United States
Environmental Protection
Agency
Toxic Substances
Washington DC 20460
\
EPA-560/13-79-010
September. 1979
Toxic Substances
EPA
ANALYTICAL PROTOCOLS FOR
MAKING A PRELIMINARY
ASSESSMENT OF
HALOGENATED ORGANIC
COMPOUNDS IN MAN AND
ENVIRONMENTAL MEDIA
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ANALYTICAL PROTOCOLS FOR MAKING A PRELIMINARY ASSESSMENT OF
HALOGENATED ORGANIC COMPOUNDS IN MAN AND
ENVIRONMENTAL MEDIA
by
Edo D. Pellizzari, Mitchell D. Erickson and R. A. Zweidinger
Contract No. 68-01-4731
Project Officer
Dr. Joseph Breen
Office of Toxic Substances
Washington, DC 20460
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, DC 20460
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DISCLAIMER
This report has been reviewed by the Surveillance and Analysis Division
of the Office of Toxic Substances, U. S. Environmental Protection Agnecy,
and approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U. S. Environmental protec-
tion Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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ABSTRACT
This comprehensive report presents the methods which will be used in
Phase II of this program. Analytical methods for halogenated hydrocarbons in
air, water, soil, breath, blood, urine, and tissue have been validated. A
radioimmuneassay procedure for carcinoembryonic antigen (CEA) was validated.
The data collection instruments (participant consent form, questionnaire,
etc.) are also presented here.
iii
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iv
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CONTENTS
Abstract iii
Figures vii
Tables xi
Acknowledgements xx
List of Abbreviations xxi
1. Conclusions 1
2. Recommendations 2
3. Program Objectives 3
4. Introduction 4
5. Collection and Analysis of Vapor-Phase Halogenated Organics . . 7
6. Collection and Analysis of "Purgeable" Halogenated Organics
in Aqueous Environmental and Human Samples 9
7. Collection and Analysis of "Extractable" Halogenated Organics
in Environmental and Human Samples 47
8. Collection and Analysis of Blood Samples for CEA 62
9. Collection and Analysis of Halogenated Organics in Human Breath 64
10. Field Operations 69
Appendices
A. Analytical Protocol: Sampling and Analysis of Volatile Organic
Compounds in Ambient Air 73
B. Analytical Protocol: Sampling and Analysis of Purgeable
Halogenated Organics in Water 102
C. Analytical Protocol: Sampling and Analysis of Purgeable
Halogenated Hydrocarbons in Blood 114
D. Analytical Protocol: Sampling and Analysis of Purgeable
Halogenated Organics in Urine 126
E. Analytical Protocol: Sampling and Analysis of Purgeable
Halogenated Organics in Tissue 138
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CONTENTS (cont'd.)
F. Analytical Protocol: Sampling and Analysis of Extractable
Halogenated Organics in Blood 149
G. Analytical Protocol: Sampling and Analysis of Extractable
Halogenated Organics in Water 164
H. Analytical Protocol: Sampling and Analysis of Extractable
Halogenated Organics in Tissue 178
I. Analytical Protocol: Sampling and Analysis of Extractable
Halogenated Organics in Soil and Sediment 193
J. Analytical Protocol: Carcinoembryonic Antigen Assay 206
K. Analytical Protocol: Sampling and Analysis Procedure for
Breath Samples 218
L. GC/MS/COMP Limit of Detection Data: Mass Spectra, Ion
Tracings, and SIM Plots 222
M. GC/MS/COMP Single Ion Plots (SIP) of Extractable Halogenated
Organics Used in RMR Determinations 277
N. Data Collection Instruments Utilized in Pretest 284
0. Data Collection Instruments Proposed for Main Study 292
VI
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FIGURES
Number Page
4-1 Schematic flow diagram demonstrating interlocking relation-
ship between environment, man and association with
incidence of cancer 5
6-1 Dynamic purge apparatus for water samples 15
6-2 Dynamic purge apparatus with magnetic stirring capability ... 16
6-3 Headspace purge apparatus for blood, urine, and tissue samples. 17
6-4 Tenax cartridge loading and desorption apparatus for use with
radiolabelled volatile compound 20
6-5 Schematic of permeation tube flow system for synthesizing
air/vapor mixtures 26
6-6 Percent recovery as a function of purge time 35
A-l Vapor collection and analytical systems for analysis of organic
vapors in ambient air 75
A-2 Sampling head for housing cartridge sampling train 83
A-3 Profile of ambient air pollutants obtained using high resolu-
tion gas chromatography/mass spectrometry/computer .... 90
A-4 Background profile for Tenax GC cartridge blank 91
A-5 Schematic diagram of GC-MS computer system 92
A-6 Mass fragmentogram of ions characteristic of internal standards
(perfluorobenzene, m/z 186 and perfluorotoluene, m/z 236
with a fragment at m/z 186) used in ambient air samples. . 97
A-7 Mass fragmentograms of characteristic ions representing
chlorotoluene (m/z^ 91, 126, 128), dichlorotoluene (m/z
125, 127, 160), chlorobenzaldehyde (m/z 139, 140) and
standards perfluorobenzene (m/z 186) and perfluoro-
toluene (m/z 236) 98
B-l Purge apparatus for water samples 107
vii
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FIGURES (cont'd.)
Number Page
C-l Headspace purge apparatus for blood, urine, and tissue samples 119
D-l Headspace purge apparatus for blood, urine, and tissue samples 131
E-l Headspace purge apparatus for blood, urine, and tissue samples 142
J-l Typical inhibition curve of CEA standard indirect assay. . . . 217
K-l Schematic of Spirometer for collection of breath samples . . . 220
L-l Mass spectrum of trifluralin near LOD (MW = 335) 223
L-2 Mass spectrum of atrazine near LOD (MW = 216) 224
L-3 Mass spectrum of y-BHC near LOD (MW = 291) 225
L-4 Mass spectrum of heptachlor near LOD (MW = 373) 226
L-5a Mass spectrum of chlordane near LOD (MW = 410) 227
L-5b Mass spectrum of chlordane near LOD (MW = 410) 228
L-6 Mass spectrum of £,p_'-DDE near LOD (MW = 318) 229
L-7 Mass spectrum of 2-chlorobiphenyl near LOD (MW = 188) 230
L-8a Mass spectrum of hexachlorobiphenyl near LOD (MW = 358).... 231
L-8b Mass spectrum of hexachlorobiphenyl near LOD (MW = 358).... 232
L-9 Mass spectrum of decachlorobiphenyl near LOD (MW = 494). . '. . 233
L-10 Ion tracings for trifluralin, atrazine, lindane and heptachlor 234
L-ll Ion tracings for lindane and heptachlor 235
L-12 Ion tracings for chlordane 236
L-13 Ion tracings for p_,p_'-DDE 237
L-14 Ion tracings for 2-chlorobiphenyl and hexachlorobiphenyl . . . 238
L-15 Ion tracing for decachlorobiphenyl 239
L-16 Analysis of Y-BHC (40 m SCOT capillary, SE-30) ; m/z 219 chosen
for SIM analysis 240
L-17 Analysis of chlordane (40 m SCOT capillary, SE-30); m/z 373
chosen for SIM analysis 241
L-18 Analysis of heptachlor (40 m SCOT capillary, SE-30); m/z 272
chosen for SIM analysis 242
L-19 Analysis of p_,p_'-DDE (40 m SCOT capillary, SE-30); m/z 246
chosen for SIM analysis 243
L-20 Analysis of trifluralin (40 m SCOT capillary, SE-30); m/z 306
chosen for SIM analysis 244
viii
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FIGURES (cont'd.)
Number Page
L-21 Analysis of trazine (40 m SCOT capillary, SE-30); m/z 215
chosen for SIM analysis 245
L-22 Analysis of pesticide mixture, full scan mode; ViOO ng . . . . 246
L-23 Analysis of pesticide mixture, full scan mode; V>00 ng . . . . 247
L-24 Analysis of pesticide mixture, full scan mode; ^500 ng . . . . 248
L-25 Analysis of pesticide mixture, full scan mode; ^500 ng . . . . 249
L-26 Analysis of pesticide mixture, full scan mode; ^100 ng . . . . 250
L-27 Analysis of pesticide mixture, full scan mode; ^100 ng . . . . 251
L-28 Analysis of pesticide mixture, full scan mode; ^100 ng . . . . 252
L-29 Analysis of pesticide mixture, full scan mode; -^100 ng . . . . 253
L-30 Analysis of pesticide mixture, full scan mode; ^20 ng 254
L-31 Analysis of pesticide mixture, full scan mode, "^20 ng 255
L-32 Pesticide analysis, SIM mode 257
L-33 Pesticide analysis, SIM mode 258
L-34 Pesticide analysis, SIM mode 259
L-35 Analysis of d, --pyrene 260
L-36 Analysis of polychlorobiphenyls (40 m glass SCOT capillary,
200°C, isothermal) 261
L-37 Analysis of polychlorinated biphenyls (40 m glass SCOT capil-
lary, 220°C isothermal) 262
L-38 Analysis of PCB mixture (SID-100) 264
L-39 Analysis of PCB standard mixture (STD-20) 267
L-40 Representative Output from Analysis of PCB mixtures, selected
ion monitoring mode 269
L-41 Analysis of arochlor mixture, full scan mode 272
M-l SIP for PCB Mix: 104 ng/pl of 2-chlorobiphenyl, m/z 188; 570
ng/|Jl hexachlorobiphenyl, m/z 360; 1156 ng/pl decachloro-
biphenyl, m/z 498 278
M-2 SIP for PCB mix - 10.4 ng/|Jl of hexachlorobiphenyl, m/z 360;
8.4 ng-d10-pyrene, m/z 212 279
M-3a SIP for m/z 183 of lindane 100 ng/fjl 280
M-3b SIP for m/z 181 of lindane 100 ng/fJl 280
ix
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FIGURES (cont'd.)
Number Page
M-3c SIP for m/z 202 of atrazine 100 ng/|Jl 280
M-3d SIP for m/z 200 of atrazine 100 ng/^l 281
M-3e SIP for m/z 212 of d1()-pyrene „ 281
M-3f SIP for m/z 373 of chlordane 100 ng/fJl 281
M-3g SIP for m/z 246 of p_,£-DDE 100 ng/pl 282
M-3h SIP for m/z 264 of trifluralin 100 ng/|jl 282
M-3i SIP for m/z 272 of heptachlor 100 ng/pl 283
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TABLES
Number Page
6-1 [ C] Compounds for Recovery Study 13
6-2 Recovery of C-Labeled Compounds from Tenax GC by Thermal
Desorption 22
6-3 Recovery of Halogenated Hydrocarbons from Distilled Water
Spiked in Methanol and Toluene Solutions 24
6-4 Recovery of Halogenated Hydrocarbons from Distilled Water
Spiked from Gas Permeation System 28
6-5 Recovery of Halogenated Hydrocarbons from Distilled Water
Spiked from Gas Mixing Bulb 29
6-6 RMR Values of Standards for 100 m Capillary on the LKB 2091
GC/MS 30
14
6-7 Recovery of C-Labeled Model Compounds from Water 32
6-8 Percent Recovery at Various Purge Times 34
14
6-9 Percent Recovery of C-Labeled Compound from Blood ...... 37
6-10 Recovery of Halogenated Hydrocarbons from Human Blood Spiked
from Gas Mixing Bulb 39
14
6-11 Percent Recovery of C-Labeled Compounds from Urine 41
6-12 Recovery of Halogenated Hydrocarbons from Human Urine Spiked
from Gas Mixing Bulb 42
6-13 Recovery of Halogenated Hydrocarbons from Human Adipose Tissue 44
7-1 Estimated Limits of Detection for Extractable Halogenated
Organic Analysis 50
7-2 RMRs for PCBs and Pesticides of Interest to This Program. . . 52
7-3 RMR Factors for Standard PCB Solutions, Selected Ion
Monitoring Mode 53
7-4 Recovery of Extractable Halogenated Organics from Whole Blood
and Plasma 54
xi
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TABLES (cont'd.)
Number Page
7-5 Recovery Studies of Extractable Halogenated Hydrocarbons
from Human Plasma Equilibrated for 19 Hr 55
7-6 Recovery Studies of Extractable Halogenated Hydrocarbons
from Water 58
7-7 Recovery of Extractable Halogenated Hydrocarbons from Human
Tissue Extracts 59
8-1 Plasma CEA Levels of Individuals Residing in the "Old Love"
Canal Site of Niagara Falls, NY 63
9-1 Preliminary Results of Pilot Study of Benzene Body Burden. . . 65
9-2 Estimated Levels of Halogenated Compounds in Human Breath From
"Old Love" Canal in Niagara Falls, NY 67
9-3 Estimated Levels of Benzene in Human Breath from "Old Love"
Canal in Niagara Falls, NY. . . . 68
A-l Overall Theoretical Sensitivity of High Resolution Gas Chroma-
tography/Mass Spectrometry/Computer Analysis for Atmos-
pheric Pollutants 76
A-2 Tenax GC Breakthrough Volumes for Several Atmospheric Pollutants 84
A-3 Operating Parameters for GLC-MS-COMP System 89
14
B-l Recovery of C-Labeled Model Compounds from Water 104
B-2 Recovery of Halogenated Hydrocarbons from Distilled Water
Spiked from Gas Mixing Bulb 105
B-3 Instrumental Operating Conditions 108
14
C-l Percent Recovery of C-Labelled Compound from Blood 116
C-2 Recovery of Halogenated Hydrocarbons from Human Blood Spiked
from Gas Mixing Bulb 117
C-3 Instrumental Operating Conditions 120
14
D-l Percent Recovery of C-Labeled Compounds from Urine 128
D-2 Recovery of Halogenated Hydrocarbons from Human Urine Spiked
from Gas Mixing Bulb 129
D-3 Instrumental Operating Conditions 132
E-l Recovery of Halogenated Hydrocarbon from Human Adipose Tissue. 140
xii
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TABLES (cont'd.)
Number
E-2 Instrumental Operating Conditions 144
F-l Recovery Studies of Extractable Halogenated Hydrocarbons
from Human Plasma 151
F-2 Estimated Limits of Detection for Extractable Halogenated
Organics Analysis 156
F-3 RMR for PCBs and Pesticides of Interest to This Program. ... 158
F-4 RMR Factors for Standard PCB Solutions, Selected Ion
Monitoring Mode 159
F-5 Semi-Volatile Halogenated Hydrocarbons in Methanol Spiking
Solution 160
G-l Recovery Studies of Extractable Halogenated Hydrocarbons from
Water 166
G-2 Estimated Limits of Detection for Extractable Halogenated
Organics Analysis 171
G-3 RMRs for PCBs and Pesticides of Interest to This Program . . . 172
G-4 RMR Factors for Standard PCB Solutions, Selected Ion
Monitoring Mode 173
G-5 Semi-Volatile Halogenated Hydrocarbons in Methanol Spiking
Solution • 175
H-l Recovery of Extractable Halogenated Hydrocarbons from Human
Tissue Extracts 180
H-2 Estimated Limits of Detection for Extractable Halogenated
Organics Analysis 186
H-3 RMRs for PCBs and Pesticides of Interest to This Program . . . 187
H-4 RMR Factors for Standard PCB Solutions, Selected Ion
Monitoring Mode 188
H-5 Semi-Volatile Halogenated Hydrocarbons in Methanol Spiking
Solution 190
1-1 Estimated Limits of Detection for Extractable Halogenated
Organics Analysis 198
1-2 RMRs for PCBs and Pesticides of Interest to This Program . . . 200
1-3 RMR Factors for Standard PCB Solutions, Selected Ion
Monitoring Mode 201
xiii
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TABLES (cont'd.)
Number Page
1-4 Semi-Volatile Halogenated Hydrocarbons in. Methanol Spiking
Solution 202
L-l Standard Solutions of Polychlorinated Biphenyls 263
xix
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ACKNOWLEDGEMENTS
The authors wish to thank the following RTI individuals for their parti-
cipation and assistance in the program: Dr. J. Bursey, Dr. K. Tomer, Larry
Michael, Doris Smith, Steve Cooper, Nora Castillo, Joseph Davis, Jane Barclay,
Margaret Ray, Sandra Parks and Neal Williams. Also Drs. Joseph Breen and
Vincent DeCarlo and Ms. Cindy Stroup of the Office of Toxic Substances provided
assistance and guidance in the preliminary studies, method validation, and
preparation of this report.
xx
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LIST OF ABBREVIATIONS
CEA
dpm
GC
GC/ECD
GC/FID
GC/MS/COMP
LOD
OMB
PCB
PCF
RIA
RMR
SCOT
SIP
SIM
WCOT
carcinoembryonic antigen assay
disintegrations per minute
gas (liquid) chromatography
gas chromatography with electron capture detection
or gas chromatograph with electron capture detector
gas chromatography with flame ionization detection
or gas chromatograph with flame ionization detector
gas chromatography/mass spectrometry/computer or
gas chromatograph/mass spectrometer/computer
limit of detection
Office of Management and Budget
polychlorinated biphenyl
Participant Consent Form
radioimmunoassay
relative molar response
support-coated open tubular (glass capillary GC
column)
single ion plot
selected ion monitoring
wall-coated open tubular (glass capillary GC
column)
xxx
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SECTION 1
CONCLUSIONS
Methods have been assembled for (1) chemical analysis of halogenated
organics in a variety of media, (2) carcinoembryonic antigen assay (CEA), and
(3) data collection. The halogenated hydrocarbons of interest to this study
include both volatiles (purgeable compounds such as dichloroethylene and
dichlorobenzene) and semi-volatiles (extractable compounds such as tetrachloro-
benzene and selected pesticides).
Methods have been validated for the sampling and analysis of purgeables
and semi-volatiles in air, water, blood, urine, tissue, and soil. For most
of the matrices, the compounds of interest are removed from the sample matrix,
cleaned up, and analyzed by GC/MS/COMP.
Several clinical assays were considered but only CEA held any potential
for yielding useful information. It is hoped that the CEA will provide an
additional dimension to the correlation of the environmental exposure with
health effects. CEA levels will be compared to body burden data to test the
hypothesis that halogenated hydrocarbons elevate CEA.
The data collection instruments were developed, communication established
with responsible authorities in the study areas, and a pretest completed.
This report will serve as a laboratory manual for those involved in
subsequent phases of this project. The consolidation of all of the methods
in a standard format will help assure the quality of the data. The use of
these standard methods will also permit comparison of data collected in
different areas and seasons.
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SECTION 2
RECOMMENDATIONS
Future programs which undertake a comparative analysis between pollu-
tants in the environment and in man should be extended to other chemicals
which may be potentially responsible for the increase incidence of cancer in
the Continental U.S. It is recommended however that a pilot program be
instituted which provides the following information: (1) environmental
levels of chemicals in air, water, soil, sediment and the food chain; (2)
levels of the pollutants in the human biological fluids and tissue; (3)
biological testing of chemicals for carcinogenic and mutagenic activity
which is needed for the selection of the more important compounds for
epidemiological studies; and (4) production and emission levels for chemicals
during the past 10-15 years.
These data are required and are considered as the key ingredients in
designing a program which makes an assessment of the organic pollutants in
man and environmental media.
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SECTION 3
PROGRAM OBJECTIVES
The general thrust of this program is to conduct environmental sampling
and related epidemiological studies concerning halogenated organic compounds
in five metropolitan locations within the Continental U.S. A comparative
analysis is to be made of environmental levels (air, water, soil, sediment
and aquatic and terrestrial species) with levels of chemicals found in the
population and cancer mortality rates (in the metropolitan areas). The
selected halogenated hydrocarbon chemicals are to be measured in breath,
human blood, urine and/or fat tissues.
Under the first phase of this program, identification and quantification
of the halogenated hydrocarbons occurring in air, water, etc. are to be
performed for the sampling areas selected. The ultimate objective is to
acquire information which will allow a statistical correlation of the levels
of halogenated organic compounds in the environmental samples with those in
human tissue and with the incidence of mortality.
The second phase is a comprehensive monitoring program of the five
study areas over two seasons each. It is imperative that all sampling and
analysis be conducted using standardized protocols so that the data may be
properly correlated. This report presents these analytical protocols.
The third phase is an epidemiological study of the Phase II data.
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SECTION 4
INTRODUCTION
GENERAL PROGRAM CONCEPT
The general program concept which has been developed attempts to furnish
a comprehensive and systematic approach to making a comparative analysis of
selected organic compounds in man and the environmental media. For the
purpose of presenting an overview of the concept, Figure 4-1 depicts a flow
diagram demonstrating the interlocking relationship between the environment,
man and a potential association with the incidence of cancer. This schematic
presents a number of prerequisite components of the current program which
need examination to demonstrate a potential associational relationship
between halogenated hydrocarbons and the incidence of cancer in man. The
general program concept has been simplistically divided into three basic
levels. The first demonstrates the dosage of man with halogenated hydrocar-
bons via environmental media such as air, water and food. The second demon-
strates the degree of body-burden in man via examination of urine, blood,
breath and tissue for these compounds. The last phase of the program attempts
to demonstrate an associational relationship (i..£. , response) between body-
burden and the incidence of cancer.
METHODOLOGY
In order that data collected from the various study areas and sampling
times be comparable, it is imperative that all of the methodology be thoroughly
standardized and validated. Some of the methods to be used are already in
use at RTI and required only calibration or validation for the specific
compounds of interest. Other methods have been either significantly altered
from previous in-house procedures or were adapted from literature methods,
and thus required thorough validation. It was the goal of the validation
studies to define the precision and accuracy of each method and eliminate
artifacts where possible.
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INDUSTRIAL DISCHARGE
SOURCES OF HALOCENATED
HYDROCARBONS
D*ta on
Specific
Industrial
Plants and
^Processes .
ANALYSIS OF ENVIRONMENTAL
MATRICES REPRESENTING PORTALS
OF ENTRY TO MAN
ANALYSIS
OF
ENVIRONMENTAL
SINKS
Hyd
Hetcorologic
Topographic
LEVEL 1: DOSE
LEVEL 2: BODY BURDEN-/Urine\-fBloodl- Sliaiud - fBreacM —
Historical
Etiology
LEVEL 3: RESPONSE-
BACK
OR FORWARD
EXTRAPOLATIO
OF
AIR/WATER
QUALITY
FORMULATION OF
HYPOTHESES
HYPOTHESES TESTING
CONCLUSION
Figure 4-1.
Schematic flow diagram demonstrating interlocking rela-
tionships between environment, man and association with
incidence of cancer.
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In addition to the chemical sampling and analysis and the use of morbi-
dity and mortality statistics, clinical blood chemistry and enzyme analyses
were considered as potential indications of health effects on the study
populations. A number of assays were considered, including serum glutaraic-
oxaloacetic transaminase, y-glutamyl transpeptidase, ornithine carbamyl
transferase, and serum aryl hydrocarbon hydroxylase, which were not sufficien-
tly specific or sensitive for detection of low level pathological damage by
halogenated organics. This is discussed in detail elsewhere (4-1).
The data collection instruments must be carefully designed to obtain
all pertinent information, maintain confidentiality, follow legal guidelines
and be amenable to computer data entry.
Reference
4-1 Pellizzari, E. D., M. D. Erickson and R. A. Zweidinger, Formulation of
a Preliminary Assessment of Halogenated Organic Compounds in Man and
Environmental Media, Annual Report, Contract No. 68-01-4731, July,
1979.
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SECTION 5
COLLECTION AND ANALYSIS OF VAPOR-PHASE HALOGENATED ORGANICS
INTRODUCTION
The methodology (Appendix A) for collection and analysis of vapor-phase
halogenated organics has been previously described (5-1-5-8). These techni-
ques have been successfully employed in Phase I of this program without any
necessary modifications (5-8) and will be used in the final phase of this
program.
QUALITY CONTROL AND ASSURANCE
In addition to the procedures described in Appendix A, the following
guidelines will be employed:
(1) 10% of all samples acquired will include "blank" sampling devices
for determining background contributions,
(2) 10% of all samples will include "spiked" sampling cartridges for
determining recovery of halogenated compounds selected for study
(3) Spiked samples will be generated from a permeation system, using
gravimetrically calibrated permeation tubes
(4) During analysis every tenth sample will be a blank sampling cart-
ridge followed by a spiked sample. The set of spiked samples will
include external standards for calculating relative molar response
ratio for quantification of unknowns.
(5) All samples will include a sampling protocol and chain of custody
sheet indicating the history of the sample.
(6) Quality Assurance (QA) will include independent checks of collec-
tion and analysis procedures by an individual not involved in the
procedure. QA by an independent laboratory may be included.
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REFERENCES
5-1 Pellizzari, E. D., Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres", EPA-650/2-74-121, Contract No. 6802-1228, 148
pp., July, 1974.
5-2 Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors, EPA-600/2-75-076, Contract No.
68-02-1228, 186pp., November, 1975.
5-3 Pellizzari, E. D., The Measurement of Carcinogenic Vapors in Ambient
Atmospheres, EPA-600/7-77-055, Contract No. 68-02-1228, 288 pp., June,
1977-
5-4 Pellizzari, E. D., Measurement of Carcinogenic Vapors in Ambient
Atmospheres, EPA-600/7-78-062, Contract No. 68-02-1228, 224 pp., April,
1978.
5-5 Pellizzari, E. D., Improvement of Methodologies for the Collection
and Analysis of Carcinogenic Vapors, EPA Contract No. 68-02-2764, in
press.
5-6 Pellizzari, E. D., Analysis of Organic Air Pollutants by Gas Chroma-
tography and Mass Spectroscopy", EPA-600/2-77-100, Contract No. 68-02-
2262, 104 pp., June, 1977.
5-7 Pellizzari, E. D., Analysis of Organic Air Pollutants by Gas Chroma-
tography and Mass Spectroscopy, EPA-600/2-79-057, Contract No. 68-02-
2262, 243, pp., March, 1979.
5-8 Pellizzari, E. D., M. D. Erickson and R. A. Zweidinger, Formulation
of a Preliminary Assessment of Halogenated Organic Compounds in Man and
Environmental Media, EPA Contract No. 68-01-4731, Annual Report, in
prep.
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SECTION 6
COLLECTION AND ANALYSIS OF "PURGEABLE" HALOGENATED ORGANICS
IN AQUEOUS ENVIRONMENTAL AND HUMAN SAMPLES
The purgeable halogenated organics are those compounds which are removed
from liquid samples (water, urine, blood, etc.) by passing an inert gas
stream through or over the surface of the sample and then collecting the
evolved halogenated organics on a polymeric sorbent trap (e.g., Tenax GC).
The technique, in varied forms, is routinely applied to water analysis.
However, because of foaming and other problems, it has not been widely applied
to biological matrices. Alternate techniques (e.g. solvent extraction) for
extraction of volatile organics from blood and urine were not applicable due
to a lack of sensitivity and the time required for analysis.
Under this contract, methods have been validated for analysis of purgeable
halogenated organics in water, urine, blood, tissue, and soil. The methods
are presented in Appendices B-E. Briefly, the sample is collected in a
vapor-tight inert container (e.g. glass bottle with teflon-lined cap). The
purgeable compounds are purged from an aliquot of the sample (either through
solution or headspace purge) and trapped on a Tenax GC cartridge. The Tenax
GC cartridge is then analyzed by GC/MS/COMP in a manner analogous to that for
cartridges from air sampling. Appropriate quality control and quality assurance
procedures are used. The purgeable halogenated organics range from gases up
to the di- and trichlorobenzenes in volatility. It should be noted that the
compound range for "purgeables" overlaps with that for "extractables" so
there is no gap between the two procedures.
Two purging techniques, gas stripping and dynamic headspace analysis,
have been validated. The former is used to purge water samples and the
latter for the biological samples.
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The approach to the validation of these techniques involved comple-
mentary, parallel studies: 1) recovery studies with radiolabeled compounds
and 2) recovery studies with non-radiolabeled substances. The experiments
with radiolabeled compounds addressed recovery on the basis of the percentage
of fortified material detected on the sorbent cartridge and also on the
basis of how much remained in the sample following analysis. This experimen-
tal approach permitted construction of a "mass balance" for the analysis.
Experiments with non-radiolabeled compounds involved compounds comprising a
range of volatility to simulate the procedure to be employed in the analysis
of actual samples. The use of two independent validation procedures assured
not only the precision and accuracy of the analytical results but also
guarded against systematic errors.
The sections below discuss the validation of the methods to be used for
sampling and analysis of "purgeable" halogenated organics in detail.
BACKGROUND OF PURGE AND TRAP PROCEDURE
A number of techniques developed for the determination of volatile
organic compounds in water, particularly drinking water, were considered for
application to water and human tissue samples. The methods fall into six
basic categories: 1) solution purge (gas stripping) (6-1,6-2,6-3,6-4); 2)
dynamic headspace purge (6-5,6-6); 3) solvent extraction (6-7); 4) liquid
phase adsorption on polymeric sorbents (6-8,6-9,6-10); 5) direct aqueous
injection (6-11,6-12) and 6) static headspace analysis (6-13). Solvent
extraction, using high- or low-boiling solvents, generally suffers from poor
sensitivity due to the large volume of solvent relative to the amount
injected. Direct aqueous injection lacks adequate sensitivity for these
analyses due to injection volume limitations and in addition would present
large matrix interferences. Adsorption of volatile organics from a tissue
matrix directly onto polymeric sorbents such as XAD or polyurethane foam is
also highly susceptible to matrix interferences. Static headspace analysis
requires knowledge of the equilibrium coefficients and rigid control of the
sample temperature, headspace volume, and other parameters. In addition,
except for compounds which strongly favor the gas phase (e.g. highly volatile,
water-insoluble substances), static headspace analysis lacks sufficient
sensitivity. In light of these difficulties, actual experimental
10
-------
investigations were performed only for gas stripping and dynamic headspace
techniques in combination with vapor collection on Tenax GC.
Gas stripping analysis involves bubbling an inert gas through the
solution to purge the volatile organic materials from the sample, followed
by collection of the vapor on a polymeric sorbent. This technique has been
shown to be extremely sensitive for water-insoluble organics with boiling
points less than approximately 150°C (6-1). Dynamic headspace analysis is
similar to gas stripping in its dependence on the partition coefficient of
the solute between the aqueous and gas phases except the inert gas passes
over, rather than through the solution.
Tenax GC was chosen as the sorbent for these experiments based on
previous successful use in purge and trap analyses of water (6-1) its low
affinity for water (6-14), its low background compared with other sorbents
(6-15) and our previous work to determine breakthrough volumes and thermal
desorption conditions (6-16).
BACKGROUND OF GC/MS ANALYTICAL PROCEDURE
Gas Chromatography/Mass Spectrometry/Computer (GC/MS/COMP) has been
selected as the analytical technique for this program. This selection was
based on previous knowledge of the complexity of the purgeable organic
fraction of water and biological samples. Other methods of chromatographic
detection (electron capture, electrolytic conductivity, etc.) do not provide
sufficient identification to verify the presence of the halogenated organics
of interest.
In addition to the need for mass spectrometric detection, it is neces-
sary to use high resolution gas chromatography to separate the complex
mixtures which are purged from the water and biological samples. High
resolution GC is achieved by the use of capillary columns.
In order to limit the scope of the program and concentrate on the
compounds of highest concentration and highest carcinogenic potential, the
number of compounds of interest has been restricted. One problem with this
approach is the potential exclusion of a compound which may later be of
interest. Therefore, where possible, full spectral data are collected and
archived on magnetic tape. If this data is subsequently needed it may be
retrieved.
11
-------
Even though full scan spectra are collected, their printout and inter-
pretation are time-consuming. A more efficient technique, mass fragmento-
graphy, consists of printing out on paper or scope display the chromatogram
of an ion(s) selective for certain compounds. If the retention time matches
that of the standard, the peak may be quantitated using the Relative Molar
Response (RMR). Identifications may be verified by comparing intensities of
fragmentograms of different ions for the same chromatographic peak to see if
the intensity ratio matches that of a standard. Details of the approach may
be found in Appendices B-E.
GENERAL METHOD VALIDATION PROCEDURES
The validation of the methods for analysis of purgeable halogenated
organics in various matrices required similar procedures. Rather than
repeat the general experimental protocols for each medium, they are discussed
in the sections below.
Materials and Methods
All chemical compounds in this study were reagent grade (various sources)
and were used without further purification. Carbon-14 labeled chloroform
(California Bionuclear Corp.), carbon tetrachloride (Amersham Corp.), chloro-
benzene (Amersham Corp.) and bromobenzene (New England Nuclear) were quantita-
tively transferred to 50 ml of redistilled methanol in screw cap vials.
Solvents were exclusively "distilled-in-glass" grade (Burdick and Jackson
Laboratories). Working solutions were prepared at the concentrations listed
in Table 6-1. Safety precautions were observed while handling radioisotopes,
solutions thereof, Tenax GC cartridges containing isotopically-substituted
compounds, and contaminated glassware.
Tenax GC was purified before use by Soxhlet extraction for 24 hours each
in redistilled methanol and pentane. After vacuum drying at 60°C for 18 hr
and sizing into 35/60 mesh, 1.5 cm i.d. x 6.0 cm cartridges were packed and
thermally desorbed at 270° in a stream of helium for 30 min.
Gas chromatographic analyses were performed on either a Varian 3700 or
a Varian 1400 gas chromatograph with flame-ionization detection (GC/FID).
Exposed sample cartridges and standard cartridges were analyzed by GC/FID on
a 0.35 mm i.d. x 80 m, 1% SE-30 glass SCOT column which was temperature
programmed from 30 to 220°C at 4°/min. Compounds were removed from the
12
-------
Table 6-1. [ C] COMPOUNDS FOR RECOVERY STUDY
Compound
[14C]-CHC13
[14c]-cci4
[ Cl-chlorobenzene
14
[ Cj-bromobenzene
Source
California Blonuclear Corp.
Amersham
California Blonuclear Corp.
New England Nuclear
Radlochemlcal
Purity
98%
97%
98%
not specified
Sneclflc
Activity
3.4 i»Ci/nraol
6.94 mCl/mmol
4.1 mCl/mmol
5 mCl/mmol
MW (g/mol)
119.
157.
112.
157.
38
82
56
02
Methanol Solution
bp,°C Concentration (dpra/ml)
62
76
132
156
44,400
22,200
44,400
55,500
a 14
All compounds universally labeled with C.
dpra = disintegrations per minute.
-------
Tenax GC cartridge for injection into the GC by thermal desorption at 250°C
for 5 min in a nitrogen stream (30 ml/min) and trapped in a liquid nitrogen-
cooled, nickel capillary. Upon heating this trap to 250°C, the desorbed
compounds were injected onto the column in a minimum volume. This procedure
has been previously described in detail (6-17). Scintillation counting was
achieved on a Packard-TriCarb Model 3255 liquid scintillation counter.
Counting cocktail was prepared by mixing toluene (3.0 1), Triton X (1.0 1)
and Omnifluor (15 g, New England Nuclear).
The purgeable halogenated organics were analyzed by GC/MS/COMP using an
LKB 2091 GC/MS with an LKB 2031 data system equipped with a desorption
chamber ~ . The operating conditions for the thermal desorption unit and
the GC/MS system for the analysis of Tenax GC cartridges were as follows:
Desorption chamber temperature = 270°C
Desportion chamber He flow = 20 ml/min
Desorption time =5.0 min
Capillary trap temperature during desorption = -196°C
Temperature of capillary trap during injection onto
column = -196°C to 250°C then held at 190°C
Time of He flow through capillary trap = 12 3/4 min
He flow through column (sweep time) =9.5 min after injection
Capillary column = 100m SE-30 Column temperature = 30°/2 min;
increased at 4°/min; to 240°
Scan range = 5 •* 490 daltons
Scan cycle time =2.4 sec
Scan mode = parabolic
Trap current = 50 pA
Ionizing energy = 70 eV
Accelerating voltage = 3.5 kV
Purge and Trap Apparatus
Three apparatuses, shown in Figures 6-1, 6-2, and 6-3 have been used.
The gas stripping apparatus for water samples (Figure 6-1) combines a uniform
dispersion of fine helium bubbles throughout the solution with a minimum of
headspace. This apparatus has proven satisfactory in repeated applications.
14
-------
TIIERMOMETEI
ERLENMEYER FLASK
125 ml
FRITTED DISK
TEHAX CAflTRlOGE
TEFLON ADAPTER
GLASS WOOL PLUG
. — HELIUM PURGE
7mm 0.0., I mm 1.0
10 mm O.D.
Figure 6-1. Dynamic purge apparatus for water samples.
15
-------
TENAX CARTRIDGE
THERMOMETER
-20 to I50°c
THERMOMETER ADAPTER
with 0-ring
$10/18
HELIUM
PURGE
HELIUM INLET
TUBE
LIQUID LEVEL
00ml ROUND BOTTOM FLASK
GAS DISPERSION TUBE (coarse)
MAGNETIC STIRRING BAR
Figure 6-2. Dynamic purge apparatus with magnetic stirring capability,
16
-------
TENAX CARTRIDGE
THERMOMETER
-20tol50°c
THERMOMETER ADAPTER
with 0—ring
¥ 10/18
HELIUM
"PURGE
HELIUM INLET
TUBE
LIQUID LEVEL
100 ml ROUND BOTTOM FLASK
MAGNETIC STIRRING BAR
Figure 6-3. Headspace purge apparatus for blood, urine, and tissue
samples.
17
-------
The biological samples tend to precipitate upon heating and must be
stirred to adequately mix during purging. This precluded the use of the
apparatus in Figure 6-1, since a stirring bar would wear through the glass
frit. On this basis, the dynamic purge apparatus was redesigned as shown in
Figure 6-2. It consisted of a round-bottom flask containing a fritted tube
for purge gas inlet, a thermometer and a Teflon-coated magnetic stir bar.
The flask was topped with a short glass tube containing a small plug of glass
wool to trap water aerosols, followed by a glass cartridge packed with 1.5 x
6.0 cm 35/60 mesh Tenax GC (Applied Science Laboratories, Inc.). A flow of
pre-purified helium was bubbled through the stirred, heated solution and the
entrained volatile materials adsorbed by the Tenax GC.
Attempts to purge volatile organics from blood and urine at 90°C with
helium at 25 ml/min by gas stripping using the apparatus shown in Figure 6.2
resulted in a severe foaming problem. Reduction of the temperature to 30°C
and flow to 15 ml/min did not significantly reduce foaming. The use of
either a loose glass wool plug in the neck of the flask to break the bubbles
or a "foam trap" similar to that described by Bellar and Lichtenberg (6-1),
were also unsuccessful.
Failure to physically obstruct or dissipate the foam prompted use of
chemical agents. Ammonium sulphate, which has been used to denature protein
material, had no effect. Dow Corning Antifoam A spray was quite effective in
controlling foaming; unfortunately the hydrocarbon and fluorocarbon content
of the spray contributed significantly to the background on the Tenax GC
sorbent, making data interpretation extremely difficult. Octanol was modera-
tely successful as an antifoaming agent but was too volatile for this applica-
tion.
Dynamic headspace purge analysis of volatile compounds in biological
media, like gas stripping analysis, depends on the partition between the
aqueous and gas phases and the surface area of the interface. The partition
is affected by a number of factors including temperature and solubility of
the compounds of interest in water. Although the gas-water interfacial area
is significantly smaller in the headspace technique than in gas stripping and
hence the rate of transport of volatile materials from the liquid to gas
phase is diminished, several parameters can be adjusted to optimize the purge
18
-------
efficiency in headspace analysis. Higher temperatures are possible since the
gas does not flow through the solution and foaming is not a concern. Further-
more, vortexing the solution with rapid stirring increases the interfacial
area.
In order to prevent foaming, the apparatus shown in Figure 6-2 was
modified as shown in Figure 6-3. This apparatus for dynamic headspace
purging performed satisfactorily with blood, urine, tissue, and other biologi-
cal samples during preliminary tests. Accordingly, it was used in the valida-
tion studies discussed below.
Recovery Studies With Radiolabeled Compounds
Recovery studies using carbon-14-labeled chloroform, carbon tetrachlo-
ride, chlorobenzene and bromobenzene were performed to assess the percentages
of material collected by the Tenax GC trap, loss through connections in the
apparatus and remaining in the sample matrix. This allowed construction of
a mass balance for the analysis. Recoveries were assessed for the sorption/
desorption process alone, the gas stripping technique, and the dynamic
headspace technique.
Residue samples were oxidized using the Packard Tri-Carb Combustion
Analyzer. A 1 ml aliquot was completely oxidized to H00 and CCL; the CO- was
®
trapped in an absorbing solution and a scintillation "cocktail" (Permafluor
in 1.0 1 toluene) in a second series of scintillation vials.
To isolate the purging efficiency from the sorption/desorption process,
recoveries of compounds loaded directly onto Tenax GC cartridges were evaluated
The apparatus illustrated in Figure 6-4 was used to load the model compounds
onto the cartridge. Methanol solutions of the carbon-14 labeled compounds
were injected through the septum of the loading tube into a heated (106°C)
chamber where they were vaporized and swept into the Tenax cartridge in a N~
stream of 20 ml/min. Approximately 15 minutes were required to vaporize and
load up to 67.6 |Jl (0.75- 1.42 Mg). Loaded cartridges were assayed by thermal
desorption for 10 minutes in a desorption chamber (6-17) at 270°C under a He
flow of 30 ml/min. Effluent gas containing the desorbed compounds was bubbled
into two vials in tandem containing 15 ml of scintillation counting cocktail.
These samples were subsequently analyzed by liquid scintillation counting.
Triplicate standards of each compound were prepared by injecting known amounts
19
-------
TENAX GC CARTRIDGE
CARBON CARTRIDE
to
o
TEFLON UNIONS
LOADING TUBE
(wrapped with heating tape)
RUBBER STOPPER:
TO CARBON
CARTRIDGE
SCINTILLATION COUNTER
VIALS
Nt FLOW OOml/mln)
0=
—DESORPT10N CHAMBER
LIQUID LEVEL
Figure 6-4. Tenax cartridge loading and desorption apparatus for use with radiolabeled volatile
compound.
-------
of radiolabeled material directly into counting vials containing 15 ml of
cocktail. Carbon cartridges backed up both the Tenax GC cartridge during
loading and the scintillation vials during desorption and trapping to retain
any radioactive material which escaped. Appropriate safety precautions were
observed in the handling of radioisotopes.
Thermal desorption of radiolabeled compounds from Tenax GC cartridges
directly into scintillation cocktail was found to be very efficient as shown
in Table 6-2. The low recovery of carbon tetrachloride was attributed to
incomplete loading of this relatively large amount of material rather than
incomplete desorption. Less than 1% of the total amount of each compound was
found in the second impinger, indicating that the compounds were not breaking
through the scintillation cocktail. A second desorption of a loaded Tenax
cartridge, identical to the first desorption after loading, extracted an
average of 0.06% of the total dpm loaded on the cartridge. To investigate
the possibility of breakthrough of compounds from the cartridge during
loading, a second cartridge was placed behind several of the cartridges and
was desorbed into Triton X cocktail, which was scintillation counted. The
second cartridge contained from 0.9 to 3.0% of the total dpm loaded.
When large volumes of radioisotope solutions (160-689 |Jl) were injected
into the loading tube, recoveries decreased in direct proportion to the total
volumes of solution injected. Apparently this quantity of methanol competed
with chloroform and carbon tetrachloride for absorption sites on the Tenax
GC, causing the labeled compounds to pass through the sorbent bed without
being adsorbed.
In summary, thermal desorption of the labeled compounds from Tenax GC
was sufficiently reproducible and efficient to be a technique useful for
completing recovery studies of the purging of volatile compounds from various
sample media.
Combusted samples, Tenax eluent samples, blanks and standards were
counted for 5 min periods on a Packard scintillation counter. Data was
recorded via teletype and paper-tape punch. Computer programs at TUCC
(Triangle Universities Computation Center) were used to reduce the data.
21
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Table 6-2. RECOVERY OF "^C-LABELED COMPOUNDS FROM TENAX GC
BY THERMAL DESORPTION
l-o
to
Compound
Loaded
chloroform
carbon tetrachloride
chlorobenzene
bromobenzene
Volume
Loaded (yl)
16.1
67.6
29.7
12.6
Average
Loaded (yl)
0.56
0.75
1.24
1.42
Average
dpm
87436.5
73764.2
85076.9
80074.8
% Recovery (+ Std.
97.1 + 1.91
89.6 + 8.25
98.9 + 0.59
96.5 + 2.08
dev. )
Based on three cartridges.
First cartridge, first impinger only.
"Nine replicates.
-------
Recovery Studies with Non-Radiolabeled Compounds
The recovery efficiencies of model compounds were determined in a manner
analogous to that used with radiolabeled compounds except for the sample
fortification technique and the analysis of compounds desorbed from the Tenax
GC cartridge.
Standard mixtures of methylene chloride, chloroform, bromodichloro-
methane, tetrachloroethylene, chlorobenzene and m-dichlorobenzene were
prepared in dry, pre-purified nitrogen gas at approximately 75°C by injecting
small amounts (1-5 |Jl) of the pure liquid into a 2.0 1 spherical glass bulb.
Mixtures were equilibrated with stirring for one hour. Using a gas tight
syringe (Precision Sampling) a 1.0 ml aliquot of the vapor standard was
injected into the sample contained in a septum-capped bottle at 4°C. Other
means of loading standards were investigated, as discussed in the following
section. Quantitation standards were prepared by loading 1.0 ml aliquots of
the vapor standard directly into Tenax GC cartridges. After allowing the
spiked water samples to eqilibrate at 4°C for one hour, they were transferred
to the apparatus shown in Figure 6-1. The solution itself was purged for 90
minutes with 25 ml He/min. Exposed Tenax GC cartridges were desiccated for
12 hours by storing in capped culture tubes containing approximately 2 g of a
calcium sulfate. This effectively removed the large amount of adsorbed water
vapor from the cartridge which would have interfered with thermal desorption
of the cartridges and would have degraded gas chromatographic columns. The
cartridges were analyzed by thermal desorption/GC/FID.
Investigation of Methods for Loading Standards
Several procedures for loading standards to the aqueous samples were
attempted. The vaporization of liquid samples in a glass bulb described
above does not take into account wall adsorption, incomplete volatilization,
and syringe leaks which may reduce the measured recovery. The other proced-
ures tried, spiking with solutions in methanol and toluene and the use of gas
permeation tubes were unsuccessful as discussed below.
The use of methanol solutions for spiking water samples is routine
(6-18). This procedure was used to spike distilled water samples. The
results of the analyses of four samples spiked with methanol solution are
presented in Table 6-3. The results indicate a wide range of recoveries
23
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Table 6-3. RECOVERY OF HALOGENATED HYDROCARBONS FROM DISTILLED WATER SPIKED IN
METHANOL AND TOLUENE SOLUTIONS
Ni
Amount
Compound Spiked
(l-lg)
methylene chloride
chloroform
2-chloro-l,3-butadiene
1,1, 1- trichloroethane
1, 2-dichloroethane
bromodichloromethane*)
fc 2.50
1,2-dichloropropane J
tetrachloroethylene 1 . 30
chlorobenzene 0 . 884
m-dichlorobenzene 1.03
Boiling
Point
40
61.7
59.4
74.1
83.5
90
96.4
121
132
173
% Recovery
1234
b
b
b
b
b
12.8 14.8 36.4 36.4
139 161 211 217
67.9 76.9 95.0 95.0
52.4 56.3 96.1 87.4
Average
25.1
182
83.7
73.0
The first 5 spiked in toluene solution; the last 5 spiked in methanol solution.
Analysis of Tenax cartridges unsuccessful because of trap freezing with toluene.
-»
"Not separated by glc.
-------
from about 25% to 182%. Due to the imprecision found and the interference of
the methanol peak in the gas chromatographic analysis, this technique was
abandoned.
Analysis of Tenax cartridges loaded by purging distilled water spiked
with standards in toluene was unsuccessful as shown in Table 6-3. The high
breakthrough volume of toluene on Tenax resulted in quantitative adsorption
of the 10 |jl used to spike the standards. Thermal desorption of these
toluene-laden cartridges immediately prior to GC/FID analysis plugged the
cryogenic capillary trap with the large volume of solidified toluene.
Consequently the transfer of desorbed materials from the Tenax to the trap
ceased. Methanol, having a very low breakthrough volume on Tenax, did not
pose this problem. However, it could not be used as a solvent for the more
volatile compounds.
Another procedure for fortifying aqueous samples with halogenated
hydrocarbons was attempted using gas permeation tubes. A stream of purified
N_ passing over a group of permeation tubes in a thermostated chamber entrains
a low concentration of vapor as shown in Figure 6-5. Permeation rates,
determined gravimetrically by weighing each tube at periodic intervals,
ranged from 1 x 10 to * 1 x 10 g/min/cm using tubes constructed of TFE,
FEP and polyethylene.
Fortification of distilled water by placing the purge flask (Figure 6-1)
with 100 ml water in-line in the gas permeation system (Figure 6-5) appeared,
on the surface, to be the most straight forward and accurate procedure.
Unfortunately the backpressure created by the sintered-glass frit and the 100
ml of water was sufficient to stop the gas flow in the permeation system.
Leakage of gas from the many fittings in the system was not correctable and
the procedure was not investigated further.
An alternate fortification procedure involved withdrawing aliquots of
gas from a septum port of the permeation system with a gas-tight syringe.
These gas aliquots were then injected into 60 ml of distilled water samples
contained in septum-capped, amber-glass bottles. Tenax cartridges were
loaded similarly and were used as standards. After equilibration for approxi-
mately 1 hr at 4°C, the solution was quickly transferred to a purge flask
topped with a Tenax cartridge and diluted to 100 ml with distilled water.
25
-------
3-«oy TEFLON-P'JJG
STOPCOCKS
NEECLE VALVES
MIXING CHAMBER
PERMEATION ChAMSES
JACKET
TKESMOSTAT , HEATER , AND
CIRCULATING PUMP
CARRIER GAS LiNS
THERMOSTAT FLUID LINES
Figure 6-5. Schematic of permeation tube flow system for
synthesizing air/vapor mixtures.
26
-------
The solution was heated to 90°C and purged for 90 rain at 25 ml He/min.
Vapors collected on the Tenax were detected and quantitated by GC/FID interfa-
ced with a thermal desorption/injection system. The results appear in Table
6-4.
The fourth method of fortification involved preparation of vapor standards
in a gas mixing bulb. Two microliters of each compound were injected into
the bulb at 75°C and the vapor mixture stirred for 30 min. Aliquots (1 ml)
were withdrawn with a gas-tight syringe and injected into 60 ml of distilled
water contained in a septum-capped, amber glass bottle. The solutions were
analyzed according to the procedure in the previous paragraph. The results
are presented in Table 6-5.
Comparison of the results of the last two spiking procedures reveals
similar recovery. However, the precision shown when spikes were made from
the gas mixing bulb is approximately a factor of 2 better than that shown
when spikes were made by withdrawing aliquots from the permeation system.
Consequently fortification of samples for recovery studies was accomplished
from the gas mixing bulb.
VALIDATION OF GAS CHROMATOGRAPHY/MASS SPECTROMETRY/COMPUTER ANALYSIS PRO-
CEDURES
The GC/MS/COMP procedures to be used for the purgeable halogenated
organics are routine in this laboratory and do not require extensive valida-
tion. One area which requires ongoing investigation is the determination of
Relative Molar Response (RMR) factors for use in quantification. Details on
the use and determination of RMR may be found in the Appendices. Due to
instrumental variations, RMRs may change slightly with time. Therefore, RMR
values are periodically checked and updated if necessary.
As part of this program, RMRs were obtained for many of the purgeable
halogenated organics of interest. Using the LKB 2091 GC/MS/COMP system, a
series of Tenax GC cartridges were loaded with standards for the determina-
tion of Relative Molar Response factors in order to calibrate this instrument
for subsequent quantification of the purgeable halogenated organics in
water, urine and blood samples. The RMRs for standard compounds (determined
in replicate) were calculated relative to the m/z 186 ion of perfluorobenzene.
These data are given in Table 6-6.
27
-------
Table 6-4. RECOVERY OF HALOGENATED HYDROCARBONS FROM DISTILLED WATER
SPIKED FROM GAS PERMEATION SYSTEM
ro
oo
% Recovery
Amount
Compound spiked (ug) 123456 Average + Std. Dev.
trans-l,2-dichloroethylene 1.00 82.4 30.5 92.4 75.0 42.1 85.3
benzene 6.09 121 137 79.8 57.4 85.8 39.4
1,1,2-trichloroethane 1.70 143 92.4 95.3 78.3 69.1 42.5
1,1,2,2-tetrachloroethane 1.61 75.0 60.2 61.7 51.1 54.5 10.8
1,2-dlchlorobenzene 1.80 157 93.7 110 89.4 98.4 46.8
Mean
59.6 + 26.7
86.7 + 37.0
86.8 + 33.5
52.2 + 21.9
99.2 + 35.6
78.6 + 33.5
-------
Table 6-5. RECOVERY OF HALOGENATED HYDROCARBONS FROM DISTILLED WATER
SPIKED FROM GAS MIXING BULB
Compound
methylene chloride
chloroform
bromodlchloromethane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
Mean
Amount
spiked (vig)
1.33
1.48
1.98
1.70
1.11
1.35
% Recovery
1234 Average + Std. Dev.
a a a a
88.5 73.7 47.9 55.0 66.3 + 18.4
98.1 121 84.4 85.8 97.3 + 16.9
71.4 65.9 64.2 101 75.6+17.2
81.7 81.7 79.7 89.7 83.2+4.4
82.3 110 68.5 102 90.7 + 18.8
79.2 + 23.9
Not quantifiable because of background interferences.
-------
Table 6-6. RMR VALUES OF STANDARDS FOR 100 m CAPILLARY
ON THE LKB 2091 GC/MS
Compound
trichloroethylene
chloroform
1,2-dichloroethane
1, 1, 1-trichloroethane
methylene chloride
tetrachloroethylene
1 , 1-dichloroethane
m-dichlorobenzene
o-dichlorobenzene
bromodichlorome thane
carbon tetrachloride
chlorobenzene
MW
130
118
98
132
84
164
98
146
146
162
152
112
m/z
95
130
83
62
97
49
84
166
63
65
146
146
83
129
117
119
112
RMR
.79 + 11%
.78 + 13%
1.41 + 8%
.431 + 3%
1.93 + 10%
.475 + 5%
.294 + 5%
.819 + 3%
.646 + 5%
.204 + 17,
.648 + 7%
.351 + 17%
1.855 + 13%
.302 + 20%
1.77 + 7%
1.70 + 7%
1.82 + 14%
30
-------
VALIDATION OF PROCEDURES FOR WATER SAMPLES
Sampling
Water samples are to be collected directly from the drinking water
tap. This is a standard procedure and no problems were encountered in the
preliminary sampling trips. No further validation was done.
Purge Procedure
Radiolabeled Recovery Experiments —
The recoveries of radiolabeled model compounds from distilled water
are shown in Table 6-7. The observed losses were attributed to leaks in
the glassware. Breakthrough on the Tenax has been shown to be negligible
in previous experiments (see "Efficiency and Reproducibility of Tenax
Desorptions", above) and in validation of air sampling methods. Incomplete
purging of the water was eliminated as a possibility by counting the residual
radioactivity in the purged water samples. Less than 1% of the added
radioactivity was found. Within the precision requirements of this study,
the radiolabeled compound recoveries indicate the method is essentially
quantitative.
Non-Radiolabeled Recovery Experiments --
The recoveries of five model compounds were determined as part of the
experiments to evaluate spiking methods (see "Investigation of Methods for
Loading Standards", above). These results are reported in Table 6-5.
In addition, a study was undertaken for determining the optimum purge
time. The procedure involved spiking each of a number of distilled water
samples with five halogenated hydrocarbons from a gas mixing bulb. These
spiked samples were then purged with helium onto Tenax GC cartridges for
times ranging from five minutes to ninety minutes. Duplicate loaded Tenax
cartridges were then analyzed by FID and the percent recoveries calculated.
The gas mixing bulb for spiking water samples was prepared by heating
to approximately 200°C and flushing with nitrogen (^100 ml/min) for one
hour. The temperature was then reduced to ^70°C, the bulb sealed, and
known amounts (approximately 2 pg) of chloroform, bromodichloromethane,
tetrachloroethylene, chlorobenzene, and m-dichlorobenzene were injected.
After equilibration for one hour, 1.0 ml aliquots were withdrawn with a
gas-tight syringe and injected into 60 ml distilled water at -4°C contained
31
-------
Table 6-7. RECOVERY OF 14C-LABELED MODEL COMPOUNDS FROM WATER3
Compound
1
2
3
4
5
6
7
8
Mean
+ S.D.
RSD (%)
Chloroform
85.0
67.6
91.7
85.9
91.0
47.9
92.8
88.1
81.3
15.7
19.3
Carbon tetrachloride
96.2
83.8
98.6
70.9
65.2
70.9
67.4
-
79.0
14.0
17.7
Chlorobenzene
84.2
90.3
89.0
92.5
90.4
87.1
94.0
90.3
89.7
3.1
3.4
Bromobenzene
71.9
88.7
93.7
69.1
87.8
82.4
87.2
90.3
83.9
8.9
10.6
100 ml water spiked with 70,000-90,000 dpm of the model compound (vL yg)
SD = standard deviation
RSD = (SD/Mean) x 100
-------
in 60 ml amber bottles covered with septum caps. The gas aliquots were
added to the distilled water one bubble at a time in order to allow maximum
dissolution of the standard. After the standards were added to the distilled
water, the samples were allowed to equilibrate for approximately 1 hr at
~4°C. In all, fourteen distilled water samples were prepared. Two distilled
water blanks were also prepared. The spiked water samples and blanks were
added to the purge flasks (Figure 6-1) and heated to 90°C. After the
samples and the blank had reached 90°C, helium flow at 25 ml/min was begun.
Purged compounds were collected on Tenax GC cartridges. Individual samples
were purged for 5, 10, 15, 30, 45, 60 and 90 minutes. The loaded cartridges
were desiccated overnight with anhydrous CaSO, in a Teflon-lined, screw
cap culture tube.
Loaded Tenax cartridges were analyzed by thermal desorption GC/FID.
Peaks were identified by comparison of retention times to individual stan-
dards. Recovery was determined by comparison of peak heights with calibra-
tion standards prepared by injecting 1 ml vapor aliquots from a gas mixing
bulb directly onto Tenax GC cartridges.
The results of this analysis are presented in Table 6-8 and Figure 6-
6. These results indicate that the recovery of all of the four halogenated
hydrocarbons that could be quantified reaches a maximum at a purge time of
approximately thirty minutes. Chloroform could not be quantified because of
difficulty in peak identification.
Summary
Based on the experiments summarized in Tables 6-5, 6-7 and 6-8, the
expected recoveries of purgeable halogenated organics for water are about
80% or better. Within the precision requirements of this study, these
recovery values indicate that the method is essentially quantitative. The
entire analytical protocol has been tested during the preliminary field
sampling studies. No significant problems are anticipated with execution
of the protocol.
VALIDATION OF PROCEDURE FOR BLOOD SAMPLES
Sampling
Blood samples are collected in replicate 10 ml vacutainer tubes contain-
ing an anticoagulant. Using a qualified phlebotomist, the samples are
33
-------
Table 6-8. PERCENT RECOVERY AT VARIOUS PURGE TIMES
Purge Time (min)
Compound
Bromodichloromethane
Tetrachloroethylene
Chlorobenzene
m-Dichlorobenzene
Mean for all Compounds
Trial
I
II
I
II
I
II
I
II
5
a
66.1
14.2
60.2
46.0
71.8
42.1
32.4
51.3
10
61.6
106.8
29.7
73.8
42.9
58.9
39.5
38.4
75.1
15
a
74.7
102.4
74.7
83.9
30
100.0
76.7
80.7
65.1
106.2
82.6
91.3
85.3
86.0
45
a
67.3
77.3
59.7
112.9
66.3
86.1
99.4
79.5
60
72.6
74.9
73.8
55.4
95.8
58.9
68.9
69.8
71.3
90
89.7
152.9
70.4
41.1
93.1
55.8
78.8
57.1
79.9
Q
Interferences prevented quantification.
-------
DATA FROM TRIAL 1 ONLY
100 P
90
80
70
60
x
ut
I
111
ee
50
40
30
20
10
"5 10 15 20 25 30 3S 4045 50Sfe 60 65 70 75 5o 55"
PURGE TIME (min)
Figure 6-6. Percent recovery as a function of purge time.
35
-------
collected by brachial venipuncture. Glass syringes represent the optimal
collection device, since no polymeric material which may contaminate the
sample comes in contact with the blood. However, sterilization of large
numbers of glass syringes in the field is not practical, so vacutainers
will be used.
The possible contamination or loss by permeation through the rubber
septum caps of the vacutainers was a cause for concern. Teflon-lined
vacutainers are not available but the manufacturers recommended special
vacutainers "suitable for GC" (Venoject L428, Kimble). Validation experi-
ments for benzene under a separate contract (EPA No. 68-01-3849, Task I)
found the background of these tubes to be acceptable.
Following collection, shipping and storage procedures must assure that
the purgeable halogenated organics remain intact in the blood sample.
Leakage of the vacutainer caps has been observed and permeation through
the cap material is suspected. Accordingly, these containers are not
suitable for storage. In the procedure used for this project, the blood
sample is chilled and transferred to a clean shell vial with a Teflon-lined
screw cap. The cap is then taped shut. This procedure has been validated
and field tested on EPA Contract No. 68-01-3849, Task I.
Radiolabeled Recovery Experiments--
The recoveries of radiolabeled model compounds for blood are shown in
Table 6-9. The observed losses were attributed to leaks in the glassware.
Breakthrough on the Tenax has been shown to be negligible in previous
experiments (see "Efficiency and Reproducibility of Tenax Desorption",
above) and in validation of air sampling methods. Incomplete purging of
the blood was eliminated as a possibility by counting the residual radioacti-
vity in the purged blood sample. Aliquots of blood samples were retained
after purging and a 1.0 ml portion of each aliquot was oxidized in a Packard
Tri-Carb Combustion Analyzer. The combustion analyzer converted C-labeled
14
compounds to CC- and trapped the labeled carbon dioxide in a solution to
be scintillation counted. Of the twelve blood samples which had been
purged, only the samples which had been spiked with C-bromobenzene were
radioactive. These three samples contained 7.7%, 12.7% and 4.9% (8.4%
average) of the C-bromobenzene which had been added initially (77.1%,
36
-------
Table 6-9. PERCENT RECOVERY OF 14C-LABELED COMPOUND FROM BLOODS
Mass
Compound Loading (yg)
chloroform 1.46
carbon tetrachloride 0.785
chlorobenzene 1.12
bromobenzene 1 . 21
dpm
Loaded % Recovery
91755.1 94.2
93.1
46. 5b
77902.3 92.6
92.2
83.5
89538.1 43. 5b
88.0
92.2
84493.9 77.1
74.4
90.3
Average
% Recovery
93.7
89.4
90.1
80.6
aHeadspace purge of whole blood (25 ml) diluted 1:1 with distilled
water and purged in 100 ml 3-neck purge flasks at 50°C for 90 min
with helium at 25 ml/min.
Leaking desorption chamber cap; not included in average.
37
-------
74.4% and 90.3%, respectively, of the bromobenzene had been recovered on
the Tenax GC cartridge). Since the boiling point of bromobenzene is 156°C,
it seems reasonable that a headspace purge at 50°C may not completely
remove this compound from the sample matrix.
Non-radiolabeled Recovery Experiments—
Blood was spiked with halogenated compounds by injecting a 1.0 ml
aliquot of a vapor standard prepared in a gas mixing bulb into a septum-
capped vial containing 25 ml of cold, outdated, whole human blood and 25 ml
of distilled water. Aliquots (1.0 ml) of the vapor standards were also
loaded onto Tenax GC cartridges. These cartridges served as quantitation
standards. After allowing the spiked blood mixtures to equilibrate for 1
hr at 4°C, they were transferred to round-bottom three-necked flasks (100
ml capacity) topped with Tenax GC cartridges (Figure 6-2). The solution
was heated to 50°C and the headspace above the liquid purged for 90 min at
25 ml He/min. Loaded cartridges were desiccated over CaSO, overnight and
analyzed by GC/FID. The results are shown in Table 6-10.
Summary
Based on the experiments summarized in Tables 6-9 and 6-10, the expected
recoveries of purgeable halogenated organics are at least 80%. Within the
precision requirements of this study, these recovery values indicate that
the method is essentially quantitative. The entire analytical procedure
has been tested during preliminary field sampling studies. No significant
problems are anticipated with execution of the protocol.
VALIDATION OF PROCEDURE FOR URINE SAMPLES
Sampling
During the preliminary sampling trips, each participant was provided
with a clean 120 ml.(4 oz) bottle and asked to collect an early morning
urine sample. In addition, spot urine samples were collected from selected
participants at the time breath and blood samples were collected. No
problems were encountered with this procedure and it will be followed in
the future.
38
-------
Table 6-10. RECOVERY OF HALOGENATED HYDROCARBONS FROM HUMAN BLOOD
SPIKED FROM GAS MIXING BULB
CO
Compound
methylene chloride
chloroform
bromodichlorome thane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
mean
Amount
spiked (yg)
7.96
8.16
10.4
8.52
5.53
6.44
% Recovery
1
105
84.6
105
108
108
85.1
2
116
145
159
121
120
101
3 4 Average + Std. Dev.
a a 1 1 1 -i- 7 ft
X Li. i / . o
a
1 T Q 1 9 1 1 IT 1
J.jy J-iJ 1 JJ.-L
94.9 94.9 113 + 30.7
82.8 97.5 99.8 + 17.9
85.6 78.6 98.1 + 19.3
74.6 84.5 86.3 + 10.9
104.3 + 22,7
Not quantified because of background interferences.
-------
Purge Procedure
Radiolabeled Recovery Experiments --
The recoveries of radiolabeled model compounds from urine are shown in
Table 6-11. The observed losses were attributed to leaks in the glassware.
Breakthrough on the Tenax has been shown to be negligible in previous
experiments (see "Efficiency and Reproducibility of Tenax Desorption",
above) and in validation of air sampling methods. Incomplete purging of
the water was eliminated as a possibility by counting the residual radioacti-
vity in the purged water samples. Less than 1% of the added radioactivity
was found. For C-bromobenzene, recoveries greater than 100% are attributed
to quenching by methanol in the standard, reducing the dpm counted in the
standards. Methanol quenching was not observed in the desorbed samples
because of the low breakthrough volume for methanol on Tenax. Since the
bromobenzene standards and samples were much more dilute, this quenching
effect was more significant than for the other model compounds.
Non-Radiolabeled Recovery Experiments --
The recoveries of six model compounds from urine are summarized in
Table 6-12.
Summary
Based on the experiments summarized in Tables 6-11 and 6-12, the
expected recoveries of purgeable halogenated organics are about 80%.
Within the precision requirements of this study, these recovery values
indicate that the method is essentially quantitative. The entire analytical
procedure has been tested during the preliminary field sampling studies.
No significant problems are anticipated with execution of the protocol.
VALIDATION PROCEDURE FOR TISSUE SAMPLES
Sampling
It is anticipated that tissue samples collected from cadavers or
surgery will be obtained from a pathologist. Personnel from RTI will work
with pathologists advising them of proper sample handling procedures. To
be of use for purgeable halogenated organics a tissue sample must be
collected a short time following death and immediately frozen in a cleaned
glass container with as small a "headspace" as possible. Any handling or
40
-------
Table 6-11. PERCENT RECOVERY OF 14C-LABELED COMPOUNDS FROM URINE
Compound
Mass dpm Average
Loaded (yg) Loaded % Recovery % Recovery
chloroform
chlorobenzene
br omob enz ene
1.50
carbon tetrachloride 0.809
1.12
0.0127
94237.0
80278.6
89589.9
885.4
83.9
84.3
85.2
57.2
66.7
67.1
89.9
87.8
90.2
147
114
107
84.5
63.7
89.3
123
41
-------
Table 6-12. RECOVERY OF HALOGENATED HYDROCARBONS FROM HUMAN URINE
SPIKED FROM GAS MIXING BULB
Amount
Compound spiked (yg)
methylene chloride 6.96
chloroform 8.16
bromodichloromethane 10.2
tetrachloroethylene 8.44
chlorobenzene 5 . 58
m-dichlorobenzene 7.54
mBan
% Recovery
1 2 3 4 Average + Std. Dev.
ft
Qfl A /i IT 917 A7Q4- Ifi fl
OU . O 1-L . J <£-L./ H / . 7 T JU.U
107 72.6 89.5 44.8 78.5 + 26.5
123 88.2 126 80.8 105 + 23.3
85.2 61.2 84.4 57.1 72.0+14.9
103 77.1 92.6 71.9 86.2 + 14.2
73.6 70.5 90.1 80.7 78.7+8.7
79.2 + 24.3
a
Not quantified because of background interferences.
-------
storage in contact with, polymeric materials represents potential contamina-
tion.
Samples received from Niagara Falls, New York during the preliminary
sampling period were not handled as above and were useless for purgeable
halogenated organics analysis. Among the improper handling procedures
were: storage in plastic bags, treatment with formalin, refrigeration (not
freezing) of the samples for at least one week postmortem, and thawing of
the sample to cut off portions for shipment to RTI.
Purge Procedure
Radiolabeled Recovery Experiments —
No radiolabeled recovery experiments were conducted with tissue.
Non-radiolabeled Recovery Experiments --
Human adipose tissue specimens were obtained from pathology departments
of local hospitals, fortified with halogenated compounds and purged to
assess recovery. Samples (5 g) of frozen human fat were cut from a larger
mass with a scalpel, sectioned and transferred to a 100 ml, round-bottom,
three-necked flask. Aliquots of distilled water (60 ml) were fortified with
1 (Jg each of methylene dichloride, chloroform, bromodichloromethane, tetra-
chloroethylene, chlorobenzene and m-dichlorobenzene using a gas mixing bulb.
The water was added to the tissue in the purge flask and the mixture macera-
ted in an ice bath using a Virtis tissue homogenizer. The purge apparatus
was immediately assembled, heated to 50°C and the headspace purged for 30
minutes at 25 ml He/min. The exposed cartridges were desiccated and analyzed
by GC/FID as described in the "Materials and Methods" section.
The recovery of halogenated hydrocarbons from human adipose tissue
(Table 6-13) vary significantly. Great difficulty was encountered in quanti-
tative introduction of a representative fortified sample into the container
for analysis. Consequently variations in recovery may be attributed to
losses during tissue maceration and transfer.
Summary
The tissue sample analysis procedure has been partially validated. Due
to some low recovery values listed in Table 6-13, the difficulties in handling
tissue during the purge, and (most significantly) the lack of control over
43
-------
Table 6-13. RECOVERY OF HALOGENATED HYDROCARBONS
FROM HUMAN ADIPOSE TISSUE
Compound
methylene chloride
chloroform
bromodichloromethane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
mean
% Recovery
123
a
70 o c.n o
/ o . f. oy . o
a
f. 9 O 1 A P
0
-------
sampling and storage prior to receipt by RTI; the analysis of tissue samples
of a purgeable halogenated organics must be regarded as semi-quantitative.
REFERENCES .
6-1 Bellar, T. A. and J. J. Lichtenberg, The Determination of Volatile
Organic Compounds at the [Jg/1 Level in Water by Gas Chromatography, U.
S. Environmental Protection Agency, EPA-670/4-74-009, 33 pp (1974).
6-2 Pellizzari, E. D., R. A. Zweidinger and M. D. Erickson, Environmental
Monitoring Near Industrial Sites: Brominated Chemicals, Part I, U. S.
Environmental Protection Agency, EPA-560/6-78-002, 296 pp (1978).
6-3 Pellizzari, E. D., Identification of Compounds of Energy Related Wastes
and Effluents, U. S. Environmental Protection Agency, EPA-600/7-78-004
(1978).
6-4 Kopfler, F. C., R. G. Melton, R. D. Lingg and W. E. Coleman in Identi-
fication and Analysis of Organic Pollutants in Water, L. H. Keith,
Ed.; Ann Arbor Science, Ann Arbor, MI, 1976; Chapter 6.
6-5 Sievers, R. F., R. H. Shapiro, H. F. Walton, G. A. Eiceman, and R.
Barley, presented in part at the 1977 Annual Meeting, Amer. Chem. Soc.,
Chicago, IL., September, 1977.
6-6 Mieure, J. P. and J. Dietrich, J. Chromatogr. Sci., U, 550 (1973).
6-7 Deetman, A. A. and P. Demeulemeester, Anal. Chim. Acta, 82, 1 (1976).
6-8 Suffet, I. H., L. Brenner and J. V. Radziul, in Identification and
Analysis of Organic Pollutants in Water, L. H. Keith, Ed., Ann Arbor
Science, Ann Arbor, MI, p. 375 (1976).
6-9 Aue, W. A., S. A. Kapila and C. R. Hastings, J. Chromatogr., 7_3, 99
(1972).
6-10 Weliber, A., W. M. D'Angelo, and S. W. Bigelow, Evaluation of High
Performance Liquid Chromatographic Packing for Concentration and Separa-
tion of Organic Materials in Water, NTIS, AD-A053,683-4ST.
6-11 McKinney, J. D., R. R. Maurer, J. R. Hass and R. 0. Thomas, in Iden-
tification and Analysis of Organic Pollutants in Water, L. H. Keith,
ed., Ann Arbor Science, Ann Arbor, MI, p. 417 (1976).
6-12 Nicholson, A. A., B. Meresz and B. Lemyk, Anal. Chem., 4£; 814 (1976).
45
-------
6-13 Budde, W. L., and J. W. Eichelburger, Organic Analysis Using Gas
Chromatography/Mass Spectrometry—A Technique and Procedures Manual,
Ann Arbor Science, Ann Arbor, MI, 1979, p. 57-
6-14 Janak, J. , J. Ruzickova, J. Novak, J. Chromatogr. , 9_9, 689 (1974).
6-15 Chester, S. N., B. H. Gump, H. S. Hertz, W. E. May, S. M. Dyszel, and
D. P. Enagonio, Trace Hydrocarbon Analysis: The National Bureau of
Standards Prince William Sound/Northeastern Gulf of Alaska Baseline
Study, NBS Tech. Note, 889 (January, 1976).
6-16 Pellizzari, E. D., Analysis of Organic Air Pollutants by Gas Chromatp-
graphy and Mass Spectrometry, US Environmental Protection Agency, EPA-
600/2-77-100 (1977).
6-17 Pellizzari, E. D., B. H. Carpenter, J. H. Bunch and E. Sawicki, Environ
Sci. Technol., 9, 556-560 (1975).
6-18 Sampling and Analysis Procedures for Screening of Industrial Effluents
for Priority Pollutants, USEPA, Cincinnati, OH, 1977-
46
-------
SECTION 7
COLLECTION AND ANALYSIS OF "EXTRACTABLE" HALOGENATED ORGANICS
IN ENVIRONMENTAL AND HUMAN SAMPLES
The "extractable" halogenated organics, also referred to as "semi-
volatiles" and "pesticides and PCBs" are those compounds which may be sol-
vent-extracted and concentrated for analysis. At the lower end, volatile
compounds such as tetrachloroethylene, chlorobenzene and lighter compounds
are either lost in solvent evaporation and/or are not chromatographically
separated from the solvent peak. At the upper end, nonvolatile (high boiling)
and/or polar compounds do not elute from the chromatography column under the
conditions to be used. Compounds which would not elute include decabromobi-
phenyl and tris(2,3-dibromopropyl)phosphate.
The compounds of interest in this class include halogenated aromatics,
halogenated pesticides, and PCBs. Their polarity, volatility and reactivity
all make them amenable to standard pesticides residue methods. Therefore,
the collection, extraction, cleanup, concentration, and analysis procedures
are modifications of these standard methods. Briefly, semi-volatile halogena-
ted hydrocarbons are extracted from the matrix with organic solvents, dried,
concentrated, and, where necessary, subjected to chromatographic cleanup on
Florisil to remove lipids and other non-gas chromatographable impurities.
The sample is analyzed by GC/ECD. Qualitative identifications are based
upon retention times using two columns and quantification is achieved by
comparison of detector response to that of standards. Identifications are
confirmed by GC/MS/COMP when sufficiently concentrated.
GENERAL METHOD VALIDATION
Materials and Methods
®
The pesticides and PCB mixtures (Aroclor ) used were obtained from the
Pesticides Repository of the Quality Assurance Section, Analytical Chemistry
47
-------
Branch, EPA, HERL, ETC (MD-69), Research Triangle Park, NC 27711. Indivi-
dual PCS isomers were obtained from RFR Corp., Hope, RI, and d,Q-pyrene from
Merck and Co., Inc., Isotopes, St. Louis, MO. All solvents were "Distilled
in Glass" (Burdick and Jackson, Muskegon, MI) and redistilled prior to use.
Extraction and cleanup validation samples were analyzed using a Fisher/-
Victoreen 4400 gas chromatograph equipped with a microcell electron capture
detector (GC/ECD). The primary analytical column was a 0.38 mm i.d. x 38 m
1% SE-30, 0.32% Tullanox SCOT capillary column. Operating conditions were:
column temperature, 220°C; injection temperature, 265°C; detector temperature,
285°C; carrier gas, 2.5 ml/min nitrogen, with nitrogen makeup gas adjusted
to a total flow of 25 ml/min. A column used to verify extractable halo-
genated organics in samples at concentrations below the GC/MS/COMP detection
limit was a 180 cm x 2.0 mm i.d. glass column packed with 1.5% OV-17/1.95%
QF-1 on 80/100 Chromosorb W(HP). The operating conditions were: column
temperature, 220°C isothermal; detector temperature, 285°C; injector tempera-
ture, 265°C; carrier gas 18 ral/min nitrogen.
The GC/MS/COMP systems used were a Finnigan 3300 GC/MS/COMP and an 1KB
2091 GC/MS equipped with an 1KB 2031 data system. Operating conditions for
the Finnigan 3300 were: 20 m x 0.38 mm i.d., 1% SE-30 SCOT capillary operated
isothermally at 235°C and a flow rate of 2.5 ml/min helium. Splitless
injection (0.2 - 0.3 (Jl) was used, with standard electron impact (70 eV)
ionization conditions.
The 1KB 2091 was operated using a 18 m 1% SE-30/BaCO. SCOT capillary
column at 240°, isothermal for PCBs and a 40 m x 0.38 mm i.d. 1% SE-30 SCOT
capillary column at 230° isothermal for the pesticides. In both cases, the
column flow rate was 2 ml/min with 20 ml/min split off at the injector. The
mass spectrometer was operated under standard electron impact conditions as
described in Section 6.
The Finnigan 3300 and the LKB 2091 systems were operated in both the
full scan and single ion monitoring (SIM) modes. In the full scan mode
spectra are repetitively collected. Spectra or mass fragmentograms (single
ion plots) may be plotted for interpretation. In the SIM mode, only a small
number (up to 9 for the Finnigan 3300 and up to 16 for the LKB 2091) of ions
are monitored. Full spectra are not collected. The advantage of this
48
-------
method is that the detector spends more time "looking" at the selected ions
and therefore better (generally 10-50 times) sensitivity is obtained.
Validation of Gas Chromatographic Procedures
Due to the selectivity and sensitivity of GC/ECD, this will be the
primary procedure for analysis of extractable halogenated organics. Identi-
fications by GC/ECD will be confirmed on a second chromatographic column
and, where the sample concentration is sufficient, by GC/MS/COMP-
Using both the SE-30 SCOT capillary column and the OV-17/QF-1 packed
column, retention times were determined for the standards used in recovery
studies (vide infra) and others as needed. Using the operating conditions
appropriate for pesticides analysis, the linear response of the ECD was
found to be from about 186 to <2 pg for trifluralin and 220 to <2 pg for
lindane. For the purposes of routine analysis, an upper detection limit of
160 pg will be asssumed.
Validation of GC/MS/COMP Analysis Procedures
The chromatography conditions were similar to those used for GC/ECD,
The samples for this study to be screened by GC/ECD were to be confirmed (if
sufficiently concentrated) by GC/MS/COMP. Therefore, the retention times of
the two techniques must be similar - GC/ECD must operate isothermally, so the
GC/MS/COMP conditions reflect this restriction.
The basic areas of validation for the GC/MS/COMP analysis procedure
were to determine conditions which provide the lowest limit of detection
(LOD) and to determine Relative Molar Response (RMR) factors for compounds
of interest to permit quantitation in samples.
Determination of Limits of Detection for GC/MS/COMP --
Standard solutions of selected pesticides and PCB isomers were analyzed
on the Finnigan 3300 and 1KB 2091. In the full scan mode the limit of
detection was the amount of compound required for an interpretable spectra.
In the SIM mode, the limit of detection was the amount of compound required
to yield a peak 2-4 times the noise level. The estimated limits of detection
for the Finnigan 3300 and LKB 2091 are presented in Table 7-1.
49
-------
Table 7-1. ESTIMATED LIMITS OF DETECTION FOR EXTRACTABLE HALOGENATED ORGANIC ANALYSIS'
Compound
trifluralin
atrazine
Y-BHC (lindane)
heptachlor
chlordane
_p_,£' -DDE
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
o
See text for conditions
15:1 split at injector,
°0.2 yl injected with no
Full Scat
ng/yl
12
>12<20
>12<20
12
•^30°
12
*1
<1
12
gc only
split.
LKB 2091b
SIM
i ji
m/z ng/yl
264 0.4
200 0.4
181 0.18-0.4
272 0.18-0.4
375 5
246 >0.3
188 0.004
360 ^0.016
498 0.42
1/15 of injection is on column.
ull Scan
ng/yl
5-10
<;50
5-10
10-20
25-50
5-10
*2.5
25-50
150
Finnigan
3300°
SIM
m/z
264
200
181
272
375
246
188
360
498
ng/yl
<0.5
5-10
1
1-1.5
5-10
0.5-1
^0.025
M).15
M).3
-------
Representative mass spectra, single ion plots and reconstructed gas
chromatograms are included in Appendix L to illustrate the limits of detec-
tion.
Determination of Relative Molar Response Factors (RMRs) for GC/MS/COMP--
Tables 7-2 and 7-3 present the RMRs for PCBs and pesticides determined
on the LKB-2091 system. The high limits of error for the early eluting
components (through lindane) are due to the fact that only three scans were
acquired across the peak (7.2 sec wide peaks). Attempts were made to degrade
the performance of the column by lowering the column temperature or by
lowering the flow rate. This, however, led to poor intensities for the
later eluting components without increasing the peak width significantly.
Typical single ion plots used for the RMR determinations are presented
in Appendix M.
VALIDATION OF PROCEDURES FOR BLOOD
The procedure to be used to work up the extractable halogenated organics
in blood was modified from that of Thompson (7-1). Two major validation
efforts were: determination of whether whole blood or plasma should be
extracted and validation of the Florisil cleanup procedure. These efforts
are discussed below.
Validation of Extraction Procedure
During the validation procedure it was determined that no pesticides
were extractable from the cells. This indicated that only the plasma frac-
tion which is much easier to work with than whole blood, need be analyzed.
Briefly, when whole blood was spiked with pesticides, recoveries from the
plasma fraction were variable and low and almost no pesticide was found in
the cells. When plasma was spiked, recoveries were both higher and less
variable. These results are summarized in Table 7-4. However, with longer
equilibration (19 hr vis 2 hr), as shown in Table 7-5, the recoveries from
plasma were significantly lower. The paragraphs below describe the validation
experiments in detail.
Eight pesticides at the 500 ng concentration level were added to four 7
ml aliquots of whole blood. Each tube was incubated at 37°C for two hours
and centrifuged for ten minutes. The plasma was separated from whole blood
cells by pipetting off all possible plasma and submitting the whole cell
51
-------
Table 7-2. RMRs FOR PCBs AND PESTICIDES OF INTEREST TO THIS PROGRAM'
Compound
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
trif luralin
atrazine
lindane
heptachlor
£,£'-DDE '
chlordane (peak 1)
chlordane (peak 2)
Concentration
104 ng/ul
3.8 ng/ul
570 ng/ul
10.4 ng/ul
1156 ng/ul
8.4 ng/ul
100 ng/ul
100 ng/ul
100 ng/ul
100 ng/ul
100 ng/ul
100 ng/ul
100 ng/ul
Ion
188
360
498
264
200
202
181
183
272
246
373
375
373
375
RMR
elutes with solvent
and was two scans wide -
not determinable
.38 + 3%
.35 + 10%
.34 + 7%
not determinable
1.32 + 20%
.74 + 7%
.25+8%
.74 + 9%
.62 + 12%
.74+6%
.45 + 6%
.71 + 5%
.65+5%
.051 + 6%
.045 + 13%
Standard is d.,0-pyrene (m/z = 212)
52
-------
Table 7-3. RMR FACTORS FOR STANDARD PCB SOLUTIONS, SELECTED ION MONITORING MODE*
Ln
(jj
RMR RMR RMR
m/z 188 m/z 358 m/z 498
Standard 2-Chlorobiphenyl Hexachlorobiphenyl Decachlorobiphenyl
I PCB-STD-20 0.60
11 PCB-STD-2 0.620"^
n'^J > °-640 + -171
0.466 I
0.643J
III PCB-STD-0.2 0.566"^
0.840 1
0.637 )0.699 +.171
0.597 j
0.705 J
IV PCB-STD-0.04 1.020~\
0.692 \ 0.763 + .257
0.576J
0.257
0.291^
0*319 }0'325 ± -009
0.321 J
•^
0 . 3oo i
0.293 1
0.301 } 0.294 + .072
0.239 I
0.273J
0.528 V0.459 + .072
0.528 J
0.341
0.430^
0.474 ln .,, .
0.462 >°'456±
0.431 J
0.372*^
0.361 1
0.373 ) 0.361 +
0.303 [
0.394J
0.287"^
0.543 ) 0.401 +
0.372 J
.018
.033
.142
Standard is d -pyrene (m/z = 212)
-------
Table 7-4. RECOVERY OF EXTRACTABLE HALOGENATED ORGANICS FROM WHOLE BLOOD AND PLASMA
Ui
Percentage Recoveries + Std. Dev.
Compound
Trifluralin
Lindane
Heptachlor
Heptachlor epoxide
Endosulfan
Dieldrin
£,£'-DDT
Mean
Whole Blood
Plasma
36.0 + 7.5
62.8 + 12.7
29.0 + 9.0
31.3 + 7.6
37.7 + 8.5
31.6 + 10.4
82.9 + 24.8
44.4 + 20.4
(^500 ng spike)
Cellb
Not det.
<1.0
Not det.
Not det.
Not det.
Not det.
Not det.
<1
Q
Plasma
^500 ng spike
87.3 + 7.7
106.8 + 4.9
106.3 + 8.1
63.1 + 1.48
75.0 + 4.3
79.6 + 4.2
101.4 + 15. 8h
88.5 + 17.0
Plasma
VLO ng spike
e
100.6 + 9.2
88.5 + 5.0f
59.4 + 11.6
73.4 + 5.4
79.6 + 6.4
78.8 + 10. I1
80.1 + 13.9
Data based on mean of four samples, each of which is quantified twice against a standard. Whole
, blood was spiked and separated. Extractions were for a short time (^45 min).
Based on injection of two of four samples—Not Det. = not detected.
Data based on mean of three samples, each of which is quantified twice against a standard. Whole
,blood spiked; plasma fraction extracted over long time (^24 hr).
Plasma spiked ane extracted with hexane 24 hr, then 12 hr with hexane. Data based on mean of two
samples, each of which is twice quantified against a standard.
Compound elutes with solvent; not quantified.
Based only on standard deviation of standard.
^Data based only on one sample. Precision between samples very poor.
.47 ng spiked.
XBased only on standard deviation of sample.
-------
Table 7-5. RECOVERY STUDIES OF EXTRACTABLE HALOGENATED HYDROCARBONS
FROM HUMAN PLASMA EQUILIBRATED FOR 19 HR
•a
Recovery from Plasma (%)
Compound
Trifluralin
a-BHC
B-BHC
Y-BHC
2,4, 5-Trichlorobiphenyl
Heptachlor
Aldrin
Heptachlor Epoxide
Endosulfan
£,_p_'-DDE
Dieldrin
Mean + Std. Dev.
ng
spiked
14.8
14.0
14.5
15.2
14.0
13.6
14.8
14.2
13.1
11.6
15.8
Trial
One
87.6
61.9
60.0
57.1
48.2
50.2
b
35.2
53.2
51.4
58.6
Trial
Two
92.3
56.2
60.1
53.3
b
42.2
40.3
43.1
59.1
b
b
Trial
Three
83.3
57.6
43.8
52.4
b
51.1
37.2
b
44.3
48.8
58.5
Mean
85.7
58.6
50.8
54.3
48.2
47.8
38.8
39.2
52.2
50.1
58.6
53.1 + 12.6
Experimental conditions similar to those described in Footnote d, Table 7-1,
except spiked plasma was equilibrated 19 hr vs. 2 hr.
Not quantifiable due to instrumental problems.
55
-------
fraction to four successive 0.85% NaCl washes. Layers were again separated
by centrifugation. Each fraction was successively extracted with 4 ml of
hexane for 45 minutes; a 4 ml aliquot was then removed, 4 ml of fresh hexane
added, extraction continued for 45 minutes, and another 4 ml aliquot of
hexane removed. The samples were then dried over anhydrous Na.SO,, concentra-
ted to the proper linear response range, and quantified by GC/ECD. Table 7-
4 shows the partitioning of pesticides between whole blood and plasma.
Virtually no detectable pesticides were detected in whole cell extracts, and
recoveries from plasma were poor.
Eight pesticides at the 500 ng concentration level were added to three
2 ml aliquots of human plasma. Each tube was incubated at 37°C for two
hours and extracted with 7.0 ml hexane for 24 hours. A 6.0 ml aliquot of
solvent was removed from each, 6.0 ml fresh hexane was added, extraction
continued 12 hours, and 6.0 ml again removed. Each sample was dried over
Na SO,, concentrated to the proper linear response range, and analyzed by
GC/ECD. Table 7-4 shows the percentage recovery by this methodology.
The experiment above was repeated at spiking levels near the limit of
detection (about 10 ng) to determine if the recovery is concentration-
dependent. The results presented in Table 7-4 indicate that good recoveries
are achieved even at low limits.
The recoveries were again determined with a longer equilibration time
(19 hr) at a 11-16 ng spiking level. All other conditions were the same.
The recoveries, shown in Table 7-5, are significantly lower than the analogous
data in Table 7-4 (mean 53.1% vs 80.1%). These data indicate that the
model compounds may be binding to the plasma matrix to form unextractable
complexes.
Summary
Based on the experiments summarized in Table 7-4 and 7-5, the expected
recoveries of extractable halogenated organics from blood are about 40-100%.
Because of the apparent low recoveries, this method must be regarded as
semi-quantitative.
VALIDATION OF PROCEDURE FOR WATER
The water solubilities of most of the compounds of interest are very
low and the probability of finding any extractable halogenated organics in
56
-------
water is remote. Nevertheless, an extraction procedure was validated for
water samples so that at least some of the samples may be run to confirm
this assumption.
The recoveries of selected pesticides from water, listed in Table 7-6,
indicate that the procedure is, in general, satisfactory. These pesticides
were chosen as representative of the range of polarity and volatility expected
in the compounds to be studied.
Based on the experiments summarized in Table 7-6, the expected recoveries
of extractable halogenated organics from water are about 70% or better.
Within the precision requirements of this study, these recovery values indi-
cate that the method is essentially quantitative. The entire analytical
procedure has been tested during preliminary field sampling studies. No
significant problems are anticipated with the execution of the protocol.
VALIDATION OF PROCEDURE FOR TISSUE
The procedure for tissue followed that of Thompson (7-1) with minor
modifications. Recovery studies were done on human samples received from
the Buffalo/Niagara Falls area. The recoveries, listed in Table 7-7 indicate
that the method works not only for adipose tissue (for which it was designed)
but also for other tissue samples. The paragraphs below detail the analytical
procedure and the rationale behind the individual steps.
Tissue fractions were analyzed for semi-volatile halogenated hydrocarbons
using a modified procedure by Thompson (7-1). Approximately five grams of
each tissue was ground in a large glass beaker with a glass rod with acid-
washed, sea sand and sodium sulfate (both cleaned up prior to use by extrac-
tion in a Soxhlet extractor with hexane) until a dry, granular mass was
obtained. Aldrin, 198 ng in 100 (Jl hexane was added to the sample as an
internal standard. Each tissue was then extracted, with vigorous grinding,
with three 50 ml aliquots of hexane for approximately 5 min each. The ex-
tracts were then filtered, concentrated in a Kuderna-Danish (KD) apparatus
to approximately 10 ml, blown down under nitrogen to dryness at room tempera-
ture and weighed.
The high lipid content of the adipose tissue extract necessitated
partitioning of the extract into acetonitrile and back-partitioning of the
halogenated hydrocarbons into hexane. Approximately 2.5 grams of fat extract
57
-------
Table 7-6. RECOVERY STUDIES OF EXTRACTABLE HALOGENATED HYDROCARBONS FROM WATER
Recovery (%)
Compound
Trifluralin
a-BHC
6-BHC
Y-BHC
2,4, 5-Trichlorob ipheny 1
Heptachlor
Aldrin
Heptachlor epoxide
Endosulfan
£,£* -DDE
Dieldrin
Low
Trial
One
55.4
57.8
54.7
65.6
62.4
99.1
58.3
87.4
78.7
73.8
82.0
Fortification3
Trial
Two
48.1
57.9
65.1
70.6
73.1
93.6
47.8
76.1
d
80.3
88.6
Average
51.8
57.8
65.1
68.1
67.8
96.4
53.0
81.8
78.7
77.0
85.3
High Fortification
Trial Trial Trial Average
One Two Three
68.3 ± 0.8 69.9 ± 4.3 126.8 ± 14.0 88.3 ± 11.2
93.2 ± 5.0 98.7 ± 6.2 82.2 ± 1.2 91.4 ± 7.7
c
_
_
71.0 ± 6.0 66.4 ± 4.3 83.8 ± 2.0 73.7 ± 8.0
_
_
81.1 ± 1.46 103.1 ± 8.7 90.9 ± 7.9 91.7 ± 11.2
_
57.5 ± 4.7 67.9 ± 8.7 63.5 ± 4.9 63.0 ± 10.7
00
Mean ± Std. Dev.
70.7 ± 14.4
74.0 ± 21.7
100 ml distilled/deionized water fortified with 11-16 ng of model compound in 100
according to protocol.
3Same as (a) except fortification level about 500 ng.
^Not included in this experiment.
Not quantifiable due to instrumental problems.
BOnly one standard injection.
methanol, extracted
-------
Table 7-7- RECOVERY OF EXTRACTABLE HALOGENATED HYDROCARBONS
FROM HUMAN TISSUE EXTRACTS
Tissue
Adipose
Brain
Liver
Kidney
Spleen
Lung
Percent
Extr actable
Material3
84.3
4.4
1.1
1.1
1.3
0.4
Percent,
Recovery
76.6
69.7
85.1
64.5
91.2
87.5
a
Also described as "percent fat" in some procedures.
Recovery of 198 ng aldrin added to tissues after maceration and
before extraction as an internal standard. Percent recovery deter-
mined after all analytical manipulations. Aldrin represents a suit-
able standard, since it is metabolized to endrin and has not been
found in tissues. Mean recovery for adipose tissues = 79.1 ± 10.6%.
£
Analytical workup includes acetonitrile partitioning to reduce fat
content.
59
-------
was dissolved in 12 ml hexane and extracted two rain each with four 30 ml
aliquots of redistilled acetonitrile. The extracts were combined with 250
ml 2% aqueous sodium chloride and back-extracted with four 30 ml aliquots of
hexane. The hexane extracts were combined, dried over Na^SO,, concentrated
in a KD to about 5 ml, blown down under nitrogen to approximately 2 ml, and
subjected to Florisil cleanup as described below.
Florisil (60/100 mesh, activated at 130°C overnight) columns (2.2 cm
i.d. x 10 cm) with glass frits or glass wool plugs were packed in hexane
solvent. Each tissue extract was transferred to the surface of the column
in approximately 2 ml of solvent, and the column walls were washed with
approximately 4 ml of hexane. The halogenated hydrocarbons were then eluted
with 200 ml each of 6% ether/hexane and 15% ether/hexane, respectively.
Each fraction was concentrated in a KD apparatus to approximately 5 ml.
After storage in the refrigerator overnight, a precipitate was found in
the concentrated 15% eluant of the adipose and brain extracts. Consequently,
these fractions were stored in the freezer prior to further cleanup and
analysis. The 6% eluants were blown down under ambient nitrogen to appro-
priate volumes and analyzed by GC/ECD.
Previous studies by Thompson suggest the following halogenated hydro-
carbons should be found in the 6% eluant: BHC isomers, p_,p_'-DDE, p_,p'-DDT,
heptachlor, heptachlor epoxide, mirex, PCB, hexachlorobenzene, and trifluralin
(7-1). These represent the same general polarity of the halogenated compounds
of interest in tissue samples, so this fraction is of primary interest for
analysis.
Based on the experiments summarized in Table 7-7, the expected recoveries
of extractable halogenated organics from tissue are 60-90%. This procedure
has only been partially validated so, depending upon the quality control
studies carried out with field samples, the data may need to be corrected
for low recoveries. The entire analytical procedure has been tested during
preliminary field sampling studies. No significant problems are anticipated
with the execution of the protocol.
VALIDATION OF PROCEDURE FOR URINE
Based on literature data (7-2) the concentrations of most of the extract-
able halogenated organics of interest is very low. Therefore, significant
60
-------
results are not expected from urine samples. The procedure to be used,
described by Thompson (7-1) has not been validated. Quality control samples
analyzed with the field samples will serve to determine recoveries. If
recoveries are less than quantitative, correction factors will be used.
VALIDATION OF PROCEDURE FOR SOIL AND SEDIMENT
The procedure to be used, described in Thompson (7-1) has not been
validated. Quality control samples analyzed with the field samples will
serve to determine the recoveries. If recoveries are less than quantitative,
correction factors will be used.
REFERENCES
7-1 Thompson, J. F., Analysis of Pesticide Residues in Human and Environmen-
tal Samples, A Compilation of Methods Selected for Use in Pesticide
Monitoring Programs, Environ. Toxicol. Div., Health Effects Research
Lab., USEPA, RTP, NC, December, 1974.
7-2 Pellizzari, E. D., MTPR No. 15, EPA Contract No. 68-01-4731, February
1979, Appendix B.
61
-------
SECTION 8
COLLECTION AND ANALYSIS OF BLOOD SAMPLES FOR CEA
The selection of the CEA-Roche radioimmunoassay (RIA) was in part pre-
dicated on the large body of data on normal and abnormal populations in this
test (8-1 - 8-4). However the validation of the accuracy of the test must be
performed in each laboratory with standards. In order to test the overall
procedure from sample collection to data analysis the CEA analysis (Appendix
J) was performed on the 9 participants in the "Love Canal" pilot study. The
results of the analysis of these participants in the pilot study are given in
Table 8-1. The CEA-Roche procedure indicates standard deviations are roughly
±0.5 ng/ml in the 0 to 5 ng/ml range.
CEA levels are not considered elevated until they exceed 2.5 ng/ml by
this procedure. Three individuals in this group fall into that category. In
a normal healthy population of non-smokers the predicted frequency of elevated
CEA is 3 in 100 and 19 per 100 for smokers. Of the 9 participants in this
study, 5 were smokers. By using a weighted average incidence of elevated CEA
levels for smokers and non-smokers, the 3 elevated CEA levels in this pilot
was of borderline significance (barely significant at the 90% confidence
level). This is a very small population to try to analyze statistically, but
this indicates the approach to be taken.
REFERENCES
8-1 Go VLW: Data on file, Hoffmann-La Roche Inc., Nutley, NJ.
8-2 Hansen, H. J., et al. , J. Clin. Res., 19, 143 (1971).
8-3 Data available on request from Hoffmann-La Roche Inc., Nutley, NJ.
8-4 Hansen, H. J., et al., Human Pathology, 5, 139-147 (1974).
62
-------
Table-8-1. PLASMA CEA LEVELS OF INDIVIDUALS RESIDING IN THE
"OLD LOVE" CANAL SITE OF NIAGARA FALLS, NY
Sample No.
10009
10017
10025
10033
10066
10074
10041
10058
10090
Mean ng/ml
1.91
1.18
1.11
2.29
1.40
1.98
3.02b
2.74b
2.58°
Standard Error ng/ml
0.30
0.21
0.66
0.27
0.46
0.21
0.69
0.50
-
O O I/O
The standard error was computed from (s , , + s , /n)
standards samples
Initial assay resulted in loss of one sample. A separate assay was
made after the expiration date of the kit.
CInitial assay resulted in loss of one sample and-insufficient plasma
remained for a repeat.
63
-------
SECTION 9
COLLECTION AND ANALYSIS OF HALOGENATED ORGANICS IN HUMAN BREATH
The apparatus used for the collection of halogenated organics was
developed under Contract No. 68-01-3849 for the analysis of benzene in human
breath. The performance of the sampling apparatus was validated for benzene
in a pilot study using filling station attendants, fuel truck drivers and
laboratory personnel. These validation experiments with benzene serve as a
model for the volatile halogenated hydrocarbons. Individuals for both work
and non-work exposures, where the responses of the breath analysis to exposure
could be estimated. In the case of benzene, smoking constitutes a substantial
exposure.
It was our original intent to use all non-smokers. However, of the
relatively limited population available to us for this pilot study, a very
high percentage of them were smokers. We were, therefore, only able to
obtain participation of one non-smoker filling station attendant.
Each participant was supplied with a personnel sampler and a Tenax
cartridge 6-8 hours preceding the breath sample collection. A blood sample
was drawn (~10 ml) and the breath sample collected immediately thereafter.
All of the work exposure samples were collected on a Friday afternoon after a
minimum of five consecutive days of working 10-12 hours per day. The off-day
sampling was performed either on Sunday afternoon or before work on Monday
morning. In the case of participant E, there was a 2-week delay between the
Friday of the work-day sample .and Sunday off-day sample. Otherwise, the
sampling was done on consecutive Fridays and Sundays. In order to obtain
baseline information, two non-smoking laboratories workers were sampled on
their work days.
Table 9-1 summarizes the results from the analysis of all of the parti-
cipants in the breath analysis. The breath sampling followed the protocol
for breath analysis in Appendix K.
-------
Table 9-1. PRELIMINARY RESULTS OF PILOT STUDY OF BENZENE BODY BURDEN
UI
Sample
A
B
C
D
E
F
G
H
I
Occupation
Laboratory worker
Shop worker
Filling station
attendant
Filling station
attendant
Filling station
attendant
Laboratory worker
Laboratory worker
Filling station
attendant
Gasoline tanker
driver
Smoker
or Non-
smoker
smoker
smoker
smoker
smoker
non-
smoker
non-
smoker
non-
smoker
smoker
non-
smoker
Benzene Levels Found
Sex Weight
(kg)
M 100
M 60
M 60-70
M 80-90
M 90-100
M 60
M 70-80
M 60-70
M 60-70
Worked or Off
Preceding Test
Worked
Worked
Workedb
Off
Worked
Off
Worked
Off
Worked
Worked
Worked
Off
Worked
Off
Air
dig/i
a
a
153
-
260
13
c
13
a
a
190
1.
54
85
Monitor
3) (ppb)
a
a
48
-
82
4
c
4
a
a
60
4 0.4
17
27
(Ug/m3)
9.3
19.0
432.0
6.8
17.0
11.0
7.2
1.3
1.1
3.7
27
15
58
28
Breath
(ppb)
3.1
6.5
136.0
2.1
5.4
3.5
2.3
0.4
0.4
1.2
8.4
4.7
18
8.4
(ng/mln)
87
110
2300
60
98
57
40
8.4
7.3
10.4
200
130
513
246
Blood Urine
Mg/A Pg/1
-
-
18.6 + 1.4
0.42 + 0.07
1.88
<0.35
0.28 + 0.06
<0.34
0.1
d 0.72 + 0.14e
1.14 0.93 + 0.14
0.40 + 0.06 0.41 + 0
1.30 + 0.34 2.4 + 0.4
1.93 + 0.12 1.6 + 0
a 3
The individuals were not monitored; however, air monitoring in the vicinity of their work areas indicated 8.2 Ug/m of benzene in
the air.
Subject C had been repairing a fuel line on an automobile. It is probable that he was exposed to more benzene than Indicated by the personnel
monitor.
GCartridge lost due to breakage.
Interfering peak on the gas chromatograra.
Sample taken from the same subject at a later date.
-------
Breath levels of benzene were consistently higher on "working" days than
on "off" days. Smokers had consistently higher benzene levels in their
breath than non-smokers.
A similar pilot study was performed on a group of nine residents of the
"Love Canal" to confirm the extension of this technique to halogenated
organics. Here halogenated organics were measured (Table 9-2) as well as
benzene (Table 9-3). The benzene levels reflected the individuals smoking
habits as in the earlier study. Eighteen halogenated organics were found in
breath samples and at least some of the compounds appear to be site-specific
(i^.e. chlorobenzotrifluoride, dichlorotoluene, hexachlorobutadiene, trichloro-
benzene, pentachlorobenzene, chlorotoluene).
66
-------
Table 9-2 . ESTIMATED LEVELS OF HALOGENATED COMPOUNDS IN HUMAN BREATH FROM
"OLD LOVE" CANAL IN NIAGARA FALLS, NY&
Halogenated Compounds
1 , 2-dichloroethane
carbon tetrachloride
trichloroethylene
1,1, 1- tr Ichloroethane
tetrachloroethylene
1,1-dichloroethane
dichlorobenzene lsomer(s)C
chlorobenzene
chloroform
1, 3-hexachlorobutadlene
trichlorobenzene isomer(s)
tetrachlorobenzene isomer(s)
pentachlorobenzene isomer(s)
chlorotoluene Isomer(s)
dlchlorotoluene isomer(s)
bromotoluene Isomer(s)
chloronaphthalene Isomer(s)
chlorobenzotrif luoride isomer(s)
10009
_b
90 + 36
-
108 + 36
658 + 63
-
459 + 72
-
36,576
T
T
180
-
261 + 45
-
-
-
-
10017
T
796 + 135
T
286 + 122
1,224 + 285
T
755 + 204
T
94,510
-
-
-
-
-
-
-
-
T
10025 10033
243 + 108
622 689 + 378
-
351 329 + 106
1,904 1,152 + 129
-
513 5,294
-
45,892 37,447
-
-
27
-
338
-
-
-
T
Participant No.
10066 10041
136 + 75
167 + 75
T
2,812 394 + 121
750 667 + 75
-
T
-
3,896 23,530
-
-
-
-
-
-
-
-
-
10058 10074
_ _
T T
T T
T T
4,469 1,753
-
T
-
25,591 20,424
-
-
-
-
-
-
-
-
T T
10090
73 + 12
88 + 22
-
264 + 44
632 + 294
-
58 + 0
-
23,529 + 11,764
-
88
-
73
-
1,220
T
T
852 + 441
a ~3~
Values are in ng/m , duplicate values were obtained where indicated.
- equals not detected.
Q
Values are summed for all isomers when more than one observed.
-------
Table 9-3 . ESTIMATED LEVELS OF BENZENE IN HUMAN BREATH FROM
"OLD LOVE" CANAL IN NIAGARA FALLS, NY
Participant No.
Benzene (ng/m )
Comment
10009
10017
10025
10033
10066
10041
10058
10074
10090
1,837 + 117
2,571 + 1,551
905
6,776 + 776
687
6,954 + 1,651
898
4,877
735 + 132
smoker
smoker
smoker
smoker
smoker
68
-------
SECTION 10
FIELD OPERATIONS
Field operations activities have been centered in A areas:
1. Establishing communications regarding study activities with
appropriate regional, state and local agencies;
2. Developing appropriate instrumentation;
3. Conducting a pretest of data collection instruments and proce-
dures in Niagara Falls, New York; and
4. Preparing the submission for Office of Management and Budget
(OMB) review.
More details on these activities is presented in the following sections.
CONTACT WITH REGIONAL, STATE AND LOCAL AGENCIES
The RTI Project Survey Director has met with representatives of the
following agencies to discuss study activities:
Office of Toxic Substances, EPA Region II, Edison, New Jersey
New York State Health Department, Albany, New York
New Jersey State Health Department, Trenton, New Jersey
Niagara County Health Department, Niagara Falls, New York
EPA Region VI, Dallas, Texas
Division of Epidemiology, Texas Department of Health, Austin, Texas
Texas Air Control Board, Austin and Bellaire, Texas
EPA Laboratory, Houston, Texas
Harris County Health Department, Houston, Texas
Harris County Pollution Control Department, Pasadena, Texas
City of Houston Health Department, Houston, Texas
Department of Public Health, Pasadena, Texas
Louisiana Department of Health and Human Resources, New Orleans, LA
69
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The purpose of these meetings was to discuss the study protocol. State
and local officials were solicited regarding support for the study, particula-
rly an appropriate individual's name and telephone number to appear on the
participant consent form (PCF) should participants have questions or wish to
verify the study. Publicity was also discussed, as well as the possibility
of certain forms of assistance at the local level, specifically a van to
serve as a central data collection facility in sampling areas remote from
public buildings and an experienced phlebotomist to assist in the collection
of blood samples.
All individuals contacted indicated interest and support for, the
study. The general consensus of local officials in Texas was that the study
should be low-key without widespread publicity. The Texas Department of
Health indicates that they might be of some assistance in securing tissue
samples when and if that time comes. They noted that Texas may present some
special problems with regard to mortality records and the definition of
death. EPA Region II has indicated that a mobile laboratory van will be at
RTI's disposal in lieu of renting a Winnebago for the main study activites in
Region II, if RTI so desires; a similar vehicle may be available from the EPA
Laboratory in Houston. Such arrangements would improve EPA's visibility and
reduce RTI's field costs.
PRETEST
In July 1978, a limited pretest of data collection instruments and
procedures was executed in the Love Canal area of Niagara Falls, New York,
with the cooperation and assistance of EPA Region II, the New York State
Health Department, and the Niagara County Health Department. Potential
participants were selected from two sources: (1) a list of individuals who
had indicated to the New York State Health Department, during that agency's
health status screening in the Love Canal area in June 1978, that they would
be interested in participating in further study; and (2) a list of households
whose basements were sampled by RTI early in 1978. Nine participants were
purposely selected so that (1) both residential blocks of the Love Canal
area were represented, and (2) the pretest stayed within limits imposed by
OMB. Two participants resided in the northernmost block and 7 in the southern-
most block, and 2 participants resided in the same household (but were
70
-------
studied over different time periods - see below). In addition, 7 participants
were female, 2 male; all were adults. Six participants were selected from
the State list, 2 from the list of households monitored earlier by RTI, and 1
participant appeared on both lists. There were no refusals; one participant
did not recall telling the State that she would participate in further
studies, but she participated anyway. RTI had originally planned to offer a
$15 incentive for participation; however, due to the close temporal relation-
ship of the RTI pretest to the State study, which offered no incentive, RTI
did not offer an incentive either, on the recommendation of EPA Region II.
EPA Region II arranged with the Niagara Falls school system for RTI to
use space at the school in the Love Canal area as a central data collection
facility; RTI was granted access to the health room at the school, ideally
suited for use as a central data collection facility, replete with refrigera-
tor and sink. The Niagara County Health Department provided the services of
one experienced R.N. phlebotomist to assist with the collection of blood
samples.
For each participant, a questionnaire (see below) was administered; air
monitoring devices were set up inside the house and in the yard; two 120 ml
tap water samples were collected before and after flushing the lines for 3
minutes; and arrangements were made for them to come to the school at a
specified time where a breath sample was obtained and three 10 ml Vacutainer
tubes (two containing sodium heparin as an anticoagulant, one containing
liquid EDTA) were filled by brachial venipuncture. At the time of initial
contact, each participant was provided with a 120 ml bottle and asked to
collect an early morning urine sample, which all did. The indoor and outdoor
air monitoring was terminated shortly after the collection of the blood and
breath samples. Specific gravity determinations were performed on all urine
samples using a protometer.
The first 5 participants were interviewed early on the morning of July
6, and were processed at the central data collection facility that afternoon.
The other 4 participants were interviewed that afternoon and processed at the
central data collection facility on the morning of July 7. Spot urine
samples were collected from participants 1-3 and 5 the afternoon of July 6
71
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at the time the breath and blood samples were collected; however, no attempt
was made to collect a spot urine sample from participants 6-9 due to the
short time elapsed between the early morning sample and their appointments at
the school. For the 2 participants who resided in the same household, only
one set of tap water samples was collected at that residence.
Following the pretest, several discussions took place among RTI project
principals and between the RTI Project Survey Director and the EPA/OTS
Project Officer regarding the release and dissemination of data. Summary
data which did not identify individual participants were provided to EPA/OTS,
and through them to EPA Region II and the New York State Health Department.
There was concern that direct release of data to participants could
result in gross misinterpretation and unnecessary anxiety. Therefore, the
decision was made to provide the participant-data linkage to the New York
State Health Department only for appropriate action. That agency had con-
sent/release forms from virtually every resident of the Love Canal area which
had been the basis of their providing RTI with the list of names which yielded
7 of the 9 participants in the RTI pretest. RTI obtained copies of those
consent forms for all 9 participants, then discussed both consent forms, New
York's and RTI's (the RTI consent form specifically mentions cooperation with
the New York State Health Department - see below), with the Director of RTI's
Survey Operations Center and Mr. Kenneth R. Wing, Assistant Professor of
Health Law at the University of North Carolina and an RTI consultant. These
individuals saw no problems with the RTI-New York State Health Department
interaction.
INSTRUMENTATION
Appendix N presents a copy of the PCF and Questionnaire used in the
pretest. Following the pretest, these instruments were revised and in March
1979 were submitted to EPA for review and submission to OMB. The final
version of the Household Screening Questionnaire, the PCF, and the Question-
naire appear in Appendix 0.
72
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APPENDIX A
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF VOLATILE ORGANIC
COMPOUNDS IN AMBIENT AIR
73
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ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF VOLATILE ORGANIC COMPOUNDS
IN AMBIENT AIR
1.0 Principle of Method
Volatile organic compounds are concentrated from ambient air onto Tenax
GC in a short glass tube (A1-A3). Recovery of the volatile organics is
accomplished by thermal desorption and purging with helium into a liquid
nitrogen cooled nickel capillary trap (A1-A2,A4) and then the vapors are
introduced into a high resolution glass gas chromatographic column where the
constituents are separated from each other (A2,A5). Characterization and
quantification of the constituents in the sample are accomplished by mass
spectrometry either by measuring the intensity of the total ion current
signal or by mass fragmentography (A2,A6). The collection and analysis
systems are shown in Figure A-l.
2.0 Range and Sensitivity
The linear range for the analysis of volatile organic compounds depends
upon two principal features. The first is a function of the breakthrough
volume of each specific compound which is trapped on the Tenax GC sampling
cartridge and the second is related to the inherent sensitivity of the mass
spectrometer for each organic (A2,A7). Thus, the range and sensitivity are
a direct function of each compound which is present in the original ambient
air. The linear range for quantitation on the gas chromatograph/mass spectro-
meter/computer (GC/MS/COMP) is generally three orders of magnitude. Table
A-l lists the overall theoretical sensitivity for some examples of volatile
organics which is based on these two principles (A7).
3.0 Interferences
The potential difficulties with this technique are primarily associated
with those cases where isomeric forms of a particular substance cannot be
resolved by the high resolution chromatographic column and when the mass
spectral fragmentation patterns of each of the isomers are identical. An
example of such a problem is seen with the C^-alky! aromatics of which there
are 53 isomers. As the number of carbon atoms increases in the hydrocarbons
and aromatics, the number of potential isoraers becomes increasingly large
74
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FLOW
METER
PUMP
CARTRIDGE
NEEDLE
VALVE
GLASS
FIBE*
FILTER
VAPOR COLLECTION SYSTEM
PURGE
GAS
ION
CURRENT
RECORDER
GLASS
JET
SEPARATOR
TWO
POSITION
VALVE
THERMAL
DESORPTION
CHAMBER
CAPILLARY
GAS
CHROMATOGRAPH
CARRIER
GAS
HEATED
BLOCKS
EXHAUST
CAPILLARY
TRAP
ANALYTICAL SYSTEM
Figure A-l. Vapor collection and analytical systems for analysis
of organic vapors in ambient air.
75
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Table A-l. OVERALL THEORETICAL SENSITIVITY OF HIGH RESOLUTION
GAS CHROMATOGRAPHY/MASS SPECTROMETRY/COMPUTER ANALYSIS
FOR ATMOSPHERIC POLLUTANTS
Estimated Detection
Limit
Chemical
Class
Halogenated
hydrocarbon
Compound
Vinyl bromide
Bromoform
Bromodichlorome thane
Dibromochlorome thane
l-Bromo-2-chloroethane
Allyl bromide
1-Bromopropane
l-Chloro-3-bromopropane
l-Chloro-2 , 3-dibromopropane
1 , l-Dibromo-2-chloropropane
1 , 2-Dibromoethane
1 , 3-Dibromopropane
Epichlorohydrin
(l-Chloro-2 , 3-epoxypropane)
Epibromohydrin
(l-Bromo-2 , 3-epoxypropane)
Bromobenzene
Methyl bromide
Methyl chloride
Vinyl chloride
Methylene chloride
Chloroform
Carbon tetrachloride
ng/m
250
0.340
1.300
0.667
1.00
5.00
5.200
0.150
M).100
MK100
0.530
MK100
9.600
0.300
0.100
500
2000
800
700
200
250
PPt
57
0.03
0.22
0.07
0.67
1.04
1.06
0.01
<0.01
<0.01
0.07
•^0.01
2.50
0.05
0.02
135
1000
333
200
420
400
(continued)
-------
Table A-l (cont'd.)
-vl
-vl
Estimated Detection
Limit
Chemical
Class
Halogenated
hydrocarbon
(cont'd)
Halogenated
ethers
Nitrosamines
Oxygenated
hydrocarbons
Compound
1 ,2-Dichloroethane
1,1, 1-Trichloroethane
Tetrachloroethylene
Trichloroethylene
l-Chloro-2-methylpropene
3-Chloro-2-methylpropene
3-Chloro-l-butene
Allyl chloride
4-Chloro-l-butene
l-Chloro-2-butene
Chlorobenzene
o-Dichlorobenzene
m-Dichlorobenzene
Benzylchloride
2-Chloroethyl ethyl ether
Bis-(chloromethyl)ether
N-Nitrosodimethylamine
N-Nitrosodiethylamine
Acrolein
Glycidaldehyde
Propvlene oxide
ng/m
32
66
2.5
1°
62
62
83
83
38
13
2.10
1.00
0.75
0.65
4.15
1.0
5.0
3.0
vLOO
•v-59
•^60
ppt
8.15
12.45
0.38
1.92
21.5
21.5
28.8
28.8
13.2
4.5
0.47
0.06
0.01
0.01
0.97
1.10
1.67
0.74
56.5
9.5
25.5
(continued)
-------
Table A-l (cont'd.)
oo
Acetophenone
p-Propiolactone
Estimated Detection
Limit
Chemical
Class
Oxygenated
hydrocarbons
(cont'd)
Compound
Butadiene diepoxide
Cyclohexene oxide
Styrene oxide
. 3
ng/m
VLO
2
PPt
6.7
2.5
0.415
•v-3
VL.2
Nitrogenous
Compounds
Sulfur
Compounds
Nitromethane
Aniline
Diethyl sulfate
Ethyl methane sulfate
8
3.0
-x.50
0.78
-
**T-T : J 1 . 1 *. 4 A 4-l~A V «»»-:« « £ 4- l~ A l.-u-AAlrt-l-lw..'*!-! »n 1 11M** -pAV 1 O rt. «-t 'Pnv^Avr (T* f r. 4- ~i f\QT?\
capillary column performance and sensitivity of the mass spectrometer to that compound in the
mass fragmentography mode of most intense ion.
-------
and difficult to completely resolve by gas chromatography and/or by their
corresponding mass spectral fragmentation patterns. However, differentiation
between the hydrocarbons, that is, alkanes, alkenes, aromatics, oxygenated,
etc., can be accomplished.
4.0 Reproducibility
The reproducibility of this method has been determined to range from
+10 to +30% for different substances when replicate sampling cartridges are
examined (A5). The inherent analytical errors are a function of several
factors: [1] the ability to accurately determine the breakthrough volume
for each of the organic compounds identified; [2] the accurate measurement
of the ambient air volume sampled; [3] the percent recovery of the organic
from the sampling cartridge after a period of storage; [4] the reproducibility
of thermal desorption for a compound from the cartridge and its introduction
into the analytical system; [5] the accuracy of determining the relative
molar response ratios between the identified substance and the external
standard used for calibrating the analytical system; [6] the reproducibility
of transmitting the sample through the high resolution gas chromatographic
column; and [7] the day-to-day reliability of the MS/COMP system (A1-A8).
The accuracy of analysis is generally +30% but depends on the chemical
and physical nature of the compound (A2,A8).
5.0 Advantages and Disadvantages of the Method
The gas chromatograph/mass spectrometer interfaced with a jet separator
is extremely sensitive and specific for the analysis of many volatile organic
compounds in ambient air. High resolution gas chromatographic separation
provides adequate resolution of the substances found in ambient air for
their subsequent quantification. The combination of the high resolution gas
chromatographic column and the selection of specific or unique ions represent-
ing the various compounds of interest identified in the air samples yields a
relatively specific assay method for these compounds (A1-A8).
Collected samples can be stored up to one month with less than 10%
losses for most of the chemical classes (A2,A8). Because some of the compounds
of interest may be hazardous to man, it is extremely important to exercise
safety precautions in the preparation and disposal of liquid and gas standards,
cleaning of used glassware, etc. in the analysis of air samples.
79
-------
The efficiency of air sampling increases as the ambient air temperature
decreases (i.e., sensitivity increases) (A8).
The retention of water by Tenax is low; its thermal stability is high
and its background is negligible, allowing sensitive analysis
(A1,A2,A5,A8).
6.0 Apparatus
6.1 Sampling Cartridges
The sampling tubes are prepared by packing a ten centimeter long by
1.5 cm i.d. glass tube containing 6 cm of 35/60 mesh Tenax GC with glass
wool in the ends to provide support (A2,A5). Tenax is extracted in a Soxhlet
apparatus for a minimum of 24 hours each time with methanol and pentane
prior to preparation of cartridge samplers (A2,A5). After purification of
the Tenax GC sorbent and drying in a vacuum oven at 100°C for 3 to 5 hours
at 28 inches of water. All the sorbent material is meshed to provide a
35/60 particle size range. Cartridge samplers are then prepared and condi-
tioned at 270°C with helium flow at 30 ml/min for 30 min. The conditioned
(5\
cartridges are transferred to Kimax (2.5 cm x 150 cm) culture tubes, immedia-
tely sealed using Teflon-lined caps and cooled. This procedure is performed
in order to avoid recontamination of the sorbent bed (A2,A5).
Cartridge samplers with longer beds of sorbent may be prepared using a
proportionally increased amount of Tenax in order to achieve a larger break-
through volume for compounds of interest, and thus increasing the overall
sensitivity of the technique (A8).
6.2 Gas Chromatographic Column
A 0.35 mm i.d. x 100 ra glass SCOT capillary column coated with SE-30
stationary phase is used for effecting the resolution of the volatile organic
compounds (A5). The capillary column is conditioned for 48 hrs. at 245° at
2.25 ml/min of helium flow.
A glass jet separator is employed to interface the glass capillary
column to the mass spectrometer on the Varian MAT CH-7 GC/MS/COMP system and
on the Finnigau 3300 system. The glass jet separators are maintained at
240°C (A2,A5). A metal jet separator, maintained at 210°C, is employed to
interface the LKB 2091 GC/MS/COMP system to the glass capillary column.
80
-------
6.3 Inlet Manifold
An inlet manifold for thermally recovering vapors trapped on Tenax
sampling cartridges is used and is shown in Figure A-l (A1,A2,A4,A5).
6.4 Gas Chromatograph
A Varian 1700 gas chromatograph is used to house the glass capillary
column and is interfaced to the inlet manifold on the Varian MAT CH-7 system.
A Finnigan 9500 gas chromatograph houses the glass capillary column and is
interfaced to the thermal desorption inlet manifold on the Finnigan 3300
system. A Pye-Unicam 104 gas chromatograph is used to house the glass
capillary column and is interfaced to the thermal desorption inlet manifold
on the 1KB 2091 system. These analytical systems are presented schematically
in Figure A-l.
6.5 Mass Spectrometer/Computer
A Varian MAT CH-7 mass spectrometer capable of a resolution of 2,000
equipped with single ion monitoring capability is used in tandem with the
Varian 1700 gas chromatograph and interfaced to a Varian 620/L computer
(Figure A-l). A Finnigan 3300 mass spectrometer (unit resolution over a
mass range of 1,000 daltons) equipped with selected ion monitoring capability
(nine channels available) is used in tandem with the Finnigan 9500 gas
chroraatograph and interfaced to a PDP-12 computer (Figure A-l). An LKB 2091
mass spectrometer capable of a resolution of 1,000 equipped with selected
ion monitoring capability (sixteen channels available) is used in tandem
with the Pye-Unicam 104 gas chromatograph and interfaced to a PDP-11/04
computer (Figure A-l).
7.0 Reagents and Materials
All reagents used are analytical reagent grade.
8.0 Procedure
8.1 Cleaning of Glassware
All glassware, sampling tubes, cartridge holders, etc. are washed in
Isoclean/water, rinsed with deionized distilled water, acetone and air
dried. Glassware is heated to 450-500°C for 2 hours to insure that all
organic material has been removed prior to its use.
81
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8.2 Collection of Volatile Organics in Ambient Air
Continuous sampling of ambient air is accomplished using a Nutech Model
221-A portable sampler (Nutech Corp., Durham, NC, see Figure A-l, Reference
2). Flow rates between 1-10 1/min are available with this sampling system.
Flow rates are generally maintained at 1 1 using critical orifices and the
total flow is monitored through a calibrated flow meter. The total flow is
also registered by a dry gas meter. This portable sampling unit operates on
a 12 volt storage battery and is capable of continuous operation up to a
period of 24 hours. However, in most cases, at the rates which are employed
in the field, the sampling period is generally 1-3 hours. This portable
sampling unit is generally utilized for obtaining "high volume" samples.
Duplicate cartridges are deployed on each sampling unit utilizing a sampling
head as shown in Figure A-2.
Concomitant with these parameters the temperature is continuously
recorded with a Meterological Research, Inc. Weather Station since the
breakthrough volume is important in order to obtain quantitative data on the
volatile organics.
In addition to the Nutech samplers, personnel samplers (DuPont and MSA)
are also used to acquire "low volumes" of ambient air as well as long-term
integrated samples (12-36 hrs.) Identical Tenax GC sampling cartridges are
employed in this case, and the sampling is conducted in duplicate. The flow
rate is balanced between duplicate cartridges using critical orifices to
maintain a rate of 25-100 ml/min per cartridge.
For large sample volumes, it is important to realize that the total
volume of air may cause the elution of compounds through the sampling tube
if their breakthrough volume is exceeded. The breakthrough volumes of some
of the volatile organics are shown in Table A-2 (A2,A4,A8). These break-
through volumes have been determined by a previously described technique
(A2). The breakthrough volume is defined as that point at which 50% of a
discrete sample introduced into the cartridge is lost. For cases in which
the identity of a volatile organic compound is not known until after GC/MS
analysis, the breakthrough volume may be subsequently determined.
Previous experiments have shown that the organic vapors collected on
Tenax GC sorbent are stable and can be quantitatively recovered from the
82
-------
Figure A-2. Sampling -head for housing cartridge sampling train.
83
-------
Table A-2. TENAX GC BREAKTHROUGH VOLUMES FOR SEVERAL ATMOSPHERIC POLLUTANTS
00
Temperature (°F)
Chemical
Class
Halogenated
hydrocarbon
Compound
methyl chloride
methyl bromide
vinyl chloride
methylene chloride
chloroform
carbon tetrachloride
1 , 2-dichloroethane
1 , 1 , 1-trichloroethane
tetrachloroethylene
trichloroethylene
l-chloro-2-methylpropene
3-chloro-2-methylpropene
1 , 2-dichloropropane
1 , 3-dichloropropane
epichlorohydrin (1-chloro-
2 , 3-epoxypropane )
3-chloro-l-butene
allyl chloride
4-chloro-l-butene
l-chloro-2-butene
chlorobenzene
o-dichlorobenzene
m-dichlorobenzene
b.p.
C°c)
-24
3.5
13
41
61
77
83
75
121
87
68
72
95
121
116
64
45
75
84
132
181
173
50
8
3
2
11
42
34
53
23
361
90
26
29
229
348
200
19
21
47
146
899
1,531
2,393
60
6
2
1.5
9
31
27
41
18
267
67
20
22
162
253
144
15
16
36
106
653
1,153
1,758
70
5
2
1.25
7
24
21
31
15
196
50
16
17
115
184
104
12
12
27
77
473
867
1,291
80
4
1
1.0
5
18
16
23
12
144
38
12
13
81
134
74
9
9
20
56
344
656
948
90
3
1
0.8
4
13
13
18
9
106
28
9
10
58
97
54
7
6
15
40
249
494
697
100
2.5
0.9
0.6
3
10
10
14
7
78
21
7
8
41
70
39
6
5
12
29
181
372
510
(continued)
-------
Table A-2 (cont'd.)
00
Cn
Temperature (°F)
Chemical
Class
Halogenated
hydrocarbons
(cont'd)
Halogenated
Ethers
Nitrosamines
Oxygenated
hydrocarbons
Nitrogenous
Hydrocarbons
Sulfur
Compounds
Compound
benzyl chloride
bromoform
ethylene dibromide
bromobenzene
2-chloroethyl ethyl ether
Bis-(chloromethyl)ether
N-nitrosodimethylamine
N-nitrosodiethylamine
acrolein
glycida Idehyde
propylene oxide
butadiene diepoxide
cyclohexene oxide
styrene oxide
phenol
acetopheonone
p-propiolactone
nitromethane
aniline
diethyl sulfate
ethyl methane sulfate
b.p.
179
149
131
155
108
-
151
177
53
—
34
-
132
194
183
202
57
101
184
208
86
50
2,792
507
348
2,144
468
995
385
2,529
19
364
35
1,426
2,339
5,370
2,071
3,191
721
45
3,864
40
5,093
60
2,061
386
255
1,521
336
674
280
1,836
14
247
24
1,009
1,644
3,926
1,490
2,382
514
34
2,831
29
3,681
70
1,520
294
188
1,079
241
456
204
1,330
10
168
17
714
1,153
2,870
1,072
1,778
366
25
2,075
21
2,564
80
1,125
224
138
764
234
309
163
966
8
114
11
506
811
2,094
769
1,327
261
19
1,520
15
1,914
90
830
171
101
542
124
209
148
700
6
77
8
358
570
1,531
554
991
186
14
1,114
11
1,384
100
612
131
74
384
89
142
107
508
4
52
5
253
400
1,119
398
740
132
11
817
8
998
(continued)
-------
Table A-2 (cont'd.)
00
o\
Temperature (°F)
Chemical
Class
Amines
Ethers
Esters
Ketones
Aldehydes
Alcohols
Compound
dimethylamine
isobutylamine
t-butylamine
di-(n-butyl)amine
pyridine
aniline
diethyl ether
propylene oxide
ethyl acetate
methyl acrylate
methyl methacrylate
acetone
methyl ethyl ketone
methyl vinyl ketone
acetophenone
acetaldehyde
benzaldehyde
methanol
n-propanol
allyl alcohol
b.p.
(°C)
7.4
69
89
159
115
184
34.6
35
77
80
100
56
80-2
81
202
20
179
64.7
97.4
97
50
9
71
6
9,506
378
8,128
29
13
162
164
736
25
82
84
5,346
3
7,586
1
27
32
60
6
47
5
7,096
267
5,559
21
9
108
111
484
17
57
58
3,855
2
5,152
1
20
23
70
4
34
4
4,775
189
3,793
15
7
72
75
318
12
39
40
2,767
2
3,507
0.8
14
16
80
3
23
3
3,105
134
2,588
11
5
48
50
209
8
27
28
2,000
1
2,382
0.6
10
11
90
2
16
2
2,168
95
1,766
8
4
32
34
137
6
19
19
1,439
0.9
1,622
0.4
7
8
100
1
11
1
1,462
67
1,205
5
3
22
23
90
4
13
14
1,037
0.7
1,101
0.3
5
6
(continued)
-------
Table A-2 (cont'd.)
oo
Temperature (°F)
Chemical
Class
Aromatics
Hydrocarbons
Inorganic
gases
Compound
benzene
toluene
ethylbenzene
cumene
n-hexane
n-heptane
1-hexene
1-heptene
2 , 2-dimethylbutane
2 ,4-dimethylpentane
4-methyl-l-pentene
cyclohexane
nitric oxide
nitrogen dioxide
chlorine
sulfur dioxide
water
b.p.
(°c)
80.1
110.6
136.2
152.4
68.7
98.4
63.5
93.6
49.7
80.5
53.8
80.7
-
-
100
50
108
494
1,393
3,076
32
143
28
286
0.5
435
14
49
0
0
0
0.06
0.06
60
77
348
984
2,163
23
104
20
196
0.4
252
10
36
0
0
0
0.05
0.05
70
54
245
693
1,525
17
75
15
135
0.3
146
8
26
0
0
0
0.03
0.04
80
38
173
487
1,067
12
55
11
93
0.2
84
6
19
0
0
0
0.02
0.03
90
27
122
344
750
9
39
8
64
0.2
49
4
14
0
0
0
0.02
0.01
100
19
86
243
527
6
29
6
44
0.1
28
3
10
0
0
0
0.01
0
Breakthrough volume is given in SL/2.2 g Tenax GC used in sampling cartridges.
-------
cartridge samplers up to 4 weeks after sampling when they are tightly closed
in cartridge holders and placed in a second container that can be sealed,
protected from light and stored at 0°C (A1,A2).
8.3 Analysis of Samples
The instrumental conditions for the analysis of volatile organics on
the sorbent Tenax GC sampling cartridge is shown in Table A-3. The thermal
desorption chamber and the six port Valco valve are maintained at 270° and
240°, respectively. The helium purge gas through the desorption chamber is
adjusted to 15-20 ml/min. The nickel capillary trap on the inlet manifold
is cooled with liquid nitrogen. In a typical thermal desorption cycle, a
sampling cartridge is placed in the preheated desorption chamber and the
helium gas is channeled through the cartridge to purge the vapors into the
liquid nitrogen capillary trap [the inert activity of the trap has been
shown in a previous study (A5)]. After the desorption has been completed,
the six-port valve is rotated and the temperature on the capillary loop is
rapidly raised (greater than 100°/min); the carrier gas then introduces the
vapors onto the high resolution GC column. The glass capillary column is
temperature programmed from ambient to 240°C at 4°C/min and held at the
upper limit for a minimum of 10 min. After all the components have eluted,
the column is cooled to ambient temperature and the next sample is processed
(A2).
An example of the analysis of volatile organics in ambient air is shown
in Figure A-3 and the background from a blank cartridge is shown in Figure
A-4. The high resolution glass capillary column was coated with SE-30
stationary phase which is capable of resolving a multitude of compounds to
allow their subsequent identification by MS/COMP techniques; in this case
over 110 compounds were identified in this chromatogram.
8.3.1 Operation of the MS/COMP System (Figure A-5)
8.3.1.1 Varian MAT CH-7
Typically the Varian MAT CH-7 mass spectrometer is first set to operate
in the repetitive scanning mode. In this mode the magnet is automatically
scanned exponentially upward from a preset low mass to a high mass value.
Although the scan range may be varied depending on the particular sample,
typically the range is set from approximately m/z 20 to m/z 350. The scan
88
-------
Table A-3. OPERATING PARAMETERS FOR GLC-MS-COMP SYSTEM
Parameter
Setting
Inlet-manifold
desorption chamber and valve
capillary trap - minimum
maximum
thermal desorption time
He purge flow
GLC
270°C
195°C
240°C
8 min
15 ml/rain
100 m glass SCOT SE-30
carrier (He) flow
MS
Varian MAT CH-7
25-240°C, 4°C/min
**3 ml/min
scan range
scan cycle, automatic-cyclic
filament current
multiplier
ion source vacuum
Finnigan 3300
scan range
scan cycle, automatic-cyclic
filament current
multiplier
ion source vacuum
m/z 20 -> 350
1 sec/decade
300 pA
4.0 _6
^4 x 10 torr
m/z 20 •* 350
2 sec/cycle
0.50 mA
1.7 kV
1 x 10* torr
LKB 2091
mass range
scan cycle, automatic-cyclic
filament current
multiplier
ion source vacuum
m/z 5 -» 492
2.4 sec/cycle (parabolic scan)
4A
300 ,
3 x 10 torr
89
-------
i—j—|
Mass Spectrum No.
Figure A-3. Profile of ambient air pollutants obtained using high resolution gas chromatography/
' mass spectrometry/computer.
-------
V£)
90
80-
TO-
P
1
K
8
90-
40-
20-
44 56 68 80 92
TEMPERATURE CO
104
116
128
192
Figure A-4. Background profile for Tenax GC cartridge blank.
-------
Terminal
GC column
(capillary)
Sample
inlet/
manifold
Separator
10
Random
access
disk
9-track
magnetic
tape
Electrostatic
printer/plotter
Figure A-5. Schematic diagram of GC-MS computer system.
-------
is completed in approximately 1.8 seconds. At this time the instrument
automatically resets itself to the low mass position in preparation for the
next scan, and the information is accumulated by an on-line 620/L computer
and written onto magnetic tapes. The reset period requires approximately
2.0 seconds. Thus, a continuous scan cycle of 3.8 seconds/scan is maintained
and repetitively executed throughout the chromatographic run. The result is
the accumulation of a continuous series of mass spectra throughout the
chromatographic run in sequential fashion.
Prior to analysis of unknown samples, the system is calibrated by
introducing a standard substance, perfluorokerosene, into the instrument and
determining the time of appearance of the known standard peaks in relation
to the scanning magnetic field. The calibration curve thus generated is
stored in the 620/L computer memory. This calibration serves only to cali-
brate the ions over the scanning range.
While the magnet is continuously scanning, the sample is injected and
automatic data acquisition is initiated. As each spectrum is acquired by
the computer, each peak which exceeds a preset threshold is recognized and
reduced to centroid time and peak intensity. This information is stored in
the computer core while the scan is in progress. In addition, approximately
30 total ion current values and an equal number of Hall probe signals are
stored in the core of the computer as they are acquired. During the two-
second period between scans this spectral information, along with the spectrum
number, is written sequentially on magnetic tape, and the computer is reset
for the acquisition of the next spectrum.
This procedure continues until the entire GC run is completed. By this
time there are from 800-1400 spectra on the magnetic tape which are then
subsequently processed. Depending on the information required, they may
then either be processed immediately or additional samples may be run,
stored on magnetic tape and the results examined at a later time.
The mass spectral data are processed in the following manner. First,
the original spectra are scanned and the total ion current (TIC) information
is extracted. Then the TIC intensities are plotted against the spectrum
number on the plotter. The information will generally indicate whether the
run is suitable for further processing, since it provides some idea of the
93
-------
number of unknowns in the sample and the resolution obtained using the
particular GC column conditions.
The next stage of the processing involves the conversion of the spectral
peak times to peak masses which is done directly via the dual disk system.
The mass conversion is accomplished by use of the calibration table obtained
previously using perfluorokerosene. Normally one set of the calibration
data is sufficient for an entire day's data processing since the characteris-
tics of the Hall probe are such that the variation in calibration is less
than 0.2 daltons/day. A typical time required for this conversion process
for 1,000 spectra is approximately 30 rain.
After the spectra are obtained in mass-converted form, processing
proceeds either manually or by computer. In the manual mode, the full
spectrum of scans for the GC run is recorded on the States 31 plotter. The
TIC information available at this time is most useful for deciding which
spectra are to be analyzed. At the beginning of the runs where peaks are
very sharp nearly every spectrum must be inspected individually to determine
the identity of the component. Later in the chromatographic run when the
peaks are broader only selected scans need to be analyzed.
8.3.1.2 Finnigan 3300
The Finnigan 3300 mass spectrometer is set to operate in the repetitive
scanning mode, scanning the quadrupole from low mass to high mass. A typical
range is from m/z 20 to m/z 350. The scan requires approximately one second;
the instrument then rests at the high end of the mass scale and then
automatically re-initiates a scan cycle at the low mass. During the rest/re-
set period, data from the previous scan are accumulated by an on-line PDP-12
computer and written on disk. The scan cycle of 2.0 seconds is repetitively
executed throughout the chromatographic run, resulting in the accumulation
of a continuous series of mass spectra.
The quadrupole mass spectrometer acquires data as masses and intensities.
A calibration table is obtained by use of perfluorotributylamine (FC-43) to
correlate voltages and masses. Normally one set of calibration data will
suffice for a day of data acquisition. The data can then be presented as
TIC information (obtained by summing ion intensities over the entire spectrum)
with chromatographic peak intensities plotted against time. This information
94
-------
is most useful to decide which spectra should be output and analyzed. At
the beginning of the analysis where chromatographic peaks are very sharp
nearly every spectrum must be plotted and inspected individually to identify
every component. At longer elution times the peaks broaden and only selected
scans must be analyzed.
8.3.1.3 1KB 2091
The 1KB 2091 mass spectrometer is operated under computer control. The
start time and scan interval time are selected by the operator. Typically
the magnet is scanned from m/z_ 5 to m/z 490 at a scan speed of 2 sec full
scan. The scan cycle time is typically 2.4 sees. Mass vs. intensity data
is acquired by the computer and is stored on a disk. The mass values are
determined by the 1KB mass marker and modified as necessary by a computer
program which corrects for deviations due to scan speed. The mass calibration
tables are stable for over a six month period of time. The data can then be
plotted as total ion current (over an operator determined mass range-usually
m/z 33 •* m/z 492) versus spectrum number.
This information is useful in deciding which spectra should be plotted
and interpreted. Normally all spectra are plotted. Data are stored archi-
vally on 9-track IBM-compatible magnetic tapes.
8.3.2 Spectral Interpretation
Identification of resolved components from data produced by all the
mass spectrometer systems is achieved by comparing the fragmentation patterns
of the unknown mass spectra to an eight major peak index of mass spectra
(A9). Individual difficult unknowns are searched by the use of the Cornell
University STIRS and PBM systems. Unknowns are also submitted to MSSS
system for identification. When feasible, the identification of unknowns is
confirmed by comparing the fragmentation pattern and elution temperatures
for two different chromatographic columns (SE-30 and Carbowax SCOT capillaries)
for the unknown and authentic compounds. ' The relationship between the
boiling point of the identified compound and the elution temperature on a
non-polar column (the order of elution of constituents is predictable in
homologous series since the SE-30 SCOT capillary separates primarily on the
basis of boiling point) is carefully considered in making structure assign-
ments .
95
-------
Mass spectral search programs are operational at the Triangle Universi-
ties Computation Center (TUCC). RTI maintains twice daily service to TUCC,
which is a one-quarter mile distance from the RTI campus. Additional infor-
mation about each magnetic tape containing mass spectra is entered directly
into the TUCC job stream using a remote job entry processing. This is
normally done at TUCC using one of the five terminals located within the
Analytical Sciences Laboratory. The control information contains selected
spectrum numbers with instructions to process entire GC runs. The computer
program systems simultaneously compare either the entire library of 25,000
compounds or some subset of this library. The complete reports showing the
best fits for each of the unknowns are produced at TUCC and printed out at
the high speed terminals located on the RTI campus or TUCC.
8.3.3 Quantitative Analysis
In many cases the estimation of the level of pollutants by capillary
gas chromatography in combination with mass spectrometry is not feasible
utilizing only the total ion current monitor (See Figure A-3, for example).
Since baseline resolution between peaks is not always achieved, we employ
the techniques which have previously been developed under contract whereby
full spectra are obtained during the chromatographic separation step and the
selected ions are presented as mass fragmentograms using computer software
programs which allow the possibility of deconvoluting constituents which
were not resolved in the total ion current chromatogram (A6). Examples are
depicted in Figures A-6 and A-7 which represent an ambient air sample with a
TIC profile as in Figure A-3.
In our GC/MS/COMP systems we request from the dedicated computer mass
fragmentograms for any combination of ions when full mass spectra have been
obtained during chromatography. Thus selectivity is obtained by choosing
characteristic ions for a particular organic substance. The ion intensity
is plotted and the ion intensity vs. time is subsequently used for quantifi-
cation. Also quantification with external standards is easily achieved
using the intensity of the total ion current monitor or a characteristic ion
in the mass spectrum of the external standard. Thus, we use mass fragmento-
graphy for the quantification of organics in ambient air when the total ion
96
-------
8
VD
186
o
o
o
o
MASS SPECTRUM NO.
Figure A-6.
Mass fragmentogram of Ions characteristic of internal standards
(perfluorobenzene, m/£ 186 and perfluorotoluene, in/z^ 236 with
a fragment at mjz_ 186) used in ambient air samples.
-------
1M
MASS SPECTRUM NO.
Figure A-7. Mass fragmentograms of characteristic ions representing chlorotoluene (m/£ 91,126,128), dichloro-
toluene (m/£ 125,127,160), chlorobenzaldehyde (m/£ 139,140) and standards perfluorobenzene
(W?_ 186) and perfluorotoluene (ra/z 236).
-------
current monitor is inadequate because of the lack of complete resolution
between components in the mixture.
As described previously, the quantitation of constituents in ambient
air samples is accomplished either by utilizing the total ion current monitor
or, where necessary, mass fragmentograms. In order to eliminate the need to
obtain complete calibration curves for each compound for which quantitative
information is desired, we use the method of relative molar response (RMR)
factors (A10). Successful use of this method requires information on the
exact amount of standard added and the relationship of RMR (unknown) to the
RMR (standards). The method of calculations is as follows:
A , /Moles
(1) RMR. - unk
'unknown/standard A ,/Moles ,
A = system response, height or area determined by integration
or triangulation.
The value of RMR is determined from at least three independent
analyses.
A ./g ./GMW .
(2) RMR unk/sunk unk
unk/std A . ./g ,,/GMW . ,
1 std'6std std
A = system response, as above
g = number of grams present
GMW = gram molecular weight
A . «GMW . -g . ,
_ unk unk °std
8unk ~ A ,,'GMW , ,-RMR , . .,
std std unk/std
The standard can be added as an internal standard during sampling.
Since, however, the volume of air taken to produce a given sample is accu-
rately known, it is also possible and more practical to use an external
standard where the standard is introduced into the cartridge prior to its
analysis. Two standards, hexafluorobenzene and octafluorotoluene, are used
99
-------
for the purpose of calculating RMRs. From previous research it has been
determined that the retention times for these two compounds are such that
they elute from the glass capillary column (SE-30) at a temperature and
retention time which does not interfere with the analysis of unknown com-
pounds in ambient air samples.
Since the volume of air taken to produce a given sample is accurately
known and an external standard is added to the sample, then the weight per
cartridge and hence the concentration of the unknown can be determined. The
approach for quantitating ambient air pollutants requires that the RMR be
determined for each constituent of interest (before or after sample analy-
sis) . In this manner it is possible to obtain qualitative and quantitative
information on the same sample with a minimum of effort.
9.0 References
Al. Pellizzari, E. D., Development of Method for Carcinogenic Vapor
Analysis in Ambient Atmospheres. Publication No. EPA-650/2-74-121,
Contract No. 68-02-1228, 148 pp., July, 1974.
A2. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA-600/2-75-
075, Contract No. 68-02-1228, 187 pp., November, 1975.
A3. Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki, Environ.
Sci. Technol., 9, 552 (1975).
A4. Pellizzari, E. D., B. H. Carpenter, J. E. Bunch and E. Sawicki, Environ.
Sci. Technol., 9, 556 (1975).
A5. Pellizzari, E. D., The Measurement of Carcinogenic Vapors in Ambient
Atmospheres. Publication No. EPA-600-7-77-055, Contract No. 68-02-
1228, 288 p., June, 1977.
A6. Pellizzari, E. D., J. E. Bunch, R. E. Berkley and J. McRae, Anal.
Chem., 48, 803 (1976).
A7. Pellizzari, E. D., "Analysis of Organic Air Pollutants by Gas Chroma-
tography and Mass Spectrometry", Publication No. EPA-600/2-77-100, 114
pp., June 1977-
A8. Pellizzari, E. D., J. E. Bunch, R. E. Berkley and J. McRae, Anal.
Lett., 9, 45 (1976).
100
-------
A9. "Eight Peak Index of Mass Spectra", Vol. I, (Tables 1 and 2) and II
(Table 3), Mass Spectrometry Data Centre, AWRE, Aldermaston, Reading,
ARF74PR, OT, 1970.
A10. Pellizzari, E. D. , "Measurement of Carcinogenic Vapors in Ambient
Atmospheres", Publication No. EPA-600/7-77-055, 302 pp., June, 1977.
Written analytical protocol prepared 1/24/77, revised 4/79.
101
-------
APPENDIX B
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE
HALOGENATED ORGANICS IN WATER
102
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE
HALOGENATED ORGANICS IN WATER
1.0 Principle of the Method
Volatile compounds are recovered from an aqueous sample by warming the
sample and purging an inert gas through the warm sample. The vapors are
trapped on a Tenax cartridge and are then analyzed by thermal desorption
interfaced to a GC/MS.
2.0 Range and Sensitivity
For a typical organic compound approximately 30 ng are required for
mass spectral identification using high resolution glass capillary GC/MS
analysis. A 100 ml water aliquot is used, so the limit is ^0.3 Mg/1- The
4
dynamic range for a purged sample is ^10 , however, smaller samples may be
purged and the range increased commensurately.
3.0 Interferences
Two possible types of interference must be considered: (1) material
present in the sample which physically prevents the effective purge of the
sample, and (2) a material which interferes with the analysis of the purged
sample. In the former case, several techniques have been developed to
handle such problems (e.g., foaming) by diluting and stirring the sample.
The second case is minimized by the use of GC/MS for the analysis since
unique combinations of m/z^ and retention time can be selected for most
compounds. This permits the analysis of compounds even though chromato-
graphic resolution is not obtained.
4.0 Precision and Accuracy
14
The purge and trap technique was validated using both C-labeled model
compounds and "cold" model compounds. Results of these recovery studies are
presented in Tables B-l and B-2.
Based on these data, expected recoveries of purgeable halogenated
organics from water are about 80% or better. Within the precision require-
ments of this study, these recovery values indicate that the method is
essentially quantitative.
103
-------
Table B-l. RECOVERY OF C-LABELED MODEL COMPOUNDS FROM WATER*
Compound
Chloroform Carbon Tetrachloride Chlorobenzene Bromobenzene
1
2
3
4
5
6
7
8
Mean
+S.D.
RSD (%)
85.0
67.6
91.7
85.9
91.0
47.9
92.8
88.1
81.3
15.7
19.3
96.2
83.6
98.6
70.9
65.2
70.9
67.4
-
79.0
14.0
17.7
84.2
90.3
89.0
92.5
90.4
87.1
94.0
90.3
89.7
3.1
3.4
71.9
88.7
93.7
69.1
87.8
82.4
87.2
90.3
83.9
8.9
10.6
«a
100 ml water spiked with 70,000-90,000 dpm of the model compound
SD = standard deviation
RSD = (SD/Mean) x 100
104
-------
Table B-2. RECOVERY OF HALOGENATED HYDROCARBONS FROM DISTILLED WATER
SPIKED FROM GAS MIXING BULB
Compound
methylene chloride
chloroform
bromodichloromethane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
mean
Amount
spiked (yg)
1.33
1.48
1.98
1.70
1.11
1.35
% Recovery
12 34 Average + Std. Dev.
a a a a
88.5 73.7 47.9 55.0 66.3+18.4
98.1 121 84.4 85.8 97.3 + 16.9
71.4 65.9 64.2 101 75.6+17.2
81.7 81.7 79.7 89.7 83.2+4.4
82.3 110 68.5 102 90.7+18.8
79.2 + 23.9
Not quantifiable because of background interferences.
-------
5.0 Apparatus
5.1 Purge Apparatus
The apparatus required is shown in Figure B-l.
5.2 Sampling Cartridges
The sampling tubes are prepared by packing a 10 cm long x 1.5 cm i.d.
glass tube containing 6.0 cm of 35/60 mesh Tenax GC (VL.6 g) using glass
wool in the ends to provide support (B1-B5). Tenax GC is extracted in a
Soxhlet extractor for a minimum of 24 hours each with methanol and then with
pentane prior to preparation of cartridge samplers (B5). After purification,
the Tenax GC sorbent is air dried, vacuum dried and then was meshed to
provide a 35/60 particle size range. Cartridge samplers were then prepared
and conditioned at 270°C with helium flow at 30 ml/min for 20 minutes. The
conditioned cartridges were transferred to Kimax (2.5 cm x 150 cm) culture
tubes containing calcium sulfate desiccant which are immediately sealed
using Teflon-lined caps to prevent contamination.
5.3 GC/MS/COMP
The volatile halogenated hydrocarbons purged from water are analyzed on
either an LKB 2091 GC/MS with an LKB 2031 data system or a Varian MAT CH-7
GC/MS with a Varian 620/i data system. The sample, concentrated on a Tenax
GC cartridge, is thermally desorbed using an inlet manifold system (B1-B3,
B5). The operating conditions for the thermal desorption unit and the
analysis of Tenax GC cartridges are given in Table B-3.
6.0 Procedure
6.1 Collection of Samples
Samples (120 ml) are obtained in triplicate from residential homeowners
or other appropriate sources. The first sample is collected immediately and
is analyzed for purgeable components. The second and third samples are
taken after two minutes of continuous, moderate flow. One of these is
analyzed for extractables and the other serves as a backup sample for either
purgeables or extractables. Larger samples are optionally obtained from
water and wastewater treatment plants.
106
-------
THERMOMETER
ERLENMEYER FLASK
125 ml
FRITTED DISK-
TENAX CAnrntOGE
TEFLON ADAPTER
GLASS WOOL PLUG
. — HELIUM PUOGE
7mm 0.0., I mm 1.0
—~IO mm O.D.
Figure B-l. Purge apparatus for water samples.
107
-------
Table B-3. INSTRUMENTAL OPERATING CONDITIONS
LKB 2091
Varian MAT CH-7
o
00
Desorptlon chamber temperature
Desorptlon chamber He flow
Desorption time
Capillary Trap Temperature during desorption
Temperature of capillary trap during injection
onto column
Time of He flow through capillary trap
He flow through column [sweep time]
Carrier flow
Capillary column
Column temperature
Scan range
Scan rate
Scan cycle time
Scan mode
Trap current
Filament current
Accelerating volatage
270
20 ml/min
5.0 min
-196°C
-196°C to 250°C
265
10 ml/min
8.0 min
-196°C
then held at 190°C
12 3/4 min 12 3/4 min
9. 5 min 5 min
2.0 ml/min 2.5 ml/min
100 m SE-30 SCOT
30°C for 2 min,
then 4°/min to 240°
5-490 dalton
1 sec full scale
2.4 sec
parabolic
4A
50 yA
3.5 kV
20 -> 240° at 4°/min
20 -+ 500 dalton
1 sec/decade
4.5 sec
exponential
200 yA
2 kV
-------
6.2 Purge of Volatile Organics
The water sample is cooled to ^4°C and a 100 ml aliquot transferred to
the purge apparatus. The apparatus is assembled as depicted in Figure B-l
including the Tenax GC cartridge (1.5 cm diameter x 6.0 cm length). A
carbon cartridge, 1.5 cm diameter x 4.0 cm length is connected to the ef-
fluent end of the Tenax cartridge to prevent contamination of the cartridge
by laboratory vapors. The flask is wrapped with heating tape and the sample
heated to 90°C. The sample is purged at 25 ml helium/min and 90°C for 30
minutes. The loaded cartridge is removed and stored in a culture tube con-
taining 1-2 g of CaSO, desiccant for at least two hr. The desiccant is
removed from the culture tube and the dry, loaded cartridge stored at -20°C.
6.3 Analysis of Sample Purged on Cartridge
The instrumental conditions for the analysis of halogenated hydrocarbons
on the sorbent Tenax GC sampling cartridge is shown in Table B-3. The ther-
mal desorption chamber and six-port valve are maintained at 270° and 200°C,
respectively. The helium purge gas through the desorption chamber is adjusted
to 15-20 ml/min. The nickel capillary trap at the inlet manifold is cooled
with liquid nitrogen. In a typical thermal desorption cycle a sampling
cartridge is placed in the preheated desorption chamber and helium gas is
channeled through the cartridge to purge the vapors into the liquid nitrogen
cooled nickel capillary trap. After desorption the six-port valve is rotated
and the temperature on the capillary loop is rapidly raised; the carrier gas
then introduces the vapors onto the high resolution GLC column. The glass
capillary column is temperature programmed from 20° to 240°C at 4°/min and
held at the upper limit for a minimum of 10 min. After all of the components
have eluted from the capillary column the analytical column is then cooled
to ambient temperature and the next sample is processed.
6.4 Quantitation
All data are acquired in the full scan mode. Quantitation of the
halogenated compounds of interest is accomplished by utilizing selected ion
plots, SIPs, which are plots of the intensity of specific ions (obtained
from full scan data) vs. time. Using SIPs of ions characteristic of a given
109
-------
compound in conjunction with retention times permits quantitation of com-
ponents of overlapping peaks. Two external standards, perfluorobenzene and
perfluorotoluene, were added to each Tenax GC cartridge in known quantities
just prior to analysis. In order to eliminate the need to construct com-
plete calibration curves for each compound quantitated, the method of rela-
tive molar response (RMR) is used. In this method the relationship of the
RMR as the unknown to the RMR of the standard is determined as follows:
A ./Moles ,
_ unk7 unk
unknown/standard ~ Astd/Molesstd
A . /g , /GMW ,
unk7 5unk7 unk
where A = peak response of a selected ion,
g = number of grams present, and
GMW = gram molecular weight
Thus, in the sample analyzed:
"unknown ~ (Agtd) (GMWgtd
The value of an RMR is determined from at least three independent analyses of
standards of accurately known concentration prepared using a gas permeation
system (B5). The precision of this method has been determined to be generally
±10 percent when replicate sampling cartridges are examined.
7 .0 Quality Assurance Program
In addition to the validation procedures described above, an ongoing
quality assurance program is required to assure the data quality. Quality
control procedures determine artifacts, losses, etc. through a system of
blanks and controls. Quality assurance procedures monitor the execution of
the procedure and check data interpretation and calculations .
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks
110
-------
consist of 100 ml of prepurged, distilled water in the same type of sampling
container as is used in the field. Controls consist of 100 ml of prepurged,
distilled water spiked at 100-500 ng with the compounds listed in Table B-2.
These blanks and controls are carried to the field and receive the same
handling as the field samples. Workup and analysis of field blanks and con-
trols is interspersed with the field samples on a regular basis. This
method allows assessment of sample storage stability.
7.1.2 Procedural Blanks and Controls
7.1.2.1 Tenax GC
The purity of each batch of Tenax GC cartridges is checked by thermal
desorption/GC/FID. An acceptable background is a judgement based upon the
backgrounds obtained previously which exhibited no GC/MS artifacts.
7.1.2.2 Purge Blanks
With each set of purge samples, a procedural blank is run. This
consists of 100 ml of prepurged, distilled water which is purged under the
same conditions as the samples. These blanks are designed to detect
artifacts from dirty glassware, laboratory atmosphere intrusion and storage.
7.1.2.3 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol, 2,6-
dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor the
capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks, and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a Tenax GC cartridge directly loaded with stan-
dards is analyzed each day to serve as a procedural control and also to
update the RMR value. Thus, in a typical Working day, 4 field samples, 1
blank or control, and 1 RMR standard are run.
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may affect the
111
-------
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in receipt, operations performed, and transmittal
of the sample. This record is important for tracing a contaminant, bad
standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, location, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent infor-
mation. Where appropriate, a map is made to precisely identify the location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
protocol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data has been
produced.
8.0 References
El. Pellizzari, E. D., Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres, Publication No. EPA-650/2-74-121, Contract No.
68-02-1228, 148 pp., July, 1974.
112
-------
B2. Pellizzari, E. D., Development of Analytical Technqiues for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA-600/2-76-
076, Contract No. 68-02-1228, 185 pp., November, 1975.
B3. Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki, Environ.
Sci. Tech., 9, 552 (1975).
B4. Pellizzari, E. D., B. H. Carpenter, J. E. Bunch and E. Sawicki, Environ.
Sci. Tech., 9, 556 (1975).
B5. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA 600/7-77-
055, 288 pp., June, 1977.
Revised April, 1979
113
-------
APPENDIX C
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE HALOGENATED
HYDROCARBONS IN BLOOD
114
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE HALOGENATED
HYDROCARBONS IN BLOOD
1.0 Principle of the Method
Volatile compounds are recovered from a blood sample by warming the
sample and purging an inert gas over the warm sample. The vapors are then
trapped on a Tenax cartridge which is analyzed by thermal desorption inter-
faced to GC/MS.
2.0 Range and Sensitivity
For a typical organic compound approximately 30 ng are required for
a mass spectral identification using high resolution glass capillary
GC/MS analysis. Based on a 10 ml blood sample, the limit of detection is
4
about 3 ng/ral or 3 ppb. The dynamic range for a purged sample is ^10 ,
however, smaller samples may be purged and the range increased
commensurately.
3.0 Interferences
Two possible types of interference must be considered: (1) material
present in the sample which physically prevents the effective purge of the
sample, and (2) 'a material which interferes with the analysis of the purged
sample. In the former case, several techniques have been developed to
handle such problems (e.g.-, foaming) by diluting and stirring the sample.
The second case is minimized by the use of GC/MS for the analysis since
unique combinations of m/z and retention time can be selected for most
compounds. This permits the analysis of compounds even though
chromatographic resolution is not obtained.
4.0 Precision and Accuracy
14
The purge and trap technique was validated using both C-labeled model
compounds and "cold" model compounds. Results of these recovery studies are
presented in Tables C-l and C-2. Based on these data, expected recoveries
of purgeable halogenated organics from blood are about 80% or better.
Within the precision requirements of this study, these recovery values
indicate that the method is essentially quantitative.
115
-------
Table C-l. PERCENT RECOVERY OF 14C-LABELED COMPOUND FROM BLOOD3
Mass dpm
Compound Loading (yg) Loaded
chloroform 1.46 91755.1
carbon tetrachloride 0.785 77902.3
chlorobenzene 1.12 89538.1
bromobenzene 1.21 84493.9
% Recovery
94.2
93.1
46. 5b
92.6
92.2
83.5
43. 5b
88.0
92.2
77.1
74.4
90.3
Average
% Recovery
93.7
89.4
90.1
80.6
Headspace purge of whole blood (25 ml) diluted 1:1 with distilled
water and purged in 100 ml 3-neck purge flasks at 50°C for 90 min
with helium at 25 ml/min.
Leaking desorption chamber cap; not included in average.
116
-------
Table C-2. RECOVERY OF HALOGENATED HYDROCARBONS FROM HUMAN BLOOD
SPIKED FROM GAS MIXING BULB
Compound
methylene chloride
chloroform
bromodichlorome thane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
mean
Amount
spiked (ug)
7.96
8.16
10.4
8.52
5.53
6.44
1
105
84.6
105
108
108
85.1
%
2
116
145
159
121
120
101
Recovery
3
a
139
94.9
82.8
85.6
74.6
4
a
a
94.9
97.5
78.6
84.5
Average + Std. Dev.
Ill + 7.8
123 + 33.1
113 ± 30.7
99.8 + 17.9
98.1 + 19.3
86.3 + 10.9
104.3 ± 22.7
Not quantified because of background interferences.
-------
5.0 Apparatus
5.1 Sampling Apparatus
Vacutainers "suitable for GC", Venoject L 428 (Kimble, or other high
purity vacutainers; ice bath; disposable Pasteur pipettes and bulbs, and
cleaned and oven-treated shell vials with Teflon-lined screw caps.
5.2 Purge Apparatus
The apparatus required is shown in Figure C-l.
5.3 Tenax Cartridges
The sampling tubes are prepared by packing a 10 cm long x 1.5 cm i.d.
glass tube containing 6.0 cm of 35/60 mesh Tenax GC (VL.6 g), using glass
wool in the ends to provide support (C1-C5). Tenax GC is extracted in a
Soxhlet extractor for a minimum of 24 hours each with methanol and then with
pentane prior to preparation of cartridge samplers (Cl). After purifica-
tion, the Tenax GC sorbent is air-dried, vacuum dried and then meshed to
provide a 35/60 particle size range. Cartridge samplers are then prepared
and conditioned at 270°C with helium flow at 30 ml/min for 20 minutes. The
conditioned cartridges are transferred to Kimax (2.5 cm x 150 cm) culture
tubes which are immediately sealed using Teflon-lined caps to prevent contami-
nation.
5.4 GC/MS/COMP
The volatile halogenated hydrocarbons purged from water are analyzed on
either an LKB 2091 GC/MS with an LKB 2031 data system or a Varian MAT CH-7
GC/MS with a Varian 620/i data system. The sample, concentrated on a Tenax
GC cartridge is thermally desorbed using an inlet manifold system (C1-C3,
C5). The operating conditions for the thermal desorption unit and the
analysis of Tenax GC cartridges are given in Table C-3.
6.0 Procedure
6.1 Collection of Samples
Blood samples are collected in replicate 10 ml vacutainer tubes contain-
ing an anticoagulant. Using a qualified phlebotomist, the samples are
collected by brachial venipuncture. Glass syringes represent the optimal
collection device, since no polymeric material which may contaminate the
sample comes in contact with the blood. However, sterilization of large
118
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TENAX CARTRIDGE
THERMOMETER
-20to«SO°C
THERMOMETER ADAPTER
with 0—ring
f 10/18
HELIUM
PURGE
HELIUM INLET
TUBE
I LIQUID LEVEL
« 100 ml ROUND BOTTOM FLASK
MAGNETIC STIRRING BAR
Figure C-l. Headspace purge apparatus for blood, urine, and tissue
samples.
119
-------
Table C-3. INSTRUMENTAL OPERATING CONDITIONS
LKB 2091
Varlan MAT CH-7
Desorption chamber temperature
Desorptlon chamber He flow
Deso rp t ion t ime
Capillary Trap Temperature during desorption
Temperature of capillary trap during injection
onto column
Time of He flow through capillary trap
He flow through column [sweep time]
Carrier flow
Capillary column
Column temperature
Scan range
Scan rate
Scan cycle time
Scan mode
Trap current
Filament current
Accelerating volatage
270
20 ml/min
5.0 min
-196°C
-196°C to 250°C
265
10 ml/min
8.0 min
-196°C
then held at 190°C
12 3/4 min 12 3/4 min
9.5 min 5 min
2.0 ml/min 2.5 ml/min
100 m SE-30 SCOT
30°C for 2 min,
then 4°/min to 240C
5-490 dalton
1 sec full scale
2.4 sec
parabolic
4A
50 yA
3.5 kV
20 + 240° at 4°/min
20 -> 500 dalton
1 sec/decade
4.5 sec
exponential
200 yA
2 kV
-------
numbers of glass syringes in the field is not practical, so vacutainers will
be used.
Possible contamination by permeation through the rubber septum caps of
the vacutainers is a cause for concern. Teflon-lined vacutainers are not
available but manufacturers recommended special vacutainers "suitable for
GC" (Venoject L 428, Kimble). Validation experiments for benzene under a
separate contract (EPA No. 68-01-3849, Task I) found the background of these
tubes to be acceptable.
Following collection, shipping and storage procedures must assure that
the purgeable halogenated organics remain intact in the blood sample.
Leakage of the vacutainer caps has been observed and permeation through the
cap material is suspected. Accordingly, these containers are not suitable
for storage. In the procedure used for this project, the blood sample is
chilled and transferred to a clean shell vial with a Teflon-lined screw cap.
The cap is then taped shut. This procedure has been validated and field
tested on EPA Contract No. 68-01-3849, Task I.
6.2 Purge of Volatile Organics
An aliquot of whole blood, chilled to 4°C is measured into the purge
flask in Figure C-l. Prepurged, distilled water is added to the sample to
dilute it to about 50 ml; and a stirring bar added. The apparatus is
assembled, stirring started, the temperature raised to 50°C, and the helium
flow started at 25 ml/min. After 90 min, the purge is terminated, the
apparatus disassembled, and the Tenax cartridge stored in a Kimax culture
tube with calcium sulfate desiccant in a freezer until it is ready for
analysis.
6.3 Analysis of Sample Purged on Cartridge
The instrumental conditions for the analysis of halogenated hydrocarbons
of the sorbent Tenax GC sampling cartridge is shown in Table C-3. The
thermal desorption chamber and six-port valve are maintained at 270° and
200°C, respectively. The helium purge gas through the desorption chamber is
adjusted to 15-20 ml/min. The nickel capillary trap at the inlet manifold
is cooled with liquid nitrogen. In a typical thermal desorption cycle a
sampling cartridge is placed in the preheated desorption chamber and helium
121
-------
gas is channeled through the cartridge to purge the vapors into the liquid
nitrogen cooled nickel capillary trap. After desorption the six-port valve
is rotated and the temperature on the capillary loop is rapidly raised; the
carrier gas then introduces the vapors onto the high resolution GLC column.
The glass capillary column is temperature programmed from 20° to 240°C at
4°/min and held at the upper limit for a minimum of 10 min. After all of
the components have eluted from the capillary column the analytical column
is then cooled to ambient temperature and the next sample is processed (C2).
6.4 Quantitation
All data are acquired in the full scan mode. Quantitation of the
halogenated compounds of interest is accomplished by utilizing selected ion
plots, SIPs, which are plots of the intensity of specific ions (obtained
from full scan data) vs. time. Using SIPs of ions characteristic of a given
compound in conjunction with retention times permits quantitation of compon-
ents of overlapping peaks. Two external standards, perfluorobenzene and
perfluorotoluene, were added to each Tenax GC cartridge in known quantities
just prior to analysis. In order to eliminate the need to construct complete
calibration curves for each compound quantitated, the method of relative
molar response (RMR) is used. In this method the relationship of the RMR of
the unknown to the RMR of the standard is determined as follows:
WMolesunk,
^unknown = Astd/Molesstd
RMR , , _ , =
unk/std
where A = peak response of a selected ion,
g = number of grams present, and
GMW = gram molecular weight.
Thus, in the sample being analyzed:
(A . )(GMW ,)(g ,)
unk unk'V5std
cr ~
^unknown
122
-------
The value of an RMR is determined from at least three independent analyses
of standards of accurately known concentration prepared using a gas
permeation system (C5). The precision of this method has been determined to
be generally ±10 percent when replicate sampling cartridges are examined.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an ongoing
quality assurance program is required to assure the data quality. Quality
control procedures determine artifacts, losses, etc. through a system of
blanks and controls. Quality assurance procedures monitor the execution of
the procedure and check data interpretation and calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks
consist of 10 ml of locally obtained blood plasma in the same type of
sampling container as is used in the field. Controls consist of 10 ml of
locally obtained blood plasma spiked with 10 ng each of the compounds listed
in Table C-2. These blanks and controls are carried to the field and receive
the same handling as the field samples. Workup and analysis of field
blanks and controls is interspersed with the field samples on a regular
basis. This method allows assessment of sample storage stability.
7.1.2 Procedure of Blanks and Controls
7.1.2.1 Tenax GC
The purity of each batch of Tenax GC cartridges is checked by thermal
desorption/GC/FID. An "acceptable background" is a judgement based upon the
backgrounds obtained previously which exhibited no GC/MS artifacts.
7.1.2.2 Purge Blanks
With each set of purge samples, a procedural blank is run. This
consists of 50 ml of prepurged distilled water which is purged under the
same conditions as the samples. These blanks are designed to detect artifacts
from dirty glassware, laboratory atmosphere intrusion and storage.
7.1.2.3 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
123
-------
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a Tenax GC cartridge directly loaded with standards
is analyzed each day to serve as a procedural control and also to update the
RMR value. Thus, in a typical working day, 4 field, samples, 1 blank or
control, and 1 RMR standard are run.
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a Sampling Protocol Sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, location, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
124
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7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
protocol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data have been
produced.
8.0 References
Cl. Pellizzari, E. D. , Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres. Publication No. EPA-650/2-74-121, Contract No.
68-02-1228, 148 pp., July, 1974.
C2. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA-600/2-76-076,
Contract No. 68-02-1228, 185 pp., November, 1975.
C3. Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki, Environ.
Sci. Tech., 9, 552 (1975).
C4. Pellizzari, E. D., B. H. Carpenter, J. E. Bunch and E. Sawicki, Environ.
Sci. Tech., 9. 556 (1975).
C5. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors, Publication No. EPA-600/7-77-
055, 288 pp., June 1977.
Revised April, 1979.
125
-------
APPENDIX D
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE
HALOGENATED ORGANICS IN URINE
126
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE
HALOGENATED ORGANICS IN URINE
1.0 Principle of the Method
Volatile compounds are recovered from a urine sample by warming the
sample and purging an inert gas over the warm sample. The vapors are then
trapped on a Tenax cartridge and subsequently can be analyzed by thermal
desorption interfaced to GC/MS.
2.0 Range and Sensitivity
For a typical organic compound approximately 30 ng are required for
mass spectral identification using high resolution glass capillary GC/MS
analysis. Based on a 25 ml urine sample, a limit of detection of about 1.2
(Jg/1 (1.2 ppb) may be expected. The dynamic range for a purged sample is
^•10 , however, smaller samples may be purged and the range increased commen-
surately.
3.0 Interferences
Two possible types of interference must be considered: (1) material
present in the sample which physically prevents the effective purge of the
sample, and (2) a material which interferes with the analysis of the purged
sample. In the former case, several techniques have been developed to
handle such problems (e.g., foaming) by diluting and stirring the sample.
The second case is minimized by the use of GC/MS for the analysis since
unique combinations of m/z and retention time can be selected for most
compounds. This permits the analysis of compounds even though chromato-
graphic resolution is not obtained.
4.0 Precision and Accuracy
14
The purge and trap technique was validated using both C-labeled model
compounds and "cold" model compounds. Results of these recovery studies are
presented in Tables D-l and D-2.
Based on these data, expected recoveries of purgeable halogenated
organics from urine are about 80% or better. Within the precision require-
ments of this study, these recovery values indicate that the method is
essentially quantitative.
127
-------
Table D-l. PERCENT RECOVERY OF 14C-LABELED COMPOUNDS FROM URINE
Mass
Compound Loaded (ug)
chloroform 1.50
carbon tetrachloride 0.809
chlorobenzene 1.12
bromobenzene 0.0127
dpm
Loaded % Recovery
94237.0 83.9
84.3
85.2
80278.6 57.2
66.7
67.1
89589.9 89.9
87.8
90.2
885.4 147
114
107
Average
% Recovery
84.5
63.7
89.3
123
128
-------
Table D-2. RECOVERY OF HALOGENATED HYDROCARBONS FROM HUMAN URINE
SPIKED FROM GAS MIXING BULB
VO
Compound
methylene chloride
chloroform
bromodichloromethane
tetrachloroethylene
chlorobenzene
m-dichlorobenzene
mean
Amount
spiked (ug)
6.96
8.16
10.2
8.44
5.58
7.54
% Recovery
1
80.6
107
123
85.2
103
73.6
2
a
72.6
88.2
61.2
77.1
70.5
3
41.3
89.5
126
84.4
92.6
90.1
4
21.7
44.8
80.8
57.1
71.9
80.7
Average + Std. Dev.
47.9 + 30.0
78.5 + 26.5
105 +23.3
72.0 + 14.9
86.2 + 14.2
78.7 + 8.7
79.2 + 24.3
Not quantified because of background interferences.
-------
5.0 Apparatus
5.1 Sampling
Urine samples are collected in 120 ml cleaned and oven-treated glass
bottles.
5.2 Purge Apparatus
The apparatus required is shown in Figure D-l.
5.3 Tenax Cartridges
The sampling tubes are prepared by packing a 10 cm long x 1.5 cm i.d.
glass tube containing 6.0 cm of 35/60 mesh Tenax GC (VL.6 g) using glass
wool in the ends to provide support (D1-D5). Tenax GC is extracted in a
Soxhlet extractor for a minimum of 24 hours each with methanol and then with
pentane prior to preparation of cartridge samplers (D5). After purification,
the Tenax GC sorbent is air dried, vacuum dried and then was meshed to
provide a 35/60 particle size range. Cartridge samplers were then prepared
and conditioned at 270°C with helium flow at 30 ml/min for 20 minutes. The
conditioned cartridges were transferred to Kimax (2.5 cm x 150 cm) culture
tubes which are immediately sealed using Teflon-lined caps to prevent contami-
nation.
5.4 GC/MS/COMP
The volatile halogenated hydrocarbons purged from water are analyzed on
either an LKB 2091 GC/MS with an LKB 203.1 data system or a Varian MAT CH-7
GC/MS with a Varian 620/i data system. The sample, concentrated on a Tenax
GC cartridge, is thermally desorbed using an inlet manifold system (D1-D3,
D5). The operating conditions for the thermal desorption unit and the
analysis of Tenax GC cartridges are given in Table D-3.
6.0 Procedure
6.1 Collection of Samples
Each participant is provided with a clean 120 ml (4 oz.) bottle and
asked to collect the first urine sample in the morning. In addition, spot
urine samples are collected from selected participants when tissue, breath
and blood samples are collected. These samples serve both as backups and
to determine individual variability.
130
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THERMOMETER
-20tol50°c
THERMOMETER ADAPTER
with 0—ring
J 10/18
TEN AX CARTRIDGE
HELIUM
PURGE
HELIUM INLET
TUBE
LIQUID LEVEL
100 ml ROUND BOTTOM FLASK
MAGNETIC STIRRING BAR
Figure D-l.
Headspace purge apparatus for blood, urine, and tissue
samples.
131
-------
Table D-3. INSTRUMENTAL OPERATING CONDITIONS
LKB 2091
Varlan MAT CH-7
to
Desorption chamber temperature
Desorptlon chamber He flow
Desorption time
Capillary Trap Temperature during desorptlon
Temperature of capillary trap during injection
onto column
Time of He flow through capillary trap
He flow through column [sweep time]
Carrier flow
Capillary column
Column temperature
Scan range
Scan rate
Scan cycle time
Scan mode
Trap current
Filament current
Accelerating volatage
270
20 ml/min
5.0 min
-196°C
-196°C to 250°C
265
10 ml/min
8.0 min
-196°C
then held at 190°C
12 3/4 min 12 3/4 min
9. 5 min 5 min
2.0 ml/min 2.5 ml/min
100 m SE-30 SCOT
30°C for 2 min,
then 4°/min to 240°
5-490 dalton
1 sec full scale
2.4 sec
parabolic
4A
50 uA
3.5 kV
20 -> 240° at 4°/min
20 -> 500 dalton
1 sec/decade
4.5 sec
exponential
200 uA
2 kV
-------
6-2 Purge of Volatile Organics
A 25 ml aliquot of urine, chilled to 4°C is measured into the purge
flask in Figure D-l; purged, distilled water is added to the sample to
dilute it to about 50 ml; and a stirring bar added. The apparatus is
assembled, stirring started, the temperature raised to 50°C, and the helium
flow started at 25 ml/min. After 90 min, the purge is terminated, the
apparatus disassembled, and the Tenax cartridge stored in a Kimax culture
tube with calcium sulfate desiccant in a freezer until it is ready for
analysis.
6.3 Analysis of Sample Purged on Cartridge
The instrumental conditions for the analysis of halogenated hydrocarbons
of the sorbent Tenax GC sampling cartridge is shown in Table D-3. The
thermal desorption chamber and six-port valve are maintained at 270° and
200°C, respectively. The helium purge gas through the desorption chamber is
adjusted to 15-20 ml/min. The nickel capillary trap at the inlet manifold
is cooled with liquid nitrogen. In a typical thermal desorption cycle a
sampling cartridge is placed in the preheated desorption chamber and helium
gas is channeled through the cartridge to purge the vapors into the liquid
nitrogen cooled nickel capillary trap. After desorption the six-port valve
is rotated and the temperature on the capillary loop is rapidly raised; the
carrier gas then introduces the vapors onto the high resolution GLC column.
The glass capillary column is temperature programmed from 20° to 240°C at
4°/min and held at the upper limit for a minimum of 10 min. After all of
the components have eluted from the capillary column the analytical column
is then cooled to ambient temperature and the next sample is processed.
6.4 Quantitation
All data are acquired in the full scan mode. Quantitation of the
halogenated compounds of interest is accomplished by utilizing selected ion
plots, SIPs, which are plots of the intensity of specific ions (obtained
from full scan data) vs. time. Using SIPs of ions characteristic of a given
compound in conjunction with retention times permits quantitation of compo-
nents of overlapping peaks. Two external standards, perfluorobenzene
and perfluorotoluene, were added to each Tenax GC cartridge in known
133
-------
quantities just prior to analysis. In order to eliminate the need to cons-
truct complete calibration curves for each compound quantitated, the method
of relative molar response (RMR) is used. In this method the relationship
of the RMR as the unknown to the RMR of the standard is determined as follows:
A , /Moles ,
_ unk' _ unk
^unknown/ standard ~ Astd/Molesst
-------
with 100-500 ng each of the compounds listed in Table D-2. These blanks and
controls are carried to the field and receive the same handling as the field
samples. Workup and analysis of field blanks and controls is interspersed
with the field samples on a regular basis. This method allows assessment of
sample storage stability.
7.1.2 Procedural Blanks and Controls
7.1.2.1 Tenax GC
The purity of each batch of Tenax GC cartridges is checked by thermal
desorption/GC/FID. An acceptable background is a judgement based upon the
backgrounds obtained previously which exhibited no GC/MS artifacts.
7.1.2.2 Purge Blanks
With each set of purge samples, a procedural blank is run. This
consists of 50 ml of prepurged, distilled water which is purged under the
same conditions as the samples. These blanks are designed to detect arti-
facts from dirty glassware, laboratory atmosphere intrusion and storage.
7.1.2.3 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol, 2,6-
dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor the
capillary GC column performance. This also serves to check the mass spectro-
meter tuning.
Field samples, field controls, field blanks, and procedural blanks are
queued up for GC/MS analysis such that at least one GC sample is run each
working day. In addition, a Tenax GC cartridge directly loaded with stan-
dards is analyzed each day to serve as a procedural control and also to
update the RMR value. Thus, in a typical working day, 4 field samples, 1
blank or control, and 1 RMR standard are run.
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may affect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
135
-------
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, location, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
protocol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data has been
produced.
8.0 References
Dl. Pellizzari, E. D., Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres, Publication No. EPA-650/2-74-121, Contract No.
68-02-1228, 148 pp., July, 1974.
136
-------
B2. Pellizzari, E. D., Development of Analytical Technqiu.es for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA-600/2-76-
076, Contract No. 68-02-1228, 185 pp., November, 1975.
D3. Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki, Environ.
Sci. Tech., 9, 556 (1975).
D4. Pellizzari, E. D., B. H. Carpenter, J. E. Bunch and E. Sawicki, Environ.
Sci. Tech., 9, 556 (1975).
D5. Pellizzari, E. D. , Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA 600/7-77-
055, 288 pp., June, 1977-
Revised April, 1979
137
-------
APPENDIX E
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE
HALOGENATED ORGANICS IN TISSUE
138
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF PURGEABLE HALOGENATED
ORGANICS IN TISSUE
1.0 Principle of the Method
Volatile compounds are recovered from a tissue sample by macerating
with water, warming the sample and purging an inert gas over the warm
sample. The vapors are then trapped on a Tenax cartridge which is analyzed
by thermal desorption interfaced to GC/MS.
2.0 Range and Sensitivity
For a typical organic compound approximately 30 ng are required for
mass spectral identification using high resolution glass capillary GC/MS
analysis. Based on a 5 g tissue sample, the limit of detection of about 6
ng/g (6 ppb) would be typical. The dynamic range for a purged sample is
VLO , however, smaller samples may be purged and the range increased commen-
surately.
3.0 Interferences
Two possible types of interference must be considered: (1) material
present in the sample which physically prevents the effective purge of the
sample, and (2) a material which interferes with the analysis of the purged
sample. In the former case, several techniques have been developed to
handle such problems (e.g., foaming) by diluting and stirring the sample.
The second case is minimized by the use of GC/MS for the analysis since
unique combinations of m/z and retention time can be selected for most
compounds. This permits analysis of compounds even though chromatographic
resolution is not obtained.
4.0 Precision and Accuracy
The purge and trap technique was validated using "cold" model compounds
Results of these recovery studies are presented in Table E-l. Based on
these data, expected recoveries of purgeable halogenated organics from
tissue are about 50%.
Great difficulty was encountered in quantitative introduction of a
representative fortified sample into the container for analysis. Consequen-
tly variations in recovery may be attributed to losses during tissue macera-
tion and transfer.
139
-------
Table E-l. RECOVERY OF HALOGENATED HYDROCARBON FROM HUMAN ADIPOSE TISSUE.
Compound 1
*»
methylene chloride
chloroform
bromodichlorome thane 64 . 4
tetrachloroethylene 39.4
chlorobenzene 21.5
m-dlchlorobenzene 82.1
Mean
% Recovery
2 3
78.2 69.8
62.3 14.8
15.5 13.4
15.2 112
6.8 7.3
3 56.5
4
92.8
59.7
49.4
40.6
17.9
30.8
Average + S.D.
80.3 + 11.6
45.6 + 26.7
35.7 ± 25.3
51.8 + 41.8
13.4 + 7.5
56.5 -1- 25.7
45.3 ± 30.8
Not quantified due to peak interference.
-------
The tissue sample analysis procedure has been partially validated. Due
to some low recovery values listed in Table E-l, the difficulties in handling
tissue during the purge, and (most significantly) the lack of control over
sampling and storage prior to receipt by RTI; the analysis of tissue samples
of a purgeable halogenated organics must be regarded as semiquantitative.
5.0 Apparatus
5.1 Sampling Apparatus
Samples must be collected and stored with a minimum potential for con-
tamination or loss of volatile components. Plastic or "rubber" containers
can leach contaminents into the sample. In addition, purgeable organics can
permeate through plastic, either representing a loss or an artifact from air
pollutants (e.g. freons from a freezer compressor).
Samples should be collected with minimum exposure to plastic or rubber.
They should be stored in cleaned, oven-treated glass jars sealed with
either Teflon or foil-lined caps.
5.2 Purge Apparatus
The apparatus required is shown in Figure E-l.
5.3 Tenax Cartridges
The sampling tubes are prepared by packing a 10 cm long x 1.5 cm i.d.
glass tube containing 6.0 cm of 35/60 mesh Tenax GC (^1.6 g), using glass
wool in the ends to provide support (E1-E5). Tenax GC is extracted in a
Soxhlet extractor for a minimum of 24 hours each with methanol and then with
pentane prior to preparation of cartridge samplers (E5). After purifi-
cation, the Tenax GC sorbent is air-dried, vacuum dried and then meshed to
provide a 35/60 particle size range. Cartridge samplers are then prepared
and conditioned at 270°C with helium flow at 30 ml/min for 20 minutes. The
®
conditioned cartridges are transferred to Kimax (2.5 cm x 150 cm) culture
tubes which are immediately sealed using Teflon-lined caps to prevent contami-
nation.
5.4 GC/MS/COMP
The volatile halogenated hydrocarbons purged from water are analyzed on
either an 1KB 2091 GC/MS with an 1KB 2031 data system or a Varian MAT CH-7
GC/MS with a Varian 620/i data system. The sample, concentrated on a Tenax
GC cartridge, is thermally desorbed using an inlet manifold system (E1-E3,
141
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THERMOMETER
-20tol50°c
THERMOMETER ADAPTER
with 0-ring
I 10/18
TENAX CARTRIDGE
HELIUM
PURGE
HELIUM INLET
TUBE
LIQUID LEVEL
100 ml ROUND BOTTOM FLASK
MAGNETIC STIRRING BAR
Figure E-l. Headspace purge apparatus for blood, urine, and tissue
samples.
142
-------
E5). The operating conditions for the thermal desorption unit and the
analysis of Tenax GC cartridges are given in Table E-2.
6.0 Procedure
6.1 Collection of Samples
It is anticipated that tissue samples collected from cadavers or
surgery will be obtained from a pathologist. Personnel from RTI will work
with pathologists advising them of proper sample handling procedures. To be
of use for purgeable halogeanted organics a tissue sample must be collected
a short time following death and immediately frozen in a cleaned glass
container with as small a "headspace" as possible. Any handling or storage
in contact with polymeric materials represents potential contamination.
6.2 Purge of Volatile Organics
An aliquot (5 g) of frozen tissue is cut from a larger mass, sectioned
and transferred to the purge flask in Figure E-l. Purged, distilled water
is added to the sample to dilute it to about 50 ml; and the mixture macerated
in an ice bath using a Virtis tissue homogenizer. The purging apparatus is
immediately assembled, stirring started, the temperature raised to 50°C, and
the helium flow started at 25 ml/min. After 30 min, the purge is terminated,
the apparatus disassembled, and the Tenax cartridge stored in a Kimax
culture tube with calcium sulfate desiccant in a freezer until it is ready
for analysis.
6.3 Analysis of Sample Purged on Cartridge
The instrumental conditions for the analysis of halogenated hydrocarbons
of the sorbent Tenax GC sampling cartridge is shown in Table E-2. The
thermal desorption chamber and six-port valve are maintained at 270° and
200°C, respectively. The helium purge gas through the desorption chamber is
adjusted to 15-20 ml/min. The nickel capillary trap at the inlet manifold
is cooled with liquid nitrogen. In a typical thermal desorption cycle a
sampling cartridge is placed in the preheated desorption chamber and helium
gas is channeled through the cartridge to purge the vapors into the liquid
nitrogen cooled nickel capillary trap. After desorption the six-port valve
is rotated and the temperature on the capillary loop is rapidly raised; the
carrier gas then introduces the vapors onto the high resolution GLC column.
The glass capillary column is temperature programmed from 20° to 240°C at
143
-------
Table E-2. INSTRUMENTAL OPERATING CONDITIONS
LKB 2091
Varian MAT CH-7
Desorptlon chamber temperature
Desorptlon chamber He flow
Desorptlon time
Capillary Trap Temperature during desorption
Temperature of capillary trap during injection
onto column
Time of He flow through capillary trap
He flow through column [sweep time]
Carrier flow
Capillary column
Column temperature
Scan range
Scan rate
Scan cycle time
Scan mode
Trap current
Filament current
Accelerating volatage
270
20 ml/min
5.0 min
-196°C
-196°C to 250°C
265
10 ml/min
8.0 min
-196°C
then held at 190°C
12 3/4 min 12 3/4 min
9.5 min 5 min
2.0 ml/min 2.5 ml/min
100 m SE-30 SCOT
30°C for 2 min,
then 4%nin to 240°
5-490 dalton
1 sec full scale
2.4 sec
parabolic
4A
50 pA
3.5 kV
20 + 240° at 4°/min
20 -*• 500 dalton
1 sec/decade
4.5 sec
exponential
200 uA
2 kV
-------
4°/min and held at the upper limit for a minimum of 10 min. After all of
the components have eluted from the capillary column the analytical column
is then cooled to ambient temperature and the next sample is processed (E2) .
6.4 Quant i tat ion
All data are acquired in the full scan mode. Quantitation of the
halogenated compounds of interest is accomplished by utilizing selected ion
plots, SIPs, which are plots of the intensity of specific ions (obtained
from full scan data) vs. time. Using SIPs of ions characteristic of a given
compound in conjunction with retention times permits quantitation of compo-
nents of overlapping peaks. Two external standards, perfluorobenzene and
perfluorotoluene, are added to each Tenax GC cartridge in known quantities
just prior to analysis. In order to eliminate the need to construct com-
plete calibration curves for each compound quantitated, the method of
relative molar response (RMR) is used. In this method the relationship of
the RMR of the unknown to the RMR of the standard is determined as follows:
A , /Moles .
unk _ unk,
RMR . = A ,/Moles ,
unknown std' std
A . /g . /GMW .
unk °unk unk
where A = peak response of a selected ion,
g = number of grams present, and
GMW = gram molecular weight.
Thus, in the sample being analyzed:
(A . )(GMW
y v
g . = (A 4.,)(GMW . ,)(RMR . , .,)
^unknown std' std' v unk/std
The value of an RMR is determined from at least three independent analyses
of standards of accurately known concentration prepared using a gas permea-
tion system (E5). The precision of this method has been determined to be
generally ±10 percent when replicate sampling cartridges are examined.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an ongoing
quality assurance program is required to assure the data quality. Quality
control procedures determine artifacts, losses, etc. through a system of
145
-------
blanks and controls. Quality assurance procedures monitor the execution of
the procedure and check data interpretation and, calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks
consist of 5 g of tissue or 50 ml water in the same type of sampling contai-
ner as is used in the field. Controls consist of 5 g of tissue or 50 ml
water spiked with 100-500 ng of each of the compounds listed in Table E-l.
These blanks and controls are carried to the field and receive the same
handling as the field samples. Workup and analysis of field blanks and
controls is interspersed with the field samples on a regular basis. This
method allows assessment of sample storage stability.
7.1.2 Procedure of Blanks and Controls
7.1.2.1 Tenax GC
The purity of each batch of Tenax GC cartridges is checked by thermal
desorption/GC/FID. An "acceptable background" is judgement based upon the
backgrounds obtained previously which exhibited no GC/MS artifacts.
7.1.2.2 Purge Blanks
With each set of purge samples, a procedural blank is run. This
consists of 50 ml of prepurged distilled water which is purged under the
same conditions as the samples. These, blanks are designed to detect arti-
facts from dirty glassware, laboratory atmosphere intrusion and storage.
7.1.2.3 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a Tenax GC cartridge directly loaded with stan-
dards is analyzed each day to serve as a procedural control and also to
update the KMR value. Thus, in a typical working day, 4 field samples, 1
blank or control, and 1 RMR standard are run.
146
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7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparations of a sample container through reporting
of the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a Sampling Protocol Sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, location, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
protocol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data have been
produced.
147
-------
8.0 References
El. Pellizzari, E. D., Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres. Publication No. EPA-650/2-74-121, Contract No.
68-02-1228, 148 pp., July, 1974.
E2. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Publication No. EPA-600/2-76-076,
Contract No. 68-02-1228, 185 pp., November, 1975.
E3- Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki, Environ.
Sci. Tech., 9, 552 (1975).
E4. Pellizzari, E. D., B. H. Carpenter, J. E. Bunch and E. Sawicki, Environ.
Sci. Tech., 9, 556 (1975).
E5. Pellizzari, E. D., Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors, Publication No. EPA-600/7-77-055,
288 pp., June 1977.
Revised April, 1979.
148
-------
APPENDIX F
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE
HALOGENATED ORGANICS IN BLOOD
149
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE HALOGENATED
ORGANICS IN BLOOD
1.0 Principle of Method
Semi-volatile halogenated hydrocarbons are extracted from blood plasma
with organic solvents, dried, and concentrated to an appropriate volume for
quantification using gas chromatograph/electron capture detector (GC/ECD).
Identifications are confirmed by GC/ECD using a second column and, when
sufficiently concentrated, by GC/MS/COMP. Blood samples are optionally
subjected to liquid chromatographic cleanup on Florisil if severe inter-
ferences are encountered. This procedure was adapted from that of Thompson
(Fl).
2.0 Range and Sensitivity
The sensitivity of response to GC/ECD is a function of the instrument,
the compound, and the matrix from which it is extracted. Acceptable recover-
ies from human plasma at approximately 5 ppb (parts per billion) have been
achieved.
3.0 Interferences
Interferences in sample analysis and quantification using GC/ECD are
manifested in the electron capturing ability of the given contaminant.
Blood extracts which have not been cleaned up contain rapidly eluting compo-
nents which exhibit high electron capturing properties; these interferences
can largely be removed by gradient liquid-liquid chromatography on 2% aqueous
deactivated Florisil, as discussed below.
4.0 Precision and Accuracy
Recovery studies were initiated with a wide variety of model halogenated
hydrocarbons (e.g., trifluralin, a-BHC, p-BHC, y-BHC, 2,4,5-trichlorobiphenyl,
heptachlor, aldrin, heptachlor epoxide, endosulfan, p_,p_'-DDE and dieldrin).
For 2.0 ml aliquots of human plasma spiked with 14 ng of the above halogenated
hydrocarbons and equilibrated for 19 hr at 4°C, a mean recovery of 53.1 ±
12.6% was obtained. The recovery data is reproduced in Table F-l.
150
-------
Table F-l. RECOVERY STUDIES OF EXTRACTABLE HALOGENATED
HYDROCARBONS FROM HUMAN PLASMA
Recovery Plasma (%)
Compound
Trifluralin
a-BHC
g-BHC
Y-BHC
2, 4, 5-Trichlorobiphenyl
Heptachlor
Aldrin
Heptachlor epoxide
Endosulf an
£.,£* -DDE
Dieldrin
Mean + Std. Dev.
ng
spiked
14.8
14.0
14.5
15.2
14.0
13.6
14.8
14.2
13.1
11.6
15.8
Trial
One
87.6
61.9
60.0
57.1
48.2
50.2
a
35.2
53.2
51.4
58.6
Trial
Two
92.3
56.2
60.1
53.3
a
42.2
40.3
43.1
59.1
a
a
Trial
Three
83.3
57.6
43.8
52.4
a
51.1
37.2
a
44.3
48.8
58.5
Mean
85.7
58.6
50.8
54.3
48.2
47.8
38.8
39.2
52.2
50.1
58.6
53.1 + 12.6
quantifiable due to instrumental problems.
151
-------
5.0 Apparatus
5.1 Sampling Apparatus
Blood samples are obtained in 10 ml vacutainer tubes "suitable for GC"
(Venoject L428, Kimble) containing 15 mg tripotassium salt of EDTA and 20 |Jg
potassium sorbate.
5.2 Extraction Apparatus
Glass culture tubes (16 x 125 mm) and caps equipped with Teflon liners,
reciprocal shaker (ca. 40 oscillations/minute), 500 ml Kuderna-Danish evapora-
tors (or 10 ml microevaporators) and receiving tubes, three ball Snyder
®
columns, glass bottles and caps equipped with Teflon liners, reactivials ,
centrifuge, and 22 mm i.d. chromatography columns. Solvent: pentane,
distilled in glass and redistilled prior to use. Reagents: anhydrous
sodium sulfate, (extracted with pentane in Soxhlet extractor for 24 hr and
stored in an oven at 140°C), 60/100 mesh reagent Florisil.
6.0 Procedure
6.1 Collection of Samples
Blood is obtained from volunteers by a qualified phlebotomist using
vacutainers. Samples are inverted to ensure dispersal of the anticoagulant
and are then centrifuged. Plasma is then pipetted off, stored in a shell
vial with teflon-lined screw caps and frozen as soon as possible.
6.2 Extraction, Cleanup, and Concentration
Exactly 2.0 ml of plasma is transferred to a 16 x 125 mm culture tube.
Hexane (7.0 ml) is then pipetted in, the culture tubes capped, and the
samples placed on the shaker bath at 40 oscillations/minute for 24 hours.
The samples are removed from the bath, 5.0 ml of organic extract is withdrawn
and transferred to glass bottles with Teflon liners, and the samples are
returned to the shaker bath for 24 hours with 5.0 ml additional fresh hexane.
Again, 5.0 ml are withdrawn, and the combined extracts are dried a minimum
of 30 minutes over about 0.5 g anhydrous Na.SO,. The extract is transferred
to a 500 ml KD flask (or micro KD), topped with a Synder column, concentrated
to ca. 2-4 ml, and cooled to ambient temperature. The sides of the KD are
rinsed with about 1.5 ml hexane and the extract is then blown down under
(R)
nitrogen to about 1 ml, transferred to a reactivial previously calibrated
to a specific volume, and further concentrated.
152
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6.3 Instrumental
The detection and quantification of semi-volatile halogenated hydrocar-
bons is made using a Series 4400 Fisher/Victoreen Gas Chroamtograph equipped
with a tritium foil electron capture detector. Separation is effected on a
40 m, 0.38 mm i.d., glass SCOT capillary column coated with 1% SE-30 on
0.32% Tullanox (F2,F3). Maximum efficiency is obtained with a flow rate of
2.5 ml/min of nitrogen gas with makeup nitrogen gas adjusted to a total flow
of 25.0 ml/min, column 220°C (isothermal), and detector 285°C.
As a confirmatory column a 190 cm x 0.2 cm i.d. 1.5% OV-17/1.95% QF-1
on 80/100 Chromosorb W-HP packing is employed. Efficient responses are
obtained for flow rates of 18 ml/min at identical column and detector tempera-
tures .
Final confirmation of the identity of the components of sufficiently
concentracted extracts (generally greater than 10 ng/|Jl) can be made using
gas chromatography/mass spectrometry/computer (GC/MS/COMP) (Finnigan 3300).
The GC/MS/COMP systems used are a Finnigan 3300 GC/MS/COMP and an LKB
2091 GC/MS equipped with an LKB 2031 data system. Chromatographic conditions
for the Finnigan 3300 are 20 m x 0.38 mm i.d., 1% SE-30 SCOT capillary
operated isothermally at 235°C and a flow rate of 2.0 ml/min helium. Split-
less injection (0.2-0.3 |Jl) is used, with standard electron impact (70 eV)
ionization conditions.
The LKB 2091 is operated using a 18 m 1% SE-30/BaCO. WCOT capillary
column at 240°, isothermal for PCBs and a 40 m x 0.38 mm i.d. 1% SE-30 SCOT
capillary column at 230° isothermal for the pesticides. In both cases, the
column flow rate is 2 ml/min with 20 ml/min split off at the injector. The
mass spectrometer is operated under standard electron impact conditions.
6.4 Qualitative and Quantitative Analysis
6.4.1 Qualitative Analysis
Alternate single injection of extracts and standard solutions is the
routine procedure for processing samples. If the retention time of a given
component of an extract suggests the presence of a standard compound, a
repetitive injection is then made. Tentative identification is made if the
deviation between the two respective means is no greater than three percent.
A similar criterioq is then applied to the retention times of both extract
153
-------
and standard component upon a second confirmatory column. Qualitative
identification of a component is made if both criteria are satisfied.
6.4.2 Quantitative Analysis
A mean linear response range of 5-160 pg/fJl has been established for
the compounds trifluralin and y-BHC on a 1% SE-30/0.32% Tullanox 40 m, 0.38
mm i.d. SCOT capillary column installed in a Series 4400 Fisher/Victoreen
Gas Chromatograph. Quantification of a given component is made by a com-
parison of the means of recorder trace areas of two extract and two standard
solutions within this linear response range. The precision of the concentra-
tion of a given component is normally less than ten percent of the mean
concentrations and is obtained by propagation of the standard deviations of
the responses of both the extract and standard solutions. The effective
concentration multiplied by the volume of extract results in the total
amount of extracted material.
The areas employed in the quantification of blood extracts must be
normalized to compensate for the volume of extract that is not analyzed.
The removal of the first 5.0 ml aliquot from the 7.0 ml extract leaves 2/7
of the extracted material behind. Since the partition coefficients for
semi-volatiles in human plasma are large, one approximates that the second
extraction contributes more as a dilution gradient than as an extracting
solvent. Under this approximation, 5/7 of the original 2/7 remaining in the
organic extract would be obtained from the second aliquot. One would then
have 5/7 + (2/7)-(5/7) = 45/49 of the total initial extract to analyze, and
the area responses for the extracts would necessarily require normalization
by 49/45 = 1.089 prior to comparison to standard solutions. This methodology
was initiated due to the formation of complex emulsions between the plasma
and organic solvent which prevented the easy removal of all of the organic
extract. This procedure assumes an infinite partition coefficient and that
the second extraction does not influence the total recovery.
If the extracts are colored, the presence of lipids may interfere with
either analysis or concentration of the extracts due to precipitation. In
this case, the sample should be transferred to a 22 mm i.d. column contain-
ing 1.6 g of 2% aqueous-deactivated Florisil and eluted with 10 ml each of
hexane, 5% MeCl2/hex, 10% MeCl2/hex, 15% MeCl2/hex, 20% MeCl2/hex, 30%
154
-------
MeCl2/hex, 50% MeCl^/hex, and MeCl-. The extracts are concentrated and
analyzed. Most semi-volatile halogenated hydrocarbons are expected to
appear in the first five fractions. This estimate is based upon elution
data of pesticides on Florisil (F4) and has not been subjected to full
experimental verification.
6.4.3 GC/MS/COMP Confirmation
The chromatography conditions are similar to those used for GC/ECD.
The samples for this study are to be screened by GC/ECD and confirmed (if
sufficiently concentrated) by GC/MS/COMP. Therefore, the retention times of
the two techniques must be similar. GC/ECD must operate isothermally, so
the GC/MS/COMP conditions reflect this restriction.
The Finnigan 3300 and the 1KB 2091 systems may be operated in both the
full scan and selected ion monitoring (SIM) modes. In the full scan mode,
full spectra are collected. Spectra or mass fragmeatograms (single ion
plots) may be plotted for interpretation. In the SIM mode, only a small
number (up to 9 for the Finnigan 3300 and up to 16 for the 1KB 2091) of ions
are monitored. Full spectra are not collected. The advantage of this
method is that the detector spends more time "looking" at the selected ion
and therefore better (generally 10-50 times) sensitivity is obtained.
To determine the limits of detection, standard solutions of selected
pesticides and PCB isomers have been analyzed on the Finnigan 3300 and LKB
2091. In the full scan mode, the limit of detection was the amount of
compound required for an interpretable spectrum. In the SIM mode, the limit
of detection was the amount of compound required to yield a peak 2-4 times
the noise level.
The estimated limits of detection for the Finnigan 3300 and LKB 2091
are presented in Table F-2.
Quantitation using GC/MS/COMP is achieved by comparing the computer-
calculated integrated area of the unknown with the integrated response for a
known amount of standard. To compensate for differences in ionization
cross-section, the relative molar response of authentic compounds is obtained
The calculation of the relative molar response (RMR) factor allows the
estimation of the levels of sample components without establishing a cali-
bration curve. The RMR is calculated as the integrated peak area of a known
155
-------
Table F-2. ESTIMATED LIMITS OF DETECTION FOR EXTRACTABLE HALOGENATED
ORGANICS ANALYSIS3
Compound
trifluralin
atrazine
Y-BHC (lindane)
heptachlor
chlordane
£,p_'-DDE
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
LKB
Full Scan
ng/yl
12
>12<20
>12<20
12
^30°
12
.1
<1
12
2091b
m/z
264
200
181
272
375
246
188
360
498
Finnigan 3300a
SIM
ng/pl
0.4
0.4
0.10-0.4
0.10-0.4
5
>0.3
0.004
^0.016
0.42
Full Scan
ng/yl
5-10
<50
5-10
10-20
25-50
5-10
^2.5
25-50
150
m/z
264
200
181
272
375
246
188
360
498
SIM
ng/ul
<0.5
5-10
1
1-1.5
5-10
0.5-1
^0.025
M).15
^0.3
See text for conditions.
15:1 split at injection, only 1/15 of injection is on column.
•»
"0.2 yl injected with no split.
156
-------
amount of the compound, A° , , with respect to the integrated peak area of a
known standard-, A° , (in this case d,0-pyrene), according to the equation
- . _
' ' " C ^
From this calculated value, the concentration of an identified compound in
a sample is calculated by rearranging Equation 1 to give
. (Aunk) t
unk - (A) (mw) (RMR)
The use of RMR for quantitation by GC/MS has been successful in repea-
ted applications to similar research problems.
The RMRs for the compounds were calculated from the numerical inte-
grations of peaks observed in the appropriate MID channel. Typical RMRs
listed in Table F-3 and F-4 are mean values of three injections of each of
three replicate standard mixtures.
The RMRs given here are to be regarded as typical values . Not only
must they be determined for each instrument, but day-to-day variations are
sometimes large enough to require daily calibration.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an on-going
quality assurance program is required to assure the data quality. Quality
control (QC) procedures determine artifacts, losses, etc. through a system
of blanks and controls. Quality assurance (QA) procedures monitor the
execution of the procedure and check data interpretations and calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks con-
sist of 10 ml of water in the same type of sampling container as is used in
the field. Controls consist of 10 ml of plasma spiked at 10-15 ng with the
compounds listed in Table F-5. These blanks and controls are carried to the
field and receive the same handling as the field samples. Workup and
157
-------
Table F-3. RMRs FOR PCBs AND PESTICIDES OF INTEREST
TO THIS PROGRAM3
Compound
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
trifluralin
atrazine
lindane
heptachlor
2,2* -DDE
chlordane (peak 1)
chlordane (peak 2)
Concentration
104 ng/yl
3.8 ng/yl
570 ng/yl
10.4 ng/yl
1156 ng/yl
8.4 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
Ion
188
360
498
264
200
202
181
183
272
246
373
375
373
375
RMR
elutes with solvent
and was two scans wide -
not determinable
.38 + 3%
.35 + 10%
.14 + 7%
not determinable
1.32 + 20%
.74 + 7%
.25 + 8%
.74 + 9%
.62 + 12%
.74+6%
.45+6%
.71 + 5%
.65+5%
.051 + 6%
.045 + 13%
3Standard is d -pyrene (m/jz = 212)
10
158
-------
Table F-4. RMR FACTORS FOR STANDARD PCB SOLUTIONS,
SELECTED ION MONITORING MODE
RMR RMR RMR
m/z 188 m/z 358 m/z 498
Standard 2-Chlorobiphenyl Hexachlorobiphenyl Decachlorobiphenyl
I PCB-STD-20 0.60
II PCB-STD-2 0.620""\
n'?^ > 0-640 + .171
0.466 f —
0.643J
III PCB-STD-0.2 0.566*"N
0.840 1
0.637 )0.699 +.171
S 0.597 I
0.705 J
IV PCB-STD-0.04 1.020"^
0.692 SO. 763 + .257
0.576 J
0.257
0.291"^
0 334 \
/ 0.325 + .009
0.321 J
^^
0 • 3oo |
0.293 1
0.301 > 0.294 + .072
0.239 f
0.273J
0.320">|
0.528 } 0.459 + .072
0.528 J
0.341
0.430^
0.474 (n
0.462 >°'456±
0.431 J
0.372*^
0.361 1
0.373 ) 0.361 +
0.303 [
0.394J
0.287*^
0.543 > 0.401 +
0.372 J
.018
.033
.142
Standard is d.. -pyrene (m/^ = 212).
-------
Table F-5. SEMI-VOLATILE HALOGENATED HYDROCARBONS IN
METHANOL SPIKING SOLUTION
Compound An:t. spiked, ng
4-Chlorobiphenyl 13.2
Trifluralin 14.8
tt-BHC 14.0
e-BHC 14.5
Y-BHC 15.2
4,4'-Dichlorobiphenyl 15.8
2,4,5-Trichlorobiph'enyl 14.0
Heptachlor 13.6
Aldrin 14.8
Heptachlor epoxide 14.2
Endosulfan 13.1
Dieldrin 15.8
p,p'-DDE 11.6
p.p'-DDT 12.5
Endrin 11.5
160
-------
analysis of field blanks and controls is interspersed with the field samples
on a regular basis. This method allows assessment of sample storage stability.
7.1.2 Procedural Blanks and Controls
7.1.2.1 Extraction Blanks.
With each set of samples, a procedural blank is run. This consists of
10 ml of prepurged distilled water which is extracted under the same condi-
tions as the samples. These blanks are designed to detect artifacts from
dirty glassware, laboratory atmosphere intrusion, and other sources.
7.1.2.2 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6rdimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks, and procedural blanks are .
queued up for GC/MS analysis such that at least one QC sample is run each
working day. j. In addition, a standard solution is analyzed each day to serve
as a procedural control and also to update the RMR value. Thus, in a typical
working day, 4 field samples, 1 blank or.control, and 1 RMR standard are
run.
The Finnigan 3300 GC/MS is a quadrupole mass spectrometer which requires
frequent tuning. Daily tuning is achieved using FC-43 and decafluorotriphenyl-
phosphine (DFTPP).
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At-the .second level, the chemist's
supervisor monitors their daily activities, reviews-the notebook, checks
i
data and calculations, and assists in "troubleshooting" problems. At the
T - '
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
161
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7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contami-
nant, bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, locations, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
procotol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data has been
produced.
8.0 References
Fl. Thompson, J. F., Analysis of Pesticide Residues in Human and Environ-
mental Samples, A Compilation of Methods Selected for Use in Pesticide
Monitoring Programs, Environ. Toxicol. Div., Health Effects Research
Lab., USEPA, RTF, NC, December, 1974.
F2. Hines, J. W., R. Shapiro, E. Pellizzari and A. Schwartz, HRC and
CC, submitted for publication (1978).
F3. Hines, J. R., R. Shapiro, A. Schwartz, and E. D. Pellizzari, HRC and
CC, submitted for publication (1978).
162
-------
F4. Sherma, J., Manual of Analytical Quality Control for Pesticides and
Related Compounds, EPA-600/1-76-017, 1976, Table 7-1.
Revised, April, 1979
163
-------
APPENDIX G
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE
HALOGENATED ORGANICS IN WATER
164
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE HALOGENATED
ORGANICS IN WATER
1.0 Principle of Method
Semi-volatile halogenated hydrocarbons are extracted from water samples
with organic solvents, dried, and concentrated to an appropriate volume for
quantification using GC/ECD. Results are confirmed by GC/MS/COMP if the
sample is sufficiently concentrated. This procedure was adapted from that
of Thompson (Gl).
2.0 Range and Sensitivity
The sensitivity of response to GC/ECD is a function of the instrument,
the compound, and the matrix from which it is extracted. Detection limits
of approximately 5 ppt (parts per trillion) have been established for water
extracts (120 ml aliquots) concentrated to 100 |Jl.
3.0 Interferences
Interferences in sample analysis and quantification using GC/ECD are
manifested in the electron capturing ability of the given contaminant. The
relative purity of the water matrices (i^.e. , residential drinking water)
minimizes large interferences and largely eliminates the necessity of sample
cleanup prior to analysis.
4.0 Precision and Accuracy
Recovery studies were initiated with a wide variety of model halogenated
hydrocarbons (e.g., trifluralin, a-BHC, p-BHC, y-BHC, 2,4,5-trichlorobiphenyl,
heptachlor, aldrin, heptachlor epoxide, endosulfan, p_,p_'-DDE and dieldrin).
For 100 ml of deionized H_0 spiked and equilibrated for 19 hours at 4°C with
about 14 ng of the above halogenated hydrocarbons, a mean recovery of 70.7 ±
14.4% was obtained, while at 500 ng spiking, the recovery was 74.0 ± 21.7%.
These data are shown in Table G-l.
5.0 Apparatus
5.1 Sampling Apparatus
Water samples are taken in either 120 ml or 1.0 1 prewashed glass
bottles which were baked in an oven at 500°C and sealed with prewashed
plastic caps and Teflon liners.
165
-------
Table G-l. RECOVERY STUDIES OF EXTRACTABLE HALOGENATED HYDROCARBONS FROM WATER
Os
o\
Compound
Trifluralin
cx-BHC
B-BHC
|Y-BHC
t
2,4, 5-Tr ichlorobiphenyl
Heptachlor
Aldrin
Heptachlor epoxide
Endosulf an
£,£*-DDE
Dieldrin
Mean + Std. Dev.
Low
Trial
One
55.4
57.8
54.7
65.6
62.4
99.1
58.3
87.4
78.7
73.8
82.0
o
Fortification
Trial
Two
48.1
57.9
65.1
70.6
73.1
93.6
47.8
76.1
76.1
80.3
88.6
Average
51.8
57.8
65.1
68.1
67.8
96.4
53.0
81.8
78.7
77.0
85.3
70.7 + 14.4
Recovery (%)
High Fortification
Trial One Trial Two Trial Three Average
68.3+0.9 69.9+4.3 126.8+14.0 88.3+11.2
93.2 + 5.0 98.7 + 6.2 82.2 + 1.2 91.4 + 7.7
_
_
_
71.0+6.0 66.4+4.3 83.8+2.0 73.7+8.0
- - - -
81.1 + 1.4d 103.1 + 8.7 90.9 + 7.9 91.7 + 11.2
_
57.5+4.7 67.9+8.7 63.5+4.9 63.0+10.7
74.0 + 21.7
a!00 ml distilled/deionized water fortified with 11-15 ng of model compound in 100
, ted according to protocol.
Samples as (a) except fortification level about 500 ng.
^|Mot quantifiable due to instrumental problem.
Qnl.y one standard injection.
metVianol, extrac-
-------
5.2 Extraction Apparatus
Cylindrical separatory funnels (250 ml) equipped with glass or Teflon
stoppers and Teflon stopcocks, reciprocal shaker bath (40 oscillations/minute),
500 ml Kuderna-Danish (KD) evaporators and receiving tubes, three ball
(§
Snyder columns, reactivials . Solvents: pentane and methylene chloride,
distilled in glass. Solvents are redistilled prior to use. Reagent:
anhydrous sodium sulfate.
6.0 Procedure
6.1 Collection of Samples
Samples (120 ml) are obtained in triplicate from residential homeowners
or other appropriate sources. The first sample is collected immediately and
is analyzed for purgeable components. The second and third samples are
taken after two minutes of continuous, moderate flow. One of these is
analyzed for extractables; the other serves as a backup sample for either
purgeables, or extractables. Larger samples (1.0 1) are optionally obtained
from water and wastewater treatment plants.
6.2 Extraction, Cleanup, and Concentration
Samples are transferred to 250 ml glass cylindrical separatory funnels.
Fifteen ml of 15% methylene chloride/hexane (MeCl2/hex) is pipetted in the
funnel, the stopper fitted, and the funnel vented. Samples are placed on
the shaker bath for fifteen minutes at about 40 oscillations/minute. The
organic phase is then transferred into prewashed glass bottles with Teflon
liners. The extraction procedure is repeated once, as before, and then once
with pure hexane (no methylene chloride). Approximately 0.8 g anhydrous
Na SO, is added to the organic extract. A minimum of 30 minutes is allowed
for drying. Extracts are then transferred to 500 ml Kuderna-Danish (KD)
receiving flasks and are topped with Snyder columns. The extracts are then
concentrated to approximately 2-4 ml over a steam bath in a well ventilated
hood. The extracts and KD's are allowed to come to ambient temperature, and
the sides of the flasks are washed down with approximately 1.5 ml hexane.
The receiving vials are then removed, and the extracts are blown down with
nitrogen to about 1 ml, transferred to a reactivial previously calibrated
to a specific volume, and further concentrated to 100-300 |Jl.
167
-------
6.3 Instrumental
The detection and quantification of semi-volatile halogenated hydrocar-
bons is made using a Series 4400 Fisher/Victoreen Gas Chromatograph equipped
with a tritium foil electron capture detector. Separation is effected on a
40 m, 0.38 mm i.d., glass SCOT capillary column coated with 1% SE-30 on
0.32% Tullanox (G2,G3).. Maximum efficiency is obtained with a flow rate of
2.5 ml/min of nitrogen gas with makeup nitrogen gas adjusted to a total flow
of 25.0 ml/min, column 220°C (isothermal), and detector 285°C.
As a confirmatory column a 190 cm x 0.2 cm i.d. 1.5% OV-17/1.95% QF-1
on 80/100 Chromosorb W-HP packing is employed. Efficient responses are
obtained for flow rates of 18 ml/min at identical column and detector tempera-
tures .
Final confirmation of the identity of the components of sufficiently
concentrated extracts (generally greater than 10 ng/|Jl) can be made using
GC/MS/COMP.
The GC/MS/COMP systems used are a Finnigan 3300 GC/MS/COMP and an 1KB
2091 GC/MS equipped with an LKB 2031 data system. Chromatographic conditions
for the Finnigan 3300 are 20 m x 0.38 mm i.d., 1% SE-30 SCOT capillary
operated isothermally at 235°C and a flow rate of 2.0 ml/min helium. Split-
less injection (0.2-0.3 (Jl) is used, with standard electron impact (70 eV)
ionization conditions.
The LKB 2091 is operated using a 18 m 1% SE-30/BaCO WCOT capillary
column at 240°, isothermal for PCBs and a 40 m x 0.38 mm i.d. 1% SE-30 SCOT
capillary column at 230° isothermal for the pesticides. In both cases, the
column flow rate is 2 ml/min with 20 ml/min split off at the injector. The
mass spectrometer is operated under standard electron impact conditions.
6.4 Qualitative and Quantitative Analysis
6.4.1 Qualitative Analysis
Alternate single injections of extracts and standard solutions is the
routine procedure for processing samples. If the retention time of a given
component of an extract suggests the presence of a standard compound, a
repetitive injection is then made. Tentative identification is made if the
deviation between the two respective means is no greater than three percent.
A similar criterion is then applied to the retention times of both extract
168
-------
and standard component upon a second confirmatory column. Qualitative
identification of a component is made if both criteria are satisfied.
6.4.2 Quantitative Analysis
A mean linear response range of 5-160 pg/jjl has been established for
the compounds trifluralin and v-BHC on a 1% SE-30/0.32% Tullanox 40 m, 0.38
mm i.d. SCOT capillary column installed in a Series 4400 Fisher/Victoreen
Gas Chromatograph. Quantification of given component is made by a comparison
of the means of recorder trace areas of two extract and two standard solutions
within this linear response range. The precision of the concentration of a
given component is normally less than ten percent of the mean concentrations
and is obtained by propagation of the standard deviations of the responses
of both the extract and standard solutions. The effective concentration
multiplied by the volume of extract results in the total amount of extracted
material.
6.4.3 GC/MS/COMP Confirmation
The chromatography conditions are similar to those used for GC/ECD.
The samples for this study are to be screened by GC/ECD and confirmed (if
sufficiently concentrated) by GC/MS/COMP. Therefore, the retention times of
the two techniques must be similar. GC/ECD must operate isothermally, so
the GC/MS/COMP conditions reflect this restriction.
The Finnigan 3300 and the 1KB 2091 systems may be operated in both the
full scan and selective ion monitoring (SIM) modes. In the full scan mode,
full spectra are collected. Spectra or mass fragmentograms (single ion
plots) may be plotted for interpretation. In the SIM mode, only a small
number (up to 9 for the Finnigan 3300 and up to 16 for the LKB 2091) of ions
are monitored. Full spectra are not collected. The advantage of this
method is that the detector spends more time "looking" at the selected ion
and therefore better (generally 10-50 times) sensitivity is obtained.
To determine the limits of detection, standard solutions of selected
pesticides and PCB isomers have been analyzed on the Finnigan 3300 and LKB
2091. In the full scan mode, the limit of detection was the amount of
compound required for an interpretable spectrum. In the SIM mode, the limit
of detection was the amount of compound required to yield a peak 2-4 times
the noise level.
169
-------
The estimated limits of detection for the Finnigan 3300 and LKB 2091
are presented in Table G-2.
Quantitation using GC/MS/COMP is achieved by comparing the computer-
calculated integrated area of the unknown with the integrated response for a
known amount of standard. To compensate for differences in ionization
cross-section, the relative molar response of authentic compounds is obtai-
ned.
The calculation of the relative molar response (RMR) factor allows the
estimation of the levels of sample components without establishing a cali-
bration curve. The RMR is calculated as the integrated peak area of a known
amount of the compound, A° , , with respect to the integrated peak area of a
unK
known standard, A° , (in this case d, -pyrene) , according to the equation
A°unk/molesunk (A°unk} ^un^ (gstd)
From this calculated value, the concentration of an identified compound in
a sample is calculated by rearranging Equation 1 to give
«
(Aunk) fr^unk3 (gstd)
unk - (A) (mw) (RMR)
The use of RMR for quantitation by GC/MS has been successful in repea-
ted applications to similar research problems.
The RMRs for the compounds were calculated from the numerical inte-
grations of peaks observed in the appropriate SIM channel. Typical RMRs
listed in Table G-3 and G-4 are mean values of three injections of each of
three replicate standard mixtures.
The RMRs given here are to be regarded as typical values. Not only
must they be determined for each instrument, but day-to-day variations are
sometimes large enough to require daily calibration.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an on-going
quality assurance program is required to assure the data quality. Quality
control (QC) procedures determine artifacts, losses, etc. through a system
170
-------
Table G-2. ESTIMATED LIMITS OF DETECTION FOR EXTRACTABLE HALOGENATED
ORGANICS ANALYSIS3
Compound
trifluralin
atrazine
Y-BHC (lindane)
heptachlor
chlordane
p_,p_'-DDE
2-chlorob iphenyl
hexachlorobiphenyl
decachlorob iphenyl
LKB
Full Scan
ng/yl
12
>12<20
>12<20
12
^30°
12
*1
<1
12
2091b
m/z
264
200
181
272
375
246
188
360
498
Finnigan 3300a
SIM
ng/yl
0.4
0.4
0.10-0.4
0.10-0.4
5
>0.3
0.004
M3.016
0.42
Full Scan
ng/yl
5-10
<50
5-10
10-20
25-50
5-10
^2.5
25-50
150
SIM
m/ z
264
200
181
272
375
246
188
360
498
ng/yl
<0.5
5-10
1
1-1.5
5-10
0.5-1
MD.025
M3.15
-------
Table 0-3. RMRs FOR PCBs AND PESTICIDES OF INTEREST
TO THIS PROGRAM3
Compound
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
trif luralin
atrazine
lindane
heptachlor
£,2'-DDE
chlordane (peak 1)
chlordane (peak 2)
Concentration
104 ng/yl
3.8 ng/yl
570 ng/yl
10.4 ng/yl
1156 ng/yl
8.4 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
Ion
188
360
498
264
200
202
181
183
272
246
373
375
373
375
RMR
elutes with solvent
and was two scans wide -
not determinable
.38+3%
.35 + 10%
.14 + 7%
not determinable
1.32 + 20%
.74+7%
.25 + 8%
.74 + 9%
.62 + 12%
.74 + 6%
.45 + 6%
.71 + 5%
.65 + 5% .
.051 + 6%
.045 + 13%
Standard is d.. -pyrene (m/z = 212),
xu
172
-------
Table G-4. RMR FACTORS FOR STANDARD PCB SOLUTIONS,
SELECTED ION MONITORING MODE
Standard
RMR
m/z 188
2-Chlorobiphenyl
RMR
m/z 358
Hexachlorobiphenyl
RMR
m/z 498
Decachloroblphenyl
I PCB-STD-20
II PCB-STD-2
0.60
III PCB-STD-0.2
~j
CO
IV PCB-STD-0 .04
0.620"^
0.811 \nfrn i 1-1-1
f\ /c.a / °-6^° + -171
0.466 f —
0.643J
0.566"*^
0.840
0.637
0.597
0.705
M).699 +.171
1 . 020
0.692
0.576
}
0.763 + .257
0.257
0.291"^
\
i
0.325 + .009
0.321J
0.366*
0.293
0.301 > 0.294 + .072
0.239
0.273
0.320^
0.528 >0.
0.528J
459 + .072
0.341
0.430
0.474
0.462
0.
456 + .018
0.361 + .033
n 9R7"*\
°-287 \
0.543 > 0.401 + .142
0.372J
Standard is d.. _-pyrene
212).
-------
of blanks and controls. Quality assurance (QA) procedures monitor the
execution of the procedure and check data interpretations and-calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks con-
sist of 120 ml of prepurged distilled water in the same type of sampling
container as is used in the field. Controls consist of 100 ml of water
spiked at 10-15 ng with the compounds listed in Table G-5. These blanks and
controls are carried to the field and receive the same handling as the field
samples. Workup and analysis of field blanks and controls is interspersed
with the field samples on a regular basis. This method allows assessment of
sample storage stability.
7.1.2 Procedural Blanks and Controls
7.1.2.1 Extraction Blanks
With each set of samples, a procedural blank is run. This consists of
100 ml of prepurged distilled water which is extracted under the same condi-
tions as the samples. These blanks are designed to detect artifacts from
dirty glassware, laboratory atmosphere intrusion, and other sources.
7.1.2.2 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks, and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a standard solution is analyzed each day to serve
as a procedural control and also to update the RMR value. Thus, in a typical
working day, 4 field samples, 1 blank or control, and 1 RMR standard are
run.
The Finnigan 3300 GC/MS is a quadrupole mass spectrometer which requires
frequent tuning. Daily tuning is achieved using FC-43 and decafluorotriphenyl-
phosphine (DFTPP).
174
-------
Table G-5. SEMI-VOLATILE HALOGENATED HYDROCARBONS IN
METHANOL SPIKING SOLUTION
Compound Anit. spiked, ng
4-Chlorobiphenyl 13.2
Trifluralin 14.8
a-BHC 14.0
6-BHC 14.5
Y-BHC 15.2
4,4'-Dichlorobiphenyl 15.8
2,4,5-Trichlorobiphenyl 14.0
Heptachlor 13.6
Aldrin 14.8
Heptachlor epoxide 14.2
Endosulfan 13.1
Dieldrin 15.8
p,p'-DDE 11.6
p.p'-DDT 12.5
Endrin 11•5
175
-------
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, locations, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
procotol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data has been
produced.
176
-------
8.0 References
Gl. Thompson, J. F., Analysis of Pesticide Residues in Human and Environ-
mental Samples, A Compilation of Methods Selected for Use in Pesticide
Monitoring Programs, Environ. Toxicol. Div., Health Effects Research
Lab., USEPA, RTP, NC, December, 1974.
G2. Hines, J. W., R. Shapiro, E. Pellizzari and A. Schwartz, HRC and
CC, submitted for publication (1978).
G3. Hines, J. R., R. Shapiro, A. Schwartz, and E. D. Pellizzari, HRC and
CC, submitted for publication (1978).
177
-------
APPENDIX H
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE
HALOGENATED ORGANICS IN TISSUE
178
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE HALOGENATED
ORGANICS IN TISSUE
1.0 Principle of Method
Semi-volatile halogenated hydrocarbons are extracted from tissue samples
with organic solvents, dried, and concentrated to an appropriate volume for
quantification using GC/ECD. Identifications are confirmed by GC/ECD using
a second column and, when sufficiently concentrated, by GC/MS/COMP, Samples
are optionally subjected to liquid chromatographic cleanup on Florisil to
remove lipids if severe interferences are encountered. This procedure was
adapted from that of Thompson (HI).
2.0 Range and Sensitivity
The sensitivity of response to GC/ECD is a function of the instrument,
the compound, and the matrix from which it is extracted. The detection
limit for GC/ECD analysis is 1-5 ng/g (ppb), depending on compound and
instrumental conditions.
3.0 Interferences
Interferences in sample analysis and quantification using GC/ECD are
manifested in the electron capturing ability of the given contaminant.
Blood extracts which have not been cleaned up contain interferences which
can largely be removed by gradient liquid-liquid chromatography on 2% aqueous
deactivated Florisil and/or back partitioning with acetonitrile, as discussed
below.
4.0 Precision and Accuracy
Preliminary recovery results, shown in Table H-l indicate that recoveries
are from 60-90% for several types of tissue. Further recoveries will be
determined with individual samples as they are analyzed throughout the
program.
5.0 Apparatus
5.1 Sampling Apparatus
Samples must be collected and stored with a minimum potential for con-
tamination or loss of more volatile components. The primary cause of
contamination is from plasticizers (e.g., phthalates) in plastic and rubber.
Therefore contact with these materials must be minimized or eliminated.
179
-------
Table H-l. RECOVERY OF EXTRACTABLE HALOGENATED HYDROCARBONS
FROM HUMAN TISSUE EXTRACTS
Tissue
Adipose
Brain.
Liver
Kidney
Spleen
Lung
Percent
Extractable
Material3
84.3
4.4
1.1
1.1
1.3
0.4
Percent,
Recovery
76.6
69.7
85.1
64.5
91.2
87.5
a
Also described as "percent fat" in some procedures.
Recovery of 198 ng aldrin added to tissues after maceration and before
extraction as an internal standard. Percent recovery determined after
all analytical manipulations. Aldrin represents a suitable standard,
since it is metabolized to endrin and has not been found in tissues.
Mean recovery for adipose tissues = 79.1 + 10.6%.
Q
Analytical workup includes acetonitrile partitioning to reduce fat
content.
180
-------
Samples should be stored in glass jars with foil-lined or (preferably)
teflon-lined screw caps. The bottles must be thoroughly cleaned and oven-
treated prior to use.
5.2 Extraction Apparatus
Beakers (500-1000 ml), 500 ml Kuderna-Danish evaporators (or 10 ml
microevaporators) and receiving tubes, three ball Snyder columns, glass
@
bottles and caps equipped with Teflon liners, reactivials , centrifuge, and
22 mm i.d. chromatography columns. Solvent: hexane, distilled in glass and
redistilled prior to use. Reagents: sea sand, anhydrous sodium sulfate,
(extracted with pentane in Soxhlet extractor for 24 hr and stored in an oven
at 140°C), 60/100 mesh reagent Florisil.
6.0 Procedure
6.1 Collection of Samples
It is anticipated that tissue samples collected from cadavers or surgery
will be obtained from a pathologist. Personnel from RTI will work with
pathologists advising them of proper sample handling procedures. To be of
use for extractable halogenated organics a tissue sample must be collected a
short time following death and immediately frozen in a cleaned glass container
with as small a "headspace" as possible. Any handling or storage in contact
with polymeric materials represents potential contamination.
6.2 Extraction, Cleanup, and Concentration
Tissue fractions are analyzed for extractable halogenated organics
using a modified procedure by Thompson (HI). Approximately five grams of
each tissue is ground in a large glass beaker with acid-washed sea sand and
sodium sulfate (both cleaned up prior to use by extraction in a Soxhlet
extractor with hexane) using a glass rod until a dry, granular mass is
obtained. Aldrin, 198 ng in 100 |Jl hexane, is added to the sample as an
internal standard. Each tissue is then extracted, with vigorous grinding,
with three 50 ml aliquots of hexane for approximately 5 min each. The
extracts are then filtered, concentrated in a Kuderna-Danish (KD) apparatus
to approximately 10 ml, blown down under nitrogen to dryness at room tempera-
ture and weighed to obtain a value for the percent extractable material
("percent fat").
181
-------
The high lipid content of adipose tissue extract necessitates partition-
ing of the extract into acetonitrile and back-partitioning of the halogenated
hydrocarbons into hexane. Approximately 2.5 grams of fat extract is dissolved
in 12 ml hexane and extracted two rain each with four 30 ml aliquots of
redistilled acetonitrile. The extracts are combined with 250 ml 2% aqueous
sodium chloride and back-extracted with four 30 ml aliquots of hexane. The
extracts are combined, dried over Na9SO,, concentrated in a KD to about 5
ml, blown down under nitrogen to approximately 2 ml, and subjected to Florisil
cleanup as described below.
Florisil (60/100 mesh, activated at 130°C overnight) columns (2.2 cm x
10 cm) with glass frits or glass wool plugs are packed in hexane solvent.
Each tissue extract is transferred to the surface of the column in approxi-
mately 2 ml of solvent, and the column walls are washed with approximately 4
ml of hexane. The halogenated hydrocarbons are then eluted with 200 ml each
of 6% ether/hexane and 15% ether/hexane respectively. Each fraction is
concentrated in a KD apparatus to approximately 5 ml. The 6% eluants are
blown down under ambient nitrogen to appropriate volumes and immediately
analyzed by GC/ECD.
Previous studies by Thompson suggest the following halogenated hydro-
carbons should be found in the 6% eluant: BHC isomers, p_,p_'-DDE, p_,p_'-DDT,
heptachlor, heptachlor epoxide, mirex, PCB, hexachlorobenzene, and trifluralin
(HI). these represent the same general polarity of the halogenated compounds
of interest in tissue samples, so this fraction is of primary interest for
analysis. Extracts are dried a minimum of 30 minutes over about 0.5 g anhy-
drous Na2SO,. The extract is transferred to a 500 ml KD flask (or micro
KD), topped with a Snyder column, concentrated to ca. 2-4 ml, and cooled to
ambient temperature. The sides of the KD are rinsed with about 1.5 ml
hexane and the extract is then blown down under nitrogen to about 1 ml,
®
transferred to a reactivial previously calibrated to a specific volume, and
further concentrated.
6.3 Instrumental
The detection and quantification of semi-volatile halogenated hydrocar-
bons is made using a Series 4400 Fisher/Victoreen Gas Chroraatograph equipped
with a tritium foil electron capture detector. Separation is effected on a
182
-------
40 m, 0.38 mm i.d., glass SCOT capillary column coated with 1% SE-30 on
0.32% Tullanox (H2,H3). Maximum efficiency is obtained with a flow rate of
2.5 ml/min of nitrogen gas with makeup nitrogen gas adjusted to a total flow
of 25.0 ml/min, column 220°C (isothermal), and detector 285°C.
As a confirmatory column a 190 cm x 0.2 cm i.d. 1.5% OV-17/1.95% QF-1
on 80/100 Chromosorb W-HP packing is employed. Efficient responses are
obtained for flow rates of 18 ml/min at identical column and detector tempera-
tures .
Final confirmation of the identity of the components of sufficiently
concentracted extracts (generally greater than 10 ng/pl) can be made using
GC/MS/COMP.
The GC/MS/COMP systems used are a Finnigan 3300 GC/MS/COMP and an 1KB
2091 GC/MS equipped with an 1KB 2031 data system. Chromatographic conditions
for the Finnigan 3300 are 20 m x 0.38 mm i.d., 1% SE-30 SCOT capillary
operated isothermally at 235°C and a flow rate of 2.0 ml/min helium. Split-
less injection (0.2-0.3 |Jl) is used, with standard electron impact (70 eV)
ionization conditions.
The 1KB 2091 is operated using a 18 m 1% SE-30/BaC03 WCOT capillary
column at 240°, isothermal for PCBs and a 40 m x 0.38 mm i.d. 1% SE-30 SCOT
capillary column at 230° isothermal for the pesticides. In both cases, the
column flow rate is 2 ml/min with 20 ml/min split off at the injector. The
mass spectrometer is operated under standard electron impact conditions.
6.4 Qualitative and Quantitative Analysis
6.4.1 Qualitative Analysis
Alternate single injections of extracts and standard solutions is the
routine procedure for processing samples. If the retention time of a given
component of an extract suggests the presence of a standard compound, a
repetitive injection is then made. Tentative identification is made if the
deviation between the two respective means is no greater than three percent.
A similar criterion is then applied to the retention times of both extract
and standard component upon a second, confirmatory, column. Qualitative
identification of a component is made if both criteria are satisfied.
183
-------
6.4.2 Quantitative Analysis
A mean linear response range of 5-160 pg/pl has been established for
the compounds trifluralin and y-BHC On a 1% SE-30/0.32% Tullanox 40 m, 0.38
mm i.d. SCOT capillary column installed in a Series 4400 Fisher/Victoreen Gas
Chromatograph. Quantification of given component is made by a comparison of
the means of recorder trace areas of two extract and two standard solutions
within this linear response range. The precision of the concentration of a
given component is normally less than ten percent of the mean concentrations
and is obtained by propagation of the standard deviations of the responses
of both the extract and standard solutions. The effective concentration
multiplied by the volume of extract results in the total amount of extracted
material.
If the extracts are deep yellow, the presence of lipids may interfere
with either analysis or concentration of the extracts due to precipitation.
In this case, the sample should be transferred to a 22 mm i.d. column contain-
ing 1.6 g of 2% aqueous-deactivated Florisil and eluted with 10 ml each of
hexane, 5% MeCl2/hex, 10% MeCl2/hex, 15% MeCl2/hex, 20% MeCl2/hex, 30%
MeCl2/hex, 50% MeCl /hex, and MeCl-. The extracts are concentrated and
analyzed. Most semi-volatile halogenated hydrocarbons are expected to
appear in the first five fractions. This estimate is based upon elution
data of pesticides on Florisil (H4) and has not been subjected to full
experimental verification.
6.4.3 GC/MS/COMP Confirmation
The chromatography conditions are similar to those used for GC/ECD.
The samples for this study are to be screened by GC/ECD and confirmed (if
sufficiently concentrated) by GC/MS/COMP. Therefore, the retention times of
the two techniques must be similar. GC/ECD must operate isothermally, so
the GC/MS/COMP conditions reflect this restriction.
The Finnigan 3300 and the 1KB 2091 systems may be operated in both the
full scan and selective ion monitoring (SIM) modes. In the full scan mode,
full spectra are collected. Spectra or mass fragmentograms (single ion
plots) may be plotted for interpretation. In the SIM mode, only a small
number (up to 9 for the Finnigan 3300 and up to 16 for the LKB 2091) of ions
are monitored. Full spectra are not collected. The advantage of this
184
-------
method is that the detector spends more time "looking" at the selected ion
and therefore better (generally 10-50 times) sensitivity is obtained.
To determine the limits of detection, standard solutions of selected
pesticides and PCB isomers have been analyzed on the Finnigan 3300 and 1KB
2091. In the full scan mode, the limit of detection was the amount of
compound required for an interpretable spectrum. In the SIM mode, the limit
of detection was the amount of compound required to yield a peak 2-4 times
the noise level.
The estimated limits of detection for the Finnigan 3300 and LKB 2091 are
presented in Table H-2.
Quantitation using GC/MS/COMP is achieved by comparing the computer-
calculated integrated area of the unknown with the integrated response for a
known amount of standard. To compensate for differences in ionization
cross-section, the relative molar response of authentic compounds is obtai-
ned.
The calculation of the .relative molar response (RMR) factor allows the
estimation of the levels of sample components without establishing a cali-
bration curve. The RMR is calculated as the integrated peak area of a known
amount of the compound, A° , , with respect to the integrated peak area of a
known standard, A° , (in this case d,^-pyrene), according to the equation
_ A° ./moles , .A0 , ) (raw ,) (g _,) ,_ ,,
R = unk' unk = ( unk v unky vsstd' (Eq. 1)
A° . ,/moles . . (A° . ,) (mw , ,) (g , )
std std std std 6unk
From this calculated value, the concentration of an identified compound in a
sample is calculated by rearranging Equation 1 to give
(A ) (mw ) (g ,) (Eq. 2)
g , = unk unk std
(BWStd) (RMR)
The use of RMR for quantitation by GC/MS has been successful in repeated
applications to similar research problems.
The RMRs for the compounds were calculated from the numerical inte-
grations of peaks observed in the appropriate SIM channel. Typical RMRs
listed in Table H-3 and H-4 are mean values of three injections of each of
three replicate standard mixtures.
185
-------
Table H-2. ESTIMATED LIMITS OF DETECTION FOR EXTRACTABLE HALOGENATED
ORGANICS ANALYSIS3
LKB
2091b
Finnigan 3300a
Full Scan SIM
Compound
trifluralin
atrazine
Y-BHC (lindane)
heptachlor
chlordane
£,£*-DDE
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
ng/pl
12
>12<20
>12<20
12
.30°
12
*1
<1
12
m/z
264
200
181
272
375
246
188
360
498
ng/pl
0.4
0.4
0.10-0.4
0.10-0.4
5
>0.3
0.004
M3.016
0.42
Full Scan SIM
ng/pl
5-10
<50
5-10
10-20
25-50
5-10
.2.5
25-50
150
m/ z
264
200
181
272
375
246
188
360
498
ng/pl
<0.5
5-10
1
1-1.5
5-10
0.5-1
MD.025
.0.15
.0.3
See text for conditions.
15:1 split at injection, only 1/15 of injection is on column.
•«
"0.2 yl injected with no split.
186
-------
Table H-3. RMRs FOR PCBs AND PESTICIDES OF INTEREST
TO THIS PROGRAM^
Compound
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
trifluralin
atrazine
lindane
heptachlor
£,£' -DDE
chlordane (peak 1)
chlordane (peak 2)
Concentration
104 ng/yl
3.8 ng/yl
570 ng/yl
10. A ng/yl
1156 ng/yl
8.4 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
Ion
188
360
498
264
200
202
181
183
272
246
373
375
373
375
RMR
elutes with solvent
and was two scans wide -
not determinable
.38+3%
.35 + 10%
.14 + 7%
not determinable
1.32 + 20%
.74+7%
.25 + 8%
.74+9%
.62 + 12%
.74+6%
.45 + 6%
.71+5%
.65+5%
.051 + 6%
.045 + 13%
Standard is d1Q-pyrene (m/jz = 212)
187
-------
Table H-4. RMR FACTORS FOR STANDARD PCB SOLUTIONS,
SELECTED ION MONITORING MODE
Standard
RMR
m/z 188
2-Chlorobiphenyl
RMR
m/z 358
Hexachlorobiphenyl
RMR
m/z 498
Decachlorobiphenyl
I PCB-STD-20
II PCB-STD-2
III PCB-STD-0.2
c»
00
IV PCB-STD-0.04
0.60
0.620"^
0.811 \
0.466 /
0.643J
0.257
0.341
0.640 + .171
0.699 +.171
1.020
0.692
0.576
763 + .257
0.325 + .009
0.294 + .072
0.320
0.528
0.528
459 + .072
0.372
0.361
0.373
0.303
0.394
0.456 + .018
0.361 + .033
0.287"^
0.543 } 0.401 + .142
0.372J
Standard Is d.. -pyrene (m/z = 212).
J.U
-------
The RMRs given here are to be regarded as typical values. Not only
must they be determined for each instrument, but day-to-day variations are
sometimes large enough to require daily calibration.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an on-going
quality assurance program is required to assure the data quality. Quality
control (QC) procedures determine artifacts, losses, etc. through a system
of blanks and controls. Quality assurance (QA) procedures monitor the
execution of the procedure and check data interpretations and calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks consist
of 50 ml of distilled water in the same type of sampling container as is
used in the field. Controls consist of 50 ml of plasma spiked at 10-15 ng
with the compounds listed in Table H-5. These blanks and controls are
carried to the field and receive the same handling as the field samples.
Workup and analysis of field blanks and controls is interspersed with the
field samples on a regular basis. This method allows assessment of sample
storage stability.
7.1.2 Procedural Blanks and Controls
7.1.2.1 Extraction Blanks
With each set of samples, a procedural blank is run. This consists of
5 ml of prepurged distilled water which is extracted under the same conditions
as the samples. These blanks are designed to detect artifacts from dirty
glassware, laboratory atmosphere intrusion, and other sources.
7.1.2.2 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks, and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a standard solution is analyzed each day to serve
189
-------
Table H-5. SEMI-VOLATILE HALOGENATED HYDROCARBONS IK
METHANOL SPIKING SOLUTION
Compound Acit. spiked, ng
4-Chlorobiphenyl 13.2
Trifluralin 14.8
a-BHC 14.0
g-BHC 14.5
Y-BHC 15.2
4,4'-Dichlorobiphenyl 15.8
2,4,5-Trichlorobiphenyl 14.0
Heptachlor 13.6
Aldrin 14.8
Heptachlor epoxide 14.2
Endosulfan 13.1
Dieldrin 15.8
p,p'-DDE 11.6
p,p'-DDT 12.5
Endrin 11.5
190
-------
as a procedural control and also to update the RMR value. Thus, in a typical
working day, 4 field samples, 1 blank or control, and 1 RMR standard are
run.
The Finnigan 3300 GC/MS is a quadrupole mass spectrometer which requires
frequent tuning. Daily tuning is achieved using FC-43 and decafluorotriphenyl-
phosphine (DFTPP).
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, locations, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
191
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analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
procotol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data are archived, and what hardcopy data has been
produced.
8.0 References
HI. Thompson, J. F., Analysis of Pesticide Residues in Human and Environ-
mental Samples, A Compilation of Methods Selected for Use in Pesticide
Monitoring Programs, Environ. Toxicol. Div., Health Effects Research
Lab., USEPA, RTP, NC, December, 1974.
H2. Hines, J. W., R. Shapiro, E. Pellizzari and A. Schwartz, HRC and
CC, submitted for publication (1978).
H3. Hines, J. R., R. Shapiro, A. Schwartz, and E. D. Pellizzari, HRC and
CC, submitted for publication (1978).
H4. Sherma, J., Manual of Analytical Quality Control for Pesticides and
Related Compounds, EPA-600/1-76-017, 1976, Table 7-1.
Revised, April, 1979
192
-------
APPENDIX I
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE
HALOGENATED ORGANIGS IN SOIL AND SEDIMENT
193
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS OF EXTRACTABLE HALOGENATED
ORGANICS IN SOIL AND SEDIMENT
1.0 Principle of Method
Semi-volatile halogenated hydrocarbons are extracted from soil and
sediment samples with organic solvents, dried, and concentrated to an
appropriate volume for quantification using GC/ECD. Results are confirmed
by GC/MS/COMP. This procedure is adapted from that described for PCBs, PCNs
and pesticides (11,12).
2.0 Range and Sensitivity
The sensitivity of response to GC/ECD is a function of the instrument,
the compound, and the matrix from which it is extracted. Detection limits
of approximately 10 pg/g (ppt) are expected for soil extracts (50 g aliquots)
concentrated to 100 pi.
3.0 Interferences
Interferences in sample analysis and quantification using GC/ECD are
manifested in the electron capturing ability of the given contaminant. Any
interferences will be removed using silica gel column chromatography (11,12).
4.0 Precision and Accuracy
Recoveries from soil on a related project (13) ranged from 52-162%.
Precision and accuracy will be determined for this study using the quality
control samples during field sampling.
5.0 Apparatus
5.1 Sampling Apparatus
A garden bulb planter, trowel, or small shovel.
5.2 Extraction Apparatus
Cylindrical separatory funnels (250 ml) equipped with glass or Teflon
stoppers and Teflon stopcocks, reciprocal shaker (ca. 40 oscillations/
minute), 500 ml Kuderna-Danish evaporators and receiving tubes, three ball
Snyder columns, 1.0 1 glass bottles and caps equipped with Teflon liners,
®
precleaned glass wool reactivials . Solvents: pentane and methylene chloride,
194
-------
distilled in glass and redistilled prior to use. Reagents: anhydrous
sodium sulfate.
6.0 Procedure
6.1 Collection of Samples
Soil samples are collected by taking cores with a garden bulb planter.
The cores are placed in one liter wide mouth jars, sealed with foil lined
caps and taped shut. The sample is labeled and its location and other
pertinent data recorded on a protocol sheet.
6.2 Extraction, Cleanup, and Concentration
Composite 1/2 of top 2.5 cm of soil core and place in wide mouth jar
with other samples to be composited and cap with foil-lined cap. Agitate
vigorously to produce a homogeneous sample. A 50 g portion of the composite
is then extracted in a 1.0 1 wide mouth jar with 50 ml of diether ether
(shake for 30 minutes). Decant the ether extract through glass wool into a
Kuderna-Danish (K-D) apparatus and combine with subsequent extracts.
The soil residue is then treated with acetone (40 ml) and shaken for 20
minutes. Toluene (80 ml) is added and shaken for 10 minutes. The extract
is decanted through a glass wool plug into a 500 ml round bottom flask. The
above acetone-toluene extraction is repeated a total of 2 times and the
extracts concentrated by heating the round bottom flask with a 3 ball Synder
column attached. Final concentration (100 |Jl) is achieved under a stream of
dry nitrogen. All procedures are conducted under minimum light and samples
are stored in the dark at 5°C.
6.3 Instrumental
The detection and quantification of semi-volatile halogenated hydrocar-
bons is made using a Series 4400 Fisher/Victoreen Gas Chroamtograph equipped
with a tritium foil electron capture detector. Separation is achieved on a
40 m, 0.38 mm i.d., glass SCOT capillary column coated with 1% SE-30 on
0.32% Tullanox (14,15). Maximum efficiency is obtained with a flow rate of
2.5 ml/min of nitrogen gas with makeup nitrogen gas adjusted to a total flow
of 25.0 ml/min, column 220°C (isothermal), and detector 285°C.
As a confirmatory column a 190 cm x 0.2 cm i.d. 1.5% OV-17/1.95% QF-1
on 80/100 Chromosorb W-HP packing is employed. Efficient responses are
obtained for flow rates of 18 ml/min at identical column and detector tem-
peratures .
195
-------
Final confirmation of the identity of the components of sufficiently
concentracted extracts (generally greater than 10 ng/|Jl) can be made using
GC/MS/COMP.
The GC/MS/COMP systems used are a Finnigan 3300.GC/MS/COMP and an 1KB
2091 GC/MS equipped with an 1KB 2031 data system. Chromatographic conditions
for the Finnigan 3300 are 20 m x 0.38 mm i.d., 1% SE-30 SCOT capillary
operated isothermally at 235°C and a flow rate of 2.0 ml/min helium. Split-
less injection (0.2-0.3 fJl) is used, with standard electron impact (70 eV)
ionization conditions.
The LKB 2091 is operated using a 18 m 1% SE-30/BaC03 WCOT capillary
column at 240° isothermal for PCBs and a 40 m x 0.38 mm i.d. 1% SE-30 SCOT
capillary column at 230° isothermal for the pesticides. In both cases, the
column flow rate is 2 ml/min with 20 ml/min split off at the injector. The
mass spectrometer is operated under standard electron impact conditions.
6.4 Qualitative and Quantitative Analysis
6.4.1 Qualitative Analysis
Alternate single injections of extracts and standard solutions is the
routine procedure for processing samples. If the retention time of a given
component of an extract suggests the presence of a standard compound, a
repetitive injection is then made. Tentative identification is made if the
deviation between the two respective means is no greater than three percent.
A similar criterion is then applied to the retention times of both extract
and standard component upon a second, confirmatory, column. Qualitative
identification of a component is made if both criteria are satisfied.
6.4.2 Quantitative Analysis
A mean linear response range of 5-160 pg/(Jl has been established for
the compounds trifluralin and y-BHC on a 1% SE-30/0.32% Tullanox 40 m, 0.38
mm i.d. SCOT capillary column installed in a Series 4400 Fisher/Victoreen
Gas Chromatograph. Quantification of a given component is made by a com-
parison of the means of recorder trace areas of two extract and two standard
solutions within this linear response range. The precision of the concentra-
tion of a given component is normally less than ten percent of the mean
concentrations and is obtained by propagation of the standard deviations of
the responses of both the extract and standard solutions. The effective
196
-------
concentration multiplied by the volume of extract results in the total
amount of extracted material.
6.4.3 GC/MS/COMP Confirmation
The chromatography conditions are similar to those used for GC/ECD.
The samples for this study are to be screened by GC/ECD and confirmed (if
sufficiently concentrated) by GC/MS/COMP- Therefore, the retention times of
the two techniques must be similar. GC/ECD must operate isothermally, so
the GC/MS/COMP conditions reflect this restriction.
The Finnigan 3300 and the 1KB 2091 systems may be operated in both the
full scan and selective ion monitoring (SIM) modes. In the full scan mode,
full spectra are collected. Spectra or mass fragmentograms (single ion
plots) may be plotted for interpretation. In the SIM mode, only a small
number (up to 9 for the Finnigan 3300 and up to 16 for the 1KB 2091) of ions
are monitored. Full spectra are not collected. The advantage of this
method is that the detector spends more time "looking" at the selected ion
and therefore better (generally 10-50 times) sensitivity is obtained.
To determine the limits of detection, standard solutions of selected
pesticides and PCB isomers have been analyzed on the Finnigan 3300 and 1KB
2091. In the full scan mode, the limit of detection was the amount of
compound required for an interpretable spectrum. In the SIM mode, the limit
of detection was the amount of compound required to yield a peak 2-4 times
the noise level.
The estimated limits of detection for the Finnigan 3300 and LKB 2091
are presented in Table 1-1.
Quantitation using GC/MS/COMP is achieved by comparing the computer-
calculated integrated area of the unknown with the integrated response for a
known amount of standard. To compensate for differences in ionization
cross-section, the relative molar response of authentic compounds is obtained
The calculation of the relative molar response (RMR) factor allows the
estimation of the levels of sample components without establishing a cali-
bration curve. The RMR is calculated as the integrated peak area of a known
amount of the compound, A° ,, with respect to the integrated peak area of a
1U1K
known standard, A° , (in this case d,~-pyrene), according to the equation
197
-------
Table 1-1. ESTIMATED LIMITS OF DETECTION FOR EXTRACTABLE HALOGENATED
ORGANICS ANALYSIS3
LKB
2091b
Finnigan 3300a
Full Scan SIM
Compound
trifluralin
atrazine
Y-BHC (lindane)
heptachlor
chlordane
_p_,_p_'-DDE
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
ng/yl
12
>12<20
>12<20
12
^30°
12
^1
<1
12
m/z
264
200
181
272
375
246
188
360
498
ng/yl
0.4
0.4
0.10-0.4
0.10-0.4
5
>0.3
0.004
M).016
0.42
Full Scan SIM
ng/yl
5-10
<50
5-10
10-20
25-50
5-10
^2.5
25-50
150
m/z
264
200
181
272
375
246
188
360
498
ng/yl
<0.5
5-10
1
1-1.5
5-10
0.5-1
MD.025
MK15
M).3
See text for conditions.
15:1 split at injection, only 1/15 of injection is on column.
•»
"0.2 yl injected with no split.
198
-------
A° . /moles . (A° . ) (mw .) (g „,)
_ unk _ unk _ v unk^ unk V6std , x
- L)
_
A° _ /moles «, ~ (A° «. .) (mw _) (g . )
std' std v std' v std' V6unk
From this calculated value, the concentration of an identified compound in
a sample is calculated by rearranging Equation 1 to give
(mwunk)
(RMR)
The use of RMR for quantitation by GC/MS has been successful in repea-
ted applications to similar research problems.
The RMRs for the compounds were calculated from the numerical inte-
grations of peaks observed in the appropriate SIM channel. Typical RMRs
listed in Tables 1-2 and 1-3 are mean values of three injections of each of
three replicate standard mixtures.
The RMRs given here are to be regarded as typical values. Not only
must they be determined for each instrument, but day-to-day variations are
sometimes large enough to require daily calibration.
7.0 Quality Assurance Program
In addition to the validation procedures described above, an on-going
quality assurance program is required to assure the data quality. Quality
control (QC) procedures determine artifacts, losses, etc. through a system
of blanks and controls. Quality assurance (QA) procedures monitor the
execution of the procedure and check data interpretations and calculations.
7.1 Quality Control
7.1.1 Field Blanks and Controls
Prior to a field sampling trip, enough blanks and controls are prepared
to equal 10% each of the anticipated number of field samples. Blanks con-
sist of 50 g of soil in the same type of sampling container as is used in
the field. Controls consist of 50 g of soil spiked at 10-15 ng with the
compounds listed in Table 1-4. These blanks and controls are carried to the
field and receive the same handling as the field samples . Workup and analy-
sis of field blanks and controls is interspersed with the field samples on a
regular basis. This method allows assessment of sample storage stability.
199
-------
Table 1-2. RMRs FOR PCBs AND PESTICIDES' OF INTEREST
TO THIS PROGRAM3
Compound
2-chlorobiphenyl
hexachlorobiphenyl
decachlorobiphenyl
trif luralin
atrazine
lindane
heptachlor
£,£'-DDE
chlordane (peak 1)
chlordane (peak 2)
Concentration
104 ng/yl
3.8 ng/ul
570 ng/yl
10.4 ng/ul
1156 ng/yl
8.4 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
100 ng/yl
Ion
188
360
498
264
200
202
181
183
272
246
373
375
373
375
RMR
elutes with solvent
and was two scans wide -
not determinable
.38+3%
.35 + 10%
.14 + 7%
not determinable
1.32 + 20%
.74 + 7%
.25 + 8%
.74+9%
.62 + 12%
.74+6%
.45+6%
.71 + 5%
.65+5%
.051 + 6%
.045 + 13%
3Standard is d -pyrene (m/2 = 212)
J.U
200
-------
Table 1-3. RMR FACTORS FOR STANDARD PCB SOLUTIONS,
SELECTED ION MONITORING MODE
RMR RMR RMR
m/z 188 m/z 358 m/z 498
Standard 2-Chlorobiphenyl Hexachloroblphenyl Decachloroblphenyl
I PCB-STD-20 0.60
II PCB-STD-2 0.620"^
0*811 \ n f/n i 111
r\ IC.L / 0-640 + .171
0.466 f —
0.643J
III PCB-STD-0.2 0.566*^
0.840 1
0.637 )0.699 +.171
> 0.597 f
0.705 J
IV PCB-STD-0.04 1.020"^
0.692 } 0.763 + .257
0.576J
0.257
0.291^
°Q'mll* V 0.325 ±.009
0.321 J
0.366"^
0.293 1
0.301 > 0.294 + .072
0.239 f
0.273J
0.320"^
0.528 i 0.459 + .072
0.528 J
0.341
0.430"^
0.474 ln .,, .
0.462 >°'456±
0.431 J
0.372"^
0.361 1
0.373 ) 0.361 +
0.303 f
0.394J
0.287*^
0.543 ) 0.401 +
0.372 J
.018
.033
.142
Standard is d.. n~pyrene
= 212).
-------
Table 1-4. SEMI-VOLATILE HALOGENATED HYDROCARBONS IN
METHANOL SPIKING SOLUTION
Compound Amt. spiked, ng
4-Chlorobiphenyl 13.2
Trifluralin 14.8
a-BHC 14.0
B-BHC 14.5
Y-BHC 15.2
4,4'-Dichlorobiphenyl 15.8
2,4,5-Trichlorobiphenyl 14.0
Heptachlor 13.6
Aldrin 14.8
Heptachlor epoxide 14.2
Endosulfan 13.1
Dieldrin 15.8
p.p'-DDE 11.6
p.p'-DDT 12.5
Endrin 11.5
202
-------
7.1.2 Procedural Blanks and Controls
7.1.2.1 Extraction Blanks
With each set of samples, a procedural blank is run. This consists of
50 ml of prepurged distilled water which is extracted under the same condi-
tions as the samples. These blanks are designed to detect artifacts from
dirty glassware, laboratory atmosphere intrusion, and other sources.
7.1.2.2 GC/MS Procedural Control
At the start of each working day, a mixture of 2,6-dimethylphenol,
2,6-dimethylaniline, and acetophenone (PA mixture) is analyzed to monitor
the capillary GC column performance. This also serves to check the mass
spectrometer tuning.
Field samples, field controls, field blanks, and procedural blanks are
queued up for GC/MS analysis such that at least one QC sample is run each
working day. In addition, a standard solution is analyzed each day to serve
as a procedural control and also to update the RMR value. Thus, in a typical
working day, 4 field samples, 1 blank or control, and 1 RMR standard are
run.
The Finnigan 3300 GC/MS is a quadrupole mass spectrometer which requires
frequent tuning. Daily tuning is achieved using FC-43 and decafluorotriphenyl-
phosphine (DFTPP).
7.2 Quality Assurance
7.2.1 Supervision and Monitoring of Activities
There are three levels of quality assurance (QA). The primary quality
assurance is the person conducting the sampling and/or analysis. This
person must be aware of their actions, observe events which may effect the
data, and maintain appropriate records. At the second level, the chemist's
supervisor monitors their daily activities, reviews the notebook, checks
data and calculations, and assists in "troubleshooting" problems. At the
tertiary level, a QA coordinator interviews all personnel on the project.
The interviews cover the operations they perform (precisely), the data they
obtain, a spot-check of their calculations, and any problems they have had.
203
-------
7.2.2 Documentation
7.2.2.1 Chain of Custody
From the initial preparation of a sample container through reporting of
the analytical results, each sample is accompanied by a chain of custody
sheet. Each person signs in the time of receipt, operations performed, and
transmittal of the sample. This record is important for tracing a contaminant,
bad standard, or some other problem.
7.2.2.2 Sampling Protocol Sheets
When a sample is collected, a sampling protocol sheet is filled in
which contains a discrete sample code which identifies project number, area,
site, locations, trip number, sampling period, and sample type. Also included
are sample times, volumes, addresses, meteorology, and other pertinent
information. Where appropriate, a map is made to precisely identify the
location.
7.2.2.3 Sample Log
Upon return from a sampling trip, each sample code is entered into a
sample log book. This log is updated as samples proceed through workup and
analysis. Thus, at a glance, project personnel can tell the status of each
sample and find out how many are at different stages in the analytical
procotol.
7.2.2.4 GC/MS Log
Each sample run by GC/MS is logged into a notebook, detailing analysis
conditions, where the data is archived, and what hardcopy data has been
produced.
8.0 References
II. Goerlitz, D. F. and L. M. Law, J. Assoc. Offic. Anal. Chem., 5J7, 176
(1974).
12. Erickson, M. D., R. A. Zweidinger, L. C. Michael and E. D. Pellizzari,
Environmental Monitoring Near Industrial Sites: EPA 560/6-77-019
(July 1977), 266 pp.
13. Pellizzari, E. D., R. A. Zweidinger and M. D. Erickson, Environmental
Monitoring Near Industrial Sites: Brominated Chemicals. Part II:
Appendix, EPA 560/6-78-002, p. 99.
204
-------
14. Hines, J. W., R. Shapiro, E. Pellizzari and A. Schwartz, HRC and
CC, submitted for publication (1978).
15. Hines, J. R. , R. Shapiro, A. Schwartz, and E. D. Pellizzari, HRC and
CC, submitted for publication (1978).
Revised, April, 1979
205
-------
APPENDIX J
ANALYTICAL PROTOCOL: CARCINOEMBRYONIC ANTIGEN ASSAY
206
-------
ANALYTICAL PROTOCOL: CARCINOEMBRYONIC ANTIGEN ASSAY
1.0 Principle of the Method
A radioimmunoassay (RIA) based on the indirect CEA-Roche assay is
presented. In order to detect the low titres of CEA in plasma a perchloric
acid extraction is used to remove interferences . The perchloric acid is
dialyzed against a buffer before the RIA is performed. The RIA procedure
125
consists of sequential addition of CEA-antiserum, I labeled CEA and
zirconyl phosphate gel (Z-gel). The Z-gel binds the antiserum-CEA complex
125
(CEA plasma + CEA [I ]) and determination of the I in the Z-gel by
gamma scintillation counting can be related to the plasma CEA by calibration
standards.
2.0 Range and Sensitivity
The indirect assay is sensitive from M3.5 ng/ml to <20 ng/ml. At
higher titres a direct assay may be used (see manufacturers literature).
3.0 Interferences
The indirect assay was designed to contend with interferences. Interpre-
tation of the origin of elevated CEA levels is a separate issue.
4.0 Precision and Accuracy
The precision and accuracy are to some extent a function of the level
since response is non-linear. Typically precision is ±0.5 ng/ml from 0-3
ng/ml and ^±1 ng from 3 to 12 ng/ml.
5.0 Apparatus
Gamma Scintillation Spectrometer
2.5 ml Dispenser: available from Roche Diagnostics for dispensing of
Z-gel
Mixer: Vortex-type mixer
Centrifuge: Capable of generating at least 1000 x g with horizontal-
head centrifuge; do not use angle-head type.
6.0 Materials
Vacutainer tubes (Becton, Dickinson and Co. Nos. 4759 or 4727) or
equivalent.
207
-------
CEA-Roche Kit
The CEA-Roche Kit contains the following reagents in excess to assure
sufficient material for at least 100 tubes or for approximately 40 patient
plasma samples assayed in duplicate by the indirect assay with necessary
controls.
This kit supplies:
Item Contents Quantity
CEA Antiserum
CEA Standard
125
I-CEA
Zirconyl Phos-
phate Gel
(Z-gel)
EDTA Buffer
goat antiserum adsorbed with blood
group A human plasma and saliva
from a blood group A positive secre-
tor diluted in sodium borate buffer
containing thimerosal 1:10,000
125 ng of CEA activity/ml in sodium
borate buffer containing 10% blood
group A human plasma and thimero-
sal 1:10,000
approximately 50 ng of activity/ml
in sodium borate buffer containing
10% blood group A human plasma
and thimerosal 1:10,000
in ammonium acetate (0.1 M acetate)
solution and thimerosal 1:10,000,
pH 6.25. Do not use Z-gel which
has been frozen since the material
will .not be uniformly dispersed and
will settle rapidly
stock solution with bovine serum
albumin and sodium azide 0.17% as
preservative. NOTE: This is to
be diluted 1:10 in distilled or
deionized water just prior to use
(final pH should be 6.5 ± 0.1).
It is important to check quality of
water before dilution, since if pH
is not 6.5 (±0.1), solution must
be discarded.
One 2.5 ml vial
(sterile)
One 1.25 ml vial
(sterile)
One 2.5 ml vial
(not sterile)
One 250 ml bottle
(not sterile)
One 25 ml vial
(sterile)
Other materials required:
Test Tubes:
Disposable test tubes
glass or plastic test tubes can be used; however,
user should be consistent. Do not mix glass and
plastic as inconsistency of results might occur;
1.5 x 15 cm or 1.2 x 12 cm test tubes are recom-
mended. If a smaller tube is used, it will be
necessary to cap the tubes to avoid spillage
during the vortex-mixing step, especially after
the addition of Z-gel.
208
-------
Pipettes:
10 pi, 25 pi, 50 Ml,
100 Ml, 500 Ml micro-
pipettes
Automatic pipetter
Dialysis Bags:
Reagents:
Item
12 M Perchloric acid:
Use: add cold to pre-
pared plasma for
precipitating plasma
proteins
Normal Saline
Solution (0.85% or
0.9%):
Use: to dilute
plasma samples
0.01 M Acetate
Buffer, pH 6.5
(±0.2):
Use: in the final
step of dialysis
with an accuracy of ±1%
0.1 M Acetate Buffer,
pH 6.25 (±0.05):
Use: final 0.1 M solu-
tion is used in the wash
step of assay
2.5 ml volume - 2 required for perchloric acid
and Z-gel.
available presealed from Roche Diagnostics
Pore size 15,000 mw; at least 2 cm diameter
and cut to 12" length
Approximate
Quantity
Needed/Kit
0.25 liter
0.25 liter
1.0 liter
0.5 liter
Directions For
Making
add 108 ml concentrated (70
to 72%) perchloric acid to
892 ml distilled or de-
ionized water; mix carefully
and store at 4°C for not
longer than one month
add 8.5 or 9 gm NaCl to
distilled or deionized
water; bring volume up to
1 liter and store at room
temperature
add 1 ml of stock ammonium
acetate solution to 250 ml
deionized water (final con-
centration 0.01 M ammonium
acetate); after at least 3
hours to assure mixing and
equilibrium of dialysis,
final pH of both samples and
dialysate should be 6.5
(±0.2).
dissolve 2.85 ml glacial
acetic acid in approximately
400 ml of distilled or
deionized water; add concen-
trated ammonium hydroxide
until pH 6.25 is obtained;
add distilled or deionized
water to bring volume to
500 ml; store for 6 weeks
at room temperature
209
-------
Approximate
Quantity Directions For
Item Needed/Kit Making
Distilled or deionized 250 ml for each
water (of not less than dialysis bag
1 x 106 ohms):
Directions for Making 2.5 M Ammonium Acetate Stock Solution:
Add 142.3 ml concentrated acetic acid to 600 ml distilled or deionized
water; gradually add 140.0 ml of concentrated ammonium hydroxide while
stirring constantly. Upon cooling, add concentrated ammonium hydroxide
until pH is 6.8 to 7.0; add distilled or deionized water to make 1 liter and
recheck pH; store in air-tight container at room temperature not longer than
six weeks. Observe visually for growth before each use.
Important: 1 ml of this solution diluted 1:250 should be checked to be
certain that a final pH of 6.5 ± 0.2 will be obtained in the dialysis bath.
If the pH of the 0.01 M acetate falls below 6.5, adjust pH of stock ammonium
acetate with concentrated ammonium hydroxide - do not exceed 7.0 pH. If pH
of stock solution exceeds 7.0, discard. Do Not adjust with acetic acid.
7.0 Procedure
7.1 Sample Collection, Storage and Shipping
Blood specimens to be analyzed for presence of carcinoembryonic antigen
are collected in 7 ml or 10 ml standard Vacutainer tubes (Becton, Dickinson
and Company, catalog numbers 4759 or 4727) or equivalent, containing 15 mg
EDTA (ICJ and 20 |jg potassium sorbate in a 0.1 ml solution. The tubes
should be filled to exhaustion of vacuum to ensure proper ratio of anti-
coagulant to blood. Mix by gentle inversion several times.
The blood-must be centrifuged at 1000 x g for 30 minutes within six
hours after collection if left at room temperature and within 12 hours if
refrigerated at 4°C. The supernate plasma must be immediately withdrawn
using a disposable Pasteur pipette. This plasma must be refrigerated at 4°C
to 8°C until the time of shipment to the laboratory. If the plasma will not
be analyzed within two weeks, the sample should be frozen.
210
-------
7.2 CEA-ROCHE Indirect Assay
When setting up a run of the CEA-ROCHE indirect assay, patient specimen
should be processed in duplicate. Each run should be accompanied by four
(4) control specimens and five (5) concentrations of standards.
Example Number of Tubes
20 patient samples run in duplicate 40
4 control specimens run in duplicate
0 to 2.5 ng/ml 2
2.6 to 5.0 ng/ml 2
5.1 to 10.0 ng/ml 2
>10 ng/ml 2
5 concentrations of standards run in duplicate
0 ng of CEA activity 2
1.25 ng of CEA activity 2
3.125 ng of CEA activity 2
6.25 ng of CEA activity 2
12.5 ng of CEA activity 2
Total Number of Tubes Run 58
7.2.1 Perchloric Acid Extraction of Plasma
Pipette 0.5 ml aliquots of plasma sample or control specimen (in dup-
licate) into 1.5 x 15 cm or 1.2 x 12 cm test tubes. Add 2 ml of 0.85% of
NaCl solution to each tube and mix contents thoroughly on Vortex-type mixer.
Add 2.5 ml of 1.2 M cold perchloric acid (PCA) to each tube to precipitate
the protein. This reagent should be pipetted only with an automatic pipet-
ter. It should not be dispensed with a mouth-type pipette.
Immediately after the addition of perchloric acid (PCA), mix the con-
tents for 30 seconds vigorously on a Vortex-type mixer. Any time delay
between PCA addition and mixing can result in an inconsistent denaturation
process. Allow tubes to stand for 5 minutes before centrifuging. Centri-
fuge tubes for 20 minutes at 1000 x g. Occasionally a turbid sample may be
encountered in the perchloric acid extracts, particularly from patients
having liver malfunctions. Samples should be processed in a normal manner.
If all samples are turbid, centrifuge for an additional 10 minutes.
211
-------
After centrifugation, a white pellet of precipitated protein at the
bottom of the tube and a supernatant fluid are obtained.
7.2.2 Dialysis
Dialysis bags should be separated and allowed to soak in deionized
water for a minimum of one hour before use. Dialysis bags held for over 18
hours in deionized water before use should be discarded.
Decant supernatant fluids into prewetted dialysis bags. Squeeze excess
water from the inner and outer surfaces of the dialysis bag prior to decanting
the extracted sample. Tie dialysis bags using double knots, and label. To
prevent floating, avoid trapping air in the dialysis bag before knotting.
Air can also retard osmosis while dialyzing. The tagged dialysis bag contain-
ing the supernatant fluid is now ready to be placed in the dialysis unit.
Load dialysis bag into the dialysis unit. The bags must be submerged
below the top knot throughout the entire dialysis procedure. During dialysis,
some device should be used to hold each bag in place or label strings will
entangle.
Important: At no time during dialysis should metal be allowed to come
in contact with the dialysis water as ions would be added thus affecting the
results of the assay.
To perform dialysis step without the automatic dialysis unit, one
method may be to use an appropriate size beaker with a magnetic stirrer to
assure circulation of water and buffer solution. Routine laboratory methods
of dialysis are acceptable as long as the volume of dialysis fluid is 50
times the total volume of extracts within the container (250 ml of water is
required per bag for each dialysis step). The water should be circulated
during the dialysis. If air is used to agitate the water, it should be
filtered to remove oil or solids.
Four complete changes of water are required with a minimum of 3 hours
between changes. (Dialysis may be run for longer periods without adverse
reactions). Dialysis should be performed with water changes as recommended.
After being dialyzed against deionized water, dialyze bags using 50
volumes of 0.01 M ammonium acetate solution having a pH of 6.5 for a minimum
of 3 hours and not more than 24 hours. A suggested method for performing
this final dialysis is to make an accurate measurement of the dialysis bath
212
-------
fluid volume. Fill to the appropriate volume with water and add a predeter-
mined quantity of 2.5 M buffer to the bath so that dilution of the concentra-
ted buffer with the bath water will equal 0.01 M. (4 ml of 2.5 M ammonium
acetate is required for each 1000 ml of water). If the final pH of the
dialysis bath, after addition of the buffer, does not read pH 6.5 ± 0.2, DO
NOT adjust the bath with concentrated acid or base since this will only
increase the ionic strength above 0.01 M. A dialysis bath that does not
give a pH of 6.5 ± 0.2 should be discarded and samples redialyzed with the
buffer (stock) readjusted to give a final pH of 6.5 ± 0.2 in the dialysis
bath. Any pH adjustments must be made to the 2.5 M stock buffer only.
Concentrated ammonium hydroxide may be used to adjust the 2.5 M buffer.
Acetic acid cannot be used; if the pH of the 0.01 M buffer is above the
desired level, the 2.5 M buffer must be discarded and remade.
If the ionic strength or pH of the dialysis buffer is too high, falsely
elevated plasma CEA values will be obtained. Dialysis buffer of too low
ionic strength or low pH will result in plasma CEA values which are too low.
Remove dialysis bags from the dialysis unit and cut open. The pH of
the final dialysis bath should be checked and a pH check of random samples
made after dialysis with a pH meter. The pH of the sample must be 6.5
(±0.2). If samples do not have this pH, they should be redialyzed in the
final buffer. The difference between the pH of the dialysate and dialysis
bath should not vary more than 0.2 pH units. If the variation is greater
the dialysis should be continued.
Transfer entire contents of dialysis bags to 1.5 x 15 cm or 1.2 x 12 cm
test tubes for determination of CEA content. A low or high volume in the
test tube following transfer from the dialysis bag may be indicative of a
leaking dialysis bag. When this is encountered, one should be alert to the
possibility of a low value.
NOTE: Assays should be performed the same day the contents are removed from
the dialysis bags.
7.2.3 RIA Assay of CEA
These extracts are now ready to be assayed for CEA content. In order
to determine the CEA content of the plasma extract, it is necessary to
prepare a standard inhibition curve utilizing the CEA standard in the
213
-------
CEA-Roche kit. This curve must be prepared at the same time that each bath
of plasma extracts is assayed.
Preparation of Standards:
1. Dilute an aliquot of stock EDTA buffer (kit component) 1:10 (1 volume +
9 volumes) with distilled or deionized water just prior to use - final
pH should be 6.5 (±0.1). It is important to check quality of deionized
water before dilution, since if pH is not 6.5 (±0.1) solution should be
discarded. A second aliquot should then be prepared with deionized
water. Keep tightly closed. No need to adjust pH of reconstituted
versene buffer.
2. Label 5 pairs of 1.5 x 15 cm or 1.2 x 12 cm test tubes with CEA stan-
dard dose level to be used in each tube. (It is suggested that 0, 10,
25, 50 and 100 |Jl of the standard, which is equivalent to 0, 1.25,
3.125, 6.25 and 12.5 ng of CEA activity, be used).
3. Add 5 ml of diluted stock EDTA buffer to each tube.
4. Add proper amount of CEA standard (10, 25, 50 and 100 |Jl) to each tube,
and mix contents of tubes on Vortex-type mixer. From this point the
tubes are treated the same as the tubes containing the plasma extracts.
Assay of Extracts for CEA Content:
Treat both sets of tubes - those containing the plasma extracts and
those set up for preparation of the standard curve - in exactly the same
manner.
In the following steps (addition of antiserum, isotope-labeled CEA, and
Z-gel) it is essential that the following sequence be maintained: (1) the
time required for the addition of any reagent must be the same for all [no
more than 60 seconds]; (2) the sequence of adding reagent to the tubes in
the rack must be consistent. The most critical step is the incubation time
following the addition of the isotope-labeled CEA. Tubes should be batched
as one person cannot handle a large number of tubes at one time since the
time for dispensing reagent and mixing is limited to one minute. The addition
of Z-gel will stop most of the reaction if dispensed in a manner to intermix
with the contents in the tubes. This allows adding Z-gel to all the tubes
before vortex mixing. Low plasma CEA values will result if the material for
the inhibition assay is incubated for a shorter period than the extracts of
214
-------
plasma. High plasma CEA values will result if the material for the inhibition
assay is incubated for a longer period than the plasma extracts.
Add 25 pi of CEA antiserum to each tube. Either an automatic diluter
or pipette may be used. Mix contents on a Vortex type mixer. Place tubes
in a 45°C (±1°) circulating water bath for 30 (±1) minutes. If water bath
is covered, stopper tubes to prevent water from condensing inside tubes.
12S
Remove tubes from 45°C water bath and add 25 |Jl of I-CEA to each.
Mix contents with a Vortex-type mixer. Return tubes to water bath for
another 30 (±1) minutes. Remove tubes and immediately add 2.5 ml of zirconyl
phosphate gel (Z-gel) to each tube. It is important that the Z-gel be
shaken vigorously before use to assure homogeneity and added rapidly with an
automatic dispensing instrument so that the reaction is kept constant in all
125
tubes. At this point the I-CEA bound to the antibody is separated from
125
the excess free I-CEA. Vortex and centrifuge immediately at 1000 x g for
5 minutes (tubes smaller than 1.2 x 12 cm must be covered before mixing).
Gently decant supernatants into an isotope waste container, and blot
tube tops with paper towels before returning tubes to upright position.
Add 5 ml of pH 6.25 ammonium acetate (0.1 M acetate) solution, to each tube.
Completely disperse gel by using a Vortex-type mixer. Careful attention to
mixing at this time is necessary to assure proper washing. Centrifuge again
at 1000 x g for 5 minutes. Again gently decant supernatants into an isotope
waste container and blot tube tops with paper towels.
When gently decanting the supernatant fluid from the Z-gel pellet, care
should be taken so that once the tube is inverted during decanting it remains
in the inverted position for blotting. This single inversion preserves the
Z-gel pellet configuration. Do not shake the tubes since this will force
the Z-gel up the sides of the tube and cause scintillation counting errors.
Wash outside of tubes by dipping rack of tubes into a clean water bath.
Wipe tubes individually prior to positioning in the counter. This step pre-
vents erroneous counts and counter contamination by any isotope contamination
on the outside tube wall. The Z-gel pellet is now ready to be counted.
Counting:
125
Determine the amount of bound I-CEA by placing tube in a gamma
scintillation spectrometer and counting for one minute. This information
215
-------
may be recorded by computer. A typical print-out shows the specimen number
in the first column, the time it was counted in the second column, and the
counts per minute in the third column. This information is used to construct
125
a standard curve - plot the mean CPM of I-CEA bound to the Z-gel against
the antigen dose in each tube (see Figure J-l). Duplicates should be within
5% of the mean CPM.
8.0 Calculations and Interpretation
The CEA content of each extract is determined from a graph prepared by
plotting CPMs vs. ng of CEA in standards. Values must be multiplied by two
to convert to ng of CEA/ml of plasma. CPM values greater than the 0 antigen
dose are reported as 0 ng of CEA/ml of plasma. Values above 20 ng/ml obtained
by this indirect assay are not quantitatively accurate.
References
Jl. "Procedure Manual for CEA-ROCHE Carcinoembryonic Antigen Assay" Roche
Diagnostics, Division of Hoffmann-La Roche Inc., Nutley, NJ 07110.
216
-------
45
E
I 40
a
a 35-]
25H
20
»
TYPICAL INHIBITION CURVE OF CEA STANDARD
INDIRECT ASSAY
90,000 CPM/TUBE (25 >.! ml-CEA)
Counter Efficiency 47% (CPM must
be a strictly asymptotically decreas-
ing function of CEA antigen)
Unknown plasma extract
- 6.25 ng/0.5 ml plasma x 2
= 12.5 ng/ml plasma
Do not use in lieu
of standard curve
prepared at time
of assay
1.25 3.125 6.25
ng CEA STANDARD/TUBE
12.5
Sample calculation:
0.5-ml sample result 23,500 CPM = 6.25 ng CEA
6.25 ng CEA/0.5 ml x 2 = 12.5 ng CEA/ml
Figure J-l. Typical inhibition curve of CEA standard indirect assay.
217
-------
APPENDIX K
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS PROCEDURE
FOR BREATH SAMPLES
218
-------
ANALYTICAL PROTOCOL: SAMPLING AND ANALYSIS PROCEDURE
FOR BREATH SAMPLES
1.0 Principle of the Method
The breath sample is collected on Tenax GC using a specially designed
spirometer and low hydrocarbon air "ultrapure" or equivalent. The Tenax
cartridge is then dried over CaSO, and analyzed by thermal desorption into a
gas chromatograph with a SCOT capillary column and mass spectrometry (GC/MS).
2.0 Apparatus
Spirometer - as diagrammed in Figure K-l. The valves in the Douglas
mouth piece must be replaced with Tedlar or similar material. A bubbler
filled with distilled deionized water is placed inline with the air tank to
humidify the air for subject comfort.
The gas chromatograph/mass spectrometer/computer and thermal desorption
are described in Appendix A.
3.0 Materials
Tenax GC cartridges
Culture tubes
CaSO, anhydrous and hydrocarbon free (treated in a muffle furnace
at 400°C for 2 hrs)
Ultrapure air
4.0 Procedure
The subject is seated in a comfortable chair and the mouthpiece height
adjusted to a convenient level. A long spring clamp is used to seal the air
flow from Bag A to the mouthpiece. A plug is placed in the mouthpiece
opening until the test begins to prevent room air contamination. Air flow
from the air tank is started and when the 50 1 Bag A is about half full, the
mouthpiece is attached, the clamp and plug removed and the subject may begin
to breathe on the apparatus. The nose clips must also be in place at this
time. After a minute or two, the Nutech sampler pump is started with the
flow at approximately 7 1/min. The flow may be adjusted to match the indivi-
dual subject's respiration rate. It is useful to furl Bag B using spring
clips to avoid using this bag as an exponential dilutor but retaining the
safety factor of the 50 1 bag capacity. After a predetermined volume (75 1)
219
-------
0.
a.
NJ
10
ULTRAPURE
AIR TANK
TEFLON
CONNECTORS
TEDLAR BAG A
^
TENAX GC
CARTRIDGES
TEDLAR BAG B
DOUGLAS
VALVE AND
MOUTHPIECE
Figure K-l. Schematic of Spirometer for collection of breath samples.
-------
of breath has been sampled, the test is terminated. The subject is removed
from the apparatus and the nose clips removed from the subject. The Tenax
cartridges are removed and stored in culture tubes. The entire apparatus is
then flushed with pure air to decontaminate it for the next use. This is
best done by successively filling and evacuating the bags. The mouthpiece
is sterilized by placing in alcohol after each use. The Tenax cartridges
are desiccated over CaSO, before analysis by placing "*2 g in the bottom of a
culture tube and covering with glass wool. The cartridge is then sealed in
the culture tube for a minimum of 1 hr.
Analysis of the Tenax cartridge performed as for air samples (Appendix
A).
221
-------
APPENDIX L
GC/MS/COMP LIMIT OF DETECTION DATA: MASS SPECTRA,
ION TRACINGS, AND SIM PLOTS
222
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chosen for SIM analysis.
240
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se-
48-
30-
28-
jaa.
tee-
aa-
ei 9.59 MINUTES
eOCKCTOUND SUBTSOCTCO
48B
CHLORDDNE.
0 388
-9. l.BXV..3nCL. 85-83-78
98-
88-
re-
38-
29-
In.
338
MISS SPECTRUM 248 7.89 MINUTES
BDCKGROUND SUBTRACTED
4>a 438 sae
CHLORMNE.SE3B-48n-228>C.-9.1.8KV..3HCL.83-83-78
Figure L-17. Analysis of chlordane (40 m SCOT capillary, SE-30);
m/z_ 373 chosen for SIM analysis.
241
-------
iea.8-1
80. fl-
9.8-
28.8-
• 378* 5 '
TIC*IB
H£PTftCHLOR.SE38-4Bn-22B»C.-9.1.8KV, .2.85-03-78
1 158
MPSS SPECTRUM 148 4.93 MINUTES
BftCKGROUND SUBTRflCTEO
200 230 300
HEPTSCHLOR.SE30-40n-22B*C.-9.1.8KV..2MCU 05-03-78
Figure L-18.
Analysis of heptachlor (40 m,SCOT capillary- SE-30);
m/_z 272 chosen for SIM analysis.
242
-------
iee.a-i
89.8-
se.a-
40.8-
28. B-
.8-
a s IB
P. P-DDE. SE30-48rt-220»C.-9.1.8KV..2MCL.8S-83-?8
- 318*
... TIC*
100-,
9B-
8B-
7B-
68-
SB-
48-
3B-
29-
10-
80-
ILlLl
JL
.I,,..
IBB itea 20B 2 a 36
MftSS SPECTRUM 288 9.59 MINUTES P.P-DDE.S638-48M-228»C.-9.1.8KV..2rXL.BS-83-ra
BACKGROUND SUBTRACTED
Figure L-19. Analysis of £»£'-DDE (40 m SCOT capillary, SE-30);
m/_z 246 chosen for SIM analysis.
243
-------
468,9*1
88. fl-
a, a-
48.B-
28. B-
- 335»
- TlC*l
TRIFLORALINE.SE38-48M-228*.-9. 1.8. .2.85-83-78
. 188-
98-
88-
78-
68-
sa-
38-
28-
18-
88-
1
MASS
188-
98-
88-
78-
68-
58-
48-
38-
28-
18-
88-
1
,Ml
iiiiiii
•
iii.jii.i
II ll
1
IP .
III I III
1. Jlll.ll, . ...IN.I
18 ' ' 158 ' ' 2a'0
SPECTRUM 74 2.39 MINUTES TRIFLORALIN
BACKGROUND SUBTRACTED
III
illlj
'9
LpUl
111,11
' 158
1
(null li
I""1"'!
(l7,|tl|, |r|l|(
aa'e
, ,!!l|i"i i ,, n •
Ui....niilli.
1 . 1 ...
1, 1
258 388 358
.SE38-48M-228»C>-9. 1 .BKV. .2MCL. 85-83-78
1 i - • - i
'"i|""l""i^"i i i r ' i ' i i ' i ' i
388 350
OSES SPECTRUM 75 2.43 MINUTES
BACKGROUND SUBTRACTED
TR!FLORAL IN.SE38-48M-228*C.-9.1.BKV..2MCL,85-B3-7B
Figure L-20. Analysis of trifluralin (40 m SCOT capillary, SE-30);
m/_z 306 chosen for SIM analysis.
244
-------
IBB. 8-1
68.8-
28.8-
- 213* 1
- TIC* 1
ftTRflZINE.SE38-48M-228*C.-B.2KV..2MCL.85-B2-7B
IB91
9B-
80-
78-
60-
5B-
3B-
28-
00.
a-"- -r-^-,--^-!-- , ...-
IBB 150
nftSS SPECTRUM 84 2.BS niNUTES
BACKGROUND SUBTRACTED
288 258
STRflZ IME. SE38-4Btt-22B*C.-8. 2KV.. 2HCL. 35-82-78
Figure L-21. Analysis of trazine (40. m SCOT capillary, SE-30); m/z 215
chosen for SIM analysis.
245
-------
tea. e-,
SB.9-
40.0-
20.0-
III
II •
IV
I Trifluralin
II Atrazine
III Y-BHC
IV Heptachlor
V-VI Chlordane
VII £,2.'-DDE
VII
v vi.';
- 318« 1
.8
a
•^ i^M
' ^« i mi
* ^f *^w*
5
1568-STD-5aa . SE30-48n-22a». -9
180-
98-
88-
78-
sa-
58-
43-
38-
28-
18-
00-
1
100-
98-
80-
78-
60-
58-
48-
38-
28-
18-
88-
aa"
,
,.|ll,
Illi H
ill
>V
••'•Y i '
IB
.1.8. .2.05-83-78
In ill llllil i, illlll Hill,
288
1 tlll|llll, III! till nil
250
Trifluralin
300
358
MftSS SPECTRUM 73 2.43 MINUTES
BACKGROUND SUBTRACTED
9B 450 508
1S6B-STD-500.SE3e-48M-22B»C, -9.1.B..3MCL.85-83-78
Figure L-22. Analysis of pesticide mixture, full scan mode;
^500 ng.
246
-------
78-
68-
3B-
Atrazine
.s
238
360
1BB-
98-
88-
78-
CB-
sa-
«-
38-
28-
18-
mss SPEcroun as 2.86 CIINUTES
BOCKCROUND SUBTRACTED
488 ' "^
156B-STB-HB.SE3'
156 388
•228«C.-9. I.e. .3rCU83-83-76
38-
28-
1B-
68'
Y-BHC
86-
76-
mSS SPECTRUM 97 3.23 MINUTES
BOCKCROUND SUBTROCTEO
1MB-STP-5W. ItCJB
-------
INr
ie-
m-
78-
68-
SB-
IUII ln.,,1, Ilii
Heptachlor
78-
68-
58-
48-
38-
28-
ee-U-r
'3V ' ' '" U. 45. ' ' ',88
SPECTRUM 147 4.89 MINUTES l56e-STD-5e8.SE38-«8M-Z2e«C.-9. 1.8. .3nCL.85-83-78
BACKGROUND SUBTRACTED
189-
98-
88-
78-
68-
58-
48-
38-
28-
18-
1
1,1
II,
i
1
llnl
il
1
lie
|
,|
1,11
288
'
,11 i
'
Chlordane
il
,11 ill mil
2ia ' ' 388
88-
78-
68-
58-
48-
38-
28-
te-
88'
11 I,,.
i SPECTRUH 236 7.96 MINUTES
1 SUBTKOCTEO
' 'J.' ' ' '' 588
1568-STD-5Be.SE38-4en-228>C.-5.1.8..3TCL.85-83-78
Figure L-24. Analysis of pesticide mixture, full scan mode;
'•^500 ng.
248
-------
M-
II-
Chlordane
IBB
zis
3BB
M-
18-
ross specmm 259 1.63 nixuTEs
•OCKGItaUNO SUBTRBCTEC
li«B-STV-»8.SE3B-4Bn-22B«.-9. 1.8..
sie
I
,l .i
,.!. [l ,. .ll. .|..l
' lie '
' -DDE
3BB
M-
Ifr-
srccntun 291 >.» MINUTES
•ncxaiouNB sumtncTED
Figure L-25. Analysis of pesticide mixture, full scan mode;
^500 ng.
249
-------
Trifluralin
188-
98-
88-
6B-
5B-
38-
28-
i fl-
ee-
i
188-
se-
88-
78-
68-
58-
48-
38-
28-
18-
ii'l jiii in ' •]• -'I'1 ']"• • -'|' "
ie
1
ill ill
158
1 1
1 II , II i,|l|l M In- l'11'l - ' 'f' 1
' ' ' ' 288
,
LL
, ,
, — U—
38a
F ' ' J,'"' ' '458 588
t«SS SPECTSUn 78 2.33 nlNUTES l568-ST»-IB8.S63B-4Bi1-22B>C.-9.2KV..2rii:L.85-84-78
SU8TCOCTCD
'"1
38-
28-
18-
Atrazine
188-
98-
78-
se-
58-
48-
38-
28-
18-
I.I .ill . ,1. .1 1 ... I li I I i..i. . . . .1. I. i.l .1 . . . 1. . 1,. . ,1 .
358 488 45B 568
mss SRECTDun sr 2.89 HIHUTES i!6e-sm-i8B.SE3a-4er^22a«c.-9.2Kv. ,2rcL.e5-M-7a
SUBTPOCTHD
Figure L-26. Analysis of pesticide mixture, full scan mode;
^100 ng.
250
-------
76-
68-
56-
46-
36-
26-
y-BHC
lie
366
56-
40-
38-
26-
^ ' ' 3S6 '
MUSS SPEC7KUM 95 3. IS HINUTES
BOCKGRDUNO SUBTRBCTEO
1S68-STD-18B. SE3B-«n-22il«:. -9. 2KV. ,
76-
68-
56-
46-
38-
28-
18-
Heptachlor
' — •!•
256
166-
98-
86-
76-
68-
56-
46-
38-
28-
16-
' 356 46,
MASS SPECTRUM 142 4.73 MINUTES 19CB-STD-166.SE:
9ACKGROUND SUBTRACTED
'456 '•'•'•' !8B
Z2e»C.-9.2KV..2rCL. 65-84-76
Figure L-27. Analysis of pesticide mixture, full scan mode;
VLOO ng.
251
-------
BBCK-JR3
188-
98-
ae-
78-
68-
58-
48-
38-
28-
18-
aa-Wi
IBB
188-
•90-
80-
70-
60-
S8-
40-
30-
20-
10-
. .
1
III |lll
.llnl,
lie
I.,,,!
.ill
.
1 |lll il h t •! ' n 1
.
' 1 ! I' l> 1 1 1 1 1 i 1
_p_,_p_'-DDE
1 !,,,,! 1, ,1 ijlll I ,111 [ .til
2SB 388
1 f
MflSS SPECTRUM 273 9.83 MINUTES
BACKGROUND SUBTRACTED
4SB 583
1SS8-STD-1B8.SE30-4BM-220*C. -9.2KV. .2MCL. 8S-84-7S
18B.8n
3.8-
68.8-
156B-STD-1BB
246
23. a-
.8-
272
vf^j"-^'-r'f-^^
, ' ' ' ' 4 ' '
- VKO*
Y33J3
i-^*C **s *w»vi-%™:.-
'•"••" • " • ' ••••!- • • ' 1
18
- 373*10
TIC* 5
- 272* 1
1
Figure L-28. Analysis of pesticide mixture, full scan mode;
vLOO ng.
252
-------
lee-.
M-
88-
78-
68-
58-
48-
38-
28-
18-
1
J
ill
1
ll
Chlordane
1
lllll
1 1
tl ,|| 1
8 lie
I I
288
1 1 1.1,1 ll,ll,i.. hiil. .1'
' ' ' ' ' ' ' '* ' '
III
„ ||
,!, llllll.ll III ,.|J
' 38. ' ' '
88-
78-
68-
58-
48-
38-
28-
18-
88
rnss SPECTWJtl 2S1 8.3S tllNUTCS
BDCKGROUNI SU8TROCTED
156e-STD-ieO.SE3B-«n-22e-C.-9.«V. .2
lee-
9B-
88-
78-
68-
M-
38-
28-
18-
|||l. ill | . |,||
,
llll.,i..l...l . ll nl 1
,1 ill
'Ml I 1 1 1 1 Lilt 1 1
MASS SPECTPUTI 239 7.SS nlNUTES
BAdCGROUND SUCTMC1ED
lS68-5n-188.SE38-48n-228
-------
88,8-
sa. e-
48.a-'
III IV
20.0-
- 246*10
8 5 18
1 56S-STD-23. SE30-4BM-220*C, -9.2KV. . 2MCL. flfl Y 4
Trifluralin
130-
90-
80-
70-
60-
50-
08-
33-
20-
IB-
80-
1
, H 1
I/'
. ltil--.lt. r
- T ' • " i
,
I4*T*™
ll Jill
1 "
158
|||
'!' •'••• -
III
1 ' '
III,
1 1 ilii! ii , 'ill hi
zea
nil1)' M i
ll III 1
250
M
. , 1 It
--,---,--,
306
ii 1 >
Ii I)!,,,
300
iaa-
90-
80-
70-
60-
sa-
40-
38-
20-
1P-
I I
, , ^ , • , ,..-.
MftSS SPECTRUM 78 2.33 MINUTES
BflCKGROUHD SUBTRACTED
ia 4Sa see
lSSS-STD-28.SE3a-48M-220xC.-9.2KV..2rCL.B5-04-78
Figure L-30. Analysis of pesticide mixture, full scan mode;
^20 ng.
254
-------
78-
68-
58-
«-
38-
28-
11 Hull,
Heptachlor
272
288
' ' 388
iae-
96-
38
20
IB
80
MBSS
168-
98-
88-
7a-
66-
58-
48-
38-
28-
18-
1
188-
98-
SB-
78-
1
1
, . .^. ,.,.,.,
SPECTRUM 1« ^.73 MINUTES
BPCKGROUND SUBTRflrTED
n II
1,
a
1
1 '1
,, 1
1
1 | 1 1 1 1
8
II
1 1
-------
246
1B0-
90-
80-
70-
60-
50-
40-
30-
20-
10-
00-
1
iee-
90-
80-
7B-
60-
50-
40-
30-
20-
10-
aa-
mss
Ao
,|
SFtCTRUtl
||
K*-T«
JLL
||
' I'"11
|
^"slo ' " '
271 9.83
,S
||
ll
e
III
MINUTES
ll
2
1
400
1568-STD-20
|||
MM*
00
llll
'— i' 1 — — r
£,£!-DDE
LL . . i i
S50 300
) ' '1 ' I ' f ' f ' •( • -' [
450 S00
. SE30-40M-220*C. -9, 2KV..2MCL. 05-04-78
BACKGROUND SUBTRflCTEP
Figure L-31 (cont'd.)
256
-------
FILE 1569-1 NO. 2
IS6e-ST>-l8e.SE3e-4en-228»C.-9.2KV. .2.83-84-78
FILE 1563-1 NO. 2
1568-5TI-iea.SE36-48M-238-C.-9.2KV..2.89-84-76
FILE 1569-1 NO. 2
IS6B-STS-1W.SE3B-4Bf1-22B»C.-3.2KV. .2.83-64-78
FILE 1KB-1 NO. 2
i:69-STD-18«.SE3e-4Kr-228>C.-9.2KV. .2.89-«4-»
Figure L-32. Pesticide analysis, SIM mode.
257
-------
FILE ISSB-1 NO. 2
156B-STT-JOT.SE3e-«BH-22e><.-9.2KV..2.83-84-78
FILE 1568-1 40. 7
15S0-STD-.4 .SE3e-4arv22B«C.-9.2.5..5.63-94-78
PILE 1569-1 NO. 7
15S9-STD-.4 ,5E3B-4Bn-228<.-9.2.3. .5.BS-fl4-79
FILE 1SS8-1 NO. 7
1SS8-STTI-.4 .SE3e-4erV22B*C.-9.2.S..5.05-64-79
Figure L-33. Pesticide analysis, SIM mode.
258
-------
PILE 1566-1 NO. 7
IS6B-STD-.4 .SC3e-4en-22*»C.-9.2.3. .3.BS-B4-78
FILE 1568-1 NO. 8
TOLUENE .SE3B-«n-22B*.-9.2.J. .5.B5-B4-78
FILE 1568-1 NO. 8
TOLUENE .!E38-«n-22««:.-9.2.5..S.B5-«4-7a
FILE 15S8-1 HO.
TOLUENE .SE3B-.WV;
Figure L-34. Pesticide analysis, SIM mode.
259
-------
108.8-1
68. fl-
ea. 0-
20.0-
- JI2* I
- TIC» 1
Dt8-PYRENE.SE3B-4BM-228*C.-8.2KV..2MCL.5-2-78
IBfl-i
90-
88-
78-
S0-
sa-
2B-
I I I I'" I"" I"
198
MRSS SPECTRUM 358 B.39 MINUTES
BACKGROUND SUBTRACTED
I T '''I I I"'1 1 T"
D1B-PYRENE. SE38-»n-228*C. -8. 2KV,
"I T
"I"11 1 —T"
,85-82-78
Figure L-35. Analysis of d ' -pyrene.
260
-------
; 88.8
ee.e-
se.B-
4G.B-
28.8-
.8
b
156."
iee-
38-
88-
78-
68-
58-
48-
38-
28-
18-
,1
188
188-
92-
ee-
78-
68-
58-
48-
38-
28-
i F ii
1 ' • • • | • r---
5 18
*—^
'
•P6B-STB. S£38-228*C. -=. 2KV. . 2fCL . 85-84- 7d
1 , ,,,|il- III Ilii •
1
ll |,| h. 1, , 1 |ll!l| .| l(l , .{
158 ' ' ' 288
Ii . i . I. . 1 Ii, _
III
258 388
C12H4C16
I,'
• i • i • i • i • i • i • i • i • i • i • i • i • i • i
WSS SPECTRUM 379 12.63 MINUTES PBB-STD.SE38-4an-22B^C.-9.2KV. .SrCL.B5-84-78
BACKGROUND SUBTRRCTED
Figure L-36. Analysis of polychlorobiphenyls (40 m glass SCOT
capillary, 200°C, isothermal).
261
-------
78-
68-
se-
28-
18-
137
M Ml
188
TST
68-
se-
48-
38-
28-
T- I I I I1"
338
i SPECTRUM 55 1.83 n
t SUBTRACTED
188-
98-
SB-
78-
68-
38-
28-
18-
PBB-STt.SE3e-4en-Z2e*C.-9.2KV. .SnCL.eS-94-r6
35.
SPECTWn 234 7.79 tll
BACKGROUND SUBTRACTED
a ' ' ' 'J.' ..... ' ' '
PBB-STt.SE36-4an-2aa
-------
Table L-l. STANDARD SOLUTIONS OF POLYCHLORINATED BIPHENYLS
2-Chlorobiphenyl, Hexachlorobiphenyl, Decachlorobiphenyl,
Solution ng/yl ng/yl ng/yl
STD-100
STD-20
STD-2
STD-0.2
STD-0.04
97.9
20.8
2
0.2
0.04
536
114
11.4
VL
MD.2
1088
231
23
^2.3
^0.45
263
-------
188.8-1
•u 88.8-
*^
CO
B
01
•M 69.8-
C
j>48.8-
Integrated Ion Current Chromatogram
0 i
' 1 ' • ' 1 •
18 15
/\
' ' 1 Tn'
28
Time (min)
- 496* 1
PCB-STIl.SE38-2aM-23S*C.-9. I.8KV. .3nCL.8S-89-?8
zensity
d
M
CU
•H
ca
OJ
&
188-
98-
80-
70-
60-
50-
40-
30-
20-
10-
00-
1
.......11 1
Mass Spectrum of Peak No. 1
1 1 |iiii|iiii|iiii|iiii|iiii|iui | | ) | M — | | i i 1 i — ' — i — ,—-,.••-,•— i •••-. | . i
88 158 200 258 380
m/z ->•
>,
4->
•H
CO
c
cu
4J
c
M
0)
•H
4J
CS
r-t
CU
&
100-
90-
80-
70-
60-
50-
40-
38-
28-
18-
00-
MBSS SPECTRUM 26 8.83 MINUTES
BflCKGROUND SUBTRPCTED
1 r...,-..-! , I' I |----.--
08 458 SB
PCB-STB.SE30-2BM-235*C,-9.1.8KV..3MCL.85-89-78
m/z ->
Figure L-38. Analysis of PCB mixture (SID-100),
264
-------
ft im-
4-1
•H s«-
s »-
0) ?e_
1 ^
C 68-
58-
•H 38-
4J
c8 M~
"oj ">-
Oi! „
i
1 ll
• 1
Mass Spectrum of Peak No
B 150 2D6 23B 30B
m/z ->
>,188]
•U 90-
"^
CD 88-
S '«-
•y 68-
c
M 58-
OJ 48-
•H 3B"
4J 28-
ca
I— 1 18-
3 B,
i il
MOSS SPECTRUM 97 3.19 MINUTES
BACKGROUND !
PCB-STB.SE38-28n-235«C.-9.1.8KV..3MCL.85-89-78
m/z -»•
>1 188-,
•H
i 88H
C
' ' ,58
' 2B8 ' ' '
m/z ->
250
i) 18«-
•H 98-
CO
C BB-
M 68'
58-
OJ
•H ..
J-l ™"
Cd 28-
01 18-
1
IL (Ii
III,
niSS SPECTRUM 144 4.76 MINUTES PCB-STB.SE3B-28M-235«C.-9.1.8KV..3nCL.85-e9-7B
BRCKCROUNB SUBTRDCTED
m/z
Figure L-38 (cont'd.)
265
-------
100-
90-
CD '
§ 68-
4-1
(3 50-
- 30-
W .
td <
0) 10-
Mass spectrum of Peak No. 4
280
m/z -»•
250
380
9B-
•H _.
co 7E)-
0) 60-
IJ
C 50-
M
OJ
> 30-
4J
m 20-
01 10-
es
00-
4wt, ,„ TTT» 1 1
350
1 |
480
1
i ||
" • '•• -|-- ••.••••! i •-' ,-.. -I..-., ,..111111
4se 5
""' i
00
mss spECTRun 194 20.25 MINUTES
BflCKGROUM SLIBTRftCTED
PCa-STD.SE30-20rl-235*C.-5. 1.3KV. .3nCL.05-09-78
m/ z
Figure L-38 (cont'd.)
266
-------
••••!
•H
to
48. e-
28.8-
01
Pi
Integrated Ion Current Chromatogram
.V
/ VfJ'f
\
e s IB is
Time (min)
PCB-SH-2B.SE3e-28n-235-.-9.2nv. .3rCL.B5-B9-78
- TIC«tC
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Mass Spectrum of Peak No. 1
i i , ...
1 "• i - " i -—-I — ^j — ' — i — " — i — • — i — " — i — ' — 1 — ' — i — ' — i — ' — ; — ^~T — — i — ' — r — ' — i — •• — i — ' — r— — i — " — i — ' — r—
ee isa zee zse see
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tee-
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8.83 niNUItS
-88 «Se 568
PC8-S7B-Z8. SE38-2BO-233..-».2KV. .3TCL.85-85-71
m/z ->
Figure L-39. Analysis of PCB Standard Mixture (STD-20)
267
-------
l^r-
-------
188.8-1
80.8-
5 60.0-
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-------
8.0-
6.0-
ca
S 4.0-
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File 1568-1 f|0- 13
PCB-STD-.2.SE30-20M-235*C.-9.2.5KV.,30.5-9-78
28
20.0-,
16.0-
12.0-
8.0-
12 pg
60 pg
I
r«^-^v 212
Time (min)
FILE 1568-1 NO. 14
PC8-STD.Q4.SE30-29ri-235*C--9,2.5KV. .30.5-3-73
10
Figure L-40 (cont'd.)
270
-------
s.a-
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•H B8.8-
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0)
ti 68.8-
> 48.8-
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•U
(0
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0) 28.0-
Integrated Ion Current Chromatogram
'"s"° '*-%.' '-My-•
>»*^*%-*«'.*»*W
.e-) , , H 1 r
3 5
Time (min)
fiROCHL1232+I254,SE30-20M-235*C.-9.2..3.5-10-78
BflCK-20
100-
90-
CO
fi 68-
CU
•P 50-
M 40-
4J 20-
Cfl
i-l 18-
OJ
.
1,
ill
15
1
1
ll
I
1
3 200 250 300 330 400
WSS SPECTRUn 28 B.B9niN\JTES SRQCHLOR-1232-i-1254,SE38-2Bn-235*C.-9,2, .3nCL,nfiY18
BftCKGRQUND SUBTRflCTED
BBCK-2B m/Z *
100-
90-
^ 80-
4-t
•H 70-
CO
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al
t! 50-
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. 1
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r ' ' '"''•' T"'""1""'""1""1"'1 ' 1 ' ' ' ' 1 1" " '' '""""'I ' 1 1 ' " l""'"'1 1 '
200 250 300 350 400
3 8.93 MINUTES flROCHLOR-1232+1254.SE38-20M-235*C.-9.2. .3MCL.nflY18
HD SUBTRflCTED
m/z -»•
Figure L-41. Analysis of arochlor mixture, full scan mode.
272
-------
BACK-28
>-. 18e"
il ""
CO BB-
c
-
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M 58-
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« " ,58 ' '288 258 ' ' ' ' " ' "J, 358 4l '
MASS SPECTRUM 26 6.83 MINUTES AHOCHLOR-I232-H234.SE3B-2BM-233X.-9.2. .3MCL.nBY16
•U 98-
•H
CO B8-
c ~.
OJ 7e-
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., I.I 1., .illlll ..ll .11 11,1 ,, .. .. ill, .
156 288 2S6
, ••'•!, • • • ' 1 • , •,-,-, 'I ,
MRSS SPECTRUM 56 1.63 MINUTES ARaCHLOR-1232->-1254.SE38-28M-23S>C.-9.2. .3MCL.r»Y16
BACKGROUND SUBTRACTED
BBCK-Z8 m/ Z •*"
JJ 186-
•H ««_
M
C B8-
Figure L-41 (cont'd.)
273
-------
BBCK-20
188-
90-
« SB-
CO 7B_
2 60-
• 80-
^
c
(U 60-
4J
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•
Figure L-41 (cont'd.)
274
-------
CO
B TS-
UI
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X lie see
n«SS SPECTRUM 117 3.96 MINUTES
BACKGROUND SUBTRACTED
2Se 388
AROCHL•
Wse-
0)48-
mss spECTRun let 3.43 MINUTES
OflCKWOUHD SUBTRACTED
AROCHLOR-1232+1254. SE3e-29n-23!<-
m/z -»-
0)
-------
s
Olre-
4J
(-60-
0>48-
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1 1 i jiiiii I iiiini| i | i i| ....... |n ''I'li
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1
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1
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MflSS SPECTTJUn 133 4.39 MINUTES
BACKGROUND SUBTRACTED
cu
« 78H
4J 30-
-------
APPENDIX M
GC/MS/COMP SINGLE ION PLOTS (SIP) OF EXTRACTABLE HALOGENATED ORGANICS
USED IN RMR DETERMINATIONS
277
-------
00
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to
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f FILE NOME fififi242
J* SPECTRfl 1 TO 151
ION 188 INT. FflCTOR
|0 w ION 360 INT. FflCTOR
I^-Q ION 212 INT. FfiCTOR
CO H> _ _^
o\ o 0 20
O M •
CO O
*-
3
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(D 3
0 00
RELflTIVE INTENSITY
4Q 60
80
100"
to
o
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a:
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-------
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oc
oo
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a
M~3e
a z
«r >-
a
UJ C->
-Jill Z
•— O- O
U.U1 —
M-3f
Figure M-3d.
SIP for m/z 200 of atrazine 100 ng/yl; M-3e. SIP for m/z^ 212 of d1Q-pyrene;
M-3f. SIP for m/z 373 of chlordane 100 ng/yl.
-------
00
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in si
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-------
APPENDIX N
DATA COLLECTION INSTRUMENTS UTILIZED IN PRETEST
284
-------
RESEARCH TRIANGLE INSTITUTE
ASSESSMENT OF ORGANIC COMPOUNDS IN MAN AND ENVIRONMENTAL MEDIA
PARTICIPANT CONSENT FORM
I understand that the Research Triangle Institute is engaged in a study of the exposure and absorption of selected
organic compounds by persons living in areas having various levels of selected organic compounds in the environ-
ment. I understand that the survey is being conducted in order to help measure the levels of exposure and absorp-
tion of selected organic compounds in populations environmentally exposed, and is limited to the purpose stated. I
further understand that the survey is being conducted under the auspices of the United States Environmental Pro-
tection Agency in cooperation with the New York State and Niagara County Health Departments.
I do hereby freely consent to participate in this study of exposure and absorption of selected organic compounds
and understand that my participation will consist of providing answers to a questionnaire related to environmental
exposure and the following environmental and biological samples: (1) three four ounce samples of cold tap water
from a source commonly used for drinking and cooking, (2) a sample of environmental exposure collected by a small
device which I will keep with me for a short time, (3) a small (approximately 25 cc) blood sample to be taken from
an arm vein, (4) a breath sample, and (5) two small (approximately four ounce) urine samples, one to be taken early
in the morning and one at the time of the blood sample. I understand that an agent of the Research Triangle Institute
will administer the questionnaire in my home and at the same time collect the tap water samples, instruct me regar-
ding the exposure monitoring device, and make arrangements regarding collection of the breath, blood, and urine
samples.
I understand that my name will not be voluntarily disclosed, and that my name will not be referred to in any way
when compiling and evaluating the results of the study. I understand that participation in this study may result in no
direct benefits to me, and that I am free to withdraw from this study at any time. It has been explained to me that
there are no significant risks to me from participation in this study. I further understand that while participating in
the study I will be free to ask any questions concerning the study; if I have any further questions about the project. I
know that I am free to contact Mr. Benjamin S. H. Harris, III, Survey Operations Center, Research Triangle Institute,
Research Triangle Park, North Carolina 27709, telephone number 800-334-8571, extension 6055.
One:
Participant's Name.
(Month) IDtyl (Yuri
n
Sin Number: Am Number:
SIGNATURES:
Pirticipent: Wltntw
Addtwi:
IPrint)
Pirticipint Number:
Interviewer Number:
285
-------
ASSESSMENT OF HALOGENATEO ORGANIC COMPOUNDS
IN MAN AND ENVIRONMENTAL MEDIA
Spoiuaradby:
OffTot of Toxic Subsonen
Emironimnal Protection Agwicy
Wttitnomn, O.C. 20460
Conduetid by:
Roarch Trunflto Innitut*
P.O. Box 12194
RtMVCti Tri»ngl« Park, North Carolina 27709
QUESTIONNAIRE
THE RESEARCH TRIANGLE INSTITUTE OF RESEARCH TRIANGLE PARK, NORTH CAROLINA,
IS UNDERTAKING A RESEARCH STUDY FOR THE U.S. ENVIRONMENTAL PROTECTION
AGENCY TO ASSESS LEVELS AND RELATIONSHIPS OF HALOGENATED ORGANIC COM-
POUNDS IN MAN AND ENVIRONMENTAL MEDIA. THE INFORMATION RECORDED IN THIS
QUESTIONNAIRE WILL BE HELD IN STRICT CONFIDENCE AND WILL BE USED SOLELY FOR
RESEARCH INTO THE EFFECTS OF ENVIRONMENTAL FACTORS ON PUBLIC HEALTH. ALL
RESULTS WILL BE SUMMARIZED FOR GROUPS OF PEOPLE; NO. INFORMATION ABOUT IN-
DIVIDUAL PERSONS WILL BE RELEASED WITHOUT THE CONSENT OF THE INDIVIDUAL.
THIS QUESTIONNAIRE IS AUTHORIZED BY LAW (P.L 94-469). WHILE YOU ARE NOT RE-
QUIRED TO RESPOND, YOUR COOPERATION IS NEEDED TO MAKE THE RESULTS OF THIS
SURVEY COMPREHENSIVE, ACCURATE, AND TIMELY.
Study number
Site number
D
Am number
Participant number:
286
-------
•2-
Hnt, I would tlka to ask ioma ganaral quastions about you.
1. Sax fay oottnmtionl: [jj Mila |__J Famala
Rao:
1 Amarlan Indian/
I Hack, not of f~~l A_an/Pacrf Ic
I Hlapante orlfln | * 11 dandar
f—Iwmlta. notof I——lOthar
4. What it your birthdata?
(Uomht IBtyl
lYnrl
X What wa» your aga in yaan at lait birthday?
5. What i> your approximata waight in poundi?
KM. I 1 I Do not know
8. What if your approximata haight in faat and inehai?
| [ Faat ___ __ Incha*
Maxt, | would lika to ask soma questions about your occupation.
7. ATI you araaanily ampleyad in any capacity? | 1 [ Y« ICominutl
a, HOM long hava you baan amployad by your praaant amployar?
11)
Unita
[_2J
9. Ooai your occupation utually taka you away from homa? | 1 | Yaa (Caminu»l
10. What iitha natura and location (oraat addraa) of tha company for which you work?
Monthi |
a No /So ro OL
IZtp Coaal
11. If net pnsmtly •mpk)v«d. which of »• following b«t daeribii your jtatui?
HouMwita
Student
(Go to O. tSI
3 I Unimo(oy«d
« Oloblid
12. Whn i«/w«i your usual occupation? ISpKifyi _____
13. An you pnaamly «mploy«d in tnif occupation? jjj Yat a No
14. If yaa to abOM quaction, how long hav« you baan amptoyad in that occupation?
IQuationi 13*nd 14 /nay ft» Aioatd (or umnployfd,
normi. mi almoM pfrmu.)
Unit! P| 0»Vf [_*] Montfn Qj Year:
16. Do you work at or in any of ttia following occupation or acabliihmants? (One* til ttift tpalyJ
Qr"~7 I~"I Sarvlemtlon/ f~~l
Painring | »| Chamical plant | »| g_^g«/«ngln« rvair | ' | Nona of ttv
HI—T •umtan ra«nl_ilng I 1
Ptntaumplant |_JJ orrapair | "|
2 Dry daaning
Otfiar (Sanfyl
16. Ha«* you workad at any of *a following oceuoationa/bu«inaaaa at any tima during tha pan waak? IOi*dt ill Out tpoly.t
mP""l n Swtaa nation/ r~~
•aiming |_3j Chamicat plant | «| gara»a/an»lna r^alr |_JJ Nonaof thaaa
Hr~~1 (""I 'umitura raflntoilna, r~~]
Orydaaning |_4j Pairolaum plant [_«J orraoair | « | Ottiar (Spmfyl
287
-------
Not, I would Ilk* to «k Joma question* regarding your panonal habits.
17. Oo you nnoka?
Yss ICentimnl
18. How old wara you whan you first starttd smoking?
19. On tha a»araga, how many tigarama do you smotea paf day?
|Tj Laa than X pack (1-4 dgarama) !_*] About 1K packs (25-34 dgaranss)
Q] About X pack (5-14dgw«m) [_»] About 2 ptcki (35-48 eiaMRa)
|a| About 1 pjck (15-24 dyumm) [T] Men ttan 2 pidu (50 or mora efgvnra)
NOTE: /' BX pwtteicwnr um (OOKB in ie/n* ortw form (attar tfan dgtrnnt-tj., aym. pip*, or snuff).
20. VWin iith*iwng*nume haw you mm in Kit paa 48 noun?
25. Oo you purmw any of *t following hobbw? tOudt ttl ttat mply.l
LjJ Fumitura rafinnhing [jj Painting Qj Scala medals Qj Gardaning \s] Nonaofthna
Ifffrdtnittf a imttaud, inquin tbout ptttiddfhl and: H ocncr noao/at art intilaad, inquin JOOUT sUKitl lo/twra. ISpterfyi
2& Oo you ounua any aaivitv tnat indudat ragular uaa of tonwnt glua or modal airplana c*mant?
Q] Yai 0 No
288
-------
Next. I would Ok* to ask Jome question! regarding your hwtth.
27. Whn do you consider tna currtnt ntui of your health? IChfdc ant.]
j Excellent j Good j Wr j Poor
28. Are you currently taking any preemption nwdication(s) on a regular daily ban*? (jj Yu | » | No
SfywiapMriy.- - __ ___ _ _
29. HIM you taken any non-prescription mediation* In tna put 48 houra? Qj Yts [T] No
30. Are you presently under a decWi cere? M Yes I a I No
m
It ytt, ipwifV nuon:
31. An you prmmly luffcring from «iry n»irnory problwra CucA uaoH, cough, an ttiratt. flu, lahim, Aronctirtti, ttiermttt of
trmtti. Itryngrtn. plfunty. *tej?
32. Hm« you mr b*m tnnad for (mrnis? 1 Y« » | No
[ 1 J
Lastly, I would like to ask some questions about your residanea and household.
33. How many years have you lived in this area? |
Ye
34. How long have you lived at your current addrea? Units [__J Days | 2 | Month* | 3 [ Yean
35. Do you cool your home with any of the following appliances? tO>»dt til Oitttuplyj
I 1 I Central air conditioning [ * \ Window fan(s) __J None of than
|__j Window air conditioner(i) [__] Ceiling aurieuR fan(s) [__J Do not know
\3\ Evaporative coolerd) \»\ Circulating fan
-------
40. What a th. primary aura of your watar for cooking?
[ il Bottled watar | 3 | Tap • eommunitv wall | » | Tap - ciftarn
| 3 | Tap - munidpaf nipply | 4 [• -Tap • privaw-wall | * | Oo not know
41. Doai anyone alia in your houithold amoka? | 1 |
H m, &** ill Out wptY: I 1 [ Ogaranaa
ouwhold work ai
Oiamial plant
No
j Coin [»| Plpi 1*1
Oo not know
Marappw | ' | Cigarattaa \ * | ugara | - | rv* | - | UOTW u»»u.r/
42. Don anyona d«a In your houwhold work at any of dw following occupatfora/bus!n«*aH? IChtelt til ttttt tpe/y.l
[7] Painting |a| Oiamial plant [riwoln.rap.lr *" \1 \ Nonaoftnna
. . .,. m , , ,— - i_5J wflin. f^.lr
ml—I f—I Fumttur* raflnWiIng
Dry dawilng | * \ Pttrolaum plant | * | or m*r
43. Dow anyom alaa In your hounnoM punua any of.tha following bobbin?
[jj Mnting [ » | Fumitura raflnithing | » | Sal* modab | * | Gardaning | » | Nona of th«sa
INTERVIEWER INFORMATION
(Month!
(Dtyl
/Yuri
44. dam nittM muvbtr.
48. Dan of inttrvitw:
COMMENTS
290
-------
SAMPLE INFORMATION
Pomonnvl monitor nunvtr
n
COMMENTS:
On
0«
Dm
Mo<
mil
On
Yeer
Time
Noun: Mlnutei
'.
'•
•low Mete
(ml/nrin)
Interviewer
Number
47.
enheunpta
Collected
Yet
1
NO
3
One
Momn
Oev
Yeer
Oea on Collect
edSemple
Time.
•ten
HourcMlfiutee
;
Stop
Houn : Mlnutei
;
Temp.
C
Volume Meter
Heeding
(cubic feet)
If net callecwd. reeean:
""N-uTr
Svnp4t No* 1
Caltoend
Y«i
1
No
2
S«T»l»No.J
Colleead
Y«
1
No
2
Simple No. 3
Caltacnd
Y«i
1
No
2
All Sempled)
If Collected, Dm
Mamh
Oey
Yeir
If Not Collected.
Renon
t iiceivieMer
Number
Evlv Momlnf Semple
OMlected
Yee
1
NO
2
If Celleend. Oete
Mom*
Oev
Yeer
If NOT Collect*!.
ItaMon
Ifl^wfew^
NumBer
Spat Simole
Callectid
fm
1
No
3
If Collected. Oete
Mom*
Oev
"
Yeir
If Not Collected,
Reuon
SO. Saedfiegmvity:
SI. Soedflcgnvity:
TepMtereunpled) SOURC
SempleNo. 1
Colloeud
Y«
1
No
2
SempJeNo.2
Calliatf
Y«
1
No
2
Simple No. 3
Collected
Ye»
1
No
2
All Sempledl
If Collected. One
htaitrii
Oev
Yeir
If Not Callectid,
Rieean
Number
COMMENT'S
291
-------
APPENDIX 0
DATA COLLECTION INSTRUMENTS PROPOSED FOR MAIN STUDY
292
-------
RESEARCH TRIANGLE INSTITUTE
ASSESSMENT OF HALOGENATED ORGANIC COMPOUNDS
IN MAN AND ENVIRONMENTAL MEDIA
PARTICIPANT CONSENT FORM
I understand that in* Research Triangle Institute it engaged in a study of the exposure and absorption of selected
halogeneted organic compounds by persons living in areas having various levels of selected helogenated organic compounds
in the environment. I understand that the survey is being conducted in order to help measure the levels of exposure and
absorption of selected halogenated organic compounds in populations environmentally exposed, and is limited to the
purpose stated. I further understand that the survey is being conducted under the auspices of the United States Environ-
mental Protection Agency in cooperation with the
(San ind loe*l />«/*> dm*rm»nal.
I do hereby freely consent to participate in this study of exposure and absorption of selected halogenated organic
compounds and understand that my participation will consist of providing answers to a questionnaire related to environ-
mental exposure and the following environmental and biological samples: (1) two four ounce samples of cold tap water
from a source commonly used for drinking and cooking, (2) a small (approximately 25 cc) blood sample to be taken from
an arm vein, (3) a breath sample, and (4) a small (approximately four ounce) urine sample to be taken early in the morning.
I understand that an agent of the Research Triangle Institute will administer the questionnaire in my home and at the same
time collect the tap water samples and make arrangements regarding collection of the breath, blood, and urine samples. I
understand that after the collection of the breath and blood samples I will receive an incentive of ten dollars for my full
participation in the study. I further understand that a small sample of participants will be requested to provide an addi-
tional 25 cc blood sample at a different time and that, if I am so selected and agree to provide this additional sample, I
will receive an additional incentive of five dollars for my assistance. I understand that a small number of households and
individuals will be selected for reinterview and the collection of duplicate tap water and blood (10 cc additional to be
collected at the same time as the original sample) samples, but that such selection would not entitle me to further
compensation.
I understand that my name will not be voluntarily disclosed, and that my name will not be referred to in any way
when compiling and evaluating the results of the study. I understand that participation in this study may result in no
direct benefits to me, other than those described herein, and that I am free to withdraw from this study at any time.
It has been explained to me that there are no significant risks to me from participation in this study. I further under-
stand that while participating in the study I will be free to ask any questions concerning the study; if I have any further
questions about the project, I know that I am free to contact
(Leal hnlth dm*rm*nt npn*nativ»l nltphorw numbtr
(San htmKh dfpmrmtm nentmntnint tilephoiw number
or Mr. Benjamin S. H. Harris, III, Survey Operations Center, Research Triangle Institute, Research Triangle Park, North
Carolina 27709, telephone number 800-334-8571, extension 60s5.
Date: — - P»rticip»nt'i N«mt:
IPrintl
Stgnwnt Number: Houahold Number:
SIGNATURES:
Pfcrtl ClPAHt ! MM^
Participant Number:
lnwrvltw»r Number:
293
-------
ASSESSMENT OF HALOGENATED ORGANIC COMPOUNDS
IN MAN AND ENVIRONMENTAL MEDIA
Spoiuond by:
Offlei of Toxic Subninen
Environnwnul Prenetion Aamcy
Vtehington. O.C. 20460
Conduend by:
Rnureh Tritngli Iratitun
P.O. Box 12194
Rnureh Trcmnglt Pirk. North Carolina 27709
QUESTIONNAIRE
THE RESEARCH TRIANGLE INSTITUTE OF RESEARCH TRIANGLE PARK, NORTH CAROLINA,
IS UNDERTAKING A RESEARCH STUDY FOR THE U.S. ENVIRONMENTAL PROTECTION
AGENCY TO ASSESS LEVELS AND RELATIONSHIPS OF HALOGENATED ORGANIC COMPOUNDS
IN MAN AND ENVIRONMENTAL MEDIA. THE INFORMATION RECORDED IN THIS QUESTION-
NAIRE WILL BE HELD IN STRICT CONFIDENCE AND WILL BE USED SOLELY FOR RESEARCH
INTO THE EFFECTS OF ENVIRONMENTAL FACTORS ON PUBLIC HEALTH. ALL RESULTS WILL
BE SUMMARIZED FOR GROUPS OF PEOPLE; NO INFORMATION ABOUT INDIVIDUAL PERSONS
WILL BE RELEASED WITHOUT THE CONSENT OF THE INDIVIDUAL. THIS QUESTIONNAIRE IS
AUTHORIZED BY LAW (P.L. 94-*69). WHILE YOU'ARE NOT REQUIRED TO RESPOND, YOUR
COOPERATION IS NEEDED TO MAKE THE RESULTS OF THIS SURVEY COMPREHENSIVE,
ACCURATE, AND TIMELY.
Study number:
ATM number: | | Site number: | | Segment number:
Household number:
Participant number:
294
-------
First, I would like to a*fc MUM general quutioiu ibout you.
1. Sex toy ota*man):
1 | Male [ 2 | Female
4. Whit ii your birthdata?
(Month) IDtyl
HI American Indian/
Hispanic | 2 [ Alaskan Native
-
-
[71 Hiapanieforigin fT] WwUder*""" 8. What ii your approximate weight in pounds?
m Whin, not of t~— i Other
Hispanic origin 6 ISpfdfvl —
' '
3. What waa your age in yean at left birthday?
8. What
YMT,
iiyo
D
Ib
f. | 1 | Do
not km
ur approximate height in feet and n
Feet
)W
:hei?
Inches
Nixt, I would Ilk* to aik torn* questions about your occupation.
7. Ara you presently amptoyad in any capacity? | 1 | Yti (Contlnutl \ 2 | No IGo m Q. 111
8. How long hava you baan amployad by your present amplovar? | Unitt |_1 |
9. DOM your occupation usually taka you away from home? | 1 [ Yes (Conenutl
10. What it tha natura and location (nraat address) of tha company for which you work?
Days | 2 | Monthi | 3 | Y.ari
2 No IGo m a 121
IGa a a 12!
(Zip Codel
11. If not praaantty amptoyad. which of tha following ban daicribas) your tntui?
161
\ 3 1 Unamptayad
| 4 | Ratirad
| 5 | Disabled
(Continu*!
Houasmifa
2 I Studant
12. What i*/wa> your usual occupation
IQutalaia 13 wd 14 may t» tHipptd for untmploywo, ratircc/, tntt dittbltd pirsont.1
13. Ara you praatndy amployad in this occupation? | 1 | Yai (Coattnaml [ 2 | No (Go to Q. 151
14. If yat to abova quattion. how long hava you baan amployad in that occupation?
| | | Unrn | 1 | Oayi | 2 | Months | 3 | Yaars
IE. Have you avar workad at or in any of tha following occupation! or anablishmann? IChfdc til tnar tpply.)
I 1 I Painting | 3 |
mr—i (—~| Fumitura rafinlihing (~~|
Dry daanfng [ 4 [ Patrolaum plant | 6 | or rapalr | 8 | Taxtila mill | §
16. Hava you workad at any of tha following occupationt/businanat at any tima during tha past waak? IChtek ill Hut tpply.l
ml | I I Sarviea station/ I I Plinics manufacture I I
Painting [ 3 [ Chemical plant | 5 [ garage/engine repair | 7 | or formulation | 9 \
ml I I ] Furniture refinMiing I I I I
Dry daaning | 4 | Patrolaum plant | 6 | or repair [ 8 | Textile mill | 0 | None of these
Plastics manufacture
or formulation
—•] Wood processing
9 | plant
"""] None of these
0 \ (Go to 0. 171
Wood processing
plant
295
-------
Ntxt, I would liki to ufc jorrw questions regirding your personal habio.
17. Have you ever smoked a many as 5 packs of eigarettes-that i», at many at 100 cigarettes during your antira lifa?
| 1 | Yes tContinufl \ 2 | No (Go a Q. 221
18, Do YOU now moka cigarettes? | 1 | Yes IComlnutl \ 2 | No ISe Co O. 201
19. How old war* you whan you tint itartid smoking?
Years (Go a a 211
20. If you no longer smoke, how old wan you whan you latt gave up smoking?
Yaari
21. On ttia avaraga, how many eigaranai do (did) you unoka par day?
| 1 | Laa than 'A pack (1-4 cigaratm) [ 4 | About 154 Packs (25-34 cigarette! I
|2 I About 'A peek (5-14 cigarettes) | 8 | About 2 packs (35-49 cigarenM)
| 3 I About 1 pack (15-24 cigarattas) | 8 | Mora than 2 packs (50 or mora cigarette!)
22. Oo you uta tobacco in any other form (*.g.. a'ytn. pip*, muff, dinning mAaecoJ? 1 Yes | 2 | No
Hy«. Indian form: \ 1 | Cigars | 2 | Snuff | 3 | Chawing tobacco
I 4 I Pipe I 5 I Othar ISpteifY)
23. What is tna average number of hours that you spend out of doon each day?
Hours
24. How many hours of the day, on the avenge, do you normally spend away from home? MMragf t*p*rtaly for wnkdays ami
waevlrena'fJ
Hours
Weekdays
Hour.
Weekends
25. Oo you pump your own gas? | 1 | Yes | 2 | No
Ifyn. have you pumped your own gas in the last 24 hours? Ill Yas | 2 | No
28. Oo you do your own dry cleaning? | 1 | Yes | 2 [ No
27. Hava you been to a dry cleaning establishment in the pan 24 hours? | 1 | Yes [ 2| No
296
-------
28. Oo you puraua wy of tha following hobbia*? IChtOt til Out app/c.1
I 1 I Furnltura raflniihlng | 3 | Piloting | 3\ Scala modtli | 4 [ Qardaning | 5 | Nonaofthaia
29. Oo you work with or am innctleidn, paatickjtt, or harbicidai, n in farming, gardening, or •xnrrninitlon?
fi"i v«a JTJ NO
a. How ofttn would you lav that you work with or u» such ubnaneat? | 1 | Rarely | 2 | Occasionally | 3 \ Oftan
tfym.
Whan did you lair work with tuch wbnaneat?
(MemHI IDtyl
lYurl
Naxt, I would Ilk* to ask som* quaitiont regarding your hurth.
3D. What do you eontfdartha currant itatui of your haaltfi? (Ctifdc on*.l
| 1 | Excallam [ 2 | Good | 3 | Fair | * | Poor
31. Ara you currently taking any pmcription madicationd) on a ragular daily baiit? |_1_| Yti [ 2 | No
It ya, tpfcify: _^______^_____^____^^__^_____^___^_______
32. Haw you takan any non-preteriptlon madieatiom in ttx pan 48 hours? | 1 | Yat | 2 |
No
33. Ara you pmantly undar a doctor"! cam? | 1 | YM | 2 [ No
If ytt. vtcify naonftl: __^-^^_____^^.^_^_^___
34. An you praaantly tuffaring from any raipiratory problami Isucti a cold, cough, ton tnroaf, flu, itttimt, bronchitis, stiormu of bntth,
Itryngnit, plturiiy. ne.1?
El
YM 2 No
35. Hava you avar baan traatad for any of tha following condition!?
I 1 I Anamia | 2 | Liwrdiaaaia | 3 | Kidnaydimn
36. How would you rata your ganaral recreational activity ?
| 1 | Haavy | 2 | Light | 3 | Sadantary
37. How would you rata your activity on tha lob?
| 1 | Haavy [ 2 | Ught [_a| Sadantary | 4 | Not applicabla tHrimt. untmployttl, or diabltdl
297
-------
None
Next. I would Ilk* to «k »nv question! regarding your diet.
38. Which meald) do you u«-ally eat at home? CCftec* *// ttef tpply.l
| 1 | Braakfaft | 2 | Lunch | 3 | Dinner
39. When you do not eat at home, where do you eat? «*ee* til Oinvply.l
[T[ Meel preperad at home but eaten eliewhen \2\ School \3\ Work | 4 ]
| 6 [ Other (Spfafyl
Renaurant
| 5 I Never eat anywhera but at home
40. An you presently following any of the following dietary regimeni? ICtndt til tfitt ipply.)
| 1 | Bland food ulcer diet | 4 [ Organic foods | 7 |
| S | Vegetarian
2 Diabetic diet
I 3 | Reducing diet | 6 | None of these
41. On the average, how often do you eat the following foodi? CCnec* eeeft appropriate box.)
Foodftuff
a. Beef
b. Fish
c. Pork
d. Poultry
e. Fmh fruit
f. Frozen fruit
g. Canned fruit
h. Fresh vegetables
L Frozen vegetable]
|. Canned vegetable!
a
1
,
|i
ii
2
h
ii
3
I;
H
On
4
€
!
i
0
5
S
c
Jl
H
6
Uitty, I would like to ak torn* questions about your rasidenca and houuhold.
42. How many yean have you lived In thi« area? | Yean
42.
43. How long have you lived at your current addreu?
k^^^.^^^^
44. Do you cool your home with any of the following appliances?
| 1 I Centnl eir conditioning | 4 | Window fand)
Unitj
| 1 | Days | 2 | Months [ 3 |
I 7 [ None of these
Year.
I 2 I Window air condltionerd) | 5 | Ceiling exhaust fan(s) | 8 | Do not know
I 3 I evaporative cooler(s) | 8 | Circulating fand) | 9 | Other ISpfdfyl
298
-------
"46. Don your houaihold grow my of In own food in t horn* gardan? | 1 | Ya» la
No
3 Do not know
H yn. &feify locttion of gtnitn.-
46. Whara don your houaahold obuin fraih fruit ind/or vagatatalat? ISetcityl ______
47. What ittna primary Burca of your watar for drinking?
I * I Bottladw«ar | 3 I T» - community wall | S | Tap-dfttm
I 2 | Tip • municipal wpply | 4 | Tap - privata Mil
I 6 I Oo not know
m
4i li that tha ama primary nurea of watar for drink mixat wen at eoffaa, taa, Kool-Aid, ate?
I 1 I Vat I 3 | No If no. how doat it dlffar? iSpte/fyl
49. What it tha primary tourca of your watar for cooking?
I 1 I Bortad watar | 3 | Tap • oommunity wall | 5 | Tap-dttarn
I 3 I Tap • municipal mioply | 4 | Tap - prnata wall | 6 | Oo not know
50. Ooa* anyona (ato) in your houahold nnoka? [ 1 |
r: [ 1 | Cigaranai [ 2 |
Ya«
2 No
3 pf.
[ 3 | Oo not know
| 4 |
51. Ooaianyona (alia) in your houaihok) work at any of tha following oeeupation>/buain«aa>? (CtiKic til th*t mply.l
H_ . . I , I _. . . . I , I Sarvica nation /garaga/ PTI Plajtia manufactura PT"! Wood proceaing
Pamting [_3j Chamieal plant [_SJ ,ngjn. f^j, " " [JJ or formulation I 9 I plant
| 2 I Dry daaning | 4 | Patrolaum plant [ 6 | er rtpair ' ' ' [ 8 | Taxtila mill | 0 | Nona of tfie«a
53. Don anyena did) in your houahold punua any of tha following hobbiai? ICtitdr ill Outwply.1
I 1 I Painting | 1 \ rrttolfrinQ I 3 I SeilamodaU | 4 | Gardaning | 5 | Nonaofthasa
53. Iniauiawai number:
INTERVIEWER INFORMATION
Month)
IDtYl
(Yuri
54. Data of intarviaw:
COMMENTS
,299
-------
SAMPLE INFORMATION
'OR EACH SAMPLE COLLECTED FOR A GIVEN HOUSEHOLD OH INDIVIDUAL. ATTACH THE APPROPRIATE LABEL TO THE APPROPRIATE
CONTAINER BEFORE COLLECTING THE SAMPLE.
, ORIGINAL SAMPLE COLLECTION
1. Ouoidt lir (houaahold) monitor numbar
D
Contend
YM
1
No
2
Data on Collactad Sampla
On
Off
Dan
Month
Day
Yaar
Tlnw
Hours : MlnutM
:
:
Flow
Rna
Iml/min)
IntMvimwr
Numbar
If not contend, r«*jon:
COMMENTS:
2. SoHsampIt
Original Sampla(s)
Collactad
Y«
1
No
2
If Contend, Data
Month
Day
Yaar
If Not Collactad,
Raaaan
Intarviawar
Numbar
Duplicra Sampla
Salaend
Yai
1
No
2
Collactad
Ya»
1
No
2
If Salaend.
But Not Centered,
Rfcton
3. Tap watar sampla
Original Samplad)
Contend
Ya«
1
No
2
If Collactad, Data
Month
Day
Yaar
If Not Collactad,
Raaaan
Intarviawar
Numbar
Ouplicatt Sampla
Salactad
Ya«
1
No
2
Collactad
Yai
1
No
2
If Salaend,
But Not Colltcted,
Raaion
Sourea:
(Manthl IDtyl
(Yuri
Appointmant dan:
And timt:
impla
Contend
Yai
1
No
2
Oat* on Collactad Sampla
Data
Month
Day
If not collactad, n
Yaar
won:
Tima
Start
Hours : Minutas
Stop
Hours : Minutes
:
Tw
C
F
"P.
i
Voluma Matar
Raading
(cubic faat)
i
Numbar
Apparatus numbar
S. Blood «mplt
n
Original Samola
Collacnd
Yaa
1
No
2
If Collactad, Data
Month
Oay
Yaar
If Net Collactad,
Raaaon
Imarviawar
Numbar
Oupllata Sampla
Salaend
Ya«
1
No
2
Collacnd
Yt»
1
No
2
If Salaend.
But Not Collaeiad.
Rtaton
300
-------
6. Urine sunpleU)
7.
Early Morning Sample
Contend
Yei
1
No
2
Specific gravity:
If Colltcnd. Dan
Month
1
Day
Year
1
If Not Contend.
Reeeofl
Innrvie
Numb
•ver
tr
Other Samplt (Expltin btloiml
Contend
Yei
1
No
2
If Contend. Dtn
Moi
ith
Day
8.
Year
If Not Contend,
Rtaton
Sptcffie gravity:
4
. ADDITIONAL SAMPLE
(Man*! (Owl
Appointmmt dan: | [ | —
And tint*: I I
2 | No
Ytt ("Tl No
1. HIM you pumped your own get in the Ian 24 houn? Ml Yei _
Z Hm you bttn to a dry ctaning trablltfinwit in tht I«R 24 houn? | 1 |
3. Havt you worktd with or und any iiutetiejdn, ptnieida, or htrtaicidti in tht pan 24 hours? | 1 |
4. Havt you nktn any non-pmeription mtdieitiont in tht put 24 houn? | 1 | Y« | 2 | No
Yn
2 No
5. Blood omplt
Original Samplt
Colltcwd
Ya>
1
No
2
If Contend, Dan
Month
Day
Yttr
If Not Contend.
Raaaon
Innrvitwtr
Number
Duplion Samplt
Stltend
Ytt
1
No
2
Contend
Y«
1
No
2
If Stltend,
But Not Contend.
Retson
COMMENTS
301
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing) '
1. REPORT NO.
EPA-560/13-79-010
2.
3. RECIPIENT'S ACCESSIOONO.
4. TITLE AND SUBTITLE
ANALYTICAL PROTOCOLS FOR MAKING A PRELIMINARY ASSESS-
MENT OF HALOGENATED ORGANIC COMPOUNDS IN MAN AND
ENVIRONMENTAL MEDIA
5. REPORT DATE
July 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
E. D. Pellizzari, M. D. Erickson and R. A. Zweidinger
8. PERFORMING ORGANIZATION REPORT NO.
S. PERFORMING ORGANIZATION NAME AND ADDRESS
Research Triangle Institute
P. 0. Box 12194
Research Triangle Park, NC 27709
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-4731
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
Washington, DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Annual
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This comprehensive report presents the methods which will be used in Phase II
of this program. Analytical methods for halogenated hydrocarbons in air, water,
soil, breath, blood, urine, and tissue have been validated. A radioimmunoassay
procedure for carcinoembryonic antigen (CEA) was validated. The data collection
instruments (participant consent form, questionnaire, etc.) are also presented here.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
3alogenated hydrocarbons
Air
tfater
Soil
Breath
Blood
Urine
Ti'ggiio
Analytical
methods
CEA
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
317
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
302
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