United States
Environmental Protection
Agency
Office of
Toxic Substances
Washington, DC 20460
EPA 560/5-83-001
September 1983
Office of Toxic Substances
cxEPA Hispanic HANES Pilot Study
Measurement of Volatile
and Semivolatile Organic
Compounds in Blood and
Urine Specimens
3.1
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EPA 560/5-83-001
September 1983
HISPANIC HANES PILOT STUDY
Measurement of Volatile and Semivolatile Organic Compounds
in Blood and Urine Specimens
by
S. Pierson
R. Lucas
Research Triangle Institute
Research Triangle Park, NC 27709
EPA Contract Number: 68-01-5848
Task Manager: Cindy Stroup
Project Officer: Joseph Carra
Office of Toxic Substances
Washington, DC 20460
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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DISCLAIMER
This report was prepared under contract to an agency of the
United States Government. Neither the United States Government
nor any of its employees, contractors, subcontractors, or their
employees makes any warranty, expressed or implied, or assumes
any legal liability or responsibility for any third party's use
or the results of such use of any information, apparatus, prod-
uct, or process disclosed in this report, or represents that its
use by such third party would not infringe on privately owned
rights.
Publication of the data in this document does not signify
that the contents necessarily reflect the joint or separate views
and policies of each sponsoring agency. Mention of trade names
or commercial products does not constitute endorsement or recom-
mendation for use.
11
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TABLE OF CONTENTS
Page
LIST OF TABLES iv
LIST OF FIGURES v
I. INTRODUCTION AND BACKGROUND 1
II. SUMMARY OF RESULTS 5
A. Introduction 5
B. Analysis of Blood for Volatiles 5
C. Analysis of Serum for Semivolatiles 6
D. Analysis of Urine for Semivolatiles 7
E. Conclusion 7
III. SAMPLE SELECTION AND DATA COLLECTION OVERVIEW .... 9
IV. QUALITY ASSURANCE PLAN OVERVIEW 11
A. Field Controls 11
B. Replicate Specimen Analysis 11
C. Spiked-Split Duplicate Analysis 12
V. ANALYSIS OF BLOOD FOR VOLATILE COMPOUNDS 13
A. Summary of Results 13
B. Specimen Collection, Storage, and Shipping ... 14
C. Analytical Methodology 14
D. Analytical Results 15
1. Limits for Detection and
Reporting Level 15
2. Primary Laboratory Results 15
3. External Reference Laboratory Results ... 16
E. Quality Assurance Procedures and Results .... 16
1. Field Controls 16
2. Overall Study Precision 21
3. Chemical Analysis Precision Estimates
Using Field Spikes 22
111
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TABLE OF CONTENTS (continued)
Page
4. Comparison of Interlaboratory
Chemical Analysis 22
5. Total Accuracy Estimates for
Chemical Analysis 22
6. Compound Degradation 24
VI. ANALYSIS OF SERUM FOR SEMIVOLATILES 27
A. Summary of Results 27
B. Specimen Collection, Storage, and Shipping ... 28
C. Analytical Methodology 28
D. Analytical Results 28
1. Detection and Reporting Level Limits .... 28
2. Results ........... 29
E. Quality Assurance Procedures and Results .... 31
1. Field Controls 31
2. Plan for Set Structure and Analysis .... 31
3. Procedures for Replicate Specimen
Analysis 34
4. Analytical Procedures for Spiked-Split
Specimens 34
5. Precision Estimates 35
a. Overall Measurement Precision 35
b. Precision of Chemical Analysis .... 35
6. Chemical Analysis Percent Recovery 40
7. Total Error Estimates 40
VII. ANALYSIS OF URINE FOR SEMIVOLATILES 43
A. Summary of Results 43
B. Specimen Collection, Storage, and Shipping ... 44
C. Analytical Methodology 44
D. Analytical Results 44
1. Detection and Reporting Level Limits .... 44
2. Results 44
IV
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TABLE OF CONTENTS (continued)
Page
E. Quality Assurance Procedures and Results 45
1. Field Controls 45
2. Plan for Set Structure and Analysis .... 45
3. Procedures for Replicate Specimen
Analysis 49
4. Procedures for Spiked-Split Specimen
Analysis 49
5. Chemical Analysis Precision Estimates ... 49
a. Overall Estimate 49
b. Within- and Among-Set Precision .... 51
6. Chemical Analysis Percent Recovery 51
7. Total Error Estimates 51
VIII. REFERENCES 57
APPENDIX A: VOLATILES .ANALYTICAL METHODOLOGY
APPENDIX B: TAC-SPIKED BLOOD SERUM RECOVERIES
APPENDIX C: SEMIVOLATILE ANALYTICAL METHODOLOGY FOR URINE
APPENDIX D: GLOSSARY
REPORT DOCUMENTATION PAGE
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LIST OF TABLES
Number Pag<
1 Hispanic HANES (EPA Component) Pilot Study
Target Volatile Compounds 2
2 Hispanic HANES (EPA Component) Pilot Study
Target Semivolatile Compounds 3
3 Number of Specimens Collected by Type in
the El Paso, Texas, Pilot Study Site 10
4 Analysis of Blood for Volatiles: Summary
of Chloroform Results 17
5 Analysis of Blood for Volatiles: Summary
of Bromoform and Dibromochloromethane Results ... 18
6 Analysis of Blood for Volatiles: Summary
of External Reference Laboratory Results ...... 19
7 Analysis of Blood for Volatiles: Results
of Analyzing Field and Matching Laboratory-
Spiked Specimens 20
8 Analysis of Blood for Volatiles: Estimates
of Chemical Analysis Precision From Field
and Laboratory Spikes 23
9 Analysis of Blood for Volatiles: Estimates
of Chemical Analysis Total Error Using
Field Spikes 25
10 Analysis of Serum for Semivolatiles:
Summary of pp'-DDT, pp'-DDE, and
p-BHC Results 30
11 Analysis of Serum for Semivolatiles:
Results of Field-Spiked Specimens 32
12 Analysis of Serum for Semivolatiles:
Set Structure and Composition 33
13 Analysis of Serum for Semivolatiles:
Estimates of Overall Measurement Precision .... 36
14 Analysis of Serum for Semivolatiles:
Precision Estimate by Duplicate Type 37
VI
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LIST OF TABLES (continued)
Number
15
Analysis of Serum for Semivolatiles:
Estimates of Chemical Analysis Precision
26 Analysis of Urine Semivolatiles:
Estimates of Chemical Analysis Total Error
Pac
38
16 Analysis of Serum for Semivolatiles:
Estimates of Within- and Among-Set
Chemical Analysis Precision 39
17 Analysis of Serum for Semivolatiles:
Mean Percent Recovery by Compound 41
18 Analysis of Serum for Semivolatiles:
Estimates of Chemical Analysis Total Error .... 42
19 Analysis of Urine for Semivolatiles:
Frequency of Detection by Compound 46
20 Analysis of Urine Semivolatiles:
Summary of PCP Results 47
21 Analysis of Urine for Semivolatiles:
Results of Analyzing Field-Spiked Specimens .... 48
22 Analysis of Urine for Semivolatiles:
Set Structure and Composition 50
23 Analysis of Urine Semivolatiles:
Chemical Analysis Precision Estimates 52
24 Analysis of Urine Semivolatiles:
Within- and Among-Set Chemical Analysis
Precision Estimates 53
25 Analysis of Urine Semivolatiles: Mean
Percent Recovery by Compound 54
55
FIGURE
Number
Stability of Chloroform Levels in Pooled
Specimens Over Time
Page
26
VI1
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Vlll
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ACKNOWLEDGMENTS
The authors would like to recognize the participation of the
many persons and organizations required to conduct this activity.
Research Triangle Institute: The authors thank Dr. Robert
Handy and Dr. David Myers for their assistance in developing the
quality assurance protocols, Dr. Linda Sheldon for supervising
the preparation of the field QA specimens and chemical analyses
done at the Institute, and Dr. Edo Pellizzari and Mr. Hu Burnett
for their comments and assistance in revising the draft report.
National Center for Health Statistics: The authors thank
Mr. Robert Murphy and Ms. Trena Ezatti for their cooperation in
the specimen collection activities.
Chemical Epidemiology Division, Department of Epidemiology
and Public Health, University of Miami (Florida): The authors
thank Dr. Carl Phaffenburger and Ms. Anita Peoples for their
cooperation in implementing the QA protocols and performing the
chemical analyses of blood serum for volatiles.
Toxicant Analysis Center, U.S. Environmental Protection
Agency: The authors thank Drs. Aubry Dupuy, William Mitchell,
and Joseph Yonan for their cooperation in implementing the QA
protocols and performing the chemical analyses for semivolatiles
in serum and urine.
Office of Toxic Substances, U.S. Environmental Protection
Agency: The authors thank Ms. Linda Greenberg, Task Manager, and
Mr. Brion Cook for their assistance in coordinating the activ-
ities of the task. Ms. Cindy Stroup succeeded Ms. Greenberg as
Task Manager and supervised- the completion of this final report.
IX
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I. INTRODUCTION AND BACKGROUND
The Hispanic Health and Nutritional Examination Survey
(HANES) is one in a series of related studies carried out over
the past 20 years by the National Center for Health Statistics
(NCHS). These studies, authorized by Congress under the National
Health Survey Act of 1956, are characteristically national in
scope, based on probability sampling, and are used to collect a
broad range of morbidity data and related health and nutrition
information. In this latest study, NCHS will focus on the U.S.
Hispanic population, conducting medical examinations and adminis-
tering health-related questionnaires over a 2-year period to a
probability sample of Hispanic residents in the United States.
The study sites will include the southwestern States, portions of
Florida, and the New York/New Jersey metropolitan area. The data
collection period will extend from July 1982 through December
1984. During that time period, data will be collected from
approximately 12,000 Hispanic participants in approximately 30
county sites.
EPA's objective in this study is to assess the Hispanic
population's exposure to environmental pollutants (primarily
pesticides) by measuring the concentrations of selected pesti-
cides and toxic substances in body fluids, and evaluating the
approximate amount and type of exposure as reported by the re-
spondents. As part of a cooperative agreement, NCHS will provide
EPA with blood and urine specimens and interview results from a
subsample of study participants. EPA will chemically analyze the
specimens, statistically analyze the interview and chemical
analysis results, and .provide estimates of body fluid residue
levels and environmental exposure for the Hispanic participants
living in the study sites.
In preparation for data collection, NCHS conducted a pilot
study in 'El Paso, Texas, during January through March, 1982.
During the pilot study, EPA received 171 blood, serum, and urine
specimens from a subsample of study participants. These speci-
mens were shipped to (1) the Chemical Epidemiology Division,
Department of Epidemiology and Public Health, University of
Miami, and (2) the EPA Toxicant Analysis Center (TAG), for vola-
tile and semivolatile analyses, respectively. A total of 161
specimens were analyzed; 51 blood specimens were analyzed for
volatiles, and 59 serum and 51 urine specimens were analyzed for
semivolatiles. Tables 1 and 2 present lists of the pilot study
target compounds. During the follow-on national study, the list
of urine compounds will be expanded to include malathion metabo-
lites and carbamates.
EPA's primary objective in participating in the pilot study
was to assess the quality of the measurement procedures and data,
and to set data quality objectives for the national study. To
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Table 1. Hispanic HANES (EPA Component) Pilot Study
Target Volatile Compounds
Bromodichloromethane
Bromoform
Carbon tetrachloride
Chlorobenzene
Chloroform
Dibromochloromethane
1,2-Dichloroethane
Tetrachloroethylene
1,1,1-Trichloroethane
Trichloroethylene
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Table 2. Hispanic HANES (EPA Component) Pilot Study
Target Semivolatile Compounds
Serum
Urine
a-BHC
p-BHC
6-BHC
y-BHC
op'-DDD
pp'-DDD
op'-DDE
pp'-DDE
op'-DDT
pp'-DDT
Dieldrin
Endrin.
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Mirex
Oxychlordane
PCB's (Polychlorinated .
biphenyl)(Arochlor 1254)
trans-Nonachlor
2,4-D (2,4-dichlorophenoxy acetic
acid)
Dicamba
p_-Ni tr opheno 1
PCP (pentachlorophenol)
Silvex
2,4,5-T (2,4,5-Trichlorophenoxy
acetic acid)
2,4,5-Trichlorophenol
3,5,6-Trichloro-2-pyridinol
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this end, RTI was engaged to develop a quality assurance plan for
the pilot study and to assess the quality of the analytical re-
sults. This report describes EPA's participation in and results
from the pilot study, including the QA procedures and their re-
sults. Section II contains a summary of the results. Sections
III through IV provide overviews of the sample selection, data
collection, and quality assurance procedures. In sections V
through VII, the analytical methodologies, the analytical re-
sults, and the quality assessment data are presented in detail.
Because of the uniqueness of the quality assurance proce-
dures implemented in this study, a unique terminology was created
to facilitate distinguishing among the types of specimens and
specimen groups. A glossary of selected terms is included as
appendix D to assist the reader in understanding the terminology
used in this report.
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II. SUMMARY OF RESULTS
A. Introduction
The results of EPA's participation in the pilot study
are summarized in the following subsections. In all, over 300
specimens from 171 subjects ("sample persons") were chemically
analyzed over a 6-month period. A quality assurance plan was
developed and implemented in order to assess data quality. This
plan involved field controls to assess field contamination and
degradation, replicate chemical analyses to assess precision, and
spiked-split duplicate chemical analyses to assess chemical
analysis bias through compound percent recovery.
" A note of caution is in order regarding the interpretation
of the pilot study results. Estimates were calculated for a
number of parameters relating to these: percent detected; per-
cent recovery; averages, percentiles, and variances for detected
values; and variances of the measurement process. These param-
eters were all calculated from relatively small sample sizes of
fewer than 60 specimens, mainly because field problems resulted
in EPA being provided with a fewer number of specimens than was
originally planned. A second, and perhaps more critical problem
was the lack of data from which to base data quality estimates;
of the 37 total target compounds, only 5 compounds were detected
in a significant number of specimens. To estimate data quality
for the compounds which were seldom endogenous* in the specimens,
it was necessary to use substitute measurements such as field-
spiked and spiked-split duplicate specimens for chemical analysis
precision. These sample sizes were even fewer (e.g., only three
to nine field spikes per matrix). Using field spikes and spiked-
split specimens for making estimates for which they were not
intended is inappropriate and yields data of unknown validity.
The estimates in this report are' the best that could be made
based on the available data; however, because of the few speci-
mens available and lack of specimens with endogenous compounds,
most of these results should be considered preliminary. Analysis
of these data does point to potential problems with the analyt-
ical methodologies that warrant further investigation.
B. Analysis of Blood for Volatiles
Blood specimens from 51 sample persons were analyzed
for the presence of volatile compounds at the Chemical Epidemi-
ology Division, Department of Epidemiology and Public Health,
University of Miami ("Miami lab"), using a purge/trap/desorb
*The word "endogenous" as used in this report refers to compounds
that are found in the specimens naturally, as opposed to com-
pounds that are spiked in the specimens.
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procedure based on that of Bellar and Lichtenberg (see appen-
dix A). A subsample of 20 specimens was also analyzed by an
external reference laboratory, the Research Triangle Institute
(RTI), using the identical procedure. Of the 10 target com-
pounds, only chloroform was detected in a significant number of
specimens (100 percent) at the primary laboratory. The median
chloroform value was 8.3 ppb, and the highest value was 4,000
ppb. At the external reference laboratory, chloroform also had
the highest percent detected and highest mean concentration. Of
the 10 target compounds, positive detections were obtained for 6
compounds at the external reference laboratory, while interfer-
ences prevented analysis of the other 4 compounds. Different
instrumentation allowed RTI to set lower minimum reporting levels
than could the Miami lab. An interlaboratory chemical analysis
comparison showed that the Miami lab obtained mean chloroform
values approximately 183 percent higher than did RTI.
An assessment of total error for chemical analysis based
upon a combination of percent recovery and precision showed that
only chloroform had a total error estimate (36 percent) within
the EPA guidelines (USEPA 1979) for acceptability (below 50 per-
cent). The other 9 compounds (excluding chloroform) had unac-
ceptable total errors, ranging up to 256 percent for dibromo-
chloromethane. The estimates for these 9 compounds were wholly
obtained, however, from a small sample of field spikes, due to
the lack of endogenous-compounds data. The high total errors and
the large interlaboratory differences suggest possible problems
with the analytical method.
Replicate analyses over time from a specimen pool showed
that no degradation of chloroform levels occurred over a 2^-month
time period.
C. Analysis of Serum for Semivolatiles
Specimens from a total of 59 sample persons were ana-.
lyzed for the presence of 19 semivolatile compounds at the EPA
Toxicant Analysis Center (TAG) using the EPA standard electron
capture gas chromatography method. Positive detections occurred
for only 6 of the 19 tested compounds: trans-nonachlor; pp'-DDT;
pp'-DDE; p-BHC; dieldrin, and 6-BHC. Of these, only 3 were
detected in a significant number of specimens: in 44 percent of
specimens, pp'-DDT was detected with a mean positive value* of
3.2 ppb; in 100 percent of specimens, pp'-DDE was detected with a
mean positive value of 34.2 ppb; and in nearly 85 percent of
specimens, p-BHC was detected with a mean positive value of
2.4 ppb.
*When the adjective "positive" is used, it means that zero values
were excluded from the calculation.
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An assessment of chemical analysis total error was made for
6 of the 19 target compounds. Due to the lack of endogenous-
compounds data, substitute measurements for precision were used
for 3 of these 6 compounds, while estimates could not be made at
all for the other 13 compounds. The total error estimates for
each of the 6 compounds were within the EPA guidelines for accep-
tability (below 50 percent). Based on limited data, it appears
that data of acceptable quality can be produced with the analy-
tical procedures used for the compounds for which estimates could
be made.
D. Analysis of Urine for Semivolatiles
Urine specimens from a total of 51 sample persons were
analyzed at TAG fdr the presence of eight phenol compounds using
the electron capture gas chromatpgraphy method of T. M. Shafik
(see appendix C). Four of the eight tested compounds were de-
tected. Only pentachlorophenol (PCP) was detected in a signifi-
cant number of specimens; PCP had a 75-percent detection rate and
a mean positive value of 4.3 ppb. Three other compounds (3,5,6-
trichloro-2-pyridinol; p_-nitrophenol; and 2,4,5-trichlorophenol)
were detected, but in less than 4 percent of the specimens.
An assessment of chemical analysis total error (defined in
section V.E.5) showed that of the eight target compounds, only
two compounds, 3,5,6-trichloro-2-pyridinol and 2,4,5-trichoro-
phenoxy acetic acid (2,4,5-T) were within the EPA guidelines
criterion of below 50-percent total error. Because of the lack
of endogenous-compounds data, chemical analysis precision had to
be estimated from the percent coefficient of variation of the
spiked-split duplicates for every compound except PCP. The total
error for PCP was 83 percent, based upon a 9.1 percent coeffi-
cient of variation of duplicate measurements and a 45-percent
chemical analysis percent recovery.
E. Conclusion
The great majority of target compounds (32 of 37) were
not detected. For volatiles, only chloroform was consistently
detected, and only chloroform showed an acceptable total error
rate. For semivolatiles in serum, only three compounds were
consistently detected: pp'-DDT; pp'-DDE; and trans-nonachlor.
Total error rates for these three compounds were acceptable, as
were the rates for three additional compounds for which sub-
stitute data quality estimates were made. Only one semivolatile
compound, PCP, was consistently detected in urine. Substitute
measurements permitted the estimation of total error for all
eight compounds; however, of these, only two compounds had total
error estimates within acceptable limits. The total error esti-
mates for the PCP analytical data were not within the acceptable
limits.
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Judging from the high total error estimates made, it appears
that problems may exist with the analytical methods for volatiles
in blood and for semivolatiles in urine. The method for semi-
volatiles in serum appears to produce data of acceptable quality.
Again, it should be stressed that because of the small sample
sizes and lack of endogenous-compounds data, these results should
be considered preliminary. Further study is needed to more
accurately assess data quality from these methods.
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III. SAMPLE SELECTION AND DATA COLLECTION OVERVIEW
The pilot study site, El Paso, Texas, was selected by NCHS
because of its high proportion of Hispanic residents. An area
probability sample of El Paso households was selected, and study
participants were chosen from eligible households within the
sample. Households were considered eligible if at least one
member was of Hispanic descent. Selected participants were
administered a health history questionnaire in the home and then
scheduled for a detailed physical examination at a mobile exam-
ination center.
As part of the physical examination process, blood and urine
specimens were collected from each participant. NCHS collected
specimens for EPA from a systematic subsample of study partici-
pants who were 12-74 years of age. Whole blood was collected for
volatile analysis, and serum and urine were collected for semi-
volatile analysis. Table 3 depicts the number of specimens that
EPA received within each age group for each matrix. The response
rate for the EPA component was significantly lower than expected
within each category, in most cases lower than 50 percent.
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Table 3. Number of Specimens Collected by Type
in the El Paso, Texas, Pilot Study Site
Number of EPA sample persons
from whom specimens were
Matrix Age group collected
Serum 20-74 years 59
Urine 12-74 years 54
Blood 12-19 years 58
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IV. QUALITY ASSURANCE PLAN OVERVIEW
A quality assurance plan was implemented by EPA during the
pilot study in order to assess and set objectives for data qual-
ity and provide a method for identifying specific problems or
factors affecting data quality. The plan was designed to assess
overall measurement error and chemical analysis precision and
accuracy, and to identify specific biases such as specimen con-
tamination or degradation. The quality assurance procedures that
were implemented in the pilot are summarized below. Specific
procedural details and the data assessment results are presented
in sections V through VII.
A. Field Controls
Field controls were used to monitor specimen contami-
nation and degradation occurring in the field and during ship-
ping. The controls, which consisted of both spiked and blank
(unspiked) specimens, were prepared from a large homogenous
matrix pool on a weekly basis at RTI and shipped overnight to the
collection site. At the collection site, the controls were
stored with the survey specimens and then included in the ship-
ment of specimens to the laboratories. The arrival of controls
at the collection site was timed so that each set of controls
would remain there for the same length of time as the longest
holding time for survey specimens, in order to provide data on a
worst case storage-time basis.
Two blanks and two spikes (spiked specimens) of each matrix
were prepared to accompany each specimen shipment to the labora-
tories. RTI also prepared matching laboratory controls for each
field control set and shipped these directly to the laboratories.
Upon receipt at the laboratories, one-half of the field
controls—one spike and one blank from each shipment—were chem-
ically analyzed. The remaining field controls and the matching
laboratory controls were held in reserve for analysis in case
problems were detected in the analysis of the initial field
controls. The identity of field controls was not known to the
chemists at TAG; however, at the Miami lab, it was not feasible
to prevent the chemist from knowing the identity of the contents
to be analyzed because of limited staff.
B. Replicate Specimen Analysis
In order to obtain estimates of measurement error, a
subsample of specimens was independently analyzed in duplicate.
Various types of duplicates were used in order, to provide dif-
ferent levels of precision estimates. Due to the differences in
procedures among blood, serum, and urine collection and proces-
sing, the procedures for collecting and analyzing duplicates were
not identical for each matrix. The general types and purposes of
11
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replicate analyses are listed below, and the specific procedures
for each matrix are described in sections V-VII.
Field Duplicates - Blood was drawn into
separate vacuum tubes and processed and
analyzed independently. This type of repli-
cate analysis provided the best estimates of
overall study precision. It was not possible
to collect this type of duplicate for urine
due to the nature of urine collection.
Field Splits - For serum semivolatile speci-
mens, duplicate aliquots were prepared. This
type of duplicate provided an estimate of
precision from the point of specimen split-
ting through chemical analysis.
Lab-Split Duplicates - Specimens, either sin-
gle or duplicate halves, were split in the
laboratory prior to chemical analysis. This
provided the best estimate of chemical analy-
sis precision.
External Reference Duplicate Analyses - A
subsample of blood specimens was sent to an
external reference laboratory, RTI, for
volatile analysis. These specimens were the
field duplicates of specimens analyzed at the
Miami lab. The results of the two analyses
were compared to provide an estimate of
interlaboratory chemical analysis precision.
All chemical analyses were performed within "sets" consist-
ing of groups of other specimens analyzed at the same time. In
order to estimate the chemical analysis precision both within and
among sets, some duplicates had both halves analyzed in the same
set, and other duplicates had the halves analyzed in separate
sets.
C. Spiked-Split Duplicate Analysis
Spiked-split specimens were used in order to estimate
the compound percent recovery being obtained in the semivolatile
chemical analyses. Spiked-split specimens were created by spik-
ing only one of the two duplicate specimens. Upon chemical
analysis, the measured difference between the spiked and unspiked
specimens was divided by the known spiking amount to obtain the
fractional recovery.
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V. ANALYSIS OF BLOOD FOR VOLATILE COMPOUNDS
Blood specimens were shipped to the Miami Lab and chemically
analyzed for the presence of selected volatile compounds. A
quality assurance plan involving replicate analyses was developed
and followed. As an external quality assurance procedure, a sub-
sample of 20 duplicate specimens was analyzed by RTI. In order
to determine if compound levels were changing, samples from two
specimen pools were repeatedly analyzed over a 24-month time
period. The analytical and quality assurance procedures and
results are discussed below.
A. Summary of Results
The results at the primary laboratory show only chloro-
form being detected consistently in levels above the minimum
reporting level of 1 ppb. The median chloroform detection was
8.3 ppb. Bromoform and dibromochloromethane were detected by the
primary laboratory in fewer than 10 percent of the specimens,
with median positive values of 6.0 and 6.5 ppb, respectively. In
the external reference laboratory (RTI), six of the ten target
compounds were detected. Interferences and a resolution problem
prevented data from being obtained for the remaining four com-
pounds. RTI also found that the highest concentrations measured
were those of chloroform. An interlaboratory chemical analysis
comparison, however, showed that the Miami lab obtained mean
chloroform values that were approximately 183 percent higher than
did RTI.
In order to assess the quality of the chemical analysis
data, a total error estimate was calculated from a combination of
the percent recovery and precision estimates of the chemical
analyses. Due to the infeasibility of using spiked-split dupli-
cates, the field spikes were used to estimate chemical analysis
percent recovery. Since there were no endogenous-compounds
precision data, field spikes were also used to estimate precision
for every compound except chloroform, for which duplicate speci-
mens were used. Of the ten target compounds, only the chloroform
total error (36 percent) was within the EPA guidelines (USEPA
1979) for acceptability (below 50 percent). The remaining nine
compounds all had unacceptable total error estimates, ranging up
to 256 percent for dibromochloromethane. The small number of
specimens and limited endogenous-compounds data limit the con-
clusions that may be drawn from the data. The high total error
rates and large interlaboratory differences suggest that problems
exist with the analytical method.
Analyses of the field blanks showed that chloroform was
present at a mean concentration of 11.3 ppb. No other compounds
were detected. The analysis of the field spikes showed a percent
recovery for chloroform of 101 percent; however, the percent
13
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recoveries for the other compounds ranged from 0 to 290 percent,
indicating that there were problems either with contamination and
degradation in the field, or, more likely, with the analytical
method. Field contamination seems unlikely since basically only
chloroform was detected in the field blanks and survey specimens.
Results from the degradation study showed that no changes in
levels occurred over the 2^-month time period.
B. Specimen Collection, Storage, and Shipping
Blood specimens for volatile analysis were collected
via brachial venipuncture into Becton-Dickinson green-top vacuum
tubes. After the specimens were drawn, the containers were
inverted several times in order to mix the anticoagulant present
in the container. Specimens were labeled and immediately stored
in the refrigerator in order to minimize the loss of volatile
compounds.
Specimens were collected from a total of 58 sample persons.
Duplicate specimens were obtained from 39 of these sample persons
by drawing blood into separate vacuum tubes.
Specimens were shipped from the collection site to the Miami
lab by overnight mail service. Insulated boxes with cold packs
were used for shipping in order to maintain a cool temperature;
however, it is known that the first several shipments of speci-
mens arrived at the Miami lab at an ambient temperature.
C. Analytical Methodology
The method of Peoples and coworkers (appendix A), a
purge/trap/desorb method based on that of Bellar and Lichtenberg,
was used by both the Miami lab and RTI to determine the concen-
tration of purgeable halogenated hydrocarbons in blood plasma.
The procedure involves heating the specimen while ' purging the
volatiles from the solution with a flow of an inert gas. The
purged compounds are directed to an absorbent trap. After the
purge/trap period is completed, the volatile analytes are therm-
ally desorbed from the absorbent trap to a gas chromatograph
programmed to provide complete resolution of all the compounds of
interest. Other incidental compounds may cause interference
problems.
The Miami lab analyzed each specimen on two GC columns:
N-octane and SP-1000. Peak heights were manually measured, and
quantitations were based on the response of a single standard-
analyte mixture.
RTI analyzed each of 20 specimens once on an N-octane GC
column. An integrator was used to measure peak area, and quanti-
tation was based on a five-point standard calibration curve.
14
-------
D. Analytical Results
1. Limits for Detection and Reporting Level
The minimum reporting level set by the Miami lab
was 1 ppb. Compounds detected at levels between 0 and 1 ppb were
reported as not detected (zero).
RTI reported all compounds detected in amounts above the
calculated quantitation limits:
RTI minimum reporting levels
Quantitation PPB for 1 mL
Compound limit (ng) analysis volume
_2
carbon tetrachloride 4.3 x 10 .043
_2
chloroform 3.4 x 10 .034
_2
1,1,1-trichloroethane 5.5 x 10 .055
_2
trichloroethylene 4.6 x 10 .046
_2
bromodichloromethane 5.9 x 10 .059
_2
tetrachloroethylene 3.8 x 10 .038
RTI also reported trace values that occurred.
2. Primary Laboratory Results
Specimens from a total of 51* sample persons were
analyzed by the primary laboratory. Since the majority of speci-
mens were analyzed in duplicate, a total of 90 independent analy-
ses were performed.
Of the ten target compounds, only three were detected in
levels above 1 ppb: chloroform, bromoform, and dibromochloro-
methane. Of these, only chloroform was consistently detected in
levels above 1 ppb, with 100 percent of the specimens containing
concentrations between 2 and 4,000 ppb. The specimen! with the
highest detected chloroform value, 4,000 ppb, was substantially
higher than any amounts found in the other specimens, the next
highest detected value was 165 ppb. Due to the great influence
that this extreme value has on the mean and variance, these sta-
*The remaining 7 specimens were either not analyzable or were
used in the degradation study pool.
tRTI analyzed this specimen also and reported a concentration of
120 ppb.
15
-------
tistics were calculated both with and without this value. Table
4 presents a summary of the chloroform data.
The proportions above the 1 ppb detection level for bromo-
form and dibromochloromethane were 5.9 and 7.8 percent, respec-
tively. Table 5 presents a summary of the results for both
compounds. Because the detection rate was so low, only the
nonzero values were used in calculating statistics.
3. External Reference Laboratory Results
No data were obtained by RTI for four of the ten
target compounds. Due to unidentified late eluting general
interferences, data were not obtained for three compounds:
chlorobenzene, bromoform, and dibromochloromethane. The fourth
compound, 1,2-dichloroethane, could not be quantitated because of
a resolution problem with tetrachloroethylene under the N-octane
column. Like the Miami lab,. RTI obtained the highest concentra-
tions for chloroform. Of the six compounds for which results
were obtained, three compounds other than chloroform were de-
tected in amounts exceeding 1 ppb. These were 1,1,1-trichloro-
ethane, tetrachloroethylene, and carbon tetrachloride. The
values reported for these three compounds ranged up to 4.6 ppb.
A summary of the RTI results is presented in Table 6.
E. Quality Assurance Procedures and Results
1. Field Controls
Field controls were prepared at RTI, shipped to
and stored at the collection site, and then included in the
specimen shipments to the Miami lab (sec. IV.C). Matching lab-
oratory controls were prepared at RTI and shipped directly to the
Miami lab. Upon receipt at the Miami lab, approximately one-half
of the field controls and several of the matching lab controls
were chemically analyzed.
A total of eight blanks were analyzed: six field blanks and
two matching lab blanks. Both types of blanks were found to
contain similar levels of chloroform. The two lab blanks con-
tained 11 and 9 ppb, and the six field blanks contained a mean
chloroform concentration of 11.3 ppb. No other compounds were
detected in either type of blank.
Table 7 presents a summary of overall mean percent recov-
eries, and percent recoveries for field and matching lab spikes.
Seven field spikes and two matching lab spikes were analyzed,
each spiked at varying levels. Because the spiking levels varied
among the specimens, they are not included in the table. The de-
tected levels were adjusted for the inherent presence of chloro-
form in blood by subtracting out the mean detected level of
chloroform found in the field and matching lab blanks.
16
-------
Table 4. Analysis of Blood for Volatiles:
Summary of Chloroform Results*
Number of specimens analyzed 51
Number of positive detections 51
Percent positive detections 100%
Mean (ppb)a 18.7
Mean (ppb)b 96.8
Variance3 929
Variance13 3.12 x io5
Percent coefficient of variation3 163%
Percent coefficient of variation 577%
Minimum value (ppb) 2
Maximum value (ppb) 4,000
Second highest value (ppb) 165
Median (ppb)a 8.3
Twenty-fifth percentile (ppb)a 6.0
Seventy-fifth percentile (ppb)a 17.0
*Based on results reported by the Chemical Epidemiology Division,
Department of Epidemiology and Public Health, University of
Miami.
aExcludes the high value of 4,000 ppb.
Includes the high value of 4,000 ppb.
17
-------
Table 5. Analysis of Blood for Volatiles:
Summary of Bromoform and Dibromochloromethane Results*
Bromoform Dibromochloromethane
Number of specimens analyzed
Number of positive detections3
Percent positive detections
Mean positive values (ppb)
Median positive values (ppb)
Minimum reported value (ppb)
Maximum value (ppb)
Variance of the positive values
Coefficient of variation of
the positive values
51
3
5 Qa/
J • y/o
5.8
6
4.5
7
1.6
21.6%
51
4
*7 Q°/
/ .O/o
6.5
6.5
3
10
16.3
62.2%
*Based on results reported by the Chemical Epidemiology Division,
Department of Epidemiology and Public Health, University of
Miami.
aAbove the minimum reporting level of 1 ppb.
18
-------
Table 6. Analysis of Blood for Volatiles:
Summary of External Reference Laboratory Results*
Compound'
Bromo-
dichloro-
methane
Trichloro-
ethylene
1,1,1- Tetra-
Trichloro- chloro-
ethane ethylene
Carbon
Chloro- tetra-
form chloride
Number and percent
positive detections
(excluding trace
detections)
Number and percent
trace detections
Number and percent
2(10%)
2(10%)
7(35%)
3(15%)
14(70%) 14(70%) 19(95%) 2(10%)
0 (0%)
0 (0%)
0 (0%)
2(10%)
of values > 1 ppb
Mean (ppb)
Mean positive
value (ppb)
Median positive
value, (ppb)
Maximum value (ppb)
0 (0%)
.02
.2
.2
.3
0 (0%)
.1
.3
.3
.5
3(15%)
.6
.9
.6
4.6
4(20%)
.9
1.2
.7
4.4
18(90%)
14.5
15.2
2.7
120
1 (5%)
.1
.8
.2
1.5
*Based on analyses of 20 specimen analyses reported by the Analytical Sciences
Division, Research Triangle Institute.
aDue to interferences, no data were obtained for chlorobenzene, bromoform, dibromo-
chloromethane, and 1,2-dichloroethane.
Trace is defined as below the minimum quantitation levels defined in section V.C.I.
-------
Table 7. Analysis of Blood for Volatiles: Results of
Analyzing Field and Matching Laboratory-Spiked Specimens*
Mean percent recovery
Matching
Compounds Overall Field spikes lab spikes
Bromodichloromethane
Bromoform
Carbon tetrachloride
Chlorobenzene
Chloroform
Dibromochloromethane
1 , 2-Dichloroethylene
Tetrachloroethylene
1,1, 1-Trichloroethane
Trichloroethylene
155
100
87
56
101
134
121
86
156
115
177
95
93
72
102
130
121
77
118
108
80
119
67
0
98
149
119
90
290
141
Note: The mean positive values have been adjusted by subtracting
the mean concentration found in the blanks; chloroform was
the only compound affected.
*Based on results of analyzing 7 field spikes and 2 matching lab-
oratory spikes at the Chemical Epidemiology Division, Department
of Epidemiology and Public Health, University of Miami.
The relative percent bias is estimated by the difference of the
mean percent recovery and 100 percent.
20
-------
The overall mean percent recovery for chloroform was excel-
lent, approximately 101 percent. The mean percent recoveries for
the other compounds ranged from 0 to 290 percent, indicating
either problems with contamination and degradation in the field,
or, more likely, problems with the analytical method. Field
contamination seems unlikely, however, since only chloroform was
detected in any frequency in the field blanks and survey
specimens.
2. Overall Study Precision
The majority of specimens (39 of 58) were collec-
ted in duplicate in the field (blood drawn in separate vacutain-
ers) and independently analyzed. In addition to the field dupli-
cates, ten specimens were split and independently analyzed at the
Miami lab. Approximately one-half of all duplicates were ana-
lyzed in the same set, and approximately one-half were analyzed
in separate sets.* Due to a logistical problem at the primary
laboratory that prevented specimen renumbering and relabeling,
the identities of duplicate specimens was known to the chemist
performing the analyses.
An estimate of overall study precision was obtained by
comparing the results obtained from the analyses of both field
duplicates (blood collected and processed in separate vacutain-
ers), and laboratory-split duplicates at the primary laboratory.
Both types of duplicates were analyzed both within and among
sets. A total of 39 duplicate specimen pairs were analyzed at
the Miami lab; 10 pairs were laboratory splits and the remaining
pairs were field duplicates.
2
A pooled estimate of variance, s , was calculated using
T ^ / TT ^T \ ™
2
where s denotes estimate of variance, X. . represents the jth
measurement on the ith pair, X. is the mean of the ith pair, and
2
n is total number of duplicate pairs (39). For chloroform, s
was equal to 5.76. The coefficient of j/ariation, 17.7 percent,
was obtained by dividing s by X where X is the overall mean of
the 39 pairs.
The variance was not calculated for bromoform or dibromo-
chloromethane because of the low frequency of detection. Bromo-
form was detected in only one duplicate pair; the values obtained
*A set consisted of all specimens analyzed on the same day,
usually 1-3 specimens.
21
-------
were 4 and 5 ppb. Dibromochloromethane was detected in two
duplicate pairs; in one pair the values were the same, and in the
other pair the values were 6 and 14 ppb.
3. Chemical Analysis Precision Estimates
Using Field Spikes
As noted in the preceding sections, not much data
were obtained on compounds endogenous to the blood. Only chloro-
form was detected with sufficient frequency to obtain reliable
estimates of the chemical analysis precision. In order to sup-
plement the limited endogenous-compounds data available, the
field spikes and matching lab spikes were used to provide esti-
mates of the chemical analysis precision for all ten compounds.
The coefficient of variation was calculated separately for field
and matching lab spikes and overall. The overall coefficients of
variation ranged from 23 percent for 1,2-dichloroethylene to 111
percent for dibromochloromethane. .Only dibromochloromethane was
higher than 41 percent. Table 8 presents these estimates.
4. Comparison of Interlaboratory Chemical Analysis
An estimate of the chemical analysis precision
between the primary and reference laboratories was calculated
from the results of the 20 specimens that were analyzed by both
laboratories. Chloroform is the only compound for which both
laboratories obtained positive results, and it is the only com-
pound for which precision was estimated.
The mean and variance of the differences in detected values
between laboratories were calculated both including and excluding
the specimen for which the Miami lab obtained a value of 4,000
ppb, since RTI obtained a much lower value (120 ppb) for the same
specimen. The Miami lab obtained higher chloroform values than
did RTI for each of the 20 specimens. When the 4,000 ppb speci-
men was excluded, the mean difference was 16.37 and the variance
of the differences was 623.45; the mean was found to be signif-
icant at the .01 level. When the 4,000 ppb specimen was exclud-
ed, the Miami lab was 183 percent higher than was RTI. After
including the 4,000 ppb specimen, the mean difference was 209,
and the variance of the differences was 746,974. After including
the 4,000 ppb specimen, the Miami lab was 1,447 percent higher
than was RTI.
5. Total Accuracy Estimates for Chemical Analysis
Chemical analysis total error was estimated for
each compound by using both the chemical analysis bias (percent
recovery) and precision information. Due to the infeasibility of
using spiked-split duplicates, the percent recovery of the field
spikes was used to estimate chemical analysis percent recovery.
22
-------
Table 8. Analysis of Blood for Volatiles: Estimates of
Chemical Analysis Precision From Field and Laboratory Spikes*
Compounds
Bromodichlorome thane
Bromoform
Carbon tetrachloride
Chlorobenzene
Chloroform
Dibromochloromethane
1 , 2-Dichloroethylene
Tetrachloroethylene
1,1, 1-Trichloroethane
Trichloroethylene
Coefficient
Field
spikes
39
28
19
51
36
110
24
31
50
36
of variation
Lab
spikes
0
21
40
0
11
113
21
0
8
32
(percent)
Overall
30
26
24
40
30
111
23
24
41
35
*Based on results from the analysis of 7 field spikes and 2
matching laboratory spikes at the Chemical Epidemiology
Division, Department of Epidemiology and Public Health,
University of Miami.
23
-------
Since there were no endogenous precision data, the coefficient of
variation of the field spikes was used to estimate precision for
every compound except chloroform, for which the coefficient of
variation of duplicate analyses was used. Two estimates were
calculated for each compound. The Total Error was calculated by:
percent recovery - 100| + 2 (percent coefficient
of variation) .
The twice Root Mean Square Error was calculated by:
2 [(percent recovery - 100)2 + (percent coefficient
2 i-
of variation) ] 2 .
The estimates for both Total Error and twice the Root Mean
Square Error are presented in Table 9. Only one compound, chlor-
oform, had either Total Error or Root Mean Square Error less than
50 percent, the EPA guidelines acceptance criterion. The Total
Error for chloroform was 36 percent. The remaining nine com-
pounds had Total Errors or Root Mean Square Errors of unaccept-
able proportions, ranging up to 256 percent for dibromochloro-
methane.
6. Compound Degradation
In order to determine if changes in compound
levels were occurring in specimens over time, samples from two
specimen pools were repeatedly analyzed over a 2H-month time
period. The plasma pools were created by combining several study
specimens, each pool containing a different composite.
One specimen from each pool was analyzed approximately every
2 weeks over the 2V-month period. Chloroform was the only com-
pound detected in either pool. The results, depicted in the
graph in Figure 1, show no apparent trends in the levels of
chloroform over time.
24
-------
Table 9. Analysis of Blood for Volatiles: Estimates of
Chemical Analysis Total Error Using Field Spikes*
2 RMSE
Compound (percent)
Bromodichloromethane
Bromoform
Carbon tetrachloride
Chlorobenzene
Chloroform
Dibromochlorome thane
1 , 2-Dichloroethylene
Tetrachloroethylene
1,1, 1-Trichloroethane
Trichloroethylene
125
52
55
119
36C
232
62
62
139
76
Total error
(percent)
115
52
61
124
36C
256
67
68
138
85
*Based on results from the Chemical Epidemiology Division,
Department of Epidemiology and Public Health, University of
Miami.
aTwo Root Mean Square Error (RMSE)
o
= 2 [(percent recovery - 100)
2 l*
+ (percent coefficient of variation) ]%
Total Error
= I percent recovery - 1001
+ 2 (percent coefficient of variation).
The percent coefficient of variation of the duplicate analyses
was used for chloroform.
25
-------
26
24
22
20
18
16
14
O)
o
'^
(0
I
§ 12
o
•o
. Q>
en
10
. 8
6
4
2
Pool 1
Pool 2
J I
I
I
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Day
Figure 1. Stability of Chloroform Levels in
Pooled Specimens Over Time.
26
-------
VI. ANALYSIS OF SERUM FOR SEMIVOLATILES
Serum specimens were shipped to the EPA Toxicant Analysis
Center (TAG) and analyzed for the presence of selected semivola-
tile compounds (Table 2). A quality assurance plan involving
replicate analyses and spiked-split duplicate analyses was devel-
oped and followed. The analytical and quality assurance proce-
dures and results are discussed below.
A. Summary of Results
Only 6 of the 19 tested compounds were detected: trans-
nonachlor; pp'-DDT; pp'-DDE; p-BHC; dieldrin; and 6-BHC. Of
these, only three yielded reporting levels in a significant
number of specimens: in 44 percent of the specimens, pp'-DDT was
measured with a mean positive value of 3.2 ppb; in 100 percent of
the specimens, pp'-DDE was measured with a mean value of 34.2
ppb; and in nearly 85 percent of the specimens, p-BHC was meas-
ured with a mean positive value of 2.4 ppb.
In order to assess the quality of the chemical analytical
data, total error estimates were calculated based upon a combina-
tion of the chemical analysis percent recovery and analytical
precision estimates. Chemical analysis percent recovery was
estimated from the spiked-split duplicates. The percent coeffi-
cient of variation of the lab-split duplicates was used to esti-
mate precision for the three compounds frequently detected:
pp'-DDE, pp'-DDT, and p-BHC. Precision for trans-nonachlor,
dieldrin, and oxychlordane was estimated from the percent coeffi-
cient of variation of the spiked-split duplicates. Due to the
lack of endogenous-compounds data, total error estimates could be
made for only 6 of the 19 target compounds. The accuracy esti-
mates were within the EPA guidelines (USEPA 1979) for acceptabil-
ity (below 50 percent) for each of the six compounds. It there-
fore appears that the analytical procedures resulted in data of
acceptable quality for the six compounds, although the small
sample sizes and the use of substitute precision measurements
limit the conclusions that can be drawn from the data.
After comparing duplicates analyzed within the same set to
duplicates analyzed in separate sets, much better precision was
obtained from within-set analyses, suggesting large measurement
differences from set to set.
The analysis of the field blanks showed moderate levels of
pp'-DDE and hexachlorobenzene. Analysis of the field spikes
showed that the majority of percent recoveries fell within the 75
to 125 percent range. Two compounds, heptachlor and oxychlor-
dane, had very high percent recovery estimates of 161 and 130
percent, respectively. Field contamination seems unlikely,
however, since the two compounds were not detected in either
27
-------
field blanks or survey specimens. Three compounds were detected
that were not present in the spiking solution: op'-DDT; 6-BHC;
and dieldrin. This suggests that false positives may be produced
using the analytical method since none of the three compounds
were detected in the field blanks.
B. Specimen Collection, Storage, and Shipping
Blood specimens were drawn via brachial venipuncture
into red top Becton-Dickinson vacuum tubes. Several specimens
were drawn from each participant. After collection, the tubes
were stored upright at room temperature until clots formed, and
then centrifuged. After centrifuging, the serum from each tube
was drawn off by suction into a temporary storage container, and
EPA's subspecimens were removed using a disposable pipette.
Specimens were labeled and stored in the freezer until shipment
to the laboratory. Specimens were shipped to the laboratory in
insulated containers packed with dry ice in order to maintain the
frozen state.
C. Analytical Methodology
Serum specimens were extracted and the analytes of
interest were determined by electron capture gas chromotography.
The procedure in the EPA reference manual (EPA-600/8-80-030, June
1980, Analysis of Human Blood or Serum in Analysis of Pesticide
Residues in Human and Environmental Samples, RTP, NC) was fol-
lowed exactly.
D. Analytical Results
1. Detection and Reporting Level Limits
The minimum reporting levels set by TAG for this
study are listed below. Compounds detected in amounts below
these levels were reported as zero, except in the case of spiked-
split specimens, where trace values were reported.
Minimum reporting
Compound level (ppb)
a-BHC 1
P-BHC 2
6-BHC 1
y-BHC 1
op'-DDD 2
pp'-DDD 2
op'-DDE 2
pp'-DDE 2
28
-------
Minimum reporting
Compound level (ppb)
opf-DDT 2
pp'-DDT 2
dieldrin 2
endrin 2
heptachlor epoxide 1
heptachlor 1
hexachlorobenzene 1
mirex 4
oxychlordane 2
PCB's (Arochlor 1254) 20
trans-nonachlor 1
2. Results
Specimens from a total of 59 sample persons were
analyzed for the presence of 19 separate compounds. Six of the
19 target compounds had levels measured above the minimum re-
porting level:
p-BHC;
6-BHC;
pp'-DDE;
pp'-DDT;
dieldrin; and
trans-nonachlor.
Of these, only three yielded reporting levels in a significant
number of specimens. Table 10 presents a summary of these
results: in 44 percent of the specimens, pp'-DDT was measured
with a mean positive value of 3.2 ppb; in 100 percent of the
specimens, pp'-DDE was measured with a mean positive value of
34.2 ppb and the values ranged from 105 to 10 ppb; in nearly 85
percent of the specimens, p-BHC was measured with a mean positive
value of 2.4 and the median positive value was 2 ppb; in two
specimens, trans-nonachlor was measured with a mean positive
value of 1.2 and the median positive value was 0.7 ppb; in only
one specimen, dieldrin was measured at 1 ppb; and in one speci-
men, 6-BHC was also only measured at 0.9 ppb.
29
-------
Table 10. Analysis of Serum for Semivolatiles:
Summary of pp'-DDT, pp'-DDE, and p-BHC Results*
pp'-DDT pp'-DDE 0-BHC
Number of
specimens analyzed
Number of
positive detections
Percent
positive detections
Mean of the
positive values (ppb)
Mean overall value (ppb)
Median of the detected
value (ppb)
Median overall value (ppb)
Minimum reported positive
value (ppb)
Maximum value (ppb)
59
26
44.1
3.2
1.4
2.8
0
1.5
8
59
59
100
34.2
34.2
29.7
29.7
9.5
105
59
50
84.8
2.4
2
2
1.9
.6
5.8
Variance of the positive
values 2.4 414 1.6
Coefficient of variation
of the positive
values (Percent) 47.8 59.5 52.9
*Based on results reported by the EPA Toxicant Analysis Center.
Above the minimum reporting level of 2 ppb.
30
-------
E. Quality Assurance Procedures and Results
1. Field Controls
Field Controls were prepared at RTI, shipped to
and stored at the collection site, and then included in the
specimen shipments to the primary analysis laboratory (see sec.
IV.C). Matching laboratory controls were prepared at RTI and
shipped directly to the laboratory.
Upon receipt at the primary laboratory, four field spikes
and four field blanks were analyzed. All four field blanks
showed levels of pp'-DDE, with a mean level of 7 ppb. Two blanks
showed levels of hexachlorobenzene with a mean positive value of
0.6 ppb.
The results of the field-spike analyses are presented in
Table 11. Mean percent recoveries ranged from 0 for PCB's
(Arochlor 1254) and mirex to 161 percent for heptachlor. Most
compounds fell within the 75 to 125 percent recovery range.
Endrin's mean percent recovery was 64 percent, while heptachlor
and oxychlordane had recoveries of 161 and 130 percent, respec-
tively. PCB's and mirex were spiked but not detected.
It would be appropriate to compare the field-spike percent
recoveries to the spiked-split duplicate percent recoveries;
however, data are only available for five compounds. Of these
five compounds, the pp'-DDT, pp'-DDE, and p-BHC data are compar-
able. Trans-nonachlor had a field-spike recovery of 115 percent
and a lab-spike recovery of 80 percent, and oxychlordane had a
field-spike recovery of 130 percent and a lab-spike recovery of
90 percent. Field contamination of heptachlor and oxychlordane
seems unlikely since the two compounds were not detected in
either field blanks or survey specimens.
2. Plan for Set Structure and Analysis
All specimens, including field spikes and blanks,
were analyzed in analytical sets, a set consisting of a group of
specimens analyzed at the same time under the same conditions. A
randomization scheme was used to assign specimens to sets and to
distribute duplicates both within and among sets. Specimens were
renumbered so that the identity of duplicates would not be known
to the chemist.
In addition to the specimens, three standards, a method
blank, and a standard reference material (SRM) were run with
every set. Table 12 outlines the set structure and composition.
Prior to analyzing any field specimens, TAG analyzed a number of
SRM's and calculated the standard deviation from the resulting
data. A control chart was constructed using these data, with the
31
-------
Table 11. Analysis of Serum for Semivolatiles:
Results of Field-Spiked Specimens*
Mean Coefficient
Spiking positive Mean of
level value Standard percent variation
Compound (ppb) (ppb) deviation recovery (percent)
Hexachloro-
benzene
trans-
0.9
0.9
0.3
106
29
Nonachlor
pp ' -DDT
op ' -DDE
pp ' -DDE
pp ' -DDD
a-BHC
P-BHC
Y-BHC
6-BHC
Aldrin
Dieldrin
Endrin
Heptachlor
Heptachlor
epoxide
PCB's
Oxychlordane
Mirex
1.7
5.8
0
2.6
3.3
1
1.2
1.3
0
1.2
0
1
1.1
1.5
3.5
1.2
1.1
2
5.8
0.5
0.6
3.2
1.2
1.2
1.1
0.8
0.9
0.7
0.7
1.7
1.5
NRt
1.5
NR
0.1
0.6
0.5
1.3
0.4
0.4
0.2
0.1
0.2
0.2
0.1
0.4
0.3
0.4
—
0.2
—
115
99
—
21
96
123
98
91
—
77
--
64
161
101
—
130
' --
7
10
116
238
13
28
16
12
26
27
14
68
20
24
--
11
—
Note: The mean positive values have been adjusted by subtracting
the mean compound level detection in the field blanks.
*Based on the results of analyzing 4 field-spiked specimens at
the EPA Toxicant Analysis Center.
tNR denotes not reported.
32
-------
Table 12. Analysis of Serum for Semivolatiles:
Set Structure and Composition
Order
1
2
3
4
5
6
7
8
9
10
11
12
13
Type
Standard3
Method Blank
SRM
Field Specimen
Field Specimen
Field Specimen
Field Specimen
Standard
Field Specimen
Field Specimen
Field Specimen
Field Specimen
Standard
Additional standards were run at the discretion
of the analyst.
Field specimens included serum specimens
collected in the field and field spikes and
blanks.
33
-------
upper and lower control limit lines being set at + 2 standard
deviations. An SRM was run with every set and plotted on the
control chart in order to provide information on both precision
and accuracy. The SRM results are contained in appendix B.
3. Procedures for Replicate Specimen Analysis
Various types of serum duplicates were analyzed in
order to provide estimates of overall study and chemical analysis
precision. In the original plans for the pilot, EPA was to
receive two types of serum field duplicates: true field dupli-
cates, created by collecting and processing blood in separate
containers; and field-split duplicates, obtained by taking two
specimens from a common serum pool after centrifuging (sec.
IV.C). In addition to the two types of field duplicates, a third
type of duplicate was to be created by splitting specimens in the
laboratory just prior to chemical analysis.
The three types of duplicates, created at three separate
stages in the collection/processing/analysis process, were to
provide some comparison as to the different levels of precision
obtained at different stages in the study. Problems at the
collection site, however, prevented EPA from obtaining as many
field duplicates as originally planned. Additionally, no records
were kept in the field to distinguish the two types of field
duplicates, and many of the specimens that were received at TAG
were of too low a volume to split again, thereby creating a
shortage of lab-split duplicates. In light of these problems, it
was decided (1) to treat all field duplicates as of the same type
and (2) to create substitute lab-split duplicates (pseudo-
duplicates) by compositing two single specimens from separate
sample .persons and then resplitting these specimens for inde-
pendent duplicate analyses.
In all, four types of duplicates were analyzed. Field
duplicates were analyzed either within the same set (internal
duplicate), or in different sets (external duplicate). Lab-split
duplicates that were created from pseudo-duplicates were also
analyzed either within the same set (internal-split specimen), or
in different sets (external-split specimen). These duplicates
were analyzed both within and among sets in order to determine if
precision was greater for specimens analyzed at the same time
than for specimens analyzed during different time periods.
4. Analytical Procedures for Spiked-Split Specimens
A subsample of lab-split duplicates was used to
create spiked-split duplicate specimens in order to provide an
estimate of the chemical analysis percent recovery. Spiked-split
specimens were created by spiking one-half of a duplicate, and
not spiking the other half. Both halves were independently ana-
34
-------
,2 .
n
I
_ i=l
2
I (X
j=l
.. . - X.
3
>2
lyzed, either within the same set (internal spiked-split dupli-
cate) or in different sets (external spiked-split duplicate).
5. Precision Estimates
a. Overall Measurement Precision
Overall measurement precision was estimated
for each compound by calculating a pooled estimate of variance
for all field duplicates with a mean value greater than zero.
Table 13 presents these estimates. The variance was calculated
as follows:
(1)
LJ j-
n
2
where s denotes estimate of variance, X^. represents the jth
measurement on the ith pair, X. is the mean of the ith pair, and
n is the total number of duplicate pairs with a mean value
greater than zero.
Table 14 presents precision estimates by compound for each
of the four duplicate types: internal duplicate, external dupli-
cate, internal split specimen, and external split specimen. The
poorest precision was obtained with external field duplicates.
b. Precision of Chemical Analysis
Chemical analysis precision was estimated by
two different methods. In the first method, a pooled estimate of
variance for all lab-split duplicates with a mean greater than
zero was calculated for each compound using eq. (1). The coeffi-
cient of variation was obtained by dividing the square root of
this variance by the overall mean for lab-split duplicates. In
the second method, spiked-split duplicates were used to estimate
precision by taking the coefficient of variation of the differ-
ence between the spiked and nonspiked halves. Table 15 presents
the two chemical analysis precision estimates for each compound.
The difference between the chemical analysis precision
obtained in within-set analyses and that obtained in among-set
analyses was estimated by comparing internal and external field
and lab-split duplicates. Again, precision was estimated from
the coefficient of variation, obtained by dividing the square
root of the pooled estimate of variance for internal and external
duplicates with a mean greater than zero by the overall mean for
each type of duplicate. As can be seen from Table 16, internal
field and lab-split duplicates had better precision than external
field and lab-split duplicates, indicating potential large set-
to-set differences in the measurement process.
35
-------
Table 13. Analysis of Serum for Semivolatiles:
Estimates of Overall Measurement Precision*
Compound Coefficient of variation (C.V.) (percent)a
trans-Nonachlor 141
pp'-DDT 60.9°
pp'-DDE 20.2
P-BHC 77.2
6-BHC 141b
*Based on the analyses of 19 field duplicate pairs at the EPA
Toxicant Analysis Center.
aThe coefficient of variation was calculated by dividing the
square root of the pooled estimate of variance for all field
duplicates (internal and external) with a mean > 0 by the
overall mean for these specimens.
Measured in only one specimen from one duplicate pair; because
of this, the percent C.V. always equals the square root of
2 times 100.
c
Measured in specimens from 44 percent of the sample persons
overall.
36
-------
Table 14. Analysis of Serum for Semivolatiles:
Precision Estimate by Duplicate Type*
Coefficient of variation (C.V.) (percent)3
Compound
trans-Nonachlor
pp ' -DDT
pp ' -DDE
P-BHC
6-BHC
Internal
field
duplicate
NDe
2.2d
14.7
21.8
141C
Internal
lab
split
11. lb
8.8d
16.8
13.9
ND6
External
field
duplicate
141C
62. 8d
22.8
92
NDe
External
lab
split
NDe
1.0d
9
22.8
ND6
*Based on the analyses of 9 internal field duplicate specimens,
4 internal lab splits, 10 external field duplicates, and 4 ex-
.ternal laboratory splits at the EPA Toxicant Analysis Center.
aThe coefficient of variation was calculated by dividing the
square root of the pooled estimate of variance for all dupli-
cates (internal and external) with a mean > 0 by the overall
mean for these specimens.
Measured in only one duplicate pair (in both specimens).
Q
Measured in only one specimen from one duplicate pair; because
of this, the percent C.V. always equals the square root of
2 times 100.
Measured in specimens from 44 percent of the sample persons
overall.
eNot detected.
37
-------
Table 15. Analysis of Serum for Semivolatiles:
Estimates of Chemical Analysis Precision*
Coefficient of variation (C.V.) (percent)
trans-Nonachlor
pp ' -DDT
pp ' -DDE
P-BHC
Dieldrin
Oxychlordane
C.V. for all lab
split duplicates
11.1°
6.8d
14.8
17.3
NDe
NDe
C.V. for differences
between spiked
and nonspiked
11
15.3
67.5
8.2
8.9
9.1
*Based on the analyses of 8 lab-split duplicate pairs and 12
spiked-split duplicate pairs at the EPA Toxicant Analysis
Center.
aThe coefficient of variation was calculated by dividing the
square root of the pooled estimate of variance for all lab-split
duplicates with a mean > 0 (internal and external) by the over-
all mean for these specimens. The results from 8 specimen pairs
(16 analyses) were used in the calculations.
The coefficient of variation of the difference between the
spiked and unspiked halves of spiked-split specimens. Results
from 12 specimen pairs (24 analyses) were used in the calcu-
lations.
°Measured in only one duplicate pair (in both specimens).
Measured in specimens from only 44 percent of the sample persons
overall.
eNot detected.
38
-------
Table 16. Analysis of Serum for Semivolatiles: Estimates of
Within- and Among-Set Chemical Analysis Precision*
Coefficient of variation (C.V.) (percent)
Compound
trans-Nonachlor
pp ' -DDT
pp ' -DDE
P-BHC
6-BHC
Internal duplicates
11. ld
8.1f
15.9
18.5
1416
External duplicates0
141e
55. 8f
20.7
85.2
NDg
*Based on the analyses of 14 external duplicate pairs and 13
internal duplicate pairs at the EPA Toxicant Analysis Center.
The coefficient of variation was calculated by dividing the
square root of. the pooled estimate of variance for all specimens
with a mean > 0 by the overall mean for these specimens.
Field duplicates and lab-split duplicates with both specimens
analyzed in the same set.
°Field duplicates and lab-split duplicates with both specimens
analyzed in separate sets.
"Treasured in only one duplicate pair (in both specimens).
eMeasured in only one specimen from one duplicate pair; because
of this, the percent C.V. always equals the square root of
2 times 100.
Measured in specimens from 44 percent of sample persons overall.
%ot detected.
39
-------
6. Chemical Analysis Percent Recovery
Accuracy of the analytical method was estimated by
calculating the chemical analysis percent recovery for each
compound. Spiked-split duplicate analyses were used for this
purpose. Percent recovery was estimated by dividing the mean
difference between the spiked and unspiked halves by the spiked
amount. Table 17 presents the estimated percent recoveries for
each spiked compound. The percent recoveries ranged from 83
percent for pp'-DDT to 99 percent for dieldrin.
7. Total Error Estimates
Total analytical error was estimated from six
compounds using both the chemical analysis bias (percent recov-
ery) and precision information. Chemical analysis percent recov-
ery was estimated from the spiked-split duplicates, as discussed
in section VI.D.4. The percent coefficient of variation of the
lab-split duplicates was used to estimate precision for the three
compounds frequently detected: pp'-DDE (100 percent); pp'-DDT
(44 percent); and p-BHC (85 percent). Due to the lack of
endogenous-compounds data, precision estimates for trans-
nonachlor, dieldrin, and oxychlordane was estimated from the
percent coefficient of variation of the difference between the
spiked and nonspiked halves of spiked-split duplicates. Table 15
presents both types of precision estimates.
Two estimates of chemical analysis total error were calcu-
lated for each of the six compounds. The Total Error was calcu-
lated by:
percent recovery - 100 + 2 (percent coefficient
of variation) .
The twice Root Mean Square Error was calculated by:
2 [(percent recovery - 100)2 + (percent coefficient
2 \,
of variation) ] 2 .
The estimates for both Total Error and twice Root Mean
Square Error are presented in Table 18. With the analytical
procedures, data of acceptable quality were produced for the six
compounds; the total error estimates are all under 50 percent,
which is the EPA guidelines acceptability level.
40
-------
Table 17. Analysis of Serum for Semivolatiles:
Mean Percent Recovery by Compound*
Compound
trans -Nonachlor
pp ' -DDT
pp ' -DDE
P-BHC
Dieldrin
Oxychlordane
Spiked
amount
(ppb)
20
40
20
20
20
20
Mean
difference
17.1
33.0
18.5
17.5
19.8
17.9
Mean percent
recovery
86
83
93
88
99
90
*Based on the analysis of 12 spiked-split duplicate specimen
pairs at the EPA Toxicant Analysis Center.
41
-------
Table 18. Analysis of Serum for Semivolatiles:
Estimates of Chemical Analysis Total Error*
Compound
trans-Nonachlor
pp ' -DDT
pp ' -DDE
P-BHC
Dieldrin
Oxychlordane
Percent
2 RMSE
36
37
33
42
18
27
Percent ,
total error
36
31
37
47
19
28
*Based on the analyses of 12 spiked-split specimens at the EPA
Toxicant Analysis Center.
aTwo Root Mean Square Error (RMSE)
2
= 2 [(percent recovery - 100)
2 4-
+ (percent coefficient of variation) ] 2.
Total Error
percent recovery - 1001
+ 2 (percent coefficient of variation).
42
-------
VII. ANALYSIS OF URINE FOR SEMIVOLATILES
Urine specimens were shipped to the EPA Toxicant Analysis
Center (TAG) and chemically analyzed for the presence of selected
semivolatile compounds (phenols). A quality assurance plan
involving field controls, replicate analyses, and spiked-split
duplicate analyses was developed and followed. Table 2 presents
the list of target compounds. The analytical results and quality
assurance procedures are discussed below.
A. Summary of Results
Four of the eight target compounds were detected; how-
ever, only one compound, pentachlorophenol (PCP), was measured in
a significant number of sample persons (74.5 percent positive).
The mean positive value was 4.3 ppb and the variance was 5.7.
The compounds 3,5,6-trichloro-2-pyridinol, p_-nitrophenol, and
2,4,5-trichlorophenol were measured in less than 4 percent of the
specimens.
In order to assess the quality of the chemical analysis
data, a total error estimate was calculated from the chemical
analysis percent recovery and precision estimates. Percent
recovery was estimated from the spiked-split duplicates. Because
of the lack of endogenous-compounds data for every compound
except PCP, precision was estimated from the percent coefficient
of variation of the spiked-split duplicates, except for PCP,
where the percent coefficient of variation of all duplicates was
used. The total error estimates show that only two of the target
compounds, 3,5,6-trichloro-2-pyridinol and trichlorophenoxy
acetic acid (2,4,5-T), were within the EPA guidelines criterion
(USEPA 1979) of less than 50 percent total error. The remaining
six compounds, including PCP, had unacceptable error rates. The
chemical analysis precision for PCP was fairly good, 9.1 percent;
however, the mean percent recovery was only 45 percent. The
total error estimates suggest that problems may exist with the
analytical method for six of the eight target compounds, includ-
ing PCP; however, the small sample sizes and lack of endogenous-
compounds data limits the conclusions that may be drawn from the
data.
The analysis of the three field blanks showed the presence
of PCP, the mean value being 5.5 ppb. Of the three field spikes
analyzed, two compounds, p_-nitrophenol and 2,4,5-trichlorophenol,
appeared to have abnormal recoveries, indicating that degradation
or contamination may have occurred in the field. However, no firm
conclusions can be drawn from a sample size of only three field
spikes.
43
-------
B. Specimen Collection, Storage, and Shipping
EPA received urine specimens from a subsample of study
participants. Each EPA specimen was an aliquot drawn from a
larger urine sample. Upon collection, specimens were labeled and
stored in the freezer until shipment to the laboratory. Speci-
mens were then shipped to the laboratory in insulated containers
packed with dry ice in order to maintain the frozen state.
C. Analytical Methodology
Urine specimens were extracted and the analytes of
interest determined by electron capture gas chromotography. The
procedural details are described in "Multiresidue Procedure for
Halo- and Nitrophenols. Measurement of Exposure to Biodegradable
Pesticides Yielding These Compounds as Metabolites," (T. M.
Shafik et al.). A copy of the procedure is contained in appen-
dix C.
D. Analytical Results
1. Detection and Reporting Level Limits
The minimum reporting levels set by TAG for this
study are listed below. Compounds detected in amounts less than
these levels were reported as zero, except in the case of spiked-
split specimens, for which the actual values were reported.
Minimum Reporting
Compound . Level (ppb)
pyridinol 3
2,4,5-trichlorophenol 5
pentachlorophenol (PCP) 2
p_-nitrophenol 10
dicamba 2
dichlorophenoxy acetic acid (2,4-D) 10
silvex 5
trichlorophenoxy acetic acid (2,4,5-T) 5
2. Results
*
Specimens from a total of 51 sample persons were
analyzed for the presence of 8 phenol compounds. Only four of
*Three urine specimens were not analyzed successfully due to
laboratory technical problems.
44
-------
the tested compounds were measured:
3,5,6-trichloro-2-pyridinol;
pentachlorophenol (PCP) ;
p_-nitrophenol; and
2,4,5-trichlorophenol.
Table 19 presents the percent positive measurements for each
compound.
Only one compound, pentachlorophenol (PCP), was detected in
a significant number of sample persons (75 percent). The mean
positive value was 4.3 ppb, and the variance was 5.7. Table 20
presents a summary of PCP results.
The compound 3,5,6-trichloro-2-pyridinol was measured in
specimens from one sample person, in both halves of duplicate
analyses. The mean of the two duplicate halves was 5.1 ppb.
P-nitrophenol was measured in two specimens, one with a value of
12.4 ppb and another with a value of 27.1 ppb. In one specimen,
2,4,5-trichlorophenol was measured with a value of 15.4 ppb.
E. Quality Assurance Procedures and Results
1. Field Controls
Field controls were prepared at RTI, shipped to
and stored at the collection site, and then included in the
specimen shipments to the laboratory (sec. IV.C). Matching
laboratory controls were prepared at RTI and shipped directly to
the laboratory.
Upon receipt at the laboratory, three field blanks and three
field spikes were analyzed. All three field blanks showed levels
of PCP, the mean value being 5.5 ppb. No other compounds were
measured in the blanks analyzed.
Table 21 presents the results from the field spikes: Two
compounds had unusual recovery levels. The percent recovery for
p_-nitrophenol was only 39 percent, and the percent recovery for
2,4,5-trichlorophenol was at 135 percent. The percent recoveries
obtained from the spiked-split specimens, by comparison, were 78
and 97 percent respectively, well within the expected range.
2. Plan for Set Structure and Analysis
All specimens, including field spikes and blanks,
were analyzed in analytical sets, a set consisting of a group of
specimens analyzed at the same time under the same conditions. A
randomization scheme was used to assign specimens to sets and to
distribute duplicates both within and among sets. Specimens were
45
-------
Table 19. Analysis of Urine for Semivolatiles:
Frequency of Detection by Compound*
Compound
3,5, 6-Trichloro-2-pyridinol
Dicamba
2,4-dichlorophenoxy acetic
acid (2,4-D)
Pentachlorophenol (PCP)
p_-Nitrophenol
2,4,5-trichlorophenoxy acetic
acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
Sample
persons
tested
51
51
51
51
51
51
51
51
Number
with
positive
detection
1
0
0
38
2
0
0
1
Percent
with
positive
detection
2.0
0
0
74.5
3.9
0
0
2.0
*Based on results reported by the EPA Toxicant Analysis Center.
46
-------
Table 20. Analysis of Urine Semivolatiles:
Summary of PCP Results*
Number of specimens analyzed . 51
Number of positive detections 38
Percent positive detections 74.5
Mean of the.positive values (ppb) 4.3
Mean overall value (ppb) 3.2
Median of the positive values (ppb) 3.5
Minimum reported positive value (ppb) 2.0
Maximum value (ppb) 14.6
Variance of the positive values 5.7
Coefficient of variation of the
• positive values 56%
*Based on results reported by the EPA
Toxicant Analysis Center.
47
-------
•Table 21. Analysis of Urine for Semivolatiles:
Results of Analyzing Field-Spiked Specimens*
Compound
3,5, 6-Trichloro-2-pyridinol
Dicamba
2 , 4-dichlorophenoxy acetic
acid (2,4-D)
Pentachlorophenol (PCP)
p_-Nitrophenol
2,4, 5-trichlorophenoxy acetic
acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
Spiking
level
(ppb)
26.
10.
19.
129
11.
11.
13.
16.
1
9
7
5
7
7
8
Mean
detected
level
(ppb)
23.1
10
15.7
117
4.5
10
14
22.8
Measurement
standard
deviation
4
2
2
1
7
0
4
2
.0
.3
.2
.7
.7
.2
.2
.8
Mean
percent
recovery
87
92
80
90
39
85
102
135
Percent
coefficient
of
variation
17
23
14
11
173
2
30
12
Note: The mean positive levels have been adjusted by subtracting the mean levels meas-
ured in the field blanks.
*Based on the analyses of 3 field-spiked specimens at the EPA Toxicant Analysis Center.
-------
renumbered so that the identity of duplicate specimens would not
be known to the chemist.
In addition to the specimens, three standards, a method
blank, and a lab spike were run with every set. Table 22 out-
lines the set structure and composition. Prior to analysis of
any field specimens, TAG analyzed a number of lab spikes and
calculated the standard deviation. A control chart was con-
structed using these data, with the upper and lower control
limits set at the. mean ±2 standard deviations. A lab spike was
run with every set and plotted on the control chart to provide
information on both precision and accuracy.
3. Procedures for Replicate Specimen Analysis
In order to provide estimates of chemical analysis
precision, urine specimens were split in the laboratory and
independently analyzed. Duplicates were analyzed both within the
same set (internal duplicates) and in separate sets (external
duplicates ) .
4. Procedures for Spiked-Split Specimen Analysis
A subsample of duplicates was used to create
spiked-split specimens in order to estimate chemical analysis
percent recovery. Spiked-split specimens were created by spiking
one-half of a duplicate; the other half was not spiked. Both
halves were independently analyzed, either within the same set
(internal spiked-split specimen) or in different sets (external
spiked-split specimens).
5 . Chemical Analysis Precision Estimates
a. Overall Estimate
Overall chemical analysis precision was
estimated for each compound by two different methods. In the
first method, a pooled estimate of variance for all duplicates
with a mean greater than zero was calculated using
n 2
I I (X±. - X±)2
2 _ j=l j=i H x
s
n
2
where s denotes estimate of variance, X. . represents the jth
measurement on the ith pair, X. is the mean of the ith pair, and
n is the total number of duplicate pairs with mean value greater
than zero. The coefficient of variation was obtained by dividing
the square root of this variance by the overall mean for all
duplicates with mean greater than zero.
49
-------
Table 22. Analysis of Urine for Semivolatiles:
Set Structure and Composition
Order
1
2
3
4
5
6
7
8
9
10
11
12
13
Type
Standard3
Method Blank
Lab Control (Spike)
Field Specimen
Field Specimen
Field Specimen
Field Specimen
Standard
Field Specimen
Field Specimen
Field Specimen
Field Specimen
Standard
aAdditional standards were run at the discre-
tion of the analyst.
Field specimens included serum specimens
collected in the field and field spikes and
blanks.
50
-------
In the second method, spiked-split specimens were used to
estimate precision by taking the coefficient of variation of the
differences between the spiked and nonspiked halves. Table 23
presents precision estimates for each compound by each method.
b. Within- and Among-Set Precision
The precision obtained between specimens
analyzed in the same set and the precision obtained when speci-
mens are analyzed in separate .sets was estimated by the same
methods as was used to estimate overall chemical analysis preci-
sion. These estimates are given in Table 24.
6. Chemical Analysis Percent Recovery
The chemical analysis percent recovery of each
compound from spiked-split duplicates was estimated by dividing
the mean difference between the spiked and unspiked halves by the
spiked amount. Table 25 contains the estimated percent recov-
eries for each spiked compound. The recoveries range from a low
of 45 percent for pentachlorophenol (PCP) to 97 percent for
2,4,5-trichlorophenol.
7. Total Error Estimates
Total error was estimated for each compound using
both the bias (percent recovery) and precision information.
Percent recovery was estimated from the spiked-split duplicates,
as presented in Table 25. Because of the lack of endogenous-
compounds data for every compound except for PCP, precision was
estimated from the percent coefficient of variation of the
spiked-split duplicates except for PCP, where the percent coeffi-
cient of variation of all duplicates was used.
Two estimates were calculated for each compound. The
Total Error was calculated by:
percent recovery - 100 | + 2 (percent coefficient
of variation) .
The twice Root Mean Square Error was calculated by:
2 [(percent recovery - 100)2 + (percent coefficient
2 \,
of variation) ] 2 .
The estimates for both Total Error and twice Root Mean
Square Error are presented in Table 26. Of the eight target
compounds, only two compounds, 3,5,6-trichloro-2-pyridinol and
trichlorophenoxy acetic acid (2,4,5-T) had Total Error or twice
Root Mean Square Errors within the EPA guidelines criterion
(below 50 percent total error). The remaining six compounds,
51
-------
Table 23. Analysis of Urine Semivolatiles:
Chemical Analysis Precision Estimates*
Coefficient of
variation (C.V.) (percent)
Compound
3,5, 6-Trichloro-2-pyridinol
Dicamba
Dichlorophenoxy acetic
acid (2,4-D)
Pentachlorophenol ( PCP )
p_-Nitrophenol
Trichlorophenoxy acetic
acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
C.V. fora
all duplicates
.4C
ND
ND
9.1
141d
ND
ND
141d
C.V. for .
all spiked-
nonspiked
differences
10.3
28.5
13.1
26.8
26.5
10.9
27.3
84.9
*Based on analyses of analyzing 24 duplicate pairs and 25 spiked-
split duplicate pairs at the EPA Toxicant Analysis Center.
The coefficient of variation was calculated by dividing the
square root of the pooled estimate of variance for all dupli-
cates with a mean > 0 by the overall mean for these specimens.
The coefficient of variation of the difference between the
spiked and unspiked halves of the spiked-split specimens.
£
Measured in specimens from only one sample person, in both
halves of duplicate analyses.
Measured in only one specimen from one duplicate pair; because
of this, the percent C.V. always equals the square root of
2 times 100.
52
-------
Table 24. Analysis of Urine Semivolatiles:
Within- and Among-Set Chemical Analysis Precision Estimates*
Coefficient
of variation (C.V.]
Internal
(within-set)
Compound
3,5, 6-Trichloro-
2-pyridinol
Dicamba
Dichlorophenoxy acetic
acid (2,4-D)
Pentachlorophenol ( PCP )
p_-nitrophenol
Trichloirophenoxy acetic
acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
C.V.(%)C
NDg
NDg
NDg
10.6
NDg
NDg
NDg
NDg
C.V.(%)d
9
18.6
10.7
26.1
36.2
13.4
15.2
29.4
1 (percent)
External ,
(among-set)
C.V.(%)C
.4e
NDg
NDg
7.9
141f
NDg
NDg
141f
C.V.(%)d
11.8
36.2
15.2
28.4
18
9
11.4
11
*Based on results reported by the EPA Toxicant Analysis Center.
Duplicates with both specimens analyzed in the same set; 11 in-
ternal spiked-split duplicates and 13 internal duplicates were
analyzed.
Duplicates with specimen halves analyzed in separate sets;
14 external spiked-split duplicates and 15 external duplicates
were analyzed.
GThe coefficient of variation was calculated by dividing the
square root of the pooled estimate of variance for all dupli-
cates with a mean > 0 by the overall mean for these specimens.
The coefficient of variation of the difference between the
spiked and unspiked halves of the spiked-split specimens.
eMeasured in only one sample person, in both halves of duplicate
analyses.
Measured in only one specimen from one duplicate pair; because
of this, the percent C.V. always equals the square root of
2 times 100.
detected.
53
-------
Table 25. Analysis of Urine Semivolatiles:
Mean Percent Recovery by Compound*
Compound
3,5, 6-Trichloro-2-pyridinol
Dicamba
dichlorophenoxy acetic
acid (2,4-D)
pentachlorophenol (PCP)
p_-Nitrophenol
Trichlorophenoxy acetic
acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
Spiked
amount
(ppb)
10
10
60
10
50
20
20
20
Mean
difference
8.2
6.7
45.1
4.5
39.2
15.8
16.4
19.4
Mean
percent
recovery
82
67
75
45
78
79
82
97
*Based on the analysis of 25 spiked-split duplicates at the
EPA Toxicant Analysis Center.
54
-------
Table 26. Analysis of Urine Semivolatiles:
Estimates of Chemical Analysis Total Error*
Percent3
Compound 2 RMSE
3,5, 6-Trichloro-2-pyridinol
Dicamba
Dichlorophenoxy acetic acid (2,4-D)
Pentachlorophenol ( PCP )
p_-Nitrophenol
Trichlorophenoxy acetic acid (2,4,5-T)
Silvex
2,4, 5-Trichlorophenol
42
87
56
66C
69
47
65
170
Percent
total error
39
90
51
83C
75
43
73
173
*Based on results reported by the EPA Toxicant Analysis Center.
Chemical analysis percent recovery and precision was estimated
from spiked-split duplicates.
aTwo Root Mean Square Error (RMSE)
2
= 2 [(percent recovery - 100)
2 i-
+ (percent coefficient of variation) ] 2.
Total Error
= I percent recovery - 100|
+ 2 (percent coefficient of variation).
cUsing field-spike percent recoveries, the RMSE drops to 14 per-
cent and the Total Error drops to 28 percent; however, the
spiked-split duplicates most likely provide a better estimate
of chemical analysis percent recovery.
55
-------
including PCP, had unacceptable error levels. The chemical
analysis precision for PCP was fairly good, 9.1 percent; how-
ever, the spiked-split duplicate mean percent recovery was only
45 percent. As an alternative percent recovery estimate, the
field-spike percent recovery for PCP, 90 percent, could be used,
yielding a Total Error of 28 percent and Root Mean Square Error
of only 14 percent. As previously stressed, however, field
spikes are not ideal for estimating chemical analysis precision
because of possible biases introduced in the field, and only
three field spikes were analyzed, compared to 24 spiked-split
duplicates analyzed. Spiked-split duplicates provide a better
estimate of chemical analysis percent recovery. Judging from the
limited data available, it seems appropriate to suggest that
problems may exist with the analytical method for six of the
eight target compounds, including PCP.
56
-------
REFERENCES
"Multiresidue Procedure for Halo- and Nitrophenols. Measurement
of Exposure to Biodegradable Pesticides Yielding these Compounds
as Metabolites," J_._ Agric. and Food Chemistry. 21(2) :295-298.
1973.
USEPA. 1979. U.S. Environmental Protection Agency. Manual for
Analyzing Quality Control for Pesticides and Related Compounds in
Human and Environmental Samples. Section 2K~! First Revision.
Washington, DC: USEPA. EPA-600/1-79-008.
57
-------
58
-------
APPENDIX A
VOLATILES ANALYTICAL METHODOLOGY
-------
Bull. Environm. Contain. ToxicoL 23,244-249 (1979)
Determination of Volatile Purgeable Halogenated Hydrocarbons'
in Human Adipose Tissue and Blood Serum
A. J. Peoples, C. D. Pfaffenberger, T. M. Shafik*. and H. F. Enos
University of Miami School of Medicine, Department of Epidemiology and Public Health,
Chemical Epidemiology Division, 15655 S.W. 127th Avenue, Miami, Fla. 33177
Organohalogens have been detected in virtually all chlori-
nated drinking vaters (ROOK 197**, 3ELLAR et_ al. 1971*, SYMONS et.
al. 1975, THOMASOI? et_ al_. 1978). During a nationwide study
(SCIONS et_ al,. 1975), concentration levels in the finished drink-
ing vater of 79 cities of the United States vere established for
six volatile purgeable halocenated hydrocarbons (VPKH's): chloro-
form (CHCl^), bromodichloromethane (BDCM), dibronochloronethane
(DBCM), bronofona (d-IBro), carbon tetrachloride (CCli,) and 1,2-
dichloroethane (DCE). The health implications of these findings
stimulated the development of a project to determine if any of
these six substances or trichloroethylene (TCZ) could be detected
in relatively snail samples of human adipose tissue (250 ag) or
blood serum (0.5 ml) obtained from residents of Dade County,
Florida, an area in vhich chloroform levels in excess of 300
Ug/1 (ppb) have been reported. Accordingly, the puree/trap/
desorb method of 3ELLAR and LICIITZirEERG (1971*) ^as modified to
accomplish these objectives. The procedure reported here requires
no extraction or clean-up step and is relatively inexpensive to
perform. Each analysis is completed in about 30 minutes .
MATERIALS AND METHODS
Apparatus. A Tekmar Model LSC-1 liquid sample concentrator
vas interfaced to a Tracer Model 222 cas chromatorjraph (CC)
equipped vith a Hall electrolytic conductivity detector vhich
vas operated in the halide specific =ode. The chromatographic
column vas a 6-ft x 0.25-in I.D. c-ass U-tube containing n_-octane
on 100-120 mesh Porasil C packing. .The CC operating conditions
included: a nitrogen carrier gas flow-rate of 30 rJ./rnin, an inlet
temperature of 1UO°, and a transfer line temperature of 210°. The
Hall detector furnace vas maintained at 900° vith a hydrogen flov-
rate of 1*0 ml/min and a solvent (1:1 n_-propanol: distilled vater)
flov of 0,1* ml/min.
A Finnigan Model bOOO gas chronatograph/nass spectrometer
(CC/f-E) analytical system interfaced to a Tekcar liquid sar.ple
concentrator vas used to confirm the identities of the compounds
quantified by the gas chromatographic procedure.
•Present address : Cyanamid, Cyan amid Agricultural Research Cta'-ion,
Cyanor.id Overseas Corp., P.O. Box 1071, Alexandria, Egypt
0007-4861/79/0023-024) $01.20
© 1979 Springcr-Vcrlag New York Inc.
-------
Both the GC and the GC/MS systems utilized a hot plate
stirrer and a glycerol bath to heat the sample in the Teknar
purging device.
Solvents and Reagents,. Chloroform, carbon tetrachloride and
hexane vere Pesticide Grade from Fisher Scientific Co. Trichloro-
ethylene, 1,2-dichloroethane and bromofora vere from Aldrich
Chemical Co.; bronodichloromethane and dibromochloronethane, from
Columbia Organic Chemical Co. Dov Corning antifoam emulsion B vas
from Fisher Scientific Co. and the n_-octane on 100-120 mesh
Porasil C chronatographic packing vas purchased froa Supelco, Inc.
Preparation of Standards. Tvo al each of carbon tetrachlo-
ride, dibromochloromethane and bromoform and 1 ml each of chloro-
form, tricnloroethylene, bromodichloronethane and 1,2-dichloro-
ethane are diluted vith hexane to a final volune of 100 al (solu-
tion l). The concentration of each component is calculated by
using the respective specific gravities. One ml of solution 1 is
quantitatively diluted to 100 ml vith hexane (solution 2), and a
convenient vorking standard is prepared by diluting 0.1 ml of
solution 2 to 25 al vith hexane. Use of 5 Hi of this solution
leads to acceptable peak heights vhen the Hall detector attenua-
tion is 10 x 8.
A standard curve nay be obtained by using three hexane
dilutions of solution 2: the vorking standard, 0.1 ml diluted to
50 ml (for 10 x k attenuation) and 0.2 ml diluted to 25 al (for
10 x 16 attenuation). (Although solutions 1 and 2 are stable at
room temperature, fresh vorking ^-standards must be riade daily.)
Procedure for Blood Serum. One nl of 1* aqueous antifoam is
added to a 5-al purging device and the sample concentrator is
operated in the Trap Bake Mode for 20 min vhile the trap temper-
ature is 200°. (This procedure purges the system of all poten-
tially interfering volatile compounds; hovever, a blank run say
be nade at this time to be certain that the system is uncontami-
nated.) The trap is then cooled to the ambient temperature. 3y
means of a gas tight syringe, 0.5 ml of serum is introduced into
the purging device and a purge flov-rate of 10 al/min is started.
The lover portion of the purging device is immersed in a 115°
stirred glycerol bath for 30 min.* To prevent steam contamination
of the Tenax/silica gel trap, a small glass vapor trap or inter-
ceptor is placed betveen the purging device and the adsorbent
trap. After the pur^e/trap period is cor.plote, the adsorbed
compounds are desorbed and transferred to the analytical column
(60°) by heating the trap at 150° for 6 min. The GC column is
then temperature programmed 7°/min to
*Eoth serum and fat appear to have an inherent binding capacity
for chloroform vhich can be overcome by purging at elevated
temperatures.
-------
Procedure for Adipose Tissue. Betvcen 200 and 500 mg of
frozen adipose tissue is cut into thin strips and pushed to the
bottom of a pre-purged, 5-ml TeJonar purging device. The purge
flov-rate is adjusted to 10 ml/min, and the lover portion of
the device is immersed in a 115° stirred glycerol "bath for 20
min. After the volatile components have been purged from the
liquefied fat, the analytical procedure given above for serum
analysis is folloved. Hexane is added to the purging device
containing the purged adipose tissue, and the residue is quanti-
tatively extracted to separate the fat component from residual
connective tissue. Values are reported in ng VPHH/g of hexane
extractable fat.
GC/MS Component Confirmation. The Identities of the conpo-
nents quantified by the LSC/GC method are confirmed by using an
LSC/GC/MS analytical system. Any confirmation is based on both
relative retention values (GC data) and mass fragmentation data
(m/e_ values and isotopic ratios).
RESULTS AND DISCUSSION
Chloroform is the major volatile purgeable halogenated hydro-
carbon identified during the analysis of human adipose tissue and
blood serum vhen this methodology is used.
Reproducibility. One serum sample vas analysed ten times
over a tvo-day period. The chloroform concentration ranged from
23 to 36 pg/1 vith a mean value of 27 yg/1 and a standard deviation
of U.
One fat sample vas analyzed ten times over a three-day
period. The chloroform values ranged from 101* to 1^0 ng per gram
of hexane extractable fat vith a mean value of 122 ng/g and a
standard deviation of 6.
Recovery. Because of the volatility of the compounds in
this study, recovery studies vere performed vithin the purging
device. Serum vas pre-purged, then analyzed to insure that no
halogenated compounds vere present, for each replication, a 5-ul
aliquot of the vorking standard vas added to the serum in the
purging, device. The components of the standard vere alloved to
nix vith the serum for several minutes; then the serum vas
treated like an unknovn sample. Table 1 indicates hov cuch of
each VPHH vas added, the average per cent recovered and the
recovery range for ten replications.
-------
TABLE 1
Recovery of VPHH's from Human Blood Serum
Amount Average Recovery
Added Recovery Range
(Kg/1) (*) (?)
CC1U 1.3 112 108-121*
CHC1, 0.6 100 100
TCE 0.6 98 83-100
3DCM 0.8 92 88-100
DCE 1.0 100 93-110
DBCM 2.1 8? 78-100
CHBr3 2.3 90 79-100
A heated sample of human adipose tissue vas pvurged for 30
minutes to remove all volatile purgeable halogenated compounds.
For each replication, a. 5-pl aliquot of the working standard vas
added to the fat in the purging device and alloved to mix for
several minutes. Then the fat vas treated as an urLknovn sample.
Table 2 indicates hov much of each VTIIH vas added, the average
per cent recovered and the recovery range for ten replications.
TABLE 2
Recovery of VPHH's from Human Adipose Tissue
Amount Average Recovery
Added Recovery Range
(Jig/1) (?) (?)
CC1|, 1.3 96 90-100
CHC1, 0.6 92 88-100
TCE 0.6 101 100-110
BDCM 0.8 109 100-125
DCE 1.0 98 93-100
DBCM 2.1 105 90-118
CHBr3 2.3 110 83-137
Blood Serum Analyses. Ten serum samples vere collected from
healthy human subjects and analyzed vithin 21* hours of collection.
The chloroform values ranged from 13 to 1»9 ug/1 as indicated in
Table 3.
Sample S-l vas analyzed several times over a period of tvo
months; each result vas vithin 2 yg/1 of the initial value.
Adipose Tissue Analyses. Ten fat samples, taken from
near the anterior abdominal vail at autopsy, vere analyzed.
Chloroform concentrations ranged from 20 to ^60 ng per gram of
hexane extractable fat as indicated in Table U.
-------
TABLE 3 TABLE k
Human Blood Serum Euman Adipose Tissue
Chloroform Levels Chloroform Levels
Sample pg/1 Sample pg/1 Sample ng/g* Sample ng/g*
S-l 13 S-6 25 F-l 80 7-6 20
S-2 13 S-7 26 F-2 230 7-7 28
S-3 1*9 S-8 30 7-3 *»60 7-5 lUd
S-i 13 S-9 30 7-k 65 F-9 95
S-5 U5 S-10 13 F-5 2UO F-10 2t»0
*hexane extractable fat
The identities of the reference compounds and the chloroform
in human serum and fat vere confirmed by LSC/CC/!-S methods . When
coupled vith the appropriate GC retention data, the cluster having
m/e_ = 83, 85 and 87 corresponding to the respective positively
charged fragments CHCl|5, CHC13SC137 and CHCl|7 vas particularly
useful to confirm the presence of chloroform in the biological
samples.
The exact source of the chloroform detected in hunan fat and
serum by this procedure is presently unknovu. Possible sources
include municipal drinking vater and chronic exposure to trichloro-
ethylene (TCE) and/or tetrachloroethylene (perchloroethylene, PCS).
Municipal drinking vater contains both residual chlorine and
chloroform generated during the ch-lorination process from reactions
between humic substances in r»v vater and either dissolved chlorine
or hypochlorous acid (MORRIS 1978). It has also been proposed that
"intermediate bonding states" betveen halogen and various organic
molecules can exist (GLAZE et_ al. 1977; NICHOLSOJI et al. 1977).
If this latter situation occurs to any great extent, ingestion of
municipal drinking vater could conceivably lead to in vivo genera-
tion of chloroform from the precursors. Moreover, the residual
chlorine in finished drinking vater could also form chloroform
precursors after the vater has been consumed. A clear explanation
of the origin of chloroform in human tissue is thus not currently
possible.
A satisfactory explanation is even aore difficult if exposure
to TCE and/or PCE is considered. During an earlier study and from
periodic vater analysis reports, neither of these compounds has
been detected in amounts comparable to the chloroform levels
usually found in Miami drinking vater. Hovever, both TCE and PCE
are metabolic precursors of chloroform (3UTLZK 19^9, IKEDA 1977).
and FISHBEIN (1976) has revieved tvo additional routes of exposure,
air and food (McCOiniELL et_ al_. 1975, CAMISA 1975). McCOIIKELL et_ al_.
(1975) reported tissue ranees of TCE and PCE for humans betveen
less than 0.5 ug/kg and 29 yg/V.g '-/et tissue. These values are far
belov the chloroform levels reported here for adipose tissue.
Although it Is impossible to rule out the possibility that some of
our experimental subjects had been exposed to these tvo organc—
chlorides, it is reasonable to assume that they were not. Hone of
-------
the subjects vere involved in either dry cleaning or degreasing
occupations ncr did any of them work near these types of estab-
lishments. If a portion of the serum chloroform reported in
this investigation is traceable to metabolized TCE or PCS, the
intact chlorinated ethylene(s) vill be present in corresponding
samples of adipose tissue from the individuals. Such a study is
in progress.
ACKNOWLEDGEMENTS
The authors vish to thank Dr. JC. P. Cantor, Mr. J. Guerra
and Mr. J. J. Freal, III for their assistance. This vork was
supported by EPA grant R8Qi*6ll.
REFERENCES
BELLAR, T.A., and J.J. LICHTENBERG: J. Acer. Water Works Assoc.
66_, 739 (197>0.
BELLAR, T.A., J.J. LICHTEN3ERG, and R.C. KROIJER: J. Amer. Water
Works Assoc. 66, 703 (197^).
BUTLER, T.: J. Pharmacol. Exp. Ther. 97,, 3k (191»9) .
CAMISA, A.G.: J. Water Pollut. Control Fed. kj_, 1021 (1975).
FISHBEIN, LAWRENCE: Mutation Ses. 32., 267 (1976).
GLAZE, W.H., G.R. PEYTON, and R. RAWLEY: Environ. Sci. Techn. 11,.
685 (1977).
IKEDA, MASAYUKI: Environ. Health Perspect. 21, 239 (1977).
McCONHELL, G., O.M. FERGUSON, and C.R. PEARSON: Endeavor 3^, 13 (1975)
MORRIS, J.C.: Water chlorination, environmental impact and health
effects, 1 ed. Michigan: Ann Arbor Science Publishers 1978.
NICHOLSON, A.A., 0. MERESZ, and B. LEMYK: Anal. Chen. ^9_, 8ll* (1977).
ROOK, J.J.: Water Treatn. Exanin. 23_, 23^ (197«»).
SYMONS, J.M., T.M,'BELLAR, J.K. CARS'-'ELL, J. DE-1AP.CO, K.L. KROPP,
G.G. ROBECK, D.R. SEZGER, C.J. SLOCUM, B.L. S7-IITH, and
A.A. STEVENS: J. Amer. Water Works Assoc. 13^ 631* (1975).
THOMASON, M., M. SHOULTS, W. BERTSCH, and G. HOLZZR: J. Chronatogr.
158. U37 (1978),
-------
APPENDIX B
TAC-SPIKED BLOOD SERUM RECOVERIES
-------
Appendix B. TAC-Spiked Blood Serum Recoveries
SPRM
107*
Compound
Hexachloro-
benzene
p-BHC
Oxychlordane
Heptachlor
epoxide
trans-
Nonachlor
pp ' -DDE
Dieldrin
pp ' -DDT
Hexachloro-
benzene
P-BHC
Oxychlordane
Heptachlor
epoxide
trans-
Nonachlor
pp ' -DDE
Dieldrin
pp ' -DDT
level
2
4
6
5
9
28
5
4
2
4
6
5
9
28
5
4
.4
.6
.5
.7
.7
.7
.7
.7
.4
.6
.5
.7
.7
.7
.7
.7
#1
2
3
6
5
9
28
5
4
1
3
5
4
7
15
4
3
.22
.94
.11
.07
.28
.60
.35
.36
.94
.60
.37
.33
.05
.67
.55
.08
#2
2
3
5
5
9
28
5
4
2
4
6
5
9
23
5
4
.25
.86
.90
.18
.16
.30
.35
.00
.17
.20
.92
.68
.90
.67
.86
.00
#3
2
4
5
5
9
28
5
4
2
4
6
5
9
22
5
4
.33
.03
.90
.07
.03
.00
.17
.00
.00
.13
.61
.41
.31
.51
.68
.55
#4
2.23
4.00
5.30
5.21
6.97
20.81
5.30
3.27
1.99
4.00
6.36
5.22
9.09
22.69
5.68
4.80
#5
2.15
4.08
5.20
5.21
6.63
20.41
5.47
3.27
1.82
3.64
5.97
4.93
8.81
21.40
5.25
4.30
PPB Recovered — TAC-spiked blood serum
#6 #7 #8 #9 #10 #11 #12 #13 X
2.13 2.31 2.14 2.25 2.16 2.22 2.18 2.31 2
3.92 4.08 4.00 4.08 3.95 4.11 3.87 4.11 4
4.90 5.29 5.48 5.86 5.35 5.25 5.63 5.53 5
5.10 5.43 5.12 4.91 5.10 5.30 5.00 5.30 5
6.63 7.78 7.56 7.67 7.53 7.42 7.97 7.86 7
19.66 23.42 22.97 23.27 22.85 22.42 23.86 23.29 23
5.12 5.54 5.19 5.19 5.27 5.27 5.27 5.44 5
2.91 3.48 3.83 3.83 3.48 3.48 3.48 3.48 3
1
3
6
5
8
21
5
4
.22
.00
.52
.15
.81
.68
.30
.61
.98
.91
.25
.11
.83
.19
.40
.15
SD
0.
0.
0.
2.
0.
0.
0.
0.
.0.
0.
1.
3.
0.
0.
07
09
35
14
88
92
13
38
13
28
60
50
07
19
53
67
RSD (%)
3.04
2.21
6.31
2.69
11.27
12.34
2.37
10.6
6.38
7.12
9.58
9.80
12.16
15.05
9.77
16.04
*SPRM = standard pesticide reference material.
Fort, level = fortification level.
-------
APPENDIX C
SEMIVOLATILE ANALYTICAL METHODOLOGY FOR URINE
-------
HALO- AND NITKOI'HKNOl.S
Multiresidue Procedure for Halo- and Nitrophenols. Measurement of Exposure to
Biodegradable Pesticides Yielding these Compounds as Metabolites
Talaat M. Shafik,* Hazel C. Sullivan, and Henry R. Enos
The urinary level of phenolic compounds may be
the key for establishing an index of exposure to
pesticides containing this moiety as an easily hy-
drolyzed or metabolized portion of the molecule.
A method has been developed for quantitating
ten halo- and nitrophenols in rat urine which
could result from exposure to and subsequent
metabolism and excretion of a broad spectrum of
pesticides. The procedure involves acid hydroly-
sis, extraction, derivatization, silica gel chroma-
tography, and electron-capture gas chromatogra-
phy. Male rats fed pesticidal compounds contain-
ing halo- and nitrophenol moieties at levels vary-
ing from factors of 10~5 to 10"2 of the LD5o were
used to establish the usefulness of this procedure
for determining the extent of exposure to the bio-
degradable pesticides.
Determination of the urinary excretion of halo- and ni-
trophenol metabolites of biodegradable pesticides is of in-
creasing interest to those involved in pesticide epidemiol-
ogy. Metabolism studies (Menzie, 1969) of the biode-
gradable pesticides EPN, fenitrothion, dicapthon, methyl
bromophos, C-9491, Dursban, DNOC, PCP, VC-13, and
ronnel, which contain halo- or nitrophenol substituent
groups in their molecular structures, indicate that these
phenols are the major urinary metabolites. The purpose of
this investigation was to develop a multiresidue method
for the determination of low levels of halo- and nitrophe-
nols in urine. The method is based on electron-capture gas
chromatography of ethyl ether derivatives of the phenols
(Bradway and Shafik, 1971; Shafik et at., 1971a). Such a
procedure may be of value in developing an exposure
index to biodegradable pesticides based on levels of these
urinary phenols.
EXPERIMENTAL SECTION
Apparatus and Equipment. A Micro-Tek 220 gas chro-
matograph equipped with tritium foil electron-capture
detector was used. A glass U-shaped column, 6 ft x y4 in.,
was packed with 4% SE-30/6% QF-1 on 80/100 mesh
Chromosorb W (HP). The gas chromatographic column
was operated under the following parameters: nitrogen
carrier gas flow rate, 60 ml/min; column temperature,
175°; inlet, 210°; detector, 210°; transfer line, 240°. Chro-
matographic columns were size 22, Kontes K-420100. Con-
centrator tubes were 25 m!. Kontes K-570050. Condensers
were Kontes K-286810. Nitrogen evaporator was equipped
with a water bath maintained at 40°. N-Evap was ob-
tained from Organomation Associates, Worcester, Mass.
Reagents. The following were used: iV-ethyl-/V'-nitro-
iV-nitrosoguanidine (Aldrich Chemical Co., Milwaukee,
Wis.); ethylating reagent (Stanley, 1966; Shafik et ai,
1971a); and silica gel, Woelm, activity grade I (Waters As-
sociates, Inc., Framingham, Mass.). Dry adsorbent for 48
hr at 170° and store in a desiccator. Prepare daily 2 g of
deactivated silica gel for each chromatographic column by
adding 40 n\ of benzene-extracted deionized or distilled
water for each 2 g of dried silica gel in a tightly stoppered
container. Rotate container until the water is evenly dis-
tributed throughout the adsorbent. Allow to equilibrate
for 2 to 3 hr with periodic shaking. Prepare the chromato-
graphic columns just before use. Deionized or distilled
water used for deactivating the silica gel must be extract-
ed twice with benzene. Prepare each day the anticipated
amount of deactivated silica gel to be used. Larger
amounts may be prepared by using the same ratio of
water to dried silica gel.
Perrine Primate Laboratory. Environmental
Agency, Perrine. Florida 33157.
-rotection
Preparation of Standard Solutions. The phenolic
compounds were 95+% pure and a mixture of the ten
compounds was prepared at the final concentrations as
follows: 2,4-dichlorophenol (2,4-DCP), 0.4 Mg/ml; 2,4,5-
trichlorophenol (2,4,5-TCP), 0.1 Mg/ml; and 3,5,6-tri-
chloro-2-pyridinol (3,5,6-TCPyridinol), 0.1 Mg/ml, Dow
Chemical Co., Midland, Mich.; 3,6-dichloro-4-iodophenol
(3,6-DCIP), 0.05 Mg/ml, Ciba Agrochemicals Co., Sum-
mit, N. J.; 2,5-dichloro-4-bromophenol (2,5-DCBrP), 0.02
Mg/ml, Cela, Ingelheim, West Germany; pentachlorophe-
nol (PCP), 0.01 Mg/ml, and p-nitrophenol (PNP), 0.2 Mg/
ml, Aldrich Chemical Co., Milwaukee, Wis.; p-nitro-m-
cresol (PNC), 1.2 Mg/ml, Chemagro Corp., Kansas City,
Mo.; 2-chloro-4-nitrophenol (2-C-4NP), 0.08 Mg/ml, Ameri-
can Cyanamid Co., Princeton. N. J.; 2,4-dinitro-6-methyl-
phenol (DNOC), 0.4 Mg/ml, Chemical Insecticide Corp.,
Edison, N. J. These compounds are the corresponding
phenolic metabolites of pesticides listed in Column 1,
Table II.
Weigh 10 mg of each of the ten analytical standards
into separate 10-ml volumetric flasks. Add 5 ml of ben-
zene to each flask and swirl the flask until the compound
dissolves. In a well-ventilated hood, wearing disposable
gloves, add diazoethane dropwise with a disposable pipet
until a definite yellow color persists. Allow the solution to
stand 15 min, and then bubble dry nitrogen through the
solution until the yellow color disappears (5-10 min). Di-
lute to volume with benzene. From these ethylated stock
standards, prepare a mixture of the ten compounds at the
final concentrations listed above.
Chromatographic Behavior of the Ten Ethyl Ethers
on a Silica Gel Column. Pipet an aliquot of the mixture
of the ten ethylated compounds into a 15-ml graduated
centrifuge tube. Evaporate the benzene to a volume of 0.3
ml using the nitrogen evaporator, and add 1.7 ml of hex-
ane. To a chromatographic column lightly plugged with
glass wool, add the 2.00 g of the partially deactivated sili-
ca gel and top with 1.5 g of anhydrous sult'ate. Prewash
the column with 10 ml of hexane and discard the hexane
eluate. Transfer the standard mixture quantitatively to
the column using 8 ml of the 20% benzene-in-hexane solu-
tion and collect this fraction in a 15-ml centrifuge tube
(fraction I). Elute the column with 10 ml of 40% benzene-
in-hexane (fraction II), followed by 10 ml of 60% benzene-
in-hexane and 10 ml of 80% benzene-in-hexane, collecting
the 20 ml in a 25-ml concentrator tube (fraction III).
Elute the column with 10 ml of benzene and collect this
fraction in a 15-ml centrifuge tube (fraction IV). Inject
5-10 M' from each fraction into the gas ehromatograph to
establish the clulion paitern of the ten ethyl ethers from
the silica gel column.
Under our laboratory conditions, fraction 1 (20% ben-
zene-in-hexanel contained the ethyl ethers of 2.4-DCI'.
2.4.5-TCI'. HAivTCPyridinol. :t.li-l)< MI-1. 2.:VDrBrP. and
-------
SHAFIK. SULLIVAN. ENDS
6
5
f '
* 3
I
* 2
Fortifiid Rat Urine
itrol Rat Urine
4 8 12
Minutes
16
Figure 1. Chromatogram of the 20% benzene-in-hexane fraction
of fortified and control rat urine. A. 2,4-DCP, 0.8 ppm; B. 3,5,6-
TCPyridinol, 0.05 ppm; C. 2,4,5-TCP, 0.03 ppm; D. 2,5-DCBrP,
0.02 ppm; E. 3,6-DCIP, 0.03 ppm; F. PCP, 0.02 ppm.
PCP. The ethyl ethers of PNP, PNC, 2-C-4NP, and
DNOC eluted in fraction HI (60 and 80% benzene-in-hex-
ane). Occasionally, traces of DNOC are found in fraction
IV (benzene). Fraction II (40% benzene-in-hexane) did not
contain any of the derivatives.
Elution patterns may vary from one laboratory to an-
other, depending on the temperature and relative humidi-
ty. It is therefore necessary to establish an elution pattern
under local conditions before attempting to analyze sam-
ples.
Analysis of Urine. Pipet 1 to 5 ml (the actual volume
to be determined by the anticipated residue level) of urine
into a 25-ml concentrator tube. Add dropwise a volume of
concentrated HC1 equal to one-fifth the amount of urine,
and mix well. Fit a stoppered reflux condenser to the tube
and heat in a boiling water bath for 1 hr while cooling the
condenser with circulating ice water. Remove from the
bath, cool, and rinse the sides and tip of the condenser
with a total of 2 ml 0.1 N NaOH. Add 3 ml of anhydrous
ethyl ether to the tube and mix contents vigorously on a
Vortex mixer for 2 min; then centrifuge and transfer ethyl
ether layer to a 15-ml centrifuge tube with a disposable
pipet. Repeat the extraction with an additional 3-ml vol-
ume of ethyl ether and add the second ethyl ether extract
to the centrifuge tube.
Add diazoethane dropwise with a disposable pipet until
the yellow color persists. Let the solution stand 15 min;
Table I. Electron-Capture Detector Sensitivities,a Limits of
Detection and Recovery Data" for Ten Halo- and Nitrophenol
Ethyl Ethers
Limits of
Compound
2.4-DCP
2,4,5-TCP
3,5,6-TCPyridinol
3,6-DCIP
2,5-DCBrP
PCP
PNP
PNC
2-C-4NP
DNOC
" Based on 15% scale
method
Sensitivity,
ng
0.2
0.02
0.05
0.02
0.02
0.01
0.1
0.3
0.05
0.1
deflection "
Detection.
ppm
0.1
0.01
0.01
0.01
0.01
0.01
0.02
0.05
0.01
0.05
Based on use
Recovery.
%
87-96
85-95
91-97
88-94
88-96
92-96
85-98
88-98
85-92
• 86-96
of the described
Control Rot Urine
8 12
Minutes
16
20
Figure 2. Chromatogram of the 60-80% benzene-in-hexane
fraction of fortified and control rat urine: A. PNP, 0.1 ppm; B.
PNC, 0.4 ppm; C. 2-C-4NP, 0.04 ppm, D. DNOC, 0.16 ppm; E.
2,4-D; f. 2,4,5-TP; G. 2,4,5-T.
then bubble clean dry nitrogen through the solution to re-
move excess reagent. Concentrate the ethylated urine ex-
tract to approximately 0.3 ml, using the nitrogen evapora-
tor. Add 2 ml of hexane and continue evaporating the
ether-hexane solution to 0.3 ml.
Prepare a silica gel chromatographic column as pre-
viously described. Prewash the column with 10 ml of hex-
ane and discard the washing. Transfer the concentrated
urine extract quantitatively to the column using 2 ml of
the 20% benzene-in-hexane. As soon as the solvent sinks
in the sodium sulfate, add 8 ml of the 20% benzene-in-
hexane to the column and collect the total volume of the
20% benzene-in-hexane (10 ml). This fraction contains the
halogenated phenols. Continue eluting with 10 ml of 40%
benzene-hexane and discard this fraction. Add 10 ml of
60% benzene-hexane, followed by 10 ml of 80% benzene-
hexane and collect these fractions in a single tube. The
combined 60-80% benzene-hexane fractions will contain
the nitrophenols. Add 10 ml of benzene to column and
collect eluate. Frequently a small amount of the DNOC is
found in the benzene fraction. The urinary impurities
eluted by the benzene solvent do not interfere with the
gas chromatographic determination of DNOC, which has
a relatively long retention time. The elution pattern of
spiked control urine extracts must be established before
the analysis of actual samples is undertaken.
RESULTS AND DISCUSSION
Control rat urine samples were fortified with the sodi-
um salts of the phenols. The samples were analyzed and
aliquots of the 20% B-H and 60 + 80% fractions were in-
jected separately into the gas chromatograph. Figure 1 il-
lustrates chromatograms of the 20% B-H fraction (halo-
genated phenols) of spiked and control rat urine samples,
and Figure 2 shows chromatograms of the 60 + 80% frac-
tion (nitrophenols) of control and fortified rat urine sam-
ples. An average of 0.01 ppm of pentachlorophenol was
routinely found in all control urine samples. The percent
recovery, limits of detection in ppm. and detector sensi-
tivity in nanograms (based on 15% scale deflection) are
shown in Table I.
Kthylation of the phenols proceeds rapidly at room tem-
perature, producing ethyl ether derivatives which are gas
chrnmai.ographable and stable to silica gel column chro-
malography. Better gas chromatographic resolution of the
more volatile phenols was achieved by preparing the ethyl
-------
HAI.O- AND NITROPHENOLS
Table II. The Relation between Total Dosage of Biodegradable Pesticides and Urinary Excretion ol Halo- and Nltrophenols
Excretion of phenolic-type metabolites
Compound
VC-13
Ronnel
Dursban
C-9491
Bromophos
PCP
EPN
Fenltrothion
Dicapthon
DNOC
Dose
level
LDjo
io-2
io-3
io-4
10-»
io-3
io-4
io-4
10-5
io-4
10-s
io-4
io-5
10-2
io-3
10-'
10-3
10-*
10-3
10~2
10-'
nnvil
lee
5140
514
234
23.4
232
23.2
314
31.4
617
61.7
40. S
4.06
670
67.0
5430
543
8700
810
910
91.0
Metabolite
2,4-DCP
2,4,5-TCP
3,5,6-TCPyridlnol
3,6-DCIP
2,5-DCBrP
PCP
PNP
PNC
2-C-4NP
DNOC
nmol
excreted
3470
359
124
7.4
157
29.5
7.6
0.834
305
41.4
9.03
1.05
108
9.35
3610
472
3570
20
0
0
%ol
dose
excreted
67
70
53
32
68
100+
2.4
2.7
49
67
22
26
16
14
66
87
41
2.5
0
0
Days lor
complete
excretion
3
1
2
1
4
4
2
1
3
3
2
1
3
3
2
1
1
1
derivatives instead of the more commonly used methyl
ethers.
Silica gel column chromatography serves two purposes:
it provides a clean sample for gas chromatographic analy-
sis and it conveniently separates the halogenated phenols,
thus simplifying the gas chromatographic analysis.
A mixture of the ten phenols and three phenoxy acids,
namely 2,4-D, 2,4,5-T, and silvex, can be determined in
one sample. All of the halogenated phenols involved in
this study are eluted with 20% benzene-hexane, while the
nitrophenols and the phenoxy acids are eluted in the 60
and 80% benzene-hexane fractions. These herbicides are
included in this report because they are detected as intact
excreted residues in the urine using the present procedure.
2,4-DCP and 2,4,5-TCP, potential mammalian metabo-
lites of 2,4-D and 2,4,5-T, are also determined in this pro-
cedure. The analysis of these herbicides and their metabo-
lites has been described in detail in a previous report
(Shafik et at., 1971a). In the gas chromatographic step, the
retention time of the ethyl ether derivative of 2-ch!oro-4-
nitrophenol is almost identical to that of the ethyl ester of
2,4-D. In addition, these two derivatives are eluted in the
60 + 80% benzene-hexane fractions from silica gel. Con-
firmation of identity can be accomplished using the clas-
sical NaHCOa and acid extraction steps which separate
carboxylic acids from phenols (Bakke and Scheline, 1969).
In order to determine if a correlation exists between ex-
posure to intact pesticides and excretion of urinary pheno-
lic metabolites, male Charles River rats weighing 190 to
220 g were dosed by gavage with peanut oil solutions of
eight organophosphorus compounds, PCP, and DNOC in
concentrations ranging from 10~2 through 10~5 of the
LD30 (Kenaga and Allison, 1969), as indicated in Table II.
The doses were administered daily for 3 days to two rats
at each dose level. The animals were maintained in stain-
less steel metabolism cages with the two rats adminis-
tered the same dose regimen maintained in the same
cage. Urine samples were collected at 24-hr intervals and
stored in a freezer until analysis was performed.
Urine samples were analyzed for several days following
the third dose until no detectable levels of the phenolic
type metabolites were observed. This established the
number of days required for total excretion of the metabo-
lites. The percent of the total dose excreted as the pheno-
lic metabolite was calculated from the sum of the amount
of phenolic metabolite excreted each day and the total
amount of pesticide fed in the 3-day period.
As indicated in Table II, the amount of urinary metabo-
lites excreted is proportional to the dose of parent com-
pound administered. The percent of the dose excreted in
the urine as a phenolic metabolite of the pesticide fed in-
dicates that low level animal exposure to VC-13, ronnel,
Dursban, Bromophos, PCP, EPN, and fenitrothion can be
detected. The low excretion rates of the phenols of C-9491
and dicapthon in urine indicate that the method cannot
detect low-level exposure to these compounds. DNOC was
not detected in the'urine of rats fed 10~2 and 10~3 of the
LD50ofDNOC.
There are other pesticide chemicals not included in this
investigation which may produce the same phenolic me-
tabolites reported in this study. 2,4,5-TCP can be a uri-
nary metabolite of ronnel, Gardona, lindane, 2,4,5-T, and
Silvex. PNP in urine can result from exposure to EPN,
ethyl parathion, and methyl parathion. The method is not
capable of distinguishing between the sources of the uri-
nary 2,4,5-TCP and PNP. Wherl such a distinction is es-
sential, the corresponding alkyl phosphate of the organo-
phosphorous pesticide can be determined in the urine
(Shafik et at., 1971a). The free' phenoxy acids of 2,4,5-T
and Silvex can also be determined in the urine (Shafik et
al., 1971a). It must be emphasized that the rat-feeding ex-
periment was designed only for the purpose of evaluating
the method and not as a study of the metabolism of these
compounds.
In conclusion, a multiresidue method has been devel-
oped for the determination of low levels of halo- and ni-
trophenols and phenoxy acids in rat urine which undoubt-
edly can be extended to environmental samples. Such a
method should be useful, if properly evaluated on a moni-
toring and surveillance scale, in establishing human expo-
sure to low levels of a large number of biodegradable pes-
ticides.
LITERATURE CITED
Bakke, 0. M., Scheline, R. R., Anal. Biochvm. 27, 451 (1969).
Bradway, D., Shafik, M. T., "A Gas Chromatographic Method
for the Determination of Low Levels of p-Nitrophenol in
Human and Animal Urine," presented at the 16'2nd National
Meeting of the American Chemical Society. Washington, D. ('..
September 12-17. 1971.
-------
MOOKHERJEE, THKNKLK
Kcnaga. K. K.. Allison, VV. Iv. reprinted from Hull Knlomul. .S'w Shalik. M. I'., Hradwav. I).. Kims. II. !•'., J. ,-li/r I'mxl Chi'm.. I!),
Amur. 15. 8!) (1969). rtH.r> ( l»7lb).
Menzie. C. M., Metabolism of 1'eslicides. Hurciiu of Sport Kish Sliinli'v. (' W..-/ A#r h'unil ('h<-m 11, :i'2l I HMilil.
cries & Wildlife. Special Scientific Report. Wildlife No. 127.
1969. Kectivfd for review Septeinln'r I, 1972. Accepted I)ecenil>er 11,
Shafik, M. T., dullivan. H. C., Knos, H. K.. .) Environ Anal. 1972. Presented at the Mi2nd National Meet in); of the American
Chem. 1, 23 (1971a). Chemical Society, Washington. I). 0.. September 1971.
-------
MULTIRESIDUE PROCEDURE FOR HALO- AND NITROPHENOLS
1. Pipet 5 mL urine into a 50-mL culture tube.
2. Add 1.25 mL cone HCl, cap tube securely, and place in a
59° C oven overnight.
3. Remove from oven and allow to cool.
4. Transfer contents to a 12- to 15-mL centrifuge tube.
5. Rinse the 50-mL culture tube with 3 mL of anhydrous ethyl
ether and add to the centrifuge tube.
6. Mix contents vigorously on a Vortex mixer for 2 min; then
centrifuge and transfer ethyl ether layer to a 12- to 15-mL
centrifuge tube with a disposable pipet.
7. Repeat the extraction with an additional 3-mL volume of
ethyl ether and add the second ethyl ether extract to the
centrifuge tube.
8. Add diazoethane dropwise with a disposable pipet until a
yellow color persists, then mix gently (1 mL).
9. Let the solution stand 15 min; then bubble clean dry nitro-
gen through the solution to remove excess reagent.
10. Concentrate the ethylated urine extract to ca. 0.3 mL, using
the nitrogen evaporator.
11. Add 2 mL of hexane, and continue evaporating the ether-
hexane solutions to 0.3 mL.
12. Prepare a silica gel column using 2 grams of silica gel
(Woelm) that has been dried for 48 hr at 170° C and deacti-
vated by adding 40 microliters of benzene-extracted dis-
tilled H20 and allowed to equilibrate for 2 to 3 hr with
periodic shaking. Top with 1.5 g of anhydrous Na2S04.
13. Prewash the column with 10 mL of hexane and discard.
14. Transfer the concentrated urine extract quantitatively to
the column using 2 mL of the 20 percent-benzene-in-hexane.
15. As soon as the solvent sinks into the sodium sulfate, add
8 mL of the 20-percent-benzene-in-hexane to the column, and
collect the total volume of the 20-percent-benzene-in-hexane
(10).
-------
16. Continue elution with 10 mL of 40-percent-benzene-in-hexane,
and discard this fraction.
17. Add 10 mL of 60-percent-benzene-in-hexane, followed by 10 mL
of 80-percent-benzene-in-hexane, and collect these fractions
in a single tube.
18. Add 10 mL of benzene to column, and collect eluate.
19. Concentrate fractions to 5 mL.
-------
MULTIRESIDUE PROCEDURE FOR HALO- AND NITROPHENOLS AND HERBICIDES
Color Code—Orange
1. Pipet 5 mL of urine into a 15-mL screw-top centrifuge tube.
2. Add 1.25 mL of cone. HC1, cap tube securely, mix, and place
in a 59° C oven overnight.
3. Remove from oven and allow to cool.
4. Add 3 mL of anhydrous ethyl ether to the centrifuge tube.
5. Mix contents vigorously on a Vortex mixer for 2 min, then
centrifuge and transfer ethyl ether layer to a 12- to 15-mL
centrifuge tube with a disposable pipet.
6. Repeat the extraction with an additional 3-mL volume of
ethyl ether and add the second ethyl ether extract to the
first.
7. Using the glove box, add 1 mL of diazoethane to the combined
extracts.
8. Let the solution stand for 15 min, then remove from glove
box and place tubes on the concentrator and bubble nitrogen
through the solution for 10 min to remove the excess
reagent.
9. Raise the needles above the surface of the wolution, and
concentrate the extract to ca. 0.3 mL.
10. Add 2 mL of hexane, mix, and continue evaporating the ether-
hexane solution to 0.3 mL.
11. Prepare a silica gel column using 2 grams of silica gel
(Woelm) that has been dried for 48 hr at 170° C and deacti-
vated by adding 40 microliters of benzene-extracted dis-
tilled water and allowed to equilibrate for a minimum of
2 hours with periodic shaking. Top with 1.5 g of anhydrous
Na2S04.
12. Prewash the column with 10 mL of hexane and discard.
13. Transfer the concentrated urine extract quantitatively to
the column using 2 mL of the 20-percent-benzene-in-hexane.
14. As soon as the extract sinks into the sodium sulfate, add
8 mL of the 20-percent-benzene-in-hexane to the column, and
collect the total volume of the 20-percent-benzene-in-hexane
(10 mL).
-------
15. Continue eluting with 10 mL of 40-percent-benzene-in-hexane,
and discard this fraction.
16. Add 10 mL of 60-percent-benzene-in-hexane, followed by 10 mL
of 80-percent-benzene-in-hexane, and collect these fractions
in a single tube.
17. Add 10 mL of benzene to the column, and collect eluate.
18. Concentrate fractions to 5 mL.
TUBES REQUIRED FOR THIS PROCEDURE*
12 15-mL screw-top centrifuge tubes
12 12- to 15-mL glass-stoppered centrifuge tubes for
extracts
3 rows of 12 12- to 15-mL glass-stoppered centrifuge
tubes for fractions
12 25- to 50-mL centrifuge tubes for the combined
eluate from step 16.
19. The 5.0-mL fractions are analyzed on GC equipped with
tritium foil electron-capture detector, a glass U-shaped
column, 6 ft x \ in. packed with 35 SE 30/4.5 QFL on 80/100
mesh Gas chrom Q. The gas chromatographic column was oper-
ated under the following parameters: nigrogen carrier gas
flow rate 70 mL/min; column temperature 168°; inlet, 225°,
detector, 210°; transfer line, 235°. Standards: 3,5,6-Tc
Pyr, 30 pg/pL; 2,4,5-TCP, 30 Pg/pL; PNP, 100 pg/pL; silvex
40 pg/pL; 2,4,5-T, 50 pg/(jL; 2,4-D, 200 pg/pL; dicamba,
25 pg/nL; PCP, 7 pg/nL.
*Required when analyzing 10 samples, 1 blank, and 1 spike.
"Multiresidue Procedure for Halo- and Nitrophenols. Measurement
of Exposure to Biodegradable Pesticides Yielding these Compounds
as Metabolites," J_._ Agric. and Food Chemistry. 21(2) ;295-298.
1973.
-------
APPENDIX D
GLOSSARY OF SELECTED TERMS
-------
GLOSSARY OF SELECTED TERMS
duplicate: either of two things (e.g., specimens) that exactly
resemble or correspond to each other.
endogenous-compound: a compound found naturally in specimens as
opposed to compounds added for quality assurance purposes.
endogenous-compounds data: measurements reported for endogenous
compounds.
external reference duplicate analysis: chemical analysis per-
formed by an external reference laboratory on one of the dupli-
cate specimens; used to compare chemical analysis results of the
corresponding duplicate specimen obtained by the primary labora-
tory .
field blank: one specimen of a large homogenous matrix pool that
was shipped to the collection site and handled identically to
specimens collected from sample persons (subjects).
field control: either a field blank or field spike.
field duplicate: either of two specimens that were collected in
an identical manner from sample persons.
field spike: a field blank fortified (spiked) with selected
target compounds.
field split: one of two duplicate specimens obtained from a
large matrix pool.
lab-split duplicate: one of two aliquots obtained in the labora-
tory by splitting specimens from sample persons.
spiked-split duplicates: a pair of duplicate specimens, one of
which was fortified with selected target compounds, and the other
of which was not.
-------
5027? -101
REPORT DOCUMENTATION .i._REPORT NO.
PAGE
3. Recipient's Accession No.
! t. Title and Suotitle -
HISPANIC HANES PILOT STUDY—Measurement of Volatile
S. Report Date
September 1983
and Semivolatile Organic Compounds in Blood and Urine j6-
Specimens I
7. Author(s)
S. Pierson and R. Lucas
j 8. Performing Organization Reot. No
I RTI/1864/38-7
9. Performing Organization Name and Address
Research Triangle Institute
P.O.. Box 12194
Research Triangle Park, NC 27709-2194
10. Project/Task/Work Unit No.
11. Contract(C) or Graot(G) No.
!(oEPA 68-01-5848
(G)
12~ Sponsoring Organization Name and Address
U.S. Environmental Protection Agency
Office of Toxic Substances
401 M Street, SW
Washington, D.C. 20460
i 13. Type of Report & Period Covered
I FINAL
14.
IS. Supplementary Notes
16. Abstract (Limit: 200 words)
Specimens of serum and urine were selected as part of the Hispanic HANES
Pilot Study. Statistically designed quality assurance protocols were used
to permit estimation of the field procedures and chemical analysis quality,
The quality of field procedures was assessed by the use of field QA speci-
mens, both spiked and unspiked to estimate the levels of contamination and
degradation. The quality of the chemical analysis was assessed using
duplicates and split samples (spiked and unspiked) to estimate chemical
analysis precision .and bias. The results of the quality assessment of the
analysis of volatiles in serum and semivolatiles in serum and urine are
summarized.
17. Document Analysis a. Descriptors
6. Identifiers/Open-Ended Terms
:. %C05AT1 Fielc/G.-cup
13. Availability Statement
19. Security Class (This Report)
UNCLASSIFIED
1 21. No. of Pages
20. Security Class (This Page)
22. Price
(See ANS!-Z39.:S)
See Instruction* on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
r>nartment of Commerce
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