United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-84-062 Aug. 1984
<&EPA Project Summary
EPA Method Study 15
Method 605 — Benzidines
Glenn Kinzer, Ralph Riggin, Thomas Bishop, and Cory C. Howard
The U.S. Environmental Protection
Agency (USEPA) sponsored an interla-
boratory study in which 17 laboratories
participated to provide precision and
accuracy statements for the proposed
EPA Method 605 for the Category 7
chemicals benzidine and 3,3'-dichloro-
benzidine (DCB) in municipal and
industrial aqueous discharges. Method
605 involves extraction of benzidine and
DCB from the aqueous sample at pH
7-8 with chloroform. The extract is then
back extracted into acid, reextracted
into chloroform at a neutral pH and
concentrated. The benzidines are deter-
mined in the final extract using high
performance liquid chromatography
(HPLC) with electrochemical detection.
If interference in the measurement of
benzidine is encountered, the method
provides additional detector settings to
increase the selectivity of the analytical
system.
The study design was based on
Youden's nonreplicate design for colla-
borative tests of analytical methods.
Three Youden pair samples of the test
compounds were spiked into six test
waters and analyzed. The test waters
were distilled water, tap water, a
surface water, and three different
industrial wastewater effluents. The
resulting data were statistically analyzed
using USEPA's computer program,
Interlaboratory Method Validation
Study (IMVS).
This Project Summary was prepared
by USEPA's Environmental Monitoring
and Support Laboratory, Cincinnati,
OH, 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
USEPA first promulgated guidelines
establishing test procedures for the
analysis of pollutants in 1973 following
the passage of the Federal Water Pollu-
tion Control Act in 1972 by Congress.
Pursuant to the amendment and publica-
tion of these guidelines, USEPA entered
into a Settlement Agreement — the
Consent Decree—requiring it to study
and, if necessary to regulate, 65 "priority"
pollutants and classes of pollutants of
known or suspected toxicity to the biota.
Subsequently, Congress passed the
Clean Water Act of 1977 mandating the
control of toxic pollutants discharged into
ambient waters by industry.
To facilitate the implementation of the
Clean Water Act, USEPA selected 113
organic and 16 inorganic toxic pollutants
for initial study. The organic pollutants
were divided into 12 categories based on
their chemical structure. Analytical
methods were developed for these 12
categories by USEPA through in-house
and contracted research. The use of these
analytical methods currently under
review by USEPA and industry may
eventually be required for the monitor-
ing of the 113 toxic pollutants in
industrial wastewater effluents as speci-
fied by the Clean Water Act of 1977.
Method 605 was developed in the
Battelle-Columbus Laboratories under a_
contract with the Physical and Chemical
Methods Branch, Environmental Moni-
toring and Support Laboratory-Cincinnati.
It is described in the Federal Register, Vol.
44, No. 233, December 3, 1979.
Procedure
The study design was patterned after
Youden's nonreplicate plan for collabor-
ative evaluation of analytical methods.
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in which samples are analyzed in pairs,
each member of a pair having a slightly
different concentration of the constituent
of interest. The analyst is directed to do a
single analysis and to report one value for
each sample, as for a normal routine
sample. Three Youden pair samples used
in this study contained low, medium, and
high concentrations of the Category 7
compounds which were spiked into each
of six different waters and analyzed.
Prior to the interlaboratory method
study, participants analyzed one trial pair
of Youden samples to obtain experience
with the analytical method and sample
handling procedures. Upon completion of
the trial analyses, the participants
attended '.a prestudy conference to
resolve method interpretation and analy-
tical problems. Finally, participating
laboratories were supplied with the test
materials required for the formal study
and instructed to begin the analyses.
The test waters were:
a. Distilled water
b. A municipal drinking water
c. A surface water, for example, a river,
vulnerable to synthetic chemical
contamination
d. Three industrial wastewaters from
industries that were potential sources
of benzidines.
Analyses were conducted on distilled
water to evaluate the proficiency of the
analyst to use the method on a sample
free of interference. Since municipal
drinking and surface waters are subject
to contamination, it was considered
important to obtain information about the
performance of Method 605 in such
matrices as well as in industrial waste-
water effluents.
Statistical analyses of the data were
performed using USEPA's IMVS compu-
ter program. The IMVS program devel-
oped at Battelle's Columbus Laboratories
is a revised version of the USEPA's
COLST program and is designed to output
the raw data in tabular form and compile
summary statistics including:
Number of data points
True value
Mean recovery
Accuracy as percent relative error
Overall standard deviation
Overall percent relative standard
deviation
Single-analyst standard deviation ,
Single-analyst percent relative stan-
dard deviation,
The overall standard deviations indi-
cate the dispersion expected among
values generated from multiple labora-
tories. The single-analyst standard
deviations indicate the dispersion expect-
ed among replicate determinations with-
in a single laboratory.
Results and Discussion
The data collected during this interla-
boratory study were statistically analyzed
in order to establish the relationship
between precision and the mean recovery,
and between mean recovery and the true
concentration. These relationships are
summarized by the linear regression
equations presented in Table 1 for (1)
overall standard deviation and mean
recovery, (2) single-analyst precision and
mean recovery, and (3) true concentra-
tion and mean recovery.
The slopes of the regression equations
can be used to estimate the percentage
recovery, and overall and single-analyst
percent relative standard deviations at
concentrations exceeding 20 pg/L since
ignoring the intercepts introduces an
error of less than 11 percent in the
estimated value. Below 20 pg/L, some
regression equations, for example, ben-
zidine in tap water, have intercepts which,
are rather significant and care should be
exercised in using the slopes as an
estimate of accuracy and precision.
Based upon the slopes of the equations
at concentrations above 20pg/L, percent-
age recoveries for benzidine and DCB
ranged from 52-70 and 48-66, respec-
tively, for all water types. The overall
percent relative standard deviation for
benzidine and DCB ranged from 40 69
and 38-58 perceht, respectively, for all
water types. The single-analyst percent
relative standard deviation for benzidine
and DCB ranged from 24-40 and 26-40,
respectively, for all water types.
Examination of the raw data from each
of the laboratories reveals that several of
the laboratories did not obtain a recovery
greater than 30% for benzidine in any.of
the matrices, even at the highest level,
while many of the other laboratories
obtained recoveries consistently above
50% for benzidine. A similar pattern was
not found for DCB. This observation
indicates that systematic error at several
of the laboratories was responsible for
low recoveries and high standard devia-
tions of benzidine and^DCB.-
Oxidants (e.g., chlorine) present in
some of the reagent water-sources may
have contributed .to the low recoveries.
Reagent water used for blanks as well as
for preparing reagents for sample workup
and HPLC mobile phase must be free from
even traces of oxidant, as demonstrated
by obtaining good recovery of benzidine
and DCB from spiked process blanks.
Reagent water purified by a multi-
cartridge systerri such as Millipore's
"Milli-Q" system, which utilizes reverse
Table 1. Regression Equations for A ccuracy and Precision of Method 60S by Compound and
Water Type
Water Type
Applicable Cone. Range
Distilled Water
Single-Analyst Precision
Overall Precision
Accuracy
Tap Water
Single-Analyst Precision
Overall Precision
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-74)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater fC-75)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-76J
Single-Analyst Precision
Overall Precision
Accuracy
Benzidine
(1.00 -55.00 (jg/L)
SR=0.28X + O.J9
S = 0.40X + 0. 18
X =0.70C + 0.06
SR = 0.32X + 0.42
S = O.SOX + 0.21
X = 0.64C + O.32
SR = 0.30X + 0.09
S = 0.56X + 0.06
X = 0.57C + 0.01
SR = 0.40X + 0.04
S =O.69X-0.13
X = 0.55C + 0.10
SR = 0.34X - 0.09
S =0.6OX-O.07
X =0.62C-O.18
SR = 0.24X + O.JO
S =0.64X-0.01
X = 0.52C - 0.02
3,3-Dichlorobenzidine
; (1 .00 - 70.00 ug/L)
SR = 0.39X - O.O5
S = 0.38X + 0.02
X =0.66C + 0.23
• SR = 0.29X + 0.06
S = 0.38X + 0. 16
: X =0.64C + 0.12
' SR = 0.26X + 0. 12
S = 0.40X + 0.10
X =0.59C + O.14
SR = 0.27X + 0.28
S =0.56X + 0.13
' X = 0.48C + 0.03
SR = 0.26X + 0.26
S = 0.58X + 0.09
: X =0.62+0.19
SR = 0.40X + 0.05
S = O.SOX + 0. 10
X =0.53C + 0.08
X = Mean Recovery
C = True Value lor the Concentration
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osmosis, ion-exchange, and carbon
filtration purification steps, is recom-
mended. Carbon filtration is particularly
important because it removes residual
chlorine. Recoveries for benzidine and
DCB from finished drinking water and
distilled water were similar, indicating
that the dechlorination procedure was
effective to prevent oxidation of benzidine
spiked into drinking water.
HPLC separation efficiency reported by
several of the laboratories was low, in
some instances less than 1000 theoreti-
cal plates per 25-cm column. Resolution
of analytes from interferences becomes a
significant problem when such low sepa-
ration efficiencies are achieved. Probably
the method should specify that the mini-
mum separation efficiency greater than
3000 theoretical plates per 25-cm
column be achieved before sample analy-
ses are conducted.
Several of the laboratories objected to
the large number of manipulations re-
quired in the method. This is a valid
consideration, although elimination of
steps in the procedure does not appear
feasible at present.
A statistically significant effect due to
water type was established for DCB in the
case of wastewater C-74 which yields
significantly lower mean recoveries than
distilled water. However, this effect was
not determined to be of practical impor-
tance. There were no other discernible
differences due to water types, among
mean recoveries, overall precisions or
single-analyst precisions.
Conclusions and
Recommendations
Based on the results of the interlabora-
tory method study. Method 605 can be
used for measuring concentrations of the
Category 7 chemicals, benzidine and
DCB, in various water types. Use of
Method 605 by experienced analysts
should enable industries to meet the
permit requirements of the National
Pollutant Discharge Elimination System.
Both benzidine and DCB are chemically
labile and, hence, the following recom-
mendations should be followed in the
workup and analysis of samples.
• Benzidine and DCB are readily oxi-
dized and sources of oxidant must be
acrefully avoided.
• The basic character of the compounds
requires careful pH control through-
out the workup procedure in order to
obtain maximum extraction efficien- .
ces from aqueous solutions.
• A minimum separation efficiency
greater than 3000 theoretical plates
per 25-cm column should be achieved
before starting sample analyses.
1 The analyst must be well versed in
the operational characteristics of the
electrochemical detector, which,
while relatively straight-forward, is
not as widely used as the UV or other
common HPLC detectors.
Glenn Kinzer, Ralph Riggin, Thomas Bishop and Cory C. Howard are withBattelle
Columbus Laboratories, Columbus, OH 432O1-2693.
Edward Berg is the EPA Project Officer (see below).
The complete report, entitled"EPA Method Study 15. Method 605—Berizidines "
(Order No. PB 84-211 176; Cost: $10.0O, 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 and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
r U.S. GOVERNMENT PRINTING OFFICE; 1984—759-015/7767
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United States
Environmental Protection
Agency
Official Business
Penalty for Private Use $300
Center for Environmental Research
Information
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