o'A1'*
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-84-018 Apr. 1984
&ERA Project Summary
EPA Method Study 19, Method
609 (Nitroaromatics and
Isophorone)
Glenn Kinzer, Ralph Riggin, Thomas Bishop, Michelle A. Birts, Cory C.
Howard, and Robert Iden
An Intel-laboratory study in which 18
laboratories participated was conducted
to provide precision and accuracy
statements for the proposed EPA
Method 609 for measuring concentra-
tions of the Category 4 chemicals nitro-
benzene, isophorone, 2,4-dinrtrotoluene,
and 2,6-dinitrotoluene in municipal and
industrial aqueous discharges. Method
609 involves solvent extraction of the
pollutants with methylene chloride,
followed by Florisil cleanup and subse-
quent gas chromatographic analysis of
the four subject compounds, using
flame ionization and electron capture
detection techniques.
The study design was based on
Youden's plan for collaborative tests of
analytical methods. Three Youden pair
samples of the test compounds were
spiked into six types of test waters and
then 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 the
computer program entitled "Interlabora-
tory Method Validation Study" (IMVS).
Mean recoveries of the subject com-
pounds were in the range of 49-75
percent. Overall precision was in the
range of 26-60 percent and single-
analyst precision was in the range of
13-45 percent. In general, mean recov-
eries, overall standard deviations (S),
and the single-analyst standard devia-
tions (SR) were directly proportional to
the true concentration levels. There were
no discernible differences due to water
types among mean recoveries, overall
precisions, and single-analyst precisions.
This Project Summary was developed
by EPA'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
EPA first promulgated guidelines
establishing test procedures for the
analysis of pollutants in 1973, following
the passage of the Federal Water Pollution
Control Act in 1972 by Congress. Pur-
suant to the amendment and publication
of these guidelines, EPA entered into a
Settlement Agreement—the so-called
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.
In order to facilitate the implementation
of the Clean Water Act, EPA selected for
initial study 129 specific toxic pollutants,
113 organic and 16 inorganic. The
organic pollutants were divided into 12
categories based on their chemical
structure. Analytical methods were
developed for these 12 categories by EPA
through in-house and contracted research.
The use of these analytical methods may
eventually be required for the monitoring
of the 113 toxic pollutants in industrial
wastewater effluents, as specified by the
Clean Water Act of 1977.
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Method 609 was developed in the
Battelle-Columbus Laboratories under a
contract with the Physical and Chemical
Methods Branch, Environmental Moni-
toring and Support Laboratory of EPA.
The interim Method 609 is described in the
Federal Register, Vol. 44, No. 233,
December 3, 1979. The method requires
extraction of the pollutants with methylene
chloride, Kurderna Danish Concentration,
Florisil cleanup, and subsequent gas
chromatographic analysis of the four
subject compounds. Nitrobenzene and
isophorone are measured using flame
ionization detection and the 2,4- and 2,6-
dinitrotoluenes are measured using
electron capture detection.
Procedure
The study design was patterned after
Youden's plan for collaborative evaluation
of precision and accuracy for analytical
methods 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. Samples of three
Youden pairs used in this study contained
low, medium, and high concentrations of
the Category 4 compounds which were
spiked into each of six different water
types and then analyzed.
Prior to the start of the interlaboratory
method study, participants were familiar-
ized with both the study design and the
analytical procedure by analyzing one trial
Youden pair sample followed by attend-
ance at a prestudy conference. After
resolving method interpretation and
analytical problems there, participating
laboratories were supplied with the test
materials required by the study design
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 chem-
ical contamination
d. Three industrial wastewaters from
industries that were potential candi-
dates for priority pollutant control
under the National Pollutant Dis-
charge Elimination System (NPDES)
program.
Analyses were conducted on distilled
water to evaluate the analyst's proficiency.
Municipal drinking and surface waters
were included as test waters since these
water types are subject to contamination.
Hence, it was considered important to
obtain information about the performance
of Method 609 in such matrices, as well
as those found in industrial wastewater
effluents.
Statistical analyses of the data were
performed using the IMVS computer
program. The IMVS program which was
developed at Battelle's Columbus Labora-
tories is a revised version of the EPA
COLST program. The program 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
standard deviation.
The overall standard deviations indicate
the dispersion expected among values
generated from multiple laboratories. This
. represents the broad error in any mass of
data collected in a collaborative study.
The single-analyst standard deviations
indicate the dispersion expected among
replicate determinations within a single
laboratory.
Results and Discussion
The data collected during this inter-
laboratory study were statistically ana-
lyzed in order to establish the relationship
between precision and the true concen-
trations, and between accuracy and the
true concentration. Those relationships
are summarized by the linear regression
equations presented in Table 1.
The results of the regression analyses
indicate apparent linear relationships
between (1) overall standard deviation
and mean recovery; (2) single-analyst
precision and mean recovery; and (3) true
concentration and mean recovery.
The percent recoveries of isophorone
and the nitroaromatic compounds were
in the range 49 - 75 percent. The overall
relative standard deviation ranged from
25 to 60 percent and single-analyst
relative standard deviations varied from
13 to 45 percent.
Evaluation of the data in Table 1
indicates that if a laboratory performs
well with the method using distilled
water, it should also be able to obtain
comparable results with surface waters
and industrial wastewaler, provided that
the level of interferences does not
overwhelm the components of interest.
However, it is important to recognize that
about 15 percent of the laboratories were
unable to achieve good results. A major
contributing factor was the experience of
the laboratory in applying the method
(i.e., better data will be obtained as a
laboratory gains experience with the
method). Also, certain experimental steps
in the method may contribute errors in
the data. For example, improperly activated
Florisil results in selectively low recovery
for nitrobenzene, while concentration
problems would tend to selectively
decrease the recoveries for both nitro-
benzene and isophorone. But, it is not
obvious from the data set which factors
are major contributors to the analytical
errors.
All of the laboratories were able to
achieve satisfactory chromatographic
performance. Several laboratories indi-
cated problems with "bumping" of the
extract in the Kuderna-Danish evaporator,
especially at low extract volume, which
can result in relatively uniform losses for
all four analytes. This phenomenon may
explain some of the low recoveries
observed, since recoveries were similar
for all four analytes. A few laboratories
reported the formation of air bubbles in
the Florisil cleanup columns, but this did
not appear to affect the results and was
not a widespread problem.
One of the questions of interest in this
study was whether water types affected
the precision and accuracy of the method.
An analysis of variance procedure
(ANOVA) was used to test for the effect of
water type on precision and accuracy.
Based on the results of this analysis there
was no indication that water type had a
significant affect on the precision or
accuracy of the method.
Conclusions and
Recommendations
Based on the results of the interlabora-
tory method study. Method 609 is a viable
analytical method for measuring concen-
trations of the Category 4 chemicals in
industrial wastewaters. Use of Method
609 by experienced analyst should
enable industries to meet the require-
ments of the NPDES program for dis-
charging the subject pollutants into the
environment-
Certain laboratory operations in Method
609 have a primary impact on method
performance. They are:
a. Solvent extraction of the water
sample.
b. Activation of the Florisil adsorbent
and subsequent preparation of the
cleanup column.
c. Concentration and exchange of the
solvent extract and Florisil fractions.
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Table 1. Regression Equations for Accuracy and Precision of Method 609 by Compound and Water Type
Water Type Nitrobenzene Isophorone 2,6-Dinitrotoluene
2,4-dinitrotoluene
Distilled Water
Single-Analyst Precision
Overall Precision
Accuracy
Tap Water
Single Analyst Precision
Overall Precis/on
Accuracy
Surface Water
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (C-44)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (0-45)
Single-Analyst Precision
Overall Precision
Accuracy
Wastewater (0-46)
Single-Analyst Precision
Overall Precision
Accuracy
(25-425 fjg/L]a
SR = 0.25X + 2.53
S=0.37X-O.78
X = 0.06C + 2.00
SR = 0.30X + 1.63
S=0.45X - 1.24
X = 0.710-0.34
SR = 0.37X - 2.20
S = 0.43X + 2.87
X = 0.690-0.99
SR = 0.18X + 2.10
S = 0.26X + 0.54
X = 0.680 -0.38
SR = O.20X + 3.10
S=0.38X-0.58
X = 0.610+ 2.25
SR = 0.16X + 2.48
S = 0.26X + 1.88
X = 0.75C- 1.15
(25-475 fjg/Lf
SR = 0.28X + 2.77
S=0.46X + O.31
X = 0.490+ 2.93
SR = 0.45X - 3.07
S = 0.60X-3.27
X = 0.660 + 1.76
SR=0.37X+ 1.67
S = 0.46X - 0.06
X = 0.590 -0.03
SR = 0.27X + 2.06
S=0.33X + 3.75
X = 0.670 - 1.23
SR = 0.22X + 7.15
S = 0.52X - 1.16
X = 0.620 + 1.87
SR = 0.26X + 6.86
S=0.54X + 1.64
X = 0.62C+ 12.63
(a) Concentration range of compound for which regression equations are generally applicable.
X = Mean Recovery
0 = True Value for the Concentration
(1-60ng/L]T
SR = 0.19X + 0.06
S = 0.36X - 0.00
X = 0.66C + 0.20
SR = 0.23X - 0.02
S = 0.37X - 0.06
X = 0.66C + 0.14
SR = 0.24X + 0.00
S = 0.34X + 0.03
X = 0.63C + 0.28
SR = 0.15X + 0.03
S = 0.25X + 0.01
X = 0.67C+0.18
SR = 0.13X + 011
S = 0.25X + 0.17
X = 0.68C + 0.09
SR = 0.20X - 0.01
S = 0.29X + 0.04
X = O.67C +O.12
(1-55 ug/Lf
SR = 0.20X + 0.08
S = 0.37X - 0.07
X = 0.650+ 0.22
SR = 0.25X + 0.03
S = 0.35X - 0.06
X = 0.650 +0.17
SR = 0.27X + 0.08
S =0.34X + 0.21
X = 0.590 + 0.25
SR = 0.18X + 0.09
S = 0.28X + 0.04
X = 0.640+ 0.21
SR = 0.16X + 0.09
S = 0.32X + 0.09
X = 0.6OC + 0.01
SR = 0.22X - 0.05
S = 0.29X + 0.03
X = 0.640 + 0.16
d. Operation of the gas chromato-
graphic flame ionization detection
and gas chromatographic electron
capture detection systems.
The user of the method must exercise
care in conducting these operations in
order to obtain accurate and reproducible
data.
Glenn Kinzer. Ralph Riggin, Thomas Bishop. Michelle A. Bins. Cory C. Howard.
and Robert Iden are with Battelle-Columbus Laboratories. Columbus, OH
43201,
Edward L. Berg and Robert L. Graves are the EPA Project Officers (see below).
The complete report, entitled "EPA Method Study 19. Method 609 (Nitroaromatics
and Isophorone)." (Order No. PB84-176 908; Cost: $11.50, 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 Officers can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
•A U S. GOVERNMENT PRINTING OFFICE, 1984 — 759-015/7693
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
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