United States
Environmental Protection
Agency
Environmenta/ Monitoring
Systems Laboratory
Las Vegas NV 89193-3478
Research and Development
EPA/540/SR-93/518 September 1993
EPA Project Summary
Superfund Innovative
Technology Evaluation (SITE)
Program Evaluation Report For
Antox BTX Water Screen (BTX
Immunoassay)
R.W. Gerlach, R.J. White, N.F.D. O'Leary, and J.M. Van Emon
The results of a demonstration of a
portable immunoassay for the detec-
tion of benzene, toluene, and xylene(s)
(BTX) are described in this report The
BTX immunoassay was developed by
Antox, Inc., (South Portland, ME) and
is intended as a screening technology.
Seventy-nine field samples were ob-
tained from monitoring wells at several
sites with gasoline-contaminated
ground water. Sample splits were ana-
lyzed on-site by the BTX immunoassay
and in the laboratory by gas chroma-
tography (GC) using EPA Method 8020.
The BTX immunoassay was rapid and
simple to use. It performed well in iden-
tifying high-level contamination and
gasoline-contaminated samples having
BTX concentrations greater than 100
ppb. It did not fully meet the claims of
the developer of identifying contamina-
tion levels down to 25 ppb BTX. Two
field samples determined by GC to have
between 25 and 100 ppb BTX failed to
be classified correctly by the immu-
noassay. Results from quality assur-
ance samples with BTX concentrations
of 2.5, 25, and 100 ppb also showed
that false negative results would be
expected at higher than a 5 percent
rate when BTX contamination levels
were between 25 and 100 ppb. How-
ever, for samples with higher BTX lev-
els, the immunoassay gave excellent
results. Two field samples yielded false
positive results compared to GC val-
ues, but these samples showed signs
of low-level gasoline contamination.
This technology could provide cost-ef-
fective screening or monitoring func-
tions at sites with ground water con-
taminated by gasoline or other fuels. It
could also be used in a laboratory set-
ting to select samples for further analy-
sis.
This Project Summary was developed
by EPA's En vironmental Monitoring Sys-
tems Laboratory, Las Vegas, NV, to an-
nounce 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
The performance of an immunoassay
field kit technique was evaluated during a
U.S. Environmental Protection Agency
(EPA) Superfund Innovative Technology
Evaluation (SITE) demonstration. This
semi-quantitative method was developed
by Antox Inc. (South Portland, ME), to
detect benzene, toluene, and xylene (BTX)
contaminant levels in environmental water
samples. The evaluation compared results
from BTX immunoassays performed in the
field to quantitative results obtained by
laboratory-based gas chromatography
(GC) using EPA Method 8020.
Technology Description
The BTX immunoassay is a competitive
enzyme-linked immunosorbent assay
(ELISA), which uses an antibody-coated
polystyrene cuvette as the solid phase. A
hapten-enzyme conjugate mimics the free
analyte (BTX compounds) and competes
with aromatic compounds in the sample
for binding sites on the immobilized anti-
body. The test is performed by placing
Printed on Recycled Paper
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reference and test solutions in separate
coated cuvettes in which competition for
binding to the antibody occurs. After a 10-
minute Incubation step, substrate and chro-
mogen are added, and a colored
enzymatic reaction product is formed. The
optical density (OD) of the reference and
sample cuvettes Is determined using a
portable colorimeter, and the ratio of
sample to reference OD values is deter-
mined. The color intensity is inversely pro-
portional to the BTX hydrocarbon level in
the test sample. Low sample-to-reference
ratios are associated with high BTX con-
centrations and high ratios with low BTX
concentrations. The developer had se-
lected a ratio of 0.85 as the decision level,
with all samples having a ratio less than
0.85 being labeled as having 25 ppb BTX
or greater.
The laboratory confirmatory method was
gas chromatography following EPA Method
8020 using the purge-and-trap sample
preparation technique in EPA Method
5030.
Demonstration Design
Seventy-nine field samples were ob-
tained from four separate monitoring well-
fields in the Las Vegas valley during the
first quarter of 1992. Three of these sites
represented gasoline sources and the
fourth site had wells that might also be
contaminated with other hydrocarbon mix-
tures, such as jet fuel or diesel fuel. Each
sample well was purged of three well-
volumes prior to sampling to remove stag-
nant water from the well and surrounding
aquifer. Sample splits were placed in clear
40-mL volatile organic analysis (VOA) vi-
als with Teflon septums. The field immu-
noassay sample split was run in duplicate
on-site and the other sample splits were
maintained at 4°C until analysis. In addi-
tion to sample blanks, a variety of quality
assurance samples were prepared having
concentrations from 2.5 to 100 ppb BTX.
These samples were used to check for
sample stability, transportation and stor-
age problems, and to verify the perfor-
mance characteristics of the GC method.
Results and Discussion
Of the 79 field samples, 36 were deter-
mined to be above 25 ppb BTX by GC
analysis while 43 samples had concentra-
tions below the 25 ppb criteria of the im-
munoassay. Two (six percent) of the
positive samples gave false negative re-
sults on both replicate analyses. One of
these samples had 35.5 ppb and the other
had 74.8 ppb BTX. Two (five percent) of
the negative samples gave false positive
results on both replicate analyses. How-
ever, each of these samples was associ-
ated with minor levels of gasoline
contamination.
A plot of sample to reference OD ratios
vs. BTX concentration as determined from
GC analysis revealed the expected
sigmoidal curve shape. However, there
was significant variability in the S/R ratio.
for a given concentration level. In the re-
gion of 25 to 100 ppb BTX, this variability
hindered the immunoassay from perform-
ing up to the developer's expectations.
Evaluation of quality assurance samples
having concentrations from 2.5 to 100 ppb
BTX also showed that a greater than five
percent chance of false negatives would
be expected up to approximately 100 ppb
BTX. For higher level samples, the immu-
noassay worked quite well.
Conclusions and
Recommendations
The BTX immunoassay performed well
for most samples evaluated in this study.
The immunoassay was portable, easy to
use, inexpensive, and provided a rapid
estimate of BTX concentration that would
prove useful in certain applications. For
instance, this method could prove useful
in mapping the distribution of contami-
nants at a site or for monitoring changes
in contaminant levels over time. Samples
with BTX concentrations above 100 ppb
were accurately identified. Only five per-
cent of the samples were misclassified
based on replicate determinations. The
false negative and quality assurance re-
sults suggest that the current formulation
of the BTX immunoassay is riot robust
enough to accurately deal with samples in
the range of 25 to 100 ppb BTX (as the
manufacturer claims). If one wants to be
very accurate in this range, a more sensi-
tive format should be developed. An addi-
tional area for development that was
identified by this demonstration was the
need for a stable low-level performance
standard or a simple procedure for gener-
ating these types of standards.
The information in this document has
been funded wholly or in part by the U.S.
Environmental Protection Agency under
Contract Number 68-CO-0049 to Lockheed
Engineering & Sciences Company. It has
been subjected to the Agency's peer and
administrative review, and it has been ap-
proved for publication as an EPA docu-
ment. Mention of trade names and
commercial products does not constitute
endorsement or recommendation for use.
•fru.S. GOVERNMENT PRINTING OFFICE: I9t3 - 75O-07I/8O078
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R.W. Gerlach, R.J. White, and N.F.D. O'Leary are with Lockheed Engineering &
Sciences Company, Las Vegas, NV 89119. The EPA author, J.M. Van Emon,
(also the EPA Project Officer, see below) is with Environmental Monitoring
Systems Laboratory, Las Vegas NV 89193-3478.
Ths complete report, entitled "Superfund Innovative Technology Evaluation (SITE)
Program Evaluation Report For AntoxBTX Water Screen (BTX immunoassay),"
(Order No. PB93-218337; Cost: $27.00; 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:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas NV 89193-3478
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
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