  SEQ CHAPTER \h \r 1 Addendum to the Method 1668A Interlaboratory
Validation Study Report

March 2010

Pub Number EPA-820-R-10-003

Summary

	This addendum to the Method 1668A Interlaboratory Validation Study
Report (dated November 2008) revises Table 4-1, “Congener Detection
Rates and Concentrations in Study Samples (by Matrix and Level of
Chlorination),” and revises the section on quality control (QC)
acceptance criteria in the report, including revision of the QC
acceptance criteria in Table 5-1 of the report.  The criteria were
reassessed in response to laboratory feedback that some of the criteria
in the report were unrealistically restrictive, and were revised based
on an assessment of recently submitted data.  The revised QC acceptance
criteria in this addendum were developed based on the combination of a
statistical analysis and a holistic view of the data.

This March 2010 revision to the addendum made the following changes:

Footnote 1 to Table 4-1 was corrected to show that the biosolids
concentration is in wet-weight units (because not all laboratories
provided % solids and dry-weight results).

Footnote 2 to Table 4-1 was revised to clarify that the mean, median,
and maximum results are based on any detected congener in an LOC, and
expanded to further explain that concentrations for coeluted congeners
are for combined concentrations of all congeners within that coelution.

The section on revision of QC acceptance criteria was completely revised
to take into account calibration verification data received from AXYS
Analytical and TestAmerica-Knoxville and to make the criteria consistent
across all performance tests.

Table A-1 presents the revised QC acceptance criteria.

A section was added at the end of the addendum to explain that the
revised QC acceptance criteria will appear in Revision C to Method 1668.

Background

EPA initially published Method 1668A in 1999.  Since that time, the
Agency has: 

Revised the method in 2000 and 2003 to reflect user suggestions and peer
reviews, 

Published a study plan for the Method 1668A interlaboratory validation
study in 2003,

Conducted the interlaboratory validation study in 2003-2004, 

 Published the peer reviewed validation study report, and 

Published a revised method that reflects peer review and user
suggestions and data.  

Additional details regarding the method revisions, the study, and the
peer review are available in Revisions A and B of Method 1668, and the
study plan, peer review report, validation study report, and validation
study peer review report listed above.

	After Revision B was published, AXYS Analytical Services, Ltd. (AXYS)
informed EPA that the revised initial precision and recovery (IPR) and
ongoing precision and recovery (OPR) QC acceptance criteria for some
congeners could not be met during routine laboratory operations because
these criteria did not allow recoveries exceeding 100% for many
congeners.  (In developing the Method 1668B criteria, EPA set the upper
limit on recovery to 100 percent for congeners for which a statistical
analysis resulted in recoveries less than 100 percent.)  EPA responded
to this concern by using recently submitted QC data to examine the QC
acceptance criteria published in Section 5 of the November 2008
interlaboratory study report and in Method 1668 Revision B.  This
examination resulted in revised criteria, as presented below.  The
examination also identified errors in Table 4-1 of the November 2008
report.  These changes are documented in this addendum.

Corrections to Table 4-1 of the Interlaboratory Validation Study Report

	The following is a corrected table.  The corrections result from
conversion of biosolids samples from dry weight to wet weight, so that
the results from all four laboratories were reported on the same basis. 
To allow quick comparison with the values in Table 4-1 of the original
validation study report, the corrected values in the table below are
shown in boldface type.

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

Matrix	LOC	# Labs	# Congeners Analyzed	# Congeners Detected	% Congeners
Detected	Concentration (detects only)1,2







Mean	Median	Maximum

Biosolids	1	4	24	23	96	71	74	94

	2

88	64	73	259	140	967

	3

160	134	84	514	387	2370

	4

240	195	81	667	277	4130

	5

237	166	70	1090	488	4720

	6

254	196	77	602	224	4450

	7

169	129	76	362	181	1670

	8

81	72	89	195	140	583

	9

24	23	96	161	91	630

	10

8	8	100	166	161	193

Tissue	1	6	36	26	72	4	3	12

	2

131	90	69	47	27	188

	3

232	181	78	267	150	1610

	4

352	288	82	402	130	3330

	5

347	258	74	418	128	15700

	6

362	270	75	429	108	10700

	7

240	182	76	276	120	3560

	8

114	105	92	157	108	709

	9

35	35	100	162	137	390

	10

12	12	100	200	201	236

Water	1	6	36	25	69	27	20	106

	2

128	118	92	533	505	1460

	3

233	223	96	1100	946	3430

	4

356	356	100	2850	2170	15300

	5

344	344	100	2660	1750	21800

	6

362	362	100	2190	1660	11800

	7

235	235	100	1750	1420	7370

	8

116	116	100	2410	1740	9560

	9

35	35	100	1760	1520	3350

	10

12	12	100	1740	1510	3170

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

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

Revision of QC Acceptance Criteria

	In response to the information from AXYS, the IPR and OPR QC acceptance
criteria were re-evaluated using more OPR data than were available from
the Method 1668A interlaboratory validation study.  Specifically, AXYS
and TestAmerica-Knoxville (TestAmerica) provided EPA with large sets of
OPR data they had generated as part of their routine sample analysis
activities.  Both sets of additional data were from analyses performed
after completion of the Method 1668A interlaboratory study.  AXYS
provided 113 sets of results from aqueous and solid OPR samples, and
TestAmerica provided 112 sets of results from aqueous and solid OPR
samples.

	When these recent data were compared to QC acceptance criteria in Table
5-1 of the interlaboratory validation study report (and the identical
Method 1668B criteria), EPA observed that: 

failure rates were notably higher than expected for high chlorination
level labeled analogs, and

the failure rate was higher than anticipated when assessed on a
per-sample basis (i.e., if at least one congener would fail, the OPR
would fail and, therefore, the OPR and associated batch of samples would
have to be reanalyzed for all congeners).  

As a result, EPA used these data, along with the OPR data from the
method validation study, to revise the QC acceptance criteria that were
published in the 2008 validation study report and in Method 1668B.

	When determining QC acceptance criteria, it is assumed that all data
used in the calculation are representative of the population of results
from laboratories performing the method properly, and that any extreme
results produced would be due to analytical variability, and not to
laboratory issues.  When using existing data to establish QC acceptance
criteria, this assumption can be problematic because it cannot easily be
determined whether a result is unusually high or low due to chance or
due to a problem with the sample preparation or analysis.  However, the
large number of PCB congeners tested in an OPR sample (27 native and 27
labeled congeners) allows an assessment of the consistency of each
individual sample with the overall dataset.  An OPR sample for which the
recoveries for many congeners are consistently higher or lower than
those for other samples gives an indication that there may have been an
issue with the analysis of that sample, whereas an OPR sample for which
only one or two congeners yielded unusual recoveries is more likely to
be failing by chance alone.  Therefore, each OPR sample submitted by the
two laboratories was assessed for a high frequency of unusually high or
low recoveries.  Based on this assessment, five OPR samples were removed
from the dataset.

	After removal of the five OPR samples, all remaining data from the two
laboratories were combined, and the distribution of recoveries was
examined for each native and labeled congener.  Based on this
examination, three subgroups were identified that yielded similar
distributions.  These subgroups were defined as follows:

All native congeners

Labeled congeners 1 to 54

Labeled congeners 77 to 209

	A revised set of OPR recovery criteria was chosen for each of these
subgroups that would result in an approximate 5% failure rate on a
per-sample basis.  To further assess these chosen criteria, OPR results
from the two laboratories were combined with the Method 1668A validation
study data.  This approach allowed the chosen criteria to be assessed
using data with a larger between-laboratory variance component.  Because
there were many more OPR results from the two post-study laboratories
than from the validation study laboratories, 100 sets of data were
simulated.  For each simulation run, four OPR results per congener were
chosen randomly (such that the same sample would not be chosen for all
congeners) from each of the two post-study laboratories.  For each of
these simulation runs, OPR criteria were calculated using the same
formulas used to produce Method 1668B criteria from the method
validation study.  Because the resulting simulated QC criteria tended to
be tighter than the nominal criteria, it was concluded that the added
between-laboratory variability was not large enough to necessitate
widening the chosen criteria. 

	After choosing the OPR criteria, IPR criteria were chosen based on the
OPR criteria.  Unlike OPRs, which are evaluated on an individual sample
basis, IPRs are analyzed and evaluated in sets of four.  Because means
of four measurements are less variable than single measurements, IPR
recovery criteria tend to be tighter (by approximately 10-15%) than OPR
recovery criteria.  Based on this assumption, nominal IPR criteria were
chosen for each of the three congener subsets that were approximately
10-15% tighter than the corresponding OPR recovery criteria.  These
criteria then were evaluated using the data submitted by the two
post-study laboratories.  Because these data comprised OPR samples only,
IPR sets needed to be simulated to assess the IPR criteria.  To do this,
100 sets of 4 OPRs were chosen randomly in order to simulate an IPR
“set.”  The number of occurrences where an IPR set failed the
nominal criteria was then determined for each congener.  It would be
expected that the failure rate could be slightly larger than the target
5%, because the simulated sets included a larger amount of temporal
variability than a set of IPRs analyzed by a laboratory in practice
(which are usually run within a single batch).  However, the observed
failure rates were well within the expected 5%, and therefore supported
the chosen nominal criteria.

Revision of QC Acceptance Criteria for Calibration Verification

 	QC acceptance criteria for calibration verification were not revised
based on the 2003-2004 interlaboratory study because calibration and
calibration verification data were not gathered in the study.  After
completion of the interlaboratory study, calibration verification data
were gathered from AXYS and TestAmerica.  AXYS supplied results of
analysis of 686 calibration verification samples and TestAmerica
supplied results of 1160 calibration verification samples.  Using a
similar approach to that described for the OPR criteria modification,
the results from the two laboratories were assessed to arrive at
calibration verification criteria with an approximate per-sample failure
rate of 5%.  

The criteria for all native congeners were set to 75 - 125%, vs. 70 -
130% in Method 1668A

The criteria for labeled congeners 1L to 209L were set to 50 - 145%, vs.
50 - 150% in Method 1668A

The criterion for labeled cleanup standard 28L was set to 65 - 135%, vs.
60 - 130% in Method 1668A, and 

The criteria for labeled cleanup standards 111L and 178L were set to 75
- 125% vs. 60 - 130% in Method 1668A.  

For labeled compounds 1L - 209L and the cleanup standards, the observed
per-sample failure rate for the AXYS data was 5.3 percent and the
observed per-sample failure rate for the TestAmerica data was 3.0
percent.  The adjusted criteria are shown in Table A-1.

Adjustment of QC Acceptance Criteria for OPR and Labeled Compound
Recovery from Samples

When the revised QC acceptance criteria for calibration verification,
IPR, OPR, and labeled compound recovery from samples were compared, the
upper limit of the calibration verification criteria was less
restrictive than the upper limits of the OPR and the labeled compound
recovery from samples.  To make all criteria consistent, upper limits of
criteria for OPR and labeled compound recovery from samples were
increased to be greater than or equal to the upper limits of the
calibration verification criteria. For example, for labeled congeners 1L
- 54L, OPR criteria for recovery, based on a 5% failure rate, were 15 -
140%, necessitating an increase in the upper limit to 145%.  The upper
limits on IPR recovery for labeled compounds 1L - 54L and 77L - 209L
were not increased to be greater than or equal to calibration
verification criteria because an IPR consists of the average of results
of 4 tests, whereas calibration verification is a single test.  The
adjusted criteria are shown in Table A-1.  These revised criteria
replace the criteria in Section 5 and Table 5-1 of the November 2008
report.

Table A-1. 	Revised QC Acceptance Criteria for Calibration Verification,
Initial Precision and Recovery (IPR), On-going Precision and Recovery
(OPR), and Labeled Compound Recovery from Samples

Congener set	Calibration

Verification	IPR

Recovery	IPR

Precision	OPR

Recovery	Labeled Compound

Recovery from Samples

Native Toxics and LOCs	75-125%	70-130%	25%	60-135%	NA

Labeled congeners





	1L to 54L	50-145%	20-135%	70%	15-145%	5-145%

77L to 209L	50-145%	45-135%	50%	40-145%	10-145%

Cleanup standards





	28L	65-135%	20-135%	70%	15-145%	5-145%

111L and 178L	75-125%	45-135%	50%	40-145%	10-145%

NA = Not applicable

Revision C to Method 1668

	To accommodate the changes to the QC acceptance criteria presented in
this addendum, EPA had the option of revising Method 1668B, or creating
Revision C to Method 1668.  The advantage to creating Method 1668C is
that it minimizes confusion associated with multiple versions of
Revision B.  EPA has therefore chosen to create Method 1668C to include
the revised QC acceptance criteria presented in this addendum.

Addendum to the Method 1668A Interlaboratory Validation Study Report

March 2010	  PAGE   \* MERGEFORMAT  1 

