United Slates
Environmental Protection
Agency
Office of
Toxic Substances
Washington DC 20460
EPA-560/5-86-038
December, 1986
Toxic Substances
xvEPA
BROAD SCAN ANALYSIS OF THE FY82
NATIONAL HUMAN ADIPOSE TISSUE SURVEY
SPECIMENS
VOLUME IV -
POLYCHLORINATED DIBENZO-p-DIOXINS
(PCDD) AND
POLYCHLORINATED DIBENZOFURANS
(PCDF)
West North
Central
East North
Centra! •
West South Centra
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BROAD SCAN ANALYSIS OF HUMAN ADIPOSE TISSUE
VOLUME IV POLYCHLORINATED DIBENZO-p_-DIOXINS (PCDDs)
AND POLYCHLORINATED DIBENZOFURANS (PCDFs)
By
John S. Stanley
FINAL REPORT
EPA Prime Contract No. 68-02-4252
Work Assignment 21
MRI Project No. 8821-A(01)
December 31, 1986
Prepared for
National Human Monitoring Program
Field Studies Branch (TS-798)
Design and Development Branch
Office of Toxic Substances
U.S. Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460
Attn: Ms. Janet Remmers and Mr. Philip Robinson, Work Assignment Managers
Dr. Joseph Breen and Ms. Cindy Stroup, Program Managers
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
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DISCLAIMER
This document has been reviewed and approved for publication by the
Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S.
Environmental Protection Agency. The use of trade names or commercial prod-
ucts does not constitute Agency endorsement or recommendation for use.
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PREFACE
This report is the fourth of a five-volume series that details the
broad scan chemical analysis of composite adipose tissue samples. These com-
posite samples were prepared from individual specimens obtained from the En-
vironmental Protection Agency's (EPA) National Human Adipose Tissue Survey
(NHATS) fiscal year 1982 (FY82) repository.
This volume summarizes data generated from the analysis of the com-
posited samples for polychlorinated dibenzo-p_-dioxins (PCDD) and polychlori-
nated dibenzofurans (PCDF). Volume I, the executive summary, presents a
synopsis of all analysis efforts completed under the broad scan program.
Volumes II, III, and V deal specifically with the chemical analysis of the
NHATS composites, volatile organics, semivolatile organics, and trace ele-
ments, respectively. The statistical analyses of the data reported in these
volumes will be reported separately by the EPA's Office of Toxic Substances
(OTS) Design and Development Branch contractor, Battelle Columbus Laboratories.
The entire series of reports are referenced as follows:
Stanley JS. 1986. Broad scan analysis of human adipose tissue:
Volume I: Executive summary. EPA 560/5-86-035.
Stanley JS. 1986. Broad scan analysis of human adipose tissue:
Volume II: Volatile organic compounds. EPA 560/5-86-036.
Stanley JS. 1986. Broad scan analysis of human adipose tissue:
Volume III: Semivolatile organic compounds. EPA 560/5-86-037.
Stanley JS. 1986. Broad scan analysis of human adipose tissue:
Volume IV: Polychlorinated dibenzo-p_-dioxins (PCDDs) and poly-
chlorinated dibenzofurans (PCDFs). EPA 560/5-86-038.
Stanley JS, Stockton RA. 1986. Broad scan analysis of human adi-
pose tissue: Volume V: Trace elements. EPA-560/5-86-039.
These method development, sample analyses, and reporting activities
were completed for the EPA/OTS Field Studies Branch (FSB) broad scan analysis
of human adipose tissue program (EPA Prime Contract Nos. 68-02-3938 and 68-02-
4252, Work Assignments 8 and 21, respectively, Ms. Janet Remmers, Work Assign-
ment Manager, and Dr. Joseph Breen, Project Officer).
The samples were prepared with the assistance of Ms. Leslie Moody
and Mr. Steven Turner. The HRGC/MS methods development and sample analyses
were conducted by Ms. Kathy Boggess, Mr. Jon Onstot, and Dr. Thomas Sack.
The compositing scheme used to prepare the samples from the NHATS repository
was provided by Dr. Gregory Mack, Battelle Columbus Laboratories, under con-
tract to the EPA/OTS Design and Development Branch (Mr. Philip Robinson, Task
Manager, and Ms. Cindy Stroup, Program Manager).
Appy'ov'
MIDWEST RESEARCH INSTITUTE
Jo^rn E. Going, Director
Chemical Sciences Department
'Paul
Program Manager
C. Constant "
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TABLE OF CONTENTS
Executive Summary xi
I. Introduction 1
A. Broad Scan Analysis Strategy 1
B. Work Assignment Objectives 1
II. Recommendations 2
III. Experimental 2
A. Extraction 4
B. Cleanup 4
1. Gel Permeation Chromatography 4
2. Florisil Fractionation 5
C. Isolation of PCDDs and PCDFs 5
1. Amoco PX-21/Glass Fiber Adsorbent 6
2. Carbopak C/Celite Adsorbent Column Cleanup . . 7
D. Instrumental Analysis 8
E. Quality Assurance/Quality Control (QA/QC) 8
F. Data Interpretation 13
1. Qualitative 13
2. Quantitation 13
IV. Results 15
V. Quality Assurance/Quality Control (QA/QC) 44
A. Method Blanks ..... 44
B. Column Resolution 44
C. Instrument Performance 48
D. Internal Standard Recovery 48
E. Confirmation of 2,3,7,8-TCDD 53
VI. References 53
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LIST OF FIGURES
Page
Flow scheme for analysis of semivolatile organic
compounds in human adipose tissue
2 PCDD and PCDF detected in the NHATS FY82 composite
specimens from the Northeast Census Region 16
3 PCDD and PCDF detected in the NHATS FY 82 composite
specimens from the West Census Region 17
4 PCDD and PCDF detected in the NHATS FY82 composite
specimens from the South Census Region 18
5 PCDD and PCDF detected in the NHATS FY82 composite
specimens from the North Central Census Region 19
6 HRGC/MS chromatograms of a sample extract (composite 1 of
the 15-44 age group, Middle Atlantic (MA) Census divi-
sion) taken through (a) GPC/Carbopack C cleanup (equiva-
lent to 2.0 g tissue sample) and (b) GPC/Florisil/Amoco
PX-21 cleanup (equivalent to 20 g tissue sample) 20
7 PCDD and PCDF distribution in the general U.S. population
by age group 43
8 Comparison of PCDD and PCDF concentration (based on wet
tissue weight) profiles for Sweden, the general U.S.
population, and upstate New York 46
9 Selected ion current profile from the analysis of the
TCDD column performance mixture on a 60 m DB-5 column . . 47
10 Examples of response factor summary control charts for
2,3,7,8-tetrachlorodibenzo-p_-dioxin and 2,3,7,8-
tetrachlorodibenzofuran 49
Vll
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LIST OF TABLES
Table Page
1 Mass Ranges and Dwell Times for Analysis of Tetra- and
Pentachloro PCDDs and PCDFs at 2500 Resolution 9
2 Mass Ranges and Dwell Times for Analysis of Hexa-, Hepta-,
and Octachloro PCDDs and PCDFs at 2500 Resolution .... 10
3 Mass Ranges and Dwell Times for Analysis of Tetra- Through
Octachloro PCDDs and PCDFs in a Single Determination at
2500 Resolution 11
4 Specific PCDD and PCDF Congeners Available for Calibration
and Isomer Specific Measurements 12
5 Data Summary for 2,3,7,8-Tetrachlorodibenzo-p_-dioxin
[1746-01-6] - FY82 Composite Adipose Tissue Samples ... 22
6 Data Summary for l,2,3,7,8-Pentachlorodibenzo-p_-dioxin
[40321-76-4] - FY82 Composite Adipose Tissue Samples. . . 24
7 Data Summary for Hexachlorodibenzo-p_-dioxin [34465-46-8] -
FY82 Composite Adipose Tissue Samples 26
8 Data Summary for l,2,3,4,7,8,9-Heptachlorodibenzo-p_-dioxin
[35822-46-9] - FY82 Composite Adipose Tissue Samples. . . 28
9 Data Summary for Octachlorodibenzo-p_-dioxin [3268-87-9] -
FY82 Composite Adipose Tissue Samples 30
10 Data Summary for 2,3,7,8-Tetrachlorodibenzofuran
[51207-31-9] - FY82 Composite Adipose Tissue Samples. . . 32
11 Data Summary for 2,3,4,7,8-Pentachlorodibenzofuran
[57117-31-4] - FY82 Composite Adipose Tissue Samples. . . 34
12 Data Summary for Hexachlorodibenzofuran [55684-94-1] -
FY82 Composite Adipose Tissue Samples 36
13 Data Summary for 1,2,3,4,6,7,8-Heptachlorodibenzofuran
[67562-39-4] - FY82 Composite Adipose Tissue Samples. . . 38
14 Data Summary for Octachlorodibenzofuran [39001-02-0] -
FY82 Composite Adipose Tissue Samples 40
15 Wet Tissue Weight Concentration of PCDDs and PCDFs in
the NHATS FY82 Composite Specimens 42
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LIST OF TABLES (concluded)
Table Page
16 Lipid-Adjusted Concentration of PCDDs and PCDFs in the
NHATS FY82 Composite Specimens 45
17 Relative Response Factor (RRF) Summary for TCDD Versus
13C12-TCDD 50
18 Relative Response Factor (RRF) Summary for TCDF Versus
13C12-TCDD 51
19 Relative Response Factor (RRF) Summary for PCDD and PCDF
Calibration Standards 52
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EXECUTIVE SUMMARY
The U. S. Environmental Protection Agency's Office of Toxic Sub-
stances (EPA/OTS) maintains a unique program for monitoring human exposure
to potentially toxic substances. The National Human Adipose Tissue Survey
(NHATS) is a statistically designed annual program to collect and analyze a
nationwide sample of adipose tissue specimens for toxic compounds. The pri-
mary focus for NHATS has been to document trends in human exposure to en-
vironmentally persistent contaminants, specifically, organochlorine pesti-
cides and polychlorinated biphenyls (PCBs).
EPA/OTS has recognized the need to provide a more comprehensive
assessment of toxic substances that accumulate in adipose tissues. The
NHATS specimens collected during fiscal year 1982 (FY82) were designated for
"broad scan analysis" to detect volatile and semivolatile organic compounds
and trace elements.
This volume of the final report deals with the measurement of poly-
chlorinated dibenzo-£-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF)
in composited adipose tissue samples from the FY82 NHATS repository. The
objective of this study was (1) to identify analytical methods based on high
resolution gas chromatography/mass spectrometry (HRGC/MS) detection that are
capable of achieving detection limits in the low parts per trillion (picogram/
gram, pg/g) concentration range for individual PCDD and PCDF congeners and
(2) to complete the analysis of composite adipose tissue samples representing
the general U.S. population for the tetra- through octachloro-PCDD and PCDF
congeners.
Forty-six composite samples were prepared from the FY82 NHATS re-
pository according to a study design prepared by the EPA/OTS Design and De-
velopment Branch contractor, Battelle Columbus Laboratories. The composite
samples represent the nine U.S. census division stratified by three age
groups (0-14, 15-44, and 45 plus).
The sample preparation was completed using techniques that isolate
PCDD and PCDF congeners from potential interferences. The isolation of the
PCDD and PCDF was achieved using carbon-based chromatography columns. Two
different carbon materials were used to complete the analysis for the full
range of tetra- through octachloro-PCDD and PCDF congeners. The HRGC/MS op-
erated in the selected ion monitoring (SIM) mode was required for determina-
tion of the compounds at concentrations ranging from less than 5 pg/g (for
tetra- and pentachloro congeners) to greater than 1,000 pg/g for the octa-
chloro dibenzo-p_-dioxin.
The results of this study demonstrate that the EPA NHATS program
is an effective vehicle for documenting the exposure of the general U.S.
population to PCDD and PCDF. The analysis of the 46 composite samples es-
tablishes the presence of the 2,3,7,8-substituted tetra- through octachloro-
PCDD and PCDF congeners in the adipose tissues collected from the general
U.S. population.
XI
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The data from this study are comparable to work that has been re-
ported for other studies on adipose tissue samples from the United States
(specifically, upstate New York), Sweden, and Canada. Specifically, 2,3,7,8-
TCDD was detected in 35 of the 46 composites with an average 1 ipid-adjusted
concentration of 6.2 ± 3.3 pg/g. The average concentration of the other PCDD
compounds ranged from 33.5 pg/g for pentachlorodibenzo-£-dioxin (detected in
91% of the composites) up to 554 pg/g for octachlorodibenzo-p-dioxins which
was detected in all samples.
The data presented in this volume demonstrate some differences in
PCDD levels for the three age groups (0-15, 15-44, and 45 plus) evaluated.
The PCDF congeners were generally detected less frequently and were present
at lower concentration than the PCDD congeners. Obvious trends in the levels
of the PCDF congeners with respect to age were not observed. The PCDD and
PCDF congeners were detected in the composites representing each of the nine
U.S. census divisions.
The quantitative data for the PCDD and PCDF congeners presented in
this report have been submitted along with all supporting quality control
data to Battelle Columbus Laboratories for statistical analysis. This data
will be analyzed to determine the significance of differences in PCDD and
PCDF levels based on various demographic factors.
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I. INTRODUCTION
The National Human Adipose Tissue Survey (NHATS) is the main opera-
tive program of the National Human Monitoring Program (NHMP). The NHMP was
first established by the U.S. Public Health Service in 1967 and was subse-
quently transferred to the U. S. Environmental Protection Agency in 1970.
During 1979 the program was transferred within EPA to the Exposure Evaluation
Division (EED) of the Office of Toxic Substances (OTS).
NHATS is an annual program to collect a nationwide sample of adi-
pose tissue specimens and to chemically analyze them for the presence of
toxic compounds. The objective of the NHATS program is to detect and quantify
the prevalences of the compounds in the general population. The NHATS data
are used to address part of OTS's mandate under the Toxic Substances Control
Act (TSCA) to assess chemical risk to the U.S. population. The specimens are
collected from autopsied cadavers and surgical patients according to a sta-
tistical survey design (Lucas, Handy 1981). The survey design ensures that
specified geographical regions and demographic categories are appropriately
represented to permit valid and precise estimates of baseline levels, time
trends, and comparisons across subpopulations. Historically, organochlorine
pesticides and PC8 residues have been selected for evaluation.
A. Broad Scan Analysis Strategy
EPA/OTS has recognized the need to provide a more comprehensive as-
sessment of the toxic substances that accumulate in adipose tissue. An ag-
gressive strategy to assess TSCA-related substances that persist in the adi-
pose tissue of the general U.S. population has been developed by EED. The
NHATS specimens collected during fiscal year 1982 (FY82) were selected for a
broad scan analysis of volatile and semivolatile organic TSCA-related chem-
icals and trace elements (Mack, Stanley 1984).
The initiative to achieve a more comprehensive assessment necessi-
tated either the development of new methods or the modification of the exist-
ing analytical procedures, specifically high resolution gas chromatography/
mass spectrometry (HRGC/MS). Data on organochlorine pesticides and PCBs re-
ported for the NHATS specimens up to the FY82 collection are based on packed
column gas chromatography/electron capture detector (PGC/ECD) analysis.
B. Work Assignment Objectives
Growing concern about exposure to polychlorinated dibenzo-p_-dioxins
(PCDD) and dibenzofurans (PCDF) and reports of endogenous levels in human
adipose tissues from upstate New York, (Schecter et al. 1985; Schecter, Ryan
1985) Canada, (Ryan et al. 1985; Ryan et al. 1985), and Sweden (Nygren et al.
1985) prompted analysis for these compounds using composite adipose tissue
specimens from the FY82 repository.
The objectives of this phase of the work assignment were (a) to
identify appropriate analytical methods to determine the presence of PCDD and
PCDF congeners in human adipose tissue based on HRGC/MS detection; (b) to
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conduct preliminary evaluation of the analytical procedures; and (c) to com-
plete the sample workup and HRGC/MS analysis of 46 composite samples prepared
from the NHATS specimens collected during FY82.
Following this introductory section, recommendations for improving
the analytical method are presented in Section II. Experimental procedures,
detailed results of sample analysis, and a summary of QA/QC activities are
presented in Sections III to V. Pertinent references are cited in Section VI.
Appendix A provides a glossary of terms used throughout this text.
II. RECOMMENDATIONS
The methods described for PCDD and PCDF analysis were developed in
conjunction with the HRGC/MS broad scan analysis method (Stanley 1986c) for
detecting general semi volatile organic compounds in human adipose tissues. A
continued effort in following the trends of PCDD and PCDF will require that
the analytical method with the modifications discussed below be fully vali-
dated through intra- and interlaboratory studies.
Certified standards other than the 2,3,7,8-TCDD are not currently
available. It is imperative that the additional 2,3,7,8-substituted PCDD and
PCDF congeners be made available as certified materials for future studies to
make accurate comparisons of residue levels in the general population.
The analytical method also should be modified to include additional
carbon-13 labeled internal standards to improve the accuracy of the quantita-
tion of the tetra- through octachloro-PCDDs and PCDFs.
The time required for preparation of 10- to 20-g tissue samples by
the method described in this report is time intensive as a result of bulk
lipid removal by gel permeation chromatography (GPC). This procedure was
necessary to achieve the objective of the overall broad scan analysis pro-
gram. However, future studies that focus on PCDD and PCDF levels will re-
quire developing techniques that result in more expedient sample preparation.
III. EXPERIMENTAL
This section describes the procedures used to qualitatively and
quantitatively determine the PCDD and PCDF compounds present in human adipose
tissue. Figure 1 presents a schematic of the analytical methods for the broad
scan analysis for semivolatile organic compounds that are present in human
adipose tissue at concentrations of greater than 10 ng/g (parts per billion).
The method required compositing specified adipose tissue specimens from the
NHATS repository. The compositing scheme was prepared by Battelle Columbus
Laboratories under contract to EPA/OTS Exposure Evaluation Division, Design
and Development Branch.
The collection, handling, and storage of the FY82 NHATS specimens,
as well as the composite design and compositing procedures have been described
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Composite FY82 NHATS
Specimens (BCL Protocol)
Add Stable Isotope Labeled
Surrogate Compounds
Extraction - Tissumizer
Bulk Lipid Removal
Get Permeation Chromatography
10%
90%
Florisil Fractionation
HRGC/MS (Scanning)
0.01-O.litg/g
(PCBs. OCI Pesticides, Etc.)
AMOCO PX-21/
Glass Fiber
1 Florisil
HRGC/MS (SIM) for
Specific Compound Class
(Toxaphene PCDD, PCDF)
Quantitation/Dara Transfer
to BCL
Figure 1 - Flow scheme for analysis of semivolatile organic
compounds in human adipose tissue.
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previously. (Stanley 1986a; Stanley 1986b). The composite samples described
in this report consisted of aliquots of 5 to 26 individual adipose tissue
specimens. The composites were prepared to represent the general vs. popu-
lation stratified by the nine U.S. census divisions and three age groups.
Several stable isotope labeled compounds were added to the tissue
as surrogates. The spiked adipose tissue sample was extracted with methylene
chloride using a Tekmar® Tissumizer. The extracts were filtered through an-
hydrous sodium sulfate. Extractable lipid was determined using approximately
1% of the resulting extract. The extract was concentrated and the lipid was
separated from organic analytes using GPC. Approximately 10% of each sample
extract (1.0 to 2.0 g) was reserved for additional cleanup on a carbon based
(Carbopak C/Celite) chromatography column. The GPC-cleaned extracts (90% of
the original sample) were concentrated and then fractionated using Florisil.
The Florisil fractions were concentrated, spiked with an internal quantita-
tion standard, and analyzed by HRGC/MS. Before proceeding with the analysis
of PCDD and PCDF, the fractions were combined and subjected to further cleanup
on a charcoal/glass fiber column.
A. Extraction
Frozen composited adipose tissue samples (^ 20 g) were placed in a
2.2 x 15 cm culture tube. Each composite was spiked with several surrogate
compounds including naphthalene-d8 (2 ug), chrysene-d12 (2 pg), 1,2,4,5-tetra-
chlorobenzene-13C6 (2 (.jg), 3,3',4,4'-tetrachlorobiphenyl-13C12 (10 ug),
2,2',3,3',5,5',6,6'-octachlorobiphenyl-13C12 (8 pg), decachlorobiphenyl-13Ci2
(10 pg), 2,3,7,8-tetrachlorodibenzo-p_-dioxin-13C12 (1 ng), and octachlorodi-
benzo-p_-dioxin-13C12 (5 ng). The spiked adipose tissues were brought to room
temperature and homogenized for approximately 1 min with a Tekmar® Tissumizer
(Tekmar 18-EN probe) with successive aliquots (10 ml) of methylene chloride
(Burdick & Jackson, distilled in glass). The methylene chloride extracts
were dried by passage through anhydrous sodium sulfate. The sodium sulfate
column was rinsed with enough methylene chloride to bring the final extract
volume to 100 ml. The extractable lipids were determined by removing a I-mL
aliquot from the final extract. This aliquot was placed in a preweighed
2-dram vial, and solvent was removed using purified nitrogen. The vial was
reweighed and the lipid content was determined using the weight difference.
B. Cleanup
1. Gel Permeation Chromatography
The sample extracts were concentrated by Kuderna-Danish evapora-
tion. The final volumes were adjusted such that the solutions contained ap-
proximately 0.3 g of lipid per milliliter. An ABC autoprep GPC with an auto-
mated sampling valve was used for all bulk lipid separations. GPC columns
were prepared with approximately 60 g of Biobeads SX-3 swelled in methylene
chloride and packed as a slurry. The GPC was operated using methylene chloride
at 5 mL/min under a pressure of 7 to 15 psi. The GPC columns were calibrated
using a solution of vitamin E-acetate. Collection of the GPC effluent for
the semivolatile organic compounds was initiated as the response to the
vitamin E-acetate returned to baseline. Approximately 1 g of lipid material
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was added to each sampling loop of the GPC system. A total of 160 ml_ of GPC
effluent was collected after elution. The GPC effluents for a single sample
(3,200 mL/20 g of lipid) were combined, concentrated, and taken through the
GPC procedure a second time to remove residual lipid materials.
2. Florisil Fractionation
Florisil columns (12.5 g, 60/100 mesh, activated at 130°C) were
packed in hexane. Anhydrous sodium sulfate was added to the top of each col-
umn. The GPC extracts were concentrated and exchanged to hexane (final vol-
ume approximately 5 mL). This extract was added to the top of the Florisil
column and eluted with 200 ml each of 6%, 15%, and 50% diethyl ether in hex-
ane. The 6% fraction was collected separately from the 15% and 50% diethyl
ether fractions which were combined. The fractions were concentrated and
solvent exchanged to hexane using Kuderna-Danish evaporation. When the
eluents had concentrated to approximately 5 mL, they were further concen-
trated to 1 ml under a gentle stream of dry nitrogen. The fractions were
transferred to 1-mL conical vials and concentrated again to a final volume of
200 uL using nitrogen. All extracts were stored in a refrigerator until
analyzed by HRGC/MS. The 6% Florisil fractions were analyzed for general
semivolatile organics (particularly organochlorine pesticides, PCBs, chloro-
benzenes, polynuclear aromatic hydrocarbons, etc.). The more polar Florisil
fractions were analyzed for compounds such as phthalates, phosphate triestes
and additional organochlorine pesticide residues (Stanley 1986c).
C. Isolation of PCDD and PCDF
Following the broad scan HRGC/MS analysis for general semivolatile
organics, the Florisil column fractions were recombined and the sample ex-
tracts were taken through additional cleanup for isolation of the PCDDs and
PCDFs prior to analysis by HRGC/MS selected ion monitoring (SIM) techniques.
It was necessary to combine the Florisil fractions due to partial separation
of the PCDD and PCDF congeners in that chromatographic.
The isolation of the PCDD and PCDF congeners was accomplished using
one of two carbon-based (Amoco PX-21 or Carbopak C) chromatography procedures.
These cleanup techniques are modified procedures that isolate polychlorinated
aromatics from biological samples (Smith 1984) and soils. (USEPA 1983) The
Amoco PX-21 was used for samples that had been taken through the GPC and
Florisil fractionation procedures. Analysis of these samples yielded informa-
tion only for the tetra- and pentachloro dioxins and furans. The higher
chlorinated PCDD and PCDF congeners, especially OCDD and OCDF were retained
on Florisil using the elution procedure designated for the general semivola-
tile organic analysis.
The Carbopak C/Celite column was selected over the Amoco PX-21/glass
fiber column for the cleanup of the smaller sample aliquots since it resulted
in a less labor intensive procedure. The Carbopak C/Ceiite column was used
for the aliquot (1.0-2.0 g) of the original sample that had been taken only
through the GPC cleanup steps. These aliquots were used for analysis of the
hexa- through octachloro congeners.
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1. Amoco PX-21/G1ass Fiber Adsorbent
This carbon based chromatography column was used to prepare the
extracts that were analyzed for the tetra- and pentachloro PCDD and PCDF
congeners. Whatman GF/D fiber filters (600 mg) were cut into small pieces,
suspended in approximately 70 ml of methylene chloride, and shredded with a
Tekmar® Tissumizer. Amoco PX-21 carbon (50 mg), provided by Dr. L. Smith,
U.S. Fisheries and Wildlife, Columbia, Missouri, was added to this mixture.
The grinding was continued until the carbon was uniformly distributed on the
fibers. This mixture yielded the packing required for a single adsorbent
column.
a. Preparation of the Adsorbent Column
Thick-walled, 1.0 cm i.d. precision bore glass tubes (6-cm
lengths) were custom fit with Teflon® plugs. These plugs were bored to ac-
commodate 1/16 in. o.d. stainless steel tubing. This stainless steel tubing
was used to connect the columns to the solvent reservoir. Both ends of the
column were equipped with stainless steel tubing to allow disconnection of
the column and inversion to change the direction of the solvent flow. To
pack the column, one end was fitted with a Teflon® plug. Four disks of glass
fiber filters (Whatman GF/D 1.0 cm diameters) were placed flush against the
Teflon® plug. The carbon/glass fiber mixture consisting of 600 mg of glass
fibers and 50 mg of carbon was added to the column in methylene chloride. A
glass rod was used to pack the mixture. When this mixture was added to the
column, four disks of Whatman GF/D 1.0 cm diameter glass fiber filters were
gently packed on top of the carbon/glass fiber adsorbent. The second Teflon®
plug was pushed into place, compressing the adsorbent. The column bed height
measured 3 to 4 cm.
b. Column Cleanup
Prior to sample cleanup, the column was washed with 100 ml of
toluene, then 100 mL of methanol, and then 100 mL of toluene again. The re-
sidual toluene was displaced with 150 ml of cyclohexane/methylene chloride,
50/50. After this solvent had eluted through the column in reverse flow, the
column was inverted for forward flow. Immediately before sample application,
an additional 50 ml of the 50/50 solvent was eluted through the column with
nitrogen pressure to remove any air pockets. To maintain a flow of 3 to
5 mL/min, a slight nitrogen pressure was necessary.
c. Cleanup of Composite Sample Extracts
Following the broad scan HRGC/MS analysis, Florisil fraction
extracts for the selected composites were combined and diluted with 5 ml of
the cyclohexane/methylene chloride, 50/50 (v/v), solvent. This sample was
added to the column reservoir and allowed to drain onto the column. The sam-
ple vial and column reservoir were rinsed with two 5-mL portions of the cyclo-
hexane/methylene chloride (50/50) solvent. The flow rate was adjusted to 3
to 5 mL/min. After the last rinse, 75 mL of the cyclohexane/methylene chloride
(50/50) solvent was added to the reservoir. This was followed by 50 mL of
methylene chloride/methanol/benzene (75/20/5). The flow of the column was
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reversed by inversion of the column. The reservoir was filled with 40 mL of
toluene. This fraction was collected from the column at a rate no greater
than 3 to 4 mL/min. A positive pressure on the system with nitrogen was
necessary to achieve this flow rate.
Each solvent that eluted through the carbon/glass fiber adsor-
bent was collected separately. The toluene fraction collected in the reverse
elution sequence was reserved for analysis of PCDD and PCDF.
Concentration of the toluene fraction was achieved using a
gentle stream of prepurified nitrogen. The extract was transferred to a
1.0-mL conical vial and concentrated just to dryness. The collection tube
was rinsed with additional 1.0-mL aliquots of toluene that were also concen-
trated to dryness in the conical vial.
2. Carbopak C/Celite Adsorbent Column Cleanup
An alternate cleanup procedure was necessary to achieve analytical
data for the hexa- through octachloro-PCDD and PCDF. The method evaluation
studies for the broad scan analysis procedure of general semivolatile organ-
ics demonstrated that the higher chlorinated PCDD and PCDF, especially the
octachloro congeners were inefficiently recovered from the Florisil frac-
tionation column. (Stanley 1986) To overcome this problem approximately 10%
(1 to 2 g original weight) of each sample was reserved following the GPC-
cleanup of the original extract. This aliquot was taken through a carbon
cleanup column consisting of 18% Carbopak C on Celite 545®.
a. Preparation of the Carbon Adsorbent Column
This material was prepared by mixing 3.6 g of Carbopak C
(Supelco, 80/100 mesh) and 16.4 g of Celite 545® (Fisher Scientific). The
mixture was activated at 150°C for at least 6 h and then stored in a desic-
cator. Chromatographic columns were prepared using 5-mL disposable pipettes
fitted with small plugs of glass wool. The Carbopak C/Celite mixture was
packed using a vacuum aspirator until a 2-cm (340 mg) length of packing was
obtained.
b. Column Cleanup
The columns were preeluted with 2 mL of toluene followed by
I ml of methylene chloride/methanol/benzene (75/20/5), 1 mL of 1/1 cyclo-
hexane in methylene chloride, and 2 mL of hexane.
c. Cleanup of Composite Sample Extracts
The sample extracts were added to the Carbopak C/Celite col-
umns with several rinses of hexane. The columns were eluted with 1 mL of
cyclohexane/methylene chloride (50/50), 1 mL of methylene chloride/methanol/
benzene (75/20/5), and 20 mL of toluene. The toluene fraction was collected
in a culture tube and concentrated under flowing prepurified nitrogen. Final
concentration was achieved in a 1-mL conical vial. The sample was taken just
to dryness and submitted for HRGC/MS-SIM analysis for PCDDs and PCDFs.
-------
D. Instrumental Analysis
The PCDD and PCDF analyses for the tetra- through octachloro con-
geners were completed using a Kratos MS-50 double focusing mass spectrometer.
The ion source of the MS-50 was interfaced with a Carlo Erbra gas chromato-
graph equipped with a Grob type split/splitless injector. Analysis of the
PCDD and PCDF by congener group was achieved using either a 15 m or 60 m x
0.25 Durabond DB-5 fused silica column. Twenty microliters of isooctane was
added to the conical vials containing the sample extract from the Amoco PX-21/
glass fiber adsorbent column. Ten microliters of isooctane was added to the
residues from the Carbopak C/Celite column. The resulting extract was soni-
cated for at least 30 s in an ultrasonic bath before proceeding with the analy-
sis.
The analyses for the tetra- to octachloro-PCDD and PCDF congeners
for the composite of the 45-plus age category was achieved using two injec-
tions. Two ions characteristic of the molecular cluster for each PCDD and
PCDF congener, the internal standards, and a reference compound (perfluoro-
kerosene, PFK) were monitored. Tables 1 and 2 summarize the masses mon-
itored. The two values for each entry indicate the narrow mass ranges that
were monitored for a specified time at a mass resolution of 2500. All other
sample analyses (0-14 and 15-44 age composites) were completed in a single
injection using the data acquisition parameters listed in Table 3. The dif-
ferences in the instrumental analyses for the 45 plus versus the 0-14 and
15-44 age groups resulted from modification of the data acquisition programs.
This modification allowed the MS operator to automatically switch acquisition
parameters to monitor first the tetra- and pentachloro congeners and then the
hexa- through octachloro congeners.
One-microliter aliquots were injected and the HRGC column was held
isothermally at 100°C for 4 min, programmed rapidly to 270°C, held for 15 min,
and then programmed at 10°C/min to a final temperature of 325°C and held for
20 min. Table 4 summarizes the analytical standards used for calibration
standards and isomer specific measurements. Quantitation was achieved by the
internal standard method using 2,3,7,8-TCDD-13C12 for the tetra- and penta-
chloro congeners and OCDD-13C12 for the hexa- through octachloro congeners.
E. Quality Assurance/Quality Control
The QA/QC procedures included the daily verification of response
factors and instrument sensitivities, analysis of method blanks, and estimat-
ing recoveries of the internal quantitation standards. Additional QA/QC pro-
cedures included establishing criteria for qualitative identification and
quantisation. These criteria, including the relative retention time of spe-
cific congeners, ion ratios, and definition of limits of detection are de-
scribed below in the discussion on data interpretation.
-------
Table 1. Mass Ranges and Dwell Times for Analysis of Tetra- and Pentachloro
PCDD and PCDF at 2500 Resolution
Analyte Mass range (amu) Dwell time (s)
TCDF 303.801 - 304.001 0.069
TCDF 305.798 - 305.998 0.069
TCDD 319.796 - 319.996 0.062
TCDD 321.793 - 321.993 0.062
13C12-TCDD 331.836 - 332.036 0.059
13C12-TCDD 333.833 - 334.033 0.057
PeCDF 337.762 - 337.962 0.056
PeCDF 339.759 - 339.959 0.056
PeCDD 353.757 - 353.957 0.051
PeCDD 355.754 - 355.954 0.051
PFK 330.879 - 331.079 0.059
-------
Table 2. Mass Ranges and Dwell Times for Analysis of Hexa-, Hepta-,
and Octachloro PCDD and PCDF at 2500 Resolution
Analyte
HxCDF
HxCDF
HxCDD
HxCDD
HpCDF
HpCDF
HpCDD
HpCDD
OCDF
OCDF
OCDD
OCDD
13C12-OCDD
13C12-OCDD
PFK
Mass range
373.720 -
375.717 -
389.715 -
391.712 -
407.682 -
409.678 -
423.676 -
425.674 -
441.593 -
443.590 -
457.587 -
459.642 -
469.627 -
471.624 -
430.875 -
(amu)
373.920
375.917
389.915
391.912
407.882
409.878
423.876
425.874
441.893
443.890
457.887
459.826
469.927
471.925
431.075
Dwell time (s)
0.056
0.056
0.051
0.052
0.048
0.046
0.043
0.043
0.061
0.059
0.056
0.069
0.052
0.052
0.043
10
-------
Table 3. Mass Ranges and Dwell Times for Analysis of Tetra- Through
Octachloro PCDD and PCDF in a Single Determination
at 2500 Resolution
Analyte
Mass range (amu)
Dwell time (s)
Descriptor 1
TCDF
TCDF
13C12-TCDF
13C12-TCDF
TCDD
TCDD
13C12-TCDD
13C12-TCDD
PeCDF
PeCDF
PeCDD
PeCDD
PFK
Descriptor 2
HxCDF
HxCDF
PFK
HxCDD
HxCDD
HpCDF
HpCDF
HpCDD
HpCDD
37Cl4-HpCDD
37Cl4-HpCDD
OCDF
OCDF
OCDD
OCDD
13C12-OCDD
13C12-OCDD
303.806 -
305.801 -
315.802 -
317.798 -
319.795 -
321.791 -
331.832 -
333.820 -
337.756 -
339.752 -
335.742 -
357.738 -
380.855 -
373.702 -
375.698 -
380.855 -
389.692 -
391.689 -
407.653 -
409.649 -
423.643 -
425.639 -
429.632 -
431.628 -
441.603 -
443.600 -
457.593 -
459.590 -
469.589 -
471.586 -
303.998
305.995
316.001
317.999
319.997
321.995
332.041
334.039
337.969
339.967
355.967
357.964
381.095
373.938
375.936
381.095
389.938
391.936
407.910
409.907
423.910
425.907
429.903
431.900
441.882
443.879
457.881
459.879
469.885
471.883
0.033
0.033
0.033
0.032
0.031
0.031
0.030
0.030
0.029
0.030
0.029
0.056
0.052
0.014
0.014
0.013
0.013
0.013
0.025
0.025
0.024
0.025
0.024
0.023
0.023
0.023
0.022
0.022
0.022
0.022
11
-------
Table 4. Specific PCDD and PCDF Congeners Available for Calibration
and Isomer Specific Measurements
Compound
Source
Lot/code
Use
2,3,7,8-TCDF
2,3,4,8-TCDF
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,4,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,7,8,9-HpCDF
OCDD
OCDF
2,3,7,8-TCDD-13C12
2,3,7,8-TCDF-13C12
1,2,3,4,6,7,8-
HpCDD-37Cl4
13C12-OCDD
Cambridge Isotope Laboratories
C Rappe, Univ. Umea, Sweden
EPA QA Materials Branch
KOR Isotopes
C Rappe, Univ. Umea, Sweden
C Rappe, Univ. Umea, Sweden
KOR Isotopes
C Rappe, Univ
KOR Isotopes
C Rappe, Univ
Ultra Scientific
Ultra Scientific
Cambridge Isotope
Cambridge Isotope
KOR Isotopes
Umea, Sweden
Umea, Sweden
Laboratories
Laboratories
EF-903
T16
20603
AA-8-185
P8
P26
JB-II-65
Hx4
JB-II-64
Hp4
R00208
AWN-1203-T2
SSY-4-32
Cambridge Isotope Laboratories
quantitative
qua!itative
quantitative
quantitative
qua!itative
qua!itative
quantitative
qua!itative
quantitative
qualitative
quantitative
quanti tative
quantitative
quantitative
quantitative
quantitative
12
-------
F. Data Interpretation
1. Qual 1 tati ve
The HRGC/MS elation profiles of the tetra- through octachloro-PCDD
and PCDF congeners were established through the analysis of environmental
sample extract (fly ash from a municipal waste incinerator). The character-
istic ions for each homolog were plotted within the retention window es-
tablished using this mixture. The coincidental response of the characteristic
ions monitored within the established retention window and within ± 20% of
the theoretical ion ratio were the qualitative criteria that were used to
identify a response as a PCDD or PCDF congener. The identification of the
specific congeners required the response to be within ± 5 s of the retention
of the authentic standard relative to the specific internal standard.
2. Quantisation
Quantisation of the PCDD and PCDF congeners was achieved using the
carbon-13 (13C) labeled internal standards, 13C12-2,3,7,8-TCDD (13C12-TCDD) ,
and 13C12-OCDD. The tetra- and pentachloro homologs were quantitated versus
13C12-TCDD, while the levels of the hexa- through octachloro compounds were
calculated versus the 13C12-OCDD response. The recoveries of the internal
quantitation standards 13C12-2,3,7,8-TCDD and 13C12-QCDD were achieved by
comparing the relative responses to the internal recovery standards, 13C12-
2,3,7,8-TCDF and 37Cl4-l,2,3,4,6,7,8-HpCDD, respectively. These internal
recovery standards were added only to the extracts from the 0-14 and 15-44
age groups.
Relative response factors (RRF) were calculated for each homolog
using the PCDD compounds listed in Table 4. Except for 2,3,7,8-TCDF and
OCDF, the PCDF congeners were available as qualitative standards only. The
RRF values for the PCDF homologs, therefore, were assumed to be the same as
the respective quantitative PCDD congener. The RRF values were calculated as
shown in Equation 1.
RRf = Eq
MIS X
where A-Tr, = the sum of the area responses for the two characteristic ions
of the standard compound;
= the sum of the area responses for the two characteristic ions
of the internal standard;
= concentration of the internal standard (pg/uL); and
= Concentration of the standard compound (pg/jjL).
A calibration curve was established using three concentration levels
of standards; for example, the calibration curve for 2,3,7,8-TCDD was initially
established with standards at concentrations of 1, 10, and 100 pg/uL. The
1 and/or 10 pg/uL standards were analyzed daily to verify response factors
and method sensitivity.
13
-------
The concentration of a PCDD or PCDF congener in a composite sample
was calculated as shown in Equation 2.
sample x
WT A x RRF x Wt
r _ sample x IS F
q'
where CWT = wet tissue concentration of the PCDD or PCDF congener in each
tissue (pg/g);
A , = sum of the area responses for the two characteristic ions of
sample Qf thg RCDD Qr pCQf congener;
A,<- = sum of the area responses for the two characteristic ions of
the respective internal standard;
C,,. = concentration of the internal standard added to the sample
(1,000 pg of 13C12-TCDD or 5,000 pg of 13C12-OCDD);
RRF = the relative response factor for the PCDD or PCDF congener
from Equation 1; and
Wt = mass of the composited FY82 NHATS specimens (grams).
The 1 ipid-adjusted concentration was calculated by dividing the wet tissue
weight concentration by the extractable lipid (%) value.
All data were qualified to reflect whether the compound was a posi-
tive quantifiable parameter, present as a trace value only, or was not de-
tected. Positive quantifiable values were identified for responses greater
than 10 times the average background signal-to-noise. Trace (Tr) values were
assigned to responses that were in the range of 2.5 to 10 times the average
background signal-to-noise. A value of not detected (ND) was used to reflect
that a response was not detected at greater than 2.5 times the average signal-
to-noise. The definition of 2.5 times the average background signal-to-noise
for the LOD measurement was selected to maintain consistency with the existing
protocols for the determination of 2,3,7,8-TCDD (EPA 1983). A limit of detec-
tion (LOD) was calculated for all trace and not detected values using the
peak height response of the respective internal standard and the average mea-
sured signal-to-noise for the characteristic ions of the PCDD and PCDF con-
geners.
14
-------
IV. RESULTS
A qualitative summary of the HRGC/MS-SIM analyses of the sample ex-
tracts of FY82 NHATS composite samples is presented in Figures 2 to 5. Ident-
ification of compounds at trace or positive quantifiable values are desig-
nated by a plus. Compounds that were not detected are designated by a minus.
The number of plus and minus symbols under each age group represents the total
number of composites analyzed. As noted, the predominant compounds in the
adipose tissue composites were the 2,3,7,8-substituted congeners. Congener
designations are presented based on the corresponding retention times of the
observed responses with the available standards and on the basis of informa-
tion of the specific congeners present in adipose tissue from previous studies
(Graham et al. 1985; Nygren et al. 1985; Patterson et al. 1985; Rappe et al.
1985; Ryan et al. 1985a; Ryan et al. 1985b; Shecter, Ryan 1985). Although
the elution patterns of the HxCDD and HxCDF congeners were similar to those
reported in other studies (Schecter et al. 1985; Schecter, Ryan 1985; Ryan et
al. 1985a; Ryan et al 1985b; Nygren et al. 1985; Rappe et al. 1985; Graham et
al. 1985; Patterson et al 1985; Ryan 1985), the exact congeners cannot be
assigned due to availability of standards at the time of analysis.
Figure 6 provides examples of the HRGC/MS chromatograms observed
for the two different extract cleanup procedures. The lower reconstructed
ion chromatogram (RIC) illustrates the response noted for an extract taken
through the Amoco PX-21/glass fiber column. The upper RIC was achieved for
the analysis of an aliquot of the same sample taken through the Carbopak C/
Celite column. As noted from the chromatogram (Figure 6) of the Amoco PX-21/
glass fiber column, there was no response noted for the OCDD and 13C12"OCDD.
This is a result of the retention of the higher chlorinated compounds on the
Florisil column used for preparation of the samples for broad scan analysis
of semivolatile organic compounds by HRGC/MS scanning techniques. (Stanley
1986). This is the reason it was necessary to use a second aliquot of the
extract that was taken only through GPC and a carbon based column to deter-
mine the hexa- through octachloro congeners. The abrupt change in baseline
observed for each chromatogram at approximately 1,750 scans (-v 35 min) is due
to the change in descriptors from monitoring the tetra- and pentachloro con-
geners to the hexa- through octachloro congeners.
The large response at approximately 1300 scans can be attributed to
13C12-3,3',4,4'-tetrachlorobiphenyl. This compound was added as a surrogate
to the initial sample to monitor method recoveries in the broad scan analysis.
Retention of this particular PCB on the charcoal column is a result of non-
ortho.ortho'-substitution. (Stalling et al. 1979) The responses for the other
peaks in the HRGC/MS chromatograms can be assigned to PCDD congeners and in-
ternal standards. The responses to PCDF congeners are obvious when looking
at the extracted ion current plots of the characteristic ions.
15
-------
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-------
A) GPC/Corfeop,ik C-C
•'CIJ-OCDD/OCDD,
8) GPC/Floriill/Amoco PX- 21
/I3C|3-TCDD/TCDD
500
10-00
1000
20-00
1500
30-00
Figure 6. HRGC/MS chromatograms of a sample extract (composite 1 of the
15-44 age group, Middle Atlantic (MA) Census division) taken
through (a) GPC/Carbopak C cleanup (equivalent to 2.0 g
tissue sample) and (b) GPC/Florisil/Amocol PX-21
cleanup (equivalent to 20 g tissue sample).
20
-------
The results of the PCDD and PCDF analyses for the specific congen-
ers or homologs are summarized in Tables 5 to 14. These data are reported as
both wet tissue weight and 1ipid-adjusted concentrations. As discussed in
the experimental section of this report, the data for the tetra- and penta-
chloro-PCDD and PCDF compounds were determined in the sample aliquot (approx-
imately 20 g), that was taken through the entire cleanup procedure (GPC,
Florisil, and Amoco PX-21). However, as noted in Tables 5, 6, 10, and 11,
the data for the tetra- and pentachloro congeners for several of the compos-
ites were reported for sample aliquots (1.0 to 2.0 g) that were taken only
through the GPC separation and Carbopak C/Celite cleanup. This was done be-
cause of reanalysis requirements of the larger sample aliquot for general
semivolatile organics or as a result of irrecoverable loss of the larger sam-
ple aliquot during final cleanup. The analysis of the smaller aliquot for
the tetra- and pentachloro homologs was compromised only with respect to the
achievable limit of detection.
The data for the hexa- through octachloro congeners (Tables 7-9 and
12-15) are generally reported for sample aliquots of 1.0 to 2.0 g that had
been taken through the GPC and Carbopak C/Celite procedures. However, the
results of the analysis of the larger sample aliquot for two composites (sam-
ples 82042 and 82083) are reported in the data tables. Quantitative data are
reported for sample 82042 while entries for 82083 indicate that the hexa-
through octachloro congeners were detected but not quantitated due to the low
recovery of the internal standard. Quantitative data have been reported for
the hexa- through octachloro-PCDDs and PCDFs for sample 82042 but the data
may be considered suspect based on the possible differences in the recoveries
of these compounds in the GPC, Florisil and Amoco PX-21 cleanup steps as com-
pared to the GPC and Carbopak C/Celite procedure used for the smaller sample
aliquots.
Figure 7 summarizes the average PCDD and PCDF congener levels in
the composite specimens based on the age categories. The data are plotted to
indicate both the average wet tissue weight concentrations and the 1ipid-
adjusted concentrations. The average concentrations were determined from
composites that resulted in trace or positive quantifiable values for each
PCDD or PCDF congener. The estimated limits of detection for the not de-
tected responses were not included in the calculation of these averages. A
simple Q-test was used to determine data points that were qualified as out-
liers for each age group. The values determined to be outliers by this test
were not included in the values for the average PCDD and PCDF levels given in
Figure 7. These data points are also identified by a footnote in Tables 5-14.
As noted in Figure 7, the average concentrations of specific PCDD
congeners, with the exception of PeCDD, generally show an increase with re-
spect to age. But no trends are noted for the PCDF data with respect to age.
This possibly resulted from the low levels of PCDFs in the composite samples,
indicating that potential exposure to these compounds is considerably less
than that of PCDDs. The large difference noted for the OCDF concentration in
the 45-plus age category is that a positive response was detected in only 3
of the 16 composites. The OCDF concentration of these samples ranged from
240 to 360 pg/g (wet tissue weight concentration).
21
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Table 15. Wet Tissue Weight Concentration of PCDDs and PCDFs in
the NHATS FY82 Composite Specimens
Frequency of Mean concentration Range of
Compound detection (%)a (pg/g) detection (pg/g)
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
HxCDDC
1,2,3,4,7,8,9-HpCDD
OCDD
2,3,7,8-TCDF
2,3,4,7,8-PeCDF
HxCDFb
1,2,3,4,6,7,8-HpCDF
OCDF
^Percentage of composites
76
91
98
98
100
26
89
72
93
39
in which
5.0 ± 2.7
33.5 ± 37.4
69.4 ± 68.5
81.9 ± 77.8
554 ± 291
10.1 ± 9.2
28.7 ± 17.1
19.1 ± 9.5
16.5 ± 11.8
60.1 ± 110
the compound(s) was
ND
ND
ND
ND
10
ND
ND
ND
ND
ND
detected.
- 12
- 4,300
- 500
- 900
- 2,950
- 460
- 77
- 51
- 55
- 620
positive quantifiable values.
Reference compounds not available for specific isomers.
42
-------
800
700
600
500
400
300
'
100
50
PCDDs from NHATS FY82
Composite Specimens
Wet Tissue Concentration
HI 0-14 years
f"j 15 - 44 years
r~j 45+ years
Lipid Adjusted Concentration
250
o>
O>
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O
is so
1
o
o
O
PCDFs from NHATS FY82
Composite Specimens
TCDF
PeCDF
HxCDF
HpCDF
OCDF
Figure 7. PCDD and PCDF distribution in the general
U.S. population by age group.
43
-------
The data used to prepare the illustration in Figure 7 were also
used to calculate an average concentration across all age groups. Tables 15
and 16 provide these average concentrations based on wet weight and lipid
bases, respectively. These tables indicate the number of samples for which
positive responses were noted as well as the range of concentrations observed.
Figure 8 graphically demonstrates the average PCDD and PCDF data
(based on wet tissue weight) for the FY82 NHATS composite samples as compared
to the data reported for Swedish (Nygren et al. 1985; Rappe et al. 1985) and
upstate New York human adipose tissue samples. (Schecter et al. 1985; Schecter,
Ryan 1984) The profiles observed from each of these studies are very similar
with the exception of the OCDF response in the NHATS specimens. This increase
in the OCDF average value, as explained previously, is the result of high
concentrations observed for a minimum number of samples observed as positive
values. These data reported for the FY82 NHATS composite specimens also are
consistent with the general trends reported for human adipose samples col-
lected in Canada (Ryan et al. 1985a; Ryan et al. 1985b; Ryan 1985) and other
specific regions within the United States. (Graham et al 1985; Peterson et al.
1985)
V. QUALITY ASSURANCE/QUALITY CONTROL (QA/QC)
As discussed in the experimental section of this report, the analy-
sis of the composite samples was completed with various QA/QC efforts. These
included the analysis of method blanks, verification of column resolution for
2,3,7,8-TCDD from other TCDD isomers, daily verification of response factors
and method sensitivity, estimation of the absolute recovery of the internal
standards, and verification of 2,3,7,8-TCDD using fragment ions and high reso-
lution MS.
A. Method Blanks
Method blanks were handled exactly as samples and were analyzed
along with the actual composite sample extracts. The PCDD and PCDF congeners
were not detected in the method blanks. The analysis of these method blanks
documented that the response to the PCDD and PCDF congeners noted in sample
extracts were, in fact, due to the endogenous levels in the composited speci-
mens from the FY82 NHATS repository.
B. Column Resolution
The resolution of the 60-m DB-5 column for separation of 2,3,7,8-
TCDD from the other TCDD isomers, specifically the 1,4,7,8-, 1,2,3,4-, 1,2,3,7-,
and 1,2,3,8- isomers, was verified on a daily basis. Figure 9 is an example
of a daily column resolution check using the DB-5 column. Resolution is de-
fined as the height of the valley (x) between the 2,3,7,8-TCDD isomer and the
closest eluting TCDD isomers divided by the height of the 2,3,7,8-TCDD re-
sponse (y) times 100%. As noted in this figure, the analysis of the isomer
mixture demonstrated that the column achieved a resolution of 29% using the
322 response from the other TCDD isomers. The column performance varied up
to 50% through the analysis of these samples but was considered acceptable.
44
-------
Table 16. Li pid-Adjusted Concentration of PCDDs and PCDFs in
the NHATS FY82 Composite Specimens
Compound
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
HxCDDC
1,2,3,4,7,8,9-HpCDD
OCDD
2,3,7,8-TCDF
2,3,4,7,8-PeCDF
HxCDFb
1,2,3,4,6,7,8-HpCDF
OCDF
Frequency of
detection (%)
76
91
98
98
100
26
89
72
93
39
Mean concentration
8 (pg/g)
6.2 ± 3.3
43.5 ± 46.5
86.9 ± 83.8
102 ± 93.5
694 ± 355
15.6 + 16.5
36.1 ± 20.4
23.5 ± 11.6
20.9 ± 15.0
73.4 ± 134
Range of
detection (pg/g)
ND -
ND -
ND -
ND -
19 -
ND -
ND -
ND -
ND -
ND -
14.2
5,000
620
1,300
3,700
660
90
60
79
890
.Percentage of composites in which the compound(s) was detected.
Mean concentration (± one standard deviation) calculated using trace and
positive quantifiable values.
Reference compounds not available for specific isomers.
45
-------
420
140
120
100
80
60
40
20
0
Sweden, 1985
Nygren et al., 1985
TCDF PeCDF HxCDF HpCDF OCDF
TCDD PeCDD HxCDD HpCDD OCDD
a
O
General U.S. population
EPA NHATS FY 82 Composite Specimens
TCDF PeCDF HxCDF HpCDF OCDF
TCDD PeCDD HxCDD HpCDD OCDD
c
a
o
c
O
O
600
580
140
120
100
80
60
40
20
0
Upstate New York
Schecter, A. and J.J. Ryan, 1985
TCDF PeCOF HxCDF HpCDF OCDF
TCDD PeCDD HxCDD HpCDD OCDD
Figure 8. Comparison of PCDD and PCDF concentration (based on
wet tissue weight) profiles for Sweden, the general
U.S. population, and upstate New York.
(Source: Schecter, Ryan 1985; Nygren et al. 1985.)
46
-------
8
ID
CN
CN
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to m
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00
>> S
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4-> O
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IX
47
-------
However, it is recommended that future studies focusing on the analysis of
2,3,7,8-TCDD in human adipose tissue require a minimum column resolution of
25% to maintain conformance with other protocols developed during the course
of this study. (USEPA 1983) This will require a specific analysis for
2,3,7,8-TCDD using a polar column such as 60 m SP2330, SP2340, or a 50 m
CP-Sil 88.
C. Instrument Performance
The instrument sensitivity and consistency of response factors were
documented through the routine analysis of calibration standards. Figure 10
provides a plot of the response factors for 2,3,7,8-TCDD and 2,3,7,8-TCDF
over the course of the analysis of the 0-14 and 15-44 age categories. The
target limits on the variability of the average relative response factors
were ± 20% for TCDD and TCDF and ± 30% for the penta- through octachloro-PCDD
and PCDF.
Tables 17 and 18 present the summaries of the actual response factors
measured for calibration standards ranging from 1 to 100 pg/uL for 2,3,7,8-TCDD
and 2,3,7,8-TCDF. The data represent the calibration summaries completed
during the analysis of the 0-14 and 15-44 age group composites. The average
relative response factor (RRF) was updated daily, and this mean value was
used to calculate the residue levels in the composite samples on each analy-
sis day. The composites representing the 45-plus age group were analyzed
several months before the 0-14 and 15-44 age groups. Hence, a calibration
curve for each analyte was prepared at the start of each analysis.
Table 19 presents a summary of the daily calibration data that was
generated during the analysis of the 0-14 and 15-44 age groups. This table
includes the calibration range, the average RRF, and the observed variability
for each of the PCDD and PCDF congeners included in the calibration standards.
The ratio of the average RRF values for 2,3,7,8-TCDF/2,3,7,8-TCDD is 1.7 com-
pared to 1.4 for the ratio of the average RRF values for OCDF/OCDD. This
indicates that the actual RRF values for the penta- through heptachlorodibenzo-
furan congeners might be expected to be somewhat greater than measured for
the dibenzo-£-dioxin congeners with the same degrees of chlorination. Thus,
the true concentrations of the PCDF congeners may be less than reported in
Tables 1-13. This potential difference in the relative response factors re-
flects the need to develop analytical standards representative of each PCDD
and PCDF congener group in future programs.
D. Internal Standard Recovery
The recovery of the internal quantitation standards (13C12~2,3,7,8-
TCDD and 13C12-OCDD) were measured using two additional internal recovery
standards, (13C12-2,3,7,8-TCDF and 37C14-1,2,3,4,6,7,8-HpCDD). These recovery
standards were added just prior to the HRGC/MS analysis. These compounds
were also included in the calibration standards to establish the necessary
response ratios. The average recovery of the 13C12-2,3,7,8-TCDD was deter-
mined to be 86 ± 26% for extracts taken through the Amoco PX-21/glass fiber
column. As noted previously, the 13C12-OCDD was not detected in these ex-
tracts due to its retention on Florisil that was used as part of the broad
scan analysis preparation scheme. The 13C12-TCDD and 13C12-OCDD recoveries
averaged 50 ± 17% and 90 ± 50%, respectively, for the composite extracts
eluted through the Carbopak C/Celite column.
48
-------
TETRACHLORODIBENZO-p-DIOXIN
k.
O
4-
I?
a
O
a
V
i- .0 -
2.4 -
o n _
n
1 .8 -
1 .6 -
1 .4 -
1 .2 -
1 1
0.8 -
0.6 -
0.4 -
0.2 -
•
a a
D a
j D n an
j-j LJ __ pt LJ
n nannnDu a a
a n a o i
u D a
DO
,
I I 1 1 I 1 1 1 | 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ( 1 1 1 1 t
11 -MAP-8514-MAP-8519-MAP-851O-APR-8519-APR-85 29-APP-85
of Analysis
o
£
a
n
TETRACHLORODIBENZOFURAN
3.5 -
3 -
2.5
1 .5 -
1
0.5 -
0
a
an
_a_
r~r" n—r-rn i r i i i i i i i i-r—r~r~r ~r ~ T-i~T~T~r~r~r i i i i r
1 1 -MAP-8514-MAR-8519-MAR-8511-APP-8522-APP-8529-APP-85
Dot* of Analysis
Figure 10. Examples of resnonse factor summary control
charts for 2,3,7,8-tetrachlorodibenzo-p_-dioxin
and 2,3,7,8-tetrachlorodibenzofuran.
49
-------
Table 17. Relative Response Factor (RRF)
Summary for TCDD Versus 13C12-TCDD
Date
ll-March-1985
ll-March-1985
ll-March-1985
Average RRF
Standard Deviation
12-March-1985
13-March-1985
13-March-1985
14-March-1985
15-March-1985
15-March-1985
18-March-1985
18-March-1985
19-March-1985
19-March-1985
20-March-1985
21-March-1985
21-March-1985
22-March-1985
10-Apri 1-1985
11-Apri 1-1985
18-Apri 1-1985
18- April -1985
19-Apri 1-1985
19-Apri 1-1985
19-Apri 1-1985
22-Apri 1-1985
24-Apri 1-1985
25-Apri 1-1985
25- April -1985
25-Apri 1-1985
29-Apri 1-1985
29-Apri 1-1985
30-Apri 1-1985
30-Apri 1-1985
Ol-May-1985
Ol-May-1985
Average RRF
Standard Deviation
RRF
0.953
0.724
0.816
0.831
0.094
0.717
0.987
0.987
0.736
0.787
0.684
0.776
0.799
0.640
0.881
0.830
0.832
0.670
0.809
0.805
0.845
0.913
0.869
1.119
0.799
0.876
0.983
1.114
0.983
1.059
0.904
1.199
1.180
0.790
0.521
0.496
0.724
0.850
0.168
Concentration
(ng/(jL)
0.100
0.010
0.001
0.010
0.010
0.010
0.010
0.010
0.001
0.010
0.001
0.001
0.010
0.010
0.010
0.001
0.010
0.010
0.010
0.001
0.010
0.001
0.001
0.010
0.100
0.010
0.100
0.010
0.100
0.001
0.010
0.001
0.001
0.001
O.G10
50
-------
Table 18. Relative Response Factor (RRF)
Summary for TCDF Versus 13C12-TCDD
Date
ll-March-1985
ll-March-1985
ll-March-1985
Average RRF
Standard Deviation
12-March-1985
13-March-1985
13-March-1985
14-March-1985
15-March-1985
15-March-1985
18-March-1985
18-March-1985
18-March-1985
19-March-1985
20-March-1985
21-March-1985
21-March-1985
22-March-1985
10-Apri 1-1985
11-Apri 1-1985
18- April -1985
18-Apri 1-1985
19-Apri 1-1985
19-Apri 1-1985
19-Apri 1-1985
22-Apri 1-1985
24-Apri 1-1985
25-Apri 1-1985
25-Apri 1-1985
25-Apri 1-1985
25-Apri 1-1985
29-Apri 1-1985
29-Apri 1-1985
30-Apri 1-1985
30-Apri 1-1985
Ol-May-1985
Ol-May-1985
Average RRF
Standard Deviation
RRF
1.726
1.356
1.307
1.463
0.187
1.434
1.708
1.262
1.456
1.383
1.135
1.692
1.768
1.280
1.580
1.508
1.583
1.100
1.561
1.386
1.316
1.513
1.484
1.347
1.179
1.476
1.853
1.860
1.082
1.656
1.794
1.361
1.476
1.846
1.098
1.128
1.043
1.215
1.442
0.249
Concentration
(ng/uL)
0.100
0.010
0.001
0.010
0.010
0.010
0.010
0.010
0.001
0.010
0.010
0.001
0.010
0.010
0.010
0.001
0.010
0.010
0.010
0.001
0.010
0.001
0.001
0.010
0.100
0.010
0.001
0.100
0.010
0.100
0.001
0.010
0.001
0.001
0.001
0.010
51
-------
Table 19. Relative Response Factor (RRF) Summary
for PCDD and PCDF Calibration Standards3
2,3,7
2,3,7
1,2,3
1,2,3
1,2,3
OCDD
OCDF
Compound
,8-TCDD
,8-TCDF
,7,8-PeCDD
,4,7,8-HxCDD
,4,6,7,8-HpCDD
Cal ibration
range (pg/pL)
1-100
1-100
1-100
5-500
5-500
10-1,000
10-1,000
Average
0.
1.
0.
1.
1.
0.
0.
850
442
259
953
410
656
927
RRFb
Standard
0.
0.
0.
0.
0.
0.
0.
deviation
168
249
071
603
340
164
225
These data represent a summary of calibration events completed from March 11,
b!985 through May 1, 1985.
The RRF values reported are a compilation of all calibration standard analy-
ses rather than a summary of the initial calibration curve data.
52
-------
E. Confirmation of 2,3,7,8-TCDD
Qualitative confirmation of 2,3,7,8-TCDD in selected extracts was
achieved using the following criteria: (1) retention time of the character-
istic mass (m/z 320 and 322) corresponding to the 2,3,7,8-TCDD, (2) proper
response ratios of m/z 320/322 (0.67 to 0.90), (3) response of fragment ions
corresponding to a loss of COC1 (m/z 257 and 259), and (4) HRMS (R = 10,000)
analysis using the characteristic ions 319.897 and 321.894.
A second chromatographic peak was noted for several of the compos-
ite sample analyses that eluted 8 to 10 scans earlier than the 2,3,7,8-TCDD.
This peak exhibited response within the acceptable ion ratios for m/z 320 and
m/z 322 for the low resolution MS analyses. This peak also exhibited response
to these ions for the high resolution MS analyses and the fragment ion cor-
responding to loss of COC1 in a separate analysis. However, this second peak
could not be confirmed as a TCDD isomer as a result of a coincidental response
noted at m/z 358. Further evaluation of this response is necessary before an
identification can be established. This response has not been noted in other
studies dealing with PCDD and PCDF in human adipose tissue. This response
may be due to some interferent that might be effectively removed by frac-
tionating the sample extract on alumina prior to MS analysis.
VI. REFERENCES
Graham M, Hileman FD, Wendling J, Wilson JD. Chlorocarbons in adipose tissue
samples. 5th International Symposium on Chlorinated Dioxins and Related Com-
pounds, Bayreuth, FRG, September 16-19, 1985.
Mack GA, Stanley JS. 1984. Preliminary strategy on the National Human
Adipose Tissue Survey. Washington, DC: Office of Toxic Substances. Con-
tracts 68-07-6221 (Task 21) and 68-02-3938 (Work Assignment 8). NHATS-ST-01.
Nygren M, Hansson M, Rappe C, Domellof L, Hardell L. 1985. Analysis of
polychlorinated dibenzo-p_-dioxins and dibenzofurans in adipose tissue from
soft-tissue sarcoma patients and controls. 189th National ACS Meeting Sym-
posium on Chlorinated Dioxins and Dibenzofurans in the Total Environment III,
Miami, Florida, 1985. Preprint Division of Environmental Chemistry, ACS 25:
160-163, Paper No. 55.
Patterson DG, Holler JS, Smith SJ, Liddle JA, Sampson EJ, Needham LL. Human
tissue data in certain U.S. populations. 5th International Symposium on
Chlorinated Dioxins and Related Compounds, Bayreuth, FRG, September 16-19,
1985.
Rappe C, Nygren M, Lindstrom G, Hanson H. Dioxins and dibenzofurans in human
tissues and milk of European origin. 5th International Symposium on Chlor-
inated Dioxins and Related Compounds, Bayreuth, FRG, September 16-19, 1985.
Ryan JJ. Variation of dioxins and furans in humans with age and organ by
country. 5th International Symposium on Chlorinated Dioxins and Related Com-
pounds, Bayreuth, FRG, September 16-19, 1985.
53
-------
Ryan JJ, Williams DT, Lau BPY, Sakuma T. 1985a. Analysis of human fat tissue
for 2,3,7,8-tetrachlorodibenzo-p_-dioxin and chlorinated dibenzofuran residues
in dioxins and dibenzofurans in the total environment II. Keith LH, Rappe C,
Choudhary G, eds. Butterworth Publishers, pp. 205-214.
Ryan JJ, Schecter A, Lizotte R, Sun W-F, Miller L. 1985b. Tissue distri-
bution of dioxins and furans in humans from the general population.
Chemosphere 14: 929-932.
Schecter A, Ryan JJ. 1985. Dioxin and furan levels in human adipose tissue
from exposed and control populations. 189th National ACS Meeting Symposium
on Chlorinated Dioxins and Dibenzofurans in the Total Environment III, Miami,
Florida. Preprint Division of Environmental Chemistry, ACS 25:160-163, Paper
No. 56.
Schecter A, Tiernan TO, Taylor ML, VanNess GF, Garrett JH, Wagel DJ, Gitlitz
G, and Bogdasarian M. 1985. Biological markers after exposure to polychlor-
inated dibenzo-p_-dioxins, dibenzofurans, biphenyls, and biphenylenes. Part
I: Findings using fat biopsies to estimate exposure in chlorinated dioxin
and dibenzofurans in the total environment II. Keith LH, Rappe C, Choudhary,
G, eds. Butterworth Publishers, pp. 215-246.
Smith LM, Stalling DL, Johnson JJ. 1984. Determination of part per trillion
levels of polychlorinated dibenzofurans and dioxins in environmental samples.
Anal Chem 56:1830-1842.
Stalling DL, Smith LM, Pethy JD. 1979. An expanded approach to the study
and measurement of PCBs and selected planar halogenated aromatic environ-
mental pollutants. Ann. N.Y. Acad. Sci. 320, 48-59.
Stanley JS. 1986a. Broad scan analysis of human adipose tissue: Volume I
Executive summary. EPA 560/5-86-035.
Stanley JS. 1986b. Broad scan analysis of human adipose tissue: Volume II
Volatile organic compounds. EPA 506/5-86-036.
Stanley JS. 1986c. Broad scan analysis of human adipose tissue: Volume III
Semivolatile organic compounds. EPA 560/5-86-037.
Stanley JS. 1986d. Broad scan analysis of human adipose tissue: Volume IV
Polychlorinated dibenzo-£-dioxins (PCDD) and polychlorinated dibenzofurans
(PCDF). EPA 560/5-86-038.
Stanley JS, Stockton RA. 1986. Broad scan analysis of human adipose tissue:
Volume V Trace elements. EPA 560/5-86-039.
USEPA. 1983. 2,3,7,8-Tetrachlorodibenzo-p_-dioxin in soil and sediment by
high resolution gas chromatography/low resolution mass spectrometry. Con-
tract laboratory protocol. Solicitation WA-84-A002.
54
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APPENDIX A
GLOSSARY OF TERMS
55
-------
FY82 Fiscal year 1982
HpCDD Heptachlorodibenzo-p_-dioxin
HpCDF Heptachlorodibenzofuran
HRGC High resolution gas chromatography
HxCDD Hexachlorodibenzo-p-dioxin
HxCDF Hexachlorodibenzofuran
MS Mass spectrometry
NHATS National Human Adipose Tissue Survey
NHMP National Human Monitoring Program
OCDD Octachlorodibenzo-£-dioxin
OCDF Octachlorodibenzofuran
OTS Office of Toxic Substances
PCDD Polychlorinated dibenzo-p_-dioxin
PCDF Polychlorinated dibenzofuran
PeCDD Pentachlorodibenzo-p_-dioxin
PeCDF Pentachlorodibenzofuran
SIM Selected ion monitoring
TCDD Tetrachlorodibenzo-p_-dioxin
TCDF Tetrachlorodibenzofuran
56
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1 REPORT NO."
EPA-560/5-86-038
3. RECIPIENT'S ACCESSlOr»NO.
4 TITLE AND SUBTITLE
Broad Scan Analysis of Human Adipose Tissue
Volume 4 - Polychlorinated Dibenzo-£-dioxins (PCDD) and
Polychlorinated Dibenzofurans (PCDF)
5. REPORT DATE
December 1986
6. PERFORMING ORGANIZATION CODE
Midwest Research Institute
7 AUTHOR(S)
John S. Stanley
8. PERFORMING ORGANIZATION REPORT NO.
8821-4(01
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri, 64110
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-02-3938
68-02-4252
12 SSJDNSJDHIMG AGENCY NAME AND ADDRESS
U.i. Environmental Protection Aqency
Office of Toxic Substances
Field Studies Branch (TS-798)
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
siqn
01, ?
a c n t n
ent Branch/Exposure Evaluation Division
20160
15." sGPP'UETWEN'T ARY"NoTES '
J. Remmers and P. Robinson, Work Assignment Managers
J. Breen and C. Stroun. Program Manaaers
16. ABSTRACT
The U.S. EPA's Office of Toxic Substances (OTS) maintains a unique capability
for monitoring human exposure to potential toxic substances through the National Human
Adipose Tissue Survey (NHATS). The primary focus for NHATS has been to document trends
in human exposure to environmentally persistent contaminants, specifically, organochlor
ine pesticides and polychlorinated biphenyls (PCBs).
EPA/OTS has recognized a need to expand the use of the NHATS program to pro-
vide a more comprehensive assessment of toxic substances that are accumulated in adi-
pose tissues. This report deals specifically with the measurement of polychlorinated
dibenzo-jj-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) in composited adipose
tissue samples from the FY82 NHATS repository.
The results of this study demonstrate that the EPA NHATS program is an effec-
tive vehicle for documenting the exposure of the general U.S. population to PCDDs and
PCDFs. The analysis of the 46 composite samples prepared from the fiscal year 1982
NHATS repository establishes the prevalence of the 2,3,7,8-substituted tetra- through
octachloro-PCDD and PCDF congeners in the U.S. population. The PCDD and PCDF levels
are comparable to data presented from other studies that focus on samples collected in
upstate New York, Canada, and Sweden.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c COSATl Field/Group
Human Adipose Tissue
Polychlorinated Dibenzo-£-dioxins (PCDD) and
Polychlorinated Dibenzofurans (PCDF)
HRGC/MS
Selected ion monitoring
Parts per trill ion
Analysis
Determination
13. ;.3TPI3UT,GN STATEMENT
Release unlimited
19 SECURITY CLASS I This Report)
Unclassified
21 NO. OF PAGES
20 SECURITY CLASS ,
Unclassified
22 PRICE
EPA ?arm 2220-1 (9-73)
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