United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas, NV 89193-3478
Research and Development
EPA/600/SR-92/032 June 1992
EPA Project Summary
Superfund Innovative
Technology Evaluation (SITE)
Report for the Westinghouse
Bio-Analytic Systems
Pentachlorophenol
Immunoassays
M.E. Silverstein, R. J. White, R.W. Gerlach, and J. M. Van Emon
The results of the demonstration of two
Westinghouse Bio-Analytic Systems
(WBAS) immunoassay technologies are
described in this report. The immunoas-
says measure parts per billion concentra-
tions of pentachlorophenol in environmen-
tal water samples. The study was con-
ducted under the Superfund Innovative
Technology Evaluation (SITE) Program
and designed to evaluate the ruggedness
and utility of a semiquantitative immuno-
assay field kit. Results obtained from the
field kit were compared to those obtained
from a quantitative, high-sample-capacity
plate immunoassay. Both techniques were
compared to a standard U.S. Environmen-
tal Protection Agency (EPA) gas chroma-
tography/mass spectrometry (GC/MS) pro-
cedure (EPA Method 8270) for pentachlo-
rophenol determination.
The results of the WBAS immunoassay
demonstration support the conclusion that
the field immunoassay is a useful screen-
ing tool. The demonstration verified that
the method can provide qualitative or
semiquantitative screening information.
Although the results were more variable
than had been anticipated, the incorpora-
tion of additional procedural precautions
and carefully chosen quality control ac-
ceptance criteria for on-site analysis could
improve performance substantially. Both
immunoassays produced results biased
high compared to the GC/MS results, but
the tendency was not large and may have
been partly due to loss during sample
extraction (EPA Method 3510) prior to
analysis by GC/MS. The detection of
structurally related compounds by the im-
munoassays may have also contributed
to the high bias. The results indicate that
the plate immunoassay is an accurate and
precise method for quantitating pentachlo-
rophenol in water.
This Project Summary was developed
by EPA's Environmental Monitoring
Systems Laboratory, Las Vegas, NV,
to announce key findings of the re-
search project that is fully documented
in a separate report of the same title
(see Project Report ordering informa-
tion at back).
Introduction
This evaluation report presents the re-
sults of a demonstration designed to as-
sess the capabilities of two immunoassay
technologies to measure pentachlorophe-
nol (PCP) in water. The technologies, a
semiquantitative field kit immunoassay and
a quantitative plate immunoassay, were
both developed by Westinghouse Bio-Ana-
lytic Systems (WBAS)* of Rockville, Mary-
land. The demonstration was conducted
under the Monitoring and Measurement
Technologies Program as part of the U.S.
Environmental Protection Agency (EPA)
Superfund Innovative Technology Evalua-
tion (SITE) Program. The demonstration
was conducted under the guidance of the
EPA Environmental Monitoring Systems
Laboratory in Las Vegas, Nevada (EMSL-
LV).
Description of Technologies
Immunoassays are analytical techniques
based on protein molecules (antibodies).
* Mention of trade names or commercial products does
not constitute an endorsement or reccomendation for
use.
Printed on Recycled Paper
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The binding of a specific antibody to its
target analyte can be used to quantita-
tively or qualitatively determine the extent
of contamination in environmental samples.
Specific antibodies have been developed
to detect single analytes or groups of re-
lated compounds. The WBAS kit immu-
noassay, based on rabbit polyclonal anti-
sera adsorbed on 8-weil, polystyrene,
mlcrotiter strips, has a reported detection
limit for PGP of 3 parts per billion (ppb), a
linear dynamic range of 3 to 40 ppb, and
a total analysis time of 30 minutes per
sample. It requires minimal logistical re-
quirements for on-site analyses. The
\AfBAS 96-well plate immunoassay, based
on a rat monoclonal antibody, has a re-
ported detection limit of 30 ppb and a
linear dynamic range of 50 to 400 ppb. It
requires 3 hours of hands-on analysis time
per plate (10 to 20 samples run in tripli-
cate) and involves certain logistical con-
siderations (e.g., a mobile laboratory) for
on-site analyses. A previous evaluation
by the EMSL-LV compared the plate im-
munoassay to GC results for PCP analy-
sis; data from this SITE immunoassay
demonstration complement the previous
study.
The WBAS kit immunoassay was dem-
onstrated under field (on-site) and labora-
tory (off-site) conditions to determine its
ruggedness, reliability, and potential for
use as a rapid, on-site, analytical tool in
the Superfund Program. The results ob-
tained from the kit immunoassay analyses
performed on-site and off-site were com-
pared to those generated off-she by the
plate immunoassay; both immunoassay
techniques were compared to standard
EPA gas chromatography/mass spectrom-
etry (GC/MS) methods for the analysis of
PCP (EPA Method 3510 sample extrac-
tion followed by EPA Method 8270 analy-
sis by GO/MS).
PCP Immunoassay
Demonstration Design
The on-site demonstration took place in
July and August, 1989, at the MacGillis &
Gfobs Superfund Site in New Brighton,
Minnesota, a National Priorities List site
known to have ground water contaminated
with PCP. The immunoassay demonstra-
tion was coordinated through RREL and
conducted jointly with a SITE demonstra-
tion of a bioremediation technology de-
signed by BioTrol, Inc. (Chaska, Minne-
sota), to biodegrade PCP in aqueous
waste streams. The design of the immu-
noassay SITE demonstration involved sev-
eral planning components: predemon-
stration tests, an experimental design, a
sampling and analysis design, quality as-
surance and quality control (QA/QC) plan-
ning, and data base management.
Field samples for the immunoassay
demonstration were obtained from three
sampling points in the bioreactor system:
influent samples collected before pretreat-
ment (nutrient addition and pH adjustment),
influent samples collected after pretreat-
ment, and effluent samples collected be-
fore filtration. Samples were collected
over three 1 -week periods which coincided
with a 1-, 3-, and 5-gallon-per-minute flow
rate of the ground water through the
bioreactor. Composite and grab samples
were collected, homogenized, split, and
analyzed on-site with the kit immunoas-
say. Sample splits were analyzed at the
WBAS and EMSL-LV laboratories with the
kit and plate immunoassays and with GC/
MS by Science Applications International
Corporation in San Diego, California.
Comparison of the analysis results by each
method and analysis site was a critical
component in the evaluation of the immu-
noassays.
A rigorous QA project plan was imple-
mented at all sites involved in the study.
This plan included the analysis of a bat-
tery of QA/QC samples, including dupli-
cate, split, matrix spike, QA audit, QC
performance, field blank, negative control
(NC) samples and cross-calibration stan-
dards. The QA/QC samples were used to
assess the performance characteristics of
the two immunoassay methods and to test
the capabilities of the technologies to meet
the stated data quality objectives (DQOs)
of the demonstration; the most critical DQO
was that the immunoassay sample results
had to be within a factor of two (50 to
200%) of the GC/MS results. Traditional
methods such as GC/MS have interlabor-
atory biases of 30 % or more in addition
to other sources of variability. Thus, the
use of a factor of two for the immunoas-
say implies a slightly greater (but quite
useable) variability than one might expect
from the more traditional methods. All
bioreactor and QA/QC sample data from
all analysis locations were subjected to
EMSL-LV QA review and verification. The
data were then entered and stored in a
documented data base.
Method Results and
Comparisons
The immunoassay technologies were
assessed by comparing the analyses of
the bioreactor influent and effluent
samples. Because of the differences in
sample ranges of the influent and the ef-
fluent samples, results from these sample
types were treated separately in data
evaluation. The most critical method and
analysis site comparisons were: (1) the
on-site kit immunoassay to the GC/MS,
(2) the on-site kit immunoassay to the
plate immunoassay, and (3) the plate im-
munoassay to the GC/MS.
The on-site kit immunoassay performed
well as a semiquantitive screening method.
When compared to the results from the
GC/MS analysis, there was good relative
(rank order) agreement between the two
methods. Fourteen of the 16 influent
samples analyzed on-site were within the
factor-of-two DQO over a concentration
range of approximately 1 to 60 ppm PCP.
The effluent samples analyzed by the two
methods were in the same general con-
centration range (kit immunoassay = 0.2
to 2.3 ppm; GC/MS = 0.008 to 0.9 ppm).
Results of influent and effluent samples
indicated a consistent tendency for the kit
immunoassay data to have a high bias
when compared to the GC/MS data. This
bias may be due to extraction inefficiency
of EPA Method 3510, cross-reactivity of
tetrachlorophenol in the immunoassay, or
a combination of these and other factors.
Kit immunoassay results for influent
samples averaged from 65 to 119 % higher
than GC/MS results, depending on analy-
sis site. Effluent sample bias was small in
practical (ppm) terms. The positive bias
suggests that the kit immunoassay has a
minimal tendency to generate false nega-
tive responses.
The kit immunoassay results were com-
pared to the plate immunoassay results to
detect differences between the methods
and to provide insight for interpreting the
performances of the immunoassays com-
pared to the GC/MS. There was reason-
able agreement between the two immu-
noassay techniques; 27 of 38 (71 %) on-
site kit immunoassay influent sample re-
sults were within a factor of two of the
plate immunoassay results (WBAS and
EMSL-LV analysis sites combined). For
both immunoassays, effluent samples were
in the same general range (0.20 to 2.74
ppm; n = 38). Although no significant bias
was observed between the two immuno-
assay techniques, a significant amount of
scatter (variability) was observed.
Overall, the plate immunoassay results
compared more favorably to the GC/MS
than did the kit immunoassay results. At
one analysis site (EMSL-LV), 17 of 18 (94
%) effluent sample results were within a
factor of two of the corresponding GC/MS
results, while at the other site (WBAS), 12
of 18 samples were within this limit. The
results from various QA/QC samples sug-
gest that WBAS had unusual site- or op-
erator-specific factors affecting the quality
of their analyses. As with the kit immuno-
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assay, the plate immunoassay exhibited a
high bias when compared to the GC/MS,
although the bias was much smaller (17
to 40 % for influent samples, depending
on analysis site).
Quality Assurance and Quality
Control Results
Data derived from the QA and QC
samples provided insight into the intra-
and intermethod performance assessment
in terms of the accuracy and precision of
the kit and plate immunoassays. Sev-
enty-six percent of the audit samples and
74 percent of the bioreactor samples ana-
lyzed using the kit immunoassay met the
accuracy DQOs. The false negative rate
was 2.6 percent (based on 76 effluent
and influent sample analyses), and the
false positive rate was 19 percent (based
on 98 NC samples). However, the matrix
spike recoveries were unsatisfactory (-166
to +313 %), a fact that may be attributed
to a poorly developed matrix spike proto-
col. Precision for the kit immunoassay
was not as good as expected. The coeffi-
cients of variability for QC performance
and QA audit samples exceeded the DQO
of ±50 % in most cases; however, results
of the duplicate and split sample analyses
were reasonably good for a semiquantit-
ative method.
Ninety-five percent of the audit samples
and 81 percent of the bioreactor samples
met the accuracy DQO for the plate im-
munoassay. There were no false nega-
tives (based on 78 effluent and influent
sample analyses) and no false positives
(based on 21 NC samples). The matrix
spike recoveries were less than satisfac-
tory (41 to 169 %), but were considerably
better than for the kit immunoassay spike
recovery results. Overall, precision for
the plate immunoassay method was bet-
ter than the kit immunoassay method.
Better precision and accuracy for the plate
immunoassay was not surprising because
the kit immunoassay was designed to be
a semiquantitative method while the plate
immunoassay was expected to be quanti-
tative.
Conclusions and
Recommendations
The WBAS kit immunoassay proved to
be a useful and promising technology that
can provide on-site, real-time, cost-effec-
tive, semiquantitative data with a low risk
of generating false negative responses.
The kit immunoassay, which is easy to
learn and perform in the field, can be an
effective field screening method at
Superfund sites known to have PCP-con-
taminated water. The plate immunoassay
exhibited better precision and accuracy
than the kit immunoassay, with quantita-
tive results closer to those generated by
the GC/MS. The plate immunoassay can
be readily set up in a field laboratory, and
its sample output is greater than that of
the kit immunoassay. The SITE demon-
stration indicated that the WBAS kit and
plate immunoassay technologies can pro-
vide effective screening capabilities in the
field and can be used to complement con-
ventional laboratory methods for measur-
ing PCP in aqueous samples. The dem-
onstration also underscored the need for
continued QA/QC guidelines and protocol
development to improve and fully charac-
terize the quality of immunoassay data.
Both WBAS immunoassays evaluated in
this report showed promise as measure-
ment and monitoring tools at hazardous
waste sites.
This evaluation is submitted in partial
fulfillment of Contract Nos. 68-03-3249 and
68-CO-0049 by Lockheed Engineering &
Sciences Company under the sponsor-
ship of the EPA.
•U.S. Government Priming Office: 1992— 648-080/60032
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The EPA author JM VanEmon (also the EPA Project Officer, see below) is with
the Environmental Monitoring Systems Laboratory, Las Vegas, NV 89193-3478,
and M.E. Silverstein, R. J. White, and R.W. Gerlach, are with Lockheed
Engineering & Sciences Co., Las Vegas, NV 89119.
The complete report, entitled "Superfund Innovative Technology Evaluation (SITE)
Report forthe Westlnghouse Bio-Analytic Systems Pentachlorophenol Immune-
assays,* (Order No. PB92-188 713/AS; Cost: $26.00; 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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