United States
Environmental Protection
Agency
National Exposure
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/SR-97/1Q9 November 1997
of
for
and
in Air,
Soil, and Diet
Jane C. Chuang, Ying-Liang Chou, Marcia Nishioka, Kimberlea Andrews, Mary
Pollard, and Ronald Menton
The objectives of this work assign-
ment were to evaluate ELISA screening
methods and determine whether these
methods indicate effectively those mi-
croenvironments where high exposure
to polycyclic aromatic hydrocarbons
(PAH) or other semivolatile organic com-
pounds (SVOC) is likely.
Four commercially available assay
kits for PAH, carcinogenic PAH (C-PAH),
2,4-D, and pentachlorophenol (PCP)
were evaluated. The testing procedures
were refined based on the evaluation
results. The overall method precision
and assay precision of each ELISA test-
ing method were determined. The dust/
soil samples as well as sample extracts
of air and food samples collected from
13 low-income homes in the summer of
1995 were analyzed by PAH and C-PAH
assays. These sample extracts were
also analyzed by gas chromatography/
mass spectrometry (GC/MS) to deter-
mine alkyl PAH and phthalates. The
dust/soil samples from 13 low-income
homes collected during the spring of
1996 were analyzed by PAH, C-PAH,
2,4-D, and PCP assays. Different
aliquots of these samples were ana-
lyzed by conventional (GC/MS) meth-
ods for PAH and by GC with electron
capture detection (GC/ECD) for 2,4-D
and PCP. The ELISA data were com-
pared with GC/MS data or GC/ECD data.
For PAH measurements, there is no
strong relationship between the ELISA
results and GC/MS results when data
of similar types of samples were com-
bined from different field studies. The
ELISA data (C-PAH) and GC/MS (B2
PAH) data showed stronger relation-
ships for dust/soil collected from 22
NHEXAS homes. The ELISA screening
for PAH can indicate the likely pres-
ence of high levels of PAH in dust/soil
samples. There is a positive but weak
relationship between GC/ECD data and
ELISA data for 2,4-D and PCP.
This Project Summary was developed
by EPA's National Exposure Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
In 1992, the National Academy of Sci-
ences identified polycyclic aromatic hy-
drocarbons (PAH) and other semivolatile
organic compounds (SVOC) as among
the highest priorities for exposure re-
search, in part because these compounds
are frequently constituents of fine aerosol
and some of them are mutagens and
probable human carcinogens. Addition-
ally, several of the PAH and other SVOC,
including phthalates, pentachlorophenol,
and 2,4-D, are likely to be endocrine dis-
rupters or have other quasi-hormonal or
reproductive effects. Therefore, it is im-
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perative that the identities, concentrations,
and distributions of these compounds in
the environment be investigated. Deter-
mining exposure to PAH and SVOC is
still a new area of research. It is still
largely unknown in what ways they are
distributed among the vapor and particu-
late phases in air or the aqueous and
nonaqueous phases in water. Likewise,
their distributions and levels in other me-
dia, such as food or soil, are largely un-
known. Because of the extensive and
costly sampling and analysis efforts that
are required to obtain complete informa-
tion on these levels and distributions, it is
desirable to apply fast, inexpensive
screening methods to indicate those en-
vironments and media that are most likely
to be significant sources of human or
ecological exposure to PAH and SVOC.
Enzyme-linked immunosorbent assay
(ELISA) techniques are currently available
commercially for analysis of water and
soil for PAH and for other SVOC. For
example, Ohmicron Environmental Diag-
nostics, Inc., and the Immunosystems di-
vision of Millipore, Inc., currently market
immunoassay testing kits intended for field
screening applications. The test kits from
Ohmicron utilize the suspended magnetic
particle competition assay format, as op-
posed to a well-coated competition assay
format from Millipore. These immunoas-
says are formatted to be used only for
determining whether a given sample con-
tains PAH at a concentration above or
below a set threshold value.
The objectives of this work were to
evaluate low-cost ELISA screening meth-
ods and determine whether application of
these methods indicates effectively those
microenvironments where high exposure
to PAH and other SVOC is likely.
ELISA techniques were evaluated for
applicability to screening of air particle
sample extracts and food sample extracts
generated from EPA Cooperative Agree-
ment CR822073. Simplified and cost ef-
fective sample preparation methods for
dust/soil samples were also evaluated for
ELISA. Two different ELISA systems, one
for total PAH and one for carcinogenic
PAH (C-PAH), were included in this study.
In addition, two other ELISA systems were
evaluated for screening pentachlorophe-
nol (PCP) and 2,4-dichlorophenoxyacetic
acid (2,4-D) in dust/soil samples.
This work was done simultaneously with
a portion of the National Human Expo-
sure Assessment Survey (NHEXAS) Ari-
zona pilot study, which is being conducted
jointly by the University of Arizona, Battelle,
and the Illinois Institute of Technology.
Samples of dust/soil from 22 homes of
the NHEXAS study and from 13 homes of
low-income families in North Carolina
were tested by both PAH and C-PAH
ELISA systems. Different aliquots of these
samples were analyzed conventionally by
gas chromatography/mass spectrometry
for PAH. The results of the ELISA screen-
ing and conventional measurements were
compared to determine the ability of the
ELISA techniques to predict microenvi-
ronmental levels of PAH and other SVOC
in house dust and soil.
It is desirable to know whether high
PAH levels in the dust/soil are indicators
of high levels of other SVOC in the same
environmental media, because of the
costly and extensive sampling and analy-
sis efforts that are required to obtain com-
plete information on the levels of pollutants
in multimedia samples. We therefore re-
analyzed the sampled extracts of air, dust,
soil, and food generated from the EPA
Cooperative Agreement (CR822073) by
GC/MS for alkyl PAH and phthalates.
Results and Conclusions
The procedures from the commercial
testing kits for PAH and C-PAH assays
were revised to provide adequate extrac-
tion efficiency of PAH from dust/soil. The
overall precision of these revised methods
expressed as percent relative standard de-
viation (%RSD) of triplicate real-world dust/
soil samples was within + 30% for PAH
ELISA and + 25% for C-PAH ELISA. The
overall method accuracy for the PAH and
C-PAH assays cannot be assessed for
real-world dust/soil samples, which con-
tain multiple components of PAH, because
the spike recovery procedures are based
on single component spiking: phenan-
threne for PAH ELISA and benzo[a]py-
rene (BaP) for C-PAH ELISA. The
recoveries of phenanthrene and BaP from
dust/soil samples ranged from 68 to 150%
and from 110 to 130%, respectively.
The sample extracts of indoor and out-
door air samples collected from 13 low-
income homes in previous studies were
analyzed by GC/MS for alkyl-PAH and
phthalates. Among these 13 homes there
were 9 nonsmokers' homes and 4 smok-
ers' homes. Approximately half of the
homes were located in the inner city (5
nonsmokers and 2 smokers) and half of
these homes were located in rural areas
(4 nonsmokers and 2 smokers). Levels of
2- to 3-ring alkyl PAH in indoor air from
these homes were higher than those in
the corresponding outdoor air. Similar con-
centrations of most 4- to 6-ring alkyl PAH
were observed in indoor and outdoor air
for nonsmokers' households, whereas
higher concentrations were in indoor air
for smokers' households. Higher outdoor
concentrations were observed in the in-
ner city as compared to the rural area.
The sums of alkyl PAH concentrations
ranged from 369 to 3,270 ng/m3 in indoor
air and from 49.9 to 702 ng/m3 in outdoor
air. With few exceptions, the relative con-
centrations trend for alkyl PAH found in
dust/soil samples from these homes was
house dust > entryway dust > pathway
soil, as was also observed for their parent
PAH. The sums of alkyl PAH concentra-
tions in these samples ranged from 0.092
to 3.32 ppm. Concentrations of alkyl PAH
found in the 24-h food composite samples
ranged from 0.866 to 15.6 ppb.
Indoor phthalate concentrations were
higher than the corresponding outdoor lev-
els. Total target phthalate concentrations
ranged from 1,160 to 5,330 ng/m3 in in-
door air and from 64.2 to 1,070 ng/m3 in
outdoor air. The general concentration
trend for phthalates in dust/soil samples
was similar to those of PAH and alkyl
PAH. Concentrations of total target ph-
thalates found in the 24-hr liquid and solid
composite food samples ranged from 0.09
to 245 ppb.
The dust and soil samples collected
from 13 low-income homes and 22
NHEXAS homes were extracted and ana-
lyzed by GC/MS for 19 target PAH. The
B2 PAH (probable human carcinogens)
included among the target PAH are benz-
[a]anthracene, chrysene, benzo[b]fluoran-
thene, benzo[k]-fluoranthene, benzo-
[ajpyrene, indeno-[1,2,3-c,d]pyrene, and
dibenz[a,h]-anthracene. The levels of the
sums of these B2 PAH correlated well
(correlation coefficient >0.90) with total
target PAH (the sums of 19 target PAH)
in dust/soil samples collected from the
low-income homes and NHEXAS homes.
The results from GC/MS analysis showed
that levels of the sums of B2 PAH ac-
count for approximately half of the total
PAH. There were positive but weak rela-
tionships of PAH among different sample
media (dust, soil, and air). Stronger rela-
tionships between dust and soil samples
collected from the NHEXAS homes were
observed. Thus, house dust may be used
as a potential indicator for other sample
media for PAH exposure. More studies
are needed to test this hypothesis.
Different aliquots of the above dust and
soil samples were extracted and analyzed
by PAH and C-PAH assays. Statistical
analysis showed that PAH data in dust/
soil samples generated from ELISA and
GC/MS methods are significantly differ-
ent. In general, PAH ELISA responses
were higher than PAH GC/MS responses.
The regression analyses showed that the
linear relationship between ELISA and GC/
MS measurements is not strong. This re-
lationship became stronger when the data
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from each type of samples were treated
separately. This finding suggested that
the results of ELISA depend strongly on
the sample matrices. The screening per-
formance of ELISA was evaluated based
on the frequency distribution of ELISA
and GC/MS data. The results indicated
that PAH and C-PAH ELISA can be used
as a screening tool, but not as a quantita-
tive analytical method for total PAH and
B2 PAH in real-world dust and soil
samples.
The precision for the 2,4-D assay was
better than for the PCP assay in both
dust and soil matrices. The average as-
say precision was within 20% for the 2,4-
D assay and greater than 60% for the
PCP assay. There was a positive but weak
relationship between GC/ECD and the
ELISA method for 2,4-D data as well as
for PCP data. Positive biases for 2,4-D
and PCP in most house dust samples
were observed by ELISA as compared to
GC/ECD.
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J. C. Chuang, Y.-L.Chou, M. Nishioka, K. Andrews, M. Pollard, and R. Menton
are with Battelle, Columbus, OH 43201-2693.
Nancy K. Wilson is the EPA Project Officer (see below).
The complete report, entitled "Field Evaluation of Screening Techniques for
Polycyclic Aromatic Hydrocarbons, 2,4-Diphenoxyacetic Acid, and Pentachlo-
rophenol in Air, House Dust, Soil, and Total Diet," (Order No. PB98-103195;
Cost: $31.00, subject to change) will be available only from
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at
National Exposure Research Laboratory
U. S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
Official Business
Penalty for Private Use
$300
EPA/600/SR-97/1Q9
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