48394 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 tetrathrough 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

§ 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 Longterm 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 HCldigested 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.
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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, §§ 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, §§ 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-pertrillion (ppt) range for solid and semisolid sample matrices. EPA believes

Method 1613 is adequate and applicable

for the measurement of solid and semisolid 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
48396 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 realworld 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 mediumconcentration 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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 remixing, 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 preeluted 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
48398 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 backextracted 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 backextracted 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°C and

preserved as described above, aqueous

samples may be stored for up to one

year. Similarly, if stored in the dark at

<¥10°C, solid, semi-solid, and tissue

samples may be stored for up to one

year. Sample extracts are stored in the

dark at <¥10°C until analyzed. If stored

in the dark at <¥10°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 isomerspecific 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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 nondetectable 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.
48400 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 singlelaboratory 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 realworld samples because the Agency felt

that domestic and international

shipment of large volumes of dioxincontaining 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 realworld 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-ofthe-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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 startup 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.
48402 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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-ofthe-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 costeffective 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-ofinformation requirement to the Director

of the Office of Management and Budget

(OMB) for review and approval. This
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 § 136.3(a), Table 1C.—List of

Approved Test Procedures for Nonpesticide 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:

§ 136.3 Identification of test procedures.

* * * * *

TABLE 1C.—LIST OF APPROVED TEST PROCEDURES FOR NON-PESTICIDE ORGANIC COMPOUNDS

Parameter 1 GC

EPA method number 2, 7

ASTM Other

GC/MS HPLC Standard methods

18th ed.

* * * * * * *

60. 1,2,3,4,6,7,8-

Heptachlorodibenzofuran.

............ 1613 ....................... ............

61. 1,2,3,4,7,8,9-

Heptachlorodibenzofuran.

............ 1613 ....................... ............

62. 1,2,3,4,6,7,8-Heptachlorodibenzo-pdioxin............. 1613 ....................... ............

63. Hexachlorobenzene ......................... 612 625, 1625 .............. ............ 6410B

64. Hexachlorobutadiene ....................... 612 625, 1625 .............. ............ 6410B

65. Hexachlorocyclopentadiene ............. 612 625, 1625 5 ............ ............ 6410B

66. 1,2,3,4,7,8-Hexachlorodibenzofuran ............ 1613 ....................... ............

67. 1,2,3,6,7,8-Hexachlorodibenzofuran ............ 1613 ....................... ............

68. 1,2,3,7,8,9-Hexachlorodibenzofuran ............ 1613 ....................... ............

69. 2,3,4,6,7,8-Hexachlorodibenzofuran ............ 1613 ....................... ............

70. 1,2,3,4,7,8-Hexachlorodibenzo-pdioxin............. 1613 ....................... ............

71. 1,2,3,6,7,8-Hexachlorodibenzo-pdioxin............. 1613 ....................... ............

72. 1,2,3,7,8,9-Hexachlorodibenzo-pdioxin............. 1613 ....................... ............

73. Hexachloroethane ............................ 616 625, 1625 .............. ............ 6410B

74. Ideno(1,2,3-cd)pyrene ..................... 610 625, 1625 .............. 610 6410B, 6440B D4657–87

75. Isophorone ....................................... 609 625, 1625 .............. ............ 6410B

76. Methylene chloride .......................... 601 624, 1624 .............. ............ 6230B Note 3, p. 130.

77. 2-Methyl-4,6-dinitrophenol ............... 604 625, 1625 .............. ............ 6420B, 6410B

78. Naphthalene .................................... 610 625, 1625 .............. 610 6410B, 6440B

79. Nitrobenzene ................................... 609 625, 1625 .............. ............ 6410B D4657–87

80. 2-Nitrophenol ................................... 604 625, 1625 .............. ............ 6410B, 6420B

81. 4-Nitrophenol ................................... 604 625, 1625 .............. ............ 6410B, 6420B

82. N-Nitrosodimethylamine .................. 607 625, 1625 .............. ............ 6410B

83. N-Nitrosodi-n-propylamine ............... 607 625, 1625 5 ............ ............ 6410B

84. N-Nitrosodiphenylamine .................. 607 625, 1625 5 ............ ............ 6410B

85. Octachlorodibenzofuran ................... ............ 1613 ....................... ............

86. Octachlorodibenzo-p-dioxin ............. ............ 1613 ....................... ............

87. 2,2-Oxybis(1-chloropropane) ........... 611 625, 1625 .............. ............ 6410B

88. PCB–1016 ....................................... 608 625 ......................... ............ 6410B Note 3, p. 43.

89. PCB–1221 ....................................... 608 625 ......................... ............ 6410B Note 3, p. 43.

90. PCB–1232 ....................................... 608 625 ......................... ............ 6410B Note 3, p. 43.

91. PCB 1242 ........................................ 608 625 ......................... ............ 6410B Note 3, p. 43.

92. PCB–1248 ....................................... 608 625 ......................... ............

93. PCB–1254 ....................................... 608 625 ......................... ............ 6410B Note 3, p. 43.

94. PCB–1260 ....................................... 608 625 ......................... ............ 6410B, 6630B Note 3, p. 43.

95. 1,2,3,7,8-Pentachlorodibenzofuran .. ............ 1613 ....................... ............

96. 2,3,4,7,8-Pentachlorodibenzofuran .. ............ 1613 ....................... ............
48404 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

TABLE 1C.—LIST OF APPROVED TEST PROCEDURES FOR NON-PESTICIDE ORGANIC COMPOUNDS—Continued

Parameter 1 GC

EPA method number 2, 7

ASTM Other

GC/MS HPLC Standard methods

18th ed.

97. 1,2,3,7,8-Pentachlorodibenzo-pdioxin............. 1613 ....................... ............

98. Pentachlorophenol 604 625, 1625 .............. ............ 6410B, 6630B Note 3, p. 140.

99. Phenanthrene .................................. 610 625, 1625 .............. 610 6410B, 6440B D4657–87

100. Phenol ............................................ 604 625, 1625 .............. ............ 6420B, 6410B

101. Pyrene ........................................... 610 625, 1625 .............. 610 6410B, 6440B D4657–87

102. 2,3,7,8-Tetrachlorodibenzofuran .... ............ 1613 ....................... ............

103. 2,3,7,8-Tetrachlorodibenzo-pdioxin............. 613, 1613 5 ............ ............

104. 1,1,2,2-Tetrachloroethane ............. 601 624, 1624 .............. ............ 6230B, 6210B Note 3, p. 130.

105. Tetrachloroethene .......................... 601 624, 1624 .............. ............ 6230B, 6410B Note 3, p. 130.

106. Toluene .......................................... 602 624, 1624 .............. ............ 6210B, 6220B

107. 1,2,4-Trichlorobenzene .................. 612 625, 1625 .............. ............ 6410B Note 3, p. 130.

108. 1,1,1-Trichloroethane ..................... 601 624, 1624 .............. ............ 6210B, 6230B

109. 1,1,2-Trichloroethane ..................... 601 624, 1624 .............. ............ 6210B, 6230B Note 3, p. 130.

110. Trichloroethene .............................. 601 624, 1624 .............. ............ 6210B, 6230B

111. Trichlorofluoromethane .................. 601 624 ......................... ............ 6210B, 6230B

112. 2,4,6-Trichlorophenol ..................... 604 625, 1625 .............. ............ 6410B, 6240B

113. Vinyl chloride ................................. 601 624, 1624 .............. ............ 6210B, 6230B

* * * * * * *

1All parameters are expressed in micrograms per liter (μg/L) except for Method 1613 in which the parameters are expressed in picograms per

liter (pg/L). 2The 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 § 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:

§ 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 § 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, 39–43,

45–47, 56, 76, 104, 105, 108–111,

113. Purgeable Halocarbons.

G, Teflon-lined septum ...... Cool, 4°C, 0.008% Na2S2O3 5. ................ 14 days.

6, 57, 106. Purgeable aromatic hydrocarbons.......do ................................. Cool, 4°C, 0.008% Na2S2O3,5 HCl to

pH29.

Do.

3, 4. Acrolein and acrylonitrile .......... ......do ................................. Cool, 4°C, 0.008% Na2S2O3,5 adjust pH

to 4–510.

Do.

23, 30, 44, 49, 53, 77, 80, 81, 98,

100, 112. Phenols 11.

G, Teflon-lined cap. ........... Cool, 4°C, 0.008% Na2S2O3 5 ................. 7 days until extraction; 40

days after extraction.

7, 38. Benzidines 11 ........................... ......do ................................. ......do ....................................................... 7 days until extraction.13

14, 17, 48, 50–52. Phthalate

esters 11.

......do ................................. Cool, 4°C ................................................. 7 days until extraction; 40

days after extraction.

82–84. Nitrosamines 11 14 .................. ......do ................................. Cool, 4°C, 0.008% Na2S2O3,5 store in

dark.

Do.

88–94. PCBs 11 ................................. .....do .................................. Cool, 4°C ................................................. Do.

54, 55, 75, 79. Nitroaromatics and

isophorone 11.

......do ................................. Cool, 4°C, 0.008% Na2S2O3,5 store in

dark.

Do.

1, 2, 5, 8–12, 32, 33, 58, 59, 74, 78,

99, 101. Polynuclear aromatic hydrocarbons 11.

......do ................................. ......do ....................................................... Do.

15, 16, 21, 31, 87. Haloethers 11 ...... ......do ................................. Cool, 4°C, 0.008% Na2S2O3 5 ................. Do.
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48405

TABLE II.—REQUIRED CONTAINERS, PRESERVATION TECHNIQUES, AND HOLDING TIMES—Continued

Parameter No./name Container 1 Preservation 2 3 Maximum holding time 4

29, 35–37, 63–65, 73, 107.

Chlorinated hydrocarbons 11.

......do ................................. Cool, 4°C ................................................. Do.

60–62, 66–72, 85, 86, 95–97, 102,

103. CDDs/CDFs 11

aqueous: field and lab preservation..G ........................................ Cool, 0–4°C, pH<9, 0.008% Na2S2O3 5 .. 1 year.

Solids, mixed phase, and tissue:

field preservation..

......do ................................. Cool, <4°C ............................................... 7 days.

Solids, mixed phase, and tissue: lab

preservation.

......do ................................. Freeze, <¥10°C ...................................... 1 year.

* * * * * * *

* * * * *

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-tetrachlorodibenzofuran (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 (partsper-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 ‘‘performancebased’’. 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 multiphase 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, 37Cl4-labeled

2,3,7,8-TCDD is added to each extract to

measure the efficiency of the cleanup

process. Sample cleanups may include
48406 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 ionabundance 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 tetraisomers.

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 preextracted 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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 highboiling 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 μg per wipe;

analysis using this method can achieve

an even lower detection limit. Less than

0.1 μg per wipe indicates acceptable

cleanliness; anything higher warrants

further cleaning. More than 10 μg 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
48408 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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°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°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 × 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 roundbottom 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—Wristaction 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—Solventextracted 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 glasswool 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

× 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 μL

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 highperformance 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 μL sample loop.

6.7.2.3 Column—Two 6.2 mm × 250

mm Zorbax-ODS columns in series
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48409

(DuPont Instruments Division,

Wilmington, DE, or equivalent),

operated at 50°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 × 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 × 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 × 15 mm ID,

with coarse-glass frit or glass-wool plug

and 250 mL reservoir.

6.7.4.3 300 mm long × 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 widemouth clear glass.

6.7.6 Oven—For baking and storage

of adsorbents, capable of maintaining a

constant temperature (±5°C) in the range

of 105–250°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°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°C,

installed in a fume hood.

6.8.3 Nitrogen blowdown

apparatus—Equipped with water bath

controlled in the range of 30–60°C (NEvap, 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—60±5 m long × 0.32±0.02 mm ID;

0.25 μm 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—30±5 m

long × 0.32±0.02 mm ID; 0.25 μm

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 BackExtraction.

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°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, lotcertified 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°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°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 screwcap.

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
48410 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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°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°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°C for a minimum of 12

hours.

7.5.2.2 Basic alumina—Supelco

19944–6C (or equivalent). Activate by

heating to 600°C for a minimum of 24

hours. Alternatively, activate by heating

in a tube furnace at 650–700°C under an

air flow rate of approximately 400 cc/

minute. Do not heat over 700°C, as this

can lead to reduced capacity for

retaining the analytes. Store at 130°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°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°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°C for a

minimum of four hours.

7.6.3 Paper reference matrix—Glassfiber 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 screwcapped 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,
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 μL 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 isomerspecificity 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°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°C from the time of collection until

receipt at the laboratory.

Store aqueous samples in the dark at

0–4°C. Store solid, semi-solid, oily,

mixed-phase, and tissue samples in the

dark at <¥10°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°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°C until prepared.

Maintain unused sample in the dark at

<¥10°C.

8.4 Holding Times.

8.4.1 There are no demonstrated

maximum holding times associated with

CDDs/CDFs in aqueous, solid, semisolid, tissues, or other sample matrices.

If stored in the dark at 0–4°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°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°C until analyzed. If stored

in the dark at <¥10°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 onethird 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:
48412 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

(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

13C12-1,2,3,4-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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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°C

Interface temperature: 290°C

Initial temperature: 200°C

Initial time: Two minutes

Temperature program:

200–220°C, at 5°C/minute

220°C for 16 minutes

220–235°C, at 5°C/minute

235°C for seven minutes

235–330°C, at 5°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

μL or 2 μL, consistent with the

capability of the HRGC/HRMS

instrument. Inject a 1 μL or 2 μL 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 retentiontime 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-
48414 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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:

RR

A A C

A A C

n n l

l l n

= ( ) +

( ) +

1 2

1 2

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 fivepoint 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:

RF

A A C

A A C

s s is

is is s

= ( ) +

( ) +

1 2

1 2

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

μL or 2.0 μL 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 fivepoint 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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 multiphase 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.

% solids = − × weight of sample aliquot after drying (g) weight of filter (g)

10 g

100

11.2.1.2 Filter 10.0±0.02 mL of wellmixed sample through the filter.11.2.1.3 Dry the filter a minimum of

12 hours at 110±5•Ψ ανδ ψοολ ιν α

δεσσιψατορ.

11.2.1.4 Calculate percent solids as

follows:

% solids = − × weight of sample aliquot after drying (g) weight of filter (g)

10 g

100

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 110±5°C, and cool in a dessicator.

11.2.2.3 Calculate percent solids as

follows:

% solids = × weight of sample aliquot after drying 

weight of sample aliquot before drying

100

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
48416 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 matrixdependent. 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 °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 °C. Return

any tissue that was not homogenized to

its original container and store at <¥10

°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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48417

reference matrix to a 500–600 mL widemouth 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 widemouth 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 onethird 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 glassfiber 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.
48418 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 preextracted 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°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:

Percent lipid = Weight of residue(g)

Weight of tissue (g)

×100

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 macroconcentration 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°C. Weigh the

receiver, record the weight, and return

the receiver to the blowdown apparatus,

concentrating the residue until a

constant weight is obtained.
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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. Reconcentrate 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 KudernaDanish 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°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
48420 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 backextraction 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 backextraction, transfer the extract to a

blowdown vial using two to three rinses

of solvent. Proceed with microconcentration 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°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 μL.

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 μL. 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 μL. Add

10 μL 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°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 backextraction (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 Biobeads (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 reextracted 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 μL

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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 glasswool 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/μL in methylene

chloride.

13.6.1.2 Inject 30 μL 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 octaisomers.

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 μL 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 reextracted 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 μL of extract. If the

extract cannot be concentrated to less

than 30 μL, 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 μL of methylene chloride

and reduce to 30 μL with the

evaporation apparatus (Section 12.7).
48422 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

13.6.2.2 Inject the 30 μL 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 μL 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 μL 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 backextraction’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 oncolumn 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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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, reprepare, 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
48424 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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:

C ng mL

A A C

A A RR ex

n n l

l l

( / ) = ( ) +

( ) +

1 2

1 2

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 non2,3,7,8-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 13Clabeled 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:

C ng mL

A A C

A A RF ex

s s is

is is

( / ) = ( ) +

( ) +

1 2

1 2

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 37Cllabeled 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:

Recovery (%) = Concentration found ( /mL)

Concentration spiked ( /mL)

μ

μ

g

g

×100

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:

Concentration in solid (ng/kg) = ( ) Cex × V

W

ex

s

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:

Concentration in (pg/ ) =

Cex aqueous phase LV

V

ex

s

( ) ×

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/μL in the extract, and analyze an

aliquot of this diluted extract by the

internal standard method.
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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 μL (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 μL 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°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
48426 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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 Dibenzop-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, Multiconcentration 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 .................................. 37Cl4-2,3,7,8-TCDD ...................................76523–40–5

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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48427

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)—Continued

CDDs/CDFs 1 CAS registry Labeled analog CAS registry

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

CDD/CDF Retention time and quantitation

reference

Relative retention time

Minimum level 1

Water

(pg/L;

ppq)

Solid (ng/

kg; ppt)

Extract

(pg/μL;

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 ................ ................ ................
48428 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

TABLE 2.—RETENTION TIME REFERENCES, QUANTITATION REFERENCES, RELATIVE RETENTION TIMES, AND MINIMUM

LEVELS FOR CDDS AND DCFS—Continued

CDD/CDF Retention time and quantitation

reference

Relative retention time

Minimum level 1

Water

(pg/L;

ppq)

Solid (ng/

kg; ppt)

Extract

(pg/μL;

ppb)

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 ................ ................ ................

1The 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. 2The retention time reference for 1,2,3,7,8,9-HxCDD is 13C12-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is quantified using the averaged responses for 13C12-1,2,3,4,7,8-HxCDD and 13C12-1,2,3,6,7,8-HxCDD.

TABLE 3.—CONCENTRATION OF STOCK AND SPIKING SOLUTIONS CONTAINING CDDS/CDFS AND LABELED COMPOUNDS

CDD/CDF

Labeled

compound

stock solution 1

(ng/mL)

Labeled

compound

spiking solution 2

(ng/mL)

PAR stock

solution 3

(ng/mL)

PAR spiking

solution 4

(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

37Cl4-2,3,7,8-TCDD ............................................................................................... 0.8 .................... .................... ......................

Internal Standards 6

13C12-1,2,3,4-TCDD ............................................................................................... 200 .................... .................... ...................... 13C12-1,2,3,7,8,9-HxCDD ....................................................................................... 200 .................... .................... ......................

1Section 7.10—prepared in nonane and diluted to prepare spiking solution.

2Section 7.10.3—prepared in acetone from stock solution daily.

3Section 7.9—prepared in nonane and diluted to prepare spiking solution.

4Section 7.14—prepared in acetone from stock solution daily.

5Section 7.11—prepared in nonane and added to extract prior to cleanup.

6Section 7.12—prepared in nonane and added to the concentrated extract immediately prior to injection into the GC (Section 14.2).
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48429

TABLE 4.—CONCENTRATION OF CDDS/CDFS IN CALIBRATION AND CALIBRATION VERIFICATION SOLUTIONS 1 (SECTION

15.3)

CDD/CDF CS2

(ng/mL)

CS3

(ng/mL)

CS4

(ng/mL)

CS5

(ng/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

CDD/CDF Test conc.

(ng/mL)

IPR 2 3

OPR

(ng/mL)

VER

s (ng/mL)

(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
48430 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

TABLE 6.—ACCEPTANCE CRITERIA FOR PERFORMANCE TESTS WHEN ALL CDDS/CDFS ARE TESTED 1—Continued

CDD/CDF Test conc.

(ng/mL)

IPR 2 3

OPR

(ng/mL)

VER

s (ng/mL)

(ng/mL)

X

(ng/mL)

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 13C12-2,3,7,8-TCDD .......................................................................... 100 37 28–134 20–175 82–121 13C12-2,3,7,8-TCDF ........................................................................... 100 35 31–113 22–152 71–140 13C12-1,2,3,7,8-PeCDD ..................................................................... 100 39 27–184 21–227 62–160 13C12-1,2,3,7,8-PeCDF ..................................................................... 100 34 27–156 21–192 76–130 13C12-2,3,4,7,8-PeCDF ..................................................................... 100 38 16–279 13–328 77–130 13C12-1,2,3,4,7,8-HxCDD .................................................................. 100 41 29–147 21–193 85–117 13C12-1,2,3,6,7,8-HxCDD .................................................................. 100 38 34–122 25–163 85–118 13C12-1,2,3,4,7,8-HxCDF .................................................................. 100 43 27–152 19–202 76–131 13C12-1,2,3,6,7,8-HxCDF .................................................................. 100 35 30–122 21–159 70–143 13C12-1,2,3,7,8,9-HxCDF .................................................................. 100 40 24–157 17–205 74–135 13C12-2,3,4,6,7,8,-HxCDF ................................................................. 100 37 29–136 22–176 73–137 13C12-1,2,3,4,6,7,8-HpCDD ............................................................... 100 35 34–129 26–166 72–138 13C12-1,2,3,4,6,7,8-HpCDF ............................................................... 100 41 32–110 21–158 78–129 13C12-1,2,3,4,7,8,9-HpCDF ............................................................... 100 40 28–141 20–186 77–129 13C12-OCDD ...................................................................................... 200 95 41–276 26–397 96–415 37Cl4-2,3,7,8-TCDD ........................................................................... 10 3.6 3.9–15.4 3.1–19.1 7.9–12.7

1All specifications are given as concentration in the final extract, assuming a 20 μL volume. 2 s = standard deviation of the concentration.

3X = average concentration.

TABLE 6A.—ACCEPTANCE CRITERIA FOR PERFORMANCE TESTS WHEN ONLY TETRA COMPOUNDS ARE TESTED 1

CDD/CDF Test Conc.

(ng/mL)

IPR 2 3 OPR

(ng/mL)

VER

(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

1All specifications are given as concentration in the final extract, assuming a 20 μL volume. 2 s = standard deviation of the concentration.

3X = average concentration.

TABLE 7.—LABELED COMPOUNDS RECOVERY IN SAMPLES WHEN ALL CDDS/CDFS ARE TESTED

Compound Test conc.

(ng/mL)

Labeled compound

recovery

(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
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48431

TABLE 7.—LABELED COMPOUNDS RECOVERY IN SAMPLES WHEN ALL CDDS/CDFS ARE TESTED—Continued

Compound Test conc.

(ng/mL)

Labeled compound

recovery

(ng/mL) 1 (%)

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

1Specification given as concentration in the final extract, assuming a 20-μL volume.

TABLE 7A.—LABELED COMPOUND RECOVERY IN SAMPLES WHEN ONLY TETRA COMPOUNDS ARE TESTED

Compound Test conc.

(ng/mL)

Labeled compound

recovery

(ng/mL) 1 (%)

13C12-2,3,7,8-TCDD ................................................................................................................................ 100 31–137 31–137 13C12-2,3,7,8-TCDF ................................................................................................................................ 100 29–140 29–140 37Cl4-2,3,7,8-TCDD ................................................................................................................................ 10 4.2–16.4 42–164

1Specification given as concentration in the final extract, assuming a 20 μL volume.

TABLE 8.—DESCRIPTORS, EXACT M/Z’S, M/Z TYPES, AND ELEMENTAL COMPOSITIONS OF THE CDDS AND CDFS

Descriptor Exact M/Z 1 M/Z type Elemental composition Substance 2

1 ........................ 292.9825 Lock C7F11 .................................................................................................................... PFK

303.9016 M C12H4

35Cl4O ......................................................................................................... TCDF

305.8987 M+2 C12H4

35Cl3

37ClO ..................................................................................................... TCDF

315.9419 M 13C12H4

35Cl4O ...................................................................................................... TCDF 3

317.9389 M+2 13C12H4

35Cl337ClO ................................................................................................. TCDF 3

319.8965 M C12H4

35Cl4O2 ........................................................................................................ TCDD

321.8936 M+2 C12H4

35Cl337ClO2 ................................................................................................. TCDD

327.8847 M C12H4

37Cl4O2 ........................................................................................................ TCDD 4

330.9792 QC C7F13 .................................................................................................................... PFK

331.9368 M 13C12H4

35Cl4O2 ..................................................................................................... TCDD 3

333.9339 M+2 13C12H4

35Cl337ClO2 .............................................................................................. TCDD 3

375.8364 M+2 C12H4

35Cl537ClO ................................................................................................... HxCDPE

2 ........................ 339.8597 M+2 C12H3

35Cl437ClO ................................................................................................... PeCDF

341.8567 M+4 C12H3

35Cl337Cl2O ................................................................................................. PeCDF

351.9000 M+2 13C12H3

35Cl437ClO ............................................................................................... PeCDF

353.8970 M+4 13C12H3

35Cl337Cl2O .............................................................................................. PeCDF 3

354.9792 Lock C9F13 .................................................................................................................... PFK

355.8546 M+2 C12H3

35Cl437ClO2 ................................................................................................. PeCDD

357.8516 M+4 C12H3

35Cl337Cl2O2 ................................................................................................ PeCDD

367.8949 M+2 13C12H3

35Cl437ClO2 .............................................................................................. PeCDD 3

369.8919 M+4 13C12H3

35Cl337Cl2O2 ............................................................................................. PeCDD 3

409.7974 M+2 C12H3

35Cl637ClO ................................................................................................... HpCDPE

3 ........................ 373.8208 M+2 C12H2

35Cl537ClO ................................................................................................... HxCDF

375.8178 M+4 C12H2

35Cl437Cl2O ................................................................................................. HxCDF

383.8639 M 13C12H2

35Cl6O ...................................................................................................... HxCDF 3

385.8610 M+2 13C12H2

35Cl537ClO ............................................................................................... HxCDF 3

389.8157 M+2 C12H2

35Cl537ClO2 ................................................................................................. HxCDD

391.8127 M+4 C12H2

35Cl437Cl2O2 ................................................................................................ HxCDD

392.9760 Lock C9F15 .................................................................................................................... PFK

401.8559 M+2 13C12H2

35Cl537ClO2 .............................................................................................. HxCDD 3

403.8529 M+4 13C12H2

35Cl437Cl2O2 ............................................................................................. HxCDD 3

430.9729 QC C9F17 .................................................................................................................... PFK

445.7555 M+4 C12H2

35Cl637Cl2O ................................................................................................. OCDPE

4 ........................ 407.7818 M+2 C12H35Cl6

37ClO .................................................................................................... HpCDF

409.7789 M+4 C12H35Cl5

37Cl2O ................................................................................................... HpCDF

417.8253 M 13C12H35Cl7O ....................................................................................................... HpCDF 3

419.8220 M+2 13C12H35Cl6

37ClO ................................................................................................. HpCDF 3

423.7766 M+2 C12H35Cl6

37ClO2 ................................................................................................... HpCDD

425.7737 M+4 C12H35Cl5

37Cl2O2 ................................................................................................. HpCDD

430.9729 Lock C9F17 .................................................................................................................... PFK

435.8169 M+2 13C12H35Cl6

37ClO2 ............................................................................................... HpCDD 3

437.8140 M+4 13C12H35Cl5

37Cl2O2 .............................................................................................. HpCDD 3

479.7165 M+4 C12H35Cl7

37Cl2O ................................................................................................... NCDPE
48432 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

TABLE 8.—DESCRIPTORS, EXACT M/Z’S, M/Z TYPES, AND ELEMENTAL COMPOSITIONS OF THE CDDS AND CDFS—

Continued

Descriptor Exact M/Z 1 M/Z type Elemental composition Substance 2

5 ........................ 441.7428 M+2 C1235Cl7

37ClO ....................................................................................................... OCDF

442.9728 Lock C10F17 ................................................................................................................... PFK

443.7399 M+4 C1235Cl6

37Cl2O ..................................................................................................... OCDF

457.7377 M+2 C1235Cl7

37ClO2 ..................................................................................................... OCDD

459.7348 M+4 C1235Cl6

37Cl2O2 .................................................................................................... OCDD

469.7779 M+2 13C1235Cl7

37ClO2 .................................................................................................. OCDD3

471.7750 M+4 13C1235Cl6

37Cl2O2 ................................................................................................. OCDD3

513.6775 M+4 C1235Cl8

37Cl2O ..................................................................................................... DCDPE

1 Nuclidic masses used:

H = 1.007825.

O = 15.994915.

C = 12.00000.

35Cl = 34.968853. 13C = 13.003355. 37Cl = 36.965903.

F = 18.9984. 2TCDD = 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.

4There is only one m/z for 37Cl4-2,3,7,8,-TCDD (cleanup standard).

TABLE 9.—THEORETICAL ION ABUNDANCE RATIOS AND QC LIMITS

Number of chlorine atoms M/Z’s forming ratio Theoretical

ratio

QC limit 1

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

1QC 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:
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48433

TABLE 10.—SUGGESTED SAMPLE QUANTITIES TO BE EXTRACTED FOR VARIOUS MATRICES 1—Continued

Sample Matrix 2 Example Percent solids Phase Quantity

extracted

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 ........................ ............................................

1The 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. 2The 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. 3Aqueous 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.

BILLING CODE 6560–50–P
48434 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48435
48436 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48437
48438 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 48439
48440 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

BILLING CODE 6560–50–C
Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations 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:

°C—degrees Celsius

μL—microliter

μm—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 tetrathrough octa-chlorodibenzo-p-dioxin.

CDF—Chlorinated Dibenzofuran—

The isomers and congeners of tetrathrough 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 methodspecified 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.
48442 Federal Register / Vol. 62, No. 178 / Monday, September 15, 1997 / Rules and Regulations

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