Environmental Monitoring Series
Tentative Procedure
Analyzing
Pesticide Residues in
Solid Waste
National Environmental Research Center
Office of Research and Monitoring
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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EPA-R4-72-002
August 1972
Tentative Procedure
Analyzing Pesticide Residues In
Solid Waste
Richard A. Carnes
Solid Waste Research Laboratory
National Environmental Research Center
Cincinnati, Ohio 45268
Program Element 1D2311
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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REVIEW NOTICE
The Solid Waste Research Laboratory of the National Environmental Research
Center, Cincinnati has reviewed this report and approved its publication. Mention of
trade names or commercial products does not constitute endorsement or recommendation
for use.
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ABSTRACT
Because of the concern about pesticide residues in the environment, a procedure to
analyze solid waste samples for pesticides was developed. Nine samples of solid waste
from municipal refuse; incinerator emissions, residue, and fly ash; and compost were
prepared, extracted, and cleaned up with column and thin layer chromatography for
analysis by gas liquid chromatography. The cleanup methods were carefully selected
to yield reproducible results upon final GLC analyses.
When the chromatograms from the solid waste samples were compared with stand-
ards, lindane, o,p'-DDD, p,p'-DDT, chlordane, o,p'-DDE, and p,p'-DDE were identified
as possible contaminants of municipal refuse and the products of its disposal.
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FOREWORD
To find, through research, the means to protect, preserve, and improve our environ-
ment, we need a focus that accents the interplay among the components of our physical
environment-the air, water, and land. The missions of the National Environmental
Research Centers-in Cincinnati, Research Triangle Park, N. C., and Corvallis Ore.~
provide this focus. The research and monitoring activities at these centers reflect multi-
disciplinary approaches to environmental problems; they provide for the study of the
effects of environmental contamination on man and the ecological cycle and the search
for systems that prevent contamination and recover valuable resources.
Man and his supporting envelope of air, water, and land must be protected from the
multiple adverse effects of pesticides, radiation, noise, and other forms of pollution as
well as poor management of solid waste. These separate pollution problems can receive
interrelated solutions through the framework of our research programs-programs
directed to one goal, a clean livable environment.
This publication, published by the National Environmental Research Center, Cin-
cinnati, reports on work from this center. The problems created by pesticide residues in
the environment are of concern to everyone. We, with publication of Tentative
Procedure: Analyzing Pesticide Residues in Solid Waste, offer researchers a starting
point for future research in this particular area of solid waste.
ANDREW W. BREIDENBACH, Ph. D.
Director, National Environmental
Research Center, Cincinnati
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TABLE OF CONTENTS
Page
CONCLUSIONS viii
INTRODUCTION 1
EXPERIMENTAL
A. Sample Preparation 1
B. Extraction 2
C. Cleanup - Column Chromatography 2
D. Cleanup - Thin Layer Chromatography 2
E. Analysis - Gas-Liquid Chromatography 3
DISCUSSION
A. Samples 3
B. Sample Preparation 5
C. Extraction 5
D. Column Chromatography 5
E. Thin Layer Chromatography 5
F. Analysis 5
SUMMARY
A. Sample Preparation 5
B. Extraction 5
C. Cleanup - Column Chromatography 5
D. Cleanup - Thin Layer Chromatography 6
E. Analysis - Gas-Liquid Chromatography 6
ACKNOWLEDGEMENTS 15
REFERENCES 15
VII
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CONCLUSIONS
By employing the tentative procedures developed here to analyze solid waste for
pesticide residues, the results indicated the presence of various pesticides in the solid
waste samples.
With the use of these procedures as a basis for future research, it is hoped that
further refinements will be developed for more exacting systems.
vui
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TENTATIVE PROCEDURE:
ANALYZING PESTICIDE RESIDUES IN SOLID WASTE
INTRODUCTION
Public concern has grown recently over the spread
of pesticides throughout the world. The persistence of
DDT and its metabolites in the ecosystem is the cause
of most concern. Pesticide sales increased 84% in the
United States from 1955 to 1965. The production in
1965 was 875 million pounds, which amounted to $250
million in sales. With the population of the United
States now increasing at the rate of 3 million persons
each year, past experience has indicated that we may
expect a correspondingly large increase in production of
pesticides. This increased production will increase pesti-
cide residues throughout the environment and conse-
quently in solid wastes (1).
The main objective of this project undertaken at
the Solid Waste Research Laboratory of the National
Environmental Research Center, Cincinnati, was the
development of a procedure for the analysis of pesticides
in solid waste samples. The complex and heterogeneous
nature of solid waste samples makes pesticide analysis
an extremely challenging task. The cleanup methods
utilized were carefully selected to yield reproducible
results upon final analysis by gas-liquid chromatography.
EXPERIMENTAL
A. Sample Preparation
Nine solid waste samples were selected for analysis
to determine pesticide residues in the waste and waste
byproducts. Table 1 lists the sample source and size.
After collection, the municipal refuse (Sample 1)
was ground in a large hammermill to approximately a
1- by 1-inch size. Following this, a grab sample was
ground in a Wiley Mill until it could pass through a
2-inm screen. The sample was then dried in a laboratory
oven at 105 C for several hours. After drying, the
TABLE 1. SOLID WASTE SAMPLES ANALYZED
Sample No.
1
2
3
4
5
6
7
8
9
Source
Municipal refuse
Incinerator stack condensate
Trapped gaseous incinerator emissions
Incinerator residue
Incinerator residue
Incinerator fly ash
Incinerator fly ash
0-Day compost
42-Day compost
Amount extracted
50.9 grams
250 ml
300 ml*
90.1 grams
254.0 grams
115.7 grams
87.8 grams
50.1 grams
69.3 grams
*This 300 ml was the collecting agent for almost 19 cubic feet of gaseous
emissions.
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sample was weighed into preextracted soxhlet thimbles
for pesticide extraction.
The condensate and trapped gaseous emissions
(Samples 2 and 3) were obtained from an experimental
high-temperature (maximum temperature of 2,500 F),
low-capacity combustion unit. A sample probe was
placed in the stack, and a portion of the gaseous
emissions was passed through this probe. These emissions
were routed through a series of water-cooled condensers,
then through a gas-diffusion bubbler immersed in ethyl-
ene glycol. The purpose of the ethylene glycol was to
extract any organic material, especially pesticides, that
had been volitalized in the combustion process. Even-
tually a water condensate began building in the con-
densers; this was collected and utilized as a solid waste
processing sample along with the ethylene glycol mate-
rial. These two samples were extracted with the use of
the procedure described by FWPCA(2).
The residue and fly ash samples (Samples 4-7)
were prepared according to the method described by
Cohen and Allen (3) and stored in the laboratory at
room temperature until analyzed. The procedure of
Cohen and Allen involves various sorting, grinding,
quartering, and drying procedures to reduce the sample
volume and particle size to useful working dimensions.
In final treatment, the material used in this study was
pulverized so the particles would pass a 60-mesh sieve.
The compost samples (Samples 8 and 9) were
initially ground, mixed, and dried at 100 C to a con-
stant weight at the compost plant in Johnson City,
Tennessee. On arrival, the large pieces of glass, ceramics,
metals, and rocks were removed manually, and the
samples were then ground in a Wiley Mill until they
could pass a 2-mm screen. All of the compost samples
originated from the same windrow, and each sample
represented a composite of many small grab samples
collected at various sites along the windrow.
B. Extraction
All samples, except 2 and 3, were solid and were
extracted with the use of a soxhlet apparatus that
contained a preextracted 43- by 123-mm thimble that
was attached to a 300-ml, round-bottom flask. The
extracting solvent was 9 parts hexane to one part
acetone. After all of these solid samples were extracted
for at least 12 hours, the solvent was evaporated to
dryness in a vacuum oven at room temperature.
Treatment of samples 2 and 3 involved two ex-
tractions, one with 150 ml of 15% ethyl ether in hexane
and the other with 150 ml of hexane only. These ex-
tracts were combined and then were passed through a
column of anhydrous sodium sulfate and evaporated to
dryness in a vacuum oven at room temperature.
C. Cleanup - Column Chromatography
After evaporation of the extracts, the residues
were redissolved in a small quantity of hexane. A 15-
gram charge of Florisil (which had been stored in an
airtight container at 130 C) was added over a /2-inch
layer of anhydrous sodium sulfate in a 300-mm-long
Pyrex glass column with a 25-mm O.D. After tapping
the Florisil into the column, another layer of sodium
sulfate (%-inch deep) was added to the top. The column
was allowed to cool to room temperature and was pre-
eluted with about 75 ml of hexane. This 75 ml was
discarded, and just prior to exposure of the top layer of
sodium sulfate by the eluting solvent, the sample extract
was quantitatively transferred into the column by
decantation and subsequent hexane washings. The elu-
tion rate was adjusted so that it did not exceed 5 ml
per minute. The complete column cleanup procedure
involved two separate solvent systems: the first elution
was performed with 200 ml of 6% ethyl ether in hexane,
the second, with 200 ml of 15% ethyl ether in hexane(2).
These eluates were collected and evaporated to dryness
for thin layer chromatography cleanup.
D. Cleanup—Thin Layer Chromatography (TLC)
The residues of the evaporated eluates from the
column cleanup were redissolved in 1 ml of hexane for
cleanup by TLC. The TLC system described by Lichten-
stein et at (4) was utilized at the final cleanup prior to
gas-liquid chromatography (GLC). The TLC system
employed activated aluminum oxide plates developed
in a solvent system 99% heptane and 1% acetone.
Standards and at least 100 u\ of the sample were put
on the plate. The 20- by 20-cm plate was then developed
to 15 cm and allowed to air dry. The standards were
then sprayed with a solution of Rhodamine B (O.lmg/ml
in ethanol), with the sample portion being covered so
that it was not sprayed. The sprayed portion was
allowed to thoroughly dry (about 5 minutes) and was
then viewed under short wavelength UV light.
The standards were marked, and the area of
sample corresponding to the area of standard was
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collected from the plate. This material was placed in a
15-ml centrifuge tube and extracted three times with
10-ml portions of hexane. After the hexane was added,
the material was thoroughly mixed and then centrifuged
to yield a clear supernatant. This liquid was collected
and evaporated to 2 ml volume. One ml was removed
from each sample, transferred to a separate vial, evap-
orated to dryness, and sent to the Perrine Primate
Laboratory for GLC analysis. The remaining 1 ml was
analyzed at Analytical Quality Control Laboratory
(1014 Broadway, Cincinnati). Figure 1 shows the TLC
plate for standards and samples.
E, Analysis-Gas-Liquid Chromatography
Four instruments were employed for analysis: A,
B, C, and D.
Instrument A was operated with the following
parameters:
G. C., Barber Colman Series 5000
floor model
Column, 6-foot U-tube, 4 mm I.D.,
glass
Packing, 10% DC-200 on Anakrom
ABS 80/90 mesh
Detector, tritium foil
Gas flow, 100 ml/min N2
Column temperature, 200°C
Detector temperature, 212°C
Injector temperature, 211°C
Instrument B was operated with the following
parameters:
G. C., Micro Tek 160
Column, 8-foot tube, /4-inch O.D.,
aluminum
Packing, 3% DC-200 + 5% QF-1 on Gas
Chrom 0 80/100 mesh
Detector, tritium foil
Gas flow, 80/min N2
Column temperature, 202°C
Detector temperature, 202°C
Injector temperature, 220°C
Instrument C was operated with the following
parameters:
G. C., Micro Tek 220
Column, 6-foot U-tube, 4 mm I. D., glass
Packing, 1.5% OV-17 + 1.95% QF-1 on
Chromosorb W. H. P., 100/
120 mesh
Detector, tritium foil
Gas flow, 100 ml/min N2
Column temperature, 188°C
Detector temperature, 200 C
Injector temperature, 225 C
Instrument D was operated with the following
parameters:
G. C., Micro Tek 220
Column, 6-foot U-tube, 4 mm I. D.,
glass
Packing, Chromosorb W. H. P., 80/100
mesh
Detector, tritium foil
Gas flow, 65 ml/min N2
Column temperature, 195 C
Detector temperature, 205 C
Injector temperature, 225°C
DISCUSSION
A. Samples
The municipal refuse (Sample 1) was a grab
sample from a 10-cu-yd packer truck collecting from
residential sources in St. Bernard, Ohio, during the
summer when lawn clippings are high.
The condensate and trapped gaseous emissions
(Samples 2 and 3) were collected from an experimental,
high-temperature, low-capacity incinerator and repre-
sented approximately 19 cu ft of gaseous emissions
trapped.
The incinerator residue and fly ash samples were
collected while personnel from Federal solid waste
activities evaluated municipal incinerators in Medina,
Pennsylvania (Samples 4 and 6), in Greenwood, South
Carolina (Sample 5), and in New Orleans, Louisiana
(Sample 7).
Samples 8 and 9 came from the joint U.S. Public
Health Service-Tennessee Valley Authority Composting
Project at Johnson City, Tennessee. Both samples came
from the same windrow: Sample 8 represented 0-day
compost, closely resembling raw refuse, and Sample 9
represented the end product of the composting process,
42-day-old compost.
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Area Eluted
Aroclor 1254
Dieldrin
P,P' -DDT r
s /r
tf &
' * l^-
p,p'-DDD
Sample Extract
Figure 1. Thin layer chromatogram of pesticide standards and sample showing
area eluted for further analysis. (Reduced 20%.)
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B. Sample Preparation
For all samples (except 2 and 3), a complex
process of preparation was followed. Care was taken to
produce a homogeneous sample, and the samples were
closely examined to see that large pieces of glass, wood,
metal, ceramics, etc., had been removed before grinding.
The sample used in the laboratory was the end result of
many hours of tedious labor, both in collection and prep-
aration, and was representative of a large volume of
solid waste and solid waste disposal processes.
C. Extraction
A search of the literature yields many possible
extracting solvents, any of which would perform ad-
equately. The 9:1 hexane-acetone solution was selec-
ted because it had previously been used on solid
samples, particularly bottom muds and soil, and because
time did not permit experimenting with various solvents.
D. Column Chromatography
The activated Florisil column was selected after a
column of Florisil :Celite was studied and found un-
acceptable for this project. A review of the literature
indicates activated Florisil is widely accepted for this
E. Thin Layer Chromatography
The immediate goal of the TLC system was to
remove the polychlorinated biphenyls (PCB's) that inter-
fere with pesticide analysis. The selected TLC system did
this and also removed some residual color from the
extracts, along with oils and grease that came through
during the column cleanup step.
A TLC absorbent of silica gel was studied with the
use of standards; it was found that PCB's interfered
with the pesticides because both traveled similar dis-
tances during development.
F. Analysis
An alternative source of final analysis was sought
because of the constant operational problems encoun-
tered with Instrument A. Instruments B, C, and D are
frequently used for pesticide analysis and yield highly
reproducible results. Because the detectors in instru-
ments B, C, and D do not become contaminated as
easily as that of Instrument A, fewer operational
problems are encountered.
Schematically, the sample extraction and cleanup
procedures before GLC are
Soxhlet
extraction
—
Evaporation
Column
Chromatography
Evaporation
TLC
Area
elution
Hexane
extraction
—
Evaporation
—
GLC
SUMMARY
A. Sample Preparation
A method for preparing a large number of solid
waste samples previously investigated by the Solid
Waste Research Laboratory was found to be satisfactory.
B. Extraction
The solvent system of hexane—acetone recov-
ered more than 95% of p,p'-DDT when added to a
refuse sample similar to Sample 1; however, some
research is needed to investigate other systems that
could possibly be oriented to yield an extract lower in
oils and greases that will still yield the desired pesticides.
In the literature, many systems are given for
extracting pesticides. Alternative solvent systems were
not investigated, primarily because of time limitations.
C. Cleanup — Column Chromatography
Although the column cleanups were grossly over-
loaded because of the large amount of extractables
from the soxhlet extractive step, they did remove much
of the color, oil, and grease. We believed that activated
Florisil would suffice here because most investigators of
pesticides use the Florisil system.
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D. Cleanup —Thin Layer Chromatography
Again, time did not permit the extensive investi-
gation of TLC systems. A system, found in the literature,
that separated PCB's from pesticide residues was used
because this was considered the most important function
of this cleanup as far as pesticide analysis was concerned.
Since new systems are continually being added to
the literature, we suggested that in the future other TLC
systems be investigated; they may yield better results
than those obtained in this project.
E. Analysis-Gas-Liquid Chromatography
The last step of the experimental procedure is the
GLC analysis. The need for a good sound instrument
becomes imperative when dealing with pesticide residue
analysis. The chromatograms obtained from instrument B
(Figures 2-4) show the results of a mixed pesticide stand-
ard sample, a chlordane standard, and the results of
0-day compost sample. By comparing the results obtained
from the standards with those from the 0-day compost
sample, we concluded the compost sample contained
lindane, p,p'-DDT, and chlordane. Other peaks were
observed, but identification could not be made at that
time.
When a more sensitive instrument (C) was used
with the same comparison procedure, the results indica-
ted the possible presence of lindane, p,p'-DDE, o,p'-DDE,
p,p'-DDT, and chlordane (Figure 5).
The chromatograms obtained from Instrument D
(Figures 6 and 7) show the many chlordane peaks
(Figure 6) and the many peaks obtained from the 0-day
compost. Some possible identifications in Figure 7 are
lindane, o,p'-DDD, p,p'-DDT, and chlordane. Once again,
the identifications .were made by comparing pesticide
standards.
Instrument A failed to function during most of
this project; thus, the final analyses were performed on
instrumentation better suited for pesticide analysis.
The choice of columns and column packing
(liquid phase and solid support) is very important for
accurate separation of pesticide residues; therefore, we
recommend that more research be conducted in this
particular phase of final analysis.
The results of all the chromatographic findings are
shown in Tables 2, 3, and 4. As indicated in these
tables, no pesticides of any significance were identified
from the types of samples analyzed. Table 2 lists
standards for the several pesticides run on each instru-
ment. The calculated relative retention time for each
standard relative to an aldrin standard, the various
samples, and the instrument that detected the possible
presence of each pesticide are also listed.
Tables 3 and 4 list, by instrument and sample,
which pesticides were indicated from the chromatograms
obtained from each instrument for each sample. As can
be seen, at least two of the three instruments indicate
the presence of chlordane for several samples. The
standard for chlordane has peaks that can interfere with
other pesticides and thus complicate the interpretation
of the chromatogram. Table 4 indicates the presence of
dieldrin for several samples, but the concentration of this
compound was in trace amounts in most cases. Quanti-
tation of the observed compounds was not performed.
The procedures presented here can serve as a
starting point for future research concerning pesticides
in solid waste. When analyzing for pesticides with these
procedures in the future, however, the literature should
be further screened for TLC systems and solvent systems
for extracting pesticides.
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Ll
Lindane
Figure 2. Chromatogram of mixed pesticide standards using Instrument B. Final
concentration was 0.2 ng for lindane, aldrin, heptachlor epoxide, and
o,p'-DDE; 0.14 ng for o,p'-DDT; and 0.64 ng for p,p'-DDT. (Reduced
20%.)
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Figure 4. Chromatogram of 0-day compost from Instrument B showing evidence
of lindane, chlordane (see multiple peaks in Fig. 3), and p,p'-DDT.
(Reduced 20%.)
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Retention time (mm)
Figure 5. Chromatogram of 0-day compost from Instrument C showing evidence
of lindane, p,p'-DDE, o,p'-DDE, p,p'-DDT, chlordane. See Table 2.
(Reduced 20%.)
10
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Retention time (mm)
Figure 7. Chromatogram of 0-day compost from Instrument D showing
evidence of lindane, o,p'-DDD, p,p'-DDT, and chlordane. See Table 2.
(Reduced 20%.)
12
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TABLE 2. RA* VALUES FOR PESTICIDE STANDARDS AND SAMPLES
Standard
and
samples
Standard
Sample 1A^
2A
3A
4A
5A
6A
7A
8A
9A
and
samples
Standard
Sample 1A'
2A
3A
4A
5A
6A
7A
8A
9A
Pesticide
Lindane o,p'-DDE
Instrument Instrument
BCD BCD
0.58 0.68 0.57 1.85 1.91 1.53
1.91
1.91 1.51
1.85 1.94 1.50
1.85 1.51
1.91
0.58 0.69 0.57 1.94
1.94 1.51
Pesticide
o,p'-DDD p,p'-DDT
Instrument Instrument
BCD BCD
2.84 1.98 3.22 4.55 3.19
1.94
1.98
3.22 4.55 3.19
1.96
1.94 3.17 3.15
1.98
1.94 3.18 4.55 3.26
1.94
p,p'-DDE
Instrument
BCD
2.36 1.83
1.89
1.84
2.36 1.83
1.82
2.36 1.82
2.36
Dieldrin
Instrument
B C
2.15 2.54
IBti" 2.16 2.53
2B
3B 2.15
4B 2.15 2.52
5B 2.15
6B
7B
8B 2.54
9B 2.52
D
2.16
2.15
2.16
2.17
2.17
2.19
2.20
2.14
2.19
*RA> Relative Retention Time (relative to aldrin).
^From 6% fraction.
15% fraction.
13
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TABLE 3. PESTICIDES INDICATED FROM THE 6% FRACTION* BY VARIOUS INSTRUMENTS*
Instrument
Sample B
1A chlordane
2A chlordane (trace)
3A
4A chlordane, p,p'-DDT
o.p'-DDE
5A chlordane, o,p'-DDE
6A chlordane
7A chlordane
8A chlordane, lindane
p,P'-DDT
9A
C*
o,p'-DDE (trace)
o.p'-DDE (trace)
p,p'-DDT, o,p'-DDE
P,p,-DDE
chlordane
o.p'-DDE (trace)
p,p'-DDE
lindane
p,p'-DDE, p,p'-DDT
o.p'-DDE
DDA(ME) [possible]
D
chlordane, p,p'-DDE
p,p'-DDE, o,p'-DDD,
o.p'-DDE
o,p'-DDD, chlordane
chlordane, p,p'-DDT,
o,p'-DDE
chlordane, o,p'-DDD
p,p'-DDE
chlordane, p,p'-DDE
o,p'-DDD, p,p'-DDT
chlordane, o,p'-DDD
chlordane, o,p'-DDD
p,p'-DDT, lindane
o,p'-DDD (trace)
o,p'-DDE
*First eluate with 200 ml of 6% ethyl ether in hexane.
'The R A values did not correspond for instrument D as well as they did for instruments B
and C. Therefore, there remains some doubt as to positive identification.
tNo chlordane standard run on this instrument.
TABLE 4. PESTICIDES INDICATED
FROM THE 15% FRACTION* BY VARIOUS INSTRUMENTS
Instrument
Sample
IB
2B
3B
4B
5B
6B
7B
8B
9B
B
dieldrin
dieldrin (trace)
dieldrin
dieldrin (trace)
C D
- - - - dieldrin
dieldrin
dieldrin
dieldrin
dieldrin
dieldrin dieldrin
dieldrin
^Second eluate with 200 ml of 15% ethyl ether in hexane.
14
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REFERENCES
1. Floyd, E. P. Occurrence and Significance of
Pesticides in Solid Waste. Bureau of Solid Waste
Management (1970).
2. FWPCA Method for Chlorinated Hydrocarbon
Pesticides in Water and Wastewater. U. S. Depart-
ment of the Interior (1969).
3. Cohen, I. R. and Allen, R. L. Sampling and Sample
Preparation of Solid Refuse and Incinerator Resi-
dues. To be published.
4. Lichtenstein, E. P., Schultz, K. R., Fuhremann,
T. W., and Liang, T. T. Biological interaction
between plasticizers and insecticides. Journal of
Economic Entomology. 4:761-765 (1969).
ACKNOWLEDGEMENTS
The author wishes to acknowledge the Office of
Water Quality, Organic Section, 1014 Broadway, Cin-
cinnati, Ohio, for permitting the use of their Micro Tek
160 Gas Chromatograph (Instrument B); S. Poznanski
of the Perrine Primate Laboratory, Perrine, Florida, for
his analysis of the final extract using instrumentation
available to him (Instruments C and D); J. U. Doerger
for technical assistance during the progress of this
project; Dr. D. F. Bender and H. Johnson for their
encouragement and contributions to this project; I. R.
Cohen for his preparation of residue and fly ash
samples; and C. Wiles for securing, preparing, and
forwarding the compost samples to this office.
15
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UNITED STATES
ENVIRONMENTAL PROTECTION AGENCY
NATIONAL ENVIRONMENTAL RESEARCH CENTER
Cincinnati, Ohio 45268
OFFICIAL BUSINESS
PENALTY FOR PRIVATE USE. S3OO
AN EQUAL OPPORTUNITY EMPLOYER
POSTAGE AND FEES PAID
U.S. ENVIRONMENTAL PROTECTION AGENCY
EPA-335
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