[Federal Register Volume 62, Number 178 (Monday, September 15, 1997)]
[Rules and Regulations]
[Pages 48394-48442]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-23841]
[[Page 48393]]
_______________________________________________________________________
Part III
Environmental Protection Agency
_______________________________________________________________________
40 CFR Part 136
Guidelines Establishing Test Procedures for the Analysis of Pollutants;
EPA Method 1613; Final Rule
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 /
Rules and Regulations
[[Page 48394]]
ENVIRONMENTAL PROTECTION AGENCY
40 CFR Part 136
[FRL-5889-3]
RIN 2040-AC64
Guidelines Establishing Test Procedures for the Analysis of
Pollutants; EPA Method 1613
AGENCY: Environmental Protection Agency (EPA).
ACTION: Final rule.
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SUMMARY: Today's final regulation amends the ``Guidelines Establishing
Test Procedures for the Analysis of Pollutants'' under section 304(h)
of the Clean Water Act to approve EPA Method 1613 for determination of
tetra-through octa-chlorinated, 2,3,7,8-substituted, dibenzo-p-dioxins
and dibenzofurans (CDDs/CDFs) by high resolution gas chromatography
(HRGC) coupled with high resolution mass spectrometry (HRMS). This
regulation makes available at 40 CFR part 136 an additional, more
sensitive test procedure for CDDs/CDFs. Method 1613 is the most
sensitive analytical test procedure approved under the Clean Water Act
for the analysis of CDDs/CDFs because it measures into the low part-
per-quadrillion (ppq) range. Use of approved test procedures is
required whenever the discharge constituent specified is required to be
measured for: a National Pollutant Discharge Elimination System (NPDES)
permit application; discharge monitoring reports; state certification;
and other requests from the permitting authority for quantitative or
qualitative effluent data. Use of approved test procedures also is
required for the expression of pollutant amounts, characteristics, or
properties in effluent limitations guidelines and standards of
performance and pretreatment standards, unless otherwise specifically
noted or defined.
EFFECTIVE DATE: This regulation is effective October 15, 1997. In
accordance with 40 CFR 23.2, this rule shall be considered issued for
the purposes of judicial review September 29, 1997, at 1 p.m. eastern
daylight time. Under section 509(b)(1) of the Clean Water Act, judicial
review of these amendments can be obtained only by filing a petition
for review in the United States Court of Appeals within 120 days after
they are considered issued for the purposes of judicial review. Under
section 509(b)(2) of the Clean Water Act, the requirements of these
amendments may not be challenged later in civil or criminal proceedings
to enforce these requirements.
ADDRESSES: Documents that support this final rule are in the Water
Docket and are available for public inspection from 9 a.m. to 4 p.m. in
Room M2616, 401 M Street, SW., Washington, D.C. 20460, phone: (202)
260-3027. The Docket staff request that interested parties call for an
appointment before visiting the Docket. The EPA regulations at 40 CFR
Part 2 provide that a reasonable fee may be charged for copying.
FOR FURTHER INFORMATION CONTACT: Mr. Ben Honaker at (202) 260-2272,
USEPA Office of Science and Technology, Engineering and Analysis
Division (4303), 401 M Street, SW., Washington, DC 20460.
SUPPLEMENTARY INFORMATION:
Regulated Entities
This action approves a test procedure for the determination of
tetra- through octa-chlorinated, 2,3,7,8-substituted, CDDs/CDFs in
wastewater by HRGC/HRMS. Regulatory authorities may, at their
discretion, require use of this method in NPDES permits. Entities
potentially regulated by this action are listed in the table below.
------------------------------------------------------------------------
Category Examples of regulated entities
------------------------------------------------------------------------
Public................................. Government laboratories that
develop or employ analytical
methods for use in
demonstrating compliance with
the CWA.
Private................................ Commercial laboratories,
consensus methods
organizations, instrument
manufacturers, vendors, and
other entities that develop or
employ analytical methods for
use in demonstrating
compliance with the CWA.
------------------------------------------------------------------------
This table is not intended to be exhaustive, but rather provides a
guide for readers regarding entities likely to be regulated by this
action. This table lists the types of entities that EPA is now aware
could potentially be regulated by this action. Other types of entities
not listed in the table could also be regulated. To determine whether
your organization is regulated by this action, you should carefully
examine the applicability language of today's rule at Sec. 136.3. If
you have questions regarding the applicability of this action to a
particular entity, consult the person listed in the preceding FOR
FURTHER INFORMATION CONTACT section.
Outline of Preamble
I. Authority
II. Background and History
A. Analytical Methods Under 40 CFR Part 136, Including Method
1613
B. Promulgation of Method 1613 Under EPA's Drinking Water Rules
C. Proposal of Method 1613 for Monitoring in Pulp, Paper, and
Paperboard Industry Wastewaters
III. Summary of the Final Rule Amending Part 136
A. Purpose
B. Summary of Improvements Since Proposal
1. Development of Improved Quality Control Acceptance Criteria
(a) Interlaboratory Method Validation Study
(i) Simulated Sample Extracts
(ii) Sample Processing
(iii) Data Submission by Laboratories
(b) Data from the Pulp and Paper Long-term Variability Study
(c) Statistical Analysis
2. Procedures for Fish and Other Tissues
(a) Extraction Procedures
(i) Dehydration and Soxhlet Extraction
(ii) Hydrochloric Acid Digestion and Extraction
(b) Bulk Lipid Removal Procedures for Soxhlet Extracts
(i) Anthropogenic Isolation Column
(ii) Acidified Silica Gel
(c) Sulfuric Acid Back-extraction for HCl-digested Extracts
(d) Further Cleanup of Tissue Extracts
3. Solid-phase Extraction of Aqueous Samples
4. Sample Preservation and Holding Times
5. Other Improvements
C. Method Detection Limit (MDL) Studies
IV. Public Participation and Response to Comments
A. Duplication of Methods
B. Method Flexibility
C. Feasibility-Instrumentation and Cost Issues
1. Waste
2. Instrumentation
D. Insufficient Validation-General Comments
E. Insufficient Validation of the Matrices Specified in the
Federal Register Document
F. Interlaboratory Study
G. Method Detection Limit Studies
H. Detection/Quantitation Levels
I. Quality Assurance/Quality Control (QA/QC)
J. Miscellaneous
K. Technical Comments
V. Regulatory Analysis
A. Executive Order 12866
B. Unfunded Mandates Reform Act
C. Regulatory Flexibility Act
D. Paperwork Reduction Act
E. Submission to Congress and the General Accounting Office
I. Authority
Today's final rule is promulgated under the authority of sections
301, 304(h), 307, 308 and 501(a) of the Clean Water Act (CWA), 33
U.S.C. 1251 et seq. (the Federal Water Pollution Control Act Amendments
of 1972 as amended by the Clean Water Act of 1977 and the Water Quality
Act of 1987), 33 U.S.C. 1311, 1314(h), 1328, 1329, 1361(a); 86 Stat.
[[Page 48395]]
816, Pub. L. 92-500; 91 Stat. 1567, Pub. L. 95-217; 100 Stat. 7, Pub.
L. 100-4 (the ``Act''). Section 301 of the Act prohibits the discharge
of any pollutant into navigable waters unless the discharge complies
with an NPDES permit issued under section 402 of the Act. Section 301
also specifies levels of pollutant reductions to be achieved by certain
dates. Section 304(h) of the Act requires the EPA Administrator to
``promulgate guidelines establishing test procedures for the analysis
of pollutants that shall include the factors which must be provided in
any certification pursuant to section 401 of this Act or permit
applications pursuant to section 402 of this Act.'' These test
procedures for the analysis of pollutants also assist in the
implementation of section 301. Section 501(a) of the Act authorizes the
Administrator to prescribe such regulations as are necessary to carry
out her function under the Act.
The Administrator has also made these test procedures (methods)
applicable to monitoring and reporting of NPDES permit applications and
permits (40 CFR part 122, Secs. 122.21, 122.41, 122.44, 122.48, and
123.25), and implementation of the pretreatment standards issued under
section 307 of CWA (40 CFR part 403, Secs. 403.10 and 402.12).
II. Background and History
A. Analytical Methods Under 40 CFR Part 136, Including Method 1613
The Agency provided a history of analytical methods under 40 CFR
part 136 on February 7, 1991 (56 FR 5090) when EPA proposed the rule
being promulgated today. The preamble to today's final rule updates
that history with technical changes to EPA Method 1613 between proposal
and promulgation. These technical changes are described below in
Section III.B., ``Summary of Improvements Since Proposal.''
B. Promulgation of Method 1613 Under EPA's Drinking Water Rules
Under the Safe Drinking Water Act, EPA proposed Method 1613 for the
measurement of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also known
as dioxin, in support of the National Primary Drinking Water Regulation
for that contaminant. See 55 FR 30426 (July 25, 1990). EPA also
discussed plans to conduct an interlaboratory method validation study
to determine whether the detection and quantitation values derived by
EPA for Method 1613 represented a reasonable expectation for different
laboratories. EPA solicited comments on the appropriate level to be
used to set the maximum contaminant level (MCL) for the drinking water
rule. EPA further discussed Method 1613 for determination of dioxin in
drinking water in a ``Notice of Availability with Request for Comment''
on November 29, 1991, at 56 FR 60949.
On December 5, 1994, EPA promulgated Method 1613 for measurement of
dioxin in drinking water at 40 CFR parts 141 and 142 (59 FR 62455). In
section I.B.3.b of the preamble to that rulemaking, EPA responded to
general and specific comments on the application of EPA Method 1613 to
drinking water. EPA stated in the preamble that the Agency had
previously solicited and received comments on the proposal of Method
1613 for application to wastewater, that some of these same comments
had been received in response to the proposal of Method 1613 for
application to drinking water, and that EPA would restrict its
responses to general issues covering the application of Method 1613 to
both drinking water and wastewater and to issues specific to drinking
water. In today's preamble, EPA is responding to all comments received
on the proposal of Method 1613 for application to wastewater (56 FR
5090), including general comments that were duplicated in comments
received on the drinking water notice (56 FR 60949).
The December 5, 1994, revision to Method 1613 (for application to
drinking water) is consistent with the version of the Method in today's
rule. Therefore, with today's rulemaking, the same version of EPA
Method 1613 applies to analysis of wastewater and drinking water.
C. Proposal of Method 1613 for Monitoring in Pulp, Paper, and
Paperboard Industry Wastewaters
On December 17, 1993, EPA proposed national effluent limitations
and guidelines, pretreatment standards, and new source performance
standards for the Pulp, Paper, and Paperboard industrial point source
category. See 58 FR 66078. In the proposal, EPA referenced a compendium
titled ``Analytical Methods for the Determination of Pollutants in Pulp
and Paper Industry Wastewater.'' This compendium contained methods that
had not been promulgated at 40 CFR part 136, but would be applicable
for monitoring compliance with the numerical limitations and standards
proposed in the Pulp, Paper, and Paperboard rule. These methods were
proposed for promulgation at 40 CFR part 430 to support the proposed
regulation and were included in the docket for the proposed pulp and
paper rule.
The methods proposed for monitoring under the proposed pulp and
paper rule included an earlier version of Method 1613 than the version
EPA is promulgating today. To further conform analytical methods, NPDES
permits issued after the effective date of today's rule will require
use of today's promulgated revision of Method 1613 for determining
compliance with the final rule for the Pulp, Paper, and Paperboard
category.
III. Summary of the Final Rule Amending Part 136
A. Purpose
This rule allows the use of Method 1613 for determination of
seventeen tetra-through octa-chlorinated, 2,3,7,8-substituted dibenzo-
p-dioxins and dibenzofurans (CDDs/CDFs) in effluent samples by isotope
dilution high resolution gas chromatography (HRGC) combined with high
resolution mass spectrometry (HRMS). Method 1613 was developed to lower
the measurable range of minimum levels for the CDDs/CDFs, specifically,
into the low part per quadrillion (ppq) range for aqueous samples and
into the low part-per-trillion (ppt) range for solid and semi-solid
sample matrices. EPA believes Method 1613 is adequate and applicable
for the measurement of solid and semi-solid sample matrices, such as
biosolids and fish tissue, but today's rule does not amend test
procedures for sewage sludge regulations at 40 CFR 503.8 and does not
constitute rulemaking for measurement of fish tissue. Today's
rulemaking at 40 CFR part 136 applies for measurement of aqueous
samples.
The promulgation of Method 1613 provides a test procedure
(analytical method) for compliance monitoring under the National
Pollutant Discharge Elimination System (CWA section 402) and CWA
section 401 certifications. Method 1613 is also available for:
Development of and monitoring compliance with effluent limitations
guidelines, pretreatment standards, and new source performance
standards in EPA's water programs; ambient water quality monitoring;
and general laboratory use. By today's action, however, EPA is not
withdrawing approval of the existing method, Method 613, which also
measures 2,3,7,8-TCDD , albeit with limited sensitivity. Method 613 is
still applicable for those NPDES permits that require that this method
be used and thus existing permits do not need to be modified prior to
expiration. In addition, Method 613 remains available
[[Page 48396]]
for screening purposes. However, NPDES permits issued after
promulgation of today's rule must include Method 1613 if the permit
contains effluent limitations for dioxin.
B. Summary of Improvements Since Proposal
EPA proposed Method 1613 on February 7, 1991. See 56 FR 5090. At
the time of proposal, EPA had initiated (but had not completed) an
Interlaboratory Method Validation Study (IMVS) and was considering
other improvements to Method 1613 to increase the utility of the Method
and make the Method more efficient and cost-effective. EPA proceeded
with proposal of Method 1613 before completion of the IMVS because:
Method 1613 had been validated in single-laboratory
studies and in data gathering by EPA. The data gathering consisted of
over 500 analyses of real-world environmental samples to support
regulation development in EPA's effluent guidelines and other programs.
EPA desired to make Method 1613 available for reporting of
CDDs/CDFs under the NPDES permit regulations at 40 CFR parts 122 and
123, and the pretreatment regulations at 40 CFR part 403. At that time,
the only method approved for the determination of 2,3,7,8-
tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) under 40 CFR part 136 was
Method 613. Method 613 is 200 times less sensitive than Method 1613 for
2,3,7,8-TCDD and does not measure other CDDs/CDFs.
EPA was developing regulations for the Pulp, Paper, and
Paperboard industrial category. A high sensitivity method for 2,3,7,8-
TCDD and 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF) was required
for development of these regulations.
EPA desired to collect comments on proposed Method 1613 to
improve the Method and learn of deficiencies that needed to be
addressed before promulgation.
Since proposal, EPA has received a considerable number of
suggestions on improving the utility of Method 1613, both as described
below in Section IV, ``Public Participation and Response to Comments,''
and in technical meetings and informal and formal discussions with
laboratories, academicians, and the regulated industry. Based on the
IMVS and these discussions, EPA has made technical revisions to Method
1613 to improve the usability of the method for water and other sample
matrices. This section of the preamble describes how EPA developed some
of these improvements in response to public comment.
1. Development of Improved Quality Control Acceptance Criteria
As proposed, Method 1613 contained performance specifications in
the form of quality control (QC) acceptance criteria that were based
upon data gathered by EPA during the development of Method 1613 between
1988 and 1991. EPA developed improved QC acceptance criteria using data
from EPA's IMVS and data from the paper industry and EPA's Pulp and
Paper Long-term Variability Study (LTVS). EPA has revised the QC
acceptance criteria in the version of Method 1613 being promulgated
today. The IMVS and LTVS studies are described below. A more detailed
description of the IMVS and development of the revised QC acceptance
criteria is given in the report titled ``Results of the International
Interlaboratory Validation Study of USEPA Method 1613'' (1613 Report).
The 1613 Report is included in the docket for today's final rule.
(a) Interlaboratory Method Validation Study. In February 1990, EPA
began its interlaboratory validation of Method 1613 for the
determination of CDDs/CDFs by HRGC/HRMS. The study was international in
scope, ultimately involving receipt of data from 20 laboratories in
five countries. The purpose of the study was to further characterize
Method 1613 and to gather additional data to support today's
promulgation.
Details of the IMVS study design are given in the ``Study Plan for
the Evaluation of Method 1613'' (Study Plan). The Study Plan was
included in the docket at proposal, and the results of the study are
summarized in the 1613 Report included in the docket for this final
rule. The pertinent specifics of the IMVS are summarized below.
(i) Simulated Sample Extracts
Each laboratory participating in the IMVS received two concentrated
extracts prepared from a large-volume sample of industrial wastewater.
This large-volume sample was extracted with benzene, and the benzene
extract was concentrated. The concentrate was highly colored and
contained small amounts of solids derived from the bulk extraction of
the original sample.
The extract concentrate was split into three portions: low, medium,
and high. The low concentration extract was not fortified with any
additional CDDs/CDFs, and contained 2,3,7,8-TCDD and 2,3,7,8-TCDF at
approximately 60 and 300 ppq, respectively. The medium extract was
fortified with most of the CDDs/CDFs not already present at
concentrations in the 100- to 500-ppq range. The high extract was
fortified with most of the CDDs/CDFs in the 250-to 1000-ppq range.
After spiking, each of the three portions was further split and sealed
into glass ampules.
Two ampules of the same concentration were submitted to each
laboratory as a single blind duplicate sample, i.e., the laboratory did
not know which, if any, CDDs/CDFs were in the ampules and did not know
the concentrations of the CDDs/CDFs that were present in the ampules.
The ampules were shipped to the laboratories over a period of four
months, as additional participants joined the study.
The study design formed an incomplete block, i.e., not all
laboratories were sent each of the three different concentrates. Under
the incomplete block design used in this study, eight laboratories were
sent two low-concentration ampules each, seven laboratories were sent
two medium-concentration samples each, and the seven remaining
laboratories were sent two high-concentration ampules each. At each
laboratory, each concentrate was withdrawn from its ampule, further
concentrated, and solvent-exchanged to acetone to ensure that the
extract would be water miscible. Each acetone solution was then spiked
into a one-liter volume of reagent water to produce a simulated
effluent sample.
(ii) Sample Processing
Each simulated effluent sample was processed through the sample
extraction procedure in the proposed version of Method 1613. Although
all but one of the laboratories were experienced in performing CDD/CDF
analyses using HRGC/HRMS, less than one-third of the 22 laboratories
had direct experience with Method 1613. Therefore, laboratories were
given time to familiarize themselves with the details of the Method,
and each laboratory was required to demonstrate its general proficiency
with the Method through the analysis of four initial precision and
recovery (IPR) aliquots, as described in the Method.
In addition to demonstrating method proficiency and analyzing the
simulated effluent samples according to Method 1613, the participating
laboratories were required to perform all other QC procedures described
in the Method. These QC requirements were described in Section III.D.
of the proposal (56 FR 5092-5093).
For each sample and quality control analysis, the laboratories were
to
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provide the concentration of each analyte detected and the recovery of
each labeled standard. All supporting raw data, including selected ion
current profiles, were to be reported for all analyses.
(iii) Data Submission by Laboratories
A total of 22 laboratories in 6 countries agreed to participate in
the study on a voluntary basis. The list of laboratories is given in
the 1613 Report. After two years, data were received from a total of 20
laboratories in 5 countries. Data from each laboratory were reviewed
thoroughly and, after resolution of data problems with the
laboratories, the data were entered into a data set and combined with
data from the LTVS to construct the final QC acceptance criteria for
Method 1613 being promulgated today. EPA wishes to publicly thank the
laboratories that participated in the study, particularly those that
took the time to submit additional data and suggestions for improvement
of Method 1613.
(b) Data from the Pulp and Paper Long-term Variability Study. Data
gathering in the LTVS is described in detail in Section 7.5.2 of the
Technical Support Document for the rule proposed for the Pulp, Paper,
and Paperboard category (58 FR 66078). The procedures for validation of
these data were developed in discussions between EPA and
representatives of the paper industry. These validation procedures
included detailed examination of all QC data associated with each field
sample result. Specifically, the QC data were used to determine if the
field sample results should be included in or excluded from the LTVS
database that was used during development of the proposed pulp and
paper industry effluent limitations guidelines and standards. Both the
QC and the field sample data were maintained by EPA in a separate
database intended for method development purposes. This included QC
data for Method 1613, which were used to develop the final QC
acceptance criteria in the version of the Method being promulgated
today. The statistical procedures used to develop these final
acceptance criteria are summarized below.
(c) Statistical Analysis. QC limits were calculated by constructing
statistical prediction intervals for future observations of a quantity
of interest using statistical estimates from data from the IMVS and
LTVS. The statistical methods used are the same as those used to
develop QC limits for EPA Method 1625 (49 FR 43234).
In other EPA method validation studies, compound-specific
performance specifications usually have been determined at individual
test levels with a probability of 0.05 (i.e., based on 95 percent
confidence limits for a single future observation). Using such
specifications, each compound measured would have a five percent chance
of falling outside its QC limit. Because of the large number of
compounds simultaneously tested in the quality control tests for Method
1613, it would be extremely likely that one or more criteria on each
test would be failed simply by random chance if the tests were all
performed at individual test levels of p = .05. It was deemed
desirable, instead, to specify test limits such that the global test
level (i.e., the chance of failing on any one or more of the CDDs/CDFs
out of the whole list) was held to five percent. This was done by
adjusting the significance level used on each compound such that the
overall Type I error rate would be 0.05 for each test situation.
Details of the binomial calculations for these considerations are given
in appendix A to the 1613 Report.
QC acceptance criteria were developed for tests of calibration
linearity, calibration verification (VER), precision of relative
retention time (RRT), IPR, ongoing precision and recovery (OPR), and
labeled compound recovery in field samples and blanks.
Separate QC acceptance criteria were developed for the instances in
which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-
tetrachlorodibenzofuran (TCDF) are determined independently of the
other CDDs/CDFs. The purpose of creating these separate criteria is to
support those regulations, such as drinking water rules and the
proposed rule for the Pulp, Paper, and Paperboard category, in which
only TCDD and/or TCDF are regulated.
2. Procedures for Fish and Other Tissues
Procedures for the homogenization, preparation, extraction, and
cleanup of fish and other tissues have been included in the revision of
Method 1613 being promulgated today to increase the applicability of
Method 1613 to these sample matrices. EPA is including these tissue
extraction procedures based on tissue sample data gathered by EPA's
Duluth laboratory, Dow Chemical Company, and commercial laboratories
performing tests for EPA and other entities. See the docket for today's
rule for references 21 through 28 cited in section 22.0 of Method 1613.
(a) Extraction Procedures. Two extraction procedures are in common
use for the extraction of the CDDs/CDFs from tissue: Dehydration and
Soxhlet Extraction, and Hydrochloric Acid Digestion and Extraction.
Both of these procedures have been incorporated into the version of
Method 1613 that is being promulgated today.
(i) Dehydration and Soxhlet Extraction
In this procedure, a 10-gram aliquot of homogenized tissue is mixed
with powdered, anhydrous sodium sulfate and allowed to stand for 12-24
hours so that the sodium sulfate can adsorb most of the moisture in the
tissue. After re-mixing, the tissue is placed in a Soxhlet extractor
and extracted for 18-24 hours with methylene chloride:hexane (1:1). The
organic extract containing the CDDs/CDFs and lipids is evaporated to
dryness, and the lipid content of the residue is determined. The
residue is dissolved in hexane and subjected to one of the two bulk
lipid removal procedures associated with the Soxhlet extraction that
are described below.
(ii) Hydrochloric Acid Digestion and Extraction
In this procedure, a 10-gram aliquot of homogenized tissue is
digested with hydrochloric acid and simultaneously extracted with
methylene chloride:hexane (1:1) in a glass bottle. The organic extract
containing the CDDs/CDFs and lipids is decanted and evaporated to
dryness, and the lipid content of the residue is determined. The
residue is dissolved in hexane, and lipids are removed using the
sulfuric acid back-extraction procedure described below.
(b) Bulk Lipid Removal Procedures for Soxhlet Extracts. Two
procedures are in common use for removal of lipids from extracts
produced by the Soxhlet extraction procedure. Both of these procedures
have been incorporated into the version of Method 1613 that is being
promulgated today.
(i) Anthropogenic Isolation Column
This column contains, in order from bottom to top, neutral silica
gel, potassium silicate, anhydrous sodium sulfate, acid silica gel, and
anhydrous sodium sulfate. The column is pre-eluted with hexane, and the
extract from the Soxhlet extraction is placed on the column and eluted
with 200 mL of hexane. Fats, lipids, and other materials are retained
on the column while the CDDs/CDFs elute in the hexane.
(ii) Acidified Silica Gel
In this bulk cleanup procedure, 30-100 grams of acidified silica
gel are stirred for 2-3 hours with the extract from the Soxhlet
extraction. After stirring, the solution is filtered to remove the
silica gel. Fats, lipids, and other materials are retained on the
silica
[[Page 48398]]
gel while the CDDs/CDFs remain in solution in the hexane.
(c) Sulfuric Acid Back-extraction for HCl-digested Extracts. In
this cleanup procedure, the re-dissolved residue from the hydrochloric
acid digestion is back-extracted with concentrated sulfuric acid for a
maximum exposure time of 45 seconds. The sulfuric acid severs the bonds
in the lipidic material during this period but there is insufficient
contact time for the acid to attack the CDDs/CDFs. After back-
extraction with sulfuric acid, the extract is further back-extracted
with potassium hydroxide solution to remove residual lipidic material
and to neutralize any residual acid that may be present.
(d) Further Cleanup of Tissue Extracts. After each of the
procedures for extraction and bulk cleanup described above, the extract
is further cleaned up using any or all of the cleanup procedures in
Method 1613. For further cleanup of tissues (and for general use), a
Florisil cleanup procedure has been added to the revision
of Method 1613 being promulgated today. The Florisil
cleanup is intended primarily for removal of chlorodiphenylethers, a
common contaminant in tissues. Though Florisil is a trade
name for a specific adsorbent, EPA does not endorse any specific
product or manufacturer; equivalent products may be substituted.
After cleanup, the extract is reconcentrated, internal standards
are added, and an aliquot is injected into the HRGC/HRMS, as in the
proposed version of Method 1613.
3. Solid-phase Extraction of Aqueous Samples
An optional solid-phase extraction (SPE) procedure has been added
to the revision of Method 1613 being promulgated today. This SPE
procedure allows laboratories to minimize solvent usage and is
therefore consistent with EPA's objectives for source reduction of
pollutants and pollution prevention. The SPE procedure is for use with
water samples containing less than one percent suspended solids and is
therefore applicable to drinking water, river water, ocean water, and
relatively clean wastewaters.
In this optional SPE procedure, an SPE disk is placed on a fritted
glass disk on top of a vacuum flask. A glass-fiber filter is placed on
top of the SPE disk, and a glass container is placed on top of the
stack of disks. The assembly is clamped to prevent leakage.
Particles in a 1-L aqueous sample are allowed to settle. The disk
is wetted with organic solvents and water, and is kept wet during the
extraction. The aqueous sample is poured through the disks. Vacuum is
used to increase the flow rate of sample through the disks, if desired.
The particles remaining in the bottle are poured in last to minimize
plugging of the disks. The sample bottle is rinsed and the rinsate is
added to the container on top of the disks.
After all of the sample has been processed through the disks, the
disks are extracted using the SDS procedures given in Method 1613 and
described at proposal (56 FR 5094).
4. Sample Preservation and Holding Times
Dechlorination, pH reduction below pH=9, and refrigeration or
freezing (depending on the sample matrix) are the only techniques
required to stabilize the CDDs/CDFs against degradation during storage.
There are no demonstrated maximum holding times associated with
CDDs/CDFs in aqueous, solid, semi-solid, tissue, or other sample
matrices. If stored in the dark at 0-4 deg.C and preserved as described
above, aqueous samples may be stored for up to one year. Similarly, if
stored in the dark at <-10 deg.c,="" solid,="" semi-solid,="" and="" tissue="" samples="" may="" be="" stored="" for="" up="" to="" one="" year.="" sample="" extracts="" are="" stored="" in="" the="" dark="" at=""><-10 deg.c="" until="" analyzed.="" if="" stored="" in="" the="" dark="" at=""><-10 deg.c,="" sample="" extracts="" may="" be="" stored="" for="" up="" to="" one="" year.="" the="" version="" of="" method="" 1613="" that="" is="" being="" promulgated="" today="" reflects="" these="" findings.="" in="" addition,="" today's="" rule="" revises="" table="" ii="" of="" 40="" cfr="" part="" 136="" to="" reflect="" the="" changes="" in="" sample="" preservation="" and="" holding="" times="" in="" method="" 1613="" being="" promulgated="" today.="" 5.="" other="" improvements="" other="" significant="" improvements="" include:="" addition="" of="" an="" optional="" rotary="" evaporation="" procedure="" for="" concentration="" of="" extracts;="" simplification="" of="" test="" solutions="" for="" demonstration="" of="" isomer-specific="" separation="" of="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf;="" and="" the="" addition="" of="" flow="" charts="" to="" illustrate="" procedures="" for="" aqueous,="" solid,="" tissue,="" and="" multi-="" phase="" samples.="" with="" the="" improvements="" described="" above,="" epa="" believes="" that="" the="" flexibility="" within="" method="" 1613="" has="" been="" increased="" and="" the="" costs="" of="" performing="" analyses="" using="" method="" 1613="" have="" potentially="" been="" reduced,="" thereby="" resulting="" in="" an="" overall="" benefit="" to="" the="" regulated="" and="" analytical="" communities.="" c.="" method="" detection="" limit="" (mdl)="" studies="" at="" the="" time="" of="" proposal,="" epa="" had="" conducted="" an="" initial="" ``method="" detection="" limit''="" (mdl)="" study="" and="" determined="" that="" method="" 1613="" could="" achieve="" an="" mdl="" of="" 5.6="" ppq="" for="" 2,3,7,8-tcdd.="" epa="" used="" this="" mdl="" to="" support="" a="" minimum="" level="" (ml)="" of="" 10="" ppq="" in="" method="" 1613.="" in="" section="" iv,="" ``public="" participation="" and="" response="" to="" comments,''="" in="" this="" preamble,="" epa="" responds="" to="" comments="" about="" this="" initial="" mdl="" study.="" since="" proposal,="" epa="" has="" conducted="" four="" additional="" mdl="" studies="" to="" confirm="" the="" mdl="" for="" 2,3,7,8-tcdd="" (tcdd)="" and="" to="" measure="" mdls="" and="" confirm="" mls="" for="" the="" other="" cdds/cdfs.="" the="" four="" studies="" were="" conducted="" in="" reagent="" water="" and="" in="" final="" effluent="" and="" bleach="" plant="" effluent="" from="" a="" pulp="" and="" paper="" industry="" facility.="" the="" studies="" of="" reagent="" water="" resulted="" in="" mdls="" of="" 5.1="" and="" 1.0="" ppq="" for="" tcdd="" and="" mdls="" for="" the="" other="" cdds/cdfs="" that="" are="" consistent="" with="" the="" mls="" in="" method="" 1613.="" for="" the="" final="" effluent,="" the="" mdl="" for="" tcdd="" was="" 4.2="" ppq="" and="" the="" mdls="" for="" the="" other="" cdds/cdfs="" were="" consistent="" with="" the="" mls="" in="" method="" 1613,="" except="" for="" one="" hexachloro-="" dioxin,="" one="" heptachlorofuran,="" heptachlorodioxin,="" and="" ocdd,="" which="" were="" higher="" than="" expected.="" for="" the="" bleach="" plant="" effluent,="" the="" mdls="" were="" consistent="" with="" the="" mls="" in="" method="" 1613="" except="" for="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf,="" which="" did="" not="" meet="" the="" mdl="" procedure="" criteria="" because="" of="" high="" background="" concentrations="" of="" these="" compounds="" in="" the="" sample.="" the="" results="" of="" the="" four="" mdl="" studies="" are="" included="" in="" the="" docket="" for="" this="" final="" rule.="" collectively,="" the="" four="" mdl="" studies="" demonstrate="" that="" the="" mdls="" and="" mls="" for="" the="" cdds/cdfs="" can="" be="" attained="" in="" reagent="" water="" and="" in="" wastewaters="" from="" a="" pulp="" and="" paper="" industry="" facility.="" iv.="" public="" participation="" and="" response="" to="" comments="" condensed="" significant="" comments="" and="" responses="" are="" presented="" below.="" the="" full="" text="" of="" summarized="" comments="" and="" responses="" are="" contained="" in="" the="" docket="" in="" the="" document="" titled="" ``detailed="" responses="" to="" comments="" on="" proposal="" of="" method="" 1613.''="" comments="" and="" responses="" are="" presented="" by="" the="" following="" subject="" areas:="" a.="" duplication="" of="" methods="" b.="" method="" flexibility="" c.="" feasibility--instrumentation="" and="" cost="" issues="" 1.="" waste="" 2.="" instrumentation="" d.="" insufficient="" validation--general="" comments="" e.="" insufficient="" validation="" of="" the="" matrices="" specified="" in="" the="" federal="" register="" notice="" f.="" interlaboratory="" study="" g.="" method="" detection="" limit="" studies="" h.="" detection/quantitation="" levels="" i.="" quality="" assurance/quality="" control="" j.="" miscellaneous="" k.="" technical="" comments="" [[page="" 48399]]="" a.="" duplication="" of="" methods="" comment:="" proposed="" method="" 1613="" and="" office="" of="" solid="" waste="" sw-846="" method="" 8290="" are="" significantly="" different.="" another="" commenter="" stated="" that="" methods="" 1613="" and="" 8290="" are="" similar="" and="" that="" the="" efforts="" by="" ow="" and="" osw="" are="" duplicative.="" response:="" epa="" agrees="" that="" the="" two="" methods="" are="" different="" in="" exact="" technical="" detail,="" but="" asserts="" that="" the="" principle="" of="" the="" two="" methods="" is="" the="" same.="" epa="" is="" in="" the="" process="" of="" consolidating="" methods="" for="" dioxin="" measurement="" in="" air,="" water,="" and="" solid="" waste.="" however,="" the="" agency's="" intention="" for="" such="" a="" merger="" for="" all="" of="" these="" matrices="" should="" not="" preclude="" prompt="" development="" and="" promulgation="" of="" this="" method="" for="" the="" water="" matrix.="" method="" 1613="" is="" a="" test="" method="" specifically="" designed="" to="" support="" revisions="" of="" the="" effluent="" guidelines="" for="" the="" pulp,="" paper,="" and="" paperboard="" category="" under="" the="" cwa.="" epa="" used="" method="" 1613="" in="" the="" development="" of="" those="" regulations,="" specifically="" for="" the="" water="" matrix.="" therefore,="" epa="" is="" promulgating="" method="" 1613="" for="" evaluation="" of="" matrices="" regulated="" under="" the="" cwa,="" notwithstanding="" any="" technical="" differences="" in="" the="" method="" used="" to="" evaluate="" matrices="" evaluated="" under="" the="" resource="" conservation="" and="" recovery="" act.="" epa="" also="" notes="" that="" today's="" action="" does="" not="" promulgate="" a="" test="" method="" for="" measurement="" of="" dioxin="" in="" sewage="" sludge,="" even="" though="" the="" agency="" used="" method="" 1613="" to="" measure="" dioxin="" concentrations="" in="" the="" ``national="" sewage="" sludge="" survey.''="" in="" the="" future,="" epa="" intends="" to="" propose="" and="" invite="" comment="" on="" the="" use="" of="" method="" 1613="" (or="" the="" consolidated="" multi-matrix="" method)="" for="" the="" measurement="" of="" dioxin="" in="" sewage="" sludge.="" b.="" method="" flexibility="" comment:="" flexibility="" in="" sample="" preparation="" and="" tailoring="" of="" the="" procedure="" to="" the="" matrix="" type="" are="" desirable,="" but="" allowing="" the="" analyst="" the="" flexibility="" to="" modify="" the="" method="" may="" adversely="" affect="" method="" performance="" on="" real-world="" samples.="" response:="" flexibility="" is="" permitted="" only="" in="" discretionary="" elements="" of="" the="" test="" procedures="" indicated="" by="" use="" of="" the="" terms="" ``may''="" and="" ``can.''="" all="" data="" generated="" must="" meet="" all="" performance="" criteria="" (quality="" control="" acceptance="" criteria)="" in="" the="" method.="" applicability="" of="" the="" qc="" performance="" criteria="" will="" preclude="" adverse="" effects="" of="" any="" modifications="" allowable="" under="" the="" flexibility="" in="" the="" method.="" c.="" feasibility--instrumentation="" and="" cost="" issues="" 1.="" waste="" comment:="" substantial="" volumes="" of="" cdd/cdf-contaminated="" lab="" wastes="" will="" be="" created="" that="" cannot="" be="" disposed="" of="" or="" treated.="" the="" use="" of="" isotope="" dilution="" instead="" of="" external="" standard="" techniques="" will="" result="" in="" the="" generation="" of="" more="" hazardous="" waste="" because="" each="" sample="" is="" spiked="" with="" labeled="" analogs.="" response:="" any="" analytical="" method="" that="" employs="" analytical="" standards="" for="" calibration="" and="" quality="" control="" (qc)="" purposes="" will="" generate="" a="" certain="" amount="" of="" laboratory="" waste.="" epa="" believes="" that="" there="" are="" environmental="" benefits="" associated="" with="" using="" isotope="" dilution="" techniques,="" namely="" better="" monitoring="" and="" regulation="" of="" cdds/cdfs="" at="" very="" low="" levels.="" these="" benefits="" outweigh="" any="" possible="" disadvantage="" of="" creating="" relatively="" small="" amounts="" of="" laboratory="" waste.="" 2.="" instrumentation="" comment:="" high="" resolution="" mass="" spectrometer="" (hrms)="" instruments="" are="" expensive="" and="" there="" are="" no="" u.s.="" manufacturers.="" response:="" hrms="" instrumentation="" represents="" state-of-the-art="" technology="" that="" allows="" detection="" of="" cdd/cdf="" compounds="" at="" far="" lower="" levels="" in="" less="" time="" and="" with="" greater="" certainty="" than="" lrms="" instrumentation="" and="" is="" therefore="" worth="" the="" added="" cost.="" currently,="" there="" are="" several="" u.s.="" manufacturers="" of="" hrms="" instruments.="" moreover,="" method="" 1613="" will="" not="" be="" the="" only="" applicable="" method="" for="" dioxin="" in="" all="" instances;="" approval="" of="" method="" 613="" is="" not="" being="" withdrawn="" by="" today's="" action.="" comment:="" method="" 1613="" is="" not="" very="" practical="" since="" it="" requires="" at="" least="" two="" separate="" analytical="" runs="" on="" two="" different="" gc="" columns,="" resulting="" in="" considerable="" instrument="" down-time="" to="" switch="" columns="" and="" data="" collection="" criteria.="" response:="" epa="" disagrees="" with="" the="" commenters'="" conclusion="" that="" the="" separate="" analytical="" runs="" will="" be="" required="" in="" all="" circumstances.="" the="" use="" of="" a="" second="" gc="" column="" is="" routinely="" used="" in="" many="" analytical="" laboratories="" for="" confirmatory="" purposes.="" an="" analytical="" run="" on="" the="" second="" column="" is="" not="" required="" unless="" 2,3,7,8-tcdf="" is="" found="" or="" if="" ambiguities="" exist="" about="" the="" identification="" of="" other="" cdd/cdf="" congeners="" on="" the="" first="" column.="" comment:="" the="" soxhlet/dean-stark="" (sds)="" extraction="" procedure="" for="" solids="" has="" only="" been="" tested="" to="" a="" limited="" extent="" on="" one="" municipal="" sludge.="" response:="" since="" proposal="" of="" method="" 1613,="" epa,="" ncasi,="" and="" others="" have="" extracted="" many="" samples="" using="" the="" sds="" technique.="" although="" some="" data="" show="" that="" some="" of="" the="" higher="" isomers="" and="" congeners="" of="" dioxin="" may="" not="" be="" extracted="" as="" efficiently="" with="" the="" sds="" techique="" as="" with="" other="" extraction="" techniques,="" epa="" has="" not="" yet="" confirmed="" these="" results.="" the="" originators="" of="" the="" application="" of="" the="" sds="" technique="" at="" the="" dow="" chemical="" company="" tested="" the="" technique="" on="" many="" samples="" prior="" to="" the="" time="" that="" epa="" adopted="" the="" technique="" and="" showed="" that="" the="" technique="" was="" able="" to="" extract="" certain="" cdds/cdfs="" from="" samples="" believed="" to="" contain="" non-detectable="" levels="" of="" these="" compounds.="" in="" one="" of="" the="" single-laboratory="" tests,="" epa="" confirmed="" dow's="" findings="" that="" certain="" isomers/congeners="" were="" extracted="" more="" efficiently="" with="" the="" sds="" procedure="" than="" with="" the="" soxhlet="" extractor.="" epa="" reported="" the="" results="" of="" its="" sds="" extraction="" study="" in="" its="" proposal="" of="" method="" 1613="" (56="" fr="" 5094).="" epa="" therefore="" believes="" that="" the="" sds="" extractor="" represents="" the="" best="" available="" technique="" for="" a="" diversity="" of="" sample="" matrices.="" most="" importantly,="" however,="" by="" today's="" rulemaking,="" epa="" is="" not="" promulgating="" a="" test="" procedure="" for="" measurement="" of="" solid="" matrix="" samples,="" only="" waste="" water="" samples.="" comment:="" the="" procedures="" proposed="" for="" extraction="" of="" solids="" are="" inappropriate="" for="" use="" on="" process="" pulps,="" dried="" pulps,="" or="" fiber-="" containing="" sludges.="" response:="" epa="" is="" aware="" that="" dried="" pulp="" and="" similar="" samples="" present="" a="" formidable="" extraction="" problem.="" pulp="" swells="" when="" wet,="" allowing="" exchange="" of="" the="" extraction="" solvent="" with="" the="" water="" in="" the="" interstices="" of="" the="" pulp.="" low="" molecular="" weight="" alcohols="" also="" seem="" to="" swell="" the="" pulp="" fibers="" and="" are="" an="" alternative="" to="" the="" use="" of="" nonpolar="" solvents="" for="" the="" extraction="" of="" cdds/cdfs="" from="" dry="" pulp.="" epa="" believes="" that="" if="" the="" dry="" pulp="" or="" similar="" material="" is="" completely="" swollen="" in="" reagent="" water,="" however,="" the="" sds="" extractor="" will="" reliably="" extract="" cdds/cdfs="" from="" this="" matrix.="" epa="" has="" included="" instructions="" for="" dealing="" with="" dried="" pulp="" and="" similar="" materials="" in="" the="" version="" of="" method="" 1613="" being="" promulgated="" today.="" d.="" insufficient="" validation--general="" comments="" comment:="" epa="" is="" premature="" in="" proposing="" method="" 1613="" under="" section="" 304(h)="" of="" cwa="" since="" it="" was="" not="" completely="" and="" thoroughly="" subjected="" to="" intra-="" and="" interlaboratory="" testing="" according="" to="" accepted="" scientific="" standards.="" response:="" prior="" to="" proposal="" of="" epa="" method="" 1613,="" epa="" had="" completed="" a="" single-laboratory="" validation="" of="" the="" sds="" extraction="" technique="" in="" municipal="" sewage="" sludge="" and="" a="" single-laboratory="" method="" detection="" limit="" (mdl)="" study.="" [[page="" 48400]]="" since="" proposal,="" epa="" has="" completed="" a="" total="" of="" four="" additional="" mdl="" studies="" and="" the="" imvs="" described="" in="" this="" preamble="" and="" in="" greater="" detail="" in="" the="" 1613="" report="" that="" is="" included="" in="" the="" docket="" for="" today's="" rule.="" the="" four="" additional="" mdl="" studies="" were="" performed="" in="" reagent="" water="" and="" in="" bleach="" plant="" effluent="" and="" final="" effluent="" from="" a="" pulp="" and="" paper="" industry="" facility.="" epa="" conducted="" the="" international="" imvs="" with="" the="" express="" purpose="" of="" further="" characterizing="" method="" 1613="" and="" developing="" qc="" acceptance="" criteria.="" epa="" believes="" that="" the="" results="" of="" these="" studies="" provide="" more="" than="" sufficient="" validation="" to="" confirm="" that="" method="" 1613="" is="" suitable="" for="" use="" as="" a="" test="" procedure="" in="" accordance="" with="" the="" requirements="" of="" the="" clean="" water="" act.="" these="" data="" confirmed="" epa's="" conclusions="" about="" achievable="" mdls,="" which="" were="" based="" on="" intralaboratory="" validation="" studies.="" therefore,="" epa="" does="" not="" believe="" it="" is="" premature="" to="" promulgate="" method="" 1613="" at="" this="" time="" because="" the="" interlaboratory="" validation="" data="" merely="" confirms="" epa's="" earlier="" conclusions.="" comment:="" epa="" has="" failed="" to="" publish="" performance="" information="" for="" method="" 1613,="" whereas="" such="" performance="" information="" has="" been="" published="" for="" the="" organic="" methods="" already="" incorporated="" into="" 40="" cfr="" part="" 136,="" appendix="" a.="" this="" commenter="" urges="" epa="" to="" include="" interlaboratory="" and="" intralaboratory="" performance="" data="" in="" any="" final="" rule="" it="" publishes="" for="" method="" 1613.="" response:="" epa="" has="" included="" performance="" information="" in="" the="" 1613="" report="" and="" in="" the="" results="" of="" the="" mdl="" studies="" conducted="" between="" proposal="" and="" this="" promulgation.="" these="" performance="" data="" are="" included="" in="" the="" docket="" that="" supports="" today's="" final="" rule.="" e.="" insufficient="" validation="" of="" the="" matrices="" specified="" in="" the="" federal="" register="" document="" comment:="" there="" has="" been="" insufficient="" intralaboratory="" testing="" and="" validation="" using="" the="" sample="" matrices="" for="" which="" epa="" claims="" applicability="" for="" method="" 1613.="" response:="" epa="" has="" collected="" single-laboratory="" data="" on="" several="" matrices,="" including="" reagent="" water,="" treated="" and="" untreated="" wastewater,="" paper="" pulp,="" sludge,="" soil,="" and="" fish="" tissue,="" but="" has="" not="" undertaken="" complete="" intra-="" and="" interlaboratory="" validation="" of="" each="" matrix.="" epa="" will="" perform="" intra-="" and="" interlaboratory="" validations="" of="" method="" 1613="" and="" other="" methods="" on="" those="" matrices="" for="" which="" the="" agency="" believes="" that="" such="" validation="" is="" necessary="" and="" appropriate.="" however,="" epa="" believes="" that="" it="" is="" unnecessary="" to="" perform="" extensive="" validation="" studies="" of="" method="" 1613="" or="" any="" other="" method="" on="" every="" sample="" matrix="" to="" which="" the="" method="" is="" to="" be="" applied.="" for="" example,="" epa="" regulates="" more="" than="" 600="" subcategories="" of="" wastewater="" discharge.="" epa="" believes="" that="" interlaboratory="" validation="" of="" method="" 1613="" on="" each="" discharge="" not="" only="" would="" be="" costly="" and="" impose="" an="" enormous="" administrative="" burden,="" but="" would="" not="" be="" likely="" to="" yield="" any="" more="" improvements="" in="" the="" method="" than="" would="" be="" gained="" by="" single-="" laboratory="" tests="" on="" a="" few="" such="" representative="" discharges,="" particularly="" for="" aqueous="" samples="" from="" every="" conceivable="" type="" of="" industrial="" facility.="" most="" importantly,="" however,="" though="" epa="" believes="" that="" method="" 1613="" is="" adequate="" and="" applicable="" for="" the="" measurement="" of="" solid="" matrices,="" such="" as="" sewage="" sludge="" and="" fish="" tissue,="" today's="" action="" does="" not="" promulgate="" a="" method="" for="" measurement="" of="" those="" solid="" matrices.="" in="" the="" future,="" epa="" does="" intend="" to="" propose="" and="" invite="" public="" comment="" on="" use="" of="" method="" 1613="" for="" measurement="" of="" dioxin="" in="" sewage="" sludge.="" f.="" interlaboratory="" study="" comment:="" several="" commenters="" stated="" that="" epa="" had="" not="" completed="" its="" interlaboratory="" study="" at="" time="" of="" proposal="" and="" that="" epa="" is="" premature="" in="" proposing="" method="" 1613="" without="" validating="" it="" first.="" response:="" the="" international="" imvs="" has="" been="" completed="" and="" data="" from="" the="" study="" were="" combined="" with="" data="" from="" the="" pulp="" and="" paper="" ltvs="" to="" produce="" the="" final="" qc="" acceptance="" criteria="" in="" method="" 1613="" being="" promulgated="" today.="" comment:="" the="" use="" of="" extracts="" rather="" than="" real-world="" mill="" effluents="" in="" the="" interlaboratory="" study="" will="" not="" provide="" the="" necessary="" validation="" of="" method="" 1613.="" response:="" epa="" used="" extracts="" of="" real-world="" samples="" because="" the="" agency="" felt="" that="" domestic="" and="" international="" shipment="" of="" large="" volumes="" of="" dioxin-containing="" water="" would="" create="" too="" great="" a="" risk="" to="" human="" health="" and="" the="" environment.="" the="" agency="" also="" felt="" that="" it="" would="" be="" too="" difficult="" to="" produce="" a="" homogeneous="" mixture="" of="" cdds/cdfs="" in="" such="" large="" water="" volumes.="" g.="" method="" detection="" limit="" studies="" comment:="" the="" mdls="" in="" method="" 1613="" have="" not="" been="" demonstrated="" and="" it="" is="" not="" possible="" for="" even="" the="" best="" laboratories="" to="" attain="" the="" mdl="" developed="" by="" epa.="" the="" 5="" ppq="" mdl="" for="" 2,3,7,8-tcdd="" in="" method="" 1613="" was="" calculated="" from="" a="" single-shot="" experiment="" that="" was="" not="" conducted="" properly="" and="" does="" not="" represent="" a="" real-world="" estimate="" of="" the="" mdl="" because="" it="" was="" not="" conducted="" in="" pulp="" and="" paper="" industry="" wastewater.="" response:="" epa="" disagrees.="" epa="" had="" demonstrated="" an="" mdl="" of="" 5="" ppq="" using="" method="" 1613,="" as="" described="" at="" proposal.="" epa="" conducted="" the="" iterative="" mdl="" procedure="" according="" to="" the="" procedures="" specified="" in="" 40="" cfr="" part="" 136="" appendix="" b.="" since="" proposal,="" epa="" has="" conducted="" a="" total="" of="" four="" additional="" mdl="" studies="" in="" reagent="" water="" and="" in="" in-process="" and="" final="" effluents="" from="" the="" pulp="" and="" paper="" industry.="" the="" results="" of="" these="" mdl="" studies="" confirm="" results="" from="" the="" reagent="" water="" mdl="" study="" described="" in="" the="" method="" proposal.="" h.="" detection/quantitation="" levels="" comment:="" method="" 1613="" will="" not="" ensure,="" or="" even="" come="" close="" to="" ensuring,="" that="" dioxin="" concentrations="" at="" or="" below="" epa's="" water="" quality="" criterion="" will="" be="" achieved.="" the="" proposed="" method="" will="" not="" be="" capable="" of="" detecting="" effluent="" dioxin="" levels="" that="" exceed="" the="" in-stream="" water="" quality="" criterion,="" yet="" are="" less="" than="" 10="" ppq.="" response:="" epa="" agrees.="" epa's="" water="" quality="" criterion="" for="" 2,3,7,8-="" tcdd="" is="" 13="" parts="" per="" quintillion="" (ppqt),="" while="" the="" method="" 1613="" minimum="" level="" is="" 10="" ppq.="" method="" 1613="" is="" the="" product="" of="" an="" extensive="" method="" development="" effort="" to="" produce="" a="" method="" that="" utilizes="" state-of-the-art="" technology="" to="" reliably="" achieve="" the="" lowest="" level="" of="" detection="" possible="" with="" one-liter="" water="" samples.="" while="" method="" 1613="" is="" not="" capable="" of="" achieving="" the="" water="" quality="" criterion="" of="" 13="" ppqt,="" it="" is="" at="" least="" 200="" times="" more="" sensitive="" than="" the="" only="" currently="" approved="" 304(h)="" dioxin="" method,="" method="" 613.="" epa="" will="" continue="" to="" explore="" new="" measurement="" techniques="" to="" develop="" methods="" that="" yield="" mdls="" that="" will="" allow="" determination="" of="" 2,3,7,8-tcdd="" at="" the="" ambient="" criteria="" level.="" in="" the="" meantime,="" however,="" epa="" must="" regulate="" effluent="" discharges="" at="" levels="" lower="" than="" those="" in="" method="" 613,="" and="" therefore="" must="" move="" forward="" with="" promulgation="" of="" method="" 1613="" for="" such="" purposes.="" comment:="" the="" term="" ``minimum="" level''="" (ml)="" as="" defined="" in="" the="" proposed="" rule="" is="" inconsistent="" with="" previous="" epa="" definitions="" of="" ml.="" epa="" equates="" the="" ml="" with="" the="" american="" chemical="" society's="" limit="" of="" quantitation="" (loq),="" which="" is="" different="" from="" other="" epa="" documents="" in="" which="" epa="" appears="" to="" equate="" the="" ml="" to="" a="" limit="" of="" detection="" not="" a="" limit="" of="" quantitation.="" epa="" also="" stated="" that="" the="" ml="" is="" to="" be="" calculated="" based="" on="" interlaboratory="" analyses="" of="" the="" analyte="" in="" the="" matrix="" of="" concern.="" epa="" should="" develop="" scientifically="" sound="" conventions="" for="" limits="" of="" detection="" and="" quantitation,="" allow="" public="" review="" and="" comment,="" and="" apply="" those="" limits="" consistently="" to="" avoid="" confusion="" in="" the="" interpretation="" of="" test="" data.="" response:="" epa="" believes="" that="" the="" definitions="" of="" the="" ml="" in="" methods="" are="" [[page="" 48401]]="" consistent.="" epa="" agrees,="" however,="" that="" there="" is="" a="" need="" for="" greater="" consensus="" on="" the="" definition="" of="" terms="" among="" methods="" from="" all="" epa="" offices="" and="" federal="" and="" state="" analytical="" programs.="" epa="" is="" currently="" addressing="" these="" issues="" through="" internal="" communications="" and="" meetings="" with="" stakeholders.="" the="" mls="" for="" method="" 1613="" have="" been="" verified="" in="" every="" laboratory="" that="" uses="" the="" method="" by="" requiring="" calibration="" at="" the="" ml.="" mls="" can="" be="" verified="" by="" single="" laboratory="" studies="" or="" by="" use="" since="" laboratories="" must="" calibrate="" at="" the="" ml.="" epa="" will="" continue="" to="" examine="" the="" issues="" of="" detection="" and="" quantitation="" and="" will="" involve="" the="" public="" on="" these="" issues="" when="" an="" improved="" concept="" is="" developed.="" i.="" quality="" assurance/quality="" control="" (qa/qc)="" comment:="" the="" instrument="" calibration="" procedure="" outlined="" in="" method="" 1613="" is="" much="" more="" involved="" than="" procedures="" for="" the="" 600="" series="" methods.="" it="" would="" be="" extremely="" difficult="" to="" meet="" the="" 12-hour="" calibration="" requirements="" after="" running="" a="" few="" ``dirty''="" samples.="" response:="" epa="" agrees="" that="" the="" calibration="" procedure="" in="" method="" 1613="" may="" be="" somewhat="" more="" complicated="" than="" the="" procedures="" in="" the="" 600="" series="" methods.="" however,="" the="" calibration="" procedure="" in="" method="" 1613="" is="" virtually="" identical="" to="" the="" procedures="" in="" method="" 1624="" and="" 1625,="" the="" isotope="" dilution="" variants="" of="" methods="" 624="" and="" 625.="" as="" to="" the="" statement="" that="" it="" would="" be="" extremely="" difficult="" to="" meet="" the="" 12-hour="" calibration="" requirements="" after="" running="" a="" few="" dirty="" samples,="" laboratories="" under="" contract="" to="" epa="" have="" not="" reported="" that="" verifying="" calibration="" is="" a="" problem.="" these="" laboratories="" have="" analyzed="" in="" excess="" of="" 1,000="" samples="" for="" epa="" using="" method="" 1613.="" comment:="" no="" other="" method="" in="" 40="" cfr="" part="" 136="" has="" a="" requirement="" for="" initial="" demonstration="" of="" laboratory="" capability="" (iprs,="" section="" 8.2="" of="" method="" 1613)="" and="" method="" 1613="" should="" not="" either.="" the="" existing="" methods="" approved="" for="" measurement="" under="" the="" cwa="" and="" the="" sdwa="" already="" require="" determination="" of="" mdls="" in="" accordance="" with="" 40="" cfr="" part="" 136,="" which="" should="" be="" sufficient="" for="" method="" 1613.="" response:="" the="" use="" of="" ipr="" analyses,="" which="" are="" also="" referred="" to="" as="" the="" start-up="" test,="" are="" not="" new="" requirements="" in="" cwa="" and="" sdwa="" methods.="" all="" 600="" and="" 1600="" series="" methods="" promulgated="" at="" 40="" cfr="" part="" 136="" appendix="" a="" include="" a="" requirement="" for="" demonstration="" of="" analyst/laboratory="" capability.="" these="" ipr="" tests="" are="" not="" equivalent="" to="" mdl="" determinations,="" nor="" are="" they="" intended="" to="" be.="" although="" many="" of="" the="" cwa="" and="" sdwa="" methods="" specify="" mdls,="" few="" require="" determination="" of="" these="" mdls="" as="" proofs="" of="" performance.="" comment:="" method="" 1613="" calls="" for="" instrument="" calibration="" to="" be="" verified="" at="" a="" high="" level,="" but="" calibration="" should="" be="" verified="" instead="" at="" the="" ml="" because="" of="" uncertainties="" at="" that="" level.="" response:="" epa="" disagrees="" that="" calibration="" should="" be="" verified="" at="" the="" ml.="" in="" method="" 1613,="" calibration="" is="" verified="" at="" the="" mid-point="" of="" the="" analytical="" range.="" this="" verification="" is="" common="" and="" accepted="" practice="" for="" analytical="" methods.="" comment:="" method="" 1613="" relies="" on="" the="" use="" of="" reagent="" water="" for="" tests="" to="" determine="" initial="" precision="" and="" recovery="" (ipr)="" and="" ongoing="" precision="" and="" recovery="" (opr).="" this="" practice="" is="" inappropriate="" for="" methods="" that="" must="" rely="" on="" extensive="" cleanup.="" response:="" epa="" disagrees="" that="" reagent="" water="" is="" inappropriate="" for="" use="" in="" the="" determination="" of="" ipr,="" opr,="" and="" other="" tests="" because="" the="" primary="" purpose="" of="" these="" tests="" is="" to="" demonstrate="" laboratory="" performance="" rather="" than="" performance="" on="" a="" sample="" matrix.="" in="" addition,="" method="" 1613="" requires="" that="" if="" the="" method="" is="" to="" be="" applied="" to="" a="" sample="" matrix="" other="" than="" water="" (e.g.,="" soil,="" filter="" cake,="" compost,="" tissue),="" the="" most="" appropriate="" alternate="" matrix="" is="" substituted="" for="" the="" reagent="" water="" matrix="" in="" these="" performance="" tests.="" alternate="" matrices="" are="" listed="" in="" section="" 7="" of="" method="" 1613.="" further,="" method="" 1613="" requires="" that="" all="" steps="" used="" for="" processing="" samples,="" including="" preparation,="" extraction,="" and="" cleanup,="" shall="" be="" included="" in="" the="" performance="" tests.="" this="" requirement="" assures="" that="" performance="" problems="" will="" be="" found="" prior="" to="" application="" of="" the="" method="" to="" analysis="" of="" an="" environmental="" sample.="" j.="" miscellaneous="" comment:="" for="" samples="" containing="" less="" than="" one="" percent="" solids,="" the="" sample="" preparation="" procedure="" in="" method="" 1613="" (which="" is="" designed="" for="" liquids="" and="" solids)="" could="" take="" twice="" as="" long="" as="" the="" method="" 613="" preparation="" procedure="" (which="" is="" designed="" for="" liquids="" only),="" and="" for="" samples="" with="" more="" than="" one="" percent="" solids,="" it="" could="" take="" 3-4="" times="" as="" long="" as="" the="" method="" 613="" preparation="" procedure.="" response:="" epa="" agrees="" that="" the="" sample="" preparation="" procedures="" in="" method="" 1613="" will="" be="" more="" time-consuming="" than="" those="" in="" method="" 613.="" since="" cdds/cdfs="" are="" known="" to="" be="" strongly="" associated="" with="" any="" particles="" in="" the="" sample,="" epa="" believes="" that="" the="" additional="" filtration="" and="" extraction="" steps="" are="" necessary="" to="" accurately="" measure="" cdds/cdfs="" in="" environmental="" samples="" at="" low="" concentrations.="" to="" reduce="" the="" time="" required="" for="" extraction="" of="" aqueous="" samples="" containing="" less="" than="" 1="" percent="" solids,="" and="" to="" reduce="" costs="" and="" the="" amount="" of="" solvent="" used="" in="" extraction="" in="" the="" interest="" of="" pollution="" prevention,="" epa="" has="" added="" a="" procedure="" for="" solid-phase="" extraction="" (spe)="" to="" the="" version="" of="" method="" 1613="" being="" promulgated="" today.="" epa="" believes="" that="" this="" procedure="" will="" reduce="" the="" time="" required="" for="" extraction="" to="" levels="" commensurate="" with="" those="" required="" for="" extraction="" using="" method="" 613.="" comment:="" the="" proposed="" rulemaking="" provides="" an="" insufficient="" basis="" for="" a="" thorough="" discussion="" and="" consideration="" of="" wet="" weight/dry="" weight="" issues="" for="" permits.="" response:="" nothing="" in="" the="" promulgation="" of="" this="" method="" requires="" the="" use="" of="" dry="" weight="" values="" in="" establishing="" effluent="" limitations="" in="" npdes="" permits.="" comment:="" the="" proposal="" does="" not="" require="" the="" use="" of="" method="" 1613="" for="" any="" npdes="" permits,="" but="" permittees="" should="" not="" presume="" that="" the="" npdes="" permitting="" authority="" would="" not="" require="" use="" of="" method="" 1613="" if="" the="" authority="" determines="" that="" pollutants="" of="" concern="" in="" the="" effluent="" can="" only="" be="" measured="" at="" the="" level="" of="" concern="" by="" method="" 1613.="" response:="" epa="" agrees="" and="" intends="" for="" method="" 1613="" to="" be="" specified="" in="" npdes="" permits="" at="" the="" discretion="" of="" the="" npdes="" permitting="" authority.="" k.="" technical="" comments="" comment:="" table="" 3="" should="" have="" one="" additional="" chlorinated="" diphenyl="" ether="" monitored="" (pecdpe,="" hxcdpe,="" hpcdpe,="" ocdpe,="" and="" ncpdpe).="" the="" commenter="" suggested="" a="" specific="" modification="" to="" sections="" 15.1="" through="" 15.4="" in="" those="" cases="" when="" a="" chlorodiphenyl="" ether="" may="" interfere="" with="" the="" determination="" of="" certain="" cdds="" and="" cdfs.="" response:="" epa="" agrees="" in="" principle="" with="" the="" commenter's="" suggestion="" but="" instead="" has="" incorporated="" requirements="" that="" meet="" the="" spirit="" of="" the="" suggestion="" into="" section="" 18.3="" of="" method="" 1613.="" the="" method="" states="" that="" if="" chromatographic="" peaks="" are="" detected="" at="" the="" retention="" time="" of="" cdds/cdfs="" in="" any="" of="" the="" m/z="" channels="" being="" monitored="" for="" the="" chlorodiphenyl="" ethers,="" cleanup="" procedures="" must="" be="" employed="" until="" these="" interferences="" are="" removed.="" this="" statement="" encompasses="" all="" the="" chlorodiphenyl="" ethers="" that="" may="" interfere="" in="" the="" analysis.="" comment:="" methylene="" chloride="" is="" a="" poor="" extraction="" solvent="" because="" the="" solubility="" of="" cdds/cdfs="" in="" it="" is="" less="" than="" that="" of="" other="" readily="" available="" solvents.="" benzene="" or="" toluene="" should="" be="" used="" instead.="" [[page="" 48402]]="" response:="" epa="" believes="" that="" methylene="" chloride="" is="" the="" solvent="" of="" choice="" for="" the="" aqueous="" filtrates="" because="" its="" higher="" than="" water="" density="" simplifies="" the="" extraction="" procedure.="" similarly,="" epa="" believes="" that="" toluene="" is="" most="" suitable="" for="" the="" sds="" extraction="" of="" particulate="" sample="" matter.="" finally,="" epa="" believes="" that="" safety="" concerns="" over="" the="" use="" of="" a="" carcinogen="" such="" as="" benzene="" preclude="" the="" use="" of="" this="" traditional="" solvent="" in="" new="" analytical="" methods.="" comment:="" epa="" is="" correct="" in="" pointing="" out="" the="" significant="" importance="" of="" handling="" particulates="" from="" aqueous="" samples,="" but="" further="" study="" of="" the="" methodology="" is="" needed="" to="" demonstrate="" that="" it="" can="" produce="" true="" quantitative="" and="" accurate="" values="" which="" can="" be="" used="" for="" compliance="" monitoring.="" response:="" the="" sds="" extraction="" technique="" that="" is="" used="" in="" method="" 1613="" is="" based="" on="" widely="" published="" uses="" of="" the="" technique.="" ample="" data="" to="" support="" its="" use="" are="" available="" in="" the="" open="" literature.="" for="" example,="" see="" references="" 6="" and="" 7="" cited="" in="" section="" 22.0="" of="" method="" 613.="" further,="" epa="" has="" now="" tested="" the="" sds="" procedure="" on="" hundreds="" of="" aqueous="" environmental="" samples="" containing="" particulates="" (e.g.,="" the="" databases="" for="" the="" imvs="" and="" ltvs)="" and="" believes="" that="" sds="" is="" the="" preferred="" procedure="" for="" such="" samples.="" comment:="" the="" method="" should="" include="" a="" statement="" that="" indicates="" the="" expected="" analytical="" range="" of="" the="" method.="" response:="" epa="" agrees="" in="" principle="" with="" the="" comment,="" however,="" the="" analytical="" range="" is="" constrained="" on="" the="" low="" end="" by="" the="" calibration="" range,="" the="" sample="" size,="" and="" the="" ability="" to="" take="" a="" representative="" aliquot="" of="" a="" sample.="" the="" analytical="" range="" is="" not="" constrained="" on="" the="" upper="" end="" because="" the="" sample="" may="" be="" diluted="" to="" bring="" the="" concentrations="" of="" cdds/cdfs="" within="" the="" calibration="" range,="" as="" described="" in="" sections="" 17.5="" and="" 18.2="" of="" method="" 1613.="" comment:="" ncasi="" included="" with="" its="" comments="" approximately="" 40="" pages="" of="" suggested="" technical="" modifications="" to="" method="" 1613="" to="" improve="" the="" reliability="" of="" the="" method.="" response:="" epa="" appreciates="" ncasi's="" suggestions.="" ncasi="" has="" participated="" in="" epa's="" validation="" studies,="" conducted="" validation="" studies="" of="" its="" own,="" scrutinized="" the="" details="" of="" method="" 1613,="" and="" provided="" many="" valuable="" suggestions="" for="" improvements="" to="" the="" method.="" epa="" has="" adopted="" most="" of="" these="" suggestions,="" as="" well="" as="" the="" suggestions="" of="" others,="" as="" described="" in="" the="" ``detailed="" responses="" to="" comments="" on="" the="" proposal="" of="" epa="" method="" 1613''="" included="" in="" the="" docket="" for="" today's="" rule.="" epa="" will="" continue="" to="" work="" with="" all="" interested="" parties="" to="" ensure="" that="" method="" 1613="" and="" other="" analytical="" methods="" are="" as="" state-of-the-art="" as="" possible.="" v.="" regulatory="" analysis="" a.="" executive="" order="" 12866="" under="" executive="" order="" 12866,="" 58="" fr="" 51,735="" (oct.="" 4,="" 1993),="" the="" agency="" must="" determine="" whether="" the="" regulatory="" action="" is="" ``significant''="" and="" therefore="" subject="" to="" omb="" review="" and="" the="" requirements="" of="" the="" executive="" order.="" the="" order="" defines="" ``significant="" regulatory="" action''="" as="" one="" that="" is="" likely="" to="" result="" in="" a="" rule="" that="" may:="" (1)="" have="" an="" annual="" effect="" on="" the="" economy="" of="" $100="" million="" or="" more="" or="" adversely="" affect="" in="" a="" material="" way="" the="" economy,="" a="" sector="" of="" the="" economy,="" productivity,="" competition,="" jobs,="" the="" environment,="" public="" health="" or="" safety,="" or="" state,="" local,="" or="" tribal="" governments="" or="" communities;="" (2)="" create="" a="" serious="" inconsistency="" or="" otherwise="" interfere="" with="" an="" action="" taken="" or="" planned="" by="" another="" agency;="" (3)="" materially="" alter="" the="" budgetary="" impact="" of="" entitlements,="" grants,="" user="" fees,="" or="" loan="" programs="" or="" the="" rights="" and="" obligations="" of="" recipients="" thereof;="" or="" (4)="" raise="" novel="" legal="" or="" policy="" issues="" arising="" out="" of="" legal="" mandates,="" the="" president's="" priorities,="" or="" the="" principles="" set="" forth="" in="" the="" executive="" order.''="" this="" regulation="" is="" not="" major="" because="" it="" approves="" a="" testing="" procedure="" for="" use="" in="" compliance="" monitoring="" and="" data="" gathering="" but="" does="" not="" itself="" require="" these="" activities.="" therefore,="" this="" regulation="" would="" not="" result="" in="" a="" cost="" to="" the="" economy="" of="" $100="" million="" or="" more;="" would="" not="" result="" in="" a="" major="" increase="" in="" costs="" or="" prices="" for="" consumers="" or="" individual="" industries;="" and="" would="" not="" have="" significant="" adverse="" effects="" on="" competition,="" investment,="" innovation,="" or="" international="" trade.="" it="" has="" been="" determined="" that="" this="" rule="" is="" not="" a="" ``significant="" regulatory="" action''="" under="" the="" terms="" of="" executive="" order="" 12866="" and="" is="" therefore="" not="" subject="" to="" omb="" review.="" b.="" unfunded="" mandates="" reform="" act="" title="" ii="" of="" the="" unfunded="" mandates="" reform="" act="" of="" 1995="" (umra),="" pub.="" l.="" 104-4,="" establishes="" requirements="" for="" federal="" agencies="" to="" assess="" the="" effects="" of="" their="" regulatory="" actions="" on="" state,="" local,="" and="" tribal="" governments="" and="" the="" private="" sector.="" under="" section="" 202="" of="" the="" umra,="" epa="" generally="" must="" prepare="" a="" written="" statement,="" including="" a="" cost-benefit="" analysis,="" for="" proposed="" and="" final="" rules="" with="" ``federal="" mandates''="" that="" may="" result="" in="" expenditures="" to="" state,="" local,="" and="" tribal="" governments,="" in="" the="" aggregate,="" or="" to="" the="" private="" sector,="" of="" $100="" million="" or="" more="" in="" any="" one="" year.="" before="" promulgating="" an="" epa="" rule="" for="" which="" a="" written="" statement="" is="" needed,="" section="" 205="" of="" the="" umra="" generally="" requires="" epa="" to="" identify="" and="" consider="" a="" reasonable="" number="" of="" regulatory="" alternatives="" and="" adopt="" the="" least="" costly,="" most="" cost-effective="" or="" least="" burdensome="" alternative="" that="" achieves="" the="" objectives="" of="" the="" rule.="" the="" provisions="" of="" section="" 205="" do="" not="" apply="" when="" they="" are="" inconsistent="" with="" applicable="" law.="" moreover,="" section="" 205="" allows="" epa="" to="" adopt="" an="" alternative="" other="" than="" the="" least="" costly,="" most="" cost-effective="" or="" least="" burdensome="" alternative="" if="" the="" administrator="" publishes="" with="" the="" final="" rule="" an="" explanation="" why="" that="" alternative="" was="" not="" adopted.="" before="" epa="" establishes="" any="" regulatory="" requirements="" that="" may="" significantly="" or="" uniquely="" affect="" small="" governments,="" including="" tribal="" governments,="" it="" must="" have="" developed="" under="" section="" 203="" of="" the="" umra="" a="" small="" government="" agency="" plan.="" the="" plan="" must="" provide="" for="" notifying="" potentially="" affected="" small="" governments,="" enabling="" officials="" of="" affected="" small="" governments="" to="" have="" meaningful="" and="" timely="" input="" in="" the="" development="" of="" epa="" regulatory="" proposals="" with="" significant="" federal="" intergovernmental="" mandates,="" and="" informing,="" educating,="" and="" advising="" small="" governments="" on="" compliance="" with="" the="" regulatory="" requirements.="" epa="" has="" determined="" that="" this="" rule="" does="" not="" contain="" a="" federal="" mandate="" that="" may="" result="" in="" expenditures="" of="" $100="" million="" or="" more="" for="" state,="" local,="" and="" tribal="" governments,="" in="" the="" aggregate,="" or="" the="" private="" sector="" in="" any="" one="" year.="" this="" rule="" makes="" available="" a="" testing="" procedure="" for="" use="" in="" compliance="" monitoring="" and="" data="" gathering="" but="" does="" not="" require="" these="" activities.="" thus,="" today's="" rule="" is="" not="" subject="" to="" the="" requirements="" of="" sections="" 202="" and="" 205="" of="" the="" umra.="" epa="" has="" determined="" that="" this="" rule="" contains="" no="" regulatory="" requirements="" that="" might="" significantly="" or="" uniquely="" affect="" small="" governments.="" this="" rule="" simply="" approves="" a="" test="" procedure="" to="" be="" available="" for="" use="" by="" testing="" laboratories.="" c.="" regulatory="" flexibility="" act="" pursuant="" to="" section="" 605(b)="" of="" the="" regulatory="" flexibility="" act,="" 5="" u.s.c.="" 605(b),="" the="" administrator="" certifies="" that="" this="" rule="" will="" not="" have="" a="" significant="" economic="" impact="" on="" a="" substantial="" number="" of="" small="" entities.="" this="" regulation="" simply="" approves="" a="" test="" procedure="" to="" be="" available="" for="" use="" by="" testing="" laboratories.="" d.="" paperwork="" reduction="" act="" in="" accordance="" with="" the="" paperwork="" reduction="" act="" of="" 1980,="" 44="" u.s.c.="" 3501="" et="" seq.,="" epa="" must="" submit="" a="" copy="" of="" any="" rule="" that="" contains="" a="" collection-of-information="" requirement="" to="" the="" director="" of="" the="" office="" of="" management="" and="" budget="" (omb)="" for="" review="" and="" approval.="" this="" [[page="" 48403]]="" rule="" contains="" no="" additional="" information="" collection="" requirements="" beyond="" those="" already="" required="" by="" 40="" cfr="" parts="" 122,="" 141,="" 142,="" 403,="" and="" 425,="" and="" approved="" by="" omb="" (40="" cfr="" part="" 9).="" the="" relevant="" omb="" control="" numbers="" are="" 2040-0086,="" 2040-0170,="" 2040-0068,="" 2040-0110,="" 2040-0004,="" 2040-0090,="" and="" 2040-0009.="" therefore,="" preparation="" of="" an="" information="" collection="" request="" to="" accompany="" this="" rule="" is="" unnecessary.="" e.="" submission="" to="" congress="" and="" the="" general="" accounting="" office="" under="" 5="" u.s.c.="" 801(a)(1)(a)="" as="" added="" by="" the="" small="" business="" regulatory="" enforcement="" fairness="" act="" of="" 1996,="" epa="" submitted="" a="" report="" containing="" this="" rule="" and="" other="" required="" information="" to="" the="" u.s.="" senate,="" the="" u.s.="" house="" of="" representatives,="" and="" the="" comptroller="" general="" of="" the="" general="" accounting="" office,="" prior="" to="" publication="" of="" the="" rule="" in="" today's="" federal="" register.="" this="" rule="" is="" not="" a="" ``major="" rule''="" as="" defined="" by="" 5="" u.s.c.="" 804(2).="" list="" of="" subjects="" in="" 40="" cfr="" part="" 136="" environmental="" protection,="" reporting="" and="" recordkeeping="" requirements,="" water="" pollution="" control.="" dated:="" september="" 2,="" 1997.="" carol="" m.="" browner,="" administrator.="" in="" consideration="" of="" the="" preceding,="" usepa="" amends="" 40="" cfr="" part="" 136="" as="" set="" forth="" below.="" part="" 136--[amended]="" 1.="" the="" authority="" citation="" for="" part="" 136="" continues="" to="" read="" as="" follows:="" authority:="" secs.="" 301,="" 304(h),="" 307,="" and="" 501(a)="" pub.="" l.="" 95-217,="" stat.="" 1566,="" et="" seq.="" (33="" u.s.c.="" 1251,="" et="" seq.)="" (the="" federal="" water="" pollution="" control="" act="" amendments="" of="" 1972="" as="" amended="" by="" the="" clean="" water="" act="" of="" 1977="" and="" the="" water="" quality="" act="" of="" 1987),="" 33="" u.s.c.="" 1314="" and="" 1361;="" 86="" stat.="" 816,="" pub.="" l.="" 92-500;="" 91="" stat.="" 1567,="" pub.="" l.="" 92-="" 217;="" stat.="" 7,="" pub.="" l.="" 100-4="" (the="" ``act'').="" 2.="" in="" sec.="" 136.3(a),="" table="" 1c.--list="" of="" approved="" test="" procedures="" for="" non-pesticide="" organic="" compounds,="" is="" amended="" by="" revising="" entries="" 60="" through="" 97,="" by="" adding="" new="" entries="" 60="" through="" 113,="" and="" by="" revising="" table="" ic="" notes="" \1\="" and="" \2\="" as="" follows:="" sec.="" 136.3="" identification="" of="" test="" procedures.="" *="" *="" *="" *="" *="" table="" 1c.--list="" of="" approved="" test="" procedures="" for="" non-pesticide="" organic="" compounds="" --------------------------------------------------------------------------------------------------------------------------------------------------------="" epa="" method="" number="">2, 7
Parameter \1\ GC ------------------------------------------------------------------ ASTM Other
GC/MS HPLC Standard methods 18th ed.
--------------------------------------------------------------------------------------------------------------------------------------------------------
* * * * * * *
60. 1,2,3,4,6,7,8- ....... 1613...................... .......
Heptachlorodibenzofuran.
61. 1,2,3,4,7,8,9- ....... 1613...................... .......
Heptachlorodibenzofuran.
62. 1,2,3,4,6,7,8- ....... 1613...................... .......
Heptachlorodibenzo-p-dioxin.
63. Hexachlorobenzene......... 612 625, 1625................. ....... 6410 B
64. Hexachlorobutadiene....... 612 625, 1625................. ....... 6410 B
65. Hexachlorocyclopentadiene. 612 625, 1625 \5\............. ....... 6410 B
66. 1,2,3,4,7,8- ....... 1613...................... .......
Hexachlorodibenzofuran.
67. 1,2,3,6,7,8- ....... 1613...................... .......
Hexachlorodibenzofuran.
68. 1,2,3,7,8,9- ....... 1613...................... .......
Hexachlorodibenzofuran.
69. 2,3,4,6,7,8- ....... 1613...................... .......
Hexachlorodibenzofuran.
70. 1,2,3,4,7,8- ....... 1613...................... .......
Hexachlorodibenzo-p-dioxin.
71. 1,2,3,6,7,8- ....... 1613...................... .......
Hexachlorodibenzo-p-dioxin.
72. 1,2,3,7,8,9- ....... 1613...................... .......
Hexachlorodibenzo-p-dioxin.
73. Hexachloroethane.......... 616 625, 1625................. ....... 6410 B
74. Ideno(1,2,3-cd)pyrene..... 610 625, 1625................. 610 6410 B, 6440 B D4657-87
75. Isophorone................ 609 625, 1625................. ....... 6410 B
76. Methylene chloride........ 601 624, 1624................. ....... 6230 B Note 3, p. 130.
77. 2-Methyl-4,6-dinitrophenol 604 625, 1625................. ....... 6420 B, 6410 B
78. Naphthalene............... 610 625, 1625................. 610 6410 B, 6440 B
79. Nitrobenzene.............. 609 625, 1625................. ....... 6410 B D4657-87
80. 2-Nitrophenol............. 604 625, 1625................. ....... 6410 B, 6420 B
81. 4-Nitrophenol............. 604 625, 1625................. ....... 6410 B, 6420 B
82. N-Nitrosodimethylamine.... 607 625, 1625................. ....... 6410 B
83. N-Nitrosodi-n-propylamine. 607 625, 1625 \5\............. ....... 6410 B
84. N-Nitrosodiphenylamine.... 607 625, 1625 \5\............. ....... 6410 B
85. Octachlorodibenzofuran.... ....... 1613...................... .......
86. Octachlorodibenzo-p-dioxin ....... 1613...................... .......
87. 2,2-Oxybis(1- 611 625, 1625................. ....... 6410 B
chloropropane).
88. PCB-1016.................. 608 625....................... ....... 6410 B Note 3, p. 43.
89. PCB-1221.................. 608 625....................... ....... 6410 B Note 3, p. 43.
90. PCB-1232.................. 608 625....................... ....... 6410 B Note 3, p. 43.
91. PCB 1242.................. 608 625....................... ....... 6410 B Note 3, p. 43.
92. PCB-1248.................. 608 625....................... .......
93. PCB-1254.................. 608 625....................... ....... 6410 B Note 3, p. 43.
94. PCB-1260.................. 608 625....................... ....... 6410 B, 6630 B Note 3, p. 43.
95. 1,2,3,7,8- ....... 1613...................... .......
Pentachlorodibenzofuran.
96. 2,3,4,7,8- ....... 1613...................... .......
Pentachlorodibenzofuran.
[[Page 48404]]
97. 1,2,3,7,8- ....... 1613...................... .......
Pentachlorodibenzo-p-dioxin.
98. Pentachlorophenol 604 625, 1625................. ....... 6410 B, 6630 B Note 3, p. 140.
99. Phenanthrene.............. 610 625, 1625................. 610 6410 B, 6440 B D4657-87
100. Phenol................... 604 625, 1625................. ....... 6420 B, 6410 B
101. Pyrene................... 610 625, 1625................. 610 6410 B, 6440 B D4657-87
102. 2,3,7,8- ....... 1613...................... .......
Tetrachlorodibenzofuran.
103. 2,3,7,8- ....... 613, 1613 \5\............. .......
Tetrachlorodibenzo-p-dioxin.
104. 1,1,2,2-Tetrachloroethane 601 624, 1624................. ....... 6230 B, 6210 B Note 3, p. 130.
105. Tetrachloroethene........ 601 624, 1624................. ....... 6230 B, 6410 B Note 3, p. 130.
106. Toluene.................. 602 624, 1624................. ....... 6210 B, 6220 B
107. 1,2,4-Trichlorobenzene... 612 625, 1625................. ....... 6410 B Note 3, p. 130.
108. 1,1,1-Trichloroethane.... 601 624, 1624................. ....... 6210 B, 6230 B
109. 1,1,2-Trichloroethane.... 601 624, 1624................. ....... 6210 B, 6230 B Note 3, p. 130.
110. Trichloroethene.......... 601 624, 1624................. ....... 6210 B, 6230 B
111. Trichlorofluoromethane... 601 624....................... ....... 6210 B, 6230 B
112. 2,4,6-Trichlorophenol.... 604 625, 1625................. ....... 6410 B, 6240 B
113. Vinyl chloride........... 601 624, 1624................. ....... 6210 B, 6230 B
* * * * * * *
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ All parameters are expressed in micrograms per liter (g/L) except for Method 1613 in which the parameters are expressed in picograms per
liter (pg/L).
\2\ The full text of Methods 601-613, 624, 625, 1624, and 1625, are given at Appendix A, ``Test Procedures for Analysis of Organic Pollutants,'' of this
Part 136. The full text of Method 1613 is incorporated by reference into this Part 136 and is available from the National Technical Information
Services as stock number PB95-104774. The standardized test procedure to be used to determine the method detection limit (MDL) for these test
procedures is given at Appendix B, ``Definition and Procedures for the Determination of the Method Detection Limit,'' of this Part 136.
* * * * * * *
5 5a, and 7 unchanged.
3. In Sec. 136.3(b), the listing titled References, Sources, Costs,
and Table Citations is amended by revising the first sentence of
paragraph (1) to read as follows:
Sec. 136.3 Identification of test procedures.
* * * * *
References, Sources, Costs, and Table Citations
(1) The full texts of Methods 601-613, 624, 625, 1613, 1624, and
1625 are printed in appendix A of this part 136. * * *
4. In Sec. 136.3(e), Table II--Required Containers, Preservation
Techniques, and Holding Times, is amended by revising Table IC--Organic
Tests to read as follows:
Table II.--Required Containers, Preservation Techniques, and Holding Times
--------------------------------------------------------------------------------------------------------------------------------------------------------
Parameter No./name Container \1\ Preservation \2\ \3\ Maximum holding time \4\
--------------------------------------------------------------------------------------------------------------------------------------------------------
* * * * * * *
Table IC--Organic Tests \8\
13, 18-20, 22, 24-28, 34-37, 39- G, Teflon-lined septum................... Cool, 4 deg.C, 0.008% 14 days.
43, 45-47, 56, 76, 104, 105, Na2S2O3 \5\..
108-111, 113. Purgeable
Halocarbons.
6, 57, 106. Purgeable aromatic ......do................................. Cool, 4 deg.C, 0.008% Do.
hydrocarbons. Na2S2O3,\5\ HCl to pH2\9\.
3, 4. Acrolein and acrylonitrile ......do................................. Cool, 4 deg.C, 0.008% Do.
Na2S2O3,\5\ adjust pH to 4-
5\10\.
23, 30, 44, 49, 53, 77, 80, 81, G, Teflon-lined cap...................... Cool, 4 deg.C, 0.008% 7 days until extraction; 40 days after
98, 100, 112. Phenols \11\. Na2S2O3 \5\. extraction.
7, 38. Benzidines \11\.......... ......do................................. ......do.................... 7 days until extraction.\13\
14, 17, 48, 50-52. Phthalate ......do................................. Cool, 4 deg.C............... 7 days until extraction; 40 days after
esters \11\. extraction.
82-84. Nitrosamines \11\ \14\... ......do................................. Cool, 4 deg.C, 0.008% Do.
Na2S2O3,\5\ store in dark.
88-94. PCBs \11\................ .....do.................................. Cool, 4 deg.C............... Do.
54, 55, 75, 79. Nitroaromatics ......do................................. Cool, 4 deg.C, 0.008% Do.
and isophorone \11\. Na2S2O3,\5\ store in dark.
1, 2, 5, 8-12, 32, 33, 58, 59, ......do................................. ......do.................... Do.
74, 78, 99, 101. Polynuclear
aromatic hydrocarbons \11\.
15, 16, 21, 31, 87. Haloethers ......do................................. Cool, 4 deg.C, 0.008% Do.
\11\. Na2S2O3 \5\.
[[Page 48405]]
29, 35-37, 63-65, 73, 107. ......do................................. Cool, 4 deg.C............... Do.
Chlorinated hydrocarbons \11\.
60-62, 66-72, 85, 86, 95-97,
102, 103. CDDs/CDFs \11\
aqueous: field and lab G........................................ Cool, 0-4 deg.C, pH<9, 1="" year.="" preservation..="" 0.008%="">2S2O3 \5\.
Solids, mixed phase, and tissue: ......do................................. Cool, <4 deg.c..............="" 7="" days.="" field="" preservation..="" solids,="" mixed="" phase,="" and="" tissue:="" ......do.................................="" freeze,=""><-10 deg.c..........="" 1="" year.="" lab="" preservation.="" *="" *="" *="" *="" *="" *="" *="" --------------------------------------------------------------------------------------------------------------------------------------------------------="" *="" *="" *="" *="" *="" note:="" the="" footnotes="" remain="" unchanged.="" 4.="" in="" part="" 136,="" appendix="" a="" is="" amended="" by="" adding="" method="" 1613="" to="" read="" as="" follows:="" method="" 1613,="" revision="" b="" tetra-="" through="" octa-chlorinated="" dioxins="" and="" furans="" by="" isotope="" dilution="" hrgc/hrms="" 1.0="" scope="" and="" application="" 1.1="" this="" method="" is="" for="" determination="" of="" tetra-="" through="" octa-="" chlorinated="" dibenzo-p-dioxins="" (cdds)="" and="" dibenzofurans="" (cdfs)="" in="" water,="" soil,="" sediment,="" sludge,="" tissue,="" and="" other="" sample="" matrices="" by="" high="" resolution="" gas="" chromatography/high="" resolution="" mass="" spectrometry="" (hrgc/="" hrms).="" the="" method="" is="" for="" use="" in="" epa's="" data="" gathering="" and="" monitoring="" programs="" associated="" with="" the="" clean="" water="" act,="" the="" resource="" conservation="" and="" recovery="" act,="" the="" comprehensive="" environmental="" response,="" compensation="" and="" liability="" act,="" and="" the="" safe="" drinking="" water="" act.="" the="" method="" is="" based="" on="" a="" compilation="" of="" epa,="" industry,="" commercial="" laboratory,="" and="" academic="" methods="" (references="" 1-6).="" 1.2="" the="" seventeen="" 2,3,7,8-substituted="" cdds/cdfs="" listed="" in="" table="" 1="" may="" be="" determined="" by="" this="" method.="" specifications="" are="" also="" provided="" for="" separate="" determination="" of="" 2,3,7,8-tetrachloro-dibenzo-p-dioxin="" (2,3,7,8-tcdd)="" and="" 2,3,7,8-tetrachloro-dibenzofuran="" (2,3,7,8-tcdf).="" 1.3="" the="" detection="" limits="" and="" quantitation="" levels="" in="" this="" method="" are="" usually="" dependent="" on="" the="" level="" of="" interferences="" rather="" than="" instrumental="" limitations.="" the="" minimum="" levels="" (mls)="" in="" table="" 2="" are="" the="" levels="" at="" which="" the="" cdds/cdfs="" can="" be="" determined="" with="" no="" interferences="" present.="" the="" method="" detection="" limit="" (mdl)="" for="" 2,3,7,8-tcdd="" has="" been="" determined="" as="" 4.4="" pg/l="" (parts-per-quadrillion)="" using="" this="" method.="" 1.4="" the="" gc/ms="" portions="" of="" this="" method="" are="" for="" use="" only="" by="" analysts="" experienced="" with="" hrgc/hrms="" or="" under="" the="" close="" supervision="" of="" such="" qualified="" persons.="" each="" laboratory="" that="" uses="" this="" method="" must="" demonstrate="" the="" ability="" to="" generate="" acceptable="" results="" using="" the="" procedure="" in="" section="" 9.2.="" 1.5="" this="" method="" is="" ``performance-based''.="" the="" analyst="" is="" permitted="" to="" modify="" the="" method="" to="" overcome="" interferences="" or="" lower="" the="" cost="" of="" measurements,="" provided="" that="" all="" performance="" criteria="" in="" this="" method="" are="" met.="" the="" requirements="" for="" establishing="" method="" equivalency="" are="" given="" in="" section="" 9.1.2.="" 1.6="" any="" modification="" of="" this="" method,="" beyond="" those="" expressly="" permitted,="" shall="" be="" considered="" a="" major="" modification="" subject="" to="" application="" and="" approval="" of="" alternate="" test="" procedures="" under="" 40="" cfr="" 136.4="" and="" 136.5.="" 2.0="" summary="" of="" method="" flow="" charts="" that="" summarize="" procedures="" for="" sample="" preparation,="" extraction,="" and="" analysis="" are="" given="" in="" figure="" 1="" for="" aqueous="" and="" solid="" samples,="" figure="" 2="" for="" multi-phase="" samples,="" and="" figure="" 3="" for="" tissue="" samples.="" 2.1="" extraction.="" 2.1.1="" aqueous="" samples="" (samples="" containing="" less="" than="" 1%="" solids)--="" stable="" isotopically="" labeled="" analogs="" of="" 15="" of="" the="" 2,3,7,8-substituted="" cdds/cdfs="" are="" spiked="" into="" a="" 1="" l="" sample,="" and="" the="" sample="" is="" extracted="" by="" one="" of="" three="" procedures:="" 2.1.1.1="" samples="" containing="" no="" visible="" particles="" are="" extracted="" with="" methylene="" chloride="" in="" a="" separatory="" funnel="" or="" by="" the="" solid-phase="" extraction="" technique="" summarized="" in="" section="" 2.1.1.3.="" the="" extract="" is="" concentrated="" for="" cleanup.="" 2.1.1.2="" samples="" containing="" visible="" particles="" are="" vacuum="" filtered="" through="" a="" glass-fiber="" filter.="" the="" filter="" is="" extracted="" in="" a="" soxhlet/="" dean-stark="" (sds)="" extractor="" (reference="" 7),="" and="" the="" filtrate="" is="" extracted="" with="" methylene="" chloride="" in="" a="" separatory="" funnel.="" the="" methylene="" chloride="" extract="" is="" concentrated="" and="" combined="" with="" the="" sds="" extract="" prior="" to="" cleanup.="" 2.1.1.3="" the="" sample="" is="" vacuum="" filtered="" through="" a="" glass-fiber="" filter="" on="" top="" of="" a="" solid-phase="" extraction="" (spe)="" disk.="" the="" filter="" and="" disk="" are="" extracted="" in="" an="" sds="" extractor,="" and="" the="" extract="" is="" concentrated="" for="" cleanup.="" 2.1.2="" solid,="" semi-solid,="" and="" multi-phase="" samples="" (but="" not="" tissue)--the="" labeled="" compounds="" are="" spiked="" into="" a="" sample="" containing="" 10="" g="" (dry="" weight)="" of="" solids.="" samples="" containing="" multiple="" phases="" are="" pressure="" filtered="" and="" any="" aqueous="" liquid="" is="" discarded.="" coarse="" solids="" are="" ground="" or="" homogenized.="" any="" non-aqueous="" liquid="" from="" multi-phase="" samples="" is="" combined="" with="" the="" solids="" and="" extracted="" in="" an="" sds="" extractor.="" the="" extract="" is="" concentrated="" for="" cleanup.="" 2.1.3="" fish="" and="" other="" tissue--the="" sample="" is="" extracted="" by="" one="" of="" two="" procedures:="" 2.1.3.1="" soxhlet="" or="" sds="" extraction--a="" 20="" g="" aliquot="" of="" sample="" is="" homogenized,="" and="" a="" 10="" g="" aliquot="" is="" spiked="" with="" the="" labeled="" compounds.="" the="" sample="" is="" mixed="" with="" sodium="" sulfate,="" allowed="" to="" dry="" for="" 12-24="" hours,="" and="" extracted="" for="" 18-24="" hours="" using="" methylene="" chloride:hexane="" (1:1)="" in="" a="" soxhlet="" extractor.="" the="" extract="" is="" evaporated="" to="" dryness,="" and="" the="" lipid="" content="" is="" determined.="" 2.1.3.2="" hcl="" digestion--a="" 20="" g="" aliquot="" is="" homogenized,="" and="" a="" 10="" g="" aliquot="" is="" placed="" in="" a="" bottle="" and="" spiked="" with="" the="" labeled="" compounds.="" after="" equilibration,="" 200="" ml="" of="" hydrochloric="" acid="" and="" 200="" ml="" of="" methylene="" chloride:hexane="" (1:1)="" are="" added,="" and="" the="" bottle="" is="" agitated="" for="" 12-24="" hours.="" the="" extract="" is="" evaporated="" to="" dryness,="" and="" the="" lipid="" content="" is="" determined.="" 2.2="" after="" extraction,="">4-labeled 2,3,7,8-TCDD is
added to each extract to measure the efficiency of the cleanup process.
Sample cleanups may include
[[Page 48406]]
back-extraction with acid and/or base, and gel permeation, alumina,
silica gel, Florisil and activated carbon chromatography. High-
performance liquid chromatography (HPLC) can be used for further
isolation of the 2,3,7,8-isomers or other specific isomers or
congeners. Prior to the cleanup procedures cited above, tissue extracts
are cleaned up using an anthropogenic isolation column, a batch silica
gel adsorption, or sulfuric acid and base back-extraction, depending on
the tissue extraction procedure used.
2.3 After cleanup, the extract is concentrated to near dryness.
Immediately prior to injection, internal standards are added to each
extract, and an aliquot of the extract is injected into the gas
chromatograph. The analytes are separated by the GC and detected by a
high-resolution (10,000) mass spectrometer. Two exact m/z's
are monitored for each analyte.
2.4 An individual CDD/CDF is identified by comparing the GC
retention time and ion-abundance ratio of two exact m/z's with the
corresponding retention time of an authentic standard and the
theoretical or acquired ion-abundance ratio of the two exact m/z's. The
non-2,3,7,8 substituted isomers and congeners are identified when
retention times and ion-abundance ratios agree within predefined
limits. Isomer specificity for 2,3,7,8-TCDD and 2,3,7,8-TCDF is
achieved using GC columns that resolve these isomers from the other
tetra-isomers.
2.5 Quantitative analysis is performed using selected ion current
profile (SICP) areas, in one of three ways:
2.5.1 For the 15 2,3,7,8-substituted CDDs/CDFs with labeled
analogs (see Table 1), the GC/MS system is calibrated, and the
concentration of each compound is determined using the isotope dilution
technique.
2.5.2 For 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds, the
GC/MS system is calibrated and the concentration of each compound is
determined using the internal standard technique.
2.5.3 For non-2,3,7,8-substituted isomers and for all isomers at a
given level of chlorination (i.e., total TCDD), concentrations are
determined using response factors from calibration of the CDDs/CDFs at
the same level of chlorination.
2.6 The quality of the analysis is assured through reproducible
calibration and testing of the extraction, cleanup, and GC/MS systems.
3.0 Definitions
Definitions are given in the glossary at the end of this method.
4.0 Contamination and Interferences
4.1 Solvents, reagents, glassware, and other sample processing
hardware may yield artifacts and/or elevated baselines causing
misinterpretation of chromatograms (References 8-9). Specific selection
of reagents and purification of solvents by distillation in all-glass
systems may be required. Where possible, reagents are cleaned by
extraction or solvent rinse.
4.2 Proper cleaning of glassware is extremely important, because
glassware may not only contaminate the samples but may also remove the
analytes of interest by adsorption on the glass surface.
4.2.1 Glassware should be rinsed with solvent and washed with a
detergent solution as soon after use as is practical. Sonication of
glassware containing a detergent solution for approximately 30 seconds
may aid in cleaning. Glassware with removable parts, particularly
separatory funnels with fluoropolymer stopcocks, must be disassembled
prior to detergent washing.
4.2.2 After detergent washing, glassware should be rinsed
immediately, first with methanol, then with hot tap water. The tap
water rinse is followed by another methanol rinse, then acetone, and
then methylene chloride.
4.2.3 Do not bake reusable glassware in an oven as a routine part
of cleaning. Baking may be warranted after particularly dirty samples
are encountered but should be minimized, as repeated baking of
glassware may cause active sites on the glass surface that will
irreversibly adsorb CDDs/CDFs.
4.2.4 Immediately prior to use, the Soxhlet apparatus should be
pre-extracted with toluene for approximately three hours (see Sections
12.3.1 through 12.3.3). Separatory funnels should be shaken with
methylene chloride/toluene (80/20 mixture) for two minutes, drained,
and then shaken with pure methylene chloride for two minutes.
4.3 All materials used in the analysis shall be demonstrated to be
free from interferences by running reference matrix method blanks
initially and with each sample batch (samples started through the
extraction process on a given 12-hour shift, to a maximum of 20
samples).
4.3.1 The reference matrix must simulate, as closely as possible,
the sample matrix under test. Ideally, the reference matrix should not
contain the CDDs/CDFs in detectable amounts, but should contain
potential interferents in the concentrations expected to be found in
the samples to be analyzed. For example, a reference sample of human
adipose tissue containing pentachloronaphthalene can be used to
exercise the cleanup systems when samples containing
pentachloronaphthalene are expected.
4.3.2 When a reference matrix that simulates the sample matrix
under test is not available, reagent water (Section 7.6.1) can be used
to simulate water samples; playground sand (Section 7.6.2) or white
quartz sand (Section 7.3.2) can be used to simulate soils; filter paper
(Section 7.6.3) can be used to simulate papers and similar materials;
and corn oil (Section 7.6.4) can be used to simulate tissues.
4.4 Interferences coextracted from samples will vary considerably
from source to source, depending on the diversity of the site being
sampled. Interfering compounds may be present at concentrations several
orders of magnitude higher than the CDDs/CDFs. The most frequently
encountered interferences are chlorinated biphenyls, methoxy biphenyls,
hydroxydiphenyl ethers, benzylphenyl ethers, polynuclear aromatics, and
pesticides. Because very low levels of CDDs/CDFs are measured by this
method, the elimination of interferences is essential. The cleanup
steps given in Section 13 can be used to reduce or eliminate these
interferences and thereby permit reliable determination of the CDDs/
CDFs at the levels shown in Table 2.
4.5 Each piece of reusable glassware should be numbered to
associate that glassware with the processing of a particular sample.
This will assist the laboratory in tracking possible sources of
contamination for individual samples, identifying glassware associated
with highly contaminated samples that may require extra cleaning, and
determining when glassware should be discarded.
4.6 Cleanup of tissue--The natural lipid content of tissue can
interfere in the analysis of tissue samples for the CDDs/CDFs. The
lipid contents of different species and portions of tissue can vary
widely. Lipids are soluble to varying degrees in various organic
solvents and may be present in sufficient quantity to overwhelm the
column chromatographic cleanup procedures used for cleanup of sample
extracts. Lipids must be removed by the lipid removal procedures in
Section 13.7, followed by alumina (Section 13.4) or Florisil (Section
13.8), and carbon
[[Page 48407]]
(Section 13.5) as minimum additional cleanup steps. If chlorodiphenyl
ethers are detected, as indicated by the presence of peaks at the exact
m/z's monitored for these interferents, alumina and/or Florisil cleanup
must be employed to eliminate these interferences.
5.0 Safety
5.1 The toxicity or carcinogenicity of each compound or reagent
used in this method has not been precisely determined; however, each
chemical compound should be treated as a potential health hazard.
Exposure to these compounds should be reduced to the lowest possible
level.
5.1.1 The 2,3,7,8-TCDD isomer has been found to be acnegenic,
carcinogenic, and teratogenic in laboratory animal studies. It is
soluble in water to approximately 200 ppt and in organic solvents to
0.14%. On the basis of the available toxicological and physical
properties of 2,3,7,8-TCDD, all of the CDDs/CDFs should be handled only
by highly trained personnel thoroughly familiar with handling and
cautionary procedures and the associated risks.
5.1.2 It is recommended that the laboratory purchase dilute
standard solutions of the analytes in this method. However, if primary
solutions are prepared, they shall be prepared in a hood, and a NIOSH/
MESA approved toxic gas respirator shall be worn when high
concentrations are handled.
5.2 The laboratory is responsible for maintaining a current
awareness file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of material safety
data sheets (MSDSs) should also be made available to all personnel
involved in these analyses. It is also suggested that the laboratory
perform personal hygiene monitoring of each analyst who uses this
method and that the results of this monitoring be made available to the
analyst. Additional information on laboratory safety can be found in
References 10-13. The references and bibliography at the end of
Reference 13 are particularly comprehensive in dealing with the general
subject of laboratory safety.
5.3 The CDDs/CDFs and samples suspected to contain these compounds
are handled using essentially the same techniques employed in handling
radioactive or infectious materials. Well-ventilated, controlled access
laboratories are required. Assistance in evaluating the health hazards
of particular laboratory conditions may be obtained from certain
consulting laboratories and from State Departments of Health or Labor,
many of which have an industrial health service. The CDDs/CDFs are
extremely toxic to laboratory animals. Each laboratory must develop a
strict safety program for handling these compounds. The practices in
References 2 and 14 are highly recommended.
5.3.1 Facility--When finely divided samples (dusts, soils, dry
chemicals) are handled, all operations (including removal of samples
from sample containers, weighing, transferring, and mixing) should be
performed in a glove box demonstrated to be leak tight or in a fume
hood demonstrated to have adequate air flow. Gross losses to the
laboratory ventilation system must not be allowed. Handling of the
dilute solutions normally used in analytical and animal work presents
no inhalation hazards except in the case of an accident.
5.3.2 Protective equipment--Disposable plastic gloves, apron or
lab coat, safety glasses or mask, and a glove box or fume hood adequate
for radioactive work should be used. During analytical operations that
may give rise to aerosols or dusts, personnel should wear respirators
equipped with activated carbon filters. Eye protection equipment
(preferably full face shields) must be worn while working with exposed
samples or pure analytical standards. Latex gloves are commonly used to
reduce exposure of the hands. When handling samples suspected or known
to contain high concentrations of the CDDs/CDFs, an additional set of
gloves can also be worn beneath the latex gloves.
5.3.3 Training--Workers must be trained in the proper method of
removing contaminated gloves and clothing without contacting the
exterior surfaces.
5.3.4 Personal hygiene--Hands and forearms should be washed
thoroughly after each manipulation and before breaks (coffee, lunch,
and shift).
5.3.5 Confinement--Isolated work areas posted with signs,
segregated glassware and tools, and plastic absorbent paper on bench
tops will aid in confining contamination.
5.3.6 Effluent vapors--The effluents of sample splitters from the
gas chromatograph (GC) and from roughing pumps on the mass spectrometer
(MS) should pass through either a column of activated charcoal or be
bubbled through a trap containing oil or high-boiling alcohols to
condense CDD/CDF vapors.
5.3.7 Waste Handling--Good technique includes minimizing
contaminated waste. Plastic bag liners should be used in waste cans.
Janitors and other personnel must be trained in the safe handling of
waste.
5.3.8 Decontamination
5.3.8.1 Decontamination of personnel--Use any mild soap with
plenty of scrubbing action.
5.3.8.2 Glassware, tools, and surfaces--Chlorothene NU Solvent is
the least toxic solvent shown to be effective. Satisfactory cleaning
may be accomplished by rinsing with Chlorothene, then washing with any
detergent and water. If glassware is first rinsed with solvent, then
the dish water may be disposed of in the sewer. Given the cost of
disposal, it is prudent to minimize solvent wastes.
5.3.9 Laundry--Clothing known to be contaminated should be
collected in plastic bags. Persons who convey the bags and launder the
clothing should be advised of the hazard and trained in proper
handling. The clothing may be put into a washer without contact if the
launderer knows of the potential problem. The washer should be run
through a cycle before being used again for other clothing.
5.3.10 Wipe tests--A useful method of determining cleanliness of
work surfaces and tools is to wipe the surface with a piece of filter
paper. Extraction and analysis by GC with an electron capture detector
(ECD) can achieve a limit of detection of 0.1 g per wipe;
analysis using this method can achieve an even lower detection limit.
Less than 0.1 g per wipe indicates acceptable cleanliness;
anything higher warrants further cleaning. More than 10 g on a
wipe constitutes an acute hazard and requires prompt cleaning before
further use of the equipment or work space, and indicates that
unacceptable work practices have been employed.
5.3.11 Table or wrist-action shaker--The use of a table or wrist-
action shaker for extraction of tissues presents the possibility of
breakage of the extraction bottle and spillage of acid and flammable
organic solvent. A secondary containment system around the shaker is
suggested to prevent the spread of acid and solvents in the event of
such a breakage. The speed and intensity of shaking action should also
be adjusted to minimize the possibility of breakage.
6.0 Apparatus and Materials
Note: Brand names, suppliers, and part numbers are for
illustration purposes only and no endorsement is implied. Equivalent
performance may be achieved using apparatus and materials other than
those specified here. Meeting the performance requirements of this
method is the responsibility of the laboratory.
6.1 Sampling Equipment for Discrete or Composite Sampling
[[Page 48408]]
6.1.1 Sample bottles and caps
6.1.1.1 Liquid samples (waters, sludges and similar materials
containing 5% solids or less)--Sample bottle, amber glass, 1.1 L
minimum, with screw cap.
6.1.1.2 Solid samples (soils, sediments, sludges, paper pulps,
filter cake, compost, and similar materials that contain more than 5%
solids)--Sample bottle, wide mouth, amber glass, 500 mL minimum.
6.1.1.3 If amber bottles are not available, samples shall be
protected from light.
6.1.1.4 Bottle caps--Threaded to fit sample bottles. Caps shall be
lined with fluoropolymer.
6.1.1.5 Cleaning
6.1.1.5.1 Bottles are detergent water washed, then solvent rinsed
before use.
6.1.1.5.2 Liners are detergent water washed, rinsed with reagent
water (Section 7.6.1) followed by solvent, and baked at approximately
200 deg.C for a minimum of 1 hour prior to use.
6.1.2 Compositing equipment--Automatic or manual compositing
system incorporating glass containers cleaned per bottle cleaning
procedure above. Only glass or fluoropolymer tubing shall be used. If
the sampler uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used in the pump only. Before use, the
tubing shall be thoroughly rinsed with methanol, followed by repeated
rinsing with reagent water to minimize sample contamination. An
integrating flow meter is used to collect proportional composite
samples.
6.2 Equipment for Glassware Cleaning--Laboratory sink with
overhead fume hood.
6.3 Equipment for Sample Preparation
6.3.1 Laboratory fume hood of sufficient size to contain the
sample preparation equipment listed below.
6.3.2 Glove box (optional).
6.3.3 Tissue homogenizer--VirTis Model 45 Macro homogenizer
(American Scientific Products H-3515, or equivalent) with stainless
steel Macro-shaft and Turbo-shear blade.
6.3.4 Meat grinder--Hobart, or equivalent, with 3-5 mm holes in
inner plate.
6.3.5 Equipment for determining percent moisture
6.3.5.1 Oven--Capable of maintaining a temperature of 110
5 deg.C.
6.3.5.2 Dessicator.
6.3.6 Balances
6.3.6.1 Analytical--Capable of weighing 0.1 mg.
6.3.6.2 Top loading--Capable of weighing 10 mg.
6.4 Extraction Apparatus
6.4.1 Water samples
6.4.1.1 pH meter, with combination glass electrode.
6.4.1.2 pH paper, wide range (Hydrion Papers, or equivalent).
6.4.1.3 Graduated cylinder, 1 L capacity.
6.4.1.4 Liquid/liquid extraction--Separatory funnels, 250 mL, 500
mL, and 2000 mL, with fluoropolymer stopcocks.
6.4.1.5 Solid-phase extraction
6.4.1.5.1 One liter filtration apparatus, including glass funnel,
glass frit support, clamp, adapter, stopper, filtration flask, and
vacuum tubing (Figure 4). For wastewater samples, the apparatus should
accept 90 or 144 mm disks. For drinking water or other samples
containing low solids, smaller disks may be used.
6.4.1.5.2 Vacuum source capable of maintaining 25 in. Hg, equipped
with shutoff valve and vacuum gauge.
6.4.1.5.3 Glass-fiber filter--Whatman GMF 150 (or equivalent), 1
micron pore size, to fit filtration apparatus in Section 6.4.1.5.1.
6.4.1.5.4 Solid-phase extraction disk containing octadecyl
(C18) bonded silica uniformly enmeshed in an inert matrix--
Fisher Scientific 14-378F (or equivalent), to fit filtration apparatus
in Section 6.4.1.5.1.
6.4.2 Soxhlet/Dean-Stark (SDS) extractor (Figure 5)--For filters
and solid/sludge samples.
6.4.2.1 Soxhlet--50 mm ID, 200 mL capacity with 500 mL flask (Cal-
Glass LG-6900, or equivalent, except substitute 500 mL round-bottom
flask for 300 mL flat-bottom flask).
6.4.2.2 Thimble--43 x 123 to fit Soxhlet (Cal-Glass LG-6901-122,
or equivalent).
6.4.2.3 Moisture trap--Dean Stark or Barret with fluoropolymer
stopcock, to fit Soxhlet.
6.4.2.4 Heating mantle--Hemispherical, to fit 500 mL round-bottom
flask (Cal-Glass LG-8801-112, or equivalent).
6.4.2.5 Variable transformer--Powerstat (or equivalent), 110 volt,
10 amp.
6.4.3 Apparatus for extraction of tissue.
6.4.3.1 Bottle for extraction (if digestion/extraction using HCl
is used)'' 500-600 mL wide-mouth clear glass, with fluoropolymer-lined
cap.
6.4.3.2 Bottle for back-extraction--100-200 mL narrow-mouth clear
glass with fluoropolymer-lined cap.
6.4.3.3 Mechanical shaker--Wrist-action or platform-type rotary
shaker that produces vigorous agitation (Sybron Thermolyne Model LE
``Big Bill'' rotator/shaker, or equivalent).
6.4.3.4 Rack attached to shaker table to permit agitation of four
to nine samples simultaneously.
6.4.4 Beakers--400-500 mL.
6.4.5 Spatulas--Stainless steel.
6.5 Filtration Apparatus.
6.5.1 Pyrex glass wool--Solvent-extracted by SDS for three hours
minimum.
Note: Baking glass wool may cause active sites that will
irreversibly adsorb CDDs/CDFs.
6.5.2 Glass funnel--125-250 mL.
6.5.3 Glass-fiber filter paper--Whatman GF/D (or equivalent), to
fit glass funnel in Section 6.5.2.
6.5.4 Drying column--15-20 mm ID Pyrex chromatographic column
equipped with coarse-glass frit or glass-wool plug.
6.5.5 Buchner funnel--15 cm.
6.5.6 Glass-fiber filter paper--to fit Buchner funnel in Section
6.5.5.
6.5.7 Filtration flasks--1.5-2.0 L, with side arm.
6.5.8 Pressure filtration apparatus--Millipore YT30 142 HW, or
equivalent.
6.6 Centrifuge Apparatus.
6.6.1 Centrifuge--Capable of rotating 500 mL centrifuge bottles or
15 mL centrifuge tubes at 5,000 rpm minimum.
6.6.2 Centrifuge bottles--500 mL, with screw-caps, to fit
centrifuge.
6.6.3 Centrifuge tubes--12-15 mL, with screw-caps, to fit
centrifuge.
6.7 Cleanup Apparatus.
6.7.1 Automated gel permeation chromatograph (Analytical
Biochemical Labs, Inc, Columbia, MO, Model GPC Autoprep 1002, or
equivalent).
6.7.1.1 Column--600-700 mm long x 25 mm ID, packed with 70 g of
SX-3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
6.7.1.2 Syringe--10 mL, with Luer fitting.
6.7.1.3 Syringe filter holder--stainless steel, and glass-fiber or
fluoropolymer filters (Gelman 4310, or equivalent).
6.7.1.4 UV detectors--254 nm, preparative or semi-preparative flow
cell (Isco, Inc., Type 6; Schmadzu, 5 mm path length; Beckman-Altex
152W, 8 L micro-prep flow cell, 2 mm path; Pharmacia UV-1, 3
mm flow cell; LDC Milton-Roy UV-3, monitor #1203; or equivalent).
6.7.2 Reverse-phase high-performance liquid chromatograph.
6.7.2.1 Column oven and detector--Perkin-Elmer Model LC-65T (or
equivalent) operated at 0.02 AUFS at 235 nm.
6.7.2.2 Injector--Rheodyne 7120 (or equivalent) with 50 L
sample loop.
6.7.2.3 Column--Two 6.2 mm x 250 mm Zorbax-ODS columns in series
[[Page 48409]]
(DuPont Instruments Division, Wilmington, DE, or equivalent), operated
at 50 deg.C with 2.0 mL/min methanol isocratic effluent.
6.7.2.4 Pump--Altex 110A (or equivalent).
6.7.3 Pipets.
6.7.3.1 Disposable, pasteur--150 mm long x 5-mm ID (Fisher
Scientific 13-678-6A, or equivalent).
6.7.3.2 Disposable, serological--10 mL (6 mm ID).
6.7.4 Glass chromatographic columns.
6.7.4.1 150 mm long x 8-mm ID, (Kontes K-420155, or equivalent)
with coarse-glass frit or glass-wool plug and 250 mL reservoir.
6.7.4.2 200 mm long x 15 mm ID, with coarse-glass frit or glass-
wool plug and 250 mL reservoir.
6.7.4.3 300 mm long x 25 mm ID, with 300 mL reservoir and glass
or fluoropolymer stopcock.
6.7.5 Stirring apparatus for batch silica cleanup of tissue
extracts.
6.7.5.1 Mechanical stirrer--Corning Model 320, or equivalent.
6.7.5.2 Bottle--500-600 mL wide-mouth clear glass.
6.7.6 Oven--For baking and storage of adsorbents, capable of
maintaining a constant temperature (5 deg.C) in the range
of 105-250 deg.C.
6.8 Concentration Apparatus.
6.8.1 Rotary evaporator--Buchi/Brinkman-American Scientific No.
E5045-10 or equivalent, equipped with a variable temperature water
bath.
6.8.1.1 Vacuum source for rotary evaporator equipped with shutoff
valve at the evaporator and vacuum gauge.
6.8.1.2 A recirculating water pump and chiller are recommended, as
use of tap water for cooling the evaporator wastes large volumes of
water and can lead to inconsistent performance as water temperatures
and pressures vary.
6.8.1.3 Round-bottom flask--100 mL and 500 mL or larger, with
ground-glass fitting compatible with the rotary evaporator.
6.8.2 Kuderna-Danish (K-D) Concentrator.
6.8.2.1 Concentrator tube--10 mL, graduated (Kontes K-570050-1025,
or equivalent) with calibration verified. Ground-glass stopper (size
19/22 joint) is used to prevent evaporation of extracts.
6.8.2.2 Evaporation flask--500 mL (Kontes K-570001-0500, or
equivalent), attached to concentrator tube with springs (Kontes K-
662750-0012 or equivalent).
6.8.2.3 Snyder column--Three-ball macro (Kontes K-503000-0232, or
equivalent).
6.8.2.4 Boiling chips.
6.8.2.4.1 Glass or silicon carbide--Approximately 10/40 mesh,
extracted with methylene chloride and baked at 450 deg.C for one hour
minimum.
6.8.2.4.2 Fluoropolymer (optional)--Extracted with methylene
chloride.
6.8.2.5 Water bath--Heated, with concentric ring cover, capable of
maintaining a temperature within 2 deg.C, installed in a
fume hood.
6.8.3 Nitrogen blowdown apparatus--Equipped with water bath
controlled in the range of 30-60 deg.C (N-Evap, Organomation
Associates, Inc., South Berlin, MA, or equivalent), installed in a fume
hood.
6.8.4 Sample vials.
6.8.4.1 Amber glass--2-5 mL with fluoropolymer-lined screw-cap.
6.8.4.2 Glass--0.3 mL, conical, with fluoropolymer-lined screw or
crimp cap.
6.9 Gas Chromatograph--Shall have splitless or on-column injection
port for capillary column, temperature program with isothermal hold,
and shall meet all of the performance specifications in Section 10.
6.9.1 GC column for CDDs/CDFs and for isomer specificity for
2,3,7,8-TCDD--605 m long x 0.320.02 mm ID;
0.25 m 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase
fused-silica capillary column (J&W DB-5, or equivalent).
6.9.2 GC column for isomer specificity for 2,3,7,8-TCDF--
305 m long x 0.320.02 mm ID; 0.25 m
bonded-phase fused-silica capillary column (J&W DB-225, or equivalent).
6.10 Mass Spectrometer--28-40 eV electron impact ionization, shall
be capable of repetitively selectively monitoring 12 exact m/z's
minimum at high resolution (10,000) during a period of
approximately one second, and shall meet all of the performance
specifications in Section 10.
6.11 GC/MS Interface--The mass spectrometer (MS) shall be
interfaced to the GC such that the end of the capillary column
terminates within 1 cm of the ion source but does not intercept the
electron or ion beams.
6.12 Data System--Capable of collecting, recording, and storing MS
data.
7.0 Reagents and Standards
7.1 pH Adjustment and Back-Extraction.
7.1.1 Potassium hydroxide--Dissolve 20 g reagent grade KOH in 100
mL reagent water.
7.1.2 Sulfuric acid--Reagent grade (specific gravity 1.84).
7.1.3 Hydrochloric acid--Reagent grade, 6N.
7.1.4 Sodium chloride--Reagent grade, prepare at 5% (w/v) solution
in reagent water.
7.2 Solution Drying and Evaporation.
7.2.1 Solution drying--Sodium sulfate, reagent grade, granular,
anhydrous (Baker 3375, or equivalent), rinsed with methylene chloride
(20 mL/g), baked at 400 deg.C for one hour minimum, cooled in a
dessicator, and stored in a pre-cleaned glass bottle with screw-cap
that prevents moisture from entering. If, after heating, the sodium
sulfate develops a noticeable grayish cast (due to the presence of
carbon in the crystal matrix), that batch of reagent is not suitable
for use and should be discarded. Extraction with methylene chloride (as
opposed to simple rinsing) and baking at a lower temperature may
produce sodium sulfate that is suitable for use.
7.2.2 Tissue drying--Sodium sulfate, reagent grade, powdered,
treated and stored as above.
7.2.3 Prepurified nitrogen.
7.3 Extraction.
7.3.1 Solvents--Acetone, toluene, cyclohexane, hexane, methanol,
methylene chloride, and nonane; distilled in glass, pesticide quality,
lot-certified to be free of interferences.
7.3.2 White quartz sand, 60/70 mesh--For Soxhlet/Dean-Stark
extraction (Aldrich Chemical, Cat. No. 27-437-9, or equivalent). Bake
at 450 deg.C for four hours minimum.
7.4 GPC Calibration Solution--Prepare a solution containing 300
mg/mL corn oil, 15 mg/mL bis(2-ethylhexyl) phthalate, 1.4 mg/mL
pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
7.5 Adsorbents for Sample Cleanup.
7.5.1 Silica gel.
7.5.1.1 Activated silica gel--100-200 mesh, Supelco 1-3651 (or
equivalent), rinsed with methylene chloride, baked at 180 deg.C for a
minimum of one hour, cooled in a dessicator, and stored in a precleaned
glass bottle with screw-cap that prevents moisture from entering.
7.5.1.2 Acid silica gel (30% w/w)--Thoroughly mix 44.0 g of
concentrated sulfuric acid with 100.0 g of activated silica gel in a
clean container. Break up aggregates with a stirring rod until a
uniform mixture is obtained. Store in a bottle with a fluoropolymer-
lined screw-cap.
7.5.1.3 Basic silica gel--Thoroughly mix 30 g of 1N sodium
hydroxide with 100 g of activated silica gel in a clean container.
Break up aggregates with a stirring rod until a uniform mixture is
obtained. Store in a bottle with a fluoropolymer-lined screw-cap.
7.5.1.4 Potassium silicate.
7.5.1.4.1 Dissolve 56 g of high purity potassium hydroxide
(Aldrich, or
[[Page 48410]]
equivalent) in 300 mL of methanol in a 750-1000 mL flat-bottom flask.
7.5.1.4.2 Add 100 g of silica gel and a stirring bar, and stir on
a hot plate at 60-70 deg.C for one to two hours.
7.5.1.4.3 Decant the liquid and rinse the potassium silicate twice
with 100 mL portions of methanol, followed by a single rinse with 100
mL of methylene chloride.
7.5.1.4.4 Spread the potassium silicate on solvent-rinsed aluminum
foil and dry for two to four hours in a hood.
7.5.1.4.5 Activate overnight at 200-250 deg.C.
7.5.2 Alumina--Either one of two types of alumina, acid or basic,
may be used in the cleanup of sample extracts, provided that the
laboratory can meet the performance specifications for the recovery of
labeled compounds described in Section 9.3. The same type of alumina
must be used for all samples, including those used to demonstrate
initial precision and recovery (Section 9.2) and ongoing precision and
recovery (Section 15.5).
7.5.2.1 Acid alumina--Supelco 19996-6C (or equivalent). Activate
by heating to 130 deg.C for a minimum of 12 hours.
7.5.2.2 Basic alumina--Supelco 19944-6C (or equivalent). Activate
by heating to 600 deg.C for a minimum of 24 hours. Alternatively,
activate by heating in a tube furnace at 650-700 deg.C under an air
flow rate of approximately 400 cc/minute. Do not heat over 700 deg.C,
as this can lead to reduced capacity for retaining the analytes. Store
at 130 deg.C in a covered flask. Use within five days of baking.
7.5.3 Carbon.
7.5.3.1 Carbopak C--(Supelco 1-0258, or equivalent).
7.5.3.2 Celite 545--(Supelco 2-0199, or equivalent).
7.5.3.3 Thoroughly mix 9.0 g Carbopak C and 41.0 g Celite 545 to
produce an 18% w/w mixture. Activate the mixture at 130 deg.C for a
minimum of six hours. Store in a dessicator.
7.5.4 Anthropogenic isolation column--Pack the column in Section
6.7.4.3 from bottom to top with the following:
7.5.4.1 2 g silica gel (Section 7.5.1.1).
7.5.4.2 2 g potassium silicate (Section 7.5.1.4).
7.5.4.3 2 g granular anhydrous sodium sulfate (Section 7.2.1).
7.5.4.4 10 g acid silica gel (Section 7.5.1.2).
7.5.4.5 2 g granular anhydrous sodium sulfate.
7.5.5 Florisil column.
7.5.5.1 Florisil--60-100 mesh, Floridin Corp (or equivalent).
Soxhlet extract in 500 g portions for 24 hours.
7.5.5.2 Insert a glass wool plug into the tapered end of a
graduated serological pipet (Section 6.7.3.2). Pack with 1.5 g (approx
2 mL) of Florisil topped with approx 1 mL of sodium sulfate (Section
7.2.1) and a glass wool plug.
7.5.5.3 Activate in an oven at 130-150 deg.C for a minimum of 24
hours and cool for 30 minutes. Use within 90 minutes of cooling.
7.6 Reference Matrices--Matrices in which the CDDs/CDFs and
interfering compounds are not detected by this method.
7.6.1 Reagent water--Bottled water purchased locally, or prepared
by passage through activated carbon.
7.6.2 High-solids reference matrix--Playground sand or similar
material. Prepared by extraction with methylene chloride and/or baking
at 450 deg.C for a minimum of four hours.
7.6.3 Paper reference matrix--Glass-fiber filter, Gelman Type A,
or equivalent. Cut paper to simulate the surface area of the paper
sample being tested.
7.6.4 Tissue reference matrix--Corn or other vegetable oil. May be
prepared by extraction with methylene chloride.
7.6.5 Other matrices--This method may be verified on any reference
matrix by performing the tests given in Section 9.2. Ideally, the
matrix should be free of the CDDs/CDFs, but in no case shall the
background level of the CDDs/CDFs in the reference matrix exceed three
times the minimum levels in Table 2. If low background levels of the
CDDs/CDFs are present in the reference matrix, the spike level of the
analytes used in Section 9.2 should be increased to provide a spike-to-
background ratio in the range of 1:1 to 5:1 (Reference 15).
7.7 Standard Solutions--Purchased as solutions or mixtures with
certification to their purity, concentration, and authenticity, or
prepared from materials of known purity and composition. If the
chemical purity is 98% or greater, the weight may be used without
correction to compute the concentration of the standard. When not being
used, standards are stored in the dark at room temperature in screw-
capped vials with fluoropolymer-lined caps. A mark is placed on the
vial at the level of the solution so that solvent loss by evaporation
can be detected. If solvent loss has occurred, the solution should be
replaced.
7.8 Stock Solutions.
7.8.1 Preparation--Prepare in nonane per the steps below or
purchase as dilute solutions (Cambridge Isotope Laboratories (CIL),
Woburn, MA, or equivalent). Observe the safety precautions in Section
5, and the recommendation in Section 5.1.2.
7.8.2 Dissolve an appropriate amount of assayed reference material
in solvent. For example, weigh 1-2 mg of 2,3,7,8-TCDD to three
significant figures in a 10 mL ground-glass-stoppered volumetric flask
and fill to the mark with nonane. After the TCDD is completely
dissolved, transfer the solution to a clean 15 mL vial with
fluoropolymer-lined cap.
7.8.3 Stock standard solutions should be checked for signs of
degradation prior to the preparation of calibration or performance test
standards. Reference standards that can be used to determine the
accuracy of calibration standards are available from CIL and may be
available from other vendors.
7.9 PAR Stock Solution
7.9.1 All CDDs/CDFs--Using the solutions in Section 7.8, prepare
the PAR stock solution to contain the CDDs/CDFs at the concentrations
shown in Table 3. When diluted, the solution will become the PAR
(Section 7.14).
7.9.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined,
prepare the PAR stock solution to contain these compounds only.
7.10 Labeled-Compound Spiking Solution.
7.10.1 All CDDs/CDFs--From stock solutions, or from purchased
mixtures, prepare this solution to contain the labeled compounds in
nonane at the concentrations shown in Table 3. This solution is diluted
with acetone prior to use (Section 7.10.3).
7.10.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined,
prepare the labeled-compound solution to contain these compounds only.
This solution is diluted with acetone prior to use (Section 7.10.3).
7.10.3 Dilute a sufficient volume of the labeled compound solution
(Section 7.10.1 or 7.10.2) by a factor of 50 with acetone to prepare a
diluted spiking solution. Each sample requires 1.0 mL of the diluted
solution, but no more solution should be prepared than can be used in
one day.
7.11 Cleanup Standard--Prepare 37Cl4-
2,3,7,8-TCDD in nonane at the concentration shown in Table 3. The
cleanup standard is added to all extracts prior to cleanup to measure
the efficiency of the cleanup process.
7.12 Internal Standard(s).
7.12.1 All CDDs/CDFs--Prepare the internal standard solution to
contain 13C12-1,2,3,4-TCDD and
13C12-1,2,3,7,8,9-HxCDD in nonane at the
concentration shown in Table 3.
7.12.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined,
[[Page 48411]]
prepare the internal standard solution to contain
13C12-1,2,3,4-TCDD only.
7.13 Calibration Standards (CS1 through CS5)--Combine the
solutions in Sections 7.9 through 7.12 to produce the five calibration
solutions shown in Table 4 in nonane. These solutions permit the
relative response (labeled to native) and response factor to be
measured as a function of concentration. The CS3 standard is used for
calibration verification (VER). If only 2,3,7,8-TCDD and 2,3,7,8-TCDF
are to be determined, combine the solutions appropriate to these
compounds.
7.14 Precision and Recovery (PAR) Standard--Used for determination
of initial (Section 9.2) and ongoing (Section 15.5) precision and
recovery. Dilute 10 L of the precision and recovery standard
(Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix
for each sample batch. One mL each are required for the blank and OPR
with each matrix in each batch.
7.15 GC Retention Time Window Defining Solution and Isomer
Specificity Test Standard--Used to define the beginning and ending
retention times for the dioxin and furan isomers and to demonstrate
isomer specificity of the GC columns employed for determination of
2,3,7,8-TCDD and 2,3,7,8-TCDF. The standard must contain the compounds
listed in Table 5 (CIL EDF--4006, or equivalent), at a minimum. It is
not necessary to monitor the window-defining compounds if only 2,3,7,8-
TCDD and 2,3,7,8-TCDF are to be determined. In this case, an isomer-
specificity test standard containing the most closely eluted isomers
listed in Table 5 (CIL EDF-4033, or equivalent) may be used.
7.16 QC Check Sample--A QC Check Sample should be obtained from a
source independent of the calibration standards. Ideally, this check
sample would be a certified reference material containing the CDDs/CDFs
in known concentrations in a sample matrix similar to the matrix under
test.
7.17 Stability of Solutions--Standard solutions used for
quantitative purposes (Sections 7.9 through 7.15) should be analyzed
periodically, and should be assayed against reference standards
(Section 7.8.3) before further use.
8.0 Sample Collection, Preservation, Storage, and Holding Times
8.1 Collect samples in amber glass containers following
conventional sampling practices (Reference 16). Aqueous samples that
flow freely are collected in refrigerated bottles using automatic
sampling equipment. Solid samples are collected as grab samples using
wide-mouth jars.
8.2 Maintain aqueous samples in the dark at 0-4 deg.C from the
time of collection until receipt at the laboratory. If residual
chlorine is present in aqueous samples, add 80 mg sodium thiosulfate
per liter of water. EPA Methods 330.4 and 330.5 may be used to measure
residual chlorine (Reference 17). If sample pH is greater than 9,
adjust to pH 7-9 with sulfuric acid.
Maintain solid, semi-solid, oily, and mixed-phase samples in the
dark at <4 deg.c="" from="" the="" time="" of="" collection="" until="" receipt="" at="" the="" laboratory.="" store="" aqueous="" samples="" in="" the="" dark="" at="" 0-4="" deg.c.="" store="" solid,="" semi-="" solid,="" oily,="" mixed-phase,="" and="" tissue="" samples="" in="" the="" dark="" at=""><-10 deg.c.="" 8.3="" fish="" and="" tissue="" samples.="" 8.3.1="" fish="" may="" be="" cleaned,="" filleted,="" or="" processed="" in="" other="" ways="" in="" the="" field,="" such="" that="" the="" laboratory="" may="" expect="" to="" receive="" whole="" fish,="" fish="" fillets,="" or="" other="" tissues="" for="" analysis.="" 8.3.2="" fish="" collected="" in="" the="" field="" should="" be="" wrapped="" in="" aluminum="" foil,="" and="" must="" be="" maintained="" at="" a="" temperature="" less="" than="" 4="" deg.c="" from="" the="" time="" of="" collection="" until="" receipt="" at="" the="" laboratory.="" 8.3.3="" samples="" must="" be="" frozen="" upon="" receipt="" at="" the="" laboratory="" and="" maintained="" in="" the="" dark="" at=""><-10 deg.c="" until="" prepared.="" maintain="" unused="" sample="" in="" the="" dark="" at=""><-10 deg.c.="" 8.4="" holding="" times.="" 8.4.1="" there="" are="" no="" demonstrated="" maximum="" holding="" times="" associated="" with="" cdds/cdfs="" in="" aqueous,="" solid,="" semi-solid,="" tissues,="" or="" other="" sample="" matrices.="" if="" stored="" in="" the="" dark="" at="" 0-4="" deg.c="" and="" preserved="" as="" given="" above="" (if="" required),="" aqueous="" samples="" may="" be="" stored="" for="" up="" to="" one="" year.="" similarly,="" if="" stored="" in="" the="" dark="" at=""><-10 deg.c,="" solid,="" semi-solid,="" multi-phase,="" and="" tissue="" samples="" may="" be="" stored="" for="" up="" to="" one="" year.="" 8.4.2="" store="" sample="" extracts="" in="" the="" dark="" at=""><-10 deg.c="" until="" analyzed.="" if="" stored="" in="" the="" dark="" at=""><-10 deg.c,="" sample="" extracts="" may="" be="" stored="" for="" up="" to="" one="" year.="" 9.0="" quality="" assurance/quality="" control="" 9.1="" each="" laboratory="" that="" uses="" this="" method="" is="" required="" to="" operate="" a="" formal="" quality="" assurance="" program="" (reference="" 18).="" the="" minimum="" requirements="" of="" this="" program="" consist="" of="" an="" initial="" demonstration="" of="" laboratory="" capability,="" analysis="" of="" samples="" spiked="" with="" labeled="" compounds="" to="" evaluate="" and="" document="" data="" quality,="" and="" analysis="" of="" standards="" and="" blanks="" as="" tests="" of="" continued="" performance.="" laboratory="" performance="" is="" compared="" to="" established="" performance="" criteria="" to="" determine="" if="" the="" results="" of="" analyses="" meet="" the="" performance="" characteristics="" of="" the="" method.="" if="" the="" method="" is="" to="" be="" applied="" to="" sample="" matrix="" other="" than="" water="" (e.g.,="" soils,="" filter="" cake,="" compost,="" tissue)="" the="" most="" appropriate="" alternate="" matrix="" (sections="" 7.6.2="" through="" 7.6.5)="" is="" substituted="" for="" the="" reagent="" water="" matrix="" (section="" 7.6.1)="" in="" all="" performance="" tests.="" 9.1.1="" the="" analyst="" shall="" make="" an="" initial="" demonstration="" of="" the="" ability="" to="" generate="" acceptable="" accuracy="" and="" precision="" with="" this="" method.="" this="" ability="" is="" established="" as="" described="" in="" section="" 9.2.="" 9.1.2="" in="" recognition="" of="" advances="" that="" are="" occurring="" in="" analytical="" technology,="" and="" to="" allow="" the="" analyst="" to="" overcome="" sample="" matrix="" interferences,="" the="" analyst="" is="" permitted="" certain="" options="" to="" improve="" separations="" or="" lower="" the="" costs="" of="" measurements.="" these="" options="" include="" alternate="" extraction,="" concentration,="" cleanup="" procedures,="" and="" changes="" in="" columns="" and="" detectors.="" alternate="" determinative="" techniques,="" such="" as="" the="" substitution="" of="" spectroscopic="" or="" immuno-assay="" techniques,="" and="" changes="" that="" degrade="" method="" performance,="" are="" not="" allowed.="" if="" an="" analytical="" technique="" other="" than="" the="" techniques="" specified="" in="" this="" method="" is="" used,="" that="" technique="" must="" have="" a="" specificity="" equal="" to="" or="" better="" than="" the="" specificity="" of="" the="" techniques="" in="" this="" method="" for="" the="" analytes="" of="" interest.="" 9.1.2.1="" each="" time="" a="" modification="" is="" made="" to="" this="" method,="" the="" analyst="" is="" required="" to="" repeat="" the="" procedure="" in="" section="" 9.2.="" if="" the="" detection="" limit="" of="" the="" method="" will="" be="" affected="" by="" the="" change,="" the="" laboratory="" is="" required="" to="" demonstrate="" that="" the="" mdl="" (40="" cfr="" part="" 136,="" appendix="" b)="" is="" lower="" than="" one-third="" the="" regulatory="" compliance="" level="" or="" one-third="" the="" ml="" in="" this="" method,="" whichever="" is="" higher.="" if="" calibration="" will="" be="" affected="" by="" the="" change,="" the="" analyst="" must="" recalibrate="" the="" instrument="" per="" section="" 10.="" 9.1.2.2="" the="" laboratory="" is="" required="" to="" maintain="" records="" of="" modifications="" made="" to="" this="" method.="" these="" records="" include="" the="" following,="" at="" a="" minimum:="" 9.1.2.2.1="" the="" names,="" titles,="" addresses,="" and="" telephone="" numbers="" of="" the="" analyst(s)="" who="" performed="" the="" analyses="" and="" modification,="" and="" of="" the="" quality="" control="" officer="" who="" witnessed="" and="" will="" verify="" the="" analyses="" and="" modifications.="" 9.1.2.2.2="" a="" listing="" of="" pollutant(s)="" measured,="" by="" name="" and="" cas="" registry="" number.="" 9.1.2.2.3="" a="" narrative="" stating="" reason(s)="" for="" the="" modifications.="" 9.1.2.2.4="" results="" from="" all="" quality="" control="" (qc)="" tests="" comparing="" the="" modified="" method="" to="" this="" method,="" including:="" [[page="" 48412]]="" (a)="" calibration="" (section="" 10.5="" through="" 10.7).="" (b)="" calibration="" verification="" (section="" 15.3).="" (c)="" initial="" precision="" and="" recovery="" (section="" 9.2).="" (d)="" labeled="" compound="" recovery="" (section="" 9.3).="" (e)="" analysis="" of="" blanks="" (section="" 9.5).="" (f)="" accuracy="" assessment="" (section="" 9.4).="" 9.1.2.2.5="" data="" that="" will="" allow="" an="" independent="" reviewer="" to="" validate="" each="" determination="" by="" tracing="" the="" instrument="" output="" (peak="" height,="" area,="" or="" other="" signal)="" to="" the="" final="" result.="" these="" data="" are="" to="" include:="" (a)="" sample="" numbers="" and="" other="" identifiers.="" (b)="" extraction="" dates.="" (c)="" analysis="" dates="" and="" times.="" (d)="" analysis="" sequence/run="" chronology.="" (e)="" sample="" weight="" or="" volume="" (section="" 11).="" (f)="" extract="" volume="" prior="" to="" each="" cleanup="" step="" (section="" 13).="" (g)="" extract="" volume="" after="" each="" cleanup="" step="" (section="" 13).="" (h)="" final="" extract="" volume="" prior="" to="" injection="" (section="" 14).="" (i)="" injection="" volume="" (section="" 14.3).="" (j)="" dilution="" data,="" differentiating="" between="" dilution="" of="" a="" sample="" or="" extract="" (section="" 17.5).="" (k)="" instrument="" and="" operating="" conditions.="" (l)="" column="" (dimensions,="" liquid="" phase,="" solid="" support,="" film="" thickness,="" etc).="" (m)="" operating="" conditions="" (temperatures,="" temperature="" program,="" flow="" rates).="" (n)="" detector="" (type,="" operating="" conditions,="" etc).="" (o)="" chromatograms,="" printer="" tapes,="" and="" other="" recordings="" of="" raw="" data.="" (p)="" quantitation="" reports,="" data="" system="" outputs,="" and="" other="" data="" to="" link="" the="" raw="" data="" to="" the="" results="" reported.="" 9.1.3="" analyses="" of="" method="" blanks="" are="" required="" to="" demonstrate="" freedom="" from="" contamination="" (section="" 4.3).="" the="" procedures="" and="" criteria="" for="" analysis="" of="" a="" method="" blank="" are="" described="" in="" sections="" 9.5="" and="" 15.6.="" 9.1.4="" the="" laboratory="" shall="" spike="" all="" samples="" with="" labeled="" compounds="" to="" monitor="" method="" performance.="" this="" test="" is="" described="" in="" section="" 9.3.="" when="" results="" of="" these="" spikes="" indicate="" atypical="" method="" performance="" for="" samples,="" the="" samples="" are="" diluted="" to="" bring="" method="" performance="" within="" acceptable="" limits.="" procedures="" for="" dilution="" are="" given="" in="" section="" 17.5.="" 9.1.5="" the="" laboratory="" shall,="" on="" an="" ongoing="" basis,="" demonstrate="" through="" calibration="" verification="" and="" the="" analysis="" of="" the="" ongoing="" precision="" and="" recovery="" aliquot="" that="" the="" analytical="" system="" is="" in="" control.="" these="" procedures="" are="" described="" in="" sections="" 15.1="" through="" 15.5.="" 9.1.6="" the="" laboratory="" shall="" maintain="" records="" to="" define="" the="" quality="" of="" data="" that="" is="" generated.="" development="" of="" accuracy="" statements="" is="" described="" in="" section="" 9.4.="" 9.2="" initial="" precision="" and="" recovery="" (ipr)--to="" establish="" the="" ability="" to="" generate="" acceptable="" precision="" and="" recovery,="" the="" analyst="" shall="" perform="" the="" following="" operations.="" 9.2.1="" for="" low="" solids="" (aqueous)="" samples,="" extract,="" concentrate,="" and="" analyze="" four="" 1="" l="" aliquots="" of="" reagent="" water="" spiked="" with="" the="" diluted="" labeled="" compound="" spiking="" solution="" (section="" 7.10.3)="" and="" the="" precision="" and="" recovery="" standard="" (section="" 7.14)="" according="" to="" the="" procedures="" in="" sections="" 11="" through="" 18.="" for="" an="" alternative="" sample="" matrix,="" four="" aliquots="" of="" the="" alternative="" reference="" matrix="" (section="" 7.6)="" are="" used.="" all="" sample="" processing="" steps="" that="" are="" to="" be="" used="" for="" processing="" samples,="" including="" preparation="" (section="" 11),="" extraction="" (section="" 12),="" and="" cleanup="" (section="" 13),="" shall="" be="" included="" in="" this="" test.="" 9.2.2="" using="" results="" of="" the="" set="" of="" four="" analyses,="" compute="" the="" average="" concentration="" (x)="" of="" the="" extracts="" in="" ng/ml="" and="" the="" standard="" deviation="" of="" the="" concentration="" (s)="" in="" ng/ml="" for="" each="" compound,="" by="" isotope="" dilution="" for="" cdds/cdfs="" with="" a="" labeled="" analog,="" and="" by="" internal="" standard="" for="" 1,2,3,7,8,9-hxcdd,="" ocdf,="" and="" the="" labeled="" compounds.="" 9.2.3="" for="" each="" cdd/cdf="" and="" labeled="" compound,="" compare="" s="" and="" x="" with="" the="" corresponding="" limits="" for="" initial="" precision="" and="" recovery="" in="" table="" 6.="" if="" only="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf="" are="" to="" be="" determined,="" compare="" s="" and="" x="" with="" the="" corresponding="" limits="" for="" initial="" precision="" and="" recovery="" in="" table="" 6a.="" if="" s="" and="" x="" for="" all="" compounds="" meet="" the="" acceptance="" criteria,="" system="" performance="" is="" acceptable="" and="" analysis="" of="" blanks="" and="" samples="" may="" begin.="" if,="" however,="" any="" individual="" s="" exceeds="" the="" precision="" limit="" or="" any="" individual="" x="" falls="" outside="" the="" range="" for="" accuracy,="" system="" performance="" is="" unacceptable="" for="" that="" compound.="" correct="" the="" problem="" and="" repeat="" the="" test="" (section="" 9.2).="" 9.3="" the="" laboratory="" shall="" spike="" all="" samples="" with="" the="" diluted="" labeled="" compound="" spiking="" solution="" (section="" 7.10.3)="" to="" assess="" method="" performance="" on="" the="" sample="" matrix.="" 9.3.1="" analyze="" each="" sample="" according="" to="" the="" procedures="" in="" sections="" 11="" through="" 18.="" 9.3.2="" compute="" the="" percent="" recovery="" of="" the="" labeled="" compounds="" and="" the="" cleanup="" standard="" using="" the="" internal="" standard="" method="" (section="" 17.2).="" 9.3.3="" the="" recovery="" of="" each="" labeled="" compound="" must="" be="" within="" the="" limits="" in="" table="" 7="" when="" all="" 2,3,7,8-substituted="" cdds/cdfs="" are="" determined,="" and="" within="" the="" limits="" in="" table="" 7a="" when="" only="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf="" are="" determined.="" if="" the="" recovery="" of="" any="" compound="" falls="" outside="" of="" these="" limits,="" method="" performance="" is="" unacceptable="" for="" that="" compound="" in="" that="" sample.="" to="" overcome="" such="" difficulties,="" water="" samples="" are="" diluted="" and="" smaller="" amounts="" of="" soils,="" sludges,="" sediments,="" and="" other="" matrices="" are="" reanalyzed="" per="" section="" 18.4.="" 9.4="" recovery="" of="" labeled="" compounds="" from="" samples="" should="" be="" assessed="" and="" records="" should="" be="" maintained.="" 9.4.1="" after="" the="" analysis="" of="" five="" samples="" of="" a="" given="" matrix="" type="" (water,="" soil,="" sludge,="" pulp,="" etc.)="" for="" which="" the="" labeled="" compounds="" pass="" the="" tests="" in="" section="" 9.3,="" compute="" the="" average="" percent="" recovery="" (r)="" and="" the="" standard="" deviation="" of="" the="" percent="" recovery="" (sr)="" for="" the="" labeled="" compounds="" only.="" express="" the="" assessment="" as="" a="" percent="" recovery="" interval="" from="">R to R+2SR for each matrix. For example,
if R = 90% and SR = 10% for five analyses of pulp, the
recovery interval is expressed as 70-110%.
9.4.2 Update the accuracy assessment for each labeled compound in
each matrix on a regular basis (e.g., after each 5-10 new
measurements).
9.5 Method Blanks--Reference matrix method blanks are analyzed to
demonstrate freedom from contamination (Section 4.3).
9.5.1 Prepare, extract, clean up, and concentrate a method blank
with each sample batch (samples of the same matrix started through the
extraction process on the same 12-hour shift, to a maximum of 20
samples). The matrix for the method blank shall be similar to sample
matrix for the batch, e.g., a 1 L reagent water blank (Section 7.6.1),
high-solids reference matrix blank (Section 7.6.2), paper matrix blank
(Section 7.6.3); tissue blank (Section 7.6.4) or alternative reference
matrix blank (Section 7.6.5). Analyze the blank immediately after
analysis of the OPR (Section 15.5) to demonstrate freedom from
contamination.
9.5.2 If any 2,3,7,8-substituted CDD/CDF (Table 1) is found in the
blank at greater than the minimum level (Table 2) or one-third the
regulatory compliance level, whichever is greater; or if any
potentially interfering compound is found in the blank at the minimum
level for each level of chlorination given in Table 2 (assuming a
response factor of 1 relative to the \13\C12-1,2,3,4-TCDD
internal standard for compounds not listed in Table 1), analysis of
samples is halted until the blank associated with the sample batch
shows no evidence of contamination at
[[Page 48413]]
this level. All samples must be associated with an uncontaminated
method blank before the results for those samples may be reported for
regulatory compliance purposes.
9.6 QC Check Sample--Analyze the QC Check Sample (Section 7.16)
periodically to assure the accuracy of calibration standards and the
overall reliability of the analytical process. It is suggested that the
QC Check Sample be analyzed at least quarterly.
9.7 The specifications contained in this method can be met if the
apparatus used is calibrated properly and then maintained in a
calibrated state. The standards used for calibration (Section 10),
calibration verification (Section 15.3), and for initial (Section 9.2)
and ongoing (Section 15.5) precision and recovery should be identical,
so that the most precise results will be obtained. A GC/MS instrument
will provide the most reproducible results if dedicated to the settings
and conditions required for the analyses of CDDs/CDFs by this method.
9.8 Depending on specific program requirements, field replicates
may be collected to determine the precision of the sampling technique,
and spiked samples may be required to determine the accuracy of the
analysis when the internal standard method is used.
10.0 Calibration
10.1 Establish the operating conditions necessary to meet the
minimum retention times for the internal standards in Section 10.2.4
and the relative retention times for the CDDs/CDFs in Table 2.
10.1.1 Suggested GC operating conditions:
Injector temperature: 270 deg.C
Interface temperature: 290 deg.C
Initial temperature: 200 deg.C
Initial time: Two minutes
Temperature program:
200-220 deg.C, at 5 deg.C/minute
220 deg.C for 16 minutes
220-235 deg.C, at 5 deg.C/minute
235 deg.C for seven minutes
235-330 deg.C, at 5 deg.C/minute
Note: All portions of the column that connect the GC to the ion
source shall remain at or above the interface temperature specified
above during analysis to preclude condensation of less volatile
compounds.
Optimize GC conditions for compound separation and sensitivity.
Once optimized, the same GC conditions must be used for the analysis of
all standards, blanks, IPR and OPR aliquots, and samples.
10.1.2 Mass spectrometer (MS) resolution--Obtain a selected ion
current profile (SICP) of each analyte in Table 3 at the two exact m/
z's specified in Table 8 and at 10,000 resolving power by
injecting an authentic standard of the CDDs/CDFs either singly or as
part of a mixture in which there is no interference between closely
eluted components.
10.1.2.1 The analysis time for CDDs/CDFs may exceed the long-term
mass stability of the mass spectrometer. Because the instrument is
operated in the high-resolution mode, mass drifts of a few ppm (e.g., 5
ppm in mass) can have serious adverse effects on instrument
performance. Therefore, a mass-drift correction is mandatory and a
lock-mass m/z from PFK is used for drift correction. The lock-mass m/z
is dependent on the exact m/z's monitored within each descriptor, as
shown in Table 8. The level of PFK metered into the HRMS during
analyses should be adjusted so that the amplitude of the most intense
selected lock-mass m/z signal (regardless of the descriptor number)
does not exceed 10% of the full-scale deflection for a given set of
detector parameters. Under those conditions, sensitivity changes that
might occur during the analysis can be more effectively monitored.
Note: Excessive PFK (or any other reference substance) may cause
noise problems and contamination of the ion source necessitating
increased frequency of source cleaning.
10.1.2.2 If the HRMS has the capability to monitor resolution
during the analysis, it is acceptable to terminate the analysis when
the resolution falls below 10,000 to save reanalysis time.
10.1.2.3 Using a PFK molecular leak, tune the instrument to meet
the minimum required resolving power of 10,000 (10% valley) at m/z
304.9824 (PFK) or any other reference signal close to m/z 304 (from
TCDF). For each descriptor (Table 8), monitor and record the resolution
and exact m/z's of three to five reference peaks covering the mass
range of the descriptor. The resolution must be greater than or equal
to 10,000, and the deviation between the exact m/z and the theoretical
m/z (Table 8) for each exact m/z monitored must be less than 5 ppm.
10.2 Ion Abundance Ratios, Minimum Levels, Signal-to-Noise Ratios,
and Absolute Retention Times--Choose an injection volume of either 1
L or 2 L, consistent with the capability of the HRGC/
HRMS instrument. Inject a 1 L or 2 L aliquot of the
CS1 calibration solution (Table 4) using the GC conditions from Section
10.1.1. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the
operating conditions and specifications below apply to analysis of
those compounds only.
10.2.1 Measure the SICP areas for each analyte, and compute the
ion abundance ratios at the exact m/z's specified in Table 8. Compare
the computed ratio to the theoretical ratio given in Table 9.
10.2.1.1 The exact m/z's to be monitored in each descriptor are
shown in Table 8. Each group or descriptor shall be monitored in
succession as a function of GC retention time to ensure that all CDDs/
CDFs are detected. Additional m/z's may be monitored in each
descriptor, and the m/z's may be divided among more than the five
descriptors listed in Table 8, provided that the laboratory is able to
monitor the m/z's of all the CDDs/CDFs that may elute from the GC in a
given retention-time window. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are
to be determined, the descriptors may be modified to include only the
exact m/z's for the tetra-and penta-isomers, the diphenyl ethers, and
the lock m/z's.
10.2.1.2 The mass spectrometer shall be operated in a mass-drift
correction mode, using perfluorokerosene (PFK) to provide lock m/z's.
The lock-mass for each group of m/z's is shown in Table 8. Each lock
mass shall be monitored and shall not vary by more than 20%
throughout its respective retention time window. Variations of the lock
mass by more than 20% indicate the presence of coeluting interferences
that may significantly reduce the sensitivity of the mass spectrometer.
Reinjection of another aliquot of the sample extract will not resolve
the problem. Additional cleanup of the extract may be required to
remove the interferences.
10.2.2 All CDDs/CDFs and labeled compounds in the CS1 standard
shall be within the QC limits in Table 9 for their respective ion
abundance ratios; otherwise, the mass spectrometer shall be adjusted
and this test repeated until the m/z ratios fall within the limits
specified. If the adjustment alters the resolution of the mass
spectrometer, resolution shall be verified (Section 10.1.2) prior to
repeat of the test.
10.2.3 Verify that the HRGC/HRMS instrument meets the minimum
levels in Table 2. The peaks representing the CDDs/CDFs and labeled
compounds in the CS1 calibration standard must have signal-to-noise
ratios (S/N) greater than or equal to 10.0. Otherwise, the mass
spectrometer shall be adjusted and this test repeated until the minimum
levels in Table 2 are met.
10.2.4 The absolute retention time of 13C12-
1,2,3,4-TCDD (Section 7.12) shall exceed 25.0 minutes on the DB-5
column, and the retention time of 13C12-
[[Page 48414]]
1,2,3,4-TCDD shall exceed 15.0 minutes on the DB-225 column; otherwise,
the GC temperature program shall be adjusted and this test repeated
until the above-stated minimum retention time criteria are met.
10.3 Retention-Time Windows--Analyze the window defining mixtures
(Section 7.15) using the optimized temperature program in Section
10.1. Table 5 gives the elution order (first/last) of the window-
defining compounds. If 2,3,7,8-TCDD and 2,3,7,8-TCDF only are to be
analyzed, this test is not required.
10.4 Isomer Specificity.
10.4.1 Analyze the isomer specificity test standards (Section
7.15) using the procedure in Section 14 and the optimized conditions
for sample analysis (Section 10.1.1).
10.4.2 Compute the percent valley between the GC peaks that elute
most closely to the 2,3,7,8-TCDD and TCDF isomers, on their respective
columns, per Figures 6 and 7.
10.4.3 Verify that the height of the valley between the most
closely eluted isomers and the 2,3,7,8-substituted isomers is less than
25% (computed as 100 x/y in Figures 6 and 7). If the valley exceeds
25%, adjust the analytical conditions and repeat the test or replace
the GC column and recalibrate (Sections 10.1.2 through 10.7).
10.5 Calibration by Isotope Dilution--Isotope dilution calibration
is used for the 15 2,3,7,8-substituted CDDs/CDFs for which labeled
compounds are added to samples prior to extraction. The reference
compound for each CDD/CDF compound is shown in Table 2.
10.5.1 A calibration curve encompassing the concentration range is
prepared for each compound to be determined. The relative response (RR)
(labeled to native) vs. concentration in standard solutions is plotted
or computed using a linear regression. Relative response is determined
according to the procedures described below. Five calibration points
are employed.
10.5.2 The response of each CDD/CDF relative to its labeled analog
is determined using the area responses of both the primary and
secondary exact m/z's specified in Table 8, for each calibration
standard, as follows:
[GRAPHIC] [TIFF OMITTED] TR15SE97.002
Where:
A1n and A2n = The areas of the primary and
secondary m/z's for the CDD/CDF.
A1l and A2l = The areas of the primary and
secondary m/z's for the labeled compound.
Cl = The concentration of the labeled compound in the
calibration standard (Table 4).
Cn = The concentration of the native compound in the
calibration standard (Table 4).
10.5.3 To calibrate the analytical system by isotope dilution,
inject a volume of calibration standards CS1 through CS5 (Section 7.13
and Table 4) identical to the volume chosen in Section 10.2, using the
procedure in Section 14 and the conditions in Section 10.1.1 and Table
2. Compute the relative response (RR) at each concentration.
10.5.4 Linearity--If the relative response for any compound is
constant (less than 20% coefficient of variation) over the five-point
calibration range, an averaged relative response may be used for that
compound; otherwise, the complete calibration curve for that compound
shall be used over the five-point calibration range.
10.6 Calibration by Internal Standard--The internal standard
method is applied to determination of 1,2,3,7,8,9-HxCDD (Section
17.1.2), OCDF (Section 17.1.1), the non 2,3,7,8-substituted compounds,
and to the determination of labeled compounds for intralaboratory
statistics (Sections 9.4 and 15.5.4).
10.6.1 Response factors--Calibration requires the determination of
response factors (RF) defined by the following equation:
[GRAPHIC] [TIFF OMITTED] TR15SE97.003
Where:
A1s and A2s = The areas of the primary and
secondary m/z's for the CDD/CDF.
A1is and A2is = The areas of the primary and
secondary m/z's for the internal standard.
Cis = The concentration of the internal standard (Table 4).
Cs = The concentration of the compound in the calibration
standard (Table 4).
Note: There is only one m/z for 37Cl4-
2,3,7,8-TCDD. See Table 8.
10.6.2 To calibrate the analytical system by internal standard,
inject 1.0 L or 2.0 L of calibration standards CS1
through CS5 (Section 7.13 and Table 4) using the procedure in Section
14 and the conditions in Section 10.1.1 and Table 2. Compute the
response factor (RF) at each concentration.
10.6.3 Linearity--If the response factor (RF) for any compound is
constant (less than 35% coefficient of variation) over the five-point
calibration range, an averaged response factor may be used for that
compound; otherwise, the complete calibration curve for that compound
shall be used over the five-point range.
10.7 Combined Calibration--By using calibration solutions (Section
7.13 and Table 4) containing the CDDs/CDFs and labeled compounds and
the internal standards, a single set of analyses can be used to produce
calibration curves for the isotope dilution and internal standard
methods. These curves are verified each shift (Section 15.3) by
analyzing the calibration verification standard (VER, Table 4).
Recalibration is required if any of the calibration verification
criteria (Section 15.3) cannot be met.
10.8 Data Storage--MS data shall be collected, recorded, and
stored.
10.8.1 Data acquisition--The signal at each exact m/z shall be
collected repetitively throughout the monitoring period and stored on a
mass storage device.
10.8.2 Response factors and multipoint calibrations--The data
system shall be used to record and maintain lists of response factors
(response ratios for isotope dilution) and multipoint calibration
curves. Computations of relative standard deviation (coefficient of
variation) shall be used to test calibration linearity. Statistics on
initial performance (Section 9.2) and ongoing performance (Section
15.5) should be computed and maintained, either on the instrument data
system, or on a separate computer system.
11.0 Sample Preparation
11.1 Sample preparation involves modifying the physical form of
the sample so that the CDDs/CDFs can be extracted efficiently. In
general, the samples must be in a liquid form or in the form of finely
divided solids in order for efficient extraction to take place. Table
10 lists the phases and suggested quantities for extraction of various
sample matrices.
For samples known or expected to contain high levels of the CDDs/
CDFs, the smallest sample size representative of the entire sample
should be used (see Section 17.5).
For all samples, the blank and IPR/OPR aliquots must be processed
through the same steps as the sample to check for contamination and
losses in the preparation processes.
11.1.1 For samples that contain particles, percent solids and
particle size are determined using the procedures in Sections 11.2 and
11.3, respectively.
11.1.2 Aqueous samples--Because CDDs/CDFs may be bound to
suspended
[[Page 48415]]
particles, the preparation of aqueous samples is dependent on the
solids content of the sample.
11.1.2.1 Aqueous samples visibly absent particles are prepared per
Section 11.4 and extracted directly using the separatory funnel or SPE
techniques in Sections 12.1 or 12.2, respectively.
11.1.2.2 Aqueous samples containing visible particles and
containing one percent suspended solids or less are prepared using the
procedure in Section 11.4. After preparation, the sample is extracted
directly using the SPE technique in 12.2 or filtered per Section
11.4.3. After filtration, the particles and filter are extracted using
the SDS procedure in Section 12.3 and the filtrate is extracted using
the separatory funnel procedure in Section 12.1.
11.1.2.3 For aqueous samples containing greater than one percent
solids, a sample aliquot sufficient to provide 10 g of dry solids is
used, as described in Section 11.5.
11.1.3 Solid samples are prepared using the procedure described in
Section 11.5 followed by extraction via the SDS procedure in Section
12.3.
11.1.4 Multiphase samples--The phase(s) containing the CDDs/CDFs
is separated from the non-CDD/CDF phase using pressure filtration and
centrifugation, as described in Section 11.6. The CDDs/CDFs will be in
the organic phase in a multiphase sample in which an organic phase
exists.
11.1.5 Procedures for grinding, homogenization, and blending of
various sample phases are given in Section 11.7.
11.1.6 Tissue samples--Preparation procedures for fish and other
tissues are given in Section 11.8.
11.2 Determination of Percent Suspended Solids.
Note: This aliquot is used for determining the solids content of
the sample, not for determination of CDDs/CDFs.
11.2.1 Aqueous liquids and multi-phase samples consisting of
mainly an aqueous phase.
11.2.1.1 Dessicate and weigh a GF/D filter (Section 6.5.3) to
three significant figures.
[GRAPHIC] [TIFF OMITTED] TR15SE97.004
11.2.1.2 Filter 10.00.02 mL of well-mixed sample
through the filter.
11.2.1.3 Dry the filter a minimum of 12 hours at
1105\
.
11.2.1.4 Calculate percent solids as follows:
[GRAPHIC] [TIFF OMITTED] TR15SE97.004
11.2.2 Non-aqueous liquids, solids, semi-solid samples, and multi-
phase samples in which the main phase is not aqueous; but not tissues.
11.2.2.1 Weigh 5-10 g of sample to three significant figures in a
tared beaker.
11.2.2.2 Dry a minimum of 12 hours at 1105 deg.C, and
cool in a dessicator.
11.2.2.3 Calculate percent solids as follows:
[GRAPHIC] [TIFF OMITTED] TR15SE97.005
11.3 Determination of Particle Size.
11.3.1 Spread the dried sample from Section 11.2.2.2 on a piece of
filter paper or aluminum foil in a fume hood or glove box.
11.3.2 Estimate the size of the particles in the sample. If the
size of the largest particles is greater than 1 mm, the particle size
must be reduced to 1 mm or less prior to extraction using the
procedures in Section 11.7.
11.4 Preparation of Aqueous Samples Containing 1% Suspended Solids
or Less.
11.4.1 Aqueous samples visibly absent particles are prepared per
the procedure below and extracted directly using the separatory funnel
or SPE techniques in Sections 12.1 or 12.2, respectively. Aqueous
samples containing visible particles and one percent suspended solids
or less are prepared using the procedure below and extracted using
either the SPE technique in Section 12.2 or further prepared using the
filtration procedure in Section 11.4.3. The filtration procedure is
followed by SDS extraction of the filter and particles (Section 12.3)
and separatory funnel extraction of the filtrate (Section 12.1). The
SPE procedure is followed by SDS extraction of the filter and disk.
11.4.2 Preparation of sample and QC aliquots.
11.4.2.1 Mark the original level of the sample on the sample
bottle for reference. Weigh the sample plus bottle to 1.
11.4.2.2 Spike 1.0 mL of the diluted labeled-compound spiking
solution (Section 7.10.3) into the sample bottle. Cap the bottle and
mix the sample by careful shaking. Allow the sample to equilibrate for
one to two hours, with occasional shaking.
11.4.2.3 For each sample or sample batch (to a maximum of 20
samples) to be extracted during the same 12-hour shift, place two 1.0 L
aliquots of reagent water in clean sample bottles or flasks.
11.4.2.4 Spike 1.0 mL of the diluted labeled-compound spiking
solution (Section 7.10.3) into both reagent water aliquots. One of
these aliquots will serve as the method blank.
11.4.2.5 Spike 1.0 mL of the PAR standard (Section 7.14) into the
remaining reagent water aliquot. This aliquot will serve as the OPR
(Section 15.5).
11.4.2.6 If SPE is to be used, add 5 mL of methanol to the sample,
cap and shake the sample to mix thoroughly, and proceed to Section 12.2
for extraction. If SPE is not to be used, and the sample is visibly
absent particles, proceed to Section 12.1 for extraction. If SPE is not
to be used and the sample
[[Page 48416]]
contains visible particles, proceed to the following section for
filtration of particles.
11.4.3 Filtration of particles.
11.4.3.1 Assemble a Buchner funnel (Section 6.5.5) on top of a
clean filtration flask. Apply vacuum to the flask, and pour the entire
contents of the sample bottle through a glass-fiber filter (Section
6.5.6) in the Buchner funnel, swirling the sample remaining in the
bottle to suspend any particles.
11.4.3.2 Rinse the sample bottle twice with approximately 5 mL
portions of reagent water to transfer any remaining particles onto the
filter.
11.4.3.3 Rinse any particles off the sides of the Buchner funnel
with small quantities of reagent water.
11.4.3.4 Weigh the empty sample bottle to 1 g.
Determine the weight of the sample by difference. Save the bottle for
further use.
11.4.3.5 Extract the filtrate using the separatory funnel
procedure in Section 12.1.
11.4.3.6 Extract the filter containing the particles using the SDS
procedure in Section 12.3.
11.5 Preparation of Samples Containing Greater Than 1% Solids.
11.5.1 Weigh a well-mixed aliquot of each sample (of the same
matrix type) sufficient to provide 10 g of dry solids (based on the
solids determination in Section 11.2) into a clean beaker or glass jar.
11.5.2 Spike 1.0 mL of the diluted labeled compound spiking
solution (Section 7.10.3) into the sample.
11.5.3 For each sample or sample batch (to a maximum of 20
samples) to be extracted during the same 12-hour shift, weigh two 10 g
aliquots of the appropriate reference matrix (Section 7.6) into clean
beakers or glass jars.
11.5.4 Spike 1.0 mL of the diluted labeled compound spiking
solution (Section 7.10.3) into each reference matrix aliquot. One
aliquot will serve as the method blank. Spike 1.0 mL of the PAR
standard (Section 7.14) into the other reference matrix aliquot. This
aliquot will serve as the OPR (Section 15.5).
11.5.5 Stir or tumble and equilibrate the aliquots for one to two
hours.
11.5.6 Decant excess water. If necessary to remove water, filter
the sample through a glass-fiber filter and discard the aqueous liquid.
11.5.7 If particles >1mm are present in the sample (as determined
in Section 11.3.2), spread the sample on clean aluminum foil in a hood.
After the sample is dry, grind to reduce the particle size (Section
11.7).
11.5.8 Extract the sample and QC aliquots using the SDS procedure
in Section 12.3.
11.6 Multiphase Samples.
11.6.1 Using the percent solids determined in Section 11.2.1 or
11.2.2, determine the volume of sample that will provide 10 g of
solids, up to 1 L of sample.
11.6.2 Pressure filter the amount of sample determined in Section
11.6.1 through Whatman GF/D glass-fiber filter paper (Section 6.5.3).
Pressure filter the blank and OPR aliquots through GF/D papers also. If
necessary to separate the phases and/or settle the solids, centrifuge
these aliquots prior to filtration.
11.6.3 Discard any aqueous phase (if present). Remove any non-
aqueous liquid present and reserve the maximum amount filtered from the
sample (Section 11.6.1) or 10 g, whichever is less, for combination
with the solid phase (Section 12.3.5).
11.6.4 If particles >1mm are present in the sample (as determined
in Section 11.3.2) and the sample is capable of being dried, spread the
sample and QC aliquots on clean aluminum foil in a hood. After the
aliquots are dry or if the sample cannot be dried, reduce the particle
size using the procedures in Section 11.7 and extract the reduced
particles using the SDS procedure in Section 12.3. If particles >1mm
are not present, extract the particles and filter in the sample and QC
aliquots directly using the SDS procedure in Section 12.3.
11.7 Sample grinding, homogenization, or blending--Samples with
particle sizes greater than 1 mm (as determined in Section 11.3.2) are
subjected to grinding, homogenization, or blending. The method of
reducing particle size to less than 1 mm is matrix-dependent. In
general, hard particles can be reduced by grinding with a mortar and
pestle. Softer particles can be reduced by grinding in a Wiley mill or
meat grinder, by homogenization, or in a blender.
11.7.1 Each size-reducing preparation procedure on each matrix
shall be verified by running the tests in Section 9.2 before the
procedure is employed routinely.
11.7.2 The grinding, homogenization, or blending procedures shall
be carried out in a glove box or fume hood to prevent particles from
contaminating the work environment.
11.7.3 Grinding--Certain papers and pulps, slurries, and amorphous
solids can be ground in a Wiley mill or heavy duty meat grinder. In
some cases, reducing the temperature of the sample to freezing or to
dry ice or liquid nitrogen temperatures can aid in the grinding
process. Grind the sample aliquots from Section 11.5.7 or 11.6.4 in a
clean grinder. Do not allow the sample temperature to exceed 50 deg.C.
Grind the blank and reference matrix aliquots using a clean grinder.
11.7.4 Homogenization or blending--Particles that are not ground
effectively, or particles greater than 1 mm in size after grinding, can
often be reduced in size by high speed homogenization or blending.
Homogenize and/or blend the particles or filter from Section 11.5.7 or
11.6.4 for the sample, blank, and OPR aliquots.
11.7.5 Extract the aliquots using the SDS procedure in Section
12.3.
11.8 Fish and Other Tissues--Prior to processing tissue samples,
the laboratory must determine the exact tissue to be analyzed. Common
requests for analysis of fish tissue include whole fish--skin on, whole
fish--skin removed, edible fish fillets (filleted in the field or by
the laboratory), specific organs, and other portions. Once the
appropriate tissue has been determined, the sample must be homogenized.
11.8.1 Homogenization.
11.8.1.1 Samples are homogenized while still frozen, where
practical. If the laboratory must dissect the whole fish to obtain the
appropriate tissue for analysis, the unused tissues may be rapidly
refrozen and stored in a clean glass jar for subsequent use.
11.8.1.2 Each analysis requires 10 g of tissue (wet weight).
Therefore, the laboratory should homogenize at least 20 g of tissue to
allow for re-extraction of a second aliquot of the same homogenized
sample, if re-analysis is required. When whole fish analysis is
necessary, the entire fish is homogenized.
11.8.1.3 Homogenize the sample in a tissue homogenizer (Section
6.3.3) or grind in a meat grinder (Section 6.3.4). Cut tissue too large
to feed into the grinder into smaller pieces. To assure homogeneity,
grind three times.
11.8.1.4 Transfer approximately 10 g (wet weight) of homogenized
tissue to a clean, tared, 400-500 mL beaker. For the alternate HCl
digestion/extraction, transfer the tissue to a clean, tared 500-600 mL
wide-mouth bottle. Record the weight to the nearest 10 mg.
11.8.1.5 Transfer the remaining homogenized tissue to a clean jar
with a fluoropolymer-lined lid. Seal the jar and store the tissue at
<-10 deg.c.="" return="" any="" tissue="" that="" was="" not="" homogenized="" to="" its="" original="" container="" and="" store="" at=""><-10 deg.c.="" 11.8.2="" qc="" aliquots.="" 11.8.2.1="" prepare="" a="" method="" blank="" by="" adding="" approximately="" 10="" g="" of="" the="" oily="" liquid="" reference="" matrix="" (section="" 7.6.4)="" to="" a="" 400-500="" ml="" beaker.="" for="" the="" alternate="" hcl="" digestion/extraction,="" add="" the="" [[page="" 48417]]="" reference="" matrix="" to="" a="" 500-600="" ml="" wide-mouth="" bottle.="" record="" the="" weight="" to="" the="" nearest="" 10="" mg.="" 11.8.2.2="" prepare="" a="" precision="" and="" recovery="" aliquot="" by="" adding="" approximately="" 10="" g="" of="" the="" oily="" liquid="" reference="" matrix="" (section="" 7.6.4)="" to="" a="" separate="" 400-500="" ml="" beaker="" or="" wide-mouth="" bottle,="" depending="" on="" the="" extraction="" procedure="" to="" be="" used.="" record="" the="" weight="" to="" the="" nearest="" 10="" mg.="" if="" the="" initial="" precision="" and="" recovery="" test="" is="" to="" be="" performed,="" use="" four="" aliquots;="" if="" the="" ongoing="" precision="" and="" recovery="" test="" is="" to="" be="" performed,="" use="" a="" single="" aliquot.="" 11.8.3="" spiking="" 11.8.3.1="" spike="" 1.0="" ml="" of="" the="" labeled="" compound="" spiking="" solution="" (section="" 7.10.3)="" into="" the="" sample,="" blank,="" and="" opr="" aliquot.="" 11.8.3.2="" spike="" 1.0="" ml="" of="" the="" par="" standard="" (section="" 7.14)="" into="" the="" opr="" aliquot.="" 11.8.4="" extract="" the="" aliquots="" using="" the="" procedures="" in="" section="" 12.4.="" 12.0="" extraction="" and="" concentration="" extraction="" procedures="" include="" separatory="" funnel="" (section="" 12.1)="" and="" solid="" phase="" (section="" 12.2)="" for="" aqueous="" liquids;="" soxhlet/dean-stark="" (section="" 12.3)="" for="" solids,="" filters,="" and="" spe="" disks;="" and="" soxhlet="" extraction="" (section="" 12.4.1)="" and="" hcl="" digestion="" (section="" 12.4.2)="" for="" tissues.="" acid/base="" back-extraction="" (section="" 12.5)="" is="" used="" for="" initial="" cleanup="" of="" extracts.="" macro-concentration="" procedures="" include="" rotary="" evaporation="" (section="" 12.6.1),="" heating="" mantle="" (section="" 12.6.2),="" and="" kuderna-danish="" (k-d)="" evaporation="" (section="" 12.6.3).="" micro-concentration="" uses="" nitrogen="" blowdown="" (section="" 12.7).="" 12.1="" separatory="" funnel="" extraction="" of="" filtrates="" and="" of="" aqueous="" samples="" visibly="" absent="" particles.="" 12.1.1="" pour="" the="" spiked="" sample="" (section="" 11.4.2.2)="" or="" filtrate="" (section="" 11.4.3.5)="" into="" a="" 2="" l="" separatory="" funnel.="" rinse="" the="" bottle="" or="" flask="" twice="" with="" 5="" ml="" of="" reagent="" water="" and="" add="" these="" rinses="" to="" the="" separatory="" funnel.="" 12.1.2="" add="" 60="" ml="" methylene="" chloride="" to="" the="" empty="" sample="" bottle="" (section="" 12.1.1),="" seal,="" and="" shake="" 60="" seconds="" to="" rinse="" the="" inner="" surface.="" transfer="" the="" solvent="" to="" the="" separatory="" funnel,="" and="" extract="" the="" sample="" by="" shaking="" the="" funnel="" for="" two="" minutes="" with="" periodic="" venting.="" allow="" the="" organic="" layer="" to="" separate="" from="" the="" aqueous="" phase="" for="" a="" minimum="" of="" 10="" minutes.="" if="" an="" emulsion="" forms="" and="" is="" more="" than="" one-third="" the="" volume="" of="" the="" solvent="" layer,="" employ="" mechanical="" techniques="" to="" complete="" the="" phase="" separation="" (see="" note="" below).="" drain="" the="" methylene="" chloride="" extract="" through="" a="" solvent-rinsed="" glass="" funnel="" approximately="" one-half="" full="" of="" granular="" anhydrous="" sodium="" sulfate="" (section="" 7.2.1)="" supported="" on="" clean="" glass-fiber="" paper="" into="" a="" solvent-rinsed="" concentration="" device="" (section="" 12.6).="" note:="" if="" an="" emulsion="" forms,="" the="" analyst="" must="" employ="" mechanical="" techniques="" to="" complete="" the="" phase="" separation.="" the="" optimum="" technique="" depends="" upon="" the="" sample,="" but="" may="" include="" stirring,="" filtration="" through="" glass="" wool,="" use="" of="" phase="" separation="" paper,="" centrifugation,="" use="" of="" an="" ultrasonic="" bath="" with="" ice,="" addition="" of="" nacl,="" or="" other="" physical="" methods.="" alternatively,="" solid-phase="" or="" other="" extraction="" techniques="" may="" be="" used="" to="" prevent="" emulsion="" formation.="" any="" alternative="" technique="" is="" acceptable="" so="" long="" as="" the="" requirements="" in="" section="" 9="" are="" met.="" experience="" with="" aqueous="" samples="" high="" in="" dissolved="" organic="" materials="" (e.g.,="" paper="" mill="" effluents)="" has="" shown="" that="" acidification="" of="" the="" sample="" prior="" to="" extraction="" may="" reduce="" the="" formation="" of="" emulsions.="" paper="" industry="" methods="" suggest="" that="" the="" addition="" of="" up="" to="" 400="" ml="" of="" ethanol="" to="" a="" 1="" l="" effluent="" sample="" may="" also="" reduce="" emulsion="" formation.="" however,="" studies="" by="" epa="" suggest="" that="" the="" effect="" may="" be="" a="" result="" of="" sample="" dilution,="" and="" that="" the="" addition="" of="" reagent="" water="" may="" serve="" the="" same="" function.="" mechanical="" techniques="" may="" still="" be="" necessary="" to="" complete="" the="" phase="" separation.="" if="" either="" acidification="" or="" addition="" of="" ethanol="" is="" utilized,="" the="" laboratory="" must="" perform="" the="" startup="" tests="" described="" in="" section="" 9.2="" using="" the="" same="" techniques.="" 12.1.3="" extract="" the="" water="" sample="" two="" more="" times="" with="" 60="" ml="" portions="" of="" methylene="" chloride.="" drain="" each="" portion="" through="" the="" sodium="" sulfate="" into="" the="" concentrator.="" after="" the="" third="" extraction,="" rinse="" the="" separatory="" funnel="" with="" at="" least="" 20="" ml="" of="" methylene="" chloride,="" and="" drain="" this="" rinse="" through="" the="" sodium="" sulfate="" into="" the="" concentrator.="" repeat="" this="" rinse="" at="" least="" twice.="" set="" aside="" the="" funnel="" with="" sodium="" sulfate="" if="" the="" extract="" is="" to="" be="" combined="" with="" the="" extract="" from="" the="" particles.="" 12.1.4="" concentrate="" the="" extract="" using="" one="" of="" the="" macro-="" concentration="" procedures="" in="" section="" 12.6.="" 12.1.4.1="" if="" the="" extract="" is="" from="" a="" sample="" visibly="" absent="" particles="" (section="" 11.1.2.1),="" adjust="" the="" final="" volume="" of="" the="" concentrated="" extract="" to="" approximately="" 10="" ml="" with="" hexane,="" transfer="" to="" a="" 250="" ml="" separatory="" funnel,="" and="" back-extract="" using="" the="" procedure="" in="" section="" 12.5.="" 12.1.4.2="" if="" the="" extract="" is="" from="" the="" aqueous="" filtrate="" (section="" 11.4.3.5),="" set="" aside="" the="" concentration="" apparatus="" for="" addition="" of="" the="" sds="" extract="" from="" the="" particles="" (section="" 12.3.9.1.2).="" 12.2="" spe="" of="" samples="" containing="" less="" than="" 1%="" solids="" (references="" 19-="" 20).="" 12.2.1="" disk="" preparation.="" 12.2.1.1="" place="" an="" spe="" disk="" on="" the="" base="" of="" the="" filter="" holder="" (figure="" 4)="" and="" wet="" with="" toluene.="" while="" holding="" a="" gmf="" 150="" filter="" above="" the="" spe="" disk="" with="" tweezers,="" wet="" the="" filter="" with="" toluene="" and="" lay="" the="" filter="" on="" the="" spe="" disk,="" making="" sure="" that="" air="" is="" not="" trapped="" between="" the="" filter="" and="" disk.="" clamp="" the="" filter="" and="" spe="" disk="" between="" the="" 1="" l="" glass="" reservoir="" and="" the="" vacuum="" filtration="" flask.="" 12.2.1.2="" rinse="" the="" sides="" of="" the="" filtration="" flask="" with="" approx="" 15="" ml="" of="" toluene="" using="" a="" squeeze="" bottle="" or="" syringe.="" apply="" vacuum="" momentarily="" until="" a="" few="" drops="" appear="" at="" the="" drip="" tip.="" release="" the="" vacuum="" and="" allow="" the="" filter/disk="" to="" soak="" for="" approx="" one="" minute.="" apply="" vacuum="" and="" draw="" all="" of="" the="" toluene="" through="" the="" filter/disk.="" repeat="" the="" wash="" step="" with="" approx="" 15="" ml="" of="" acetone="" and="" allow="" the="" filter/disk="" to="" air="" dry.="" 12.2.1.3="" re-wet="" the="" filter/disk="" with="" approximately="" 15="" ml="" of="" methanol,="" allowing="" the="" filter/disk="" to="" soak="" for="" approximately="" one="" minute.="" pull="" the="" methanol="" through="" the="" filter/disk="" using="" the="" vacuum,="" but="" retain="" a="" layer="" of="" methanol="" approximately="" 1="" mm="" thick="" on="" the="" filter.="" do="" not="" allow="" the="" disk="" to="" go="" dry="" from="" this="" point="" until="" the="" end="" of="" the="" extraction.="" 12.2.1.4="" rinse="" the="" filter/disk="" with="" two="" 50-ml="" portions="" of="" reagent="" water="" by="" adding="" the="" water="" to="" the="" reservoir="" and="" pulling="" most="" through,="" leaving="" a="" layer="" of="" water="" on="" the="" surface="" of="" the="" filter.="" 12.2.2="" extraction.="" 12.2.2.1="" pour="" the="" spiked="" sample="" (section="" 11.4.2.2),="" blank="" (section="" 11.4.2.4),="" or="" ipr/opr="" aliquot="" (section="" 11.4.2.5)="" into="" the="" reservoir="" and="" turn="" on="" the="" vacuum="" to="" begin="" the="" extraction.="" adjust="" the="" vacuum="" to="" complete="" the="" extraction="" in="" no="" less="" than="" 10="" minutes.="" for="" samples="" containing="" a="" high="" concentration="" of="" particles="" (suspended="" solids),="" filtration="" times="" may="" be="" eight="" hours="" or="" longer.="" 12.2.2.2="" before="" all="" of="" the="" sample="" has="" been="" pulled="" through="" the="" filter/disk,="" rinse="" the="" sample="" bottle="" with="" approximately="" 50="" ml="" of="" reagent="" water="" to="" remove="" any="" solids,="" and="" pour="" into="" the="" reservoir.="" pull="" through="" the="" filter/disk.="" use="" additional="" reagent="" water="" rinses="" until="" all="" visible="" solids="" are="" removed.="" 12.2.2.3="" before="" all="" of="" the="" sample="" and="" rinses="" have="" been="" pulled="" through="" the="" filter/disk,="" rinse="" the="" sides="" of="" the="" reservoir="" with="" small="" portions="" of="" reagent="" water.="" 12.2.2.4="" allow="" the="" filter/disk="" to="" dry,="" then="" remove="" the="" filter="" and="" disk="" and="" place="" in="" a="" glass="" petri="" dish.="" extract="" the="" filter="" and="" disk="" per="" section="" 12.3.="" 12.3="" sds="" extraction="" of="" samples="" containing="" particles,="" and="" of="" filters="" and/or="" disks.="" [[page="" 48418]]="" 12.3.1="" charge="" a="" clean="" extraction="" thimble="" (section="" 6.4.2.2)="" with="" 5.0="" g="" of="" 100/200="" mesh="" silica="" (section="" 7.5.1.1)="" topped="" with="" 100="" g="" of="" quartz="" sand="" (section="" 7.3.2).="" note:="" do="" not="" disturb="" the="" silica="" layer="" throughout="" the="" extraction="" process.="" 12.3.2="" place="" the="" thimble="" in="" a="" clean="" extractor.="" place="" 30-40="" ml="" of="" toluene="" in="" the="" receiver="" and="" 200-250="" ml="" of="" toluene="" in="" the="" flask.="" 12.3.3="" pre-extract="" the="" glassware="" by="" heating="" the="" flask="" until="" the="" toluene="" is="" boiling.="" when="" properly="" adjusted,="" one="" to="" two="" drops="" of="" toluene="" will="" fall="" per="" second="" from="" the="" condenser="" tip="" into="" the="" receiver.="" extract="" the="" apparatus="" for="" a="" minimum="" of="" three="" hours.="" 12.3.4="" after="" pre-extraction,="" cool="" and="" disassemble="" the="" apparatus.="" rinse="" the="" thimble="" with="" toluene="" and="" allow="" to="" air="" dry.="" 12.3.5="" load="" the="" wet="" sample,="" filter,="" and/or="" disk="" from="" section="" 11.4.3.6,="" 11.5.8,="" 11.6.4,="" 11.7.3,="" 11.7.4,="" or="" 12.2.2.4="" and="" any="" nonaqueous="" liquid="" from="" section="" 11.6.3="" into="" the="" thimble="" and="" manually="" mix="" into="" the="" sand="" layer="" with="" a="" clean="" metal="" spatula,="" carefully="" breaking="" up="" any="" large="" lumps="" of="" sample.="" 12.3.6="" reassemble="" the="" pre-extracted="" sds="" apparatus,="" and="" add="" a="" fresh="" charge="" of="" toluene="" to="" the="" receiver="" and="" reflux="" flask.="" apply="" power="" to="" the="" heating="" mantle="" to="" begin="" refluxing.="" adjust="" the="" reflux="" rate="" to="" match="" the="" rate="" of="" percolation="" through="" the="" sand="" and="" silica="" beds="" until="" water="" removal="" lessens="" the="" restriction="" to="" toluene="" flow.="" frequently="" check="" the="" apparatus="" for="" foaming="" during="" the="" first="" two="" hours="" of="" extraction.="" if="" foaming="" occurs,="" reduce="" the="" reflux="" rate="" until="" foaming="" subsides.="" 12.3.7="" drain="" the="" water="" from="" the="" receiver="" at="" one="" to="" two="" hours="" and="" eight="" to="" nine="" hours,="" or="" sooner="" if="" the="" receiver="" fills="" with="" water.="" reflux="" the="" sample="" for="" a="" total="" of="" 16-24="" hours.="" cool="" and="" disassemble="" the="" apparatus.="" record="" the="" total="" volume="" of="" water="" collected.="" 12.3.8="" remove="" the="" distilling="" flask.="" drain="" the="" water="" from="" the="" dean-="" stark="" receiver="" and="" add="" any="" toluene="" in="" the="" receiver="" to="" the="" extract="" in="" the="" flask.="" 12.3.9="" concentrate="" the="" extract="" using="" one="" of="" the="" macro-="" concentration="" procedures="" in="" section="" 12.6="" per="" the="" following:="" 12.3.9.1="" extracts="" from="" the="" particles="" in="" an="" aqueous="" sample="" containing="" less="" than="" 1%="" solids="" (section="" 11.4.3.6).="" 12.3.9.1.1="" concentrate="" the="" extract="" to="" approximately="" 5="" ml="" using="" the="" rotary="" evaporator="" or="" heating="" mantle="" procedures="" in="" section="" 12.6.1="" or="" 12.6.2.="" 12.3.9.1.2="" quantitatively="" transfer="" the="" extract="" through="" the="" sodium="" sulfate="" (section="" 12.1.3)="" into="" the="" apparatus="" that="" was="" set="" aside="" (section="" 12.1.4.2)="" and="" reconcentrate="" to="" the="" level="" of="" the="" toluene.="" 12.3.9.1.3="" adjust="" to="" approximately="" 10="" ml="" with="" hexane,="" transfer="" to="" a="" 250="" ml="" separatory="" funnel,="" and="" proceed="" with="" back-extraction="" (section="" 12.5).="" 12.3.9.2="" extracts="" from="" particles="" (sections="" 11.5="" through="" 11.6)="" or="" from="" the="" spe="" filter="" and="" disk="" (section="" 12.2.2.4)--concentrate="" to="" approximately="" 10="" ml="" using="" the="" rotary="" evaporator="" or="" heating="" mantle="" (section="" 12.6.1="" or="" 12.6.2),="" transfer="" to="" a="" 250="" ml="" separatory="" funnel,="" and="" proceed="" with="" back-extraction="" (section="" 12.5).="" 12.4="" extraction="" of="" tissue--two="" procedures="" are="" provided="" for="" tissue="" extraction.="" 12.4.1="" soxhlet="" extraction="" (reference="" 21).="" 12.4.1.1="" add="" 30-40="" g="" of="" powdered="" anhydrous="" sodium="" sulfate="" to="" each="" of="" the="" beakers="" (section="" 11.8.4)="" and="" mix="" thoroughly.="" cover="" the="" beakers="" with="" aluminum="" foil="" and="" allow="" to="" equilibrate="" for="" 12-24="" hours.="" remix="" prior="" to="" extraction="" to="" prevent="" clumping.="" 12.4.1.2="" assemble="" and="" pre-extract="" the="" soxhlet="" apparatus="" per="" sections="" 12.3.1="" through="" 12.3.4,="" except="" use="" the="" methylene="" chloride:hexane="" (1:1)="" mixture="" for="" the="" pre-extraction="" and="" rinsing="" and="" omit="" the="" quartz="" sand.="" the="" dean-stark="" moisture="" trap="" may="" also="" be="" omitted,="" if="" desired.="" 12.4.1.3="" reassemble="" the="" pre-extracted="" soxhlet="" apparatus="" and="" add="" a="" fresh="" charge="" of="" methylene="" chloride:hexane="" to="" the="" reflux="" flask.="" 12.4.1.4="" transfer="" the="" sample/sodium="" sulfate="" mixture="" (section="" 12.4.1.1)="" to="" the="" soxhlet="" thimble,="" and="" install="" the="" thimble="" in="" the="" soxhlet="" apparatus.="" 12.4.1.5="" rinse="" the="" beaker="" with="" several="" portions="" of="" solvent="" mixture="" and="" add="" to="" the="" thimble.="" fill="" the="" thimble/receiver="" with="" solvent.="" extract="" for="" 18-24="" hours.="" 12.4.1.6="" after="" extraction,="" cool="" and="" disassemble="" the="" apparatus.="" 12.4.1.7="" quantitatively="" transfer="" the="" extract="" to="" a="" macro-="" concentration="" device="" (section="" 12.6),="" and="" concentrate="" to="" near="" dryness.="" set="" aside="" the="" concentration="" apparatus="" for="" re-use.="" 12.4.1.8="" complete="" the="" removal="" of="" the="" solvent="" using="" the="" nitrogen="" blowdown="" procedure="" (section="" 12.7)="" and="" a="" water="" bath="" temperature="" of="" 60="" deg.c.="" weigh="" the="" receiver,="" record="" the="" weight,="" and="" return="" the="" receiver="" to="" the="" blowdown="" apparatus,="" concentrating="" the="" residue="" until="" a="" constant="" weight="" is="" obtained.="" 12.4.1.9="" percent="" lipid="" determination--the="" lipid="" content="" is="" determined="" by="" extraction="" of="" tissue="" with="" the="" same="" solvent="" system="" (methylene="" chloride:hexane)="" that="" was="" used="" in="" epa's="" national="" dioxin="" study="" (reference="" 22)="" so="" that="" lipid="" contents="" are="" consistent="" with="" that="" study.="" 12.4.1.9.1="" redissolve="" the="" residue="" in="" the="" receiver="" in="" hexane="" and="" spike="" 1.0="" ml="" of="" the="" cleanup="" standard="" (section="" 7.11)="" into="" the="" solution.="" 12.4.1.9.2="" transfer="" the="" residue/hexane="" to="" the="" anthropogenic="" isolation="" column="" (section="" 13.7.1)="" or="" bottle="" for="" the="" acidified="" silica="" gel="" batch="" cleanup="" (section="" 13.7.2),="" retaining="" the="" boiling="" chips="" in="" the="" concentration="" apparatus.="" use="" several="" rinses="" to="" assure="" that="" all="" material="" is="" transferred.="" if="" necessary,="" sonicate="" or="" heat="" the="" receiver="" slightly="" to="" assure="" that="" all="" material="" is="" re-dissolved.="" allow="" the="" receiver="" to="" dry.="" weigh="" the="" receiver="" and="" boiling="" chips.="" 12.4.1.9.3="" calculate="" the="" lipid="" content="" to="" the="" nearest="" three="" significant="" figures="" as="" follows:="" [graphic]="" [tiff="" omitted]="" tr15se97.006="" 12.4.1.9.4="" it="" is="" not="" necessary="" to="" determine="" the="" lipid="" content="" of="" the="" blank,="" ipr,="" or="" opr="" aliquots.="" 12.4.2="" hcl="" digestion/extraction="" and="" concentration="" (references="" 23-="" 26).="" 12.4.2.1="" add="" 200="" ml="" of="" 6="" n="" hcl="" and="" 200="" ml="" of="" methylene="" chloride:hexane="" (1:1)="" to="" the="" sample="" and="" qc="" aliquots="" (section="" 11.8.4).="" 12.4.2.2="" cap="" and="" shake="" each="" bottle="" one="" to="" three="" times.="" loosen="" the="" cap="" in="" a="" hood="" to="" vent="" excess="" pressure.="" shake="" each="" bottle="" for="" 10-30="" seconds="" and="" vent.="" 12.4.2.3="" tightly="" cap="" and="" place="" on="" shaker.="" adjust="" the="" shaker="" action="" and="" speed="" so="" that="" the="" acid,="" solvent,="" and="" tissue="" are="" in="" constant="" motion.="" however,="" take="" care="" to="" avoid="" such="" violent="" action="" that="" the="" bottle="" may="" be="" dislodged="" from="" the="" shaker.="" shake="" for="" 12-24="" hours.="" 12.4.2.4="" after="" digestion,="" remove="" the="" bottles="" from="" the="" shaker.="" allow="" the="" bottles="" to="" stand="" so="" that="" the="" solvent="" and="" acid="" layers="" separate.="" 12.4.2.5="" decant="" the="" solvent="" through="" a="" glass="" funnel="" with="" glass-="" fiber="" filter="" (sections="" 6.5.2="" through="" 6.5.3)="" containing="" approximately="" 10="" g="" of="" granular="" anhydrous="" sodium="" sulfate="" (section="" 7.2.1)="" into="" a="" macro-="" concentration="" apparatus="" (section="" 12.6).="" rinse="" the="" contents="" of="" the="" bottle="" with="" two="" 25="" ml="" portions="" of="" hexane="" and="" pour="" through="" the="" sodium="" sulfate="" into="" the="" apparatus.="" 12.4.2.6="" concentrate="" the="" solvent="" to="" near="" dryness="" using="" a="" macro-="" concentration="" procedure="" (section="" 12.6).="" 12.4.2.7="" complete="" the="" removal="" of="" the="" solvent="" using="" the="" nitrogen="" blowdown="" apparatus="" (section="" 12.7)="" and="" a="" water="" bath="" temperature="" of="" 60="" deg.c.="" weigh="" the="" receiver,="" record="" the="" weight,="" and="" return="" the="" receiver="" to="" the="" blowdown="" apparatus,="" concentrating="" the="" residue="" until="" a="" constant="" weight="" is="" obtained.="" [[page="" 48419]]="" 12.4.2.8="" percent="" lipid="" determination--the="" lipid="" content="" is="" determined="" in="" the="" same="" solvent="" system="" [methylene="" chloride:hexane="" (1:1)]="" that="" was="" used="" in="" epa's="" national="" dioxin="" study="" (reference="" 22)="" so="" that="" lipid="" contents="" are="" consistent="" with="" that="" study.="" 12.4.2.8.1="" redissolve="" the="" residue="" in="" the="" receiver="" in="" hexane="" and="" spike="" 1.0="" ml="" of="" the="" cleanup="" standard="" (section="" 7.11)="" into="" the="" solution.="" 12.4.2.8.2="" transfer="" the="" residue/hexane="" to="" the="" narrow-mouth="" 100-200="" ml="" bottle="" retaining="" the="" boiling="" chips="" in="" the="" receiver.="" use="" several="" rinses="" to="" assure="" that="" all="" material="" is="" transferred,="" to="" a="" maximum="" hexane="" volume="" of="" approximately="" 70="" ml.="" allow="" the="" receiver="" to="" dry.="" weigh="" the="" receiver="" and="" boiling="" chips.="" 12.4.2.8.3="" calculate="" the="" percent="" lipid="" per="" section="" 12.4.1.9.3.="" it="" is="" not="" necessary="" to="" determine="" the="" lipid="" content="" of="" the="" blank,="" ipr,="" or="" opr="" aliquots.="" 12.4.2.9="" clean="" up="" the="" extract="" per="" section="" 13.7.3.="" 12.5="" back-extraction="" with="" base="" and="" acid.="" 12.5.1="" spike="" 1.0="" ml="" of="" the="" cleanup="" standard="" (section="" 7.11)="" into="" the="" separatory="" funnels="" containing="" the="" sample="" and="" qc="" extracts="" from="" section="" 12.1.4.1,="" 12.3.9.1.3,="" or="" 12.3.9.2.="" 12.5.2="" partition="" the="" extract="" against="" 50="" ml="" of="" potassium="" hydroxide="" solution="" (section="" 7.1.1).="" shake="" for="" two="" minutes="" with="" periodic="" venting="" into="" a="" hood.="" remove="" and="" discard="" the="" aqueous="" layer.="" repeat="" the="" base="" washing="" until="" no="" color="" is="" visible="" in="" the="" aqueous="" layer,="" to="" a="" maximum="" of="" four="" washings.="" minimize="" contact="" time="" between="" the="" extract="" and="" the="" base="" to="" prevent="" degradation="" of="" the="" cdds/cdfs.="" stronger="" potassium="" hydroxide="" solutions="" may="" be="" employed="" for="" back-extraction,="" provided="" that="" the="" laboratory="" meets="" the="" specifications="" for="" labeled="" compound="" recovery="" and="" demonstrates="" acceptable="" performance="" using="" the="" procedure="" in="" section="" 9.2.="" 12.5.3="" partition="" the="" extract="" against="" 50="" ml="" of="" sodium="" chloride="" solution="" (section="" 7.1.4)="" in="" the="" same="" way="" as="" with="" base.="" discard="" the="" aqueous="" layer.="" 12.5.4="" partition="" the="" extract="" against="" 50="" ml="" of="" sulfuric="" acid="" (section="" 7.1.2)="" in="" the="" same="" way="" as="" with="" base.="" repeat="" the="" acid="" washing="" until="" no="" color="" is="" visible="" in="" the="" aqueous="" layer,="" to="" a="" maximum="" of="" four="" washings.="" 12.5.5="" repeat="" the="" partitioning="" against="" sodium="" chloride="" solution="" and="" discard="" the="" aqueous="" layer.="" 12.5.6="" pour="" each="" extract="" through="" a="" drying="" column="" containing="" 7-10="" cm="" of="" granular="" anhydrous="" sodium="" sulfate="" (section="" 7.2.1).="" rinse="" the="" separatory="" funnel="" with="" 30-50="" ml="" of="" solvent,="" and="" pour="" through="" the="" drying="" column.="" collect="" each="" extract="" in="" a="" round-bottom="" flask.="" re-concentrate="" the="" sample="" and="" qc="" aliquots="" per="" sections="" 12.6="" through="" 12.7,="" and="" clean="" up="" the="" samples="" and="" qc="" aliquots="" per="" section="" 13.="" 12.6="" macro-concentration--extracts="" in="" toluene="" are="" concentrated="" using="" a="" rotary="" evaporator="" or="" a="" heating="" mantle;="" extracts="" in="" methylene="" chloride="" or="" hexane="" are="" concentrated="" using="" a="" rotary="" evaporator,="" heating="" mantle,="" or="" kuderna-danish="" apparatus.="" 12.6.1="" rotary="" evaporation--concentrate="" the="" extracts="" in="" separate="" round-bottom="" flasks.="" 12.6.1.1="" assemble="" the="" rotary="" evaporator="" according="" to="" manufacturer's="" instructions,="" and="" warm="" the="" water="" bath="" to="" 45="" deg.c.="" on="" a="" daily="" basis,="" preclean="" the="" rotary="" evaporator="" by="" concentrating="" 100="" ml="" of="" clean="" extraction="" solvent="" through="" the="" system.="" archive="" both="" the="" concentrated="" solvent="" and="" the="" solvent="" in="" the="" catch="" flask="" for="" a="" contamination="" check="" if="" necessary.="" between="" samples,="" three="" 2-3="" ml="" aliquots="" of="" solvent="" should="" be="" rinsed="" down="" the="" feed="" tube="" into="" a="" waste="" beaker.="" 12.6.1.2="" attach="" the="" round-bottom="" flask="" containing="" the="" sample="" extract="" to="" the="" rotary="" evaporator.="" slowly="" apply="" vacuum="" to="" the="" system,="" and="" begin="" rotating="" the="" sample="" flask.="" 12.6.1.3="" lower="" the="" flask="" into="" the="" water="" bath,="" and="" adjust="" the="" speed="" of="" rotation="" and="" the="" temperature="" as="" required="" to="" complete="" concentration="" in="" 15-20="" minutes.="" at="" the="" proper="" rate="" of="" concentration,="" the="" flow="" of="" solvent="" into="" the="" receiving="" flask="" will="" be="" steady,="" but="" no="" bumping="" or="" visible="" boiling="" of="" the="" extract="" will="" occur.="" note:="" if="" the="" rate="" of="" concentration="" is="" too="" fast,="" analyte="" loss="" may="" occur.="" 12.6.1.4="" when="" the="" liquid="" in="" the="" concentration="" flask="" has="" reached="" an="" apparent="" volume="" of="" approximately="" 2="" ml,="" remove="" the="" flask="" from="" the="" water="" bath="" and="" stop="" the="" rotation.="" slowly="" and="" carefully="" admit="" air="" into="" the="" system.="" be="" sure="" not="" to="" open="" the="" valve="" so="" quickly="" that="" the="" sample="" is="" blown="" out="" of="" the="" flask.="" rinse="" the="" feed="" tube="" with="" approximately="" 2="" ml="" of="" solvent.="" 12.6.1.5="" proceed="" to="" section="" 12.6.4="" for="" preparation="" for="" back-="" extraction="" or="" micro-concentration="" and="" solvent="" exchange.="" 12.6.2="" heating="" mantle--concentrate="" the="" extracts="" in="" separate="" round-="" bottom="" flasks.="" 12.6.2.1="" add="" one="" or="" two="" clean="" boiling="" chips="" to="" the="" round-bottom="" flask,="" and="" attach="" a="" three-ball="" macro="" snyder="" column.="" prewet="" the="" column="" by="" adding="" approximately="" 1="" ml="" of="" solvent="" through="" the="" top.="" place="" the="" round-bottom="" flask="" in="" a="" heating="" mantle,="" and="" apply="" heat="" as="" required="" to="" complete="" the="" concentration="" in="" 15-20="" minutes.="" at="" the="" proper="" rate="" of="" distillation,="" the="" balls="" of="" the="" column="" will="" actively="" chatter,="" but="" the="" chambers="" will="" not="" flood.="" 12.6.2.2="" when="" the="" liquid="" has="" reached="" an="" apparent="" volume="" of="" approximately="" 10="" ml,="" remove="" the="" round-bottom="" flask="" from="" the="" heating="" mantle="" and="" allow="" the="" solvent="" to="" drain="" and="" cool="" for="" at="" least="" 10="" minutes.="" remove="" the="" snyder="" column="" and="" rinse="" the="" glass="" joint="" into="" the="" receiver="" with="" small="" portions="" of="" solvent.="" 12.6.2.3="" proceed="" to="" section="" 12.6.4="" for="" preparation="" for="" back-="" extraction="" or="" micro-concentration="" and="" solvent="" exchange.="" 12.6.3="" kuderna-danish="" (k-d)--concentrate="" the="" extracts="" in="" separate="" 500="" ml="" k-d="" flasks="" equipped="" with="" 10="" ml="" concentrator="" tubes.="" the="" k-d="" technique="" is="" used="" for="" solvents="" such="" as="" methylene="" chloride="" and="" hexane.="" toluene="" is="" difficult="" to="" concentrate="" using="" the="" k-d="" technique="" unless="" a="" water="" bath="" fed="" by="" a="" steam="" generator="" is="" used.="" 12.6.3.1="" add="" one="" to="" two="" clean="" boiling="" chips="" to="" the="" receiver.="" attach="" a="" three-ball="" macro="" snyder="" column.="" prewet="" the="" column="" by="" adding="" approximately="" 1="" ml="" of="" solvent="" through="" the="" top.="" place="" the="" k-d="" apparatus="" in="" a="" hot="" water="" bath="" so="" that="" the="" entire="" lower="" rounded="" surface="" of="" the="" flask="" is="" bathed="" with="" steam.="" 12.6.3.2="" adjust="" the="" vertical="" position="" of="" the="" apparatus="" and="" the="" water="" temperature="" as="" required="" to="" complete="" the="" concentration="" in="" 15-20="" minutes.="" at="" the="" proper="" rate="" of="" distillation,="" the="" balls="" of="" the="" column="" will="" actively="" chatter="" but="" the="" chambers="" will="" not="" flood.="" 12.6.3.3="" when="" the="" liquid="" has="" reached="" an="" apparent="" volume="" of="" 1="" ml,="" remove="" the="" k-d="" apparatus="" from="" the="" bath="" and="" allow="" the="" solvent="" to="" drain="" and="" cool="" for="" at="" least="" 10="" minutes.="" remove="" the="" snyder="" column="" and="" rinse="" the="" flask="" and="" its="" lower="" joint="" into="" the="" concentrator="" tube="" with="" 1-2="" ml="" of="" solvent.="" a="" 5="" ml="" syringe="" is="" recommended="" for="" this="" operation.="" 12.6.3.4="" remove="" the="" three-ball="" snyder="" column,="" add="" a="" fresh="" boiling="" chip,="" and="" attach="" a="" two-ball="" micro="" snyder="" column="" to="" the="" concentrator="" tube.="" prewet="" the="" column="" by="" adding="" approximately="" 0.5="" ml="" of="" solvent="" through="" the="" top.="" place="" the="" apparatus="" in="" the="" hot="" water="" bath.="" 12.6.3.5="" adjust="" the="" vertical="" position="" and="" the="" water="" temperature="" as="" required="" to="" complete="" the="" concentration="" in="" 5-10="" minutes.="" at="" the="" proper="" rate="" of="" distillation,="" the="" balls="" of="" the="" column="" will="" actively="" chatter="" but="" the="" chambers="" will="" not="" flood.="" 12.6.3.6="" when="" the="" liquid="" reaches="" an="" apparent="" volume="" of="" 0.5="" ml,="" remove="" the="" [[page="" 48420]]="" apparatus="" from="" the="" water="" bath="" and="" allow="" to="" drain="" and="" cool="" for="" at="" least="" 10="" minutes.="" 12.6.3.7="" proceed="" to="" 12.6.4="" for="" preparation="" for="" back-extraction="" or="" micro-concentration="" and="" solvent="" exchange.="" 12.6.4="" preparation="" for="" back-extraction="" or="" micro-concentration="" and="" solvent="" exchange.="" 12.6.4.1="" for="" back-extraction="" (section="" 12.5),="" transfer="" the="" extract="" to="" a="" 250="" ml="" separatory="" funnel.="" rinse="" the="" concentration="" vessel="" with="" small="" portions="" of="" hexane,="" adjust="" the="" hexane="" volume="" in="" the="" separatory="" funnel="" to="" 10-20="" ml,="" and="" proceed="" to="" back-extraction="" (section="" 12.5).="" 12.6.4.2="" for="" determination="" of="" the="" weight="" of="" residue="" in="" the="" extract,="" or="" for="" clean-up="" procedures="" other="" than="" back-extraction,="" transfer="" the="" extract="" to="" a="" blowdown="" vial="" using="" two="" to="" three="" rinses="" of="" solvent.="" proceed="" with="" micro-concentration="" and="" solvent="" exchange="" (section="" 12.7).="" 12.7="" micro-concentration="" and="" solvent="" exchange.="" 12.7.1="" extracts="" to="" be="" subjected="" to="" gpc="" or="" hplc="" cleanup="" are="" exchanged="" into="" methylene="" chloride.="" extracts="" to="" be="" cleaned="" up="" using="" silica="" gel,="" alumina,="" carbon,="" and/or="" florisil="" are="" exchanged="" into="" hexane.="" 12.7.2="" transfer="" the="" vial="" containing="" the="" sample="" extract="" to="" a="" nitrogen="" blowdown="" device.="" adjust="" the="" flow="" of="" nitrogen="" so="" that="" the="" surface="" of="" the="" solvent="" is="" just="" visibly="" disturbed.="" note:="" a="" large="" vortex="" in="" the="" solvent="" may="" cause="" analyte="" loss.="" 12.7.3="" lower="" the="" vial="" into="" a="" 45="" deg.c="" water="" bath="" and="" continue="" concentrating.="" 12.7.3.1="" if="" the="" extract="" is="" to="" be="" concentrated="" to="" dryness="" for="" weight="" determination="" (sections="" 12.4.1.8,="" 12.4.2.7,="" and="" 13.7.1.4),="" blow="" dry="" until="" a="" constant="" weight="" is="" obtained.="" 12.7.3.2="" if="" the="" extract="" is="" to="" be="" concentrated="" for="" injection="" into="" the="" gc/ms="" or="" the="" solvent="" is="" to="" be="" exchanged="" for="" extract="" cleanup,="" proceed="" as="" follows:="" 12.7.4="" when="" the="" volume="" of="" the="" liquid="" is="" approximately="" 100="" l,="" add="" 2-3="" ml="" of="" the="" desired="" solvent="" (methylene="" chloride="" for="" gpc="" and="" hplc,="" or="" hexane="" for="" the="" other="" cleanups)="" and="" continue="" concentration="" to="" approximately="" 100="">L. Repeat the addition of solvent and
concentrate once more.
12.7.5 If the extract is to be cleaned up by GPC, adjust the
volume of the extract to 5.0 mL with methylene chloride. If the extract
is to be cleaned up by HPLC, further concentrate the extract to 30
L. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.6,
respectively).
12.7.6 If the extract is to be cleaned up by column chromatography
(alumina, silica gel, Carbopak/Celite, or Florisil), bring the final
volume to 1.0 mL with hexane. Proceed with column cleanups (Sections
13.3 through 13.5 and 13.8).
12.7.7 If the extract is to be concentrated for injection into the
GC/MS (Section 14), quantitatively transfer the extract to a 0.3 mL
conical vial for final concentration, rinsing the larger vial with
hexane and adding the rinse to the conical vial. Reduce the volume to
approximately 100 L. Add 10 L of nonane to the vial,
and evaporate the solvent to the level of the nonane. Seal the vial and
label with the sample number. Store in the dark at room temperature
until ready for GC/MS analysis. If GC/MS analysis will not be performed
on the same day, store the vial at <-10 deg.c.="" 13.0="" extract="" cleanup="" 13.1="" cleanup="" may="" not="" be="" necessary="" for="" relatively="" clean="" samples="" (e.g.,="" treated="" effluents,="" groundwater,="" drinking="" water).="" if="" particular="" circumstances="" require="" the="" use="" of="" a="" cleanup="" procedure,="" the="" analyst="" may="" use="" any="" or="" all="" of="" the="" procedures="" below="" or="" any="" other="" appropriate="" procedure.="" before="" using="" a="" cleanup="" procedure,="" the="" analyst="" must="" demonstrate="" that="" the="" requirements="" of="" section="" 9.2="" can="" be="" met="" using="" the="" cleanup="" procedure.="" if="" only="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf="" are="" to="" be="" determined,="" the="" cleanup="" procedures="" may="" be="" optimized="" for="" isolation="" of="" these="" two="" compounds.="" 13.1.1="" gel="" permeation="" chromatography="" (section="" 13.2)="" removes="" high="" molecular="" weight="" interferences="" that="" cause="" gc="" column="" performance="" to="" degrade.="" it="" should="" be="" used="" for="" all="" soil="" and="" sediment="" extracts="" and="" may="" be="" used="" for="" water="" extracts="" that="" are="" expected="" to="" contain="" high="" molecular="" weight="" organic="" compounds="" (e.g.,="" polymeric="" materials,="" humic="" acids).="" 13.1.2="" acid,="" neutral,="" and="" basic="" silica="" gel="" (section="" 13.3),="" alumina="" (section="" 13.4),="" and="" florisil="" (section="" 13.8)="" are="" used="" to="" remove="" nonpolar="" and="" polar="" interferences.="" alumina="" and="" florisil="" are="" used="" to="" remove="" chlorodiphenyl="" ethers.="" 13.1.3="" carbopak/celite="" (section="" 13.5)="" is="" used="" to="" remove="" nonpolar="" interferences.="" 13.1.4="" hplc="" (section="" 13.6)="" is="" used="" to="" provide="" specificity="" for="" the="" 2,3,7,8-substituted="" and="" other="" cdd="" and="" cdf="" isomers.="" 13.1.5="" the="" anthropogenic="" isolation="" column="" (section="" 13.7.1),="" acidified="" silica="" gel="" batch="" adsorption="" procedure="" (section="" 13.7.2),="" and="" sulfuric="" acid="" and="" base="" back-extraction="" (section="" 13.7.3)="" are="" used="" for="" removal="" of="" lipids="" from="" tissue="" samples.="" 13.2="" gel="" permeation="" chromatography="" (gpc).="" 13.2.1="" column="" packing.="" 13.2.1.1="" place="" 70-75="" g="" of="" sx-3="" bio-beads="" (section="" 6.7.1.1)="" in="" a="" 400-500="" ml="" beaker.="" 13.2.1.2="" cover="" the="" beads="" with="" methylene="" chloride="" and="" allow="" to="" swell="" overnight="" (a="" minimum="" of="" 12="" hours).="" 13.2.1.3="" transfer="" the="" swelled="" beads="" to="" the="" column="" (section="" 6.7.1.1)="" and="" pump="" solvent="" through="" the="" column,="" from="" bottom="" to="" top,="" at="" 4.5-5.5="" ml/minute="" prior="" to="" connecting="" the="" column="" to="" the="" detector.="" 13.2.1.4="" after="" purging="" the="" column="" with="" solvent="" for="" one="" to="" two="" hours,="" adjust="" the="" column="" head="" pressure="" to="" 7-10="" psig="" and="" purge="" for="" four="" to="" five="" hours="" to="" remove="" air.="" maintain="" a="" head="" pressure="" of="" 7-10="" psig.="" connect="" the="" column="" to="" the="" detector="" (section="" 6.7.1.4).="" 13.2.2="" column="" calibration.="" 13.2.2.1="" load="" 5="" ml="" of="" the="" calibration="" solution="" (section="" 7.4)="" into="" the="" sample="" loop.="" 13.2.2.2="" inject="" the="" calibration="" solution="" and="" record="" the="" signal="" from="" the="" detector.="" the="" elution="" pattern="" will="" be="" corn="" oil,="" bis(2-ethyl="" hexyl)phthalate,="" pentachlorophenol,="" perylene,="" and="" sulfur.="" 13.2.2.3="" set="" the="" ``dump="" time''="" to="" allow="">85% removal of the corn
oil and >85% collection of the phthalate.
13.2.2.4 Set the ``collect time'' to the peak minimum between
perylene and sulfur.
13.2.2.5 Verify the calibration with the calibration solution
after every 20 extracts. Calibration is verified if the recovery of the
pentachlorophenol is greater than 85%. If calibration is not verified,
the system shall be recalibrated using the calibration solution, and
the previous 20 samples shall be re-extracted and cleaned up using the
calibrated GPC system.
13.2.3 Extract cleanup--GPC requires that the column not be
overloaded. The column specified in this method is designed to handle a
maximum of 0.5 g of high molecular weight material in a 5 mL extract.
If the extract is known or expected to contain more than 0.5 g, the
extract is split into aliquots for GPC, and the aliquots are combined
after elution from the column. The residue content of the extract may
be obtained gravimetrically by evaporating the solvent from a 50
L aliquot.
13.2.3.1 Filter the extract or load through the filter holder
(Section 6.7.1.3) to remove the particles. Load the 5.0 mL extract onto
the column.
13.2.3.2 Elute the extract using the calibration data determined
in Section
[[Page 48421]]
13.2.2. Collect the eluate in a clean 400-500 mL beaker.
13.2.3.3 Rinse the sample loading tube thoroughly with methylene
chloride between extracts to prepare for the next sample.
13.2.3.4 If a particularly dirty extract is encountered, a 5.0 mL
methylene chloride blank shall be run through the system to check for
carry-over.
13.2.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for
further cleanup or injection into the GC/MS.
13.3 Silica Gel Cleanup.
13.3.1 Place a glass-wool plug in a 15 mm ID chromatography column
(Section 6.7.4.2). Pack the column bottom to top with: 1 g silica gel
(Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g silica
gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g
granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to
settle the adsorbents.
13.3.2 Pre-elute the column with 50-100 mL of hexane. Close the
stopcock when the hexane is within 1 mm of the sodium sulfate. Discard
the eluate. Check the column for channeling. If channeling is present,
discard the column and prepare another.
13.3.3 Apply the concentrated extract to the column. Open the
stopcock until the extract is within 1 mm of the sodium sulfate.
13.3.4 Rinse the receiver twice with 1 mL portions of hexane, and
apply separately to the column. Elute the CDDs/CDFs with 100 mL hexane,
and collect the eluate.
13.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for
further cleanup or injection into the HPLC or GC/MS.
13.3.6 For extracts of samples known to contain large quantities
of other organic compounds (such as paper mill effluents), it may be
advisable to increase the capacity of the silica gel column. This may
be accomplished by increasing the strengths of the acid and basic
silica gels. The acid silica gel (Section 7.5.1.2) may be increased in
strength to as much as 44% w/w (7.9 g sulfuric acid added to 10 g
silica gel). The basic silica gel (Section 7.5.1.3) may be increased in
strength to as much as 33% w/w (50 mL 1N NaOH added to 100 g silica
gel), or the potassium silicate (Section 7.5.1.4) may be used.
Note: The use of stronger acid silica gel (44% w/w) may lead to
charring of organic compounds in some extracts. The charred material
may retain some of the analytes and lead to lower recoveries of
CDDs/CDFs. Increasing the strengths of the acid and basic silica gel
may also require different volumes of hexane than those specified
above to elute the analytes off the column. Therefore, the
performance of the method after such modifications must be verified
by the procedure in Section 9.2.
13.4 Alumina Cleanup.
13.4.1 Place a glass-wool plug in a 15 mm ID chromatography column
(Section 6.7.4.2).
13.4.2 If using acid alumina, pack the column by adding 6 g acid
alumina (Section 7.5.2.1). If using basic alumina, substitute 6 g basic
alumina (Section 7.5.2.2). Tap the column to settle the adsorbents.
13.4.3 Pre-elute the column with 50-100 mL of hexane. Close the
stopcock when the hexane is within 1 mm of the alumina.
13.4.4 Discard the eluate. Check the column for channeling. If
channeling is present, discard the column and prepare another.
13.4.5 Apply the concentrated extract to the column. Open the
stopcock until the extract is within 1 mm of the alumina.
13.4.6 Rinse the receiver twice with 1 mL portions of hexane and
apply separately to the column. Elute the interfering compounds with
100 mL hexane and discard the eluate.
13.4.7 The choice of eluting solvents will depend on the choice of
alumina (acid or basic) made in Section 13.4.2.
13.4.7.1 If using acid alumina, elute the CDDs/CDFs from the
column with 20 mL methylene chloride:hexane (20:80 v/v). Collect the
eluate.
13.4.7.2 If using basic alumina, elute the CDDs/CDFs from the
column with 20 mL methylene chloride:hexane (50:50 v/v). Collect the
eluate.
13.4.8 Concentrate the eluate per Sections 12.6 and 12.7 for
further cleanup or injection into the HPLC or GC/MS.
13.5 Carbon Column.
13.5.1 Cut both ends from a 10 mL disposable serological pipet
(Section 6.7.3.2) to produce a 10 cm column. Fire-polish both ends and
flare both ends if desired. Insert a glass-wool plug at one end, and
pack the column with 0.55 g of Carbopak/Celite (Section 7.5.3.3) to
form an adsorbent bed approximately 2 cm long. Insert a glass-wool plug
on top of the bed to hold the adsorbent in place.
13.5.2 Pre-elute the column with 5 mL of toluene followed by 2 mL
of methylene chloride: methanol:toluene (15:4:1 v/v), 1 mL of methylene
chloride:cyclohexane (1:1 v/v), and 5 mL of hexane. If the flow rate of
eluate exceeds 0.5 mL/minute, discard the column.
13.5.3 When the solvent is within 1 mm of the column packing,
apply the sample extract to the column. Rinse the sample container
twice with 1 mL portions of hexane and apply separately to the column.
Apply 2 mL of hexane to complete the transfer.
13.5.4 Elute the interfering compounds with two 3 mL portions of
hexane, 2 mL of methylene chloride:cyclohexane (1:1 v/v), and 2 mL of
methylene chloride:methanol:toluene (15:4:1 v/v). Discard the eluate.
13.5.5 Invert the column, and elute the CDDs/CDFs with 20 mL of
toluene. If carbon particles are present in the eluate, filter through
glass-fiber filter paper.
13.5.6 Concentrate the eluate per Sections 12.6 and 12.7 for
further cleanup or injection into the HPLC or GC/MS.
13.6 HPLC (Reference 6).
13.6.1 Column calibration.
13.6.1.1 Prepare a calibration standard containing the 2,3,7,8-
substituted isomers and/or other isomers of interest at a concentration
of approximately 500 pg/L in methylene chloride.
13.6.1.2 Inject 30 L of the calibration solution into the
HPLC and record the signal from the detector. Collect the eluant for
reuse. The elution order will be the tetra- through octa-isomers.
13.6.1.3 Establish the collection time for the tetra-isomers and
for the other isomers of interest. Following calibration, flush the
injection system with copious quantities of methylene chloride,
including a minimum of five 50 L injections while the detector
is monitored, to ensure that residual CDDs/CDFs are removed from the
system.
13.6.1.4 Verify the calibration with the calibration solution
after every 20 extracts. Calibration is verified if the recovery of the
CDDs/CDFs from the calibration standard (Section 13.6.1.1) is 75-125%
compared to the calibration (Section 13.6.1.2). If calibration is not
verified, the system shall be recalibrated using the calibration
solution, and the previous 20 samples shall be re-extracted and cleaned
up using the calibrated system.
13.6.2 Extract cleanup--HPLC requires that the column not be
overloaded. The column specified in this method is designed to handle a
maximum of 30 L of extract. If the extract cannot be
concentrated to less than 30 L, it is split into fractions and
the fractions are combined after elution from the column.
13.6.2.1 Rinse the sides of the vial twice with 30 L of
methylene chloride and reduce to 30 L with the evaporation
apparatus (Section 12.7).
[[Page 48422]]
13.6.2.2 Inject the 30 L extract into the HPLC.
13.6.2.3 Elute the extract using the calibration data determined
in Section 13.6.1. Collect the fraction(s) in a clean 20 mL
concentrator tube containing 5 mL of hexane:acetone (1:1 v/v).
13.6.2.4 If an extract containing greater than 100 ng/mL of total
CDD or CDF is encountered, a 30 L methylene chloride blank
shall be run through the system to check for carry-over.
13.6.2.5 Concentrate the eluate per Section 12.7 for injection
into the GC/MS.
13.7 Cleanup of Tissue Lipids--Lipids are removed from the Soxhlet
extract using either the anthropogenic isolation column (Section
13.7.1) or acidified silica gel (Section 13.7.2), or are removed from
the HCl digested extract using sulfuric acid and base back-extraction
(Section 13.7.3).
13.7.1 Anthropogenic isolation column (References 22 and 27)--Used
for removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).
13.7.1.1 Prepare the column as given in Section 7.5.4.
13.7.1.2 Pre-elute the column with 100 mL of hexane. Drain the
hexane layer to the top of the column, but do not expose the sodium
sulfate.
13.7.1.3 Load the sample and rinses (Section 12.4.1.9.2) onto the
column by draining each portion to the top of the bed. Elute the CDDs/
CDFs from the column into the apparatus used for concentration (Section
12.4.1.7) using 200 mL of hexane.
13.7.1.4 Concentrate the cleaned up extract (Sections 12.6 through
12.7) to constant weight per Section 12.7.3.1. If more than 500 mg of
material remains, repeat the cleanup using a fresh anthropogenic
isolation column.
13.7.1.5 Redissolve the extract in a solvent suitable for the
additional cleanups to be used (Sections 13.2 through 13.6 and 13.8).
13.7.1.6 Spike 1.0 mL of the cleanup standard (Section 7.11) into
the residue/solvent.
13.7.1.7 Clean up the extract using the procedures in Sections
13.2 through 13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section
13.8) and carbon (Section 13.5) are recommended as minimum additional
cleanup steps.
13.7.1.8 Following cleanup, concentrate the extract to 10
L as described in Section 12.7 and proceed with the analysis
in Section 14.
13.7.2 Acidified silica gel (Reference 28)--Procedure alternate to
the anthropogenic isolation column (Section 13.7.1) that is used for
removal of lipids from the Soxhlet/SDS extraction (Section 12.4.1).
13.7.2.1 Adjust the volume of hexane in the bottle (Section
12.4.1.9.2) to approximately 200 mL.
13.7.2.2 Spike 1.0 mL of the cleanup standard (Section 7.11) into
the residue/solvent.
13.7.2.3 Drop the stirring bar into the bottle, place the bottle
on the stirring plate, and begin stirring.
13.7.2.4 Add 30-100 g of acid silica gel (Section 7.5.1.2) to the
bottle while stirring, keeping the silica gel in motion. Stir for two
to three hours.
Note: 30 grams of silica gel should be adequate for most samples
and will minimize contamination from this source.
13.7.2.5 After stirring, pour the extract through approximately 10
g of granular anhydrous sodium sulfate (Section 7.2.1) contained in a
funnel with glass-fiber filter into a macro contration device (Section
12.6). Rinse the bottle and sodium sulfate with hexane to complete the
transfer.
13.7.2.6 Concentrate the extract per Sections 12.6 through 12.7
and clean up the extract using the procedures in Sections 13.2 through
13.6 and 13.8. Alumina (Section 13.4) or Florisil (Section 13.8) and
carbon (Section 13.5) are recommended as minimum additional cleanup
steps.
13.7.3 Sulfuric acid and base back-extraction'Used with HCl
digested extracts (Section 12.4.2).
13.7.3.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into
the residue/solvent (Section 12.4.2.8.2).
13.7.3.2 Add 10 mL of concentrated sulfuric acid to the bottle.
Immediately cap and shake one to three times. Loosen cap in a hood to
vent excess pressure. Cap and shake the bottle so that the residue/
solvent is exposed to the acid for a total time of approximately 45
seconds.
13.7.3.3 Decant the hexane into a 250 mL separatory funnel making
sure that no acid is transferred. Complete the quantitative transfer
with several hexane rinses.
13.7.3.4 Back extract the solvent/residue with 50 mL of potassium
hydroxide solution per Section 12.5.2, followed by two reagent water
rinses.
13.7.3.5 Drain the extract through a filter funnel containing
approximately 10 g of granular anhydrous sodium sulfate in a glass-
fiber filter into a macro concentration device (Section 12.6).
13.7.3.6 Concentrate the cleaned up extract to a volume suitable
for the additional cleanups given in Sections 13.2 through 13.6 and
13.8. Gel permeation chromatography (Section 13.2), alumina (Section
13.4) or Florisil (Section 13.8), and Carbopak/Celite (Section 13.5)
are recommended as minimum additional cleanup steps.
13.7.3.7 Following cleanup, concentrate the extract to 10 L as
described in Section 12.7 and proceed with analysis per Section 14.
13.8 Florisil Cleanup (Reference 29).
13.8.1 Pre-elute the activated Florisil column (Section 7.5.3)
with 10 mL of methylene chloride followed by 10 mL of hexane:methylene
chloride (98:2 v/v) and discard the solvents.
13.8.2 When the solvent is within 1 mm of the packing, apply the
sample extract (in hexane) to the column. Rinse the sample container
twice with 1 mL portions of hexane and apply to the column.
13.8.3 Elute the interfering compounds with 20 mL of
hexane:methylene chloride (98:2) and discard the eluate.
13.8.4 Elute the CDDs/CDFs with 35 mL of methylene chloride and
collect the eluate. Concentrate the eluate per Sections 12.6 through
12.7 for further cleanup or for injection into the HPLC or GC/MS.
14.0 HRGC/HRMS Analysis
14.1 Establish the operating conditions given in Section 10.1.
14.2 Add 10 uL of the appropriate internal standard solution
(Section 7.12) to the sample extract immediately prior to injection to
minimize the possibility of loss by evaporation, adsorption, or
reaction. If an extract is to be reanalyzed and evaporation has
occurred, do not add more instrument internal standard solution.
Rather, bring the extract back to its previous volume (e.g., 19 L) with
pure nonane only (18 L if 2 L injections are used).
14.3 Inject 1.0 L or 2.0 L of the
concentrated extract containing the internal standard solution, using
on-column or splitless injection. The volume injected must be identical
to the volume used for calibration (Section 10). Start the GC column
initial isothermal hold upon injection. Start MS data collection after
the solvent peak elutes. Stop data collection after the OCDD and OCDF
have eluted. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be
determined, stop data collection after elution of these compounds.
Return the column to the initial temperature for analysis of the next
extract or standard.
15.0 System and Laboratory Performance
15.1 At the beginning of each 12-hour shift during which analyses
are performed, GC/MS system performance and calibration are verified
for all CDDs/CDFs and labeled compounds. For these tests, analysis of
the CS3 calibration verification (VER) standard (Section
[[Page 48423]]
7.13 and Table 4) and the isomer specificity test standards (Section
7.15 and Table 5) shall be used to verify all performance criteria.
Adjustment and/or recalibration (Section 10) shall be performed until
all performance criteria are met. Only after all performance criteria
are met may samples, blanks, IPRs, and OPRs be analyzed.
15.2 MS Resolution--A static resolving power of at least 10,000
(10% valley definition) must be demonstrated at the appropriate m/z
before any analysis is performed. Static resolving power checks must be
performed at the beginning and at the end of each 12-hour shift
according to procedures in Section 10.1.2. Corrective actions must be
implemented whenever the resolving power does not meet the requirement.
15.3 Calibration Verification.
15.3.1 Inject the VER standard using the procedure in Section 14.
15.3.2 The m/z abundance ratios for all CDDs/CDFs shall be within
the limits in Table 9; otherwise, the mass spectrometer shall be
adjusted until the m/z abundance ratios fall within the limits
specified, and the verification test shall be repeated. If the
adjustment alters the resolution of the mass spectrometer, resolution
shall be verified (Section 10.1.2) prior to repeat of the verification
test.
15.3.3 The peaks representing each CDD/CDF and labeled compound in
the VER standard must be present with S/N of at least 10; otherwise,
the mass spectrometer shall be adjusted and the verification test
repeated.
15.3.4 Compute the concentration of each CDD/CDF compound by
isotope dilution (Section 10.5) for those compounds that have labeled
analogs (Table 1). Compute the concentration of the labeled compounds
by the internal standard method (Section 10.6). These concentrations
are computed based on the calibration data in Section 10.
15.3.5 For each compound, compare the concentration with the
calibration verification limit in Table 6. If only 2,3,7,8-TCDD and
2,3,7,8-TCDF are to be determined, compare the concentration to the
limit in Table 6a. If all compounds meet the acceptance criteria,
calibration has been verified and analysis of standards and sample
extracts may proceed. If, however, any compound fails its respective
limit, the measurement system is not performing properly for that
compound. In this event, prepare a fresh calibration standard or
correct the problem causing the failure and repeat the resolution
(Section 15.2) and verification (Section 15.3) tests, or recalibrate
(Section 10).
15.4 Retention Times and GC Resolution.
15.4.1 Retention times.
15.4.1.1 Absolute--The absolute retention times of the
13C12-1,2,3,4-TCDD and
13C12-1,2,3,7,8,9-HxCDD GCMS internal standards
in the verification test (Section 15.3) shall be within 15
seconds of the retention times obtained during calibration (Sections
10.2.1 and 10.2.4).
15.4.1.2 Relative--The relative retention times of CDDs/CDFs and
labeled compounds in the verification test (Section 15.3) shall be
within the limits given in Table 2.
15.4.2 GC resolution.
15.4.2.1 Inject the isomer specificity standards (Section 7.15) on
their respective columns.
15.4.2.2 The valley height between 2,3,7,8-TCDD and the other
tetra-dioxin isomers at m/z 319.8965, and between 2,3,7,8-TCDF and the
other tetra-furan isomers at m/z 303.9016 shall not exceed 25% on their
respective columns (Figures 6 and 7).
15.4.3 If the absolute retention time of any compound is not
within the limits specified or if the 2,3,7,8-isomers are not resolved,
the GC is not performing properly. In this event, adjust the GC and
repeat the verification test (Section 15.3) or recalibrate (Section
10), or replace the GC column and either verify calibration or
recalibrate.
15.5 Ongoing Precision and Recovery.
15.5.1 Analyze the extract of the ongoing precision and recovery
(OPR) aliquot (Section 11.4.2.5, 11.5.4, 11.6.2, 11.7.4, or 11.8.3.2)
prior to analysis of samples from the same batch.
15.5.2 Compute the concentration of each CDD/CDF by isotope
dilution for those compounds that have labeled analogs (Section 10.5).
Compute the concentration of 1,2,3,7,8,9-HxCDD, OCDF, and each labeled
compound by the internal standard method (Section 10.6).
15.5.3 For each CDD/CDF and labeled compound, compare the
concentration to the OPR limits given in Table 6. If only 2,3,7,8-TCDD
and 2,3,7,8-TCDF are to be determined, compare the concentration to the
limits in Table 6a. If all compounds meet the acceptance criteria,
system performance is acceptable and analysis of blanks and samples may
proceed. If, however, any individual concentration falls outside of the
range given, the extraction/concentration processes are not being
performed properly for that compound. In this event, correct the
problem, re-prepare, extract, and clean up the sample batch and repeat
the ongoing precision and recovery test (Section 15.5).
15.5.4 Add results that pass the specifications in Section 15.5.3
to initial and previous ongoing data for each compound in each matrix.
Update QC charts to form a graphic representation of continued
laboratory performance. Develop a statement of laboratory accuracy for
each CDD/CDF in each matrix type by calculating the average percent
recovery (R) and the standard deviation of percent recovery
(SR). Express the accuracy as a recovery interval from
R-2SR to R+2SR. For example, if R=95% and
SR=5%, the accuracy is 85-105%.
15.6 Blank--Analyze the method blank extracted with each sample
batch immediately following analysis of the OPR aliquot to demonstrate
freedom from contamination and freedom from carryover from the OPR
analysis. The results of the analysis of the blank must meet the
specifications in Section 9.5.2 before sample analyses may proceed.
16.0 Qualitative Determination
A CDD, CDF, or labeled compound is identified in a standard, blank,
or sample when all of the criteria in Sections 16.1 through 16.4 are
met.
16.1 The signals for the two exact m/z's in Table 8 must be
present and must maximize within the same two seconds.
16.2 The signal-to-noise ratio (S/N) for the GC peak at each exact
m/z must be greater than or equal to 2.5 for each CDD or CDF detected
in a sample extract, and greater than or equal to 10 for all CDDs/CDFs
in the calibration standard (Sections 10.2.3 and 15.3.3).
16.3 The ratio of the integrated areas of the two exact m/z's
specified in Table 8 must be within the limit in Table 9, or within
10% of the ratio in the midpoint (CS3) calibration or
calibration verification (VER), whichever is most recent.
16.4 The relative retention time of the peak for a 2,3,7,8-
substituted CDD or CDF must be within the limit in Table 2. The
retention time of peaks representing non-2,3,7,8-substituted CDDs/CDFs
must be within the retention time windows established in Section 10.3.
16.5 Confirmatory Analysis--Isomer specificity for 2,3,7,8-TCDF
cannot be achieved on the DB-5 column. Therefore, any sample in which
2,3,7,8-TCDF is identified by analysis on a DB-5 column must have a
confirmatory analysis performed on a DB-225, SP-2330, or equivalent GC
column. The operating conditions in Section 10.1.1 may be adjusted to
optimize the analysis on the second GC column, but the GC/MS must meet
the mass resolution
[[Page 48424]]
and calibration specifications in Section 10.
16.6 If the criteria for identification in Sections 16.1 through
16.5 are not met, the CDD or CDF has not been identified and the
results may not be reported for regulatory compliance purposes. If
interferences preclude identification, a new aliquot of sample must be
extracted, further cleaned up, and analyzed.
17.0 Quantitative Determination
17.1 Isotope Dilution Quantitation--By adding a known amount of a
labeled compound to every sample prior to extraction, correction for
recovery of the CDD/CDF can be made because the CDD/CDF and its labeled
analog exhibit similar effects upon extraction, concentration, and gas
chromatography. Relative response (RR) values are used in conjunction
with the initial calibration data described in Section 10.5 to
determine concentrations directly, so long as labeled compound spiking
levels are constant, using the following equation:
[GRAPHIC] [TIFF OMITTED] TR15SE97.007
Where:
Cex = The concentration of the CDD/CDF in the extract, and
the other terms are as defined in Section 10.5.2.
17.1.1 Because of a potential interference, the labeled analog of
OCDF is not added to the sample. Therefore, OCDF is quantitated against
labeled OCDD. As a result, the concentration of OCDF is corrected for
the recovery of the labeled OCDD. In instances where OCDD and OCDF
behave differently during sample extraction, concentration, and cleanup
procedures, this may decrease the accuracy of the OCDF results.
However, given the low toxicity of this compound relative to the other
dioxins and furans, the potential decrease in accuracy is not
considered significant.
17.1.2 Because 13C12-1,2,3,7,8,9-HxCDD is
used as an instrument internal standard (i.e., not added before
extraction of the sample), it cannot be used to quantitate the
1,2,3,7,8,9-HxCDD by strict isotope dilution procedures. Therefore,
1,2,3,7,8,9-HxCDD is quantitated using the averaged response of the
labeled analogs of the other two 2,3,7,8-substituted HxCDD's:
1,2,3,4,7,8-HxCDD and 1,2,3,6,7,8-HxCDD. As a result, the concentration
of 1,2,3,7,8,9-HxCDD is corrected for the average recovery of the other
two HxCDD's.
17.1.3 Any peaks representing non-2,3,7,8-substituted CDDs/CDFs
are quantitated using an average of the response factors from all of
the labeled 2,3,7,8-isomers at the same level of chlorination.
17.2 Internal Standard Quantitation and Labeled Compound Recovery.
17.2.1 Compute the concentrations of 1,2,3,7,8,9--HxCDD, OCDF, the
13C-labeled analogs and the 37C-labeled cleanup
standard in the extract using the response factors determined from the
initial calibration data (Section 10.6) and the following equation:
[GRAPHIC] [TIFF OMITTED] TR15SE97.008
Where:
Cex = The concentration of the CDD/CDF in the extract, and
the other terms are as defined in Section 10.6.1.
Note: There is only one m/z for the 37Cl-labeled
standard.
17.2.2 Using the concentration in the extract determined above,
compute the percent recovery of the 13C-labeled compounds
and the 37C-labeled cleanup standard using the following
equation:
[GRAPHIC] [TIFF OMITTED] TR15SE97.009
17.3 The concentration of a CDD/CDF in the solid phase of the
sample is computed using the concentration of the compound in the
extract and the weight of the solids (Section 11.5.1), as follows:
[GRAPHIC] [TIFF OMITTED] TR15SE97.010
Where:
Cex = The concentration of the compound in the extract.
Vex = The extract volume in mL.
Ws = The sample weight (dry weight) in kg.
17.4 The concentration of a CDD/CDF in the aqueous phase of the
sample is computed using the concentration of the compound in the
extract and the volume of water extracted (Section 11.4 or 11.5), as
follows:
[GRAPHIC] [TIFF OMITTED] TR15SE97.011
Where:
Cex = The concentration of the compound in the extract.
Vex = The extract volume in mL.
Vs = The sample volume in liters.
17.5 If the SICP area at either quantitation m/z for any compound
exceeds the calibration range of the system, a smaller sample aliquot
is extracted.
17.5.1 For aqueous samples containing 1% solids or less, dilute
100 mL, 10 mL, etc., of sample to 1 L with reagent water and re-
prepare, extract, clean up, and analyze per Sections 11 through 14.
17.5.2 For samples containing greater than 1% solids, extract an
amount of sample equal to \1/10\, \1/100\, etc., of the amount used in
Section 11.5.1. Re-prepare, extract, clean up, and analyze per Sections
11 through 14.
17.5.3 If a smaller sample size will not be representative of the
entire sample, dilute the sample extract by a factor of 10, adjust the
concentration of the instrument internal standard to 100 pg/L
in the extract, and analyze an aliquot of this diluted extract by the
internal standard method.
[[Page 48425]]
17.6 Results are reported to three significant figures for the
CDDs/CDFs and labeled compounds found in all standards, blanks, and
samples.
17.6.1 Reporting units and levels.
17.6.1.1 Aqueous samples--Report results in pg/L (parts-per-
quadrillion).
17.6.1.2 Samples containing greater than 1% solids (soils,
sediments, filter cake, compost)--Report results in ng/kg based on the
dry weight of the sample. Report the percent solids so that the result
may be corrected.
17.6.1.3 Tissues--Report results in ng/kg of wet tissue, not on
the basis of the lipid content of the sample. Report the percent lipid
content, so that the data user can calculate the concentration on a
lipid basis if desired.
17.6.1.4 Reporting level.
17.6.1.4.1 Standards (VER, IPR, OPR) and samples--Report results
at or above the minimum level (Table 2). Report results below the
minimum level as not detected or as required by the regulatory
authority.
17.6.1.4.2 Blanks--Report results above one-third the ML.
17.6.2 Results for CDDs/CDFs in samples that have been diluted are
reported at the least dilute level at which the areas at the
quantitation m/z's are within the calibration range (Section 17.5).
17.6.3 For CDDs/CDFs having a labeled analog, results are reported
at the least dilute level at which the area at the quantitation m/z is
within the calibration range (Section 17.5) and the labeled compound
recovery is within the normal range for the method (Section 9.3 and
Tables 6, 6a, 7, and 7a).
17.6.4 Additionally, if requested, the total concentration of all
isomers in an individual level of chlorination (i.e., total TCDD, total
TCDF, total Paced, etc.) may be reported by summing the concentrations
of all isomers identified in that level of chlorination, including both
2,3,7,8-substituted and non-2,3,7,8-substituted isomers.
18.0 Analysis of Complex Samples
18.1 Some samples may contain high levels (>10 ng/L; >1000 ng/kg)
of the compounds of interest, interfering compounds, and/or polymeric
materials. Some extracts will not concentrate to 10 L (Section
12.7); others may overload the GC column and/or mass spectrometer.
18.2 Analyze a smaller aliquot of the sample (Section 17.5) when
the extract will not concentrate to 10 L after all cleanup
procedures have been exhausted.
18.3 Chlorodiphenyl Ethers--If chromatographic peaks are detected
at the retention time of any CDDs/CDFs in any of the m/z channels being
monitored for the chlorodiphenyl ethers (Table 8), cleanup procedures
must be employed until these interferences are removed. Alumina
(Section 13.4) and Florisil (Section 13.8) are recommended for removal
of chlorodiphenyl ethers.
18.4 Recovery of Labeled Compounds--In most samples, recoveries of
the labeled compounds will be similar to those from reagent water or
from the alternate matrix (Section 7.6).
18.4.1 If the recovery of any of the labeled compounds is outside
of the normal range (Table 7), a diluted sample shall be analyzed
(Section 17.5).
18.4.2 If the recovery of any of the labeled compounds in the
diluted sample is outside of normal range, the calibration verification
standard (Section 7.13) shall be analyzed and calibration verified
(Section 15.3).
18.4.3 If the calibration cannot be verified, a new calibration
must be performed and the original sample extract reanalyzed.
18.4.4 If the calibration is verified and the diluted sample does
not meet the limits for labeled compound recovery, the method does not
apply to the sample being analyzed and the result may not be reported
for regulatory compliance purposes. In this case, alternate extraction
and cleanup procedures in this method must be employed to resolve the
interference. If all cleanup procedures in this method have been
employed and labeled compound recovery remains outside of the normal
range, extraction and/or cleanup procedures that are beyond this scope
of this method will be required to analyze these samples.
19.0 Pollution Prevention
19.1 The solvents used in this method pose little threat to the
environment when managed properly. The solvent evaporation techniques
used in this method are amenable to solvent recovery, and it is
recommended that the laboratory recover solvents wherever feasible.
19.2 Standards should be prepared in volumes consistent with
laboratory use to minimize disposal of standards.
20.0 Waste Management
20.1 It is the laboratory's responsibility to comply with all
federal, state, and local regulations governing waste management,
particularly the hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations.
Compliance is also required with any sewage discharge permits and
regulations.
20.2 Samples containing HCl to pH <2 are="" hazardous="" and="" must="" be="" neutralized="" before="" being="" poured="" down="" a="" drain="" or="" must="" be="" handled="" as="" hazardous="" waste.="" 20.3="" the="" cdds/cdfs="" decompose="" above="" 800="" deg.c.="" low-level="" waste="" such="" as="" absorbent="" paper,="" tissues,="" animal="" remains,="" and="" plastic="" gloves="" may="" be="" burned="" in="" an="" appropriate="" incinerator.="" gross="" quantities="" (milligrams)="" should="" be="" packaged="" securely="" and="" disposed="" of="" through="" commercial="" or="" governmental="" channels="" that="" are="" capable="" of="" handling="" extremely="" toxic="" wastes.="" 20.4="" liquid="" or="" soluble="" waste="" should="" be="" dissolved="" in="" methanol="" or="" ethanol="" and="" irradiated="" with="" ultraviolet="" light="" with="" a="" wavelength="" shorter="" than="" 290="" nm="" for="" several="" days.="" use="" f40="" bl="" or="" equivalent="" lamps.="" analyze="" liquid="" wastes,="" and="" dispose="" of="" the="" solutions="" when="" the="" cdds/cdfs="" can="" no="" longer="" be="" detected.="" 20.5="" for="" further="" information="" on="" waste="" management,="" consult="" ``the="" waste="" management="" manual="" for="" laboratory="" personnel''="" and="" ``less="" is="" better--laboratory="" chemical="" management="" for="" waste="" reduction,''="" available="" from="" the="" american="" chemical="" society's="" department="" of="" government="" relations="" and="" science="" policy,="" 1155="" 16th="" street="" n.w.,="" washington,="" d.c.="" 20036.="" 21.0="" method="" performance="" method="" performance="" was="" validated="" and="" performance="" specifications="" were="" developed="" using="" data="" from="" epa's="" international="" interlaboratory="" validation="" study="" (references="" 30-31)="" and="" the="" epa/paper="" industry="" long-="" term="" variability="" study="" of="" discharges="" from="" the="" pulp="" and="" paper="" industry="" (58="" fr="" 66078).="" 22.0="" references="" 1.="" tondeur,="" yves.="" ``method="" 8290:="" analytical="" procedures="" and="" quality="" assurance="" for="" multimedia="" analysis="" of="" polychlorinated="" dibenzo-p-dioxins="" and="" dibenzofurans="" by="" high="" resolution="" gas="" chromatography/high="" resolution="" mass="" spectrometry,''="" usepa="" emsl,="" las="" vegas,="" nevada,="" june="" 1987.="" 2.="" ``measurement="" of="" 2,3,7,8-tetrachlorinated="" dibenzo-p-dioxin="" (tcdd)="" and="" 2,3,7,8-tetrachlorinated="" dibenzofuran="" (tcdf)="" in="" pulp,="" sludges,="" process="" samples="" and="" wastewaters="" from="" pulp="" and="" paper="" mills,''="" wright="" state="" university,="" dayton,="" oh="" 45435,="" june="" 1988.="" 3.="" ``ncasi="" procedures="" for="" the="" preparation="" and="" isomer="" specific="" analysis="" of="" pulp="" and="" paper="" industry="" samples="" for="" 2,3,7,8-tcdd="" and="" 2,3,7,8-tcdf,''="" national="" council="" of="" the="" paper="" industry="" for="" air="" and="" stream="" [[page="" 48426]]="" improvement="" inc.,="" 260="" madison="" avenue,="" new="" york,="" ny="" 10016,="" technical="" bulletin="" no.="" 551,="" pre-release="" copy,="" july="" 1988.="" 4.="" ``analytical="" procedures="" and="" quality="" assurance="" plan="" for="" the="" determination="" of="" pcdd/pcdf="" in="" fish,''="" usepa,="" environmental="" research="" laboratory,="" 6201="" congdon="" boulevard,="" duluth,="" mn="" 55804,="" april="" 1988.="" 5.="" tondeur,="" yves.="" ``proposed="" gc/ms="" methodology="" for="" the="" analysis="" of="" pcdds="" and="" pcdfs="" in="" special="" analytical="" services="" samples,''="" triangle="" laboratories,="" inc.,="" 801-10="" capitola="" dr,="" research="" triangle="" park,="" nc="" 27713,="" january="" 1988;="" updated="" by="" personal="" communication="" september="" 1988.="" 6.="" lamparski,="" l.l.="" and="" nestrick,="" t.j.="" ``determination="" of="" tetra-,="" hexa-,="" hepta-,="" and="" octachlorodibenzo-p-dioxin="" isomers="" in="" particulate="" samples="" at="" parts="" per="" trillion="" levels,''="" analytical="" chemistry,="" 52:="" 2045-2054,="" 1980.="" 7.="" lamparski,="" l.l.="" and="" nestrick,="" t.j.="" ``novel="" extraction="" device="" for="" the="" determination="" of="" chlorinated="" dibenzo-p-dioxins="" (pcdds)="" and="" dibenzofurans="" (pcdfs)="" in="" matrices="" containing="" water,''="" chemosphere,="" 19:27-31,="" 1989.="" 8.="" patterson,="" d.g.,="" et.="" al.="" ``control="" of="" interferences="" in="" the="" analysis="" of="" human="" adipose="" tissue="" for="" 2,3,7,8-tetrachlorodibenzo-p-="" dioxin,''="" environmental="" toxicological="" chemistry,="" 5:355-360,="" 1986.="" 9.="" stanley,="" john="" s.="" and="" sack,="" thomas="" m.="" ``protocol="" for="" the="" analysis="" of="" 2,3,7,8-tetrachlorodibenzo-p-dioxin="" by="" high="" resolution="" gas="" chromatography/high="" resolution="" mass="" spectrometry,''="" usepa="" emsl,="" las="" vegas,="" nevada="" 89114,="" epa="" 600/4-86-004,="" january="" 1986.="" 10.="" ``working="" with="" carcinogens,''="" department="" of="" health,="" education,="" &="" welfare,="" public="" health="" service,="" centers="" for="" disease="" control,="" niosh,="" publication="" 77-206,="" august="" 1977,="" ntis="" pb-277256.="" 11.="" ``osha="" safety="" and="" health="" standards,="" general="" industry,''="" osha="" 2206,="" 29="" cfr="" 1910.="" 12.="" ``safety="" in="" academic="" chemistry="" laboratories,''="" acs="" committee="" on="" chemical="" safety,="" 1979.="" 13.="" ``standard="" methods="" for="" the="" examination="" of="" water="" and="" wastewater,''="" 18th="" edition="" and="" later="" revisions,="" american="" public="" health="" association,="" 1015="" 15th="" st,="" n.w.,="" washington,="" dc="" 20005,="" 1-35:="" section="" 1090="" (safety),="" 1992.="" 14.="" ``method="" 613--2,3,7,8-tetrachlorodibenzo-p-dioxin,''="" 40="" cfr="" 136="" (49="" fr="" 43234),="" october="" 26,="" 1984,="" section="" 4.1.="" 15.="" provost,="" l.p.="" and="" elder,="" r.s.="" ``interpretation="" of="" percent="" recovery="" data,''="" american="" laboratory,="" 15:="" 56-83,="" 1983.="" 16.="" ``standard="" practice="" for="" sampling="" water,''="" astm="" annual="" book="" of="" standards,="" astm,="" 1916="" race="" street,="" philadelphia,="" pa="" 19103-1187,="" 1980.="" 17.="" ``methods="" 330.4="" and="" 330.5="" for="" total="" residual="" chlorine,''="" usepa,="" emsl,="" cincinnati,="" oh="" 45268,="" epa="" 600/4-79-020,="" march="" 1979.="" 18.="" ``handbook="" of="" analytical="" quality="" control="" in="" water="" and="" wastewater="" laboratories,''="" usepa="" emsl,="" cincinnati,="" oh="" 45268,="" epa-600/4-="" 79-019,="" march="" 1979.="" 19.="" williams,="" rick.="" letter="" to="" bill="" telliard,="" june="" 4,="" 1993,="" available="" from="" the="" epa="" sample="" control="" center="" operated="" by="" dyncorp="" viar,="" inc.,="" 300="" n="" lee="" st,="" alexandria,="" va="" 22314,="" 703-519-1140.="" 20.="" barkowski,="" sarah.="" fax="" to="" sue="" price,="" august="" 6,="" 1992,="" available="" from="" the="" epa="" sample="" control="" center="" operated="" by="" dyncorp="" viar,="" inc.,="" 300="" n="" lee="" st,="" alexandria="" va="" 22314,="" 703-519-1140.="" 21.="" ``analysis="" of="" multi-media,="" multi-concentration="" samples="" for="" dioxins="" and="" furans,="" pcdd/pcdf="" analyses="" data="" package'',="" narrative="" for="" episode="" 4419,="" mri="" project="" no.="" 3091-a,="" op.cit.="" february="" 12,="" 1993,="" available="" from="" the="" epa="" sample="" control="" center="" operated="" by="" dyncorp="" viar="" inc,="" 300="" n="" lee="" st,="" alexandria,="" va="" 22314="" (703-519-1140).="" 22.="" ``analytical="" procedures="" and="" quality="" assurance="" plan="" for="" the="" determination="" of="" pcdd/pcdf="" in="" fish'',="" u.s.="" environmental="" protection="" agency,="" environmental="" research="" laboratory,="" duluth,="" mn="" 55804,="" epa/600/3-="" 90/022,="" march="" 1990.="" 23.="" afghan,="" b.k.,="" carron,="" j.,="" goulden,="" p.d.,="" lawrence,="" j.,="" leger,="" d.,="" onuska,="" f.,="" sherry,="" j.,="" and="" wilkenson,="" r.j.,="" ``recent="" advances="" in="" ultratrace="" analysis="" of="" dioxins="" and="" related="" halogenated="" hydrocarbons'',="" can="" j.="" chem.,="" 65:="" 1086-1097,="" 1987.="" 24.="" sherry,="" j.p.="" and="" tse,="" h.="" ``a="" procedure="" for="" the="" determination="" of="" polychlorinated="" dibenzo-p-dioxins="" in="" fish'',="" chemosphere,="" 20:="" 865-872,="" 1990.="" 25.="" ``preliminary="" fish="" tissue="" study'',="" results="" of="" episode="" 4419,="" available="" from="" the="" epa="" sample="" control="" center="" operated="" by="" dyncorp="" viar,="" inc.,="" 300="" n="" lee="" st,="" alexandria,="" va="" 22314,="" 703-519-1140.="" 26.="" nestrick,="" terry="" l.="" dow="" chemical="" co.,="" personal="" communication="" with="" d.r.="" rushneck,="" april="" 8,="" 1993.="" details="" available="" from="" the="" u.s.="" environmental="" protection="" agency="" sample="" control="" center="" operated="" by="" dyncorp="" viar="" inc,="" 300="" n="" lee="" st,="" alexandria,="" va="" 22314,="" 703-519-1140.="" 27.="" barnstadt,="" michael.="" ``big="" fish="" column'',="" triangle="" laboratories="" of="" rtp,="" inc.,="" sop="" 129-90,="" 27="" march="" 27,="" 1992.="" 28.="" ``determination="" of="" polychlorinated="" dibenzo-p-dioxins="" (pcdd)="" and="" dibenzofurans="" (pcdf)="" in="" environmental="" samples="" using="" epa="" method="" 1613'',="" chemical="" sciences="" department,="" midwest="" research="" institute,="" 425="" volker="" boulevard,="" kansas="" city,="" mo="" 44110-2299,="" standard="" operating="" procedure="" no.="" cs-153,="" january="" 15,="" 1992.="" 29.="" ryan,="" john="" j.="" raymonde="" lizotte="" and="" william="" h.="" newsome,="" j.="" chromatog.="" 303="" (1984)="" 351-360.="" 30.="" telliard,="" william="" a.,="" mccarty,="" harry="" b.,="" and="" riddick,="" lynn="" s.="" ``results="" of="" the="" interlaboratory="" validation="" study="" of="" usepa="" method="" 1613="" for="" the="" analysis="" of="" tetra-through="" octachlorinated="" dioxins="" and="" furans="" by="" isotope="" dilution="" gc/ms,''="" chemosphere,="" 27,="" 41-46="" (1993).="" 31.="" ``results="" of="" the="" international="" interlaboratory="" validation="" study="" of="" usepa="" method="" 1613'',="" october="" 1994,="" available="" from="" the="" epa="" sample="" control="" center="" operated="" by="" dyncorp="" viar,="" inc.,="" 300="" n="" lee="" st,="" alexandria,="" va="" 22314,="" 703-519-1140.="" 23.0="" tables="" and="" figures="" table="" 1.--chlorinated="" dibenzo-p-dioxins="" and="" furans="" determined="" by="" isotope="" dilution="" and="" internal="" standard="" high="" resolution="" gas="" chromatography="" (hrgc)/high="" resolution="" mass="" spectrometry="" (hrms)="" ----------------------------------------------------------------------------------------------------------------="" cdds/cdfs="" \1\="" cas="" registry="" labeled="" analog="" cas="" registry="" ----------------------------------------------------------------------------------------------------------------="" 2,3,7,8-tcdd................="" 1746-01-6="">13C12-2,3,7,8-TCDD................................ 76523-40-5
37Cl4-2,3,7,8-TCDD................................ 85508-50-5
Total TCDD.................. 41903-57-5
2,3,7,8-TCDF................ 51207-31-9 13C12-2,3,7,8-TCDF................................ 89059-46-1
Total-TCDF.................. 55722-27-5
1,2,3,7,8-PeCDD............. 40321-76-4 13C12-1,2,3,7,8-PeCDD............................. 109719-79-1
Total-PeCDD................. 36088-22-9
1,2,3,7,8-PeCDF............. 57117-41-6 13C12-1,2,3,7,8-PeCDF............................. 109719-77-9
[[Page 48427]]
2,3,4,7,8-PeCDF............. 57117-31-4 13C12-2,3,4,7,8-PeCDF............................. 116843-02-8
Total-PeCDF................. 30402-15-4
1,2,3,4,7,8-HxCDD........... 39227-28-6 13C12-1,2,3,4,7,8-HxCDD........................... 109719-80-4
1,2,3,6,7,8-HxCDD........... 57653-85-7 13C12-1,2,3,6,7,8-HxCDD........................... 109719-81-5
1,2,3,7,8,9-HxCDD........... 19408-74-3 13C12-1,2,3,7,8,9-HxCDD........................... 109719-82-6
Total-HxCDD................. 34465-46-8
1,2,3,4,7,8-HxCDF........... 70648-26-9 13C12-1,2,3,4,7,8-HxCDF........................... 114423-98-2
1,2,3,6,7,8-HxCDF........... 57117-44-9 13C12-1,2,3,6,7,8-HxCDF........................... 116843-03-9
1,2,3,7,8,9-HxCDF........... 72918-21-9 13C12-1,2,3,7,8,9-HxCDF........................... 116843-04-0
2,3,4,6,7,8-HxCDF........... 60851-34-5 13C12-2,3,4,6,7,8-HxCDF........................... 116843-05-1
Total-HxCDF................. 55684-94-1
1,2,3,4,6,7,8-HpCDD......... 35822-46-9 13C12-1,2,3,4,6,7,8-HpCDD......................... 109719-83-7
Total-HpCDD................. 37871-00-4
1,2,3,4,6,7,8-HpCDF......... 67562-39-4 13C12-1,2,3,4,6,7,8-HpCDF......................... 109719-84-8
1,2,3,4,7,8,9-HpCDF......... 55673-89-7 13C12-1,2,3,4,7,8,9-HpCDF......................... 109719-94-0
Total-HpCDF................. 38998-75-3
OCDD........................ 3268-87-9 13C12-OCDD........................................ 114423-97-1
OCDF........................ 39001-02-0 Not used..........................................
----------------------------------------------------------------------------------------------------------------
\1\ Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans.
TCDD = Tetrachlorodibenzo-p-dioxin.
TCDF = Tetrachlorodibenzofuran.
PeCDD = Pentachlorodibenzo-p-dioxin.
PeCDF = Pentachlorodibenzofuran.
HxCDD = Hexachlorodibenzo-p-dioxin.
HxCDF = Hexachlorodibenzofuran.
HpCDD = Heptachlorodibenzo-p-dioxin.
HpCDF = Heptachlorodibenzofuran.
OCDD = Octachlorodibenzo-p-dioxin.
OCDF = Octachlorodibenzofuran.
Table 2.--Retention Time References, Quantitation References, Relative Retention Times, and Minimum Levels for CDDS and DCFS
--------------------------------------------------------------------------------------------------------------------------------------------------------
Minimum level \1\
----------------------------------
CDD/CDF Retention time and quantitation reference Relative Extract (pg/
retention time Water (pg/ Solid (ng/ L;
L; ppq) kg; ppt) ppb)
--------------------------------------------------------------------------------------------------------------------------------------------------------
Compounds using 13 C12-1,2,3,4-TCDD as the Injection Internal Standard
--------------------------------------------------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDF........................... \13\ C12-2,3,7,8-TCDF....................................... 0.999-1.003 10 1 0.5
2,3,7,8-TCDD........................... \13\ C12-2,3,7,8-TCDD....................................... 0.999-1.002 10 1 0.5
1,2,3,7,8-Pe........................... \13\ C12-1,2,3,7,8-PeCDF.................................... 0.999-1.002 50 5 2.5
2,3,4,7,8-PeCDF........................ \13\ C12-2,3,4,7,8-PeCDF.................................... 0.999-1.002 50 5 2.5
1,2,3,7,8-PeCDD........................ \13\ C12-1,2,3,7,8-PeCDD.................................... 0.999-1.002 50 5 2.5
\13\ C12-2,3,7,8-TCDF.................. \13\ C12-1,2,3,4-TCDD....................................... 0.923-1.103 ......... ......... ...........
\13\ C12-2,3,7,8-TCDD.................. \13\ C12-1,2,3,4-TCDD....................................... 0.976-1.043 ......... ......... ...........
\13\ C12-2,3,7,8-TCDD.................. \13\ C12-1,2,3,4-TCDD....................................... 0.989-1.052 ......... ......... ...........
\13\ C12-1,2,3,7,8-PeCDF............... \13\ C12-1,2,3,4-TCDD....................................... 1.000-1.425 ......... ......... ...........
\13\ C12-2,3,4,7,8-PeCDF............... \13\ C12-1,2,3,4-TCDD....................................... 1.001-1.526 ......... ......... ...........
\13\ C12-1,2,3,7,8-PeCDF............... \13\ C12-1,2,3,4-TCDD....................................... 1.000-1.567 ......... ......... ...........
--------------------------------------------------------------------------------------------------------------------------------------------------------
Compounds using 13 C12-1,2,3,7,8,9-HxCDD as the Injection Internal Standard
--------------------------------------------------------------------------------------------------------------------------------------------------------
1,2,3,4,7,8-HxCDF...................... \13\ C12-1,2,3,4,7,8-HxCDF.................................. 0.999-1.001 50 5 2.5
1,2,3,6,7,8-HxCDF...................... \13\ C12-1,2,3,6,7,8-HxCDF.................................. 0.997-1.005 50 5 2.5
1,2,3,7,8,9-HxCDF...................... \13\ C12-1,2,3,7,8,9-HxCDF.................................. 0.999-1.001 50 5 2.5
2,3,4,6,7,8-HxCDF...................... \13\ C12-2,3,4,6,7,8-HxCDF.................................. 0.999-1.001 50 5 2.5
1,2,3,4,7,8-HxCDD...................... \13\ C12-1,2,3,4,7,8-HxCDD.................................. 0.999-1.001 50 5 2.5
1,2,3,6,7,8-HxCDD...................... \13\ C12-1,2,3,6,7,8-HxCDD.................................. 0.998-1.004 50 5 2.5
1,2,3,7,8,9-HxCDD...................... (\2\)....................................................... 1.000-1.019 50 5 2.5
1,2,3,4,6,7,8-HpCDF.................... \13\ C12-1,2,3,4,6,7,8-HpCDF................................ 0.999-1.001 50 5 2.5
1,2,3,4,7,8,9-HpCDF.................... \13\ C12-1,2,3,4,7,8,9-HpCDF................................ 0.999-1.001 50 5 2.5
1,2,3,4,6,7,8-HpCDD.................... \13\ C12-1,2,3,4,6,7,8-HpCDD................................ 0.999-1.001 50 5 2.5
OCDF................................... \13\ C12-OCDD............................................... 0.999-1.001 100 10 5.0
OCDD................................... \13\ C12-OCDD............................................... 0.999-1.001 100 10 5.0
1,2,3,4,6,7,8,-HxCDF................... \13\ C12-1,2,3,7,8,9-HpCDD.................................. 0.949-0.975 ......... ......... ...........
\13\ C121,2,3,7,8,9-HxCDF.............. \13\ C12-1,2,3,7,8,9-HpCDD.................................. 0.977-1.047 ......... ......... ...........
\13\ C122,3,4,6,7,8,-HxCDF............. \13\ C12-1,2,3,7,8,9-HpCDD.................................. 0.959-1.021 ......... ......... ...........
\13\ C121,2,3,4,7,8,-HxCDF............. \13\ C12-1,2,3,7,8,9-HpCDD.................................. 0.977-1.000 ......... ......... ...........
[[Page 48428]]
\13\ C121,2,3,6,7,8,-HxCDF............. \13\ C12-1,2,3,7,8,9-HpCDD.................................. 0.981-1.003 ......... ......... ...........
\13\ C121,2,3,4,6,7,8-HxCDF............ \13\ C12-1,2,3,7,8,9-HpCDD.................................. 1.043-1.085 ......... ......... ...........
\13\ C121,2,3,4,7,8,9-HxCDF............ \13\ C12-1,2,3,7,8,9-HpCDD.................................. 1.057-1.151 ......... ......... ...........
\13\ C121,2,3,4,6,7,8-HxCDF............ \13\ C12-1,2,3,7,8,9-HpCDD.................................. 1.086-1.110 ......... ......... ...........
\13\ C12OCDD........................... \13\ C12-1,2,3,7,8,9-HpCDD.................................. 1.032-1.311 ......... ......... ...........
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ The Minimum Level (ML) for each analyte is defined as the level at which the entire analytical system must give a recognizable signal and acceptable
calibration point. It is equivalent to the concentration of the lowest calibration standard, assuming that all method-specified sample weights,
volumes, and cleanup procedures have been employed.
\2\ The retention time reference for 1,2,3,7,8,9-HxCDD is \13\C12-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is quantified using the averaged responses
for \13\C12-1,2,3,4,7,8-HxCDD and \13\C12-1,2,3,6,7,8-HxCDD.
Table 3.--Concentration of Stock and Spiking Solutions Containing CDDS/CDFS and Labeled Compounds
----------------------------------------------------------------------------------------------------------------
Labeled Labeled
compound compound PAR stock PAR spiking
CDD/CDF stock spiking solution solution \4\
solution \1\ solution \3\ (ng/mL) (ng/mL)
(ng/mL) \2\ (ng/
------------------------------------------------------------------------------mL)-------------------------------
2,3,7,8-TCDD.............................................. ............ ........... 40 0.8
2,3,7,8-TCDF.............................................. ............ ........... 40 0.8
1,2,3,7,8-PeCDD........................................... ............ ........... 200 4
1,2,3,7,8-PeCDF........................................... ............ ........... 200 4
2,3,4,7,8-PeCDF........................................... ............ ........... 200 4
1,2,3,4,7,8-HxCDD......................................... ............ ........... 200 4
1,2,3,6,7,8-HxCDD......................................... ............ ........... 200 4
1,2,3,7,8,9-HxCDD......................................... ............ ........... 200 4
1,2,3,4,7,8-HxCDF......................................... ............ ........... 200 4
1,2,3,6,7,8-HxCDF......................................... ............ ........... 200 4
1,2,3,7,8,9-HxCDF......................................... ............ ........... 200 4
2,3,4,6,7,8-HxCDF......................................... ............ ........... 200 4
1,2,3,4,6,7,8-HpCDD....................................... ............ ........... 200 4
1,2,3,4,6,7,8-HpCDF....................................... ............ ........... 200 4
1,2,3,4,7,8,9-HpCDF....................................... ............ ........... 200 4
OCDD...................................................... ............ ........... 400 8
OCDF...................................................... ............ ........... 400 8
13C12-2,3,7,8-TCDD........................................ 100 2 ........... ............
13C12-2,3,7,8-TCDF........................................ 100 2 ........... ............
13C12-1,2,3,7,8-PeCDD..................................... 100 2 ........... ............
13C12-1,2,3,7,8-PeCDF..................................... 100 2 ........... ............
13C12-2,3,4,7,8-PeCDF..................................... 100 2 ........... ............
13C12-1,2,3,4,7,8-HxCDD................................... 100 2 ........... ............
13C12-1,2,3,6,7,8-HxCDD................................... 100 2 ........... ............
13C12-1,2,3,4,7,8-HxCDF................................... 100 2 ........... ............
13C12-1,2,3,6,7,8-HxCDF................................... 100 2 ........... ............
13C12-1,2,3,7,8,9-HxCDF................................... 100 2 ........... ............
13C12-2,3,4,6,7,8-HxCDF................................... 100 2 ........... ............
13C12-1,2,3,4,6,7,8-HpCDD................................. 100 2 ........... ............
13C12-1,2,3,4,6,7,8-HpCDF................................. 100 2 ........... ............
13C12-1,2,3,4,7,8,9-HpCDF................................. 100 2 ...