[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.

-----------------------------------------------------------------------

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

[[Page 48397]]

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% Na2S2O3 \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, \37\Cl4-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-2SR 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  ...........  ............
13C12-OCDD................................................         200              4  ...........  ............
Cleanup Standard \5\                                                                                            
    \37\Cl4-2,3,7,8-TCDD..................................           0.8  ...........  ...........  ............
Internal Standards \6\                                                                                          
    13C12-1,2,3,4-TCDD....................................         200    ...........  ...........  ............
    13C12-1,2,3,7,8,9-HxCDD...............................         200    ...........  ...........  ............
----------------------------------------------------------------------------------------------------------------
\1\ Section 7.10--prepared in nonane and diluted to prepare spiking solution.                                   
\2\ Section 7.10.3--prepared in acetone from stock solution daily.                                              
\3\ Section 7.9--prepared in nonane and diluted to prepare spiking solution.                                    
\4\ Section 7.14--prepared in acetone from stock solution daily.                                                
\5\ Section 7.11--prepared in nonane and added to extract prior to cleanup.                                     
\6\ Section 7.12--prepared in nonane and added to the concentrated extract immediately prior to injection into  
  the GC (Section 14.2).                                                                                        


[[Page 48429]]


  Table 4.--Concentration of CDDS/CDFS in Calibration and Calibration Verification Solutions \1\ (Section 15.3) 
----------------------------------------------------------------------------------------------------------------
                                                                            CS3  (ng/    CS4  (ng/    CS5  (ng/ 
                                                   CDD/CDF    CS2 (ng/mL)      mL)          mL)          mL)    
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDD..................................           0.5            2           10           40          200
2,3,7,8-TCDF..................................           0.5            2           10           40          200
1,2,3,7,8-PeCDD...............................           2.5           10           50          200         1000
1,2,3,7,8-PeCDF...............................           2.5           10           50          200         1000
2,3,4,7,8-PeCDF...............................           2.5           10           50          200         1000
1,2,3,4,7,8-HxCDD.............................           2.5           10           50          200         1000
1,2,3,6,7,8-HxCDD.............................           2.5           10           50          200         1000
1,2,3,7,8,9-HxCDD.............................           2.5           10           50          200         1000
1,2,3,4,7,8-HxCDF.............................           2.5           10           50          200         1000
1,2,3,6,7,8-HxCDF.............................           2.5           10           50          200         1000
1,2,3,7,8,9-HxCDF.............................           2.5           10           50          200         1000
2,3,4,6,7,8-HxCDF.............................           2.5           10           50          200         1000
1,2,3,4,6,7,8-HpCDD...........................           2.5           10           50          200         1000
1,2,3,4,6,7,8-HpCDF...........................           2.5           10           50          200         1000
1,2,3,4,7,8,9-HpCDF...........................           2.5           10           50          200         1000
OCDD..........................................           5.0           20          100          400         2000
OCDF..........................................           5.0           20          100          400         2000
\13\ C12-2,3,7,8-TCDD.........................         100            100          100          100          100
\13\ C12-2,3,7,8-TCDF.........................         100            100          100          100          100
\13\ C12-1,2,3,7,8-PeCDD......................         100            100          100          100          100
\13\ C12-PeCDF................................         100            100          100          100          100
\13\ C12-2,3,4,7,8-PeCDF......................         100            100          100          100          100
\13\ C12-1,2,3,4,7,8-HxCDD....................         100            100          100          100          100
\13\ C12-1,2,3,6,7,8-HxCDD....................         100            100          100          100          100
\13\ C12-1,2,3,4,7,8-HxCDF....................         100            100          100          100          100
\13\ C12-1,2,3,6,7,8-HxCDF....................         100            100          100          100          100
\13\ C12-1,2,3,7,8,9-HxCDF....................         100            100          100          100          100
\13\ C12-1,2,3,4,6,7,8-HpCDD..................         100            100          100          100          100
\13\ C12-1,2,3,4,6,7,8-HpCDF..................         100            100          100          100          100
\13\ C12-1,2,3,4,7,8,9-Hp CDF.................         100            100          100          100          100
\13\ C12-OCDD.................................         200            200          200          200          200
Cleanup Standard:                                                                                               
    \37\ C14-2,3,7,8-TCDD.....................           0.5            2           10           40          200
Internal Standards:                                                                                             
\13\ C12-1,2,3,4-TCDD.........................         100            100          100          100          100
\13\ C12-1,2,3,7,8,9-HxCDD....................         100            100          100          100          100
----------------------------------------------------------------------------------------------------------------


    Table 5.--GC Retention Time Window Defining Solution and Isomer Specificity Test Standard (Section 7.15)    
----------------------------------------------------------------------------------------------------------------
                             DB-5 column GC retention-time window defining solution                             
-----------------------------------------------------------------------------------------------------------------
               CDD/CDF                                First eluted                          Last eluted         
----------------------------------------------------------------------------------------------------------------
TCDF.................................  1,3,6,8-..................................  1,2,8,9-                     
TCDD.................................  1,3,6,8-..................................  1,2,8,9-                     
PeCDF................................  1,3,4,6,8-................................  1,2,3,8,9-                   
PeCDD................................  1,2,4,7,9-................................  1,2,3,8,9-                   
HxCDF................................  1,2,3,4,6,8-..............................  1,2,3,4,8,9-                 
HxCDD................................  1,2,4,6,7,9-..............................  1,2,3,4,6,7-                 
HpCDF................................  1,2,3,4,6,7,8-............................  1,2,3,4,7,8,9-               
HpCDD................................  1,2,3,4,6,7,9-............................  1,2,3,4,6,7,8-               
----------------------------------------------------------------------------------------------------------------


               DB-5 Column TCDD Specificity Test Standard               
                                                                        
                          1,2,3,7+1,2,3,8-TCDD                          
                              2,3,7,8-TCDD                              
                              1,2,3,9-TCDD                              
                                                                        
           DB-225 Column TCDF Isomer Specificity Test Standard          
                                                                        
                              2,3,4,7-TCDF                              
                              2,3,7,8-TCDF                              
                              1,2,3,9-TCDF                              
                                                                        


              Table 6.--Acceptance Criteria for Performance Tests When All CDDS/CDFS Are Tested \1\             
----------------------------------------------------------------------------------------------------------------
                                                                    IPR 2 3                                     
                  CDD/CDF                     Test conc. ---------------------------- OPR  (ng/mL)  VER  (ng/mL)
                                               (ng/mL)     s  (ng/mL)    X  (ng/mL)                             
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDD...............................           10           2.8  8.3-12.9      6.7-15.8      7.8-12.9    

[[Page 48430]]

                                                                                                                
2,3,7,8-TCDF...............................           10           2.0  8.7-13.7      7.5-15.8      8.4-12.0    
1,2,3,7,8-PeCDD............................           50           7.5    38-66         35-71         39-65     
1,2,3,7,8-PeCDF............................           50           7.5    43-62         40-67         41-60     
2,3,4,7,8-PeCDF............................           50           8.6    36-75         34-80         41-61     
1,2,3,4,7,8-HxCDD..........................           50           9.4    39-76         35-82         39-64     
1,2,3,6,7,8-HxCDD..........................           50           7.7    42-62         38-67         39-64     
1,2,3,7,8,9-HxCDD..........................           50          11.1    37-71         32-81         41-61     
1,2,3,4,7,8-HxCDF..........................           50           8.7    41-59         36-67         45-56     
1,2,3,6,7,8-HxCDF..........................           50           6.7    46-60         42-65         44-57     
1,2,3,7,8,9-HxCDF..........................           50           6.4    42-61         39-65         45-56     
2,3,4,6,7,8-HxCDF..........................           50           7.4    37-74         35-78         44-57     
1,2,3,4,6,7,8-HpCDD........................           50           7.7    38-65         35-70         43-58     
1,2,3,4,6,7,8-HpCDF........................           50           6.3    45-56         41-61         45-55     
1,2,3,4,7,8,9-HpCDF........................           50           8.1    43-63         39-69         43-58     
OCDD.......................................          100          19     89-127        78-144        79-126     
OCDF.......................................          100          27     74-146        63-170        63-159     
\13\C12-2,3,7,8-TCDD.......................          100          37     28-134        20-175        82-121     
\13\C12-2,3,7,8-TCDF.......................          100          35     31-113        22-152        71-140     
\13\C12-1,2,3,7,8-PeCDD....................          100          39     27-184        21-227        62-160     
\13\C12-1,2,3,7,8-PeCDF....................          100          34     27-156        21-192        76-130     
\13\C12-2,3,4,7,8-PeCDF....................          100          38     16-279        13-328        77-130     
\13\C12-1,2,3,4,7,8-HxCDD..................          100          41     29-147        21-193        85-117     
\13\C12-1,2,3,6,7,8-HxCDD..................          100          38     34-122        25-163        85-118     
\13\C12-1,2,3,4,7,8-HxCDF..................          100          43     27-152        19-202        76-131     
\13\C12-1,2,3,6,7,8-HxCDF..................          100          35     30-122        21-159        70-143     
\13\C12-1,2,3,7,8,9-HxCDF..................          100          40     24-157        17-205        74-135     
\13\C12-2,3,4,6,7,8,-HxCDF.................          100          37     29-136        22-176        73-137     
\13\C12-1,2,3,4,6,7,8-HpCDD................          100          35     34-129        26-166        72-138     
\13\C12-1,2,3,4,6,7,8-HpCDF................          100          41     32-110        21-158        78-129     
\13\C12-1,2,3,4,7,8,9-HpCDF................          100          40     28-141        20-186        77-129     
\13\C12-OCDD...............................          200          95     41-276        26-397        96-415     
\37\Cl4-2,3,7,8-TCDD.......................           10           3.6  3.9-15.4      3.1-19.1      7.9-12.7    
----------------------------------------------------------------------------------------------------------------
\1\ All specifications are given as concentration in the final extract, assuming a 20 L volume.        
\2\ s = standard deviation of the concentration.                                                                
\3\ X = average concentration.                                                                                  


           Table 6a.--Acceptance Criteria for Performance Tests When Only Tetra Compounds are Tested 1          
----------------------------------------------------------------------------------------------------------------
                                                                    IPR 2 3                                     
                   CDD/CDF                     Test Conc. --------------------------- OPR  (ng/mL)  VER  (ng/mL)
                                                (ng/mL)     s (ng/mL)     X (ng/mL)                             
----------------------------------------------------------------------------------------------------------------
2,3,7,8-TCDD................................           10          2.7  8.7-12.4      7.314.6       8.2-12.3    
2,3,7,8-TCDF................................           10          2.0  9.1-13.1      8.0-14.7      8.6-11.6    
13C12-2,3,7,8-TCDD..........................          100           35   32-115        25-141        85-117     
13C12-2,3,7,8-TCDF..........................          100           34    35-99        26-126        76-131     
37Cl4-2,3,7,8-TCDD..........................           10          3.4  4.5-13.4      3.7-15.8      8.3-12.1    
----------------------------------------------------------------------------------------------------------------
1 All specifications are given as concentration in the final extract, assuming a 20 L volume.          
2 s = standard deviation of the concentration.                                                                  
3 X = average concentration.                                                                                    


 Table 7.--Labeled Compounds Recovery in Samples When all CDDS/CDFS are 
                                 Tested                                 
------------------------------------------------------------------------
                                                    Labeled compound    
                                   Test conc.           recovery        
            Compound                (ng/mL)   --------------------------
                                                 (ng/mL) 1       (%)    
------------------------------------------------------------------------
13C12-2,3,7,8-TCDD..............          100   25-164            25-164
13C12-2,3,7,8-TCDF..............          100   24-169            24-169
13C12-1,2,3,7,8-PeCDD...........          100   25-181            25-181
13C12-1,2,3,7,8-PeCDF...........          100   24-185            24-185
13C12-2,3,4,7,8-PeCDF...........          100   21-178            21-178
13C12-1,2,3,4,7,8-HxCDD.........          100   32-141            32-141
13C12-1,2,3,6,7,8-HxCDD.........          100   28-130            28-130
13C12-1,2,3,4,7,8-HxCDF.........          100   26-152            26-152
13C12-1,2,3,6,7,8-HxCDF.........          100   26-123            26-123
13C12-1,2,3,7,8,9-HxCDF.........          100   29-147            29-147

[[Page 48431]]

                                                                        
13C12-2,3,4,6,7,8-HxCDF.........          100   28-136            28-136
13C12-1,2,3,4,6,7,8-HpCDD.......          100   23-140            23-140
13C12-1,2,3,4,6,7,8-HpCDF.......          100   28-143            28-143
13C12-1,2,3,4,7,8,9-HpCDF.......          100   26-138            26-138
13C12-OCDD......................          200   34-313            17-157
37Cl4-2,3,7,8-TCDD..............           10  3.5-19.7           35-197
------------------------------------------------------------------------
1 Specification given as concentration in the final extract, assuming a 
  20-L volume.                                                 


     Table 7a.--Labeled Compound Recovery in Samples When Only Tetra    
                          Compounds are Tested                          
------------------------------------------------------------------------
                                                    Labeled compound    
                                   Test conc.           recovery        
            Compound                (ng/mL)   --------------------------
                                                (ng/mL) \1\      (%)    
------------------------------------------------------------------------
\13\C12-2,3,7,8-TCDD............          100   31-137            31-137
\13\C12-2,3,7,8-TCDF............          100   29-140            29-140
\37\Cl4-2,3,7,8-TCDD............           10  4.2-16.4           42-164
------------------------------------------------------------------------
\1\ Specification given as concentration in the final extract, assuming 
  a 20 L volume.                                               


         Table 8.--Descriptors, Exact M/Z's, M/Z Types, and Elemental Compositions of the CDDs and CDFs         
----------------------------------------------------------------------------------------------------------------
                            Exact M/Z                                                                           
        Descriptor             \1\               M/Z type            Elemental composition       Substance \2\  
----------------------------------------------------------------------------------------------------------------
1........................     292.9825  Lock                       C7F11....................  PFK               
                              303.9016  M                          C12H4\35\Cl4O............  TCDF              
                              305.8987  M+2                        C12H4\35\Cl3\37\ClO......  TCDF              
                              315.9419  M                          \13\C12H4\35\Cl4O........  TCDF \3\          
                              317.9389  M+2                        \13\C12H4\35\Cl3\37\ClO..  TCDF \3\          
                              319.8965  M                          C12H4\35\Cl4O2...........  TCDD              
                              321.8936  M+2                        C12H4\35\Cl3\37\ClO2.....  TCDD              
                              327.8847  M                          C12H4\37\Cl4O2...........  TCDD \4\          
                              330.9792  QC                         C7F13....................  PFK               
                              331.9368  M                          \13\C12H4\35\Cl4O2.......  TCDD \3\          
                              333.9339  M+2                        \13\C12H4\35\Cl3\37\ClO2.  TCDD \3\          
                              375.8364  M+2                        C12H4\35\Cl5\37\ClO......  HxCDPE            
2........................     339.8597  M+2                        C12H3\35\Cl4\37\ClO......  PeCDF             
                              341.8567  M+4                        C12H3\35\Cl3\37\Cl2O.....  PeCDF             
                              351.9000  M+2                        \13\C12H3\35\Cl4\37\ClO..  PeCDF             
                              353.8970  M+4                        \13\C12H3\35\Cl3\37\Cl2O.  PeCDF \3\         
                              354.9792  Lock                       C9F13....................  PFK               
                              355.8546  M+2                        C12H3\35\Cl4\37\ClO2.....  PeCDD             
                              357.8516  M+4                        C12H3\35\Cl3\37\Cl2O2....  PeCDD             
                              367.8949  M+2                        \13\C12H3\35\Cl4\37\ClO2.  PeCDD \3\         
                              369.8919  M+4                        \13\C12H3\35\Cl3\37\Cl2O2  PeCDD \3\         
                              409.7974  M+2                        C12H3\35\Cl6\37\ClO......  HpCDPE            
3........................     373.8208  M+2                        C12H2\35\Cl5\37\ClO......  HxCDF             
                              375.8178  M+4                        C12H2\35\Cl4\37\Cl2O.....  HxCDF             
                              383.8639  M                          \13\C12H2\35\Cl6O........  HxCDF \3\         
                              385.8610  M+2                        \13\C12H2\35\Cl5\37\ClO..  HxCDF \3\         
                              389.8157  M+2                        C12H2\35\Cl5\37\ClO2.....  HxCDD             
                              391.8127  M+4                        C12H2\35\Cl4\37\Cl2O2....  HxCDD             
                              392.9760  Lock                       C9F15....................  PFK               
                              401.8559  M+2                        \13\C12H2\35\Cl5\37\ClO2.  HxCDD \3\         
                              403.8529  M+4                        \13\C12H2\35\Cl4\37\Cl2O2  HxCDD \3\         
                              430.9729  QC                         C9F17....................  PFK               
                              445.7555  M+4                        C12H2\35\Cl6\37\Cl2O.....  OCDPE             
4........................     407.7818  M+2                        C12H\35\Cl6\37\ClO.......  HpCDF             
                              409.7789  M+4                        C12H\35\Cl5\37\Cl2O......  HpCDF             
                              417.8253  M                          \13\C12H\35\Cl7O.........  HpCDF \3\         
                              419.8220  M+2                        \13\C12H\35\Cl6\37\ClO...  HpCDF \3\         
                              423.7766  M+2                        C12H\35\Cl6\37\ClO2......  HpCDD             
                              425.7737  M+4                        C12H\35\Cl5\37\Cl2O2.....  HpCDD             
                              430.9729  Lock                       C9F17....................  PFK               
                              435.8169  M+2                        \13\C12H\35\Cl6\37\ClO2..  HpCDD \3\         
                              437.8140  M+4                        \13\C12H\35\Cl5\37\Cl2O2.  HpCDD \3\         
                              479.7165  M+4                        C12H\35\Cl7\37\Cl2O......  NCDPE             

[[Page 48432]]

                                                                                                                
5........................     441.7428  M+2                        C12\35\Cl7\37\ClO........  OCDF              
                              442.9728  Lock                       C10F17...................  PFK               
                              443.7399  M+4                        C12\35\Cl6\37\Cl2O.......  OCDF              
                              457.7377  M+2                        C12\35\Cl7\37\ClO2.......  OCDD              
                              459.7348  M+4                        C12\35\Cl6\37\Cl2O2......  OCDD              
                              469.7779  M+2                        \13\C12\35\Cl7\37\ClO2...  OCDD\3\           
                              471.7750  M+4                        \13\C12\35\Cl6\37\Cl2O2..  OCDD\3\           
                              513.6775  M+4                        C12\35\Cl8\37\Cl2O.......  DCDPE             
----------------------------------------------------------------------------------------------------------------
\1\ Nuclidic masses used:                                                                                       
  H = 1.007825.                                                                                                 
  O = 15.994915.                                                                                                
  C = 12.00000.                                                                                                 
  \35\Cl = 34.968853.                                                                                           
  \13\C = 13.003355.                                                                                            
  \37\Cl = 36.965903.                                                                                           
  F = 18.9984.                                                                                                  
\2\ TCDD = Tetrachlorodibenzo-p-dioxin.                                                                         
  PeCDD = Pentachlorodibenzo-p-dioxin.                                                                          
  HxCDD = Hexachlorodibenzo-p-dioxin.                                                                           
  HpCDD = Heptachlorodibenzo-p-dioxin.                                                                          
  OCDD = Octachlorodibenzo-p-dioxin.                                                                            
  HxCDPE = Hexachlorodiphenyl ether.                                                                            
  OCDPE = Octachlorodiphenyl ether.                                                                             
  DCDPE = Decachlorodiphenyl ether.                                                                             
  TCDF = Tetrachlorodibenzofuran.                                                                               
  PeCDF = Pentachlorodibenzofuran.                                                                              
  HxCDF = Hexachlorodibenzofuran.                                                                               
  HpCDF = Heptachlorodibenzofuran.                                                                              
  OCDF = Octachlorodibenzofuran.                                                                                
  HpCDPE = Heptachlorodiphenyl ether.                                                                           
  NCDPE = Nonachlorodiphenyl ether.                                                                             
  PFK = Perfluorokerosene.                                                                                      
\3\ Labeled compound.                                                                                           
\4\ There is only one m/z for \37\Cl4-2,3,7,8,-TCDD (cleanup standar                                      


                            Table 9.--Theoretical Ion Abundance Ratios and QC Limits                            
----------------------------------------------------------------------------------------------------------------
                                                                                              QC limit \1\      
        Number of chlorine atoms                M/Z's forming ratio        Theoretical -------------------------
                                                                              ratio        Lower        Upper   
----------------------------------------------------------------------------------------------------------------
4 \2\...................................  M/(M+2)........................         0.77         0.65         0.89
5.......................................  (M+2)/(M+4)....................         1.55         1.32         1.78
6.......................................  (M+2)/(M+4)....................         1.24         1.05         1.43
6 \3\...................................  M/(M+2)........................         0.51         0.43         0.59
7.......................................  (M+2)/(M+4)....................         1.05         0.88         1.20
7 \4\...................................  M/(M+2)........................         0.44         0.37         0.51
8.......................................  (M+2)/(M+4)....................         0.89         0.76        1.02 
----------------------------------------------------------------------------------------------------------------
\1\ QC limits represent 15% windows around the theoretical ion abundance ratios.                    
\2\ Does not apply to 37Cl4-2,3,7,8-TCDD (cleanup standard).                                                    
\3\ Used for 13C12-HxCDF only.                                                                                  
\4\ Used for 13C12-HpCDF only.                                                                                  


                                     Table 10.--Suggested Sample Quantities To Be Extracted for Various Matrices \1\                                    
--------------------------------------------------------------------------------------------------------------------------------------------------------
          Sample Matrix \2\                       Example             Percent solids                     Phase                      Quantity extracted  
--------------------------------------------------------------------------------------------------------------------------------------------------------
Single-phase:                                                                                                                                           
    Aqueous..........................  Drinking water...............              <1  (\3\).....................................  1000 mL.              
                                       Groundwater                    ..............  ..........................................  ......................
                                       Treated wastewater             ..............  ..........................................  ......................
    Solid............................  Dry soil.....................             >20  Solid.....................................  10 g.                 
                                       Compost                        ..............  ..........................................  ......................
                                       Ash                            ..............  ..........................................  ......................
    Organic..........................  Waste solvent................              <1  Organic...................................  10 g.                 
                                       Waste oil                      ..............  ..........................................  ......................
                                       Organic polymer                ..............  ..........................................  ......................
    Tissue...........................  Fish.........................  ..............  Organic...................................  10 g.                 
                                       Human adipose                  ..............  ..........................................  ......................
Multi-phase:                                                                                                                                            

[[Page 48433]]

                                                                                                                                                        
    Liquid/Solid:                                                                                                                                       
        Aqueous/Solid................  Wet soil.....................            1-30  Solid.....................................  10 g.                 
                                       Untreated effluent...........                                                                                    
                                       Digested municipal sludge....                                                                                    
                                       Filter cake..................                                                                                    
                                       Paper pulp...................                                                                                    
        Organic/solid................  Industrial sludge............           1-100  Both......................................  10 g.                 
                                       Oily waste                     ..............  ..........................................  ......................
    Liquid/Liquid:                                                                                                                                      
        Aqueous/organic..............  In-process effluent..........              <1  Organic...................................  10 g.                 
                                       Untreated effluent             ..............  ..........................................  ......................
                                       Drum waste                     ..............  ..........................................  ......................
        Aqueous/organic/solid........  Untreated effluent...........              >1  Organic and solid.........................  10 g.                 
                                       Drum waste                     ..............  ..........................................  ......................
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous samples containing 1% solids will   
  contain 10 g of solids. For aqueous samples containing greater than 1% solids, a lesser volume is used so that 10 g of solids (dry weight) will be    
  extracted.                                                                                                                                            
\2\ The sample matrix may be amorphous for some samples. In general, when the CDDs/CDFs are in contact with a multiphase system in which one of the     
  phases is water, they will be preferentially dispersed in or adsorbed on the alternate phase because of their low solubility in water.                
\3\ Aqueous samples are filtered after spiking with the labeled compounds. The filtrate and the materials trapped on the filter are extracted           
  separately, and the extracts are combined for cleanup and analysis.                                                                                   


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[[Page 48441]]

24.0  Glossary of Definitions and Purposes

    These definitions and purposes are specific to this method but have 
been conformed to common usage as much as possible.
    24.1  Units of weight and Measure and Their Abbreviations.
    24.1.1  Symbols:

 deg.C--degrees Celsius
L--microliter
m--micrometer
<--less than
>--greater than
%--percent

24.1.2  Alphabetical abbreviations:

amp--ampere
cm--centimeter
g--gram
h--hour
ID--inside diameter
in.--inch
L--liter
M--Molecular ion
m--meter
mg--milligram
min--minute
mL--milliliter
mm--millimeter
m/z--mass-to-charge ratio
N--normal; gram molecular weight of solute divided by hydrogen 
equivalent of solute, per liter of solution
OD--outside diameter
pg--picogram
ppb--part-per-billion
ppm--part-per-million
ppq--part-per-quadrillion
ppt--part-per-trillion
psig--pounds-per-square inch gauge
v/v--volume per unit volume
w/v--weight per unit volume

    24.2  Definitions and Acronyms (in Alphabetical Order).
    Analyte--A CDD or CDF tested for by this method. The analytes are 
listed in Table 1.
    Calibration Standard (CAL)--A solution prepared from a secondary 
standard and/or stock solutions and used to calibrate the response of 
the instrument with respect to analyte concentration.
    Calibration Verification Standard (VER)--The mid-point calibration 
standard (CS3) that is used in to verify calibration. See Table 4.
    CDD--Chlorinated Dibenzo-p-ioxin--The isomers and congeners of 
tetra-through octa-chlorodibenzo-p-dioxin.
    CDF--Chlorinated Dibenzofuran--The isomers and congeners of tetra-
through octa-chlorodibenzofuran.
    CS1, CS2, CS3, CS4, CS5--See Calibration standards and Table 4.
    Field Blank--An aliquot of reagent water or other reference matrix 
that is placed in a sample container in the laboratory or the field, 
and treated as a sample in all respects, including exposure to sampling 
site conditions, storage, preservation, and all analytical procedures. 
The purpose of the field blank is to determine if the field or sample 
transporting procedures and environments have contaminated the sample.
    GC--Gas chromatograph or gas chromatography.
    GPC--Gel permeation chromatograph or gel permeation chromatography.
    HPLC--High performance liquid chromatograph or high performance 
liquid chromatography.
    HRGC--High resolution GC.
    HRMS--High resolution MS.
    IPR--Initial precision and recovery; four aliquots of the diluted 
PAR standard analyzed to establish the ability to generate acceptable 
precision and accuracy. An IPR is performed prior to the first time 
this method is used and any time the method or instrumentation is 
modified.
    K-D--Kuderna-Danish concentrator; a device used to concentrate the 
analytes in a solvent.
    Laboratory Blank--See method blank.
    Laboratory Control sample (LCS)--See ongoing precision and recovery 
standard (OPR).
    Laboratory Reagent Blank--See method blank.
    May--This action, activity, or procedural step is neither required 
nor prohibited.
    May Not--This action, activity, or procedural step is prohibited.
    Method Blank--An aliquot of reagent water that is treated exactly 
as a sample including exposure to all glassware, equipment, solvents, 
reagents, internal standards, and surrogates that are used with 
samples. The method blank is used to determine if analytes or 
interferences are present in the laboratory environment, the reagents, 
or the apparatus.
    Minimum Level (ML)--The level at which the entire analytical system 
must give a recognizable signal and acceptable calibration point for 
the analyte. 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.
    MS--Mass spectrometer or mass spectrometry.
    Must--This action, activity, or procedural step is required.
    OPR--Ongoing precision and recovery standard (OPR); a laboratory 
blank spiked with known quantities of analytes. The OPR is analyzed 
exactly like a sample. Its purpose is to assure that the results 
produced by the laboratory remain within the limits specified in this 
method for precision and recovery.
    PAR--Precision and recovery standard; secondary standard that is 
diluted and spiked to form the IPR and OPR.
    PFK--Perfluorokerosene; the mixture of compounds used to calibrate 
the exact m/z scale in the HRMS.
    Preparation Blank--See method blank.
    Primary Dilution Standard--A solution containing the specified 
analytes that is purchased or prepared from stock solutions and diluted 
as needed to prepare calibration solutions and other solutions.
    Quality Control Check Sample (QCS)--A sample containing all or a 
subset of the analytes at known concentrations. The QCS is obtained 
from a source external to the laboratory or is prepared from a source 
of standards different from the source of calibration standards. It is 
used to check laboratory performance with test materials prepared 
external to the normal preparation process.
    Reagent Water--Water demonstrated to be free from the analytes of 
interest and potentially interfering substances at the method detection 
limit for the analyte.
    Relative Standard Deviation (RSD)--The standard deviation times 100 
divided by the mean. Also termed ``coefficient of variation.''
    RF--Response factor. See Section 10.6.1.
    RR--Relative response. See Section 10.5.2.
    RSD--See relative standard deviation.
    SDS--Soxhlet/Dean-Stark extractor; an extraction device applied to 
the extraction of solid and semi-solid materials (Reference 7).
    Should--This action, activity, or procedural step is suggested but 
not required.
    SICP--Selected ion current profile; the line described by the 
signal at an exact m/z.
    SPE--Solid-phase extraction; an extraction technique in which an 
analyte is extracted from an aqueous sample by passage over or through 
a material capable of reversibly adsorbing the analyte. Also termed 
liquid-solid extraction.

[[Page 48442]]

    Stock Solution--A solution containing an analyte that is prepared 
using a reference material traceable to EPA, the National Institute of 
Science and Technology (NIST), or a source that will attest to the 
purity and authenticity of the reference material.
    TCDD--Tetrachlorodibenzo-p-dioxin.
    TCDF--Tetrachlorodibenzofuran.
    VER--See calibration verification standard.

[FR Doc. 97-23841 Filed 9-12-97; 8:45 am]
BILLING CODE 6560-50-P