gy)
PB88-199526
Guidelines for the Determination of Halogenated
Dibenzo-p-Dioxins and Dibenzofurans in
Commercial Products (RE-ANNOUNCEMENT of
PB88-101050 - see notes field for explanation)
Midwest Research Inst. , Kansas City, MO
Prepared for
Environmental Protection Agency, Washington, DC
Sep 87
EJBD
ARCHIVE
EPA
560-
5-
87-
007
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
-------
United States Office of Pesticides EPA-560/5-87/007
Environmental Protection and Toxics Substances September 1987
Agency Washington DC 20460
6- __ P3d8-199526
§EPA Guidelines for the
Determination of Halogenated
Dlbenzo-p-Dioxins
and Dibenzofurans
in Commercial Products
3:
CD
US EPA
Headquarters and Chemical Libraries
EPA West Bldg Room 3340
Mailcode 3404T
1301 Constitution Ave NW
Washington DC 20004
202-566^556 Permanent Collection
REPRODUCED BY
U.S. DEPARTMENT OF COMMERCE
SerViCe
-------
TECHNICAL REPORT DATA
(Pleau read laurucnara on :ne mint snare eomntetmfi
I flS.aa«TNC.
EPA 560/5-87-007
AC=sssi
,. TITUS ANO :
Guidelines for the Determination of Halogenated
Dibenzo-o-dioxins and Dibenzofurans in Commercial
Products .^__
9.
3ATS
a.
:=cs
7. -I.THOHIS)
Oavid H. Stsale and Jonn S. Stanley
.3.'
8833A-01
9. "SPOH.vii.NG ORGANISATION .MA.VI6 ANO AOOHESS
Midwest Research Institute
*25 Volker Boulevard
Kansas City, MO 5-1110
sLsME.N
63-02-1252
12. jPCNSOfING AG
MAMS AAIO AOOHESS
.13.
Field Studies Sranch, TS-798
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington. DC 20*60
Final
14.S7CNSOPING AG£NCV ;3 = 6
Office of Toxic iuostances
U.S. Environ, '"-ot,
IS. SUPfM-SMBNTAav .NOTHS T^sie'C IV
-------
GUIDELINES FOR THE DETERMINATION OF HALOGENATED OIBENZO-fi-OIOXINS
AND OIBENZOFURANS IN COMMERCIAL PRODUCTS
by
David H. Staele
John S. Stanley
FINAL REPORT
Work Assignment No. 33
EPA Prime Contract No. 68-02-4252
MRI Project No. 8833-A01
June 19, 1987
Prepared for:
U.S. Environmental Protection Agency
Office of Pesticides and Toxic Substances
Field Studies Branch
401 M Street, S.W.
Washington, DC 20460
Attn: j^ Janet C. Remmers, Work Assignment Manager
Mr. Tom Murray, Work Assignment Manager
Dr. Joseph Breen, Program Manager
-------
DISCLAIMER
This document has been revised «*<«««*'?»?";£"*
-------
PREFACE
This document provides guidelines for the determination of haloge-
nated dibenzo-g-dioxins (HDDs) and dibenzofurans (HDFs) in commercial products
using instrumental methods of analysis. This report includes a brief review
of analytical requirements and existing methods for the analysis of HDDs and
HDFs, guidelines for sampling and instrumental analysis for these compounds,
and a quality assurance plan.
The analytical guidelines focus on high resolution gas chromatog-
raphy (HRGC) with either mass spectrometry (MS) or electron capture detectors
(ECD) for qualitative identification and quantisation. Bioanalytical tech-
niques may be a more cost-effective alternative, but these procedures are not
sufficiently validated at this time to be useful. Therefore, guidelines for
bioanalytical methods have not been included in this document.
Preparation of the document was initiated under MRI Project No. 8201
and completed under MRI Project No. 8800 for the U.S. Environmental Protection
Agency's (EPA) Office of Toxic Substances, Field Studies Branch, EPA Prime
Cor)^r|ct. ^gs.rs6jQp2-3938 and 68-02-4252, Work Assignments 41 and 33, respec-
tively,^. Tom Murray, Work Assignment Manager*, and Or. Joseph Breen, Project
Officer. A draft version of this report was submitted for EPA review and
approval on June 3, 1985, and a revised draft submitted on June 27, 1985.
The main body and Appendix A (analytical guidelines) of this document
were prepared by Mr. David H. Steele and Dr. John S. Stanley, Work Assignment
Leader, with input from Dr. Mitchell D. Erickson, Mr. Richard D. Brown, and
Ms. LeAnn L. Timmons. Appendices B, C, and D, containing the quality assurance
project plan and approaches for sampling commercial products for analysis,
were prepared by Dr. John Smith of EPA's Office of Toxic Substances Chemical
Regulations Branch.
MIDWEST RESEARCH INSTITUTE
•-P
aul C. Constant
Program Manager
Approved:
\_'>*- Co o>" •>
John E. Going, Director /
Chemical Sciences Department
n
-------
TABLE OF CONTENTS
Page
I. Introduction 1
II. Scope of Problem 1
A. Diversity of Sample Matrices 1
B. Potential Number of HDD and HDF Isomers 1
C. Potential Interferences 2
III. • General Analytical Method Considerations 3
A. Sample Extraction and Cleanup 3
B. Detection Method 3
C. Method Sensitivity a
0. Analytical Standards 6
E. Quality Assurance/Quality Control (QA/QC) 6
IV. Existing Methods 6
A. Instrumental Methods 11
B. Bioanalytical Methods 11
V. Proposed Method 12
Bibliography 14
Appendix A - Guidelines for the Analysis of Halogenated Dibenzo-
g-dioxins and Dibenzofurans in Commercial Products A-l
Appendix B - Quality Assurance Project Plan for Measurement of
Halogenated Dibenzo-g-dioxins and Dibenzofurans 8-1
Appendix C - Guidance for Sampling Halogenated Oibenzo-g-dioxins
and Dibenzofurans ' C-l
Appendix D - Guidance for a Sequential Approach to the Sampling of
Halogenated Dibenzo-g-dioxins and Dibenzofurans Q-l
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I. INTRODUCTION
The Environmental Protection Agency was petitioned (October 22,
1984) by the Environmental Defense Fund (EDF) and the National Wildlife
Federation (NWF), pursuant to Section 21 of the Toxic Substances Control Act
(TSCA), to issue rules and orders to prevent and reduce environmental contam-
ination by halogenated (specifically brominated and/or chlorinated) dibenzo-
g-dioxins and dibenzofurans (HDDs and HOFs). The Agency has reviewed the
Petition and has granted certain aspects. In addition, there are a number of
relevant Agency activities, planned or currently underway, which address the
Petitioners' concerns. The Agency feels that in many cases it is necessary
to gather preliminary information before the Agency can make regulatory find-
ings. As part of the preliminary assessments, appropriate analytical metnoa-
ologies must be identified or developed.
The objective of this work assignment is to identify and/or develoo
appropriate analytical methodologies to quantitata HOOs and HOFs -in commercial
products. This report identifies the scope of the problem (Section II) and
general considerations for analytical methods (Section III) dealing with the
determination of HOOs and HOFs. Existing analytical methods for the deter-
mination of chlorinated dioxins and furans are discussed (Section IV), and
general guidelines for the sampling and analysis of HDDs and HOFs in commer-
cial products are provided in Section V and Appendix A. A quality assurance
project plan and guidance for sampling are contained in Appendices 8, C, and
0.
II. SCOPE OF PROBLEM
The development of analytical methodologies for the determination
of HOOs and HOFs in commercial products is complicated by the diverse range
of sample matrices, the potentially large number of brominated, chlorinated,
and mixed brominated/chlorinated congeners of the HDDs and HOFs, and by the
potential interferences which can be expected to be encountered. The follow-
ing subsections discuss these considerations individually.
A. Diversity of Sample Matrices
It is anticipated that commercial products which will be analyzed
under this program will range in volatility from chlorinated solvents to neavy
metal salts and dyes. Acids, bases, and neutral compounds with a wide range
of molecular geometries can be expected.
B. Potential Number of HDD and HDF Isomers
Both the dibenzodioxin and the dibenzofuran molecules have eight
positions where bromine or chlorine can substitute. These positions are
numbered as shown for the dibenzo-£-dioxin and dibenzofuran molecules below.
-------
The total number of unique positional isotners has been calculated
(MRI Interim Report, "Analytical Methodologies for Halogenated Oioxins and
Dibenzofurans in Commercial Products, May 1, 1985) at 1,700 HDD and 3,320 HDF
for bromo, chloro and bromo/chloro-substitution. Within these compounds there
are 351 HDDs and 666 HDFs with substitution in the 2,3,7,8-positions. The
breakdown of the exact number of positional isomers with degree of halogena-
tion are provided in the general method guidelines (Appendix A, Tables 1 and
2). Although the number of theoretical isomers is high, the actual number of
HDDs and HDFs in commercial products may be limited to relatively few. The
analyst must first determine the probability of the presence of bromo-, chloro-,
or bromo/chloro-HODs and HDFs in specific commercial products. - Clearly, a
chlorinated commercial product that has had no contact with bromine needs only
to be analyzed for PCDDs and PCDFs. Likewise, a brominated material produced
with no intermediate chlorinated materials, needs only to be analyzed for
brominated HDDs and HDFs (PBDDs and PBOFs). It is estimated that the bromo/
chloro-commercial products requiring analyses of all HDDs and HDFs are very
few.
C. Potential Interferences
Interferences are expected to arise from contaminants in solvents,
reagents, and glassware, as well as contaminants (halogenated diphenyl ethers,
halobenzyl phenyl ethers, bis(chlorophenoxy)methanes, and halogenated phenoxy-
phenols) which are coextracted with the HDDs and HDFs.
Halogenated diphenyl ethers yield mass spectral fragment ions with
exactly the same mass and number of halogen atoms as the corresponding HDFs.
Buser (1975) and Mieure et al. (1977) have demonstrated that basic alumina
column chromatography will separate the chlorodiphenyl ethers from the PCDDs
(polychlorinated dibenzo-g-dioxins) and PCDFs (polychlorinated dibenzofurans).
Likewise, Huckins et al. (1978) have demonstrated that a graphitized charcoal
column will remove the chlorodiphenyl ether interferences while selectively
isolating and concentrating chlorinated dibenzo-p_-dioxins.'
The halobenzyl phenyl ethers may cause mass spectral interferences
to HDDs with mass spectrometry selected ion monitoring (SIM) techniques. In-
terferences arise with these compounds when using electron capture detectors;
however, these compounds are distinguishable from PCDDs by mass spectrometry.
These compounds can also be removed from sample extracts by further cleanup
on alumina or carbon columns. The bis(chlorophenoxy)methanes have similar
gas chromatographic retention characteristics to PCDDs.
The halogenated phenoxyphenols can dehydrohalogenate in the hot
inlet of the gas chromatograph to form HDDs. This has been reported for the
chlorinated phenoxyphenols (Baker et al. 1981) and for 6-hydroxy-2,2',4,4',5,5'-
hexabromobiphenyl (Gardner et al. 1979). It is expected that analogous reac-
tions will occur for the mixed bromo/chloro phenoxyphenols". In order to avoid
false positives, these species must be excluded from the final extracts, or
the extracts must be methylated to prevent ring closure during injection.
In addition to interferences caused by contaminants in the sample
extracts, interferences may be caused by the HDDs and HDFs themselves. This
-------
will occur due to the potentially large number of isomers and to the ion
clusters produced by the isotopes of bromine and chlorine. The potential for
overlap of the isotope clusters becomes greater as the potential for mixed
bromo/chloro-HDDs and HDFs increases. For example, some interferences might
be anticipated for determination of the tetrabromodibenzo-£-dioxin (TBDD)
using ions m/z 500 and m/z 502. The interference may result as contribution
from dibromopentachlorodibenzofurans or bromoheptachlorodibenzo-g-dioxins
which exhibit the same ions in the molecular clusters (Appendix A, Table 5).
Again, it should be noted that the probability of this occurring may be small
and is undoubtedly a concern only for the mixed bromo/chloro-commercial prod-
ucts. Since little is known regarding the chemical and physical nature of
bromo and bromo/chloro-HDDs and HDFs, it is not clear whether these interfer-
ences will be resolved by HRGC retention time. The masses and relative aoun-
dances of the ions in the molecular ion clusters of the HDDs and HDFs are pre-
sented as part of the method guidelines (Appendix A). Possible interferences
from the molecular ion clusters of the HDDs and HDFs are also presented in
the method guidelines.
III. GENERAL ANALYTICAL METHOD CONSIDERATIONS
The analytical procedures necessary for the quantitative measurement
of HDDs and HDFs in commercial products include (1) the quantitative extraction
or partitioning of the analytes from the commercial product, (2) separation of
the HDDs and HDFs from interferences present in the extract, and (3) separation
and quantitation of homologous series or specific congeners by gas chromatog-
raphy with mass spectral or electron capture detection. Some general consid-
erations concerning these procedures are discussed below.
A. Sample Extraction and Cleanup
The most significant difference in the analysis of HDDs and HDFs in
commercial products in comparison with environmental and biological samples
will be the extraction and cleanup procedures. The physical and chemical
properties of environmental and biological matrices are typically different
enough from the properties of the analytes to allow relative ease of separa-
tion. In contrast, the commercial products in most cases may be structurally
similar to the analytes, complicating the separation and necessitating the
complete removal of the matrix to avoid interferences in the final determina-
tion.
B. Detection Method
Most of the analyses of commercial products for polychlorinated
dioxins (PCDDs), dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and
polybrominated biphenyls (PBBs) that have been reported in-.the literature have
employed mass spectrometry (MS) detection. Mass spectrometry will provide
both qualitative and quantitative data for HDDs and HOFs. In addition, its
primary advantage over other detectors is provided by confirmation of the
presence of a specific analyte. Mass spectrometry is a proven tool for pro-
viding quantitative information at concentration levels ranging from micro-
grams per gram to picograms per gram.
-------
Several options are available for mass spectrometry analyses includ-
ing (1) full mass scan analysis (FMS), which provides identification based on
complete mass spectra of individual components; (2) limited mass scan (LMS),
which allows the analyst to monitor small mass ranges, possibly full molecular
ion clusters; and (3) selected ion monitoring (SIM), which permits the analyst
to select specific ions characteristic of the target compounds. The mass spec-
trometery method sensitivity is increased by decreasing the range of masses
or number of ions monitored during an analysis. Thus the SIM method is more
sensitive than the LMS or FMS procedures.. Full scan mass spectrometry is
useful for measurements for the 50 ng/g and higher concentration levels, while
the SIM technique will allow measurements as low as 1-10 pg/g. Some caution
must be exercised in approaching the final analysis since the lower detection
levels require that sample extracts be relatively free of interference.
Another option that is available is high resolution mass spectrom-
etry (HRMS). This approach can be used with any of the techniques discussed
above. The advantage of HRMS is that it allows the analyst to lower the con-
tribution of signal due to background and interferences by monitoring the
exact masses of the analytes. This usually results in an observed increase
of signal to noise and corresponding lower limit of detection (LOD). The
major drawbacks of HRMS involve the availability of instrumentation, experi-
enced analysts, cost, and sample turnaround time.
Electron capture detection (ECO) has proven to be effective for
monitoring PCDD and PCDF contaminants at the microgram-per-gram (ppm) level
in some commercial products (pentachlorophenol, Mieure et al. 1977). It is
expected that this technique may be suitable for the analysis of HDDs and HDFs
in commercial products. The ECD analysis technique is typically employed on
samples on which extensive extraction and cleanup procedures have been per-
formed, producing extracts of acceptable quality for final analysis.
The electron capture detector is highly sensitive to compounds with
halogen substitution and provides an attractive, cost-effective procedure com-
pared to mass spectrometry. However, this detector provides qualitative in-
formation only, based on a response at a certain retention time. The inci-
dence of false positives will increase as the analyst presses the detection
limit from the micrograms-per-gram level to the low nanograms-per-gram level
(parts-per-million to parts-per-billion in commercial product). The decision
to pursue analysis based on ECD will require that the analyst comply with a
well-validated analytical procedure and stringent QA/QC program to minimize
the number of false positives during sample analysis.
C. Method Sensitivity
One of the major concerns in using any of the analytical techniques
is the ability to achieve the desired limits of detection. . Table 1 provides
a summary of the reported detection limits for various PCDDs and PCDFs in phen-
oxyalkanoic acid herbicides (Baker et al. 1981). These detection limits range
from 5 to 500 ng/g (ppb). A detection limit of 50 ng/g can be achieved for
specific isomers of TCDD and TCDF assuming a conservative instrument sensi-
tivity (quadrupole mass spectrometer) and a 1-uL injection from a 1-mL final
extract of a 1-g sample. This detection limit can be lowered by (1) increas-
ing the initial sample size (~ 10 g), (2) decreasing the final extract volume
-------
fable 1. A Summary of the Reported Analytical Limits of Detection for Polychlorodibeiwo-g-dioxins (PCDDs)
and Polyclilurodibenzofurans (PCOFs) in Phenoxydlkanojc Acid Herbicides
Reference
Storherr et al.
Brenner et al.
(1972)
Vo.jel (1976)
Rdiiiblad et al.
(1077)
Elvidge (1971)
Wool son et al.
(1972)
Edmunds et al.
(1973)
Crunimelt and
SUIil (1973)
Du&er et al.
(1974)
lluckins et al.
(197ft)
Hdppe et al.
(1978)
Pul/hofer (1979)
Cdi el lo et al
Applicability
2.4.5-T
2.4-D. dichlorprop, mecoprop and
2.4.5-T
2.4.5-T
Butoxypropyl esters of 2.4,5-T
and of fenoprop
2.4,5-T, ethylhexyl ester of 2,4,5-T
and formulations containing the
ethylhexyl ester of 2,4,5-T
2.4-0, 2,4-DB. dichlorprop, 2,4,5-T.
fenoprop and dicamba
2.4.5-T. the butyl and octyl
esters of 2,4,5-T and formulations
of the butyl ester of 2.4.5-T
(50% in mineral oil)
Esters of 2.4.5-T and of fenoprop.
Herbicide Orange
2,4,5-T, formulations of esters of
2,4.5-r and of amine salts of 2.4,5-T
Herbicide Orange
Formulations of esters of 2,4,5-T,
Herbicide Orange
2.4.5-T. alky) esters of. 2.4.5-T
and formulations
Fenoprop
Compounds delected
TCDO
TCDO
fCDD
TCDO
TCDD
Di-, tri-. teti-d-,
penta-, hexa-.
liepta. and octa-CDOs
TCOD
TCDO
TCDO
TCUO and penta-CDD
l)i-, tri-, telrd-,
penld- and hexa-CODb,
di-, tri-. letra-.
pciita- and hexa-COl s
rcoo
ICDI)
Liuiil.of detection
(my ky ' of sample)
0.05
0.01
0.03
0.005
0 05 (on 2,4,5-T
content)
0.5 (for any one
of the compounds
delected)
0 OS
0.05
0 001 (on a stan-
dard solution)
0.02
0.01-0.05
0.005 (on 2.4.5-T
content)
0 01
PC, lloodless Kll, lyltr JC. 1981. Pestle Sci 12:297-304.
-------
(~ 20 uL), OP (3) use of a more sensitive (^ 5 pg/uL) mass spectrometer (mag-
netic sector double focusing instrument). By adjusting these options, the
method limit can be lowered to 0.01 to 1 ng/g.
It should be noted that these detection limits are determined by
the sensitivity of the instrument, and that sample interferences may signifi-
cantly increase the detection limit for a given matrix. The working defini-
tion of limit of detection, as defined in EPA methods for the determination
of 2,3,7,8-TCDD in soil/sediment and water, requires that the signal for the
compound is at a minimum three times the background (signal to noise).
0. Analytical Standards
Present commercial sources of HDD and HDF analytical standards
include Cambridge Isotope Laboratories (Woburn, Massachusetts), KOR Isotopes
(Cambridge, Massachusetts), Ultra Scientific (Hope, Rhode Island), and
Wellington Laboratories (Guelph, Ontario, Canada). Table 2 provides a sum-
mary of the current availability of specific substituted unlabeled and mass-
labeled HDDs and HDFs. Table 2 is intended as a guide only, as suppliers
may add or remove reference materials from their offerings at any time.
Availability should be confirmed with the suppliers.
A limited number of the mixed bromo/chloro analogs of these compounds
are currently available (see Table 2). If analyses for other congeners are to
be conducted, standards will have to be custom synthesized and characterized
as to identity and purity. Buser (1987) has demonstrated the microsyntheses
of several of the mixed congeners by gamma irradiation of PCDDs and PCDFs in
dibromoethane. While standards of known purity were not produced, this tech-
nique may prove useful in the generation of standards for qualitative use,
for example, in the determination of gas chromatographic retention times.
E. Quality Assurance/Quality Control (QA/QC)
A strong QA/QC program is necessary to support the method guideline
approach which has been developed for this program. To ensure that the data
obtained are of known quality, the QA/QC program detailed in Appendix A (Sec-
tion 15) includes provisions for the use of carbon-13 labeled surrogates where
possible. In addition, replicate sample analysis, the routine analysis of
spiked samples, and method blanks have been incorporated. Calibration pro-
cedures for instrumentation are also clearly defined for implementation by
the analyst.
IV. EXISTING METHODS
A computer search, conducted on the CAS database.-.(1967-1984) yielded
no references concerning the analysis of HDDs and HDFs in commercial products.
However, analytical methods for the determination of polychlorinated dioxins
(PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated and poly-
brominated biphenyls (PCBs and PBBs) in various matrices have been reported.
It is anticipated that the analytical procedures for PCDDs and PCDFs, summa-
rized below, may be modified for the analysis of bromo- and bromo/chloro-HDDs
and HDFs.
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Table 2. Currently Available HOD and HDF Standards
Unlabeled
Stable isotope labeled
Chlorinated dioxins:
Mono:
Di:
Tri:
2,3
2,8
2,7/2,8e
1,2,3
1,2,4
2,3,7
Tetra: 1,2,3,4
Penta:
Hexa:
Hepta:
Octa:
1,2,3,7/1,2,3,8"
1,2,3,7/1,2,8,9!
1,2,4,7/1,2,4,8*
1,2,6,7/1,2,8,9*
1,2,7,8
1,2,8,9
1,3,6,8
1,3,6,8/1,3,7,9*
1,3,7,8
2,3,7,8
1,2,3,4',7
1,2,3,7,8
1,2,4,7,8
1,2,3,4,7,8
1,2,3,6,7,8
1,2,3,7,8,9
1,2,3,4,6,7,8
1,2,3,4,6,7,8,9
Brominated dioxins:
Mono:
Oi:
Tri:
Tetra:
Penta:
2,7
2,8
2,3,7
1,2,3,4
1,3,6,8/1,
1,3,7,8
2,3,7,8
1,2,3,7,8
1,2,4,7,8
3,7,9e
Hexa: 1,2,3,4,7,8
2,7/2,8 (u-13C12, 99%)£
1,2,3,4 (13C6, 99%)
2,3,7,8 (U-13C12> 99%)
2,3,7,8 (U-37C14, 96%)C
1,2,3,4 (U-13C12, 99%)
1,2,3,7,8 (U-13C12, 99%)
1,2,3,6,7,8 (U-13C12, 99%)
1,2,3,7,8,9 (U-13C12, 99%)
1,2,3,4,6,7,8 (Uri3C12, 99%)
1,2,3,4,6,7,8 (U-13C12, 99%)
1,2,3,6,7,8/1,2,3,7,8,9'
2,3,7,8 (U-i3C12l 99%)
1,2,3,7,8 (U-13C12, 99%)
1,2,3,4,7,8 (U-i3C12, 99%)
-------
Table 2 (continued)
Unlabeled Stable isotope labeled
Hepta: 1,2,3,4,6,7,8
Octa: 1,2,3,4,6,7,8,9
Mixed Br/Cl dioxins:
Tri: 7-9romo-2,3-dicnloro
Tetra: 2,3-Oi bromo-7,8-di ch1oro
2,8-Oi bromo-3,7-di chloro
2-8romo-3,7,8-di chloro
Penta: 2-Bromo-l,3,7,8-tetrachloro
Hexa: 3-3romo-l,2,4,7,8-pentachloro
Chlorinated dibenzofurans:
Mono:
01:
Tri:
Tetra:
2
3
4
2,3
2,6
2,7
2,8
4,6
1,2,3
1,2,4
1,3,6
1,3,7
1,6,7
2,3,4
2,3,6
2,3,8
2,4,6
2,4,7
2,4,8
2,6,7
1,2,3,4
1,2,3,6
1,2,3,7
1,2,3,8
1,2,3,9
1,2,4,6
1,2,4,8
1,2,4,9
1,2,7,3
1,3,4,6
1,3,4,7
2,3,7,8 (U-i3C12, 99%)
-------
Table 2 (continued)
Unlabeled
Stable isotope labeled
Tetra: 1,3,4,8
(cont.) 1,3,4,9
1,3,6,7
1,3,6,8
1,3,6,9
1,3,7,8
1,3,7,9
1,4,6,7
1,4,7,8
2,3,4,6
2,3,4,7
2,3,4,8
2,3,4,9
2,3,6,7
2,3,6,8
2,3,7,8
2,4,6,7
2,4,6,8
3,4,5,7
Penta:
Hexa:
1,2,3,4,6
1,2,3,4,7
1,2,3,4,8
1,2,3,4,9
1,2,3,6,7
1,2,3,7,8
1,2,3,7,9
1,2,3,8,9
1,2,4,6,7
1,2,4,7,8
1,3,4,6,7
1,3,4,7,8
2,3,4,6,7
2,3,4,6,8
2,3,4,6,9
2,3,4,7,8
2,3,4,7,9
1,2,
1,2,
1,2,
1,2,
1,2,
1.2,
1,2,
1,2.
1,3,
1,3,
2,3,
3,4,7,8
3,4,7,9
3,4,8,9
3,6,7,8
3,6,8,9
3,7,8,9
4,6,7,8
4,6,8,9
4,6,7,8
4,6,7,9
4,6,7,8
1,2,3,7,8 (U-13C12, 99%)
1,2,3,4,7,8 (U-"C12, 99%)
-------
Table 2 (concluded)
Unlabeled
Stable isotope labeled
Hepta: 1,2,3,4,6,7,8
1,2,3,4,6,8,9
1,2,3,4,7,8,9
Octa: 1,2,3,4,6,7,8,9
Brominated dibenzofurans:
D1:
Tri:
Tetra:
Penta:
Hexa:
2,7
2,8
1,3,8
2,3,7,8
1,2,3,7,8
1,2,3,4,7,8
Mixed Br/CI furans:
Tri: 8-Bromo-2,3-dichloro
Tetra: 6,8-Oibromo-2,3-dichloro
8-Bromo-2,3,4-trichloro
Penta: 6,8-Oibromo-2,3,4-trich1oro
1,2,3,4,6,7,8 (U-«C12, 99%)
1,2,3,4,6,7,8,9 (U-*3C12, 99%)
2,3,7,8 (U-13C12, 99%)
1,2,3,7,8 (U-13C12, 99%)
1,2,3,4,7,8 (U-13C12, 99%)
aMixture of Indicated isomers. The relative amounts are unknown; therefore,
these standards are not useful for quantitation calibration.
bU-13C12 indicates that the compound is universally labeled with carbon-13.
CU-37C14 indicates that the compound is universally labeled with chlorine-37.
10
-------
A. Instrumental Methods
Standard analytical methods are available for the analysis of
2,3,7,8-TCDD in soils and sediments and water/wastewater (EPA Method 613,
RCRA Method 8280, other methods under the EPA Contract Laboratory Program;
Harless et al. 1980; McMillin et al. 1983). These methods have been vali-
dated for the measurement of 2,3,7,8-TCDD to detection levels of 1 ng/g (ppb)
for soils and sediments and 1 ng/L (ppt) for water/wastewater. The soil and
sediment protocol has been used for the analysis of over 11,000 samples for
2,3,7,8-TCDD in EPA Region VII through the Contract Laboratory Program (CLP).
These methods have not been validated for the analysis of total PCDDs and
PCDFs. However, several laboratories have provided sufficient documentation
to suggest that modification of these procedures can extend the analysis for
total tetrachloro- through octachloro-PCDDs and PCDFs.
The extraction and cleanup procedures for the determination of PCDDs
and PCDFs in phenoxyalkanoic homicides has been reviewed by Baker et al.
(1981). Extraction tecnniques have included steam distillation of TCDD from
an alkaline solution of technical 2,4,5-T (Brenner et al. 1972, 1974), hexane
extraction from an alkaline solution of 2,4,5-T, liquid-liquid partitioning
of TCOD into hexane from a solution of technical 2,4,5-T in dimethylformamide/
acetonitrile/water (Vogel 1976), and separation of TCDD from isobutoxy methyl
esters of 2,4,5-T and fenoprop using a silica gel column (Ramsted et al. 1977).
Woolson et al. (1972) evaluated 17 different pesticides containing
the polychlorophenoxy group for the presence of PCDDs. Extraction was accom-
plished using hexane extraction from methanolic potassium hydroxide solution.
Cochrane et al. (1981) described the analysis of technical and formulated
products of 2,4-0 as the ester, free acid, and the amine. The 2,4-D esters
were transferred directly onto a silica column and eluted with 30% dichloro-
methane in hexane. The 2,4-0 acids were dissolved in acetonitrile/water, and
the PCOOs were partitioned into hexane. The 2,4-0 amines were dissolved in
water and partitioned into hexane.
Analysis of pentachlorophenol (PCP) for PCDOs has been reported by
several researchers (Buser 1975, Buser and Bosshardt 1976, Mieure et al. 1977,
Blaser et al. 1976, Firestone et al. 1972, Plimmer et al. 1973, Lamberton et
al. 1979, Nelsson and Renberg 1974, Pfeiffer et al. 1978, and Miles et al.
1984). The analytical methods included dissolution in a polar solvent and
partitioning the PCODs into a nonpolar solvent, separation and collection of
PCDOs by high performance liquid chromatography (Miles et al. 1984), and
separation from the commercial product using a macro silica adsorption column
(Mieure et al. 1977). Yamagishi et al. (1981) reported a similar procedure
for the determination of PCDDs and PCDFs in commercial diphenyl ether herbi-
cides such as nitrophen and chloromethoxynil.
B. Bioanalytical Methods
A possible alternative to instrumental analysis for HDDs and HDFs
may arise through the further development of existing bioanalytical or bio-
assay techniques. These bioanalytical techniques include (1) radioimmunoassay
(Albro et al. 1979, McKinney et al. 1981), (2) aryl hydrocarbon hydroxylase
11
-------
(AHH) induction assay (Bradlaw and Casterline 1979), (3) cytosol receptor
assay (Hutzinger 1979), (4) early life stage (E.L.S.) bioassay (Helder and
Seinen 1985), and (5) in vitro keratinization assay (Knutson and Poland 1980,
Gierthy et al. 1985). Each of these techniques has been evaluated as an
alternative technique for screening for the presence of PCDDs and/or PCDFs
based on biological/biochemical properties.
The radioimmunoassay, AHH, and the cytosol receptor assay have
previously been reviewed (NRCC 1981; NRCC 1984). The disadvantages of these
techniques in general are that they do not necessarily respond specifically
to PCDDs and PCDFs, they respond to other compounds such as halogenated bi-
phenyls, azoxybenzenes, and nonhalogenated polynuclear aromatic hydrocaroons,
and each technique is less sensitive than available analytical methods. The
primary advantages of these techniques are lower cost and rapidity of test
results.
The in vitro keratinization and E.L.S. bioassays more recently have
provided additional options and possibly more specificity for determining the
presence of PCODs and PCDFs in general. Both techniques have been demonstrated
to give roughly comparable results with HRGC/MS analysis of total PCDDs and
PCDFs in a PCS fire soot (Gierthy et al. 1985) and fly ash from a municipal
incinerator (Helder and Seinen 1985).-
It is important to note that each of the bioassay techniques is most
sensitive to the presence of 2,3,7,8-TCDD. It is speculated that the relative
response to other HDDs and HDFs might be dependent on halogen suostitution in
the 2,3,7,8-positions. It is also important to note that the range of com-
pounds evaluated- with each of these bioassay techniques is limited. Evalua-
tion of commercial products for the presence of HDDs and HDFs with any of
these bioassay techniques should be completed using a strong QA/QC program
(Appendix A, Section 15). It is suggested that the HDDs and HDFs be isolated
from the commercial product matrix and other potential contaminants using the
methods specified in the guidelines (Appendix) or equivalent techniques. If
bioassay techniques are used, it is advised that the analyst follow the recom-
mended QA/QC program (blanks, spikes, replicates, etc.) to avoid false nega-
tives. It may also be necessary to use an alternate technique, such as HRGC/MS,
to avoid false positives from the bioassay techniques.
V. PROPOSED METHOD
A flowchart for the general guidelines for the analysis of HDDs and
HOFs in commercial products is shown in Figure 1. This scheme, adapted from
general separation principles and techniques reported in the literature, is
based on the physical/chemical properties of the commercial product; there-
fore, the exact extraction and cleanup procedure is dependent upon the nature
of the product or formulation.
The properties utilized in this scheme are volatility, solubility,
acidity, polarity, and molecular geometry. For example, if the product is
sufficiently volatile, evaporation may be employed. Differential solubility
in immiscible solvents may also be the basis for a liquid-liquid extraction.
12
-------
Comineiclul Pioducl
PIIYSICAt/CIIEMICAL
PROPERTIES
SEPARATION
fROM MATRIX
REMOVAL Of
INTERFERENCES
QUANIIIAIJON
Valollllly
Volatile
Nan-ValallU
• 1.2 Dichloiomelliane
• Bfomobeniene
•Vlnylchlorlda
Evaporation of
Sample lo Dryneil
Solubility In
Man-Polar Salveiili
Intolubla
• Titanium
• Plgmcnli
f xliocllon with
Nan-Polar .Solvent
Soluble
Dloxld.
Acidic
Acldlly
• Peiilubioaioulienul
•Malelc Acid
•UMiloioplienol
Batlc
Extraction
Bailc Alumina
Maciocoluiim
Neuhul
Nan
•2. 4.6- «an«
liibiomouiilllne
•2.4-Dlbtoiiia-4-
iilliounlllne
•3.4-Dlililuioanlllne
Extraction
Acidic Alumina
Maciocolunm
Mine-Column Clean Up
Alumina
Giuplilllied Caibon
HPLC Pliaiei
Planailly 1
• Oclabfoa»dlpheny|oiilde
Planar
• filbrouabe
Giaplilllzed Carbon of Melliylallan
SlllcolMaciocoluain Column Cliromalon.rapliy
Silica
Alumina
Floillll
HPLC
Graplilllzed Cuiban
'
Iniliumonlal Tetlinlquei
IIRGC/MS
IIRGC/ECI)
Blaaiiulylical Icclmiquai
Kerallnliallan Anay
RaJiolnmiuno Anuy
Allll Inducllon Ai.ay
Cyloiol-Heieplor Anuy
farly llfv Sluye Aiiuy
Figure 1. Tentative scheme for the analysis of HDDs and HDFs in commercial products.
-------
For example, a salt may be dissolved in aqueous solution and the HDDs and
HDFs extracted with an immiscible nonpolar solvent such as hexane. The sol-
vent, number of extractions, solvent-to-sample ratio, and other parameters
are chosen at the analyst's discretion. The planarity of the HDDs and HDFs
may be exploited to separate them from a nonplanar matrix using graphitized
carbon column chromatography. Column chromatography with alumina, silica, or
ion exchange resins may also be applicable for use with some matrices. Other
separation techniques may also be used if validated prior to sample analysis.
Guidelines for the analysis of HDDs and HDFs in commercial products are
presented in Appendix A.
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•
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19
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APPENDIX A
GUIDELINES FOR THE ANALYSIS OF HALOGENATED DIBENZQ-a-QIOXINS
AND DIBENZOFURANS IN COMMERCIAL PRODUCTS
A-l
-------
GUIDELINES FOR THE ANALYSIS OF HALOGENATED DIBENZO-p_-OIOXINS
AND OIBENZOFURANS IN COMMERCIAL PRODUCTS
1.0 Scope and Application
1.1 These guidelines are applicable for the determination of halogenated
dioxins (HDDs) and halogenated dibenzofurans (HOFs) in commercial
products.
1.2 These guidelines are restricted to use by or under the the super-
vision of analysts experienced in the use of high resolution gas
chromatograpny (HRGC) with mass spectrometric (MS) or electron
capture detection (ECD).
1.3 The limit of detection (LOO) and limit of quantitation (LOQ) are
dependent upon the specific HDDs and/or HOFs monitored, the mass or"
sample extracted, the complexity of the sample matrix, the sensi-
tivity of the instrument, and the ability of the analyst to remove
interferences and properly maintain the analytical systems. The
method accuracy and precision should be determined by each labora-
tory for each commercial product. Specific LOQs are given in the
test rule (EPA 1985).
1.4 The validity of the results depends on equivalent recovery of tne
analytes and the surrogate compounds. If the surrogates are not
thoroughly incorporated into the matrix, the method is not applicable.
1.5 'Certain analytical parameters and equipment designs, which will
affect the validity of the analytical results, have been identified.
Proper use of the method requires that such parameters or designs
should be used as specified. An experienced analyst may make
modifications to parameters or equipment identified by the term
"recommended." Each time such modifications are made to the method,
the analyst should repeat the procedures in Section 15.2.
2.0 Summary
2.1 The product should be sampled such that the specimen collected for
analysis is representative of the whole. Statistically designed
selection of the sampling position, time, or discrete product units
should be employed. The sample should be preserved to prevent HDD
and HDF loss prior to analysis. Customary inventory storage may be
adequate for commercial products. Guidelines for sample collection,
handling, storage, etc., are provided in Appendices C and D.
2.2 The sample is mechanically homogenized and subsampled as necessary
The sample is then spiked with surrogate compounds, and the surro-
gates thoroughly incorporated by further mechanical agitation.
A-2
-------
2.3 The surrogate-spiked sample is extracted and cleaned up at the dis-
cretion of the analyst. Possible extraction techniques include
liquid-liquid partition, column chromatography, etc. The sample
volume and solvent composition are then adjusted as necessary for
final analysis.
2.4 The HDD and HDF content of the sample extract is determined by an
appropriate analytical methodology. Possibilities include high
resolution gas chromatography/electron impact mass spectrometry
(HRGC/EIMS) operated in the selected ion monitoring mode (SIM), high
resolution gas chromatography with electron capture detection (HRGC/
ECD), or other analytical procedures whicn have been validated oy
the testing laboratory.
2.5 For analysis by HRGC/MS, the HDDs and HDFs are identified by com-
parison of their retention time and mass spectral abundance ratios
or peak areas to those in the calibration standard. Identification
by HRGC/ECO is achieved by comparison of retention times.
2.6 HDDs and HDFs are quantitated against the response factors for a
mixture of HDD and HDF congeners, using the response of the surro-
gates to correct for loss of analytes during sample workup and to
determine instrument variability.
2.7 HDDs and HDFs identified by the SIM technique may be confirmed by
full mass scan HRGC/EIMS, chemical ionization mass spectrometry
(CIMS), high resolution mass spectrometry (HRMS), retention on alter-
nate GC columns, or other techniques, provided that the sensitivity
and selectivity of the techniques are demonstrated to be adequate.
2.8 The total analysis time is dependent on the sample matrix and the
extent of workup employed. The time required for a single HRGC/SIM
or HRGC/ECD analysis of a sample is estimated as approximately 1 h.
2.9 Appropriate quality control/quality assurance (QA/QC) procedures
are included to estimate the quality of the results. These QA/QC
procedures include the use of control charts, and the analysis of
blanks, replicates, and standard addition samples. A QA/QC plan
should be developed by each laboratory. QA/QC results should be
reported with the analytical results on a lot/batch basis.
3.0 Interferences
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware, leading
to discrete artifacts and/or elevated baseline iff chromatograms.
All of these materials should be routinely demonstrated to be free
from the interferences by the analysis of laboratory reagent blanks
as described in Section 16.4. These interferences are especially
noted when using electron capture detection.
A-3
-------
3.1.1 Glassware should be scrupulously cleaned. All glass should
be cleaned as soon as possible after use by rinsing with the
last solvent used. This should be followed by detergent
washing with hot water and rinses with tap water and reagent
water. The glassware should then be drained dry and heated
in a muffle furnace at 400°C for 15 to 30 min. Some ther-
mally stable materials may not be eliminated by this treat-
ment. Solvent rinses with acetone and pesticide quality
hexane may be substituted for the muffle furnace heating.
National dioxin study procedures snould be used. (Volumetric
ware should not be heated in a muffle furnace.) After it is
dry and cool, clean glassware should be stored inverted in a
contamination-free area or capped with aluminum foil to
prevent accumulation of dust or other contaminants.
3.1.2 The use of high purity reagents and solvents will help to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required. All
solvent lots should be checked for purity prior to use.
3.2 Interferences may also arise from contaminants that are coextracted
with the HDDs and HDFs from the sample. The extant of matrix
interferences will vary considerably, depending on the nature and
diversity of the samples. Examples are provided below.
3.2.1 Halogenated diphenyl ethers yield mass spectral fragment ions
with exactly the same mass and number of halogen atoms as
the corresponding HDFs. Buser (1975) and Mieure et al.
(1977) have demonstrated that basic alumina column chromatog-
raphy will separata the chlorodiphenyl ethers from the PCDDs
and PCDFs. Likewise, Huckins et al. (1978) have demonstrated
that a graphitized charcoal column will remove the chloro-
diphenyl ether interferences.
3.2.2 The halobenzyl phenyl ethers may yield mass spectral inter-
ferences to HDDs with selected ion monitoring (SIM) tech-
niques. These compounds can also be removed from sample ex-
tracts by cleanup on alumina or graphitized carbon columns.
3.2.3 The bis(chlorophenoxy)methanes have gas chromatographic re-
tention characteristics similar to PCDDs. Interferences to
analysis may arise when using electron capture detection;
however, these compounds are distinguishable from PCDOs by
mass spectrometry.
3.2.4 The halogenated phenoxyphenols can dehydro'halogenate in the
hot inlet of the gas chromatograph to form HDDs. This has
been reported for the chlorinated phenoxyphenols (Baker et al.
1981) and for S-hydroxy-2,21,4,4',5,5'-hexabromobiphenyl
(Gardner et al. 1979). It is expected that analogous reac-
tions will occur for the mixed bromo/chloro phenoxyphenols.
A-4
-------
In addition, cyclization of diphenyl ethers and hydroxy-
biphenyls to form furans has been reported (Norstrom 1976a,
1976b). In order to avoid false positives, these species
must be excluded from the final extracts, or the extracts
must be methylated to prevent ring closure during injection.
3.2.5 Interferences can be caused by the HDDs and HDFs themselves.
This occurs due to the potentially large number of congeners
(Tables 1 and 2) and to the ion clusters produced by the
isotopes of bromine and chlorine. The masses and relative
abundances of the ions in the molecular ion clusters of the
HDDs and HDFs are presented in Tables 3 and 4. Possible
interferences from the molecular ion clusters of the HDDs
and HDFs are presented in Table 5. As an example, a signal
observed at m/z 460 can arise from tribromochlorocribenzo-
dioxin, octachlorodibenzodioxin, bromohexachlorodibenzofuran,
or dibromotetrachlorodibenzofuran.
A-5
-------
Table 1. Theoretical Combinations of Bromo-, Chloro-,
and Bromo/Chloro Dibenzo-p_-dioxins
Halogen No. of theoretical No. of theoretical
Degree of substitution positional 2,3,7,8 substituted
halogenation
2
4
6
Br
0
1
0
1
2
0
1
2
3
0
1
'2
3
4
0
1
2
3
4
5
0
1
2
3
4
5
6
Cl
1
0
2
1
0
3
2
1
0
4
3
2
1
0
5
4
3
2
1
0
6
5
4
3
2
1
0
congeners
2
2
4
10
14
10
34
14
42
42
14
112
22
70
• 114
70
22
298
14
70
140
140
70
14
448
10
42
114
140
114
42
10
472
congeners
0
0
0
0
0
0
0
0
0
0
0
0
1
1
3
1
1
7
1
5
10
10
5
1
32
3
9
27
30
27
9
3
108
A-6
-------
Table 1. (concluded)
Halogen
Degree of substitution
halogenation Br
0
1
2
I
5
6
7
0
1
2
3
8 4 .
5
6
7
8
Cl
7
6
5
4
3
2
-•
0
3
7
6
5
4
3
2
1
0
No. of theoretical No. of theoretical
positional 2,3,7,8 substituted
congeners congeners
2
14
42
70
70
42
14
2
256
1
2
10
14
22
14
10
2
1
76
Total 1,700
HDDs
1
7
21
35
35
21
7
1
128
1
2
10
14
22
14
10
2
1
76
Total 351
2,3,7,8 substi-
tuted HDDs
A-7
-------
Table 2. Theoretical Combinations of Bromo-, Chloro-,
and Bromo/Chloro Dibenzofurans
Classes
No. of halogens Br
. 0
1 1
0
2 1
2
0
, 1
3 2
3
0
1
4 2
3
4
0
.1
5 2
5 3
4
5
0
1
2
6 3
4
5
6
Cl
1
0
2
1
0
3
2
1
0
4
3
2
1
0
5
4
3
2
1
0
6
5
4
3
2
1
0
No. of
positional
congeners
4
4
8
16
28
16
60
28
84
• 84
28
224
38
140
216
140
38
572
28
140
280
280
140
28
896
16
84
216
280
216
84
16
912
No. of 2,3,7,8
substituted
congeners
0
0
0
0
0
0
0
0
0
0
0
0
1
2
4
2
1
10
2
10
20
20
10
2
64
4
18
48
60
48
18
4
200
A-3
-------
Table 2. (concluded)
Classes
No. of halogens Br
0
1
2
-, 3
/
1 4
5
6
7
0
1
2
3
8 4
5
6
7
8
Cl
7
6
5
4
3
2
1
0
3
7
6
5
4
3
2
1
0 •
Total
HDFs
No. of
positional
congeners
4
28
84
140
140
84
28
4
512
1
4
16
28
38
23
16
4
1
'136
3,320
No. of 2,3,7,8
substituted
congeners
2
17
40
69
69
40
17
2
256
1
4
16
28
38
28
15
4
1
136
Total 666
2,3,7,8 sub-
stituted
HDFs
A-9
-------
3. Chdi-aclei islic Ion* and I lie I r Heldlive liotope Abundances
in llifc Molecular Ion Clusters ol HIM)*
Uuyi ee u (
lldluyeiidltiiii
UTTBr« " " 0 1
0 164: 100 262: 100
165: 13.2 263: 13 2
166. 1 2 264. 98.9
265: 12.9
266: 1.2
•
1 218. 100 296: 76 0
219. 13 2 297: 10 0
220. 33.8 296: 100
221: 4 4 299: 13.1
300: 25 4
301: 3.3
1
2 252: 100 330: 60.9
253: 13 1 331: 8 0
254. 66 4 332. 100
255. 8 6 333 13.1
256: 11 4 334. 46 5
257. 1 4 335. 6 0
336. 6 9
2
340:
341:
342:
343.
344:
345.
3/4:
375.
376:
377.
378:
379.
360:
381.
408:
409
410:
411.
412:
413:
414.
416
50.8
6.7
100
13.1
49.8
6 5
43.6
5 7
100
13.1
70.7
9.2
14.4
1.6
38.2
5.0
100
13.1
90.4
11.8
32.8
4 2
4 3
3
418:
419.
420:
421:
422:
423:
424:
425:
452:
453:
454:
455:
456:
457:
458:
459:
460:
461:
486.
487:
488:
489:
490:
491:
492:
493:
494:
495:
496:
34 0
4 4
100
13.1
98 5
12.8
32.9
4.2
25.9
3.4
84.7
11.1
100
13.0
49.6
6 4
a s
1 0
20.3
2.6
73 0
9.5
100
13 0
64.4
8 4
19. J
2 4
2 2
4
496: 17.3
497: 2.3
498: 67.9
499: 8.9
500: 100
501: 13.0
502: 65.8
503: 8 6
504: 16 6
505: 2.1
.
530: 14.2
531: l.a
532: 60.2
533: 7.8
534. 100
535: 13.0
536: 80.6
537: 10 5
548: 31 1
549: 4 0
540: 4 6
.
564: 11.8
565: 1 5
566. 54 2
567: 7 1
568. 100
569: 13.0
570. 94 6
571: 12 3
572: 46 0
573. 6 2
574- 12 J
575. 1.6
576: 1.3
5
574.
575
576.
577:
578:
579:
560.
561:
562.
503:
564.
50b.
6U8:
609.
610.
611.
612:
613:
614:
615:
616:
617.
618:
619:
620:
642.
643:
644.
645.
646.
647
648.
640.
G'JO
CM
652
653.
6b4-
10 4
1.4
51 0
6 6
100
13.0
90 3
12 6
46 6
6 3
9 8
1 2
7 9
1 0
41 5
5 4
89 0
11 6
100
13.0
61.6
6 0
19 6
2 5
2.5
6 2
< 1
J4 2
4.4
79 5
10 4
100
13 0
73 0
9.b
30 8
4 (1
6 9
6
652: b.3
653: < 1
654: 31.2
655: 4.1
656: 76.4
657: 9.9
658: 100
659: 13 0
660: 73 8
661: 96
662: 29 3
663: 3.6
664: 5 0
686: 4 2
687: < 1
668: 26 3
689: 3 4
690: 69 3
691: 9 0
692: 100
693: 13.0
694: 85 2
695. 11 0
696: 42 7
697: 5.5
698: 11.6
699: 1 5
700: 1.3
720. 3.5
721: < 1
722: 22 6
723: 2.9
724: 6 4
725: 8 2
726: 100
727: 11 0
726: 96 1
729. 12 5
730: 57 5
731. 7 4
732: 20 8
733. 2 7
734. 4 1
7
730. 3 0
731. < 1
732: 20.8
733: 2 7
734: 61.2
735: 8 0
736. 100
737: 13.0
736: 98 2
739. 12. B
740: 58.1
741: 7 5
742. 19.2
743: 2.5
744: 2.8
764: 2.3
765. < 1
766. 16.6
767: 2 2
768: 51 9
769. 6.8
770. 91 7
771: 11.9
772. 100
773 13 0
774. 68.9
775. 89 1
776. 29.2
777: 3.8
778: 6.9
a
808:
809:
810.
Bll:
812:
813:
614:
815:
816:
617.
816:
819:
020:
821:
822.
B23.
824:
1.5
< 1
12.1
1.6
41 6
5.4
81 6
10.6
100
13.0
78 6
10 2
38 8
5 0
11.0
1 4
1.4
-------
Table 3 ((.oiiUnueil)
Degree of
lid loijBiia lion
cTi Br> ' 0
3
4
S
286:
267:
268:
289:
290.
291:
292:
320:
321:
322:
323:
324:
325:
326:
327:
326:
354:
355.
356.
357:
3b8:
359.
360.
361:
362.
100
13 1
99.1
12 9
33.1
4.3
3. B
75.9
10.0
100
13.0
49.7
6 4
11.1
1 4
1.0
60.8
6 0
100 '.
13 0
66 0
6 6
21 9
2 8
3 7
364:
365:
366:
367:
366:
369:
370:
371:
372:
396:
399:
400:
401:
402:
403:
404:
405:
406:
432:
433:
434:
435:
436:
437.
438:
439.
440:
441.
442.
50.8
6.7
100
13.1
66.0
6 6
18.4
2.3
1.9
43.6
5.7
100
13.1
84.6
11.0
34.2
4.4
6.8
37.2
4.9
97.4
12.7
100
13 0
52 8
6.8
15 3
2.0
2.4
442:
443:
444:
445.
446:
447.
446:
449.
41)0:
451.
452:
476:
477:
478:
479.
400.
481:
482:
483.
484:
485:
486:
510:
511:
512.
513-
514.
515.
516.
517.
518:
519.
520.
521.
522
31.0
4.1
91.4
11.9
100
13.0
50.6
6.5
12.1
1.5
1.1
23.9
3.1
78.2
10.2
100
13.0
64.1
8.3
22.1
2.8
4.0
19.0
2.5
66.5
6.9
100
13 0
77.1
10.0
34.3
44 1
8 9
1.1
1.3
520:
521:
522:
523:
524:
525:
526:
527:
528:
529:
530:
554:
555:
556:
557:
558:
559:
560:
561:
562:
563:
564:
565:
566:
588:
589:
590:
591:
592:
593:
594:
595:
596:
597:
596:
599:
600.
16.4
2.1
6.4
6.4
100
13 0
78 4
10.2
32.6
4.2
6.9
13.5
1.8
57.6
7.5
100
13.0
91.6
11.9
48.0
61.9
14.5
1 8
2.4
10.9
1.4
49 8
6 5
95.5
12.4
100
13 0
62 7
8.1
24 2
3 1
5 7
596:
599:
600:
601:
602:
603:
604:
605:
606:
607:
606:
609:
610:
632:
633:
634:
635:
636:
637:
636:
639:
640:
641:
642:
643:
644:
645:
646:
9.3
1.2
45.6
5.9
92.5
12.0
100
13.0
62.0
8 0
22.0
2.8
4 2
7.1
< 1
37.4
4.9
82 5
10.7
100
13.0
72.7
9 4
32.4
4.2
8.7
1.1
1.3
6/6:
677.
678:
679.
680:
601:
682:
603:
684:
685:
C06:
667.
686.
689.
690:
4 9
< 1
28 7
3 7
71.9
9 4
100
13 0
83.9
10.9
43.4
5.6
13 5
1 7
2.3
-------
lable 3 (concludoil)
Degree ol
ciref^
6 JUB:
389.
390:
391:
392:
393:
394:
395:
3U6:
197.
398:
7 422.
423:
424:
425.
426.
427:
428:
429:
430:
431.
432.
8 456:
457:
458:
459.
460:
461:
462:
463.
464:
465.
466:
467.
468.
0
50.8
6.6
100
13 0
82.2
10 7
36.2
4 7
9 0
1 1
1.2
43.6
5.7
100
13.0
98.6
12.8
54.1
7.0
17.9
2.3
3.6
33.2
4.3
87.1
11 3
100
13.0
65 8
8 5
27.1
3.3
7 2
< 1
1.2
466:
467:
468:
469:
470:
471:
472:
473:
474:
475:
476:
500.
501.
502:
503:
504:
505:
506:
S07:
508:
509:
510:
511:
512:
t
\
1
28.2
3 7
83.1
10 8
100
13 0
64.6
8.4
24.7
3.2
5.6
22 2
2.9
72 6
9.4
100
13 0
76.6
9.9
36.0
4.6
10.7
1 4
2.0
544:
545:
546.
547:
548.
549:
550:
551:
552:
553:
554.
555:
556.
2345678
15.6
2 0
61.1
8.0
100
13 0
89.7
11 6
48 6
6.3
16.4
2.1
3 4
-------
Table 4 Characteristic Ions and [heir Relative Isotope Abundances
in the Molecular Ion Clusters of 111)1"s
Degree of
lldloijcnaliun
C'Tl Br. "0
0 168. 100 246:
169: 13 1 247:
248:
249.
1 202: 100 28Q:
203: 13 1 281.
204: 33 6 282.
205. 4 3 ' 283.
284:
285.
\
2 236: 100 314:
237: 13.1 315:
238: 66 2 316:
239. 8 6 317:
240- 11 3 318.
241: 1 4 319
320
1
100
13.1
98 7
12.9
76.1
10.0
100
13 0
25 2
3 2
61 0
8 0
100
13 0
46 4
6 0
6 8
2
324:
325:
326:
327:
328:
329-
358:
359:
360
361.
362.
363:
364:
365.
392
393.
394:
395.
396.
397
398
3'J'J
400
50.9
6.7
100
13 1
49.6
6 4
43.6
5.7
100
13.0
70.5
9.2
14.3
1 8
38.2
5 0
100
13.0
90.3
11.8
32 6
4.2
4 2
3
402:
403:
404:
405:
406:
407:
408:
409.
436:
437:
438:
439:
440:
441:
442:
443:
444:
445:
470:
471:
472:
473:
474:
475:
476:
477:
478.
479.
480:
34.0
4.4
100
13.0
98.4
12.8
32.7
4.2
25.9
3.4
84.8
11.0
100
13 0
49 4
6.4
8.4
1.0
20.3
2.6
73 0
9.5
100
13 0
64 3
8 3
19 2
2 4
2 2
4
480:
481:
482:
483:
484:
.485:
486:
487:
488:
489:
514:
515:
516:
517:
518:
519:
520:
521.
522:
523:
524:
548:
549:
550:
551:
552.
553.
554
555.
556.
557-
558.
559.
560.
17.3
2.3
67.9
a. a
100
13.0
65.7
8 5
16.4
2.1
14.2
1.8
60.2
7.8
100
13 0
80.5
10.4
31.0
4.0
4.5
11.8
1.5
54.2
7.0
100
13.0
94.5
12 2
47 9
6.2
12 2
1.6
1.3
5
55U:
559.
560.
561:
562.
563.
564.
565:
566:
567.
568.
569
592
593.
594.
595.
59G.
597.
598.
599.
600.
601:
602.
603:
604.
626.
627.
628:
629:
630.
631.
632
631
614.
Gib.
GJG.
637
GUI
10 4
1 4
51 0
6.6
100
13.0
98 2
12 7
48 5
6 2
9 a
1 2
8.0
1.0
41.6
5 4
89.1
11 6
100
13 0
61 5
8 0
5 4
2 5
2 5
6 2
< 1
34.2
4 4
79 6
10 3
100
13 0
7.1 0
'J 4
30 /
3'J 3
6 9
6 /
636:
637:
638:
639:
640:
641:
642:
643:
644:
645:
646:
647:
648.
670:
671:
672:
673:
674:
675:
676:
677:
£78:
679-
660:
681.
682:
683:
684:
704:
705:
706:
707:
708.
709.
710.
711
712.
713
714.
715-
716.
717
718
.5.3
< 1
31 2
4.1
76 5
19 9
100
13.0
73.7
9.5
29 2
3.7
4.9
4.2
< 1
26 4
3.4
69 3
9.0
100
13 0
65 1
11 0
42 6
5.5
11 5
1 5
1 3
3 5
< 1
22 6
2 9
63 6
8 2
100
13 0
96 0
12 4
57 4
7.4
20 /
2.G
4 1
714:
715:
716:
717.
718.
719.
720.
721:
722:
723:
724.
725.
72G:
727
720
748
749.
7bO.
/SI.
752.
753.
754.
755.
/5G.
757.
/58
759.
760.
761.
/G2.
3.0
< 1
20 8
2.7
61 2
7.9
100
12 9
98.1
12.7
57 9
7.5
19 1
2.4
2 8
2 3
< 1
1G 7
2 2
52 0
6 7
91 8
11 9
100
12.9
60 8
a 9
29 1
3 7
6 9
a
792:
793:
794.
795:
796:
797.
798.
799.
800.
001.
802:
803.
804:
805.
806:
807.
808:
1.6
< 1
12.1
1.6
41.6
5.4
81 6
10.6
100
12 9
78 5
10.1
38 7
5.0
11 0
1 4
1 4
-------
Table 4 (continued)
Decree ot
lid logem II on
Clt Br> 0
3 270:
271:
272:
273.
274:
275:
276.
4 304:
305:
306.
307:
308.
309:
310:
311.
5 338.
339:
340:
341:
342:
343:
344.
345.
346.
100
13 1
08 9
12 9
32 9
4 2
3.8
76.0
9 9
100
13 0
49.5
6.4
11.0
1.4
60.9
8 0
100 •
13 0"
65 8
8 5
21 8
2.8
3 6
348.
349.
350:
351:
352:
353:
354.
355.
356:
382:
383:
384.
385:
386:
387.
388.
389.
390.
416:
417:
418:
419:
420:
421:
422:
423:
424.
425.
426:
1
SO. 9
6 6
100
13.0
65.9
8 5
18.3
2.3
1.9
43.6
5 7
100
13 0
84.5
11 0
34.1
4 4
6.7
37.2
4.8
97.5
12 7
100
13 0
52.7
6 8
15.3
1.9
2.3
2
426.
427.
428:
429.
430.
431:
432:
433:
434:
435:
436:
460:
461:
462.
463:
464:
465:
466.
467.
46B:
469:
470:
494:
495:
496:
497:
490.
499.
500:
501.
502:
503:
504.
50b:
SOG
31.1
4.0
91 5
11.9
100
13.0
50.5
6 5
12.1
1 5
1.1
23.9
3.1
78.3
10.2
100
13.0
64 0
8 3
22.0
2.8
3.9
19.1
2.5
68.6
8 9
100
13.0
77.0
10.0
34.1
4.4
8.8
1 1
1.2
3
504:
505:
506:
507:
508:
509:
510:
511:
512:
513:
514:
538:
539:
540:
541:
542:
543:
544:
545:
546:
547:
548:
549:
550:
572:
573:
574:
575:
576:
577:
578:
579.
580:
581:
582:
583:
584:
16.4
2.1
64. J
8 4
100
13.0
78.3
10.1
32.4
4.2
6.8
13.6
1.8
57.6
7.5
100
13.0
91 6
11.8
47.9
6.1
14.4
1.8
2.3
10.9
1.4
49.9
6.5
95 6
12.4
100
12.9
62 6
8.1
24 2
3 1
5.6
4
582:
583:
584:
585:
586:
587:
588.
589:
590:
591:
592:
593:
594:
616:
617:
618:
619:
620:
621:
622:
623:
624:
625:
626:
627:
628:
629:
630:
9.3
< 1
45.7
5.9
92.5
12 0
100
13.0
61.9
8 0
21.9
2 8
4.1
7.2
< 1
37.4
4.9
8 2
10.7
100
12.9
72.6
9.3
32.3
4.1
8.7
1.1
12.9
567
660:
661.
662:
663.
664:
665.
6G6:
667.
668.
669:
670:
671:
672:
673.
674:
4 9
< 1
2H.O
3 7
72 0
9 3
100
12.9
83 8
10 8
43.3
5.6
13 4
17.0
2 3
-------
Table 4. (concluded)
Ouijrce at
lldlouenalluii
crre?*
6 372.
373:
374.
375:
376:
377.
378.
379.
380
381:
382:
7 406:
407:
408:
409.
410:
411:
412.
413.
414.
415.
416:
8 440.
441:
442:
443.
444:
445.
446:
447.
448:
449.
450:
451:
452:
0
50 8
6 6
100
13 0
82.1
10 6
36.1
4 6
9 0
1.1
1 2
43.6
5.7
100
13.0
98.4
12.7
53 9
7.0
17.8
2 2
3 6
33 3
4 3
87.2
11.3
100
12 9
65 7
B 5
27 0
3 4
7 1
< 1
1 2
450:
451:
452:
453:
454.
455:
456:
457:
458:
459:
460.
484:
485:
486:
487.
488:
489:
490.
491:
492:
493:
494:
495:
496:
*
1
2 8
3.7
83.2
10 6
100
12 9
64 7
8.4
24 6
3 1
5 6
22.2
2.9
72.7
9.4
100
12.9
76 5
9 9
36.0
4.6
10.7
1.3
2 0
528:
529.
530:
531.
532:
533.
534.
535.
536.
537:
538:
539:
540.
2345678
15.6
2.0
61.1
7.9
100
12 9
89 6
11 6
48 4
6.2
16.3
2 1
3.4
-------
Table 5. Potential Interferences from Complex Mixtures
of HDDs and HDFs
_/-
ffl/z
163
169
184
185
136
202
203
204
205
218
219
220
221
236
237
233
239
240
241
246
247
243
249
252
253
254
255
256
257
262
263
264
265
266
270
271
272
273
274
275
275
230
281
282
283
284
285
Fa
F.
n
0D
0
0
Cl-F
Cl-F
Cl-F
Cl-F
Cl-0
Cl-0
Cl-D
Cl-0
CT2-F
C12-F
C12-F
C12-F
C12-F
C12-F
Br-F
Br-F
Br-F
Br-F
C12-0
C12-0
C12-0
C12-0
C12-0
C12-0
Br2-D
Br2-0
Br2-0
Br2-0
Br2-0
C13-F
C13-F
C13-F
C13-F
C13-F
C13-F
C13-F
ClBr-F
ClBr-F
C18r-F
ClBr-F
ClBr-F
ClBr-F
A-16
-------
Table 5. (continued)
/
m/z
286
287
288
289
290
291
292
296
297
298
299
300
301
304
305
306
307
308
309
310
311
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
338
339
C13-0
Cl,-0
C13-0
C13-D
C13-D
C13-0
C13-0
ClBr-0
CIBr-0
ClBr-D
CIBr-Q
CIBr-0
CIBr-0
C14-F
C14-F
C14-F
C14-F
C14-F
C14-F
C14-F
C14-F
Cl2Br-F
Cl2Br-F
C12Br-F
C12Br-F
Cl2Br-F
Cl2Br-F
Cl2Br-F C14-0
C14-0
C14-0
C14-0
C14-0 Br2-F
C14-D Br2-F
C14-0 Br2-F
C14-0 Br2-F
C14-0 Br2-F
Br2-F
Cl2Br-0
Cl2Br-D
Cl,Br-0
Cl2Br-0
Cl2Br-0
Cl2Br-0
Cl,Br-0
C15-F
C15-F
A-17
-------
Table 5. (continued)
/
m/z
340
341
342
343
344
345
346
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
C1S-F
Clg-F
Cl.-F
CVF
C15-F
C1S-F
C15-F
Cl38r-F
Cl3Br-F
Cl3Br-F
Cl38r-F
Cl3Br-F
Cl3Br-F
Cl3Br-F
Cl3Br-F
Cl3Br-F
C15-0
ci5-o
Clg-O
ci5-o
Cls-0
C15-D
ClBr2-F
ClBr2-F
ClBr2-F
Cl3Br-D
Cl3Br-D
Cl3Br-D
Cl3Br-0
Cl3Br-0
Cl3Br-0
Cl3Br-0
C16-F
C16-F
C1S-F
C16-F
C16-F
C16-F
C16-F
C1S-F
C16-F
C16-F
Cl48r-F
Cl4Br-F
Cl4Br-F
C14Br-F
Br2-0
Br2-0
8r2-0
Br2-Q
3r2-0
Br2-0
Cls-0
ci5-o
C15-0
ClBr2-F
C1Br2-F
ClBr2-F
C1Br2-F
C1Br2-F
Cl33r-0
C13Br-0
C1S-F
C1Br2-0
C1Br2-0
C1Br2-0
ClBr2-0
ClBr2-D
C1Br2-D
ClBr2-D
ClBr2-D
C14Br-F
A-18
-------
Table 5. (continued)
-. /_
m/z
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
Cl48r-F
Cl4Br-F
Cl4Br-F
Cl48r-F
CVD
cvo
ci$-o
cvo
cvo
cvo
CVD
cvo
Cl2Br2-F
Cl28r2-F
C14Br-0
C14Br-0
Cl48r-0
Cl4Br-0
Cl4Br-D '
C14Br-0
Br3-F
Br3-F
Br3-F
C17-F
C17-F
C17-F
C17-F
C17-F
C17-F
C17-F
ClsBr-F
Cl5Br-F
Cl5Br-F
ClsBr-F
Cl5Br-F
Cls8r-F
Cl5Br-F
ClsBr-F
Cl5Br-F
ClsBr-F
C17-0
C17-0
C17-0
C17-0
C17-0
C17-0
CVD
CVO
CVO
Cl28r2-F
Cl28r2-F
Cl28r2-F
Cl2Br2-F
Cl2Br2-F
Cl,8r2-F
C12Br2-F Cl48r3-D
Cl4Br-0
Cl48r-0
Br3-F
Br3-F
Br3-F
' Br3-F
Br3-F C17.-F
• C17-F
C17-F C12Br2-0
C17-F C12Br2-D
Cl28r2-D
C12Br2-0
CT28r2-0
CT2Br2-0
C1,Br2-0
CT28r2-0
C12Br2-0 ClsBr-F
•
8r3-0
Br3-0
Br3-0
Br3-0
Br3-D C17-0
Br3-0 C17-0
8r3-0 C17-0
Br3-D C17-0
Br3-0 C17-0 " CT3Br2-
CT3Br2-F
CT3Br2-F
CT3Br2-F
Cl3Br2-F
Cl3Br2-F
Cl3Br2-F C158r-D
A-19
-------
Table 5. (continued)
_ /_
m/z
433
434
435
436
437
438
439
440
441
4*2
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
Cl5Br-D
Cl5Br-D
Cl5Br-0
Cl3Br-0
Cl58r-0
ClsBr-D
ClsBr-0
Cl5Br-D
Cl5Br-D
Cl5Br-D
C18r3-F
C1Br3-F
ClBr3-F
Cl.-F
C18-F
C18-F
C18-F
CU-F
C18-F
C18-F
Cl6Br-F
Cl6Br-F
ClsBr-F
C16Br-F
Cl6Br-F
- ClsBr-F
Cl6Br-F
Cl68r-F
ClBr3-0
C18-0
ci8-o
C18-0
C18-0
C18-0
C18-0
C18-0
Cl4Br2-F
Cl4Br2-F
Cl68r-0
Cl6Br-0
ClsBr-0
Cl6Br-D
Cl6Br-0
Cl6Br-D
Cl4Br2-Q
Cl4Br,-0
C13BP2-F
Cl3Br2-F
C138r2-F
ClBr3-F
ClBr3-F
ClBr3-F
C18r3-F
ClBr3-F
C18r3-F
C18r3-F
C18-F
C18-F
C18-F
C13Br2-0
Cl3Br2-0
Cl38r2-0
CT3Br,-0
Cl,8r;-0
C138r2-0
C13Br2-D
C1Br3-0
C18r3-0
ClBr3-D
C18r3-0
C1Br3-0
ClBr3-0
ClBr3-0
ClBr3-0
C18-0
C14Br2-F
Cl4Br2-F
C14Br,-F
Cl4Br2-F
Cl4Br2-F
Cl4Br2-F
Cl4Br2-F
Cls8r-0
ClsBr-D
C128r3-F
C12Br3-F
Cl2Br3-F
C12Br3-F
Cl2Br3-F
Cl4Br,-0
Cl28r3-F
C128r3-F
C138r,-F
CVF
C13-F
C18-F
C138r2-0
Cl3Br,-0
C"l3Br2-0
C163r-F
Cl6Br-F
C16Br-F
C18-0
C18-0
C18-0
C18-0
C18-0
.
Cl6Br-0
C1sBr-0
Cl6Br-0
C12Br3-F
Cl23r3-F
C13Br2-0
•
ClBr3-0
Cl4Br2-F
Cl48r2-F
A-20
-------
Table 5. (continued)
_ /_
m/z
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500 .
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
Cl4Br2-0
Cl4Br,-0
C148r2-0
C14Brv>-0
Cl4Br^-0
C148r2-0
Cl4Bro-0
Cl4Br2-0
Br4-F
3r4-F
Br4-F
C17Br-F
Cl78r-F
Cl7Br-F
C178r-F
C178r-F
Cl2Br3-0
Cl28r3-0
Br4-0
Br4-0
Br4-0
Br4-0
Br4-0
Br4-0
Br4-0
Br4-0
Br4-0
C1sBr2-F
Cl5Br-0
Cl7Br-D
Cl78r-0
Cl7Br-0
C17Br-0
Cl7Br-0
Cl3Br3-F
Cl3Br3-F
ClsBr2-0
ClsBr2-D
Cl5Br2-D
ClsBr2-0
ClsBr2-D
Cl5Br2-0
C15Br,-0
Cl33r2-0
Br4-F
Br4-F
Cl28r3-F
8r4-F
Br4-F
8r4-F
Br4-F
Br4-F
Br4-F
8r4-F
C178r-F
CT78r-F
Cl78r-F
Cl28r3-0
C12Br3-0
CT28r3-0
CT2Br3-0
Cl28r3-0
C158r,-F
3r4-0"
CT5Br,-F
Cl5Br2-F
Cl58r2-F
Cl5Br2-F
C15Br2-F
ClsBr2-F
C15Br2-F
Cl5Br2-F
ClsBr2-F
Cl78r-0
C13Br3-F
C13Br3-F
Cl3Br3-F
CT38r3-F
CT38r3-F
Cl3Br3-F
Cl5Br2-0
ClsBr2-0
Br4-F
Br4-F
Br4-F
Br4-F
Br4-F
Br4-F
Br4-F
Br4-F
Cl3Br3-0
Cl3Br3-0
A-21
present ------
C12Br3-F
Cl73r-F
Cl73r-F
Cl78r-F
Cl,3r3-0
CU3r,-D
C12Br3-0
Cl58r,-F
Cl38r-F
Cl7Br-0
Cl7Br-0
C17Br-0
Cl7Br-D
C17Br-0
C17Br-0
C13Br3-F
CT5Br2-D
C13Br2-D
Cl3Br2-D
ClBr4-F
C13Br3-D
Cl3Br3-0
Cl3Br3-D
Ci,8r3-0
CT3Br3-F
CT3Br3-F
-------
Table 5. (continued)
m/z
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
557
563
569
Cl38r3-0
Cl3Br3-Q
Cl3Br3-D
Cl3Br3-0
CT3Br3-D
Cl38r3-0
C16Br2-F
Cl68r2-F
C16Br2-F
ClsBr2-F
C1sBr2-F
Cl68r2-F
Cl6Br2-F
Cl6Br2-F
ClsBr2-F
ClsBr2-F
C14Br3-F
C14Br3-F
Cl48r3-F
C14Br3-F
Cl4Br3-F
Cl48r3-F
Cl4Br3-F
Cl4Br3-F
Cl4Br3-F
C14Br3-F
Cl6Br2-0
ClsBr2-0
Cl6Br2-0
Cl6Br2-0
C16Br2-D
ClsBr2-0
Cl28r4-F
C128r4-F
C12Br4-F
Cl2Br4-F
Cl4Br3-0
C14Br3-0
Cl48r3-0
Cl4Br3-D
Cl4Br3-0
Cl4Br3-0
Br3-F
Brs-F
Br3-F
C16Br2-F
Cl6Br,-F
Cl6Br2-F
ClBr4-0
ClBr4-0
C1Br4-Q
ClBr4-0
C1Br4-0
C1Br4-0
C1Br4-0
ClBr4-0
ClBr4-0
ClBr4-0-
C16Br2-0 .
C16Br2-D
Cl6Br2-0
Cl5Br,-0
Cl6Br2-0
Cl68r2-D
Cl6Br2-0
Cl2Br4-F
Cl2Br4-F
C12Br4-F
Cl2Br4-F
Cl2Br4-F
Cl2Br4-F
C148r3-0
Cl4Br3-0
Cl48r3-0
Cl4Br3-0
Brs-F
Brs-F
Brs-F
Brs-F
Brs-F
Brs-F
Cl2Br4-0
Cl28r4-0
Cl2Br4-D
Cl3Br4-0
Cl48r3-F
Cl4Br3-F "
Cl4Br3-F
Cl2Br4-F
Cl28r4-F
Cl28r4-F
Cl28r4-F
C14Br3-0
Cl4Br3-0
Cl4Br3-0
Br3-F
8r5-F
Brs-F
Cl2Br4-0
Cl28r4-0
Cl2Br4-0
Cl28r4-0
A-22
-------
Table 5. (continued)
/_
m/z
570
571
572
573
574
575
576
577
578
579
530
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
Cl2Br4-D
Cl2Br4-D
Cl28r4-D
Cl2Br4-0
Cl2Br4-D
C128r4-D
C128r4-0
3r5-0
8r5-0
Brs-0
Br5-0
Brs-D
Brs-0
Brs-0
Brs-0
Br3-0
Cl3Br4-F
C138r4-F
Cl38r4-F
Cl3Br4-F
Cl3Br4-F
Cl38r4-F
Cl3Br4-F
C138r4-F
Cl3Br4-F
Cl5Br3-0
Cl5Br3-0
ClsBr3-D
Cl58r3-0
ClsBr3-D
ClsBr3-D
C1Brs-F
C1Brs-F
ClBrs-F
ClBr5-F
Cl38r4-0
CT3Br4-0
Cl3Br4-0
Cl3Br4-0
Cl3Br4-0
Cl3Br4-0
ClBr5-D
C18r5-0
ClBr5-0
ClBr3-D
C1Br3-0
Cl5Br3-F
Cl5Br3-F
Cl58r3-F
C15Br3-F
Cl5Br3-F
C158r3-F
C15Br3-F
C15Br3-F
Cl5Br3-F
Cl5Br3-F
Cl5Br3-F
Cl5Br3-F
CT5Br3-F
Cl38r4-F
C15Br3-0
Cl5Br3-0
ClsBr3-0
ClsBr3-0
Cl5Br3-0
C153r3-0
Cl5Br3-0
ClBr5-F
C1Brs-F
ClBrg-F
C18rs-F
ClBrs-F
ClBr5-F
C138r4-0
Cl38r4-D
Cl3Br4-0
C13Br4-0
C1Brs-0
ClBrs-0
ClBrs-D
3r5-0
Brs-0
3r5-0
CT38r4-F
Cl38r4-F
Cl3Br4-F
C1Brs-F
ClBr5-F
ClBrs-F
C13Br4-0
C13Br4-0
Cl38r4-0
A-23
-------
m/z
515
517
618
519
520
621
622
523
524
525
625
627
628
629
630
631
632
533
634
635
636
637
638
639
640
641
642
643
644
645
646
647
64S
649
650
651
652
653
654
655
656
657
658
659
Table
5. (continued)
C1Brs-0
C18rs-0
ClBrs-0
C1Br5-0
C18r3-0
C148r4-F
Cl48r4-F
C14Br4-F
Cl4Br4-F
Cl4Br4-F
Cl4Br4-F
CT4Br4-F
CT4Br4-F
CT48r4-F
Cl48r4-F
Cl28r5-F
Cl28r5-F
Cl28rs-F
Cl28r5-F
CT28r5-F
Cl2Brs-F
Cl28r5-F
Cl2Brs-F
C148r4-0
Cl48r4-Q
Cl4Br4-D
Cl48r4-D
C148r4-D
Cl48r4-0
C148r4-D
Cl48r4-0
Brs-F
8r6-F
Cl28r5-0
Cl2Brs-0
C12Br5-0
Cl2Brs-0
Cl2Brs-D
C12Brs-0
Br6-D
Br6-D
Brs-0
8rs-0
8r6-0
Cl4Br4-F
Cl48r4-F
Cl4Br4-F
Cl4Br4-f
Cl43r4-F
C12Brs-F
C12Brs-F
Cl28r5-F
Cl28rs-F
Cl28r5-F
C148r4-0
Cl43r4-0
Cl4Br4-0
Cl4Br4-0
C14Br4-0
Cl4Br4-0
Cl48r4-0
Br6-F
Brs-F
Br6-F
Brs-F
8rs-F
Brs-F
Brs-F
Brs-F
C12BP5-0
Cl28rs-0
Brs-D •
Brs-D
Brs-D
•
Brs-F '
Br6-F
Brs-F
Cl28rs-0
Cl2Brs-0
C12Br3-D
Cl28rs-D
Cl2Br5-0
A-24
-------
Table 5. (continued)
_ /•»
m/z
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
577
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
704
705
706
707
708
Br6-0
Br6-0
Brs-0
3rs-0
Brs-0
Cl38r3-r
Cl38r5-F
Cl38r3-F
Cl3Br5-F
Cl3Br3-F
Cl38r5-F
Cl3Br5-F
Cl38r5-F
Cl3Brs-F
Cl38rs-F
ClBrs-F
ClBrs-F
ClBr6-F
ClBrs-F
ClBrs-F
ClBrs-F
ClBrs-F
C1Br6-F
ClBrs-F
ClBrs-F
Cl3Br5-0
Cl38rs-D
C138rs-0
C13Brs-0
Cl38rs-0
CT38r3-D
ClBrs-0
ClBrs-0
ClBrs-0
ClBrs-0
ClBrs-0
ClBrs-0
ClBrs-0
ClBr6-D
ClBrs-0
ClBr6-0
Cl2Br6-F
Cl28rs-F
Cl28r6-F
C12Brs-F
Cl2Br6-F
----- Ions present
Cl3Br5-F
Cl38r5-F
C13Srs-F
Cl3Br5-F
C13Brs-F
C1Brs-F
C1Brs-F
ClBrs-F
ClBr6-F
C1Br6-F
Cl3Br5-0
C138r3-0
Cl3Brs-0
C!3Brs-0
Cl3Brs-D
Cl3Br5-0
Cl3Brs-0
C138rs-0
Cl38r5-D -
C1Brs-0
ClBrs-0
C18r6-0
C18r6-D
ClBr6-0
A-25
-------
Table 5. (continued)
m/z
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
748
749
750
751
752
753
754
755
756
757
Cl2Brs-F
CT2Br6-F
•Cl2Brs-F
Cl2Brs-F
Cl2Br6-F
Cl2Brs-F
C12Br6-F
Cl28rs-F
C12Brs-F
CT2Br6-F
3r7-F
Br7-F
Br7-F
Br7-F
Br7-F
Br7-F
3r7-F
Br7-F
8r7-F
Br7-F
Cl2Brs-0
Cl2Brs-0
Cl2Brs-0
Cl2Brs-0
Cl2Br6-0
C12Br6-0
Br7-D
Br7-0
Br7-0
Br7-0
Br7-0
Br7-0
Br7-0
Br7-D
Br7-0
Br7-0
ClBr7-F
C18r7-F
ClBr7-F
C1Br7-F
C1Br7-F
C18r7-F
C18r7-F
ClBr7-F
C18r7-F
ClBr7-F
Br7-F
Br7-F
3r7-F
8r7-F
8r7-F
Cl28rs-0
C128rs-0
C12Br6-0
Cl23rs-0
C128rs-Q
Cl28r5-D
Cl28rs-0
Cl2Brs-0
C12Brs-0
Br7-0
Br7-0
Br7-0
Br7-D
8r7-0
A-26
-------
Table 5. (continued)
/_
m/z
758
759
760
761
762
764
765
756
767
768
769
770
771
772
773
774
775
776
111
778
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
314
815
816
ClBr7-F
C1Br7-F
ClBr7-F
ClBr7-F
ClBr7-F
C1Br7-0
ClBr7-0
ClBr7-0
ClBr7-0
C18r7-0
ClBr7-0
C18r7-0
ClBr7-0
ClBr7-0
ClBr7-0
C18r7-0
ClBr7-0
C18r7-0
C18r7-0
ClBr7-0
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F
Br8-F Br8-0
Br8-0
Br8-0
Br8-D
Br8-0
Br8-0
Br8-0
Br8-0
Br8-D
A-27
-------
Table 5. (concluded)
m/z Ions present
817 Br8-0
818 Br8-D
819 Br8-0
320 8r8-0
821 8r8-D
822 Br8-0
823 Br8-0
324 Br3-0
F = dibenzofuran.
D = dibenzo-£-dioxin.
A-28
-------
4.0
Some of the following safety practices are excerpted directly from the
EPA Method 613, "Determination of 2,3,7,8-TCDD in Water and Wastewater,"
Section 4 (October 1984 version). Although these practices were designed
for the safe handling of 2,3,7,8-tetrachlorodibenzo-g-dioxin (2,3,7,8-
TCDD), they are also applicable for other HDDs and HDFs.
4.1 2,3,7,8-TCDD has been tentatively classified as a known or suspected
human or mammalian carcinogen.
Note: Congeners substituted in the 2,3,7,3 positions are reoorted
to be the most toxic. Extreme caution should be taken when handling
these compounds as dry powders or in concentrated solutions.
The toxicity or carcinogenicity of other reagents used in these
guidelines has not been precisely defined; however, each cnenncal
compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the low-
est possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA regu-
lations regarding the safe handling of the chemicals specified in
these guidelines. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis.
4.2 If diethyl ether is used, it should be monitored regularly to deter-
mine its peroxide content. Under no circumstances should diethyl
ether be used with a peroxide content in excess of 50 ppm, as an
explosion could occur. Peroxide test strips manufactured by EM
Laboratories (available from Scientific Products Company, Cat. No
P1126-8, and other suppliers) are recommended for this test. Pro-
cedures for removal of peroxides from diethyl ether are included in
the instructions supplied with the test kit.
4.3 Each laboratory must develop a strict safety program for handling
2,3,7,8-TCDD. The following laboratory practices are recommenaed.
4.3.1 Contamination of the laboratory will be minimized by
conducting all manipulations in a hood.
4.3.2 The effluents of sample splitters for the gas chromatograph
and roughing pumps on the GC/MS should pass through either
a column of activated charcoal or be bubbled through a trap
containing oil or high-boiling alcohols.
4.3.3 Liquid waste should be dissolved in methanol or ethanol and
irradiated with ultraviolet light with a wavelength greater
than 290 nm for several days. (Use F 40 BL lamps or equiva-
lent.) Analyze liquid wastes and dispose of the solutions
when 2,3,7,8-TCDD can no longer be detected.
A-29
-------
4.4 Dow Chemical U.S.A. has issued the following precautions (revised
November 1978) for the safe handling of 2,3,7,8-TCDO in the labora-
tory:
4.4.1 The following statements on safe handling are as complete
as possible on the basis of available toxicological infor-
mation. The precautions for safe handling and use are
necessarily general in nature since detailed, specific
recommendations can be made only for the particular expo-
sure and circumstances of each individual use. Inquiries
about specific operations or uses may be addressed to the
Oow Chemical Company. Assistance in evaluating the health
hazards of particular plant conditions may be obtained from
certain consulting laboratories and from state Departments
of Health or of Labor, many of which have an industrial
health service. 2,3,7,8-TCDO is extremely toxic to labora-
tory animals. However, it has been handled for years with-
out known injury in analytical and biological laboratories.
Techniques used in handling radioactive and infectious ma-
terials are applicable to 2,3,7,8-TCDO.
4.4.1.1 Protective equipment - Throwaway plastic gloves,
apron or lab coat, safety glasses, and a lab hooo
adequate for radioactive work.
4.4.1.2 Training - Workers must be trained in the proper
method of removing contaminated gloves and cloth-
ing'without contacting the exterior*'surfaces.
4.4.1.3 Personal hygiene - Thorough washing of hands and
forearms after each manipulation ano before breaks
(coffee, lunch, and shift).
4.4.1.4 Confinement - Isolated work area, posted with
signs, segregated glassware and tools, plastic-
backed adsorbent paper on benchtops.
4.4.1.5 Waste - Good technique includes minimizing con-
taminated waste. Plastic bag liners should be
used in waste cans. Janitors must be trained in
the safe handling of waste.
4.4.1.6 Disposal of wastes - 2,3,7,8-TCDD decomposes above
800°C. Low level waste such as absorbent paper,
tissues, and plastic gloves may be burned in an
incinerator. Gross quantities -{mil 1 igrams) should
be packaged securely and disposed through commer-
cial or governmental channels which are capable
of handling extremely toxic wastes. Liquids
should be allowed to evaporate in a good hood and
in a disposable container.
A-30
-------
4.4.1.7 Decontamination • For personal decontamination, use
any mild soap with plenty of scrubbing action. For
decontamination of glassware, tools, and surfaces,
Chlorothene NU Solvent (trademark of the Dow Chem-
ical Company) is the least toxic solvent shown to
be effective. Satisfactory cleaning may be accom-
plished by rinsing with Chlorothene, then washing
with any detergent and water. Dishwater may be
disposed to the sewer. It is prudent to minimize
solvent wastes because they may require special dis-
posal through commercial sources whicn are expensive.
4.4.1.8 Laundry - Clothing known to be contaminated should
be disposed with the precautions described unaer
Section 4.4.1.6. Lab coats or other clothing worn
in 2,3,7,8-TCDD work area may be laundered. Cloth-
ing 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 the problem.
The washer should be run through a cycle before
being used again for other clothing.
4.4.1.9 Wipe tests - A useful method of determining clsan-
liness of work surfaces and tools is to wipe the
surface with a piece of filter paper. Extraction
and analysis by gas chromatography can achieve a
limit of sensitivity of 0.1 ug per wipe. Less
than 1 ug of 2,3,7,8-TCDD per sample indicates
acceptable cleanliness; anything higher warrants
further cleaning. More than 10 ug on a wipe
sample constitutes an acute hazard and requires
prompt cleaning before further use of the equip-
ment or work space. A high (> 10 |jg) 2,3,7,8-TCDD
level indicates that unacceptable work practices
have been employed in the past.
4.4.1.10 Inhalation - Any procedure that may produce air-
borne contamination must be done with gooa venti-
lation. Gross losses to a ventilation system must
not be allowed. Handling of the dilute solutions
normally used in analytical work presents no in-
halation hazards except in the case of an accident.
4.4.1.11 Accidents - Remove contaminated" clothing immedi-
ately, taking precautions not to contaminate skin
or other articles. Wash exposed skin vigorously
and repeatedly until medical attention is obtained.
A-31
-------
5.0 Apparatus and Materials
5.1 Sampling containers - Amber glass bottles or jars, 1-L or other
aopropriate volume, fitted with screw caps lined with Teflon® are
suitable for sample containment. Cleaned aluminum foil may be sub-
stituted for Teflon® if the sample is not corrosive. If amber con-
tainers are not available, samples should be protected from light
using foil or an opaque outer container. The containers should be
washed, rinsed with acetone or methylene chloride, and driea before
use in order to minimize contamination.
5.2 Glassware - Glassware requirements will be dictated by the extrac-
tion and cleanup procedures employed by each laboratory. Typical
glassware requirements are given below. All specifications are
suggestions only. Catalog numbers are included for illustration
only.
5.2.1- Volumetric flasks - Assorted sizes.
5.2.2 Pi pets - Assorted sizes, Mohr delivery.
5.2.3 Micro syringes - 1.0 uL with fused silica needle for en-
column gas chromatographic analysis.
5.2.4 Chromatography columns - Sizes dictated by method employed.
A typical column is the Chromaflex column, 400 mm x 19 mm ID
(Kontes K-420540-9011) or equivalent.
5.2.5 Kuderna-Oanish evaporative concantrator apparatus.
5.2.5.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration should
be checked. Ground glass stopper (size 519/22
joint) is used to prevent evaporative losses.
5.2.5.2 Evaporation flask - 500 ml (Kontes K-57001-0050
or equivalent). Attached to concentrator tube
with springs (Kontes K-662750-0012 or equivalent).
5.2.5.3 Snyder column - Three-ball macro (Kontes K-503000-
0121).
5.3 Balance - Analytical, capable of-accurately weighing 0.0001 g.
5.4 Gas chromatographic system.
5.4.1 _ Gas chromatograph - An analytical system complete with a
"temperature programmable gas chromatograph and all required
accessories including syringes, analytical columns, and
gases. The injection port should be designed for on-coiumn
injection when using capillary columns. Other capillary
injection techniques (split, splitless, etc.) may be used
A-32
-------
provided the performance specifications stated in Section
7.1 are met.
5.4.2 Capillary GC column - A 10 to 60 m long x 0.25 mm ID fused
silica column with a 0.25 urn thick OB-5 (Supelco Inc.,
Bellefonte, Pennsylvania) liquid phase is recommended.
Alternate liquid phases may include Silar 10-C, SP-2330,
SP-2340, 08-225, SE-54, SP-2100, or other liquid phases
which meet the performance specifications stated in Section
7.1. The Silar 10-C, SP-2330, and the SP-234Q columns are
recommended to achieve isomer specific analyses (e.g.,
2,3,7,8-TCDD from the other 21 TCDD isomers).
5.4.3 A data system should be used for manipulation of GC/ECD data.
At a minimum, electronic integration should be used. The
ideal system will allow for archiving raw chromatograohic
aata on magnetic media.
5.5 Gas chromatograph/mass spectrometer system.
5.5.1 Gas chromatograph - As described in Section 5.4.1.
5.5.2' Capillary GC column - As described in Section 5.4.2.
5.5.3 Mass spectrometer - Should be caoable of scanning a rsnge
of 400 amu in 1.5 s or less, collecting at least five aata
points per chromatographic peak, utilizing a 70-eV (nominal)
electron energy in the electron impact ionization mode, and
producing a mass spectrum which meets all the criteria in
Table 6 when 50 ng of decafluorotriphenyl phosphine [DFTPP,
bis(perfluoroohenyl)phenyl phosphine] is injected througn
the GC inlet. Any GC to MS interface that gives acceptable
calibration points at the working range and achieves all
acceptable performance criteria (Section 10) may be used.
Direct coupling of the fused silica chromatographic column
to the MS is recommended. Glass interfaces, if used, can
be deactivated by silylation with dichlorodimethylsilane
or an equivalent silylation reagent.
5.5.4 A computer system that allows the continuous acquisition
and storage on machine-readable media of all mass spectral
data obtained throughout the duration of the chromatographic
program should be interfaced to the mass spectrometer. The
data system should have the capability of integrating the
abundances .of the .selected ions between specified limits and
relating integrated abundances to concentrations using the
calibration procedures described in these guidelines. The
computer should have software that allows searching any mass
spectral data file for ions of a specific mass and plotting
such ion abundances versus time or scan number to yield an
extracted ion current profile (EICP). The software should
also allow integration of ion abundances in any EICP between
specified time or scan number limits.
A-33
-------
Table 6. OFTPP Key Ions and Ion Abundance Criteria
m/z Ion abundance criteria
197 Less than 1% of m/z 198
198 100% relative abundance
199 5-9% of m/z 198
275 10-30% of m/z 198
365 Greater than 1% of m/z 198
441 Present but less than m/z *43
442 Greater than 40% of m/z 198
443 17-23% of m/z 442
A-34
-------
6.0 Reagents
6.1 Solvents - All solvents should be pesticide residue analysis grade.
New lots should be checked for purity by concentration of an aliquot
by at least as much as will occur during the sample preparation
procedure. The concentrated solvent sample should then be analyzed
in the same manner as the samples.
6.2 Standard chlorinated and brominated congeners - Standards of the
HDD and HOP congeners, listed in Table 7, are available from Ultra
Scientific (Hope, Rhode Island), KOR Isotopes (Cambridge,
Massacnusetts), and Cambridge Isotope Laboratories (Woburn,
Massachusetts).
6.3 Standards containing mixed brominated/chlorinated congeners of the
HDDs and HDFs are not commercially available at this time. If
analyses for these congeners are to be conducted, standards must be
synthesized and characterized as to identity and purity prior to
sample analysis. One standard per homolog group may be adequate to
conduct the analysis.
6.4 Calibration standard stock solution - Primary dilutions of each of
the individual HDDs and HOFs which are to be used are preparea by
weighing approximately 1 to 10 mg of material with an accuracy of
1% or better. The standard is then diluted to 1.0 ml, or another
known volumn, with an appropriate solvent. The concentration is
calculated in milligrams per milliliter. The primary dilutions
should be stored at 4°C, or lower; in screw-cap vials with Teflon®
cap liners. The meniscus is marked on the vial wall to-monitor
solvent evaporation.
6.5 Working calibration standards - Working calibration standards,
similar in HDD or HOP composition and concentration to the samples,
are prepared by mixing and diluting the individual standard stock
solutions. The mixture is diluted to a known volume with the same
solvent that is used for the final sample extract. The concentra-
tion is calculated in nanograms per milliliter as the individual
components. Dilutions are stored at 4°C, or lower, in narrow-mouth,
screw-cap vials with Teflon® cap liners. The meniscus is marked on
the vial wall to monitor solvent evaporation. These solutions should
be labeled with the analytes and concentration, solvent, initials of
the preparer, and the date.
6.6 Alternatively, certified stock solutions, if available from a sup-
plier, may be used in lieu of the procedure described in Section 6.5.
6.7 DFTPP standard - A 50-ng/uL solution of DFTPP is"prepared in acetone
or another appropriate solvent.
6.8 Surrogate standard solutions - These solutions should be preoared
and labeled as described for the calibration standards
A-35
-------
fable 7. Currently Available HDD and HDF Standards
Unlabeled
Stable isotope labeled
Chlorinated dioxins:
Mono:
Di:
Tri:
Tetra
Penta
Hexa:
Hepta
Octa:
Bromi
Mono:
Di:
Tri:
Tetra
Penta
Hexa:
2
2,3
2,3
2,7/2,8a
1,2,3
1,2,4
2,3,7
: 1,2,3,4
1,2,3,7/1,2,3,8!
1,2,3,7/1,2,8,9?
1,2,4,7/1,2,4,8?
1,2,6, 7/l,2,8,9a
1,2,7,8
1,2,8,9
1,3,6,8
1,3,6,8/1. 3, 7,9a.
1,3,7,8
2,3,7,8
: '1,2,3,4,7
1,2,3,7,8
1,2,4,7,8
1,2,3,4,7,8
1,2,3,6,7,8
1,2,3,7,8,9
: 1,2,3,4,6,7,8
1,2,3,4,6,7,8,9
nated dioxins:
1
2,7
2,8
2,3,7
: 1,2,3,4
1,3,6, 8/1, 3, 7, 9a
1,3,7,8
2,3,7,8
: 1,2,3,7,8
1,2,4,7,3
1,2,3,4,7,8
2,7/2,8 (u-13C12, 99%)b
1,2,3,4 (13CS, 99%)
2,3,7,8 (U-13C12, 99%)
2,3,7,8 (U-37C141 96%)c
1,2,3,4 (U-13C12, 99%)
1,2,3,7,8 (U-13C12, 99%)
1,2,3,6,7,8 (IM3C12, 99%)
1,2,3,7,8,9 (U-i3C12, 99%)
1,2,3,4,6,7,8 (U-13C12, 99%)
1,2,3,4,6,7,8 (U-13C12, 99%)
2,3,7,8 (U-i.3Cl2> 99%)
1,2,3,7,8 (U-13C12, 99%)
1,2,3,4,7,8 (U-13C12, 99%)
1,2,3,6,7,8/1,2,3,7,8,9e
A-36
-------
Table 7 (continued)
Unlabeled Stable isotope labeled
Hepta: 1,2,3,4,6,7,8
Octa: 1,2,3,4,6,7,3,9
Mixed 3r/Cl dioxins:
Tri: 7-8romo-2,3-dichloro
Tetra: 2,3-Qibromo-7,8-dichloro
2,8-Oibromo-3,7-dichloro
2-3romo-3,7,3-dichloro
Penta: 2-3romo-l,3,7,3-teT:rachioro
Hexa: 3-3romo-l,2,4,7,8-pentachloro
Chlorinated dibenzofurans:
Mono: 2
3
4
Oi: 2,3
2,6
2,7
2,8
4,6
Tri: 1,2,3
1,2,4
1,3,6
1,3,7
1,6,7
2,3,4
2,3,6
2,3,8
2,4,6
2,4,7
2,4,8
. 2,6,7
Tetra: 1,2,3,4 2,3,7,8 (U-13C12, 99%)
1,2,3,6
1,2,3,7
1,2,3,8
1/2,3,9
1,2,4,6
1,2,4,8
1,2,4,9
1,2,7,3
1,3,4,5
1,3,4,7
A-37
-------
Table 7 (continued)
Unlabeled
Stable isotope labeled
Tetra: 1,3,4,8
(cont.) 1,3,4,9
1,3,6,7
1,3,6,8
1,3,6,9
1,3,7,8
1,3,7,9
1,4,6,7
1,4,7,8
2,3,4,6
2,3,4,7
2,3,4,8
2,3,4,9
2,3,6,7
2,3,6,8
2,3,7,8
2,4,6,7
2,4,6,8
3,4,6,7
Penta:
Hexa:
1,2,3,4,6
1,2,3,4,7
1,2,3,4,8
1,2,3,4,9
1,2,3,6,7
1,2,3,7,8
1,2,3,7,9
1,2,3,8,9
1,2,4,6,7
1,2,4,7,8
1,3,4,6,7
1,3,4,7,8
2,3,4,6,7
2,3,4,6,8
2,3,4,6,9
2,3,4,7,8
2,3,4,7,9
1,2,3,4
1,2,3,4
1,2,3,4
1,2,3,6
1,2,3,6
1,2,3,7
1,2,4,6
1,2,4,6
1,3,4,6
1,3,4,6
2,3,4,6
,7,8
,7,9
,8,9
,7,8
,8,9
,8,9
,7,8
,8,9
,7,8
,7,9
,7,8
1,2,3,7,8 (U-i3C12) 99%)
1,2,3,4,7,8 (U-«C12, 99%)
A-38
-------
Table 7 (concluded)
Unlabeled
Hepta:
Octa:
1,
1,
1,
1,
2
2
2
2
Brominated
01:
Tri:
Tetra:
Penta:
Hexa:
2,
2,
1,
2,
1,
1,
7
3
3
3
2
2
,3,
,3,
,3,
,3,
4
4
4
4
,6
,6
,7
,s
,7,8
,8,9
,8,9
,7,3,9
Stable
1
1
,2
,2
,3,4
,3,4
isotope labeled
,6
,6
,7,8 (U-13C12, 99%)
,7,3,9 (U-13C12, 99%)
dibenzofurans:
,3
,7,
,3,
,3,
8
7
4
,8
,7
,8
2
1
1
,3
,2
,2
,7,3
,3,7
,3,4
(U-13C12> 99%)
,8
,7
(U-13C12, 99%)
,8 (U-13C12> 99%)
Mixed 3r/Cl furans:
Tri: 8-8romo-2,3-dichloro
Tetra: 6,3-Oibromo-2,3-dichloro
8-Bromo-2,3,4-trichloro
Penta: 6,8-Oibromo-2,3,4-trichloro
Mixture of indicated isomers. The relative amounts are unknown; therefore,
.these standards are not useful for quantitation calibration.
U-13C12 indicates that the compound is universally labeled with carbon-13.
U-37C14 indicates that the compound is universally labeled with chlorine-37.
A-39
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6.9 Internal standard solution - The chosen internal-standard-is-pre-
pared at a nominal concentration of 1 to 10 mg/mL in an appropriate
solvent. The solution is further diluted to give a working standard.
6.10 Solution stability - The calibration standard, surrogate, internal
standard, and OFTPP solutions should be checked frequently for
stability. These solutions should be replaced after 6 months, or
sooner if comparison with quality control check samples indicates
compound degradation or concentration change.
7.0 Calibration
The guidelines presented in this section are based on calibration using
surrogates and internal standards. Isotope dilution techniques may also
be applicable and may be substituted if validated prior to use.
7.1 The gas chromatograph should meet the minimum operating parameters
shown in Tables 8 or 9 on a daily basis. If all criteria are not
met, the analyst should adjust conditions until all criteria are
met.
7.2 The electron capture detector-should meet the manufacturer's
specifications on a daily basis. The manufacturer's procedure for
determining detector performance should be usea. If all performance
criteria are not met, corrective action, as specifiea by the manu-
facturer, should be taken and the procedures repeated until the
detector is within specifications.
7.3 The mass spectrometer should meet the minimum operating parameters
shown in Tables 10 or 11 on a daily' basis. If all criteria are
not met, the analyst should retune the spectrometer and repeat the
test until all conditions are met.
7.4 If the analytical system (HRGC/MS, HRGC/ECD) has not been demon-
strated to yield a linear response or if the analyte concentrations
are more than one order of magnitude different from those in the
RF solution, a calibration curve should be prepared. If the ana-
lyte and RF solution concentration differ by more than two orders
of magnitude, a calibration curve must be prepared. A calibration
curve should be established with triplicate determinations at three
or more concentrations bracketing the analyte levels.
A-40
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Table 8. Recommended Operating Parameters for High Resolution Gas
Chromatographic System for Use with Mass Spectral Detection
Parameter
Recommended
Tolerance
Gas chromatograph
Column
Liquid phase
Liquid phase thickness
Carrier gas
Carrier gas velocity
Injector
Separator
Transfer line temperature
Injector temperature
Initial column temperature
Column temperature program
Finnigan 9610
15 m x 0.25 mm ID
fused silica
08-5 (J&W)
0.25 urn
Helium
30 cm/sec
On-column (J&W)C
Noned
320°C
Optimum performancec
80°C (2 min)f
80°C to 320°C at
10°C/min
Other0
Other
Other nonpolar or
semioolar
< 1 urn
Nitrogen or hydrogen
Optimum performance
Other
Glass jet or other
Optimum8
Optimum performance
Other
Other
Injection volume
Tailing factor3
Peak widthh
1.0 uLc
0.7 to 1.5
7 to 10 sec
Other
0.4 to 3
< 15 sec
.Substitutions permitted if performance criteria are met.
cMeasured by injection of air at 30°C column temperature.
For on-column injection, manufacturer's instructions should be followed
dregarding injection technique.
Fused silica columns may be routed directly into the ion source to prevent
eseparator discrimination and losses.
High enough to elute all analytes, but not high enough to degrade the column
fif routed through the transfer line.
With on-column injection, the initial temperature is dependent on the
boiling point of the solvent.
9Tailing factor is the width of the front half of the peak at 10% height
divided by the width of the back half of the peak at 10% height for single
^analytes in the calibration standard solution.
Peak width at 10% height for single analytes in the calibration standard
solution.
A-11
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Table 9. Recommended Operating Parameters for High Resolution Gas
Chromatographic/Electron Capture Detection (HRGC/ECD) System
Parameter
Recommended
Tolerance
Gas chromatograph
Detector
Column
Liquid phase
Liquid phase thickness
Carrier gas
Detector make-up gas
Carrier gas velocity
Injector
Injector temperature
Detector temperature
Initial column temperature
Column temperature program
Injection volume
Tailing factor6
Peak widthf
Varian 3700
63Ni; pulsed
15 m x 0.25 mm ID
fused silica
08-5 (J&W)
0.25 urn
Helium
Nitrogen
30 cm/sac0
On-column (J&W)
Optimum performance
350°C
80°C (2 min)d
80°C to 320°C at
10°C/min
1.0 uLC
0.7 to 1.5
7 to 10 sec
Other
Sc3H
Other
Other nonpolar or
semi polar
Nitrogen or hydrogen
Argon/methane
Optimum performance
Other
Optimum performance
Other
Other
Other
Other
0.4 to 3
< 15 sec
.Substitutions permitted if performance criteria are met.
Measured by injection of air at 30°C column temperature.
For on-column injection, manufacturer's instructions should be followed
.regarding injection technique.
v^ith on-column injection, the initial temperature is dependent on the
boiling point of the solvent.
Tailing factor is the width of the front half of the peak at 10% height
diviaed by the width of the back half of the peak at 10% height for single
^analytes in the calibration standard solution.
Peak width at 10% height for single analytes in the calibration standard
solution.
A-42
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Table 10. Recommended Operating Parameters for Quadrupole Mass
Spectrometer System
Parameter
Recommended
Tolerance
Mass spectrometer
Data system
Scan range
Scan time
Resolution
Ion source temperature
Electron energy0
Trap current
Electron multiplier voltage
Preamplifier sensitivity
Finm'gan 4043
Incos 2400
240-850 amu
1 sec
Unit
280° C
70 eV
0.2 mA
-1,600 V
10"6 A/V
Other3
Other
Other
Otherb
Optimum performance
250-350°C
70 eV
Optimum performance
Optimum
Set for desired
Corking range
bSubstitutions permitted if performance criteria are met.
Greater than five data points over a GC peak is a minimum.
Filaments should be shut off during solvent elution to improve instrument
stability and prolong filament life, especially if no separator is used.
A-43
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Table 11. Recommended Operating Parameters for Magnetic Sector
Mass Spectrometer System
Parameter
Recommended
Tolerance
Mass spectrometer
Data system
Scan range
Scan mode
Cycle time
Resolution
Ion source temperature
Electron energy
Emission current
Filament current
Electron multiplier voltage
Finnigan MAT 311A
Incos 2400
240-850 amu
Exponential (up)
1.2 sec
1,000
280°C
70 eV
1-2 mA
Optimum
-1,600 V
Other*
Other
Other
Other
Otherb
> 500
250-350°C
70 eV
Other
Optimum
Optimum
.Substitutions permitted if performance criteria are met.
Greater than five data points over a GC peak is a minimum.
Filaments should be shut off during solvent elution to improve instrument
stability and prolong filament life, especially if no separator is used.
A-44
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7.5 The HOD or HDF response factors (RFu) should be determined using
Equation 7-1 for the analyte homologs.
*F -inl^l E, 7-
R~K a v M CLi-
where:
RF. - response factor of a given HDD or HDF congener
A. = area of the HDD or HDF congener peak
M. = mass of HOD or HDF congener injected (pg)
M. = mass of internal standard injected (pg)
A. = area of internal standard peak
Using the same conditions as for RF. , the surrogate response
factors (RF.) should be determined using Equation 7-2.
A x M
RF = VL^s. - Eq. 7-2
is s
where:
A = area of the surrogate peak
M = mass of surrogate injected (pg)
Other terms are the same as defined in Equation 7-1.
If specific congeners are known to be present and if standards are
available, selected RF values may be employed.
For selected ion monitoring, the primary and secondary ions of the
molecular clusters of both the analyte and surrogate are generally
used to determine the RF values. If alternate ions are to be used
for quantisation, the RF should be determined using those charac-
teristic ions.
The RF value should be determined in a manner which ensures an
accuracy and precision of ± 30% or better. The most common pro-
cedure is to determine the RF value for at least three different
concentration levels to establish a working calibration curve.
A-45
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Other options include, but are not limited to, triplicate deter-
minations of a single concentration spaced throughout a day or a
running mean (RF) based on one value per day for seven consecutive
days.
7.6 The relative retention time (RRT) windows for the homologs and
surrogates should be determined. The windows should be set wider
than observed if all isomers are not available as standards.
8.0 Sample Collection. Handling, and Preservation
8.1 Amber glass sample containers should have Teflon®-lined screw caps.
If the sample is noncorrosive, aluminum foil liners, rinsed with
methylene chloride, may be substituted. The volume and configura-
tion of the sample containers will be determined by the amount and
physical properties of sample to be collected. For dry powders,
other containers, such as heavy-walled polyethylene bags, may be
appropriate.
8.2 Sample container preparation.
8.2.1 All sample containers and caps should be washed in deter-
gent solution, rinsed with tap water, and then with dis-
tilled water. The containers and caps are allowed to drain
dry in a contaminant-free area. The caps should be rinsed
with pesticide grade hexane and allowed to air dry.
8.2.2 Sample bottles, if used, should be heated to 400°C for
15 to 20 min or rinsed with pesticide grade acetone or
hexane and allowed to air dry.
8.2.3 The clean bottles should be stored inverted or sealed until
use.
8.3 Sample collection.
8.3.1 The primary consideration in sample collection is that the
sample collected is representative of the whole. Therefore,
sampling plans or protocols for each individual producer's
situation should be developed. The recommendations pre-
sented here describe general situations. The number of
replicates and sampling frequency should also be planned
prior to sampling. Guidelines for the collection of repre-
sentative samples are provided as Appendices C and 0.
8.3.2 Discrete product units - If the product is small enough
that one or more discrete units will be used as the analyt-
ical sample, a statistically random sampling approach is
recommended.
A-46
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8.3.3 Liquids or free-flowing solids - If possible, the source
is mixed thoroughly before collecting the sample. If mix-
ing is impractical, the sample should be collected from a
representative area of the source. If the liquid is flow-
ing through an enclosed system, sampling through a valve
should be randomly timed.
8.3.4 Solids - Larger bulk solids which should be subsampled to
get a reasonably sized analytical sample should be treated
on a case-by-case basis. A representative sample should
be obtained by designing a sampling location selection
scheme such that all parts of the whole have a finite known
probability of inclusion.
3.4 Sample preservation - Product samoles should be stored as the bulk
or packaged product inventory would be stored, or protected from
light in a cool, dry area. Intermediates, process samples, or
other nonproduct specimens should be stored protecteo from light
at 4°C or lower.
9.0 Sample Preparation
Since a wide variety of matrices may be subjected to analysis by thesa
guidelines, exact extraction/cleanup procedures cannot be specified.
Each laboratory should prepare a method document for the specific
analysis to be performed.
This section describes general guidelines for subsampling, addition of
surrogates, dilution, extraction, cleanup, extract concentration, and
other sample preparation considerations. Only those methods that deal
directly with commercial products have been presented in detail. Options
for analysis are not limited to those presented herein. Additional pro-
cedures may be referenced from several analytical protocols dealing with
analyses of chlorinated dibenzodioxins and furans in environmental
matrices.
Standard analytical methods are available for the analysis of 2,3,7,8-
TCDD in soils and sediments and water/wastewater and hazardous wastes
(EPA Method 613 1984; RCRA Method 8280, EPA/ERD-L 1984; EPA Region VII
1983; Harless et al. 1980; McMillin et al. 1983). These methods have
been validated for the measurement of 2,3,7,8-TCDD to detection levels
of 1 ng/g (ppb) for soils and sediments and 1 ng/L (ppt) for water/
wastewater. The soil and sediment protocol has been used for the anal-
ysis of over 11,000 samples for 2,3,7,8-TCDD in EPA Region VII alone.
These methods have not been validated for the analysis'- of total PCDDs
and PCDFs. However, several laboratories have provided sufficient docu-
mentation to suggest that modification of these procedures can extend
the analysis for total tetrachloro- through octachloro-PCDDs and PCDFs.
RCRA Method 8280 is currently being validated for the analysis of tetra-
through hexachloro-PCDDs and PCDFs in hazardous wastes.
A-47
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The extraction and cleanup procedures for the determination of PCDOs and
PCDFs in phenoxyalkanoic herbicides has been reviewed by Baker et al.
(1981). Extraction techniques have included steam distillation of TCOD
from an alkaline solution of technical 2,4,5-T (Brenner et al. 1972,
1974), hexane extraction from an alkaline solution of 2,4,5-T, liquid-
liquid partitioning of TCOD into hexane from a solution of technical
2,4,5-T in dimethylformamide/acetonitrile/water (Vogel 1976), and sepa-
ration of TCDD from isobutoxy methyl esters of 2,4,5-T and fenoprop using
a silica gel column (Ramsted et al. 1977).
Woolson et al. (1972) evaluated 17 different pesticides containing the
polychlorophenoxy group for the presence of PCDOs. Extraction was accom-
plished using hexane extraction from methanolic potassium hydroxide solu-
tion. Cochrane et al. (1981) described the analysis of technical and
formulated products of 2,4-0 as the ester, free acid, and the amine. The
2,4-0 esters were transferred directly onto a silica column and eluted
with 30% dichloromethane in hexane. The 2,4-D acids were dissolved in
acetonitrile/water and the PCDOs were partitioned into hexane. The 2,4-0
amines were dissolved in water and partitioned into hexane.
Analysis of pentachlorophenol (PCP) for PCODs has been reported by sev-
eral researchers (Buser 1975, Buser and Bosshardt 1976, Mieure et al.
1977, Blaser et al. 1976, Firestone et al. 1972, Plimmer et al. 1973,
Lamberton et al. 1979, Nelsson and Renberg 1974, and Pfeiffer et al.
1978). The analytical methods range from dissolution in a polar solvent
and partitioning the PCDOs into a nonpolar solvent to separation from
the commercial product using a macro siljca adsorption column (Mieure
et al. 1977). Yamagishi et al. (1981) reported a similar procedure for
the determination of PCDOs and PCDFs in commercial diphenyl ether herbi-
cides such as nitrophen and chloromethoxynil.
9.1 Sample homogenization and subsampling - The sample is homogenized
by shaking, blending, shredding, crushing, or other appropriate
mechanical techniques. A representative subsample of an appropri-
ate mass is then taken. The sample size is dependent upon the
anticipated HDD or HDF levels and difficulty of the subsequent
extraction/cleanup steps.
9.2 Surrogate addition - An appropriate volume of surrogate solution
is pipetted into the sample. The final concentration of the surro-
gates should be in the working range of the calibration curve and
well above the matrix background. The surrogates are thoroughly
incorporated by further mechanical agitation. For nonviscous
liquids, shaking for 30 s should be sufficient. For viscous
liquids or free-flowing solids, tumbling for 10 min is recommended.
In cases where adequate incorporation may be difficult, such as
solids, overnight equilibration with agitation is recommended.
Note: The volume measurement of the spiking solution is critical
to the overall method precision. The analyst should exercise cau-
tion that the volume is known with an accuracy of 13» or better.
Where necessary, calibration of the pipet is recommended.
A-48
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9.3 Sample preparation (extraction/cleanup) - After addition of the
surrogates, the sample is further treated at the discretion of the
analyst, provided that the instrumental response of the surrogates
meets the criteria listed in Section 7.0. Several possible tech-
niques are presented below for guidance only. The applicability
of any of these techniques to a specific sample matrix should be
determined by the precision and accuracy of the surrogate recover-
ies, as discussed in Section 15.2.
9.3.1 Removal of matrix - The techniques used for separation of
HDDs and HDFs from the sample matrix will depend on the
physical and chemical properties of the commercial procuct.
The properties utilized in this scneme are volatility,
solubility, acidity, polarity, and molecular geometry. If
the product is sufficiently volatile, evaporation may be
employed. Differential solubility in immiscible solvents
may be the basis for a liquid-liquid extraction. For ex-
ample, a salt may be dissolved in aqueous solution and the
HDDs and HDFs extracted with an immiscible nonpolar solvent
such as hexane. The solvent, number of extractions,
sol vent-to-sample ratio, and other parameters are chosen
at the analyst's discretion. The planarity of the HDDs
and HDFs may also be exploited to separate them from a
nonplanar matrix using grapnitized carbon column chromatog-
raphy. Column c.hromatography with alumina, silica, or ion
exchange resins may also be applicable for use with some
matrices. Other separation techniques may also be used if
validated prior to sample analysis. Several illustrative
examples, taken from the literature, are presented below.
9.3.1.1 Extraction of chlorophenols with sodium hydroxide
(Firestone 1977).
9.3.1.1.1 Weigh 25 g of sample into a 2-L
Erlenmeyer flask. Add 1 L of water
and 200 mL of 1 N sodium hydroxide.
9.3.1.1.2 If necessary, warm the flask on a
steam bath until the sample dissolves
9.3.1.1.3 Add 350 ml of petroleum ether ana
shake the flask vigorously for about
1 min.
9.3.1.1.4 Drain the lower layer and transfer
the upper layer to a' second separa-
tory funnel.
9.3.1.1.5 Repeat the extraction with petroleum
ether twice more and comoine the
extracts.
A-49
-------
9.3.1.1.5 Wash the combined extracts with 100 mL
of water by gently swirling. Repeat
the wash twice with 100-mL portions
of water. (Wash water should be
neutral.)
9.3.1.1.7 Transfer the extract to a 2-L Erlen-
meyer flask and dry by adding 200 g
of anhydous sodium sulfate. Swirl
the flask vigorously and allow it
to stand for 30 min.
9.3.1.1.8 Transfer the dry petroleum ether to
a second Erlenmeyer flask. Rinse the
first Erlenmeyer flask twice with
200-mL portions of petroleum ether
and transfer the rinsings to the
second Erlenmeyer flask.
9.3.1.1.9 Evaporate the petroleum ether to
about 500 mL.
9.3.1.1.10 Transfer to a 500-mL Kuderna-Oam'sh
concentrator and evaporate to about
10 mL.
9.3.1.2 Extraction of pentachlorophenol with lithium
hydroxide (Buser 1975, 1976).
9.3.1.2.1 Add 4.0 g of sample to 30 mL of meth-
anol in a 250-mL separatory funnel.
9.3.1.2.2 Add 10 mL.of 2.5 N lithium hydroxide
and 100 mL of water.
9.3.1.2.3 Extract with 40 mL of petroleum ether.
9.3.1.2.4 Drain the lower phase (aqueous) after
checking for alkalinity (pH > 12).
9.3.1.2.5 Wash the organic phase with 50 mL of
water. Check the last washing for
neutrality.
9.3.1.2.6 Dry the organic phase over anhydrous
sodium sulfate.
9.3.1.2.7 Concentrate a 20-mL aliquot of the
dry organic phase to about 2 mL using
a stream of nitrogen.
A-50
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9.3.1.3 Extraction of 2,4-0 amines (Cochrane 1981).
9.3.1.3.1 Transfer 2.0 to 2.5 g of sample to a
250-mL flask.
9.3.1.3.2 Add 100 ml of water and a clean mag-
netic stirring bar. Mix the solution
thoroughly for 10 min on a magnetic
stirrer.
9.3.1.3.3 Transfer the solution to a 250-mL
separatory funnel and extract three
times with 25-mL portions of hexane.
Combine the extracts.
9.3.1.3.4 Wash the hexane extracts twice with
20-mL portions of water.
9.3.1.3.5 Dry the hexane by passage through
15 g of prewashed sodium sulfate.
9.3.1.3.6 Concentrate to 1 to 2 ml.
9.3.1.4 Extraction of 2,4-0 acids (Cochrane 1381). .
9.3.1.4.1 Dissolve a 10-g sample in 400 ml of
acetonitnle-water (1:1) in a 500-mL
flask containing 40 ml of methanol.
9.3.1.4.2 Transfer the solution to a 1-L sepa-
ratory funnel using two rinses of
50 mL of hexane to wash the flask.
9.3.1.4.3 Extract three times with 100-mL por-
tions of hexane. Combine the extracts.
9.3.1.4.4 Wash the combined hexane extracts
twice with 50-mL portions of methanol-
water (1:1). Discard the rinsings.
9.3.1.4.5 Dry the hexane extract by passage
through 30 g of prewashed sodium
sulfate in a funnel.
9.3.1.4.6 Reduce the volume to 1 to 2 ml under
reduced pressure.
9.3.1.5 Extraction of 2,4-0 esters (Cochrane 1981).
9.3.1.5.1 Activate silica gel (M. Kieselgel 60;
Merck, Darmstadt, G.F.R.) overnight
at 125°C.
A-51
-------
9.3.1.5.2 Add 50 g of silica gel to a glass
chromatographic column (60 x 2.0 cm ID),
9.3.1.5.3 Transfer 2 g of sample to the head of
the column.
9.3.1.5.4 Elute the column with 150 ml of 30%
methylene chloride in hexane.
9.3.1.5.5 Discard the first 30 ml of eluent.
9.3.1.5.6 Collect the remaining eluent in a
250-mL round bottomed flask.
9.3.1.5.7 Concentrate to 1 to 2 mi. on a rotary
evaporator for subsequent cleanup.
9.3.1.6 Removal of an acidic matrix (pentachloropnenol)
with alumina (Mieure 1977).
9.3.1.6.1 Heat 190 g of alumina (Fisher Adsorp-
tion A-540, 80-200 mesh) at 400°C
for 4 h. Cool and pour i,nto a pint
bottle. Pipette 10 mL of distil lea
water into the bottle, cap the bottle,
and shake for at least 10 min. The
bottle cap should have an aluminum
liner! The deactivated alumina has
a shelf life of several weeks.
9.3.1.6.2 Place a small amount of glass wool
in the bottom of a chromatographic
column (30 cm x 1.8 cm ID Pyrex with
a Teflon® stopcock). Add benzene to
the column until it is half full.
Add 40 ml of deactivated alumina.
Allow the alumina to settle while
tapping the walls of the column. Add
1 in. of sodium sulfate to the top
of the alumina. Open the stopcock
and adjust the benzene level to the
top of the packing.
9.3.1.6.3 Dissolve a 5-g portion of sample in
benzene in a 50-mL volumetric flask.
Sonicate if necessary to effect solu-
tion. Dilute the flask to volume
with benzene.
9.3.1.6.4 Pipete a 10-mL aliquot of the sample
solution dropwise onto the head of
the column.
A-52
-------
9.3.1.6.5 El'ite the column with benzene, col-
lecting the first 150 mL of eluent.
9.3.1.6.6 Concentrate the collected eluent to
an appropriate volume for subsequent
cleanup using a Kuderna-Qanish concen-
trator.
9.3.2 Cleanup - Several cleanup techniques are described below
as examples of procedures which may be applicable to the
analysis of HDDs and HDFs in commercial products. Oeoending
on the complexity of the sample, one or more of the tech-
niques may be required to fractionate the HDDs and HDFs
from interferences. For most cleanups a concentrated
(1-5 mL) extract should be used.
9.3.2.1 Sulfuric acid cleanup (Firestone 1972).
9.3.2.1.1 Add concentrated extract (in petroleum
ether) to a 25-mL graduated cylinder.
Stopper and shake the cylinder for
about 30 s.
9.3.2.1.2 Let the layers separate and transfar
the petroleum ether layer to a 30-mL
beaker.
9.3.2.1.3 Rinse the cylinder with 5 mL of
petroleum ether and add the rinsings
to the beaker.
9.3.2.1.4 Add 1 g of sodium bicarbonate to the
petroleum ether solution. Allow to
stand for 5 min.
9.3.2.1.5 Transfer the petroleum ether solution
to a small vial and evaporate the
contents to dryness at room tempera-
ture using a gentle stream of nitrogen.
9.3.2.1.6 Dissolve the residue in an appropriate
volume of a suitable solvent for fur-
ther cleanup or for final analysis.
9.3.2.2 Alumina cleanup (Firestone 1972).
9.3.2.2.1 Activate 100-g portions of alumina
(Fisher No. A-540) by heating 4 h at
260°C. Transfer alumina, without
cooling, to a dry container and close
the container tightly. Do not store
activated alumina for more than 3
days.
A-53
-------
9.3.2.2.2 Quantitatively transfer the concen-
trated extract (in petroleum ether)
to the alumina column.
9.3.2.2.3 Elute the column with 400 mL of
petroleum ether (Fraction 1).
9.3.2.2.4 Elute the column with 200 ml of S%
diethyl ether in petroleum ether
(Fraction 2).
9.3.2.2.5 Elute the column with 400 mL of 25%
diethyl ether in petroleum ether
(Fraction 3).
9.3.2.2.6 Elute the column with 400 mL of
diethyl ether (Fraction 4).
9.3.2.2.7 Transfer Fractions 3 and 4 to Kuderna-
Oanish concentrators (keep separate)
and evaporate to about 10 ml.
9.3.2.2.8 Remove from steam bath and evaoorate
just to dryness at room temperature
under nitrogen.
9.3.2.2.9 Dissolve the residue in an appropriate
volume of a suitable solvent for fur-
ther cleanup or for final analysis.
9.3.2.3 Alumina cleanup (Buser 1975, 1976).
9.3.2.3.1 Prepare the column by adding 1.0 g of
dry alumina to a disposable Pasteur
pi pete (15 cm x 5 mm ID) containing
a plug of glass wool.
9.3.2.3.2 Quantitatively transfer the concen-
trated extract onto the head of tne
column.
9.3.2.3.3 Elute the column with 10 mL of 2%
methylene chloride in hexane. Dis-
card the eluent.
9.3.2.3.4 Elute the column wi$h 10 mL of 50%
methylene chloride in hexane.
9.3.2.3.5 Adjust the sample volume and sample
composition as needed for further
cleanup or for final analysis.
A-54
-------
9.3.2.4 Basic aluminum oxide cleanup (Mieure 1977).
9.3.2.4.1 Add 1.0 g of aluminum oxide (ICN
Aluminum Oxide W 200 basic, activity
grade Super 1, used as received) to
a disposable Pasteur pipet (15 cm x
0.5 cm 10) containing a glass wool
plug. Settle the packing by gently
tapping 15 to 20 times with a small
spatula.
9.3.2.4.2 Quantitatively transfer 0.30 mL of
benzene concentrate to the column.
9.3.2.4.3 Elute the column with 10 mL of 2%
methylene chloride in hexane. Dis-
card the eluent.
9.3.2.4.4 Elute the column with 10 ml of 50%
methylene chloride in hexane.
9.3.2.4.5 Adjust the sample volume and solvent
composition as needed for instrumental
analysis.
9.3.2.5 Basic alumina cleanup (Cochrane 1981).
9.3.2.5.1 Place a small glass wool plug in the
bottom of a glass chromatographic
column (25 x 1.5 cm ID). Add 0.5 cm
of Ottawa standard sand.
9.3.2.5.2 Add 15 g of basic alumina (Activity I;
Woelm, Eschwege, G.F.R., used as re-
ceived) to the column, then top with
0.5 cm of sodium sulfate.
9.3.2.5.3 Quantitatively transfer 1 to 2 mL of
the concentrated sample extract onto
the head of the column.
9.3.2.5.4 Elute the column with (1) 100 mL of
hexane; (2) 100 mL of 2% methylene
chloride in hexane; (3) 100 mL of 5%
methylene chloride in hexane; and
(4) 100 mL of 10% methylene chloride
in hexane.
9.3.2.5.5 Discard Fractions (1) and (2).
9.3.2.5.6 Concentrate Fractions (3) and (*) to
1 to 2 mL, transfer to a 5-mL gradu-
ated centrifuge tube with three 1-mL
A-55
-------
portions of benzene, then evaporate
to dryness.
9.3.2.5.7 Dissolve the residue In an appropriate
volume of a suitable solvent for final
analysis.
9.3.2.6 Graphltized carbon cleanup (O'Keefe 1978).
9.3.2.6.1 Transfer the extract (15 mL In meth-
ylene chloride) to a graphitized car-
bon column [40 mm x 5 mm ID, packed
with Carbopack AHT (Supelco, Inc. ,
Bellefonte, Pennsylvania)].
9.3.2.6.2 Elute the nonplanar aromatic compounds
with 50 ml of benzene/aiethyl ether
(40/60). Discard the eluent.
9.3.2.6.3 Elute the planar aromatic compounds
with 70 ml of pyridine.
9.3.2.6.4 Dilute the pyridine fraction with
140 ml of L" HC1 and extract three
times with 30-mL portions of hexane.
9.3.2.6.5 Wash the hexane extract with 100 ml
of water, then dry over sod-ium sulfate.
9.3.2.6.6 Adjust the sample volume and solvent
composition as needed for instrumental
analysis.
9.3.2.7 Graphitized carbon cleanup for TCDD (EPA 1983)
9.3.2.7.1 Prepare 18% Carbopack C on Celite
545® by thoroughly mixing 3.6 g of
Carbopack C (80/100 mesh) and 16.4
g of Celite 545® in a 40-mL vial.
Activate at 130°C for 6 h. Store
in a desiccator.
9.3.2.7.2 Prepare a column using a standard
size (5-3/4 in. long by 7.0 mm o.d.)
disposable pipet fitted with a small
plug of glass wool...-Using a vacuum
aspirator attached to the pointed
end of the pipet, add the Carbopack/
Celite mix until a 2-cm column is
obtained.
A-56
-------
9.3.2.7.3 Preelute the column with 2 ml of
toluene followed by 1 ml of 75:20:5
methylene chloride/methanol/benzene,
1 ml of 1:1 cyclohexane in methylene
chloride and 2 ml of hexane.
9.3.2.7.4 While the column is still wet with
hexane, add 50 ul_ of sample extract.
9.3.2.7.5 Elute the column sequentially with
two 1-mL aliquots of hexane, 1 mL
of 1:1 cyclohexane in methylene
chloride, and 1 ml of 75:20:5 meth-
yl ene chloride/methanol/benzene.
9.3.2.7.6 Collect tne TCDD fraction oy elution
with 2 ml of toluene.
9.3.* 7.7 Reduce the volume to near dryness
and add isooctane to obtain a final
volume of 50 uL.
9.3.2.8 Silica gel cleanup (Pfeiffer 1978).
9.3.2.8.1 This proceaure is designed to remove
chlorophenols, chlorophenoxy pnenols,
or other polar impurities extracted
from an aqueous solution.
9.3.2.8.2 Dry the silica gel (Bio-Si 1 A, 100/120
mesh; Bio-Rad Laboratories, Richmond,
California) at 180°C in a tube furnace
for 1.5 h under a 100 cc/min nitrogen
flow. Store the dried silica gel,
capped in glass bottles, in a glass
desiccator containing phosphorous
pentoxide.
9.3.2.8.3 Place a small plug of glass wool in
a disposable capillary pipet (150 x
5 mm ID; VWR Scientific, San Francisco,
California).
9.3.2.8.4 Add the silica gel to the pipet to
produce an adsorbent bed 60 mm in
height.
9.3.2.8.5 Transfer 7.5 mL of the hexane extract
onto the head of the column.
A-57
-------
9.3.2.8.6 Elate the column with 7 mL of hexane,
maintaining the flow through the
column at 2 to 3 drops per second by
applying gentle air pressure at the
top of the column using a rubber bulb.
9.3.2.8.7 Collect all of the eluent. Concen-
trate for final analysis or further
cleanup as necessary.
9.3.2.9 HPLC cleanup (Pfeiffer 1978).
9.3.2.9.1 Inject an appropriate aliquot of
extract onto a high pressure liauid
chromatographic system. An example
of an appropriate system is given in
Table 12'.
9.3.2.9.2 Collect the column eluent within
established retention windows for
the HDD and HDF homologs. Figure 1
shows a chromatogram obtained using
the system outlined in Table 12.
9.3.2.9.3 Adjust the sample volume and solvent
composition as necessary for instru-
mental analysis.
9.3.2.10 HPLC cleanup (RCRA Method 8280)
9.3.2.10.1 Operating parameters: Column tem-
perature, 30°C; column pressure,
172 atm.; flow rate, 1.0 mL/min; UV
detector, 235 nm; detector sensitiv-
ity, 0.02 UFS; isocratic operating
mode, injection via 100 |A sample
loop.
9.3.2.10.2 Elution profile for 2,3,7,8-TCDD:
An authentic standard of 2,3,7,8-
TCDD demonstrated a retention time
of 16.0 min. Under the above con-
ditions the retention time of this
standard should be verified before
proceeding. Typically, collect the
entire column effluent, eluting be-
tween 16.0 and 22.0 min following
introduction of the sample extract
onto the HPLC columns, 'into a 12.0-mL
concentrator tube.
A-58
-------
Table 12. Example HPLC Parameters for Sample Cleanup
(Pfeiffer 1978)
Pump
Injector
Detector
Analytical wavelength
Column
Column dimensions
Mobile phase
Flow rate
Column temperature
Detector sensitivity
Injection volume
Waters M-6000A
Rheodyne Model 7120
Perkin-Elmer LC-55 or LC-55T
245 nm
ODS/Zorbax (6 urn) (DuPont)
4.6 mm x 250 mm
Methanol (100%)
2.0 mL/min
Ambient
0.02 AUFS
6 uL
A-59
-------
CU-CDD
Clg-CDD
CU-CDBF
Minutes
10
12
Fiaure 1. Chromatoaram for HPLC cleanun by nentachloronhenol (?feiffer 1973)
A-60
-------
9.3.2.10.3 Solvent exchange, instrument analysis,
and sample concentration: After the
methylene chloride/hexane effluent is
taken to dryness, add 0.5 ml of meth-
ane! to the concentrator tube.
9.3.2.10.4 Using a gentle stream of prepurified
nitrogen and a 55°C sand bath evaporate
the effluent to 40 uL.
9.3.2.10.5 Using a 100-uL syringe draw the residue
solution carefully into the syringe
until an air bubble appears in the
barrel. Place a second 60-uL aliquot
of methanol into the concentrator tube,
rotate quickly for rinsing and draw
this into the syringe.
9.3.2.10.6 Inject the total volume into the HPLC.
9.3.2.10.7 At the appropriate retention time,
position a sample collection bottla to
collect the required fraction. AGO 5
mL of hexane to the sample bottle.
9.3.2.10.8 Add 2 mL of 5% (w/v) sodium carbonate
to the sample fraction collected and
shake for 1 min.
9.3.2.10.9 Quantitatively remove the hexane layer
(top layer) and transfer to a micro-
reaction vessel. Add an additional
5 ml of hexane, shake 1 min, and
transfer the solution to a concentrator
tube.
9.3.2.10.10 A Teflon boiling chip is added and
the concentrator tube is fitted with a
2-ball Snyder column.
9.3.2.10.11 The sample is concentrated to near
dryness in a 90°C water bath.
9.3.2.10.12 After cooling for 10 min, the sample
is evaporated just.-to dryness using a
gentle stream of prepurified nitrogen
and a 55°C sand bath.
A-61
-------
9.3.2.10.13 Transfer the residue to a 1.0-mL
microflex vial with a small amount
of toluene. Be very careful to
transfer the entire rinse solution.
9.3.2.10.14 Again concentrate the residue to
100 uL using a gentle stream of pre-
purified nitrogen and a 55°C sand
bath.
9.3.2.10.15 Store the microflex vial under
refrigeration until just prior to
GC/MS analyses.
9.4 Miscellaneous cleanuo procedures.
Other cleanup procedures may be employed if they are validated
prior to use. Possibilities include, but are not limited to,
extraction cartridges (e.g., Sep-Pak, Waters Associates), distil-
lation, and thin--layer chromatography.
10.0 Gas Chromatoqraphic/Electron Impact Mass Spectrometric Determination
10.1 Internal standara addition - An appropriate volume of the internal
standard solution is pipeted into the sample. The final concen-
tration of the internal standard should be in the working range
of the calibration and well above the matrix background. The in-
ternal standard is thoroughly incorporated by mechanical agitation.
Note: The volumetric measurement of the internal standard solu-
tion is critical to the overall method precision. The analyst
should exercise caution that the volume is known with an accuracy
of 1% or better. Where necessary, calibration of the pi pet is
recommended.
10.2 Tables 8, 10, and 11 summarize the recommended operation conditions
for analysis'. Figures 2 and 3 present, as examples, chromatograms
(HRGC/SIM, HRGC/FMS) obtained for an analysis of technical grade
pentachlorophenol.
10.3 While the highest available chromatographic resolution is not a
necessary objective of this protocol, good chromatographic per-
formance is recommended. With an optimized HRGC system, the
probability that the chromatographic peaks consist of single
compounds increases, making qualitative and quantitative data
reduction more reliable.
A-62
-------
a\
86.6-1
304-
100.0-
306-
49.5-
308-
332.5-
RIC-
T 1 T
A .A
III
I I I I
798720.
304.091
± 0.500
922624.
306.092
± 0.500
456704.
308.092
± 0.500
3067900.
450
12:35
500
13:59
550
15:23
I I I
I I I I
600
16:46
650
18:10
700 SCAN
19:34 TIME
Fiqure 2. HRCC/SIH determination of tetrachlorodiberizofuran in neiiLachlorooheiiol
(Midwest Research Institute).
-------
100.
RIO
5
4
M
1
j
JjC '-J
i -
u
6
200 400 600 800 1000 1200 1400 16
5:00 10:00 15:00 20:00 25:00 30:00 35:00 40
SCAN
TIME
Peak No. Impurity
Hydrocarbon Impurity
2 Hexachlorodibenzofuran
3 Hexachlorodiberrzodioxin
4 Heptachlorodibenzofuran
5 Heotachlorodibenzodioxin
f Octachlorodibenzofuran
1 Octachlorodibenzodioxin
Figure 3. HRGC/FMS analysis of PCDDs and PCDFs (C16-C18) in
pentachlorophenol (Midwest Research Institute).
A-64
-------
10.4 After performance of the system has been certified for the day
and all instrument conditions set according to Tables 3, 10, and
11, inject an aliquot of the sample onto the GC column. If the
response for any ion, including surrogates and internal standards,
exceeds the working range of the system, dilute the sample and
reanalyze. If the responses of surrogates, analytes, or internal
standard are below the working range, recheck the system perfor-
mance. If necessary, concentrate the sample and reanalyze.
10.5 Record all data on a digital storage device (magnetic disk, tape,
etc.) for qualitative and quantitative data reduction as discussed
in Sections 12 and 13.
11.0 Gas Chromatographic/Electron Capture Detection Screening
11.1 Internal standard addition - An appropriate volume of the internal
standard solution is pipeted into the sample. The final concen-
tration of the internal standard should be in the working range
of the calibration and well above the matrix background. The in-
ternal standard is thoroughly incorporated by mechanical agitation.
" Note: The volumetric measurement of the internal standard solution
is critical to the overall method precision. The analyst should
exercise caution that the volume is known with an accuracy of 1£
or better. Where necessary, calibration of the pipet is recommended.
11.2 Table 9 summarizes the recommended operating conditions for analy-
sis. Figure 4 presents, as an example, a chromatogram obtained
for an analysis of PCDOs and PCDFs in a sample of technical grade
pentachlorophenol.
11.3 While the highest available chromatographic resolution is not a
necessary objective of this protocol, good chromatographic perfor-
mance is recommended. With an optimized HRGC system, the proba-
bility that the chromatographic peaks consist of single compounds
increases, making qualitative and quantitative data reduction more
reliable.
11.4 After performance of the system has been certified for the day
and all instrument conditions are optimized, inject an aliquot of
the clean sample extract onto the GC column. If the response for
any peak, including surrogates and internal standards, exceeds the
working range of the system, dilute the sample and reanalyze. If
the responses of surrogates, analytes, or internal standard are
below the working range, recheck the system performance. If nec-
essary, concentrate and reanalyze the sample.
11.5 If possible, record all data on a digital storage device (magnetic
disk, tape, etc.) for qualitative and quantitative data reduction
as discussed in Sections 12 and 13.
A-65
-------
. JiW I.«J ,U(IU 4^.
4i-i_.., O|>
III- li.kt,..., • j>/4-
.<4u» li|MMta
, .....*,, i r-t.j-,,,
'MM t,j.4 u J, I U ^( U 4. J
... lit . |0 (A.,,. »„!( St.). I
Figure 4. HRGC/ECD profile of PCDDs and PCDFs In pentachlorophenol
(Midwest Research Institute).
-------
12.0 Qualitative Identification
12.1 Selected ion monitoring (SIM) or limited mass scan (LMS) data -
The identification of a compound as a given HOD or HDF homolog
requires that two criteria be met:
12.1.1 (1) The peak should elute within the retention time window
set for that homolog; and (2) the ratio of two ions ob-
tained by SIM (Tables 13 and 14) or by LMS (Tables 15 and
16) should match the theoretical ratio within 20%. The
analyst should search the higher mass windows to prevent
misidentification of a HOD or HOF fragment ion cluster
as the molecular ion.
12.1.2 If either of these criteria is not met, interferences may
have affectea tne results and a reanalysis using full
scan EIMS conditions is recommended.
12.2 Full mass scan (FMS) data.
12.2.1 The peak should elute within the retention time window
set for that homolog.
12.2.2 The unknown spectrum should be compared with an authentic
HDD or HDF. The intensity of the three largest ions in
the molecular ion cluster should match tne theoretical
ratio within 20%. Fragment clusters with proper abundance
ratios should also be present.
12.2.3 Alternatively, a spectral search may be used to automatic-
ally reduce the data. The criteria for acceptable identi-
fication should include a high index of similarity.
12.3 Electron capture detection data.
12.3.1 The peak should elute within the retention time window
set for that homolog.
12.3.2 Any peak which elutes within the retention time window
set for a given homolog should be identified and quanti-
tated as a HDD or HOF.
12.4 Disputes in interpretation - Where there is reasonable doubt as
to the identity of a peak as a HDD or HDF, the analyst should
either identify the peak as a HDD or HDF or proceed to a con-
firmation analysis.
A-67
-------
Table 13. Characteristic Selected Ion Monitoring (SIM) Ions for HDDs
Homokig
C12H7C102
C12HSC1202
C12H5C1302
C12H4C1402
C12H3C1502
C12H2C1602
C12HC1702
C12C1802
C12H78r02
C12H6Br202
'-12^5^r3^2
C12H4Br402
C12H3Brs02
C12H2Br602
Cl2HBr702
C128r302
C12H6CTBr02
Cl2H5Cl2Br02
C12H4Cl3Br02
C12H3Cl48r02
C12H2ClsBr02
C12HCl6Br02
C12Cl7Br02
C12H5ClBr202
C12H4Cl2Br202
C12H3C13Br202
C12H2Cl4Br20
C12HClsBr202
C12ClsBr202
Cl2H4ClBr302
C12H3Cl2Br302
C12H2Cl3Br302
C12HCl48r302
C12Cl3Br302
C12H3ClBr402
C12H2Cl28r402
C12HCl3Br402
C12Cl4Br402
Primary
218 (100)
252 (100)
286 (100)
322 (100)
356 (100)
390 (100)
424 (100)
460 (100)
262 (100)
342 (100)
420 (100)
500 (100)
578 (100)
658 (100)
736 (100)
816 (100)
298 (100)
332 (100)
366 (100)
400 (100)
436 (100)
470 (100)
504 (100)
376 (100)
410 (100)
446 (100)
480 (100)
514 (100)
548 (100)
456 (100)
490 (100)
524 (100)
558 (100)
594 (100)
534 (100)
568 (100)
604 (100)
638 (100)
Ion (relative abundance)
Secondary
220 (34)
254 (56)
288 (99)
320 (76)
358 (56)
392 (32)
426 (98)
458 (87)
264 (99)
340 (51)
422 (98)
498 (68)
580 (98)
660 (76)
738 (98)
814 (82)
296 (76)
330 (61)
368 (66)
402 (85)
434 (97)
468 (83)
506 (77)
378 (71)
412 (90)
444 (91)
478 (78)
516 (77)
550 (90)
454 (85)
488 (73)
526 (78)
560 (92)
592 (96)
536 (81) •-•
570 (95)
602 (93)
636 (83)
Tertiary
.
256 (11)
290 (33)
324 (55)
354 (51)
394 (36)
428 (54)
462 (55)
-
344 (50)
418 (34)
502 (66)
582 (49)
660 (74)
740 (58)
813 (79)
300 (25)
334 (d6)
364 (51)
398 (44)
438 (53)
472 (65)
502 (73)
374 (4*)
414 (38)
448 (51)
482 (64)
512 (68)
552 (49)
458 (50)
492 (64)
528 (33)
562 (58)
596 (63)
532 (60)
572 (48)
606 (62)
640 (73)
A-68
-------
Table 13 (concluded)
Homolog
Ion (relative abundance)
Primary
Secondary
Ternary
C12H2ClBr502
C12HCl2BrsQ2
Cl2HClBr602
C12Cl28r602
Cl2ClBr702
614 (100)
648 (100)
682 (100)
692 (100)
726 (100)
772 (100)
612 (89)
546 (80)
684 (84)
594 (85)
723 (96)
770 (92)
616 (62)
650 (73)
680 (72)
590 (69)
730 (58)
774 (70)
A-69
-------
Table 14. Characteristic Selected Ion Monitoring (SIM) Ions for HDFs
Homo log
C12H7C10
C12H6C120
C12HSC130
C12H4C140
C12H3C150
C12H,C1S0
C,,HC170
C^ClgO
C12H73rO
Cl2Hs8r20
C12HsBr30
C12H4Br40
C12H3BrsO
Cl2H28rsO
C12HBr70
C128r80
C12HsClBrO
C12H5Cl28rO
C12H4Cl3BrO
Cl2H3Cl4BrO
C12H2Cl5BrO
C12HC168rO
C12Cl7BrO
C12H5ClBr20
Cl2H4Cl2Br20
C12H3Cl3Br20
C12H2Cl48r20
C12HCls8r20
C12ClsBr20
Cl2H4ClBr30
C12H3Cl2Br30
C12H2Cl3Br30
C12HCl48r30
C12Cl58r30
C12H3ClBr40
C12H2Cl2Br40
C12HCl38r40
C12Cl4Br40
Primary
202 (100)
236 (100)
270 (100)
306 (100)
340 (100)
374 (100)
408 (100)
444 (100)
246 (100)
325 (100)
404 (100)
484 C100)
562 (100)
642 (100)
720 (100)
800 (100)
282 (100)
316 (100)
350 (100)
384 (100)
420 (100)
454 (100)
488 (100)
360 (100)
394 (100)
430 (100)
464 (100)
498 (100)
532 (100)
440 (100)
474 (100)
508 (100)
542 (100)
578 (100)
518 (100)
552 (100)
588 (100)
622 (100)
Ion (relative abundance)
Secondary
204 (34)
238 (66)
272 (99)
304 (76)
342 (66)
376 (82)
410 (98)
442 (87)
248 (99)
324 (51)
406 (98)
482 (68)
564 (98)
640 (76)
722 (98)
798 (82)
280 (76)
314 (61)
352 (66)
386 (85)
418 (98)
452 (83)
490 (76)
362 (70)
396 (90)
428 (92)
462 (78)
500 (77)
534 (90)
438 (85)
472 (73)
510 (78)
544 (92)
576 (96)
520 (80) -:
554 (94)
586 (92)
620 (83)
Tertiary
_ —
240 (11)
274 (33)
308 (56)
338 (61)
372 (51)
412 (54)
446 (56)
_
328 (50)
402 (34)
486 (66)
560 (51)
644 (74)
718 (61)
802 (78)
284 (25)
318 (*6)
348 (51)
382 (44)
422 (53)
456 (65)
486 (73)
358 (44)
392 (38)
432 (50)
466 (6^)
496 (69)
530 (61)
442 (49)
476 (64)
506 (64)
540 (58)
580 (63)
516 (60)
550 (54)
590 (62)
624 (73)
A-70
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Table 14 (concluded)
Homolog
Ion (relative abundance)
Primary
Secondary
Tertiary
C12H2ClBr50
C^HCUBrsO
C12Cl38r30
C12HC18rsO
C12Cl28rsO
Cl2ClBr70
598 (100)
632 (100)
666 (100)
576 (100)
710 (100)
756 (100)
596 (89)
630 (80)
668 (84)
678 (85)
712 (96)
754 (92)
600 (62)
634 (73)
664 (72)
574 (69)
708 (54)
758 (69)
A-71
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Table 15. Limited Mass Scanning (IMS) Ranges for HDDs
Homolog Mass range
C12H7C102 218 - 220
C12H6C1202 252 - 256
C12HSC1302 286 - 290
C12H4C1402 320 - 326
C12H3C1502 354 - 360
Cl2HoCl608 388 - 394
C12HC1702 422 - 430
C12C1302 456 - 464
C12H78r02 262 - 266
C12H68r202 340 - 344
C12HsBr30, 418 - 424
C12H43r402 495 - 504
C12H3Br302 574 - 584
C12H2Brs02 654 - 662
C12HBr702 732 - 742
C128r802 • 810 - 822
C12H6ClBr02 296 - 300
C12H5Cl23r02 330 - 334
C12H4Cl3Br02 364 - '370
C12H3Cl4Br02 398 - 404
C12H2Cl5Br02 432 - 440
C12HClsBr02 466 - 474
C12Cl78r02 500 - 510
C12H5C18r202 37* - 380
C12H4Cl2Br202 408 - 414
C12H3Cl3Br202 442 - 450
C12H2Cl4Br202 476 - 484
C12HClsBr202 510 - 518
C12Cl6Br202 544 - 554
C12H4C18r302 452 - 458
C12H3Cl28r30, 486 - 494
C12H2Cl38r302 520 - 528
C12HCl48r302 554 - 562
C12Cl5Br302 588 - 598
C12H3ClBr402 530 - 538
C12H2C128r402 564,- 574
C12HCl3Br402 598 - 608
C12Cl4Br402 634 - 640
A-72
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Table 15 (concluded)
Homolog Mass range
C12H2ClBr302 610 - 618
C12HCl28r502 644 - 652
C12Cl3Br502 678 - 638
C12HC1Br302 688 - 698
C12Cl23r602 722 - 732
C12ClBr702 764 - 776
A-73
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Table 16. Limited Mass Scanning (IMS) Ranges for HOFs
Homolog Mass range
C12H7C10 202 - 204
C12HSC120 236 - 240
C12H5C130 270 - 274
C12H4C140 304 - 310
C12H3C150 338 - 344
C12HoCl60 372 - 378
C12HC170 406 - 414
C12C130 440 - 448
C12H78rO 246 - 2*8
C12H63r20 324 - 328
Ct.,H5ar30 402 - 4Q8
C12H43r40 480 - 488
C12H3BrsO 558 - 566
C12H28rsO 638 - 646
C12HBr70 716 - 726
C12Br30 796 - 806
C12HsClBrO 280 - 28*
C12H5Cl2BrO 314 - 318
C12H4Cl3BrO 348 - 354
C12H3Cl4BrO 382 - 388
C12H2Cl5BrO 416 - 424
C12HCls8rO 452 - 458
C12Cl7BrO 484 - 494
C12H5ClBr20 358 - 364
C12H4Cl2Br20 392 - 398
C12H3Cl38r20 426 - 434
C12H2Cl4Br20 460 - 468
C12HClsBr20 494 - 502
C12ClsBr20 528 - 538
C12H4ClBr30 436 - 442
C12H3Cl2Br30 470 - 478
C12H2Cl3Br30 504 - 512
C12HCl48r30 538 - 548
C12Cl5Br30 572 - 582
C12H3ClBr40 514 - 522
C12H2Cl2Br40 548"-- 558
C12HCl3Br40 584 - 592
C12Cl4Br40 618 - 626
A-74
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Table 16 (concluded)
Homolog Mass range
C12H2ClBrsO 594 - 600
C12HCl28r50 628 - 637
C12Cl38r30 662 - 673
C12HC18rsO 672 - 537
Cl2Cl2Br60 706 - 715
C12ClBr70 750 - 760
A-75
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13.0 Quantitative Data Reduction
The guidelines presented in this section are based on quantisation using
surrogates and internal standards. Isotope dilution techniques may also
be applicable and may be substituted if validated prior tc use. The QA
record should show in detail the alternative calculation used in arriving
at final concentration values.
13.1 Once a chromatographic peak has been identified as a HDD or HDF,
the compound is quantitated based either on the integrated abun-
dance of the SIM data or EICP for the primary characteristic ion
in Tables 13 and 14. If interferences are observed for the
primary ion, use the secondary and then the tertiary ion for quan-
titation. If interferences in the parent cluster prevent quanti-
tation, an ion from a fragment cluster [e.g., M-63 (M-COC1) for
PCDOs or PCDFs] may be used. Whichever ion is used, the RF should
be determined using that ion. The same criteria should be applied
to the surrogate compounds. If electron capture detection is used,
the electronically integrated peak area is used for quantitation.
13.2 Using the appropriate analyte-internal standard pair and response
factor (RF.) as determined in Section 7.3, calculate the concen-
tration of each peak using Equation 13-1.
Concentration (ng/g) =
M
is
Ss x Me x RFh
Eq. 13-1
where:
A. = area of the characteristic ion (s) for the analyte
n HDD or HDF peak
\. = area of the characteristic ion (s) for the internal
standard peak
response factor of a given HOD or HDF congener
mass of internal standard added to extract (ng)
mass of sample extracted (g)
RF, =
Mis =
M =
13.3 If a peak appears to contain non-HDD or non-HDF interferences,
which cannot be circumvented by a secondary or-_lertiary ion,
either:
13.3.1 Reanalyze the sample on a different column which separates
the interferences; or
13.3.2 Perform additional chemical cleanup and then reanalyze
the sample; or
A-76
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13.3.3 Quantitate the entire peak as HDD or HDF.
13.3.4 Reanalyze the sample using a confirmation technique [i.e.,
high resolution mass spectrometry (HRMS), NCIMS, etc.].
13.4 Calculate the recovery of the surrogates using the appropriate
surrogate- internal standard pair and response factor (RFs) as
determined in Section 7.5 using Equation 13-2.
A x M. x 100
'
where:
A = area of the characteristic ion (s) for the surrogate
A. = area of the characteristic ion (s) for the internal
15 standard
RF = response factor for the surrogate compound with
5 respect to the internal standard (Eq. 7-1).
M. = mass of internal standard added to extract (ng)
M = mass of surrogate added to original sample (ng)
13.5 Correct the concentration of each peak using Equation 13-3. This
is the final reportable concentration.
Corrected cone. (ng/g) = *. 13-3
13.6 Sum all of the peaks for each homo log, and then sum those to yield
the total HDD and HDF concentrations in the sample. Report all
numbers in nanograms per gram. The uncorrected concentrations.
percent recovery, and corrected concentration should be reported.
13.7 Round off all numbers reported to two significant figures.
14.0 Confirmation
If there is reason to question the qualitative identification (Section
11.0), the analyst may choose to confirm that a peak is not a HDD or a
HDF. Any technique may be chosen provided that it is validated as
having adequate selectivity and sensitivity. Some candidate techniques
include alternate GC column (with EIMS detection), GC/CIMS, GC/NCIMS,
A-77
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high resolution EIMS, and MS/MS techniques. Each laboratory should
validate confirmation techniques to show adequate selectivity between
HDDs and HDFs and interferences and adequate sensitivity [limit of
quantisation (LOQ)].
If a peak is confirmed as being a non-HDD or a non-HDF, it may be
deleted from the calculation (Section 13). If a peak is confirmed as
containing both HDD or HOP and interferences, it should be quantitated
according to Section 13.3.
15.0 Quality Assurance
Each laboratory must develop a Quality Assurance Project Plan (QAPP)
and submit that plan to the Agency for review and approval. The QAPP
documents how a laboratory intends to produce data which meet the data
quality criteria specified in 40 CF3 707 and 766 (EPA 1987).
A detailed QAPP submitted to the Agency will provide the necessary
information required for the Agency to review the specific testing
protocols for a commercial product. Figure 5 provides a suggested
table of contents for a QAPP as outlined in an EPA Office of Toxic
Substances guide for preparation of QAPPs (EPA 1984). The specific
quality assurance objectives as stated in the final test rule are sum-
marized in Table 17. Guidance for the development of a QAPP is also
given in Appendix B.
16.0 Quality Control
16.1 Each laboratory that uses these guidelines should operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory capabil-
ity and the analysis of spiked samples as a continuing check on
performance. The l-aboratory should maintain performance records
to define the quality of data that are generated. After a date
specified by the Agency, ongoing performance checks should be
compared with established performance criteria to determine if
the results of analyses are within accuracy and precision limits
expected of the method. QC results should be reported with the
analytical results on a lot/batch basis.
16.2 The analysts should certify that the precision and accuracy of
the analytical results are acceptable by the following:
•.
16.2.1 The absolute precision of surrogate recovery, measured as
the RSD of the integrated EIMS or ECD peak area (A ) for
a set of samples, should be ± 30%.
16.2.2 The mean recovery (R ) of at least four replicates of a
quality control checR sample should meet Agency-specified
accuracy and precision criteria. This forms the initial
data base for establishing control limits (see Section
16.3 below).
A-78
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TABLE OF CONTENTS
1.0 Title page
2.0 Table of contents
3.0 Project description
4.0 Project organization and management
5.0 Personnel qualifications
6.0 Facilities, equipment, consumables, and services
6.1 Facilities and equipment
6.1.1 Evaluation
6.1.2 Inspections and maintenance
6.1.3 Calibration procedures and reference materials
5.2 Consumables
6.3 Services
7.0 Data generation
7.1 Experimental design
7.2 Sample collection
7.3 Sample custody
7.4 Laboratory analysis procedures
7.5 Internal quality control checks
7.6 Performance and system audits
3.0 Data processing
8.1 Collection
8.2 Validation
8.3 Storage
8.4 Transfer
8.5 Reduction
3.6 Analysis
9.0 Data quality assessment
9.1 Precision
9.2 Accuracy
9.3 Representativeness
9.4 Comparability
9.5 Completeness
10.0 Corrective action
11.0 Documentation and reporting
11.1 Documentation
11.2 Quality assurance reports to management
12.0 References
Appendices
Figure 5. Table of contents for a quality assurance project plan (QAPP)
as specified by the EPA Office of Toxic Substances (EPA 1984).
A-79
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Table 17 QC Procedures and Criteria for Analysis of Commercial Products
for 2,3,7,8-HDO and 2,3,7,8-HDF Congeners
Analysis event
QC criteria
Corrective action
Instrument
mass calibration
Calibration
standards
Samples
QC samples
Must demonstrate accurate mass
calibration daily versus OFTPP
or PFK.
Calibration standards are used
to establish the working curve,
document response factors and
mass calibration. After estab-
lishing the working curve, the
calibration standards are ana-
lyzed to bracket sample analyses.
Response factors should vary
< 30% from the working curve.
Precision of internal standards
must meet 50-150% recovery
criteria.
Precision of duplicate analyses
must meet ± 20% criteria.
The limit of quantisation (LOQ)
must meet 0.1 ppb for 2,3,7,8-
HDD congeners and 1.0 ppb for
2,3,7,8-HDF congeners. LOQ =
10 times background signal-to-
noise. Limit of detection (LOD)
must be calculated. LOD = 3 times
background signal-to-noise.
Method blanks analyzed with each
sample batch. Spiked samples
50-150% recovery.
Recalibration versus
OFTPP or PFK.
If RF values vary by
more than ± 30%,
sample analyses must
be repeated following
recalibration.
If accuracy, preci-
sion, or sensitivity
requirements are not
met, the sample must
be reanalyzed.
If method blank gives
any positive response,
repeat analysis. If
recovery results do
not meet the criteria,
the sample must be
reanalyzed.
A-80
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16.3 Control limits - The laboratory should establish control limits
using the following equations:
Upper control limit (UCL) = RC = 3 RSDC
Upper warning limit (UWL) = RC + 2 RSOC
Lower warning limit (LWL) = RC - 2 RSDC
Lower control limit (LCL) = RC - 3 RSDC
These may be plotted on control charts. If an analysis of a check
sample falls outside the warning limits, the analyst shouia be
alerted that potential problems may exist and corrective act-Ion
may be indicated. If the results for a check samole fall outside
the control limits, the laboratory should take corrective action
and recertify the performance of the method (Section 16.2) before
proceeding with analyses. The warning and control limits snould
be continuously updated as check sample replicates are added to
the data base.
16.4 Before processing any samples, the analyst should demonstrate
through the analysis of a reagent blank that all glassware and
reagent interferences are under control. Each time a set of sam-
ples is analyzed or there is a change in reagents, a laboratory
reagent blank should be processed as a safeguard against loss of
data caused by contamination.
16.5 Procedural QC - The various steps of the analytical procedure
should have quality control measures. These include but are not
limited to:
16.5.1 GC performance - See Section 7.1 for performance criteria.
16.5.2 MS performance - See Section 7.2 for performance criteria.
16.5.3 Quantisation - At least 10% of all manual calculations,
including peak area calculations, should be checked.
After changes in computer quantisation routines, the
results should be manually checked.
16.6 A minimum of 10% of all samples, one sample per month or one
sample per matrix type, whichever is greater, selected at random,
should be run in duplicate to monitor the precision of the method.
A difference of 20% or less should be achieved. If the difference
is greater than 20%, the analyst should investtgate the cause of
the imprecision and reanalyze the samples. Failure to satisfy
this requirement could invalidate the results obtained for samples
analyzed since the last precision determination.
A-81
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16.7 A minimum of 10% of all samples, one sample per mor.th, or one
sample per matrix type, whichever is greater, selected at random,
should be analyzed by the standard addition technique. Two ali-
quots of the sample are analyzed, one "as is" and one spiked with
an appropriate amount of HDD and/or HOP congeners. The samples
are analyzed together and the quantitative results calculated.
The recovery of the spiked compounds (calculated by difference)
should be 50-150%. If the sample is known to contain specific
HDD or HDF isomers, these isomers should be used for spiking.
If the aporoximate concentrations of analytes are known, the
amount added should be such that the spiking level is 1.5 to
-------
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the polybrominated biphenyl fire retardant, Firemaster FF-1. Environ Health
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APPENDIX 3
QUALITY ASSURANCE PROJECT PLAN FOR MEASUREMENT OF HALOGENATED
DIBENZO-a-OIOXINS (HMDs) AND OI3ENZQFURANS (KDFs)
Prepared by
Dr. John Smith
Environmental Protection Agency
Office of Toxic Substances
Chemical Regulation Branch
Washington, DC
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QUALITY ASSURANCE PROJECT PLAN FOR MEASUREMENT OF HALOGENATED
DIBENZO-o-OIOXINS (HMDs) AND DIBENZOFURANS (HDFs)
For quality assurance (QA) to be an effective method of measurement
validation of qualification, a Quality Assurance Project Plan (QAPP) must be
prepared and should include the following: history, management, and ultimate
location of samples; sampling and sample collection procedures; and extraction,
instrumental analysis, and data reduction procedures. The QAPP documents how
a laboratory intends to demonstrate its capability to produce data of accept-
able quality. The proposed rule is concerned with the following four classes
of compounds: brominated dibenzofurans, brominated dibenzodioxins, chlori-
nated dibenzofurans, and chlorinated dibenzodioxins. Of specific interest
within these classes are the compounds substituted with from four to seven
chlorine and/or bromine atoms per molecule. Hereafter these compounds will
collectively be referred to as halogenated dibenzofurans (HDFs) and haloge-
nated dibenzodioxins (HDDs).
An accurate trace or history of the life of a samole to be chem-
ically analyzed for HOFs and HOOs must be assembled and should contain infor-
mation beginning with a description of the system, scheme, or survey design
for sample collection. The sample collection scheme must be statistically
designed to allow measurement of variance of the sampling site. A written
record, sometimes called a traceaoility form, shall follow the sample.- Nec-
essary contents of the record are written general descriptions of what happens
to the sample, schedules and timetables, disposition, and handling. Following
final chemical analysis, the last entry in this trace should be the ultimate
location of the sample. Any further use (particularly for another activity),
movement, or examination of the sample should be added to the history.
Details of the sampling or sample collection procedures are the
second section of the QAPP. Since the history describes handling disposition,
schedules, and general descriptions of what happens in sampling, the require-
ment here is a detailed description of sampling and sample collection. Rea-
sons for using a specific or general sample selection process and reasons for
not using other processes must be included here. The rationale for any modi-
fications made to a method must also be given. Estimates of how well the
selected samples represent the material to be characterized are an essential
part of quality assurance. The greater the variability of composition of a
material, the more frequently sampling is required for estimation and charac-
terization purposes. In determining or estimating errors generated in sample
collection, control samples or blanks and HDF and HOD reinforced controls or
standards (native compounds or, preferably, isotopically labeled compounds)
must be sent to the collection site(s) and returned with the samples for
identical handling and treatment. Duplicate samples, which are collected,
documented, and handled the same as other samples, are necessary for recovery,
precision, and accuracy determinations.
The third section of the QAPP is a description of the extraction
and chemical analysis or screening test procedures. The QA record should show
in detail the mathematical calculations performed in arriving at final concen-
tration values. To determine both extraction efficiency and measurement effi-
ciency, it is necessary to use chemical ^tandards of HDF and HDD mixtures or
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individual HOP and HOD compounds in control samples. Once capabilities have
been established, the operator must determine precision and accuracy and use
those determinations to designate acceptable bounds for analytical performance.
The operator must keep control chart records to ensure that instrument read-
ings of standards fall within the range of acceptable performance. The oper-
ator must establish and describe the quantitative range of an instrument and
analytical procedure. Specific requirements are as follows:
For chemical analysis, these procedures must be demonstrated
capable to reproducibly and repeatedly quantitate HDDs and
HDFs at the required limits of quantitation. Quality control
check sample analysis begins the determination of system
capabilities.
For HDDs, at least two analyses of the same spiked standard at
the required LOQ must be quantifiable to within ± 20% of the
true value. The recovery of standard spiked into a product
material at the required LOQ and run through the entire chem-
ical analysis must be within 50-150% of the amount spiked,
with reference to the instrument standard, 99% of the time.
For HOFs, at least two analyses-of the same spiked standard at
the required LOQ must be quantifiable to within ± 20% of the
true value. The recovery of standard spiked into a product
material at the required LOQ and run through the entire chemical
analysis must be within 50-150% of the amount spiked, with
reference to the instrument standard, 99% of the time.
Qualitative requirements include: response factors for HDFs and
HDDs to be measured, instrument hardware and operating conditions (including
type and source of columns, carrier gas and flow rate, operating temperature
range, and ion source temperature), and structural assignment and/or library
matching of mass spectra. For both qualitative and quantitative measurements,
the instrument operator should be blind to the nature or source of samples,
particularly to duplicates, blanks, and brominated and spiked samples. The
limit of detection (LOD) and limit of quantitation (LOQ) shall be described
for each material. Tentative LOD and LOQ definitions are equal to three
times and ten times background noise, respectively. Details of quantitative
calibration procedures for the known and/or expected range of the HDF and HDD
levels in actual samples complete this section of the QAPP.
The last section of the QAPP must include the results of laboratory
participation in round robin analytical programs, the results of performance
audits, the results of systems audits, analytical results of performance audit
samples, persons responsible for all aspects of sampling, chemical analysis,
data analysis, corrective actions, and quality assurance/equality control
This section also must include a description of how problems are handled and
documented, and how corrections in working level notebooks are indicated and
explained.
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APPENDIX C
GUIDANCE FOR SAMPLING HALOGENATED DIBENZO-D.-DIOXINS (HDPs)
AND DIBENZOFURANS (HDFs)
Prepared by
Dr. John Smith
Environmental Protection Agency
Office of Toxic Substances
Chemical Regulation Branch
Washington, OC
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GUIDANCE FOR SAMPLING HMJ.OGENATED DIBENZO-&-DIOXINS (HDDs)
AND DIBENZQFURANS (HDFs)
The following are recommended steps for collecting samples of the
following four classes of compounds: brominated dibenzofurans, brominated
dibenzodioxins, chlorinated dibenzofurans, and chlorinated dibenzodioxins.
Of specific interest within these classes are the compounds substituted with
from four to seven chlorine and/or bromine atoms per molecule. Hereafter
these compounds will collectively be referred to as halogenated dibenzofurans
(HDFs) and halogenated dibenzodioxins (HDDs). Specific subgroups within the
collective group adaressed by the regulation will be more specifically named.
All of the following steps should be carefully recorded.
1. Determine the objectives of the samel ing exercise.
In the case of this proposed rule, some objectives might be (1) to
measure HDF and HDD levels occurring as oy-products or impurities in a product
to determine compliance with the rule, and (2) to know the limitations of
these measurements.
2. Describe the system or production process from which the sample
materials will be collected.
The-samples collected can only provide information about the product
from which they have been collected, and the completeness of this information
depends on how, at what time(s), where, and for what duration the samples are
collected. Of particular importance in determining a sampling plan for the
process are the continuity, homogeneity, and cyclic characteristics of the
production process. Compositing of samples for chemical analysis has benefits
in reducing analytical costs but risks in requiring lower instrument quanti-
fication levels and reducing the ability to identify whether HDFs and HDDs
are from uniform concentrations in all members of a composite or high concen-
trations in one member and no concentrations in the other members of the com-
posite. Each of the seven samples described in the "Guidance for a Sequential
Approach to the Sampling of Dibenzofurans and Dibenzodioxins" may be a randomly
selected composite, but the seven may not be composited unless the level of
concern is revised to one-seventh of the currently proposed levels.
3. Prepare for sample collection operations.
Professional judgment must be assisted by proven sampling methods.
To make a complete analysis, sampling should also determine the presence or
absence of HDFs and HDDs in locations or situations not necessarily judged to
be most important.
The proposed rule requires random sampling. A random sample selec-
tion system includes all possibilities and systematically chooses from among
a list or compilation of those possibilities. The way of choosing from among
the possibilities is the use of a random number table or through an automated
random number generator. A haphazard selection system, the result of selec-
tion through convenience and/or purposeful neglect, includes only a portion
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of all possibilities, has very littla estimating power, and should not be
considered a random selection procedure.
For the purpose of incorporating this sampling plan with the EPA
"Guidance for a Sequential Approach to the Sampling of Brominated and Chlori-
nated Dibenzofurans and Dibenzodioxins," the sampling unit for each process
will be a cycle. A cycle is defined as the time interval representing a
period within which the activity mix of a process is essentially repeated.
This cycle may be a batch, a shift, a day, or some other time period.
It is important to use random selection procedures in determining
the sampling time and location for each individual cycle (the details for
these determinations are given below in parts b and c). Cycles should be
sampled, one at a time, until as many as seven cycles have been sampled.
If two samples from any of the seven or fewer cycles are analyzed and show
measurement levels of HOFs and HDDs above the limit of quantitation, the
material is not in compliance with the rule. If none of the seven cycles
show HDDs above the required LOQ, no further sampling is necessary until
there are changes in the process as described below.
If there are changes known or suspected to change the generation of
HDFs and HDDs as by-products or impurities in a process or a cycle, the seven-
cycle sampling exercise must be done again. For each defined process and
independent cycle:
a. The size of a sample of a material to be analyzed will be
determined by the kind of material to be sampled and the sensitivity of the
analytical method. If the material is gaseous, a sample size of 10 m3 is
usually sufficient for analysis. If the material is aqueous or liquid, a
sample size of 1 L is usually sufficient for analysis. If the material is a
solid, a sample size of 100 g is usually sufficient for analysis. Any sample
size must have proper documentation demonstrating capability to measure HDDs
at the required limits of quantisation.
b. Location(s) of sample collection are defined as a subset
of all possible locations of storage of a given material. Physically possible
sample collection sites are listed and chosen through random selection
procedures.
c. In processes where over seven batches of a material are
produced in a year, sample collection times must be selected from a random
time period within each individual batch production cycle. The cycle time is
completely segmented into intervals long enough to collect a sample, and one
interval is randomly selected for the actual collection. For processes where
under seven batches of material are produced, random selection of seven sam-
ples from a list of production time segments for all batches is employed.
d. Quality assurance of the sampling and actual sample collec-
tion exercise includes documentation of: the sampling plan, sample collection
procedures, and any traceability records. Special notation should be made of
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any deviations from original plans and projections recorded at an earlier
stage of the sampling exercise. Especially:
dates and times of sample collection and chemical analysis
of samples;
exact location and time of sample collection;
process or product batch or lot identification: and
traceability records employed in the sample collection
and ultimate location of the samples.
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APPENDIX 0
GUIDANCE FOR A SEQUENTIAL APPROACH TO THE SAMPLING OF HALQGENATED
DIBENZO-a-QIOXINS (HDDs) AND DIBENZOFURANS (HDFs)
Prepared by
. Or. John Smith
Environmental Protection Agency
Office of Toxic Substances
Chemical Regulation Branch
Washington, DC
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GUIDANCE FOR A SEQUENTIAL ABROACH TO THE SAMPLING OF HALQGENATED
DIBENZO-a-OlOXINS CHDDs) AND DIBENZOFURANS (HDFsT
INTRODUCTION
This is the sequential sampling scheme referred to in the Proposed
Rule for Processing, Distribution, and Use of Certain Designated Chemical
Substances Suspected to Produce and/or Be Contaminated with Dioxins and Furans
(49 FR 28172, 28182, July 10, 1984).
For this proposed rule, dioxins and furans are in realitv four
classes of compounds: brominated dibenzofurans, brominated dibenzodioxins,
chlorinated dibenzofurans, and chlorinated dibenzodioxins, and specifically
within these classes the compounds substituted with from four to seven
chlorine and/or bromine atoms per molecule. Hereafter these particular com-
pounds will collectively be referred to as halogenated dibenzofurans (HDFs)
and halogenated dibenzodioxins (HDDs). Specific subgroups within the collec-
tive group addressed by the regulation will be more specifically named.
This document contains a statement of the sequential decision scheme
the rationale for its use, the details of sample selection (how much, when,
and where), and the statistical properties of the sequential decision scheme
ihis scheme is part of an overall activity to provide guidance to those who
are in a position to respond to the proposed regulations.
PROPOSED TRUNCATED SEQUENTIAL DECISION SCHEME
For a given material at the production/formulation site, select a
sample and measure the concentration of HDFs and HDDs. The sampling scheme
must be used for all tests and analytical evaluations for determination of
HDFs and HDDs. A measurement may be obtained by a screening method which has
been demonstrated by the tester to a documented degree of sensitivity and
specificity. The confirmatory method for identifying the HDFs and HDDs and
the concentrations of these compounds is high resolution gas chromatography/
high resolution mass spectrometry (HRGC/HRMS).
The product sample selection procedure is repeated until either a
product sample is found to contain HDFs and/or HDDs or seven samples have be°n
processed, in which case declare the production of the sampled material to be
free from HDFs and HDDs. If any of the concentrations of HDDs and/or HDFs in
a single product sample exceeds the required LOQ, the product will be deemed
to contain HDDs and/or HDFs. In HRGC/HRMS a resolvable chromatograohic peak
is considered a single brominated dibenzofuran compound, a single chlorinated
dibenzofuran compound, a single brominated dibenzodioxin compound, or a single
chlorinated dibenzodioxin compound. The number of samples, N, required is
not to exceed seven.
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REASON FOR NOT USING A SINGLE SAMPLE TO DETERMINE COMPLIANCE
Analysis of a material for HDFs and HDDs being generated as by-
products or impurities is subject to two main souces of variation: samples
may vary over time and/or location at the site. Secondly, the measured con-
centration of HDFs and HDDs will vary among repeated measurements on the
same sample. Therefore, the measured concentration of a particular single
screening test or single gas chromatographic peak will vary among samples
selected randomly in time and space so that a single sample would not be
representative of the underlying process except for the extreme cases.
In the following paragraphs the variable nature of ooservations
will be expressed by the symool e, the probability that a sample will have a
measured concentration below the required LOQ in a particular gas chromato-
graphic peak.
JUSTIFICATION FOR USING SEQUENTIAL SAMPLING
Given the analytical costs associated with HRGC/HRMS, it is impor-
tant to utilize statistical procedures which minimize the number of samples
which need to be analyzed. Sequential monitoring of a material allows a de-
termination of the presence of HDFs and/or HDDs to be based on fewer samoles,
on the average, than would be required under an equivalent fixed sample size
decision rule because of the large number of samples required to describe a
production having undemonstrated variability of HOF and HDD content. For eacn
such screening test or HRGC/HRMS analysis, this efficiency increases with the
number of potential HDFs and HDDs in the material and decreases as the magni-
tude of e increases. For materials containing sufficiently high concentra-
tions of even a single HDF or HDD compound (i.e., e is close to zero for
its associated screening test or peak), only one sample will be required to
declare the presence of HDFs or HDDs. For most multiple congener materials,
the use of the proposed scheme would probably result in the analysis of only
four or five samples to identify materials containing HDDs and HDFs above the
required LOQ. Even for materials containing a single congener at levels above
the required LOQ, the application of the scheme would probably result in the
analysis of fewer than seven samples to declare the presence of HDFs and HDDs
when e is no larger than 0.7.
TIMES. LOCATIONS. AND QUANTITIES
A material may be produced through a group of activities, within
one or more locations at a site. The production of the material is assumed
to be cyclic in nature, and within each cycle the distribution and concentra-
tion ranges of brominated and chlorinated dibenzofurans and dibenzodioxins
are assumed to be consistent. Each cycle, then, is a time interval represent-
ing a period within which the activity mix of the production of the material
is essentially repeated, such as a batch, shift, or day.
Samples under the proposed sequential sampling plan will be selected
from separate cycles of the production process. Further description of sample
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selection appears in "Guidance for Sampling Brominated and Chlorinated Dibenzo-
furans and Dibenzodioxins" (Appendix C). It is important to randomly select
the sampling time and location independently fop each cycle. Any deviations
from this random selection must be documented and justified. As many as seven
cycles will be required under the proposed procedure. Changes in the produc-
tion process, especially changes judged to potentially affect brominated and
chlorinated dibenzofuran and dibenzodioxin levels, require that, the entire
sequential testing effort be redone. If a single production process is the
potential source of brominated and chlorinated dibenzofuran and dibenzodioxin
exposure through several media, each medium should be separately tested by
the sequential sampling.
The size of the sample to be chemically analyzed depends on the
medium of the sample and the intended analytical method. Sample sizes are
recommended in the "Guidance for Sampling Brominated and Chlorinated Oibenzo-
furans and Dibenzodioxins."
STATISTICAL PROPERTIES OF THE PROPOSED DECISION SCHEME
In general, two decision errors are possible: (1) declaring mate-
rials in compliance to be not in compliance, and (2) declaring processes which
are not in compliance to be in compliance. Choice of 0.1 ppb as the test
threshold and requiring two samples to exceed it eliminates any significant
likelihood of committing an error of the first type. The probability of com-
mitting an error of the second type decreases with the number of potential
brominated and chlorinated dibenzofuran and dibenzodioxin congeners present
in the process and the maximum number of required samples, and increases.with
the magnitude of e for each such peak. The proposed maximum number of seven
samples was chosen because it results in an acceptable probability of the
second error without the requirement of an excessive amount of samples to be
analyzed to determine the presence of HDFs and HDDs. For the seven samples,
the error of the second type will be less than 0.25 for a typical process '
(i.e., one having five or more peaks) provided the e values for the process
are all no larger than 0.92 (assuming unconditional independence or complete
gas chromatographic resolution of individual HDF and HDD concentrations).
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