample. Because the 

participant laboratories were volunteers, EPA could not require analysis of a QC Check 

sample.

5. Please point out errors or omissions:

Comment 12: In the document “Changes to be made to EPA Method 1668A from the 8/20/03 revision 

based on the Method 1668A interlaboratory validation study and comments and 

corrections received from laboratories “, Under Quality control acceptance criteria, Table 

5-1 from the interlaboratory report should replace Table 6 not Table 5 in Method 1668A.

Comment 13: In Method 1668A, Section 13.5.1.4, page 48, typo: should read If calibration is not 

verified not If calibration is not verified.

Comment 14: In Method 1668A, Section 22.0, page 59: the title for reference 4 should be provided for 

consistency.

Comment 15: In Method 1668A, Section 22.0, page 60: the title for reference 18 should be provided 

for consistency.

Comment 16: In Method 1668A, Appendix A, Section 2.1, page 110: typo, should be individual 

congeners not individual congene4rs.

Response: We have made these corrections.

III. SPECIFIC OBSERVATIONS

These comments refer to the report on the interlaboratory study.

Comment 17: Page 14, Section 4.2, Table 4-1 - I do not feel that reporting of concentrations to up to 5 

significant figures is justifiable. The GC used must have either a splitless or on-column 

injector (Method 1668A, Section 6.9). The latter in particular does not have sufficient 

precision to justify 5 significant figures. Three or perhaps four significant figures is the 

maximum that should be present. 
Method 1668A Interlab Study Peer Review

April 2010 20

Response: We have rounded all results to three significant figures maximum.

Comment 18: Page 14, Section 4.2, Table 4-1 - In addition, while providing a mean, median, and 

maximum for concentrations of usable data is useful, it is difficult to assess the 

distribution of data. This is important, as the fairly large differences between means and 

medians suggest skewed data distributions, which are suggested by the various figures of 

variability. It would be useful to provide a box plot of this data to make assessment much 

easier for the reader.

Response: A box plot would be so busy that the reader would not be able to discern useful 

information.

Comment 19: Page 21, Section 4.6, Table 4-4 - The same comments regarding significant figures from 

my comments on Section 4.2, page 14, Table 4-1 hold here as well.

Response: We have rounded all results to three significant figures maximum. 

Comment 20: Inclusion of the interlaboratory reports Appendix A, Statistical Procedures Used to 

Develop QC Acceptance Criteria in the Method as an Appendix may be useful.

Response: This is not necessary because the interlaboratory study report has been posted on the 

CWA methods website. It would serve no useful purpose to detail how the QC 

acceptance criteria were developed in an Appendix to the method. EPA plans to provide 

the report as a part of the rulemaking package so the information would be available to 

readers interested in how EPA developed the QC acceptance criteria.

Comment 21: The following comment is suggested for improvement of Method 1688A, and not the 

interlaboratory report:

Comment 22: In Section 5.0 on Safety, pages 5-7, some explicit mention should be made about the use 

of appropriate biohazard containment for samples which are biosolids, as these are likely 

to host pathogens which are a potential danger until rendered harmless by disinfection. 

This would include preparation of raw samples in a biosafety cabinet until pathogens are 

killed (e.g., by soaking in solvents), appropriate safety garments and emergency 

procedures for accidental spills of pathogenic material, and warning about covering all 

materials containing solvents in biosafety cabinets to prevent solvent fumes that can pose 

an explosion hazard in biosafety cabinets that do not vent to exterior air.

Response: We agree and have added a sub-section on biohazards to Section 5.

IV. ADDITIONAL REFERENCES

1. Mills, S.A., III; Thal, D. A., Barney J. (2007) -A summary of the 209 PCB congener nomenclature, 

Chemosphere 60, 1603-1612.
Method 1668A Peer Review

October 2007 21

PEER REVIEW COMMENTS FROM 

KURUNTHACHALAM KANNAN 

NAME: Kurunthachalam Kannan, Ph.D.

AFFILIATION: Research Scientist. Wadsworth Center, New York State Department of Health & 

Professor, Department of Environmental Health Sciences. State University of New York at Albany. 

Empire State Plaza PO Box 509. Albany, NY 12201-0509

DATE: March 24, 2010

COMMENTS ON: Method 1668A Interlaboratory Validation Study

Review of “Method 1668A Interlaboratory Validation Study Report” and “Addendum to the 

Method 1668A Interlaboratory Validation Study Report Draft, August 2009.

Method validation is an integral part of a good analytical laboratory practice. The method 1668A 

validation study was intended to provide the Environmental Protection Agency (EPA) with sufficient 

amount of data to evaluate the performance of the method. The method 1668A was originally validated 

in 2000, in a single laboratory study. However, the guidelines published by the EPA and American 

Society for Testing and Materials (ASTM) recommend a minimum of six complete data sets for 

validating a method. The objectives of the validation study were to characterize the performance of the 

Method 1668A in multiple laboratories and matrices and to evaluate and revise the Quality Control (QC) 

acceptance criteria. The objectives of the study are sound and logical. The multi-laboratory validation 

study is a requirement to meet the national and international criteria to ensure reliability, consistency, and 

accuracy of the analytical data generated using the Method, especially when the method is used in the 

EPA’s Clean Water Act programs. The major outcome of the validation study was a revision to the 

quality control/quality acceptance (QA/QC) criteria in EPA Method 1668A.

It is agreed that method validation studies are time and resource consuming and EPA’s effort to perform 

this validation is highly commendable. Coordination of activities among several participating, volunteer, 

laboratories is a daunting task. EPA’s efforts on this method validation are significant; nevertheless, data 

generated from this study are under-utilized and with the existing data it is possible to generate additional 

information required for validation.

1. Please review EPA's Report of the Method 1668A Interlaboratory Validation Study and its 

addendum. Comment on the objectives of the study, the soundness of the technical approach, and 

the results produced.

Comment: The objectives of the study are sound and logical. The multi-laboratory validation study 

is a requirement to meet the national and international criteria to ensure reliability, 

consistency, and accuracy of the analytical data generated using the Method, especially 

when the method is used in the EPA’s Clean Water Act programs. The major outcome of 

the validation study was a revision to the quality control/quality acceptance (QAQC) 

criteria in EPA Method 1668A. 

The overall technical approach, ranging from selection of laboratories, sample selection, 

sample analysis, data reporting, data review, and validation, is good, although there are 

some potential areas for improvements. A major drawback with the report is on the 

biosolids data set. The objective of the study was to have 6 complete data sets, as per the 

EPA/ASTM guidelines, to validate the method. Only 4 complete data sets have been 

available for biosolids (Table 4-1), although validation for biosolids appears to be an 

important goal for the method, as indicated on the title. Furthermore, the data submitted 

by the 4 laboratories for biosolids are highly variable and no ‘in-depth’ statistical analysis 

of data set has been made to determine accuracy and precision of the data submitted for 
Method 1668A Interlab Study Peer Review

April 2010 22

biosolids. In other words, the Method has not adequately met the objectives to validate 

the analysis of PCBs in biosolids. Many international organizations such as the 

International Organization for Standardization (ISO) require at least eight complete data 

sets to validate the methods. Because the data set available for biosolids does not meet 

the EPA/ASTM guidelines, validation of the Method 1668A for biosolids is 

incomplete/inadequate.

Response: Reasons for receiving only four data sets for biosolid samples are in Section 3 of the 

Report. EPA believes that the high failure-to-report rate may be attributable to (1) the 

extensive effort needed to set up the extraction, concentration, cleanup, and 

HRGC/HRMS systems for determination of 209 PCB congeners, (2) the difficulty and 

lack of familiarity with the biosolids matrix, and (3) the voluntary nature of participation 

in the study. Given that four labs were able to analyze the biosolids samples successfully, 

EPA chose to use the data. Although the ASTM guideline, D2777, generally requires six 

complete data sets for method validation, ASTM has approved a means for taking 

exceptions to the requirement for six complete data sets. The Association for Official 

Analytical Chemists, AOAC, has similar requirements and exceptions.

Agency guidelines for validation of chemical methods were developed by the Forum for 

Environmental Measurement, FEM, as guidance, not requirements. These guidelines do 

not specify a minimum number of labs for a validation, and refer to D2777 as an example 

protocol. Each program office (e.g., EPA’s Office of Water) may apply the guidelines as 

appropriate to specific situations, and to the extent practicable, our validations are 

consistent with the FEM guidelines and other guidelines.

In the United States, validation principles developed AOAC and ASTM are more widely 

followed than the international ISO guidelines. EPA did not desire to validate Method 

1668 internationally, as was done with EPA Method 1613 for chlorinated dioxins and 

furans.

Note: The four lab comment is repeated as comment “m” in section 5, and comment “a” 

in section 6 of this review. 

Comment: The second major concern with the validation study results is that the data 

analysis/review is focused heavily (or only) on precision, by measuring variability in 

concentrations reported by the laboratories for PCB congeners. Little attention has been 

paid to the accuracy of the results. Only for wastewater samples, recovery values are 

presented as a measure of accuracy (only for wastewater, the spiked concentrations were 

known and therefore the recovery could be measured). Accuracy is an important criterion 

in method validation. There are several ways to measure accuracy, without increasing the 

burden on the laboratories. Accuracy could have been assessed by analyzing a sample of 

known concentration, as a laboratory control material, and comparing the measured value 

to the true value. National Institute of Standards and Technology (NIST) reference 

materials are available for PCBs and laboratories could have been supplied or asked to 

analyze NIST reference materials, along with samples supplied by the EPA. It has been 

stated that ‘to minimize the burden on volunteer participants’, laboratories were not 

required to analyze initial precision and recovery (IPR) samples. Nevertheless, accuracy 

is an important parameter for validating accuracy of a method, a well-characterized 

matrix such as NIST SRMs can be used. Alternatively, the technique of standard 

additions, which is used to determine recovery of spiked congeners from on-going 

precision and recovery (OPR) samples (sand/oil and water) could have been used (Results 

from Table 4-4) to validate the accuracy of the method.
Method 1668A Interlab Study Peer Review

April 2010 23

Response: Table 4-4 presented results for accuracy (as recovery) for the 27 labeled compounds 

spiked into all matrices (water, biosolids, and tissue) by level of chlorination (homolog). 

Mean recoveries ranged from 57 to 85 percent. We believe that these recoveries are 

reasonable for these analyses given the number of analytes and matrices covered by 

1668C. To address your concern about recoveries for the dioxin-like (Toxic) congeners, 

we have expanded Table 4-3 of the Report to list precision and recovery for the 27 

individual labeled congeners from wastewater and have expanded Table 4-4 to list 

precision and recovery for all 27 labeled congeners (including the Toxic congeners) 

spiked into all wastewater, biosolids, and tissue samples. Revised Tables 4-3 and 4-4 are 

at the end of these responses.

Regarding use of a Standard Reference Material (SRM) from NIST or a certified 

reference material (CRM) from another source, EPA did not use such materials to address 

previous criticisms for not using “real-world” sample matrices that the Agency normally 

encounters in its data-gathering activities. In addition, SRMs and CRMs are often dried 

or do not contain the mixture of pollutants found in real world samples. Therefore, we 

tested real-world wastewater, biosolid, and tissue samples, in the forms typically 

submitted to laboratories for analysis (i.e., whole-volume water samples, wet biosolids, 

and tissues).

Comment: The third issue on results: The method 1668A is intended for congener specific analysis 

of PCBs with an emphasis on toxic congeners (twelve non- and mono-ortho substituted 

PCBs). The scope of the method 1668A indicates that twelve PCB congeners designated 

as ‘toxic’ by the World Health Organization (WHO) are determined. Nevertheless, the 

validation study is focused on ten homolog groups. The data analyses involved 

summation of all isomer data within a congener group. Precision and accuracy of the 

Method 1668A for the analysis of individual toxic congeners, are not validated. In other 

words, the method is not validated for 12 toxic PCB congeners.

Response: Method 1668A was validated in wastewater for all 209 congeners, as individual 

congeners and coeluted congener groups, and in all matrices for the labeled analogs of the 

dioxin-like (“Toxic”) congeners and congeners at the earliest and latest retention time at 

each level of chlorination (27 labeled congeners total). Presenting results by level-ofchlorination (homolog) was a means of condensing results so that they could be 

understood more easily. However, to respond to your concern about presenting results 

for the Toxics, we have produced a table of recovery and precision for the 27 labeled 

compounds individually, and shall replace Tables 4-3 and 4-4 in our revision of the 

Report to show recoveries of the labeled compounds spiked into wastewater and into all 

matrices, respectively. The tables are attached for your use at the end of these responses.

Comment: Furthermore, the suitability of this method for the analysis of mono- and dichlorobiphenyls (CBs) is questionable for the reasons given below. First, the precision 

and accuracy for mono- and di-CBs are very poor, if not dismal. For example, mean 

recovery of mono- and di-CBs in wastewater samples were 3 and 54%, respectively. 

Furthermore, the concentration mono-CB found in wastewater sample is similar to what 

was found in blanks (mean for wastewater is 27 pg/L [Table 4-1] and that for water blank 

is 26 pg/L [Table 4-2]). Obviously, the QC criteria have been relaxed to reflect the poor 

recoveries (Addendum to the method validation study). Overall, the method did not 

perform well for mono- and di-CBs. If these two congener groups are removed, then the 

QC criteria for the method would be tighter and more reliable/reproducible. These two 

congeners are easily lost in the solvent evaporation steps, as has been stated in several 

places of the interlaboratory study report.
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Response: As explained in the second paragraph of Section 4.4 of the Report, low recoveries at 

levels of chlorination (LOCs) 1 and 2 for wastewater (Table 4-3) may be due to loss 

during transport from the sample preparation laboratory to the participant laboratories 

because data reported by the sample preparation laboratory showed that the congeners 

were present immediately after shipment in replicates of the samples held by the sample 

preparation lab. Because the labeled compounds at LOCs 1 and 2 were recovered in the 

normal range by the participant labs, losses during extraction and/or cleanup can be ruled 

out. Therefore, and as explained in Section 4.4, the only mechanism for loss we could 

think of is loss to the headspace of the sample bottle during shipment, with subsequent 

loss to the atmosphere when the bottle was opened. Resources were not and are not 

available to verify this theory. Also, we have added labeled compound recoveries for 

wastewater by LOC in Table 4-3, so that it is clear that the labeled compounds were 

recovered at LOCs 1 and 2, even though the native compounds were not.

We recognize that the performance of this method will vary among the 209 congeners, 

and in different matrices. This is typical of multi-analyte methods because not all 

chemicals respond identically to extraction and clean up techniques, or have identical 

instrument responses. In a study of data comparability between two laboratories on 

samples collected from the Passaic River in New Jersey, in which 151 PCB congeners 

were identified and measured, accuracy as measured by analysis of a NIST SRM was 

15% or better. Recoveries of the PCB congeners ranged from 90% to 124% and averaged 

105%; precision ranged from 4.2 to 23%.

Comment: A few other issues on the results, such as robustness, ruggedness, and limit of detection 

have not been discussed or analyzed in detail. A method validation would require that the 

above mentioned parameters are tested and validated. Some of the information presented 

on the validation report is not clear (e.g., section 4.2). For example, how were the 

outliers detected? How many outliers were found in samples? Rationale for removal of 

the outliers from data set is missing. A column showing number of outlier values should 

be presented on Table 4-1. The reproducibility variation coefficients (between-laboratory 

precision and within-laboratory precision) for the method were not reported. Overall, 

data have not been fully utilized to describe several parameters that are required in 

validating a method (based on the comment 2 above and this comment).

Response: Robustness is indicated by method performance on multiple matrices, the results of which 

are presented in revised Table 4-4 for the 27 labeled compounds spiked into all samples. 

These results show that the labeled congeners are recovered from wastewater, biosolids, 

and tissue within ranges expected for these types of analyses.

We did not examine ruggedness directly in the interlab study. Ruggedness testing is done 

in a single-laboratory study when a method is developed. As stated previously, we 

expanded the performance data gathered during the validation study to show recovery and 

precision on an individual congener basis for the 27 labeled congeners, and have included 

an updated table in the next revision to the Report. EPA provides an updated table at the 

end of these responses. In addition, evaluation of ruggedness (the effect of changes to 

variables in the sample preparation, extraction, cleanup, and instrument steps) for a 

method such as Method 1668A, which is applicable to a variety of sample matrices, can 

be counter productive. EPA’s historical experience suggests that the sample extraction 

procedures used for the various matrices are likely to have the greatest effect on the 

results. Thus, both the method and the study-specific instructions sought to minimize 

deviations from the extraction steps described in the method.
Method 1668A Interlab Study Peer Review

April 2010 25

Regarding limit of detection (LOD), evaluation of LOD was beyond the scope of the 

study. An LOD study would require 7 replicate analyses minimum of spikes at low 

concentrations to produce method detection limits (MDLs). 

Regarding outliers, as stated in the final paragraph in Section 3 of the Report, “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.” In Appendix A, we give further detail on 

removal of outliers. As stated in Appendix A, one outlier was removed for the initial and 

on-going precision and recovery test, and two outliers were removed for the test of 

labeled compound recoveries from samples. Because only three outliers were removed, 

we did not list these three values.

Regarding presentation of between- and within-laboratory precision, pooled within-pair 

RSDs were presented by LOC in Table 4-4 for the 27 labeled congeners spiked into all 

samples. We have expanded the presentation to all 27 labeled congeners in the next

revision to the Report, and have attached the revised table at the end of these responses.

2. Please render an opinion as to suitability for use of the data from the study and the addendum for 

revision of the QA/QC in Method 1668 to make it Method 1668B.

Comment: I believe that considerable effort has been put forward in validating the method 1668A. 

Nevertheless, the data from the validation study are under-utilized and more important 

validation parameters such as accuracy of the method, reproducibility, ruggedness, etc 

should be evaluated. These issues and corrections should be corrected/clarified. Once 

these revisions are made, the method may be revised to 1668B. However, I believe that 

there is lot more work to be done.

Response: As stated above, accuracy (as recovery), and reproducibility and ruggedness (as RSD) 

data were presented by LOC in Tables 4-3 and 4-4. EPA's Quality System defines 

accuracy to include precision and recovery (bias), but most use accuracy and recovery 

synonymously. Table 4-4 of the Report gave recoveries across all labs and matrices. 

Reproducibility is indicated by the RSDs presented in Table 4-4 and, although we did not 

evaluate ruggedness, per se, in the study, how rugged a method is indicated by the 

precision of the measurements (if the method isn't rugged, differences in the way 

different labs practice the method will cause great variability in results). Also as stated 

above, we have expanded the data in Tables 4-3 and 4-4 to show recovery and precision 

on an individual congener basis for the 27 labeled congeners, so that reproducibility (as 

precision) can be viewed on a per-congener basis. The revised tables are at the end of 

these responses.

3. If you have a suggestion for an improvement of the interlaboratory study report or addendum, 

please provide suggested text by section number & 

4. If you believe that a given item should be clarified, please provide the clarification by section 

number or define the clarification needed to a sufficient level of detail that we can make the 

necessary changes &

5. Please point out errors or omissions.

I feel that the charge questions 3, 4 and 5 are inter-related. The following items would help improve the 

study report and addendum

Comment:
Method 1668A Interlab Study Peer Review

April 2010 26

a. Page 3, Section 2.4 (Sample Selection): Some description of samples is needed. 

For example, does the fish tissue represent wholebody homogenate or fillet? Also, please 

specify when these fish samples were collected? I assume that these are wet samples 

(i.e., not freeze-dried). Similarly for biosolids, some sample description is needed. After 

reading the entire document, I realized that these are wet samples. It would be useful to 

indicate on page 3 (section 2.4), what biosolids really are and when they were collected; 

also state that they are wet and if possible indicate the moisture content?

Response: As stated in Section 2.4 of the Report, the tissue and biosolids samples were generated 

from excess sample collected during two unrelated EPA studies. The biosolids samples 

were collected in 2001 as part of EPA’s 2001 National Sewage Sludge Survey. The fish 

tissue samples were collected in 1999 and 2000 during the early phases of EPA’s multiyear National Lake Fish Tissue Study (NLFTS). 

All of the tissues used in the Method 1668A 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 than 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. We 

have added this information to our revision to the Report.

Regarding your concern about the fish tissue type (whole; fillet), the fish tissue 

homogenate was from a mixture of ground whole fish and ground fish fillets. Results of 

analyses of hundreds of fish tissue samples have demonstrated that the performance of 

Method 1668 is independent of tissue type. Results were reported on a wet-weight (asreceived) basis, not a lipid basis.

Both the biosolids and tissue samples had been stored in freezers at an EPA sample 

repository. In order to have a sufficient amount of each sample 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. Data for 

the PCBs from those original studies were used to select samples that contained PCB 

congeners in the range of interest for the validation study (e.g., neither the cleanest nor 

the most contaminated samples were selected, but rather samples that would contain 

PCBs in the general operating range of Method 1688A).

Because of the chance that biosolids samples could lose low-molecular weight PCB 

congeners during any drying procedure, the biosolids samples were not dried. Thus, EPA 

submitted biosolids samples to the laboratories in the form that EPA believes is typical 

for this matrix: wet, with varying moisture contents that reflect the source of the material. 

The original biosolids samples used to prepare the study samples were collected as solids, 

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 from 30 to 

40% for the two samples, amounts that are typical for many biosolids produced in the 

U.S. Results for the biosolids samples were reported on a dry-weight basis.

Comment:

b. Did the wastewater effluent samples contain suspended solids? When aliquoting 

wastewater samples, how was the homogeneity in the distribution of suspended solids 
Method 1668A Interlab Study Peer Review

April 2010 27

between aliquots ensured? Wastewater samples have been spiked with PCB congeners. 

It appears that there was no PCB present in wastewater sample prior to spiking. Was that 

true? I suspect that PCBs can be detected in wastewater samples at several tens to 

hundreds of pg/L level. The information on the spike concentrations listed on Table 2-2 

is not clear. What was the solvent that was spiked into water?

Response: The wastewater samples were prepared from a final effluent sample collected from a 

publicly owned treatment works (POTW) near the sample preparation laboratory. 

NPDES permit limits typically restrict the total suspended solids (TSS) content of final 

effluents to values around 10 mg./L. Thus, the parent wastewater did not contain 

appreciable suspended solids; and there was no difficulty in aliquoting the wastewater 

samples. The unspiked wastewater from divided into 1-L aliquot in suitable glass bottles 

and each aliquot was spiked separately (rather than spiking a bulk volume of wastewater 

and then subdividing the spiked sample into replicate aliquots). The sample preparation 

laboratory analyzed several spiked aliquots to ensure homogeneity.

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, as detailed in Method 1668A. This information has been added to the 

Report.

Regarding the presence of a PCB background in the wastewater, 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 prep lab thus flagged the 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 amount of acetone used to spike the Youden pair samples was either 0.5 or 

0.6 mL per 1-L volume of wastewater. This information has been added to the Report.

Comment:

c. Page 5, Section 2.6: Did all the participating laboratories use HRMS? The 

Method 1668A calls for the use of HRMS and the method validation requires the use 

HRMS. The deviations from the Method 1668A have been listed for certain parameters 

(section 2.7 on page 6), but not sure if all of the laboratories used HRMS for the analysis. 

Also, on page 5, it has been stated that the laboratories were instructed to follow the 

Method 1668A, it will be helpful to mention/confirm that on Section 3 (Data Review and 

Validation) that all laboratories indeed followed the Method 1668A (other than the 

deviations noted on section 2.7).

Response: All participating labs used HRMS, and all labs claimed that they followed Method 1668A 

other than for the deviations noted in Section 2.7 of the Report.

Comment:

d. Page 6, line 3: Expand “SOW”; the abbreviation is used here for the first time.
Method 1668A Interlab Study Peer Review

April 2010 28

Response: We have spelled out “SOW” to indicate Statement of Work.

Comment:

e. Page 6; Laboratory reporting of rules: One important item that is missing is the 

conversion of data from ‘wet weight’ to ‘dry weight’ and vice versa (for biosolids).

Response: Section 17 of Method 1668A states how to calculate the concentration of a congener on a 

wet- or dry-weight basis, as appropriate. 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. Other reporting requirements may be used when required 

by State or Federal regulations and it is not EPA’s intent to preclude such approaches in 

an analytical method. We have added the information on the amount used for percent 

solids determination and the basis on which results were to be reported to the Report.

Comment:

f. A major issue with the method is lies in the weight of samples used for extraction 

(especially biosolids) and final extract volume. I think the method should be modified to 

incorporate some flexibility because the weight used for extraction can vary depending on 

the concentrations, presence of interferences, etc. Also, concentration to a final volume 

of 20 uL prior to injection can introduce large variability in analytical results. I would 

prefer to use a sample weight of 15 g for biosolids and a final volume of 100 or 200 uL. I 

hope that the Method 1668B incorporates these changes to reflect some amount of 

flexibility in the weight of the samples used for extraction and the final extract volume.

Response: We believe that the most significant problem the labs encountered with the biosolids 

sample was the inability to separate the PCB congeners from the biosolids matrix using 

the extraction and cleanup procedures in the method. In part, this problem is likely 

caused by overload of the cleanup columns.

The study-specific instructions directed the laboratories to determine the percent solids 

content of the biosolids samples before any other steps, and then to use an aliquot of wet 

sample sufficient to yield about 10 g of dry biosolid material. These instructions were 

consistent with the instructions in Section 11.5.1 of the method and were based on EPA’s 

experience during the 2001 National Sewage Sludge Survey. The use of a consistent dryweight aliquot across all of the participating laboratories simplified the comparison of 

results across laboratories and between samples.

Section 11.5.1 of the method instructs the laboratory to weigh out “a well-mixed aliquot 

of each sample (of the same matrix type) sufficient to provide 10 g of dry solids,” but 

Section 11 of the method also states that “For samples known or expected to contain high 

levels of the CBs, the smallest sample size representative of the entire sample should be 

used.”

Regarding concentration of the sample extract to 20 μL, the addition of an n-nonane 

“keeper” in Section 12.7.7 of Method 1668A reduces the possibility of loss when the 

extract is concentrated to 20 μL. Moreover, EPA’s experience with Method 1613B, for 

PCDDs/PCDFs, does not suggest that the HRGC/HRMS laboratory community has 

difficulty with the 20 μL final extract volume. Finally, any option for changes to the final 

extract volume would necessitate other changes in the method to ensure that the final 

sample extract and the calibration standards contain similar amounts of the labeled 

compounds used for isotope dilution quantitation. Without clear evidence of a problem 
Method 1668A Interlab Study Peer Review

April 2010 29

with the current final extract volume, EPA does not believe that such changes are 

warranted at this time. 

Comment:

g. Page 11, First paragraph, line 9: Expand “ML”

Response: We have spelled out “minimum level of quantitation” in the Report.

Comment:

h. Table 4-1, Page 12, column #7 (Mean), third row from the bottom; 241-??? 

Something is missing here. It looks like a typo. Is there a value after ‘241’? Also, 

footnote 1 for the table: specify whether biosolid and fish data are given on wet weight 

or dry weight basis. For footnote 2; insert “sum of” between ‘represent’ and ‘all’.

Response: We have made your suggested changes to the Report. 

Comment:

i. Section 4.3.; Were the blanks analyzed similar to samples in all aspects, especially 

the concentration factor (final extract volume)? This should be mentioned in section 4.3.

Response: Yes, to the extent that the matrices could be simulated. For the soil/tissue blank, a 

mixture of playground sand and corn oil was used to simulate soil and tissue. This 

mixture was chosen to minimize the number of blanks that would need to be analyzed. 

The method requires that blanks and other QC samples be treated in the same manner as 

field samples, including concentration of extracts to 20 μL. There is no allowance in the 

method for using a different final extract volume. We have added a statement to Section 

4.3 of the Report clarifying this information. 

Comment:

j. Page 13, Table 4-2: “# Labs” for sand/oil should be “6”; “10” is a misleading 

number here because “10” is the number of datasets available, not ‘# Labs”.

Response: We have revised the header to column 2 in Table 4-2 to state #Blank samples.

Comment:

k. Section 4.4: Low recoveries of mono- and di-CBs have been reported to be due 

to loss during transport or as ‘unknown’. I would expect that it is mainly due to loss of 

these volatile congeners in the solvent evaporation steps in Method 1668A.

Response: As stated in response to the issue on results under item 1 above, the loss could not have 

occurred in the solvent evaporation steps because the mono- and di- labeled compounds 

were recovered in the normal range. We have revised Table 4-1 in the Report to show 

this information and have attached revised Table 4-1 at the end of these responses for 

your use.

Comment:

l. Section 5.5: “(Table 5-1).” Move the ‘.” Out of the parentheses.

Response: We could not find a period within parentheses in Table 5-1.

Comment:
Method 1668A Interlab Study Peer Review

April 2010 30

m. Section 6. I am little concerned about the statement that "the result of this 

interlaboratory study met our objectives to characterize......”. The results have not 

satisfied the criteria to validate the method for biosolids, if we follow the guidelines that 

we need dataset from 6 laboratories.

Response: This comment is the same as your comment in item one of section one, technical 

approach.

Comment:

n. Corrections on addendum: Table 4-1, footnote Biosolids concentrations in ng/kg, 

wet weight, not ‘ng/L’. Please correct. Also, the labeled compound recoveries are too 

low for a good method. The low recoveries are only for mono and di-CBs and if these 

two congeners are removed or reported separately, the recoveries are more than 50% for 

all other congeners. I suggest that recoveries for mono- and di-CB congeners are 

reported separately and tri-deca CB are reported separately. A 15 or 20% recovery is too 

low to consider a method to be a good method.

Response: We have made the correction to the biosolids concentrations.

Regarding low recoveries for the mono- and di-CB congeners, we responded to this issue 

in the section on results under item 1 above.

6. Please provide any additional comments you would like to make regarding the documents under 

review.

Comment:

a. Although 14 laboratories were invited to participate (on a voluntary basis), only 6 

labs have provided results for fish and wastewater and 4 labs for biosolids. Participation 

of a minimum of six labs is required for a method validation study. It is disappointing to 

note that several laboratories have expressed dissatisfaction with method and problems in 

analysis. Inviting some laboratories for participation with some (minimum) payment, and 

use of NIST SRM samples for accuracy analysis would enhance the quality of method 

validation. Because the method is intended for use in EPA’s Clean Water Act programs, I 

would strongly recommend that contract laboratories analyzing clean water act samples 

should participate in this validation study.

Response: As suggested, we have obtained data, including MDLs, from contract laboratories

experienced with this method. This data is in the current version of this method, 1668C. 

We answered your comment about the number of labs and use of SRMs in response to 

your first two comments in section 1 of this review. Again, the final data sets of 6 , 6 and 

4, respectively, for the three sample types, water, tissue and biosolids, resulted from 

review of laboratory results against our data validation guidelines and from admissions 

and narratives from the labs.

Comment:

b. I would suggest that mono and di-CBs are reported separately or removed from 

the method. The QC criteria for these two congener groups should be reported separately 

from the other congeners.

Response: As explained in the section on results under item 1 above, we believe that the low 

recoveries for wastewater may be the result of losses during sample transport and not the 

result of a method deficiency. 
Method 1668A Interlab Study Peer Review

April 2010 31

Comment:

c. Participating laboratories should report twelve toxic congeners separately and 

QC criteria for these congeners should be established; in other words, the method 

validation is needed for these 12 toxic congeners.

Response: There is no prohibition in Method 1668 to reporting results for the toxic congeners 

separately. And QC acceptance criteria were given for the toxic congeners, as shown in 

Table 5-1 of the Report and Table A-1 of the Addendum

Comment:

d. A major issue with biosolids analysis lies in the use of 30 g of sample weight for 

extraction. Similarly, concentration of samples to a final volume of 20 uL, can impose 

serious variability issues. I would recommend that the method be amended to incorporate 

some flexibility in the weight of the samples taken for extraction. For biosolids, it can be 

from 10 to 30 g (wet weight). Also, concentration of extract to 20 uL, will introduce 

large variability. I would suggest that the final volume can be from 20 uL to 200 uL. I 

would recommend using sediments for method validation.

Response: As noted in response to earlier comments, the method does not require the use of a 30-g 

sample. Section 11.5.1 of the method instructs the laboratory to weigh out “a well-mixed 

aliquot of each sample (of the same matrix type) sufficient to provide 10 g of dry solids,”

Section 11 of the method also states that “For samples known or expected to contain high 

levels of the CBs, the smallest sample size representative of the entire sample should be 

used.”

Regarding concentration of an extract to a final volume of 20 to 200 uL, a lab may 

concentrate to a higher volume under the equivalency provisions in Section 9 of Method 

1668 and under 40 CFR 136.6, provided that all QC requirements in the method are met. 

However, and as noted in an earlier response, any option for changes to the final extract 

volume would necessitate other changes in the method to ensure that the final sample 

extract and the calibration standards contain similar amounts of the labeled compounds 

used for isotope dilution quantitation. Without clear evidence of a problem with the 

current final extract volume, EPA does not believe that such changes are warranted at this 

time.

Regarding use of sediments for method validation, EPA may use sediments in future 

validation studies depending on program needs and requirements. However, at the time 

of the validation study, EPA felt that biosolids were a more likely candidate for PCB 

congener regulations than sediments. 

Revised tables 4-3 and 4-4, described in EPA’s responses above, are shown below for 

your convenience.
Method 1668A Interlab Study Peer Review

April 2010 32

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
Method 1668A Peer Review

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

LOC2 Labeled 

Congener3

Recovery (%) Within-pair RSD (%)

# 

Results

Mean Median Min. Max.

# 

Pairs Pooled4 Median Min. Max.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

9 206L 32 80.8 81.3 50.0 115 16 9.2 7.7 0 19.8
Method 1668A Interlab Study Peer Review

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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 Interlab Study Peer Review

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Section Three: Original Peer Review Comments on Draft EPA Method 1668A

Interlaboratory Validation Study

Prepared for:

U.S. Environmental Protection Agency

Office of Water

Office of Science and Technology

Health and Ecological Criteria Division

1301 Constitution Ave., N.W.

Washington, D.C. 20004

First Two Sets of Comments Prepared by:

Versar, Inc.

6850 Versar Center

Springfield, Virginia 22151

September 2007

Third Set of Comments Prepared by:

Eastern Research Group, Inc. 

110 Hartwell Avenue. 

Lexington, MA 02421

March 2010.
Method 1668A Interlab Study Peer Review

April 2010 36

PEER REVIEW COMMENTS FROM

MICHAEL C. NEWMAN

Peer Review Comments on: Draft EPA Method 1668A Interlaboratory Validation Study

NAME: Michael C. Newman, PhD

AFFILIATION: 121 York Point Dr., Seaford, VA, also Professor at the College of William and Mary, 

Virginia Institute of Marine Science

DATE: Sept. 18, 2007

I. GENERAL IMPRESSIONS

Overall, this Report of the Method 1668A Interlaboratory Validation Study is a sufficiently 

accurate, clear, and thorough analysis discussing the results of a very difficult task. The study objectives 

were to (1) characterize the performance of Method 1668A for multiple laboratories and matrices 

(wastewater, biosolid, fish tissue), and (2) evaluate and possibly revise the QC acceptance criteria. These 

objectives were met with some qualification. It is important to emphasize prior to discussing the specific 

qualifications that this effort does appear to be generally well-intended, -planned and -interpreted. Some 

challenges to applying the results do exist. The laboratory participation was disappointing and this was 

the source of numerous issues. For example, six of 14 laboratories declined to participate in the biosolids 

analysis. An additional four produced “unusable” numbers, resulting in only four of 14 potential sets of 

results being applicable to the task at hand. For wastewater and fish tissue, only six laboratories produced 

the data used in this exercise. Four laboratories successfully analyzed all three sample types and, 

therefore, have a very strong influence on the results. The candidate laboratories included government 

and private organizations which might be expected to have differences in response; however, it is unclear 

from the report if the final subset was biased toward one group or another. The very high failure-to-report 

rate for this process suggests that failure to achieve useful analyses with 1668A warrants more discussion. 

Was the high failure rate normal for such voluntary validation exercises or a feature to be anticipated for 

Method 1668A as currently written? Some laboratories deviated from 1668A for some analyses. Also 

outlier tests that require extremely thoughtful application were applied to cull away some data. Together, 

these features do not permit as high confidence in the study’s generalizations as might be desired. The 

studies conclusions are not unequivocal at this point. The resulting, central concern can be articulated 

with a quote from Stuart’s Basic Ideas of Scientific Sampling (1968): “... the credentials of a sample are 

not only important for the interpretation of results, but are in principle the only information of value we 

can have in this respect.” 

My general conclusion is that the results are adequate for meeting the study objectives but should 

be augmented in the near future in order to enhance confidence in the general validity of the conclusions. 

Detailed below are specific examples from within the report and associated recommendations.

II. RESPONSE TO CHARGE QUESTIONS

1. Please review the EPA's Report of the Method 1668A Interlaboratory Validation Study for 

the objectives of the study, the soundness of the technical approach, and the results produced.

COMMENT: The Interlaboratory Validation Study attempts to characterize the performance of 

Method 1668A in multiple laboratories and matrices (wastewater, biosolid, fish tissue), and to 

evaluate and possibly revise the QC acceptance criteria. As stated above, my overarching 

opinion is that the study met these goals but further work is required to enhance confidence in 

the generality of the conclusions. The goals for this complicated method were extremely 

challenging so the implied incremental generation of sufficient characterization/validation 
Method 1668A Interlab Study Peer Review

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information is, in my opinion, reasonable. My suggestion that more work is required is not an 

oblique inference about the work quality of the study leaders. This was a very difficult task.

2. Please render an opinion as to suitability for use of the data from the study for revision of 

the QCAC in Method 1668 to make it Method 1668B.

COMMENT: Yes. The study data are adequate, although lacking in some ways, for revision of 

the QC in Method 1668.

3. If you have a suggestion for an improvement, please provide suggested text by section 

number.

COMMENT: A wide range of suggestions and insights will be addressed below regarding the 

improvement of the study or existing study presentation.

Page 6, Top paragraph and first full paragraph: This is the first place in the report that the issue 

of sample homogeneity emerged. It is also discussed in Section 4.1. Described here is the 

process in which a “sample processing laboratory was required to perform background and 

homogeneity analyses of both the biosolids and tissue matrices.” The text does not convince me 

that the samples were “homogeneous” in a strict sense relative to the analytes. For example, 

some fatty tissues might contain more analyte than others and similar-sized pieces of fatty tissues 

might not be homogeneously distributed in a well-mixed homogenate. The biosolid to which 

sand was added might experience density-driven sample component segregation (i.e., sinking 

sand) so that the sample aliquot analyzed by the volunteer laboratory might not have been 

homogeneous (well-mixed?) relative to the analyte. There are very specific methods of 

determining this sample quality (well- or poorly-mixed) yet they are not applied by the sample 

processing laboratory. It is not clear how this might or might not have contributed to differences 

in results among laboratories. This concern is also inherently linked to sample aliquot size which 

1668 prescribes versus and the actual sample sizes used by the volunteer laboratories. (I assume 

that the recommendations of sample sizes were based only on anticipated analyte concentrations 

in typical samples and the mechanics of the sample processing.)

The point being made above can be illustrated easily using the 

conventional explanation as depicted in the figure to the left. The analyte 

(dot) is homogeneously distributed in this figure’s top panel; therefore, 

sample aliquot size illustrated in the figure as different size boxes will not 

influence the measured analyte concentration in an analyzed aliquot. 

(Homogeneity is a material quality in which the analyte is distributed in a 

completely homogeneous manner throughout the material.) In the 

bottom panel, a heterogeneous material is depicted in which sample 

aliquot size becomes critical depending on whether the heterogeneous 

material is well- or poorly-mixed. If sample aliquot sizes are too small 

for a poorly-mixed, heterogeneous material (small squares in figure 

bottom panel), variation among reported analyte concentrations will be 

higher than that derived with aliquot sizes of sufficient size (large square 

in the bottom panel) (Newman 1995). To conclude that a heterogeneous 

sample is sufficiently well-mixed to permit use of a specified aliquot size (e.g., 20 g of fish 

tissue) requires some conventional calculations. The homogeneity analyses described in this 

report do not include the associated statistics permitting such decisions. This is especially

relevant to the biosolid and tissue samples, and with sufficiently high solids content, also to 
Method 1668A Interlab Study Peer Review

April 2010 38

wastewater samples. The pertinent approach to this problem and associated statistical 

computations are described in classic papers by Visman (1969), Ingamells (1973) Ingamells and 

Switzer (1974), and Wallace and Kratochil (1987). 

Page 8, Top Bullets: The laboratories were told to report all “>nm” values as quantified 

concentrations and the statistical analysis of these measured values was treated such that all 

“>nm” values possessed the same quality of measurement. It is incorrect to assume that all 

values above “nm” had the same measurement quality (e.g., Keith et al. (1983)). The relative 

uncertainty of a measured value can be estimated relative to the standard deviation of the 

analytical process baseline noise (See also figure on page 6),

Relative Uncertainty (%)  100

Z 2

N

where Z= Z-statistic value for a confidence level of % (conventionally 95%), and N is the 

measured signal expressed in units of multiples of the standard deviation of the baseline noise, 

e.g., N=3 and 10 are associated with the signal at the detection limit and limit of quantification, 

respectively. Interpretation of the study results is very difficult if this fact is not addressed in the 

discussions and shown in graphs of results for the various samples and sample types. An 

example of this will be discussed below relative to the figures in the report.

Page 12, First paragraph after bullet list and also last sentence: Here and elsewhere, my

assumption is that the study results are intended to provide measurement quality characteristics 

anticipated for “in-control” analyses of the specified sample types by a laboratory represented by

the initial 14 laboratories. It is critical that the population for which these final QC criteria are 

generated be clearly defined given the variations in reporting, method application, and data 

selected for final QC calculations. 

Here and elsewhere, Grubbs’ single outlier test (Grubbs 1969) is applied to cull away 

apparent outliers. Outlier deletion, as discussed in Grubbs’ paper and many others, should be 

done very thoughtfully according to clear rules. For example, the “outliers” detected based on the 

assumption of a normal distribution might come from a population more appropriately described 

as lognormal. The “outlier” would be falsely removed based on the assumption of the wrong 

distribution. The rules for handling outliers in Grubbs (1969) and other papers should be 

considered prior to applying any outlier tests. The results of such application should then be 

described in the report. Classic papers that might be useful are Anscombe (1960) and Rosner 

(1983). As multiple outliers might be masking each other in these data sets, Rosner’s method 

(1983) for multiple outliers might be useful. Personally, I think that Woodworth’s (2004) 

classification of outliers and the associated approach based on interquartile ranges is more 

relevant as it is more open to intelligent application than the above hypothesis-based, parametric 

tests. This point will be repeated later in discussing the absence of quartile (Q1 and Q3) values 

in tables of summary statistics.

Page 13, Section 4.2.1, line 4: The “homogeneity ... was found acceptable...” Respectfully, my

guess would be that this statement is correct; however, I disagree that enough information was 

extracted to make a definitive statement. I base this disagreement on the material already 

presented above. 
Method 1668A Interlab Study Peer Review

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Page 14, Table 4-1: Here and elsewhere, it would be very helpful to have the first and third 

quartile values (Q1 and Q3) or, minimally, the interquartile range (|Q3-Q1|) in the table. One 

immediate use would be to apply interquartile range-based methods to explore potential outliers 

or outlying laboratories. The inclusion of these statistics is highly recommended. As an aside, 

the differences between means and medians for congeners with 3 to 8 chlorination levels 

(biosolids and tissues) suggest a skewed sample set. Are these data skewed for any reason or is 

this simply a spurious trend in the tabulated data?

Page 29, Last line of text: I generally agree but feel that a less definitive, and more conditional, 

conclusion can be reached.

Appendix A. Section 1.0., 95% Confidence Intervals: The section begins by describing 

conventional, parametric calculations for all congeners in all matrices at all concentrations. Was 

the assumption of normality tested and met, or found to be unimportant? Some discussion seems 

warranted.

Appendix A Section 1.0, Application of Grubbs’ outlier test: Again, outlier tests should be used 

very carefully and interpretation of any consequent data sets based on truncated distribution 

statistics (see Grubbs (1969) and other sources for discussion). As a simple example, what is the 

n in the equation given in Section 2.0? This is not as easy a question to answer as might be 

suspected since some observations were thrown out. Also, was the assumption of a normal 

distribution (upon which Grubbs’ test depends) appropriate? Finally, what Type I error rate was 

selected? Likely, the typical 0.05 was used yet one could argue for a more conservative error 

rate.

4. If you believe that a given item should be clarified, please provide the clarification by 

section number or define the clarification needed to a sufficient level of detail that we can 

make the necessary changes.

COMMENT: A wide range of issues will be addressed below as suggestions for clarifying 

presentation.

Section 1.2, Line 3: Change “seen” to “seven”?

Page 4, Paragraph 2: Please consider replacing “properly homogenized” with “homogenized.” 

The reasons are given above for removing the vague word from this sentence. One could easily 

argue that the samples were not shown to be “properly homogenized.”

Page 5, Paragraph 2 of Section 2.4: The term, “Youden pairs” is first used here but not defined 

until Section 2.5.1. Please move the definition up to the first use of the term.

Table 2-1 (page 4) and Table 3-1 (page 12): It is extremely important in judging the 

generalizability of the report conclusions that the laboratory participation in generating the final 

QC criteria is very clearly discussed. At present, it is not easy to follow. Generally, one starts 

with 14 laboratories (government and private) and ends up with data from 4 to 6 labs. It isn’t 

easy to judge how (1) withdrawal of the various laboratories from providing results, (2) the study 

author’s judgment about reported data usefulness, and (3) the final data inclusion influences the 

statistical population of laboratories for which inferences about QC are being made. Several 

issues to be addressed include the following. Are the QC conclusions relevant to only the best or 
Method 1668A Interlab Study Peer Review

April 2010 40

best equipped laboratories? Are they representative of primarily government laboratories, private 

laboratories, or both? A very clear and concise summary addressing this linchpin issue is needed. 

In developing this summary, discussion of the influence of outlier rejection is also warranted.

Page 8, Section 2.7, first paragraph: The requirements were given for calibration curves but the 

laboratories interpreted the requirements differently. I saw no obvious reason for the 

misinterpretation; however, some thought should be put into rewriting the associated text so it is 

even clearer. Also, relative to Section 2.7.1. and 2.7.2, were the consequences of any of the 

various deviations quantifiable? For example, the differences due to different aliquot weights 

used by the various laboratories might be quantified with the methods cited above for “Page 6, 

Top paragraph and first full paragraph.”

Page 9, Section 2.7.2. Different problems were encountered by different laboratories. Different 

weight normalizations were done by the different laboratories. It isn’t clear to me how this 

directly influenced statistical estimates and associated inferences. Also, the numerous deviations 

suggest that even more clarity needs to be placed into 1668A. Perhaps more discussion is 

needed about what deviations should not be considered.

Page 11, Section 3, first paragraph: This section begins by describing three laboratories that did 

not submit data. It is important to know if non-reporters are considered a biased or random 

subset of the population of possible responders. Was there a risk of biased sampling of possible 

laboratories using this method as a result of biased non-response?

Page 11, Section 3, fourth bullet: How was this issue handled during the analysis of results? 

Would omission of an outlier laboratory create a biased sample of laboratories?

Page 15, Table 4-2 and all other tables: Univariate statistics are applied throughout these tables. 

Some graphs suggest skewed distributions for some data. Was the assumption of normality or 

the robustness of conclusions to violations of this assumption assessed?

Page 16, first lines: This discussion is too speculative in my opinion.

Page 17, Figure 4-1 (and also 4-3, 4-5, and 4-7): As dictated by the generally-accepted equation 

given above, the relative uncertainty of a measurement will rapidly increase as concentration 

decreases. The following figure gives the general 

shape of such a relationship. Depending on the 

positions of the detection limit (LOD in this figure) 

and limit of quantitation (LOQ in this figure) (from 

Newman (1995)), the variation in the y direction as a 

function of measured concentration would be totally 

expected based on this relationship. Would it be 

possible to insert vertical lines at the LOD and LOQ 

in these figures so a reader could judge this issue? As 

a good example of how this would help, it is difficult 

to decide if the explanation for the Figure 4-5 outliers 

(Page 20, first few lines) is reasonable or whether the 

variations at low concentrations (in lower part of the Region of Less-Certain Quantitation?) are 

what you would expect for any measurement process.
Method 1668A Interlab Study Peer Review

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Page 18, Figure 4-2 (and also 4.4, 4.6): These figures do not convey any useful information. 

Relative Standard Deviations (e.g., 4-3) are much more useful for conveying the associated 

information (that variation can change with concentration).

Page 21, Table 4-4: A simple mistake was made here such that the “# pairs” column has two 

digits after the decimal point, e.g., 32.00 pairs.

Page 29: The conventional “This page intentionally blank” statement is made but, in fact, there is 

text on the page.

5. Please point out errors or omissions.

COMMENT: Please see comments above.

III. SPECIFIC OBSERVATIONS

[NONE SUBMITTED]

IV. ADDITIONAL REFERENCES

Anscombe, F.J. 1960. Rejection of outliers. Technometrics 2: 123-147.

Grubbs, F.E. 1969. Procedures for detecting outlying observations in samples. Technometrics 11: 1-21.

Ingamells, C.O. 1974. New approaches to geochemical analysis and sampling. Talanta 21: 141-155.

Ingamells, C.O. and P. Switzer. 1973. A proposed sampling constant for use in geochemical analysis. 

Talanta 20: 547-568.

Keith, L.H., W. Crummett, J. Deegan, Jr., R.A. Libby, J.K. Taylor, and G. Wentler. 1983. Principles of 

environmental analysis. Anal. Chem. 55: 2210-2218.

Newman, M.C. 1995. Quantitative Methods in Aquatic Ecotoxicology. Lewis/CRC Publishers, Boca 

Raton, FL, pp. 426. 

Rosner, B. 1983. Percentage points for a generalized ESD many-outlier procedure. Technometrics 25: 

165-172.

Stuart, A. 1968. Basic Ideas of Scientific Sampling, Hafner Publishing Co., New York, pp. 99.

Visman, J. 1969. A general sampling theory. Mat. Res. Stand. 9:9-64.

Wallace, D. and B. Kratochvil. 1987. Visman equations in the design of sampling plans for chemical 

analysis of segregated bulk materials. Anal. Chem. 59: 226-232.

Woodworth, G.G. 2004. Biostatistics. A Bayesian Introduction. John Wiley & Sons/
Method 1668A Interlab Study Peer Review

April 2010 42

PEER REVIEW COMMENTS FROM

CHARLES WONG

Peer Review Comments on: Draft EPA Method 1668A Interlaboratory Validation Study

NAME: Charles S. Wong, Ph.D.

AFFILIATION: University of Alberta, Department of Chemistry

DATE: 18 September 2007

I. GENERAL IMPRESSIONS

This document is a synopsis of the interlaboratory validation study intended to improve EPA method

1668A, a protocol for congener-specific measurement of polychlorinated biphenyls (PCBs) in water, soil, 

sediment, biosolids, and tissue by high resolution gas chromatography/high resolution mass spectrometry. 

Fourteen laboratories were invited to participate, and samples as well as instructions and required 

reporting information was provided to them. Reported data was compiled and summarized.

The document provides a useful but succinct summary of the interlaboratory validation’s very large 

dataset. Sufficient detail is provided on the background on Method 1668A and the methodology used to 

invite participating laboratories to ascertain the effectiveness of the validation study in characterizing 

measurement of PCBs in the samples chosen. Methods for selection of samples for the laboratories are 

clear and reasonable, including explicit discussion of limitations for logistical and technical purposes. A 

clear and detailed discussion of deviations from the required protocol is evident, so that the reader can 

clearly understand limitations in the reported data. For the most part, the discussion of the usable data is 

clear and pertinent to the intended goal of determining possible changes to the protocol and to acceptance 

criteria. 

1. Please review the EPA’s Report of the Method 1668 Interlaboratory Validation Study for the 

objectives of the study, the soundness of the technical approach, and the results produced.

COMMENT: The objectives of the study are sound. Should Method 1668A be revised, and if so, what 

protocol changes and/or acceptance criteria should be modified? This can only be done with an 

interlaboratory validation of a standard set of samples.

Sample selection is reasonable in my opinion. Biosolids and tissues were prepared by EPA at two distinct 

concentrations, properly homogenized, and shipped to each laboratory with appropriate safeguards 

against decompositions to ensure that that the integrity of the samples upon receipt by the participating 

laboratory was maintained. While it would have been ideal to have wastewater samples with PCBs 

shipped to the laboratories, it is reasonable to assume that testing of Method 1668 capabilities would not 

be feasible. Thus, providing instructions to spike the wastewater effluent shipped to the laboratory was 

reasonable.

As previously stated, deviations from the method were characterized in a succinct but meaningful manner. 

This allowed the flagging of suspect data as unusable for further purposes. Many of the deviations noted 

had details on the underlying rationale, e.g., problems with cleanup of samples encountered by some of 

the laboratories. However, not all such deviations were detailed in this manner, which I would have 

found useful. It is troubling that there were sufficient problems in sample processing, measurement, and 

even data reporting (e.g., no calibration reporting from one laboratory???) that less than half the data from 

the participating laboratories was usable (Table 3-1), as part of the point of having a large number of 
Method 1668A Interlab Study Peer Review

April 2010 43

independent participants was to assess the robustness of both the overall Method as well as the validation 

for improving the Method. That having been said, I feel that the most problematic deviations were 

flagged unusable, and that the usable data is sufficient for the intended purpose.

It would have been useful if the laboratories were asked to provide measurement data on QC Check 

samples (Method 1668A, Section 7.15) from standardized reference materials (e.g., NIST SRMs or 

Canadian NRC SRMs) in addition to the other QC data, e.g., OPR samples in reagent water, and blanks. 

This is important because as noted in the report, PCB concentrations in the shipped samples were 

unknown, and having this check would be useful. If this was actually done, this was not clear from the 

report.

The reported results provided a good summary of the variability observed with PCB measurement in the 

various matrices supplied to the participating laboratories. This information is most useful for resetting 

IPR/OPR guidelines. The summary and recommendations appear reasonable, e.g., using average relative 

responses under specific circumstances, noting poorer and more variable recoveries for lower chlorinated 

congeners, and somewhat tighter recovery windows particularly for heavier congeners.

2. Please render an opinion as to suitability for use of the data from the study for revision of the 

QCAC in Method 1668 to make it Method 1668B.

COMMENT: I feel that this data, despite limitations noted above and in the report, are of sufficient 

quality to revise the method for the next version of this Method.

3. If you have a suggestion for an improvement, please provide suggested text by section number.

COMMENT: In Method 1668, Section 23.0, page 61: It is useful to provide the PCB numbering system 

used, to avoid confusion. I realize that CAS registry numbers are provided, which eliminate ambiguity; 

however, most discussion of PCBs is by the numbering system, so having that clarified explicitly in the 

document is very useful. See comments below on references.

4. If you believe that a given item should be clarified, please provide the clarification by section 

number of define the clarification needed to a sufficient level of detail that we can make the necessary 

changes.

COMMENT: It would be useful to provide more details on the instructions to the participating 

laboratories as how to spike the shipped wastewaters with PCBs.

Also, as stated previously, additional QC data from the laboratories should be given to help ensure that 

the usable data was of high quality.

5. Please point out errors or omissions:

COMMENT:

In the document “Changes to be made to EPA Method 1668A from the 8/20/03 revision based on the 

Method 1668A interlaboratory validation study and comments and corrections received from 

laboratories”: Under Quality control acceptance criteria, Table 5-1 from the interlaboratory report should 

replace Table 6 not Table 5 in Method 1668A.

In Method 1668A, Section 13.5.1.4, page 48, typo: should read “If calibration is not verified” not “If 

calibration I s not verified”.

In Method 1668A, Section 22.0, page 59: the title for reference 4 should be provided for consistency.

In Method 1668A, Section 22.0, page 60: the title for reference 18 should be provided for consistency.
Method 1668A Interlab Study Peer Review

April 2010 44

In Method 1668A, Appendix A, Section 2.1, page 110: typo, should be “individual congeners” not 

“individual congene4rs”.

III. SPECIFIC OBSERVATIONS

These comments refer to the report on the interlaboratory study.

Section 4.2, page 14, Table 4-1. I do not feel that reporting of concentrations to up to 5 

significant figures is justifiable. The GC used must have either a splitless or on-column injector (Method 

1668A, Section 6.9). The latter in particular does not have sufficient precision to justify 5 significant 

figures. Three or perhaps four significant figures is the maximum that should be present. 

Section 4.2, page 14, Table 4-1. In addition, while providing a mean, median, and maximum for 

concentrations of usable data is useful, it is difficult to assess the distribution of data. This is important, 

as the fairly large differences between means and medians suggest skewed data distributions, which are 

suggested by the various figures of variability. It would be useful to provide a box plot of this data to 

make assessment much easier for the reader.

Section 4.6, page 21, Table 4-4. The same comments regarding significant figures from my 

comments on Section 4.2, page 14, Table 4-1 hold here as well.

Inclusion of the interlaboratory report’s Appendix A, Statistical Procedures Used to Develop QC 

Acceptance Criteria” in the Method as an Appendix may be useful.

The following comment is suggested for improvement of Method 1688A, and not the interlaboratory 

report:

In Section 5.0 on Safety, pages 5-7, some explicit mention should be made about the use of 

appropriate biohazard containment for samples which are biosolids, as these are likely to host pathogens 

which are a potential danger until rendered harmless by disinfection. This would include preparation of 

raw samples in a biosafety cabinet until pathogens are killed (e.g., by soaking in solvents), appropriate 

safety garments and emergency procedures for accidental spills of pathogenic material, and warning about 

covering all materials containing solvents in biosafety cabinets to prevent solvent fumes that can pose an 

explosion hazard in biosafety cabinets that do not vent to exterior air.

IV. ADDITIONAL REFERENCES

Mills, S.A., III; Thal, D. A., Barney J. (2007) “A summary of the 209 PCB congener nomenclature”, 

Chemosphere 60, 1603-1612.

#####
Method 1668A Peer Review

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PEER REVIEW COMMENTS FROM

KURUNTHACHALAM KANNAN

Peer Review Comments on: Draft EPA Method 1668A Interlaboratory Validation Study

NAME: Kurunthachalam Kannan, Ph.D.

AFFILIATION: Wadsworth Center, New York State Department of Health & Professor, 

Department of Environmental Health Sciences. State University of New York at Albany. Empire 

State Plaza PO Box 509. Albany, NY 12201-0509.

DATE: March 24, 2010

Review of “Method 1668A Interlaboratory Validation Study Report” and “Addendum to the 

Method 1668A Interlaboratory Validation Study Report Draft, August 2009.

Method validation is an integral part of a good analytical laboratory practice. The method 1668A 

validation study was intended to provide the Environmental Protection Agency (EPA) with sufficient 

amount of data to evaluate the performance of the method. The method 1668A was originally validated 

in 2000, in a single laboratory study. However, the guidelines published by the EPA and American 

Society for Testing and Materials (ASTM) recommend a minimum of six complete datasets for validating

a method. The objectives of the validation study were to characterize the performance of the Method 

1668A in multiple laboratories and matrices and to evaluate and revise the Quality Control (QC)

acceptance criteria. The objectives of the study are sound and logical. The multi-laboratory validation 

study is a requirement to meet the national and international criteria to ensure reliability, consistency, and 

accuracy of the analytical data generated using the Method, especially when the method is used in the 

EPA’s Clean Water Act programs. The major outcome of the validation study was a revision to the 

quality control/quality acceptance (QAQC) criteria in EPA Method 1668A. 

It is agreed that method validation studies are time and resource consuming and EPA’s effort to perform 

this validation is highly commendable. Coordination of activities among several participating, volunteer, 

laboratories is a daunting task. EPA’s efforts on this method validation are significant; nevertheless, data 

generated from this study are under-utilized and with the existing data it is possible to generate additional 

information required for validation. 
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April 2010 46

1. Please review EPA's Report of the Method 1668A Interlaboratory Validation Study and its 

addendum. Comment on the objectives of the study, the soundness of the technical approach, and 

the results produced.

The objectives of the study are sound and logical. The multi-laboratory validation study is a requirement

to meet the national and international criteria to ensure reliability, consistency, and accuracy of the 

analytical data generated using the Method, especially when the method is used in the EPA’s Clean Water 

Act programs. The major outcome of the validation study was a revision to the quality control/quality

acceptance (QAQC) criteria in EPA Method 1668A. 

The overall technical approach, ranging from selection of laboratories, sample selection, sample 

analysis, data reporting, data review, and validation, is good, although there are some potential areas for

improvements. A major drawback with the report is on the biosolids dataset. The objective of the study 

was to have 6 complete datasets, as per the EPA/ASTM guidelines, to validate the method. Only 4

complete datasets have been available for biosolids (Table 4-1), although validation for biosolids appears 

to be an important goal for the method, as indicated on the title. Furthermore, the data submitted by the 4

laboratories for biosolids are highly variable and no ‘in-depth’ statistical analysis of dataset has been 

made to determine accuracy and precision of the data submitted for biosolids. In other words, the Method 

has not adequately met the objectives to validate the analysis of PCBs in biosolids. Many international 

organizations such as the International Organization for Standardization (ISO) require at least eight 

complete datasets to validate the methods. Because the dataset available for biosolids does not meet the 

EPA/ASTM guidelines, validation of the Method 1668A for biosolids is incomplete/inadequate.

The second major concern with the validation study results is that the data analysis/review is focused 

heavily (or only) on precision, by measuring variability in concentrations reported by the laboratories for 

PCB congeners. Little attention has been paid to the accuracy of the results. Only for wastewater 

samples, recovery values are presented as a measure of accuracy (only for wastewater, the spiked 

concentrations were known and therefore the recovery could be measured). Accuracy is an important 

criterion in method validation. There are several ways to measure accuracy, without increasing the 

burden on the laboratories. Accuracy could have been assessed by analyzing a sample of known 

concentration, as a laboratory control material, and comparing the measured value to the true value. 

National Institute of Standards and Technology (NIST) reference materials are available for PCBs and 

laboratories could have been supplied or asked to analyze NIST reference materials, along with samples

supplied by the EPA. It has been stated that ‘to minimize the burden on volunteer participants’, 

laboratories were not required to analyze initial precision and recovery (IPR) samples. Nevertheless, 
Method 1668A Interlab Study Peer Review

April 2010 47

accuracy is an important parameter for validating accuracy of a method, a well-characterized matrix such 

as NIST SRMs can be used. Alternatively, the technique of standard additions, which is used to 

determine recovery of spiked congeners from on-going precision and recovery (OPR) samples (sand/oil 

and water) could have been used (Results from Table 4-4) to validate the accuracy of the method. 

The third issue on results: The method 1668A is intended for congener specific analysis of PCBs with an 

emphasis on toxic congeners (twelve non- and mono-ortho substituted PCBs). The scope of the method 

1668A indicates that twelve PCB congeners designated as ‘toxic’ by the World Health Organization 

(WHO) are determined. Nevertheless, the validation study is focused on ten homologue groups. The data 

analyses involved summation of all isomer data within a congener group. Precision and accuracy of the 

Method 1668A for the analysis of individual toxic congeners, are not validated. In other words, the 

method is not validated for 12 toxic PCB congeners. Furthermore, the suitability of this method for the 

analysis of mono- and di-chlorobiphenyls (CBs) is questionable for the reasons given below. First, the 

precision and accuracy for mono- and di-CBs are very poor, if not dismal. For example, mean recovery 

of mono- and di-CBs in wastewater samples were 3 and 54%, respectively. Furthermore, the 

concentration mono-CB found in wastewater sample is similar to what was found in blanks (mean for 

wastewater is 27 pg/L [Table 4-1] and that for water blank is 26 pg/L [Table 4-2]). Obviously, the QC 

criteria have been relaxed to reflect the poor recoveries (Addendum to the method validation study). 

Overall, the method did not perform well for mono- and di-CBs. If these two congener groups are 

removed, then the QC criteria for the method would be tighter and more reliable/reproducible. These 

two congeners are easily lost in the solvent evaporation steps, as has been stated in several places of the 

method validation report. 

A few other issues on the results, such as robustness, ruggedness, and limit of detection have not been 

discussed or analyzed in detail. A method validation would require that the above mentioned parameters 

are tested and validated. Some of the information presented on the validation report is not clear (e.g., 

section 4.2). For example, how were the outliers detected? How many outliers were found in samples? 

Rationale for removal of the outliers from dataset is missing. A column showing number of outlier values 

should be presented on Table 4-1. The reproducibility variation coefficients (between-laboratory 

precision and within-laboratory precision) for the method were not reported. Overall, data have not been 

fully utilized to describe several parameters that are required in validating a method (based on the 

comment 2 above and this comment).
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April 2010 48

2. Please render an opinion as to suitability for use of the data from the study and the addendum 

for revision of the QCAC in Method 1668 to make it Method 1668B. 

I believe that considerable effort has been put forward in validating the method 1668A. Nevertheless, the 

data from the validation study are under-utilized and more important validation parameters such as 

accuracy of the method, reproducibility, ruggedness, etc should be evaluated. These issues and 

corrections should be corrected/clarified. Once these revisions are made, the method may be revised to 

1668B. However, I believe that there is lot more work to be done.

3. If you have a suggestion for an improvement of the interlaboratory study report or addendum, 

please provide suggested text by section number & 

4. If you believe that a given item should be clarified, please provide the clarification by section 

number or define the clarification needed to a sufficient level of detail that we can make the 

necessary changes &

5. Please point out errors or omissions.

I feel that the charge questions 3, 4 and 5 are inter-related. The following items would help improve the 

study report and addendum

a. Page 3, Section 2.4 (Sample Selection): Some description of samples is needed. For example, 

does the fish tissue represent wholebody homogenate or fillet? Also, please specify when these 

fish samples were collected? I assume that these are wet samples (i.e., not freeze-dried). 

Similarly for biosolids, some sample description is needed. After reading the entire document, I 

realized that these are wet samples. It would be useful to indicate on page 3 (section 2.4), what 

biosolids really are and when they were collected; also state that they are wet and if possible 

indicate the moisture content? 

b. Did the wastewater effluent samples contain suspended solids? When aliquoting wastewater 

samples, how was the homogeneity in the distribution of suspended solids between aliquots 

ensured? Wastewater samples have been spiked with PCB congeners. It appears that there was 

no PCB present in wastewater sample prior to spiking. Was that true? I suspect that PCBs can be 

detected in wastewater samples at several tens to hundreds of pg/L level. The information on 

the spike concentrations listed on Table 2-2 is not clear. What was the solvent that was spiked 

into water?

c. Page 5, Section 2.6: Did all the participating laboratories use HRMS? The Method 1668A calls 

for the use of HRMS and the method validation requires the use HRMS. The deviations from the 

Method 1668A have been listed for certain parameters (section 2.7 on page 6), but not sure if all 

of the laboratories used HRMS for the analysis. Also, on page 5, it has been stated that the 
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April 2010 49

laboratories were instructed to follow the Method 1668A, it will be helpful to mention/confirm 

that on Section 3 (Data Review and Validation) that all laboratories indeed followed the Method 

1668A (other than the deviations noted on section 2.7). 

d. Page 6, line 3: Expand “SOW”; the abbreviation is used here for the first time.

e. Page 6; Laboratory reporting of rules: One important item that is missing is the conversion of 

data from ‘wet weight’ to ‘dry weight’ and vice versa (for biosolids). 

f. A major issue with the method is lies in the weight of samples used for extraction (especially 

biosolids) and final extract volume. I think the method should be modified to incorporate some 

flexibility because the weight used for extraction can vary depending on the concentrations, 

presence of interferences, etc. Also, concentration to a final volume of 20 uL prior to injection 

can introduce large variability in analytical results. I would prefer to use a sample weight of 15 g 

for biosolids and a final volume of 100 or 200 uL. I hope that the Method 1668B incorporates 

these changes to reflect some amount of flexibility in the weight of the samples used for 

extraction and the final extract volume.

g. Page 11, First paragraph, line 9: Expand “ML”

h. Table 4-1, Page 12, column #7 (Mean), third row from the bottom; 241-??? Something is missing 

here. It looks like a typo. Is there a value after ‘241’? Also, footnote 1 for the table: specify 

whether biosolid and fish data are given on wet weight or dry weight basis. For footnote 2; insert 

“sum of” between ‘represent’ and ‘all’.

i. Section 4.3.; Were the blanks analyzed similar to samples in all aspects, especially the 

concentration factor (final extract volume)? This should be mentioned in section 4.3.

j. Page 13, Table 4-2: “# Labs” for sand/oil should be “6”; “10” is a misleading number here 

because “10” is the number of datasets available, not ‘# Labs”.

k. Section 4.4: Low recoveries of mono- and di-CBs have been reported to be due to loss during 

transport or as ‘unknown’. I would expect that it is mainly due to loss of these volatile congeners 

in the solvent evaporation steps in Method 1668A.

l. Section 5.5: “(Table 5-1).” Move the ‘.” Out of the parentheses.

m. Section 6. I am little concerned about the statement that ‘the result of this interlab study met our 

objectives to characterize......”. The results have not satisfied the criteria to validate the method 

for biosolids, if we follow the guidelines that we need dataset from 6 laboratories.

n. Corrections on addendum: Table 4-1, footnote’ Biosolids concentrations in ng/kg, wet weight, 

not ‘ng/L’. Please correct. Also, the labeled compound recoveries are too low for a good 

method. The low recoveries are only for mono and di-CBs and if these two congeners are 

removed or reported separately, the recoveries are more than 50% for all other congeners. I 

suggest that recoveries for mono- and di-CB congeners are reported separately and tri-deca CB 
Method 1668A Interlab Study Peer Review

April 2010 50

are reported separately. A 15 or 20% recovery is too low to consider a method to be a good 

method. 

6. Please provide any additional comments you would like to make regarding the 

documents under review.

a. Although 14 laboratories were invited to participate (on a voluntary basis), only 6 labs have 

provided results for fish and wastewater and 4 labs for biosolids. Participation of a minimum of 

six labs is required for a method validation study. It is disappointing to note that several 

laboratories have expressed dissatisfaction with method and problems in analysis. Inviting some 

laboratories for participation with some (minimum) payment, and use of NIST SRM samples for 

accuracy analysis would enhance the quality of method validation. Because the method is 

intended for use in EPA’s Clean Water Act programs, I would strongly recommend that contract 

laboratories analyzing clean water act samples should participate in this validation study.

b. I would suggest that mono and di-CBs are reported separately or removed from the method. The 

QC criteria for these two congener groups should be reported separately from the other 

congeners.

c. Participating laboratories should report twelve toxic congeners separately and QC criteria for 

these congeners should be established; in other words, the method validation is needed for these 

12 toxic congeners.

d. A major issue with biosolids analysis lies in the use of 30 g of sample weight for extraction. 

Similarly, concentration of samples to a final volume of 20 uL, can impose serious variability 

issues. I would recommend that the method be amended to incorporate some flexibility in the 

weight of the samples taken for extraction. For biosolids, it can be from 10 to 30 g (wet weight). 

Also, concentration of extract to 20 uL, will introduce large variability. I would suggest that the 

final volume can be from 20 uL to 200 uL. I would recommend using sediments for method 

validation.