DETERMINATION OF ROTENONE IN INDUSTRIAL
AND MUNICIPAL WASTEWATSRS
by
J.S. Warner, T.M. Engel and P.J. Mondroa
BatCelie Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-03-2956
Project Officer
Thomas Press ley
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents necessarily
reflect the view and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati, conducts research to:
o Develop and evaluate methods to measure the presence and concentra-
tion of physical, chemical, and radiological pollutants in water,
wastewater, bottom sediments, and solid wastes.
o Investigate methods for the concentration, recovery, and identifi-
cation of viruses, bacteria, and other microbiological organisms in
water; and, to determine the responses of aquatic organisms to
water quality.
o Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for monitor-
ing water and wastewater.
o Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
This report is one of a series that investigates the analytical behavior
of selected pesticides and suggests a suitable test procedure for their
measurement in wastewater. The method was modeled after existing EPA
methods being specific yet as simplified as possible.
Robert L. Booth, Director
Environmental Monitoring and Support
Laboratory - Cincinnati
iii
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ABSTRACT
A method was developed for the determination of rotenone in
wastewaters. The method development program consisted of a. literature
review; determination of extraction efficiency for each compound from
water into me thylane chloride; development of a deactivated silica gel
cleanup procedure; and determination of a suitable high performance liquid
chromatographic (HPLC) method with ultraviolet (UV) detection.
The final method was applied to a .relevant industrial wastewater to
determine the precision and accuracy of the method. The wastewater was
spiked with rotenone at levels of 5.5 pg/L and 110 yg/L. Recovery for
rotenone at the 5.5 ug/L level was 85 ± 8 percent. Recovery at the
110 ug/L level was 88 ± 3 percent. The method detection limit (MDL) for
rotenone in distilled water was 1.6 ug/L. The MDL in wastewaters may be
higher due to interfering compounds.
This report was submitted in partial fulfillment of Contract No.
68-03-2956 by Battelle Columbus Laboratories under the sponsorship of the
U.S. Environmental Protection Agency. This report covers the period from
February 1, 1982 to April 30, 1982.
iv
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CONTENTS
Foreword
Abstract iv
Figures vi
1. Introduction 1
2. Conclusions. 2
Extraction and Concentration 2
Cleanup ' 2
Chromatography 2
Validation Studies - 2
3. Experimental 3
Extraction and Concentration 3
Cleanup 3
Chromatography 3
Validation Studies 3
4. Results and Discussion 5
Extraction and Concentration 5
Cleanup 5
Chromatography 5
Validation Studies 8
References • 10
Appendix
Rotenone Method 6XX 11
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FIGURES
Number Page
1 HPLC-UV Chromacogram of Standard Solution Representing
5 yg/L of Rotenone in Water (Column 1)......... 6
2 HPLC-UV Chromatogram of Standard Solution Representing
200 yg/L of Rotenone in Water (Column 2) 7
3 Analytical Curve for Rotenone 9
vi
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SECTION 1
INTRODUCTION
Rotenone (I) is a naturally occurring insecticide derived from derris
root. It is used primarily to control insects on food crops, but is also
used to eradicate undesirable fish species from lakes and streams.
OCH
The CAS registry number for rotenone is £3-79-4 and its IUPAC name is
[2R(-2a, 6a a, 12aa,)]l,2,12,12a-tetrahydro-8,9-dimethoxy-2-
(1-methylethenyl) [l] benzopyrano(3,4-V) furo[2,3-h] [l]
benzopyran-6(6a!O-one. It has a melting point of 165-166°C and an oral
LD50 in rats of 133 mg/Kg. Common synonyms for rotenone include "Derris"
and "cube". Several papers have been published which describe the
analysis of rotenone using HPLC (1-6). Most of the methods use reverse
phase liquid chromatography with UV absorbance detection. A method using
normal phase HPLC has also been reported (1). The detection wavelengths
include 254 nm (4), 280 nm (2-5), and 294 nm (1,3,4,5). Methods for trace
analysis using HPLC have been published for rotenone residues on crops
(4,5,6), in animal feed and tissues (3). Cleanup procedures using silica
gel have also been described (3,4,5,6).
Rotenone is stable in water at neutral pH and can be extracted from
water with methylene chloride. Rotenone decomposes upon exposure to light
and air; precautions should be taken to avoid excessive exposure of
rotenone containing solutions to light and air (7). The selected approach
to the determination of rotenone in water include separatory funnel
extraction from water with methylene chloride; cleanup using silica gel
chromatography; and analysis using HPLC with UV detection. Standard
concentration techniques using Kuderna-Danish (K-D) equipment were used.
The final method is included in Appendix A of this report.
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SECTION 2
CONCLUSIONS
EXTRACTION AND CONCENTRATION
Rotenone can be extracted from water into tnethylene chloride with
greater than 90 percent recovery using separatory funnel techniques. Use
of K-D concentration equipment to perform extract concentrations did not
significantly affect compound recoveries.
CLEANUP
Rotenone elutes from six percent deactivated silica gel with greater
than 90 percent recovery. This was an effective cleanup procedure for a
relevant wastewater sample.
CHROMATOGRAPHY
Two HFLC columns, Oupont Zorbax-Cyano (normal phase) and
Spherisorb-ODS (reversed phase), were found to be acceptable for this
application. The Dupont Zorbax-Cyano does not require an additional
solvent exchange step and was used as the primary column. The
Spherisorb-ODS column was designated as the alternate column.
VALIDATION STUDIES
Recoveries of rotenone from distilled water in the 5.5 to 1090
Ug/L concentration range averaged greater than 85 percent. The analytical
curve constructed from this data was linear. The MDL in distilled water
was 1.6 ug/L. Recoveries of rotenone from a pesticide,manufacturing
wastewater at the 5.5 and 110 ug/L levels were 85 ± 8 and 88 ± 3 percent,
respectively.
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SECTION 3
EXPERIMENTAL
Studies were performed to determine if extractions with separator?
funnels, cleanup by silica gel adsorption chromatography, concentration
using K-D equipment, and analysis using HPLC with UV detection would be
applicable technique for the determination of rotenone in water.
EXTRACTION AND CONCENTRATION
Extraction of rotenone from water was studied by using separatory
funnel techniques. The distilled water was spiked with rotenone at the
10 ug/L and 100 yg/L levels. The sample was adjusted to pH 7 by addition
of 1 ^ sodium hydroxide or 1 JJ sulfuric acid and extracted three times
with 60 mL each of methylene chloride. These studies were done in
duplicate. The extracts were dried by passing them through 10 cm of
anhydrous granular sodium sulfate, concentrated to five mL and analyzed by
HPLC. Column 1 was used for HPLC analyses.
CLEANUP
A 10-gram silica gel column (six percent deactivaated with water) was
prepared as follows: 10 g silica gel was slurried with 50 mL of acetone
containing 600 uL of reagent water, the slurry was transferred to a
chromatographic column and the solvent was allowed to elute and was
discarded. The column was rinsed with 100 mL of methylene chloride which
was also discarded. The rotenone, 10 or 100 us dissolved in one mL of
methylene chloride, was added to the top of the column. The column was
eluted with 50 mL portions of methylene chloride (Fl), 6 percent acetone
in methylene chloride (F2), 15 percent acetone in methylene chloride (F3),
and 25 percent acetone in methylene chloride (F4). Each fraction was
concentrated to five mL and analyzed by HPLC. Column 1 was used for HPLC
analyses.
CHROMATOGRAPHY
Two HPLC columns were evaluated for the determination of rotenone:
Dupont Zorbax-Cyano and Spheriorb-ODS.
VALIDATION STUDIES
The MDL for rotenone was determined by analyzing seven replicate
distilled water samples spiked at the 5.5 ug/L concentration levels (8).
The sample extracts were cleaned up using the silica gel cleanup procedure
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prior to analysis. The amounts recovered were determined by external
standard calibration and the MDLs were calculated from these data.
Distilled water was also spiked in duplicate at the 5.5, 22, 54,
270, and 1090 ug/L concentration levels and recoveries of the rotenone
were determined as described earlier. An analytical curve was generated
by plotting the amount spiked into the samples versus the amount recovered
from the samples.
A relevant wastewater was used for wastewater validation studies.
Seven replicates of the wastewater were analyzed to determine the
background levels. The wastewater was spiked with rotenone at the 5.5 and
110 ug/L concentration levels, processed and analyzed. Seven replicate
extractions were performed at each concentration level.
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SECTION 4
RESULTS AND DISCUSSION
EXTRACTION AND CONCENTRATION
RoCenone was extracted from water with greater than 85 percent
recovery using separatory funnel techniques. Recoveries of rotentone from
water' using separatoy funnels were 87 and 97 percent at the 10 ug/L level
and 92 and 98 percent at the 100 ug/L level. These data are results of
duplicate analyses.
CLEANUP
Rotenone eluted from six percent deactivated silica gel in the six
percent acetone in methylene chloride fraction. Recoveries of 10 ug and
100 yg of rotenone were 98 and 93 percent, respectively.
CHROMATOGRAPHY
Both the Dupont Zorbax-Cyano and Spherisorb-QDS columns were
satisfactory for the determination of rotenone. Use of the Dupont
Zorbax-Cyano column requires one less solvent exchange step in the sample
workup and was chosen as the primary column. The following conditions
were used:
Column:
Solvent:
Flow:
Detector:
Injector Volume:
Column:
Solvent:
Flow:
Detector:
Injector Volume:
Column 1
Dupont Zorbax-Cyano, 5 micron,
250 x 4.6 mm
30 percent methylene chloride,
70 percent hexane.
1 mL/min.
UV (§254 nm
10 uL
Column 2
spherisorb-ODS, 5 micron, 250 x 4.6 mm
60 percent acetonitrile, 40 percent water.
1 mL/min.
UV <§254 nm
10 UL
Chromatograms obtained under these conditions are shown in Figures 1 and
2.
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-|—i—r-'t—r f-r-i~-
1.2 2.4 3.0
—|—i—i—i—r—|-T—r—r—«-
4.0 6.0
I •
7.2
Retention Time, min.
Rotenone
_A
-|—i—r- r--r -f—?•-
8.4 9.6
10.8 12.0
Figure 1. HPLC-UV Chromatogram of Standard Solution Representing 5 ug/L
of Rotenone in Water (Column 1)
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Rotenone
L
•r~t"~t
1.5
3.0
4.5
-T-J •
6.0
I- -T— r-r-T'i~i
12.0 13.5
7.5 9.0 10.5
Retention Time, mln.
Figure 2. HPLC-UV Chromatogram of Standard Solution Representing 200 Mg/L
of Uotenone in Water (Column 2).
15.0
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VALIDATION STUDIES
Recovery of rotenone from distilled water at the 5.5 ug/L level
was 4.6 ± 0.5 Ug/L. The MDL in distilled water was calculated to be
1.6 Ug/L. Recoveries of rotenone from distilled water at the 5.5, 22,
54, 270, and 1090 ug/L levels were 4.6(17), 20(20), 45(4.4), 240(10), and
950(2.3) Ug/L, respectively. These data were the averages of duplicate
analyses. The percent relative range is given in. parentheses. The
resultant analytical curve is shown in Figure 3.
*
Recoveries of rotenone from a relevant wasCawater at the 5.5 and
110 ug/L levels were 85 ± 8 percent and" 88 ± 3 percent, respectively.
These data were the averages of seven replicate analyses. Rotenone was
not detected in the relevant wastewater.
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1250 r
1125 •
1000 -
875
Amount Recovered,
Ug/L
750
325 •
500 •
375
250
T25
I . I . I . I . I . I . I . I . I . I
125 250 375 500 625 750 875 1000 1125 1250
Amount Spiked, ug/L
Figure 3. Analytical Curve for Rotenone
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REFERENCES
f
1. Bushway, R.J., B.D., Engdahi, B.M. Colvin, and A.R. Hanks.
Separation of Rotenoids and the Determination of Rotenone
in Pesticide Formulations by High Performance Liquid
Chromatography. J. Assoc. Off. Anal. Chem., 58:965-970,
1975.
t,
2. Bushway, R.J. and A.R. Hanks, Determination of Rotenone
in Pesticide Formulations and the Separation of Six Rotenoids
by Reversed-phase High-Performance Liquid Chromatography.
J. Chromatogr. 134:210-215, 1977. •
3. Bowman, M.C., C.L., Holder, and L.I. Bone, High Pressure
Liquid Chromatographic Determination of Rotenone and Degradation
Products in Animal Chow and Tissues. J. Assoc. Off. Anal.
Chem. 16:1445-1455, 1978.
4. Moring, I.E. and J.D. McHesney. High Pressure Liquid
Chromatographic Separation of Rotenoids from Plant Extracts.
J. Assoc. Off. Anal. Chem. 62:774-781, 1979.
5. Kobayeshi, H., 0. Matano, and G. Shinko. Determination of
Rotenoids in Soil and Crops by High-Performance Liquid
Chromatography, J. Pesticide Sci. 5:89-92, 1980.
6. Newsome, W.H. and J.B. Shields. Residues of Rotenone and
Rotenolone on Lettuce and Tomato Fruit after Treatment in the
Field with Roteixone Formulations. J. Agric. 'Food. Chem.
28:722-724, 1980.
7. Roteaone In: The Pesticide Manual, C.R. Worthing (ed.),
BCPC Publications, Croydon, England, 1979. p 468.
8. Glaser, J.A. et. al. Trace Analysis for Wastewater. Environmenta1
Science and Technology 15:1426, 1981.
10
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DETERMINATION OF ROTENONE IN INDUSTRIAL AND MUNICIPAL
WASTEWATERS BY LIQUID CHROMATOGRAPHY
METHOD 635 ~
1. Scope and Application
1.1 This method covers the determination of rotenone pesticide. The
following parameter can be determined by this method:
Parameters CAS No.
Rotenone 83-79-4
1.2 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of'the compound listed above in
municipal and industrial discharges as provided under 40 CFR 136.1.
Any modification of this method beyond those expressly permitted
shall be considered a major modification subject to application and
approval of alternate test procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14)
for rotenone compound is listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in the
interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this
or muffle furnace at 400°C for 15-30 min. Some thermally
11
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1.5 When this method is used to analyze unfamiliar samples for the
compound above, compound identifications should be supported by at
least one additional qualitative technique. This method describes
analytical conditions for a second liquid chromatographic column
that can be used to confirm measurements made with the primary
column.
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, is solvent
extracted with methylene chloride using a separatory funnel.
Liquid chromatographic conditions are described which permit the
separation and measurement of the compounds in the extract by HPLC-
UV.l
3. Interferences
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts or elevated baselines in liquid
chromatograms. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in
Section 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon as possible after use by thoroughly
rinsing with the last solvent used in it. Follow by
washing with hot water and detergent and thoroughly rinsing
with tap and reagent water. Drain dry, and heat in an oven
or muffle furnace at 400°C for 15-30 min. Some thermally
12
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stable materials such as PCBs may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide
quality hexane may be substituted for the heating. After
drying and cooling, seal and store glassware in a clean
environment to prevent accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality being
sampled. The cleanup procedure in Section 11 can be used to
overcome these interferences, but unique samples may require
additional cleanup approaches to achieve the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the
lowest possible level by whatever means available. The laboratory
is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
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chemical analysis. Additional references to laboratory safety are
available and have been identified3^ for the information of the
analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined
with Teflon. Aluminum foil may be substituted for Teflon
if the sample is not corrosive. If amber bottles are not
available, protect sample's from light. The container and
cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporte glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicons rubber tubing may be used. Before use, however,
the compressible tubing should be thoroughly rinsed with
methane!, followed by repeated rinsings with distilled water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with Teflon stopcock.
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5.2.2 Drying column - Chromatographic column 400 mm long x 10 mm
ID with coarse frit.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with 250 ml
reservoir at the top and Teflon stopcock (Kontes K-420290)
or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 25-mL, graduated
(Kontes K-570050-2525 or equivalent). Calibration must be
checked at the volumes employed in the test. A ground glass
stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 250-mL (Kontes
K-570001-0250 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Snyder column, Kuderna-Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.8 Vials - Amber glass, 5 to 10 ml capacity with Teflon lined
screw-cap.
5.2.9 Volumetric flask - 10 mL.
5.2.10 Erlenmeyer flask - 250 mL.
5.2.11 Graduated cylinder - 1000 mL.
5.3 Boiling chips - approximately 10/40 mesh carborundum. Heat to
400°C for 4 hours or extract in a Soxhlet extractor with methylene
chloride.
5.4 Water bath - Heated, capable of temperature control (^2°C). The
bath should be used in a hood.
15
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5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
- 5.6 Liquid chromatograph - Analytical system complete with liquid
chromatograph and all required accessories including syringes,
analytical columns, detector and strip-chart recorder. A data
system is recommended for"measuring peak areas.
5.6.1 Pump - Isocratic pumping system, constant flow.
5.6.2 Column 1 - Normal-phase column, 5 micron Zorbax-CN,
250 x 4.6 mm or equivalent.
5.6.3 Column 2 - Reversed-phase column, 5 micron Spherisorb-ODS,
250 x 4.6 mm or equivalent.
5.6.4 Detector - Ultraviolet absorbance detector, 254 nm.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Methylene chloride, methanol, acetonitrile, acetone, hexane,
distilled-in-glass quality or equivalent.
6.3 Sodium sulfate (ACS) Granular, anhydrous; heated in a muffle
furnace at 400°C overnight.
6.4 IN. sulfuric acid.
6.5 IN sodium hydroxide.
6.6 Silica gel, Davison grade 923, 100-200 mesh, dried for 12 hours at
150°C.
6.7 Stock standard solutions (l.OOug/L) - Stock standard solutions can
be prepared from pure standard materials or purchased as certified
solutions.
16
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6.7.1 Prepare stock standard solutions by accurately weighing
about 0.0100 grams of pure material. Dissolve the material
in distilled-in-glass quality methylene chloride for
analyses performed using column 1 and methanol for analyses
performed using column 2. Dilute to volume in a 10-mL
volumetric flask. Larger volumes can be used at the
convenience of the analyst. If compound purity is certified
at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be used
at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into Teflon-sealed
screw-cap bottles. Store at 4°C and protect from light.
Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior
to preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish liquid chromatographic operating parameters equivalent to
those indicated in Table 1. The liquid chromatographic system can
be calibrated using the external standard technique (Section 7.2)
or the internal standard technique (Section 7.3).
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7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration
standards at a minimum of three concentration levels by
adding volumes of one or more stock standards to a
volumetric flask and diluting to volume with 50/50
hexane/methylene chloride for column 1 standards and
acetonitrile for column' 2 standards. One of the external
standards should be at a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the expected range of concentrations found in
real samples or should define the working range of the
detector.
7.2.2 Using injections of 5 to 20ul_ of each calibration
standard, tabulate peak height or area responses against
the mass injected. The results can be used to prepare a
calibration curve for each compound. Alternatively, the
ratio of the response to the mass injected, defined as the
calibration factor (CF), can be calculated for each compound
at each standard concentration. If the relative standard
deviation of the calibration factor is less than 10% over
the working range, linearity through the origin can be
assumed and the average calibration factor can be used in
place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any
18
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compound varies from the predicted response by more than
^10%, the test must be repeated using a fresh calibration
standard. "Alternatively, a new calibration curve or
calibration factor must be prepared for that compound.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is
not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels for each compound of interest by
adding volumes of one or more stock standards to a
volumetric flask. To each calibration standard, add a known
constant amount of one or more internal standards, and
dilute to volume with 50/50 hexane/methylene chloride for
column 1 standards and acetonitrile for column 2 standards.
One of the standards should be at a concentration near, but
above, the method detection limit. The other
concentrations should correspond to the expected range of
concentrations found in real samples, or should define the
working range of the detector.
7.3.2 Using injections of 5 to 2QuL of each calibration
standard, tabulate the peak height or area responses against
the concentration for each compound and internal standard.
19
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Calculate response factors (RF) for each compound as
follows:
RF • (AsCis)/(AisCs)
where:
As » Response for the compound to be measured.
A-jS * Response for the internal standard.
Cis = Concentration of the internal standard inug/L.
Cs = Concentration of the compound to be measured in
ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF can be used for
calculations. Alternatively, the results can be used to
pilot a calibration curve of response ratios, As/Ais
against RF.
7.3.3 The working calibration curve or RF must be verified on
each working shift by the measurement of one or more
calibration standards. If the response for any compound
varies from the predicted response by more than ^10%, the
test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve must be prepared for
that compound.
7.4- Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the
reagents.
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8. Quality control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and the
analysis of spiked samples as a continuing check on performance.
The laboratory is required to maintain performance records to define
the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must
demonstrate the ability to generate acceptable accuracy and
precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in
chromatography, the analyst is permitted certain options to
improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the
analyst is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in methylene
chloride 1000 times more concentrated than the selected
concentrations.
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8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-ml aliquots of reagent
water. A representative wastewater may *e used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test
to be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the
standard deviation of the percent recovery (s), for the
results. Wastewater background corrections must be made
before R and s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst
must review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R +• 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3. The UCL
and LCL can be used to construct control charts** that are
useful in observing trends in performance.
22
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8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater
samples. An accuracy statement for the method is defined
as R £ s. The accuracy statement should be developed by the
analysis of four aliquots of wastewater as described in
Section 8.2.2, followed by the calculation of R and s.
Alternately, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated with this method. This ability is established as
described regularly.5
8.4 The laboratory is required to collect in duplicate a portion of
»their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one sample
per month, whichever is greater. One aliquot of the sample must be
spiked and analyzed as described in Section 8.2. If the recovery
for a particular compound does not fall within the control limits
for method perfomance, the results reported for that compound in
all samples processed as part of the same set must be qualified as
described in Section 13.3. The laboratory should monitor the
frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst should demonstrate
though the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
23
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a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank should be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as liquid chromatography with a
dissimilar column, must be used. Whenever possible, the laboratory,
should perform analysis of standard reference materials and
participate in relevant performance evaluation studies.
9. Samples Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices? should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of Tygon and other potential sources of
contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 1N_ sodium hydroxide or
IN sulfuric acid immediately after sampling.
24
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10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel. Check the pH of the sample with wide
range pH paper and adjust to 7 with 1 N sodium hydroxide or 1 N
H2S04-
10.2 Add 60 ml of methylene chloride-to the sample bottle, seal, and
shake 30 seconds to rinse the inner walls. Transfer the solvent to
the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a
minimum of 10 minutes. If the emulsion interface between layers is
more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation.
The optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool,
centrifugation, or other physical methods. Collect the methylne
chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time,
collecting the extract. Perform a third extraction in the same
manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-0) concentrator by attaching a 25-mL
concentrator tube to a 250-mL evaporative flask. Other
concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
25
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10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse
the column with an additional 30 to 40 mL of methylene chloride.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and «the water temperature as required to
complete the concentration in 15 to 20 minutes. At the proper rate
of distillation, the balls of the column will actively chatter but
the chambers will not flood with condensed solvent. When the
apparent volume of liquid reaches 1 mL, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 mL of methylene
chloride. A 5-mL syringe is recommended for this operation. Add 1
or 2 clean boiling chips and attach a two-ball micro-Snyder column
to the concentrator tube. Prewet the micro-Snyder column with
methylene chloride and concentrate the solvent extract as before.
When an apparent volume of 0.5 mL is reached, or the solution stops
boiling, remove the K-D apparatus and allow it to drain and cool
26
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for 10 minutes. If analysis is being performed using column 1 or
if sample cleanup is required, proceed with Section 10.9. If column 2
is used and no sample cleanup is required, proceed with Section 10.8.
10.8 Add 10 ml of acetonitrile to the concentrator tube along with 1 or
2 clean boiling chips. Attach a two-ball micro-Snyder column to
the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract as before. When
an apparent volume of 1 ml is reached, remove the K-D apparatus and
allow it to drain and cool for 10 minutes. Transfer the liquid to
a 10-mL volumetric flask and dilute to the mark with acetonitrile.
Mix thoroughly prior to analysis. Proceed with Section 12 using
column 2.
10.9 Remove the micro-Snyder column and adjust the volume of the extract
to 1.0 ml with methylene chloride. Stopper the concentrator tube
and store refrigerated if further processing will not be performed
immediately. If the extract is to be stored longer than two days,
transfer the extract to a screw-capped vial with a Teflon-lined
cap. If the sample extract requires no further cleanup, proceed
with the liquid chromatographic analysis in Section 12 using Column 1.
If the sample requires cleanup, proceed to Section 11.
10.10 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 mL.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various clean waters and
27
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industrial effluents. If particular circumstances demand the use
of additional cleanup, the analyst must demonstrate that the
recovery of each compound of interest is no less than 85%.
11.2 Slurry 10 g of silica gel in 50 ml of acetone to which has been
added SOOuL of reagent water. Transfer the slurry to a
chromatographic column (silica gel is retained with a plug of glass
wool). Wash the column with 100 ml of methylene chloride. Use a
column flow rate of 2 to 2.5 mL/min throughout the wash and elution
profiles.-
11.3 Add the extract from Section 10.9 to the head of the column. Allow
the solvent to elute from the column until the silica gel is almost
exposed to the air. Elute the column with 50 mL of methylene
chloride. Discard this fraction.
11.4 Elute the column with 60 ml of 6% acetone in methylene chloride.
Collect this fraction in a K-D apparatus. Concentrate the column
fraction to 1 ml as described in Sections 10.6 and 10.7. If column
1 is being used, proceed with Section 11.5. If column 2 is being
used, proceed with Section 11.7
11.5 Add 5 mL of hexane to the concentrate along with 1 or 2 clean
boiling chips. Attach a three-ball micro-Snyder column to the
concentrator tube. Prewet the micro-Snyder column with hexane and
concentrate the solvent extract to an apparent volumn of 1 mL.
Allow the K-D apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 10-mL volumetric flask and dilute to the
mark with hexane. Mix thoroughly prior to analysis. If the
extracts will not be analyzed immediately, they should be
28
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transferred to Teflon sealed screw-cap vials and refrigerated.
Proceed with the liquid chromatographic analysis using column 1.
11.7 Add 10 mL of acetonitrile to the concentrate along with 1 or 2
clean boiling chips. Attach a three-ball micro-Snyder column to
the concentrator tube. Prewet the micro-Snyder column with
acetonitrile and concentrate the solvent extract to an apparent
volume of 1 mL. Allow the K-D apparatus to drain and cool for 10
minutes.
11.8 Transfer the liquid to a 10-mL volumetric flask and dilute to the
mark with acetonitrile. Mix thoroughly prior to analysis. If the
extracts will not be analyzed immediately, they should be
transferred to Teflon sealed screw-cap vials and refrigerated.
Proceed with the liquid chromatographic analysis using colunjn 2.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph. Included in this table are estimated
retention times and method detection limits that can be achieved by
this method. An example of the separations achieved by column 1 and
column 2 are shown in Figures 1 and 2. Other columns,
chromatographic conditions, or detectors may be used if the
requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in
Section 7.
12.3 If an internal standard approach is being used, the analyst must
not add the internal standard to sample extracts until immediately
before injection into the instrument. Mix thoroughly.
29
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12.4 Inject 5 to 20UL of the sample extract by completely filling the
sample valve loop. Record the resulting peak sizes in area or peak
height units.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time
variations40f standards over the course of a day. Three times the
standard deviation of a retention time for a compound can be used
to calculate a suggested window size; however, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(Vt)
Concentration, ug/L a
(V1)(VS)
where:
A = Amount of material injected, in nanograms.
V-j s Volume of extract injected inuL.
30
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Vt s Volume of total extract inuL.
Vs = Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the
response factor (RF) determined in Section 7.3.2 as
follows:
(As)(is)
Concentration, yg/L
s
(Ais)(RF)(V0)
where:
As = Response for the compound to be measured.
ATS - Response for the internal standard.
Is - Amount of internal standard added to each extract
extract i n u g.
V0 = Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected compounds must be labeled as suspect.
14. Method Performance
14.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is about zero. The MDL
concentrations listed in Table 1 were obtained using reagent
water.1 Similar results were achieved using representative
wastewaters.
31
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14.2 This method has been tested for linearity of recovery from spiked
reagent water and has been demonstrated to be applicable over the
concentration range from 10 x MDL to 1000 x MOL.
14.3 In a single laboratory, Battelle Columbus-Laboratories, using
(. j
spiked waste.^ater samples, the average recoveries presented in
Table 2 were obtained. Seven replicates of each of two different
wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 2.1
32
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U)
CJ
1.2
Rotenone
2.4
I • '
3.0
~r — | —|
6.0
4.8
Retention Time, min.
7.2
"1—r~r» f-i
8.4
T—r
9.0 10.0 12.0
FIGURE 1. IIPLC-UV CIIROHATOGRAM OF STANDARD SOLUTION REPRESENTING 5 M9/L
OF ROTENONE IN WATER (COLUMN 1).
-------
Rotenone
U>
1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time, min.
FIGURE 2. IIPLC-UV CHROMATOGRAM OF STANDARD SOLUTION REPRESENTING 200 Mq/L OF
ROTENONE IN WATER (COLUMN 2).
-------
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Retention Time (min.) Method Detection Limit
Parameter Column 1Column 2- (
Rotenone 8.6 8.0 1.6
Rotenone
Column 1 conditions: Zorbax-CN, 5 micron, 250 x 4.6 mm; 1 mL/min flow; 30/70
methylene chloride/hexane.
Column 2 conditions: Spherisorb-ODS, 5 micron, 250 x 4.6 mm; 1 mL/min flow;
60/40 acetonitrile/water.
35
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TABLE 2. SINGLE LABORATORY ACCURACY AND PRECISION^)
Parameter
Average
Percent
Recovery
Standard
Deviation,
Spike
Level
Number
of
Analyses
Matrix
Type(b)
Rotenone
85
88
8
3
5.5
109
7
7
(a) Column 1 conditions were used.
(b) 1 » __pesticide^manufacturing wastewater
36
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REFERENCES
1. "Development of Methods for Pesticides in Wastewaters," Report for EPA
Contract 68-03-2956 (In preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society
for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health, Education,
and Welfare, Public Health Service, Center for Disease Control, National
Institute for Occupational Safety and Health, Publication No. 77-206,
August, 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Adninistrati on, OSHA 2206 (Revised,
January, 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio 45268,
March, 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia,
PA, p. 76, 1980.
8. Glaser, J. A. et al, "Trace Analysis for Wastewaters," Environmental
Science and Technology. 1_5, 1426 (1981).
37
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
S. REPORT DATE
Determination of Rotenone in Industrial and
Municipal Wastewaters
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J.S. Warner, T.M. Engel, and P.O. Mondron
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battene Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
CBEBIC
11. CONTRACT/GRANT NO.
68-03-2956
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
2/82 - 4/82
14. SPONSORING AGENCY CODE
EPA 600/6
IS. SUPPLEMENTARY NOTES
16. ABSTRACT
A method was developed for the determination of rotenone in wastewaters. The
method development program consisted of a literature review; determination of
extraction efficiency for each compound from water into metnylene chloride;
development of a deactivated- silica gel cleanup procedure; and determination of a
suitable high performance liquid chromatographic (HPLC) method with ultraviolet
(UV) detection.
The final method was applied to a relevant industrial wastewater to determine
the precision and accuracy of the method. The wastewater was spiked with rotenone
at levels of 5.5 wg/L and 110 wg/L. Recovery for rotenone at the 5.5 wg/L level
was 85 * 8 percent. Recovery at the 110 yg/L level was 88 * 3 percent. The method
detection limit (MDL) for rotenone in distilled water was- 1.6'wg/L. The MDL in
wastewaters may be higher due to interfering compounds.
U.S. Environmental Protection
Region 5, Library (PL-12J)
11 West Jackson Boulevard, 12th
Chicago, IL 60604-3590
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
18. DISTRIBUTION STATEMENT
Distribute to Public
19. SECURITY CLASS (Tills Report/
Not Classified
21. NO. Or PAGES
37
20. SECURITY CLASS {Tliis page/
Not Classified
22. PRICE
EPA Form 2220-1 (R«». 4-77) PKCVIOUS SOITION is OBSOLETE
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