Environmental Monitoring Series
A QUANTITATIVE METHOD FOR
TOXAPHENE BY GC-CI-MS
SPECIFIC ION MONITORING
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, Georgia 30601
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL MONITORING series.
This series describes research conducted to develop new or improved methods
and instrumentation for the identification and quantification of environmental
pollutants at the lowest conceivably significant concentrations. It also includes
studies to determine the ambient concentrations of pollutants in the environment
and/or the variance of pollutants as a function of time or meteorological factors.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/4-76-010
March 1976
A QUANTITATIVE METHOD FOR TOXAPHENE BY
GC-CI-MS SPECIFIC ION MONITORING
by
Alfred D. Thruston, Jr.
Analytical Chemistry Branch
Environmental Research Laboratory
Athens, Georgia 30601
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
ATHENS, GEORGIA 30601
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DISCLAIMER
This report has been reviewed by the Athens Environmental
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or
recommendation for use.
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ABSTRACT
A method was developed for the identification and
quantification of toxaphene using a Specific Ion Monitoring
(SIM) program with GC-CI-MS. Interferences from DDT's and
Arochlor 1260 are eliminated or minimized. GC-CI-MS was
also used to distinguish toxaphene from strobane.
111
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CONTENTS
Page
List of Figures vi
I. Introduction 1
II. Conclusions 3
III. Materials and Methods 4
IV. Experimental and Discussion 6
V. References 19
VI. Appendix 20
v
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LIST OF FIGURES
No. Page
1. CI Mass Spectrum of 2,4,2',5' tetrachlorobiphenyl 7
2. (a) Toxaphene RGC (b) Spectrum of C,QH Cl type
(c) Spectrum of C10HXC19 type. 8
3. Standard Curve of Toxaphene Before Cleaning MS
Ion Source, 10
4. (a) SIM program Output, 0.3 ng Internal Standard
TCB, 15 ng Toxaphene (b) SIM Program Output, 0.3
ng Internal Standard TCB, 5 ng Toxaphene. 11
5. Standard Curve of Toxaphene After Cleaning MS Ion
Source. 12
6. New Orleans Water Extract SIM Program Output. 13
7- (a) Toxaphene + DDT's RGC (b) Toxaphene + DDT's
LMRGC at 307 m/e (c) Toxaphene Standard, LMRGC
at 307 m/e. 15
8. SIM Program Output, 0.3 ng Internal Standard
TCB, 5 ng Toxaphene plus 5 ng Arochlor 1260. 16
9. Toxaphene and Strobane MS Output. 18
10. Real Time Plot of SIM Program. 21
VI
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SECTION I
INTRODUCTION
Toxaphene is one of the most widely used chlorinated
hydrocarbon insecticides in the United States.1 Although it
is very effective in controlling insects in agriculture, it
also creates hazards in the environment. Several studies
are reported in the literature concerning the effects of the
pesticide on aquatic life. Recently, ng-per-liter levels of
toxaphene were shown to cause a spine defect in fish.2
Accurate quantitative methods for toxaphene analysis are
therefore needed to determine its persistence in the aquatic
environment.
Toxaphene is manufactured by chlorinating camphene, which
results in a complex mixture of approximately 177
chlorinated isomers.3 The overall average elemental
composition is C10Hi0Cl8 with the major components being
hepta-, octa-, and nonachlorobornanes. Gas chromatography
(GC) has been regarded as the most useful technique for
toxaphene residue analysis. Analysis for this complex
mixture, however, is often subject to interferences from the
pesticides DDE, TDE, or DDT or from the very common
polychlorinated biphenyls (PCB's, trade name Arochlors),
which often make necessary extensive clean-up of the extract
before GC analysis.1*'5
Gas chromatography-mass spectrometry (GC-MS), is a valuable
technique in pesticide analysis. Although in the electron
impact (El) mode, GC-MS is generally insensitive to trace
amounts of toxaphene, studies by Holmstead, et a1.3 and
Stallings and Huckins6 show that the chemical ionization
(CI) mode with GC-MS is particularly applicable to the
analysis of toxaphene, especially using Limited Mass
Reconstructed Gas Chromatograms (LMRGC) for identification.
The Specific Ion Monitoring (SIM) program, a limited mass
data acquisition program developed at Battelle Columbus
Laboratories for EPA,7 also increases the sensitivity of GC-
MS detection. It therefore is an additional useful
technique to be used along with the standard System 150
LMRGC. The program, evaluated at the Environmental Research
Laboratory (formerly Southeast Environmental Research
Laboratory) was found to be applicable to pesticide residue
analysis.8
A study was therefore undertaken 1) to develop a sensitive
quantitative GC-MS procedure (in the CI mode) using the SIM
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program for determination of toxaphene residues in
environmental samples, 2) to eliminate or minimize
interferences common in GC toxaphene analysis, such as the
DDT family or PCB's, and 3) to provide a way of
differentiating toxaphene from the similar pesticide
strobane. In parallel with this study, the Methods
Development and Quality Assurance Research Laboratory in
Cincinnati agreed to investigate the El mode of GC-MS for
toxaphene detection using LMRGC.
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SECTION II
CONCLUSIONS
This method using the Specific Ion Monitoring (SIM) program
with GC-CI-MS is rapid and accurate for the identification
and quantification of toxaphene. Interferences from DDT's
and Arochlor 1260 are eliminated or minimized. Toxaphene
can be distinguished from strobane by utilizing GC-CI-MS
techniques.
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SECTION III
MATERIALS AND METHODS
INSTRUMENTATION
Mass spectral data were obtained with a Finnigan 9500 gas
chromatograph and 1015D chemical ionization quadrupole mass
spectrometer (GC-CI-MS) equipped with a continuous dynode
multiplier and operated with the following conditions: 70 eV
electron energy, 10~7 sensitivity range, 500 mA ionizing
current, and a 2000 V electron multiplier. The
chromatograph column was a 60 cm x 2 mm (I.D.) glass column
packed with 3% SP2100 on 80/100 Supelcon AW. The methane
carrier gas, which also served as the reagent gas, was
adjusted to give a pressure of 1.0 torr in the ion source
(about 20 ml/min flow through the column). The column was
programmed from 160° to 250°C at 10°/min for each run.
SAMPLE PREPARATION
A stock solution of pesticide reference grade toxaphene (1
yg/yl) and one of the internal standard 2,4,2',5'
tetrachlorobiphenyl (TCB) (1 yg/yl) were prepared. From
these stock solutions, five mixtures were prepared,
representing three ratios of toxaphene to TCB.
Solution
1
2
3
4
5
Solution Concentration
Toxaphene
30
20
10
7.5
2.5
(ng/yl)
TCB
0.6
0.6
0.6
0.15
0.15
Toxaphene :TCB
Ratios
50:1
33:1
17:1
50:1
17:1
A standard solution (1 yg/yl) of pesticide reference grade
strobane in isooctane was also prepared.
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DATA ACQUISITION ON SIM PROGRAM
Data acquisition for the SIM program is performed as
described by Alford.8
Data acquisition parameters are specified by teletype
communications between analyst and computer. Two m/e values
for the internal standard TCB are entered as one set to be
monitored, and integration times in milliseconds for that
set are specified. Another set of m/e values and an
integration time for toxaphene are entered as a second set
to be monitored (initiated by typing "S2").
The "NO. POINTS" prompt specifies the number of times each
set of masses is to be monitored before the computer adds
the acquired data and stores the sum.
For a more detailed explanation of the procedure, see
Appendix.
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SECTION IV
EXPERIMENTAL AND DISCUSSION
The SIM program and standard System 150 were used to obtain
data for the analysis for toxaphene, showing the effects of
various interfering compounds and backgrounds. The 60-cm GC
column was chosen to condense the range of toxaphene
retention times for ease in quantification, while retaining
the characteristic "toxaphene pattern" familiar to pesticide
gas chromatographers. The analysis takes 9 minutes.
Tetrachlorobiphenyl was chosen as the internal standard
since it elutes just before toxaphene and its CI spectrum
(Figure 1) contains none of the masses of the early eluting
toxaphene peaks. To keep the chart speed the same
throughout the run, two masses were selected to be
monitored. One of these masses should provide the strongest
signal in the spectrum since the entire run is normalized to
the strongest signal. For TCB, masses 291, 293, 295, or 321
are the possibilities. The TCB concentration may then be
adjusted so that a chosen peak is the most intense peak in the
spectrum.
Several compounds that eluted after toxaphene, including
mirex and decachlorobiphenyl, were also considered as
internal standards; however reproducable intensities for
these compounds could not be obtained.
The CI-MS of toxaphene is characterized by the major
fragments [M-C1]+, [ M-Cl-HCl]+, and [ M-C1-2HC1 ] + - These ion
clusters, which reflect the substitution patterns of the
toxaphene chlorine isomers, are strongest centering around
m/e 235, 271, 307, 343* 377, and 413. Figure 2 shows a
reconstructed gas chromatogram (RGC) of toxaphene and two
typical mass spectra. Masses 307 and 343 were chosen to be
monitored by the SIM program because of their intensities
and the absence of background interferences at these masses.
These two masses also retain the familiar "toxaphene
pattern" better than the 377 m/e, which also satisfies the
interference and sensitivity criteria.
After GC conditions and the ions to be monitored were
chosen, SIM program parameters were optimized. The most
quantitative data were obtained when 2 internal standard
masses and 2 toxaphene masses were monitored for 200 msec
integration time at each mass. Three points were added
before the sum of intensities was stored.
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TETRRCHJtHQBIPHENVL
8 233
291
E>
.r..
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1SB ISO 178
8.
8.
Spectrum ^46
(b)
5
8.
8.
tsR.
Spectrum ^105
i .1 i
138 280 218 Z2B Z38 Z18 2EB 2SB ZTB 288 298 308 310 S8 338 3K) 3S8 360 378 380 398 ton 110 120 130
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The average of -the peak heights of the two highest toxaphene
peaks of each mass run gave the most reproducable
quantitative results. Both the m/e 307 and m/e 343 curves
gave similar intensities and either can be used for
quantification. In practice, the m/e with least background
interference should be used.
Two sensitivity ranges were observed for toxaphene analysis
corresponding to a normal and a freshly cleaned mass
spectrometer ion source. Figure 3 shows a standard curve
for toxaphene using 6 consecutive injections (2 yl) of 30
ng/yl solution, 5 of 20 ng/yl, and 4 of 10 ng/yl. All
instrumental conditions were kept as constant and optimum as
possible.
After this series was done, the mass spectrometer ion source
was taken apart and cleaned (a heavy load of samples caused
a gradual decrease in sensitivity). After cleaning, the
sensitivity of the mass spectrometer increased by about a
factor of 4. Figure 4(a) shows the SIM program output
(after cleaning) for 15 ng toxaphene and Figure 4 (b) shows
the output for 5 ng toxaphene. Figure 5 shows a standard
curve for toxaphene at this new sensitivity using 6
consecutive injections (2 yl) of 7.5 ng/yl solution and 4 of
2.5 ng/yl.
NEW ORLEANS DRINKING WATER SAMPLE
The New Orleans Drinking Water Survey was undertaken in July
1974 to determine the organic compounds present in the
finished water of the Carrollton Water Plant (city of New
Orleans).9 Eighty organic compounds were identified and
quantitated in the 0.05 to 10 yg/1 range. The carbon-
chloroform extract from this survey was chosen as an ideal
environmental sample to spike with toxaphene for testing the
applicability of the GC-CI-MS technique for toxaphene
determination.
Figure 6(a) shows the output for a 2 yl injection of the New
Orleans Carrollton Plant Water carbon-chloroform extract
concentrate (1 yl extract = 25 ml watetf. Figure 6(b) shows
the output for 2 yl of the New Orleans extract spiked with 5
ng toxaphene (equivalent to 0.1 yg/1 in the original water).
Figure 6 (c) is the output for 2 yl of the New Orleans
extract spiked with 15 ng toxaphene (equivalent to 0.3
yg/1). At the 5 ng toxaphene level the 307 m/e scan is
obscured by background; however, the 343 m/e scan is quite
recognizable as toxaphene (compare with Figure 4 (b)). At the
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0
Range 307 m/e
*~~,~ Rouge 343 m/e
Figure 3,
40
TOXAPHENE
Standard curve of toxaphene before cleaning
mass spectrometer ion source
10
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a
291
2SS
b
313
Figure 4. (a) SIM program output, 0.3 ng internal std,
TCB, 15 ng toxaphene
(b) SIM program output, 0.3 ng internal std,
TCB, 5 ng toxaphene
11
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Range 307 m/e
•—-~- Range 343 m/e
5 10
ng TOXAPHENE
Figure 5. Standard curve of toxaphene after cleaning
mass spectrometer ion source
12
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a
b
Figure 6. (a) New Orleans Drinking Water Extract.
1:25,000 CHCL3 solution plus 0.3 ng
standard TCB
(b) New Orleans Drinking Water Extract.
1:25,000 CHCl., solution plus 0.3 ng
standard TCB plus 5 ng toxaphene
(c) New Orleans Drinking Water Extract.
1:25,000 CHC13 solution plus 0.3 ng
standard TCB plus 15 ng toxaphene
2 yl of
internal
2 yl of
internal
2 yl of
internal
13
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15 ng toxaphene level, the toxaphene is apparent in both the
307 m/e and 343 m/e scans (compare with Figure 4 (a)).
This extract sample was not cleaned up. If regular
pesticide clean-up procedures were applied to the extract,
even lower limits of detection probably could be achieved.
INTERFERENCES—DDE, TDE, DDT, AND PCB'S
DDE, TDE, and DDT are common interferences in the GC
analysis for toxaphene. Figure 7 (a) shows the GC-CI-MS
output for a mixture of toxaphene, DDE, TDE, and DDT at
relative concentrations of 10:1:1:1. The toxaphene is
hardly recognizable. A gas chromatogram using electron
capture as a detector would be similar. Using the SIM
program, the DDT family would not interfere with toxaphene
analysis since m/e 307 and m/e 343 are not in the CI
spectrum of the DDT's. Figure 7 (b) shows that an LMRGC of
307 m/e excludes DDE, TDE, and DDT (compare with Figure
7(c), the LMRGC of a pure toxaphene standard at 307 m/e).
Similarly with the SIM program Arochlor 1260 does not
interfere. Figure 8 shows the SIM program output for a
mixture of 5 ng toxaphene and 5 ng Arochlor 1260. The
presence of the Arochlor 1260 changes the baseline slightly
(compare with Figure 4(b)), but the shape of toxaphene is
still recognizable. Any of the lower Arochlors (1254, 1248,
etc.) would interfere with quantitation since they contain
the internal standard TCB.
DISTINGUISHING TOXAPHENE FROM STROBANE
The pesticide strobane has long been difficult to
differentiate from toxaphene, although it is not as widely
used as toxaphene. Toxaphene is manufactured by
chlorination of camphene to a chlorine content of 67-69%.
Strobane is manufactured by chlorination of a mixture of
terpenes (mostly pinene) to a content of about 66% chlorine.
TOXAPHENE
STROBANE
14
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k
(7 10 20 30
SPOTTFLM
SO 60 78 B8 30 100 110 120 130 110 ISO ISO 170
R_
0 10 23 30 "K3 SO 60 70 88 X 108 110 120 130 1« ISO 168 170
0 10 ZO 30 to SO SO 70 BO 30 100 110 1ZB 139 IKS 1SS 168 17B
Figure 7. (a) Toxaphene + DDT's. RGC
(b) Toxaphene + DDT" s. LMRGC at 307 m/e
(c) Toxaphene Standard. LMRGC at 307 m/e
15
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Figure 8.
SIM Program Output, 0.3 ng Internal Standard TCB,
5 ng Toxaphene plus 5 ng Arochlor 1260
16
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Both pesticides contain many isomers of the same molecular
formula and therefore cannot be separated or distinguished
easily from each other.
Chemical ionization LMRGC offers a means for distinguishing
the two pesticides. Figure 9 shows the LMRGC's of toxaphene
and strobane at 6 specific m/e's, superimposed on the RGC's
of both. This gives a characteristic pattern for
identifying or distinguishing the two pesticides.
17
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(a) TOXAPHENE
(b) STROBANE
Figure 9. (a) Toxaphene LMRGC at 235,271,307,343,377, and
413 m/e. Shaded area is RGC
(b) Strobane LMRGC at 235,271,307,343,377, and
413 m/e. Shaded area is RGC
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SECTION V
REFERENCES
1. Guyer, G. E., P. L. Adkisson, K. DuBoiSy C. Menzie^ H.
P. Nicholson, G. Zweig, C. I. Dunn, Toxaphene Status
Report (Environmental Protection Agency* Washington,
B.C. , 1971) .
2. Chemical and Engineering News 53_s 30,, April 21, 1975.
3. Holmstead, R. L., S» Khalifa, and J. Casida. Toxaphene
Composition Analyzed by Combined Gas Chromatography--
Chemical lonization Mass Spectrometry. J. Agr. Food
Chem. 22, 939 (1974) .
4. Klein, A. K. and J. D. Link. Elimination of
Interferences in the Determination of Toxaphene
Residues. JAOAC 53g 524 (1970).
5. Stalling, D. L. and J. N. Huckins. Silicic Acid PCB-
Pesticide Separation Method. PCB Newsletter _1_-3, March
1972.
6. Stalling, D. L., and J. N. Huckins. Analysis and Gas
Chromatography-Mass Spectrometry Characterization of
Toxaphene in Fish and Water. USDA Fish and Wildlife
Service, Columbia, Missouri. Environmental Protection
Agency, Contract No. EPA-IAC-0153(D) (1975).
7. Neher, M. B., and J. R. Hoyland. Specific Ion Mass
Spectrometric Detection for Gas Chromatographic
Pesticide Analysis. Battelle Columbus Laboratories,
Columbus, Ohio. Environmental Protection Agency
Publication Number EPA-660/2-74-004, January 1974.
8. Alford, A. L. Evaluation of a Computer Program for GC-
MS Specific Ion Monitoring. Southeast Environmental
Research Laboratory, Athens^ Georgia. Environmental
Protection Agency Publication Number EPA-660/2-74-002.
June 1974.
9. Draft Analytical Report, New Orleans Area Water Supply
Study. Prepared and Submitted by Lower Mississippi
River Facility, Slidell, Louisiana, Surveillance and
Analysis Division, Region VI. US Environmental
Protection Agency, Dallas, Texas, November 1974.
Environmental Protection Agency Publication Number EPA-
906/10-74-002.
19
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SECTION VI
APPENDIX
PROCEDURE
The data aquisition is set up. The sample (2 TJ!) is
injected into the GC operating isothermally at 160°C. After
the solvent elutes (about 1 minute), the data program is
initiated and the GC temperature program of 10°/min is
initiated. The real time plot (Figure 10) appears on the
plotter; the program monitors masses 291 and 295 of TCB,
with only mass 291 showing on real time plot. Immediately
after the TCB elutes, S2 is initiated manually by teletype
and the next set of masses for toxaphene is monitored until
the end of the run (only mass 307 showing on a real time
plot). After the data acquisition is halted, data for all
masses are plotted. These plots are normalized to
the most intense signal (the internal standard). A standard
curve should be set up keeping the internal standard the
same, and varying the toxaphene. Quantitative values are
plotted using peak heights by averaging the two highest
peaks in the toxaphene plot.
20
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TCB
Figure 10.
Real time plot of 0.3 ng TCB plus 15 ng
toxaphene. 52 is initialed manually
immediately after the TCB peak
21
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-GOO/4-76-010
3. RECIPIENT'S ACCESSIOI>NO.
4. TITLE AND SUBTITLE
A Quantitative Method for Toxaphene by GC-CI-
MS Specific Ion Monitoring
5. REPORT DATE
March 1976(Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Alfred D. Thruston, Jr.
9. PERFORMING ORG \NIZATION NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, Georgia 30601
10. PROGRAM ELEMENT NO.
1BD612
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF REPORT AND PERIOD COVERED
In-house
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A method was developed for the identification and quantification of
toxaphene using a Specific Ion Monitoring (SIM) program with GC-CI-MS.
Interferences from DDT's and Arochlor 1260 are eliminated or minimized,
GC-CI-MS was also used to distinguish toxaphene from strobane.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Pesticides, Mass Spectrometry,
b.IDENTIFIERS/OPEN ENDEDTERMS
*Toxaphene, *Pesti-
cide analysis,
Analytical technique
Chlorinated pesti-
cides
c. COSATI Field/Group
05A
3. DISTRIBUTION STATEMENn
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
28
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
22
- U. S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5383 Reg ion No. 5-I I
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